Effects of estrogen and progesterone on adrenoceptors and cyclic nucleotides in rat uterus

The effects of estrogen and progesterone on adrenoceptors and cyclic nucleotides were studied in ovariectomized rat uterus. The effects on adrenoceptors were examined by measuring mechanical responses to noradrenaline and by binding site determinations with [3H]-dihydroergocryptine and [3H]-dihydroalprenolol. After acute administration of estradiol-17 beta, uterine cyclic GMP was progressively and significantly elevated, but cyclic AMP declined through it was not significant. Phentolamine suppressed this cyclic GMP elevation. Both acute and chronic treatments with estrogen increased the number of alpha-receptors. The increase in uterine cyclic GMP was related to the increase in alpha-stimulation as a result of increased alpha-receptors. Chronic treatment with estrogen increased the number of alpha-receptors. The alpha-effect induced by noradrenaline was bidirectional in the uterus treated with estrogen chronically; there was contraction in normal Tyrode's solution and relaxation in high K+ Tyrode's solution. In other hormonal group, either alpha- or beta-effects produced relaxation. Enhancement of the alpha- and beta-effects by estrogen is attributed to an increased number of the receptors. Acute treatment with estrogen decreased the responses of cyclic AMP to isoproterenol and mechanical reactivity. Progesterone also enhanced the beta-effect as a result of an increase in the number of beta-receptors.     

Pharmacological models to investigate the role of the adrenergic system on post-partum rat uterus

The adrenergic system plays a major role in the regulation of the pregnant uterine contractility. Our aim was to develop an experimental animal model to study the role of the alpha 1A-adrenergic receptor (AR) in uterine motor activity by antisense oligonucleotides (AONs). AONs were injected with DOTAP and pluronic gel into the uterine lumen of post-partum rats 2-3 hours after delivery. The decrease of the alpha 1A-AR density by AON was demonstrated by RT-PCR method, Western blot analysis and radioligand binding assay on rat uterus preparations 24 h after delivery. The changes in the contractility of the uterus were measured on isolated rat uterine tissue by electric field stimulation (EFS). The EFS investigation demonstrated that the effect of the specific alpha 1A-blocker 5-methylurapidil and WB4101 was significantly decreased in the AON-treated rat uterus as compared to the control group but the effect of the beta-mimetic terbutalin and alpha 1D-antagonist BMY7378 was unchanged. Our result suggest that the alpha 1A-ARs play a very important role in the regulation of uterine contractility, and may serve as the basis for a subsequent new group of tocolytics (uterus selective alpha 1-antagonists), which may lead to more selective therapy than currently used beta-mimetics.     

Electrophysiological analysis of the nature of adrenoceptors in the rat basilar artery during development.

The nature of adrenoceptors in basilar arteries of neonatal rats was investigated by means of electrophysiological techniques. In immature (2-6 day postnatal) rats, micro-injection of noradrenaline elicited a depolarization which consisted of two components. The initial 'fast' component (time to peak of 0.3-4s) was slightly reduced by phentolamine and was not antagonized by propranolol. The second 'slow' component (time to peak of about 50s) was not blocked by phentolamine but was antagonized by low concentrations (10(-7) M) of propranolol. In immature rats, micro-injection of isoprenaline was more potent than noradrenaline in evoking the 'slow' depolarization but less effective in eliciting the 'fast' response. The pharmacology with respect to adrenoceptor antagonists of both components of the isoprenaline- and noradrenaline-induced depolarizations was similar. There was some evidence of inhibitory beta-adrenoceptors in immature rat basilar vessels. In adult rats (6 week old) noradrenaline produced a large 'fast' depolarization which was followed by a 'slow' tail response. Both components were not antagonized by phentolamine or propranolol. It appears that in the basilar artery of neonatal rats there are excitatory alpha- and inhibitory beta-adrenoceptors but the major responses to noradrenaline and isoprenaline are mediated by gamma- and excitatory beta-receptors. In adult animals the gamma-adrenoceptor predominates. Experiments were carried out in which agonists were applied by ionophoresis. These results confirm the presence of excitatory beta-receptors in neonatal basilar vessels and show the response has slow kinetics and it is likely that the beta-receptors are distributed uniformly over the smooth muscle surface. In adult animals it was not possible to elicit an excitatory beta-receptor-mediated response. The ionophoretic application of noradrenaline never evoked a perceptible depolarization which could be attributed to gamma-adrenoceptor stimulation. This result is discussed in terms of receptor distribution with respect to synaptic function in a syncytium.     

