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Piperonyl-ranitidine: An H2-antagonist able to interact with cholinergic M3-muscarine receptors
ACTION OF PROTEIN KINASE INHIBITORS ON CALCIUM CURRENT IN INTESTINAL SMOOTH MUSCLE, CELLS A.V.Zima and A.E.Belevich. Bo~omoletz Institu{ue o f P h v s i o l o ~ y , Bo~omoletz str.4, Kiev-24, Ukraine,
VASCULAR SMOOTH MUSCLE DESENSITISATION AND THE INFLUENCE OF LENGTH OF INTERVALS ON CALCIUM CURVES. *A.C. Ugwu; +D.J. Miller, and +J.C. MeGrath *Dept. of Physiology, University of Benin, Nigeria +Institute of Physiology, University of Glasgow, U.K. Many workers had apparently recognized the progressive fall in responsiveness of vascular' smooth muscle with time or with consecutive periods of activation of the muscle by agonists (Medgeth and Langer, 1986). This had led to the neglect of the first concentration - response curve (CRC) and the choice of subsequent curve(s) for the analysis of test drugs. Lengths of the proximal segments of the rat tail artery (l-2cm) were perfused with Krebs saline c0nt~ining calcium buffers. Perfusion pressures were recorded for calcium CRCs with 3~M noradrenaline for varying incubation periods and intervals between curves. Desensitisation followed activation of the tissues but recovery occurred with time. 15min intervals appeared insufficient for complete tissue recovery so that desensitisation became cumulative. Long intervals between calcium CRCs attenuated desensitisation a n d also allowed some recovery of already desensitised tissues. Thus reduced sensitivity in shbsequent response can be revived spontaneously by long intervals between tests. Medgett, I.C. and Langer, S.Z. (1986). Schmiedeherg Arch. Pharmac. 332, 43-49.
The pole of protein kinases (PK) inhibitox's in regulation of pot,entialactivated calcium current (Ice) 'was e x a m i n a t e d i n f r e s h isolated s m o o t h muscle cells ( S M C s > F r o m ~ u i n e a pi E t a e n i a cell b y means of c o n v e n t A onal path-clamp method. N-J2- < M e t h % , l a m i n o ) e L h y i ] - S - i s o q u i n o l i n e sulfonamide (H-S) IMM, inhibitor of cyclic nucleotide dependent PK, strongly reduced lea, b 3, ~0~. T h e blockin E e f f e c t o f 11-8 w a s dose-dependent (Kd=O.21~M), reversible and volf,a ~ e independent. 8- B r o m o - c A M P %-iO0~M activated I c a ( b y 2 S ~ f o r . 1OI~M). 8 - B r o m o c(gMP 100- IO00~.,~M blocked Ic~ (by 28~ fox" 200MM). C,ell pe/~fusin~ by pipette ~ohltion containin~ H-8 O.S~M blocked effeci,~ of 8-Bl'omo-cAMP and 8-Bromo-cOMP. i-(S-isoquinolinesulfonyl)- 2- m e t h y i p i p e r a z i n e (H-T> IteM, c A M P dependent PK a n d P K C inhibitol,, als:o reduced I c a . ~helerythl~i ne O,i-iO;.sM, selective PKC inhibitor, s l i g h t ly decreased Ic~ (>lO~). T h e s e d a t e suggest t h e major" Pole o f c A M P d e p e n d e n t P K in r e ~ u l a t i o n o f basal Ica in intestinai S M C s .
Naunyn
Acknowledgements: Thanks tO Beecham, Biode, Pfizer, Welleome and Sandoz for their reagents and drugs.
PIPERONYL-RANITIDINE: AN H2-ANTAGONIST ABLE TO INTERACT WITH CHOLINERGIC M3-MUSCARINE RECEPTORS D. Barone. Istituto di Ricerche Biomediche "A.Marxer" RBM S.p.A.P.O. Box 226. 10015 - lvrea (TO)
THE MELATONIN RECEPTOR: EXPRESSION STUDIES IN
XENOPUS LAEVIS Cristina Mazzucehelli, Simona Capsoni, Deborah Angeloni, France Frasehini and Bojidar Stankov.
