[Hemolytic anemia due to silent left ventricular rupture after stent thrombosis]. / Hämolytische Anämie bei stummer gedeckter Perforation der linksventrikulären Vorderwand nach Stent-Thrombose.

A 51-year-old woman with known ischemic cardiomyopathy presented to the family doctor for investigation of mild anemia. A routinely performed computed tomography of the abdomen resulted in the suspicion of intracardiac thrombi. The subsequent cardiac examinations showed a pseudoaneurysm of the left ventricle as a result of a silent rupture of a pre-existing left ventricular aneurysm.

Expression of nitric oxide related enzymes in coronary heart disease.

Enzymes involved in the metabolism nitric oxide (NO) and reactive oxygen species (ROS) may play a role for the decreased availability of NO in atherosclerosis. We, therefore, hypothesized that the pattern of gene expression of these enzymes is altered in atherosclerosis. Myocardial tissue from patients with coronary heart disease (CHD) or without CHD (control group) was investigated. The level of enzymes related to NO/ROS metabolism was determined both at mRNA level and protein level by rt-PCR, real-time PCR, and western blot. The expression of NOS1-3 (synthesis of NO), arginase1 (reduction of L-arginine), p22phox (active subunit of NADPH oxidase), GTPCH (rate limiting enzyme for tetrahydrobiopterin), SOD1-3 (scavengers of superoxide anions), PRTMT1-3, and DDAH2 (involved in the metabolism of ADMA) was determined. All enzymes were found to be expressed in human myocardium. NOS isoforms were decreased in CHD in protein level, but only the downregulation of NOS3 expression reached statistical significance. The expression of PRMT1 and PRMT3 was increased. In addition, the expression of DDAH2 was reduced, both theoretically leading to an increase of ADMA concentration. SOD3 was downregulated in tissue from patients with CHD. Taken together, in myocardial tissue from patients with atherosclerosis, the expression of genes increasing ADMA levels is enhanced in contrast to a reduced expression of genes promoting NO synthesis. These results may contribute to the explanation of increased oxidative stress in atherosclerosis on the level of gene expression.

Upregulation of ID protein by growth and differentiation factor 5 (GDF5) through a smad-dependent and MAPK-independent pathway in HUVSMC.

GDF5 (growth and differentiation factor five), a member of the TGF-beta superfamily, binds specifically to BMPR1b, BMPR2 and ACTR2a receptors forming a heterodimeric complex, thereby inducing phosphorylation of smad1, 5, 8 and translocation to the nucleus. ID1 (inhibitor of differentiation or DNA binding) is essential for G1 to S phase transition inhibiting DNA binding thereby playing an important role in the control of differentiation, proliferation and angiogenesis. The objective of this study was, therefore, to characterize the signal transduction pathway of GDF5, especially the involvement of ID1, in human umbilical vein smooth muscle cells (HUVSMC). We observed the expression of BMPR1a, BMPR1b, BMPR2, ACTR2a, smad1, smad 5, ID1, ID2 and ID3 in HUVSMC. Application of GDF5 upregulated ID1 and ID3 expression by involvement of the smad signaling pathway. GDF5 caused phorsphorylation of smad1 followed by upregulation of ID1 and ID3. Co-incubation with anti-GDF5 prevented these effects. GDF5 significantly inhibited phosphorylation of p38 MAPK and induced phosphorylation of ERK. The specific inhibitor of p38 MAPK or ERK, SB203580 or U0126 did not induce ID protein expression. Smad1 siRNA transfection inhibited the upregulation of ID protein. GDF5 had chemotactic activity in HUVSMC; this effect was partly blocked by transfection of smad1 or ID1 siRNA. Our results indicate that GDF5 induces ID1 and ID3 in HUVSMC by a smad-dependent, MAPK-independent pathway. GDF5 binds to specific receptors, thereby inducing phosphorylation and translocation of smad1 to the nucleus where it is involved in the regulation of transcription. Since ID1 has been shown to be crucial for cell cycle control, we propose that GDF5 could be involved in the process of angiogenesis.

