Tissue and cellular distribution of the extended family of protein kinase C isoenzymes.

Polyclonal isoenzyme-specific antisera were developed against four calcium-independent protein kinase C (PKC) isoenzymes (delta, epsilon, epsilon', and zeta) as well as the calcium-dependent isoforms (alpha, beta I, beta II, and gamma). These antisera showed high specificities, high titers, and high binding affinities (3-370 nM) for the peptide antigens to which they were raised. Each antiserum detected a species of the predicted molecular weight by Western blot that could be blocked with the immunizing peptide. PKC was sequentially purified from rat brain, and the calcium-dependent forms were finally resolved by hydroxyapatite chromatography. Peak I reacted exclusively with antisera to PKC gamma, peak II with PKC beta I and -beta II, and peak III with PKC alpha. These same fractions, however, were devoid of immunoreactivity for the calcium-independent isoenzymes. The PKC isoenzymes demonstrated a distinctive tissue distribution when evaluated by Western blot and immunocytochemistry. PCK delta was present in brain, heart, spleen, lung, liver, ovary, pancreas, and adrenal tissues. PKC epsilon was present in brain, kidney, and pancreas, whereas PKC epsilon' was present predominantly in brain. PKC zeta was present in most tissues, particularly the lung, brain, and liver. Both PKC delta and PKC zeta showed some heterogeneity of size among the different tissues. PKC alpha was present in all organs and tissues examined. PKC beta I and -beta II were present in greatest amount in brain and spleen. Although the brain contained the most PKC gamma immunoreactivity, some immunostaining was also seen in adrenal tissue. These studies provide the first evidence of selective organ and tissue distributions of the calcium-independent PKC isoenzymes.

Processing of the luteinizing hormone-releasing hormone precursor in the preoptic area and hypothalamus of the rat.

The LHRH precursor is known to contain the decapeptide and a 56 amino acid peptide termed gonadotropin-releasing hormone-associated peptide (GAP). The purpose of our study was to characterize the proLHRH and its processed products from the cell body and fiber region and from the nerve terminal region of LHRH neurons. The median eminence (ME) and a tissue block containing the preoptic area and hypothalamus (POH) were dissected separately. Tissues were homogenized and peptides were separated according to mol wt. Three different LHRH antisera bound to one immunoreactive (IR) substance which eluted at approximately 1200 mol wt. Subsequently, this material coeluted with synthetic LHRH on a reversed-phase column as a single peak. There was approximately 1.6-fold more LHRH-like IR in the ME than in the POH. The four different GAP antisera recognized multiple mol wt forms of GAP-like IR at approximately 16,000 to 14,000, 8,200, 6,500, 3,500, and 2,800 mol wt. There were more of the high mol wt materials and less of the 6500 and lower mol wt materials in the POH than in the ME. The most abundant species in both regions was the 6500 mol wt form. This IR substance coeluted with synthetic rat GAP1-56 on a reversed-phase column as a single peak. These experiments demonstrate 1) that multiple IR forms of the LHRH prohormone exist in the POH of the rat and 2) that nerve terminals of the LHRH neurons contain LHRH, GAP1-56, and some lower mol wt GAP-like substances. These results provide the first information concerning the processing scheme for the LHRH prohormone in the rat brain.

Induction of promiscuous G protein coupling of the follicle-stimulating hormone (FSH) receptor: a novel mechanism for transducing pleiotropic actions of FSH isoforms.

Under physiological conditions, FSH is secreted into the circulation as a complex mixture of several isoforms that vary in the degree of glycosylation. Although it is well established that the glycosylation of FSH is important for the serum half-life of the hormone and coupling of the receptor to adenylate cyclase, little is known concerning how physiologically occurring glycosylation patterns of this hormone affect receptor signaling. In this study, we have examined the biological activity of deglycosylated human FSH (DeGly-phFSH), recombinant mammalian-expressed hFSH (CHO-hFSH), and insect cell-expressed hFSH (BV-hFSH, alternatively glycosylated) as compared with that of purified human pituitary FSH (phFSH) using a Chinese hamster ovarian cell line stably expressing the hFSH receptor (3D2 cells). Differentially glycosylated forms of FSH did not bind to the FSH receptor in the same manner as phFSH. Although all hormones showed similar potency in competing for [125I]phFSH binding to the hFSH receptor, competition curves for deglycosylated and insect cell-produced FSH were steeper. Similarly, glycosylation of FSH had a profound effect on bioactivity of the hormone. Purified hFSH produced a sigmoidal dose-dependent stimulation in cAMP production, whereas DeGly-phFSH and BV-hFSH induced biphasic (bell-shaped) dose-response curves. BV-hFSH also elicited biphasic effects on steroidogenesis in primary cultures of rat granulosa cells. The cellular response to BV-hFSH was dependent on the degree of receptor-transducer activation. BV-hFSH bioactivity was strictly inhibitory when combined with the ED80 of phFSH. Lower concentrations of phFSH resulted in a gradual shift from inhibition to a biphasic activity in the presence of the ED20 of phFSH. Biphasic responses to BV-hFSH were attributed to activation of different G protein subtypes, since treatment of 3D2 cells with cholera toxin or pertussis toxin differentially blocked the two phases of BV-hFSH bioactivity. These data suggest that alternative glycosylation of FSH leads to a functionally altered form of the hormone. Functionally different hormones appear to convey distinct signals that are transduced by the receptor-transduction system as either stimulatory or inhibitory intracellular events via promiscuous, glycosylation-dependent G protein coupling. Promiscuity in signaling of the FSH receptor, in turn, may represent a potentially novel mechanism for FSH action, whereby the gonad may respond in diverse ways to complex hormonal signals such as those presented by circulating FSH isoforms.

