Large Molecular Weight Immunoreactive Corticotrophin-Releasing Hormone has Bioactivity on Superfused Ovine Pituitary Cells

Human placental extracts fractionated with Sephadex G-50 produced three peaks of corticotrophin-releasing hormone immunoreactivity, a large molecular weight peak (M_r30,000), an intermediate peak (4,758 < M_r < 10,000) and a low molecular weight peak coeluting with the 41-residue hormone. All three peaks of immunoreactivity stimulated the release of β-endorphin-like immunoreactivity from ovine pituitary cells superfused in vitro. No response was observed from unstimulated cells superfused in parallel. Gel chromatography indicated that intermediate and small molecular weight forms of human corticotrophin-releasing hormone immunoreactivity remained intact after contact with the ovine pituitary cells, whereas the large molecular weight material dissociated to produce 41-residue hormone immunoreactivity. The secreted β-endorphin immunoreactivity was shown by gel chromatography to comprise both β-lipotrophin-like and the 31-residue β-endorphin-like immunoreactivity. The data show that the intermediate and low molecular weight forms of placental corticotrophin-releasing hormone immunoreactivity are bioactive and suggest that the intermediate form is a hormone precursor, possibly procorticotrophin-releasing hormone_125–196, and the small form is identical to the hypothalamic hormone. The results with the larger molecular weight material indicate that it is likely to be a complex of the mature 41-residue hormone and a binding protein.     

Reduced Oxytocin Release from the Neural Lobe of Lactating Rats is Associated with Reduced Pituitary Content and does not Reflect Reduced Excitability of Oxytocin Neurons

Lactating rats show reduced oxytocin release compared with virgin female rats in response to a variety of stimuli, including stress and osmotic stimulation. We sought to establish whether this is a consequence of a reduced response in the oxytocin cells, or of a change in stimulus-secretion coupling at the level of the neurosecretory terminals in the neural lobe. Blood sampling experiments in anaesthetized rats showed that systemic administration of cholecystokinin resulted in significantly less oxytocin release in lactating rats than in virgin female rats. Electrophysiological recordings of single cells in the supraoptic nucleus, however, showed no difference in the responsiveness of oxytocin cells to this stimulus. Oxytocin release evoked by electrical stimulation or by depolarization with high potassium solutions was lower in isolated neural lobes from lactating rats than in glands from non-lactating rats, whereas evoked vasopressin release was similar in the two groups. The lactating rat neural lobes had a reduced oxytocin content: to study the consequences of depletion we compared hormone release evoked by electrical stimulation in vitro in neural lobes from normal male rats, and from male rats given 2% NaCI to drink for 2 or 4 days. Saline drinking resulted in a reduction in gland content of both oxytocin and vasopressin, and the evoked release of both hormones was also significantly reduced when expressed as a percentage of the gland content, as was also seen for oxytocin release for glands from lactating rats. Finally, measurement of the extracellular potassium response to stimulation of the isolated neural lobe as an index of the excitability of neural lobe neurosecretory axons was unchanged in lactating rats compared with virgin female rats. Together, the data indicate that reduced oxytocin release observed in lactating rats is a simple consequence of reduced oxytocin content in the neural lobe rather than of a reduced excitability of the oxytocin neurons.     

Prolactin-Releasing Peptide Regulates the Cardiovascular System Via Corticotrophin-Releasing Hormone

Prolactin-releasing peptide (PrRP)-producing neurones are known to be localised mainly in the medulla oblongata and to act as a stress mediator in the central nervous system. In addition, central administration of PrRP elevates the arterial pressure and heart rate. However, the neuronal pathway of the cardiovascular effects of PrRP has not been revealed. In the present study, we demonstrate that PrRP-immunoreactive neurones projected to the locus coeruleus (LC) and the paraventricular nucleus (PVN) of the hypothalamus. The c- fos positive neurones among the noradrenaline cells in the LC, and the parvo- and magnocellular neurones in the PVN, were increased after central administration of PrRP. The arterial pressure and heart rate were both elevated after i.c.v. administration of PrRP. Previous studies have demonstrated that PrRP stimulated the neurones in the PVN [i.e. oxytocin-, vasopressin- and corticotrophin-releasing hormone (CRH)-producing neurones], which suggests that PrRP may induce its cardiovascular effect via arginine vasopressin (AVP) or CRH. Although the elevation of blood pressure and heart rate elicited by PrRP administration were not inhibited by an AVP antagonist, they were completely suppressed by treatment with a CRH antagonist. Thus, we conclude that PrRP stimulated CRH neurones in the PVN and that CRH might regulate the cardiovascular system via the sympathetic nervous system.     

