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Endothelins are potent oxytocics in the pregnant and non-pregnant rat isolated uterus
167~
Pos r presentations 13.00-14.00 Pos~s
Endothel|n~ are potent oxytocics in the pregnant and non-pregnant rat isolated uterus Rae, G.A. and Calixto, J.B. Dept. of Pharmacology, Biological Sciemces Center, Universid~_deFederal de Santa Catanno~ Fiorianopolis, 88000, $C, Brazil
Endothelins (ETs), a recently discovered class of H-residue peptides, powerfully contract vascular and nonvascular smooth muscle (Yanagisawa and Masaki, 1989). The current study compares the oxytocic effects of ET-1, ET-2 and ET-3 in the uterus isolated from 21-day pregnant and nonpregnant rats with those of other agonists including Bay K 8644. Adult Wistar rats (180-250 g) were killed by a blow on the head and their uteri removed and dissected free of a d l ~ g tissue. Uterine strips (15 mm long), obtained from virgins pretreated with estradiol benzoate (0.5 mg/kg, ip) 24 h beforehand (nonpregnant) or from 21-day pregnant animals, were suspended in 5 ml of aerated De Jalon solution (raM: NaCI 154, KCI 5.6, CaCI 2 0.3, MgCI 2 1.4, NaHCO 3 1.7 and glucose 5.5) at 30°C. Isotonic contractions were recorded on a kymograph under a I g load. Between 30 to 45 rain after setup, the tissues were contracted with K+rich solution (replacement of 80 mM NaCI by KCI) and all subsequent responses were calculated as percentages of this contraction. The results presented are each the mean of at least 5 experiments. Cumulative additions of ETs (0.1-30 nM) to nonpregnant strips caused concentration-dependent sustained contractions which subsided only slowly following washout (30 to 60 rain). ET-1 was more potent (EC50 7 nM, 95~; C.L. 5.38.8) and caused a greater maximal contraction (102 + 7~;) than ET-2 (EC50 10 nM, 70 4- 5~) or ET-3 (EC50 10 nM, 22 + 13~;). When the action of ET-1 was compared to that of other agonists, the rank order of molar potency found was: ET-1 - an~otensin II > bradykinin -- Bay K 8644 > oxytocin > serotonin > prostaglandin F 2-alpha acetylcholine. Maximal responses to these agents ranged between 100 and 120~ of that to K + 80 mM. Strips from 21-day pregnant rats displayed a 2- to 3-fold increase in sensitivity to ET-1, angiotensin II, oxytocin and prostaglandin F 2-alpha, but EC~0s to bradykinin, serotonin and acetylcholine were unaltered. In contrast, Bay K 8644 was 200-fold more potent in pregnant strips (EC50 0.1 nM, 0.04-0.3) than in nonpregnant ones. Also, maximal responses of pregnant strips were increased to angiotensin II and acetylcholine, decreased to Bay K 8644, bradykinin and serotonin and unchanged to the other agonists including the ETs. A threshold concentration of ET-1 (0.3 nM) increased responses of nonpregnant strips to submaximal concentrations of prostaglandin F 2-alpha and serotonin and contractions reduced by angiotensin lI in pregnant strips. The tonic contractions of nonpregnant strips caused by ET-1 (30 nM) were quite distinct from the phasic-rhythmic responses to Bay K 8644 (300 nM). Both agonists failed to contract strips bathed in Ca2%free solution, but graded additions of the ion (0.03 to 3 mM) revealed that ET-1 required 3-fold less extracellular Ca 2+ to cause contraction than Bay K 8644. Moreover, maximal responses to ET-1 were greater when it was added to Ca 2+-free medium before reintroducing the ion, whereas the opposite occured with the dihydrop3a-idine agonist. Prior incubation with nicardipine (10 nM) abolished responses to Bay K 8644 but ET-1 still caused a significant contraction even in presence of 100 nM of the Ca 2+-channel antagonist. It is concluded that ETs are potent oxytocic agents in the rat uterus. Indeed, ET-1 i~ one of the most potent agonists known in this tissue. Its action is susceptible to hormonal modulation, requires extracellular Ca 2+, but is clearly distinct from that of Bay K 8644. \ s a new class of endogenous oxytocics, ETs may subserve important roles in controling uterine contractility. Furthermore, considering that hypoxia, which is a strong stimulus for ET-1 synthesis by endothelial cells, occurs during menses and labour and that placental blood vessels also contain ET receptors (Fischli et al., 1989), ET-1 may be an important trigger a n d / o r mediator of dysmenorrhea and labour. This study was supported by CNPq and FINEP (Brazil).
