Rat Hypophysial Portal Blood Research Articles

Pre-exposure of rat anterior pituitary cells to somatostatin enhances subsequent growth hormone secretion.

The precise roles of GH-releasing factor (GRF) and somatostatin (SRIF) in the orchestration of pulsatile GH secretion have not yet been fully determined. We examined the interactions of rat GRF and SRIF in the concentration ranges present in rat hypophysial-portal blood, on the secretion of GH from dispersed male rat anterior pituitary cells in monolayer culture. The effects of exposing cells to GRF and/or SRIF (0.01-1.nmol/l) for 1 h were compared with the effects of preincubation of cells with SRIF before experimental incubations. As anticipated, the stimulatory effects of 0.1-1 nmol GRF/1 were abolished by concurrent incubation with SRIF at an equimolar concentration, although SRIF, at these concentrations, did not significantly inhibit basal GH secretion. Conversely, pre-exposure to 0.1 nmol SRIF/1 for 30 or 60 min, resulted in an increase in GH secretion during a subsequent 60-min incubation period, both in the absence or in the presence of GRF (0.01-1 nmol/l). Pretreatment with GRF caused increased responsivity to GRF rather than significant sensitization of the GH response to GRF. These observations demonstrate actions of SRIF, at low and probably physiological concentrations, which are more complex than those of a pure inhibitor of GH secretion. Pre-exposure of the pituitary to SRIF enhances subsequent GH secretion, suggesting that SRIF may play an additional physiological role in amplifying the GRF signal.

Corticotrophin-releasing peptides in rat hypophysial portal blood after paraventricular lesions: a marked reduction in the concentration of corticotrophin-releasing factor-41, but no change in vasopressin

Previous data show that corticotrophin-releasing factor-41 (CRF-41), arginine vasopressin (AVP) and oxytocin are released into hypophysial portal blood. It has been presumed that the CRF-41 originates mainly from parvicellular neurones of the paraventricular nuclei (PVN); however, AVP and oxytocin could also be derived as a consequence of preterminal release from magnocellular projections to the neurohypophysis. The latter has been suggested to be the case for AVP as assessed by studies of the median eminence in vitro. Here we have investigated the source of CRF-41, AVP and oxytocin in hypophysial portal blood of adult male Wistar rats 8-10 days after surgical lesioning of the PVN. In PVN-lesioned animals the output of CRF-41 into hypophysial portal blood was reduced by about 90%, and that of oxytocin by about 40%: however, the output of AVP into portal blood was reduced only by about 10%. The release of AVP into portal blood increased after adrenalectomy; this increased release could be returned to normal by treatment with dexamethasone. No change of AVP release occurred after adrenalectomy in animals in which the PVN had been lesioned. These results show (i) that most of the CRF-41 released into hypophysial portal blood is derived from the PVN, (ii) that in PVN-lesioned animals AVP and oxytocin release remains at near normal or 60% of normal respectively, suggesting that a substantial amount of both neuropeptides in portal blood is derived as a consequence of preterminal release from supraoptic nuclei projections in the median eminence, and (iii) that glucocorticoid feedback inhibition of AVP release is exerted at the level of the PVN.

Effects of Intracerebroventricular Administration of Growth Hormone-Releasing Factor and Corticotropin-Releasing Factor on Somatostatin Secretion into Rat Hypophysial Portal Blood

To clarify the regulatory mechanisms for the secretion of somatostatin (SRIF) from the hypothalamus, the effects of intracerebroventricular (i.c.v.) administration of growth hormone-releasing factor (GRF) and corticotropin-releasing factor (CRF) on SRIF secretion into hypophysial portal blood were examined in pentobarbital-anesthetized male rats. Neither the concentration of SRIF in portal plasma nor the secretion rate of SRIF was changed after i.c.v. administration of 0.9% saline. Administration of 10 ng or 5 micrograms human GRF i.c.v. produced a significant increase in the portal plasma concentration and secretion rate of SRIF. Likewise, 5 micrograms CRF significantly increased the portal plasma concentration and secretion rate of SRIF. These results suggest that the neuropeptides GRF and CRF centrally influence SRIF secretion into hypophysial portal blood.

