Role of cyclic adenosine 3',5'-monophosphate in the isoprenaline-induced relaxation of the oestrogen dominated rabbit uterus.

1. The role of cyclic adenosine 3',5'-monophosphate (cyclic AMP) in the relaxation produced by isoprenaline in muscle strips from the oestrogen dominated rabbit uterus has been investigated. 2. Isoprenaline 2 times 10- minus 8 M produced an inhibition of the mechanical activity but no increase in cyclic AMP. Isoprenaline 2 times 10- minus 6 M produced both inhibition of mechanical activity and increase in cyclic AMP. 3. The increase in cyclic AMP, but not the inhibition of mechanical activity, was blocked by propranolol 3.4 X 10-MINUS 6 M. 4. Dibutyryl-ccylic AMP produced a relaxation which mimicked that produced by isoprenaline, in that the longitudinal strips were more sensitive than the circular ones. 5. It is concluded that cyclic AMP may be a mediator of the beta-adrenergic effect in the oestrogen dominated rabbit myometrium. However, it seems not to be an obligatory link between stimulation of beta-adrenoceptors and relaxation. Other mechanisms may also exist.     

Pharmacological investigation of ritodrine hydrochloride, a beta 2-adrenoceptor stimulant

Ritodrine hydrochloride (ritodrine) has been effectively prescribed for the prevention of premature labor. The present study was carried out to investigate the mode of action of ritodrine on the uterus and heart in comparison with those of isoxsuprine and isoproterenol. 1) Ritodrine (10(-8)-10(-6) M) suppressed the spontaneous motility of pregnant rat uterus and showed positive chronotropic action at the doses of 10(-6)-10(-4) M in guinea-pig atria. 2) In the Ca2+-free, K+-rich Tyrode solution, ritodrine suppressed the Ca2+ induced contracture of pregnant rat uterus, while it potentiated the carbachol induced contraction. 3) Ritodrine increased the amount of cyclic AMP in the uterus but not in heart. This action of ritodrine was suppressed by pretreatment with propranolol (10(-6) M). 4) These results suggest that ritodrine causes actions through activation of cyclic AMP production, as in the case of isoproterenol, and it acts more selectively on beta 2-adrenoceptors than on beta 1-adrenoceptors.     

Changes in the mechanical properties of the longitudinal and circular muscle tissues of the rat myometrium during gestation.

Changes in the mechanical properties of the longitudinal and circular muscle tissues of the rat myometrium during gestation were investigated. In isolated longitudinal and circular muscles of the rat myometrium, spontaneous contractions and contractions per unit cross-sectional area induced by 128 mM K+ and 1 X 10(-5) M acetylcholine (ACh) increased with the progress of gestation. These increases appeared in longitudinal muscles to a greater extent than in circular muscles. ACh induced the largest contraction for both intact muscle tissues, at all stages of gestation. In both muscle layers, the ACh-induced contraction reached the same amplitude as the 1 X 10(-5) M Ca-induced contraction of skinned muscles, except for the longitudinal muscle at the 22nd day of gestation. In Ca-free solution containing 2 mM EGTA, ACh produced contraction in both intact muscle tissues at all stages and the amplitude was increased during the progress of gestation, whereas the K-induced and spontaneous contractions ceased. In saponin-treated skinned muscles of both layers, the free Ca concentration required to produce contraction was lowered, the maximum amplitudes of the contraction were increased and the pCa-tension relationships shifted to the left during the progress of gestation. The results indicate that during the progress of gestation, Ca sensitivity of of the contractile proteins and mechanical responses to agonists increased and that the properties of the intracellular Ca store site were also altered in both muscle layers.     

Augmentation by external Mg ions of beta-adrenoceptor-mediated actions in the longitudinal muscle of rat uterus.

