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5.HT1D BETA IS THE MAJOR 5-HT1D RECEPTOR sUBTYPE EXPRESSED IN CANINE LARGE CORONARY ARTERIES AND SAPHENOUS VEIN E,_.,,S_g~, C. Faure, and D. Graham, Synth61abo Recherche, 10 F rue des Carri~res, 92504 Rueil Malmaison, France.
5-Hydroxytryptamine (5-HT) released by aggregating platelets is believed to mediate vasospasms and vasoocclusions of human endothelium-damaged coronary arteries and recent evidence indicates that 5-HT~D.,ko receptors might play a major role in these effects. Although two different subtypes of 5-HT~o receptors (5-HT~D~,and 5-HT1Dp) have been cloned in man, one report has suggested that it is in fact the 5-HT~DI~subtype that is associated with this coronary vasoconstriction. Contraction of dog coronary arteries and saphenous vein by 5-HT is also mediated by a 5-HT~D.~ko receptor thereby offering possible paradigms of the response occurring in human coronary arteries. In the present study we used RT-PCR experiments to identify the 5-HT m receptor subtype(s) expressed in endothelium-denuded coronary arteries and saphenous vein of the beagle dog. Degenerate oligonucleotide primers, based upon the known sequences of mammalian 5-HT~D,~ and 5HT~D~receptors, were designed to amplify specifically a region of both receptor subtype cDNAs. A single identical PCR product of 535 bp was obtained from both canine coronary artery and saphenous vein and whose sequence closely resembles that of the human 5-HT~o~ receptor (98% amino acid identity). The present study provides the first report identifying a canine homologue of the 5-HT1D~ receptor and illustrates that this subtype is in fact the major 5-HT~D receptor expressed in dog coronary artery and saphenous vein. We suggest that this subtype is responsible for the vasoconstriction evoked by 5-HT and sumatriptan in these canine tissues.
CHARACTERIZATION OF THE MONOCLONAL ANTISUBSTANCE P ANTIBODY DERIVED FROM THE RAT/MOUSE HETEROHYDRIDOMA NC1/34 D.F. Biqqs and A. Jafarian Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8
I N H I B I T O R Y 5-HT R E C E P T O R S I N V O L V E D IN SYMPATHETIC PRESSOR AND CARDIAC EFFECTS / .C. Velasco, A. Mor~in, E. Mart/n, M.L. Martfn and L. San Rom~ Lab. Farmacognosia y Farmacodinamia, Facultad de Farmacia, Universidad de Salamanca. 37007 SALAMANCA, Spain. The intravenous infusion of 5-hydroxytryptamine (5HT) reduced the pressor and cardiac effects obtained by electrical stimulation of the sympathetic outflow from the spinal cord in pithed rats, at intervals of 10 min over the 1 hour that the infusion lasted. The pressor inhibitory action of 5-HT was blocked by cyproheptadine and methiothepine and did not become manifest against the effects obtained in pithed rats that had been pretreated with exogenous i.v. noradrenaline. The cardioinhibitory action was inhibited by the 5-HT2 serotonergic antagonists ritanserine and ciproheptadine and had been also obtained againts the cardioacceleration elicited by i.v. administration of isoprenaline. The 5-HT1 receptor agonist 5-carboxamidetryptamine (5-CT), caused an inhibition of pressor effect and the inhibition of the cardiac stimulating effect was also obtained by intravenous infusion of 1-(3-chlorophenyl)piperazine (m-CPP), a 5-HT2 receptor agonist. Our results suggest that 5-hydroxytryptamine interferes with sympathetic neurotransmission by inhibiting pressor effects through a m a i n l y presynaptic 5-HT1 mechanism and also inhibiting cardiac stimulating effects through a postysnaptic 5-HT2 serotonergic mechanism.
USE OF ?xNTAGONISTS TO ASSESS PAF R E C E P T O R S HETEROGENEITY C. Centemeri, M. Cereda, P. Ciceri, S. Colli*, D: Tosarello & S. Nicosia, Laboratory of Molecular Pharmacology, Institute of Pharmacological Sciences, *Center Grossi Paoletti, University of Milan, Via Balzaretti, 9 - 20133 Milan, Italy.