Gonadectomy influences the inhibitory effect of the endogenous opiate system on pulsatile gonadotropin secretion.

These studies were designed to evaluate the importance of the gonads on the inhibitory influence exerted by the endogenous opiate system on gonadotropin secretion. The effect of the opioid receptor antagonist naloxone on pulsatile LH secretion was evaluated in 1-, 2-, and 8-week orchidectomized (ORDX) rats. Freely moving rats bearing a Silastic cannula in the right jugular vein were bled every 6 min for 3 h. Saline was injected during the first 90 min, while during the last 90 min naloxone was given as an initial iv bolus injection dose of 2 mg/kg, followed by injections of 0.1 mg/kg every 6 min. In addition, another group of rats was treated, from the beginning of the 3-h bleeding period, with naloxone at an initial iv dose of 2 mg/kg, followed by injections of 0.1 mg/kg every 6 min. Analysis of pulsatile LH secretion showed enhanced mean LH levels, peak and trough values, and pulse duration in 1- and 2-week ORDX rats during the first 40 min of naloxone administration. These changes were essentially restricted to the first pulse that occurred immediately after naloxone administration. No changes in any of the parameters evaluated were observed in 8-week ORDX rats. Different regimens of testosterone replacement (capsules or sc injections) in 8-week ORDX rats significantly reduced the elevated LH value and completely suppressed pulsatile LH secretion. However, in these animals the response of LH to naloxone administration was restored in a limited number of animals among those receiving sc injections. The results indicate that the gonads play a permissive role in the inhibitory action of the endogenous opiate system on gonadotropin secretion, since a loss of sensitivity to opiate receptor blockade takes place after long term gonadectomy. The delayed loss of the response to naloxone after gonadectomy suggests that gonadal factors may have an activational and/or organizational effect on the opiate neurons involved in gonadotropin regulation. The precise mechanism(s) responsible for this loss of sensitivity is still unknown.

Estimation of endogenous adrenocorticotropin half-life using pulsatility patterns: a physiological approach to the evaluation of secretory episodes.

Several studies indicate that the disappearance rate of ACTH in different species is very rapid. The conditions under which these estimates were obtained, however, can be regarded in most cases as nonphysiological. The present studies were designed to evaluate the half-life of endogenous ACTH in rats under physiological conditions, i.e. awaken freely moving animals that were bled continuously through an indwelling iv cannula. Blood sampling at 1-min intervals allowed the detailed characterization of the ACTH pulsatile pattern. Analysis of each secretory episode indicated that many of the pulses could be used for half-life determinations, provided that the down phase of the pulse consisted of three or more points decaying in a straight line. After logarithmic transformation of the values, the declining slopes were analyzed by linear regression to determine which slopes showed a significant negative correlation. From those values the half-life estimates, in minutes, were calculated. Two groups of animals were used for these studies. In one group, blood was removed continuously without replacement. In these animals, ACTH levels rose progressively due to the hemorrhage effect, although secretion continued to be pulsatile. In a second group, blood was replaced at the same rate of withdrawal through a femoral vein, and ACTH levels remained constant throughout the experiment. The results indicate that the average ACTH half-life was 5.94 and 7.06 min for the groups without and with blood replacement, respectively. The differences between the two groups were not significant. Analysis of the frequency distribution of the calculated half-lives indicates that two populations can be separated, a fast (decays less than 8 min) and a slow one (decays greater than 8 min). Less than half of the secretory episodes showed a decay according to the half-life. This observation indicates that some secretory episodes consist of a burst of secretory activity followed by a period during which secretion is essentially turned off. Under these conditions, decays correspond to the half-life of the hormone. Other episodes show protracted secretory activity, as indicated by the slower, nonlinear decay. These observations suggest that half-life of ACTH may vary from one episode to another, and that the different types of secretory episodes recorded may reflect the multifactorial control of ACTH secretion by the hypothalamus.

Role of oxytocin on prolactin secretion during proestrus and in different physiological or pharmacological paradigms.

