Effects of substance P and neurotensin on growth hormone and thyrotropin release in vivo and in vitro

Conscious ovariectomized (OVX) rats bearing a cannula implanted in the third ventricle were injected with 2 μl of 0.9% NaCl containing varying doses of substance P (SP) or neurotensin (NT) and plasma GH and TSH levels were measured by RIA in jugular blood samples drawn through an indwelling silastic catheter. Control injections of physiologic saline iv or into the third ventricle did not modify plasma hormone levels. Intraventricular injection of SP or NT at doses of either 0.5 or 2 μg elevated plasma GH concentrations within 5 min and they remained elevated for 60 min. Third ventricular injection of similar doses of SP or NT had no effect on plasma TSH. An intermediate dose of 1 μg of SP or NT given iv had no effect on plasma GH but NT elevated plasma TSH. Incubation of hemipituitaries from OVX rats with varying doses of SP or NT did not alter GH release into the medium but TSH release was enhanced with NT at doses of 100 or more ng/ml of medium. It is suggested that SP acts centrally to stimulate growth hormone-releasing factor (GRF) or to inhibit somatostatin release and thereby enhance GH release and that NT acts directly on the pituitary to stimulate TSH release.     

Effects of a dopaminergic stimulant,amfonelic acid,on anterior pituitary hormone release in conscious rats

The response of plasma LH, Prolactin, GH and TSH levels to systematic administration of a specific central dopaminergic stimulant, amfonelic acid (AFA), by intravenous pulse injection in ovariectomized (OVX) and OVX estrogen-progesterone primed conscious rats has been evaluated. Intravenous injection of 0.2 mg/kg of AFA had no influence on plasma LH concentration until 60 min after injection when it was significantly elevated. Increasing the dose to 1 mg/kg reduced LH titers at 15 and 30 min with a return to preinjection levels by 60 min. AFA produced a dose-dependent decrease in plasma prolactin levels; the decrease occurred as early as 5 min after injection. AFA, both at 0.2 and 1 mg/kg doses, was effective in producing a sharp, dose-related rise in plasma GH levels. By contrast, TSH levels were significantly suppressed by both doses of AFA. Injection of the 1 mg/kg dose of AFA did not modify plasma LH levels in OVX-steroid-primed animals, white producing a comparable effect on plasma prolactin, GH and TSH levels to that observed in OVX animals. The present results indicate that endogenously released DA can have profound effects on pituitary hormone release, inhibiting PRL and TSH discharge, stimulating GH release and either inhibiting or stimulating LH release.     

Somatocrinin,growth hormone releasing factor,stimulates secretion of growth hormone in anesthetized rats

The synthetic replicate of a 44 amino acid peptide isolated from a human pancreatic tumor which had caused acromegaly possesses high specific activity to release growth hormone (GH) in anesthetized male rats. The GH secretion induced by this peptide is dose-dependent from 50 ng to 1 μg, with plasma GH concentrations increasing more than 10-fold within 5 min of iv administration at the higher doses. Two enzymatic degradation products of the 44 residue peptide were also isolated and consist of the first 37 and 40 amino acids. All three peptides appear to possess similar potency, on a molar basis, $\text{in}\text{vivo}$, contrary to $\text{in}\text{vitro}$ results. The specificity of these peptides on GH release was shown by their failure to alter plasma concentrations of prolactin (PRL), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and corticosterone. Based on these $\text{in}\text{vivo}$ results, the three peptides with serve as powerful tools with which to investigate the mechanisms of GH secretion.     

