Characterization and distribution of cholinesterase activity in mouse uterine horns: changes in estrous cycle

1. Both butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) are present in the mouse uterus, BChE being more abundant.2. Their molecular forms were sequentially solubilized by different extraction media obtaining three ChE fractions whose specific activity was different, depending on the stage of the estrous cycle: hydrosoluble (estrous: 75.5 ± 6.6 and diestrous: 47.9 ± 8.7 μU/mg prot); detergent-soluble or amphiphilic (estrous 26.6 ± 2.4 and diestrous 14.7 ± 3.3 mU/mg prot.), and high ionic strength-soluble (estrous: 18.7 ±4.2 and diestrous 12.8 ± 1.2 mU/mg prot.).3. Histochemical procedures demonstrated a different distribution for both ChE activities. AChE was found in nerves next to smooth muscle cells of the circular layer and blood vessels, while BChE was concentrated in the longitudinal stratum surrounding the smooth muscle cells. Under the predominance of progesterone, BChE was also found in the endometrial glands.4. Maximal contractions evoked by the addition of ACh to the isolated organ bath were concentration dependent and greater in estrous than in diestrous. Nevertheless the difference at the two stages of the estrous cycle disappeared when contractions were normalized to smooth muscle cross-sectional area.5. BChE but not AChE inhibition augmented maximal contractions elicited by ACh in longitudinal but not in circular smooth muscle.6. The effect of BChE inhibition on the contractile force developed was greater at lower concentrations of ACh and did not depend on the stage of the estrous cycle.     

Effect of consumption of sucrose and saccharin on passive avoidance learning in female Wistar rats

Influence of consumption of 32% sucrose or 0.1% saccharin solutions on passive avoidance acquisition in female Wistar rats was studied at different stages of estrous cycle. Under conditions of weak electrical shock, rats of the control and saccharin groups showed no acquisition at any stage of estrous cycle. Under the same conditions females of the sucrose group acquired the avoidance behavior if trained on the first day of diestrous and metestrous but not proestrous and estrous. The stage of estrous cycle on the second day did not influence the latency during testing session.     

Ionic mechanisms involved in the release of 3H-norepinephrine from the cat superior cervical ganglion

It has previously been reported that in the isolated cat superior cervical ganglion (SCG) labeled with tritiated norepinephrine (3H-NE), the stimulation of the preganglionic trunk at 10 Hz as well as the exposure to 100 microM exogenous acetylcholine (ACh), produced a Ca++-dependent release of 3H-NE. The present results show that a Ca++-dependent release of 3H-NE was produced also by exposure to either 50 microM veratridine or 60 mM KCl. Tetrodotoxin (0.5 microM) abolished the release of 3H-NE induced by preganglionic stimulation, ACh and veratridine but did not modify the release evoked by KCl. The metabolic distribution of the radioactivity released by the different depolarizing stimuli showed that the 3H-NE was collected mainly unmetabolized. In the cat SCG neither the release of 3H-NE evoked by KCl nor the endogenous content of NE was modified by pretreatment with 6-OH-dopamine (6-OH-DA). On the other hand, this chemical sympathectomy depleted the endogenous content of NE in the cat nictitating membrane, whose nerve terminals arise from the SCG. The data presented suggest that the depolarization-coupled release of NE from the cat SCG involves structures that are different to nerve terminals and that contain Na+ channels as well as Ca++ channels.     

In vitro effect of neuropeptide Y on melatonin and norepinephrine release in rat pineal gland

1. To study neuropeptide Y (NPY) effect on melatonin production, rat pineal explants were incubated for 6 hr with 10-1,000 nM NPY in the presence or absence of 10 microM norepinephrine (NE). Melatonin content in the pineal gland and media was measured by radioimmunoassay (RIA). 2. NPY (10-1,000 nM) increased melatonin production and, at 10 or 100 nM concentrations (but not 1,000 nM), enhanced NE stimulation of melatonin production. 3. NPY (1,000 nM) impaired 3H-labeled transmitter release induced by a K+ depolarizing stimulus in rat pineals incubated with 3H-NE. 4. These results suggest that NPY affects both pre- and postsynaptic pineal mechanisms.     