1. The longitudinal muscle isolated from the uterus of oestrogen-treated rats was not spontaneously active in Locke solution, and electrical stimulation evoked phasic contraction. Isoprenaline (3 x 10(-11) - 10(-8) M) and dibutyryl cyclic AMP (db cyclic AMP, 0.1-0.8 mM) depressed the phasic contraction; the depression was enhanced in the presence of 0.6 mM Mg. 2. The contracture generated by 40 mM K was partially relaxed by isoprenaline (10(-11) - 10(-8) M) and db cyclic AMP (0.1-0.8 mM). Mg (0.6 mM) enhanced the isoprenaline-induced relaxation, but not that induced by db cyclic AMP. 3. The membrane potential of the muscle was -61 mV, and electrical stimulation induced an action potential which consisted of spike and plateau components. Application of isoprenaline and db cyclic AMP mainly reduced the duration of the plateau potential. The effect was potentiated by 0.6 mM Mg. 4. The membrane was hyperpolarized, accompanied by a decrease in membrane resistance, when 10(-8) M isoprenaline or 0.8 mM db cyclic AMP was applied. The effects of isoprenaline were prominently augmented in the presence of 1.2 mM Mg, while those of db cyclic AMP were slightly potentiated. 5. Forskolin (0.1 microM) or papaverine (10 microM) inhibited the phasic contraction and the K-contracture. The effect on the phasic contraction was potentiated by 0.6 mM Mg, while that on the K-contracture was not affected. 6. Forskolin shortened the action potential at 0.3 microM, and hyperpolarized the membrane with a decrease in membrane resistance at 3.0 microM. The membrane effects were augmented by 0.6 and 1.2 mM Mg, respectively. 7. It was hypothesized that external Mg ions could affect at least two processes involved in actions at beta-adrenoceptors on rat myometrium; receptor-agonist interaction and cyclic AMP-mediated inhibition of membrane excitability.     

Lack of a causal relationship between the vasodilator effect of papaverine and cyclic AMP production in the dog basilar artery.

The effects of papaverine and isoprenaline on smooth muscle cells of the dog basilar artery were investigated using radioimmunoassay, electrophysiological and isometric tension recording methods. For comparative purposes, the actions of these drugs on the guinea-pig basilar artery were also examined. Papaverine and isoprenaline (1 microM and 10 microM) increased the amount of cyclic AMP in both dog and guinea-pig basilar arteries. Papaverine (up to 100 microM) and isoprenaline (up to 1 microM) had no effect on the membrane potential and membrane resistance measured by recording the amplitudes of the electrotonic potentials in smooth muscle cells of the dog and guinea-pig basilar arteries. The action potential evoked by outward current pulses after pretreatment with tetraethylammonium chloride (5-10 mM) was inhibited by papaverine (greater than 1 microM) but not by isoprenaline (up to 10 microM) in smooth muscle cells of the dog and guinea-pig basilar arteries. In the dog basilar artery, papaverine (greater than 1 microM) consistently inhibited the contractions evoked by excess concentrations of [K]o (greater than 20.2 mM) or 5-hydroxytryptamine (10 nM-10 microM), dose-dependently. Isoprenaline (1 microM) had only slight effects on the contraction evoked by low concentrations of 5-hydroxytryptamine (10 nM). In the Ca2+-free solution containing EGTA (2 mM), the contraction evoked by 5-hydroxytryptamine (10 microM) or caffeine (10 mM) was dose-dependently inhibited by papaverine (greater than 1 microM). However, isoprenaline (1 microM) had no effect on these contractions. These results indicate that the vasodilator actions of papaverine on the dog basilar artery are mainly due to inhibition of the voltage-dependent influx of Ca2+ and also to inhibition of the receptor-activated release of Ca2+ stored in the cell. Since isoprenaline increased the cyclic AMP to the same extent as papaverine but had no effect on the electrical and mechanical responses, the inhibitory actions of papaverine on this tissue may not be causally related to the increased levels of cyclic AMP induced by inhibition of phosphodiesterase.     

Chronotropic and inotropic actions of amrinone, carbazeran and isobutylmethyl xanthine: role of phosphodiesterase inhibition.

1. The chronotropic and inotropic effects of amrinone, carbazeran and 3-isobutyl-1-methyl xanthine (IBMX) were examined in isolated preparations of papillary muscle and right atria from rabbit heart. The effects of the drugs on cardiac phosphodiesterase and cyclic nucleotide content were also examined. 2. Amrinone (2.4 x 10(-4)M-2 x 10(-3) M), carbazeran (9.1 x 10(-6) M-1.2 x 10(-3) M), and IBMX (1.8 x 10(-5) M-4.5 x 10(-4) M) produced concentration-dependent positive inotropic responses of papillary muscle preparations, the rank order of potency being carbazeran = IBMX greater than amrinone. Sub-threshold positive inotropic concentrations of all three compounds potentiated the positive inotropic effects of isoprenaline; leftward shifts in the concentration-effect curves were 5 fold (IBMX), 11 fold (amrinone) and 46 fold (carbazeran). 3. Amrinone and IBMX produced concentration-dependent positive chronotropic responses in isolated right atria and showed a similar rate selectivity to isoprenaline, but carbazeran elicited a decrease in beating frequency. None of these drugs potentiated the positive chronotropic effects of isoprenaline. 4. Concentrations of amrinone, carbazeran and IBMX that produced similar positive inotropic responses were associated with different increases in papillary muscle cyclic AMP and cyclic GMP concentrations. 5. All three compounds inhibited right atrial and ventricular phosphodiesterase, with amrinone being the least potent. There was, however, a marked difference between the IC50 and EC50 values for phosphodiesterase inhibition and positive inotropy. In contrast the positive chronotropic effects of amrinone and IBMX were observed in the same concentration ranges that produced phosphodiestrease inhibition. 6. The results indicate that amrinone possesses a similar rate/force selectivity to isoprenaline and IBMX. In contrast, carbazeran exerts both positive inotropic and negative chronotropic effects. Phosphodiesterase inhibition and elevation of intracellular cyclic AMP concentration may be involved, at least in part, in the cardiac effects of these drugs.     

