Decrease in Ca(2+)-sensitizing effect of UD-CG 212 Cl, a metabolite of pimobendan, under acidotic condition in canine ventricular myocardium

We studied the influence of acidosis on the positive inotropic effect of UD-CG 212 Cl (4,5-dihydro-6-[2-(4-hydroxyphenyl)-1H-benzimidazole-5-yl]-5-methyl-3(2H)-pyridazinone), an active metabolite of pimobendan, in canine ventricular trabeculae loaded with aequorin. The positive inotropic effect of UD-CG 212 Cl was markedly suppressed under acidotic conditions. The maximal contractile response to UD-CG 212 Cl was attained at 10(-5) M in the control condition at pH 7.4, but was not achieved even at 10(-4) M during acidosis. The maximal inotropic effect of UD-CG 212 Cl was 18% of the maximal response to isoproterenol (ISO(max)) in association with an increase in Ca(2+) transients of 7% of ISO(max) in the control, while they are 8 and 6% of ISO(max) under acidosis, respectively. Acidosis abolished the increase in myofilament Ca(2+) sensitivity induced by UD-CG 212 Cl, whereas the increase in Ca(2+) transients induced by the compound was not affected by acidosis. In conclusion, UD-CG 212 Cl elicited a positive inotropic effect even under acidosis, however, UD-CG 212 Cl was much less effective as a cardiotonic agent under acidosis mainly due to a decrease in the Ca(2+)-sensitizing effect under acidotic condition.     

Differential effects of d- and l-pimobendan on cardiac myofilament calcium sensitivity

The effects is of the optical isomers of pimobendan (UD-CG 115 BS), an inotropic agent, were studied on the electrical and mechanical activity of intact and detergent-skinned preparations of cardiac muscle from guinea pig and dog. Racemic pimobendan has been shown to increase contractile force and to potentiate slow action potentials (AP) induced by stimulation of papillary muscle partially depolarized with 25 mM [K]o. These effects are shown in this study to be mainly due to the l-optical isomer of pimobendan. When slow APs were maximally stimulated by 1 microM isoproterenol, addition of either the d- or l-isomer of pimobendan did not affect the slow AP parameters. However, under these conditions, contractile force was significantly increased to 124% of control by the d-isomer and to 184% of control by the l-isomer. These results suggest that pimobendan may have direct effects on the myofilaments and that these effects are dependent on the optical isomer of the compound. To test this directly, the effects of d- and l-pimobendan were compared on Ca++-activated force developed by detergent-skinned heart muscle fibers. Submaximal force developed at constant Ca++ was increased by both optical isomers, but the l-isomer had a significantly greater Ca++-sensitizing effect. For example at pCa 6.75 force was 270% of control in the presence of the d-isomer and 400% of control in the presence of the l-isomer. At pCa 5, there was no effect of either isomer on force developed by the skinned fiber preparations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of anagrelide on platelet cAMP levels, cAMP-dependent protein kinase and thrombin-induced Ca++ fluxes

Anagrelide (BL-4162A, 6,7-dichloro-1,5-dihydroimidazo[2, 1-6] quinazolin-2[3H]one monohydrochloride hydrate) is a potent and broad spectrum inhibitor of platelet aggregation. Prior studies showed that anagrelide inhibited platelet cyclic AMP (cAMP) phosphodiesterase activity but did not appreciably elevate platelet cAMP levels. We examined the effects of anagrelide on washed human platelets and found that anagrelide caused significant elevation of cAMP levels. Anagrelide treatment also resulted in activation of the platelet cAMP-dependent protein kinase at anagrelide concentrations of 0.1 to 1 microgram/ml, which inhibited platelet aggregation but caused only small increases in platelet cAMP content. When whole platelets were incubated with radiolabeled phosphate, anagrelide increased phosphorylation of platelet proteins with relative molecular weights of 22, 26, 50 and 80 kilodaltons. The pattern of protein phosphorylation stimulated by anagrelide treatment was similar to that observed when the platelets were treated with forskolin. Anagrelide also inhibited the rise in intracellular Ca++ caused by thrombin, as measured using Fura-2-loaded platelets. The inhibition of increased intracellular Ca++ resulted from block of thrombin-induced mobilization of intracellular Ca++, as well as prevention of Ca++ influx through the plasma membrane. Anagrelide itself had no influence on inositol 1,4,5-trisphosphate-induced Caz5++ release from isolated platelet membrane vesicles. These studies suggest that anagrelide inhibits platelet phosphodiesterase activity in intact platelets resulting in an elevation in cAMP levels sufficient to activate the cAMP-dependent protein kinase and inhibit agonist-activated Ca++ fluxes.     