Stress-Induced Oxytocin Release in the Rat After Lesion of the Paraventricular Nuclei; Possible Deficiency of Corticotrophin-Releasing Factor

Oxytocin is released under stressful conditions and corticotrophin-releasing factor (CRF) is known to be involved in mediating general ‘stress responses’. We therefore examined whether CRF neurons in the paraventricular nucleus participate in the stress-induced oxytocin release in the rat. CRF (0.02 to 2 nmol) injected into the third ventricle produced a dose-dependent rise in the plasma oxytocin concentration. The oxytocin release induced by CRF occurred without a change in blood pressure, and was not affected by dexamethasone pretreatment, which prevents adrenocorticotrophin release following CRF injection. Lesioning of the paraventricular nucleus reduced oxytocin release by immobilization stress, but did not alter the release of oxytocin in response to osmotic stimulation induced by intraperitoneal injection of hypertonic saline. Anti-CRF serum injection into the third ventricle reduced delayed oxytocin response to immobilization stress. These results are consistent with the hypothesis that CRF neurons in the paraventricular nucleus are involved in the oxytocin release during immobilization stress in the rat.     

Cortisol Response to a Combined Dexamethasone-Human Corticotrophin-Releasing Hormone Challenge in Patients with Depression

We administered a combined dexamethasone-human corticotrophin-releasing hormone (hCRH) challenge test to 14 in-patients with a major depressive episode and to 14 age-matched controls. After pretreatment with 1.5 mg dexamethasone at 2300 h the day before, 100 μg hCRH was administered iv at 1500 h. Blood samples for cortisol determinations by radioimmunoassay were drawn at 1400 h, 1430 h and 1500 h before infusion of hCRH and thereafter every 15 min until 1700 h. Cortisol secretion after injection of hCRH assessed as area under the curve was significantly increased in patients with depression when compared to controls (14.5 ± 4.3 ng × min × 1,000/ml vs 3.1 ± 2.4 ng × min × 1,000/ml). Multiple regression analysis among patients revealed a significant impact of age and severity of depression upon hCRH-induced cortisol secretion, whereas in normal controls no significant influence of age on cortisol secretion after hCRH emerged.
Our data show that in depressed patients hCRH evokes an escape from dexamethasone-induced suppression of the pituitary-adrenocortical activity, whereas it fails to do so among controls. This finding suggests that at the pituitary level the action of hCRH is enhanced by a factor that is less sensitive to dexamethasone suppression in depression. We postulate that this factor is vasopressin.     

Differential Effects of Glucocorticoids on Corticotrophin-Releasing Factor in the Rat Pituitary Neurointermediate Lobe and Median Eminence

We have investigated the effects of glucocorticoid manipulation on corticotrophin-releasing factor (CRF-41) in the neurointermediate lobe (NIL) and median eminence (ME) of the rat using a radioimmunoassay specific for CRF-41. CRF-41 content in the NIL (88+/-7 fmol, mean +/- SEM) was not significantly altered by administration of dexamethasone in drinking water for 12 days (67+/-9 fmol) or adrenalectomy for 12 days (96+/-17 fmol). The saline-stimulated increase in NIL CRF-41 content (154+/-24 fmol) was not affected by dexamethasone (152+/-24 fmol) or adrenalectomy (179+/-21 fmol). In contrast, the content of CRF-41 in the ME declined following dexamethasone treatment (1247+/-92 fmol to 864+/-26 fmol), or adrenalectomy (854+/-78 fmol). Our results provide further evidence that CRF-41 in the NIL and ME can be differentially regulated.     

