Immediate release of prolactin and biphasic effects on growth hormone release following electrical stimulation of the median eminence.

The median eminence (ME) region of the hypothalamus was electrically stimulated through permanently implanted electrodes in 1 unanesthetized sheep. Plasma concentrations of GH and prolactin (PRL) were measured at 10-min intervals before, during and after 30-min periods of electrical stimulation or sham procedures. The onset of ME stimulation decreased plasma GH in all 17 cases in which prestimulation GH levels were 1.0 ng/ml or higher. In all 27 cases of ME stimulation, plasma GH never increased during the 30-min stimulation period. However, spontaneous GH increases were noted during sham procedures in 9 of 31 cases. The termination of ME stimulation was followed within 10--20 min by markedly increased GH levels in 24 of 27 cases. All 4 ewes responded to electrical stimulation with comparable biphasic effects on GH release even though electrode locations varied slightly. These results indicate localization and stimulation-induced release of endogenous neurohormones in the ME region with activities inhibiting the release of GH (GHIF). The secondary increase in plasma GH following the end of ME stimulation indicates a reversal of conditions unfavorable to GH release. This reversal may represent a rebound from the inhibitory action of GHIF or the post-inhibitory action of a hypothalamic GH-releasing factor or both. In 3 ewes with electrodes implanted in the region of the anterior ME, the onset of stimulation increased plasma PRL within 10--20 min in 17 out of 20 cases. The electrode in the fourth animal was located in the posterior ME, and when all cases were considered, stimulation through this electrode did not significantly increase or decrease plasma PRL. However, in 5 out of 7 cases stimulation of the posterior ME decreased plasma PRL. In summary, these results indicate the presence in the anterior ME of andogenous neurohormones with PRL-releasing activity (PRF). Conclusions about the posterior ME are equivocal, but this region may contain a PRL-release inhibiting compound in combination with PRF.

Prolactin secretion after hypophysial stalk transection in pigs.

Hypothalamic regulation PRL secretion was investigated in 13 7-month-old Yorkshire gilts by comparing the effects of hypophysial stalk transection and sham operation. Ovariectomized gilts were fitted with an indwelling cannula in the anterior vena cava to determine sequential serum profiles of PRL secretion before, during, and 190 h after cranial surgery. A nylon disc was inserted between severed ends of the hypophysial stalk to prevent vascular and tissue regeneration; sham operations included all surgical procedures with exception of stalk transection and insertion of the disc. During a preoperative period of 120 min, PRL concentrations in peripheral serum remained consistently low [2.3 +/- 0.3 ng/ml (mean +/- SEM)] in all gilts. During 105 min of anesthesia (induced by thiamylal sodium and maintained by halothane and oxygen), PRL increased (8.2 +/- 1.9 ng/ml) in all gilts. Peak PRL values averaged 10.7 ng/ml at hypophysial stalk transection or sham operation and then declined steadily in both groups during the last 105 min of surgery. PRL remained elevated (P less than 0.002) in hypophysial stalk-transected gilts compared with sham-operated controls throughout a postoperative period from 6-190 h (3.7 +/- 0.4 vs. 1.9 +/- 0.2 ng/ml). These results indicate that basal secretion of PRL in the pig is inhibited by the hypothalamus.

cDNA sequence and expression of bovine prodynorphin.

Prodynorphin (ProDYN) in the anterior pituitary gland appears to be processed differently from the brain, and the ProDYN-derived peptides may function differently in the anterior pituitary than in the brain. To further investigate the roles of ProDYN-derived peptides in the anterior pituitary, we have determined the nucleotide (nt) sequence of the cDNA encoding bovine ProDYN. This is the first time a complete cDNA sequence for ProDYN has been reported. The nt and deduced amino acid (aa) sequences were compared to the known ProDYN of other species. Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) combined with Southern blot analysis demonstrated that the expression of ProDYN in both the anterior and posterior pituitary glands was much lower than that in the neural tissues of the striatum and hypothalamus.

Effects of naloxone and electroacupuncture treatment on plasma concentrations of LH in sheep.

Mature ewes were injected intravenously with the opioid antagonist naloxone (1.1 mg/kg) during the breeding season. Ewes with luteal phase concentrations of plasma progesterone responded with a significant (P less than 0.05) increase in plasma LH 14-23 min after naloxone injection. In contrast, non-luteal ewes with low plasma progesterone did not respond to injection of naloxone with an LH increase. Similar treatment of castrated males (wethers) with this dosage of naloxone failed to increase plasma LH. Electroacupuncture (EA) treatment of luteal phase ewes prevented the ability of exogenous naloxone to increase plasma LH. Treatment of wethers by EA decreased significantly (P less than 0.01) their high basal concentrations of plasma LH, but similar EA treatment of intact ewes did not change their low basal concentrations of LH.