The critical period in which splenectomy causes functional disorder of the ovary in adult rats

On account of recent reports suggesting that ovulation and luteinization involve immunological reactions, we examined the effect of splenectomy in rats on the recurrence of an estrous cycle. The spleen was removed from adult female rats at various times during an estrous cycle. A delay in ovulation was specifically induced in the rats splenectomized on the metestrous morning between 0700 and 0900 h. This delay in ovulation was reversed by an injection of splenocytes obtained either from estrous or metestrous donors, but not from diestrous or proestrous donors. The isolated splenic macrophage preparation mimicked the effect of splenocytes. Measurement of progestin levels throughout the estrous cycle suggested that the delay in ovulation was caused primarily by a delay in luteolysis; progesterone levels in ovulation delayed rats were higher and 20 alpha-dihydroprogesterone levels were lower than those of intact rats on the diestrous day. These results suggest that the macrophages in the spleen under the influence of endocrine milieu probably play a role in the recurrence of an estrous cycle by controlling luteolysis. The specific time of splenectomy to cause delay in ovulation will afford a new approach in analyzing the function of immunocytes in the ovary.     

Orienting behavior of female white rats in estrus cycle and its dependence on handling

Characteristics of orienting behavior of intact and handled female white rats were studied in the "open field" test. Experimental group of animals were formed according to stages of estrous cycle: (1) diestrus, (2) proestrus, (3) estrus, and (4) metestrus. The stage of the cycle was determined by vaginal smears. Over the period of 10 days rats were handled daily for 5 minutes. No behavioral changes over the course of estrous cycle were found in intact rats. Handling revealed some behavioral features related to the levels of steroid hormones in reproductive cycle. Most prominent changes in orienting behavior were observed at transition from estrous to metestrous. At the stage of estrous motor activity was maximal and grooming was minimal. The maximal contrast in the structure of orienting behavior was observed between estrous and diestrous stages.     

Superfused pituitary cell cultures: comparative responsiveness of cells derived from various stages of the estrous cycle to LHRH stimulation administered as short duration pulses

We have reinvestigated the question of maintenance of differential LHRH sensitivity in culture and further investigated the role of pulsatile LHRH in the in vitro release of pulsatile LH and FSH at different stages of the estrous cycle. Pituitaries were collected on each day of the 4 day cycle at 0800. In addition, pituitaries were also collected at 1500 and 1900 on proestrous. The cells were dispersed and exposed 48 hrs later to short duration 4 ng LHRH pulses; this dose was optimized for LH release and was applied at a frequency of 1 pulse/60 min. In terms of absolute magnitude of LH response, observed responsiveness was ranked in the following order: proestrous 1900 greater than estrous 0800 greater than diestrous 1 0800 greater than proestrous 1500 greater than diestrous 2 0800. Responsiveness was significantly greater at proestrous 1900 (p greater than 0.01), estrous 0800 (p greater than 0.05) and diestrous 1 0800 (p greater than 0.05) when compared to either of the other stages tested. The heightened LHRH sensitivity of proestrous was therefore maintained in cell culture indicating that the system should be valid for conducting studies on the control of gonadotropin secretion during this period. FSH did not respond in pulsatile manner to the LHRH levels employed further substantiating recent evidence that LHRH seems to function somehow less directly in FSH as compared to LH secretion.     

Fluctuations of hippocampal neuronal protein levels over the estrous cycle in the rat

Hippocampal function is known to be estrous-cycle-dependent but information on estrous-cycle-dependent protein expression is limited. It was therefore the aim to study protein levels of the neuronal network over the estrous cycle in the hippocampus of female rats and in males showing protein chemical neuroanatomy in this area. Female and male OFA Sprague-Dawley rats were used and females were grouped to proestrous, estrous, metestrous and diestrous by using vaginal smears. Hippocampal tissue was taken, proteins extracted, run on two-dimensional gel electrophoresis and proteins were identified by mass spectrometry methods (MALDI-TOF-TOF and nano-LC-ESI-MS/MS). Spot volumes were quantified with specific software. A Synapsin-1 expression form was differentially regulated between proestrous and diestrous, a Synapsin IIa expression form was differentially regulated between proestrous and metestrous, the sum of ERC-2 proteins organizing the cytomatrix at the nerve terminals active zone was showing sex-dependent levels in the proestrous phase and Neurofilament triplet L protein was differentially expressed between the estrous phase and males. The findings may represent estrous-cycle-dependent hippocampal synaptic function that has been shown already in terms of electrophysiology and neuroanatomy. Neurofilament changes over the estrous cycle may reflect endoskeleton changes over the estrous cycle. We learn from this study, although increasing complexity of protein knowledge, that the estrous cycle and not only the sex per se has to be taken into account for design of future studies and interpretation of previous work at the protein level.     

