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Egypt's 1st post-Mubarak election to begin Nov. 28

CAIRO (AP) — Egypt's ruling military decreed on Tuesday that the country's first parliamentary elections since Hosni Mubarak's ouster will begin Nov. 28, ending months of speculation on the timing of the key vote.

The elections for parliament's two chambers will be staggered over several months, with the vote for the People's Assembly starting Nov. 28 and the less powerful Shura Council, the chamber's upper house, on Jan. 29. The announcement by the ruling military council, which took over from Mubarak in February, was carried on the state news agency and television.

The last parliamentary election under Mubarak was held in November and December last year, when the ousted …

It's a Wonderful Life

---

The film It's a Wonderful Life (1946) has become an important part of the Christmas season for millions of Americans. Each year, families gather around the television (see entry under 1940s—TV and Radio in volume 3) to watch the story of George Bailey (James Stewart, 1908–1997), a man who grew up yearning for a life of travel and adventure. Personal and national events thwarted George's plans to escape his home-town of Bedford Falls. He grows into one of the community's leading citizens as he runs the family Building and Loan and repeatedly tangles with Mr. Potter (Lionel Barrymore, 1878–1954), the town's miserly, mean-spirited …

Let's Do Our Part

PRESIDENTS LETTER

EVENTS IN WASHINGTON FAVOR ENGINEERING.

NEW FACULTY MEMBERS, and even some longtime members of ASEE, see the Society as an organization that focuses mostly on pedagogy and teaching methodologies. This impression makes ASEE appear to be useful only to those who want to concentrate on teaching. While ASEE certainly strives to nurture better educators, it offers many more opportunities to help members advance professionally and improve the fields of engineering and engineering technology. One avenue is our active involvement in public policies affecting federally funded research and education at all levels in science, technology, engineering, and mathematics, …

Copa Libertadores: Sao Paulo, Atletico Nacional, Nacional take last spots in last 16

Brazil's Sao Paulo, Colombia's Atletico Nacional and Uruguay's Nacional _ all former champions _ filled the last berths for the Copa Libertadores second round on Wednesday.

Sao Paulo beat Atletico 1-0 to win Group 7, and the Colombian side still advanced as the second-placed team as Chile's Audax Italiano squandered a chance to vault Atletico by losing to Sportivo Luqueno 4-1 in Asuncion.

Nacional claimed the winner-takes-all match against Peru's Cienciano with a 3-1 victory in Montevideo. It finished second in Group 4 to Flamengo, which had already qualified and defeated Peru's visiting Coronel Bolognesi 2-0.

In the round of 16 starting next …

Aretha Franklin cancels Brown U appearance

Brown University officials say Aretha Franklin will miss the school's commencement because of a family emergency.

The university announced Wednesday that a representative for the "Queen of Soul" called to say a family emergency would keep her from traveling to …

MORNINGLINE RESULTS

Yes: 70% No: 30%

The Question: Will the Bulls win their third consecutive …

German Football Summaries

BERLIN (AP) — Summary of Tuesday's quarterfinal in the German Cup (home team listed first):

Schalke 3, Nuremberg 2 (after extra …

Text of Obama's Nobel Peace Prize speech

The text of President Obama's Nobel Peace Prize acceptance speech, delivered Thursday in Oslo, Norway, as provided by the White House:

Your Majesties, Your Royal Highnesses, Distinguished Members of the Norwegian Nobel Committee, citizens of America, and citizens of the world:

I receive this honor with deep gratitude and great humility. It is an award that speaks to our highest aspirations _ that for all the cruelty and hardship of our world, we are not mere prisoners of fate. Our actions matter, and can bend history in the direction of justice.

And yet I would be remiss if I did not acknowledge the considerable controversy that your …

Notre Dame stops St. Joe in overtime

Kevin Clancy knows some people may put an asterisk next to Fridaynight's win over St. Joseph because Mark Treadwell missed the gamewith an injured back.

But the Notre Dame senior can handle the skeptics. In fact, heand his teammates have been doing that all season.

After losing four starters from an Elite Eight squad, the Donswere supposed to be humbled by a powerful Chargers team with twomarquee transfers.Instead, No. 23 Notre Dame (5-1, 2-0) is more confident thanever after a 45-42 overtime victory over visiting No. 4 St. Joe's(3-2, 1-1) in East Suburban Catholic action."They were picked to go 13-0 in conference," Clancy said. "Iknow people might take …

Honduras beats Costa Rica on PKs in Gold Cup

EAST RUTHERFORD, N.J. (AP) — Facing a penalty kick with 15 minutes left in regulation and the game tied, Honduras' Noel Valladares was willing to do anything to distract Alvaro Saborio of Costa Rica — so the goalkeeper argued about the placement of the ball.

He argued so much, referee Roberto Moreno of Panama gave him a yellow card. The delay worked.

Valladares made a diving stop on Saborio's penalty kick in the 75th minute and Honduras advanced to the Gold Cup semifinals by making all four of its tiebreaker penalty kicks in a quarterfinal victory Saturday night.

"I've felt the emotions of scoring a goal for my country, but you can't compare it to stopping a PK," …

US, Sri Lanka sites added to World Heritage list

An ocean preserve in Hawaii and Sri Lanka's central highlands are the latest additions to UNESCO's World Heritage List.

The U.N. Educational, Scientific and Cultural Organization cites Papahanaumokuakea Marine National Monument for its significance to native Hawaiian culture.

A statement Saturday says …

Swedish group to challenge for America's Cup

A Swedish syndicate with two Americans in key roles has confirmed it will challenge for the America's Cup in 2013.

Artemis Racing, backed by the Royal Swedish Yacht Club, has scheduled a news conference for Monday in Stockholm to announce its challenge.

Artemis Racing's CEO is Paul Cayard, who lives near San Francisco and is a veteran of the America's Cup, Olympics and round-the-world races. In 1998 he became the first American skipper to win the Whitbread Round the World Race.

Artemis' helmsman is Terry Hutchinson, who was the tactician for Team New Zealand in the 2007 America's Cup, won 5-2 by Alinghi of Switzerland.

Organizers are considering bids from San Francisco, Valencia, Spain, and Italy to host the 34th America's Cup.

Knee injury knocks Wendel out for rest of season

Bordeaux midfielder Wendel will miss the rest of the season after injuring his right knee in last weekend's French League Cup final victory.

The Brazilian underwent exams Monday that showed he picked up a knee sprain that will sideline him between "six and eight weeks," the club said in a statement.

Wendel scored five goals this season in all competitions and helped Bordeaux climb to second place in the French league standings. Bordeaux trails leader Marseille by five points with one game in hand.

Bordeaux won the League Cup for the third time last Saturday, defeating second division club Vannes 4-0.

Owen says Newcastle hasn't offered new contract

Michael Owen responded to speculation that he will leave Newcastle when his contract expires at the end of the season by claiming that the Premier League club has yet to offer him a new deal.

Britain's Sunday papers speculated that Owen could join clubs including Everton and Liverpool after Newcastle manager Joe Kinnear had said that he expects the striker to reject a new contract.

But Owen, speaking after he scored the opening goal in Sunday's 3-0 win at Newcastle, said the club had not yet tried to avoid him leaving on a free transfer.

"There's quite a few players who are running out of their contracts," Owen said. "None of us have been offered a contract so there's no bad guys here. I've not refused anything."

Striker Shola Ameobi, midfielder Nicky Butt and goalkeeper Steve Harper are also out of contract at the end of the season.

5to Festival de la Cultura Colombiana Rinde homenaje a las Mujeres

5to Festival de la Cultura Colombiana Rinde homenaje a las Mujeres

Las mujeres Colombianas -ayer, hoy y manana. Bajo este tema MOVING BEYOND Productions presenta el 5to Festival de la Cultura Colombiana, los dias 14, 15 y 16 de Septiembre. en el CoweIl Theater, Fort Mason Center, San Francisco. El Festival contara con la presencia del Grupo de Danzas Etnicas Colombianas (Directora: Beatriz Restrepo) con una obra original de danza-teatro, creacion colectiva de un elenco compuesto por miembros de la comunidad. A la vez queda incluida una exhibicion de artes visuales con obras de artistas Colombianos, artesanias y sabrosa comida Colombiana. El lobby abrira a las 6pm el viernes y el sabado y a las 3pm el domingo. El espectaculo se iniciara a las 8pm el viernes y el sabado y a las 5pm el domingo. Los boletos cuestan $25 para asientos de preferencia y $18 general, con descuentos del 25% para estudiantes/personas mayores/incapacitados, y del 50% para ninos menores de 12 anos. Para boletos por favor llame al FMC Box Office (taquilla) al: 415-441-3687. Para mayor informacion acerca del Festival comuniquese al 415-282-9546 o visite

www.jps.net/beretten/colfest

Este Festival recibe apoyo de la Fort Mason Foundation, del California Arts Council y del Consulado Colombiano en San Francisco.