Transcription of prolactin gene in milk secretory cells of the rat mammary gland.

In-situ hybridization and Northern blot hybridization were used to identify mRNA for pituitary prolactin in mammary tissue obtained from female rats 1 day before expected parturition, 1 day after parturition and on day 7 of lactation. Prolactin cDNA was labelled with 32P for Northern analysis and with digoxigenin for in-situ hybridization. Total and poly(A)+ RNA from pituitary, mammary and control (fat and kidney) tissues were analysed by agarose gel electrophoresis with transfer to nitrocellulose and hybridization to a cDNA for rat prolactin. Although present in much smaller amounts than the 1.0 kb transcript in pituitary RNA homogenates, mammary RNA homogenates from all three stages contained mRNA of approximately 1.0 kb which hybridized with the prolactin probe. Similar analyses of fat and kidney failed to reveal any hybridization at the 1.0 kb size. When tissue sections were hybridized to the cDNA probe, specific hybridization was observed in the milk secretory cells of the mammary alveoli and the lactotroph cells of the anterior pituitary, but not in liver cells or in RNAase-treated sections of mammary tissue. In summary, these results demonstrate that milk secretory cells of the rat mammary gland transcribe the gene for prolactin, and they raise the possibility that a primary target tissue for blood-borne prolactin may also synthesize prolactin.

Immunoreactive dynorphin-A in bovine anterior pituitary and its in vitro release.

The anterior lobe (AL) of the bovine pituitary contained and released, during in vitro culture, a form of immunoreactive dynorphin-A (ir-DYN-A) larger than that occurring in neural tissue. Bovine AL tissue from intact females contained less ir-DYN-A than did AL tissue from castrated males. Enzymatically dispersed AL cells contained and released ir-DYN-A in vitro. Preincubation of dispersed AL cells for 18 hr, rather than 1.5 hr, increased the content and release of ir-DYN-A as well as LH. Addition of gonadotropin-releasing hormone (GnRH) to tissue slices or dispersed cells stimulated release of LH, but in contrast to published observations from rat AL, GnRH had no effect on release of ir-DYN-A. Addition of estradiol-17 beta, with or without progesterone, increased release of ir-DYN-A but not LH during 2-hr cultures. In summary, bovine AL contains and releases in vitro a large molecular weight form of ir-DYN-A. Although this ir-DYN-A was not coreleased with LH, a reproductive role was suggested by in vivo and in vitro effects of gonadal hormones on ir-DYN-A in the bovine anterior pituitary.

Immunoreactive dynorphin-A in ovine anterior pituitary and effects of estradiol-17 beta administration.

Anterior lobe (AL) tissue of the ovine pituitary gland contained a form of immunoreactive dynorphin-A (ir-DYN-A) larger than that found in pituitary neurointermediate lobe. Administration of estradiol-17 beta or estradiol-17 beta plus progesterone to ovariectomized sheep decreased AL tissue concentrations of ir-DYN-A but did not affect any LH parameter. Enzymatically dispersed AL cells also contained ir-DYN-A, but specific release during in vitro incubation was too low to be detected even when cells were exposed to gonadotropin-releasing hormone.

CNS tissue levels of dynorphin-A immunoreactivity and the anorexia associated with sodium chloride imbibition in the rat.

Forced imbibition of increasing concentrations of sodium chloride (NaCl) in rats reduced daytime 2-deoxy-D-glucose (2-DG) induced feeding in a concentration dependent manner. Pituitary neurointermediate lobe (NIL) levels of immunoreactive (ir)-dynorphin-A 1-17 and 1-8 were also decreased by the NaCl regimen in a concentration dependent manner. However, there was no significant association between the reduction of NIL dynorphin levels and the suppression of 2-DG induced feeding on a within-animal basis. NaCl imbibition did not affect levels of either ir-dynorphin-A 1-17 or 1-8 in the hypothalamus, cerebral cortex, hippocampus, medulla/pons or anterior pituitary. Neither the acute changes following 2-DG administration, nor the comparison of ir-dynorphin-A 1-8/1-17 ratios appeared useful for the assessment of dynorphin-A turnover. Thus, the present results did not support the hypothesis that anorexia of NaCl treated animals results from the depletion of dynorphin-A.