Site of action for β-endorphin-induced changes in plasma luteinizing hormone and prolactin in the ovariectomized rat

A number of sites have been hypothesized as loci at which opioid substances act to alter the secretion of luteinizing hormone (LH) and prolactin (PRL) (1–8). The aim of the present study was to determine the site(s) at which the opioid peptide β-endorphin (β-END) acts to influence plasma LH and PRL levels in the ovariectomized (OVX) rat. β-END, administered into the third ventricle of conscious OVX rats fitted with jugular catheters, significantly decreased plasma LH in doses ? 50 ng and increased PRL levels at all doses administered (10, 50, 100 and 250 ng) in a dose dependent fashion. To identify possible central nervous system sites of action, 250 ng β-END was unilaterally infused into various brain sites. Plasma LH was significantly decreased and plasma PRL significantly increased by infusions into the ventromedial hypothalamic area, the anterior hypothalamic area, and the preoptic-septal area. There was no significant effect of β-END infusions into the lateral hypothalamic area, amygdala, midbrain central gray, or caudate nucleus. When hemipituitaries of OVX rats were incubated $\text{in}$$\text{vitro}$ with β-END (10^?7M to 10^?5M), there was no suppression of basal or LHRH-induced LH release, nor was there any alteration of basal PRL release. It is concluded that β-END acts at a medial hypothalamic and/or preoptic-septal site and not the pituitary, to alter secretion of LH and PRL.     

Leu-enkephalin-stimulated growth hormone and prolactin release in the rat: comparison with the effect of morphine

The effect of Leu⁵-enkephalin on growth hormone (GH) and prolactin (PRL) release was studied $\text{in vivo}$ in the infant rat and compared to that of morphine. In 10 day-old pups, intracerebroventricular injection of Leu⁵-enkephalin (50, 75 and 100 μg) resulted in a dose-related increase in plasma GH; morphine was active as GH releaser at the dose of 5 and 10 μg, but not at 2.5 μg. Pretreatment with naloxone (2 mg/kg ip) suppressed the GH-releasing effect of either Leu⁵-enkephalin (100 μg) or morphine (10 μg). Leu⁵-enkephalin (75 and 100 μg) induced a rise in plasma PRL which was neither dose-related nor antagonized by naloxone; morphine (5 and 10 μg) was active as PRL releaser and its effect was antagonized by naloxone. These results indicate that: 1) Leu⁵-enkephalin stimulates both GH and PRL release; 2) the release of GH by Leu⁵-enkephalin but likely not that of PRL involves specific opiate receptors; 3) morphine releases GH and PRL through specific opiate receptors.     

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.     

Effect of central injection of bradykinin and bradykinin potentiating factor upon release of anterior pituitary hormones in ovariectomized female rats

The effects of third ventricular (IVT) injection of 25 μg of bradykinin (BK) upon plasma levels of LH, FSH, TSH, GH and prolactin were investigated in conscious ovariectomized female rats bearing indwelling jugular cannulae. Some animals were pretreated with bradykinin potentiating factor (BPF). Intravenous administration of BK had no effect upon hormone levels. IVT injection of BK significantly depressed plasma prolactin levels at 15 and 30 min post-drug, with levels returning to control values by 60 min. Pretreatment of animals with BPF (75 μg/3 μl) prolonged the prolactin suppression induced by BK for up to two hours. Plasma LH, FSH, TSH and GH levels in BK-rats were not significantly different from those of saline-injected animals at any time point measured. Neither BPF alone nor in conjunction with BK had any effects upon plasma levels of TSH; however, BK plus BPF suppressed FSH concentrations at 75 min post-BPF, while BPF alone appeared to increase GH levels at 45 min. In vitro incubation of hemipituitaries with 0.083, 0.83 or 8.33 μg/ml BK had no effect upon the release of LH, TSH or prolactin compared to control values. However, the secretion of GH and FSH was suppressed by the lowest dose of BK tested. These results suggest that BK may play a physiological inhibitory role in the regulation of prolactin, which can be augmented by preventing its degradation, i.e. via BPF. The effect of the peptide seems to be mediated by the CNS since neither intravenous injection of BK nor in vitro incubation of pituitaries with the peptide modified prolactin release.     