Acetylcholine Synthesis in a Primary Culture of Porcine Intermediate Lobe Cells

Previous pharmacological studies with the pituitary gland have suggested that acetylcholine (ACh) might be involved in the regulation of intermediate lobe (IL) function. Whether ACh is endogenous to the IL cells or provided from an extrinsic source is unclear. The present experiments tested the possibility that the endocrine cells of the IL may be a source of ACh by measuring certain cholinergic markers in a primary culture of dissociated porcine cells. The endogenous ACh content was readily measurable in both the freshly dissociated IL cells and in 2- or 4-day primary cultures. Choline acetyltransferase activity was also measurable in the freshly dissociated and cultured IL cells and was reduced by 53% in the presence of a specific inhibitor, napthylvinylpyridine (50 μM). IL cells incubated in the presence of [¹⁴C]choline (1 μM) were able to synthesize [¹⁴C]ACh and the accumulation of the new ACh was inhibited by hemicholinium-3 (30 μM), a competitive inhibitor of high affinity choline uptake at cholinergic nerve terminals. In conclusion, these results demonstrate that the endocrine cells of the IL are capable of synthesizing and storing ACh.     

Gonadotropin-Releasing Hormone and Thyrotropin-Releasing Hormone Regulation of G Protein Function in the Rat Anterior Pituitary Lobe

In order to evaluate the role of guanine nucleotide-dependent transducer proteins (G proteins) in hormone-mediated signal transduction in the anterior pituitary lobe, we examined the effect of gonadotropin-releasing hormone (GnRH) and thyrotropin-releasing hormone (TRH) on two parameters of G protein function, namely [³⁵ S]GTP_γS binding and low K_mGTPase activity. Plasma membranes were prepared from anterior pituitary lobes of adult male rats using conventional procedures. GTP binding was determined by incubating 2 to 5 μg membrane protein with approximately 100,000 cpm [³⁵ S]GTP_γS in a buffer containing 20 mM Tris- HCl, 1 mM EDTA, 1 mM dithiothreitol, and 100 mM NaCI at a pH of 7.4 for 10 or 15 min at 37 °C GnRH agonist and TRH stimulated high affinity [³⁵ S]GTP_γS binding in a concentration-dependent manner. GTP binding was maximally stimulated by GnRH agonist (1 μM) and TRH (0.1 μM) by up to 27% and 34%, respectively. A time-course study revealed that 1 μM GnRH agonist stimulated GTP binding by 30% at 15 min; 0.1 μM TRH stimulated GTP binding by 23% at 1 min, 18% at 5 min and 25% at 10 min. A stable GTP analog, 5′-guanylylimidodiphosphate, inhibited GnRH- as well as TRH-stimulated GTP binding. GnRH antagonist did not affect GTP binding. However, in the presence of the antagonist, stimulation of GTP binding by the GnRH agonist was completely blocked. The low K_mGTPase activity (EC 3.6.1.-), another parameter of G protein function, was assayed in 2 to 5 μg membrane protein using [γ-³² P]GTP at 37 °C in an ATP-regenerating buffer containing 1 μM unlabeled GTP. GnRH agonist (0.1 μM) and TRH (1 μM) maximally stimulated this GTPase activity by up to 50% and 40%, respectively. GnRH agonist (1 μM) stimulated the GTPase activity by 30% at 10 min and 48% at 30 min. TRH (1 μM) stimulated the GTPase activity at all time points monitored; stimulation was 46% at 5 min, 49% at 20 min, and 41% at 30 min. Interestingly, the GnRH antagonist stimulated GTPase activity by about 20%, but inhibited GnRH agonist-stimulated GTPase activity in a concentration-dependent manner. These results indicate that the binding of GnRH and TRH to their receptors results in interaction of the receptor with a G protein and activation of the G protein cycle.     

