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Inhibition of calcium-dependent cellular reactions by antagonist drugs which possess common pharmacophore
THE EFFECT OF BUPIVACAINE ON C Y T O P L A S M I C CALCIUM ION MOBILIZATION IN N O N - E X C I T A B L E AND EXCITABLE CELLS N..__Kuboyama, S. Nakao, Y. Moriya, A. Scholz* and W. V o g e l * Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271, Japan, *Physiologisches Institut, Justus-LiebigUniversit&t 35392 GieSen, Germany.
T H E R O L E O F E X T R A C E L L U L A R C A L C I U M IN T H E CONTRACTILE EFFECT OF CYCLOPIASONIC ACID IN T H E C A T G A S T R I C F U N D U S G. V. Petkov and K. K. Boer ~ University of Sofia, Faculty of Biology, 1421-Sofia, Bulgaria and 1Institute of Biophysics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria In circular smooth muscle strips isolated from the cat gastric fundus, cyclopiazonic acid ( C P A ) , a specific inhibitor of sarcoplasmic reticulum CaZ-ATPase, induced a prolonged increase in mucsle tone (238 -+ 63 °/0, n=45) depending on the concentration of extracellular Ca z*. In Ca2+-deficient solution C P A ( 1 0 ~M) slightly increased the tone (20.6 -+ 14.2 °/0, n=13) but addition of 2.5 mM Ca z+ to the bath led to a very strong and rapid contraction, suggesting the enhanced Ca z+ permeability of the plasma membrane; in CaZ+-deficient solution, containing 3 l.tM nifedipine, CPA elicited only a negligible contraction (2.0 +- 0.7 %, n=4); in CaZ*-free solution, containing 2 mM E G T A , even 30"ttM C P A failed to induce contraction (n=4). In the presence of Ca z+ (2.5 mM) and nifedipine (3 [.tM), the CPA-induced increase in the tone was very slow, suggesting the important role of the L-type Ca z+ channels in the CPA-induced contraction, on the one hand, and the existence of a potential-independent mechanism, on the other. C P A shifted to the left the Ca z* concentration-response curves not only in the absence, but also in the presence of nifedipine (3 I.tM) and atropine (1 l.tM). Another evidence for the participation of the L-type Ca 2~ channels in the CPA-induced contraction was that the agonist Bay K 8644 (0.1 nM - 1 ~M)potentiated C P A effect in the presence of the antagonist nifedipine. In conclusion, the CPA-induced contraction is a result of the Ca z+ entry into the cells from the extracellular solution but not from the intracellular Ca z+ sources.
The purpose of this study was to examine the effect of Lidocaine(Lid), Bupivacaine(Bup) and Ketamine(Ket) mediated cytoplasmic Ca2+ mobilization in human neutrophils(PMNs, non-excitable cells) and dorsal root ganglia(DRG, excitable cells). Lid and Ket had no effect on the cytoplasmic Ca2+ mobilization, while Bup induced a dose-dependent elevation of the [Ca2+]i, which is observed with and without Ca2+ medium in both cells. Lid, Bup and Ket did not affect on the fMLP-stimulated increase in [Ca2+]i, but blocked Concanavalin A-stimulated increase in [Ca2+]i. In the absence of external Ca2+, Bup resulted in a small increase in [Ca2+]i. A subsequent addition of Ca2+(2mM) did not brought about response, whereas ionomycin (lone) and Thapsigargin (Tg) induced Ca2+ influx. In the absence of external Ca2+, addition of Bup to Tg-treated cells induced a small increase in [Ca2+]i in low concentration lone treated cells but did not evoked increase in high concentration. The increase "of [Ca2+]i elicited by Bup was similar to that due to lone or different intracellular Ca2+ pool. DRG was significantly inhibited Ca2+ influx through plasma membrane compared to PMNs. These results suggested that Bup alone increase the elevation of [Ca2+]i, whereas prevented Ca2+ influx across the plasma membrane.
Acknowledgements. This work is supported by grant No K-301 from the National Fund,,Scientific Research", Sofia, Bulgaria.
