Luteinizing hormone-releasing hormone and oxytocin response to hyperosmotic stimulation: in vitro study

It was shown previously that luteinizing hormone-releasing hormone (LHRH) affects the neurohypophysial oxytocin release in water-deprived rats. However, the detailed mechanisms by which LHRH modifies the oxytocin response to hyperosmotic stimulation have not been explained so far. Using the isolated hypothalamo-neurohypophysial explants obtained from euhydrated rats, the effect of LHRH on the oxytocin secretion was studied under conditions of direct osmotic (i.e., Na(+)- evoked) as well as nonosmotic (i.e., K(+)-evoked) stimulation. Additionally, the oxytocin response to LHRH was investigated using the explants obtained from animals drinking 2% saline for eight days (systemic, i. e., both direct and indirect, osmotic stimulation). LHRH significantly enhanced Na(+)- and K(+)-evoked oxytocin release from explants taken from rats drinking tap water, indicating that LHRH could affect the Na(+)/K(+)-dependent depolarization of perikarya of oxytocin neurones. In contrast, LHRH significantly diminished the K(+)-stimulated hormone release when the neurohypophysial complex was obtained from previously salt-loaded rats, suggesting that peripheral osmotic stimulation somehow modifies the sensitivity of oxytocinergic neurones to LHRH (possible mechanisms are discussed). It is concluded that LHRH may participate in the regulation of oxytocin secretion via both direct and indirect impact on magnocellular oxytocinergic neurones depending on the current functional status of the hypothalamo-neurohypophysial complex.     

Dendrotoxin and 4-aminopyridine potentiate neurohypophysial hormone secretion during low frequency electrical stimulation

The K+ channel blockers dendrotoxin (10(-9) M) and 4-aminopyridine (10(-6) M) produce similar patterns of augmentation of electrically evoked vasopressin and oxytocin secretion from isolated rat neurointermediate lobes. Both channel blockers produced a greater than two-fold increase in vasopressin release. Oxytocin release, however, was comparably increased only when endogenous opioid inhibition was abolished by the presence of naloxone. The potentiation of hormone release was dependent on the frequency of the applied stimulus. A large effect was seen with 1000 pulses at 4 Hz but not when the same number of pulses was delivered at 12 Hz. These findings suggest that a specific type of K+-channel, sensitive to dendrotoxin and low micromolar 4-aminopyridine, may play a significant role in the frequency-dependent facilitation of secretion from neurohypophysial nerve endings.     

Lamprey gonadotropin hormone-releasing hormone-III has no selective follicle-stimulating hormone-releasing effect in rats

Lamprey gonadotropin releasing-hormone (LGnRH)-III, a hypothalamic neurohormone recently isolated from sea lamprey, was reported to have a selective stimulatory effect on follicle-stimulating hormone (FSH) release in rats and suggested to be the mammalian FSH-releasing factor. In this study, we determined the relative luteinizing hormone (LH)- and FSH-releasing potency of LGnRH-III compared to mammalian gonadotropin-releasing hormone (LHRH) in normal female rats, ovariectomized (OVX) and oestrogen/progesterone substituted rats and the superfused rat-pituitary cell system. The specificity of LGnRH-III for the mammalian LHRH receptor was investigated by blocking the receptor with an LHRH antagonist, MI-1544. In vitro, LGnRH-III dose-dependently stimulated both LH and FSH secretion from rat pituitary cells at 10(-7) to 10(-5) M concentrations, while LHRH stimulated gonadotropin secretion at a 1000-fold lower doses (10(-10) to 10(-8) M). The difference between its LH- and FSH-releasing potency was similar to that of LHRH. LGnRH-III bound to high affinity binding sites on rat pituitary cells with a Kd of 6.7 nM, B(max)=113 +/- 27 fmol/mg protein. In vivo, LGnRH-III also stimulated both LH and FSH secretion in a dose-dependent manner and, similar to LHRH, induced a greater rise in the serum LH than the FSH level. In normal cycling rats, it showed 180-650-fold weaker potency than LHRH in stimulating LH secretion and 70-80-fold weaker effect in stimulating FSH secretion. In OVX rats, LGnRH-III demonstrated a similarly weak effect on both gonadotropins. It was found to be 40-210-fold less potent than LHRH regarding LH release and 50-160-fold weaker regarding FSH release. LHRH-receptor antagonist MI-1544 prevented both the LH- and the FSH-releasing effect of LGnRH-III both in vitro and in vivo. These results do not support the hypothesis that LGnRH-III might be the mammalian FSH-releasing factor but demonstrate that it is a weak agonist for the pituitary LHRH receptor and stimulates both gonadotropins in a dose-dependent fashion.     