Influence of alcohol consumption on restenosis rate after percutaneous transluminal coronary angioplasty and stent implantation.

OBJECTIVE: To disclose possible influences of alcoholic beverages on restenosis rate in men with coronary artery disease treated with percutaneous transluminal coronary angioplasty (PTCA) and stent implantation. DESIGN: Retrospective cohort study. PATIENTS: 225 consecutive male patients underwent PTCA and stent implantation. All patients had a control angiography and were contacted for a questionnaire regarding their drinking habits. MAIN OUTCOME MEASURES: Mean late loss of luminal diameter, rate of coronary restenosis of 50% or more within the stented segment, and rate of repeat angioplasty. RESULTS: 53 patients (with 80 stents) consumed < 50 g of alcohol a week and 172 (with 266 stents) consumed more (50-700 g a week). Baseline characteristics were similar in both groups except for a higher prevalence of reduced cardiac function and multivessel disease and a lower high density lipoprotein cholesterol concentration among patients who consumed little or no alcohol. Patients who consumed > or = 50 g alcohol a week had a lower mean late loss of the luminal diameter (1.1 (0.79) mm v 1.45 (0.82) mm, p = 0.002), a lower rate of coronary restenosis within the stented segment (33.7% v 48.8%, p = 0.001), and a lower rate of repeat angioplasty (23.3% v 42.5%, p = 0.002). In multivariate analysis, only alcohol consumption and diabetes were independent and significant discriminators for late loss of luminal diameter (p = 0.005 and p = 0.01, respectively), restenosis (odds ratio 0.54 and 2.08, respectively), and repeat angioplasty (odds ratio 0.39 and 2.18, respectively). CONCLUSION: Alcohol intake is associated with reduced restenosis after PTCA and stent implantation.

Comparative follow up of patients with implanted cardioverter-defibrillators after induction of sustained monomorphic ventricular tachycardias or ventricular fibrillation by programmed stimulation.

OBJECTIVE: To investigate the prognostic value of induced monomorphic ventricular tachycardia (VT) and ventricular flutter or fibrillation (VF) during programmed electrical stimulation in patients with a high risk for sudden arrhythmogenic cardiac death. DESIGN: Prospective cohort study. PATIENTS: 102 patients at high risk for arrhythmogenic sudden cardiac death who received an automated implantable cardioverter-defibrillator (AICD) were evaluated. 56 patients received the AICD for primary prevention and 46 for secondary prevention. 58 patients had induction of a monomorphic VT (VT group) and 44 had induction of a polymorphic VT, ventricular flutter, or ventricular fibrillation (VF group) during programmed electrical stimulation. Average follow up was 20 months in both groups. MAIN OUTCOME MEASURES: Appropriate AICD protocol. RESULTS: In patients who received the AICD for primary prevention, 16 of 32 patients in the VT group, compared with only four of 24 patients in the VF group, received an appropriate AICD protocol (p = 0.02). In the entire study population, 479 appropriate AICD protocols were recorded in 28 (48%) patients in the VT group and 28 appropriate protocols in 11 (25%) patients in the VF group. Cumulative Kaplan-Meier event-free survival curves were significantly different (p = 0.02). CONCLUSION: Induction of VF during programmed electrical stimulation is of no prognostic value even in high risk patients without previously documented ventricular fibrillation.

Reduction of myocardial infarct size by fluvastatin.

Statins have a variety of cardioprotective properties following chronic treatment. In contrast, little is known about the acute effects. Reperfusion acutely injures the heart by activation of neutrophils as well as endothelial cells. Because statins are known to influence the processes pathogenetically involved, we hypothesized that acute application of statins attenuates the sequelae of cardiac reperfusion. In rats, myocardial infarction (MI) was induced by ligature of the left coronary artery followed by reperfusion. Myocardial blood flow (MBF) was determined by H2 clearance and regional myocardial function (fractional thickening, FT) by pulsed Doppler. MI size was measured by triphenyltetrazolium chloride (TTC) staining, neutrophil extravasation by determination of myeloperoxidase (MPO) activity, and nitric oxide generation via measurement of cGMP. Treatment with fluvastatin, administered intravenously 20 min before the onset of ischemia, significantly attenuated the decline of FT and MBF at the end of the reperfusion period and significantly reduced MI size. Furthermore, fluvastatin induced a significant reduction of MPO activity and an increase of cGMP level compared with the control group. The effect of fluvastatin was completely abolished following pretreatment of NG-nitro-l-arginine methyl ester (l-NAME). These findings suggest that acute application of fluvastatin reduces MI size and attenuates reperfusion injury. We propose that the underlying mechanism is at least partially an inhibition of inflammation and endothelial dysfunction by preventing the activation and extravasation of neutrophils.