The rat/mouse heterohybridoma NCl/34 secretes a highly specific anti-substance P (~-SP Ab), rat, IgG1 type monoclonal antibody. Experiments, in vitro, using inhibition ELISA, indicated that the c~-SP Ab had about 50 x greater affinity for substance P (SP) than neurokinin A (NKA). Passive immunization of guinea-pigs with the ~-SP Ab prevented bronchospastic responses to SP, NKA and capsaicin (Jafarian et al. Life Sciences, in press), but there was no evidence of specificity for SP. This was attributed to differences in the molar ratio (c~-SP Ab:SP/NKA) and epitopic similarities between the C-terminals of the two tachykinins. To try and map the epitopic determinants of SP involved in binding to the ~-SP Ab, we used inhibition ELISA to compare the activities of SP fragments derived from the N- and C-terminals. N-terminal fragments were devoid of activity; all C-terminal fragments examined were active in the inhibition assay. SP ~11 and SP 7~1 were more active than SP, SP 2"~ to SP51~were as active as SP; SP 9"~ and Sp 8-~ were less active than SP. These data indicate that the c~-SP Ab is directed against epitopic determinants on amino acids 6-11 and that the integrity of the carboxyl group is crucial to binding. In guinea-pigs, in vivo, SP 6"~ was a potent NK1 receptor agonist; SP 7~ was inactive. Supported by the Alberta Lung Association
-- 189--
The application of PAF to cultured rat: peritoneal macrophages, human monocytes spontaneously differentiated to macrophages and freshly isolated human platelets elicits a biphasic increase in citosolic free calcium, [Ca2+]i. By using PAF-antagonists, SCH 37370 and WEB 2086, we investigated whether differences exist between PAF receptors coupled to [Ca2+]i elevation in different cellular types. [Ca2+]i was measured by loading the cells with Fura21AM. In platelcls, 10 laM SCH 37370 was able to completely abolish the elevation of [Ca2+]i induced by 36 nM PAF, with an IC5o=211M. On the contrary, in rat macrophages and human monocytes, the maximal inhibition observed was approximately 70% even at higher SCH 37370 concentrations (up to 140 ~M). WEB 2086 antagonized PAF-induced [Ca2+]iincrease completely both in macrophages and in platelets. In the latter cells, the inhibition was observed at /~M concentration, while macrophages requested a much higher concentration (0.1-1mM). Furthermore, in macrophages, WEB 2086 dose-response curve, spanning more than two orders of magnitude, was compatible with an heterogeneity of PAF receptors. Therefore, our results indicate that: 1. Platelets contain a homogeneous class o f receptors, fully antagonized by SCH 37370; 2. Macrophages posses a further class of receptors, antagonized by WEB 2086 and not by SCH 37370; 3. This difference between macrophages and platelets is not attributable to a species difference, because differentiated human monocytes behave as rat macrophages; 4. SCH37370 is a more selective PAF antagonist than WEB 2086, interacting with only one of the classes of PAF receptors present on macrophages and monocytes.
5-Hydroxytryptamine (5-HT) released by aggregating platelets is believed to mediate vasospasms and vasoocclusions of human endothelium-damaged coronary arteries and recent evidence indicates that 5-HT~D.,ko receptors might play a major role in these effects. Although two different subtypes of 5-HT~o receptors (5-HT~D~,and 5-HT1Dp) have been cloned in man, one report has suggested that it is in fact the 5-HT~DI~subtype that is associated with this coronary vasoconstriction. Contraction of dog coronary arteries and saphenous vein by 5-HT is also mediated by a 5-HT~D.~ko receptor thereby offering possible paradigms of the response occurring in human coronary arteries. In the present study we used RT-PCR experiments to identify the 5-HT m receptor subtype(s) expressed in endothelium-denuded coronary arteries and saphenous vein of the beagle dog. Degenerate oligonucleotide primers, based upon the known sequences of mammalian 5-HT~D,~ and 5HT~D~receptors, were designed to amplify specifically a region of both receptor subtype cDNAs. A single identical PCR product of 535 bp was obtained from both canine coronary artery and saphenous vein and whose sequence closely resembles that of the human 5-HT~o~ receptor (98% amino acid identity). The present study provides the first report identifying a canine homologue of the 5-HT1D~ receptor and illustrates that this subtype is in fact the major 5-HT~D receptor expressed in dog coronary artery and saphenous vein. We suggest that this subtype is responsible for the vasoconstriction evoked by 5-HT and sumatriptan in these canine tissues.