Lack of growth hormone-releasing activity of (pyro)Glu-Ser-Gly-NH2

Youdaev $\text{et al.}$ (1) reported that (pyro)Glu-Ser-Gly-NH₂ isolated from bovine hypothalami or made synthetically stimulates the release of growth hormone (GH). Therefore, we synthesized the tripeptide and tested it in several $\text{in vivo}$ and $\text{in vitro}$ assay systems for GH releasing activity. Our results demonstrate that (pyro)Glu-Ser-Gly-NH₂ does not stimulate the release of immunoreactive GH from rat pituitaries $\text{in vitro}$ in doses of 0.1–1000 nanog/ml. Similarly, (pyro)Glu-Ser-Gly-NH₂, injected intravenously into rats in doses of 1μg and 10μg/rat or infused into a hypophysial portal vessel in doses of 0.01 μg and 0.1 μg/rat did not increase serum GH levels as measured by radioimmunoassay. When this tripeptide was injected intravenously in doses of 500 μg into sheep it did not raise plasma GH levels. These results demonstrate that under the conditions of this investigation (pyro)Glu-Ser-Gly-NH₂ does not display any GH-releasing activity.     

Effect of substance P on thyrotropin secretion from the pituitary gland in the rat

The role of substance P (SP) on thyrotropin (TSH) secretion was investigated in ovariectomized (OVX) female, estrogen-primed OVX, and normal male rats. Third ventricular administration of SP induced a significant increase in plasma TSH levels when compared to control animals in E-primed OVX rats (p less than 0.001). The plasma TSH levels increased in a dose-related manner and reached maximum levels at 10 min after injection. In contrast, intraventricularly injected SP failed to alter plasma TSH levels in both OVX rats and normal male rats. Intravenous administration of SP dramatically stimulated TSH release in E-primed OVX rats (p less than 0.001), whereas SP had no effect on the release of TSH when injected in OVX rats and normal male rats. To investigate any direct action of SP on TSH release from the anterior pituitary gland, synthetic SP was incubated with dispersed anterior pituitary cells harvested from E-primed OVX rats and normal male rats. SP, in the dose range between 10(-8) M and 10(-6) M, failed to alter the release of TSH into the culture medium in vitro. These findings indicate that SP has a stimulatory role in the control of TSH release by an action on the hypothalamus but only in estrogen-primed rats.     

Effect of prostaglandin F2α on anterior pituitary function in man

Five healthy adult men received iv PGF_2α at dosages of 0.05, 0.20 and 2.0 μg/kg/min for 30 min. There were no significant changes in serum FSH, LH or TSH levels. Serum GH and cortisol levels were slightly increased at the highest dosage. These responses were associated with, and presumably a result of, stressful side effects. Thus, PGF_2α cannot be used as a provocative test of pituitary hormone reserve.Prostaglandins (PG's) have recently been implicated in the release of a number of hormones from the anterior pituitary gland. The stimulation of GH release by PG's of the E series from incubated rat pituitary slices has been demonstrated. $\text{In vivo}$ stimulation by PGE₁ of ACTH in rats and of GH release in man has also been shown.The present study was undertaken in order to examine the efficacy of iv administration of PGF_2α as a provocative test of anterior pituitary hormone reserve in man. The responses in circulating levels of gonadotropins, TSH, GH, and cortisol (as an index of ACTH) were measured.     