Dissociation of ACTH-Secretory Mechanisms in Rat Pituitary Cells: Evidence that Basal and Vasopressin-Stimulated Secretion Act via a Mechanism Distinct from that of Corticotrophin-Releasing Factor

To study the relationship between basal, corticotrophin-releasing factor- (CRF) and vasopressin-stimulated adrenocorticotrophic hormone (ACTH) secretion by rat anterior pituitary cells, dissociated anterior pituitary cells were seeded into tissue culture dishes and treated overnight with a cytotoxic conjugate specific for CRF-target cells. Immediately after extensive washing, or 1, 3, 6, 9 or 12 days later, cellular ACTH content, basal secretion and secretion in response to CRF or vasopressin were measured. ACTH content and basal secretion rate increased over time in both cytotoxic conjugate-pretreated and vehicle-pretreated cell populations. Compared with vehicle-pretreated cells, basal ACTH secretion was higher in cytotoxic conjugate-pretreated populations by Day 3 and reached an apparent maximum by Day 6. In such cells, net ACTH secretion post-vasopressin decreased as basal secretion increased; by Day 6 no vasopressin-stimulated secretion was seen. In cytotoxic conjugate-pretreated cells, the response to CRF was initially completely eliminated; however, as ACTH content and secretion increased with time, a small recovery of the response to CRF was observed on Days 3 and 6. In vehicle-pretreated cells, ACTH secretion in response to vasopressin increased in parallel with basal secretion. The response to CRF increased progressively over Days 1 to 6 as well; this response was more closely related to the increases observed in ACTH content. The shift in responsiveness of the cytotoxic conjugate-pretreated cells over time, from vasopressin-responsive to CRF-responsive, further demonstrates the dissociation of the mechanisms of the ACTH secretory responses to CRF and vasopressin. In addition, the increase in unstimulated secretion at the expense of the response to vasopressin in cytotoxic conjugate-treated cells is consistent with a common pathway for vasopressin-stimulated and basal release of ACTH.     

The Effect of Growth Hormone Secretagogues and Neuropeptide Y on Hypothalamic Hormone Release from Acute Rat Hypothalamic Explants

Growth hormone (GH) secretagogues (GH-releasing peptides and their non-peptide analogues) stimulate growth hormone release via specific G-protein coupled receptors both directly from the pituitary gland and through stimulation of the hypothalamus. The exact mechanism of action in the hypothalamus is not known. The presence of endogenous GH releasing hormone (GHRH) seems to be necessary for the in-vivo actions of growth hormone secretagogues (GHSs), but data suggest that further factors must be involved as well. The effect of GHSs is not entirely specific for the GH axis; they release prolactin and stimulate the hypothalamo-pituitary-adrenal axis causing elevations in circulating ACTH and cortisol levels in both animal and human studies. Recently, it has also been suggested that GHSs stimulate hypothalamic neuropeptide Y (NPY) neurones. In the present study, we have therefore investigated the direct effect of several GHSs (GHRP-6, hexarelin and the non-peptide analogues L-692, 429 and L-692, 585) on GHRH, somatostatin (SS), corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) release in vitro in an acute rat hypothalamic incubation system. We also assessed the effect of NPY on GHRH, SS and AVP release. Freshly removed hypothalami were incubated in control media for 20 min and then in 1-4 consecutive 20-min periods in each of the test substances at different concentrations. There was no significant change in either the basal or potassium-stimulated release of GHRH or SS at low concentrations of any of the secretagogues; however, at millimolar doses a paradoxical inhibition of GHRH was observed with GHRP-6, hexarelin and L-692 585 (data are expressed as the ratio of treated to preceding basal release; at 20 min control group: 0.97+/-0.02, GHRP-6: 0.55+/-0.04, P<0.001 compared to control group; hexarelin: 0. 56+/-0.06, P<0.001, L-692,585: 0.70+/-0.03, P<0.001), while SS was stimulated after 60 or 80 min (at 80 min control: 0.80+/-0.03, hexarelin: 1.23+/-0.07, P<0.05 and L-692,585: 1.37+/-0.11, P<0.05). GHSs stimulated hypothalamic AVP release (at 20 min control: 0. 99+/-0.06 ratio to basal release, 10-4 M concentration of GHRP-6: 6. 31+/-1, P<0.001, hexarelin: 1.88+/-0.4, P<0.01, L-692,429: 1.90+/-0. 5, P<0.05 and L-692,585: 2.34+/-0.96, P<0.01), while no stimulatory effect was found on CRH release. NPY significantly stimulated SS and inhibited basal and potassium-stimulated GHRH release, while potentiating potassium-evoked AVP secretion. The Y1 receptor antagonist BIBP 3226 did not inhibit the effects of NPY on SS, GHRH or AVP release. We therefore conclude that, in this in-vitro rat hypothalamic incubation model, growth hormone secretagogues stimulate the release of AVP but have no effect on either GHRH, SS or CRH at low doses; at high doses paradoxically they inhibit the hypothalamic GH axis similar to in-vivo data in the rat. We speculate that these effects might be mediated by NPY.     