The present study was designed to evaluate the possible physiological role of oxytocin (OXY) on PRL release by examining the effect of administration of potent pharmacological antagonists of OXY on the stimulation of PRL secretion observed in vitro from anterior pituitary (AP) cells in response to OXY administration or in a number of in vivo paradigms. OXY caused a dose-related increase in PRL release from dispersed AP cells and short term AP cell cultures which was blocked by administration of the OXY antagonists [1-deaminopenicillamine, 2-O-methyltyrosine, 8-ornithine]vasotocin (dPOMeOVT) or [1-(beta-mercapto-beta,beta-cyclopentamethylene propanoic acid)2-O-methyltyrosine, 8-ornithine]vasotocin (MPOMeOVT), respectively. The antagonists were given in vivo in a dose that completely blocked suckling-induced milk let-down for up to 90 min. Injection of the antagonists did not alter the 5-hydroxytryptophan-induced increase in plasma PRL or the increase associated with acute ether stress or acute suckling stimuli, suggesting that OXY is not a major component involved in the neuroendocrine mechanisms responsible for those particular increases. On the other hand, iv administration of dPOMeOVT or MPOMeOVT prevented the increase in plasma PRL normally observed on the afternoon of proestrus in the cycling female rat. The characteristic surge of LH was also blocked by high doses of these antagonists. These data demonstrate that PRL secretion undergoes a differential regulation, in that OXY appears to play a major role in regulating the increase in plasma PRL observed on the afternoon of proestrus, but apparently provides little, if any, contribution toward the neuroendocrine regulation of the increases in PRL associated with 5-hydroxytryptophan administration, acute ether stress stimulus, or acute suckling stimulus. The data also suggest that OXY receptors located in the AP that are involved in the OXY-induced increase in PRL release may be similar to those OXY receptors located in mammary and uterine tissue, since specific biological effects of OXY in those tissues are effectively blocked by the OXY antagonists dPOMeOVT and MPOMeOVT. A possible role of OXY neurons in the neural mechanisms triggering the LH surge during proestrus is also suggested.

Endogenous inhibin suppresses only basal follicle-stimulating hormone secretion but suppresses all parameters of pulsatile luteinizing hormone secretion in the diestrous female rat.

The purpose of these studies was to ascertain which parameters of pulsatile gonadotropin secretion are regulated by endogenous inhibin in the intact diestrous female rat. This was determined by examining the changes in the secretion parameters of FSH and LH that resulted from immunoneutralizing endogenous inhibin in diestrous I female rats. Passive immunoneutralization of endogenous inhibin was achieved using specific, high titer ovine antiserum generated against the alpha-subunit of the recently described inhibin molecule. The optimal times after inhibin immunoneutralization to observe the changes in FSH secretion were determined in initial experiments. Pulsatile secretion of both FSH and LH was observable in the diestrous female. Two hours after inhibin immunoneutralization, the mean trough level, mean peak level, and overall mean level of FSH began to increase. The maximal increase and plateau of these parameters were observed 5 h after antiserum injection. During the period of increase, mean FSH pulse amplitude was also increased, but returned to the level observed in control (normal sheep serum-injected) animals when the parameters of trough, peak, and overall mean FSH reached their plateau levels. FSH pulse frequency was not changed at any time. These results indicate that endogenous inhibin affects only the basal parameters of FSH secretion without affecting pulsatile FSH secretion. The transient increase in FSH pulse amplitude resulted from FSH pulses being superimposed on the increasing basal FSH secretion. In contrast, immunoneutralization of endogenous inhibin rapidly increased all parameters (i.e. pulse amplitude and frequency, mean trough and peak levels, and mean plasma levels) of LH secretion. In addition, pituitary sensitivity to an exogenous LHRH challenge was increased in inhibin-immunoneutralized females in terms of stimulated LH secretion. As a result of the already increased rate of basal secretion, the actual quantity of FSH released in response to the LHRH challenge was greatly increased in the inhibin-immunoneutralized rats compared with the normal sheep serum-injected controls; however, the increase in the rate of FSH secretion stimulated by the LHRH challenge was the same in both groups. The observations from these studies collectively demonstrate that inhibin acts endogenously to suppress those parameters of gonadotropin secretion that are regulated by LHRH.

Maturation of the prolactin and proopiomelanocortin-derived peptide responses to ether stress and morphine: neurochemical analysis.