Splenic macrophages enhance prolactin and luteinizing hormone action in rat luteal cell cultures.

We have reported that splenic macrophages play a role in the regulation of progestin secretion in rats. In this study, splenic macrophages were obtained from cycling rats at different estrous cycle stages and co-cultured with luteal cells from mid-pseudopregnant rats in the absence/presence of prolactin (PRL) or luteinizing hormone (LH). The effect of macrophages on the luteotropic action of PRL and LH was evaluated with 2 parameters, i.e. an increase in total progestin output (progesterone plus 20 alpha-hydroxyprgn-4-en-one [20 alpha-OHP]), and an increase in the progesterone to 20 alpha-OHP (P/20 alpha-OHP) secretion ratio. Splenic macrophages obtained from proestrous or metestrous rats enhanced the PRL action to increase the P/20 alpha-OHP secretion ratio, but those from estrous or diestrous donors did not. Only macrophages from proestrous donors enhanced the PRL action to increase the total progestin output. In contrast, the LH action increasing the P/20 alpha-OHP secretion ratio was enhanced by splenic macrophages regardless of the donors' estrous cycle stages. The LH action increasing the total progestin output was enhanced only by proestrous or metestrous macrophages. Therefore, if luteal cells are co-cultured with proestrous macrophages, the luteotropic actions of PRL and LH can be fully expressed. These results indicate that splenic macrophages directly act on luteal cells and enhance the luteotropic action of PRL and LH, and that this function of splenic macrophages is modified somehow according to the donors' estrous cycle stages.     

Angiotensin-(1-7) inhibits the angiotensin II-enhanced norepinephrine release in coarcted hypertensive rats

Since it has been suggested that angiotensin (Ang) (1-7) functions as an antihypertensive peptide, we studied its effect on the Ang II-enhanced norepinephrine (NE) release evoked by K+ in hypothalami isolated from aortic coarcted hypertensive (CH) rats. The endogenous NE stores were labeled by incubation of the tissues with 3H-NE during 30 min, and after 90 min of washing, they were incubated in Krebs solution containing 25 mM KCl in the absence or presence of the peptides. Ang-(1-7) not only diminished the K+-evoked NE release from hypothalami of CH rats, but also blocked the Ang II-enhanced NE release induced by K+. Ang-(1-7) blocking action on the Ang II response was prevented by [D-Ala7]Ang-(1-7), an Ang-(1-7) specific antagonist, by PD 123319, an AT2-receptor antagonist, and by Hoe 140, a B2 receptor antagonist. Ang-(1-7) inhibitory effect on the Ang II facilitatory effect on K+-stimulated NE release disappeared in the presence of Nomega-nitro-L-arginine methylester and was restored by L-arginine. Our present results suggest that Ang-(1-7) may contribute to blood pressure regulation by blocking Ang II actions on NE release at the central level. This inhibitory effect is a nitric oxide-mediated mechanism involving AT2 receptors and/or Ang-(1-7) specific receptors and local bradykinin generation.     

Physiological evidence for the possible existence of anhydrolevuglandin E2-like activity in extracts and media from uteri of rats.

Physiological evidence is presented for the possible existence of anhydrolevuglandin E2-like activity in extracts of uteri from diestrous rats and after treatment of adult rats with estradiol and progesterone. The extracts were able to inhibit contractions in rat uterine preparations stimulated by PGE2. Uteri of vaginal Stage 3 (metestrus) were quiescent and showed decreased responsiveness to PGE2 and PGF2 alpha. These uteri showed some contractility when incubation medium from diestrous uteri (Stage 5) were transferred to them and incubation medium from them inhibited the contractility of Stage 5 uteri. When incubation media were exchanged between contractile uteri from of stages other than Stage 3, there was no change in the contraction patterns. Taken together, we believe these data indicate that AnLGE2 may be a normal constituent of the rat uterus and is physiologically increased during Stage 3 (metestrus) of the estrous cycle.     