Hydroxyurea induces vasopressin release and cytokine gene expression in the rat hypothalamus

We previously showed that the cytostatic drug hydroxyurea (HU) activates the hypothalamo-pituitary-adrenal (HPA) axis in intact rats, whereas it is lethal in rats with impaired HPA function. In these animals, HU toxicity is mediated by increased circulating levels of proinflammatory cytokines, whose secretion cannot be counteracted by glucocorticoids, suggesting that HPA activation blunts HU toxicity. Here we investigated the mechanisms through which HU activates the HPA axis, looking at the direct effects of the drug on the isolated hypothalamus. We found that HU significantly increases the release of arginine vasopressin but not that of corticotrophin-releasing hormone in short-term incubation experiments. The levels of arginine vasopressin are also increased in the hypothalamus and systemic circulation 2 h after the in vivo administration of the drug. Furthermore, HU increased significantly the expression of interleukin-6 and, to a lesser extent, interleukin-1beta in the hypothalamus. Interestingly, experiments with HU on primary cultures of rat microglia and astrocytes suggested that the increase in cytokine gene expression observed in hypothalamic explants is not accounted for by glial cells. Since both vasopressin and cytokines can activate the HPA axis, our present findings provide a reasonable explanation of the HPA activation elicited by HU in vivo in the rat.     

N-Methyl-d-Aspartic Acid Stimulation of Vasopressin Release: Role in Osmotic Regulation and Modulation by Gonadal Steriods

Previous experiments demonstrated that excitatory amino acids participate in the osmotic regulation of vasopressin secretion, but the specific involvement of N -methyl- d -aspartic acid (NMDA) receptors was not evaluated. This was demonstrated in the present studies. NMDA stimulated vasopressin release from perifused explants of the hypothalamo-neurohypophyseal system (HNS), and osmotic stimulation of vasopressin release was inhibited by MK-801 (10  μM) and AP5 (100  μM) NMDA receptor antagonists. The effective concentration of NMDA was dependent upon the Mg²⁺ concentration of the perifusate with stimulation observed at 1  μM NMDA in Mg²⁺-replete compared with 5  μM in low-Mg²⁺ medium. Previous experiments also demonstrated that estradiol and dihydrotestosterone (DHT) inhibited osmotically stimulated vasopressin secretion, and a nongenomic mechanism of action was suggested by the ability of steroids conjugated to bovine serum albumin to replicate the effect. Experiments were performed to explore the potential role of NMDA receptors in this mechanism. Estradiol (50  pg/ml) and DHT (3  ng/ml) inhibited NMDA stimulated vasopressin release in perifused HNS explants. These results suggest a role of NMDA receptors in the mediation of vasopressin secretion in osmotically stimulated release. Furthermore, estradiol and DHT may exert their inhibitory effect on osmotically stimulated vasopressin release via the NMDA receptor.     

Quantitative Comparisons Between the Electrical Activity of Supraoptic Neurons and Vasopressin Release in vitro