Role of arginine vasopressin in control of ACTH and LH release during stress

To determine the role of arginine vasopressin (AVP) in stress-induced release of anterior pituitary hormones, AVP antiserum or normal rabbit serum (NRS) was micro-injected into the 3rd ventricle of freely-moving, ovariectomized (OVX) female rats. A single 3 microliter injection was given, and 24 hours later, the injection was repeated 30 min prior to application of ether stress for 1 min. Although AVP antiserum had no effect on basal plasma ACTH concentrations, the elevation of plasma ACTH induced by ether stress was lowered significantly. Plasma LH tended to increase following ether stress but not significantly so; however, plasma LH following stress was significantly lower in the AVP antiserum-treated group than in the group pre-treated with NRS. Ether stress lowered plasma growth hormone (GH) levels and this lowering was slightly but significantly antagonized by AVP antiserum. Ether stress also elevated plasma prolactin (Prl) levels but these changes were not significantly modified by the antiserum. To evaluate any direct action of AVP on pituitary hormone secretion, the peptide was incubated with dispersed anterior pituitary cells for 2 hours. A dose-related release of ACTH occurred in doses ranging from 10 ng (10 p mole)-10 micrograms/tube, but there was no effect of AVP on release of LH. The release of other anterior pituitary hormones was also not affected except for a significant stimulation of TSH release at a high dose of AVP. The results indicate that AVP is involved in induction of ACTH and LH release during stress. The inhibitory action of the AVP antiserum on ACTH release may be mediated intrahypothalamically by blocking the stimulatory action of AVP on corticotropin-releasing factor (CRF) neurons and/or also in part by direct blockade of the stimulatory action of vasopressin on the pituitary. The effects of vasopressin on LH release are presumably brought about by blockade of a stimulatory action of AVP on the LHRH neuronal terminals.     

Effect of human pancreatic polypeptide (HPP) on the release of anterior pituitary hormones

The effects of different doses of human pancreatic polypeptide (HPP) injected into the third ventricle was studied on plasma follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin (Prl) and somatotropin (GH) in freely moving ovariectomized rats. Two hundred ng of HPP produced a significant decrease in plasma LH at 15, 30, and 60 min following microinjection. The LH-lowering effect of 400 and 800 ng of HPP developed at 5 min and persisted throughout the experiment. The strongest inhibition was observed at 15 and 30 min. No change in plasma FSH was detected at any time during the experimental period. Two hundred and 400 ng of HPP failed to influence the plasma Prl, while 800 ng resulted in a moderate but significant decrease in plasma Prl levels at 15 and 30 min following injection. Intraventricular microinjection of 400 ng of HPP decreased the GH level at 15 min and 800 ng caused a more pronounced decrease which was significant at 15, 30, and 60 min after the injection. The study suggested that HPP, either from the periphery if it can pass the blood brain barriers or produced in the brain, can influence pituitary function.     

The effect of cholecystokinin octapeptide on pituitary-adrenal hormone secretion

The purpose of this investigation was to determine the influence of cholecystokinin octapeptide (CCK-OP) on pituitary-adrenal hormone secretion. CCK-OP at a dose of 5 μg/kg (i.p.) elevated plasma corticosterone from 27 to 43 μg/100 ml in one experiment and from 12 to 50 μg/100 ml in a second experiment: Lower doses of CCK-OP (0.5 μg/kg) elevated corticosterone from 12 μg/100 ml to 20 μg/100 ml. CCK-OP (1, 10, and 100 ng/ml) had no effect on ACTH-induced corticosterone released by isolated adrenal cells in vitro when tested in the presence of 50 pg of ACTH_1?24. 100 and 500 ng of CCK-OP resulted in an increased pituitary ACTH release equal to 123% (n.s.) and a 206% (P < 0.05) of control, respectively. In comparison, a $\text{3}\text{5}$ hypothalamic stalk median eminence equivalent increased ACTH release to 313% of control (P < 0.05). The exact mechanism of this CCK effect on pituitary ACTH release is unknown. Although it is likely that the direct effects on the pituitary in vitro represent a pharmacologic and not a physiologic effect of this peptide, in vivo doses are between doses used for pancreatic effects and satiety effects suggesting that there may be a physiologic stimulating action of this peptide on the hypothalamic-pituitary-adrenal axis but at a level above the adrenal and pituitary.     

Effects of thyrotropin-releasing hormone on behavioral and hormonal changes induced by beta-endorphin.