Thyroid-dependent alterations of myocardial adrenoceptors and adrenoceptor-mediated responses in the rat

Cardiovascular alterations in hypo- and hyperthyroidism have been ascribed to changes of noradrenergic neurotransmission. In the present study the influence of thyroid hormones on adrenoceptors in the rat heart was further characterized. The effect of artificial hypothyroidism (induced by feeding 6-propyl-2-thiouracil, PTU) and hyperthyroidism (induced by daily injections of triiodothyronine, T3) on myocardial adrenoceptor binding, catecholamines, some physiological responses, and their interdependence was examined. The density of myocardial beta-adrenergic binding sites (3H-dihydroalprenolol, 3H-DHA) was reduced after PTU (by 38%) and enhanced after T3 treatment (by up to 82%). The increase was dose- and time-dependent and reversible within 4 days. No changes of the affinity of 3H-DHA to its binding sites were observed. Only L-T3 and L-T4 proved to be active, D-T3 and reverse T3 had no effect. The rise in beta-adrenoceptor density caused by T3 was prevented by concomitant administration of cycloheximide, indicating its dependence on protein synthesis. The density of myocardial alpha 1-adrenergic binding sites (3H-prazosin) was significantly reduced in the PTU group (by up to 28%) and even more distinctly by T3 treatment (by up to 50%). KD values remained unaltered. The noradrenaline content and turnover of rat hearts was significantly reduced by T3-induced hyperthyroidism. PTU treatment had no influence on content and turnover of noradrenaline. Plasma noradrenaline as well as adrenaline levels in freely moving rats were increased by PTU treatment 9- and 5-fold, respectively. In T3-injected animals no significant changes were measured. The density of adrenoceptors is known to be inversely correlated with catecholamine levels in several organs. Neither alpha- nor beta-adrenoceptor changes in the myocardium of dysthyroid rats could be attributed to such a homologous regulation, since they still occurred after chemical sympathectomy with 6-hydroxydopamine and adrenalectomy.(ABSTRACT TRUNCATED AT 400 WORDS)     

老年大鼠心脏肾上腺素受体亚型的改变(英文)

目的:研究老年心脏肾上腺素受体亚型的改变.方法:制备3月龄与25月龄Wistar大鼠心脏膜标本,分别采用~(125)I-BE2254与~(125)I-Pindold作放射配基结合分析,测定α_1与β-肾上腺素受体.结果:老年大鼠心脏的α_1与β-肾上腺受体密度分别由年轻大鼠的119±4与45.9±1.9pmol/g下降至70±6与36.4±1.6 pmol/g(P<0.01),α_1-AR降低的幅度大于β-AR.此外α_(1A)与α_(1B)亚型之比由年轻大鼠的39:61下降至26:74(P<0.05).结论:老年大鼠心脏肾上腺素受体减少,且不同亚型减少的程度不同.     

The influence of hormonal and neuronal factors on rat heart adrenoceptors

1 The influence of hormonal and neuronal factors on adrenoceptors mediating increased cardiac force and rate of contraction were studied in rat isolated atria. The pharmacological properties of these receptors were deduced from the relative potencies of agonists and from the effects of selective α- and β-adrenoceptor antagonists. The numbers and affinities of α- and β-adrenoceptors were also determined by radioligand binding to ventricular membrane fragments.

2 Hypophysectomy reduced the inotropic potency of isoprenaline and increased the potency of phenylephrine and methoxamine in left atria. The effect of phenylephrine was inhibited by propranolol less effectively and by phentolamine or phenoxybenzamine more effectively in hypophysectomized than in control rats. The difference in block was smaller at low than at high antagonist concentrations. Similar but smaller changes were observed for chronotropic responses of right atria.

3 The decreased β- and increased α-receptor response after hypophysectomy was similar to that observed earlier in thyroidectomized rats (Kunos, 1977). These changes developed slowly after hypophysectomy (>2 weeks), they were both reversed within 2 days of thyroxine treatment (0.2 mg/kg daily), but were not affected by cortisone treatment (50 mg/kg every 12 h for 4 days).

