Distinct platelet thromboxane A2/prostaglandin H2 receptor subtypes. A radioligand binding study of human platelets.

Thromboxane A2 (TXA2) and prostaglandin H2 (PGH2) may aggregate platelets via a common membrane receptor(s). To further characterize this receptor, binding of the radiolabeled TXA2/PGH2 mimetic [125I]BOP to washed human platelets (WP) was investigated. [125I]BOP was competitively displaced from its platelet binding site by stable TXA2/PGH2 analogues. Competition curves were shallow with Hill coefficients of -0.73 +/- 0.05 (P less than 0.001 different from unity) (90 +/- 1% specific binding). Scatchard plots were curvilinear and most consistent with two binding sites; a high-affinity site with Kd of 234 +/- 103 pM, Bmax of 0.7 +/- 0.3 pM/mg protein (180 +/- 87 sites/WP), and a lower affinity site with Kd of 2.31 +/- 0.86 nM, Bmax of 2.2 +/- 0.3 pM/mg protein (666 +/- 65 sites/WP). [125I]BOP association and dissociation kinetics gave a Kd of 157 pM without evidence of negative cooperativity. The EC50 for I-BOP-induced initial Ca2+ increase was 209 +/- 24 pM, shape change was 263 +/- 65 pM, and aggregation was 4.4 +/- 0.5 nM. Parallel binding studies using the TXA2/PGH2 receptor antagonist [125I]PTA-OH showed a single binding site. The rank order for TXA2/PGH2 analogues to displace [125I]PTA-OH was identical to that for [125I]BOP. These studies indicate that [125I]BOP binds to two distinct sites on human platelets that may represent platelet TXA2/PGH2 receptor subtypes. The close correlation of IC50 values for I-BOP-induced platelet shape change and aggregation with the two Kds for [125I]BOP binding suggests that these platelet responses may be independently mediated by the two putative receptors.

Cardiac-specific overexpression of phospholamban alters calcium kinetics and resultant cardiomyocyte mechanics in transgenic mice.

Phospholamban is the regulator of the cardiac sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity and an important modulator of basal contractility in the heart. To determine whether all the SR Ca(2+)-ATPase enzymes are subject to regulation by phospholamban in vivo, transgenic mice were generated which overexpressed phospholamban in the heart, driven by the cardiac-specific alpha-myosin heavy chain promoter. Quantitative immunoblotting revealed a twofold increase in the phospholamban protein levels in transgenic hearts compared to wild type littermate hearts. The transgenic mice showed no phenotypic alterations and no changes in heart/body weight, heart/lung weight, and cardiomyocyte size. Isolated unloaded cardiac myocytes from transgenic mice exhibited diminished shortening fraction (63%) and decreased rates of shortening (64%) and relengthening (55%) compared to wild type (100%) cardiomyocytes. The decreases in contractile parameters of transgenic cardiomyocytes reflected decreases in the amplitude (83%) of the Ca2+ signal and prolongation (131%) in the time for decay of the Ca2+ signal, which was associated with a decrease in the apparent affinity of the SR Ca(2+)-ATPase for Ca2+ (56%), compared to wild type (100%) cardiomyocytes. In vivo analysis of left ventricular systolic function using M mode and pulsed-wave Doppler echocardiography revealed decreases in fractional shortening (79%) and the normalized mean velocity of circumferential shortening (67%) in transgenic mice compared to wild type (100%) mice. The differences in contractile parameters and Ca2+ kinetics in transgenic cardiomyocytes and the depressed left ventricular systolic function in transgenic mice were abolished upon isoproterenol stimulation. These findings indicate that a fraction of the Ca(2+)-ATPases in native SR is not under regulation by phospholamban. Expression of additional phospholamban molecules results in: (a) inhibition of SR Ca2+ transport; (b) decreases in systolic Ca2+ levels and contractile parameters in ventricular myocytes; and (c) depression of basal left ventricular systolic function in vivo.

Cardiac-specific overexpression of RhoA results in sinus and atrioventricular nodal dysfunction and contractile failure.