Introduction: Piperonyl-ranitidine (PR) is a newly marketed gastroprotective agent belonging to ranitidine's (RA) chemical and therapeutical class. It's receptogram revealed that, differently from RA, PR interacts with the cholinergic muscarine receptors that are labeled in the brain of the rat by 3H-plrenzepine (3H-PIR) and 3H-quinuclidinilbenzilate [ml- and m2-muscarine sites]. To investigate the affinity and the nature of PR interaction with peripheral muscarine receptors. 3H-PIR and 3H-4DAMP were chosen for their high affinity and selectivity for m3-muscarine sites. Materials and Methods: membrane receptors prepared from rat submaxillary salivary glands were incubated with 0.1, 0.3, 0.9 or 2.7 nM 3H-PIR or 0.03, 0.1.0.3 or 0.9 nM 3H-4DAMP in the absence (total binding) or m the presence of 3 [aM atropine (non-specific binding) or of PR. Results: Whatever the radioligand and its concentration tested, PR was able to inhibit concentration-dependently and up to 100% the specific binding of 3H-PIR and 3H-4DAMP. Non-linear fitting analysis of binding data gave parallel sigmoidal inhibition isotherms. IC50 values against 3H-PIR were: 0.23. 3.8, 10.7, 39.3 [aM and Hill numbers (nil) from 1.132 to 1.245. The corresponding figures against 3H-4DAMP were: IC50 = 1.4, 2.3, 4.9 or 18.5 gM and nH from 0.403 to 0.452. Disenssion and Conclusions: PR was recognized and bound by peripheral cholinergic m3-muscarine receptors in a pharmacological fashion since its IC50s' against the two radioligands are in the same affinity range of known muscarine-like drugs. Furthermore, the interaction of PR with m3-receptors is competitive since nH (slope of the sigmoidal inhibition isotherms) is not significantly affected by the different concentrations of radioligand displaced by PR.
Chair of Chemotherapy, Department of Pharmacology, University of Milan, 32 Via Vanvitelli, 20129 Milano, Italy. Northern analysis of poly(A) + RNA isolated from various tissues of Xenopus laevis, hybridized with a fragment of the coding region of the Xenopus melatonin receptor cDNA showed a positive signal in the ovary only. However, reverse transcription-PCR (RTPCR) amplified specific products from the total RNAs isolated from the whole brain, skin, retina and the ovary. In vitro autoradiography and in situ hybridization demonstrated that the melatonin receptor is expressed with discrete distribution in Xenopus brain, the retina and the ovary. PCR of genomic DNA resulted in amplification of a single product with a = 2 kb length, bigger than the one obtained with the same primers under RT-PCR conditions, thus suggesting the presence of an intron sequence in the
Xenopus melatonin
receptor gene. This study is the first in comparing the expresston of melatonin receptor by using molecular biology and biochemical approaches. The results strongly suggest that in Xenopus, apart from the ovary, there is no melatonin receptor expression in tissues having origin outside the CNS.
186
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VASCULAR SMOOTH MUSCLE DESENSITISATION AND THE INFLUENCE OF LENGTH OF INTERVALS ON CALCIUM CURVES. *A.C. Ugwu; +D.J. Miller, and +J.C. MeGrath *Dept. of Physiology, University of Benin, Nigeria +Institute of Physiology, University of Glasgow, U.K. Many workers had apparently recognized the progressive fall in responsiveness of vascular' smooth muscle with time or with consecutive periods of activation of the muscle by agonists (Medgeth and Langer, 1986). This had led to the neglect of the first concentration - response curve (CRC) and the choice of subsequent curve(s) for the analysis of test drugs. Lengths of the proximal segments of the rat tail artery (l-2cm) were perfused with Krebs saline c0nt~ining calcium buffers. Perfusion pressures were recorded for calcium CRCs with 3~M noradrenaline for varying incubation periods and intervals between curves. Desensitisation followed activation of the tissues but recovery occurred with time. 15min intervals appeared insufficient for complete tissue recovery so that desensitisation became cumulative. Long intervals between calcium CRCs attenuated desensitisation a n d also allowed some recovery of already desensitised tissues. Thus reduced sensitivity in shbsequent response can be revived spontaneously by long intervals between tests. Medgett, I.C. and Langer, S.Z. (1986). Schmiedeherg Arch. Pharmac. 332, 43-49.