Desde 1996 el Festival de la Cultura Colombiana ha estado brindando una voz para la interesante y variada cultura de Colombia en el Area de la Bahia de San Francisco. Tradicionalmente viene creando un punto de encuentro para la comunidad Colombiana y atrae a muchos visitantes del publico general a quienes les encantan las danzas plenas de colorido, el ritmo de la musica y la sabrosa comida de este pais. El visitante puede experimentar con todos sus sentidos la autentica cultura Colombiana, e ir mas alla de lo que nos hace creer la cobertura parcial y partidaria en los medios nacionales y locales. Demuestra ejemplos vivos de los profundos valores humanos y de las exuberantes manifestaciones artisticas que Colombia esta ofreciendo al mundo.

Millar helps Orioles beat Royals

Kevin Millar hit a three-run homer and finished with three hits to help the Baltimore Orioles beat Kansas City 6-5 on a rainy Saturday night for their 12th straight victory over the Royals.

Millar, who entered hitting just .186 in his previous 15 games, homered just inside the left-field foul pole on a 2-1 pitch from Brett Tomko (1-4) with two out in the first. Brian Roberts and Melvin Mora, both aboard after singles, scored on Millar's fifth home run.

The Orioles have hit three three-run homers in the first three games of the series after coming to Kansas City without a three-run homer this year.

Roberts suffered a left foot contusion and did not return after a rain delay of 1 hour, 52 minutes in the first inning.

Garrett Olson (2-0) went 5 1-3 innings for Baltimore, allowing three runs and eight hits. Olson, who has a 2.95 ERA, has allowed 16 hits and six earned runs in 18 1-3 innings in three starts since being called up April 29.

Mora singled in Freddie Bynum in the fifth, and the Orioles pushed across two more in the sixth to make it 6-1. Adam Jones hit an RBI triple and scored on Guillermo Quiroz's squeeze bunt.

Joey Gathright had two bunt singles and stole two bases for Kansas City, which finished with 13 hits.

Gathright and Jose Guillen collected RBIs in the sixth, cutting the lead to 6-3. David DeJesus led off the seventh with a triple against Jamie Walker and scored on Esteban German's single.

Walker failed to retire any of the three batters he faced and left with runners on the corners and no out. Jim Johnson replaced him and struck out Billy Butler and Mark Grudzielanek before retiring Guillen on a grounder to prevent further damage.

George Sherrill allowed a run in the ninth before finishing for his 13th save in 15 opportunities.

Tomko gave up six runs and nine hits in 5 1-3 innings. He struck out a season-high eight and did not walk a hitter for the first time this year.

The Royals have lost five of six to begin their 10-game homestand, their longest of the season. The Royals' 6-12 home record is the worst in the majors.

Levels of cytokines and thyroid autoantibodies in Omani patients with Graves' disease

ABSTRACT

An altered balance of pro- and anti-inflammatory cytokines is thought to play an important role in the pathogenesis of autoimmune thyroiditis. The aim of the present study is to assess the cytokine and autoantibody profiles in Omani patients with Graves' disease (GD). Cytokines and autoantibodies including interleukin (IL)-2, IL-4, tumour necrosis factor (TNF)α, interferon (IFN)γ, thyroid stimulating hormone receptor antibody (TRA) and thyroid peroxidase antibody (TPO) are measured in GD patients (n=59) before treatment (n=23) and after treatment (n=36) with ^sup 131^I-labelled iodine, and compared with normal controls (n=20). Patients with GD showed comparable serum levels of IL-2 but significantly higher levels of IL-4, TNFα, IFNγ, TRA and TPO, compared with the normal controls. There was also a significant increase in serum levels of IL-4 and TNFα, and a decrease in TRA in the treated group, compared to the untreated group. IL-4, TNFα, IFNγ, TRA and TPO showed a high prevalence in Omani patients with GD. Thus, cytokines and autoantibodies may prove useful in the diagnosis of GD and in assessing prognosis.

KEY WORDS: Autoantibodies.

Cytokines.

Graves disease.

Thyroid gland.

Introduction

Graves' disease (GD) is an organ-specific autoimmune disorder characterised by the presence of autoantibodies against thyroid-specific proteins, including the thyroid stimulating hormone (TSH) receptor, thyroglobulin and thyroid peroxidase (TPO).1-4

The TSH receptor plays an important role in the function and growth of thyroid cells.5 The pathogenesis of Graves' hyperthyroidism is characterised by humoral autoimmune responses in which hyperthyroidism is induced by a thyroid stimulating hormone antibody against the TSH receptor (TRA).6 This is a well-known marker of thyroid gland autoimmunity and may have some predictive value in the recurrence of GD after treatment with antithyroid drugs.7

A change in cytokine profile in GD has been proposed as a contributing factor in the induction and maintenance of the autoimmune process.1,8 Furthermore, serum cytokine levels appear to correlate with endocrine status, indicating that they could function as markers of thyroid activation in GD.9

T cells can be divided according to their cytokine profile into two major subsets of T helper (Th) cells. Th1 cells produce cytokines that support inflammation and cell-mediated immune responses associated with the pathogenesis of autoimmune disease (autoimmune thyroiditis), and these include interferon (IFN)γ and interleukin (IL)-2, while Th2 cells release cytokines that promote antibody-mediated immune responses and inhibit Th1 responses (e.g., IL-4).2,3

Consequently, measurement of cytokines may be of value in the differential diagnosis of hyperthyroidism and in the follow-up of GD, both during and after treatment with antithyroid drugs, ^sup 131^I-labelled iodine or surgery. The present study aims to explore the correlation between the use of antithyroid medication and the occurrence of thyroid-specific autoantibodies and cytokines.

Materials and methods

This study was approved by the Sultan Qaboos University Medical Research and Ethics Committee (Project No. MREC 155).

Subjects

A total of 59 adult Omani subjects (43 females, 16 males; age range: 16-51 years, average 29.7 years) with a clinical diagnosis of GD were included in the study. On the basis of treatment with ^sup 131^I-labelled iodine, which was the only type of treatment available, patients were allocated to the treated group (n=38) or the untreated group (n=21, newly diagnosed). The control group comprised 20 normal, healthy Omani blood donors matched for age and gender.

Measurement of cytokines

All serum samples were stored frozen at -70� C and then thawed once at the time of assay. IL-2, IL-4 and IFNγ (Quantikine enzyme-linked immunosorbent assay [ELISA], R&D Systems, Minneapolis) and tumour necrosis factor-α (TNFα) (TiterZyme ELISA, DRG International, Mountainside, NJ) were measured following the manufacturers' instructions. Assay sensitivities were 7 pg/mL for IL-2,10 pg/mL for IL-4 and TNFα, and 8 pg/mL for IFNγ. Samples were dispensed in 96-well microtitre ELISA plates and incubated at room temperature for 2 h. The plates were then rinsed (x4) with wash buffer and incubated for 2 h with anticytokine horseradish peroxidase conjugate against each of the cytokines tested. The bound enzyme was detected by incubation with tetramethylbenzidine and hydrogen peroxide as a substrate, and then quantified using a microplate reader. Sample variation was estimated at approximately 5%.

Measurement of TRA

Circulating TRA was measured with a commercial ELISA kit (Medizym TRA, Medipan Diagnostica, Germany), following the manufacturer's instructions.

In summary, the method comprised two incubation steps. During the first incubation, TRA was bound to the immobilised receptor on the solid phase of the microtitre plate. In the second incubation, the TSH complex bound to the free epitopes of the receptor.

Bound receptor-TSH complexes react specifically with horseradish peroxidase conjugate, which converts the colourless tetramethylbenzidine to a blue product. This enzyme reaction was stopped by adding H^sub 2^SO^sub 4^, which turned the solution from blue to yellow. Absorbance (A) was measured at 450 nm, and values >2 units/L were considered positive.

