Divergent prolactin and pituitary-adrenal activity in rats selectively bred for different dopamine responsiveness

The present study explores the significance of brain dopamine phenotype for individual variation in the neuroendocrine stress response of the rat. For this purpose, we used two Wistar rat lines previously selected for high or low responsiveness of the dopamine system to apomorphine using the gnawing response as the selection criterion. Systemic administration of the drug evoked in apomorphine-susceptible (apo-sus) rats a vigorous gnawing response, whereas apomorphine-unsusceptible (apo-unsus) rats did not gnaw under these conditions. These two rat lines represent individuals displaying extreme differences in gnawing behavior that otherwise coexist in a normal Wistar population. In this study basal and stress-induced hypothalamic-pituitary-adrenal activity and PRL release were measured in chronically cannulated, freely moving rats that endured a conditioned emotional response. Tyrosine hydroxylase messenger RNA (mRNA), corticosteroid receptor mRNA, and in vivo retention of [3H]corticosterone were measured in rat brain sections using in situ hybridization and in vivo autoradiography. The result show that 1) apo-sus rats had a markedly reduced PRL response to stress compared to apo-unsus animals, whereas basal levels were not significantly different. A12 dopaminergic neurons in the arcuate nucleus expressed significantly higher levels of tyrosine hydroxylase mRNA in apo-sus rats, suggesting that the reduced stress-induced PRL release could be due to an increased inhibitory control by dopaminergic neurons; 2) in apo-sus rats, stress resulted in a sustained elevation of ACTH and free corticosterone levels, whereas the total corticosterone levels were not different between the two rat lines; 3) under basal morning conditions, apo-sus rats had significantly higher plasma ACTH, but, in contrast, lower free corticosterone than apo-unsus rats; total plasma corticosterone levels were not different; 4) the basal evening ACTH level was elevated in apo-sus rats; after removal of the adrenals in the morning, this increased ACTH level in apo-sus rats persisted into the afternoon 6 h postadrenalectomy; and 5) hippocampal mineralocorticoid (MR), but not glucocorticoid (GR), receptor capacity for the ligand comparable between the groups; the MR of apo-sus rats displayed an increased retention of [3H]corticosterone in all hippocampal cell fields measured 24 h adrenalectomy; MR and GR mRNA in hippocampus as well as GR mRNA in the paraventricular nucleus were not significantly different in the two rat lines. In conclusion, the data suggest a common genetic background for individual variation in stress responsiveness and dopamine phenotype. High dopamine reactivity is linked to a reduced PRL and an increased ACTH response after stress. These high dopamine responders display a hyporesponsive adrenal cortex and corticosteroid feedback resistance associated with altered brain corticosteroid receptor properties.     

Effects of growth hormone secretagogues on prolactin release in anesthetized dwarf (dw/dw) rats

In addition to stimulating GH release, GH secretagogues such as GH-releasing peptide-6 (GHRP-6) stimulate small amounts of ACTH and PRL release. Although the effects on ACTH have recently been studied, there is little information about the effects of GHRP-6 on PRL. We have now studied GHRP-6-induced GH and PRL release and their regulation by estrogen (E2) in anesthetized male and female rats and in GH-deficient dwarf (dw/dw) rats that maintain high pituitary PRL stores and show elevated hypothalamic GH secretagogue receptor expression. Whereas GHRP-6 (0.1-2.5 microg, i.v.) did not induce PRL release in normal male or female rats, significant PRL responses were observed in dw/dw females. These responses were abolished by ovariectomy and could be strongly induced in male dw/dw rats by E2 treatment. These effects could be dissociated from GHRP-6-induced GH release in the same animals, but not from PRL release induced by TRH, which was also abolished by ovariectomy and induced in males by E2 treatment. However, the effects of GHRP-6 on PRL were unlikely to be mediated by TRH because in the same animals, TSH levels were unaffected by GHRP-6 whereas they were increased by TRH. The increased PRL response could reflect an increase in GH secretagogue receptor expression that was observed in the arcuate and ventromedial nuclei of E2-treated rats. Our results suggest that the minimal PRL-releasing activity of GHRP-6 in normal rats becomes prominent in GH-deficient female dw/dw rats and is probably exerted directly at the pituitary; these GHRP-6 actions may be modulated by E2 at both hypothalamic and pituitary sites.     

