Triiodothyronine binding in rat anterior pituitary, posterior pituitary, median eminence and brain.

In vivo studies of the exchange of tracer [125-I]-L-triiodothyronine (T3) between plasma (P), and the anterior pituitary (AP), posterior pituitary (PP), median eminence (ME) and the frontal lobes of the brain (B), in the rat show that from 2.5 h onwards the concentration of new [125I]T3 in AP, PP and B were parellel to that of the plasma, with a t1/2 of 7.4 h; the t1/2 for ME was 10.3 h. The extrapolation of these curves to zero time was assumed to indicate the relative concentration of T3 per unit weight in terms of total body T3. T3 content of these tissues was determined by radioimmunoassay. The values obtained validated the steady state parameters derived from the radio-isotopic measurements. As an indicator of the concentration gradient between tissue and plasma the organ/plasma (O/P) ratio was calculated; these data indicate that under steady state conditions, the order of T3 concentration is AP greater than PP greater than ME greater than B. Binding studies have shown that AP and PP contain "specific," saturable binders while ME and B do not. Evaluation of the binding parameters of the high affinity binders in both AP and PP gave similar association constants. These associations constants, when corrected for the binding strength of T3 to plasma proteins, resulted in values similar to those of neuclear T3 binders.     

Clusterin is expressed in the anterior and intermediate lobes, but not in the posterior pituitary of sheep.

We have examined the expression of the ovine clusterin gene in the sheep pituitary gland, with the aim of determining its site of synthesis in this tissue. Northern blotting analysis of extracted polyadenylated RNA, using a (32)P-labelled rat clusterin cDNA probe, detected the greatest amounts of clusterin mRNA in the anterior part of dissected pituitary glands. In situ hybridisation studies showed clusterin mRNA in anterior and intermediate pituitary cells, with lower amounts in vascular endothelium and posterior pituicytes. Clusterin protein, detected by immunohistochemistry, was observed in some single secretory cells, within the capillary lumen and in cells around capillaries in the anterior and intermediate lobes, but no immunoreactivity was observed in posterior pituitary tissue. The pattern of clusterin expression in anterior and intermediate pituitary cells suggests possible roles for the protein in secretory cell turnover and/or hormone secretion or lipid uptake. Clusterin does not appear to be involved in ovine posterior pituitary hormone neurosecretion.     

Ultrastructural evidence for oxytocin in the rat anterior pituitary gland

OT is synthetized in the hypothalamus. These neurons project to the posterior lobe of the pituitary and the external zone of the median eminence. In order to localize OT in the male rat anterior pituitary we have used immunocytochemistry on ultra-thin sections in target cell(s) obtained by cryoultramicrotomy. OT-like immunoreactivity was observed in lactotropes only. No immunoreactivity was observed in gonadotropes, somatotropes, corticotropes or thyrotropes. In lactotropes, immunoreactivity was localized at the plasma membrane level, in the cytoplasmic matrix and around the secretory granules, but not in the other organelles, and in the nucleus. No reaction was observed by using either non-immune serum or anti-OT serum incubated with OT. No modification of OT-like immunoreactivity was observed by using anti-OT serum incubated with heterologous peptides. These results 1) provide immunocytological evidence for the presence of OT in the anterior pituitary gland; 2) indicate the presence of this peptide in one particular cell type, and 3) support the hypothesis that OT could have a direct participation in the regulation of the PRL release.     

Adrenocorticotrophic hormone in the anterior and neurointermediate lobes of the fetal rat pituitary gland

The concentration of ACTH in the pars distalis and pars intermedia of the fetal rat hypophysis from days 17-21 of pregnancy was measured with a specific radioimmunoassay and a bioassay using isolated adrenal cells from adult rats. In both lobes of the pituitary gland, a significant correlation was observed between immunoreactive and bioreactive values, expressed as pg equivalents synthetic human 1-39 ACTH per microgram protein. In the pars distalis, ACTH concentrations increased steadily from days 17-20 and then remained unchanged to term. At this time they were tenfold higher than on day 17. In the neurointermediate lobe, ACTH was detected only from day 18; the concentration of ACTH increasing between days 18 and 19. At each of the stages of pregnancy examined, the concentration of ACTH in the pars distalis was greater than that in the pars intermedia. These data have demonstrated that ACTH is present in both anterior and neurointermediate lobes of the fetal rat hypophysis, that the functional differentiation of the pars distalis takes place earlier than that of the pars intermedia, and that the concentrations of corticotrophin in the pars distalis and in the pars intermedia have different patterns of development as gestation progresses.     

