S.M. McCann

Galanin: evidence for a hypothalamic site of action to release growth hormone.

Galanin is a 29 amino acid peptide that was isolated and characterized from porcine intestinal extracts. The presence of galanin-like immunoreactivity in neuronal elements in the hypothalamus and median eminence suggested a role for it in the hypothalamic control of anterior pituitary function. A hypothalamic site of action of galanin to stimulate growth hormone (GH) release is suggested by our observation that doses as low as 50 picomoles when infused into the third cerebroventricle of conscious, unrestrained ovariectomized rats resulted in significantly elevated plasma levels of GH. This effect was specific for GH and was dose-related. The failure of galanin to alter GH release from dispersed, cultured anterior pituitary cells in vitro further suggests that endogenous galanin plays a neuromodulatory role at the level of the median eminence, possibly affecting the release of known GH-releasing and/or inhibiting factors.

In vivo and in vitro effects of cholecystokinin on gonadotropin, prolactin, growth hormone and thyrotropin release in the rat

Abstract Varying doses of cholecystokinin (CCK) dissolved in 2 μl of 0.9% NaCl or 2 μl of saline alone were injected into the third ventricle of conscious ovariactomized (OVX) rats bearing 3rd ventricular cannulae. Plasma luteinizing hormone (LH), prolactin (PRL), growth hormone (GH), thyrotropin (TSH) and follicle stimulating hormone (FSH) levels were measured by RIA in jugular blood samples drawn through an indwelling silastic cannula. Control injections of saline i.v. or into the 3rd ventricle did not modify plasma hormone levels. Intraventricular injections of 4, 40 and 500 ng CCK produced a significant suppression of plasma LH within 5 min of injection. Injection of 4 or 500 ng doses of CCK has no effect on plasma PRL levels, but injection of the 40 ng dose produced a significant elevation of plasma PRL within 15 min. Plasma GH levels increased significantly within 15 min of the 3rd ventricular injection of each dose of CCK. The 40 ng dose of CCK caused a progressive reduction of plasma TSH which was significant by 15 min and lasted through the 60 min of experimentation. The highest dose of 500 ng reduced plasma TSH levels within 5 min. Plasma FSH was not altered by any dose of CCK. Intravenous injection of CCK caused a dose-related increase in plasma prolactin levels within 5 min, but only the highest dose of 1000 ng produced a significant decrease in plasma LH. No significant changes in GH, TSH or FSH levels were observed after i.v. injection of CCK. In vitro incubation of hemipituitaries from male rats with doses of CCK ranging from 10 ng to 5 μg had no effect on pituitary hormone release into the medium. The results indicate that CCK can alter pituitary hormone release via a hypothalamic action and suggest that it may act as t transmitter or modulator of neuronal activity controlling the release of hypothalamic releasing and/or inhibiting hormones.

Re-Evaluation of the Role of Catecholamines in Control of Gonadotropin and Prolactin Release

The effect of microinjection of catecholamines into the 3rd brain ventricle on plasma gonadotropins and prolactin (Prl) titers was evaluated in estrogen and progesterone-primed, ovariectomized (OEP) rats which are a particularly sensitive test animal for LH-RH and the responses compared to those obtained in ovariectomized (OVX), unprimed animals. Intraventricular injection of isotonic saline had no effect on plasma gonadotropins or Prl. The injection of dopamine (DA) into the 3rd ventricle elevated plasma LH in the OEP rats at doses of 4 or 20 µg, but the elevation obtained with the 10 µg dose was not significant. A greater effect was obtained with 20 µg doses of both norepinephrine (NE) and epinephrine (E), with E being the most effective of the 3 catecholamines. Apomorphine (APO) also elevated plasma LH following its injection into the 3rd ventricle in the OEP rats. Since these animals were etherized for removal of blood samples and microinjections into the ventricle, a group of animals was used with in-dwelling cannulae in the external jugular vein. In these animals, DA also elevated plasma LH. In OVX animals, the intraventricular injection of DA was without effect on plasma LH, but small transient increases in plasma LH were obtained with 20 µg doses of NE or E. The action of DA in the OEP rat was not prevented by the prior injection of diethyldithiocar-bamate (DDC) which inhibits NE synthesis. Dopamine, apomorphine, and another DA receptor-stimulating drug, Piribedil (ET-495), lowered plasma Prl following their intraventricular injection in all conditions. There were no effects of any of the drug treatments on plasma FSH. It is concluded that intraventricular DA can stimulate LH release in the steroid-primed rat but that NE and E appear to be more effective in this regard. Epinephrine was the most effective catecholamine in stimulating LH release. Dopamine clearly