Effect of isoprenaline and phenylephrine on the adenosine 3',5'-monophosphate content and mechanical activity of cold-stored and fresh taenia caecum from the guinea-pig.

1. Cold storage treatment of the guinea-pig taenia caecum had a greater inhibitory effect on the isoprenaline-induced relaxation than that induced by phenylephrine. Prolonged cold storage (12-14 days) almost abolished the effect of isoprenaline but only reduced the phenylephrine effect. The ED50 of cyclic adenosine 3',5'-monophosphate (cyclic AMP) that elicited muscle relaxation was not altered by the prolonged cold storage. 2. After cold storage treatment, tissue cyclic AMP content was decreased; however, isoprenaline still caused a dose-dependent increase in the cyclic AMP level. The threshold dose of isoprenaline for cyclic AMP accumulation was the same in fresh and cold-stored preparations. 3. In the fresh preparation, the onset of the isoprenaline (10(-6)M)-induced relaxation preceded the increase in tissue cyclic AMP. 4. Isoprenaline, phenylephrine, adrenaline and noradrenaline at doses (ED50) sufficient to induce muscle relaxation did not always increase the cyclic AMP level. 5. Similarly, the responses to papaverine and nitroglycerine were not accompanied by an increase in cyclic AMP. 6. The adenylate cyclase and phosphodiesterase (low and high Km) activities of taenia caecum were not attenuated by the prolonged cold storage. 7. Propranolol inhibited both the isoprenaline-induced relazation and cyclic AMP accumulation; however, the pA2 values were significantly different for the two events. 8. Based on these results, both the relaxation and cyclic AMP accumulation caused by isoprenaline are mediated by activation of beta-adrenoceptors but are independent phenomena.     

5-HT(7) receptor and beta(2)-adrenoceptor share in the inhibition of porcine uterine contractility in a muscle layer-dependent manner.

To compare the inhibition of uterine contractility mediated by beta-adrenoceptors and 5-HT(7) receptors, the effects of catecholamines and 5-HT on spontaneous contractions were examined in longitudinal and circular muscles isolated from three different regions (cornu, corpus and cervix) of the non-pregnant proestrus porcine uterus. In addition, the distribution of beta-adrenoceptors between muscle layers was characterized by means of adenylate cyclase activity assay, cyclic AMP assay and [(3)H]dihydroalprenolol binding studies. In the cornu, isoprenaline, adrenaline and noradrenaline inhibited the spontaneous contraction of longitudinal and circular muscles but longitudinal muscle was more sensitive to catecholamines than was circular muscle. The inhibitory response to isoprenaline was antagonized by propranolol (300 nM) or (+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol (ICI 118,551; 100 nM). The rank order of potency was isoprenaline > or =adrenaline > noradrenaline. The beta(2)-adrenoceptor-selective agonist, clenbuterol, was more potent than xamoterol (beta(1)-selective) and (+/-)-4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid (BRL 37344; beta(3)-selective) to inhibit the spontaneous contraction of longitudinal muscles. Isoprenaline increased adenylate cyclase activity in both muscle layers, but the activity in the longitudinal muscle was greater than that in the circular muscle. Cyclic AMP production by isoprenaline was also more conspicuous in the longitudinal muscle than in the circular muscle. Although both muscle layers contained a single class of [3H]dihydroalprenolol binding sites with similar K(d) values (longitudinal muscle, 3.1+/-0.94 nM, n=4; circular muscle, 2.4+/-0.73 nM, n=4), B(max) in the longitudinal muscle (175.7+/-32.8 fmol/mg protein, n=4) was significantly higher than that in the circular muscle (53.1+/-5.1 fmol/mg protein, n=4). As previously reported [Br. J. Pharmacol. 130 (2000) 79], 5-HT also inhibited the spontaneous contraction of both muscle layers from the cornu and the 5-HT(7) receptor antagonist, 2a-[4-(4-phenyl-1,2,3,6-tetrahydropyridyl)butyl]-2a,3,4,5-tetrahydro-benzo[cd]indol-2(1H)-one (DR4004; 100 nM, n=4) blocked the 5-HT-induced inhibition of spontaneous contractions in the circular muscles, and reversed the less marked inhibition in the longitudinal muscles. In other regions of the uterus (corpus and cervix), 5-HT inhibited the spontaneous contraction of the circular muscles but contracted the longitudinal muscle strips. On the other hand, isoprenaline caused muscle layer-dependent inhibition (longitudinal muscle > circular muscle) in both regions, and the responsiveness tended to increase toward the cervix. In conclusion, beta(2)-adrenoceptors are present heterogeneously in the porcine uterus (longitudinal muscle > circular muscle) and share the inhibition of uterine contractility with 5-HT(7) receptors in a layer-dependent manner (longitudinal muscle: beta(2)-adrenoceptors, circular muscle: 5-HT(7) receptors).     