Epinephrine in Rat Hypophysial Portal Blood Is Derived Mainly from the Adrenal Medulla

This study was performed to determine if epinephrine (EPI) present in hypophysial portal blood has a peripheral or a central origin. Pituitary stalk and femoral arterial EPI plasma levels were simultaneously measured in rats anesthetized with thiopental or urethane. EPI was measured by two different methods: radioenzymatic assay or HPLC followed by electrochemical detection. In thiopental-anesthetized rats, levels were higher in hypophysial portal than in femoral arterial plasma with both methods. In urethane-anesthetized rats, no difference in EPI concentrations was found between peripheral and portal plasma. A rapid blood volume depletion (15 ml/kg in 3 min) evoked a marked elevation of pituitary stalk and arterial EPI plasma concentration while norepinephrine (NE) and dopamine (DA) levels were unaffected. Under both types of anesthesia, the increase of EPI levels was higher in peripheral than in portal plasma. After removal of the adrenal glands, EPI was undetectable or barely detectable in pituitary stalk and arterial plasma. The hemorrhage-induced stimulation of EPI secretion was blunted in portal and peripheral plasma of adrenalectomized rats. These findings indicate that: (1) hypophysial portal EPI plasma concentration is highly dependent on an adrenal source; (2) hypothalamic EPI is not released into portal circulation in response to acute hemorrhage, and (3) anesthesia markedly influences catecholamines plasma levels.

Release of oxytocin but not corticotrophin-releasing factor-41 into rat hypophysial portal vessel blood can be made opiate dependent

The effects of morphine dependence and abrupt opiate withdrawal on the release of oxytocin and corticotrophin-releasing factor-41 (CRF-41) into hypophysial portal vessel blood in rats anaesthetized with urethane were investigated. Adult female Sprague-Dawley rats were made dependent upon morphine by intracerebroventricular infusion of morphine for 5 days; abrupt opiate withdrawal was induced by injection of the opiate antagonist naloxone. The basal concentrations of oxytocin in portal or peripheral plasma from morphine-dependent rats did not differ significantly from those in control, vehicle-infused rats. In rats in which the pituitary gland was not removed after stalk section, the i.v. injection of naloxone hydrochloride (5 mg/kg) resulted in a large and sustained increase in the concentration of oxytocin in both portal and peripheral plasma in control and morphine-dependent rats. The i.v. injection of naloxone resulted in a threefold increase in the secretion of oxytocin into portal blood in acutely hypophysectomized rats infused with morphine, but did not alter oxytocin secretion in vehicle-infused hypophysectomized rats. The concentration of oxytocin in peripheral plasma in both vehicle- and morphine-infused hypophysectomized rats was at the limit of detection of the assay and was unchanged by the administration of naloxone. There were no significant differences in the secretion of CRF-41 into portal blood in vehicle- or morphine-infused hypophysectomized rats either before or after the administration of naloxone. These data show that, as for oxytocin release from the neurohypophysis into the systemic circulation, the mechanisms which regulate oxytocin release into the portal vessel blood can also be made morphine dependent. The lack of effect of morphine or naloxone on the release of CRF-41 or other stress neuro-hormones suggests that the effect of opiate dependence and withdrawal is selective for oxytocin and is not simply a non-specific response to 'stress'.

Corticotropin-Releasing Factor Secretion Increases in Rat Hypophysial Portal Blood during Insulin-Induced Hypoglycemia

To study the possible involvement of hypothalamic corticotropin-releasing factor (CRF) in the stimulation of adrenocorticotropic hormone (ACTH) release caused by insulin-induced hypoglycemia (IIH), we measured CRF secretion in hypophysial portal blood (HPB) in rats anesthetized with sodium thiopental after injection of insulin. Before treatment, systemic ACTH levels (952 +/- SE 143 pg/ml; n = 12) were well above normal values, probably reflecting the anesthetic and surgical stress consecutive to the preparation for portal blood collection. Insulin injection induced a significant increase of ACTH release within 15 min (1,588 +/- 168 vs. 741 +/- 144 pg/ml; n = 6, in vehicle-injected rats) which lasted for at least 1 h. CRF levels in HPB were 857 +/- SE 168 pg/ml (n = 13) during the first-hour pretreatment collection. Vehicle injection did not modify CRF secretion (759 +/- 142 pg/ml; n = 6). Insulin injection provoked a significant increase in CRF release (1,449 +/- 257 pg/ml; n = 7). These data suggest that an increased hypothalamic CRF secretion is responsible for the stimulation of pituitary ACTH release following IIH. The possible involvement of central neuromediators in the IIH-induced CRF production is discussed.

