Angiotensin actions on the isolated rat uterus during the estrous cycle: influence of resting membrane potential and uterine morphology

The involvement of AT1 and AT2 receptor subtypes in the response of the isolated rat uterus to angiotensin II (AngII) was studied throughout the estrous cycle. The AngII potency varied during the different estrous cycle phases, as indicated by significantly different pD2 values. No significant differences were observed in AngII metabolism among different estrous phases. Morphological analysis indicated that external and internal myometrium layers were thicker during estrus. In addition, the highest resting membrane potential was also observed during this phase, when compared with the proestrus and diestrus phases. The AngII-induced uterine contractions were blocked by losartan. Different losartan pD2 values were observed. PD123319 had no effect on the contractile response to AngII. The results also indicate that estrous cycle-dependent changes in AngII potency are correlated with uterine morphological and/or membrane potential changes.     

Imipramine and tetrabenazine: effects on monoamine receptor binding sites and phosphoinositide hydrolysis

Treatment of rats for 21 days with tetrabenazine, a drug which depletes monoamines and is used behaviorally to screen for antidepressants, significantly decreased 5-HT2 receptor density, increased alpha 1-adrenoceptor density but did not alter beta-adrenoceptor density in homogenates of frontal cortices labeled with [3H]ketanserin, [3H]prazosin and [3H]dihydroalprenolol, respectively. These effects were not opposite to those of the antidepressant drug imipramine which decreased both 5-HT2 and beta-adrenoceptor density and did not alter alpha 1-adrenoceptor density. Some evidence for antagonistic interactions between the two drugs was found in that imipramine partially prevented the tetrabenazine-induced increase in alpha 1-adrenoceptor density and tetrabenazine partially prevented the imipramine-induced decrease in beta-adrenoceptor density. Neither drug altered phosphoinositide hydrolysis coupled to alpha 1-adrenoceptors. While the effects of tetrabenazine are frequently attributed to its reserpine-like action of depleting monoamines, these results provide the first indication that tetrabenazine alters 5-HT2 and beta-adrenoceptor density in a manner different from that of reserpine.     

Nicotine self-administration in rats: estrous cycle effects, sex differences and nicotinic receptor binding

RATIONALE: Research on smoking behavior and responsiveness to nicotine suggests that nicotine's effects may depend on the sex of the organism. OBJECTIVE: The present study addressed four questions: 1) Will female rats self-administer nicotine? 2) Does self-administration by females vary as a function of estrous cycle? 3) Does self-administration by females differ from that of males? 4) Does self-administration of nicotine result in up-regulation of nicotinic receptor binding and are these changes similar in males and females? METHODS: Male and female Sprague-Dawley rats were allowed to self-administer nicotine at one of four doses (0.02-0.09 mg/kg, free base) on both fixed and progressive ratio schedules of reinforcement. RESULTS: Females acquired nicotine self-administration across the entire range of doses. Acquisition of self-administration at the lowest dose was faster in females than males. However, few sex differences were found in the number of active responses, number of infusions, or total intake of nicotine during stable fixed ratio self-administration. In contrast, females reached higher break points on a progressive ratio. For both schedules, females had shorter latencies to earn their first infusion of each session and demonstrated higher rates of both inactive and timeout responding. There was no effect of estrous cycle on self-administration during either fixed or progressive ratio sessions. Self-administered nicotine resulted in average arterial plasma nicotine levels between 53 and 193 ng/ml and left hemi-brain levels between 174 and 655 ng/g, depending on dose. Nicotine self-administration produced similar up-regulation of nicotinic receptor binding sites in males and females, as reflected by increased right hemi-brain binding of [3H]-epibatidine, when compared to the brains of untreated control rats. CONCLUSIONS: These results suggest that while males and females may regulate their intake of nicotine similarly under limited access conditions, the motivation to obtain nicotine is higher in females.     

NK2 receptors mediate tachykinin-induced contractions of rat uterus during the oestrous cycle.