Vasopressin is not involved in the catecholamine-induced release of ACTH, alpha-MSH and beta-endorphin from the rat pituitary gland

The effect of catecholamines on plasma levels of immunoreactive ACTH (ACTHi), alpha-MSH (alpha-MSHi), beta-endorphin (beta-ENDi), arginine-vasopressin (AVPi) and of corticosterone (B) was studied in female rats. Intravenous infusion of the specific beta-adrenoceptor-stimulating agent l-isoproterenol in Wistar rats under pentobarbital anesthesia resulted in a dose-dependent (dose range: 10-100 ng/kg . min) increase in plasma B. At higher concentrations, l-isoproterenol also caused a dose-dependent increase in plasma AVPi (dose range: 100-1,000 ng/kg . min). In addition to isoproterenol, also intravenous infusion of l-epinephrine caused a dose-dependent increase of plasma B (dose range: 100-1,000 ng/kg . min), whereas l-epinephrine was without effect on plasma AVPi, even at the highest dose tested (1,000 ng/kg . min). The effect of l-epinephrine or l-isoproterenol on plasma B was associated with a parallel and dose-related increase in plasma ACTHi, beta-ENDi and alpha-MSHi. The increase of plasma ACTHi, B and beta-ENDi in response to l-isoproterenol (300 ng/kg . min) was identical in Wistar, Long Evans and Brattleboro rats (Long Evans rats with a hereditary lack of vasopressin). Also the responses to l-epinephrine (1,000 ng/kg . min) were identical in Wistar and Brattleboro rats. We conclude that vasopressin does not mediate the catecholamine-induced release of ACTH, beta-endorphin and alpha-MSH.     

Hormonal control of tyrosine hydroxylase in the median eminence: demonstration of a central role for the pituitary gland

In intact male rats the concentration of dopamine in hypophysial portal plasma of animals treated simultaneously with estradiol and progesterone was twice that of animals treated with the solvent vehicle. Treatment with estradiol or progesterone alone had no effect on dopamine in portal plasma. The rate of synthesis of dihydroxyphenylalanine (DOPA), the precursor of dopamine, in tuberoinfundibular dopaminergic (TID) neurites in the median eminence (ME) was 15 +/- 1.0 (mean +/- SE) pmol DOPA/ME.h in estradiol-progesterone-treated animals compared to 3.2 +/- 0.02 in vehicle-treated controls. Treatment with estradiol or progesterone alone gave a result similar to that seen in controls. In hypophysectomized animals treated with estradiol and progesterone, DOPA synthesis in the ME was greatly attenuated compared to that in intact rats. The in situ activity of tyrosine hydroxylase (TH; expressed as moles of DOPA per mol TH/h) in the ME was 178 +/- 16.5 in estradiol-progesterone-treated intact rats, but was 27 +/- 2.4, 52 +/- 4.2, and 35 +/- 2.5 in animals treated with the solvent vehicle, estradiol, and progesterone, respectively. In hypophysectomized rats the in situ activity of TH in the ME of animals treated with estradiol and progesterone was 53 +/- 8.4, which was significantly (P less than 0.01) less than that in similarly treated intact animals. The circulating PRL level in vehicle-treated animals was 35 +/- 4.6 ng/ml compared to 121 +/- 16 in estradiol-treated animals and 133 +/- 12.2 in estradiol- and progesterone-treated rats, indicating that the difference in the effects of estradiol and estradiol-progesterone on dopamine release, DOPA synthesis, and in situ TH activity was not solely due to a difference in circulating PRL levels. Maintenance for 7 days of anterior pituitary tissue as a graft in a lateral ventricle of intact rats resulted in a 2-fold increase in the synthesis of DOPA and TH activity in the ME compared to that in animals with liver implants. Results obtained in hypophysectomized animals with implants were similar to those in intact animals. The concentrations of PRL in cerebrospinal fluid of intact rats and hypophysectomized rats with anterior pituitary implants in the lateral ventricles were 96 +/- 32 and 127 +/- 35 ng/ml, respectively, which was significantly (P less than 0.001) greater than those in animals with liver implants. We suggest that a factor of pituitary origin stimulates TH activity in TID neurons. This stimulation may be due to PRL, but the existence of another stimulatory substance secreted by pituitary cells cannot be excluded.     