Molecular basis for the cardiovascular activities of amrinone and AR-L57

It has been suggested that amrinone and AR-L57 enhance cardiac contractility either by inhibiting phosphodiesterase activity or altering Ca++ homeostasis. Because these novel agents are potentially useful in the management of heart failure, it was of interest to more clearly define their mechanism(s) of action. Amrinone and AR-L57 caused concentration-dependent increases in the contractile states of either perfused guinea-pig hearts or cultured rat cardiomyocytes. To determine whether these actions might result from an increase in sarcolemmal Ca++ movement, the effects of these agents on Ca++ accumulation were studied in a simple system, dog erythrocytes. Both agents promoted erythrocyte Ca++ accumulation in time and concentration-dependent manners, effects that resulted primarily from increased Ca++ entry. However, because these effects were not measurable at inotropic drug concentrations and were apparent only after a 30-min incubation, they did not provide an explanation for the inotropic effects of these agents. Amrinone and AR-L57 inhibited dog heart phosphodiesterase activity (isozyme III) with EC50 values of 23 and 420 microM, respectively; however, only the inotropic responses to amrinone were attenuated by the muscarinic agonist, carbachol, thereby implying a cAMP (cyclic AMP)-dependent mechanism. In cultured ventricular cells, concentrations of amrinone (2 X 10(-4) M) and AR-L57 (3 X 10(-5) M) that caused maximal inotropic responses were associated with the activation of glycogen phosphorylase, but neither drug significantly increased the activation state of cAMP-dependent protein kinase. To further probe the effects of these drugs on intracellular cAMP and Ca++ metabolism, their effects on protein phosphorylation were studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that parathyroid hormone-mediated calcium transport in rat brain synaptosomes is independent of cyclic adenosine monophosphate.

In vivo PTH administration to rats resulted in increased brain synaptosomal Ca++ transport, while parathyroidectomy (PTX) resulted in decreased transport. To determine the mechanism of action of PTH on Ca++ transport in rat brain synaptosomes, we performed transport studies by the Na-Ca exchanger and also measured cAMP generation in synaptosomes from PTX rats. Ca++ transport was studied after in vivo additions of either bovine (b)PTH, cAMP, or forskolin, and adenylate cyclase activity was assessed after additions of either bPTH, forskolin, sodium fluoride (NaF), or isoproterenol. In the presence of 1-34 bPTH [10(-7) M], Ca++ uptake was significantly increased by 55% (P less than 0.001) above control, while 3-34 bPTH [10(-7) M] had no effect on uptake. Both 8br,cAMP [10(-6) M] and dibut,cAMP [10(-6) M] also significantly increased (P less than 0.001) Ca++ uptake above control by 63 and 44%, respectively. Similarly, forskolin [10(-5) M], the adenylate cyclase activator, increased Ca++ uptake by 41%. We next evaluated Ca++ efflux, and found that 1-34 bPTH [10(-7) M], 1-84 bPTH [10(-7) M], and forskolin [10(-5) M] also increased Ca++ efflux by 50, 73, and 120%, respectively, above control. Since Ca++ transport was increased by either PTH, cAMP, or forskolin, we decided to determine if PTH action on Ca++ transport in synaptosomes was dependent on cAMP. This was investigated by measuring cAMP production during the conversion of 32P-ATP to 32P-cAMP in the presence of an ATP regenerating system (30 micrograms creatine phosphokinase, 10 mM creatine phosphate), and the cyclic nucleotide phosphodiesterase inhibitor (1 mM IBMX). Whereas forskolin [10(-4) M] and NaF [100 mM] significantly increased (P less than 0.001) adenylate cyclase activity in synaptosomes by eight- and fourfold, respectively, neither 1-34 bPTH nor 1-84 bPTH increased synaptosomal cyclase activity. However, in canine renal cortical plasma membranes (CRCPM), we observed significant increases in cAMP production with either forskolin, NaF, or PTH. Finally, to determine if synaptosomes contain an intact adenylate cyclase system, we measured cAMP production in the presence of the beta adrenergic agent, isoproterenol. Isoproterenol significantly increased adenylate cyclase activity in both synaptosomes (90%) and CRCPM (50%). These data suggest that although there is an intact adenylate cyclase system in rat brain synaptosomes, PTH-stimulated calcium transport in synaptosomes appears to be independent of this system.     