Measurement of TPO

An ELISA assay (Medizym anti-TPO, Medipan Diagnostica, Germany) was used for the quantitative measurement of human TPO antibodies in serum. All procedures were performed according to the manufacturer's instructions. Values for TPO >50 lU/mL were considered positive.

Statistical analysts

Statistical significance was calculated using ANOVA for comparing means and using Pearson's for correlation. Data were presented as mean � standard deviation (SD), and were analysed using SPSS for Windows. P<0.05 was considered significant.

Results

Table 1 shows the results obtained for the cytokines tested in the treated, untreated and control groups, together with P values. No differences based on gender were observed. Table 2 shows the cytokine results with and without TRA and TPO antibodies.

Figure 1 shows the percentage of GD patients who were positive for TRA (73%; 9.6�6.5 U/L) and TPO (69%; 1190.5�1062.6 ILJ/mL). Values in the normal group for TRA and TPO were 2% (0.514�0.4 U/L) and 6% (20.7�26.2 lU/mL), respectively. Positivity for TRA and TPO antibodies was significantly more frequent (P<0.05) in the patients with GD.

When GD patients were subdivided on the basis of treatment with ^sup 131^I-labelled iodine, a significant difference (P < 0.05) was observed in TRA and TPO levels between the treated and untreated groups, with lower levels seen in the treated group compared with the untreated group (Table 1).

The results in Figure 2 show that a significant difference (P < 0.05) was observed in TRA positivity between the treated and untreated groups, with lower levels seen in the treated group (47.2%) than in the untreated group (85.9%). No significant difference was observed between TPO positivity in the treated (69%) and untreated (78.2%) groups.

To ensure that GD patients with TRA and TPO positivity did not introduce bias to the calculations, results for GD patients with and without TRA and TPO antibodies were compared, and no significant differences were seen (Table 2).

There was no correlation (r=0.13; P>0.05) between TRA and TPO antibodies. Similarly, results obtained failed to show any correlation between TRA or TPO antibodies with any of the cytokines measured (P > 0.05), either before or after treatment.

Discussion

As has been observed elsewhere,9,10 serum IL-2 values in GD patients are normal and show no difference between treated and untreated groups. Increased serum IL-4 levels have been reported by several investigators,10-12 all of whom suggested a predominance of Th2 cytokines in GD, indicating a humoral pattern of immune response. The results of the present study support these finding.

In contrast, TNFα is a multifunctional cytokine produced by macrophages and other cells, and is known to play a major role in the immunological cascade that leads to an inflammatory response, with the release of mediators such as IL-2.13,14 In the present study, serum TNFα levels in GD were increased significantly, mainly in the untreated group, and this supports earlier work.11,12

Polarisation of intrathyroidal cytokines towards a type 2 profile probably contributes to the production of pathogenic autoantibodies in GD,15 and type 1 cytokines appear to suppress their production.16 However, the fact that serum IL-4 and TNFα levels were raised in the GD group, compared with the control group, suggests a mixed Th1/Th2 response. Moreover, Th1 and Th2 cells show crossregulation; thus, cytokines produced by one Th subset can suppress the production and/or activity of the other.17

Increases in INPγ levels in GD have not been reported previously, nor have differences in levels between treated and untreated GD patients.10 However, results from the current study show higher values in the GD groups compared with the control group, with significantly higher levels in the treated GD group. This may be due to differences in the number of subjects studied or to the different methods of measurement employed,18 as well as to differences in genetic and environmental factors.

Treatment with radioactive iodine alters the production of thyroid hormones and autoantibodies in GD. Earlier investigations suggest that TRA levels fall during antithyroid drug treatment, and this may have predictive and prognostic value in the recurrence of GD after treatment.19,20 The present study supports these findings, and the post-treatment decline in TRA antibody level suggests that the thyrotoxicosis has an autoimmune aetiology.

No changes in TPO level in the treated GD group were seen, and this result is consistent with that from a recent similar study.20 However, an earlier report21 described an increase in serum TPO levels after treatment with ^sup 131^I-labelled iodine, while another study22 reported a decrease. Such inconsistency may be due to differences in immunological response as disease activity declines, or may be a direct effect of the drug.

The absence of a positive correlation between TRA and TPO in GD patients did agree with results from a previous study.22 This may have been due to differences in GD pathogenesis or to differences in the prevalence of these autoantibodies among treated and untreated groups. It may also have been due to differences in the number of subjects studied, different assays used or to genetic and environmental factors.

It is possible that interaction between genetic, environmental and endogenous factors initiates autoimmune and inflammatory responses in patients with autoimmune thyroid disease. However, whether or not serum cytokine concentration in GD is influenced by thyroid function or by autoimmune reactions remains unclear. In addition, other factors such as antithyroid therapy and age may have an effect on circulating cytokines. Clearly, further studies are needed in this area.

[Reference]

References

1 Romagnani S. Lymphokine production by human T cells in disease states. Anna Rev Immunol 1994; 12: 227-57.

2 Abbas AK, Murphy KM, Sher A. Functional diversity of helper T lymphocytes. Nature 1996; 383: 787-93.

3 Romagnani S. The Th1/Th2 paradigm. Immunol Today 1997; 18: 263-6.

4 Collins J, Gough S. Autoimmunity in thyroid disease. Eur J Nucl Med Mol Imaging 2002; 29 (Suppl 2): S417-24.

5 Tonacchera M, van Sande J, Parma J et al. TSH receptor and disease. Clin Endocrinol (Oxf) 1996; 44: 621-33.

6 Rapoport B, Chazenbalk GD, Jaume JC, McLachlan SM. The thyrotropin (TSH) receptor: interaction with TSH and autoantibodies. Endocr Rev 1998; 19: 673-716.

7 Arqueros MC, el Tamer E, el Tamer S, Houssay AB. TSH receptor antibodies as a recurrence index of Graves' disease treated with antithyroid drugs (Spanish). Medicina (B Aires) 1991; 51: 9-13.

8 Weetman AP Aijan RA, Watson PF. Cytokines and Graves' disease. Bailliere's Clin Endocrinol Metab 1997; 11: 481-97.

9 Ward LS, Fernandes GA. Serum cytokine levels in autoimmune and non-autoimmune hyperthyroid states. Braz J Med Biol Res 2000; 33: 65-9.

10 Phenekos C, Vryonidou A, Gritzapis AD, Baxevanis CN, Goula M, Papamichail M. ThI and Th2 serum cytokine profiles characterize patients with Hashimoto's thyroiditis (ThI) and Graves' disease (Th2). Neuroimmunomodulation 2004; 11 (4): 209-13.

11 Al-Humaidi MA. Serum cytokine levels in Graves' disease. Saudi Med J 2000; 21: 639-44.

12 Bossowski A, Urban M. Serum levels of cytokines in children and adolescents with Graves' disease and non-toxic goiter J Pediatr Endocrinol Metab 2001; 14: 741-7.

13 Nawroth PR Banks I, Handley D. Tumor necrosis factor/cachectin interacts with endothelial cell receptors to induce release of interleukin 1. J Exp Med 1986; 163:1363-75.

14 Bonavida B, Paubert M, Hosford D. The involvement of platelet-activating factor (PAF)-induced monocyte activation and tumor necrosis factor production in shock Prog Clin Biol Res 1989; 308: 485-9.

15 Heuer M, Aust G, Ode-Hakim S, Scherbaum WA. Different cytokine profiles in Graves' disease, Hashimoto's thyroiditis and non-autoimmune thyroid disorders determined by quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Thyroid 1996; 6: 97-106.

16 McLachlan SM, Taverne J, Atherton MC. Cytokines, thyroid autoantibody synthesis and thyroid cell survival in culture. Clin Exp Immunol 1990; 79:175-81.

17 Mosmann TR, Coffman RL. TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol 1989; 7:145-73.

18 Feldmann M, Brennen FM, Chantry D et al. Cytokine assays: role in evaluation of the pathogenesis of autoimmunity. Immunol Rev 1991; 119:105-23.

19 Feldt-Rasmussen U, Schleusenter H, Carayon E Meta-analysis evaluation of the impact of thyrotropin receptor antibodies on long-term remission after medical therapy of Graves' disease. J Clin Endocrinol Metab 1994; 78: 98-102.

20 Mankai A, Toumi D, Chadli-Chaieb M et al. Antithyroidstimulating hormone receptor antibodies determined by second-generation assay. Clin Chem Lab Med 2007; 45: 26-9.

