Further studies on possible dynorphin involvement in the ovulatory luteinizing hormone surge in the proestrous rat

A decrease in inhibitory tone of endogenous opioid peptide on the afternoon of proestrus is one event underlying generation of the ovulatory luteinizing hormone (LH) surge, since premature removal of this inhibitory tone (i.e., disinhibition) results in an early onset of the surge. Our laboratory demonstrated that blockade of κ-opioid receptors in the medial preoptic area (MPOA) advanced the onset of the LH surge on proestrus. Since dynorphin is the endogenous ligand for the κ-opioid receptor, the present studies examined the possible role of dynorphin in this disinhibition response. 1) Neutralization of endogenous dynorphin peptides, by push-pull perfusion of the MPOA with antibodies specific for dynorphin A_1–17 or A_1–8 from 1030–1355 h on proestrus, tended to prematurely advance the increase in plasma LH levels normally occurring on this day of the estrous cycle. Although this increase was not statistically significant when compared with controls, plasma LH levels in two antibody-treated rats were sufficiently elevated to cause full ovulation, a response that did not occur in controls. These data suggest that dynorphin A_1–17 and A_1–8 might have a role in the MPOA, although a minor one, in suppressing LH secretion early on proestrus. MPOA levels of prodynorphin mRNA decreased at 1700–1800 h on proestrus when plasma LH levels were high, compared with values at 1300–1400 h when plasma LH levels were low. This change did not occur on diestrous d 1 when there was no LH surge. 2) MPOA levels of κ-opioid receptor mRNA did not change on proestrus or diestrous d 1. These results suggest that a reduction in prodynorphin gene expression on the afternoon of proestrus may be one event involved in a possible decrease in dynorphin inhibitory tone on the ovulatory LH surge-generating signal.     

Species-specific processing of prodynorphin in the posterior pituitary of mammals

The steady state levels of the prodynorphin-derived opioid peptides, dynorphin A, dynorphin B, and alpha-neo-endorphin, have been extensively studied in the magnocellular/posterior pituitary system of the rat. To determine whether the rat system serves as a general model for prodynorphin processing in the mammalian posterior pituitary, we examined the steady state levels of prodynorphin-derived opioid peptides in the posterior pituitary systems of representatives of three diverse orders of mammals: guinea pig (order Rodentia), pig (order Artiodactyla), and rhesus monkey (order Primates). In each species studied there was evidence for species-specific deviations from the rat model. The most pronounced differences were observed with respect to the conversion of dynorphin A-(1-17) to dynorphin A-(1-8). In rodents, rats, and guinea pigs, under steady state conditions, the molar ratios of these forms are approximately 1:2 and 2.5:1, respectively. However, in the pig and rhesus monkey, the molar ratios of these forms are 10:1 and 14:1, respectively. Thus, under steady state conditions, the conversion of dynorphin A-(1-17) to dynorphin A-(1-8) appears to be a minor event in porcine and rhesus monkey posterior pituitary. Species-specific variations were also observed with respect to the steady state levels of alpha-neo-endorphin and dynorphin B-(1-13). In addition, the results of these studies suggest that the conversion of prodynorphin-derived opioids to leu-enkephalin probably represents a minor event in the species studied.     

Expression of the prodynorphin gene in male and female mammalian reproductive tissues

Recent studies suggest that opioid peptides may be involved in modulating the hypothalamus-pituitary-gonadal axis at a variety of levels in both males and females. We report here the presence of mRNA coding for the opioid peptide precursor prodynorphin in rat ovary, uterus, and testis. Expression of this opioid peptide precursor gene is compared to expression of two other opioid peptide precursor genes, proenkephalin and proopiomelanocortin, in mammalian reproductive tissues. Immunohistochemical analysis reveals that in the rat testis, prodynorphin-derived peptides are present in Leydig cells. The distribution of dynorphin immunoreactivity in various reproductive tissues was determined. Male reproductive tissues of the rat, rabbit, and guinea pig as well as rat ovary and uterus all contain detectable levels of dynorphin immunoreactivity. These observations suggest that prodynorphin-derived peptides may exert paracrine and/or autocrine effects in mammalian reproductive tissues.     