Effects of adrenalectomy and glucocorticoids on the peptides CRF-41, AVP and oxytocin in rat hypophysial portal blood.

1. The effects of adrenalectomy (3 weeks) and dexamethasone (3 h) treatment on the release of corticotrophin-releasing factor-41 (CRF-41), arginine vasopressin (AVP), oxytocin (OT), adrenocorticotrophin (ACTH) and corticosterone were studied in adult female Wistar rats. 2. The animals were anaesthetized with sodium pentobarbitone which, as assessed by the effects on the circadian rhythm of plasma ACTH and corticosterone, appeared to be a better anaesthetic than either urethane or alphaxalone for studies on the hypothalamic-pituitary-adrenal system. 3. Adrenalectomy increased the concentrations of ACTH in peripheral plasma and the output of CRF-41 and AVP into hypophysial portal plasma. 4. Dexamethasone administered to adrenalectomized rats significantly reduced the concentration of ACTH in peripheral plasma and the amount of AVP released into portal plasma. However, dexamethasone did not affect the output of CRF-41 into portal blood. 5. The output of OT into portal plasma was unaffected by either adrenalectomy or dexamethasone treatment. 6. Dexamethasone administered to adrenalectomized rats reduced significantly the ACTH response to CRF-41. 7. These results show that the feed-back action of glucocorticoids is mediated by two mechanisms. The increased release of ACTH which follows adrenolectomy [corrected] is produced predominantly by an increased release of both CRF-41 and AVP into hypophysial portal blood. The intermediate negative feed-back of glucocorticoids is produced by a reduction in the output of AVP but not CRF-41 into portal blood and, as well, by a significant reduction in the responsiveness of the anterior pituitary gland to CRF-41.

The corticotropin-releasing factor release in rat hypophysial portal blood is mediated by brain catecholamines.

In order to study the involvement of the hypothalamic corticotropin-releasing factor (CRF) in catecholamine-induced adrenocorticotropin (ACTH) secretion, we have measured CRF levels in rat hypophysial portal blood (HPB) after the pharmacological destruction of the ventral noradrenergic bundle (VNAB), using 6-hydroxydopamine (6-OHDA) stereotaxically injected into the VNAB. CRF levels in HPB were measured by radioimmunoassay, and the effects of 6-OHDA injection were controlled by the determination of catecholamine concentrations in the total hypothalamus. VNAB lesions induced a dramatic decrease in norepinephrine and epinephrine hypothalamic concentration. The CRF levels in HPB were also significantly reduced. These results suggest that central catecholamines exert a direct stimulatory control on the CRF release and play a major role in stress-induced ACTH secretion.

LH-RH in the rat and mouse hypothalamus and rat hypophysial portal blood: confirmation of identity by high performance liquid chromatography

The nature of the immunoreactive (IR) form of luteinizing hormone-releasing hormone (LH-RH) in rat hypophysial portal vessel blood and in extracts of hypothalamus from rats and normal and hypogonadal ( hpg) mice was investigated using high performance liquid chromatography and two highly specific anti-LH-RH sera. In rat hypophysial portal blood and in hypothalamic extracts from rats and normal mice a single immunoreactive peak was present which corresponded in retention time to synthetic LH-RH. No LH-RH-IR was detected in hypothalamic extracts from the hpg strain of mouse.

Vasoactive intestinal peptide in rat hypophysial portal blood: effects of electrical stimulation of various brain areas, the oestrous cycle and anaesthetics.