Adult male rats were injected intraventricularly either with saline or TRH (10 μg) 5 min prior to a second injection of either saline or β-endorphin (50 μg). The tripeptide produced a 100% increase of motility counts recorded over a 15 min period following the last injection, whereas β-endorphin decreased general motor activity. TRH pretreatment completely abolished the depressant effect of β-endorphin. In addition, TRH enhanced the PRL secretion induced by β-endorphin and antagonized the slight elevation of plasma GH levels observed in β-endorphin-treated rats. These results do not seem to be related to an interaction of TRH with opiate receptors since the tripeptide (10^?8, 10^?6 M) added $\text{in vitro}$ to rat brain homogenates did not alter the specific binding of ³H-naloxone nor affect the displacement by β-endorphin of such binding.     

Prostaglandin E2 (PGE2)-induced growth hormone (GH) release: Effect of intrahypothalamic and intrapituitary implants

Overiectomized rats were unilaterally implanted with a 23-gauge stainless steel cannula in different hypothalamic areas or in the pituitary gland and subsequently were treated with estrogen (sc, 10 μg estradiol benzoate, Eb). Two days after the estrogen injection, an inner cannula containing PGE₂ or PHF_2α at its tip was inserted into the cannula. Other animals were implanted with empty inner cannula. Plasma GH concentrations were measured by RIA in blood samples drawn from the jugular vein while the animals were lightly etherized before (−2) and at 20, 40, 60 and 120 min following the implantation. Plasma GH levels in control animals bearing an empty cannula in the body of the arcuate nucleus-median eminence region (BARH-ME) were signifantly depressed by the ether stress. The implantation of PGF_2α in this area was completely ineffective in preventing ether stress-induced decline in plasma GH. By contrast, PGE₂ implanted in BARH-ME or the post-chiasmatic region of the hypothalamus (HARH-ME) elevated plasma GH 20 min following its implantation and partially prevented the subsequent decrease in GH levels induced by ether stress. PGE₂ implants located in several other hypothalamic areas failed to induce GH release or to prevent the decline in GH levels induced by ether stress. However, PGE₂ implanted in the pituitary gland elicited a marked increase in plasma GH at 20 min and completely prevented the subsequent ether stress-induced decline in GH levels.The results suggest that PGE₂ can act at both hypothalamic (ARH-ME) and pituitary levels to stimulate GH release. At the hypothalamus, PGE₂ may inhibit GH-inhibiting factor (GIF) release or induce release of GH releasing factor (GHRF).     

Contrasting effects of pituitary adenylate cyclase activating polypeptide (PACAP) on in vivo and in vitro prolactin and growth hormone release in male rats.

Pituitary adenylate cyclase activating polypeptide (PACAP) is produced by hypothalamic neurons which terminate within the median eminence suggesting that it may be a hypophysiotropic hormone. However, little endocrine activity has been ascribed to the peptide. Therefore we studied the effects of PACAP on prolactin (Prl) release from dispersed cultivated rat pituitary cells in vitro using conventional cultures as well as the reverse hemolytic plaque assay (RHPA). Furthermore the effects of the peptide on in vitro GH release were assessed. In addition, the activity of the peptide on in vivo release of Prl and GH was studied in hypothalamus-lesioned animals. PACAP dose dependently inhibited Prl release form dispersed pituitary cells in both, monolayer cell cultures and the RHPA, whereas GH secretion was not affected. In hypothalamus-lesioned rats which have high Prl levels due to the absence of hypothalamic dopamine, PACAP further stimulated Prl release. Serum GH increased more than 20 fold in response to the intravenous PACAP infusion. Thus in vitro (inhibition of Prl release, no effect on GH release) and in vivo (stimulation of both hormones) experiments yielded contradicting effects of PACAP on pituitary hormone release. We suggest that PACAP may stimulate the release of a paracrine, yet unknown factor which in the intact pituitary overrides the direct inhibitory action of PACAP on the lactotropes. The same or another paracrine factor may also enhance in vivo GH release. In cell culture the paracrine factor is diluted by the medium. Therefore the peptide never reaches effective concentrations which are present within the intact pituitary tissue.     