21 Feldt-Rasmussen U, Bech K, Date J, Petersen PH, Johansen K, Madsen SN. Thyroid stimulating antibodies, thyroglobulin antibodies and serum proteins during antithyroid treatment with radioiodine or propylthiouracil of Graves' disease. Allergy 1982; 32:161-72.

22 Takamatsu J, Hosoya T, Kohno Y et al. Changes in serum autoantibodies to thyroid peroxidase during antithyroid drug therapy for Graves' disease. Endocrinol Jvn 1990; 37: 275-83.

[Author Affiliation]

A. ALNAQDY* and M. AL-MASKARI[dagger]

Departments of * Microbiology/Immunology and [dagger] Medicine, College of Medicine

and Health Sciences, Sultan Qaboos University, P.O. Box 35, Al-Khod 123,

Sultanate of Oman

Accepted: 10 October 2007

[Author Affiliation]

Correspondence to: Dr. A. Alnaady

College of Medicine and Health Sciences, P. O. Box 35, Al-Khod 123,

Sultanate of Oman. Email: aalwan@sau.edu.om

Joseph P. Lash, 77, author, friend of Eleanor Roosevelt

BOSTON (AP) Pulitzer Prize-winning author Joseph P. Lash, 77,whose works included Eleanor and Franklin, died Saturday inMassachusetts General Hospital.

Mr. Lash died of complications of a heart ailment, said ahospital official.

Mr. Lash, a close friend of the late Eleanor Roosevelt, won aPulitzer in 1971 for his book about President Franklin D. Rooseveltand his wife.

He also wrote Eleanor: The Years Alone in 1972 and Love, Eleanorin 1982.

The latter book focused on letters between Mr. Lash, Mrs.Roosevelt and Mr. Lash's wife, the former Trude W. Pratt. In it, herevealed that previously secret intelligence documents claimed he hadhad an affair with Mrs. Roosevelt in 1943. He denied the reports.

"I felt almost nauseous," he said in an interview in 1982,recalling reports of the alleged affair.

"Anyone who reads that correspondence (in the book) can see myphysical feeling was only for Trude, and whatever feeling Mrs.Roosevelt had, it was platonic."

Mr. Lash also wrote that President Roosevelt did not believe thereports and that records indicate Roosevelt was so angered by thesurveillance that his orders led to the dismantling of the Armycounterintelligence corps.

In 1943, the year of the alleged affair, Mr. Lash was a33-year-old Army Air Corps sergeant studying meteorology at ChanuteField in Illinois. He had met Mrs. Roosevelt through his work as aYoung Socialist leader and was being investigated for communistaffiliations.

He also was the author of Dag Hammarskjold: Custodian of theBrushfire Peace; From the Diaries of Felix Frankfurter; Rooseveltand Churchill, 1939-41, and Helen and Teacher, about Helen Keller.

He is survived by his wife.

A private burial is planned this week on Martha's Vineyard.

US gonorrhea rate at record low, other STDs rise

ATLANTA (AP) — A new government report on sexually spread diseases shows gonorrhea in the United States has dipped to the lowest rate ever recorded.

But chlamydia (KLAH'-mid-ee-ah) and syphilis infections continued to increase last year. That's according to a report released Monday by the Centers for Disease Control and Prevention.

The agency says there are roughly 19 million new cases of sexually transmitted diseases annually.

Gonorrhea dropped to 301,000 cases, the lowest rate since reporting began in 1941.

Chlamydia reached another record high with 1.2 million cases. It's an easily treatable infection, mostly in young women. Officials attribute the increase to more and better screening. Syphilis inched up again to 14,000 cases.

___

Online:

Report: http://www.cdc.gov/std/stats09/default.htm

School must provide prayer room for Muslims

A Berlin court ruled Tuesday that a city high school must provide a room in which Muslim students can pray or it would be violating Germany's guarantees of freedom of religion.

The Berlin administrative court, ruling on a suit brought by a 16-year-old Muslim student, said that the constitution guarantees "not only the inner freedom to believe or not to believe, but also the freedom to express this belief."

Relying on expertise from a scholar of Islam, the court ruled that specific hours of prayer had a high significance for Muslims and therefore "a devout Muslim could not be expected to pray only outside of school hours."

The ruling was a single-case decision, and is subject to appeal. School department spokesman Jens Stiller said that the city planned to take further "legal steps" but would not be more specific.

Still, court spokesman Stephan Groscurth said that Muslim students could refer to the ruling as precedent if they file suits in similar situations.

The student, who was not identified by name, sued the school after it refused to provide him with a separate room for prayer.

The school has allowed him to pray in an assigned room during breaks since March 2008, however, after the court issued an interim order while the case was pending.

Video poker player is 100 percent curious

A shuffle through the Gaming mailbag:

Dear John: My husband and I are avid 25-cent video pokerplayers. From time to time, we've read that there are 25-cent videopoker machines in Las Vegas that have a 100 percent return. We'veheard that the Desert Inn has some of these and we also have playedthem at Vegas World (which is now closed). We're students of LennyFrome's Winning Strategies for Video Poker.

Could you please inform me of any Las Vegas casinos that havethe 100 percent payback machines?

Anne Margosian, Palatine

Answer: Since you specifically mention Vegas World, I assume youmean the 10-6 and 9-7 Jacks or Better machines that were a highlightin that casino. These offered the full pay tables usually found on9-6 machines that return 99.5 percent with optimal play, except thaton the 10-6 version the full house payoff was elevated from 9-for-1to 10-for-1, and on the 9-7 version flush payoffs were elevated from6-for-1 to 7-for-1. That pushed the long-term payback with expertplay to more than 100 percent on both versions.

I know of none of these currently operating. If any readershave run across them recently, I'd like to hear about it.

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Compartmentation of cAMP Signaling in Cardiac Myocytes: A Computational Study

ABSTRACT

Receptor-mediated changes in cAMP production play an essential role in sympathetic and parasympathetic regulation of the electrical, mechanical, and metabolic activity of cardiac myocytes. However, responses to receptor activation cannot be easily ascribed to a uniform increase or decrease in cAMP activity throughout the entire cell. In this study, we used a computational approach to test the hypothesis that in cardiac ventricular myocytes the effects of β^sub 1^i-adrenergic receptor (β^sub 1^ AR) and M^sub 2^ muscarinic receptor (M^sub 2^R) activation involve compartmentation of cAMP. A model consisting of two submembrane (caveolar and extracaveolar) microdomains and one bulk cytosolic domain was created using published information on the location of β^sub 1^ARs and M^sub 2^Rs, as well as the location of stimulatory (G^sub s^) and inhibitory (G^sub i^) G-proteins, adenylyl cyclase isoforms inhibited (AC5/6) and stimulated (AC4/7) by G^sub i^, and multiple phosphodiesterase isoforms (PDE2, PDE3, and PDE4). Results obtained with the model indicate that: 1), bulk basal cAMP can be high (~1 �M) and only modestly stimulated by β^sub 1^AR activation (~2 �M), but caveolar cAMP varies in a range more appropriate for regulation of protein kinase A (~100 nM to ~2 �M); 2), M^sub 2^R activation strongly reduces the β^sub 1^AR-induced increases in caveolar cAMP, with less effect on bulk cAMP; and 3), during weak β^sub 1^AR stimulation, M^sub 2^R activation not only reduces caveolar cAMP, but also produces a rebound increase in caveolar cAMP following termination of M^sub 2^R activity. We conclude that compartmentation of cAMP can provide a quantitative explanation for several aspects of cardiac signaling.

INTRODUCTION

It is well accepted that receptor-mediated changes in cAMP production play an essential role in autonomie regulation of cardiac function, β^sub 1^-adrenergic receptors (β^sub 1^ARs) increase cAMP production through stimulatory G-protein (G^sub s^)-dependent activation of adenylyl cyclase (AC), and M^sub 2^ muscarinic receptors (M^sub 2^Rs) antagonize β^sub 1^ AR responses through inhibitory G-protein (G^sub i^)-dependent inhibition of AC activity. M^sub 2^Rs also produce a delayed stimulatory response that involves an increase in cAMP production (1).