Role of human GTP cyclohydrolase I and its regulatory protein in tetrahydrobiopterin metabolism.

OBJECTIVE: GTP cyclohydrolase I (GTPCH I) catalyzes the de novo biosynthesis of tetrahydrobiopterin (BH(4)), an essential cofactor of NO-synthase. The enzyme underlies negative feedback regulation by the end product BH(4). This feedback inhibition is mediated through complex formation with the GTP cyclohydrolase I feedback regulatory protein (GFRP). To further classify the mechanism involved in the regulation of BH(4) synthesis, we measured expression of GTPCH I and GFRP in different human tissues. Furthermore, we looked for the influence of phenylalanine that is known to reverse BH(4)-mediated feedback inhibition of GTPCH I, and of immunostimulation with interferon gamma on the expression of GTPCH I and GFRP. METHODS AND RESULTS: Using RT-PCR and northern blot technique, coexpression of GFRP and GTPCH I could be demonstrated in a number of different tissues such as endothelial cells and peripheral blood cells. Following stimulation of human umbilical vein endothelial cells (HUVEC) with phenylalanine (1 mM), there was no change of GFRP mRNA. In contrast, the mRNA level of GTPCH I was significantly upregulated with a maximum after 6 hours (p = 0.04). Incubation of HUVEC with interferon-gamma (100 U/ml) showed an increase of GTPCH I mRNA and a significant downregulation of GFRP mRNA after 24 hours (p = 0.03). CONCLUSION: This study shows for the first time the expression of GFRP in different human tissues. The biosynthesis of BH(4) is not only regulated on the substrate level but also through transcription of the interacting proteins. Phenylalanine stimulates the biosynthesis of BH(4) not only by reversing the negative feedback inhibition of GTPCH I but also by increasing the mRNA level of GTPCH I. Immunostimulation alters protein expression of GTPCH I and GFRP in a way that favors BH(4) synthesis.

Electrophysiological characteristics and outcome in patients with idiopathic right ventricular arrhythmia compared with arrhythmogenic right ventricular dysplasia.

BACKGROUND: Idiopathic right ventricular arrhythmias (IRVA) are responsive to medical and ablative treatment and have a benign prognosis. Arrhythmias caused by right ventricular dysplasia (ARVD) are refractory to treatment and may cause sudden death. It is difficult to distinguish between these two types of arrhythmia. OBJECTIVE: To differentiate patients with IRVA and ARVD by a conventional electrophysiological study. METHODS: 56 patients with a right ventricular arrhythmia were studied. They had no history or signs of any cardiac disease other than right ventricular dysplasia. They were classified as having IRVA (n = 41) or ARVD (n = 15) on the basis of family history, ECG characteristics, and various imaging techniques. They were further investigated by standard diagnostic electrophysiology. RESULTS: The two groups were clearly distinguished by the electrophysiological study in the following ways: inducibility of ventricular tachycardia by programmed electrical stimulation with ventricular extrastimuli (IRVA 3% v ARVD 93%, p < 0.0001); presence of more than one ECG morphology during tachycardia (IRVA 0% v ARVD 73%, p < 0.0001); and fragmented diastolic potentials during ventricular arrhythmia (IRVA 0% v ARVD 93%, p < 0.0001). Data from the clinical follow up in these patients supported the diagnosis derived from the electrophysiological study. CONCLUSIONS: Patients with IRVA or ARVD can be distinguished by specific electrophysiological criteria. A diagnosis of ARVD can be made reliably on the basis of clinical presentation, imaging techniques, and an electrophysiological study.