Characterization of Opioid Binding Sites in the Neural and Intermediate Lobe of the Rat Pituitary Gland by Quantitative Receptor Autoradiography

Previous studies have suggested an involvement of enkephalins in regulation of oxytocin (OXT) and vasopressin (AVP) release, which seems to disagree with the very low affinities of Met- and Leu-enkephalin for the kappa opioid receptor. As opioid receptors in the neural lobe exclusively exist of kappa receptors, we studied the binding characteristics of larger pro-enkephalin derived peptides for opioid binding sites in the neural lobe by means of light microscopic receptor autoradiography. In addition, the pharmacological characteristics of opioid binding sites in the neural lobe were compared with those in other parts of the pituitary. In the neural as well as the intermediate lobe both high and low affinity ³H-bremazocine binding sites were present. Binding to these sites was completely displaceable by both naloxone and nor-binaltorphimine, suggesting that these sites represent kappa opioid receptors. Also with regard to selectivity and affinity characteristics to other ligands, opioid binding sites in the neural and intermediate lobe were quite similar. In the anterior lobe a very low level of bremazocine binding was present, which could not be displaced by nor-binaltorphimine. Displacement studies with pro-enkephalin and pro-dynorphin derived peptides showed that both groups of peptides could bind to opioid binding sites in the neural and intermediate lobe. Especially the relatively large pro-dynorphin and pro-enkephalin derived peptides, such as dynorphin 1–17 and BAM22, appeared to be very potent ligands for these opioid binding sites and were much more potent than smaller fragments, such as dynorphin 1–8, and Met- and Leu-enkephalin. These results contradict the existence of a mismatch in the neural (and intermediate) lobe with regard to the local type of opioid peptides and receptors present.     

Effects of Different Opioid Receptor Antagonists on the Electrically-Evoked Release of Endogenous Dopamine from the Isolated Neural Lobe of the Rat Pituitary Gland in vitro

Isolated neural lobes of the rat pituitary gland were incubated in Krebs-HEPES solution which contained the dopamine uptake inhibitor GBR 12921 and in some experiments additionally pargyline. The release of endogenous dopamine evoked by electrical stimulation of the pituitary stalk was determined by high-performance liquid chromatography with electrochemical detection. (±)- Naloxone increased the evoked dopamine release maximally by 440% (EC₅₀ 209 nM). The (+)-enantiomer of naloxone (up to 10 μM) did not affect the release of dopamine. The preferential κ-opioid receptor antagonist MR 2266 increased the evoked dopamine release maximally by 135% (EC₅₀ 7 nM). MR 2267, the inactive (+)-enantiomer of MR 2266, had no effect on dopamine release. The δ-opioid receptor selective antagonist ICI 174864 increased the release of dopamine maximally by 120% (EC₅₀ 10 nM). The non-selective opioid receptor agonist etorphine up to 10 μM had no effect on the evoked dopamine release. In conclusion, endogenous opioids in the neurohypophysis strongly inhibit the release of endogenous dopamine from this gland. Activation of κ- and δ-opioid receptors appears to be involved in the inhibitory action of the endogenous opioids on the neurohypophysial release of dopamine.     

