Adrenoceptors involved in the contractile activity of islated pregnant rat uterus.

Cumulative log dose-response curves of soterenol, isoproterenol, phenylephrine and norepinephrine on isolated pregnant rat uterus at different days of gestation, were investigated. Soterenol produced a sustained inhibition of spontaneous motility during the whole pregnancy and this effect was blocked by butoxamine. The affinity of myometrium for the beta 2-adrenoceptor agonist was parallel to the concentration of progesterone in plasma during pregnancy. Isoproterenol, norepinephrine and phenylephrine caused dual, alpha- and beta-mediated responses, their relative dominance varied with the concentration and the days of pregnancy, alpha-Adrenoceptor effects of the amines coincided with increased plasma concentrations of estrogens, whereas beta ones were in parallel with the increment of plasma progesterone. It is concluded that: (a) there exist in the pregnant rat uterus beta 2-receptor-mediated responses influenced by the length of gestation; and (b) the concentration-pregnancy-dependent biphasic actions of isoproterenol, norepinephrine or phenylephrine suggest that their variable hormone-modulated ability interacts with both alpha- and beta-adrenoceptive uterine sites.     

Characterization of an alpha 1D-adrenoceptor mediating the contractile response of rat aorta to noradrenaline.

1. The affinities of a number of alpha 1-adrenoceptor antagonists were determined by displacement of [3H]-prazosin binding from cloned human alpha 1A-adrenoceptors (previously designated cloned alpha 1c subtype), alpha 1B alpha 1D and rat alpha 1D-adrenoceptors, stably expressed in rat-1 fibroblasts. Functional affinity estimates for these compounds were also determined from noradrenaline-mediated contractions of rat aorta. 2. BMY 7378 displayed high affinity for cloned human alpha 1D-adrenoceptors (pKi = 8.2 +/- 0.10) and was selective over alpha 1A (pKi = 6.2 +/- 0.10) and alpha 1B subtypes (6.7 +/- 0.11). WB 4101, benoxathian and phentolamine displayed high affinity for alpha 1A and alpha 1D adrenoceptors compared to the alpha 1B subtype. Spiperone displayed high affinity and selectivity for alpha 1B adrenoceptors (pKi 8.8 +/- 0.16). 5-Methyl-urapidil was selective for cloned alpha 1A adrenoceptors. 3. Comparative binding affinities (pKi) for compounds at cloned human and rat1D adrenoceptors were almost identical (r = 0.99, slope = 1.08). 4. Prazosin, doxazosin and 5-methyl-urapidil were potent, competitive antagonists of noradrenaline-mediated contractions of rat aorta (pA2 values of 9.8, 8.8 and 7.8 respectively). The selective alpha 1D antagonist BMY 7378 was also a potent antagonist on rat aorta (pKB = 8.3 +/- 0.1) but the interaction of this compound was not consistent with competitive antagonism at a single population of receptors. 5. Functional affinities for compounds determined against noradrenaline-mediated contractions of rat aorta correlated well with binding affinities at cloned alpha 1D-adrenoceptors (r = 0.96), but not with alpha 1A (r = 0.61) or alpha 1B (r = 0.46) subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein kinase C-mediated contractile response of the rat vas deferens.

The role of protein kinase C (PKC) in mediating contractile responses in the rat vas deferens was studied. Phorbol-12,13-diacetate (PDA) in the presence of 20 mM K+ elicited a concentration-dependent response with an EC50 of 190 nM. The non-PKC activator 4 alpha-phorbol (2 microM) was unable to elicit contraction in 20 mM K+ buffer. Incubation of rat vas deferens with the PKC inhibitor iso-H7 (30 microM) attenuated the response to norepinephrine (NE) and neurokinin A, with maximal effects depressed to 42 and 39% of control, respectively. Responses to 60 mM K+ and 2 microM PDA (20 mM K+) was also significantly inhibited by iso-H7. In the presence of 2 microM PDA and 20 mM K+, the NE concentration-effect curve was shifted 3.6-fold to the right of the control curve in a parallel manner. 4 alpha-Phorbol (20 mM K+) at the same concentration did not produce this effect. These results suggest a significant role for PKC in the contractile response of the rat vas deferens.     