Consequences of prenatal morphine exposure on the hypothalamo-pituitary-adrenal axis in the newborn rat: effect of maternal adrenalectomy

The hypothalamo-pituitary-adrenal axis is already functional in rat fetuses in late gestation. We have reported previously that prenatal morphine exposure induced a severe atrophy of the adrenals and a decrease of corticosterone release in newborn rats at birth and during the early postnatal period. The first aim of the present study was to determine the effects of prenatal morphine exposure (1) on corticotrophin releasing factor (CRF) content of the hypothalamus, CRF immunofluorescence in the median eminence, CRF mRNA in the paraventricular nucleus (PVN) and pro-opiomelanocortin (POMC) mRNA in the anterior pituitary gland; (2) on CRF-induced ACTH release from the anterior pituitary gland in vitro; and (3) on ACTH-induced corticosterone release by the adrenals in vitro. Moreover, as morphine is a hepatotoxic factor, we determined the effects of prenatal morphine on liver weight and plasma corticosteroid binding globulin (CBG) binding capacity in newborn rats. Since acute administration of morphine stimulates corticosterone secretion in adult rats and since maternal corticosterone can cross the placental barrier, we also measured both adrenal weight and glucocorticoid activity in newborns from adrenalectomized mothers treated with morphine. The present results show that prenatal morphine given to intact mothers induced adrenal atrophy and hypoactivity in newborns but did not affect the responsiveness of the anterior pituitary gland to CRF or that of the adrenal gland to ACTH. Prenatal morphine reduced both CRF content in the newborn hypothalamus and CRF immunofluorescence in the median eminence without a significant effect on CRF mRNA expression in the PVN. Moreover, morphine induced a significant decrease of POMC mRNA in the anterior pituitary gland. However, morphine did not significantly affect the weight of the liver, or the plasma CBG binding capacity for corticosterone, in rat pups. In contrast, morphine treatment of the adrenalectomized mothers did not induce adrenal atrophy in newborns and did not impair adrenal activation during the early postnatal period. Maternal adrenalectomy also prevented the effects of prenatal morphine on hypothalamic content of CRF, CRF immunofluorescence in the median eminence, and POMC mRNA in the anterior pituitary gland. However, adrenal atrophy was observed at term in newborns of adrenalectomized mothers treated with both morphine and corticosterone or only corticosterone. In conclusion, morphine given to pregnant rats induced inhibition of the hypothalamo-pituitary-adrenal axis in pups at term. As maternal adrenalectomy prevented these effects, we speculate that an adrenal factor of maternal origin, probably corticosterone, mediated these drug effects on newborns.     

Calcium modulation of adrenocorticotropin levels in women--a clinical research center study

In vitro calcium modulation of anterior pituitary hormone secretion has been well described. In addition, several investigations performed in human subjects have documented modulation of the circulating levels of pituitary hormones by supraphysiological calcium concentrations. Recent data from our laboratory document the existence of an extracellular calcium-sensing receptor that is thought to mediate the effects of variations in extracellular calcium on the secretion of PTH and calcitonin. We have also demonstrated the presence of this receptor in pituitary-derived, ACTH-secreting AtT-20 cells as well as in the anterior pituitary of rats and mice. In the present study we investigated the effect on anterior pituitary hormone levels of variations in serum calcium within the physiological range. We serially measured serum levels of ionized calcium (Cai), ACTH, cortisol, TSH, and PRL during 90-min iv infusions (on separate days) of calcium, citrate, and dextrose in 10 healthy women with a mean age of 55 +/- 5 yr. During the calcium infusion, the serum Cai level increased significantly from 4.32 +/- 0.10 mg/dL at baseline to 4.86 +/- 0.08 mg/dL at completion (P = 0.002), and this change was accompanied by a significant increment in the serum ACTH level from 9.87 +/- 1.32 to 16.31 +/- 2.84 pg/mL (P = 0.0008). There was no change in the serum ACTH level during the citrate infusion despite significant decrements in serum Cai, nor were there changes in either Cai or ACTH during the dextrose infusion. Finally, changes in Cai did not alter TSH or PRL levels. In summary, our dynamic studies are the first to demonstrate an increase in baseline serum ACTH levels in response to physiological increments in Cai (i.e. increments within the normal range). This effect was specific for increments and not decrements in serum Cai and was selective for ACTH, as TSH and PRL levels did not change with any of the infusions.     

Murine leukemia inhibitory factor gene disruption attenuates the hypothalamo-pituitary-adrenal axis stress response