The firing rate of antidromically identified neurons in the tuberal portion of the rat supraoptic nucleus and vasopressin release were compared in an in vitro preparation of the hypothalamo-neurohypophysical system. Extracellular potentials were isolated and held while the perifusing medium was collected for radioimmunoassay of vasopressin. Neurons in the tuberal portion of the supraoptic nucleus were induced to first bursts of action potentials by injecting supra-threshold concentrations (0.01, 0.1 and 1 mM) of the α₁-agonist phenylephrine into the perifusing line. Phenylephrine caused a dose-dependent release of vasopressin into the perifusate and a dose-dependent increase in the peak firing rate, initial burst duration and the total number of action potentials (compared to a 10 min control period) of the recorded neurons. Peak firing rate and peak vasopressin release both increased linearly with the log of the phenylephrine dose, but the efficiency ratio of the increase in vasopressin release to the increase in firing rate was greater from the middle to the highest dose than it was for the lowest to the middle dose, indicating a possible facilitation of hormone release with higher firing frequencies. The total amount of vasopressin released in 10 min post-injection and the total number of evoked action potentials in the same period also increased linearly, but in this case there was no change in the efficiency ratio between the low to middle and the middle to high doses of phenylephrine. Release of vasopressin to phenylephrine was dependent on an intact neural stalk, and in a separate group of isolated neural lobes, 1 mM phenylephrine did not significantly alter the peak or total amount of vasopressin released to an electrical stimulus given in the pattern of an evoked burst. In another group of explants the temporal relationship between firing rate and vasopressin release was examined after a single dose of 1 mM phenylephrine. In these explants the true time-course of release was estimated using a deconvolution procedure which corrected for diffusion of the hormone. Mean vasopressin release and spike activity were still elevated above baseline 4 to 5 min after the first elicited burst, suggesting that the latter parts of the initial burst and/or the after-discharges contribute to the prolonged vasopressin release. However, there was a dramatic drop in the efficiency ratio (vasopressin release/peak firing rate) from the first to the second minute following stimulation. Thus, changes in the frequency of action potentials generated by supraoptic neurons can be directly related to simultaneous changes in the rate of vasopressin release in the same preparation. The results suggest that elevations in firing rate are accompanied by an increased rate of vasopressin release, but as has been demonstrated in isolated neural lobes, this relationship is probably not constant across different stimulation strengths or within a single burst.     

Galanin and galanin-like peptide modulate vasopressin and oxytocin release in vitro: The role of galanin receptors

Galanin (Gal) and galanin-like peptide (GALP) may be involved in the mechanisms of the hypothalamo-neurohypophysial system. The aim of the present in vitro study was to compare the influence of Gal and GALP on vasopressin (AVP) and oxytocin (OT) release from isolated rat neurohypophysis (NH) or hypothalamo-neurohypophysial explants (Hth–NH). The effect of Gal/GALP on AVP/OT secretion was also studied in the presence of galantide, the non-selective galanin receptors antagonist.Gal at concentrations of 10⁻¹⁰ M and 10⁻⁸ M distinctly inhibited basal and K⁺-stimulated AVP release from the NH and Hth–NH explants, whereas Gal exerted a similar action on OT release only during basal incubation. Gal added to the incubation medium in the presence of galantide did not exert any action on the secretion of either neurohormone from NH and Hth–NH explants.GALP (10⁻¹⁰ M and 10⁻⁹ M) induced intensified basal AVP release from the NH and Hth–NH complex as well as the release of potassium-evoked AVP from the Hth–NH. The same effect of GALP has been observed in the presence of galantide. GALP added to basal incubation medium was the reason for stimulated OT release from the NH as well as from the Hth–NH explants. However, under potassium-stimulated conditions, OT release from the NH and Hth–NH complexes has been observed to be distinctly impaired. Galantide did not block this inhibitory effect of GALP on OT secretion.It may be concluded that: (i) Gal as well as GALP modulate AVP and OT release at every level of the hypothalamo-neurohypophysial system; (ii) Gal acts in the rat central nervous system as the inhibitory neuromodulator for AVP and OT release via its galanin receptors; (iii) the stimulatory effect of GALP on AVP and OT release is likely to be mediated via an unidentified specific GALP receptor(s).     

Activation of intracellular pathways with forskolin and phorbol ester increases LHRH mRNA level in the rat hypothalamus superfused in vitro

Two intracellular signal transduction mechanisms such as cAMP-protein kinase a and phosphatidylinositol (PI) turnover-protein kinase c are known to be dually involved in the regulation of luteinizing hormone-releasing hormone (LHRH) release. However, it is not yet evident that the activation of two intracellular pathways affects the LHRH gene expression. The present study aims, therefore, to determine whether the activation of two intracellular pathways affects changes in LHRH mRNA. To this end, we took advantage of an in vitro superfusion system, where rat hypothalamic tissues were superfused with media containing forskolin (FKN) and/or phorbol-12-myristate-13-acetate (PMA). Superfusates were collected at 10-min intervals and LHRH release was determined by radioimmunoassay. After a 2-h superfusion period, the post-superfusion hypothalami were recovered and poly (A) RNA fractions were isolated. Alterations in LHRH mRNA in response to FKN and/or PMA were determined by an RNA-blot hybridization assay using a 32P-end-labeled LHRH oligonucleotide (29-mer) probe. In vitro perfusion of hypothalamic fragments with PMA and/or FKN stimulated LHRH release as well as LHRH mRNA. The combined infusion of FKN and PMA did not produce an additive effect on the LHRH mRNA levels, but it was effective in synergistically increasing LHRH secretion in vitro. These data clearly demonstrate that the biosynthetic machinery of LHRH is influenced by activation of two intracellular pathways, both cAMP-protein kinase a and phosphatidyl-inositol turnover-protein kinase c, indicating the transsynaptic regulation of hypothalamic LHRH gene expression.     