Effect of dynorphin A(1–13) on cardiomyocytes in culture: modulation of the response to increased extracellular calcium,but no effect on intrinsic cardiac contractile frequency or the response to isoproterenol or increased extracellular potassium

Summary The purpose of this study was to determine whether the endogenous opioid peptide dynorphin A(1–13) has a direct effect on the heart or acts to modulate the cardiac chronotropic response to calcium, potassium, or -adrenergic receptor stimulation. Spontaneously contracting myocardial cell aggregates were prepared from 7-day-old chick embryos and were maintained in culture for 72 h before study. Dynorphin A(1–13), 10^–8 to 10^–6 M, did not alter spontaneous contractile frequency. Increases in [Ca²⁺]₀ spontaneously suppressed cardiac contractile frequency, and dynorphin A(1–13) significantly (p<0.05) enhanced this response. Nifedipine, 10^–8 M, antagonized the effect of increased [Ca²⁺]₀ on cardiac contractile frequency, but did not block the action of dynorphin A(1–13) to accentuate the effect of increasing [Ca²⁺]₀. Dynorphin A(1–13) did not alter the significant (p<0.05) increase in contractile frequency produced by beta-adrenergic receptor stimulation by isoproterenol, or the suppression in contractile frequency produced by increases in extracellular potassium ([K⁺]₀). These data indicate that dynorphin A(1–13) does not act directly on the cardiac myocyte to alter cardiac contractile frequency or alter the response to increases in [K⁺]₀ or to isoproterenol, but that dynorphin A(1–13) does modulate the response to increases in extracellular calcium.Funded in part by a grant from the Medical Research Council of Canada     

Role of endogenous opioid peptides in mediating progesterone-induced disruption of the activation and transmission stages of the GnRH surge induction process.

How progesterone blocks the E2-induced GnRH surge in females is not known. In this study we assessed whether the endogenous opioid peptides (EOPs) that mediate progesterone negative feedback on pulsatile GnRH secretion also mediate the blockade of the GnRH surge. We treated ovariectomized ewes with physiological levels of E2 and progesterone to stimulate and block the GnRH surge, respectively, using LH secretion as an index of GnRH release. A pilot study confirmed that blocking opioidergic neurotransmission with the opioid receptor antagonist, naloxone (NAL; 1 mg/kg.h, i.v.), could prevent the suppression of pulsatile LH secretion by progesterone in our model. By contrast, antagonizing EOP receptors with NAL did not restore LH surges in ewes in which the E2-induced GnRH surge was blocked by progesterone treatment during the E2-dependent activation stage (Exp 1) of the GnRH surge induction process. However, in ewes treated with progesterone during the E2-independent transmission stage (Exp 2), NAL partially restored blocked LH surges, as indicated by increased fluctuations in LH that, in some cases, resembled LH surges. We conclude, therefore, that the EOPs that mediate progesterone negative feedback on pulsatile GnRH secretion are not involved in blockade of activation of the E2-induced GnRH surge by progesterone, but do appear to be part of the mechanism by which progesterone disrupts the transmission stage.     

Opposing effects on infarction of delta and kappa opioid receptor activation in the isolated rat heart: implications for ischemic preconditioning

δ-Opioid receptors are known to participate in the protection found following ischemic preconditioning (IPC), but the role of κ-receptors in IPC is currently controversial. Langendorff-perfused rat hearts received 35 min regional ischemia and 2 h reperfusion. PC (2 cycles 5 min global ischemia) substantially reduced infarct size. Pharmacological PC with the δ-agonist DADLE (10 nmol/L) had similar protective effects. However, higher dose DADLE (1 μmol/L) had a less beneficial effect, and in conjunction with the δ-antagonist naltrindole unexpectedly increased infarct size (61.5 ± 2.0%, p < 0.05 v 45.9 ± 2.3% in controls) sugggesting a non-δ effect. The universal κ-opioid agonist bremazocine (30 nmol/L) increased infarct size (61.3 ± 1.6%, p < 0.05 v controls), an effect abrogated by the selective κ₁-antagonist nor-binaltorphimine (BNI). Since opiates are known to have anti-adrenergic effects, which hypothetically may help to mediate IPC, cyclic AMP levels were measured in DADLE and in bremazocine-treated hearts. Decreased levels of cyclic AMP at the start of the regional ischemic period were found in low dose DADLE hearts (0.485 ± 0/020, n = 8, vs controls, 0.654 ± 0.025 nmol/g wet weight, p < 0.001), but not in high dose DADLE nor in bremazocine treated hearts. Thus, in the isolated rat heart κ₁-opioid receptor activation exacerbates infarct size through an as yet unknown mechanism, suggesting that there could be an “anti-preconditioned state”. In contrast, δ-activity mediates protection which may be associated with a reduction of tissue cyclic AMP levels. Received: 16 November 1999, Accepted: 7 December 1999     