Recently, we have shown that human fetal pituitary, mouse corticotroph AtT20 cells, and murine hypothalamus and pituitary express leukemia inhibitory factor (LIF). LIF knockout mice (LIFKO), heterozygous and wild type (wt), of B6D2F1 genetic background were used to examine whether LIF may play a role in the regulation of the hypothalamo-pituitary-adrenal axis in vivo. Resting levels of plasma ACTH and corticosterone were similar in all three genotypes. However, LIFKO mice did not respond to 30-min restraint and 45-min immobilization stress with increased plasma ACTH. Increased circulating ACTH was only observed in LIFKO mice after very short immobilization stress (15 min), but this ACTH level was lower than in wt animals (P < 0.05). Injection of mycobacterial adjuvant resulted in a 2-fold increase of corticosterone levels 7 days after treatment in wt, but not LIFKO, mice (P < 0.05). Pituitary POMC messenger RNA (mRNA) levels were very low in LIFKO animals. Although 15 and 45 min of immobilization stress resulted in enhanced POMC mRNA content in all three groups, this elevation was lowest in LIFKO mice. Injection of 12 microg murine LIF to LIFKO and normal C57BL/6 animals resulted in increased plasma ACTH and corticosterone levels and elevated pituitary POMC mRNA levels in both LIF-repleted and LIF-depleted mice. Thus, LIF appears to play an important role in activating the hypothalamo-pituitary-adrenal axis during stress and inflammation.     

A phospholipase A2-related snake venom (from Crotalus durissus terrificus) stimulates neuroendocrine and immune functions: determination of different sites of action

Immune neuroendocrine interactions are vital for the individual's survival in certain physiopathological conditions, such as sepsis and tissular injury. It is known that several animal venoms, such as those from different snakes, are potent neurotoxic compounds and that their main component is a specific phospholipase A type 2 (PLA2). It has been described recently that the venom from Crotalus durissus terrificus [snake venom (SV), in the present study] possesses some cytotoxic effect in different in vitro and in vivo animal models. In the present study, we investigated whether SV and its main component, PLA2 (obtained from the same source), are able to stimulate both immune and neuroendocrine functions in mice, thus characterizing this type of neurotoxic shock. For this purpose, several in vivo and in vitro designs were used to further determine the sites of action of SV-PLA2 on the hypothalamo-pituitary-adrenal (HPA) axis function and on the release of the pathognomonic cytokine, tumor necrosis factor alpha (TNF alpha), of different types of inflammatory stress. Our results indicate that SV (25 microg/animal) and PLA2 (5 microg/animal), from the same origin, stimulate the HPA and immune axes when administered (i.p.) to adult mice; both preparations were able to enhance plasma glucose, ACTH, corticosterone (B), and TNF alpha plasma levels in a time-related fashion. SV was found to activate CRH- and arginine vasopressin-ergic functions in vivo and, in vitro, SV and PLA2 induced a concentration-related (0.05-10 microg/ml) effect on the release of both neuropeptides. SV also was effective in changing anterior pituitary ACTH and adrenal B contents, also in a time-dependent fashion. Direct effects of SV and PLA2 on anterior pituitary ACTH secretion also were found to function in a concentration-related fashion (0.001-1 microg/ml), and the direct corticotropin-releasing activity of PLA2 was additive to those of CRH and arginine vasopressin; the corticotropin-releasing activity of both SV and PLA2 were partially reversed by the specific PLA2 inhibitor, manoalide. On the other hand, neither preparation was able to directly modify spontaneous and ACTH-stimulated adrenal B output. The stimulatory effect of SV and PLA2 on in vivo TNF alpha release was confirmed by in vitro experiments on peripheral mononuclear cells; in fact, both PLA2 (0.001-1 microg/ml) and SV (0.1-10 microg/ml), as well as concavalin A (1-100 microg/ml), were able to stimulate TNF alpha output in the incubation medium. Our results clearly indicate that PLA2-dependent mechanisms are responsible for several symptoms of inflammatory stress induced during neurotoxemia. In fact, we found that this particular PLA2-related SV is able to stimulate both HPA axis and immune functions during the acute phase response of the inflammatory processes.     

131I therapy for pediatric thyroid cancer

Azido-3'-deoxy-thymidine (AZT) is a drug extensively used in the treatment of AIDS. AZT was incubated in vitro either with the pituitary-hypothalamus complex (PHc) or the intact pituitary (PI) of male rats. The PHc is comprised of the hypothalamus and the attached pituitary gland. After a preincubation period, the PHc or PI was incubated for 1 or 2 h with Krebs-Ringer bicarbonate buffer or either of two different concentrations of AZT (1 and 10 microM). In the control incubations, the PHc released less prolactin (PRL) and more follicle-stimulating hormone (FSH) and luteinizing hormone (LH) than the PI, indicating that hypothalamic control of the pituitary was exerted in vitro, presumably by diffusion of releasing and inhibiting hormones from the neurohypophysis to the anterior lobe of the hypophysis. Both concentrations of AZT evoked a significant increase in release of PRL and a decreased release of LH and FSH from the PHc. In the case of LH, the higher concentration of AZT partially suppressed LH release within 1 h. The other effects were not dose-related and were observed after incubating the tissue with AZT for 2 h. However, incubation of the PI with AZT failed to alter anterior pituitary hormone release, illustrating that the site of action of AZT is in the hypothalamus. We hypothesize that AZT blocks DNA synthesis resulting in suppression of synthesis and consequent release of hypothalamic hormones that control release of pituitary hormones in vitro. The results raise the possibility that AZT may alter hypothalamic-pituitary function in vivo.     