Mechanism of the contractile response to Platelet-Activating Factor (PAF) of the rat stomach fundus. I. PAF-induced contractile response and calcium mobilization

• 1.1. Platelet-activating factor (PAF) caused contraction of the rat stomach fundus in a concentration-dependent manner in the presence of atropine, guanethidine, chlorpheniramine, methysergide, indomethacin, nordihydroguaiaretic acid and tetrodotoxin.
• 2.2. PAF produced phasic contraction followed by tonic contraction. The PAF-induced tonic contraction was significantly reduced by treatment with CV-6209, an antagonist of PAF, but phasic contraction induced by PAF was rather resistant to CV-6209.
• 3.3. The contraction induced by PAF was markedly reduced when tissues were previously exposed to PAF (desensitization).
• 4.4. Nicardipine reduced the PAF-induced phasic contraction but not of the tonic contraction.
• 5.5. PAF-induced contractions were almost abolished in Ca²⁺-free medium.
• 6.6. The Ca²⁺-contraction in Ca²⁺-free solution was significantly augmented by PAF, whereas the Ca²⁺-contraction in Ca²⁺-free, isotonic high K⁺ (60 mM) medium was unaffected by PAF.
• 7.7. These results suggest that PAF-induced contractile response in the rat stomach fundus is due to an influx of Ca²⁺ through voltage-dependent Ca²⁺-channels (VDC) and receptor-operated Ca²⁺-channels (ROC). It is further suggested that PAF may depolarize the stomach fundus and this depolarization may open the VDC, whereas PAF may not act directly on the VDC.

     

Effects of ouabain on contractile response to norepinephrine in isolated rat aorta.

1. Inhibition of sodium(Na+), potassium(K+)-ATPase activity was dependent on the concentration of ouabain and inverse to the concentration of potassium ([K+]) in the reaction mixture. 2. Contractility of isolated rat aorta preparation in response to norepinephrine was augmented by ouabain and low [K+]. 3. Results suggest that inhibition of Na+,K(+)-ATPase activity will be correlated with augmentation of vascular contractility maybe through activation of Na(+)-calcium exchange system.     

Influence of aging on the contractile response to endothelin of rat thoracic aorta.

Age-related changes in the contractile response to endothelin-1 and ACh were assessed in thoracic aortas isolated from 2-, 6- and 24-month-old male Fischer 344 rats. In aortic strips with an intact endothelium, the maximal contractile response to endothelin-1 decreased with development to maturity. Removal of the endothelium did not affect the contractile response to endothelin-1. Endothelin-1 did not elicit a relaxant response in phenylephrine-precontracted strips. The ACh-induced relaxation decreased in senescent rats. These results indicate that the contractile response of aortic smooth muscle to endothelin-1 decreases with age, and that the endothelial vasorelaxant factors do not contribute to this age-induced modulation.     

Pharmacological modulation of the contractile response to toluene diisocyanate in the rat isolated urinary bladder.

1. Toluene diisocyanate produced concentration-dependent contractions of the rat isolated urinary bladder. 2. The contractions were tetrodotoxin-resistant and were abolished by previous exposure of the strips to capsaicin. 3. Indomethacin (5 microM) and ruthenium red (30 microM) inhibited toluene diisocyanate-induced contractions. Responses expressed as a percentage of the response obtained with substance P, 30 nM, were respectively 141.6 +/- 24.8% and 20.1 +/- 5.1% in control and indomethacin-treated strips (P less than 0.005); 123.0 +/- 30.2% and 14.0 +/- 6.5% in control and ruthenium red-treated strips (0.01 less than P less than 0.05). 4. These results suggest that toluene diisocyanate-induced contractions of the rat isolated bladder are the result of the release of cyclo-oxygenase products which may act by activating the capsaicin receptor.     