Expression and Regulation of mRNA for Inhibin/Activin α- and βA-Subunits in the Granulosa Layer of the Two Largest Preovulatory Follicles during the Hen Ovulatory Cycle

In order to understand the role of inhibin and activin in regulating follicular development in the hen, the steady-state mRNA levels of inhibin/activin α- and β_A-subunits in the granulosa layer of the largest (F₁) and second largest (F₂) follicles of the hen were investigated at 4-hr intervals throughout the ovulatory cycle. In addition, because it was hypothesized that luteinizing hormone (LH) regulated β_A-subunit expression, the effect ofin vivoadministration of ovine LH (oLH) on the expression of these subunits during the early- and mid-ovulatory cycle was examined. Northern blot analysis, using³²P-labeled cDNA probes of chicken inhibin/activin α- and β_A-subunits and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, internal control), revealed that in the F₁follicle, the relative level of β_A-mRNA (n = 3) was low at 23.5 hr and increased (P < 0.05) at 19.5, 15.5, and 11.5 hr before the next predicted ovulation. It then decreased (P < 0.05) at 7.5 hr and was further reduced at 3.5 and 0.5 hr prior to ovulation. In the F₂follicle, β_A-mRNA was maintained at a basal level throughout the sampling period except for a brief increase (P < 0.05) at 0.5 hr before ovulation. In contrast to the β_A-subunit, inhibin α-mRNA was abundantly expressed with no significant variations throughout the ovulatory cycle in either the F₁or the F₂follicle. When oLH was injected at 18 hr before ovulation, 200 but not 100 or 50 μg/kg (n = 3 hens per dose) significantly (P < 0.05) reduced the β_A-mRNA level in the F₁follicle by 2 hr after injection compared to the control (saline). The experiment was repeated at 12 hr before ovulation and both 100 and 200 but not 50 μg/kg oLH significantly (P < 0.05) reduced the expression of β_A-subunit mRNA with no significant difference between 100 and 200 μg/kg oLH. In contrast to the β_A-subunit, inhibin α-subunit mRNA was abundantly expressed and not affected by oLH treatment. Our data indicate that the expression of inhibin/activin β_A- but not α-subunit mRNA is developmentally regulated in the granulosa layer of the two largest follicles during the hen ovulatory cycle. In addition, LH may participate, directly or indirectly, in negative regulation of the β_A-subunit.     

Cardioprotective and antiarrhythmic effect of U50,488H in ischemia/reperfusion rat heart