Experimental dissociation of food intake and plasma beta-endorphin following 2-deoxy-D-glucose in rats.

The present studies were undertaken to further assess the role of plasma beta-endorphin (beta-EP) in the hyperphagia induced by the glucose antimetabolite, 2-deoxy-D-glucose (2-DG). Plasma concentrations of immunoreactive beta-EP (ir-beta-EP) were measured at the end of the first hour of feeding in all animals treated with 400 mg/kg 2-DG. Previous studies had shown a consistent, positive association between 2-DG hyperphagia and plasma ir-beta-EP concentrations, but the present data revealed dissociations between hyperphagia and plasma ir-beta-EP. Dexamethasone administration blocked the 2-DG-induced rise in plasma ir-beta-EP, but had no effect on the 2-DG hyperphagia measured at 1 hour. Forced drinking of a 2% NaCl solution decreased 2-DG hyperphagia, but not the 2-DG induced rise in plasma ir-beta-EP. Thus, elevations in plasma ir-beta-EP are not necessary for the full expression of 2-DG-induced hyperphagia in dexamethasone-treated rats. Furthermore, decreased feeding responses to 2-DG could coexist with increased levels of plasma ir-beta-EP in NaCl-treated normal rats. Elevations in plasma ir-beta-EP do not appear to be the critical opiate link in 2-DG induced hyperphagia.

Plasma cortisol and beta-endorphin in horses subjected to electro-acupuncture for cutaneous analgesia.

Electro-acupuncture (EA) treatment of horses to induce cutaneous analgesia also increased plasma concentrations of beta-endorphin (beta-EP) and cortisol. The magnitude of these increases did not relate consistently to the degree of EA-induced analgesia. Respiration and heart rates were also markedly increased during EA treatment. Intact female horses had higher packed cell volume and plasma beta-EP as well as lower plasma total protein than castrated male horses. Plasma cortisol, heart rate, and respiration rate did not differ significantly between sexes. None of the parameters measured before or during EA treatment provided an explanation for the differential cutaneous analgesia which depended on sex of subject and locus of stimulation as reported elsewhere.

Relationship between plasma concentrations of immunoreactive beta-endorphin and food intake in rats.

Periods of increased food intake in male rats were characterized by significant elevations in the plasma concentrations of immunoreactive beta-endorphin (beta-ep). Administration of 2-deoxy-D-glucose (400 mg/kg) produced rapid and concurrent increases in both food intake and plasma beta-ep. Administration of insulin (10 units/kg) produced large delayed increases in food intake but only modest delayed increases in plasma beta-ep. Spontaneous nocturnal feeding was associated with increased plasma beta-ep. Increases in daytime food intake in rats subjected to 24 hr of food deprivation were also characterized by elevated plasma beta-ep. In all cases examined, those feeding behaviors in male rats which were subject to inhibition by naloxone were characterized by elevated concentration of plasma beta-ep.

Plasma luteinizing hormone in ovariectomized rats following pharmacologic manipulation of endogenous brain serotonin.

Ovariectomized rats were treated with pharmacologic agents to manipulate endogenous brain serotonin (5-HT). Neither the magnitude nor the timing of luteinizing hormone (LH) pulses in plasma were affected by drug-induced decreases in 5-HT. Acute increases in extraneuronal 5-HT resulting from fluoxetine administration to inhibit 5-HT uptake into neurons, decreased the magnitude of LH pulses, but did not affect the interval between successive LH pulses.

Chronic recording of multiple-unit activity from the brain of conscious sheep.

A technique is described for recording chronic multiple-unit activity from the brain of conscious sheep. Four to six prefabricated arrays of 6 flexible 70 micrometer wires were implanted in each of 16 sheep. During implantation the electrode tips were protected with a crystalline glucose sheath, and following implantation the flexible electrode wires allowed the electrode tips to "float" with movements of the brain tissue. The electrodes were connected to a thin recording cable via a "switchboard" mounted on the skull which allowed the experimenter to determine which 4 of the 24-36 electrodes to monitor at any one time.

Effects of intracerebral administration of neuropeptide-Y on secretion of luteinizing hormone in ovariectomized sheep.