Tachykinin receptors involved in the contractile effect of the natural tachykinins in the rat gastric fundus

1 The receptors involved in mammalian tachykinin-induced contractions of longitudinal smooth muscle strips of the rat gastric fundus were characterized pharmacologically. 2 Substance P (SP), neurokinin A, neurokinin B and senktide contracted the strips in a concentration-dependent manner with a potency order of neurokinin A senktide > neurokinin B > substance P. The contractions were not influenced by tetrodotoxin and atropine. 3 L 659877, a NK_2B-receptor-preferring antagonist reduced neurokinin A- and neurokinin B-induced contractions (estimated pK_B 6.9 and 6.3, respectively) but had less pronounced effects on SP-induced contractions and none on contractions induced by senktide. MEN 10376, an NK_2A-receptor-preferring antagonist, reduced the neurokinin A-induced contractions (estimated pK_a 5.2), while dactinomycin, reduced the neurokinin A-induced contractions only to a minor extent at 10^?4 M. 4 CP 96345, an NK₁-receptor antagonist, reduced substance P- and neurokinin A-induced responses, but also reduced the contractions induced by KC1 and methacholine. RP 67580, another non-peptide NK₁-receptor antagonist had no effect on the substance P-, neurokinin A- and neurokinin B-induced contractions up to a concentration of 3 × 10^?6 M. 5 These results suggest that the mammalian tachykinins induce contractions of the longitudinal smooth muscle strip of the rat gastric fundus by direct action at muscular NK_2B- and NK₃-receptors.     

Characterization of the leukotriene D4 receptor in hyperreactive rat lung.

A [3H]leukotriene D4 radioreceptor binding assay has been established in rat lung and has been used to fully characterize the leukotriene D4 receptor in lung membranes from an inbred strain of rats displaying non-specific bronchial hyperreactivity. [3H]leukotriene D4 specific binding in this tissue is of high affinity (KD 0.12 nM), saturable (Bmax 42 fmol/mg protein), inhibited by both guanine nucleotide analogues and sodium ions and increased by divalent cations. In addition, Ki values show that agonists, but not antagonists, compete for [3H]leukotriene D4 binding in rat lung with the same potency as they compete for [3H]leukotriene D4 binding in guinea-pig lung, the classical tissue for leukotriene D4 receptor studies. Finally, [3H]leukotriene D4 binding in hyperreactive rat lung has been compared with [3H]leukotriene D4 binding in lung tissue from Fischer rats, which are a less responsive strain.     

Effect of aclarubicin on contractile response of rat aorta

The effect of aclarubicin on vasocontractility was investigated using aortic strips isolated from rats. The aortic strips from rats injected i.p. with aclarubicin (4 mg/kg body weight per day for 5 consecutive days) showed diminished contractile responses to KCl and phenylephrine in comparison with the controls injected with 0.9% saline. In vitro preincubation of rat aorta with aclarubicin (20-80 micrograms/ml) attenuated the aortic contractile responses to KCl and phenylephrine compared with the control preincubated with 0.9% saline. These results suggest that aclarubicin reduces the contractility of vascular smooth muscle directly.     

Effect of strychnine on the contractile response of rat vas deferens

Strychnine at concentrations of 0.1 approximately 1.0 microM induced supersensitivity of rat vas deferens to alpha-agonists but not to other stimulants. Strychnine-treatment resulted in an increase in the number of alpha-adrenoceptors, determined by measuring the binding of [3H]prazosin. Glycine did not have any significant effect on the contracture and did not antagonize the action of strychnine. Thus, specific supersensitivity of alpha-adrenergic system was induced by treatment with low concentrations of strychnine.     

L-methionine enhances the contractile response to norepinephrine of rat vas deferens

Under Ca-depleted conditions, the contractile responses of rat vas deferens in the presence of norepinephrine were not elicited until the addition of CaCl2. L-Methionine enhanced the contractile response of vas deferens in the presence of methylation blockers under these conditions. The enhancing effect of L-methionine on some other smooth muscles could not be determined because under Ca-depleted conditions, these muscles showed 60-80% of the maximal contractile response on addition of CaCl2 alone. These findings suggested that L-methionine has an enhancing effect on contraction of the rat vas deferens as it does on rat uterine muscle.     