4 Treatment of hypophysectomized rats for 2 days with thyroxine increased the density of [³H]-dihydroalprenolol ([³H]-DHA) binding sites from 27.5 ± 2.7 to 45.5 ± 5.7 fmol/mg protein and decreased the density of [³H]-WB-4101 binding sites from 38.7 ± 3.1 to 18.7 ± 2.5 fmol/mg protein. The affinity of either type of binding site for agonists or antagonist was not significantly altered by thyroxine treatment and the sum total of α₁- and β-receptors remained the same.

5 Sympathetic denervation of thyroidectomized rats by 6-hydroxydopamine increased the inotropic potency of isoprenaline and noradrenaline and the blocking effect of propranolol, and decreased the potency of phenylephrine and the blocking effect of phenoxybenzamine to or beyond values observed in euthyroid controls. The density of [³H]-DHA binding sites was higher and that of [³H]-WB-4101 binding sites was lower in the denervated than in the innervated hypothyroid myocardium. Depletion of endogenous noradrenaline stores by reserpine did not significantly alter the adrenoceptor response pattern of the hypothyroid preparations and did not influence the density or affinity of [³H]-DHA and [³H]-WB-4101 binding sites.

6 These results indicate that thyrotropin or steroids do not contribute to the reciprocal changes in the sensitivity of cardiac α₁- and β-adrenoceptors in altered thyroid states. These thyroid hormone-dependent changes are probably due to a parallel, reciprocal change in the numbers but not the affinities of α₁- and β-adrenoceptors. Reciprocal regulation of cardiac α₁- and β-adrenoceptors by thyroid hormones requires intact sympathetic innervation but not the presence of normal stores of the neurotransmitter.

     

The role of adrenoceptors in the mechanism of reserpine-induced stimulation of gastric acid secretion in the rat

The role of alpha- and beta-adrenoceptors in the mechanism of reserpine-induced stimulation of gastric acid secretion in the rat has been examined. After 6 h reserpine (0.1 mg kg-1 i.p.) significantly stimulated acid secretion relative to control values (176 +/- 4 vs 60 +/- 3 mumol, mean +/- s.e.m., n = 10, P less than 0.001). Neither coeliac ganglionectomy nor propranolol (5-15 mg kg-1) influenced this action. Vagotomy prevented acid stimulation by reserpine and was associated with H+ output similar to that of vagotomy controls (13 +/- 1 vs 14 +/- 1 mumol, mean +/- s.e.m., n = 10). Dose-dependent inhibition of the reserpine-induced acid secretion was produced by phenoxybenzamine or phentolamine; an inhibition similar to that achieved by vagotomy was noted with the 15 mg kg-1 dose (13 +/- 1 and 15 +/- 1 mumol, respectively, vs 176 +/- 4 mumol, mean +/- s.e.m., n = 10, P less than 0.001). The similarity in action between vagotomy and large doses of phenoxybenzamine or phentolamine suggests that, in the rat, vagal alpha-adrenoceptor stimulation is directly involved in the mechanism of reserpine-induced stimulation of gastric acid secretion.     

The selective action of beta-adrenoceptor blocking drugs and the nature of beta1 and beta2 adrenoceptors.

1 Purified membranes retaining a catecholamine responsive adenylate cyclase have prepared from rabbit heart, lung and (pseudo-pregnant) uterus. 2 These preparations have the characteristics of plasma membranes and both heart and lung respond to beta-adrenoceptor agonists in the order: (+/-)-isoprenaline greater than (-)-noradrenaline greater than (-)-adrenaline greater than (+)-isoprenaline greater than salbutamol. The sensitivity of the adenylate cyclase to beta-adrenoceptor stimulation is improved by pre-treatment of the animals with reserpine and syrosingopine. 3 Dose-ratios for several concentrations of propranolol (non-selective beta-adrenoceptor blocker), practolol and atenolol (cardio-selective beta-adrenoceptor blockers) have been measured on all three membrane preparations. Schild plots of log (dose ratio -1) vs. log dose were virtually coincident for heart and lung with a dissociation constant (Kb) for propranolol very close to the pharmacological value. The ratio of Kb values was 0.65 for practolol and 1.23 for atenolol compared with pharmacological cardio-selectivity ratios (measured on isolated atria and tracheal chain) of 67.6 and 110 respectively. The uterus/heart Kb ratio was 51.5 for atenolol. Inhibition of the uterus by practolol gave a Schild plot with slope significantly less than 1, indicating a different mechanism of action from the heart. 4 Kb values obtained by measuring adenylate cyclase stimulation in chopped tissue (including preparations of bronchial tree and alveolar tissue as well as whole lung) resembled the membrane values rather than those found in whole organs. 5 The results show that the pharmacological selectivity of practolol and atenolol is maintained at the receptor-adenylate cyclase level, at least as far as heart and uterus are concerned, though the smaller selectivity ratios in the biochemical system suggest that receptor differences is not the only factor and that phase distribution of the drug may also be important. Membranes prepared from whole lung show that phase distribution of the drug may also be important. Membranes prepared from whole lung show an overall beta1 response which may simply reflect the predominance of beta1 cell types containing beta1-adrenoceptors over bronchial smooth muscle.     