1673
References Fischli, W., Clozel, M. and Guilly, C., 1989, Life Sci. 44, 1429. Yanagisawa, M. and Masaki, T., 1989, Biochem. Pharmacol. 38, 1877.
P.th.002 ]
Endogenous urea(U): a candidate to be qualified as an autocoid (in vivo and in vitro data from animal and human studies) Zhelyazkov, D. and Temnyalov, N. Department of Pharmacology, Higher Institute of Medicine, "Marin Drinov'"str. No. 55, 9002 Varna, Bulgaria
The idea of "autopharmacology" (Zhelyazkov, 1961) and those of "autocoids", proposed by Sch'~fer, 1916 and Douglas, 1985, are identical in many aspects. The starting position of this idea according to Lal et al., 1985, is that "the most versatile drug producer in the nature may be the human body" and that an "endocoid may represent a previously unrecognized endogenous ligand" with neurotransmitter/neuromodulator activity. The purpose of the present review is to bring under this idea the results of our in vivo/in vitro experimental and clinical studies, permitting to qualify the U as a bioregulatory endmetabolite, whose activity, based on its physicochemical properties, characterizes it as an autocoid. In our long term studies we paid attention to some extrarenal U functions, using three methodological approaches: 1) in vitro studies with vascular and nonvascular smooth muscle preparations; 2) in vivo series of acute experiments on anaesthetized cats, dogs, rats on a single U rapid infusion (1,0 g/'t:g) with adrenergic agonists and antagonists as refferent drugs; and 3) clinicopharmacological study with patients on periodical haemodialysis immediately before and after a single haemodialysis procedure in comparison with matched in sex and age healthy persons, constructing dose-response curves with isoproterenol (ISO) infusions 2,5-5-10-20 ng/kg/min. The results showed that U is able to act as: 1) nons,oecific, nonselective and reversible cardiovascular/]l~-adrenergic antagonist; 2) a promoter of Ca 2+ entry in K+-depolarized smooth muscle organs; 3) an activator of the vascular Ca2+-stimulated-Mg-ATP-ase, but not of the basal one. Further U per se destablizes vascular/32-AR-complex ss judged by the values of K s in comparison with butoxamine according to Furchgott method; U enhances noadrenaline contraction in cDRC without changing of K s of phenolamine (isometric regimen- "Ugo Basicle equipment). Urea in 0,0005-50 mmol/l releases noradrenaline in perfusates from rat thoracic aortic rings. The osmolality of the Krebs solution used was not changed till 5 mmol/I U and increased after 50 mmol/l (Osmometer "Knaner", BRDeutschland). Typically for endogenous modulators (Zhelyazkov, 19~8) U in 5-50 mmol/l shows a predominant noncompetitive type of drug antagonism, respectively sensitization, plotted by Line-weaver-Burk method. In vivo U, in four-fold elevated blood U concentrations above control level, antagonizes ISO (+)-chronotropic and vas0depressor effects, -.'r. cats and dogs. In patients on periodic haemodialysis immediately before a single procedure these cardiovascular effects of ISO are reduced, but immediately after the procedure a restoration of ISO pl = adrenoreceptor effects is observed. In conclusion, in our opinion the data principally described above, are reasonable points U to be included in the fist autocoids. At the same time they represent a support of the hypotheses of Kostoyanz-Turpaev-Mannkhin (1950-1968) and of van Rossum, 1964 concerning the action of U on protein structure initiating change(s) of its native st.~te.
References Tenmyalov, N.D., Tzekov, T.T. and Zhelyazkov, D.K. Urea as an endofenous metabolic factor with a behaviour of a nonselective beta-adrenergic blocking agent. In Abstracts of First Bulgarian Congress of Pharmacology, Sofia, 234 p, 1978 (in Bulgarian and in
English).
Zhelyazkov, D.K. Regulatory mechanisms in the organism (Autopharmacology of the organism), Sofia, State pubiisin8 house "Medizina and fiskultura", Sofia, 1961 (in Bulgarian with abstract in English).