The objective of this study was to investigate the protective effect of U50,488H, a selective κ-opioid receptor agonist, in the ischemia/reperfusion (I/R) rat and to delineate the underlying mechanism. Rat heart I/R injury was induced by occluding the left anterior descending coronary artery for 45 min and restoring perfusion for 120 min. U50,488H or vehicle was intravenously injected before ischemia. Electrocardiogram, heart rate (HR), arterial blood pressure (ABP), left ventricular pressure (LVP), systolic function (+dp/dt _max), and diastolic function (−dp/dt _max) were monitored in the course of the experiment. Myocardial infarction size was evaluated. Plasma concentrations of cardiac troponin T (cTnT), creatine kinase (CK), and lactate dehydrogenase (LDH) were measured. Single rat ventricular myocyte was obtained by enzymatic dissociation method. The potassium currents (I _K) of isolated ventricular myocytes were recorded with the whole-cell configuration of the patch-clamp technique. Compared with the sham control group, no significant change was found in HR, while ABP, LVP and ±dp/dt _max were significantly reduced in the I/R group. Administration of U50,488H significantly lowered HR in both control and I/R groups. Compared with the vehicle-treated I/R group, administration of U50,488H had no significant effect on I/R-induced reduction in ABP, LVP, and ±dp/dt _max. However, this treatment significantly reduced the myocardial infarction size, and markedly decreased the contents of plasma cTnT, CK and LDH. During ischemia and reperfusion, the incidence of ventricular arrhythmia in U50,488H-treated rats was significantly reduced. These effects were independent of the bradycardia induced by U50,488H, as the reducing infarct size and antiarrhythmic effect of U50,488H were still observed in animals in which heart rate was kept constant by electrical pacing. U50,488H and BRL-52537 still produced an antiarrhythmic effect when the rat heart was subjected to a shorter ischemic period of 10 min occlusion of coronary artery, which produced no infarction. I _K of the myocytes were inhibited by U50,488H in a dose-dependent manner in normal and hypoxic rat ventricular myocytes. However, the effects of U50,488H on I _K did not show any significant difference in normal and hypoxic myocytes. The above-described effects of U50,488H were totally blocked by nor-Binaltorphimine, a selective κ-opioid receptor antagonist. The results suggest that κ-opioid agonist U50,488H exerts its direct cardioprotective and antiarrhythmic effects against I/R via κ-opioid receptor, which participates in the regulation of potassium channels in normal and hypoxic ventricular myocytes. The first two authors contributed equally to this project.     

Further evidence of an opposite effect of dorsal and median raphe nuclei on the proestrous surge of LH

The effect of stimulation or lesions of either the dorsal or the median raphe nucleus on the proestrous surge of LH and on ovulation was studied in rats kept under constant illumination. Electrochemical stimulation (anodic DC of 100 microA during 30 sec) was applied at noon on the day of proestrus through chronically implanted electrodes. Lesions of the raphe nuclei were made by passing a cathodic current of 1 mA for 20 sec through nichrome electrodes stereotaxically implanted. Blood samples from freely behaving rats were obtained hourly through cannulae inserted into the jugular vein. Rats under constant light from diestrous day 1 or diestrous day 2 showed a delay in the onset of the LH surge of the next proestrus. Stimulation applied into the median raphe nucleus inhibited proestrous LH release and blocked ovulation, whereas stimulation of the dorsal raphe nucleus resulted in enhanced LH release in rats under constant light from diestrous day 2 but not from diestrous day 1. In turn, in rats bearing lesions in the dorsal raphe nucleus LH surges were decreased and ovulation was blocked, but rats with lesions in the median raphe nucleus exhibited enhanced LH release. It is concluded that the dorsal and the median raphe nuclei exert opposite effects on the proestrous surge of LH and on ovulation.     

Alterations in plasma and ovarian immunoreactive inhibin during reproductive aging in the female rat

Previously, we showed that ovarian inhibin α- and β;_A-subunit mRNAs are elevated in middle-aged and old persistent-estrous (PE) female rats. To determine whether higher inhibin subunit mRNA expressions result in increased circulating inhibins during reproductive aging, plasma immunoreactive inhibin α (ir-inh α) and gonadotropins were measured in young, middle-aged and PE rats. Plasma LH profiles were distinctly divergent in the middle-aged rats with some showing LH surges indistinguishable from young rats and others showing significantly attenuated LH surges. Plasma ir-inh α in middle-aged rats with LH surges were similar to those of young rats. However, animals with attenuated LH surges had higher peak ir-inh α levels than young and middle-aged animals with LH surges. Immunohistochemistry revealed increased levels of ovarian inhibin α staining in those animals with attenuated LH surges. Overall, the highest plasma and ovarian inhibin α were found in PE rats which lack LH surges. However, significant decreases of plasma and ovarian inhibin α were seen after reinstatement of estrous cyclicity with progesterone implant treatment. Thus, increases in both plasma and ovarian inhibin α appear to be closely associated with attenuation or loss of the preovulatory gonadotropin surge that occurs during aging.     