Ovariectomized ewes received unilateral infusions of 20 micrograms neuropeptide-Y (NPY) at a total of 13 intracerebral sites. Episodic secretion of luteinizing hormone (LH) was transiently suppressed on more than one occasion by daily infusions at a total of five intracerebral sites. Four of the effective sites were located within the third ventricle (two sites) and the rostral and ventral part of a lateral ventricle (two sites). The precise neural site of action of exogenous NPY cannot be determined from intraventricular administration, but it indicates a neural rather than pituitary site of NPY action to inhibit LH-releasing hormone (LHRH) in sheep. The only tissue infusion site (ventromedial nucleus) at which NPY also suppressed LH/LHRH also supports a neural action on LHRH, but this single result is insufficient to establish the neural area at which NPY acted. It is known from other work that the production of endogenous NPY in neural tissue of underfed animals is increased, and if endogenous NPY exerts effects on LH/LHRH similar to the suppression presently observed following exogenous NPY this neuropeptide might serve as one neuroendocrine factor that suppresses reproduction in underfed animals.

Serum thyroxin levels during hyperthermia evoked in the monkey by intrahypothalamic injection of PGE1, 5-HT or pyrogen.

Serotonin (5-HT), prostaglandin E1 (PGE1) or a bacterial pyrogen (E. coli or S. typhosa) was microinjected in a volume of 1.0--1.5 microliter into the hypothalamus of the unanesthetized monkey to evoke a long-term hyperthermia. Samples of venous blood collected every 15 min, before, during and after each fever were analyzed by radioimmunoassay for plasma thyroxin levels. There was no statistically significant correlation between plasma thyroxin values and a given phase of the hyperthermic episode induced by the microinjections of 5--HT, PGE1 or bacteria. The possibility that an enhanced release of the thyroid hormone serves to sustain a long-term elevation in temperature evoked by a centrally acting pyrogenic substance is not supported.

Transient effects of MK-801 administration on secretion of luteinizing hormone and prolactin in ovariectomized and estradiol-treated sheep.

The neuroendocrine role of endogenous ligands for the excitatory amino acid receptor subtype known as the NMDA receptor was investigated by administering the NMDA receptor antagonist MK-801 to ovariectomized (ovx) and estradiol-treated sheep. Repetitive administration of MK-801 at intravenous (iv) dosages of 0.1 mg/kg to untreated ovx ewes did not affect the episodic profiles of luteinizing hormone (LH) release, but each injection of MK-801 abruptly stimulated release of prolactin (PRL) demonstrating the effectiveness of the dosage. Injection of estradiol-17beta (50 microg/ewe) to ovx ewes produced the expected biphasic LH response; an initial suppression followed by a surge-like LH increase together with an elevated basal secretion of PRL. Injections of MK-801 occurring 9 and 11 h after estradiol-17beta injection rapidly and transiently increased serum LH in a very unexpected response. However, these same MK-801 injections also resulted in decreased serum LH 14-17 h after estradiol-17beta by delaying the onset of the surge-like release of LH. Estradiol-17beta administration itself elevated basal release of PRL to serum concentrations observed after repetitive MK-801, and further injection of MK-801 no longer transiently increased serum PRL as it had done prior to estradiol-17beta treatment. In summary, antagonizing the endogenous excitatory amino acid ligands of the NMDA receptor with MK-801 did not alter either the timing or magnitude of the putative episodic discharges of gonadotropin-releasing hormone (GnRH) which in turn cause the episodic releases of pituitary LH in ovx sheep. Estrogen administration created a transient neuroendocrine environment in which antagonism of these endogenous ligands was stimulatory to abrupt discharge of GnRH and thereby to acute release of LH. Antagonism of NMDA receptors in untreated ovx ewes stimulated release of PRL suggesting that the endogenous NMDA ligands were probably stimulatory to the release of a PRL-inhibiting neurohormone such as dopamine.

Direct opioid regulation of pituitary release of bovine luteinizing hormone.

