Desensitization and resensitization of human platelets to 5-hydroxytryptamine at the level of signal transduction.

Desensitization of platelets to 5-hydroxytryptamine (5HT) (1 microM), during active removal of the agonist by the platelet 5HT-uptake system, was studied at the level of signal transduction. Desensitization to 5HT was dose-dependent and homologous. Without occupation of the 5HT2 receptor, neither an increase in cytosolic [Ca2+] (30 nM ionomycin), nor a separate or simultaneous activation of protein kinase C (by 10 microM 1-oleoyl-2-acetylglycerol), could induce desensitization to 5HT (1 microM). During the early phase of desensitization, the 5HT2 receptor was coupled to phospholipase C, whereas during the late phase of desensitization this coupling was disconnected. However, after disappearance of the agonist, the coupling in the resting platelet recovered quickly, and was nearly complete (82%) after 30 min. During this resensitization, the 5HT-inducibility of activation of phospholipase C, of the increase in cytosolic [Ca2+] and of stimulation of protein kinase C were restored in parallel. The time course for resensitization of the 5HT-induced increase in cytosolic [Ca2+] was independent of the presence of extracellular Ca2+. It is concluded that, after dissociation of 5HT from the platelet 5HT2-receptor, 5HT-induced responses rapidly resensitize. Because of its short duration and the parallelism in recovery between the different 'down-stream phospholipase C' intracellular transduction signals, it is considered that desensitization arises from a reversible change in the transduction mechanism at a step up to or including the activation of phospholipase C. Neither desensitization nor resensitization to 5HT is dependent on the presence of extracellular Ca2+.

Cyclic AMP-phosphodiesterase IIIA1 inhibitors decrease cytosolic Ca2+ concentration and increase the Ca2+ content of intracellular storage sites in human platelets.

The effect of cyclic AMP-phosphodiesterase (cAMP-PDE) inhibitors on Ca2+ homeostasis in human platelets was studied using both quin-2 (2-(bis-(acetylamino)-5-methyl-phenoxy)methyl-6-methoxy-8-bis-(acetylami no) quinoline) and chlorotetracycline (CTC) to measure changes in cytosolic Ca2+ as well as changes in the amount of Ca2+ accumulated in intracellular storage sites. At therapeutic concentrations (1 microM) milrinone and R 80 122 but not enoximone decreased the cytosolic Ca2+ concentration in the resting platelet while the Ca2+ content in intracellular stores was increased. These observations are in accord with the proposed mechanism of action of cAMP-PDE inhibitors on cardiomyocites and highlight the particular role of cAMP in regulation of Ca2+ homeostasis.

The synergistic effect of 5-hydroxytryptamine and epinephrine on the human platelet is related to the activation of phospholipase C.

Simultaneous addition of 5-hydroxytryptamine (5-HT) and epinephrine synergistically elevated cytosolic free Ca2+ concentration ([Ca2+]cyt) and activated phospholipase C. These phenomena occurred in an agonist concentration-dependent manner and reflected an increase in maximal response rather than a, shift in affinity for either of the stimuli. Ketanserin, a 5HT2 receptor antagonist completely blocked the response of 5-HT plus epinephrine and this in the nanomolar concentration range in which the drug selectively antagonizes 5-HT2 receptor-mediated responses. The synergistic effect was not a consequence of the activation by the platelet-release products ADP and 5-HT nor of influx of extracellular Ca2+. It is concluded that an increased activation of phospholipase C plays a major role in provoking the synergistic effects between 5-HT and epinephrine at the level of signal transduction.

Cardiac effects of R 79595 and its isomers (R 80122 and R 80123) in an acute heart failure model. A new class of cardiotonic agents with highly selective phosphodiesterase III inhibitory properties.