Annexin 1 (lipocortin 1) mimics inhibitory effects of glucocorticoids on testosterone secretion and enhances effects of interleukin-1beta

Annexin 1 is an important mediator of glucocorticoid action in the hypothalamo-pituitary axis; however, little is known of its role in mediating glucocorticoid actions in the peripheral endocrine organs. Accordingly, we have carried out a preliminary study to investigate the effects of annexin 1 in vitro on the testicular secretion of testosterone, a process inhibitied by both glucocorticoids and interleukin-1β (IL-1β). Luteinizing hormone (LH) and forskolin stimulated the release of testosterone from dispersed murine testicular cells in vitro. Their effects were reduced in cells from mice pretreated with dexamethasone (DEX). Similarly, preincubation of testicular cells from untreated mice with DEX, corticosterone, or 11-dehydrocorticosterone reduced LH-stimulated testosterone release, as did the 11β-hydroxysteroid dehydrogenase inhibitors, glycyrrhetinic acid and carbenoxolone. The inhibitory actions of the steroids were mimicked by annexin 1_1–188 (ANXA1_1–188) (a stable annexin 1 analog). IL-1β produced a marked decrease in the response to LH, which was blocked by indomethacin, a nonselective cyclooxygenase inhibitor and an additive effect with DEX and ANXA1_1–188. These results confirm reports that glucocorticoids and IL-1β inhibit LH-stimulated testosterone release from mouse testicular cells. They also show, for the first time, that the effects of the steroids are mimicked by annexin 1 and that, in contrast to their mutually antagonistic effects in the neuroendocrine system, IL-1β and annexin 1 exert additive actions in the testis.     

Relative contents and concomitant release of prodynorphin/neoendorphin-derived peptides in rat hippocampus.

The contents and molecular forms of five different prodynorphin-derived opioid peptides were compared in extracts of rat hippocampus by radioimmunoassay after C18-HPLC resolution. Dynorphin (Dyn) A(1-17) immunoreactivity (ir) and Dyn B-ir were heterogeneous in form; Dyn A(1-8)-ir, alpha-neoendorphin (alpha neo)-ir and beta-neoendorphin (beta neo)-ir each eluted as single homogeneous peaks of immunoreactivity. The fraction of immunoreactivity having the same retention as the appropriate synthetic standard was used to estimate the actual hippocampal content of each peptide. Comparison of these values showed that the concentrations of Dyn B, alpha neo, and Dyn A(1-8) were nearly equal, whereas both Dyn A(1-17) and beta neo were 1/5th to 1/10th the value of the other three. Calcium-dependent K+-stimulated release of these prodynorphin-derived opioids from hippocampal slices was detected. The stimulated rates of release were highest for Dyn B-ir followed by alpha neo-ir, then beta neo-ir and Dyn A(1-8)-ir with Dyn A(1-17)-ir lowest. The relative rates of stimulated release were in agreement with the relative proportions of peptide present within the tissue. This evidence of the presence and release of these opioid peptides considerably strengthens the hypothesis that this family of endogenous opioids plays a neurotransmitter role in the hippocampus.     

Endogenous opioid peptides mediate the interleukin-1-induced inhibition of the release of luteinizing hormone (LH)-releasing hormone and LH

We have reported recently that central administration of both the alpha- and beta-subtypes of the cytokine interleukin-1 (IL-1) inhibited the estrogen-progesterone-induced LH surge in ovariectomized (ovx) rats. This inhibition was probably due to a central effect, since IL-1 alpha and IL-1 beta also suppressed the in vitro LHRH output from the hypothalami of steroid-primed ovx rats. Whether IL-1 inhibits LHRH release by a direct action or via some other neuronal system is not known. Since IL-1 reportedly stimulates the release of POMC peptides, which are known to be inhibitory to the LHRH-LH axis, we have tested the hypothesis that the inhibitory influence of IL-1 may be mediated via activation of hypothalamic opioid peptides. Ovx rats, preimplanted with cannulae in the third ventricle of the brain, were injected with 30 micrograms estradiol benzoate, followed by 2 mg progesterone 48 h later. Three hours after P injection, IL-1 alpha, IL-1 beta, or saline (SAL) was injected intracerebroventricularly (30 ng/3 microliters) at 1300 h, followed immediately by iv infusion of SAL or the opiate antagonist naloxone hydrochloride (NAL; 2 mg/0.6 ml.h) for 2 h. Plasma LH levels were measured in blood samples withdrawn hourly until 1800 h. Both IL-1 alpha and IL-1 beta blocked the afternoon LH surge. NAL infusion into control SAL-injected rats did not alter the LH surge; however, it reversed the IL-1 alpha- and IL-1 beta-induced suppression of the LH surge. To determine whether this reversal of IL-1 suppression of the LH surge was due to NAL action at the hypothalamic level, the preoptic area-medial basal hypothalamus of similarly primed ovx rats was obtained at 1300 h and incubated in vitro in the presence of 10 nM IL-1 alpha or IL-1 beta with or without 100 micrograms/ml NAL. Both subtypes of IL-1 suppressed LHRH output significantly. NAL alone did not affect LHRH release, but it completely reversed the inhibitory effects of the cytokine on LHRH release. These results suggest that IL-1 alpha and IL-1 beta inhibit LHRH-LH release by stimulating the activity of hypothalamic endogenous opioid peptide systems.     