The hormonal and neurochemical responses to acute ether stress, morphine, and/or naloxone were analyzed in infantile (13-day-old) and prepubertal (36-day-old) male CD rats in an attempt to identify a possible neurochemical correlate(s) for the previously demonstrated requisite maturation of the PRL response to ether stress. Neuronal serotonin (5-HT), norepinephrine (NE), and dopamine (DA) activities were examined in the medial preoptic hypothalamic area (MPOH), medial basal hypothalamic area (MBH), and median eminence (ME). Ether stress increased plasma PRL, ACTH, and beta-endorphin-like immunoreactivity (beta end) as well as NE metabolism in the MPOH and MBH and neuronal 5-HT activity in the MBH, and decreased neuronal DA activity in the ME of prepubertal animals. Ether stress elicited similar changes in infantile animals, with the important exceptions that plasma PRL, neuronal 5-HT activity in the MBH, and neuronal DA synthesis in the ME were not affected at this earlier age. Morphine increased plasma PRL, ACTH, and beta end levels, elevated neuronal NE and 5-HT activities in the MPOH and MBH, and decreased DA synthesis in the ME in both infantile and prepubertal animals. Naloxone administration did not alter basal hormone concentrations or neuronal monoamine activity in any brain area, but did prevent all of the morphine-induced changes as well as the ether stress-induced changes in PRL, MBH neuronal 5-HT activity, and DA synthesis in the ME of prepubertal animals. In addition, naloxone augmented the ether stress-induced increases in ACTH and beta end in prepubertal rats. Indirect stimulation of 5-HT neurons by administration of the amino acid precursor of 5-HT, 5-hydroxytryptophan, resulted in decreased DA synthesis in the ME of infantile animals and increased plasma PRL levels in that age group, indicating that this portion of the neurochemical connection is already present in infantile animals. Furthermore, the 5-hydroxytryptophan-induced increase in PRL was blocked by pretreatment with naloxone. The results demonstrate that both the ether stress- and morphine-induced increases in plasma PRL, but not in ACTH or beta end, are associated with increased neuronal 5-HT activity in the MBH and a decreased neuronal DA activity in the ME, that these are opiate receptor-mediated effects, and that infantile rats apparently lack a functional opiate-5-HT connection, which matures some time between days 13 and 36 postnatally.

Proopiomelanocortin-derived peptides in testicular interstitial fluid: characterization and changes in secretion after human chorionic gonadotropin or luteinizing hormone-releasing hormone analog treatment.

Recent studies indicate that proopiomelanocortin (POMC)-derived peptides are present in the testis and may be involved in the regulation of gonadal function, although their precise role still remains obscure. In the present study, we investigated in the rat whether testicular interstitial fluid (TIF), a functionally important compartment of the testis, contains POMC-derived peptides. Adult rats were used for all the experiments, and TIF was collected from each individual testis and analyzed for the presence of ACTH and beta-endorphin-like immunoreactivity (beta end-LI). Initial studies indicated that both ACTH and beta end-LI can be readily detected in TIF from intact rats, and that the concentrations of these peptides are severalfold higher than those in the peripheral plasma of the same animals. Similar studies in rats 3-5 days after hypophysectomy indicate that although ACTH and beta end-LI levels in plasma were undetectable, high levels of both peptides were measured in TIF. Moreover, serial dilution curves of TIF showed parallelism with the respective standard curves in the beta end and ACTH assays. Additional studies indicated that levels of POMC peptides in TIF were modified by treatments that affect the endocrine function of the testis. In that respect, hCG given sc to hypophysectomized rats increased testosterone levels in TIF and plasma, and similarly increased beta end-LI concentrations in TIF. A LHRH analog (LHRH-A; [D-Ala6,des-Gly10]LHRH-ethylamide) given sc to hypophysectomized rats resulted in increased testosterone production, but decreased beta end-LI in TIF, suggesting that the effects of hCG and LHRH-A on testicular beta end-LI secretion are not directly coupled with testosterone production. Prolonged treatment of intact rats with the LHRH-A for 2-10 days resulted in progressive declines in testicular weight and testosterone levels in TIF and in plasma, and a significant suppression of beta end-LI levels in TIF as early as 2 days after analog treatment. On the other hand, acute intratesticular injection of LHRH-A to intact rats (5 ng into the right testis) produced a short-lived increase in beta end-LI in TIF 2 h after injection, coincident with a temporary decrease in TIF volume. These results indicate that the POMC-derived peptides ACTH and beta end are present in TIF and are secreted locally into this compartment, supporting previous reports demonstrating the presence and local synthesis of POMC peptides in testicular tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Activation of gamma-aminobutyric acid B-receptors abolishes naloxone-stimulated luteinizing hormone release.