Cyclosporine Effects on in vitro Responsiveness of Anterior Pituitary Hormone Release to Dopamine and Thyrotropin-Releasing Hormone in Young Female Rats

Endocrine side effects of the immunosuppressive drug cyclosporine (CyA) include changes in anterior pituitary hormone secretion. The aim of the present study was to examine the effects of CyA on the responsiveness of in situ and ectopic anterior pituitary prolactin (PRL), growth hormone (GH) and luteinizing hormone (LH) release response to dopamine (DA) and thyrotropin-releasing hormone (TRH) treatment in young female rats, and to evaluate the possible PRL participation in these effects. Thirty day old rats were rendered hyperprolactinemic by transplanting an anterior pituitary gland of a littermate donor, under the kidney capsule, and were then injected with CyA or vehicle for 2 or 8 days. Sham-operated rats were used as controls and treated in the same way. CyA treatment prevented the increase in plasma PRL levels which occurred in controls after pituitary grafting. In vitro basal PRL release of in situ pituitaries from either sham-operated and/or pituitary-grafted animals was decreased by CyA treatment at any point studied. Basal in vitro secretion of GH was only decreased in the in situ pituitaries from grafted animals after 2 days of CyA therapy. The presence of an ectopic pituitary lead to an increase in the in vitro basal LH secretion from in situ pituitaries as compared to those from sham-operated rats. Basal LH release rates were not changed by CyA treatment, although the LH release in vitro did increase in the in situ pituitaries from sham-operated animals treated with the drug for 2 days. DA addition to the incubation media decreased the in vitro release of PRL, GH and LH from the in situ pituitaries of sham-operated and pituitary-grafted animals treated with vehicle. In CyA treated animals, DA decreased in vitro PRL release from the in situ pituitaries of animals, independently of the presence or absence of an ectopic pituitary. Reductions of the in vitro GH and LH release after DA treatment were higher in the in situ pituitaries from grafted animals on day 8 of CyA or vehicle treatment. TRH increased the in vitro release of the three hormones with differential effects related to the length of the treatment with CyA and/or the presence of an ectopic pituitary. In vitro release of PRL and GH by ectopic pituitaries was inhibited by previous treatment with CyA and this effect was decreased proportional to the duration of the treatment with the drug, while LH secretion was not modified. Addition of DA to the incubation media resulted in a marked reduction of in vitro PRL and GH release, but only at day 8 of vehicle treatment on GH release did DA addition to media further decrease the release of both hormones from ectopic pituitaries from animals treated for 2 or 8 days with the drug, whereas LH secretion was not modified. TRH addition to the incubation media of ectopic pituitaries surprisingly reduced PRL and GH secretion on day 8 of CyA treatment or after surgery. The results of these studies suggest that CyA can act directly at the hypophyseal level modifying pituitary responsiveness to external stimuli. CyA seems to exert its main effects on lactotroph activity while its effects on somatotrophs and gonadotrophs are less.     

Arachidonate Metabolism in the Anterior Pituitary: Effect of Arachidonate Inhibitors on Basal and Stimulated Secretion of Prolactin, Growth Hormone and Luteinizing Hormone. II. Hormone Release from Dispersed Pituitary Cells