S100A1: a regulator of myocardial contractility.

S100A1, a Ca(2+) binding protein of the EF-hand type, is preferentially expressed in myocardial tissue and has been found to colocalize with the sarcoplasmic reticulum (SR) and the contractile filaments in cardiac tissue. Because S100A1 is known to modulate SR Ca(2+) handling in skeletal muscle, we sought to investigate the specific role of S100A1 in the regulation of myocardial contractility. To address this issue, we investigated contractile properties of adult cardiomyocytes as well as of engineered heart tissue after S100A1 adenoviral gene transfer. S100A1 gene transfer resulted in a significant increase of unloaded shortening and isometric contraction in isolated cardiomyocytes and engineered heart tissues, respectively. Analysis of intracellular Ca(2+) cycling in S100A1-overexpressing cardiomyocytes revealed a significant increase in cytosolic Ca(2+) transients, whereas in functional studies on saponin-permeabilized adult cardiomyocytes, the addition of S100A1 protein significantly enhanced SR Ca(2+) uptake. Moreover, in Triton-skinned ventricular trabeculae, S100A1 protein significantly decreased myofibrillar Ca(2+) sensitivity ([EC(50%)]) and Ca(2+) cooperativity, whereas maximal isometric force remained unchanged. Our data suggest that S100A1 effects are cAMP independent because cellular cAMP levels and protein kinase A-dependent phosphorylation of phospholamban were not altered, and carbachol failed to suppress S100A1 actions. These results show that S100A1 overexpression enhances cardiac contractile performance and establish the concept of S100A1 as a regulator of myocardial contractility. S100A1 thus improves cardiac contractile performance both by regulating SR Ca(2+) handling and myofibrillar Ca(2+) responsiveness.

[Predictive value of frequency, duration and rate of ventricular salvos in ambulatory ECG for inducibility of sustained ventricular tachycardia]. / Prädiktiver Wert der Frequenz, der Dauer und der Häufigkeit ventrikulärer Salven im Langzeit-EKG für die Induzierbarkeit anhaltender Kammertachykardien.

Identification of high risk patients with coronary artery disease (CAD) prone to sudden cardiac death still remains a difficult issue. In 211 patients with CAD diagnosed by coronary angiography and documented non-sustained ventricular tachycardia (NSVT), programmed ventricular stimulation (PVS) was performed. NSVTs documented during Holter monitoring were analysed concerning frequency, duration and rate. To relate those parameters to the inducibility of sustained monomorphic ventricular tachycardias (MVT) during PVS, the total population was divided in different groups; patients with 1, 2-5 or > 5 salvos within 24 h; patients having salvos with a rate of > or = 150/min or < 150/min; patients with 3-5, 6-10 or > 10 consecutive extra beats. It could be demonstrated that in patients with CAD and NSVTs, induction of MVTs during PVS is more likely if the rate of the spontaneously occurring NSVT is > or = 150/min (22.1 vs 8.9%; p = 0.042). In contrast, there is apparently no correlation between the duration and incidence of NSVTs and the prevalence of MVTs during PVS. Multivariate analysis revealed the rate of documented NSVTs (odds ratio 2.98, p = 0.0314) and a decrease of left ventricular ejection fraction (odds ratio 1.69; p = 0.0013) as independent risk factors for the inducibility of MVTs. Conclusions CAD patients with fast salvos (> or = 150 beats/min) and reduced left ventricular ejection fraction are more likely to reveal inducible MVT during PVS and should, therefore, preferably be subjected to invasive risk stratification. The number of salvos per day and the number of consecutive beats, on the other hand, do not seem to be of relevant predictive value.

Increased activity of membrane-associated nucleoside diphosphate kinase and inhibition of cAMP synthesis in failing human myocardium.