Nitroglycerine- and isoprenaline-induced vasodilatation: assessment from the actions of cyclic nucleotides.

To investigate the vasodilator actions of nitroglycerine and isoprenaline, the effects of these agents, dibutyryl cyclic AMP (db cyclic AMP) and 8-bromo cyclic GMP (8-Br cyclic GMP) on intact muscle tissue, and cyclic AMP and cyclic GMP on skinned muscle of the rabbit mesenteric artery were investigated. In porcine coronary artery, nitroglycerine (greater than 0.1 microM) increased the production of cyclic GMP with no change in the amount of cyclic AMP, while isoprenaline (greater than 0.1 microM) significantly increased the production of cyclic AMP with no change in the amount of cyclic GMP. In the rabbit mesenteric artery, nitroglycerine or isoprenaline inhibited the tonic component of the 39 mM [K]o-induced contraction to a greater extent than the phasic component. Nitroglycerine and 8-Br cyclic GMP showed a stronger inhibitory action on the K-induced contraction than did isoprenaline and db cyclic AMP. The sources of Ca utilized for the generation of contraction by noradrenaline and caffeine were estimated to be the same as those determined from the amplitudes of contractions evoked in Ca-free solution by various concentrations of noradrenaline or caffeine. In intact muscle tissues, the effects of nitroglycerine or 8-Br cyclic GMP on the amount of Ca stored in cells were estimated from the caffeine-induced contraction in Ca-free solution. Both agents inhibited the contractions due to a reduction in the amount of Ca in the cells. When the effects of isoprenaline or db cyclic AMP were observed, both agents inhibited the caffeine-induced contraction but the accumulation of Ca into cells was greater than the control. In saponin skinned muscles, the pCa-tension relationship in the presence of cyclic AMP and cyclic AMP-dependent protein kinase (cyclic AMP-PK) shifted to the right and to a lower level in comparison with the control. Applications of cyclic GMP with cyclic GMP-dependent protein kinase (cyclic GMP PK) also inhibited the contraction induced by low concentrations of Ca. In skinned muscles, cyclic AMP exhibited dual actions on Ca store sites, i.e. in the presence of high concentrations of Ca or prolonged superfusion of Ca, cyclic AMP reduced the amount of Ca due to activation of the Ca-induced Ca release mechanism by excess accumulation of Ca. On the other hand, cyclic GMP consistently inhibited the amplitude of the caffeine-induced contraction due to a reduction in the amount of Ca in the store sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of cyclic AMP phosphodiesterase activity and myocardial contractility: effects of cilostamide, a novel PDE inhibitor, and methylsiobutylxanthine on rabbit and canine ventricular muscle