Reversal of beta-endorphin-induced blockade of ovulation and luteinizing hormone surge with prostaglandin E2

We examined the effects of intraventricular (Ivt) administration of beta-endorphin (beta E) on preovulatory LH release, ovulation, and the mechanism that may be involved in opioid action. Female rats were implanted with permanent cannulae in the third ventricle of the brain and were allowed to recover 4-day estrous cyclicity. Intrajugular cannulae were placed on the morning of proestrus. Thereafter, they received Ivt either saline (2 microliter) or beta E (10 micrograms/2 microliter) at 1300, 1430, and 1600 h. In addition, at 1600, 1700, and 1800 h, they were injected Ivt with either vehicle (cerebrospinal fluid or saline) or one of the following compounds: epinephrine (15.3 micrograms), norepinephrine (15.3 micrograms), or prostaglandin E2 (6 micrograms). Blood samples for LH measurements were taken 0, 10, 30, and 60 min after the additional injections at 1600 and 1700 h. beta E blocked the preovulatory LH surge and ovulation. Administration of the opiate receptor antagonist naloxone (2 mg/kg) reversed these effects. Epinephrine stimulated a small discharge of LH only after a second E injection in the beta E-treated rats, but this was insufficient to restore ovulation. On the other hand, prostaglandin E2 reversed the beta E blockade of the LH surge and ovulation. These studies suggest that beta E blocks ovulation and the LH surge primarily by suppressing either the influx or adrenergic expression of the spontaneous neurogenic stimuli responsible for the preovulatory LH discharge and not by evoking a general decrease in the secretory response of the LHRH neurons.     

Relationship between plasma atrial natriuretic factor and opioid peptide levels in healthy subjects and in patients with acute congestive heart failure

We evaluated plasma atrial natriuretic factor (ANF), ß-endorphin,met-enkephalin, dynorphin and noradrenaline levels in 20 healthysubjects and 20 acute congestive heart failure (CHF) patients.In all acute CHF patients plasma values of these hormones werehigher than in healthy subjects. The hormonal pattern differedin patients with the more severe acute CHF (group 1) from patientswith less severe acute CHF (group 2) (ANF 53.8 ± 1.0vs 34.6 ± 1.5 pg.ml^–1, noradrenaline 563.8 ±13.4 vs 202.4 ± 10.6 pg.ml^–1, met-enkephalin 41.0± 3.2 vs 17.0 ± 1.6 fmol. ml^–1, dynorphin46.8 ± 3.7 vs 25.2 ± 2.0 fmol. ml^–1, P <0.01;ß-endorphin 50.6 ± 5.2 vs 41.8 ± 4.1fmol. ml^–1, ns). Administration of an opioid antagonist(naloxone, 8 mg i.v.) did not modify ANF or noradrenaline concentrationin healthy subjects. in group 1 naloxone administration significantlyraised ANF (68.0 ± 1.4 pg. ml^–1), noradrenaline(776.6 ± 18.7 pg. ml^–1), blood pressure and heartrate, whereas in group 2 it significantly decreased ANF values(21.9 ± 0.5 pg. ml^–1)and did not modify the otherparameters. Our findings suggest that the opioid system affectsANF release in acute CHF. In patients with severe CHF opioidpeptides may attenuate ANF secretion reducing noradrenergicstimulation. On the other hand, when CHF is less severe andthe sympathetic activity is moderate, opioid peptides may directlystimulate ANF secretion.     