OBJECTIVE: Chronic heart failure is associated with a decreased responsiveness of the heart to beta-adrenergic receptor agonists. We recently demonstrated a receptor-independent activation of G proteins and modulation of cardiac adenylyl cyclase activity by sarcolemmal membrane-associated nucleoside diphosphate kinase. We wondered whether changes in the activity of nucleoside diphosphate kinase occur in heart failure and contribute to or compensate for the impairment in myocardial receptor-mediated cAMP generation. METHODS: Sarcolemmal membranes were purified from non-failing and failing human left ventricular myocardium. The protein level and activity of nucleoside diphosphate kinase were quantified. The influence of nucleoside diphosphate kinase on adenylyl cyclase activity was determined by measuring the effect of GDP on adenylyl cyclase activity in the absence and presence of nucleoside diphosphate kinase inhibitors. RESULTS: The amount and activity of nucleoside diphosphate kinase in sarcolemmal membranes from failing hearts (n=13) were increased 3- to 4-fold compared to levels in membranes from non-failing myocardium (n=5). This increase in sarcolemmal nucleoside diphosphate kinase activity resulted in a 50% inhibition of adenylyl cyclase activity over a range of GDP and ATP concentrations. CONCLUSION: The amount and activity of nucleoside diphosphate kinase are increased in sarcolemmal membranes of failing human myocardium, resulting in a substantial receptor-independent inhibition of adenylyl cyclase activity.

Fate of bacterial pathogens and indicator organisms in liquid sweeteners.

The survival of pathogenic and indicator microorganisms in liquid sweeteners was studied. Seven sweeteners--liquid sucrose, 42% high-fructose corn syrup (HFCS), 55% HFCS, 25 DE (dextrose equivalent) corn syrup (CS), 36 DE CS, 63 DE CS, 50% medium invert sucrose, and 65% high-maltose corn syrup (HMCS) were inoculated with Salmonella spp., Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and coliforms at a level of 10(5) cells per g. The inoculated products were stored both at or near their normal holding temperatures (32 to 46 degrees C) and at 26.7 degrees C (the lower limit during transportation). In most of the products the number of microorganisms fell below the detection limit in less than 3 days when the sweeteners were stored at their normal holding temperatures. However, in liquid sucrose S aureus survived up to 2 weeks. When the products were stored at 26.7 degrees C, the reduction in the number of microorganisms occurred at a slower rate. At 26.7 degrees C the fastest rates of reduction were observed in 42 and 55% HFCS and in 50% medium invert sucrose. In these products the number of bacteria fell below the detection limit in 3 to 6 days. The slowest rate of the reduction was observed in the liquid sucrose, in which S. aureus survived up to 1 month. These results indicate that incidental contamination of liquid sweeteners with microbial pathogens will not present a public health or regulatory hazard.

Receptor-independent activation of cardiac adenylyl cyclase by GDP and membrane-associated nucleoside diphosphate kinase. A new cardiotonic mechanism?

Regulation of adenylyl cyclase activity by guanine nucleoside tri- and diphosphates as well as by stimulatory and inhibitory receptors was studied in canine cardiac sarcolemmal membranes. Guanosine triphosphate (GTP) increased adenylyl cyclase activity by a maximum of 80%, with an EC50 value of 0.7 mumol/l. The addition of the beta-adrenoceptor agonist, isoprenaline (100 mumol/l), caused a further, about 100%, increase in GTP-stimulated activity. The nucleoside diphosphate (GDP) also activated cardiac adenylyl cyclase, but in a biphasic manner. At low concentrations (EC50 0.12 mumol/ l). GDP increased enzyme activity by about 80%, followed by a plateau at 0.5-2 mumol/l and a second increase to a maximum of 60% with an EC50 value of 14 mumol/l. The stable GDP analog, guanosine 5'-O-(2-thio)diphosphate (GDP beta S), also increased cardiac adenylyl cyclase activity, but in a monophasic manner, by a maximum of 150%, with an EC50 of 0.4 mumol/l. Addition of uracil diphosphate (UDP) (3 mmol/l), which completely inhibited transphosphorylation of GDP to GTP, did not reduce adenylyl cyclase stimulation by low concentrations of GDP, whereas enzyme stimulation by high GDP concentrations was almost completely attenuated. Furthermore, pretreatment of the membranes with cholera toxin led to an increased stimulation of adenylyl cyclase activity by high concentrations of GDP. These findings suggest that the second phase of adenylyl cyclase stimulation by GDP is due to transphosphorylation of GDP to GTP, associated with activation of Gs proteins, and that stimulation by GDP itself (first phase) and endogenously formed GTP (second phase) is additive. However, in contrast to exogenously added GTP, beta-adrenoceptor activation did not enhance GDP-stimulated adenylyl cyclase activity. Furthermore, in the presence of 1 mumol/l GDP, the addition of GTP did not cause any further increase in enzyme activity. On the other hand, the muscarinic acetylcholine receptor agonist carbachol inhibited both GTP- and GDP-activated adenylyl cyclase. The inhibition of GDP-stimulated activity was lost when formation of GTP from GDP was blocked. The contrasting effects of endogenously formed GTP and exogenous GTP suggest that the formation of GTP from GDP is closely linked to the activation site of adenylyl cyclase, i.e. the stimulatory Gs protein. This receptor-independent activation can apparently bypass beta-adrenoceptor-dependent activation of cardiac adenylyl cyclase.