Recent evidence suggests that the neurotransmitter gamma-aminobutyric acid (GABA) plays an important role in the control of gonadotropin secretion. The present study was conducted to identify the effect and site of action of different GABAergic drugs on LH secretion in vivo and to characterize the precise roles of different GABA receptors in these actions. Three different GABAergic drugs were used: muscimol and baclofen, which act at the level of the GABA A- and GABA B-receptors, respectively, and aminooxyacetic acid (AOAA), which increases the GABA content in the brain. The effects of these drugs were investigated in situations of enhanced LH secretion due to administration of naloxone or LHRH. In an initial experiment, adult male rats were treated ip with AOAA, followed by naloxone. AOAA treatment decreased basal LH levels and prevented naloxone-stimulated LH release. PRL levels were decreased by either AOAA or naloxone; however, the combination of these two drugs did not induce an additional or synergistic effect on the decreased PRL levels. In subsequent experiments, freely moving rats bearing Silastic cannulae in the right jugular vein received AOAA, muscimol, or baclofen a few minutes before either naloxone or LHRH administration. Baclofen and AOAA completely suppressed the naloxone-stimulated LH increase. Muscimol did not prevent the effect of naloxone. None of the three GABAergic drugs affected LH release in rats receiving LHRH. The results of these in vivo experiments suggest that the GABAergic system exerts primarily an inhibitory effect on gonadotropin secretion which is mediated at a central level, since pituitary responsiveness to LHRH is not affected by GABAergic drug treatment. GABA B-receptors are responsible for the inhibitory action of GABA.

Pulsatile follicle-stimulating hormone secretion is independent of luteinizing hormone-releasing hormone (LHRH): pulsatile replacement of LHRH bioactivity in LHRH-immunoneutralized rats.

We recently reported that passive immunoneutralization of endogenous LHRH in castrate male rats completely abolishes pulsatile LH secretion and, within 1 h, lowers mean plasma LH by 86%. While pulsatile FSH secretion, in terms of pulse amplitude and frequency, is not affected, mean plasma FSH is gradually lowered but only by 49% after 24 h. In the present study, we have examined the effect of replacing pulsatile LHRH biological activity on LH and FSH secretion in 4-week castrate male rats in which endogenous LHRH has been immunoneutralized by ovine anti-LHRH serum 772 (LHRH-AS) for 24 h. The LHRH-AS requires the 3-10 amino acid sequence of LHRH including the amidated C terminus for complete recognition. In order to circumvent the antiserum blockade, we utilized the LHRH agonist [Des Gly10]-LHRH ethyl amide (DG-LHRH) which is minimally recognized by the LHRH-AS but which possesses 2.6-fold the LH-releasing activity of LHRH. Twenty-four hours after injecting 500 microliter LHRH-AS into cannulated, castrate rats, sequential blood samples were taken every 10 min for 4 h. Bolus 3-ng injections of either DG-LHRH or saline were given iv either every 30 min during the 4-h collection period or every 30 or 60 min for 10 h before the initiation of and continuing through the 4-h collection period. Each DG-LHRH injection stimulated the release of a single pulse of LH, while pulsatile FSH secretion was unaffected. No synchrony was observed between the DG-LHRH pulses and the endogenous FSH pulses. Short term DG-LHRH treatment partially restored, and long term DG-LHRH treatment every 60 min completely restored, mean plasma FSH to the level observed in nonantiserum-treated castrate control rats. Long term DG-LHRH treatment every 30 min caused a rise in mean plasma FSH which exceeded the plasma FSH level of the nonantiserum-treated controls. The mean plasma level of LH was entirely dependent on the frequency of the DG-LHRH injection. The results of this study clearly demonstrate that pulsatile FSH secretion is independent of LHRH but that LHRH is required to elevate and/or maintain high mean plasma FSH levels. Trough levels of LH, however, are dependent on the frequency of LHRH-induced pulsatile LH secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Testosterone selectively influences protein kinase-C-coupled secretion of proluteinizing hormone-releasing hormone-derived peptides.

LHRH and GnRH-associated peptide (GAP) are two major pro-LHRH-derived peptides which are secreted from median eminence (ME) nerve terminals in vitro. The purpose of the present experiment was to determine whether manipulation of gonadal steroid levels in vivo influenced selectively the in vitro secretion of LHRH and GAP under basal or K+ and phorbol ester (PDBu) stimulation. Secretion of both peptides under each of these three conditions was reduced at least 2-fold in 2-week orchidectomized (ORDX) rats relative to the level in intact controls. Tissue stores of LHRH and GAP were also depressed in the ME of ORDX relative to control rats. When the data were expressed in terms of the percentage of peptide secreted per ME, both groups secreted similar percentages of the peptides into the medium under basal and K+-stimulated conditions. Interestingly, PDBu-activated secretion of LHRH and GAP remained depressed in ORDX animals. The nerve terminals from ORDX animals were not susceptible to a more rapid depletion of releasable peptides, since both groups secreted similar percentages of the peptides during repeated K+ depolarization. By comparison, protein kinase C (PKC)-coupled secretion from ORDX rats was selectively affected, since secretion of pro-LHRH-derived peptides became even more depressed with successive activation with PDBu. Immediate replacement with testosterone after ORDX fully restored the peptide levels in tissue and the LHRH and GAP secretory response to PKC activation. Since testosterone influenced both tissue stores and PDBu-stimulated secretion of LHRH and GAP, this steroid may selectively regulate biosynthesis and secretion of pro-LHRH-derived peptides through activation of the metabolic cascade involving the PKC system.