The effects of ionotropic agonists of excitatory amino acids on the release of arginine vasopressin in rat hypothalamic slices

The effects of ionotropic excitatory amino acids agonists on the release of vasopressin from rat hypothalamic slices were studied. Incubation with increasing doses of NMDA, kainate or AMPA decreased the release of vasopressin in a dose-dependent manner. The values of the IC50 were 1.0, 9.6, or 3.7 x 10-8 M, respectively. The inhibitory effect of the various excitatory amino acids tested was blocked by coincubation with their respective antagonists. Vasopressin secretion was stimulated to 140.3 +/- 7.6% of controls when the slices were obtained from chronically (7 days) salt-loaded rats. Addition of 1 x 10-7 M NMDA or 1 x 10-6 M kainate to the incubation medium antagonized the salt loading-induced increase in vasopressin release. Incubation with 1 x 10-4 M tetrodotoxin did not change basal vasopressin release, but it blocked the decrease in vasopressin secretion induced by 1 x 10-7 M NMDA or 1 x 10-6 M kainate or 1 x 10-6 M AMPA. Incubation with 1 x 10-5 M phaclophen (a GABAB antagonist) and 1 x 10-5 M bicuculline (a GABAA antagonist) was without effect on basal vasopressin secretion while it reversed the inhibition of vasopressin release induced by 1 x 10-7 M NMDA. Incubation with 1 x 10-6 M GABA alone decreased vasopressin secretion to 64.6 +/- 6.9% of control values. The inhibitory effect of GABA did not change when 1 x 10-7 M NMDA was added to the incubation medium. These findings demonstrate that ionotropic excitatory amino acids agonists inhibit vasopressin secretion from hypothalamic slices. They strongly suggest that this inhibitory effect is mediated through local GABAergic interneurones.     

Subcellular localization of luteinizing hormone releasing hormone degrading activity in the hypothalamus

The in vitro degradation of endogenous as well as exogenous luteinizing hormone releasing hormone (LHRH) by subcellular fractions of rat hypothalamic tissue was studied. Endogenous LHRH, localized an isolated nerve terminals (synaptosomes), was found to be resistant to enzymatic degradation (60 min, 37 degrees C) as long as the synaptosomal membrane remained intact. Endogenous LHRH was rapidly degraded by the 900 x g supernatant fluid and cytosol but not by myelin/microsomes, intact synaptosomes, or mitochondria. Lysed synaptosomes rapidly degraded exogenous LHRH. The LHRH degrading activity of synaptosomes was highly concentrated in the 'synaptosol', i.e., the cytosol of the nerve terminal. These data suggest that the LHRH degrading activity of the rat hypothalamus is a readily solubilized component of neurons, and possibly of non-neuronal cells.     

Diacylglycerol and phorbol esters enhance LHRH and prostaglandin E2 secretion from median eminence nerve terminals in vitro

The present experiments were designed to evaluate the role of protein kinase C activation on the secretion of the neural peptide, LHRH, from hypothalamic nerve terminals in vitro. Two specific protein kinase C activators, diacylglycerol (1,2-didecanoylglycerol, DiC10) and a phorbol ester (12,13-dibutyrate, PDBu) were used as probes. In addition to LHRH, secretion of prostaglandin E2 (PGE2) was also measured, since previous studies from our laboratory indicate that this arachidonic acid metabolite is intimately involved in the LHRH secretory process. PDBu at a dose of 200 nM significantly enhanced LHRH secretion from median eminence nerve terminals; in addition, a more modest but significant stimulation of PGE2 release was also observed. DiC10 (100 microM), on the other hand, enhanced PGE2 release but had no clear effect on LHRH secretion. Release of LHRH, however, was clearly stimulated when the lipoxygenase inhibitor nordihydroguaiaretic acid was added to the medium, suggesting that some arachidonic acid metabolites are inhibitory to LHRH secretion. The results indicate that protein kinase C activation leads to an enhanced secretion of LHRH. In addition, they suggest that 1,2-diacylglycerol may also activate the formation of arachidonoyl residues inhibitory to LHRH release.     