Inhibition of elastase improves myocardial function after repetitive ischaemia and myocardial infarction in the rat heart.

We investigated the potential of inhibition of elastase, a granulocyte-derived proteolytic enzyme, in ameliorating the effects of myocardial stunning caused by repetitive ischaemia (RI) and myocardial infarction (MI) for the first time in an in situ, perfused, rat heart model. The effects of the elastase-inhibitors Elafin (EL, 10 mg/kg/h) and ICI 200,880 (ICI,5 mg/kg/h) on myocardial blood flow (MBF, H2 clearance), regional myocardial function (FT, pulsed doppler) and neutrophil extravasation (myocardial myeloperoxidase activity, MPO) were investigated in RI (5x10 min ligature of the anterior descending ramus (LAD), 5x20 min reperfusion) and MI (50 min LAD ligature, 60 min reperfusion). Under control conditions, MBF and FT were significantly reduced and MPO was significantly increased after RI (n=8) and MI (n=8) in the ischaemic area compared with baseline. Pretreatment with EL (n=7) or ICI (n=7) did not improve MBF significantly and did not influence the successive attenuation of peak values of reactive hyperaemia. However, both EL and ICI significantly improved FT and significantly reduced MPO after RI and MI compared with control conditions. Additionally, both the area at risk and MI size were reduced significantly by both inhibitors. These results demonstrate that elastase inhibitors significantly improve the reduction of FT both in myocardial stunning and in myocardial infarction in the rat without significant improvement of MBF. It is concluded that elastase inhibitors exert a cardioprotective effect against reperfusion injury, probably by inhibition of leukocyte extravasation as indicated by the decrease in MPO activity.

Adaptation of myocardial blood flow to increased metabolic demand is not dependent on endothelial vasodilators in the rat heart.

OBJECTIVE: To investigate the role of endothelial vasodilating factors in adaptation of myocardial blood flow to increased metabolic demands. DESIGN: Alterations in the effects of endothelium dependent (acetylcholine) and independent (sodium nitroprusside) vasodilators and the beta 1 receptor agonist dobutamine were studied after inhibition of endothelium derived relaxing factor (EDRF) with L-NG-nitro-arginine methyl ester (L-NAME), prostanoid synthesis with indomethacin, and ATP sensitive potassium channels with glibenclamide. EXPERIMENTAL ANIMALS: Female Wistar rats, in situ perfused heart. MAIN OUTCOME MEASURES: Myocardial blood flow (H2 clearance); systolic fractional thickening (pulsed Doppler); mean arterial blood pressure. RESULTS: L-NAME reduced myocardial blood flow by 58 (12)% (mean (SD), P < 0.001) and systolic thickening fraction (FT) by 36 (9)% (P < 0.05). These effects were significantly reversed by administration of L-arginine but not D-arginine. Pretreatment with L-NAME inhibited the increase in myocardial blood flow caused by acetylcholine (control: +42 (9)%; L-NAME: -29 (7)%, P < 0.001) but did not affect the increase in myocardial blood flow caused by sodium nitroprusside (control: +44 (5)%; L-NAME: +34 (10)%, NS). Pretreatment with L-NAME did not change the effect of dobutamine on myocardial blood flow (+61 (3)%) and FT (+32 (8)%) compared with baseline values (P < 0.001). Neither pretreatment with indomethacin nor with glibenclamide reduced the dobutamine induced increase in myocardial blood flow. CONCLUSIONS: Inhibition of EDRF, prostanoid synthesis, and ATP sensitive potassium channels did not reduce the vasodilator reserve during increased metabolic demands induced by beta 1 adrenergic stimulation. Therefore, adaptation of myocardial blood flow to increased metabolic demands is independent of endothelial relaxing factors in the rat heart.