The effects of cilostamide (N-cyclohexyl-N-methyl-4-[6-carbostyriloxy]butyramide; OPC-3689), a novel cyclic AMP phosphodiesterase (PDE) inhibitor were compared with those of 1-methyl-3-isobutylxanthine (IBMX) on the rabbit and canine heart preparations. Cilostamide was about three times less potent than IBMX in inhibiting the crude PDE activity of rabbit and canine heart in the cell-free system, while it was 10 times more potent than IBMX in enhancing the positive inotropic action of isoprenaline in the rabbit and canine ventricular myocardium: 10^?6 M cilostamide shifted the concentration-response curve for isoprenaline to the left in a parellel manner to the same extent as did 10^?5 M IBMX. Thus, cilostamide enhanced β-adrenoceptor stimulation more potently than did IBMX and the substances examined previously. Accumulation of intracellular cyclic AMP caused by 10^?6 M isoprenaline in the isolated canine ventricular myocardium was significantly enhanced by 10^?6 M cilostamide and 10^?5 M IBMX; isoprenaline (10^?6 M) induced cyclic AMP accumulation was greater with IBMX (10^?5 M) than with cilostamide (10^?6 M). The threshold concentration for cilostamide itself to induce positive chronotropic and inotropic actions in the rabbit heart was lower than that for IBMX, while the intrinsic activity of IBMX was greater than that of cilostamide. In the canine ventricular myocardium, the positive inotropic actions of cilostamide were comparable to those of IBMX; the action of cilostamide in concentrations of 10^?5 M and higher was partly inhibited by a β-adrenoceptor blocking agent, pindolol (3 × 10^?8 M). During the washout period of the drugs after the maximal response to the drugs had been reached, the positive inotropic action of cilostamide disappeared more rapidly than that of IBMX. The present results suggest that cilostamide is able to permeate the myocardial cell membrane more easily than IBMX and reach the PDE in the functionally important cyclic AMP compartment. The difference in turnover rate of cyclic AMP even in the same tissue in the physiological condition may also affect the direct action of the PDE inhibitors thereon.     

Effects of porcine relaxin on contraction, membrane response and cyclic AMP content in rat myometrium in comparison with the effects of isoprenaline and forskolin.

1. The longitudinal muscle from the uterus of oestrogen-treated rats was quiescent in Mg-free Krebs solution. Electrical stimulation generated phasic contraction, which was depressed to 35% and 18% by 50 mu and 150 mu porcine relaxin, respectively. 2. The phasic contractions were more strongly depressed to 26% by 50 mu relaxin in solution containing 0.6 mM Mg, and the depression lasted for more than 4 h after the removal of relaxin. During the persisting depression, raising the external Ca to 7.5 mM did not restore the contraction, but the contraction was restored by removal of Mg. 3. The depression of the phasic contraction by relaxin, examined in Mg-free solution, was enhanced and reduced by pretreatment of the tissue with 0.6 mM Mg and 0.6 mM Mn, respectively, for about 15 min. In contrast, the depression of contraction by isoprenaline or forskolin was enhanced by pretreatment with either Mg or Mn. 4. The cellular content of cyclic AMP was measured in Krebs solution containing 0.6 mM Mg. The values were 1.24 (pmol mg-1 protein) in control solution, and 2.31 and 1.56 when the tissues were treated with 150 mu relaxin and 10(-9) M isoprenaline, respectively. 5. The cyclic AMP production in response to 10(-7) M forskolin measured in Mg-free solution was enhanced when the tissue was pretreated with either 0.6 mM Mg or Mn for 15 min. The cyclic AMP production in response to 100 mu relaxin was increased when the tissue was pretreated with 0.6 mM Mg, and was unchanged by pretreatment with Mn. The cyclic AMP production in response to 10(-9) M isoprenaline was unchanged by pretreatment with the divalent cations. 6. The membrane potential of the muscle was -60.8 mV in Krebs solution containing 0.3 mM Mg, and electrical stimulation induced an action potential which consisted of spike and plateau components. Application of 150 mu relaxin reduced the duration of the plateau; the contractions were progressively depressed. The resting membrane potential and membrane resistance were unchanged by application of 150 mu relaxin. The membrane was hyperpolarized by 2.8 mV, accompanied by a decrease in membrane resistance, when 10(-9) M isoprenaline was applied. 7. Although there were several differences between the effects of relaxin and isoprenaline, it is probable that some process, which is cyclic AMP-dependent, accelerated by Mg and depressed by Mn, is involved in the depressant action of relaxin on contraction.     