Vasoactive intestinal peptide (VIP) was measured by radioimmunoassay in hypophysial portal and peripheral blood from adult male and female Wistar rats. The results confirmed that the concentration of VIP in hypophysial portal blood was significantly greater than in peripheral blood and showed that VIP release into portal blood was not affected by removal of the gut, the largest peripheral source of VIP. Electrical stimulation of the median eminence, several hypothalamic nuclei, the amygdala or hippocampus had no significant effect on the release of VIP into portal blood, possibly because under the conditions of the experiment the spontaneous release of VIP is already at a maximum. In female rats, the VIP released into portal blood collected between 13.00 and 18.30 h of each day of the 4-day oestrous cycle varied under different anaesthetics, and there was no consistent increase in VIP release on pro-oestrus suggesting that VIP is not involved in the pro-oestrous surge of prolactin.

Oxytocin and vasopressin in rat hypophysial portal blood: experimental studies in normal and Brattleboro rats

Oxytocin (OT) and vasopressin (VP) were measured by radioimmunoassay in hypophysial portal and peripheral blood from male Wistar rats and heterozygous and homozygous Brattleboro rats anaesthetized with urethane. In Wistar rats the concentrations of OT and VP were about 50 times greater than the concentrations in peripheral blood, whether or not the pituitary gland was left in situ during collection, and also considerably greater than the reported concentrations of the peptides in the cerebrospinal fluid. The release of both peptides was increased significantly by a lesion of the supraoptico-hypophysial tract that led to diabetes insipidus, but which left intact the external layer of the median eminence (ME). Concentrations of VP were undetectable in plasma from homozygous Brattleboro rats, but the portal plasma concentrations of VP in heterozygous Brattleboro rats were not significantly lower than in Wistar rats. The concentrations of OT in portal plasma from both types of Brattleboro rat were significantly higher than in Wistar rats. The output of VP and OT into hypophysial portal blood of Wistar rats was not significantly affected by electrical stimulation of the suprachiasmatic, supraoptic or paraventricular nuclei or the ME using two types of stimuli, one of which produced an increase in peripheral plasma concentrations of VP and OT in intact rats and a significant increase in the release of LH-releasing hormone into hypophysial portal blood. The output of VP and OT into portal blood was also not significantly affected by either adrenalectomy with or without injection of dexamethasone or the injection of either the 5-hydroxytryptamine (5-HT) synthesis blocker, parachlorophenylalanine, or the 5-HT uptake blockers, alaproclate or zimelidine. These results show that large amounts of OT as well as VP are released into hypophysial portal blood from fibres of the hypothalamo-neurohypophysial system that terminate in the external layer of the ME. Although distinct from the fibres that terminate in the pars nervosa (PN), the findings in Brattleboro rats show that the VP fibres of the ME system originate in neurones with a genomic mechanism for VP synthesis similar to that of the VP neurones that project to the PN. The lack of effect of adrenalectomy and the administration of 5-HT synthesis and uptake blockers must be interpreted with caution since the results obtained with electrical stimulation suggest that when the pituitary stalk is cut the release of OT and VP into portal blood approaches a maximum and may therefore be difficult to alter by experimental manipulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of enkephalins and related peptides in rat hypophysial portal blood

Rat hypophysial portal blood, collected from the pituitary stalk, was extracted and enkephalins were assayed by different RIA. Met-Enk-IR and Leu-Enk-IR levels were1635 ± 470pg/ml and125 ± 50pg/ml, respectively. Using HPLC characterization, the presence in portal blood of Met-Enk, Leu-Enk, proenkephalins fragments and dynorphin J-17 has been demonstrated. An unidentified Met-Enk-IR peptide has also been found.

Prostaglandin D 2 stimulates vasoactive intestinal polypeptide release into rat hypophysial portal blood

The effect of prostaglandin D 2 (PGD 2) on vasoactive intestinal polypeptide (VIP) release from the hypothalamus was examined by determining plasma VIP levels in rat hypophysial portal blood. Intraventricular injection of PGD 2 (5 μg/rat) caused a 3-fold increase in the concentration of plasma VIP in hypophysial portal blood in anesthetized rats. A PGD 2 metabolite, 13,14-dihydro-15-keto PGD 2, did not affect VIP levels in portal blood. The flow rate of hypophysial portal blood was not changed after the injection of PGD 2. The intraventricular injection of PGD 2, but not PGD 2 metabolite, resulted in an increase in peripheral plasma prolactin (PRL) levels in the rat. These findings suggest that PGD 2 plays a stimulatory role in regulating VIP release from the hypothalamus into hypophysial portal blood and causes PRL secretion from the pituitary in rats.