INHIBITION OF CALCIUM-DEPENDENT CELLULAR REACTIONS BY ANTAGONIST DRUGS WHICH POSSESS COMMON PHARMACOPHORE R. Prokopenko, L Zaitsev, S. Mogilevich, G. Poda, I. Naydyenova, G. Batrak, V. Degtyar and A. Luik Biomedical Department, Institute of Bioorganic and Petroleum Chemistry, Murmanskaya 1, Kiev-94, 253660, Ukraine~
CYCLIC NUCLEOTIDES IN THE MODULATION OF A T R I A L
NATRIURETIC PEPTIDE SECRETION D. J. Church*, M Rebsamen,M. B. Vallotton and U. Lang. Division of Endocrinology, Geneva University Hospital, Geneva, Switzerlandand * Battelle Research Center, Carouge, Switzerland. Cardiomyocytescontain atrial natriuretic peptide (ANP) receptors, the activation of which leads to the formation of cGMP. In view of the negative effect that cGMP has on myocardialANP secretion, it is likely that ANP modulates its own release at the level of the cardiomyocyte itself. We investigated this hyothesis by incubating spontaneouslybeating cardiomyocytes with the adenylyl cyclase activating agonist prostaglandin E2 (PGE2) in the presence of 8-Br-cGMP or ANP, and by measuring effects in terms of Ca++fluorometry, cAMP formation, contraction frequency, cGMP production and ANP release. Incubation of cells with 0.1 IIM PGE2led to a marked increase in ANP secretion, a response accompanied by increases in cAMP formation and contraction frequency. Interestingly, the effects Of PGE2on ANP release appeared to be entirely mediated by the cAMP activation of a myocardialvoltage-operatedcalcium channel,as PGE2failed to promote Ca*+ mobilisationin this system, and ANP secretionwas abolished in the presence of 0.1 ~JM nifedipine. Consistent with this, 0.1 ~M forskolin and 10 t.tM dibutyryl cAMP fully mimicked the effects of PGE2 in these cells, responsesthat were also inhibited by 0.1 i.tM nifedipine.A role for cGMP in the negative feedback regulation of ANP secretion was also apparent, insofar as 0.1 ~tM ANP promoteda marked increasein cGMP formation in these cultures, while 1 i.tM 8-Br-cGMPabolished the totality of PGE2-induced ANP release. Furthermore, the inhibitory effect of cGMP appearedto occur via an effect on contractionfrequency itself, as PGE2-induced cAMP formation was unaffected by either 8-Br-cGMP or ANP, agents which both diminished contraction frequency. Finally, we further found that PGE2antagonizes ANP-induced cGMP formation in this system, illustrating that cAMP and cGMP have opposite effects in myocardial ANP secretion. Taken together, these results indicate that cAMP-elevating agents promote ANP release via the activation of nifedipine-sensitivecalcium channels, and that ANP modulates its own secretion via the cGMP-mediatedinhibition of contractionfrequency.
The interactions of many antagonist drugs with cell surface receptors, which inhibit adenilate cyclase cellular signalling system (AdCSS) or activate CaZ~-mobilizing phospholipid one (PLSS), and/or with nonreceptor targets such as Ca-channels, phosphodiesterases, calmodulin, protein kinase C and calmodulin kinases, phospholipase C and A2 etcl result in activation of AdCSS and/or inhibition of PLSS. This +AdCSS/PLSS b~pe of drug action causes the depression of some of the Ca2+dependent cellular processes, such as smooth muscle contraction and platelet aggregation. We studied the action of various antagonist drugs possessing +AdCSS/-PLSS type of action on rat platelet aggregation induced by thrombin, on high threshold L-type-like calcium current in snail neurones and on spontaneous and electrically stimulated contractions of isolated preparations of rat uterus and guinea pig ureter respectively. Some phenothiazine and butyrophenone neuroleptics, benzamide neuroleptic sulpiride, some tranquilizers and three-cyclic antidepressants, antihistamine and cholinolitic drugs, c~-adrenoreceptor blockers, arachidonic acid metabolism and phosphodiesterase inhibitors were compared with different Ca2+ antagonists. Using electrontopological method we found common pharmacophore in drugs which inhibit this Ca>-dependent cellular reactions in concentration not more than 100 ~tM. This pharmacophore was also found in verapamil, diltiazem but not in papaverin and dihydropyridines. We conclude that common pharmacophore revealed may be responsible for binding promiscuity of different drugs with +AdCSS/-PLSS type of action and inhibition of different Ca2+-dependent cellular reactions such as smooth muscle contraction, platelet aggregation and neuronal calcium current. More specific structure peculiarities of antagonist drugs are probably responsible for more selective inhibition of the specific cellular targets.