Changes in the response to adrenergic drugs on mouse uterine contractions during pregnancy

The effect of isoproterenol (ISO), norepinephrine (NE) and phenylephrine (PHE) on electrically-induced contractions of mice uterine horns was studied during pregnancy. At the different times of gestation adrenergic agonists always inhibited uterine contractions in the following rank order of potency: ISO greater than NE greater than PHE. Cumulative dose-response curves constructed for the effect of these amines during diestrous, and at days 3-7, 10-15, 17-21 of gestation, showed that EC50 values increased gradually as term approached, which could imply a lower capacity of the uterus to respond to adrenergic drugs. Some likely explanations for this phenomenon are proposed. It is suggested that this lower response to catecholamines at the end of pregnancy could be a cause for the reduced success of beta 2-adrenergic drugs to stop premature labor.     

Morphine decreases 3H-norepinephrine release and increases endogenous norepinephrine levels in the isolated cat superior cervical ganglion

The isolated cat superior cervical ganglion (SCG) was labeled in vitro with either 3H-norepinephrine (3H-NE) or 3H-choline and stimulated through its preganglionic trunk. The release of 3H-NE and 3H-acetylcholine (3H-ACh) elicited by the stimulation was measured under control conditions and in the presence of drugs. The incubation during 30 min with 10 microM morphine lead to a 70% decrease in the amount of 3H-NE released in response to the preganglionic stimulation (10 Hz, 80 V, during 5 min). No further decrease in 3H-NE release was produced by a 10 times higher concentration of morphine. The reduction in 3H-NE release caused by morphine was coincident with a 60% increase in the endogenous content of NE. Both effects of morphine were entirely prevented by an antagonist of opioid receptors, 1.0 microM naltrexone. The opioid antagonist did not modify by itself either the stimulation-induced release of 3H-NE or the endogenous content of NE. The basal efflux of 3H-NE was not altered by morphine. In ganglia labeled with 3H-choline, morphine (10 and 100 microM) did not modify either the basal efflux of 3H-ACh or the release of 3H-ACh evoked by stimulation of the preganglionic trunk (5 Hz, 40 V, during 5 min). These observations suggest that in the cat SCG morphine has a direct action on the dendrites of the postganglionic neuron which store and release NE. The effects of morphine in vitro on 3H-NE release and on the tissue levels of NE may be mediated through the interaction with dendritic opioid receptors.     

The effect of cold on adrenergic neurotransmission in canine saphenous arteries and veins

The effect of severe cold (5 to 10 degrees C) on adrenergic neurotransmission was compared in the isolated cutaneous (saphenous) artery and vein of the dog. The vein contracted to sympathetic nerve stimulation at temperatures as low as 10 degrees C; higher temperatures were needed for the artery to contract. Both blood vessels contracted to exogenous norepinephrine at temperatures as low as 5 degrees C. However, the contractile response to exogenous norepinephrine was less in the saphenous artery, and contractions to high K+ solution were depressed by cooling more in the artery than in the vein. During electrical stimulation of the sympathetic nerves in saphenous arteries and veins previously incubated with labeled norepinephrine, progressive cooling from 37 to 5 degrees C caused a sharp decline in overflow of [3H]norepinephrine and its metabolites. However, overflow of labeled norepinephrine in both blood vessels continued at very cold temperatures. Thus the inability of the saphenous artery to contract to sympathetic nerve stimulation at 10 degrees C can be explained by a greater sensitivity of the arterial smooth muscle to the direct depressant effect of cold, rather than to a differential release or metabolism or norepinephrine in the arterial wall or a loss of responsiveness to norepinephrine at very cold temperatures.     