We examined tachykinin-induced contractions of uteri from rats during the oestrous cycle. The potencies of substance P, neurokinin A, neurokinin B and the tachykinin NK2 receptor-selective agonist, [Lys5, MeLeu9, Nle10] neurokinin A-(4-10), and of the non-peptide tachykinin NK1, NK2 and NK3 receptor antagonists (S)1-[2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)pip eridin-3-yl]ethyl]-4phenyl-1-azonia-bicyclo[2.2.2]octane (SR 140333), (S)-N-methyl-N [4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzam ide (SR 48968) and (S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl)prop yl)-4-phenylpiperidin-4-yl)-N-methylacetamide (SR 142801), were examined. The relative agonist potencies, i.e., [Lys5, MeLeu9, Nle10] neurokinin A-(4-10) > or = neurokinin A > neurokinin B > or = substance P were similar in preparations from rats in dioestrus/metoestrus and those in proestrus/oestrus. Apparent pK(B) values for SR 48968 versus neurokinin A and [Lys5, MeLeu9, Nle10] neurokinin A-(4-10), were 9.9 and 9.2, respectively, indicating activation of an NK2 receptor. SR 140333 (10 nM) produced only a small rightward shift of the log concentration-response curve to substance P. SR 48968 (3 nM), but not SR 142801 (100-300 nM) reduced the effect of neurokinin B. These data indicate that in the rat tachykinin-induced contractions of the uteri during the oestrous cycle are mediated primarily by tachykinin NK2 receptors, and that fluctuations in ovarian hormonal levels during the oestrous cycle have little influence on the uterine response to tachykinins.     

Influence of stress and antidepressant treatment on 5-HT-stimulated phosphoinositide hydrolysis in rat brain.

The aim was to elucidate the role of 5-hydroxytryptamine (5-HT)-stimulated phosphoinositide (PI) metabolism in stress situations and in the behavioral improvement produced by chronic antidepressant treatment. Rat cerebral cortex slices were used for the purpose. Forced swimming for 15 min and longer induced changes in behavioral activities of rats associated with a significant reduction of 5-HT-stimulated PI metabolism, without any changes in density and affinity of 5-HT2 receptors. This suggests that modulation of the receptor coupling process but not of the 5-HT2 receptor binding characteristics may be responsible for the significant reduction of 5-HT-stimulated PI metabolism in stress situations. Chronic antidepressant treatment tended to reduce 5-HT-stimulated PI metabolism. This treatment improved significantly the behavioural activities during forces swimming, and prevented the forced swimming-induced reduction of 5-HT-stimulated PI metabolism. It is postulated that chronic antidepressant treatment may improve behavioral activities in relation to PI metabolism in stress situations.     

Effects of vasopressin on phosphoinositide hydrolysis and myocardial contractility.

The effects of vasopressin on phosphoinositide hydrolysis, ventricular contractility and adrenoceptor-mediated inotropy were studied in the rabbit. Vasopressin caused an accumulation of [3H]inositol monophosphate in ventricular slices prelabelled with myo-[3H]inositol, whereas it elicited a small but definite negative inotropic effect. In addition, vasopressin attenuated the positive inotropic effect elicited via alpha 1- and endothelin receptors without affecting the beta-adrenoceptor-mediated effect. The nonpeptide-selective V1 receptor antagonist OPC 21268 (a quinolinone derivative) inhibited the vasopressin-induced accumulation of [3H]inositol monophosphate. The present results indicate that vasopressin stimulates phosphoinositide hydrolysis via V1 receptors, but inhibits force and the alpha 1-mediated positive inotropic effect in the rabbit ventricular myocardium.     

Monoamine neurotransmitters and metabolites during the estrous cycle, pregnancy, and the postpartum period

A wide variety of behavioral changes in the female rat have been associated with the estrous cycle, pregnancy, and the postpartum period and their accompanying hormonal fluctuations. Since monoamine systems have been implicated in the control of these behaviors, the present study examined the tissue concentrations of norepinephrine (NE), dopamine (DA), and serotonin (5HT) and their primary metabolites dihydroxyphenylglycol (DHPG), 3-methoxy-4-hydroxyphenylglycol (MHPG), 3,4-dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5HIAA) in the anterior cerebral cortex, hippocampus, and cerebellum during the estrous cycle, late pregnancy, and the early postpartum period. Results show no significant differences in neurotransmitter or metabolite levels during the estrous cycle in any of the three brain regions examined. However, profound differences were observed between samples from late pregnancy, early postpartum, and the estrous cycle. In general, NE and 5HT levels in all three brain regions fell from normal values during late pregnancy and rose in the early postpartum period; levels of their metabolites rose in postpartum samples. Conversely, DA levels were elevated in late pregnancy and depressed in the early postpartum period in anterior cortex, while DOPAC levels were depressed in both late pregnancy and the early postpartum period. The finding of changes in monoamine metabolism associated with pregnancy and its termination could be important in understanding the increased susceptibility to affective illness in women during the postpartum period.     