Aromatic L-amino acid decarboxylase activity in the rat median eminence, neurointermediate lobe and anterior lobe of the pituitary. Physiological and pharmacological implications for pituitary regulation

Recent evidence demonstrating a direct effect of 5-hydroxytryptamine (5-HT) upon anterior pituitary (AP) hormone secretion has made the question of determining the location of possible sites that could supply 5-HT to the AP an important one. It has been assumed, based on indirect evidence, that aromatic L-amino acid decarboxylase (L-AAD), the enzyme responsible for the conversion of L-5-hydroxytryptophan (5-HTP) to 5-HT as well as L-3,4-dihydroxyphenylalanine (L-dopa) to dopamine (DA), is ubiquitously distributed in most tissues of the body including the AP. The present study examined the ability of the AP and two neural areas anatomically connected to the AP, the neurointermediate lobe (NIL) of the pituitary and the median eminence (ME), to decarboxylate 5-HTP to 5-HT or L-dopa to DA following either the in vitro incubation of the various tissues with 5-HTP or L-dopa or the in vivo administration of 5-HTP to rats treated previously with saline or a peripheral decarboxylase inhibitor, MK 486. The in vivo effects of 5-HTP, alone, or following MK 486 pretreatment were also examined on 5-HT synthesis and metabolism in AP tissues which were transplanted 5 days previously under the renal capsule and were, thus, isolated from central influences that might be regulating 5-HT and 5-hydroxyindole-3-acetic acid (5-HIAA) concentrations in the animal's own AP. In addition, the direct radioisotopic measurement of L-AAD activity in the ME, NIL, and AP was also analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thyrotrophin-releasing hormone does not accumulate glycosylated thyrotrophin, but changes heterogeneous forms of thyrotrophin within the rat anterior pituitary gland

We have investigated the effect of TRH on the accumulation of glycosylated TSH in the rat anterior pituitary gland. Hemipituitaries from adult male rats were incubated in medium containing [3H]glucosamine in the presence of TRH. [3H]Glucosamine-labelled TSH in media and pituitaries was measured by immunoprecipitation and characterized by isoelectric focusing after affinity chromatography. Incorporation of [3H]glucosamine into immunoprecipitable TSH in the media and pituitaries increased progressively with the period of incubation. Although the release of [3H]glucosamine-labelled and unlabelled TSH into media was stimulated by the addition of TRH in a time- and dose-dependent manner, TRH administration did not alter the amounts of labelled or unlabelled TSH in the anterior pituitary lobes. The anterior pituitaries were found, by isoelectric focusing analysis, to be composed of four major component peaks of [3H]glucosamine-labelled TSH. Administration of TRH caused profound changes in the radioactivity of these components and evoked new radioactive peaks, resulting in the appearance of six components in total. The present data provide evidence that TRH significantly changes the heterogeneity of glycosylated TSH in the anterior pituitary without altering the amount of the glycosylated form.     

In vitro release of vasopressin and oxytocin from rat median eminence tissue

Release of vasopressin (AVP) and oxytocin (OT) from rat median eminence and posterior pituitary tissue was studied in vitro by incubation in Krebs-56 mM KCl buffer. Both total tissue content and releasable pool of each hormone was measured in control rats, adrenalectomized rats and dexamethasone-treated rats. Adrenalectomy resulted in significantly increased release of AVP, but not OT, from median eminence tissue, whereas dexamethasone treatment failed to affect release of either hormone. Neither treatment had any effect on AVP or OT release from posterior pituitary tissue. Similarly, neither treatment caused any significant changes in total median eminence or posterior pituitary AVP and OT contents relative to controls, although dexamethasone-treated rats had a significantly lower posterior pituitary OT content than adrenalectomized rats. KCl-stimulated hormone release from median eminence tissue most likely represents an estimate of AVP and OT in zona externa terminals rather than in zona interna axons, because release was blocked by CoCl2 indicating calcium-dependent exocytosis. Immunohistochemical staining of median eminence tissue correlated well with the results of in vitro hormone release, in that increased AVP staining in the zona externa of adrenalectomized rats was also the only significant change noted using this methodology. Since increased levels of releasable AVP in the median eminence probably reflects similarly increased AVP levels in the hypothalamo-hypophyseal portal vessels of adrenalectomized rats, these results support a potential physiologic role for median eminence AVP, but not OT, in the chronic stimulation of adrenocorticotropin hormone secretion following adrenalectomy.     