Arginine vasopressin and adrenocorticotropin release: correlation between binding characteristics and biological activity in anterior pituitary dispersed cells.

The present studies were designed to evaluate the binding characteristics of arginine vasopressin (AVP), using rat anterior pituitary dispersed cells, in correlation with the biological activity of the peptide. Synthetic AVP released ACTH from dispersed anterior pituitary cells in a concentration-dependent fashion, with a minimal effective dose of 10(-10) M. [3H] AVP, the ligand for binding studies, showed full biological activity at different concentrations. Saturable and high affinity binding sites for [3H]AVP were obtained using dispersed anterior pituitary cells. Specific binding reached equilibrium by 180 min at 22 C, and rapid and complete dissociation was obtained after the addition of excess AVP. Scatchard analysis of the data indicated a single class of binding sites, with an apparent Kd of 1.63 nM and a binding capacity (Bmax) of 10.7 fmol/10(6) cells, at 37 C. When cells were incubated at 22 C, Kd (7.63 nM) and Bmax (39 fmol/10(6) cells) values were within the same order of magnitude. AVP effectively inhibited [3H]AVP binding with an IC50 of 1.5 X 10(-7) M, while oxytocin showed a somewhat higher IC50 (0.9 X 10(-6) M) and did not achieve complete inhibition of binding. An AVP analog with a ring substitution ( Asu1 ,6 AVP) showed decreased displacement capacity. Both oxytocin and the AVP analog showed weak ACTH-releasing activity compared to synthetic AVP. This indicates that modifications in the tail and/or ring portion of the AVP molecule reduce both the binding affinity and ACTH-releasing activities of these peptides. Other structurally unrelated peptides with intrinsic ACTH-releasing activity, such as rat corticotropin-releasing factor and angiotensin II, had no affinity for AVP-binding sites. The results indicate that specific AVP-binding sites in anterior pituitary cells are closely correlated with the intrinsic ability of the peptide to elicit ACTH release.

Galanin stimulates luteinizing hormone-releasing hormone secretion from arcuate nucleus-median eminence fragments in vitro: involvement of an alpha-adrenergic mechanism.

Galanin (GAL), a 29-amino acid peptide originally isolated from porcine intestine, has been shown to be widely distributed not only in the gut, but also in the central nervous system. Several studies have shown that GAL participates in the hypothalamic regulation of PRL, GH, and LH secretion. In this study, we evaluate the effects of rat GAL (rGAL) on LHRH and prostaglandin (PG) E2 release from arcuate nucleus-median eminence fragments in vitro. Fragments were incubated for 30-min periods in Krebs-Ringer bicarbonate buffer containing the different test substances. The addition to the medium of rGAL in concentrations ranging from 5-1000 nM increased the release of both LHRH and PGE2 in a concentration-dependent manner. The ED50 values were approximately 55 and 80 nM for LHRH and PGE2, respectively. rGAL-induced LHRH and PGE2 release were related, as suggested by the finding that the addition to the medium of indomethacin (10 microM), a PG synthesis blocker, completely blocked rGAL-induced LHRH release. In addition, an active catecholaminergic system appears to be necessary for obtaining the stimulatory effect of rGAL. The addition to the medium of the alpha-adrenergic blocker phentolamine or prazosin impaired the ability of rGAL to release both LHRH and PGE2. rGAL-induced stimulation of LHRH and PGE2 release was blocked by phentolamine at doses of 1-10 microM, while prazosin was able to block it at doses as low as 0.1 microM. In summary, rGAL stimulates LHRH and PGE2 release from arcuate nucleus-median eminence fragments in vitro in a dose-dependent fashion. Such an effect is blocked by both indomethacin and alpha-adrenergic blockers, indicating that rGAL-induced stimulation of LHRH secretion is exerted through alpha-adrenergic receptors and requires PGE2 as an intracellular mediator.

1,2-Didecanoylglycerol and phorbol 12,13-dibutyrate enhance anterior pituitary hormone secretion in vitro.