On the mechanism of papaverine inhibition of the voltage-dependent Ca++ current in isolated smooth muscle cells from the guinea pig trachea.

The effects of papaverine, a smooth muscle relaxant agent, on the voltage-dependent Ca++ current were examined in isolated smooth muscle cells from the guinea pig trachea. The tight-seal whole cell voltage clamp technique was used. Papaverine (1-100 microM) inhibited the Ba++ inward current (IBa) through the voltage-dependent L-type Ca++ channel in a concentration-dependent fashion. The inhibitory effect of papaverine on IBa appeared to have both tonic and use-dependent components. In addition to the reduction of the maximal conductance of IBa, papaverine (20 microM) shifted the quasi-steady-state inactivation curve of IBa to more negative membrane potentials by approximately 10 mV. These effects of papaverine on IBa were completely reversible. Although it has been suggested that papaverine inhibited phosphodiesterase to increase intracellular cyclic AMP, phosphodiesterase inhibitors (theophylline, 500 microM, and 3-isobutyl-1-methylxanthine, 500 microM), isoproterenol (2 microM) and dibutyryl cyclic AMP (1 mM) did not affect IBa. Thus, papaverine inhibits IBa in a way independent of intracellular cyclic AMP. Papaverine also had inhibitory effects on other membrane currents (i.e., the voltage-dependent transient outward K+ current and the Ca(++)-activated oscillatory K+ current), which may result in an enhancement of the excitability of the cells. These results suggest that inhibition of the voltage-dependent L-type Ca++ channel is involved in the papaverine-induced relaxation of the tracheal smooth muscle.     

Frequency modulation of the inotropic action of isoproterenol in mouse heart

In mouse right ventricular strips, field-stimulated to contract isometrically in an oxygenated bicarbonate-buffered physiological salt solution at 22--24 degrees C, the EC50 for the inotropic action of isoproterenol decreased from 37 nM in muscles stimulated at 0.2 Hz to 5 nM in muscles stimulated at 3.3 Hz. At higher rates of contraction, there was also an increased sensitivity to the inotropic actions of norepinephrine and epinephrine but not to those of Ca++ and N6,O2'-dibutyryl cyclic AMP. Increasing the Ca++ concentration further decreased the EC50 for isoproterenol at 3.3 Hz but had no effect on the EC50 at 0.2 Hz. The leftward shift of the contractile response curve at 3.3 Hz was inhibited by verapamil (0.6 microM) and Mn++ (0.25 mM). The stimulation of cyclic AMP accumulation was approximately 6-fold more sensitive to isoproterenol at 3.3 than at 0.2 Hz, but isoproterenol increased contractile force at concentrations two orders of magnitude lower than those that significantly increased cyclic AMP accumulation. Inhibition of cyclic AMP phosphodiesterase activity further increased the sensitivity to the inotropic actions of isoproterenol but did not attenuate the frequency difference. The results indicate that isoproterenol-stimulated Ca++ influx through the slow channel plays an important role in the mechanism of the increased sensitivity to the inotropic action of isoproterenol found at higher frequencies of contraction. Although cyclic AMP accumulation was also frequency dependent, its role in the inotropic action of isoproterenol in mouse heart is not clear.     

Effects on calmodulin of bepridil, an antianginal agent

Using biopharmacological techniques, we determined the effect on calmodulin of bepridil, a Ca++ channel blocker. We used two Ca++/calmodulin-dependent enzymes, Ca++/calmodulin-dependent cyclic nucleotide phosphodiesterase from bovine brain and myosin light chain kinase from chicken gizzard. Bepridil inhibited the calmodulin-induced activation of Ca++/calmodulin-dependent cyclic nucleotide phosphodiesterase and the concentration of this drug producing 50% inhibition (IC50) of this enzyme was 8 microM. There was no significant effect on unactivated Ca++/calmodulin-dependent cyclic nucleotide phosphodiesterase (in the absence of Ca++-calmodulin), up to a concentration of 100 microM. Bepridil inhibited specifically Ca++/calmodulin-dependent phosphorylation of chicken gizzard myosin light chain with an IC50 value of 18 microM. Moreover, this agent produced a marked displacement of [3H]N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, an antagonist that interacts selectively with calmodulin. The influence of bepridil on the dose-response curves of mesenteric arterial strips for CaCl2, norepinephrine and serotonin differed from the influence seen with nifedipine, another Ca++ blocker. Bepridil (100 microM) suppressed the Ca++-induced contraction of saponin-skinned mesenteric arteries and calmodulin (26 microM) reversed partly the relaxant effect of this agent. These results suggest that the effect of bepridil on the cardiovascular system is due not only to its Ca++ channel blocking action but also to a calmodulin antagonistic action.     