RhoA is a low-molecular-weight GTPase that has been implicated in the regulation of hypertrophic cardiac muscle cell growth. To study the role of RhoA in control of cardiac function in vivo, transgenic mice expressing wild-type and constitutively activated forms of RhoA under the control of the cardiac-specific alpha-myosin heavy chain promoter were generated. Transgene-positive mice expressing high levels of either wild-type or activated RhoA showed pronounced atrial enlargement and manifested a lethal phenotype, often preceded by generalized edema, with most animals dying over the course of a few weeks. Echocardiographic analysis of visibly healthy wild-type RhoA transgenic mice revealed no significant change in left ventricular function. As their condition deteriorated, significant dilation of the left ventricular chamber and associated decreases in left ventricular contractility were detected. Heart rate was grossly depressed in both wild-type and activated RhoA-expressing mice, even prior to the onset of ventricular failure. Electrocardiography showed evidence of atrial fibrillation and atrioventricular block. Interestingly, muscarinic receptor blockade with atropine did not elicit a positive chronotropic response in the transgenic mice. We suggest that RhoA regulates cardiac sinus and atrioventricular nodal function and that its overexpression results in bradycardia and development of ventricular failure.

Abnormal bone growth and selective translational regulation in basic fibroblast growth factor (FGF-2) transgenic mice.

Basic fibroblast growth factor (FGF-2) is a pleiotropic growth factor detected in many different cells and tissues. Normally synthesized at low levels, FGF-2 is elevated in various pathologies, most notably in cancer and injury repair. To investigate the effects of elevated FGF-2, the human full-length cDNA was expressed in transgenic mice under control of a phosphoglycerate kinase promoter. Overexpression of FGF-2 caused a variety of skeletal malformations including shortening and flattening of long bones and moderate macrocephaly. Comparison by Western blot of FGF-2 transgenic mice to nontransgenic littermates showed expression of human FGF-2 protein in all major organs and tissues examined including brain, heart, lung, liver, kidney, spleen, and skeletal muscle; however, different molar ratios of FGF-2 protein isoforms were observed between different organs and tissues. Some tissues preferentially synthesize larger isoforms of FGF-2 while other tissues produce predominantly smaller 18-kDa FGF-2. Translation of the high molecular weight isoforms initiates from unconventional CUG codons and translation of the 18-kDa isoform initiates from an AUG codon in the FGF-2 mRNA. Thus the Western blot data from the FGF-2 transgenic mice suggest that tissue-specific expression of FGF-2 isoforms is regulated translationally.

Calcineurin-mediated hypertrophy protects cardiomyocytes from apoptosis in vitro and in vivo: An apoptosis-independent model of dilated heart failure.

We have previously shown that the calcium-calmodulin-regulated phosphatase calcineurin (PP2B) is sufficient to induce cardiac hypertrophy that transitions to heart failure in transgenic mice. Given the rapid onset of heart failure in these mice, we hypothesized that calcineurin signaling would stimulate myocardial cell apoptosis. However, utilizing multiple approaches, we determined that calcineurin-mediated hypertrophy protected cardiac myocytes from apoptosis, suggesting a model of heart failure that is independent of apoptosis. Adenovirally mediated gene transfer of a constitutively active calcineurin cDNA (AdCnA) was performed in cultured neonatal rat cardiomyocytes to elucidate the mechanism whereby calcineurin affected myocardial cell viability. AdCnA infection, which induced myocyte hypertrophy and atrial natriuretic factor expression, protected against apoptosis induced by 2-deoxyglucose or staurosporine, as assessed by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) labeling, caspase-3 activation, DNA laddering, and cellular morphology. The level of protection conferred by AdCnA was similar to that of adenoviral Bcl-x(L) gene transfer or hypertrophy induced by phenylephrine. In vivo, failing hearts from calcineurin-transgenic mice did not demonstrate increased TUNEL labeling and, in fact, demonstrated a resistance to ischemia/reperfusion-induced apoptosis. We determined that the mechanism whereby calcineurin afforded protection from apoptosis was partially mediated by nuclear factor of activated T cells (NFAT3) signaling and partially by Akt/protein kinase B (PKB) signaling. Although calcineurin activation protected myocytes from apoptosis, inhibition of calcineurin with cyclosporine was not sufficient to induce TUNEL labeling in Gqalpha-transgenic mice or in cultured cardiomyocytes. Collectively, these data identify a calcineurin-dependent mouse model of dilated heart failure that is independent of apoptosis.