Leukemia inhibitory factor modulates interleukin-1beta-induced activation of the hypothalamo-pituitary-adrenal axis

We have shown that leukemia inhibitory factor (LIF) is expressed in corticotroph cells and stimulates POMC gene expression and ACTH secretion in vivo and in vitro. We therefore examined the regulation of in vitro and in vivo pituitary LIF expression by cytokines known to stimulate the hypothalamo-pituitary-adrenal axis. In the corticotroph cell line AtT-20/D16v-F2, recombinant murine interleukin-1beta (IL-1beta; 0.1-10.0 ng/ml) caused a 5- to 10-fold increase in LIF messenger RNA (mRNA) levels. LIF mRNA expression was induced as early as 1 h, peaked at 2 h, and still persistently elevated above the baseline after 8 h. This effect of IL-1beta on LIF mRNA expression was abolished by preincubation with human IL-1 receptor antagonist (100 ng/ml) or antimurine IL-1beta antibody (10 microg/ml). Tumor necrosis factor-alpha (20 ng/ml) only modestly increased LIF mRNA, but was synergistic with IL-1beta (up to 2.5-fold). In contrast, IL-2 and IL-6 did not alter LIF mRNA. In C57BL/6 mice, i.p. injection of 100 ng IL-1beta increased plasma ACTH and corticosterone levels after 1 h (P < 0.02). In addition, pituitary LIF mRNA content was increased for up to 2 h in response to IL-1beta. In comparison to wild-type (+/+) B6D2F1 mice, LIF knockout mice with a deleted LIF gene (-/-) exhibited decreased plasma ACTH (631 +/- 61 vs. 376 +/- 50 pg/ml; P < 0.01) and corticosterone (783 +/- 85 vs. 433 +/- 51 ng/ml; P < 0.01) levels 1 h after i.p. IL-1beta administration. In conclusion, corticotroph LIF mRNA expression is specifically stimulated by IL-1beta and tumor necrosis factor-alpha. The attenuated hypothalamo-pituitary-adrenal response to IL-1beta in LIF knockout mice indicates that the effect of IL-1beta on ACTH secretion is modulated by LIF. Thus, LIF appears to function as an immune-neuroendocrine modulator signaling the hypothalamo-pituitary-adrenal axis.     

Angiotensinergic neurons physiologically inhibit prolactin, growth hormone, and thyroid-stimulating hormone, but not adrenocorticoptropic hormone, release in ovariectomized rats

Angiotensin II (AII)-containing neurons with cell bodies in the rostral medial hypothalamus and axons project to the external layer of the median eminence, so that AII maybe released into the hypophyseal portal vessels for actions on the pituitary gland. Indeed, intrahypothalamic actions of the peptide on the release of hypothalamic hormones and direct actions on the pituitary have been reported. To determine the role of endogenously released AII in hypothalamic-pituitary hormone release, we have determined the effects of central immunoneutralization of AII upon the plasma concentrations of prolactin (PRL), growth hormone (GH), thyroid-stimulating hormone (TSH), and adrenocorticotropic hormone (ACTH). Specific antiserum directed against AII (AB-AII) or normal rabbit serum (NRS), as a control, was microinjected into third ventricular (3 V) cannulae of conscious, ovariectomized (OVX) rats. Immediately before and at various intervals after this procedure, blood samples were withdrawn through previously implanted external jugular catheters. Three hours after injection of the AB-AII, plasma PRL levels diverged from those of the NRS-injected animals and progressively increased from 4 to 24 h after administration of the antiserum. Results were similar with respect to plasma GH, except that the increase in the AB-AII animals above that in the NRS-injected controls from 4 to 6 h was not significant, but was highly significant on measurement 24 h after injection, at which time plasma GH was three times higher than in control rats. Similarly, following injection of AB-AII, plasma TSH values did not diverge significantly from those of the NRS-injected controls until 3 h after injection. From 3 to 5 h they remained constant and significantly elevated above values in the NRS-injected controls with a further nonsignificant increase at 6 h. At 24 h, there was no longer a difference between the values in both groups. In contrast to the significant elevations in plasma hormone levels observed with respect to PRL, GH, and TSH following injection of the antiserum, there was no change in plasma ACTH between the AB-AII-injected and NRS-injected animals throughout the same period of observation. Previous results by others have shown that intraventricular injection of AII has a suppressive action on the release of PRL, GH, and TSH. Consequently, we believe that the antiserum is acting intrahypothalamically to block the action of AII within the hypothalamus, resulting in the elevation of the three hormones mentioned. Therefore, the AII neurons appear to have a physiologically significant suppressive action on the release of hypothalamic neurohormones controlling the release of PRL, GH, and TSH. In contrast, there apparently is no effect of intrahypothalamically released AII on the secretion of corticotropin-releasing factors under these nonstress conditions. We cannot rule out an action of the antiserum at the pituitary level; however, in view of the fact that the actions of AII directly on the gland are to stimulate PRL, GH, TSH, and ACTH release, it appears that the antiserum was acting at the hypothalamic level.     