Inhibition of the release of growth hormone in vitro by -melanocyte stimulating hormone

A polypeptide isolated from porcine hypothalami was found to inhibit the release of growth hormone (GH) from isolated rat pituitaries. This polypeptide was identified chemically and biologically as α-MSH. Pure natural α-MSH isolated from beef posterior pituitary extracts and synthetic α-MSH also inhibit the release of GH $\text{in vitro}$. In addition, other substances not yet identified, present in porcine hypothalamic extracts, also share this property.     

Zinc may play a role in the regulation of thyrotropin function

We studied the in vitro and in vivo influence of physiologically relevant zinc concentrations on the thyrotropin function both at the pituitary and hypothalamic level. Zinc gluconate (Zn Glu) concentrations from 5 to 100 microM decreased basal TSH release from anterior pituitary gland in vitro, but did not affect TSH-stimulated release by TRH, cAMP or high K+ concentrations. Zn Glu altered neither the basal nor stimulated production of TRH by hypothalami in vitro. In vivo brain third ventricle injection of Zn Glu decreased serum TSH 30-60 min after injection. The ability of physiological concentrations of zinc to influence TSH secretion both in vitro and in vivo suggest that this trace element might be involved in the regulation of thyrotropin function.     

Effect of bromocriptine on the hypothalamo-pituitary function in adult male rats.

Daily oral administration of bromocriptine (50 μg/kg) to adult male rats, suppressed serum prolactin levels. The pituitary prolactin levels remained unaltered. Serum FSH levels as well as pituitary FSH levels showed no significant change as compared to the controls. Serum LH levels were significantly decreased in spite of the high pituitary LH levels, in bromocriptine treated rats. In the drug treated rats, $\text{in vitro}$ sensitivity of the pituitary to the exogenous LH-RH was not altered; whereas hypothalamic LH-RH content was considerably lowered. These observations suggest the possible effect of bromocriptine on the synthesis of LH-RH in the hypothalamus which leads to the accumulation of LH in the pituitary and decline of serum LH.     

Regulation of GH and TSH release from hyperplastic and ectopic pituitaries: effects of dopamine in vitro

This study was undertaken to examine the consequences of prolonged removal of the pituitary from hypothalamic control and of estrogen-induced pituitary tumors on the susceptibility of GH and TSH release to regulatory influences of dopamine (DA). Adult male Fischer 344 rats were treated with transplants of female anterior pituitaries under the renal capsule or with Silastic capsules containing diethylstilbestrol (DES). Capsules with DES remained in place until the animals were killed (DES-IN) or were removed 7 weeks prior to sacrificing the rats (DES-OUT). Both pituitary grafts and DES caused the expected elevation in plasma prolactin and suppression of plasma GH and TSH levels. Basal GH release in vitro was not affected by exposure to DES in vivo but was reduced by transplantation of the pituitary to an ectopic site. Treatment with DA in vitro suppressed GH release from the in situ pituitaries of control, DES treated and grafted rats but increased GH release from the ectopic pituitaries. Basal release of TSH in vitro was reduced in the pituitaries of DES-IN and DES-OUT animals but was not affected by the presence of pituitary transplants. No detectable TSH was released from the ectopic pituitaries in the absence of DA. DA decreased TSH release from the pituitaries of control, DES-OUT and DES-IN rats but not from the in situ pituitaries of grafted rats. In contrast, DA produced an increase in TSH release from ectopic pituitaries. These results demonstrate that somatotrophs and thyrotrophs removed from the hypothalamic influences on subjected to direct and indirect effects of DES exhibit abnormal responses to DA. We suspect that prolonged absence of normal pituitary control leads to the development of regulatory mechanism of pituitary hormone release which are different from those operating under physiological conditions.