Pos r presentations 13.00-14.00 Pos~s
Endothel|n~ are potent oxytocics in the pregnant and non-pregnant rat isolated uterus Rae, G.A. and Calixto, J.B. Dept. of Pharmacology, Biological Sciemces Center, Universid~_deFederal de Santa Catanno~ Fiorianopolis, 88000, $C, Brazil
Endothelins (ETs), a recently discovered class of H-residue peptides, powerfully contract vascular and nonvascular smooth muscle (Yanagisawa and Masaki, 1989). The current study compares the oxytocic effects of ET-1, ET-2 and ET-3 in the uterus isolated from 21-day pregnant and nonpregnant rats with those of other agonists including Bay K 8644. Adult Wistar rats (180-250 g) were killed by a blow on the head and their uteri removed and dissected free of a d l ~ g tissue. Uterine strips (15 mm long), obtained from virgins pretreated with estradiol benzoate (0.5 mg/kg, ip) 24 h beforehand (nonpregnant) or from 21-day pregnant animals, were suspended in 5 ml of aerated De Jalon solution (raM: NaCI 154, KCI 5.6, CaCI 2 0.3, MgCI 2 1.4, NaHCO 3 1.7 and glucose 5.5) at 30°C. Isotonic contractions were recorded on a kymograph under a I g load. Between 30 to 45 rain after setup, the tissues were contracted with K+rich solution (replacement of 80 mM NaCI by KCI) and all subsequent responses were calculated as percentages of this contraction. The results presented are each the mean of at least 5 experiments. Cumulative additions of ETs (0.1-30 nM) to nonpregnant strips caused concentration-dependent sustained contractions which subsided only slowly following washout (30 to 60 rain). ET-1 was more potent (EC50 7 nM, 95~; C.L. 5.38.8) and caused a greater maximal contraction (102 + 7~;) than ET-2 (EC50 10 nM, 70 4- 5~) or ET-3 (EC50 10 nM, 22 + 13~;). When the action of ET-1 was compared to that of other agonists, the rank order of molar potency found was: ET-1 - an~otensin II > bradykinin -- Bay K 8644 > oxytocin > serotonin > prostaglandin F 2-alpha acetylcholine. Maximal responses to these agents ranged between 100 and 120~ of that to K + 80 mM. Strips from 21-day pregnant rats displayed a 2- to 3-fold increase in sensitivity to ET-1, angiotensin II, oxytocin and prostaglandin F 2-alpha, but EC~0s to bradykinin, serotonin and acetylcholine were unaltered. In contrast, Bay K 8644 was 200-fold more potent in pregnant strips (EC50 0.1 nM, 0.04-0.3) than in nonpregnant ones. Also, maximal responses of pregnant strips were increased to angiotensin II and acetylcholine, decreased to Bay K 8644, bradykinin and serotonin and unchanged to the other agonists including the ETs. A threshold concentration of ET-1 (0.3 nM) increased responses of nonpregnant strips to submaximal concentrations of prostaglandin F 2-alpha and serotonin and contractions reduced by angiotensin lI in pregnant strips. The tonic contractions of nonpregnant strips caused by ET-1 (30 nM) were quite distinct from the phasic-rhythmic responses to Bay K 8644 (300 nM). Both agonists failed to contract strips bathed in Ca2%free solution, but graded additions of the ion (0.03 to 3 mM) revealed that ET-1 required 3-fold less extracellular Ca 2+ to cause contraction than Bay K 8644. Moreover, maximal responses to ET-1 were greater when it was added to Ca 2+-free medium before reintroducing the ion, whereas the opposite occured with the dihydrop3a-idine agonist. Prior incubation with nicardipine (10 nM) abolished responses to Bay K 8644 but ET-1 still caused a significant contraction even in presence of 100 nM of the Ca 2+-channel antagonist. It is concluded that ETs are potent oxytocic agents in the rat uterus. Indeed, ET-1 i~ one of the most potent agonists known in this tissue. Its action is susceptible to hormonal modulation, requires extracellular Ca 2+, but is clearly distinct from that of Bay K 8644. \ s a new class of endogenous oxytocics, ETs may subserve important roles in controling uterine contractility. Furthermore, considering that hypoxia, which is a strong stimulus for ET-1 synthesis by endothelial cells, occurs during menses and labour and that placental blood vessels also contain ET receptors (Fischli et al., 1989), ET-1 may be an important trigger a n d / o r mediator of dysmenorrhea and labour. This study was supported by CNPq and FINEP (Brazil).