Cardiotrophic effects of protein kinase C epsilon: analysis by in vivo modulation of PKCepsilon translocation.

Protein kinase C (PKC) is a key mediator of many diverse physiological and pathological responses. Although little is known about the specific in vivo roles of the various cardiac PKC isozymes, activation-induced translocation of PKC is believed to be the primary determinant of isozyme-specific functions. Recently, we have identified a catalytically inactive peptide translocation inhibitor (epsilonV1) and translocation activator (psiepsilonRACK [receptors for activated C kinase]) specifically targeting PKCepsilon. Using cardiomyocyte-specific transgenic expression of these peptides, we combined loss- and gain-of-function approaches to elucidate the in vivo consequences of myocardial PKCepsilon signaling. As expected for a PKCepsilon RACK binding peptide, confocal microscopy showed that epsilonV1 decorated cross-striated elements and intercalated disks of cardiac myocytes. Inhibition of cardiomyocyte PKCepsilon by epsilonV1 at lower expression levels upregulated alpha-skeletal actin gene expression, increased cardiomyocyte cell size, and modestly impaired left ventricular fractional shortening. At high expression levels, epsilonV1 caused a lethal dilated cardiomyopathy. In contrast, enhancement of PKCepsilon translocation with psiepsilonRACK resulted in selectively increased beta myosin heavy chain gene expression and normally functioning concentric ventricular remodeling with decreased cardiomyocyte size. These results identify for the first time a role for PKCepsilon signaling in normal postnatal maturational myocardial development and suggest the potential for PKCepsilon activators to stimulate "physiological" cardiomyocyte growth.

Increased myocardial Rab GTPase expression: a consequence and cause of cardiomyopathy.

The Ras-like Rab GTPases regulate vesicle transport in endocytosis and exocytosis. We found that cardiac Rabs1, 4, and 6 are upregulated in a dilated cardiomyopathy model overexpressing beta(2)-adrenergic receptors. To determine if increased Rab GTPase expression can contribute to cardiomyopathy, we transgenically overexpressed in mouse hearts prototypical Rab1a, the small G protein that regulates vesicle transport from endoplasmic reticulum to and through Golgi. In multiple independent mouse lines, Rab1a overexpression caused cardiac hypertrophy that progressed in a time- and transgene dose-dependent manner to heart failure. Isolated cardiac myocytes were hypertrophied and exhibited contractile depression with impaired calcium reuptake. Ultrastructural analysis revealed enlarged Golgi stacks and increased transitional vesicles in ventricular myocytes, with increased secretory atrial natriuretic peptide granules and degenerative myelin figures in atrial myocytes; immunogold studies localized Rab1a to these abnormal vesicular structures. A survey of hypertrophy signaling molecules revealed increased protein kinase C (PKC) alpha and delta, and confocal microscopy showed abnormal subcellular distribution of PKCalpha in Rab1a transgenics. These results indicate that increased expression of Rab1 GTPase in myocardium distorts subcellular localization of proteins and is sufficient to cause cardiac hypertrophy and failure.

Hearts deficient in both Mfn1 and Mfn2 are protected against acute myocardial infarction.