Somatostatin-28(1-12)-like immunoreactive substance is secreted into hypophysial portal vessel blood in the rat.

Somatostatin-28(1-12)-like immunoreactivity was measured in extracts of rat hypophysial portal blood and peripheral blood. The concentration of somatostatin-28(1-12) was higher in portal than in peripheral extracts, and its release into portal vessel blood was increased 4- to 5-fold by electrical stimulation of the median eminence. These results show that somatostatin-28(1-12) may be a physiological neurohormone and/or neurotransmitter.

Effect of prostaglandin E1 on vasoactive intestinal polypeptide release from the hypothalamus and on prolactin secretion from the pituitary in rats.

In order to elucidate the mechanisms by which prostaglandin (PG) affects PRL secretion, the effect of PGE1 on vasoactive intestinal polypeptide (VIP) release from the rat hypothalamus was examined by determining plasma VIP levels in rat hypophysial portal blood in vivo and VIP release from the perifused hypothalamus in vitro. Intraventricular injection of PGE1 (1 and 5 micrograms/rat) caused a 2- to 3-fold increase in the concentration of plasma VIP in hypophysial portal blood in anesthetized rats. The flow rate of portal blood was slightly increased after the injection of PGE1. VIP release from the perifused rat hypothalamus was stimulated by high potassium levels (56 mM). The infusion of PGE1 (10 microM) resulted in a significant increase in VIP release from the hypothalamus in vitro. Both these responses were calcium dependent. The intraventricular injection of PGE1 (1 and 5 micrograms/rat) resulted in a dose-related increase in peripheral plasma PRL levels in the rat. These findings suggest that PGE1 plays a stimulatory role in regulating VIP release from the hypothalamus into hypophysial portal blood and causes PRL secretion from the pituitary in rats.

Peptide histidine isoleucine- and vasoactive intestinal polypeptide-like immunoreactivity coexist in rat hypophysial portal blood

Plasma immunoreactive peptide histidine isoleucine (PHI) and vasoactive intestinal polypeptide (VIP) levels were measured by specific radioimmunoassays in urethane-anesthetized rats. Basal levels of plasma PHI-like immunoreactivity (PHI-LI) in the hypophysial portal blood were 414 ± 180 pmol/l (means ± S.E.), about 7 times higher than in the peripheral blood. VIP-like immunoreactivity (VIP-LI) was also high in the portal blood (399 ± 139 pmol/l). The correlation coefficient between PHI-LI and VIP-LI was 0.76. These findings suggest that PHI and VIP are co-released from the median eminence into the hypophysial portal blood in rats.

Intraventricularly injected growth hormone stimulates somatostatin release into rat hypophysial portal blood.

The effects of GH on the release of somatostatin from the hypothalamus were assessed by measuring the concentrations of immunoreactive somatostatin (IRS) in hypophysial portal blood of urethane-anesthetized male rats. A significant and dose-related increase of IRS in hypophysial portal blood was observed during 20-80 min after a single injection of rat GH (5 and 25 micrograms) into the third ventricle. An intraventricular injection of ovine LH or vehicle alone did not affect IRS values in hypophysial portal blood. When rat GH was repeatedly injected into the cerebral ventricle at 75-min intervals, IRS in hypophysial portal blood rose following each injection in a similar pattern with a latency of 30-45 min. These findings suggest that the release of somatostatin from the hypothalamus is regulated, at least in part, by GH. Furthermore, in view of the inhibitory effect of somatostatin on GH secretion, stimulation by GH of somatostatin release into hypophysial portal blood may be involved in the mechanism by which GH regulates its own secretion.

Stimulation by bombesin of immunoreactive somatostatin release into rat hypophysial portal blood.