Characterization of the central muscarinic cholinoceptors involved in the cholinergic pressor response in anesthetized dogs.

Previous reports have shown that an intracisternal (i.c.) injection of acetylcholine in the dog increases both arterial blood pressure and plasma levels of noradrenaline and vasopressin via central muscarinic receptors. The aim of the present study was to characterize the central muscarinic cholinoceptor subtypes involved in such central cholinergic responses in anesthetized male Beagle-Harrier dogs (n = 12). For this purpose, we studied the relative potency of various muscarinic receptor antagonists to block the acetylcholine-induced pressor responses (30 microg kg(-1) i.c.). The acetylcholine-induced pressor response was inhibited in a dose-dependent manner by the i.c. administration of the non-selective muscarinic receptor antagonist atropine (ID50 = 0.5 microg kg(-1)), the muscarinic M receptor antagonist pirenzepine (ID50 = 0.45 microg kg(-1)), the muscarinic M2 receptor antagonist methoctramine (ID50 = 8.5 microg kg(-1)) and the muscarinic M3 receptor antagonist para-fluoro-hexahydro-sila-difenidol (ID50) = 43.7 microg kg(-1)). The order of potency of these four muscarinic receptor antagonists was: atropine = pirenzepine > methoctramine > para-fluoro-hexahydro-sila-difenidol. In order to confirm the selectivity for muscarinic M1 receptors of this dose of pirenzepine, we checked that 40- to 50-fold higher concentrations were necessary to block a typical muscarinic M2 receptor response (bradycardia) and a typical muscarinic M3 receptor response (endothelial vasodilation) compared with methoctramine and para-fluoro-hexahydro-sila-difenidol, respectively. These results suggest that the pressor response elicited by intracisternal injection of acetylcholine in anesthetized Beagle-Harrier dogs is mediated through the activation of the muscarinic M1 cholinoceptor subtype.     

Alpha-adrenoceptors involved on the cardiovascular response induced by mianserin in the pithed rat.

1. The effects of the antidepressant drug mianserin on the cardiovascular responses induced by preganglionic electrical stimulation, and i.v. infusion of the adrenergic agonists noradrenaline (NA, alpha 1 and alpha 2), phenylephrine (alpha 1) and xylazine (alpha 2) in the pithed normotensive rat were studied. 2. Mianserin inhibited in a dose-dependent manner the pressor effect caused by electrical stimulation of spinal cord (Th7-Th9) and the infusion of NA, phenylephrine and xylazine. Cocaine increased the inhibitory effect of mianserin on the pressor effect caused by electrical stimulation and NA. 3. Mianserin blocked the xylazine-induced inhibition of cardiac nerve stimulation effect. 4. These results suggest that mianserin blocks the NA uptake, and it is more effective in blocking presynaptic alpha 2- than postsynaptic alpha-adrenoceptors.     

Characterization of the contractile activity of dopamine on the rat isolated seminal vesicle

The mechanism of the contractile effect of dopamine (DA) on the rat isolated seminal vesicle was studied. Cocaine (10 microM/1 in the organ bath, 30 min before DA) and 6-OHDA (50 mg/kg i.v. 24 hr before removal of the seminal vesicle) almost completely prevented the contractile effect of DA. Drugs known to have an affinity for DA receptors or for alpha-adrenoceptors antagonized the contractile effect of DA, the rank order of potency being: prazosin greater than phentolamine greater than yohimbine greater than clonidine greater than sulpiride greater than apomorphine greater than haloperidol. The antagonism was in each case greater against DA than against noradrenaline (NA), used for comparison; selectivity for DA being highest in the case of prazosin and sulpiride. Taken together, these findings indicate that DA makes the rat seminal vesicle contract mostly by means of an indirect mechanism, binding presynaptic DA-receptors and, in part, presynaptic alpha-adrenoceptors as well; or, alternatively, binding presynaptic DA-receptors which have some links with alpha 2-adrenoceptors; the consequence being in either case the release of NA from sympathetic nerve endings.     