Opioid receptor agonists activate pertussis toxin-sensitive G proteins and inhibit adenylyl cyclase in canine cardiac sarcolemma.

Although both opioid receptors and endogenous opioids are abundant in cardiac tissues, the signal transduction pathways of opioids in cardiac sarcolemmal membranes have yet to be identified. In highly purified canine cardiac sarcolemmal membranes, binding of the opioid receptor antagonist [3H]diprenorphine and effects of mu, delta and kappa agonists on low Km GTPase and adenylyl cyclase were measured. Equilibrium binding of [3H]diprenorphine revealed a maximal binding capacity of 7.2 pmol/mg protein and a Kd of 1.3 nmol/l. In the presence of GTP, (D-Pen2,5, p-Cl-Phe4) enkephalin and (D-Arg6) dynorphin A 1-13 fragment both inhibited adenylyl cyclase by 20-25% (from 206 +/- 30 to 164 +/- 28 pmol.min-1.mg protein-1, EC50 6 mumol/L, and from 254 +/- 109 to 204 +/- 90 pmol.min-1.mg protein-1, EC50 8 mumol/L, respectively; P < 0.001). Both substances stimulated low Km GTPase by 20% and 13%, respectively (from 12.7 +/- 3.0 to 15.2 +/- 3.7 pmol.min-1.mg protein-1, EC50 12 mumol/L, P < 0.01, and from 9.1 +/- 2.8 to 10.4 +/- 3.2 pmol.min-1.mg protein-1, EC50 6 mumol/L, P < 0.05, respectively). These effects were blocked by the opioid receptor antagonist naltrexone and by pretreatment of sarcolemmal membranes with pertussis toxin. The mu opioid receptor agonists (D-Ala2, Me Phe4, Gly-[ol]5)enkephalin and morphiceptin had no effect on either adenylyl cyclase or low Km GTPase activities. These data suggest that in cardiac sarcolemma, opioid receptors are coupled to pertussis toxin sensitive G proteins and mediate inhibition of adenylyl cyclase activity.

Stable GDP analog-induced inactivation of G(i) proteins promotes cardiac adenylyl cyclase inhibition by guanosine 5'-(beta gamma-imino)triphosphate and muscarinic acetylcholine receptor.

Low concentrations of GDP and its stable analog guanosine 5'-O-(2-thio)diphosphate (GDP beta S) have been shown to stimulate adenylyl cyclase activity in canine cardiac sarcolemmal membranes independent from a phosphate transfer reaction. The mechanism of this stimulation was further examined. The stable GTP analog guanosine 5'-(beta gamma-imino)triphosphate (Gpp(NH)p) increased basal adenylyl cyclase activity and inhibited forskolin-stimulated activity with EC50 (half-maximal effective concentration) values of 0.7 mumol/l and 10 nmol/l, respectively. In the presence of GDP beta S (5 mumol/l), which increased basal activity by about 150%, addition of Gpp(NH)p inhibited adenylyl cyclase activity by up to 50% with an EC50 value of 40 nmol/l. Activation of cardiac muscarinic acetylcholine receptors by carbachol amplified this Gpp(NH)p-induced inhibition of GDP beta S-stimulated adenylyl cyclase activity. The stimulatory effect of GDP beta S and the inhibitory effect of Gpp(NH)p on GDP beta S-stimulated adenylyl cyclase activity were both attenuated by increasing the Mg2+ concentration or substituting Mn2+ for Mg2+ in the assay. Furthermore, both effects were strongly reduced or abolished upon pretreatment of the sarcolemmal membranes with a low concentration of the SH reagent N-ethylmaleimide (10 mumol/l). These results suggest that the stimulatory effect of GDP beta S on basal adenylyl cyclase activity in canine cardiac sarcolemmal membranes is caused by inactivation of G(i) proteins, which are then rendered susceptible to activation by Gpp(NH)p and inhibitory receptors.