Intrahypothalamic action of corticotrophin-releasing factor (CRF) to inhibit growth hormone and LH release in the rat

The effects of intravenous or intraventricular injection of synthetic ovine corticotrophin-releasing factor (oCRF) on plasma levels of anterior pituitary hormones were studied in conscious, ovariectomized (OVX) female rats and compared with the actions of the peptide on dispersed anterior pituitary cells from OVX female rats incubated in the presence of CRF. Third ventricular injection of oCRF in freely moving rats caused a significant increase in plasma levels of ACTH in a dose-related manner with a minimal effective dose of less than 0.5 micrograms (0.1 nmol). The effect was observable at 5 min after injection and persisted for the 60 min duration of the experiment. In contrast, growth hormone levels were significantly depressed within 15 min with a minimal effective intraventricular dose of 0.5 micrograms. The suppression persisted for the duration of the experiment but there was no additional effect of the higher dose of 5 micrograms. Plasma LH levels were also lowered by the highest dose of 5 micrograms (1.0 nmol) of oCRF, with the first significant lowering at 30 min. Lower doses had no effect on plasma LH. Plasma TSH levels were not significantly altered. Control injections of the 0.9% NaCl diluent were without effect on the levels of any of the hormones. Intravenous injection of similar doses of oCRF had no effect on plasma levels of GH or LH. The ACTH-releasing action of the oCRF preparation was confirmed by in vitro incubation of the peptide with dispersed anterior pituitary cells for 2 h. A dose-related release of ACTH occurred in doses ranging from 0.1-10 nM, but there were no effects on the release of the other anterior pituitary hormones. The results suggest that oCRF may act within the hypothalamus to suppress the release of GH and to a lesser extent LH. The stimulation of ACTH release following intraventricular CRF is presumably related to its uptake by portal blood vessels with delivery to the pituitary and stimulation of the corticotrophs.

Effects of Drugs Modifying Catecholamine Synthesis on Plasma LH and Ovulation in the Rat

Relatively large doses of alpha methyl paratyrosine (α-MPT) (400 mg/kg, i.p.) to inhibit catecholamine synthesis were capable of blocking both the pre-ovulatory elevation in plasma LH and ovulation in proestrous rats. Similarly, large doses of diethyldithio-carbamate (DDC) to deplete brain norepinephrine stores also inhibited ovulation and LH release. The blockade induced by α-MPT could not be reversed by the administration of L-dihydroxyphenylalanine (L-dopa) or dihydroxyphenylserine (DOPS). Similarly, neither L-dopa nor DOPS was able to alter the blockade induced by DDC. An injection of progesterone (2 mg/rat, s.c.) increased the plasma LH values on the afternoon of proestrus and the stimulatory effect of progesterone was blocked by the administration of α-MPT prior to progesterone. Although L-dopa failed to reverse the blockade of α-MPT, a partial reversal was obtained by the administration of DOPS. DDC also blocked the progesterone-induced LH release and there was a slight reversal in animals given DOPS together with DDC. Except for the difficulties in reversing the blockade with drugs to augment norepinephrine synthesis, the results are consistent with the hypothesis that norepinephrine is involved in the pre-ovulatory discharge of gonadotropins.

Action of luteinizing hormone-releasing factor (lrf) in the initiation of lordosis behavior in the estrone-primed ovariectomized female rat.

In order to evaluate the precise role of luteinizing hormone-releasing factor (LRF) in mediating the onset of sexual behavior, the specificity, time-course, and dose-response relationship of LRF-facilitated lordosis behavior were determined. Ovariectomized female rats, pretreated with estrone and LRF, displayed a pattern of lordosis behavior which differed little from that produced by estrone-progesterone. Little if any lordosis behavior was observed in response to LRF alone, estrone alone, or estrone in combination with luteinizing hormone (LH), follicle-stimulating hormone (FSH), or thyrotropin-releasing factor (TRF). Furthermore, LRF-induced lordosis behavior occurred in the absence of the adrenals, thus eliminating adrenal progesterone as a factor in facilitating the appearnce of lordosis behavior. The LRF-facilitated lordosis behavior was seen 2 h after the injection of LRF and was maintained for a total of 8 h. A minimal dose of 150 ng LRF was required to initiate the first consistent appearance of lordosis behavior; the maximum response was obtained with 500 ng. It is thus suggested that LRF is not only responsible for the ovulatory discharge of LH and subsequent ovulation, but may also play a role in the initiation of the onset of mating behavior in the female rat.