Effect of sodium propionate on the contractile response of the rat ileum in situ

Effects of short-chain fatty acids (SCFA) on the contractile response of rat ileum were studied in vivo. The contractile response was estimated by means of changes in the intraluminal pressure under the isometric condition. Intravenous administration of sodium salts of propionate, butyrate, valerate or caproate produced biphasic contractions: an initial phasic contraction and a subsequent tonic contraction. The effect of propionate was studied in detail. A sigmoid dose-response curve was obtained for the phasic contraction. Atropine, hexamethonium and tetrodotoxin inhibited the phasic contraction, while neostigmine vigorously enhanced it. On the other hand, the tonic contraction was not inhibited by atropine, hexamethonium or tetrodotoxin. Repeated administration of propionate at intervals of less than 3 min led to tachyphylaxis, and this tachyphylaxis disappeared by about 10 min. These results suggest that SCFA induced the biphasic contraction of the rat ileum, probably by neurogenic and myogenic mechanisms.     

Mechanisms underlying the contractile response to endothelin-1 in the rat renal artery

We assessed the functional response and the mechanisms following receptor stimulation of endothelin-1 (ET-1) in the rat renal artery. In this study, isometric tension was recorded in renal artery rings without endothelium. Cumulative application of ET-1 from 0.1 to 100 nmol/l induced a sustained concentration-dependent contraction in the renal artery. Submaximal contraction induced by 10 nmol/l ET-1 in 2.5 mmol/l Ca(2+) and in the absence of inhibitors was used as control response (100%). The relative contribution of different sources of Ca(2+) in ET-1-induced contraction was evaluated. The contractile response to 10 nmol/l ET-1 in 2.5 mmol/l Ca(2+ )(1.2 +/- 0.2 g) was significantly inhibited either in Ca(2+)-free solution containing 100 micromol/l ethylene glycol bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (0.6 +/- 0.1 g) or after depletion of intracellular Ca(2+) stores (0.62 +/- 0.05 g). The contribution of phospholipase C and protein kinase C was evaluated by using their inhibitors 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC) and [1-(5-isoquinolinesulfonyl)-2-methylpiperazine] (H-7), respectively. The contractile response to 10 nmol/l ET-1 was inhibited by 10 micromol/l NCDC (to 80 +/- 6%) and 30 micromol/l H-7 (to 76.6 +/- 6.5%). We found that 1 micromol/l nifedipine inhibited the ET-1-induced contraction (to 48.7 +/- 6.9%), indicating the contribution of Ca(2+) influx through voltage-gated L-type Ca(2+) channels to this response. Further, the inhibitory effect of nifedipine was to a greater extent as compared with NCDC or H-7. Additive inhibition of ET-1-induced contraction was not observed in the presence of both nifedipine and NCDC. We also evaluated the role of the ionic transport system in the ET-1-induced response by using 20 nmol/l 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), an inhibitor of Na(+)-H(+) exchange, or 100 micromol/l ouabain, an inhibitor of Na(+)-K(+)-ATPase. The response to ET-1 was decreased by both EIPA (to 61.6 +/- 8.4%) and ouabain (to 62.1 +/- 8.6%). The contribution of Na(+)-Ca(2+) exchange to ouabain action was tested using the inhibitor dimethyl amiloride HCl (10 micromol/l). The decrease in ET-1-induced contraction by the combination of ouabain and dimethyl amiloride HCl was similar to that observed with ouabain alone. In view of these observations, both extra- and intracellular sources of Ca(2+) contribute to the contractile response induced by ET-1 in the renal artery. Our findings also revealed the importance of Ca(2+) influx through voltage-gated L-type Ca(2+) channels in mediating contraction to ET-1 in the renal artery, whereas a minor role of phospholipase C and protein kinase C was observed. Na(+)-H(+) exchange and Na(+)-K(+)-ATPase also play a role in the ET-1-induced contraction in renal artery. Moreover, the contribution of Na(+)-K(+)-ATPase in ET-1 contraction is not an Na(+)-Ca(2+) exchange-related process.