Evidence against a regulation of Na+/K(+)-ATPase by Gi proteins. Failure to detect an influence of G proteins on Na+/Ca(2+)-exchange in cardiac sarcolemmal membranes.

In the myocardium the inhibitory guanine nucleotide-binding regulatory proteins (Gi proteins) mediate negative chronotropic and negative inotropic effects by activation of K+ channels and inhibition of adenylyl cyclase. The concept of a uniform inhibitory action of Gi proteins on myocardial cellular activity has been questioned by the recent observations of adenosine-induced activation of the Na+/Ca(2+) exchange and a carbachol-induced inhibition of the Na+/K(+)-ATPase activity in cardiac sarcolemmal membranes. The aim of the present study, therefore, was to reinvestigate the putative regulation of Na+/Ca(2+) exchange and Na+/K(+)-ATPase activity in purified canine sarcolemmal membranes. These membranes were enriched in adenosine A1 (Maximum number of receptors, Bmax 0.033 pmol/mg) and muscarinic M2 (Bmax 2.9 pmol/mg) receptors and contained Gi2 and Gi3, two Gi protein isoforms, and G0, another pertussis toxin-sensitive G protein, as detected with specific antibodies. The adenosine A1-selective agonist, (-)-N6-(2-phenylisopropyl)-adenosine, and the muscarinic agonist, carbachol, both inhibited isoprenaline-stimulated adenylyl cyclase activity by 25% and 35% respectively, and the stable GTP analogue 5'-guanylylimidodiphosphate inhibited forskolin-stimulated adenylyl cyclase activity by 35% in these membranes. The characteristics of Na+/Ca(2+) exchange and Na+/K(+)-ATPase activity as well as those of the ouabain-sensitive, K(+)-activated 4-nitrophenylphosphatase, an ATP-independent, partial reaction of the Na+/K(+)-ATPase, were in agreement with published data with regard to specific activity, time course of activity and substrate dependency. However, none of these activities were influenced by adenosine, (-)-N6-(2-phenylisopropyl)-adenosine, carbachol, or stable GTP analogs, suggesting that Na+/Ca(2+) exchange and Na+/K(+)-ATPase are not regulated by Gi proteins in canine cardiac sarcolemmal membranes.

Phosphotransfer reactions as a means of G protein activation.

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) serve to transduce information from agonist-bound receptors to effector enzymes or ion channels. Current models of G protein activation-deactivation indicate that the oligomeric GDP-bound form must undergo release of GDP, bind GTP and undergo subunit dissociation, in order to be in active form (GTP bound alpha subunits and free beta gamma dimers) and to regulate effectors. The effect of receptor occupation by an agonist is generally accepted to be promotion of guanine nucleotide exchange thus allowing activation of the G protein. Recent studies indicate that transphosphorylation leading to the formation of GTP from GDP and ATP in the close vicinity, or even at the G protein, catalysed by membrane-associated nucleoside diphosphate kinase, may further activate G proteins. This activation is demonstrated by a decreased affinity of G protein-coupled receptors for agonists and an increased response of G protein coupled effectors. In addition, a phosphorylation of G protein beta subunits and consequent phosphate transfer reaction resulting in G protein activation has also been demonstrated. Finally, endogenously formed GTP was preferentially effective in activating some G proteins compared to exogenous GTP. The aim of this report is to present an overview of the evidence to date for a transphosphorylation as a means of G protein activation (see also refs [1 and 2] for reviews).