Bombesin was injected into the cerebral ventricle of male rats anesthetized with urethane to study its effect on plasma levels of immunoreactive somatostatin (IRS) in hypophysial portal and jugular blood. An intraventricular injection of bombesin (0.2 and 2 micrograms/rat) caused a significant and dose-related increase in plasma IRS in hypophysial portal blood but not in jugular blood. Although bombesin placed into the cerebral ventricle is known to stimulate glucagon and epinephrine release, an iv injection of glucagon (100 micrograms/100 g BW) or epinephrine (2.5 micrograms/100 g BW) did not cause any significant changes in plasma IRS levels in hypophysial portal and jugular blood, suggesting that these substances do not mediate bombesin stimulation of portal IRS release. Pretreatment with naloxone (75 micrograms/100 g BW, iv) failed to affect the portal IRS release induced by bombesin (2 micrograms/rat), indicating that the opiate receptor is not likely to be involved in this reaction. To ascertain whether IRS released by bombesin into hypophysial portal blood is biologically active, the effect of bombesin on the plasma GH level was then examined. Bombesin (2 micrograms/rat) injected intraventricularly completely suppressed the rise of plasma GH after the intraventricular injection of beta-endorphin (1 microgram/rat) or the iv injection of prostaglandin E1 (5 micrograms/100 g BW). Bombesin thus appears to stimulate the secretion of IRS, and probably biologically active somatostatin as well, from the hypothalamus into hypophysial portal blood, thereby inhibiting GH release from the anterior pituitary.

Effect of intraventricular injection of neurotensin and other various bioactive peptides on plasma immunoreactive somatostatin levels in rat hypophysial portal blood.

The role of various bioactive peptides in the control of secretion of hypothalamic somatostatin into the hypophysial portal blood was examined in anesthetized rats. Hypophysial portal blood was withdrawn at a rate of 5.0 microliter/min into a chilled tube through a cannula placed over the stump of the pituitary stalk and segmented every 20 min by air bubbles. Immunoreactive somatostatin (IRS) in the plasma was extracted with acetic acid and acetone and quantified by RIA. Basal levels (mean +/- SE) of plasma IRS in the hypophysial portal blood were 646 +/- 36 and 317 +/- 44 pg/ml in urethane- and pentobarbital-anesthetized rats, respectively. Under urethane anesthesia, injection of synthetic neurotensin into the lateral ventricle at various doses in the range of 0.016--2 microgram/rat caused a significant and dose-related increase of plasma IRS levels in the hypophysial portal blood, and this effect of neurotensin was significantly (P less than 0.05) suppressed by pretreatment with diphenhydramine (1 mg/100 g BW, iv), a histamine receptor blocker. Enhancement of IRS release by neurotensin was also observed in pentobarbital-anesthetized rats. Intraventricular injection of substance P (10 microgram/rat), beta-endorphin (1 and 5 microgram/rat), or [Met5]enkephalin had no effect on the level of somatostatin in the hypophysial portal blood of urethane-anesthetized rats. These results suggest a release of hypothalamic somatostatin into the hypophysial portal blood in response to intraventricular administration of neurotensin, probably by a histaminergic mechanism.

Plasma immunoreactive somatostatin levels in rat hypophysial portal blood: Effect of glucagon administration

Plasma somatostatin levels of the hypophysial portal blood was examined in urethane-anesthetized rats. Portal blood was withdrawn at a rate of 4.7 μl/min via a cannula placed over the stump of the pituitary stalk, collected into the ice-cold tube at 20min intervals before and after the intraventricular injection of test materials. Immunoreactive somatostatin extracted from the plasma with acid acetone was measured by a specific radioimmunoassay. Basal plasma somatostatin concentrations, 778±52pg/ml (means±SE), did not significantly change after the administration of control solution (0.1N acetic acid in physiological saline), while intraventricular injection of glucagon (2, 10 and 50 μg) caused a significant and dose-related increase in plasma somatostatin during the first 20min. These results suggested that somatostatin release from the median eminence into the hypophysial portal vessel was stimulated by glucagon, although the physiologycal significance remains to be clarified.