Effects of ritodrine hydrochloride, a beta 2-adrenoceptor stimulant, on uterine motilities in late pregnancy

Ritodrine hydrochloride (ritodrine) is a beta 2-adrenoceptor stimulant which has been effectively prescribed for the prevention of premature labor. The present studies were carried out to investigate the effects of ritodrine on uterine motility in rats and rabbits during gestation, as compared with those of isoproterenol and isoxsuprine. The results were as follows: 1) Spontaneous movements and evoked contractile responses of isolated rat uterus (19-20th days of gestation) were suppressed by 10(-9) - 10(-6) M ritodrine. The potency of ritodrine was approximately 10 times more than that of isoxsuprine and 100 - 1,000 times less than that of isoproterenol. 2) When these drugs were administered to pregnant rats or rabbits intravenously, the tocolytic potency was in the following order: isoproterenol greater than ritodrine greater than isoxsuprine. 3) Ritodrine induced hypotension and tachycardia, but these effects were less than those of isoproterenol and isoxsuprine. 4) The effects of isoproterenol and ritodrine were almost prevented by pretreatment with propranolol, but those of isoxsuprine were only partially or not affected. These results suggest that ritodrine is effective in preventing the uterine contractions in rats and rabbits and that it has less effect on the circulatory system than isoproterenol and isoxsuprine. It is also concluded that ritodrine produces these effects through activation of beta-adrenoceptors.     

Characterization of the interaction between muscarinic M2 receptors and beta-adrenoceptor subtypes in guinea-pig isolated ileum.

1. Contraction of guinea-pig ileum to muscarinic agonists is mediated by M3 receptors, even though they account for only 30% of the total muscarinic receptor population. The aim of this study was to characterize the biochemical and functional effects of stimulation of the predominant M2 muscarinic receptor (70%) and to investigate the hypothesis that M2 receptors specifically oppose beta-adrenoceptor-mediated effects in the ileum. 2. In guinea-pig ileal longitudinal smooth muscle slices, isoprenaline, a non-selective beta-adrenoceptor agonist, and BRL 37344 (sodium-4-[2-[2-hydroxy-2-(3- chlorophenyl)ethylamino]propyl]-phenoxyacetate sesquihydrate), a beta 3-adrenoceptor selective agonist, increased cyclic AMP accumulation with -log EC50 values of 6.6 +/- 0.1 and 5.8 +/- 0.1 respectively. Maximal stimulation by BRL 37344 (10 microM) was 26.4 +/- 5.2% of that observed with isoprenaline (10 microM). Isoprenaline (10 microM)-stimulated cyclic AMP accumulation was significantly, but not completely, inhibited by propranolol (5 microM), with a propranolol-resistant component of 28.2 +/- 6.8% of the maximal stimulation to isoprenaline. In contrast, basal and BRL 37344 responses were resistant to this antagonist. These data provide evidence that both beta 1- and beta 3-adrenoceptors activate adenylyl cyclase in guinea-pig ileum. 3. Isoprenaline (10 microM)-stimulated cyclic AMP accumulation was inhibited (67.4 +/- 0.9%) by the muscarinic agonist (+)-cis-dioxolane (-log EC50 = 7.3 +/- 0.1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Action of Tsoprenaline,Isoxsuprine and Ritodrine in the Human Non–pregnant Myometrium

Abstract The effects of isoprenaline, isoxsuprine and ritodrine were tested on spontaneously contracting muscle strips from non–pregnant human uterus in vitro. The strips were rather insensitive to all three drugs, the maximal inhibitions obtained being: isoxsuprine (10^–4 M) 81 %, ritodrine (10^–4 M) 45 %, isoprenaline (10^–5 M) 19%. Propranolol 3.4 × 10^–6 M did not significantly block the effects of isoxsuprine and ritodrine. It is concluded that the non–pregnant human uterus is rather insentitive to β–adrenoceptor stimulation, and that isoxsuprine and ritodrine act mainly through unspecific mechanisms.     

Cyclic AMP and Ca-binding in microsomal fractions isolated from rabbit colon smooth muscle.

From a homogenate of rabbit colon muscle two ATP dependent Ca-accumulating microsomal fractions were isolated by differential centrifugation on a sucrose density grandient at 35% and 35-45% sucrose. Adenylate cyclase and phosphodiesterase activities were found in the fractions. The Ca-accumulation and the ATPase activity of these fractions were stimulated by cyclic AMP (10(-5)M) at an ATP concentration of 0.35 mM ATP. In the presence of higher concentrations of ATP (5 mM) cyclic AMP had no effect on the Ca-binding. The higher concentration of ATP markedly increased the cyclic AMP formation in relation to the activity found at the lower concentration of ATP. Isoprenaline (2 X 10(-6)M) stimulated the Ca-accumulation in the 35-45% fraction and increased the hydrolysis of ATP. These effects were absent in the fraction isolated at 35% sucrose. In the former fraction isoprenaline also stimulated the adenylate cyclase activity at 0.35 mM but not at 5 mM ATP. Both the effect of isoprenaline on the Ca-binding and the adenylate cyclase activity were inhibited by the adrenergic beta-receptor blocking agent sotalol. In the 35-45% fraction papaverine (1 X 10(-3)M) stimulated the Ca-accumulation and inhibited the phosphodiesterase activity. It is suggested that cyclic AMP and agents which influence the cyclic AMP metabolism in the microsomes may have a regulatory role on the Ca-binding of the microsomes.     