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207
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T H E R O L E O F E X T R A C E L L U L A R C A L C I U M IN T H E CONTRACTILE EFFECT OF CYCLOPIASONIC ACID IN T H E C A T G A S T R I C F U N D U S G. V. Petkov and K. K. Boer ~ University of Sofia, Faculty of Biology, 1421-Sofia, Bulgaria and 1Institute of Biophysics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria In circular smooth muscle strips isolated from the cat gastric fundus, cyclopiazonic acid ( C P A ) , a specific inhibitor of sarcoplasmic reticulum CaZ-ATPase, induced a prolonged increase in mucsle tone (238 -+ 63 °/0, n=45) depending on the concentration of extracellular Ca z*. In Ca2+-deficient solution C P A ( 1 0 ~M) slightly increased the tone (20.6 -+ 14.2 °/0, n=13) but addition of 2.5 mM Ca z+ to the bath led to a very strong and rapid contraction, suggesting the enhanced Ca z+ permeability of the plasma membrane; in CaZ+-deficient solution, containing 3 l.tM nifedipine, CPA elicited only a negligible contraction (2.0 +- 0.7 %, n=4); in CaZ*-free solution, containing 2 mM E G T A , even 30"ttM C P A failed to induce contraction (n=4). In the presence of Ca z+ (2.5 mM) and nifedipine (3 [.tM), the CPA-induced increase in the tone was very slow, suggesting the important role of the L-type Ca z+ channels in the CPA-induced contraction, on the one hand, and the existence of a potential-independent mechanism, on the other. C P A shifted to the left the Ca z* concentration-response curves not only in the absence, but also in the presence of nifedipine (3 I.tM) and atropine (1 l.tM). Another evidence for the participation of the L-type Ca 2~ channels in the CPA-induced contraction was that the agonist Bay K 8644 (0.1 nM - 1 ~M)potentiated C P A effect in the presence of the antagonist nifedipine. In conclusion, the CPA-induced contraction is a result of the Ca z+ entry into the cells from the extracellular solution but not from the intracellular Ca z+ sources.
The purpose of this study was to examine the effect of Lidocaine(Lid), Bupivacaine(Bup) and Ketamine(Ket) mediated cytoplasmic Ca2+ mobilization in human neutrophils(PMNs, non-excitable cells) and dorsal root ganglia(DRG, excitable cells). Lid and Ket had no effect on the cytoplasmic Ca2+ mobilization, while Bup induced a dose-dependent elevation of the [Ca2+]i, which is observed with and without Ca2+ medium in both cells. Lid, Bup and Ket did not affect on the fMLP-stimulated increase in [Ca2+]i, but blocked Concanavalin A-stimulated increase in [Ca2+]i. In the absence of external Ca2+, Bup resulted in a small increase in [Ca2+]i. A subsequent addition of Ca2+(2mM) did not brought about response, whereas ionomycin (lone) and Thapsigargin (Tg) induced Ca2+ influx. In the absence of external Ca2+, addition of Bup to Tg-treated cells induced a small increase in [Ca2+]i in low concentration lone treated cells but did not evoked increase in high concentration. The increase "of [Ca2+]i elicited by Bup was similar to that due to lone or different intracellular Ca2+ pool. DRG was significantly inhibited Ca2+ influx through plasma membrane compared to PMNs. These results suggested that Bup alone increase the elevation of [Ca2+]i, whereas prevented Ca2+ influx across the plasma membrane.
Acknowledgements. This work is supported by grant No K-301 from the National Fund,,Scientific Research", Sofia, Bulgaria.