Growth hormone expression and secretion in pig pituitary and median eminence slices are not influenced by the VGF protein

Body homeostasis is maintained by a complex system that involves the brain and the periphery via many circulating hormones. In recent years the VGF protein has been indicated as an important peptide affecting the regulation of body composition. We examined the effects of VGF on growth hormone (GH) expression and secretion in porcine pituitary slices, incubated alone (group 1) or with stalk median eminence (SME) (group 2). After 2 h (time 0), medium was removed and replaced with a fresh one; tissues were challenged with VGF (10(-6) M, 10(-8) M) alone or with ghrelin (10(-8) M) or growth hormone-releasing hormone (GHRH) (10(-8) M). Medium was replaced again 2 h (+2) and 6 h (+6) later. None of the VGF concentrations influenced GH secretion in either group; the association with GHRH or ghrelin appeared ineffective in influencing GH secretion as compared with the effects of GH mRNA expression and was not influenced by VGF treatments. The presence of SME had an additive effect on GH expression. Collectively, our results confirm previous findings on GH regulation; however, further investigations are needed to establish whether the modulation of GH secretion in the absence of nutrients involves the balance of GHRH/ghrelin receptors at pituitary levels. As for VGF, a crucial aspect to clarify is whether its lack of effects depends on our experimental conditions or, alternatively, it is not effective at all.     

Comparative effects of corticotropin-releasing factor, arginine vasopressin, and related neuropeptides on the secretion of ACTH and alpha-MSH by frog anterior pituitary cells and neurointermediate lobes in vitro

The ability of corticoliberin (CRF), urotensin I, sauvagine, arginine-vasopressin (AVP), and mesotocin to stimulate ACTH release by frog anterior pituitary cells and alpha-melanotropin (MSH) by frog neurointermediate lobe was studied in vitro using a perifusion technique. CRF and AVP were found to be potent stimulators of ACTH secretion, whereas urotensin I and sauvagine were totally inactive. In opposition to recent findings in the rat. CRF did not modify alpha-MSH secretion by the frog neurointermediate lobe. Mesotocin, which is present in the parenchymal cells of the frog pars intermedia, had no effect on alpha-MSH release in vitro. No potentiation of CRF-induced ACTH release was observed when anterior pituitary cells were incubated with a combination of AVP and CRF. Together with the recent elucidation of a CRF-like molecule in the frog diencephalon, these results suggest that, in Amphibia, CRF and AVP exert their stimulatory action specifically on distal lobe corticotrophs.     

Chronic estradiol treatment decreases angiotensin II receptor density in the anterior pituitary gland and adrenal cortex but not in the mesenteric artery

Chronic estrogen treatment has been shown to produce a marked reduction in anterior pituitary angiotensin II (AII) receptor density. In order to determine whether this effect is generalized, we studied the effect of chronic estradiol treatment on AII receptor density in the anterior pituitary gland, adrenal cortex and mesenteric artery of ovariectomized (OVX) rats. Treated rats were injected daily with 25 micrograms of estradiol valerate while controls received only the vehicle. Binding affinity and density of AII receptors were measured using the AII antagonist [125I]-Sar1Ile8 AII ([125I]-SARILE). Following 7-, 14- or 28-day treatments, AII receptor density decreased by approximately 80% in the anterior pituitary; 30% in the adrenal cortex and remained the same in mesenteric artery particulate fractions. In all 3 target tissues, dissociation constants (KD) for binding of [125I]-SARILE were in the nanomolar range and were the same between control and treated rats. Using conscious rats, estradiol treatment for 7 days was also shown to block the release of aldosterone by low dose infusions of AII (10 ng/min, 30 min). Plasma AII and plasma renin activity were also the same or slightly decreased following estradiol treatments. This study suggests that estrogens may be important modulators of the AII receptor and may be directly involved in modulating target cell responsiveness to AII as expressed through differential down-regulation of AII receptors.     

Effect of dexamethasone on immunoreactive corticotropin-releasing factor in the rat median eminence and intermediate-posterior pituitary

Immunoreactive ACTH (I-ACTH) concentrations in the anterior pituitary, intermediate-posterior pituitary (IP), and plasma and immunoreactive corticotropin-releasing factor (I-CRF) concentrations in the median eminence and IP were determined in rats receiving dexamethasone for various periods from 16 h to 10 days. Plasma I-ACTH concentrations were decreased 16 h after a single injection of dexamethasone. Anterior pituitary I-ACTH concentrations did not decrease until 4 days after the start of dexamethasone medication. IP I-ACTH concentrations did not change throughout these periods. I-CRF concentrations in median eminence and IP rapidly decreased after dexamethasone administration. These results raise the possibility that the source of I-CRF in the IP is hypothalamic.