Maitotoxin, a novel activator of mediator release from human basophils, induces large increases in cytosolic calcium resulting in histamine, but not leukotriene C4, release.

Maitotoxin (MTX) is a potent marine toxin which stimulates several Ca(++)-dependent processes presumably through an increase in Ca++ permeability. We have examined the effect of MTX on the release of chemical mediators from human basophils and its mechanism of action. MTX (1-20 ng/ml) induced histamine release (37-100%) from both mixed leukocyte preparations and purified basophils. Histamine release activated by MTX was slow (t 1/2 approximately equal to 15 min), temperature and Ca++ dependent (optimal at 37 degrees C and 1-2.5 mM Ca++). Sr++ ion could substitute for Ca++ in the secretory process. Digital video microscopy analysis of purified (> 70%) basophils revealed that MTX (1-20 ng/ml) induced a slow and marked increase of cytosolic Ca++ levels that was temporally coincident with histamine release. MTX (1-20 ng/ml) stimulated the release of sulfidopeptide leukotriene C4 from mixed leukocyte preparations (approximately equal to 0.5% basophils). However, purified basophils (77 +/- 7%) showed no sulfidopeptide leukotriene C4 release even in the presence of large histamine secretion (84 +/- 14%). Two organic Ca(++)-channel entry blockers, verapamil and diltiazem (1-30 microM) inhibited the release of histamine induced by MTX, whereas the dihydropyridine nifedipine (0.1-10 microM) caused only minimal inhibition. These results suggest that MTX represents a novel stimulus useful to study the role of Ca++ in human basophil mediator release.     

Amiodarone blocks calcium current in single guinea pig ventricular myocytes

Ca++ current (lca) block by amiodarone and the underlying mechanisms thereof were investigated in guinea pig single ventricular myocytes using the single suction pipette whole cell voltage clamp method. The dose-response curve revealed a 1:1 stoichiometry for binding of amiodarone to its receptor with an apparent dissociation constant of 5.8 microM in the resting state. Amiodarone, 5 microM did not significantly alter the time course of ICa decay, but did shift the steady-state inactivation curve for lca in the hyperpolarizing direction by 9.2 +/- 3.1 mV. Development of block at depolarized potentials was voltage-dependent between -20 and 10 mV with time constants of 112 +/- 33 and 755 +/- 212 msec at 10 mV. In the presence of 0.2 microM amiodarone, recovery from inactivation was fitted by a double exponential most likely indicating rapid recovery of the drug-free Ca++ channels and slow recovery of the drug-associated Ca++ channels with time constants of 44 +/- 12 and 108 +/- 403 msec, respectively, at -80 mV. The proportion of the current recovering via the slow phase was 36 +/- 7%. By using this value, we estimated the dissociation constant in the inactivated state to be 0.36 microM. Amiodarone's marked use-dependent block of lca is explicable in terms of its high affinity for, and slow dissociation from, Ca++ channels in the inactivated state. These results suggest that amiodarone blocks lca in both the resting and inactivated states.(ABSTRACT TRUNCATED AT 250 WORDS)     

7-Bromo-1,5-dihydro-3,6-dimethylimidazo[2,1-b]quinazolin-2(3H)- one (Ro 15-2041), a potent antithrombotic agent that selectively inhibits platelet cyclic AMP-phosphodiesterase