Pre- and post-junctional actions of procaterol, a beta 2-adrenoceptor stimulant, on dog tracheal tissue.

1. The effects of procaterol, a beta 2-adrenoceptor agonist, on excitatory neuro-effector transmission in the dog trachea were investigated and the findings were compared to those seen with isoprenaline, with microelectrode, double sucrose gap and tension recording methods. 2. Procaterol (10(-10)-10(-9) M) and isoprenaline (10(-9) M) had no effect on the resting membrane potential or on the input resistance of the smooth muscle cells of dog trachea. However with increased concentrations (greater than 10(-8) M), these agents hyperpolarized the membrane and decreased the input resistance of the membrane. 3. Procaterol (10(-10)-10(-7) M) and isoprenaline (10(-9)-10(-7) M) dose-dependently reduced the amplitude of the twitch contractions evoked by field stimulation in the combined presence of indomethacin (10(-5) M) and guanethidine (10(-6) M). In parallel with actions on twitch contractions, procaterol (10(-10)-10(-7) M) and isoprenaline (10(-9)-10(-7) M) reduced the amplitude of the excitatory junction potentials (e.j.ps), evoked by single pulse field stimulation in the dog trachea. 4. Procaterol (10(-8) M) had no effect on the post-junctional response of smooth muscle cells to exogenous acetylcholine (ACh) (10(-7)-10(-6) M). 5. Pretreatment with ICI-118551, a beta 2-adrenoceptor blocking agent, reduced the inhibitory action of procaterol on the amplitude of twitch contractions evoked by field stimulations in the dog trachea.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel signal transduction pathway mediating endothelium-dependent beta-adrenoceptor vasorelaxation in rat thoracic aorta.

1. Isoprenaline (3 x 10(-8)-10(-5) M), salbutamol (3 x 10(-7)-10(-4) M) and forskolin (3 x 10(-9)-3 x 10(-7) M) relaxed rat isolated thoracic aortic rings contracted with noradrenaline (10(-7) M). Removal of the endothelium from the aortic rings abolished the effect of acetylcholine (10(-6) M) and completely prevented the vascular relaxation induced by isoprenaline, salbutamol or forskolin. 2. The isoprenaline concentration-relaxation curve was shifted in parallel to the right about 10 fold by propranolol (3 x 10(-7) M) with no change in the maximum response, showing that the relaxation was mediated by a beta-adrenoceptor. 3. The inhibitor of nitric oxide synthesis, NG-nitro-L-arginine (L-NOARG; 10(-5) M), shifted the isoprenaline relaxation curve to the right and reduced the maximum response. 4. Isoprenaline (10(-6) M) relaxed noradrenaline-induced tone by approximately 95% and at the same time increased levels of adenosine 3':5'-cyclic monophosphate (cyclic AMP) 4 fold and guanosine 3':5'-cyclic monophosphate (cyclic GMP) 12 fold in the aortic rings. Sodium nitroprusside (3 x 10(-8) M) relaxed noradrenaline-evoked tone by 82% without changing levels of cyclic AMP but raised cyclic GMP 19 fold. 5. Forskolin (10(-7) M) relaxed noradrenaline-induced tone by approximately 41% and, like isoprenaline, increased levels of cyclic AMP (2.5 fold) and cyclic GMP (12 fold) in the aortic rings. 6. Removal of the endothelium abolished the relaxant effects of isoprenaline (10(-6) M) and also the associated accumulation of cyclic AMP and cyclic GMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro characterization of the effects of endomorphin 1 and 2, endogenous ligands for mu-opioid receptors, on mouse colonic motility