CHARACTERIZATION OF THE MONOCLONAL ANTISUBSTANCE P ANTIBODY DERIVED FROM THE RAT/MOUSE HETEROHYDRIDOMA NC1/34 D.F. Biqqs and A. Jafarian Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8
I N H I B I T O R Y 5-HT R E C E P T O R S I N V O L V E D IN SYMPATHETIC PRESSOR AND CARDIAC EFFECTS / .C. Velasco, A. Mor~in, E. Mart/n, M.L. Martfn and L. San Rom~ Lab. Farmacognosia y Farmacodinamia, Facultad de Farmacia, Universidad de Salamanca. 37007 SALAMANCA, Spain. The intravenous infusion of 5-hydroxytryptamine (5HT) reduced the pressor and cardiac effects obtained by electrical stimulation of the sympathetic outflow from the spinal cord in pithed rats, at intervals of 10 min over the 1 hour that the infusion lasted. The pressor inhibitory action of 5-HT was blocked by cyproheptadine and methiothepine and did not become manifest against the effects obtained in pithed rats that had been pretreated with exogenous i.v. noradrenaline. The cardioinhibitory action was inhibited by the 5-HT2 serotonergic antagonists ritanserine and ciproheptadine and had been also obtained againts the cardioacceleration elicited by i.v. administration of isoprenaline. The 5-HT1 receptor agonist 5-carboxamidetryptamine (5-CT), caused an inhibition of pressor effect and the inhibition of the cardiac stimulating effect was also obtained by intravenous infusion of 1-(3-chlorophenyl)piperazine (m-CPP), a 5-HT2 receptor agonist. Our results suggest that 5-hydroxytryptamine interferes with sympathetic neurotransmission by inhibiting pressor effects through a m a i n l y presynaptic 5-HT1 mechanism and also inhibiting cardiac stimulating effects through a postysnaptic 5-HT2 serotonergic mechanism.
USE OF ?xNTAGONISTS TO ASSESS PAF R E C E P T O R S HETEROGENEITY C. Centemeri, M. Cereda, P. Ciceri, S. Colli*, D: Tosarello & S. Nicosia, Laboratory of Molecular Pharmacology, Institute of Pharmacological Sciences, *Center Grossi Paoletti, University of Milan, Via Balzaretti, 9 - 20133 Milan, Italy.
The rat/mouse heterohybridoma NCl/34 secretes a highly specific anti-substance P (~-SP Ab), rat, IgG1 type monoclonal antibody. Experiments, in vitro, using inhibition ELISA, indicated that the c~-SP Ab had about 50 x greater affinity for substance P (SP) than neurokinin A (NKA). Passive immunization of guinea-pigs with the ~-SP Ab prevented bronchospastic responses to SP, NKA and capsaicin (Jafarian et al. Life Sciences, in press), but there was no evidence of specificity for SP. This was attributed to differences in the molar ratio (c~-SP Ab:SP/NKA) and epitopic similarities between the C-terminals of the two tachykinins. To try and map the epitopic determinants of SP involved in binding to the ~-SP Ab, we used inhibition ELISA to compare the activities of SP fragments derived from the N- and C-terminals. N-terminal fragments were devoid of activity; all C-terminal fragments examined were active in the inhibition assay. SP ~11 and SP 7~1 were more active than SP, SP 2"~ to SP51~were as active as SP; SP 9"~ and Sp 8-~ were less active than SP. These data indicate that the c~-SP Ab is directed against epitopic determinants on amino acids 6-11 and that the integrity of the carboxyl group is crucial to binding. In guinea-pigs, in vivo, SP 6"~ was a potent NK1 receptor agonist; SP 7~ was inactive. Supported by the Alberta Lung Association
-- 189--
The application of PAF to cultured rat: peritoneal macrophages, human monocytes spontaneously differentiated to macrophages and freshly isolated human platelets elicits a biphasic increase in citosolic free calcium, [Ca2+]i. By using PAF-antagonists, SCH 37370 and WEB 2086, we investigated whether differences exist between PAF receptors coupled to [Ca2+]i elevation in different cellular types. [Ca2+]i was measured by loading the cells with Fura21AM. In platelcls, 10 laM SCH 37370 was able to completely abolish the elevation of [Ca2+]i induced by 36 nM PAF, with an IC5o=211M. On the contrary, in rat macrophages and human monocytes, the maximal inhibition observed was approximately 70% even at higher SCH 37370 concentrations (up to 140 ~M). WEB 2086 antagonized PAF-induced [Ca2+]iincrease completely both in macrophages and in platelets. In the latter cells, the inhibition was observed at /~M concentration, while macrophages requested a much higher concentration (0.1-1mM). Furthermore, in macrophages, WEB 2086 dose-response curve, spanning more than two orders of magnitude, was compatible with an heterogeneity of PAF receptors. Therefore, our results indicate that: 1. Platelets contain a homogeneous class o f receptors, fully antagonized by SCH 37370; 2. Macrophages posses a further class of receptors, antagonized by WEB 2086 and not by SCH 37370; 3. This difference between macrophages and platelets is not attributable to a species difference, because differentiated human monocytes behave as rat macrophages; 4. SCH37370 is a more selective PAF antagonist than WEB 2086, interacting with only one of the classes of PAF receptors present on macrophages and monocytes.