This study with the new analog Ro 15-2041 (7-bromo-1,5-dihydro-3,6-dimethylimidazo[2,1-b]quinazolin-2(3H)-on e) confirms and substantially extends the activity spectrum of imidazoquinazolinones as potent platelet function inhibitors. Ro 15-2041 inhibited platelet aggregation induced by all common platelet agonists in platelet-rich plasma obtained from various species including man (IC50 = 1-3 microM). The compound potentiated platelet inhibition by prostacyclin, the prostacyclin-induced increase of intraplatelet cyclic (c) AMP levels and inhibited the collagen-induced release of serotonin and beta-thromboglobulin. Ro 15-2041 reduced the increase and accelerated the normalization of cytosolic free Ca++ in thrombin-stimulated human platelets. Ro 15-2041 is a potent (IC50 = 70 nM) and selective inhibitor of platelet cAMP-phosphodiesterase activity. Whereas Ro 15-2041 caused complete inhibition of cAMP-phosphodiesterase activity in human platelet supernatants, breakdown of cAMP in cardiac homogenates was depressed to maximally 50%. In human brain and rabbit uterus Ro 15-2041 was at least 1000 times less potent. By comparison, papaverine fully inhibited phosphodiesterase activity in all four tissues with similar IC50 values of about 5 microM. Furthermore, Ro 15-2041 selectively inhibited cAMP-phosphodiesterase activity of a bovine calmodulin-independent but not of a calmodulin-dependent enzyme preparation. The compound exhibited significant p.o. activity in various ex vivo and in vivo platelet function tests.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta adrenergic receptor-stimulated prostaglandin synthesis in the isolated rabbit heart: relationship to extra- and intracellular calcium

We have investigated the contribution of extra- and intracellular Ca++ and calmodulin to beta adrenergic receptor-stimulated prostaglandin synthesis in the isolated rabbit heart perfused with Krebs-Henseleit buffer. Administration of isoproterenol (100 ng) increased the output of immunoreactive 6-keto-prostaglandin F1 alpha and prostaglandin E2 as well as heart rate and developed tension; the coronary perfusion pressure was reduced. Isoproterenol-induced output of prostaglandins was positively correlated with the extracellular Ca++ concentration (0-5 mM). Infusion of the Ca++ channel blockers diltiazem (22 microM) or nifedipine (0.27 microM) inhibited isoproterenol-stimulated output of prostaglandins and the positive inotropic but not the positive chronotropic effect of the amine. Administration of the intracellular Ca++ antagonists 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (23 microM) or ryanodine (1.6 microM) reduced the outflow of prostaglandins and the positive chronotropic and inotropic effect elicited by isoproterenol. The calmodulin inhibitors trifluoperazine (50 microM) or calmidazolium (1 microM) failed to alter isoproterenol-induced output of prostaglandins; trifluoperazine but not calmidazolium reduced the developed tension and coronary perfusion pressure without altering heart rate. The prostaglandin synthesis elicited by arachidonic acid (3 micrograms) was inhibited by indomethacin but not by alterations in extracellular Ca++, Ca++ channel blockers, intracellular Ca++ antagonists or calmodulin inhibitors. These data suggest that activation of beta adrenergic receptors promotes cardiac prostaglandin synthesis and myocardial contractility by increasing the trans-sarcolemmal flux of Ca++, which releases intracellular Ca++.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preglomerular microcirculation expresses the cAMP-adenosine pathway

The purpose of this study was to investigate whether the extracellular cAMP-adenosine pathway (i.e., transport of cAMP out of cells followed by extracellular conversion of cAMP to adenosine) exists in preglomerular microvessels (PGMVs). Incubation of PGMVs for 1 h with 30 microM cAMP increased the amount of extracellular adenosine from 163 +/- 18.6 (n = 18) to 9810 +/- 604 (n = 12) pmol/mg of protein (P < 10(-6)). The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 1 mM; n = 6) and the ecto-phosphodiesterase inhibitor 1, 3-dipropyl-8-p-sulfophenylxanthine (DPSPX; 1 mM; n = 6) significantly (P < 10(-6) and P < 10(-5), respectively) reduced the cAMP-induced increase in extracellular adenosine. Incubation of PGMVs for 1 h with isoproterenol (beta-adrenoceptor agonist; 1 microM) + IBMX (0.1 mM) increased the amount of extracellular cAMP from 0.800 +/- 0.047 to 22.3 +/- 2.20 pmol/mg of protein (P < 10(-6); n = 41). In PGMVs incubated with isoproterenol (1 microM) + IBMX (0.1 mM) for 1 h, there was a significant (P < 10(-4)) linear (r(2) = 0.6) relationship between intracellular and extracellular cAMP levels. Incubation of PGMVs for 1 h with 1 microM isoproterenol increased the amount of extracellular adenosine from 163 +/- 18.6 (n = 18) to 297 +/- 38.3 (n = 12) pmol/mg of protein (P =.002). Propranolol (beta-adrenoceptor antagonist; 1 microM; n = 7), IBMX (1 mM; n = 14), and DPSPX (1 mM; n = 12) blocked (P =.037, P =.015, and P =.026, respectively) isoproterenol-induced increases in extracellular adenosine. CONCLUSIONS: PGMVs transport endogenous cAMP to the extracellular compartment and metabolize extracellular cAMP to adenosine. This pathway can increase extracellular levels of adenosine during beta-adrenoceptor activation of adenylyl cyclase.     