Experiments were designed to evaluate the role of activators of protein kinase C, such as 1,2-diacylglycerol and phorbol esters, on the release of all the anterior pituitary (AP) hormones in vitro. Dispersed rat AP cells were incubated in the presence of 1,2-didecanoylglycerol (DiC10), a synthetic diacylglycerol, or phorbol 12,13-dibutyrate (PDBu), a tumor-promoting phorbol ester, at different concentrations and for varying periods of time. ACTH and beta-endorphin (beta-End) secretion were enhanced by DiC10 in a concentration-dependent manner, with a minimal effective concentration of 5 microM. PDBu at 5 nM produced a significant release of both ACTH and beta-End. The effect of DiC10 and PDBu was time dependent, with maximal responses occurring at 15-30 min for DiC10 and 30-60 min for PDBu. Release of GH was also enhanced significantly by DiC10 and PDBu, with minimal effective concentrations of 1 microM and 1 nM, respectively. Maximal release of GH was already attained within 15 min with DiC10 or 60 min with PDBu. In additional experiments, the effects of DiC10 and PDBu on secretion of LH, FSH, PRL, and TSH were evaluated. The results indicate that 5-25 microM DiC10 produced a concentration-dependent release of each of those hormones, and that 5 microM was the minimal effective concentration in every case. Nearly maximal stimulation was achieved within 15 min for each hormone. PDBu (50 nM) significantly enhanced LH, FSH, PRL, and TSH release within 30 min. Although qualitatively all hormones were similarly stimulated, both with respect to time and concentration, some quantitative differences were observed. ACTH and beta-End release were enhanced 100% by DiC10 and 300% by PDBu, whereas the increase in other hormones was of a lesser magnitude. The present study indicates that two specific stimulators of protein kinase C, diacylglycerol and phorbol ester, can enhance secretion of all AP hormones in a concentration- and time-dependent manner. This suggests that formation of endogenous 1,2-diacylglycerol may represent a physiological intracellular messenger in the events leading to AP peptide hormone release.

Evidence that pulsatile follicle-stimulating hormone secretion is independent of endogenous luteinizing hormone-releasing hormone.

Although LHRH can stimulate the release of both LH and FSH from the pituitary, there are a number of instances in which the secretion of LH and FSH are divergent. Previous studies from our laboratory have indicated that pulsatile LH and FSH secretion are independently regulated by gonadal factors. We have, therefore, reexamined the role of LHRH in regulating pulsatile gonadotropin secretion by evaluating the effect of passive LHRH immunoneutralization on LH and FSH secretion in castrate adult male rats. Injection of 500 microliters ovine anti-LHRH serum no. 772 (LHRH-AS) into 2-week-castrate rats caused an 85% suppression of mean plasma LH levels by 2 h, which lasted through 48 h. Mean plasma FSH, however, was reduced by only 19% after 2 h and by only 59% after 48 h. When cannulated 2-week-castrate rats were bled every 10 min, both LH and FSH were secreted in a pulsatile manner. Injection of 500 microliters LHRH-AS caused an immediate abolishment of LH pulses and a rapid reduction in mean plasma LH through 24 h. Pulsatile FSH secretion, as characterized by the parameters of pulse frequency and amplitude, was unaffected by LHRH-AS, although mean plasma FSH levels were significantly reduced. Collectively, the results suggest that pulsatile FSH secretion is regulated by a separate factor(s) distinct from LHRH, but that LHRH is required for the maintenance of elevated FSH levels.

Prostaglandin E2-induced luteinizing hormone-releasing hormone release involves mobilization of intracellular Ca+2.

The present in vitro experiments were performed to examine the involvement of Ca+2 in the mechanism by which prostaglandin E2 (PGE2) induces LHRH release from the median eminence (ME) of the hypothalamus. Stepwise decreases in the Ca+2 concentration of the incubation medium reduced the LHRH response to PGE2. Nevertheless, neither complete omission of Ca+2 (residual Ca+2 concentration, 3.5 microM) nor chelation of residual Ca+2 with EGTA prevented the stimulatory effect of the PG, suggesting that a significant portion of the PGE2 effect on LHRH release is independent of extracellular Ca+2. Blockade of Ca+2 influx with verapamil, a Ca+2 entry blocker, demonstrated that this component of the PGE2 effect is completely independent of inward Ca+2 movement. Depletion of intraterminal Ca+2 stores by exposure of the MEs to the Ca+2 ionophore A23187 in medium without Ca+2 containing EGTA almost completely obliterated the subsequent LHRH response to PGE2, indicating that normal intraterminal Ca+2 levels are important for the PGE2 effect to occur. Preloading the ME terminals with 45Ca+2 and subsequent stimulation with PGE2 demonstrated that even in the absence of extracellular Ca+2, PGE2 stimulates Ca+2 efflux from the terminals, and this Ca+2 movement occurs temporarily associated with LHRH release. Depolarization of ME terminals with 56 mM K+ in the presence of normal Ca+2 concentration resulted in massive efflux of 45Ca+2 and a greater LHRH response than that produced by PGE2, suggesting that the effect of PGE2 is not the consequence of a nonspecific general depolarization of ME nerve terminals. Thus, although a full LHRH response to (exogenous) PGE2 necessitates normal extraterminal Ca+2 concentrations, the results indicate that translocation of Ca+2 from intracellular stores is an event involved in the mechanism by which PGE2 releases LHRH.

Nitric oxide regulates luteinizing hormone-releasing hormone secretion.