R 79595 (N-cyclohexyl-N-methyl-2-[[[phenyl (1,2,3,5-tetrahydro-2 oxoimidazo [2,1-b]-quinazolin-7-yl) methylene] amin] oxy] acetamide) and its isomers represent a novel class of compounds with phosphodiesterase (PDE) inhibitory and cardiotonic (positive inotropic) actions. The cardiac effects of this class of compounds were investigated in the hexobarbital-depressed heart-lung preparation of the guinea-pig. After induction of heart failure (reduction of cardiac output to 25% of the initial value) cumulative addition of R 79595 or its isomers R 80122 (E-isomer) and R 80123 (Z-isomer) concentration-dependently reversed the cardiac depressant effects of hexobarbitone-Na. With regard to reconstitution of contractility and cardiac function R 80122 (E-isomer) was 10 fold more potent than R 79595 (1:1 mixture of the isomers) and nearly 100 fold more potent than R 80123 (Z-isomer). Furthermore, the cardiotonic action of the most potent isomer (R 80122) was compared to the effects of several positive inotropic reference compounds. The order of cardiotonic potency was as follows: (-)-adrenaline > R 80122 = adibendan > digitoxin > milrinone = enoximone > theophylline. Adibendan (EC50 value: 6.7 +/- 1.8 x 10.-8 mol/l), which showed cardiotonic effects in the same concentration range as R 80122 (EC50 value: 6.1 +/- 1.3 x 10(-8) mol/l), was significantly (p < 0.01) less effective than R 80122 with respect to the maximally induceable increase in cardiac output (CO). The cardiotonic effects of R 80122 could be observed in the low concentration range of 3 x 10(-8) to 1 x 10(-6) mol/l, whereas enoximone (EC50 value: 1.2 +/- 0.1 x 10(-5) mol/l) and milrinone (EC50 value: 8.9 +/- 3.5 x 10(-6) mol/l) elicited positive inotropic effects at 100 fold higher concentrations. Digitoxin was 10 fold less and theophylline was 300 fold less potent than R 80122 with regard to reconstitution of heart function. The cardiotonic effects of R 80122 were not accompanied by an increase in heart rate as found with milrinone, theophylline or (-)-adrenaline in this model. Furthermore, the PDE inhibitory effect of R 79595 and its E-isomer R 80122 were investigated in partially purified isoenzymes from guinea-pig ventricles. The IC50 values of R 79595 and R 80122 on PDE I-IV were compared to the IC50 values of adibendan, milrinone, enoximone and theophylline. The selectivity of an inhibitor for PDE III was evaluated by division of its IC50 values on PDE I, II and IV by the IC50 value on PDE III. R 80122 was the most potent and selective PDE III inhibitor.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparative effects of R 80122, enoximone, and milrinone on left ventricular phosphodiesterase isoenzymes in vitro and on contractility of normal and stunned myocardium in vivo in dogs.

Using different subtypes of cyclic nucleotide phosphodiesterase (PDE) isoenzymes isolated from canine left ventricle, we identified R 80122, a 1,2,3,5-tetrahydro2-oxoimidazo[2,1-b]quinazoline derivative that was a more selective and potent inhibitor of PDE type III than milrinone or enoximone. Such substances improve cardiac contraction and relaxation, elicit vasodilation, and increase cardiac output (CO). To determine the extent to which these compounds affect the contractile force of stunned myocardium, the effects of enoximone, milrinone, and R 80122 on cardiac function were compared in anesthetized dogs subjected to 15-min occlusion of the left anterior descending coronary artery (LAD) followed by reperfusion, and treated beginning 30 min after reperfusion, with the compound being studied. During occlusion, all dogs exhibited passive systolic ventricular wall bulging in the ischemic area. Thirty minutes after reperfusion, systolic wall thickening was significantly decreased in the reperfused LAD segments and remained low (at 36% of baseline) in control animals. After enoximone administration, global left ventricular (LV) function was improved with i.v. doses greater than or equal to 0.64 mg/kg. Systolic wall thickening in the ischemic myocardium was restored less than or equal to 70% of baseline at 1.25 mg/kg i.v., but this dose also induced a marked decrease in arterial pressure and an increase in heart rate (HR). Milrinone and R 80122 significantly increased global LV function and systolic wall thickening in ischemic areas at doses greater than or equal to 0.16 mg/kg i.v. At the highest doses, HR increased slightly with both compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac and hemodynamic effects of intravenous R 80122, a new phosphodiesterase III inhibitor, in anesthetized and awake dogs.

R 80122 is a newly synthesized, selective phosphodiesterase III inhibitor. The cardiac and hemodynamic effects of this compound following intravenous administration were studied in closed-chest anesthetized as well as in conscious chronically instrumented dogs. The findings in the closed-chest anesthetized dogs indicate that R 80122 has positive inotropic and possibly moderate vasodilating properties [maximum increase in LV dp/dtmax/pd: 61%, and maximum decrease in systemic vascular resistance: 29% (a 12.5% decrease for the solvent)]. As a result, cardiac output maximally increased to 149% of the control value. The most striking effect of R 80122 was its positive lusitropic effect (maximal decrease in the time constant of relaxation [T] of 46%). This pronounced lusitropic effect of R 80122 can be regarded as beneficial, because of the increasing evidence that lusitropic defects play an important role in disorders related to heart failure. These effects of R 80122 were associated with only slight changes in arterial blood pressure. The effects of R 80122 lasted about 75 min after stopping the infusion. No ventricular arrhythmias were noted during and after infusion of the compound. The positive inotropic effects seen in anesthetized dogs were confirmed in conscious nonsedated dogs. It may be concluded that R 80122 has a clinically favorable cardiovascular profile for acute applications in heart failure, because its combined positive inotropic and positive lusitropic effects, and moderate vasodilating properties lead to a pronounced increase in cardiac output and only minimal changes in aortic blood pressure.