Endocrine Actions of Central Neuropeptide Y in the Ewe: Activation of the Hypothalamo-Pituitary-Adrenal Axis by Exogenous Neuropeptide Y and Role of Endogenous Neuropeptide Y in the Secretion of Luteinizing Hormone During the Oestrous Cycle

Neurons immunoreactive for neuropeptide Y (NPY) are abundant in the hypophysiotrophic areas of the brain. In particular, there is considerable anatomical evidence for the influence of this neuropeptide on the reproductive and hypothalamo-pituitary-adrenal axes. We therefore investigated whether central administration of NPY can alter the activities of the reproductive and hypothalamo-pituitary-adrenal axes in the ewe, and whether ovarian steroids are involved in the modulation of these events. We also attempted to investigate whether endogenous NPY is important in the control of luteinizing hormone-releasing hormone/luteinizing hormone (LHRH/LH) secretion in the sheep oestrous cycle. Central injection of NPY (0.15 and 1.5 nmol in 50 μl saline), delivered by gravity flow into the third cerebral ventricle, had no effect on LH levels in ovariectomized (OVX) ewes (n = 6) or OVX ewes implanted with oestradiol (OVX/E₂) (n = 7), nor was LH secretion altered by central NPY (1.5 nmol) in intact cycling animals in either the follicular or the luteal phase (n = 5). However, central administration of 1.5 nmol NPY to intact ewes during both the follicular (P> 0.05) and the luteal phase (P> 0.01), and in OVX/E₂ ewes (P<0.5) caused a large and significant increase in plasma cortisol levels. High litre antibodies were raised to NPY in sheep and the effects of peripheral and central (intracerebroventricular) administration of anti-NPY antibodies on the timing and/or characteristics of the E₂-induced LH surge in anoestrous ewes and of the preovulatory surge of LH in cycling ewes were determined. Intravenous administration of anti-NPY antibodies (n = 6) had no effect on the oestradiol benzoate-induced LH surge, compared with the control injection of non-immune plasma (n = 6). Likewise, passive systemic immunization against NPY (n = 10) was without effect on the characteristics of the preovulatory LH surge, compared with the control group (n = 10). However, central (intracerebroventricular) administration of anti-NPY antibodies (n = 4) delayed or abolished the preovulatory LH surge when compared with non-immune plasma treatment in the same animals. In summary, tonic LHRH/LH secretion is unaffected by centrally administered NPY at the doses used in this study. However, the same doses of NPY activate the hypothalamo-pituitary-adrenal axis, thus lending clear support to the hypothesis that NPY is involved in the multifactorial regulation of adrenocorticotrophin and cortisol secretion in this species, probably by stimulating corticotrophin-releasing hormone and/or arginine vasopressin secretion within the hypothalamus. The results of the immunization experiments further suggest that hypothalamic NPY may play a part in the modulation of the tinning of the LHRH/LH surge in the ewe. In contrast to the role of NPY in this regard in the rat, this only occurs at the level of the hypothalamus. The function of NPY in the regulation of reproduction in the sheep may therefore be to provide a means whereby the efficient generation of a preovulatory LHRH/LH surge is ensured.     

Oxytocin and arginine vasopressin stimulate steroid secretion by the isolated perfused rat adrenal gland

Using the intact isolated perfused rat adrenal preparation we have shown for the first time a direct effect of oxytocin on adrenocortical steroid secretion. Oxytocin specifically stimulated aldosterone secretion in a dose-dependent manner with a threshold dose of 1 pmol. Arginine vasopressin was also shown to be a potent stimulus to aldosterone secretion and was additionally found to stimulate inner zone function. Using superfused adrenal cells, the effects of arginine vasopressin were only seen at 10,000 times higher doses than were effective in the intact perfused gland, and oxytocin had no effect at any dose. These results reinforce the hypothesis that tissue integrity is essential for full expression of steroidogenic control mechanisms. We conclude that oxytocin and vasopressin may play a role in the control of steroidogenesis.     