Mitochondria alter their shape by undergoing cycles of fusion and fission. Changes in mitochondrial morphology impact on the cellular response to stress, and their interactions with other organelles such as the sarcoplasmic reticulum (SR). Inhibiting mitochondrial fission can protect the heart against acute ischemia/reperfusion (I/R) injury. However, the role of the mitochondrial fusion proteins, Mfn1 and Mfn2, in the response of the adult heart to acute I/R injury is not clear, and is investigated in this study. To determine the effect of combined Mfn1/Mfn2 ablation on the susceptibility to acute myocardial I/R injury, cardiac-specific ablation of both Mfn1 and Mfn2 (DKO) was initiated in mice aged 4-6 weeks, leading to knockout of both these proteins in 8-10-week-old animals. This resulted in fragmented mitochondria (electron microscopy), decreased mitochondrial respiratory function (respirometry), and impaired myocardial contractile function (echocardiography). In DKO mice subjected to in vivo regional myocardial ischemia (30 min) followed by 24 h reperfusion, myocardial infarct size (IS, expressed as a % of the area-at-risk) was reduced by 46% compared with wild-type (WT) hearts. In addition, mitochondria from DKO animals had decreased MPTP opening susceptibility (assessed by Ca(2+)-induced mitochondrial swelling), compared with WT hearts. Mfn2 is a key mediator of mitochondrial/SR tethering, and accordingly, the loss of Mfn2 in DKO hearts reduced the number of interactions measured between these organelles (quantified by proximal ligation assay), attenuated mitochondrial calcium overload (Rhod2 confocal microscopy), and decreased reactive oxygen species production (DCF confocal microscopy) in response to acute I/R injury. No differences in isolated mitochondrial ROS emissions (Amplex Red) were detected in response to Ca(2+) and Antimycin A, further implicating disruption of mitochondria/SR tethering as the protective mechanism. In summary, despite apparent mitochondrial dysfunction, hearts deficient in both Mfn1 and Mfn2 are protected against acute myocardial infarction due to impaired mitochondria/SR tethering.

Response of a human megakaryocytic cell line to thrombin: increase in intracellular free calcium and mitogen release.

The CHRF-288-11 cell line has been previously shown to exhibit properties consistent with a megakaryocytic origin. The response of these cells to thrombin has now been investigated. Thrombin treatment of CHRF-288-11 cells results in both an increase in intracellular free calcium levels and secretion of mitogenic activity and beta-thromboglobulin. Cell viability is not affected. The mitogenic activity released from the cells is due primarily to the presence of basic fibroblast growth factor. Immunohistochemical data indicate a packaging of basic fibroblast growth factor into granular structures. Trypsin and phorbol 12-myristate 13-acetate also initiate release of mitogenic activity from this cell line, whereas under non-stirred conditions collagen and ADP do not. Through measurements of intracellular calcium levels it was determined that thrombin pretreatment of cells ablates a further response to thrombin, but does not block an increase in intracellular calcium levels due to trypsin. This suggests that these two agonists may act through different mechanisms. The thrombin-induced release reaction is inhibited almost completely by the reagents hirudin and dipyridamole, and only partially by indomethacin. These data indicate that the CHRF-288-11 cell line should provide an excellent model system in which to study the packaging of factors into granules which undergo regulated release.

Early and delayed consequences of beta(2)-adrenergic receptor overexpression in mouse hearts: critical role for expression level.

BACKGROUND: Transgenic cardiac beta(2)-adrenergic receptor (AR) overexpression has resulted in enhanced signaling and cardiac function in mice, whereas relatively low levels of transgenically expressed G(alphas) or beta(1)AR have resulted in phenotypes of ventricular failure. Potential relationships between the levels of betaAR overexpression and biochemical, molecular, and physiological consequences have not been reported. METHODS AND RESULTS: We generated transgenic mice expressing beta(2)AR at 3690, 7120, 9670, and 23 300 fmol/mg in the heart, representing 60, 100, 150, and 350 times background betaAR expression. All lines showed enhanced basal adenylyl cyclase activation but a decrease in forskolin- and NaF-stimulated adenylyl cyclase activities. Mice of the highest-expressing line developed a rapidly progressive fibrotic dilated cardiomyopathy and died of heart failure at 25+/-1 weeks of age. The 60-fold line exhibited enhanced basal cardiac function without increased mortality when followed for 1 year, whereas 100-fold overexpressors developed a fibrotic cardiomyopathy and heart failure, with death occurring at 41+/-1 weeks of age. Adenylyl cyclase activation did not correlate with early or delayed decompensation. Propranolol administration reduced baseline +dP/dt(max) to nontransgenic levels in all beta(2)AR transgenics except the 350-fold overexpressors, indicating that spontaneous activation of beta(2)AR was present at this level of expression. CONCLUSIONS: These data demonstrate that the heart tolerates enhanced contractile function via 60-fold beta(2)AR overexpression without detriment for a period of >/=1 year and that higher levels of expression result in either aggressive or delayed cardiomyopathy. The consequences for enhanced betaAR function in the heart appear to be highly dependent on which signaling elements are increased and to what extent.