Dysregulation of the hypothalamo-pituitary axis in rheumatoid arthritis

OBJECTIVE: To study the dynamic response of the hypothalamo-pituitary- adrenal axis and of prolactin (PRL) pituitary secretion in rheumatoid arthritis (RA). METHODS: We performed a cortisol releasing hormone (CRH) provocation test followed by determination of adrenocorticotropin hormone (ACTH), beta-endorphin, and cortisol concentration, and then a thyrotropin releasing hormone (TRH) provocation test followed by assessment of PRL pituitary secretion in 10 patients with RA and 5 control subjects. All were women under 40 years of age. Hormone concentrations were assessed by radioimmunoassay. RESULTS: Basal PRL cortisol, and ACTH concentrations were similar in patients with RA and controls. We observed a dissociation between the pituitary secretion of beta-endorphin and of ACTH in response to CRH in RA. The ACTH peak and total ACTH production (area under the curve, AUC) were similar in the 2 groups. In contrast, basal beta-endorphin was increased in RA (12.6 +/- 1.41 vs 8.29 +/- 0.144 pg/ml), and the response upregulated (AUC: 83,080 +/- 12,000 vs 54,200 +/- 2400) after CRH compared to controls (p < 0.05). Cortisol adrenal response curve was blunted, but did not reach statistical significance. In contrast, the PRL response to TRH was increased at 120 and 150 min (3461 +/- 303 vs 1897 +/- 520 muIU/ml)(p < 0.01) in patients with RA, independent of disease activity. CONCLUSION: We observed upregulated pituitary PRL secretion in RA, and a dissociation of ACTH stress. The implication concerning the neuroendocrine system in the chronic immune response in RA is discussed.     

Activated isoforms of MMP-2 are induced in U87 human glioma cells in response to beta-amyloid peptide

The effect of stress- or lipopolysaccharide (LPS) endotoxin-induced release of ACTH, beta-endorphin (beta-END) and prolactin (PRL) was investigated in two groups of conscious male rats: (1) Rats pretreated with different H3 receptor agonists, which inhibit neuronal histamine (HA) synthesis and release, and (2) rats with bilateral posterior hypothalamic lesion, which destroys the histaminergic perikarya exclusively localized in the mammillary nuclei. The H3 receptor agonists R(alpha)methyl-HA, BP 2-94 or imetit injected intraperitoneally (ip) had no effect on basal secretion of ACTH or PRL but inhibited the ACTH and PRL responses to restraint stress and the ACTH response to LPS endotoxin. LPS had no effect on PRL secretion. The inhibitory effect of the agonists was prevented by prior ip administration of the H3 receptor antagonist thioperamide. Bilateral lesion of the posterior hypothalamus inhibited the ACTH, beta-END and PRL responses to restraint stress, ether stress and LPS endotoxin, whereas sham operation had no effect compared to nonoperated control rats. In addition, posterior hypothalamic lesion inhibited the PRL response but not the ACTH and beta-END responses to activation of serotonergic neurons induced by ip administration of the 5-HT precusor 5-hydroxytryptophan in combination with the 5-HT re-uptake inhibitor fluoxetine. Thus, serotonergic pathways were not damaged by the lesions. The present results support our previous findings that inhibition of neuronal HA synthesis by alpha-fluoromethylhistidine as well as blockade of H1 or H2 receptors inhibit the ACTH, beta-END and PRL responses to stress and LPS endotoxin and further substantiate an important role of histaminergic neurons in the mediation of the stress-induced release of pituitary stress hormones. Furthermore, in accordance with our previous findings, the lesion experiments indicated the existence of an interaction between the histaminergic and serotonergic system in regulation of the stress- and LPS-induced PRL release.     