Although endogenous opioid peptides (EOP) are thought to alter pituitary release of luteinizing hormone (LH) by modifying the release of gonadotropin-releasing hormone (GnRH) from the brain, EOP may also directly affect the release of LH from pituitary cells. This hypothesis was tested using dispersed cells from the bovine anterior pituitary gland. Pituitaries were enzymatically dissociated, preincubated for 18 h and then cultured for either 2 or 24 h with GnRH, naloxone, methionine-enkephalin (Met-enk) or their combinations. Basal release of LH into media was 18.2 and 38.4 ng/100,000 cells after culture for 2 or 24 h, respectively. When cultured for 2 or 24 h with 10 nM GnRH, LH release was 296% and 131% of the basal release for each culture period. Cellular viability (75% vs 68%) and total (cells + medium) LH (128 vs 134 ng/100,000 cells) did not differ (P greater than .05) between cells cultured for 2 or 24 h. Naloxone (1 microM) increased (P less than .01) basal release of LH by 57% after 2 h of culture but not after 24 h of culture. Naloxone did not augment the amount of LH released in response to 10 nM GnRH. Addition of Met-enk (1 nM to 1 microM) suppressed (P less than .05) basal release of LH (23% to 62%) after 2 h of culture. Similar suppressive effects (8% to 49%) occurred in a dose-dependent manner (0.1 nM to 1 microM) after 24 h of culture. Met-enk (1 and 100 nM) antagonized (P less than .05) the stimulatory effect of naloxone and reduced (P less than .05) the amount of LH released in response to GnRH after 2 h of culture. In summary, the stimulatory effect of naloxone on the basal release of LH suggests that EOP may directly regulate pituitary cell function; the inhibitory effect of physiological concentrations of Met-enk on the basal in vitro release of LH suggests that EOP may directly affect the release of LH in vivo; the antagonism between the stimulatory effect of naloxone and the inhibitory effect of Met-enk is consistent with effects exerted through opioid receptors; and the stimulatory effect of GnRH may be partially reduced by Met-enk. These results are consistent with the hypothesis that opioids may directly modulate the release of LH at the pituitary level.

Endocrine and other responses to acute administration of cannabinoid compounds to non-stressed male calves.

There is an abundance of cannabinoid (CB) receptors for derivatives of cannabis plants in the brain and throughout the body, and several naturally occurring arachidonic acid derivatives can activate these receptors. The specific objective of this study was to activate these CB receptors in castrated male calves through administration of several CB agonists and to measure immediate changes in concentrations of several serum hormones, respiration rate, and sensitivity to pain. The rationale for the study was that exogenous activation of CB receptors might reveal whether the endogenous CB system (consisting of receptors and endogenous ligands) plays a role in the stress response of animals and specifically whether the activated CB system might be part of a coping mechanism to combat stress. Intravenous administration of three CB agonists (anandamide, methanandamide and WIN 55212-2) to nine castrated male calves under non-stress conditions provoked immediate increases of serum cortisol and respiration rate as well as rapidly caused hypoalgesia to cutaneous pain and thermal stimuli. Although anandamide and methanandamide did not affect serum prolactin, administration of another CB agonist (WIN 55212-2) did increase serum prolactin abruptly. None of the CB agonists affected serum growth hormone. In summary, many of the changes following administration of CB agonists were similar to a stress response in this species, but there were some agonist-specific differences, notably regarding prolactin secretion, as well as differences between calves and observations made in other species. Although CB receptors in calves may be activated by endogenous ligands during exposure to some stressors, the present results are also consistent with this CB system being part of a coping mechanism that helps animals deal with imposed stressors.

Presence of dynorphin-like immunoreactivity but not opiate binding in Walker-256 tumors.

Walker-256 tumor tissue was removed from rats on day 8 of tumor growth. An acidified methanol extract of the tumor tissue was assayed for immunoreactive (ir) dynorphin-A 1-17 (DYN-17) and ir-dynorphin-A (DYN-8). Levels of ir-DYN-17 and ir-DYN-8 were nearly 4-and 8-fold higher, respectively, in tumors versus normal muscle. However, tumor homogenates did not exhibit specific 3H-naloxone binding. These results indicate that although the Walker-256 carcinosarcoma may produce opioids, it is unlikely that these ectopic substances have direct opioid actions on the tumor itself.

Assessment of dynorphin-A depletion in the anorexia of Walker-256 tumor bearing rats.

Rats bearing the Walker-256 (W-256) tumor display an anorexic profile which resembles that of normal animals forced to drink 2% NaCl [2,24], a regimen which depletes neurohypophyseal dynorphin-A (DYN) [3,9]. As expected, the naloxone reversible feeding induced by 2-deoxy-D-glucose (2-DG) was attenuated (36%) in the W-256 tumor bearing rats (TBR). Interestingly, immunoreactive (ir) levels of dynorphin-A 1-17 (DYN-17) and its postulated breakdown product, dynorphin-A 1-18 (DYN-8), were also reduced in the neurohypophysis of W-256 TBR by 42 and 50%, respectively. However, ir-DYN levels were not reduced in TBR in those brain regions which are probably involved in the regulation of appetite (e.g., hypothalamus). 2-DG itself did not consistently affect ir-DYN levels in any tissue for either controls or TBR. The ratio of DYN-8 to DYN-17 did not change in response to any treatment, including the depletion of both peptides from the NIL of TBR. In summary, the present data do not support DYN depletion as being a factor which contributes to the anorexia of the W-256 TBR.