Acute and chronic effects of methylphenidate on cortical adrenoceptors in the rat

The effects of acute and chronic treatments with methylphenidate were examined on cortical adrenoceptors. A single dose of methylphenidate increased the affinity of [3H]prazosin and [3H]idazoxan binding sites. Acute and chronic methylphenidate treatments caused a down-regulation of alpha 1-adrenoceptors. There were no changes in cortical beta-adrenoceptors measured with [3H]dihydroalprenolol nor in endogenous monoamine levels. The alterations in alpha-adrenoceptors can be attributed to an indirect action of methylphenidate possibly through an accrued release of noradrenaline or tyramine.     

Immunolocalization of glutathione S-transferase in the rat uterus

Immunohistochemical localization of glutathione S-transferase (GST) and enzyme cytochemical staining for endogenous peroxidase (Px) activity were studied in rat uteri. Both enzymes were clearly detected in the endometrium of the uterus taken from proestrus to estrus during the estrous cycle. Based on our data, the biological significance of GST in endometrium was discussed.     

The relaxant action of BRL 34915 in rat uterus.

1 BRL 34915 (0.04-1.3 microM) caused concentration-dependent inhibition of spontaneous phasic spasms of the isolated uterus of the term pregnant rat and this effect was not antagonized by propranolol. Spasms evoked by low concentrations of KCl (less than 20 mM) were inhibited by BRL 34915 but those evoked by higher concentrations (greater than 40 mM) were unaffected. 2 In experiments using extracellular electrical recording, BRL 34915 (10 microM) selectively inhibited oxytocin-induced phasic spasms and the associated spike activity but had little effect on the tonic component of the spasms. BRL 34915, as an inhibitor of phasic spasms to oxytocin (0.2 nM), was antagonized by procaine (0.3 and 1 mM). 3 BRL 34915 (10 microM) did not inhibit Ca2+-induced spasm of saponin-skinned thin myometrial strips. 4 Intracellular microelectrode recording from myometrial strips showed that BRL 34915 (10 microM) inhibited action potentials and phasic spasms in the presence of oxytocin (0.2 nM) and produced a hyperpolarization of 5 mV. 5 In single myometrial cells under current or voltage clamp, BRL 34915 (10 microM) had no effect on action potentials and inward current in Ca2+- or Ba2+-containing media in the presence of tetraethylammonium, 4-aminopyridine and caesium chloride. In the absence of these K+-channel inhibitors, BRL 34915 had no effect on resting membrane potential, membrane resistance, action potential, inward current or outward current. 6 BRL 34915 (1 or 10 microM) had no effect on 86Rb efflux from myometrial strips. 86Rb efflux was increased by oxytocin (0.2 and 20 nM). 7 The relaxant profile of BRL 34915 in the rat uterus is similar to that described for other smooth muscles where an action to open membrane K+-channels has been proposed. BRL 34915 inhibited spike production but produced only a small hyperpolarization without a detectable increase in 86Rb efflux. Membrane resistance and transmembrane currents were unaffected. These results suggest that in the uterus the effects of BRL 34915 may be restricted to K+-channels involved in the production of pacemaker activity.     