Ouabain increases the calcium concentration in intracellular stores involved in stimulus-response coupling in human platelets.

The effect of ouabain on Ca2+ homeostasis in human platelets was studied using both quin 2 and chlorotetracycline to monitor changes in cytosolic Ca2+ as well as changes in the amount of Ca2+ accumulated in intracellular storage sites. In resting platelets, ouabain induces a concentration- and time-dependent increase in cytosolic Ca2+ concentration and a marked elevation of Ca2+ in the intracellular stores. The amount of Ca2+ mobilized from these stores upon stimulation with thrombin, as well as thrombin-induced secretion of platelet 5-hydroxytryptamine, was increased after preincubation with the glycoside (3 x 10(-6) M). These data show that ouabain induces an elevation of intracellular Ca2+ levels, most likely mediated via Na(+)-Ca2+ exchange, and that this incremental amount of Ca2+ is accumulated in an intracellular store involved in stimulus-response coupling. This may explain the enhanced functional responses of platelets to agonists in the presence of ouabain and suggests a role for Na(+)-Ca2+ exchange in Ca2+ homeostasis of the human platelet.

The role of protein kinase C in the human platelet.

Protein kinase C is a key enzyme in signal transduction systems that involve the breakdown of phospholipids. In the platelet, protein kinase C plays an important role in the induction of the aggregatory and secretory responses as well as in preventing a continuous activation of the platelet excitatory signal transducing system. Furthermore, elevation of platelet cAMP that leads to inhibition of platelet activation appears to interact with the excitatory pathway at the level of protein kinase C. A pivotal role for protein kinase C in platelet activation is proposed.

Effect of thromboxane A2 synthetase inhibition, singly and combined with thromboxane A2/prostaglandin endoperoxide receptor antagonism, on inositol phospholipid turnover and on 5-HT release by washed human platelets.

Differential effects on human platelet function of thromboxane A2 (TXA2) synthetase inhibition singly and of TXA2 synthetase inhibition combined with TXA2/prostaglandin endoperoxide receptor antagonism were revealed, using ridogrel as a probe. Ridogrel combines selective TXA2 synthetase inhibition with TXA2/prostaglandin receptor antagonism in one molecule: in washed human platelets, the compound reduces the production of TXB2 (IC50 = 1.3 X 10(-8) M) and increases that of PGF2 alpha, PGE2, PGD2 from [14C]arachidonic acid. Additionally, at higher concentrations (Ki = 0.52 X 10(-6) M), it selectively antagonizes the breakdown of inositol phospholipids, subsequent to stimulation of TXA2/prostaglandin endoperoxide receptors with U 46619. The latter happens in a competitive way with fast receptor association-dissociation characteristics. At low concentrations (1 X 10(-9)-1 X 10(-7) M) producing single TXA2 synthetase inhibition, ridogrel reduces the collagen-induced formation of TXB2 by washed platelets, but enhances [32P]phosphatidic acid (PA) accumulation and [3H]5-hydroxytryptamine (5-HT) release. At higher concentrations (1 X 10(-6)-1 X 10(-5) M) which additionally block U 46619-induced [32P]PA accumulation, ridogrel inhibits the [32P]PA accumulation and release of [3H]5-HT by human platelets stimulated with collagen. These observations, corroborated by results obtained with OKY 1581, sulotroban, indomethacin and human serum albumin, suggest a causal role for prostaglandin endoperoxides in the stimulation by TXA2 synthetase inhibition of platelet reactions to collagen. They reinforce the concept that TXA2 synthetase inhibition-induced reorientation of cyclic endoperoxide metabolism, away from TXA2 into inhibitory prostanoids, requires additional TXA2/prostaglandin endoperoxide receptor antagonism to achieve optimal anti-platelet effects.

Is there evidence of a role of the phosphoinositol-cycle in the myocardium?