Diinosine polyphosphates, a group of dinucleotides with antagonistic effects on diadenosine polyphosphate receptor

Nitric oxide synthase, an enzyme responsible for nitric oxide (NO) formation has been found in the hypothalamic paraventricular nucleus and median eminence, structures closely associated with regulation of the pituitary activity, and the pituitary gland itself. Nitric oxide modulates the stimulated release of CRH from the rat hypothalamus in vitro, which suggests its role in regulating the secretion of ACTH from the pituitary corticotrops and of corticosterone from the adrenal cortex. The purpose of the present study was to elucidate the yet unknown role of endogenous NO in the HPA response to central cholinergic stimulation in conscious rats. Neither L-arginine an NO precursor, nor the NO synthase blockers N omega-nitro-L-arginine methyl ester (L-NAME) and N omega-nitro-L-arginine (L-NNA) caused any consistent changes in the basal serum corticosterone levels. L-arginine, given in higher doses (120-150 mg/kg ip) 15 min prior to icv carbachol (2 micrograms), markedly diminished the carbachol-induced rise in corticosterone secretion. Systemic pretreatment with the nitric oxide synthase inhibitor L-NAME (5 mg/kg) significantly raised the carbachol-elicited corticosterone response, while addition of L-arginine completely blocked the effect of L-NAME. A similar increase in the carbachol-induced corticosterone response was produced by icv pretreatment with L-NAME (2 micrograms), indicating a central site of the NO interaction with cholinergic stimulation of the HPA response. L-NAME is a weak inhibitor of neuronal NOS itself, and must first be de-estrified to N omega-nitro-L-arginine to potently inhibit this enzyme. Systemic (10 mg/kg) and icv (1 microgram) pretreatment with L-NNA enhanced more effectively the carbachol-induced rise in corticosterone secretion than did pretreatment with L-NAME by either route. These results are the first direct evidence that endogenous NO significantly inhibits the HPA response to central cholinergic, muscarinic receptor stimulation under in vivo conditions.     

Evidence for two differentially regulated populations of peripheral beta-endorphin-releasing cells in humans

In the current study we tested the hypothesis that human plasma beta-endorphin (beta E) is derived from at least two subpopulations of beta E-releasing cells: one sensitive to glucocorticoids as well as to dopamine (DA; regulated analogously to the corticotrophs of the rat pituitary), and one insensitive to glucocorticoids but sensitive to DA (regulated analogously to the melanotrophs of the rat pituitary). To test this hypothesis, human plasma levels of ACTH, cortisol, and beta E-like immunoreactivity were measured at baseline and after haloperidol treatment (0.05 mg/kg, i.v.) in two experimental groups, one pretreated with dexamethasone (1.5 mg) and one pretreated with placebo. Plasma PRL levels were also measured in both groups as an indicator of DA receptor blockade. Dexamethasone partially suppressed both baseline and haloperidol-stimulated levels of human plasma beta E-like immunoreactivity, whereas it completely suppressed both basal and haloperidol-stimulated levels of ACTH and cortisol and had no statistically significant effect on either basal or haloperidol-stimulated PRL levels. These data support a negative feedback effect of glucocorticoids on one DA-sensitive cell population that releases both ACTH and beta E (corticotroph like), but not on a second cell population that releases beta E but not ACTH.     

Endocrine profile and neuroendocrine challenge tests in transgenic mice expressing antisense RNA against the glucocorticoid receptor

A transgene expressing antisense RNA complementary to a fragment of the glucocorticoid receptor cDNA was incorporated into the mouse genome and resulted in a transgenic animal that has decreased glucocorticoid receptor function. The transgenic mice showed basal plasma ACTH and corticosterone levels similar to those of the normal control animals. We have further investigated changes in HPA axis regulation by use of different neuroendocrine challenge tests including a dexamethasone suppression test (DST). In comparison to normal mice, a tenfold higher dose of dexamethasone (i.e. 20 micrograms/100 g body weight) was required to suppress the basal corticosterone levels of transgenic mice. Dexamethasone (2 micrograms/100 g body weight) produced a long-lasting suppression of plasma ACTH and corticosterone levels in control mice, whereas in transgenic animals only a short-lasting decrease in ACTH levels was apparent. Corticotropin-releasing hormone (CRH) administration resulted in an enhanced response in plasma ACTH levels in transgenic mice, whereas the corticosterone response was markedly reduced. The discrepancy between ACTH and corresponding corticosterone secretions in transgenic mice could be attributed, in part, to a reduced sensitivity of the adrenal gland to stimulation by ACTH. Pituitaries of transgenic mice contained about 50% less proopiomelanocortin (POMC) mRNA than those of control animals. No significant differences were noted in the ACTH or protein contents of normal and transgenic mice pituitary glands although a slight increase in protein content of the transgenic mouse adrenal gland was apparent. In conclusion, transgenic mice with impaired GR function show major disturbances in HPA axis regulation which seem to be caused by the primary defect in conjunction with secondary modifications in, amongst others, pituitary CRH receptor system(s), sympathetic output and adrenal development. This mouse is therefore a useful model to study the consequences of life-long defective GR function and HPA axis regulation in general.     