Dopamine and norepinephrine stimulate somatostatin release by median eminence fragments in vitro

Abstract The effect of catecholamines on somatostatin release by median eminence (ME) fragments was evaluated using an in vitro incubation system. Adult male rats were used as tissue donors. Somatostatin release was readily detected during short-term incubations (10 and 30 minutes). Dopamine (DA) significantly stimulated somatostatin release during a 30 minute incubation period at the two doses tested (0.6 and 6 μM). Under similar conditions, norepinephrine (NE) stimulated somatostatin release only at the 6 μM dose. Using a shorter incubation period (10 min) and a 6 μM dose, only DA stimulated somatostatin release. The effects of DA and NE were specifically blocked by the in vitro addition of pimozide or phentolamine, respectively, suggesting that dopaminergic and noradrenergic receptors may be present in the somatostatinergic terminals of the ME. The results indicate that both DA and NE may be involved in the regulation of somatostatin secretion.

In vitro stimulation of prolactin release by vasoactive intestinal peptide

The effect of vasoactive intestinal peptide (VIP) on anterior pituitary hormone release was examined in a variety of in vitro preparations. Synthetic VIP was capable of stimulating increased prolactin (PRL) release from male rat hemipituitaries in doses as low as 10-9M only when the enzyme inhibitor bacitracin was present in the incubation medium. Natural porcine VIP was similarly capable of stimulating PRL release, but only at higher doses (10-6M). Additionally, synthetic VIP was capable of stimulating PRL release from dispersed anterior pituitary cells harvested from adult male and lactating female rats and from an enriched population of lactotrophs obtained by unit gravity sedimentation of similar dispersed cells from infantile female rats. No effect of VIP on luteinizing hormone, growth hormone or thyroid stimulating hormone release was seen. These findings taken in concert with the presence of VIP in the hypothalamus, pituitary and hypophyseal portal plasma of the rat suggest a physiological role for VIP in the control of PRL secretion.

Ethanol and the pulsatile release of luteinizing hormone, follicle stimulating hormone and prolactin in ovariectomized rats

Conscious ovariectomized rats were administered either saline or an ethanol (ETOH)-saline solution via a permanent intragastric cannula, and plasma LH, FSH and PRL were measured by RIA of jugular blood samples drawn every 10 min through an indwelling silastic catheter. Control injections of saline into the gastric cannula did not modify any of the plasma hormone concentrations. Animals which were administered ETOH, showed marked decreases in the plasma concentrations of LH. Compared to basal levels, a significant decrease in the area under the secretion curve of LH occurred during the initial hour after ETOH administration. This decline continued with the lowest levels of plasma LH being detected at approximately 1.5 hours following the ETOH injection. Additionally, no LH pulses were detected in any of the ETOH-treated animals during the second hour after ETOH; thus, reducing the number of LH pulses observed in ETOH vs. saline-injected animals. Comparable increases in the area under the LH curve occurred following a challenge dose of LHRH in both saline and ETOH-injected rats, indicating that pituitary responsiveness was the same for both groups. In contrast to LH, ETOH did not significantly alter the pattern of FSH secretion, as represented by the area under the curve and the number of FSH pulses. In addition to the differential effects of ETOH on the pulsatile release of LH and FSH, the present data also indicate that these two gonadotropins have different secretory patterns. With regard to PRL, ETOH-injected animals showed a significant elevation in plasma PRL levels during the first hour following ETOH administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Intra- and Extrahypothalamic Luteinizing Hormone-Releasing Hormone (LHRH) Distribution in the Rat with Special Reference to Mesencephalic Sites Which Contain Both LHRH and Single Neurons Responsive to LHRH

In addition to hypothalamic sites known to contain LHRH, several extrahypothalamic structures in both male and female rats were discovered to contain the decapeptide. In the mesencephalon, the fasciculus retroflexus, interpeduncular nucleus and midbrain central grey contained readily detectable quantities of LHRH. Single neurons responsive to microiontophoretically applied LHRH were also discovered in the mesencephalon suggesting a physiological role for the decapeptide in neuronal events at the level of the midbrain.