Effects of the calcium antagonists perhexiline and cinnarizine on vascular and cardiac contractile protein function

The weakly basic, lipophilic Ca++ antagonists perhexiline and cinnarizine have been compared with the calmodulin inhibitor W-7 and the cardiotonics Vardax and APP-201-533 for the ability to modulate Ca++-dependent contractile protein interactions directly, as well as Ca++-calmodulin-mediated myosin light chain phosphorylation, in arterial actomyosin or cardiac myofibrils. Both perhexiline and cinnarizine inhibited arterial myosin P-light chain phosphorylation and superprecipitation of arterial actomyosin over the concentration range of 10 to 200 microM. Concomitant inhibition of arterial superprecipitation and phosphorylation by perhexiline (IC50 = 33 microM) and cinnarizine (IC50 = 60 microM) was similar to W-7 (IC50 = 35 microM), and was characterized by a rightward shift in the pCa superprecipitation and pCa-light chain phosphorylation relationships, depressed maximum activity and attenuation by 2 microM exogenous calmodulin. However, whereas inhibition of superprecipitation and P-light chain phosphorylation by W-7 was equal at different Mg++ concentrations, relatively greater inhibition with perhexiline and less inhibition with cinnarizine was apparent as the free Mg++ concentration was lowered. In cardiac myofibrils prepared from both bovine and canine ventricles, perhexiline stimulated Mg-adenosine triphosphatase (ATPase) activity and cinnarizine was without effect, whereas W-7 significantly depressed ATPase activity. Perhexiline was 10-fold more potent and 3-fold more efficacious than either Vardax or APP-201-533 in canine cardiac myofibrils. Whereas APP-201-533 increased Ca++ sensitivity and maximum ATPase activity (Vmax), perhexiline increased Ca++ sensitivity, but not Vmax, and W-7 depressed both parameters.(ABSTRACT TRUNCATED AT 250 WORDS)     

6-[4-(1-Cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2-(1H)quinolinone (cilostazol), a phosphodiesterase type 3 inhibitor, reduces infarct size via activation of mitochondrial Ca2+-activated K+ channels in rabbit hearts

6-[4-(1-Cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2-(1H)quinolinone (cilostazol), a phosphodiesterase type 3 (PDE III) inhibitor, activates cAMP-dependent protein kinase A (PKA). The cAMP/PKA pathway potentiates the opening of mitochondrial Ca(2+)-activated K(+) (mitoK(Ca)) channels and confers cardioprotection. Although cilostazol has been reported to directly activate sarcolemmal large-conductance Ca(2+)-activated K(+) channels, it remains unclear whether cilostazol modulates the opening of mitoK(Ca) channels. Therefore, we tested the possibility that cilostazol opens mitoK(Ca) channels and protects hearts against ischemia/reperfusion injury. Flavoprotein fluorescence in rabbit ventricular myocytes was measured to assay mitoK(Ca) channel activity. Infarct size in the isolated perfused rabbit hearts subjected to 30-min global ischemia and 120-min reperfusion was determined by triphenyltetrazolium chloride staining. Cilostazol (1, 3, 10, and 30 microM) oxidized flavoprotein in a concentration-dependent manner. The oxidative effect of cilostazol (10 microM) was antagonized by the mitoK(Ca) channel blocker paxilline (2 microM). Activation of PKA by 8-bromoadenosine 3'5'-cyclic monophosphate (0.5 mM) potentiated the cilostazol-induced flavoprotein oxidation. Treatment with cilostazol (10 microM) for 10 min before ischemia significantly reduced the infarct size from 67.2 +/- 1.3 (control) to 33.6 +/- 5.3% (p < 0.05). This infarct size-limiting effect of cilostazol was abolished by paxilline (60.3 +/- 4.9%) but not by the PKA inhibitor (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]-benzodiazocine-10-carboxylic acid hexyl ester (KT5720) (200 nM, 40.5 +/- 3.5%). On the other hand, another PDE III inhibitor, milrinone (10 microM), neither oxidized flavoprotein nor reduced infarct size. Our results suggest that cilostazol exerts a cardioprotective effect via direct activation of mitoK(Ca) channels.     

Sarcoplasmic reticulum-associated cyclic adenosine 5''-monophosphate phosphodiesterase activity in normal and failing human hearts.