Despite the large number of studies demonstrating an essential role for c AMP in mediating autonomie responses in the heart, several observations remain incompletely understood. For example, it has been estimated that the cellular concentration of cAMP in cardiac preparations is - 1 �M under unstimulated conditions (2,3). Furthermore, cAMP produces most of its acute functional responses through the activation of protein kinase A (PKA). However, the regulatory subunits of PKA bind cAMP with an affinity of 100-300 nM (4,5). This suggests that PKA is almost fully activated under resting conditions. If this is true, then how can βAR stimulation of cAMP production elicit responses that are known to involve activation of PKA? One possible explanation, which has been around for over 30 years, is that the changes in cAMP responsible for generating functional responses occur in a microdomain that is somehow isolated from the bulk of the cell (6). Support for this hypothesis comes from studies demonstrating that changes in the relative amount of cAMP and PKA activity in the membrane or paniculate fraction of cardiac preparations correlates more closely with the regulation of functional responses than changes in cAMP activity observed in the soluble orcytosolic fractions (7-9).

There are also unanswered questions concerning the mechanisms by which changes in cAMP are involved in muscarinic responses. For instance, it has not always been possible to demonstrate that muscarinic antagonism of β-adrenergic responses corresponds with a decrease in the total cAMP content of cardiac preparations (10). Furthermore, M^sub 2^R activation can produce cAMP-dependent stimulatory responses in addition to the inhibitory effects. What's more, the stimulatory effects exhibit much slower kinetics ( 11 -13 ). So how can M^sub 2^R activation produce both inhibition and stimulation of cAMP-dependent effects, and why are there differences in the kinetics of the two types of responses? At least part of the answer lies in the fact that cardiac myocytes express multiple isoforms of AC. These include ACS and AC6 as well as AC4 and AC7 (14,15). Yet, not all AC isoforms respond to G^sub i^ activation in the same manner. The activated α-subunit of G^sub i^ (G^sub i^α) directly inhibits AC5/6, but not AC4/7 (16-18). In fact, AC4/7 is actually stimulated by free βγ-subunits released upon activation of G^sub i^ (19,20).

The opposing effects of G^sub i^ activation on different types of AC activity can explain the inhibitory and stimulatory effects that M^sub 2^R activation has on cAMP-dependent responses in ventricular myocytes (11). However, it doesn't explain the disparity in the kinetics of the responses. Again, compartmentation of cAMP production may be involved. Evidence in support of this idea comes from studies demonstrating how different components of the signaling pathways are organized in the membrane. For example, AC5/6 is consistently associated with caveolar domains of the plasma membrane, whereas AC4/7 activity is associated with extracaveolar domains (21). This indicates that muscarintc inhibition and stimulation of cAMP occur in different subcellular locations. This has led us to hypothesize that compartmentation of cAMP can explain the complex temporal response produced by M^sub 2^R activation.

In this study, we used a computational approach to determine whether or not compartmentation of cAMP may be important in answering the above questions. However, earlier models of cAMP signaling in the heart did not consider compartmentation or muscarinic modulation of β-adrenergic responses (22,23). Therefore, we developed a new theoretical framework that incorporates existing information on the subcellular location of the various elements involved in cAMP production and degradation as well as the kinetics of critical reactions in both β^sub 1^ AR and M^sub 2^R signaling pathways. The resulting model was then used to predict the effects that receptor activation has on cAMP concentrations in caveolar, extracaveolar, and bulk cytoplasmic compartments. The results demonstrate that basal concentrations of cAMP in the caveolar compartment can be maintained at a level significantly lower than that of the total cell so that β^sub 1^AR activation may men regulate the activity of PKA through changes in the concentration of cAMP in this microdomain. The results also demonstrate that even when there arc only minimal changes in total cellular cAMP levels, M^sub 2^R stimulation can significantly inhibit cAMP levels in the caveolar domain. Finally, the model demonstrates that the delayed stimulatory response to M^sub 2^R activation is consistent with limited diffusion of cAMP from an extracaveolar domain to the caveolar domain, where PKA activity is regulated.

METHODS

Strategy for model design

Our goal was to determine whether a compartmenial model can quantitatively explain the changes in cAMP in cardiac ventricular myocytes in response to activation of adrenergic and muscarinic receptors. We designed the model to be realistic, in the sense of being consistent with available information on the molecules involved. In particular, the model includes multiple isoforms of AC and phosphodiesterase (PDE), which have been proposed to be critical for regulation of cAMP levels. For these reasons, the model includes many molecular entities, each of which requires several parameters to define its functional behavior. Some of the parameters are strongly constrained by existing experimental data, but others are not (as discussed further below). We manually varied the parameters, within the range consistent with existing data, to best describe experimental results on cAMP levels in basal and stimulated conditions. From a mathematical perspective, the model is underdetermined, in that there are more available parameters than are likely to be strictly necessary to produce any single desired output. Consequentially, the parameter set presented here is highly unlikely to be unique. Because our goal was not to provide quantitative estimates for any particular model parameters, we have not performed a formal sensitivity analysis, but we note in the Discussion some of the values thai strongly affected key features of the model output. In essence, we present this model as a "proof of concept", to demonstrate mat a molecularly realistic compartmemal model can account for regulation of cAMP levels under a variety of experimental conditions. We expect that this model will also provide a basis for interpreting future studies on molecular perturbations of the system (e.g., inhibition of a particular PDE isoform).

Cell size and composition

A mathematical model of β^sub 1^-radrenergic and M^sub 2^ muscarinic receptor regulation of cAMP production in a cardiac ventricular myocyte was developed for this study. As in previous theoretical studies (24), the myocyte is assumed to be a cylinder with the following characteristics: length = 100�m, radius = 10�m, surface area = 6.9 � 10^sup 4^�m^sup 2^, and volume = 38 � 10-6�l.

Existing kinetic data on the signaling pathways involved in regulating cAMP production and degradation were incorporated into a model consisting of three different compartments (Fig. 1). The first is the subsarcolemmal space reflecting me caveolar domains of the cell. The key signaling elements included in that compartment have been found in cholesterol-rich membrane fractions that are associated with caveolin 3, the muscle specific form of caveolin that is involved in creating signaling complexes necessary for producing functional responses (25-27). Major signaling components placed in the caveolar domain are β^sub 1^ARs, M^sub 2^Rs, G^sub s^, G^sub i^, ACS/6, as well as phosphodiesterase types 2 (PDE2), 3 (PDE3), and 4 (PDE4). The size of this compartment is ~1% of the cytosolic volume, and it encompasses 10% of the plasma membrane surface area (28).

The second compartment reflects the subsarcolemmal space associated with cholesterol-rich lipid rafts that do not include caveolin. In the model, this extracaveolar domain contains β^sub 1^ARs, M^sub 2^Rs, G^sub i^, G^sub s^, AC4/7, as well as PDE2 and PDE4. The size of this compartment is ~2% of the cytosolic volume, and it encompasses 20% of the plasma membrane. The final domain is the bulk cytoplasmic compartment, which makes up ~50% of the total cell volume (28,29) and is associated with plasma membrane that contains many of the components described above, but to a lesser degree.

The signaling elements included in the model are listed in Table 1. They were placed in specific domains based on studies using biochemical methods to detect the presence of protein in different cell or membrane fractions. Due to limited quantitative information, elements that have been reported to exist in more than one domain were assumed to be distributed uniformly between relevant compartments. In some instances, functional studies were used as evidence that a particular signaling element is not uniformly distributed (see below).

Most numerical values used for the parameters found in the model are either taken directly from a single experimental source or constrained by a range defined by multiple experimental sources (see Appendix I). However, no published quantitative data was available for the rate constants for G^sub i^ activation, the amount of each PDE isoform found in different subcellular compartments, or the cAMP flux rates.

To convert the amount of a specific protein described as a fraction of total membrane protein to the amount of that protein in a single ventricular myocyte, it is assumed that 1 mg of total membrane protein = 7.5 � 10^sup 5^ myocytes (30).

Computational components

Ligand/receptor/G-protein modules

The ternary complex model was used to describe the interaction between ligand, receptor, and G-protein (31). Briefly, the model characterizes the ability of the agonist to promote and stabilize the formation of a high affinity active ternary complex (LRG), starting from a low affinity binary ligand-receptor (LR) complex and G-protein (G). Due to the speed of these reactions (when compared to the other reactions present in the model), they are assumed to be at quasiequilibrium and are represented by algebraic equations. The same formalism was used to describe both β^sub 1^-adrenergic receptor and M^sub 2^ muscarinic activation in all relevant compartments.

Module input:

Ligand concentration (constant during a simulation).

Free G-protein concentration (G^sub αβg^ = Gfree).

Module output:

Receptor bound G-protein concentration (RG + LRG).