We have analyzed the role of nitric oxide (NO), an unorthodox and novel neuromodulator, on luteinizing hormone-releasing hormone (LHRH) secretion. Sodium nitroprusside (SNP), an NO donor, was used to challenge LHRH neurons using both hypothalamic explants and an immortalized neuronal cell line (GT1 cells) in vitro. In both paradigms, SNP was able to stimulate LHRH release in a dose-dependent manner. This action of SNP was accompanied by an elevation in both extra- and intra-cellular cGMP levels. In addition, exposure of LHRH cells (GT1-7 cells) to increasing concentrations of a soluble analog of cGMP (8-Br-cGMP) enhanced LHRH release in a dose-dependent manner, indicating that LHRH neurons have the intrinsic ability to respond to the intracellular messenger elicited by NO, i.e., cGMP. Furthermore, sodium nitroprusside-induced LHRH secretion from GT1-7 cells was blocked, in a dose-dependent manner, by Rp-8-Br-cGMPS, a cGMP analog which blocks cGMP-dependent protein kinase. These data clearly demonstrate that NO stimulates LHRH secretion by activating guanylate cyclase, and support a potential role of NO as a neuroactive agent involved in the control of LHRH secretion and, thereby, reproductive functions.

Transmembrane signals mediating neural peptide secretion: role of protein kinase C activators and arachidonic acid metabolites in luteinizing hormone-releasing hormone secretion.

The present experiments were designed to determine the effects of different activators of protein kinase C on the secretion of LHRH from median eminence nerve terminals incubated in vitro. The release of prostaglandin E2 (PGE2), a metabolite of arachidonic acid intimately involved in the secretion of LHRH, was also evaluated. Synthetic diacylglycerol [1,2-didecanoylglycerol (DiC10)] significantly enhanced PGE2 release in a concentration-dependent manner. Blockade of phospholipase A2 (PLA2) activity nullified this effect. LHRH release, on the other hand, was not increased by DiC10. However, in the presence of a lipoxygenase inhibitor, DiC10 produced a concentration-related increase in LHRH release, which paralleled that in PGE2. Phospholipase C (PLC) increased both PGE2 and LHRH secretion. Again, blockade of the lipoxygenase pathway enhanced the release of LHRH by PLC without affecting the stimulated secretion of PGE2. A phorbol ester, phorbol 12,13-dibutyrate (PDBu), markedly increased LHRH secretion while inducing a modest increase in PGE2 release. Both effects of PDBu were unaffected by lipoxygenase inhibition. DiC10, PDBu, and PLC significantly augmented LHRH secretion from tissues in which metabolism of arachidonic acid had been prevented by inhibition of both cyclooxygenase and lipoxygenase pathways, suggesting that activation of protein kinase C, independent of PLA2 activation, can lead to the secretion of this neural peptide. Some lipoxygenase metabolites had either no effect on [5- and 15-hydroxyeicosatetraenoic (5- and 15-HETE)] or induced a marginal stimulation of (12-HETE) LHRH release. At certain concentrations, 12-HETE enhanced the stimulatory effect of the phorbol ester on LHRH release. Our results suggest that activation of protein kinase C can stimulate LHRH secretion from nerve terminals in vitro and, further, that diacylglycerol may represent an important intracellular messenger participating in the events leading to LHRH secretion. In addition, stimulation with DiC10 and PLC uncovered inhibitory [unknown arachidonic acid metabolite(s) via lipoxygenase] and stimulatory (PGE2 via cyclooxygenase) pathways through with arachidonic acid metabolites may participate in the intracellular transduction of signals modulating neural peptide secretion.

Differential expression of gamma-aminobutyric acid receptors in immortalized luteinizing hormone-releasing hormone neurons.

gamma-Aminobutyric acid (GABA) has been shown both to stimulate and inhibit LH secretion in vivo. GABA apparently exerts these effects at the hypothalamic level by regulating the release of LHRH. In this study, we have investigated the effect of GABAergic agents on LHRH secretion from an immortalized hypothalamic neuronal cell line (GT1-7). LHRH secretion was stimulated in a dose-dependent manner with increasing concentrations of GABA. This effect was mimicked by the GABAA receptor agonist, muscimol, and was blocked by the selective antagonist, bicuculline. The stimulatory effect of muscimol on LHRH secretion was synergistic with low concentrations of [K+]. By comparison, neither activation of the GABAB receptors with baclofen nor blockade with phaclofen influenced basal LHRH secretion. Baclofen, however, did depress [K+]-induced LHRH release. Binding studies confirmed the presence of GABAA and GABAB receptors on GT1-7 cells. In addition, Northern blots with probes to the GABAA receptor alpha 1, beta 3, and gamma 2L subunits revealed that only the beta 3 messenger RNA (mRNA) was expressed in the GT1-7 cells. These data provide the first demonstration that immortalized LHRH neurons are directly responsive to GABAergic agents. To the extent that these immortalized neurons may resemble those in vivo, our results suggest that GABAergic agents may play a dual role in reproductive physiology by exerting both stimulatory and inhibitory control over LHRH release.