Assessment of pituitary function after transsphenoidal hypophysectomy in beagle dogs

Pituitary function was assessed in healthy adult beagle dogs before and after hypophysectomy. Anterior pituitary function was tested by use of the combined anterior pituitary (CAP) function test, which consisted of sequential 30-sec intravenous injections of four hypothalamic releasing hormones, in the following order and doses: 1 microgram of corticotropin-releasing hormone (CRH)/kg, 1 microgram of growth hormone-releasing hormone (GHRH)/kg, 10 micrograms of gonadotropin-releasing hormone (GnRH)/kg, and 10 micrograms of thyrotropin-releasing hormone (TRH)/kg. Plasma samples were assayed for adrenocorticotropin (ACTH), cortisol, GH, luteinizing hormone (LH), and prolactin (PRL) at multiple times for 120 min after injection. Pars intermedia function was assessed by the alpha-melanotropin (alpha-MSH) response to the intravenous injection of the dopamine antagonist haloperidol in a dosage of 0.2 mg/kg. Posterior pituitary function was assessed by the plasma vasopressin (AVP) response to the intravenous infusion of 20% saline. Basal plasma ACTH, cortisol, thyroxine, LH. PRL, and AVP concentrations were significantly lower at 10 wk after hypophysectomy than before hypophysectomy. In the CAP test and the haloperidol test, the peaks for the plasma concentrations of ACTH, cortisol, GH, LH, PRL, and alpha-MSH occurred within 45 min after injection. At 2 and 10 wk after hypophysectomy, there were no responses of plasma GH, LH, PRL, and alpha-MSH to stimulation. In four of eight hypophysectomized dogs, there were also no plasma ACTH and cortisol responses, whereas in the other four dogs, plasma ACTH and cortisol responses were significantly attenuated. The basal plasma ACTH and cortisol concentrations were significantly lower in the corticotropic nonresponders than in the responders. Plasma AVP responses were completely abolished by hypophysectomy, although water intake by the dogs was normal. Histopathological examinations at 10 wk after hypophysectomy revealed that adrenocortical atrophy was much more pronounced in the corticotropic nonresponders than in the responders. No residual pituitary tissue was found along the ventral hypothalamic diencephalon. However, in all hypophysectomized dogs that were investigated, islets of pituitary cells were found embedded in fibrous tissue in the sella turcica. A significant positive correlation was found between the number of ACTH-immunopositive cells and the ACTH increment in the CAP test at 10 wk after hypophysectomy. It is concluded that 1) stimulation of the anterior pituitary with multiple hypophysiotropic hormones, stimulation of the pars intermedia with a dopamine antagonist, and stimulation of the neurohypophysis with hypertonic saline do not cause side effects that would prohibit routine use, 2) in the routine stimulation of the anterior pituitary and the pars intermedia, blood sampling can be confined to the first 45 min, 3) the ACTH and cortisol responses to hypophysiotropic stimulation are the most sensitive indicators for residual pituitary function after hypophysectomy, 4) small islets of pituitary cells in the sella turcica, containing corticotropic cells, are the most likely source of the attenuated corticotropic response that may occur after hypophysectomy, and 5) residual AVP release from the hypothalamus after hypophysectomy is sufficient to prevent diabetes insipidus, despite the fact that the AVP response to hypertonic saline infusion is completely abolished.     

The effects of GnRH and adrenergic agents on PRL and beta-endorphin secretion by porcine pituitary cells in vitro

The direct effects of alpha- and beta-adrenergic agents on PRL and beta-endorphin (beta-END) secretion in vitro by porcine pituitary cells have been investigated. Pituitary glands were obtained from mature gilts, which were ovariectomised (OVX) one month before slaughter. Ovariectomised gilts, assigned to four groups, were primed with: (1) vehicle (OVX); (2) and (3) oestradiol benzoate (EB; 2.5 mg/100 kg b.w.) at 30-36 h (OVX+EB I) and 60-66 h (OVX+EB II) before slaughter, respectively; and (4) progesterone (P4; 120 mg/100 kg b.w.) for 5 consecutive days before slaughter (OVX+P4). Isolated anterior pituitary cells were submitted to 3.5 h incubation in the presence of GnRH, alpha- and beta-adrenergic agonists [phenylephrine (PHEN) and isoproterenol (ISOP), respectively], or alpha- and beta-adrenergic blockers [phentolamine (PHENT) and propranolol (PROP), respectively]. The culture media were assayed for PRL (exp. I) and beta-endorphin-like immunoreactivity (beta-END-LI) (experiment II). In experiment I, GnRH did not influence PRL release by pituitary cells in all experimental groups. Some of tested doses of adrenergic agonists, PHEN and ISOP, increased PRL release from pituitary cells of OVX gilts, but not from those of OVX+EB I animals. In the OVX+EB II group, PHEN alone, but ISOP with PROP, potentiated PRL secretion by the cells. In OVX+P4 animals, PHEN alone or in combination with PHENT and also ISOP alone or with PROP enhanced PRL output from the cells. In experiment II, addition of GnRH increased beta-END-LI release from pituitary cells only in the OVX+EB II group. PHEN and PHENT potentiated beta-END-LI secretion by pituitary cells in OVX+EB II and OVX+P4 groups, while ISOP and PROP increased beta-END-LI secretion by the cells of OVX and OVX+EB II animals. In turn, in the OVX+EB I group, effect of PHENT and PROP on PRL secretion by pituitary cells was inhibitory. In conclusion, our results suggest that adrenergic agents can modulate PRL and beta-END secretion by porcine pituitary cells in a manner dependent on the hormonal status of gilts.     