Effect of centrally injected allopregnanolone on sexual receptivity,luteinizing hormone release,hypothalamic dopamine turnover,and release in female rats

The effect of intracerebroventricular (icv) injection of allopregnanolone (5α-pregnan-3α-ol-20-one) on the dopaminergic and reproductive function in ovariectomized rats primed with estrogen and progesterone was investigated. Thirty minutes after icv allopregnanolone injection, the sexual receptivity, luteinizing hormone (LH) release, dopamine content, and release in the medial basal hypothalamus (MBH) and preoptic area (POA) were determined. After allopregnanolone injection, LH serum levels were reduced (p<0.001) and lordosis behavior was inhibited (p<0.005). Intracerebroventricular injection of bicuculline (a γ-aminobutyric acid_A [GABA_A] antagonist) alone was ineffective. The injection of allopregnanolone plus bicuculline blocked the effects of allopregnanolone on sexual receptivity and on LH serum levels. At the same time, endogenous dopamine concentration in both the MBH and POA was augmented (p<0.005 and p<0.006, respectively) and the turnover rate decreased in both structures. Moreover, in vitro ³H-dopamine release from MBH and POA was lower in rats injected with allopregnanolone in comparison with vehicle-treated rats. These results suggest that allopregnanolone influences the dopaminergic mechanisms in female rats, which may, in turn, be responsible for the reduced reproductive activity. Allopreganolone may exert its effects on sexual behavior through GABA_A receptor modulation and a decrease in dopaminergic activity in the MBH and POA. These actions could explain the inhibition of LH release.     

The regulation of pituitary gonadotropin release of the frontal lobe neocortex. (II). In relation to serotonergic neurons

The dorsal region of the anterior frontal lobe neocortex has an inhibitory function in the secretion of luteinizing hormone (LH) and ovulation. It is suggested that the anterior frontal lobe neocortex regulates the secretion of LH through the nerve circuit composed of the anterior median limbic area, the thalamic dorsomedial nucleus and the basolateral amygdaloid nucleus which are innervated by serotonergic neurons. In the present study, the relationship between the inhibitory effect of the frontal lobe neocortex on LH secretion and serotonergic neurons from the brain stem raphe nuclei was studied in 4-day vaginal cycle female Wistar rats. Electrochemical stimulation (DC 120 microA, 30 sec.) of the anterior frontal lobe neocortex just before the critical period of ovulation on the day of proestrus blocked the preovulatory surge of LH and ovulation. Electrochemical stimulation of the anterior frontal lobe neocortex just before the critical period but 120 min. after administration of rho-chlorophenylalanine (PCPA, 150 mg/kg, i.p.) could not block the preovulatory surge of LH and ovulation. Electrochemical stimulation of the anterior frontal lobe neocortex just before the critical period but 120 min. after excision of the median raphe nucleus blocked the preovulatory surge of LH and ovulation. Electrochemical stimulation of the anterior frontal lobe neocortex just before the critical period but 120 min. after excision of the dorsal raphe nucleus could not block the preovulatory surge of LH and ovulation. These results suggest that serotonergic neurons ascending from the dorsal raphe nucleus are involved in the inhibition of LH secretion and ovulation caused by electrochemical stimulation of the anterior frontal lobe neocortex and that the secretion of serotonin is necessary for the inhibition of LH secretion.     

Cocaine, not morphine, causes the generation of reactive oxygen species and activation of NF-κB in transiently cotransfected heart cells

This study was designed to determine levels of NF-κB reporter gene activity and free radical generation in cultured striated myocytes (H9C2 cells) exposed to cocaine or morphine in the presence of free radical scavengers. Cells were transiently transfected with a NF-κB reporter gene and changes in luciferase activity were detected, by bioluminescence. Using confocal microscopy and 2′,7′-dichlorofluorescin diacetate, cocaine-induced or morphine-induced free radicals were quantified in H9C2 cells. Cocaine and morphine (0–1×10⁻² M) were tested separately. Cocaine but not morphine significantly activated Nf-κB reporter gene, activity in H9C2 cells. Overexpression of IκB inhibited NF-κB reporter activity at low (1×10⁻⁴ M) but not high (1×10⁻² M) cocaine concentrations. Free radicals were generated in H9C2 cells stimulated with cocaine but not with morphine. The production of free radicals and NF-κB reporter gene activity could be blocked with N-acetylcysteine, glutathione, and to a lesser extent, lipoic acid. The results suggest that cocaine induces free radical production, which leads to the activation of NF-κB signal transduction and possible inflammatory responses.     