LHRH Pulse Generator is Stimulated by Naloxone in the Pentobarbital-Blocked Proestrous Rat

Previous studies by us and others led us to hypothesize that there are separate LHRH pulse and surge generators in the rat brain. The present study was designed to detect the activity of LHRH pulse generator by checking changes in LH secretion and the multiunit activity (MUA) of the arcuate-median eminence region of the hypothalamus during infusions of naloxone (NAL, 2 mg/h) in the proestrous rat in which the LHRH surge generator activity was blocked by pentobarbital sodium (PB, 32 mg/kg bw, ip). The animals were subjected to blood sampling in the morning (1000–1300 h) or afternoon (1400–1700), and injected with PB at 09.45 or 13.45, respectively. During saline infusions in the rat given PB injection at either 09.45 or 13.45, serum LH levels were low but fluctuated significantly, suggesting a pulsatile secretion in either the morning or the afternoon period. The pulse intervals were an average 28.2 min in the morning and 42.2 min in the afternoon. NAL infusions decreased the pulse interval significantly, to 22.0 min in the morning and to 27.0 min in the afternoon. In the electrophysiological experiment, characteristic increases in the MUA (volleys), which occur in association with the initiation of an LH pulse and therefore are considered to represent an increased activity of the LHRH pulse generator, appeared during NAL (5 mg/h) infusions in either the morning or the afternoon. These results strongly suggest that separate LHRH pulse and surge generators exist in the brain, and that, even during the critical period of proestrus, the activity of LHRH pulse generator is disclosed by PB, which, on the other hand, arrests the surge generator.     

The in vitro role of tumour necrosis factor-alpha and interleukin-6 in the hypothalamic-pituitary gonadal axis

The adipocyte derived hormone leptin has been implicated as an important nutritional signal to the reproductive system, but the role of other adipocyte related cytokines is not clear. Tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6 are present in adipose tissue and released into the circulation where plasma levels correlate positively with body mass index and body fat mass. These cytokines could play a role in signalling nutritional status to the hypothalamic-pituitary-gonadal axis. We investigated the effects of TNF-alpha and IL-6 on basal and luteinizing hormone releasing hormone (LHRH) stimulated luteineizing hormone (LH) release from cultured anterior pituitary cells, harvested from either proestrus female or male Wistar rats. We examined the effects of TNF-alpha and IL-6 on LHRH release from hypothalamic explants harvested from proestrus female and male rats in vitro. IL-6 significantly suppressed LHRH stimulated LH release from male dispersed pituitaries throughout the dose range, but did not influence basal LH release. IL-6 had no effect on basal or LHRH stimulated LH release in dispersed pituitaries from proestrus females. By contrast, TNF-alpha significantly suppressed LHRH stimulated LH release in dispersed pituitaries from proestrus female rats in a dose responsive manner, but did not influence basal LH release. TNF-alpha had no effect on basal or LHRH stimulated LH release in dispersed pituitaries from male rats. TNF-alpha and IL-6 had no effect on LHRH release from male hypothalamic explants in vitro. TNF-alpha and IL-6 had no effect on LHRH release from proestrus female hypothalamic explants in vitro. TNF-alpha and IL-6 have differential effects in dispersed pituitaries harvested from males and proestrus female rats. TNF-alpha and IL-6 may be important in mediating some of the nutritional effects on the reproductive axis by acting at the level of the anterior pituitary rather than the hypothalamus.     

Effect of arginine vasopressin and oxytocin on acetylcholine-stimulation of corticosteroid and catecholamine secretion from the rat adrenal gland perfused in situ

The effects of acetylcholine, arginine vasopressin (AVP) and oxytocin (OXT) on both catecholamine and steroid secretion have been investigated using the isolated rat adrenal gland perfused in situ. Significant stimulation of steroid (aldosterone and corticosterone) secretion occurred with 1 mumol/l acetylcholine; the ED50 was approximately 20-fold higher (circa 20 mumol/l) than that for catecholamine secretion. The highest concentration of acetylcholine used (100 mumol/l) stimulated aldosterone secretion eight-fold; corticosterone secretion four-fold; noradrenaline and adrenaline secretion three-fold. AVP at 100 nmol/l but not at 1 nmol/l significantly stimulated the secretion of both steroids and catecholamines. OXT had no significant effect on corticosteroid or catecholamine secretion at either concentration. The effects on aldosterone secretion of simultaneous administration of acetylcholine and AVP were additive. No similar effect was seen on corticosterone or catecholamine secretion where the degree of stimulation was the same as for acetylcholine alone. OXT (100 nmol/l) inhibited acetylcholine-stimulated aldosterone secretion but had no effect on acetylcholine-stimulated catecholamine secretion. Carbachol was equipotent with acetylcholine in stimulating steroid secretion from the perfused gland. Our results support the hypothesis that acetylcholine may play a role in the control of steroid secretion by the rat adrenal cortex. They fail to support a role for AVP and OXT in modulating catecholamine secretion by the adrenal medulla except at high concentrations.     