G-protein activation module

The ratio between the total number of G^sub i^ and G^sub s^ molecules in cardiac cells is ~2:1 (30,32,33). Experimental evidence suggests that there is approximately equal distribution of Gs between caveolar and noncaveolar membrane domains, whereas Gi is almost entirely present in the caveolar domain (34). The activation kinetics of G^sub s^ were derived from published data (35) and modified to obtain subsecond kinetics for G-protein activation, in agreement with previous studies (36). Although the absolute rate constants for G^sub i^ activation have not been determined, the rate of G^sub i^ activation has been reported to be slower than that of G^sub s^. Based on this information, it was assumed that the G^sub i^ activation rate is 50% of that determined for G^sub s^.

Module input:

Receptor bound G-protein concentration (RG + LRG).

Module output:

G-protein subunits concentration (G^sub α-GTP^, G^sub α-GDP^, G^sub βγ^).

Free G-protein concentration (G^sub αβγ^ = G^sub free^).

The following formulations are applicable both to G^sub s^ and Gi and their corresponding receptors (β^sub 1^AR and M^sub 2^R).

The output of the combined LRG and G-protein activation modules was validated by comparing the simulated concentration-response curve for isoproterenol stimulation of G^sub s^α accumulation (not shown) with published experimental data (37).

Adenylyl cyclase modules

Adenylyl cyclase activities were divided into two functionally distinct categories: AC5/6 and AC4/7. Both categories are stimulated by G^sub s^ activation, but with very different affinities. The EC^sub 50^ for G^sub s^α stimulation of AC4/7 is ~32 nM, whereas for AC5/6 it is ~200 nM (38,39). This significantly affects the sensitivity of each AC category to β^sub 1^AR stimulation. Each category of AC activity is also affected differently by G^sub i^ activation. AC5/6 is inhibited by G^sub i^α, whereas AC4/7 is not. Furthermore, AC4/7 is stimulated synergistically by G^sub i^βγ in the presence of G^sub s^α activation (39). As described below, parameter values were obtained by using a Hill equation to Rt the experimentally determined relationship between G-protein subunits and AC activity.

Adenylyl cyclase 5/6 module. The activity of AC5/6 and its regulation by G^sub s^ and G^sub i^ were simulated using a formulation to describe data obtained from isolated membrane preparations (37,40). This activity was then scaled up using an amplification factor (AF^sub 5/6^) that was defined by the ratio of the activity produced by equal amounts (I mg) of purified and membrane protein in the presence of a maximally stimulating concentration of G^sub s^α.

Module input:

Gs^sub α^ and Gi^sub α^ concentration (GS^sub α-GTP^. Gi^sub α-GTP^).

Module output:

cAMP produced by AC5/6.

Adenytyt cyclase 4/7 module. AC4/7 activity and its regulation by G^sub s^ and G^sub i^ were simulated using a formulation based on published experimental data (41,39,42). Because of their structural and functional similarities (43), the kinetic properties of AC2 were used to describe the behavior of AC4/7, where necessary. The total amount of AC4/7 present in the cell was assumed to be ~10%of AC5/6 (44,45). An amplification factor (AF,^sub 4/7^) was calculated as it was for ACS/6.

Module input:

Gs^sub α^ and Gi^sub α^ concentration (Gs^sub α-GTP^, Gi^sub βγ^).

Module output:

cAMP produced by AC4/7.

Phosphodiesterase modules

The total cellular protein content of the various PDE isoforms found in cardiac myocytes has been reported. However, the only information available on the subcellular location of the different PDE isoforms describes their presence in membrane (paniculate) and/or cytosolic (soluble) fractions. How the membrane associated PDE activity is distributed between caveolar and extracaveolar domains is not known. The available descriptive information, along with functional studies, was used as a guide in determining the relative ratio of each PDE isoform to be included in the various compartments.

Module input:

cAMP concentration for the specific compartment.

Module output:

cAMP degraded by PDEs.

cAMP flux modules

Initial rates for flux of cAMP between compartments were based on previously published estimates (46).

Module input:

cAMP concentration.

Module output:

cAMP concentration.

Implementation and validation

The model was implemented in Java (J2SE). Differential equations were solved using an iterative Euler approach. Predictions made by the model are compared to two types of previously published data. The first are cAMP measurements obtained using biochemical methods to determine the cAMP content of homogenized tissue or whole cell lysatcs. These data are assumed to reflect what is happening to cAMP at the level of the whole cell. The second are measurements of changes in cAMP activity in intact ventricular myocytes using a FRET-based biosensor (47). This probe consists of two separate proteins: the catalytic subunit of protein kinase A (PKA) labeled with yellow fluorescent protein (Cat-YFP) and the type II regulatory subunit labeled with cyan fluorescent protein (RJI-CFP). FRET occurs when PKA is in the inactive state, and Cat-YTP and RII-CFP arc bound to one another. An increase in cAMP causes molecular rearrangement of the subunits, which results in a decrease in FRET. By convention the FRET response is defined as the change in CPPfYFP fluorescence ratio relative to the baseline ratio (ΔR/R^sub o^), so that an increase in cAMP results in an increasing "FRET response", even though there is actually a decrease in FRET. These data are assumed to reflect what is happening to cAMP activity in the caveolar domain, where cAMP levels arc expected to correlate most directly with functional responses regulated by type II PKA.

RESULTS

Basal cAMP levels

We first validated the model's prediction of cAMP levels under basal conditions. In the absence of any agonist, previous studies have suggested that the basal concentration of cAMP in cardiac myocytes is - 1 �M (2,3). However, this is well above the cAMP affinity of PKA (≤300 nM), which is the primary effector for this cyclic nucleotide (4,5). This apparent discrepancy may be due to cAMP measurements having been made using traditional biochemical methods involving homogenized tissue or whole cell lysates, which represent what is happening on average throughout the whole cell, but may not accurately reflect what is happening in discrete microdomains where PKA-mediated functional responses are regulated. Consistent with this idea, the model demonstrates that under basal conditions, it is possible to maintain the average concentration of cAMP across all compartments (total cAMP) at 1 �M, while in the caveolar domain, the basal concentration is ~100 nM (see Fig. 2). Although this level of cAMP is high enough to partially activate PKA, it is still low enough to leave a concentration range over which cAMP can significantly modulate kinase activity.

Effects of β-adrenerglc receptor stimulation

We next evaluated the effect that β^sub 1^AR stimulation has on cAMP levels. As expected, the model predicts that β^sub 1^AR stimulation causes a concentration-dependent increase in cAMP in all compartments of the cell (Fig. 2). Exposure to the β^sub 1^ AR agonist isoproterenol (Iso) increases total cAMP from basal (1 �M) to a maximal level ~2.1 �M with an EC^sub 50^ of ~7 nM. This is consistent with the 50-250% increase over baseline, and EC^sub 50^ of 10-80 nM observed experimentally, when using traditional biochemical methods to measure total cAMP in homogenized preparations (7,9,48-51). The model also predicts that caveolar cAMP reaches a similar concentration following maximal β^sub 1^AR stimulation, but because of the lower baseline (100 nM), the overall change represents a >2000% increase.

Recently, we have used a fluorescence energy transfer (FRET)-based biosensor to measure changes in cAMP activity in intact cardiac myocytes (47). This biosensor consists of fluorescently labeled type II PKA, and in adult ventricular myocytes it is expressed in a striated pattern, suggesting that it is associated with T tubules, just like endogenous type II PKA (52,53). Caveolin-3, a marker for caveolae, is expressed in a similar striated pattern, in addition to being found in the peripheral sarcolemma (54). Biochemical studies have also demonstrated that type II PKA is highly enriched in caveolar membrane fractions (34). Therefore, it is assumed that this sensor responds to changes in cAMP activity occurring in a caveolar compartment. Consistent with this idea, we have previously demonstrated that this sensor detects β^sub 1^ AR stimulated changes in cAMP activity with a sensitivity that correlates directly with the β^sub 1^AR sensitivity of L-type Ca^sup 2+^ channels (47), which are located in caveolae associated with T tubules (55). The properties of this probe have also been well characterized in vitro (4). The EC^sub 50^ for cAMP activation is 300 nM, with a Hill coefficient of 1.4. Furthermore, activation by maximally effective concentrations of cAMP produces a FRET response of ~15% (see Fig. 3 B). However, when expressed in ventricular myocytes, this probe exhibits a smaller dynamic FRET response (6-12%) following maximal agonist stimulation (47,4). This can be explained if basal levels of cAMP are sufficient to have partially activated PKA, even before exposure to agonist. Consistent with this idea, PKA-dependent responses can be elicited from ventricular preparations by inhibiting basal phosphatase activity (56).