The recent findings on a more general involvement of phospholipids in signal transduction and on the different roles of inositolphospholipids in particular, thoroughly complicate research in this field. It becomes increasingly evident that measuring [3H]inositolphosphate formation alone will never provide insight into the complex machinery of cellular signalling. Certainly for the heart in which the role(s) of the inositolphospholipids is far from clarified, the novel trends provide new directions for research.

The role of endogenously formed diacylglycerol in the propagation and termination of platelet activation. A biochemical and functional analysis using the novel diacylglycerol kinase inhibitor, R 59 949.

The putative roles for the second messenger, diacylglycerol, were investigated in intact platelets using a novel diacylglycerol kinase inhibitor, R 59 949, or (3-[2-[4-[bis(4-fluorophenyl)methylene]-1-piperidinyl]ethyl]-2,3- dihydro-2-thioxo-4(1H)-quinazolinone). The compound inhibited the diacylglycerol kinase in a concentration-dependent manner (10(-8) to 10(-5) M) in isolated platelet membranes and in intact platelets. When platelets were stimulated with vasopressin in the presence of the compound, protein kinase C activity was markedly increased; the formation of inositol phosphates, the increase in intracellular Ca2+ and shape-change reaction were antagonized while the vasopressin-induced polyphosphoinositide synthesis was amplified, and this in a distinct inositolphospholipid pool. In the presence of R 59 949, vasopressin- as well as collagen-induced release reaction and aggregation was strongly increased, independently of the formation of arachidonate metabolites. It is concluded that diacylglycerol formed after receptor activation, likely by activating the protein kinase C, plays an important role in the propagation of platelet functional responses in casu aggregation and secretion and controls the termination of the primary receptor coupled responses.

R 68 070: thromboxane A2 synthetase inhibition and thromboxane A2/prostaglandin endoperoxide receptor blockade combined in one molecule--I. Biochemical profile in vitro.

R 68 070 or (E)-5-[[[(3-pyridinyl)[3-(trifluoromethyl)phenyl]- methylen]amino]oxy] pentanoic acid (Janssen Research Foundation, Belgium) combines specific thromboxane A2 (TXA2) synthetase inhibition with TXA2/prostaglandin endoperoxide receptor blockade in one molecule. In vitro, the compound specifically inhibits the production of TXB2 from [14C] arachidonic acid by washed human platelets (IC50 = 8.2 X 10(-9) M) and by platelet microsomes (IC50 = 3.6 X 10(-9) M), of MDA (IC50 = 1.91 X 10(-8) M) and of TXB2 (IC50 = 1.47 X 10(-8) M) by thrombin-coagulated human platelet-rich plasma (P.R.P.) and whole blood respectively and increases the levels of PGD2, PGE2, PGF2 alpha and 6-keto-PGF1 alpha. The activity of cyclo-oxygenase-, prostacyclin synthetase-, 5-, 12- and 15-lipoxygenase-enzymes are not affected. Additionally, R 68 070 inhibits human platelet aggregation in P.R.P. induced by U 46619 3 X 10(-7) M to 2 X 10(-6) M (IC50 = 2.08 X 10(-6) M to 2.66 X 10(-5) M, collagen 0.5 to 2 micrograms/ml (IC50 = 2.85 X 10(-6) M to 4.81 X 10(-5) M), arachidonic acid 7.5 X 10(-4) M to 2 X 10(-3) M (IC50 = 2.1 X 10(-8) M to 3.3 X 10(-8) M) and the U 46619 (1 X 10(-7) M)-induced accumulation of [32P] phosphatidic acid (IC50 = 5.24 X 10(-7) M) in washed human platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical profile of risperidone, a new antipsychotic.