Sarcoplasmic reticulum-associated cAMP phosphodiesterase activity was examined in microsomes prepared from the left ventricular myocardium of eight heart transplant recipients with end-stage idiopathic dilated cardiomyopathy and six unmatched organ donors with normal cardiac function. At cAMP concentrations less than or equal to 1.0 microM, sarcoplasmic reticulum-associated cAMP phosphodiesterase activity was functionally homogeneous. cAMP phosphodiesterase activity was inhibited competitively by cGMP (Ki = 0.031 +/- 0.008 microM) and the cilostamide derivative OPC 3911 (Ki = 0.018 +/- 0.004 microM), but was essentially insensitive to rolipram. Vmax and Km were 781.7 +/- 109.2 nmol/mg per min and 0.188 +/- 0.031 microM, respectively, in microsomes prepared from nonfailing hearts and 793.9 +/- 68.9 nmol/mg per min and 0.150 +/- 0.027 microM in microsomes prepared from failing hearts. Microsomes prepared from nonfailing and failing hearts did not differ with respect to either the ratio of cAMP phosphodiesterase activity to ATP-dependent Ca2+ accumulation activity or the sensitivity of cAMP phosphodiesterase activity to inhibition by OPC 3911. These data suggest that the diminished inotropic efficacy of phosphodiesterase inhibitors in failing human hearts does not result from changes in the level, kinetic properties, or pharmacologic sensitivity of sarcoplasmic reticulum-associated cAMP phosphodiesterase activity.     

Roles for Ca++ and cyclic AMP in mediating the cardiotonic actions of isomazole (LY175326)

The contractile state of cat papillary muscles was increased by isomazole in a concentration-dependent manner; inotropic effects of the drug were not altered by either prazosin, propranolol or cimetidine. Isomazole inhibited the peak III isozyme of dog heart phosphodiesterase with an IC50 of 100 microM; effects on isozymes I and II were less pronounced. In cat papillary muscles, carbachol (10(-5) M) shifted the relationship between contractility and concentration of isomazole to the right. These data suggest cyclic AMP (cAMP) is involved in the actions of isomazole. In order to assess the relative effects of isomazole on intracellular cAMP and Ca++, cAMP-dependent protein kinase and glycogen phosphorylase, respectively, were used as reporters of these two second messengers. The source of enzymes was either cultured cardiomyocytes or right ventricular biopsies obtained from anesthetized dogs. In the latter case, biopsies were obtained after i.v. administration of isomazole; the pure beta agonist, isoproterenol, was included for comparative purposes. A submaximal inotropic dose of isomazole (0.1 mg/kg i.v.) in dogs resulted in a pronounced increase in contractility that was associated with a 3-fold increase in phosphorylase activity (0.15 +/- 0.01 to 0.46 +/- 0.06, -5'-AMP: +5'-AMP, P less than .05); the activation state of protein kinase was not altered. By contrast, a comparably effective inotropic dose of isoproterenol (0.1 microgram/kg) caused less than a 2-fold increase in phosphorylase activity (0.15 +/- 0.01 to 0.26 +/- 0.02, -5'-AMP: +5'-AMP, P less than .05) and this was associated with a significant increase in the protein kinase activity ratio (0.36 +/- 0.01 to 0.51 +/- 0.04, -cAMP: +cAMP, P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Potentiation of penile tumescence by T-1032, a new potent and specific phosphodiesterase type V inhibitor, in dogs

We examined the mechanism underlying the potentiation of penile tumescence by methyl 2-(4-aminophenyl)-1, 2dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4, 5-trimethoxyphenyl)3-isoquinoline carboxylate sulfate (T-1032), a new potent and selective phosphodiesterase type V inhibitor. In vivo, pelvic nerve stimulation induced a penile tumescence together with increase of total nitric oxide metabolite levels within the corpus cavernosa of anesthetized dogs. Intravenous (1-100 microg/kg) and intraduodenal (3, 30, 300 microg/kg) treatment with T-1032 dose dependently potentiated the tumescence. The potency of T-1032 was equivalent to that of sildenafil. T-1032 did not influence the intracavernous pressure when the pelvic nerve stimulation was absent. The potentiation of tumescence was more pronounced by intracavernous than i.v. injection. Intracavernous N(G)-nitro-L-arginine, a nitric-oxide synthase inhibitor, but not N(G)-nitro-D-arginine diminished the effects of T-1032 on the tumescence. Furthermore, i.v. T-1032 augmented the tumescence induced by sodium nitroprusside (SNP) but not by vasoactive intestinal polypeptide (VIP). In vitro, in isolated preparations of canine corpus cavernosum precontracted with phenylephrine, SNP (0. 01-100 microM) and VIP (0.01-1 microM) produced a dose-dependent relaxation accompanied by an increase in cGMP and cAMP levels, respectively. T-1032 augmented the relaxation induced by SNP but not by VIP. These data suggest that oral treatment with T-1032 has potential to improve erectile dysfunction through the inhibition of phosphodiesterase type V in the smooth muscles of corpus cavernosa.     