Quantitative autoradiographic studies of relaxin binding in rat atria, uterus and cerebral cortex: characterization and effects of oestrogen treatment.

The binding characteristics of the relaxin receptor in rat atria, uterus and cortex were studied using a [33P]-labelled human gene 2 relaxin (B33) and quantitative receptor autoradiography. The binding kinetics of [33P]-human gene 2 relaxin (B33) were investigated in slide-mounted rat atrial sections. The binding achieved equilibrium after 60 min incubation at room temperature (23+/-1 degrees C) and dissociated slowly. The association and dissociation rate constants were 4.31+/-0.34x10(8) M(-1) x min(-1) and 1.55+/-0.38x10(-3) min(-1) respectively. Thus, the kinetic dissociation constant was 3.46+/-0.59 pM. Binding was saturable to a single population of non-interacting sites throughout atria, in uterine myometrium and the 5th layer of cerebral cortex. The binding affinities (pK(D)) of [33P]-human gene 2 relaxin (B33) were 8.92+/-0.09 in atrial myocardium and 8.79+/-0.04 in cerebral cortex of male rats, and 8.79+/-0.10 in uterine myometrium. Receptor densities in the cerebral cortex and atria were higher than in uterine myometrium, indicating that relaxin also has important roles in non-reproductive tissues. In male rats, treatment with 17beta-oestradiol (20 microg in 0.1 ml sesame oil s.c., 18-24 h) significantly decreased the density of relaxin receptors in atria and cerebral cortex. Identical treatment in female rats had no significant effect in atria and cerebral cortex, but it significantly increased the density of relaxin receptors in uterine myometrium. Relaxin binding was competitively displaced by porcine and rat native relaxins. Porcine native relaxin binds to the relaxin receptor in male rat atria (8.90+/-0.02), and cerebral cortex (8.90+/-0.03) and uterine myometrium (8.89+/-0.03) with affinities not significantly different from human gene 2 (B33) relaxin. Nevertheless, rat relaxin binds to the receptors with affinities (8.35+/-0.09 in atria, 8.22+/-0.07 in cerebral cortex and 8.48+/-0.06 in uterine myometrium) significantly less than human gene 2 (B33) and porcine relaxins. Quantitative receptor autoradiography is the method of choice for measurement of affinities and densities of relaxin receptor in atria, uterine myometrium and cerebral cortex. High densities were found in all these tissues. 17beta-oestradiol treatment produced complex effects where it increased the densities of relaxin receptors in uterus but decreased those in atria and cerebral cortex of the male rats, and had no effect on the atria and cerebral cortex of the female rats.     

Effects of prostaglandin inhibitors on angiotensin, oxytocin and prostaglandin F2α contractile effects on the rat uterus during the oestrous cycle

1 In the rat isolated uterus maximal spontaneous contractions and maximal sensitivity to angiotensin II, oxytocin and prostaglandin F(2alpha) were observed in di- and proestrus. Minimal sensitivity to the three agonists was observed in metoestrus. Maximal contractile effects of angiotensin II, oxytocin and prostaglandin F(2alpha) were thus observed when the ratio oestrogen/progesterone levels was high.2 The oestrogen-dependent sensitivity of the rat uterus is partially mediated by endogenous prostaglandins. Indomethacin suppressed the increased sensitivity to angiotensin and oxytocin present in dioestrus and proestrus but did not affect that to prostaglandin F(2alpha). Polyphloretin phosphate at a concentration of 10 mug/ml resulted in complete identity of dioestrus and metoestrus dose-response curves to angiotensin and oxytocin.3 Spontaneous uterine contractions observed when oestrogen levels are high are also dependent on intramural prostaglandins as they were inhibited by indomethacin and polyphloretin phosphate. In the metoestrus uterus, prostaglandin F(2alpha) induced the reappearance of spontaneous contractions.4 Prostaglandin F(2alpha) had a potentiating effect on angiotensin-elicited contractions which persisted after washing out prostaglandin F(2alpha).     