In the accompanying study, we reported the effects of inhibitors of arachidonic acid metabolism on the regulation of prolactin, growth hormone (GH) and luteinizing hormone secretion by male hemipituitaries. The present work extends these investigations to primary cell cultures of the same origin. Arachidonic acid metabolism was inhibited by either 5, 8, 11, 14-eicosatetraynoic acid (ETYA), a blocker of cyclooxygenase- and lipoxygenase-catalysed pathways, or the cyclooxygenase inhibitors, indomethacin and aspirin. ETYA inhibited basal GH secretion by 60%, an effect which was reversed by micromolar concentrations of exogenous arachidonic acid. ETYA was much less effective on growth hormone-releasing factor-induced GH release, a result which contrasts with data obtained on intact glands. Growth hormone-releasing factor stimulation of adenylate cyclase was not affected by ETYA. Cyclooxygenase inhibitors decreased basal secretion to a more limited extent (?30%) and were ineffective on growth hormone-releasing factor-stimulated release. Basal prolactin secretion was reduced by 30% in the presence of ETYA and unaffected by cyclooxygenase inhibitors. As with GH, the effect was reversed by exogenous arachidonic acid. However, in contrast to growth hormone-releasing factor-stimulated GH secretion, thyrotropin-releasing hormone stimulation of prolactin release was able to overcome the inhibition by ETYA in a dose-dependent manner. Again, the insensitivity of thyrotropin-releasing hormone-stimulated prolactin release to ETYA contrasts with the data obtained in intact tissue. Moreover, ETYA inhibited (?60%) prostaglandin E₂ production; thyrotropin-releasing hormone was unable to increase the prostaglandin levels in control or ETYA-treated cells. This confirms the data obtained with cyclooxygenase inhibitors, suggesting that prostaglandins are not involved in prolactin secretion. Intracellular accumulation of Ca²⁺ by the ionophore A23187 and protein kinase C stimulation by the phorbol ester 12-O- tetradecanoyl phorbol acetate (TPA), strongly stimulated GH and prolactin release. Under these conditions, ETYA was no longer able to inhibit secretion of the hormones. As with intact glands, basal and gonadotropin-releasing hormone or TPA-induced luteinizing hormone secretion were unaffected by any of the inhibitors used. It is concluded that blockade of the arachidonic acid cascade interferes with a secretory pathway involved mainly with basal release of prolactin and GH, but not luteinizing hormone. Thyrotropin-releasing hormone, a secretagogue known to trigger phospholipase C and, hence, to stimulate Ca²⁺ mobilization and protein kinase C, overcame ETYA inhibition of prolactin secretion. Growth hormone-releasing factor, a secretagogue recognized by adenylate cyclase coupled receptors, did not overcome ETYA inhibition of GH secretion. However, both secretagogues strongly stimulated hormone release from their target cells in the presence of ETYA. The arachidonic acid cascade thus seems less important in neuromediator-induced secretion coupling processes in dispersed pituitary cells, than in the intact gland. These observations suggest that eicosanoids are more likely to mediate paracrine or autocrine modulations of secretory mechanisms, rather than to function as intracellular messengers.     

Pyridostigmine, an Acetylcholinesterase Inhibitor, Stimulates Growth Hormone Release, but has no Effect on Basal Thyrotrophin or Adrenocorticotrophin Levels, or the Thyrotrophin Response to Thyrotrophin-Releasing Hormone

Pyridostigmine, an acetylcholinesterase inhibitor, stimulates growth hormone (GH) release and is thought to act by inhibiting hypothalamic somatostatin release. There are few data concerning the effect of pyridostigmine on other pituitary hormones apart from GH. We have studied the effect of pyridostigmine on basal GH, thyrotrophin (TSH), prolactin, adrenocorticotrophin and cortisol release, and thyrotrophin-releasing hormone (TRH)-stimulated TSH and prolactin release, in two studies involving nine healthy male subjects. Pyridostigmine stimulated GH release in all subjects but had no effect on adrenocortocotrophin or cortisol levels, or basal or TRH-stimulated TSH and prolactin levels. There are some data to suggest that somatostatin inhibits TRH-stimulated TSH release. Our findings, however, suggest that either endogenous somatostatin tone has little effect on the TSH response to TRH compared to its effects on GH or pyridostigmine acts through a mechanism other than altering somatostatin tone. Pyridostigmine did not alter adrenocorticotrophin or cortisol levels in the presence of a clear action on GH release, providing further evidence that the previously reported effects of cholinergic drugs on cortisol release are stress-related.     