Risperidone was compared to the 5-hydroxytryptamine2 antagonist ritanserin and to the dopamine-D2 antagonist haloperidol. The in vitro receptor binding (neurotransmitter-, peptide- and ion channel binding sites) and neurotransmitter uptake profile were investigated. Risperidone revealed, like ritanserin, a very high binding affinity for 5-hydroxytryptamine2 receptors (Ki = 0.16 and 0.30 nM, respectively) and a slow dissociation (half-time, 31 and 160 min). In accordance, risperidone (IC50 = 0.5 nM) and ritanserin (IC50 = 1.8 nM) potently blocked serotonin-induced 32P-phosphatidic acid formation in human blood platelets. Risperidone showed, like haloperidol, high binding affinity for dopamine-D2 receptors (Ki = 3.13 and 1.55 nM, respectively) and rapid dissociation (half-time, 2.7 and 5.8 min). Risperidone displayed higher binding affinity than ritanserin and haloperidol for alpha-1 adrenergic (Ki = 0.8 nM), histamine-H1 (Ki = 2.23 nM) and alpha-2 adrenergic receptors (Ki = 7.54 nM). In in vitro superfusion experiments, risperidone and haloperidol reversed at nanomolar concentrations the inhibition by LY 171555 (a dopamine-D2 agonist) and by amphetamine of potassium and electrically evoked release of [3H]acetylcholine from striatal slices (postsynaptic dopamine-D2 effects). Both drugs reversed with similar potency the inhibition by LY 171555 of electrically evoked release of [3H]dopamine (a presynaptic dopamine-D2 effect). Risperidone did not affect the activation by amphetamine of [3H]dopamine efflux from rat striatal slices. Risperidone enhanced at nanomolar concentrations the stimulated [3H]norepinephrine efflux from cortical slices and it similarly reversed the inhibition by clonidine, at concentrations corresponding to its binding affinity for alpha-2 adrenergic receptors. The in vitro biochemical properties of risperidone are in agreement with the reported in vivo pharmacological profile, the relation to clinical findings is discussed.

The signal transducing system coupled to serotonin-S2 receptors.

The signal transducing system coupled to the serotonin-S2 receptor on platelets involves metabolism of inositol-containing phospholipid, elevation of intracellular free Ca2+ and activation of protein kinase C. Evidence for coupling of the serotonin-S2 receptor to the same signal transducing system in brain and smooth muscle tissue is reviewed.

Functional expression of the amplification reaction between serotonin and epinephrine on platelets.

Human platelet reactions are enhanced when a combination of serotonin and epinephrine is used as a stimulus. The amplification of the response operates through S2-serotonergic and alpha 2-adrenergic receptor subtypes. The first wave of platelet aggregation, which is independent of platelet release products, is enhanced while the latency period for the occurrence of release reaction and prostaglandin biosynthesis is shortened. Synergism between serotonin and epinephrine on the platelet occurs at low concentration of agonists and may contribute to the formation of an arterial thrombus in vivo.

The synergistic effect of serotonin and epinephrine at the level of signal transduction.

Evidence is presented of synergistic interactions occurring between serotonin and epinephrine at the level of signal transduction. Different mechanisms that can explain these synergistic effects are discussed.

Agonist-induced desensitization of 5-HT(2) receptors on cultured calf aortic smooth muscle cells.

[(32)P]Phosphatidic acid (PA)-formation was quantified in calf aortic smooth muscle cultures for measuring the activation of the signal transducing system coupled to the 5-hydroxytryptamine(2)-(5-HT(2)) receptor. [(32)P]PA-formation was increased upon stimulation of smooth muscle cells with serotonin (5-HT) and 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM), but not with the 5-HT(1) agonists N,N-dipropyl-8-hydroxy-2-aminotetralin and RU 24969. The potency of drugs to inhibit the 5-HT induced [(32)P]PA-formation closely corresponded to their binding affinity for 5-HT(2) receptors. 24-Hour treatment of smooth muscle cultures with 5-HT or DOM resulted in a substantial decrease of 5-HT induced [(32)P]PA-formation. In contrast to the anomalous 5-HT(2) receptor regulation in vivo, 5-HT(2) receptors on smooth muscle cells appeared to be desensitized by agonist treatment.

The effect of the intracellular calcium chelator Quin-2 on the platelet phosphoinositide metabolism, protein phosphorylation and morphology.

When human platelets prelabeled with [32P] orthophosphate were loaded with Quin-2, the 32P-incorporation in phosphatidic acid, phosphatidylinositol-4 phosphate and phosphatidylinositol-4,5 bisphosphate increased, that in phosphatidylinositol decreased. These effects occurred in a Quin-2-concentration-dependent manner. On stimulation of the serotonin-S2 receptor, signal transduction, measured as changes in labeling in phospholipids and phosphoproteins, was altered in the presence of the fluorophore. Microscopic evaluation illustrated that Quin-2 affected platelet morphology as well in resting as in stimulated platelets. A correlation between platelet shape change and myosin light chain phosphorylation was apparent. The data evidence that the Quin-2 that is widely used for fluorometric determination of intracellular Ca2+, affects the metabolism of inositol-containing phospholipids whose breakdown is a key event in Ca2+-mobilization on excitatory platelet activation. These fluorophore-induced alterations might, besides the Ca2-chelating properties, play an important role in the Ca2+-dependent signalling processes in these cells.