Effects of epinephrine and oxytocin on the release of prostaglandin F from the rat uterus in vitro

Isolated uteri from rats with regular 4-day cycles were incubated in Krebs-Ringer bicarbonate buffer and the release of PGF into the medium was measured by radioimmunoassay after extraction of the incubation medium with ethyl acetate at pH 3.0-3.5. PGF was produced from endogenous precursors and accumulated in equal amounts in the medium during two successive 60 min periods on each day of cycle, but the magnitude of the production varied significantly during the cycle, being greatest in estrus. Oxytocin in doses up to 500 mU/ml had no effect on PGF accumulation in the incubation period at any stage of the cycle, while epinephrine (10(-3)) greatly stimulated PGF release from the estrous uterus but had no effect on PGF release from the diestrous uterus. Phentolamine, an alpha-blocking agent, had no effect on the epinephrine-induced release of PGF, while propranolol, a beta-blocking agent, not only prevented in increase in PGF production induced by epinephrine but also reduced the basal release of PGF by the estrous uterus. Since oxytocin contracts and epinephrine relaxes the nonpregnant rat uterus both in vivo and in vitro, it is unlikely that the effects of these two compounds on uterine contractility are mediated by the release of PGF2alpha.     

Histamine content and mast cells distribution in mouse uterus: the effect of sexual hormones, gestation and labor

The histamine content of uteri from mice was analyzed in terms of both concentration and total amount per uterine horn a) at two stages of the estrous cycle (estrous and diestrous), b) under sex hormone treatment, c) during pregnancy and after delivery. Histamine concentration and mast cell density were greater during diestrous and in mice treated with progesterone (p less than 0.001). This effect was attributed to a reduction in uterine mass weight, since the amount of histamine per uterine horn remained constant throughout the estrous cycle. During pregnancy, both concentration and amount of histamine per uterine horn were increased, values were significantly higher than in estrous (p less than 0.001) from day 14-17 until day 21 when labor occurred. After six to eight hours post-partum an abrupt reduction on histamine content was observed. Mast cells were more abundant in myometrium than in endometrium, their density followed the same pattern as histamine concentration throughout the estrous cycle.     

Further investigation of a potential calcium channel modulator isolated from rat erythrocytes

The contractile effects of a peptide isolated from rat erythrocytes were further studied in rat aortic rings. Previous data showed that preincubation of aortic tissue with the peptide had no effect on resting tension, but significantly enhanced K+ and norepinephrine (NE) induced contraction. The calcium channel antagonist verapamil noncompetitively blocked the effect of the peptide, whereas nifedipine blockage appeared to be competitive. In the present study the peptide enhanced K+, NE, and phenylephrine (PE) induced contraction in a concentration-dependent manner, with a maximum enhancement at peptide concentrations of 10(-7)-10(-6) M. At a concentration as low as 10(-9) M, the peptide significantly enhanced K(+)-induced, but not NE- or PE-induced, contraction. The magnitude of maximal enhancement was greater for K(+)-induced contraction than that for NE- or PE-induced contraction. Preincubation of the tissues with the peptide caused a leftward shift of cumulative concentration-response curves to K+ and NE. The peptide enhancement of contraction increased with increasing K+ and NE concentration. The peptide potentiated the contractile response to Ca2+ in K(+)-depolarizing medium. It also enhanced the contractile response to NE in intracellular Ca2(+)-pool-depleted tissue following the replenishment of extracellular Ca2+, but had no apparent effect on the mobilization of intracellular calcium. Addition of nifedipine caused a rightward shift of both the peptide and Bay K 8644 concentration-response curves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of micromolar concentrations of Mn, Mo, and Si on α1adrenoceptor-mediated contraction in porcine coronary artery

We studied the effects of trace elements, Mn, Mo, and Si, on vasoconstriction induced by norepinephrine (NE) or electrical field stimulation in isolated porcine right coronary arteries. α₁-Adrenoceptor (AR) antagonist prazosin dose-despondently suppressed vasoconstriction in response to NE or field stimulation indicating an α₁-AR mediated response. Mn, Mo, and Si at 0.3-3 μmol/L dosedespondently inhibited NE mediated contraction (allp < 0.05). In contrast, Mn, Mo, and Si at the same concentrations (0.3-3 μmol/L) enhanced the maximal contractile response to field stimulation in a dose-dependent manner (allp < 0.05), but these elements at 10 μmol/L suppressed the vasoconstrictive response. The results indicate that in porcine right coronary arteries, the α₁-AR-mediated vasoconstriction by NE or electrical field stimulation was affected differently by micromolar concentrations of Mn, Mo, and Si and that the elements might facilitate NE release presynaptically but inhibit the contractile response postsynaptically.