Relations between the effects of histamine,pheniramin and metiamide on spontaneous motility and the formation of cyclic AMP in the isolated rat uterus

Summary Histamine (5×10^–6 to 10^–3M) depressed the spontaneous motility of the isolated rat uterus in a dose-dependent manner. Under these conditions uterine cyclic AMP was raised up to 92%. Both effects, uterine relaxation and cyclic AMP accumulation after 2 min could be inhibited dose dependently by the H₂-antihistaminic compound metiamide (1.7×10^–6 M to 1.7×10^–4 M). By contrast, the H₁-antagonist pheniramin (4.4×10^–5 M) was ineffective. It was concluded that the histamine-induced inhibition of rat uterine motility is mediated by cyclic AMP which is formed in response to stimulation of H₂-histaminergic receptors.     

Species-related differences in inotropic effects of angiotensin II in mammalian ventricular muscle: receptors, subtypes and phosphoinositide hydrolysis.

1. Experiments were carried out to clarify the mechanisms responsible for variations in the positive inotropic effect (PIE) of angiotensin II (AII) on ventricular muscles from various mammals. We examined the density of AII receptors, the relative proportions of receptor subtypes and the acceleration of the hydrolysis of phosphoinositide that was induced by AII, as well as the PIE of AII in ventricular muscles from the rabbit, dog, rat and ferret. 2. In the rabbit, AII (1 microM) in the presence of bupranolol (0.3 microM) and prazosin (0.1 microM) elicited a concentration-dependent PIE, which was antagonized by a selective AT1 subtype antagonist, losartan, but not by an AT2 antagonist, PD123319. AII did not have any inotropic effects in ventricular muscles from the dog, rat and ferret. 3. Specific high-affinity binding of [125I]-AII, with a similar Kd value in each case (1-2 nM), was observed with membrane fractions derived from ventricular muscle of all four species tested. 4. In the rabbit, losartan and PD123319 each displaced approximately 50% of [125I]-AII specific binding having high affinity for the receptors, and indicating that AT1 and AT2 subtypes were present in equal numbers. In the other species the AT1 subtype of receptors was predominant. 5. In all four species AII caused a concentration-dependent acceleration of the hydrolysis of phosphoinositide in ventricular slices that had been prelabelled with myo-[3H]-inositol. 6. The results indicate that the signal-transduction process distal to acceleration of the hydrolysis of phosphoinositide may be responsible for the wide range of species variations in the inotropic action of AII on mammalian ventricular myocardium.     

Comparison of postnatal changes in alpha 1-adrenoceptor binding and adrenergic stimulation of phosphoinositide hydrolysis in rat cerebral cortex

Adrenergic stimulation of phosphoinositide hydrolysis is mediated by the alpha 1-adrenoceptor subtype in many tissues including the brain. We have investigated the coupling of alpha 1-adrenoceptors to phosphoinositide hydrolysis during ontogeny. Alpha 1-adrenoceptor number and affinity were measured using [3H]prazosin binding in crude membranes of cerebral cortex and compared to the ability of the adrenergic agonists norepinephrine (NE) and phenylephrine (PE) to stimulate the formation of [3H] inositol phosphates from [3H]myo-inositol in brain slices at various ages. The greatest changes in the developmental expression of both the Bmax for [3H]prazosin binding and maximal (10(-4)M) NE- or PE-stimulated [3H]inositol phosphates were observed during the period of 7-21 days of age. No changes in the KD for [3H]prazosin were observed. However, at 14 days of age the EC50 for NE but not PE stimulation of [3H]inositol phosphates was slightly but significantly lower than at later ages. To quantitatively compare these two parameters during ontogeny, data were expressed as a percentage of the adult (greater than 65 day) value. At early ages (7 and 14 days) but not at later ages (21 and 37 days) the percent expression of [3H]prazosin binding sites was significantly greater than the maximal NE-stimulated [3H]inositol phosphates. This suggests that early in neonatal development alpha 1 adrenoceptors in brain are not tightly coupled to phosphoinositide hydrolysis.     