The pole of protein kinases (PK) inhibitox's in regulation of pot,entialactivated calcium current (Ice) 'was e x a m i n a t e d i n f r e s h isolated s m o o t h muscle cells ( S M C s > F r o m ~ u i n e a pi E t a e n i a cell b y means of c o n v e n t A onal path-clamp method. N-J2- < M e t h % , l a m i n o ) e L h y i ] - S - i s o q u i n o l i n e sulfonamide (H-S) IMM, inhibitor of cyclic nucleotide dependent PK, strongly reduced lea, b 3, ~0~. T h e blockin E e f f e c t o f 11-8 w a s dose-dependent (Kd=O.21~M), reversible and volf,a ~ e independent. 8- B r o m o - c A M P %-iO0~M activated I c a ( b y 2 S ~ f o r . 1OI~M). 8 - B r o m o c(gMP 100- IO00~.,~M blocked Ic~ (by 28~ fox" 200MM). C,ell pe/~fusin~ by pipette ~ohltion containin~ H-8 O.S~M blocked effeci,~ of 8-Bl'omo-cAMP and 8-Bromo-cOMP. i-(S-isoquinolinesulfonyl)- 2- m e t h y i p i p e r a z i n e (H-T> IteM, c A M P dependent PK a n d P K C inhibitol,, als:o reduced I c a . ~helerythl~i ne O,i-iO;.sM, selective PKC inhibitor, s l i g h t ly decreased Ic~ (>lO~). T h e s e d a t e suggest t h e major" Pole o f c A M P d e p e n d e n t P K in r e ~ u l a t i o n o f basal Ica in intestinai S M C s .
Naunyn
Acknowledgements: Thanks tO Beecham, Biode, Pfizer, Welleome and Sandoz for their reagents and drugs.
PIPERONYL-RANITIDINE: AN H2-ANTAGONIST ABLE TO INTERACT WITH CHOLINERGIC M3-MUSCARINE RECEPTORS D. Barone. Istituto di Ricerche Biomediche "A.Marxer" RBM S.p.A.P.O. Box 226. 10015 - lvrea (TO)
THE MELATONIN RECEPTOR: EXPRESSION STUDIES IN
XENOPUS LAEVIS Cristina Mazzucehelli, Simona Capsoni, Deborah Angeloni, France Frasehini and Bojidar Stankov.
Introduction: Piperonyl-ranitidine (PR) is a newly marketed gastroprotective agent belonging to ranitidine's (RA) chemical and therapeutical class. It's receptogram revealed that, differently from RA, PR interacts with the cholinergic muscarine receptors that are labeled in the brain of the rat by 3H-plrenzepine (3H-PIR) and 3H-quinuclidinilbenzilate [ml- and m2-muscarine sites]. To investigate the affinity and the nature of PR interaction with peripheral muscarine receptors. 3H-PIR and 3H-4DAMP were chosen for their high affinity and selectivity for m3-muscarine sites. Materials and Methods: membrane receptors prepared from rat submaxillary salivary glands were incubated with 0.1, 0.3, 0.9 or 2.7 nM 3H-PIR or 0.03, 0.1.0.3 or 0.9 nM 3H-4DAMP in the absence (total binding) or m the presence of 3 [aM atropine (non-specific binding) or of PR. Results: Whatever the radioligand and its concentration tested, PR was able to inhibit concentration-dependently and up to 100% the specific binding of 3H-PIR and 3H-4DAMP. Non-linear fitting analysis of binding data gave parallel sigmoidal inhibition isotherms. IC50 values against 3H-PIR were: 0.23. 3.8, 10.7, 39.3 [aM and Hill numbers (nil) from 1.132 to 1.245. The corresponding figures against 3H-4DAMP were: IC50 = 1.4, 2.3, 4.9 or 18.5 gM and nH from 0.403 to 0.452. Disenssion and Conclusions: PR was recognized and bound by peripheral cholinergic m3-muscarine receptors in a pharmacological fashion since its IC50s' against the two radioligands are in the same affinity range of known muscarine-like drugs. Furthermore, the interaction of PR with m3-receptors is competitive since nH (slope of the sigmoidal inhibition isotherms) is not significantly affected by the different concentrations of radioligand displaced by PR.
Chair of Chemotherapy, Department of Pharmacology, University of Milan, 32 Via Vanvitelli, 20129 Milano, Italy. Northern analysis of poly(A) + RNA isolated from various tissues of Xenopus laevis, hybridized with a fragment of the coding region of the Xenopus melatonin receptor cDNA showed a positive signal in the ovary only. However, reverse transcription-PCR (RTPCR) amplified specific products from the total RNAs isolated from the whole brain, skin, retina and the ovary. In vitro autoradiography and in situ hybridization demonstrated that the melatonin receptor is expressed with discrete distribution in Xenopus brain, the retina and the ovary. PCR of genomic DNA resulted in amplification of a single product with a = 2 kb length, bigger than the one obtained with the same primers under RT-PCR conditions, thus suggesting the presence of an intron sequence in the
Xenopus melatonin
receptor gene. This study is the first in comparing the expresston of melatonin receptor by using molecular biology and biochemical approaches. The results strongly suggest that in Xenopus, apart from the ovary, there is no melatonin receptor expression in tissues having origin outside the CNS.
186
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