Arachidonate Metabolism in the Anterior Pituitary: Effect of Arachidonate Inhibitors on Basal and Stimulated Secretion of Prolactin, Growth Hormone and Luteinizing Hormone. I. Hormone Release from Incubated or Perifused Pituitary Fragments

The potential involvement of arachidonic acid metabolites in the regulation of adenohypophyseal secretion was analysed on pituitary glands from male rats incubated in the presence of various inhibitors with different mechanisms of action: two inhibitors of phospholipase A₂ (parabromophenacylbromide, PB and compound CB 874), an inhibitor of cyclooxygenase- and lipoxygenase-catalysed pathways (5, 8, 11, 14-eicosatetraynoic acid, ETYA) and an inhibitor of cyclooxygenase (ε-lysyl acetylsalicylate, ASP). Under conditions which minimize side effects of the drugs, all inhibitors reduced prostaglandin synthesis and release, without affecting the metabolic integrity of the tissues (assessed by their intracellular adenosine triphosphate levels). All agents tested (PB, ETYA, ASP) suppressed prolactin secretion induced either by thyrotropin-releasing hormone or vasoactive intestinal peptide. Basal prolactin secretion was sensitive to phospholipase A₂ inhibitors. Similar inhibitions were obtained with ETYA and CB 874 on growth hormone secretion under basal conditions as well as after stimulation by growth hormone-releasing factor, thyrotropin-releasing hormone, or vasoactive intestinal peptide. In contrast, luteinizing hormone secretion, stimulated or not by gonadotropin-releasing hormone, was not sensitive to any of the agents used. It is concluded that, in intact male hemipituitaries, arachidonic acid metabolism is involved in the stimulation of prolactin and growth hormone secretion by neuropeptides. In contrast, luteinizing hormone release does not seem to depend on that mechanism. It has been verified that the inhibitors of arachidonic acid metabolism do not directly interfere with adenylate cyclase, or with the activation of protein kinase C, two enzymes which are involved in the regulation of secretory mechanisms.     

Effects of Estrogen and Ectopic Pituitary Transplants on the Responsiveness of Pituitary Prolactin Release to Luteinizing Hormone-Releasing Hormone and Dopamine

The responsiveness of prolactin release to regulatory inputs depends on the functional state of the lactotrophs. In the present study, we have examined the effects of luteinizing hormone-releasing hormone (LHRH; 10^–7 or 10^–6 M) on the release of prolactin in vitro from hyperplastic pituitaries of estrogen-treated male Fischer rats, ectopic pituitary transplants and in situ pituitaries of grafted and control rats. The effects of dopamine (10^–8 or 10^–7 M) in this system were also examined. The extent of inhibition of prolactin release by dopamine was not related to the amounts of prolactin secreted under basal conditions or to plasma prolactin levels. LHRH significantly suppressed prolactin secretion in all groups but its effect was most pronounced in the ectopic pituitary transplants and in the hyperplastic pituitaries of animals after chronic exposure to estrogen followed by a period of recovery. Thus, the effects of LHRH on prolactin release appear to be related to the secretory activity and/or to the absolute or relative number of the lactotrophs.     

Central Oxytocin Stimulates Luteinizing Hormone Release in the Marmoset, a Primate which Fails to Show Lactationally-lnduced Infertility

In several species, including humans, suckling is known to suppress pulsatile gonadotrophin secretion and substantially reduce fertility (1). During suckling high titres of oxytocin are present in both plasma (2, 3) and cerebrospinal fluid (CSF) (4) and there is some evidence that oxytocin is inhibitory to gonadotrophin secretion by an action on t h e central nervous system, (5, 6). The common marmoset, however, has a different physiological response and fails to show lactational infertility; ovarian cyclicity is resumed immediately post-partum (7). We have therefore examined the effect of centrally administered oxytocin on luteinizing hormone (LH) secretion in this species.
We report that LH release was stimulated with a rapid:esponse and in a dose-dependent manner by oxytocin injected into t h e third cerebral ventricle. In contrast, oxytocin administered intravenously was without effect on LH secretion. These results clearly indicate, for the first time, a stimulatory role for oxytocin in LH secretion in the marmoset, a species which fails to demonstrate lactational infertility.