Mouse model of desmin-related cardiomyopathy.

BACKGROUND: The consequence of upregulation of desmin in the heart is unknown. Mutations in desmin have been linked to desmin-related myopathy (DRM), which is characterized by abnormal intrasarcoplasmic accumulation of desmin, but direct causative evidence that a desmin mutation leads to aberrant intrasarcoplasmic desmin accumulation, aggregation, and cardiomyopathy is lacking. METHODS AND RESULTS: Multiple transgenic mouse lines that expressed either murine wild-type desmin or a 7-amino acid deletion (R173 through E179) desmin (D7-des) mutation linked to DRM were made. The distribution of desmin protein was unchanged, and no overt phenotype was detected in the wild-type desmin transgenic mice. In contrast, the D7-des mouse heart showed aberrant intrasarcoplasmic and electron-dense granular filamentous aggregates that were desmin-positive and characteristic of human DRM. The desmin filament network was significantly disrupted, and myofibril alignment was visibly compromised. Although systolic function at the whole-organ level was substantially conserved in the young adult animals, the ability of the heart to respond to beta-agonist stimulation, as measured in the intact animal, was significantly blunted. CONCLUSIONS: Upregulation of desmin protein at moderate levels is not detrimental. However, the D7-des mutation is dominant negative, and expression of the mutant protein leads to the appearance of aggregates that are characteristic of and diagnostic for human desmin-related cardiomyopathy.

Interactions between phospholamban and beta-adrenergic drive may lead to cardiomyopathy and early mortality.

BACKGROUND: Relieving the inhibition of sarcoplasmic reticular function by phospholamban is a major target of beta-adrenergic stimulation. Chronic beta-adrenergic receptor activity has been suggested to be detrimental, on the basis of transgenic overexpression of the receptor or its signaling effectors. However, it is not known whether physiological levels of sympathetic tone, in the absence of preexisting heart failure, are similarly detrimental. METHODS AND RESULTS: Transgenic mice overexpressing phospholamban at 4-fold normal levels were generated, and at 3 months, they exhibited mildly depressed ventricular contractility without heart failure. As expected, transgenic cardiomyocyte mechanics and calcium kinetics were depressed, but isoproterenol reversed the inhibitory effects of phospholamban on these parameters. In vivo cardiac function was substantially depressed by propranolol administration, suggesting enhanced sympathetic tone. Indeed, plasma norepinephrine levels and the phosphorylation status of phospholamban were elevated, reflecting increased adrenergic drive in transgenic hearts. On aging, the chronic enhancement of adrenergic tone was associated with a desensitization of adenylyl cyclase (which intensified the inhibitory effects of phospholamban), the development of overt heart failure, and a premature mortality. CONCLUSIONS: The unique interaction between phospholamban and increased adrenergic drive, elucidated herein, provides the first evidence that compensatory increases in catecholamine stimulation can, even in the absence of preexisting heart failure, be a primary causative factor in the development of cardiomyopathy and early mortality.

Supernormal ejection performance is isolated to the ipsilateral congenitally pressure-overloaded ventricle.