If the in vitro properties of the probe described above hold true in vivo, and if we assume that the average maximal FRET response that can be elicited in adult ventricular myocytes is 9%, this then indicates that ~25% of the PKA-based probe must be in an active state under basal conditions. This corresponds to a basal level of c AMP that is close to that predicted by the model to exist in the caveolar domain. Using the relationship between Iso concentration and cAMP levels in the caveolar compartment (Fig. 2 C), we then defined the expected relationship between Iso concentration and PKA activation. According to this calculation, Iso increases PKA activity from a basal level of 25% to maximal with an ECso of 1.3 nM (Fig. 3 D). The sensitivity of this response is in good agreement with the EC^sub 50^ of 0.5 nM for Iso activation of the PKA-based probe in intact ventricular myocytes (47). With the relationship in Fig. 3 B, we can also use the model to predict the FRET response of the PKA-based probe (Fig. 3 C) and compare this to experimentally determined PKA-FRET responses (Fig. 3 B). The model's predictions are in good agreement with the experimental results. The one possible exception is the difference in the time course with which responses turn on and off. Although there may be more than one explanation, it is most likely due to the slow exchange of solutions used to add or remove drugs during the imaging experiments (47). Consistent with this conclusion, the time course of the responses predicted by the model are more in Une with the time course of cAMP-dependenl ion channel responses observed when rapid solution changes are possible (13).

Effects of M^sub 2^R activation in the presence of maximal β-adrenergic stimulation

M^sub 2^R activation is able to antagonize responses to agonists that stimulate cAMP production. Although it is now generally accepted that such inhibitory effects are due to a decrease in cAMP production, this has not always been the case (1). Many early studies measuring cAMP levels in homogenized tissue or whole cell lysates found that muscarinic inhibitory responses do not correlate with changes in cAMP. An explanation offered for this apparent paradox has been that muscarinic inhibition of cAMP activity is localized to specific subcellular compartments (10). Consistent with this idea, the model predicts that M^sub 2^R stimulation has only modest effects on total cAMP levels (Fig. 4 C). However, M^sub 2^R activation significantly reduces cAMP in the caveolar compartment (Fig. 4D). Furthermore, ACh inhibition of the Iso response is reflected in the predicted FRET response (Fig. 4 B), which correlates well with the PKA FRET response observed experimentally (Fig. 4 A). Another prediction of the model is that following the initial inhibitory effect produced by ACh, there is a gradual decrease in inhibition ("escape"), as observed experimentally (13). In the model, this is due to M^sub 2^Rs stimulating cAMP production in the extracaveolar compartment, which then spills over into the caveolar compartment.

Effects of M^sub 2^R activation In the presence of submaxlmal β-adrenerglc stimulation

Having demonstrated that the model can reproduce muscarinic inhibition of β^sub 1^AR-mediated cAMP production, we then evaluated its ability to explain muscarinic stimulatory responses. Previous studies in ventricular myocyles have demonstrated that muscarinic stimulatory effects are only observed in the presence of submaximal concentrations of agonists such as Iso (11-13). Consistent with this, the model predicts that M^sub 2^R activation would not produce a detectible rebound stimulatory response in the presence of a maximally stimulating concentration of Iso (Fig. 4 B), However, in the presence of a concentration of Iso that is near the threshold for stimulating PKA activity above basal levels, the model predicts that upon termination of M^sub 2^R activation, there should be a significant rebound stimulatory increase in cAMP concentration in the caveolar compartment, which produces a rebound increase in the predicted PKA FRET response (Fig. 5 B). This type of response correlates with the PKA-FRET response observed experimentally (Fig. 5 A). Again, the slower time course of the FRET response measured experimentally may be explained by the time required to change solutions. The model also predicts an inhibitory effect that goes below baseline during exposure to ACh, but the PKA-based probe may not be able to detect changes in cAMP below basal levels (see Appendix II).

A more detailed examination of the source of cAMP responsible for the rebound stimulatory response helps explain this complex behavior. This requires an appreciation of what is happening not only in the caveolar compartment, but also in adjacent compartments, especially the extracaveolar compartment. In the model, M^sub 2^Rs are located in both the caveolar and extracaveolar compartments. However, the caveolar compartment contains only AC5/6 activity, while the extracaveolar compartment contains only AC4/7 activity. In the presence of ACh, G^sub i^α inhibits AC5/6, rapidly reducing cAMP in the caveolar compartment (Fig. 6, A and B). However, ACh actually increases cAMP in the extracaveolar compartment, where G^sub i^βγ stimulates AC4/7 (Fig. 6 D). The resulting concentration gradient produces a slow flux of cAMP between compartments (Fig. 6 C), but the inhibitory effect of ACh on cAMP production within the caveolar compartment dominates. Upon washout of ACh, AC5/6 inhibition quickly reverses, resulting in a rapid return of cAMP production within the caveolar compartment. This cAMP, together with flux of cAMP from the extracaveolar compartment, results in a caveolar concentration exceeding that observed in the presence of Iso alone (before application of ACh). As the concentration of cAMP in the extracaveolar compartment declines, so does the flux of cAMP into the caveolar compartment. The result is that caveolar cAMP declines to its steady-state value. Based on these results, we conclude that the diffusion of cAMP from the extracaveolar compartment into the caveolar compartment is a viable explanation for the rebound stimulatory responses observed upon washout of ACh.

DISCUSSION

Computational models of cAMP signaling have recently been used to investigate cardiac myocyte responses to β^sub 1^AR stimulation (23,22). This study expands this approach by incorporating the kinetics of processes involved in β^sub 1^-adrenergic as well as M^sub 2^ muscarinic regulation of c AMP production and integrating this with information on the subcellular distribution of the various components of these signaling pathways. The model was tested by comparing cAMP responses predicted to occur in a caveolar compartment to previously published measurements of changes in cAMP activity detected by a FRET-based biosensor targeted to the corresponding subcellular location of intact cardiac ventricular myocytes (47). It was also tested by comparing total cAMP responses (using the weighted average of the cAMP concentrations in all three compartments) to previously published measurements of changes in cAMP obtained using traditional biochemical methods in ventricular tissue homogenates and/or whole cell lysates. This new theoretical framework can explain both simple and complex behaviors of cardiac ventricular myocytes evoked by both β^sub 1^-adrenergic and M^sub 2^ muscarinic signaling pathways.

The model provides a simple explanation for the observation that the total cellular cAMP level is high enough that one would expect near maximal activation of PKA, even under basal conditions. Measurements obtained using homogenized cells or tissue reflect cAMP levels averaged across the whole cell, not levels in microdomains that make up only a small traction of the total cell volume. Although compartmentalion of cAMP signaling is an obvious potential explanation, that hypothesis is difficult to evaluate experimentally. The model predicts that bulk cytoplasmic cAMP is comparable to previous estimates for the whole cell, but cAMP in the caveolar compartment operates in a range appropriate to modulate PKA activity. This obviates the need to assume that the affinity of PKA for cAMP is significantly lower in vivo than what has been measured in vitro (22,57).

The model also can explain why muscarinic inhibition of β-adrenergic functional responses has not always been found to correlate directly with changes in cAMP levels (10). Because the discrepancies have been reported in studies where cAMP levels were measured using whole cell or tissue preparations, it has been proposed that significant concentration changes actually were occurring in microdomains where PKA-dependent responses are regulated (10). In support of this idea, the model predicts that M^sub 2^R activation causes only modest changes in cAMP concentration in the bulk cytoplasmic compartment (see Fig. 4 C), whereas there are significant changes in cAMP levels in the caveolar compartment (see Fig. 4 D). Furthermore, the predicted effect of M^sub 2^R activation on PKA responses in the caveolar compartment (Fig. 4 B) correlates well with experimental results demonstrating that, in the presence of β^sub 1^AR stimulation, M2R activation reduces cAMP activity detected by the PKA-based cAMP biosensor (Fig. 4 A).