The effects of endomorphin 1 (EM1) and 2 (EM2) in colonic motility remain unknown. We investigated the effects and mechanisms of these endomorphins (EMs) on the colonic motility in vitro by applying various neural blocking agents and various opioid receptor antagonists. EMs (10(-9) to 10(-6)M) displayed significant stimulatory effects on the basal tonus or spontaneous activity of mouse colon but not of stomach and small intestine. It is noteworthy that the contractile actions of EMs varied slightly among different regions of colonic longitudinal muscle layers, whereas the contractile responses induced by EMs were significantly different among different regions of circular muscle layers. EMs-induced longitudinal or circular muscle contractions were not significantly affected by atropine, N(G)-nitro-l-arginine methyl ester, phentolamine, propranolol and methysergide. Tetrodotoxin, indomethacin and naloxone completely abolished the EMs-induced colonic contractions. Surprisingly, EMs (10(-7)M)-induced longitudinal muscle contractions were significantly attenuated by nor-binaltorphimine (3x10(-6)M). By contrast, pretreatment with naltrindole (10(-6)M) did not significantly affect EMs-induced longitudinal or circular muscle contractions. Interestingly, the circular muscle contractions in response to EM2 (10(-7)M) were not fully blocked by beta-funaltrexamine (6x10(-6)M). Naloxonazine (10(-6)M) almost fully antagonized the EMs-induced longitudinal or circular muscle contractions, and these effects could be only partially reversed by extensive washing. All the results indicated that the mechanisms and sites of actions of EMs were region-specific. Furthermore, these findings showed that the activation of multiple subtypes of opioid receptors, possibly including mu(1) (naloxonazine-sensitive), mu(2) and even other forms of muORs (beta-FNA-insensitive), was required for EMs-induced mouse colonic motility.     

Control of cyclic AMP levels in primary cultures of human tracheal smooth muscle cells.

1. [3H]-adenosine 3':5'-cyclic monophosphate ([3H]-cyclic AMP) responses were studied in primary cultures of human tracheal smooth muscle cells derived from explants of human trachealis muscle and in short term cultures of acutely dissociated trachealis cells. 2. Isoprenaline induced concentration-dependent [3H]-cyclic AMP formation with an EC50 of 0.2 microM. The response to 10 microM isoprenaline reached a maximum after 5-10 min stimulation and remained stable for periods of up to 1 h. After 10 min stimulation, 1 microM isoprenaline produced a 9.5 fold increase over basal [3H]-cyclic AMP levels. The response to isoprenaline was inhibited by ICI 118551 (10 nM), (apparent KA 1.9 x 10(9) M-1) indicating the probable involvement of a beta 2-adrenoceptor in this response in human cultured tracheal smooth muscle cells. However, with 50 nM ICI 118551 there was a reduction in the maximum response to isoprenaline. Prostaglandin E2 also produced concentration-dependent [3H]-cyclic AMP formation (EC50 0.7 microM, response to 1 microM PGE2 6.4 fold over basal). 3. Forskolin (1 nM - 100 microM) induced concentration-dependent [3H]-cyclic AMP formation in these cells. A 1.6 fold (over basal) response was also observed following stimulation with NaF (10 mM). 4. The nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) (0.1 mM) and the type IV, cyclic AMP selective, phosphodiesterase inhibitor rolipram (0.1 mM) both elevated basal [3H]-cyclic AMP levels by 1.8 and 1.5 fold respectively. IBMX (1-100 microM) and low concentrations of rolipram (< 10 microM), also potentiated the response to 1 microM isoprenaline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lymphocyte beta 2-adrenoceptors and adenosine 3':5'-cyclic monophosphate during and after normal pregnancy.

1. The beta 2-sympathomimetics, used to inhibit preterm labour, bind predominantly to beta 2-adrenoceptors, activating adenylate cyclase to form adenosine 3':5'-cyclic monophosphate (cyclic AMP), a messenger substance which inhibits the enzyme cascade triggering smooth muscle contraction. beta 2-Adrenoceptor density and cyclic AMP formation can be used as markers of beta 2-adrenergic effect. 2. The present study addresses the influence of pregnancy on the beta-adrenoceptor system. beta 2-Adrenoceptor density and cyclic AMP concentrations (basal and evoked by isoprenaline) in circulating lymphocytes were determined at three points in gestation (16, 29 and 37 weeks) and 9 weeks post partum in 22 normal pregnancies. (-)-[125Iodo]-cyanopindolol was used as the ligand to identify a homogeneous population of beta 2-adrenoceptors on lymphocytes. B- and T-cell fractions were estimated from the same samples. 3. beta 2-Adrenoceptor density decreased significantly during gestation until week 37 (P < 0.01), then increased post partum (P < 0.005). Cyclic AMP concentrations (basal and evoked by isoprenaline) were significantly lower after 16 weeks of gestation than post partum (P < 0.05). 4. The results, which cannot be explained in terms of a shift in the lymphocyte (B- and T-cell) ratio, indicate that beta-adrenoceptor density and function are reduced in normal pregnancy and only return to normal post partum. These findings may be of significance in devising future tocolytic therapy with beta 2-adrenoceptor agonists.