Circadian rhythms of vasopressin release from individual rat suprachiasmatic explants in vitro

A culture system was developed to study intrinsic oscillations within rat suprachiasmatic nuclei for long periods of time. After isolation in static organ cultures, individual rat suprachiasmatic explants released vasopressin in a circadian fashion for at least 4 cycles. This finding suggests that the suprachiasmatic nucleus contains one or more self-sustained circadian oscillators, and that this method of measuring circadian rhythms from individual suprachiasmatic organ cultures may prove to be a valuable model for elucidating the physiology and biochemistry of mammalian circadian pacemakers.     

Nitric Oxide Is Involved in the Genesis of Pulsatile LHRH Secretion from Immortalized LHRH Neurons

Neuronal networks controlling endocrine events present synchronous activity which is required for maintaining physiological functions, including reproduction. Although pulsatile hormone secretion is of paramount importance, the mechanism(s) by which secretory episodes are generated remain largely unknown. Nitric oxide (NO) has become the prototype of a new family of signaling molecules in the body. Nitric oxide diffuses from the source cell and controls activity of neighboring neurons as well as itself as a retrograde messenger. Cells of the luteinizing hormone-releasing hormone (LHRH) neuronal network, the key component in the control of reproduction, are scattered and loosely arranged in the anterior hypothalamus. A diffusible neurotransmitter could provide a means for establishing synchronous activation of the LHRH neuronal network leading to physiologically-relevant pulsatile LHRH secretion. In this study, we demonstrate that immortalized LHRH-producing neurons (GT1–7 cells) express NO synthase (NOS) mRNA and protein. Furthermore, GT1–7 cells are NADPH-diaphorase-positive (a marker of NOS activity) and the histochemical reaction can be abolished by treatment with a competitive NOS blocker. The presence of citrulline in these cells provides further evidence for the biological activity of NOS. These observations indicate that an active NO synthesizing machinery is present in immortalized LHRH neurons. In addition, we show that LHRH secretion is enhanced by NO in a cGMP-dependent manner. Since pulsatile LHRH secretion from immortalized LHRH neurons in vitro is abolished by NOS blockers and NO scavengers, it appears that NO is a unique neurotransmitter that is necessary to set LHRH neurons in phase to establish synchronized pulsatile LHRH secretion.     

Ca2+ and vasopressin release in isolated rat neurohypophysis: differential effects of four classes of Ca2+ channel ligands

In order to study the role of different types of voltage-sensitive Ca2+ channels (VSCC) in stimulus-secretion coupling in peptidergic neurons, effects of 4 major classes of pharmacological agents have been examined on evoked vasopressin release from isolated rat neurohypophyses. omega-Conotoxin GVIA (omega-CgTX), a potent blocker of N- and L-type Ca2+ channels, inhibited vasopressin release evoked electrically as well as by high K+. With maximal inhibition, release was decreased to 50% and 75% of control for electrical and 100 mM K+ stimulation, respectively. This stimulation mode-related difference in release sensitivity to omega-CgTX paralleled its stimulation mode-related sensitivity to tetrodotoxin, suggesting that the omega-CgTX-sensitive Ca2+ entry played a larger role when release was activated by action potentials invading nerve terminals. These data, and the characteristics of [125I]omega-CgTX binding to plasma membranes from bovine neurohypophyses, are consistent with N-type Ca2+ channels being responsible for the omega-CgTX-sensitive component of vasopressin release. Verapamil and diltiazem (phenylalkylamine and benzothiazepine, respectively) inhibited secretion in a pattern suggesting non-identical sets of action sites, and in a manner partly additive with inhibition by omega-CgTX. This inhibition by verapamil and diltiazem appeared at least in part to involve sites different from L channels. Several dihydropyridines known to act as agonists or antagonists at L channels did not affect vasopressin release (evoked either electrically or by high K+) in a specific manner. A significant component of neuropeptide release may depend on Ca2+ entry through omega-CgTX- and dihydropyridine-insensitive routes.