We then used the model to determine whether compartmc mat ion of cAMP can explain M^sub 2^R-mediated delayed stimulatory responses. In ventricular myocytes, this behavior is due to G^sub i^ βγ subunits stimulating AC4/7 activity (II). However, this occurs at the same time that G;a is inhibiting AC5/6. So why does the stimulatory effect produce a rebound response upon termination of M^sub 2^R activation? If AC4/7 and AC5/6 are found in different plasma membrane domains (21 ), we hypothesized that a time-dependent flux of c AMP from an extracaveolar compartment to a caveolar compartment could be responsible. The model demonstrates that this is a feasible explanation. It is interesting to note that the muscarinic stimulatory mechanism only affects functional responses (e.g., ion channels) in the presence of submaximally stimulating concentrations of Iso. The model actually predicts that the muscarinic stimulatory response affects c AMP levels even in the presence of maximally stimulating concentrations of Iso, but because this rebound response is above the level that maximally activates PKA, it is not expected to produce a detectable functional response (see Fig. 4).

Limitations and predictions

Although descriptive information dealing with compartmentation of cAMP signaling in cardiac myocytes is growing rapidly, the availability of quantitative data is limited. Where information does exist, it is not uncommon for there to be some variability in estimates between different studies. Because of this, there may be more than one set of parameters that produce the same behaviors we have described. In this respect, this version of the model should be viewed as just one potential configuration, albeit one that exhibits properties consistent with a number of experimental observations. Although a quantitative parameter analysis was beyond the scope of this study, we did conduct a more qualitative type of analysis of the importance of certain parameter in producing specific responses.

Except where noted, the equations used in the model's different modules were taken from previously published studies. This approach constrained many parameter values to those that resulted in module output consistent with experimental data. Less quantitative information was available for assigning values to other parameters. This is particularly true for the distribution of some signaling elements among subcellular compartments and the flux rates for cAMP between compartments. These were the parameters that turned out to have the greatest influence on the behaviors that were the focus of this study.

Experimental data also do noi provide a consistent view on the location of β^sub 1^ARs. Some studies have suggested that the bulk of the β^sub 1^AR population exists in an extracaveolar domain (34), whereas others have concluded that the same receplor is found predominantly in a caveolar domain (58). The present version of the model assumes that β^sub 1^ARs are found in both of these compartments. The validity of this assumption might be questioned, but it was necessary in order for β^sub 1^AR stimulation to activate both AC5/6 and AC4/7. Stimulation of AC5/6 was necessary to produce an increase in cAMP that could then be inhibited by M^sub 2^R activation. Stimulation of AC4/7 was necessary to see AChinduced rebound stimulation of cAMP production. Because these different AC isoforms are found in different membrane domains (21,27), the simplest way to model these behaviors was to include β^sub 1^ARs in those domains as well. This illustrates how the model can be used to evaluate the functional significance of biochemical data describing the location of specific elements or even differentiating between possibilities. Using this approach, our results provide support for the idea that β^sub 1^ARs actually exist in caveolar as well as extracaveolar domains.

One aspect of the model that is absolutely critical for producing the ACh-induced rebound stimulatory response is the placement of AC5/6 inside and AC4/7 outside of the caveolar compartment. Although this behavior is not lost if the distribution of AC isoforms is less strict, segregation of the majority of AC activity is important. This conclusion is consistent with biochemical studies that have looked at the distribution of AC activity in different membrane fractions (27,21).

It was not necessary to include more than one type of PDE activity to obtain the basic properties of this model. What was important was the ratio of PDE to AC activity in each compartment. Furthermore, it was critical to have much higher total PDE activity in the caveolar compartment than either of the other two compartments. This was necessary to maintain a low concentration of cAMP in the caveolar domain under basal conditions, while still allowing cAMP levels in the other compartments (and thus total cAMP) to remain high. Similarly, the transient rebound response observed following washout of ACh can be reproduced by including a single type of PDE activity in the extracaveolar domain that is much lower than that in the caveolar compartment. So one might question why we included three different types of PDE activity in the model. The primary reason is that there have been numerous studies demonstrating the functional importance of PDE2, PDE3, and PDE4 activity in regulating cAMP-dependent responses in adult cardiac myocytes (59,60), and we wanted the model to reflect what is known about PDE activity in these cells. Unfortunately, there is little information available on how the different PDE tsoforms are distributed. Biochemical studies have found evidence for all three isoforms in both soluble and paniculate fractions of cell homogenates, and while the PDE activity in particulate fractions may be membrane associated, there is little or no information on how much of that might be caveolar and/or extracaveolar. Fortunately, the behaviors described by this model did not depend critically on exactly which PDE isoforms were present in each compartment. Another reason to include multiple PDE isoforms in the model is thai each can be regulated in unique ways that may be necessary to explain other types of responses. For example, PDE4 can be activated by PKA (61). Previous work has suggested that this is involved in a feedback mechanism that can modulate the magnitude of cAMP responses detected by exogenously expressed cyclic nucleotide gated (CNG) ion channels (62). However, the subcellular location where this positive feedback activation of PDE4 may be occurring is not clear. Although it is obvious that CNG channels detect responses near the plasma membrane, they are expressed primarily in noncaveolar lipid rafts (63), and how this may relate to compartments included in this model is not known.

The parameters about which we have the least information relate to the flux of cAMP within a cell. Movement of cAMP between compartments is often thought of as being limited by functional barriers associated with PDE activity (7). In this model, the role of PDE activity in regulating the flux between compartments is actually through its contribution to the concentration of cAMP in each compartment, and therefore the concentration gradient driving cAMP movement between compartments. Flux of cAMP between compartments is also a function of physical factors affecting diffusion of cAMP down ils concentration gradient. If it is assumed that there are no physical barriers and cAMP can move between compartments at rates thai approximate free diffusion, all of the behaviors attributed to compartmentation by this model are lost. This includes the gradient of cAMP between the caveolar and extracaveolar domain under basal conditions, as well as the ACh-induced rebound stimulatory response. The complex structure of cardiac ventricular myocytes has been shown to restrict access to submembrane regions and is likely to be an important factor influencing cAMP diffusion (64), but the actual nature of the barriers between compartments proposed in the model is not known. The flux rates predicted by this model indicate that cAMP movement is far slower than free diffusion. Validation of these values awaits development of an approach to experimentally determine c AMP flux rates between specific microdomains in intact myocytes. It is noteworthy that Rich et al. (65) also found evidence for extremely slow exchange of cAMP among compartments in HEK 293 cells, indicating that limited diffusion between microdomains is not unique to cardiac myocytes. More recently, Saucerman el al. (66) have demonstrated that limited diffusion of cAMP also occurs at the macroscopic level in neonatal ventricular myocytes.

Future directions

This version of the model only contains β^sub 1^ARs, whereas most cardiac preparations express both β^sub 1^ and β^sub 2^ARs. However, β^sub 1^ARs make up ~80% of the total βAR population, and in normal hearts β^sub 2^ARs do not contribute significantly to the functional responses of ventricular myocytes to nonselective agonists such as Iso. Furthermore, the model's predictions were validated by comparison to responses obtained from guinea pig ventricular myocytes, which unlike most cardiac preparations, do not express functional β^sub 2^ARs (67). In cardiac preparations where they do exist, β^sub 2^ARs may play a more important role in regulating cardiac function under conditions such as heart failure. Interestingly, selective activation of β^sub 2^ARs produces compartmentalized cAMPdependent responses. Future incorporation of β^sub 2^ARs into the model may provide a useful means of evaluating the potential mechanism(s) responsible for that form of compartmentation. Another key future direction for the model is inclusion of signaling mechanisms downstream of cAMP.

CONCLUSIONS

This work supports the idea that compartmentation of cAMP signaling plays a critical role in shaping cAMP-dependent effects under basal conditions, as well as in response to β^sub 1^AR and M^sub 2^R activation. This includes both the inhibitory and stimulatory responses associated with M^sub 2^R activation.

[Reference]

This work was supported by grants from the National Institutes of Health Heart. Lung and Blood Institute, and the American Heart Association. R.V.I. was the recipient of a predoclonu fellowship from the Ohio Valley Affiliate of the American Heart Association.

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[Author Affiliation]

Radu V. lancu, Stephen W. Jones, and Robert D. Harvey

Department of Physiology and Biophysics, case Western Reserve University, Cleveland, Ohio

[Author Affiliation]

Submitted August 17, 2006. and accepted for publication January 17, 2007.

Address reprint requests to Robert D. Harvey, Dept. of Physiology and Biophysics, case Western Reserve University. 10900 Euclid Ave., Cleveland, OH 44106. Tel.: 216-368-5521 ; Fax: 216-368-3952; E-mail: rdh3@case.edu.