Congenital left ventricular pressure overload is associated with "excessive" hypertrophy that leads to subnormal afterload (wall stress), permitting enhanced ventricular ejection performance. Whether congenital right ventricular pressure overload is associated with a similar phenomenon is uncertain. It is also unknown whether supranormal ejection performance affects only the overloaded ventricle or is a general process affecting both ventricles. Conflicting data exist about whether the hypertrophic process associated with pressure overload is induced primarily by local loading conditions or by neuroendocrine influences. If the former postulate is true, the hypertrophic response should be confined to the overloaded ventricle; if the latter is true, one might predict that both ventricles would be affected by a less specific response to circulating catecholamines. To help resolve these issues, both right and left ventricular performance was examined in seven patients with isolated congenital pulmonary stenosis (average pulmonary pressure gradient 78 +/- 13 mm Hg), six patients with isolated congenital aortic stenosis (average gradient 80 +/- 10 mm Hg) and six normal subjects. Right ventricular ejection fraction was increased in patients with pulmonary stenosis (61 +/- 2%) compared with the value in normal subjects (53 +/- 2%, p less than 0.01) and in patients with aortic stenosis (50 +/- 3%, p = 0.007). Left ventricular ejection fraction was increased in patients with congenital aortic stenosis (84 +/- 4%) compared with the value in normal subjects (70 +/- 4%, p less than 0.01) and in patients with congenital pulmonary stenosis (65 +/- 2%, p less than 0.002).(ABSTRACT TRUNCATED AT 250 WORDS)

Gq signaling in cardiac adaptation and maladaptation.

Accumulating evidence suggests that cardiac responses to a number of circulating or locally released humoral factors contribute to adaptive responses after hemodynamic stress or myocardial injury. In particular, hormones such as angiotensin II, endothelin 1, norepinephrine and prostaglandin F2 alpha which bind to and activate cardiomyocyte membrane receptors coupled to the Gq class of GTP binding proteins have been implicated in the development and ultimate decompensation of cardiac hypertrophy. Herein we summarize recent developments in cultured cardiomyocyte and transgenic mouse systems which are defining the phenotypes resulting from Gq signaling events in cardiomyocytes, and which are elucidating the critical downstream mediators. Postulated roles for protein kinase C, p38 MAP kinase and jun-N terminal kinase are discussed in relation to Gq-mediated cardiomyocyte hypertrophy and apoptotic signaling. The evidence to date suggests that molecular targeting of Gq or its effectors has the potential to modify cardiac adaptive and maladaptive responses to stress or injury.

Divergent transcriptional responses to independent genetic causes of cardiac hypertrophy.

To define molecular mechanisms of cardiac hypertrophy, genes whose expression was perturbed by any of four different transgenic mouse hypertrophy models [protein kinase C-epsilon activation peptide (PsiepsilonRACK), calsequestrin (CSQ), calcineurin (CN), and Galpha(q)] were compared by DNA microarray analyses using the approximately 8,800 genes present on the Incyte mouse GEM1. The total numbers of regulated genes (tens to hundreds) correlated with phenotypic severity of the model (Galpha(q) > CN > CSQ > PsiepsilonRACK), but demonstrated that no single gene was consistently upregulated. Of the three models exhibiting pathological hypertrophy, only atrial natriuretic peptide was consistently upregulated, suggesting that transcriptional alterations are highly specific to individual genetic causes of hypertrophy. However, hierarchical-tree and K-means clustering analyses revealed that subsets of the upregulated genes did exhibit coordinate regulatory patterns that were unique or overlapping across the different hypertrophy models. One striking set consisted of apoptotic genes uniquely regulated in the apoptosis-prone Galpha(q) model. Thus, rather than identifying a single common hypertrophic cardiomyopathy gene program, these data suggest that extensive groups of genes may be useful for the prediction of specific underlying genetic determinants and condition-specific therapeutic approaches.

Tyrosine kinase inhibition prevents deformation-stimulated vascular smooth muscle growth.

The goal of this study was to determine the role of tyrosine phosphorylation in transducing deformation-stimulated vascular smooth muscle growth. Rat aorta-derived vascular smooth muscle cells were cultured on flexible silicone elastomer membranes and subjected to cyclic deformation (15 cycles per minute, deformed 2 seconds, relaxed 2 seconds). Deformation significantly increased proto-oncogene expression, [3H]thymidine incorporation, [3H]leucine incorporation, and cell number. Time course studies showed an 8-hour lag between initiation of cell deformation and onset of [3H]thymidine incorporation, with peak levels achieved after 18 to 24 hours. Western analysis of protein blots from deformed cells (10 minutes) demonstrated increased levels of phosphotyrosine-containing proteins having molecular weights of 110 to 130 and 70 to 80 kD. Deformation-stimulated tyrosine phosphorylation was prevented by the tyrosine kinase inhibitor Herbimycin A. Tyrosine kinase inhibition also prevented deformation-stimulated vascular smooth muscle cell growth as measured by [3H]thymidine incorporation. Cyclic deformation stimulates vascular smooth muscle proliferation through activation of tyrosine kinases. Inhibition of tyrosine phosphorylation is an effective means of preventing deformation-induced vascular smooth muscle growth in vitro.