1673
References Fischli, W., Clozel, M. and Guilly, C., 1989, Life Sci. 44, 1429. Yanagisawa, M. and Masaki, T., 1989, Biochem. Pharmacol. 38, 1877.
P.th.002 ]
Endogenous urea(U): a candidate to be qualified as an autocoid (in vivo and in vitro data from animal and human studies) Zhelyazkov, D. and Temnyalov, N. Department of Pharmacology, Higher Institute of Medicine, "Marin Drinov'"str. No. 55, 9002 Varna, Bulgaria
The idea of "autopharmacology" (Zhelyazkov, 1961) and those of "autocoids", proposed by Sch'~fer, 1916 and Douglas, 1985, are identical in many aspects. The starting position of this idea according to Lal et al., 1985, is that "the most versatile drug producer in the nature may be the human body" and that an "endocoid may represent a previously unrecognized endogenous ligand" with neurotransmitter/neuromodulator activity. The purpose of the present review is to bring under this idea the results of our in vivo/in vitro experimental and clinical studies, permitting to qualify the U as a bioregulatory endmetabolite, whose activity, based on its physicochemical properties, characterizes it as an autocoid. In our long term studies we paid attention to some extrarenal U functions, using three methodological approaches: 1) in vitro studies with vascular and nonvascular smooth muscle preparations; 2) in vivo series of acute experiments on anaesthetized cats, dogs, rats on a single U rapid infusion (1,0 g/'t:g) with adrenergic agonists and antagonists as refferent drugs; and 3) clinicopharmacological study with patients on periodical haemodialysis immediately before and after a single haemodialysis procedure in comparison with matched in sex and age healthy persons, constructing dose-response curves with isoproterenol (ISO) infusions 2,5-5-10-20 ng/kg/min. The results showed that U is able to act as: 1) nons,oecific, nonselective and reversible cardiovascular/]l~-adrenergic antagonist; 2) a promoter of Ca 2+ entry in K+-depolarized smooth muscle organs; 3) an activator of the vascular Ca2+-stimulated-Mg-ATP-ase, but not of the basal one. Further U per se destablizes vascular/32-AR-complex ss judged by the values of K s in comparison with butoxamine according to Furchgott method; U enhances noadrenaline contraction in cDRC without changing of K s of phenolamine (isometric regimen- "Ugo Basicle equipment). Urea in 0,0005-50 mmol/l releases noradrenaline in perfusates from rat thoracic aortic rings. The osmolality of the Krebs solution used was not changed till 5 mmol/I U and increased after 50 mmol/l (Osmometer "Knaner", BRDeutschland). Typically for endogenous modulators (Zhelyazkov, 19~8) U in 5-50 mmol/l shows a predominant noncompetitive type of drug antagonism, respectively sensitization, plotted by Line-weaver-Burk method. In vivo U, in four-fold elevated blood U concentrations above control level, antagonizes ISO (+)-chronotropic and vas0depressor effects, -.'r. cats and dogs. In patients on periodic haemodialysis immediately before a single procedure these cardiovascular effects of ISO are reduced, but immediately after the procedure a restoration of ISO pl = adrenoreceptor effects is observed. In conclusion, in our opinion the data principally described above, are reasonable points U to be included in the fist autocoids. At the same time they represent a support of the hypotheses of Kostoyanz-Turpaev-Mannkhin (1950-1968) and of van Rossum, 1964 concerning the action of U on protein structure initiating change(s) of its native st.~te.
References Tenmyalov, N.D., Tzekov, T.T. and Zhelyazkov, D.K. Urea as an endofenous metabolic factor with a behaviour of a nonselective beta-adrenergic blocking agent. In Abstracts of First Bulgarian Congress of Pharmacology, Sofia, 234 p, 1978 (in Bulgarian and in
English).
Zhelyazkov, D.K. Regulatory mechanisms in the organism (Autopharmacology of the organism), Sofia, State pubiisin8 house "Medizina and fiskultura", Sofia, 1961 (in Bulgarian with abstract in English).