Reanalysis of the preoptic afferents and efferents involved in the surge of LH, FSH and prolactin release in the proestrous rat

In order to elucidate neural pathways concerned with the proestrous surge of LH, FSH and prolactin (Prl) release, brain transection or lesion was made acutely under ether anesthesia between 12.00 and 14.00 h of proestrus, and electrochemical stimulation was done under anesthesia with pentobarbital sodium (31.5 mg/kg b.w.) injected at 13.45 h. Transection which interrupted the connection of septum (SEPT), diagonal band of Broca (DBB) and bed nucleus of stria terminalis (BST) with the preoptic-suprachiasmatic area interfered with ovulation and surge of release of all 3 hormones. Isolation of the basal part of the suprachiasmatic area, including the suprachiasmatic nucleus (SCH), blocked ovulation also. Bilateral lesions in the medial preoptic area (MPO) with platinium-iridium electrode blocked ovulation and the surge of LH and Prl release, but not of FSH. Lesions in the SCH blocked ovulation and the surge of LH, but not of FSH and Prl. In the rat with acute isolation of the basal part of the suprachiasmatic area and SCH, stimulation of the MPO failed to induce ovulation and LH release, but was followed by FSH release. Prl release was not inhibited as in the intact rat. When the rat had the antero-SCH cut, stimulation of the SCH induced LH release but not FSH, and the inhibition on Prl release was pronounced. These findings offer evidence that the limbic-forebrain inputs are necessary for the preoptic integration in order to stimulate the proestrous surge of LH, FSH and Prl release. Furthermore, it is possible that separate pathways from the preoptic area to the medial basal hypothalamus are concerned in the stimulation of individual hormones--a restricted route for LH which may pass through the SCH, a diffuse one for FSH which may pass through either the SCH or anterior hypothalamic area, and a relatively diffuse one for Prl which may pass outside the SCH.     

Kappa opiate receptors inhibit release of oxytocin from the magnocellular system during dehydration

Magnocellular neurons synthesize vasopressin (VP) or oxytocin (OT) and release these hormones preferentially from the neural lobe during physiological stimulation. In the rat, VP is secreted preferentially during dehydration and hemorrhage, whereas OT is released without VP by suckling, parturition, stress, and nausea. Vasopressinergic neurons also synthesize and release dynorphin-related peptides--alpha- and beta-neoendorphin, dynorphin A (1-8) or (1-17), dynorphin B--which are agonists selective for kappa opiate receptors in the neural lobe. We proposed that one mechanism for preferential secretion of neurohypophysial hormones is that a dynorphin-related peptide(s) coreleased with VP inhibits selectively OT secretion from magnocellular neurons. We tested this hypothesis in conscious adult male Sprague-Dawley rats which were stimulated by either hypertonic saline administered intraperitoneally (2.5%, 20 ml/kg) or subcutaneously (1 M, 15 ml/kg) or by 24 h of water deprivation. Two approaches were used: (1) dynorphin-related peptides (0.02-20.4 mM) were injected intracerebroventricularly 1 min before decapitating the animal, and (2) the action of endogenous opioid peptides was blocked by injecting subcutaneously or intracerebroventricularly either naloxone or a selective kappa receptor antagonist, Mr 2266 or nor-binaltorphimine. VP and OT were measured by radioimmunoassay. After 24 h of water deprivation, the elevation in plasma [OT] but not [VP] was attenuated (p less than 0.05) by alpha-neoendorphin. Dynorphin A (1-8) also inhibited the release of OT and not VP after intraperitoneal administration of hypertonic saline. Blocking the action of endogenous opioid peptides at kappa receptors with Mr 2266 given peripherally (s.c.) elevated plasma [OT] but not [VP] after stimulation with hypertonic saline administered intraperitoneally or subcutaneously.(ABSTRACT TRUNCATED AT 250 WORDS)