Effect of a prostaglandin antagonist, N-0164, on cAMP generation and hydrolysis in the rat uterus

N-0164 (sodium-p-benzyl-4-[1-oxo-2-(4-chlorobenzyl)-3-phenyl propyl] phenylphosphonate) (20–100 μM), an antagonist of the contractile effect of prostaglandins, reversed the prostaglandin E₂ (PGE₂) inhibition of isoproterenol-induced cAMP accumulation in rat uterus. N-0164, at the same concentrations, was a potent cAMP-phosphodiesterase inhibitor in broken cell preparations and potentiated the cAMP response to isoproterenol in intact tissue. The potency of N-0164 to inhibit cAMP-phosphodiesterase and to reverse the effect of PGE₂ on the cAMP response to isoproterenol were comparable (ec₅₀:50 and 60 μM respectively). In the presence of 10 mM theophylline, N-0164 did not affect the inhibitory effect of PGE₂. Furthermore, N-0164 produced similar proportional increases in the cAMP response to isoproterenol in the presence and absence of PGE₂. These results suggest that the apparent reversal by N-0164 of the PGE₂ effect on the cAMP response to isoproterenol is not due to its prostaglandin antagonistic action but to inhibition of cAMP-phosphodiesterase. N-0164, at concentrations lower than those inhibiting cAMP-phosphodiesterase, selectively inhibited the PGE₂-induced contractions of the rat uterus (ec₅₀, 4 μM), while at higher concentrations it also diminished carbachol-induced contractions. These results indicate that in the rat uterus N-0164 has at least two effects, prostaglandin antagonism and cAMP-phosphodiesterase inhibition, and suggest that the contractile effect of PGE₂ is independent of the effect of PGE₂ on the isoproterenol-induced rise in cAMP.     

Comparative effects of sodium azide and aminophylline on the rat isolated uterus during muscle activation

Sodium azide is a strong inhibitor of the tonic component of contraction produced by oxytocin, whereas aminophylline produces almost equal inhibition of all types of activation of the isolated rat uterus. Both substances inhibited the spontaneous rhythmic activity of the uterus. The effect of sodium azide is easily reversed by calcium. The results are taken to indicate a complex relation between calcium and substances which stimulate metabolism either of cGMP (sodium azide) or cAMP (aminophylline) in producing relaxation of the isolated rat uterus.     

Effects of hypothyroidism on the estrous cycle and reproductive hormones in mature female rat

The present study was undertaken to examine the systemic role of thyroid hormones by analyzing changes in reproductive functions in hypothyroid female rats. Serum concentrations of triiodo-thyronine (T3) significantly decreased 1 week after the initiation of propyl-thiouracil treatment or thyroidectomy. The estrous cycle became irregular 3 and 2 weeks after the initiation of propyl-thiouracil treatment and thyroidectomy, respectively. Serum luteinizing hormone (LH) levels significantly reduced in both groups on the day of diestrus-1 about 1 month later. Hypothyroid rat shows the high progesterone and low testosterone levels. No significant changes in inhibin and estradiol levels were detected. The serum levels of FSH decreased in the thyroidectomy group. The irregular estrous cycle and the above changes in hormone levels were recovered by administration of T4. Compensatory secretions of FSH and LH induced by ovariectomy were enhanced by thyroidectomy and suppressed by T4 treatment. These results suggest that thyroid hormones play a role in the regulation of reproductive hormones secretion in the cyclic rat ovary.     

Angiotensin II AT1 receptors in rat superior cervical ganglia: characterization and stimulation of phosphoinositide hydrolysis.

Angiotensin II receptor number was higher in superior cervical ganglia of 2-week-old when compared to 8-week-old rats. In both young and adult rats, specific binding of [125I][Sar1]angiotensin II was displaced competitively by the AT1-receptor antagonist DuP 753 but not by the AT2-receptor competitor PD 123177. In ganglia from adult rats, DuP 753 competed with an IC50 of 113 nM. The stable guanine nucleotide GTP gamma S inhibited binding of [125I][Sar1]angiotensin II in young and adult rats by approximately 50% with IC50 values of 105 and 120 nM, respectively, suggesting that the angiotensin receptor is G-protein linked. Angiotensin II at a dose of 1 microM stimulated inositol phosphate formation 58% over control values in superior cervical ganglia from 8-week-old rats. This effect was totally blocked by 10 microM DuP 753 but not by 10 microM PD 123177. Our findings demonstrate that rat superior cervical ganglia contain AT1-type angiotensin receptors that are probably G-protein linked, and their stimulation results in increased inositol phospholipid metabolism.