INHIBITION OF CALCIUM-DEPENDENT CELLULAR REACTIONS BY ANTAGONIST DRUGS WHICH POSSESS COMMON PHARMACOPHORE R. Prokopenko, L Zaitsev, S. Mogilevich, G. Poda, I. Naydyenova, G. Batrak, V. Degtyar and A. Luik Biomedical Department, Institute of Bioorganic and Petroleum Chemistry, Murmanskaya 1, Kiev-94, 253660, Ukraine~
CYCLIC NUCLEOTIDES IN THE MODULATION OF A T R I A L
NATRIURETIC PEPTIDE SECRETION D. J. Church*, M Rebsamen,M. B. Vallotton and U. Lang. Division of Endocrinology, Geneva University Hospital, Geneva, Switzerlandand * Battelle Research Center, Carouge, Switzerland. Cardiomyocytescontain atrial natriuretic peptide (ANP) receptors, the activation of which leads to the formation of cGMP. In view of the negative effect that cGMP has on myocardialANP secretion, it is likely that ANP modulates its own release at the level of the cardiomyocyte itself. We investigated this hyothesis by incubating spontaneouslybeating cardiomyocytes with the adenylyl cyclase activating agonist prostaglandin E2 (PGE2) in the presence of 8-Br-cGMP or ANP, and by measuring effects in terms of Ca++fluorometry, cAMP formation, contraction frequency, cGMP production and ANP release. Incubation of cells with 0.1 IIM PGE2led to a marked increase in ANP secretion, a response accompanied by increases in cAMP formation and contraction frequency. Interestingly, the effects Of PGE2on ANP release appeared to be entirely mediated by the cAMP activation of a myocardialvoltage-operatedcalcium channel,as PGE2failed to promote Ca*+ mobilisationin this system, and ANP secretionwas abolished in the presence of 0.1 ~JM nifedipine. Consistent with this, 0.1 ~M forskolin and 10 t.tM dibutyryl cAMP fully mimicked the effects of PGE2 in these cells, responsesthat were also inhibited by 0.1 i.tM nifedipine.A role for cGMP in the negative feedback regulation of ANP secretion was also apparent, insofar as 0.1 ~tM ANP promoteda marked increasein cGMP formation in these cultures, while 1 i.tM 8-Br-cGMPabolished the totality of PGE2-induced ANP release. Furthermore, the inhibitory effect of cGMP appearedto occur via an effect on contractionfrequency itself, as PGE2-induced cAMP formation was unaffected by either 8-Br-cGMP or ANP, agents which both diminished contraction frequency. Finally, we further found that PGE2antagonizes ANP-induced cGMP formation in this system, illustrating that cAMP and cGMP have opposite effects in myocardial ANP secretion. Taken together, these results indicate that cAMP-elevating agents promote ANP release via the activation of nifedipine-sensitivecalcium channels, and that ANP modulates its own secretion via the cGMP-mediatedinhibition of contractionfrequency.
The interactions of many antagonist drugs with cell surface receptors, which inhibit adenilate cyclase cellular signalling system (AdCSS) or activate CaZ~-mobilizing phospholipid one (PLSS), and/or with nonreceptor targets such as Ca-channels, phosphodiesterases, calmodulin, protein kinase C and calmodulin kinases, phospholipase C and A2 etcl result in activation of AdCSS and/or inhibition of PLSS. This +AdCSS/PLSS b~pe of drug action causes the depression of some of the Ca2+dependent cellular processes, such as smooth muscle contraction and platelet aggregation. We studied the action of various antagonist drugs possessing +AdCSS/-PLSS type of action on rat platelet aggregation induced by thrombin, on high threshold L-type-like calcium current in snail neurones and on spontaneous and electrically stimulated contractions of isolated preparations of rat uterus and guinea pig ureter respectively. Some phenothiazine and butyrophenone neuroleptics, benzamide neuroleptic sulpiride, some tranquilizers and three-cyclic antidepressants, antihistamine and cholinolitic drugs, c~-adrenoreceptor blockers, arachidonic acid metabolism and phosphodiesterase inhibitors were compared with different Ca2+ antagonists. Using electrontopological method we found common pharmacophore in drugs which inhibit this Ca>-dependent cellular reactions in concentration not more than 100 ~tM. This pharmacophore was also found in verapamil, diltiazem but not in papaverin and dihydropyridines. We conclude that common pharmacophore revealed may be responsible for binding promiscuity of different drugs with +AdCSS/-PLSS type of action and inhibition of different Ca2+-dependent cellular reactions such as smooth muscle contraction, platelet aggregation and neuronal calcium current. More specific structure peculiarities of antagonist drugs are probably responsible for more selective inhibition of the specific cellular targets.
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207
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