Decrease in agonist affinity for human platelet thromboxane A2/prostaglandin H2 receptors induced by a platelet-derived supernatant.

Platelets possess membrane receptors which mediate the aggregatory response to thromboxane A2 (TXA2) and prostaglandin H2 (PGH2). It has been observed recently that the affinities for a series of TXA2/PGH2 mimetics are decreased in crude human platelet membranes and solubilized membranes compared to intact washed platelets. The present study investigated the notion that platelets contain a substance that is released during platelet lysis that reduces the affinity of the TXA2/PGH2 receptor for agonists. The displacement of 9,11-dimethylmethano-11,12-methano-16-(3-iodo-4-hydroxyphenyl)-13, 14-dihydro-13 - aza-15 alpha beta-omega-tetranor-TXA2 ([125I]PTA-OH), a TXA2/PGH2 receptor antagonist, from its binding site in intact washed platelets by TXA2/PGH2 mimetics and antagonists was characterized in the presence or absence of the supernatant (50,000 g) obtained from sonicated platelets. In the presence of the supernatant, there was a significant (P less than 0.025) increase in the IC50 values for the TXA2/PGH2 mimetics U46619, SQ26655, and ONO11113. The increase in the IC50 for U46619 induced by the supernatant was abolished by either boiling or treating the supernatant with trypsin. The supernatant did not affect the Kd or Bmax of [125I]PTA-OH or the IC50 of the TXA2/PGH2 antagonist, SQ29548. Pretreatment of the platelets with the supernatant resulted in a significant (P less than 0.02) reduction in the aggregation response induced by U46619. Gel filtration (Sephacryl S200) of the supernatant revealed a fraction (molecular weight approximately 100,000 daltons) which significantly increased the IC50 for U46619 to displace [125I]PTA-OH from its binding site. Thus, human platelets appear to possess a protein(s) that is released into the supernatant upon sonication and inhibits the binding of TXA2/PGH2 agonists but not antagonists to their receptor. This protein may play a role in the regulation of platelet responses to the aggregatory stimuli TXA2/PGH2.

Left ventricular dysfunction in symptomatic mitral valve prolapse.

Idiopathic MVP is characterized by a late systolic click or murmur from myxomatous mitral valvular dysfunction. It may be complicated by atypical chest pain, ventricular arrhythmias, and ECG changes that can mimic the symptoms of coronary artery disease. We prospectively performed radionuclide cineangiograms before and after stress tests in MVP patients with chest pain compared with asymptomatic MVP patients and symptomatic normal control patients. In ten patients with MVP, chest pain, and normal coronary anatomy, the LVEF remained essentially unchanged (increase of -0.5 +/- 4 percent) after exercise. In ten patients with MVP and no chest pain and in nine with normal cardiovascular system and chest pain, the exercise LVEF increased by 11.5 +/- 2 percent (p less than 0.05) and 17.4 +/- 3 percent (p less than 0.005), respectively. The resting LVEF was significantly lower (p less than 0.02) in the symptomatic MVP patients (59 +/- 3 percent) than in the asymptomatic MVP (76 +/- 5 percent) or symptomatic normal patient control subjects (70 +/- 3 percent). Patients with MVP and chest pain had a lower resting LVEF and an abnormal left ventricular functional response to exercise compared with asymptomatic MVP patients or symptomatic normal subjects. Therefore, exercise radionuclide ventriculography may not adequately differentiate between chest pain due to MVP or coronary artery disease.