Stereoselective modulation of ryanodine-sensitive calcium channels by the delta isomer of hexachlorocyclohexane (delta-HCH).

delta-Hexachlorocyclohexane (delta-HCH) is shown to be 30-fold more potent as a positive inotropic agent with rat atrial strips compared with lindane (gamma-HCH). Threshold and ED50 values for enhanced contractile force at a pacing frequency of 0.5 Hz are less than 1 microM and 2.2 microM for delta-HCH and 40 microM and 63 microM for gamma-HCH, respectively. Contracture developed in atria exposed to greater than 4 microM delta-HCH (ED50 = 11 microM) but not in atria exposed to gamma-HCH. Uptake and release of Ca++ measured from actively loaded cardiac sarcoplasmic reticulum (SR) vesicles is measured with antipyrylazo III. Although delta-HCH (30 microM) decreases Ca(++)-dependent ATPase by 20%, it does not significantly alter Ca++ loading in the presence of ruthenium red. Addition of delta-HCH (5-50 microM) after loading is complete causes rapid, dose-dependent release of Ca++ from SR. Ca++ release induced by delta-HCH is markedly stereoselective. Compared with gamma-HCH (50 microM), delta-HCH (50 microM) induces a nearly 20-fold higher initial rate of Ca++ release (4.3 nmol of Ca++/mg/sec). Studies with [3H]ryanodine demonstrate that delta-HCH sharply inhibits Ca(++)- or daunorubicin-activated radioligand binding (IC50 = 37 and 25 microM, respectively, logit slope = 2). Inhibition of [3H]ryanodine-binding by delta-HCH is stereoselective inasmuch as IC50 values for alpha, beta and gamma isomers are greater than 100 microM. The delta-HCH modified Ca++ channel appears to proceed by a noncompetitive mechanism (reducing Bmax in equilibrium experiments) with respect to the conformationally sensitive binding site for [3H]ryanodine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Block of 45Ca uptake into synaptosomes by methylmercury: Ca++- and Na+-dependence

Block of Ca++ influx into isolated nerve terminals by the neurotoxicant methylmercury (MeHg) was studied for its dependence on extracellular Ca++ and Na+. Depolarization-independent entry of 45Ca++ was determined in rat forebrain synaptosomes incubated in 5 mM K+ solution. 45Ca++ uptake was similarly measured after 1 ("fast" phase) or 10 sec ("total") of elevated K+ (41.25 mM)-induced depolarization or after 10 sec of elevated K+-induced depolarization after synaptosomes had been predepolarized for 10 sec in Ca++- and MeHg-free solutions ("slow" phase). In 5 mM K+ solutions, MeHg concentrations of 125 microM and greater significantly reduced synaptosomal 45Ca++ uptake measured during 1 or 10 sec of incubation. In K+-depolarized synaptosomes, the estimated IC50 for block of total, fast and slow 45Ca++ uptake by MeHg is 75 microM; 250 microM MeHg reduced uptake by approximately 90%. The reversibility of block by extracellular Ca++ was tested by increasing the extracellular Ca++ concentration from 0.01 to 1.15 mM. When compared to control, 50 microM MeHg reduced total uptake of 45Ca++ by greater than or equal to 70% and reduced fast uptake by 20 to 60% at all concentrations of extracellular Ca++ tested. At Ca++ concentrations of 0.01 to 0.15 mM, MeHg (50 microM) reduced slow uptake by 75 to 90%, but did not affect slow uptake at higher Ca++ concentrations (greater than or equal to 0.30 mM). When the dependence of block of 45Ca++ uptake on extracellular Na+ was tested, equivalent levels of inhibition were caused by MeHg (25 microM) for fast uptake by synaptosomes in Na+-containing and Na+-free solutions.(ABSTRACT TRUNCATED AT 250 WORDS)