Alpha adrenoceptors in rat brain: Direct identification with prazosin

Summary Tritiated prazosin was used to characterize high affinity binding sites with characteristics similar to ₁ adrenoceptors in rat brain membranes. These sites were compared with ₂ adrenoceptors labeled with tritiated clonidine. The prazosin sites had an association constant of 2 nM^–1 and bound the ligand optimal around pH 7.0. The density of the sites was 300 fmoles per mg of protein; the half time of dissociation of prazosin was 7 min at 30° C. The order or potencies of agonists, determined from binding-inhibition experiments with labeled prazosin, was: naphazoline > clonidine > adrenaline > noradrenaline > phenylephrine > -methylnoradrenaline > dophamine. The order of potencies of antagonists was: prazosin > phenoxybenzamine > phentolamine > clozapine > yohimbine. Sodium ions and divalent cations as well as guanyl nucleotides have little or no effect on the binding of the labeled antagonist. This is in contrast to the binding of the labeled agonist clonidine (Glossmann and Presek, 1979a, 1979b). Labeled prazosin may be a useful tool to characterize ₁ adrenoceptors.This is part of the thesis of R. H. to be presented to the Fachbereich Humanmedizin, Justus Liebig-Universität Giessen, in partial fulfillment of the requirements for a Doctor of Medical Science degreee     

The effect of histamine on the electrical activity of rat uterus

Histamine produces a reduction in the spike frequency and the degree of depolarization which accompany spontaneous and induced contractions of rat uterus. Concentrations of 0.5 mug/ml can cause complete inhibition of both mechanical and electrical activity without producing any change in the resting membrane potential. Concentrations as high as 100 mug/ml cause no appreciable change in membrane potential.     

The myostimulating effect of tissue kallikrein on rat uterus

The mechanism of the myostimulating activity of rat tissue kallikrein on rat uterus was re-examined using uterus from kininogen-deficient rats and HOE 140 (D-Arg[Hyp³, This, D-Tic⁷, Oic⁸]bradykinin), a specific bradykinin receptor-B₂ antagonist. The uterus from kininogen-deficient rats was 50 times less sensitive to rat kallikrein than that from normal rats. HOE 140 (6 to 60 nM) inhibited the contracting effects of bradykinin and of rat kallikrein. Porcine kallikrein had no effect on rat uterus. Bradykinin and rat kallikrein induced a relaxation of rat duodenum. The duodenum from kininogen-deficient rats was 100 times less sensitive to rat kallikrein than the duodenum from normal rats. HOE 140 (0.6 to 3 nM) inhibited the relaxing effects of bradykinin and of kallikrein. Preincubation of rat kallikrein with aprotinin (Trasylol) abolished the effects of kallikrein on smooth muscles. HOE 140 inhibited the amidolytic activity of tissue kallikrein with a K_i value of 220 W. HOE 140, at micromolar concentrations, suppressed the kininogenase activity of tissue kallikrein. Plasma of deficient rats contained 0.7% of the normal levels of kininogens. After washing the blood vessels with saline, kininogens were present in uterine homogenates but not in duodenal homogenates from both rat strains. Uterine kininogens from deficient rats amounted to 19% of the kininogen content of the uterus of normal rats. The blood pressure of anaesthetized rats was lowered by rat tissue kallikrein but not by porcine kallikrein.We conclude that the myostimulating activity of kallikrein mostly depends on kinin formation from kininogens present in the tissue. These kininogens arise from blood contamination in the duodenum and also from a local store in the uterus. The presence of a significant amount of kininogens in the uterus from deficient rats might suggest a local synthesis of kininogens. HOE 140 inhibits the enzymatic activity of kallikrein at micromolar concentrations.     

The effect of metals on the S-S polypeptide receptor in depolarized rat uterus

1. Metals potentiate the contractile effects of S-S polypeptides in depolarized rat uterus. Their order of potency is Co(++)>/=Co(+++)>/=Mn(++)>/=Ni(++)>/= Mn(+++)>Zn(++)>/=Mg(++)>Fe(++)>Fe(+++)>/= Ca(++)>Be(++)=Sr(++)=Ba(++)=Cu(++)=O.2. S-S polypeptides with relatively weak oxytocic activity such as lys-vasopressin, arg-vasopressin and orn-vasopressin are strongly potentiated by metals while highly active polypeptides such as oxytocin are weakly potentiated.3. Potentiation by metals was specific for S-S polypeptides; other polypeptides (bradykinin, hypertensin) as well as acetylcholine and isoprenaline were unaffected.4. Potentiation by metals occurs rapidly and is fully reversible. In all cases some activity was retained by S-S polypeptides even in the complete absence of metals.5. A scheme which could account for the observed effects has been formulated. This assumes the formation of a ternary complex between receptor, metal and polypeptide leading to improved alignment between polypeptide and receptor.6. Analogies are discussed between metal enzymes and the S-S polypeptide receptor for which the term metal receptor is proposed.