Didier de Chaffoy de Courcelles

Serotonin-induced alterations in inositol phospholipid metabolism in human platelets

When human platelets were incubated for 5 min with [32P]orthophosphate and then stimulated with serotonin, the 32P content of phosphatidylinositol (PI) increased within seconds, compared with the control. The 32P content of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) only slightly increased during the first minute after addition of serotonin and became more apparent on prolonged stimulation. These changes were not caused by serotonin-induced change in the specific activity of ATP. Using inorganic phosphate determination for the chemical quantification of different inositol phospholipid pools, we found that the platelet PI content remained nearly constant; the amount of PIP increased while that of PIP2 decreased. When the platelets were first prelabeled for 80 min with [32P]orthophosphate, the changes in 32P-labeled inositol phospholipids after addition of serotonin were similar to their changes in mass. When the platelet inositol phospholipids were labeled with myo-[2-3H]inositol, serotonin induced an increase in [3H]inositol phosphates. From these data, it is concluded in addition to the earlier-reported effects on phospholipid metabolism (de Chaffoy de Courcelles, D. et al. (1985) J. Biol. Chem. 260, 7603-7608) that serotonin induces: a very rapid formation of PI; and alterations in inositol phospholipid interconversion that cannot be explained solely as a resynthesis process of PIP2.

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 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.

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 inositol-phospholipids is far from clarified, the novel trends provide new directions for research.

Agonist-induced desensitization of 5-HT2 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.

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

Abstract Differential effects on human platelet function of thromboxane A 2 (TXA 2 ) synthetase inhibition singly and of TXA 2 synthetase inhibition combined with TXA 2 /prostaglandin endoperoxide receptor antagonism were revealed. using ridogrel as a probe. Ridogrel combines selective TXA 2 synthetase inhibition with TXA 2 /prostaglandin receptor antagonism in one molecule; in washed human platelets, the compound reduces the production of TXB 2 (IC 50 =1.3 × 10 −8 M) and increases that of PGF 2α , PGE 2 , PGD 2 from [ 14 C]arachidonic acid. Additionally, at higher concentrations (K i = 0.52 × 10 −6 M), it selectively antagonizes the breakdown of inositol phospholipids, subsequent to stimulation of TXA 2 /prostaglandin endoperoxide receptors with U 46619. The latter happens in a competitive way with fast receptor association-dissociation characteristics. At low concentrations (1 × 10 −9 − × 10 −7 M) producing single TXA 2 synthetase inhibition, ridogrel reduces the collagen-induced formation of TXB 2 by washed platelets, but enhances [ 32 P]phosphatidic acid (PA) accumulation and [ 3 H]5-hydroxytryptamine (5-HT) release. At higher concentrations (1 × 10 −6 −1 × 10 −5 M) which additionally block U 46619-induced [ 32 P]PA accumulation, ridogrel inhibits the [ 32 P]PA accumulation and release of [ 3 H]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 TXA 2 synthetase inhibition of platelet reactions to collagen. They reinforce the concept that TXA 2 synthetase inhibition-induced reorientation of cyclic endoperoxide metabolism, away from TXA 2 into inhibitory prostanoids, requires additional TXA 2 /prostaglandin endoperoxide receptor antagonism to achieve optimal anti-platelet effects.

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.

Cardiac effects of R 79595 and its isomers (R 80122 and R 80123) in an acute heart failure model

SummaryR 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±1.8×10.−8 mol/l), which showed cardiotonic effects in the same concentration range as R 80122 (EC50 value: 6.1±1.3×10−8 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 3×10−8 to 1×10−6 mol/l, whereas enoximone (EC50 value: 1.2±0.1×10−5 mol/l) and milrinone (EC50 value: 8.9±3.5×10−6 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. The IC50 value was 0.017±0.001 µmol/l. IC50 ratios ranged from 2353 (PDE II/PDE III) to 7000 (PDE I/PDE III). The PDE IV/PDE III ratio for R 80122 (5118) was more than 10 times higher than the PDE IV/PDE III ratio calculated for adibendan (333) which displayed the highest PDE III selectivy and potency (IC50 0.36±0.04 µmol/l). among the reference compounds. Milrinone and enoximone also preferentially inhibited PDE III, whereas theophylline inhibited all four isoenzymes in an unselective manner with low potency.Since the EC50 values of R 80122 for cardiotonic effects and the IC50 value for PDE III inhibition were of the same order of magnitude, it can be assumed that potent and selective PDE III inhibition may be in part responsible for the cardiotonic effects of R 80122 observed in the acute heart failure model.The potent cardiotonic properties and the absence of positive chronotropy revealed by this study suggest that R 80122 might exert a beneficial effect in the treatment of acute heart failure.