Different sensitivity to sodium valproate in healthy, non-tumoral and tumoral hyperprolactinemic subjects

GABAergic drugs affect PRL secretion in both rat and man. Sodium valproate (SV) inhibits GABA transaminase so increasing the endogenous GABAergic tone. The aim of this study was to evaluate the effects of SV at low and high doses on PRL release in healthy subjects and hyperprolactinemic patients. Fifteen patients with prolactinomas, 8 patients with non-tumoral hyperprolactinemia and 10 healthy subjects were studied: in non consecutive days, all subjects received placebo and SV at the dose of 400 and 800 mg po. Serum PRL levels were assessed 30, 15 and 5 min before and every 30 min for 4 hours after administration. SV at the dose of 400 mg induced a significant decrease of serum PRL in healthy subjects (p < 0.05), whereas no effect was noted in both tumoral and non-tumoral hyperprolactinemia. The administration of 800 mg SV induced a significant decrease of PRL levels in healthy subjects and in patients with non-tumoral hyperprolactinemia (p < 0.05). Conversely, in prolactinomas a paradoxical increase of serum PRL concentration (p < 0.05) was observed 120 min after the administration of the drug. These data confirm the inhibitory activity of SV on PRL release in healthy subjects, and suggest the existence of a partial resistance to GABA in non-tumoral hyperprolactinemia. In prolactinomas, the paradoxical PRL increase after high dose of SV suggests the existence of a complete pituitary resistance to GABA. This finding might be explained by the appearance of the stimulatory effect of GABA at hypothalamic level that could have been unmasked by the lack of pituitary GABA effects on adenomatous lactotrophs.     

Ipamorelin, the first selective growth hormone secretagogue

The development and pharmacology of a new potent growth hormone (GH) secretagogue, ipamorelin, is described. Ipamorelin is a pentapeptide (Aib-His-D-2-Nal-D-Phe-Lys-NH2), which displays high GH releasing potency and efficacy in vitro and in vivo. As an outcome of a major chemistry programme, ipamorelin was identified within a series of compounds lacking the central dipeptide Ala-Trp of growth hormone-releasing peptide (GHRP)-1. In vitro, ipamorelin released GH from primary rat pituitary cells with a potency and efficacy similar to GHRP-6 (ECs) = 1.3+/-0.4nmol/l and Emax = 85+/-5% vs 2.2+/-0.3nmol/l and 100%). A pharmacological profiling using GHRP and growth hormone-releasing hormone (GHRH) antagonists clearly demonstrated that ipamorelin, like GHRP-6, stimulates GH release via a GHRP-like receptor. In pentobarbital anaesthetised rats, ipamorelin released GH with a potency and efficacy comparable to GHRP-6 (ED50 = 80+/-42nmol/kg and Emax = 1545+/-250ng GH/ml vs 115+/-36nmol/kg and 1167+/-120ng GH/ml). In conscious swine, ipamorelin released GH with an ED50 = 2.3+/-0.03 nmol/kg and an Emax = 65+/-0.2 ng GH/ml plasma. Again, this was very similar to GHRP-6 (ED50 = 3.9+/-1.4 nmol/kg and Emax = 74+/-7ng GH/ml plasma). GHRP-2 displayed higher potency but lower efficacy (ED50 = 0.6 nmol/kg and Emax = 56+/-6 ng GH/ml plasma). The specificity for GH release was studied in swine. None of the GH secretagogues tested affected FSH, LH, PRL or TSH plasma levels. Administration of both GHRP-6 and GHRP-2 resulted in increased plasma levels of ACTH and cortisol. Very surprisingly, ipamorelin did not release ACTH or cortisol in levels significantly different from those observed following GHRH stimulation. This lack of effect on ACTH and cortisol plasma levels was evident even at doses more than 200-fold higher than the ED50 for GH release. In conclusion, ipamorelin is the first GHRP-receptor agonist with a selectivity for GH release similar to that displayed by GHRH. The specificity of ipamorelin makes this compound a very interesting candidate for future clinical development.