Regulation of anterior pituitary and brain beta-adrenergic receptors by ovarian steroids

Ovariectomy of adult female rats (200-230g) resulted in an increase in beta-adrenergic receptors in the cerebral cortex, hypothalamus and anterior pituitary. The anterior pituitary had the largest overall increase as well as the most rapid increase in beta-adrenergic receptor density of the tissues examined. The increase in hypothalamic or cerebral cortical beta-adrenergic receptors became apparent only long after ovariectomy (7-14 days). Fourteen days after ovariectomy, the density of beta-adrenergic receptors was 79%, 40%, and 24% in excess of control values in crude membranes prepared from anterior pituitary, hypothalamus and cerebral cortex, respectively. Over the same interval, the plasma concentration of luteinizing hormone (LH) increased 28-fold, while the concentration of follicle-stimulating hormone (FSH) rose 5-fold compared to control levels. Estradiol replacement (20 micrograms/kg/day) in these animals for four days before sacrifice concomitantly reduced plasma levels of the gonadotropins as well as the density of beta-adrenergic receptors in both the anterior pituitary and the hypothalamus. Long-term steroid replacement during the fifth and sixth week after ovariectomy, with implants of estradiol and progesterone which released the steroids in approximately physiological concentrations, significantly reduced beta-adrenergic density in anterior pituitary, but not in the hypothalamic membranes. This treatment significantly reduced plasma LH, but not FSH. Beta-adrenergic receptor density was also found to fluctuate significantly during the 4-day estrous cycle. The highest values were found on proestrus, and the lowest on diestrus 1. These studies indicate that changes in plasma concentrations of gonadal steroids (e.g. during the estrous cycle) influence the density of beta-adrenergic receptors in tissues involved in the control and release of anterior pituitary gonadotropins.     

Somatostatin receptors in brain and pituitary

Somatostatin (SRIF) actions in the brain and pituitary are mediated by specific receptors. Using radioiodinated ligands it has been possible to characterize the kinetics of specific binding sites in the brain and pituitary, and to determine their cellular localization by autoradiography. At the pituitary level, the inhibition of growth hormone, prolactin and thyrotropin secretions induced by SRIF is mediated through a single binding site which is coupled to the inhibition of adenylate cyclase. In the brain, SRIF receptors are localized on neurons and glial cells and are also coupled to adenylate cyclase inhibition. Two sites are differentiated in the brain with an analogue of somatostatin, SMS 201995. In humans, SRIF-binding sites have been related to a number of pathologies. At the pituitary level, it has been shown that the number of binding sites was negatively correlated to growth hormone levels in acromegaly. Furthermore, SRIF-binding sites were undetectable in a patient which did not respond to SMS 201995 therapy. In the brain, meningiomas and gliomas are rich in SRIF binding sites. This suggests a possible role for SRIF on glia. In neurodegenerative diseases, cortical SRIF concentrations are decreased in Alzheimer's and Parkinson's disease associated with dementia while SRIF-binding sites are only affected in Alzheimer's disease. In conclusion, the physiological role of SRIF in the brain and pituitary can be evaluated by studying the receptors of the peptide. Such studies allow to question the implication of SRIF in endocrine and neuropathologies.     

Functional ATP receptors in rat anterior pituitary cells

The effects ofATP and other nucleotides on the cytosolicCa²⁺ concentration([Ca²⁺]_i)of single immunocytochemically typed anterior pituitary (AP) cells havebeen studied. ATP increased[Ca²⁺]_iin a large percentage (60-88%) of all five AP cell types:lactotropes, somatotropes, corticotropes, gonadotropes, andthyrotropes. Additivity experiments suggest the presence of at leasttwo different receptors, one accepting both ATP and UTP (U receptor),producing Ca²⁺ release from theintracellular stores, and the other preferring ATP (A receptor),producing Ca²⁺ (andMn²⁺) entry. The characteristicsof the U and A receptors were consistent with those ofP2Y₂ andP2X₂, respectively, and theirdistribution in the different AP cell types was not homogeneous. Thepresence of other ATP receptors suchP2Y₁ orP2X₂/P2X₃heteropolymers in a small fraction of the cells cannot be excluded.Thus functional ionophoric P2X receptors, which are typical of neuraltissue, are also present in the pituitary gland and could contribute to regulation of the gland's function.

     

Effect of ethanol on androgen receptors in the anterior pituitary, hypothalamus and brain cortex in rats

The purpose of this study was to investigate ethanol-induced changes in androgen receptor sites in the anterior pituitary, hypothalamus, and brain cortex. Young adult male King-Holtzman rats were fed for 5 months a nutritionally complete liquid diet, with ethanol or isocaloric sucrose constituting 36% of the total calories. Androgen receptor sites were measured by sucrose density gradient and charcoal assay using tritiated dihydrotestosterone (DHT). Scatchard plot analysis of the data revealed that apparent dissociation constants of DHT-receptor complex for the anterior pituitary, hypothalamus, and brain cortex from alcohol-fed animals were estimated to be 0.7 +/- 0.13, 0.6 +/- 0.16 and 0.9 +/- 0.15 nM, respectively. These values are identical to those of their isocaloric controls. The concentrations of cytosol androgen receptors of the pituitary, hypothalamus, and brain cortex from alcohol-fed rats were 8.0 +/- 1.2, 6.2 +/- 1.0 and 4.9 +/- 0.7 fmol/mg protein, respectively. This represents about a 34, 24, and 22% reduction when compared to the values of the isocaloric control animals. In contrast to control rats, neither castration nor androgen or LHRH replacement to castrated alcohol-fed rats altered an alcohol-induced reduction of androgen receptor contents. Serum LH and testosterone levels were significantly decreased in alcohol-fed rats but these hormone levels were increased by administration of LHRH or norepinephrine. Such reduction of androgen receptors, serum LH and testosterone, but enhancement of these hormone levels by treatment with neurohormone and neurotransmitter in these animals suggests that ethanol exerts an adverse effect on the hypothalamic-pituitary unit and the neurotransmitter-hypothalamic hormone relationship, resulting in impairment of the androgen-induced sexual events and a suppression of the pituitary gonadotropin secretion.     

GABAergic control of anterior pituitary hormone secretion

Anatomical and biochemical studies have identified a hypothalamic tubero-infundibular GABAergic system, which plays a functional role on anterior pituitary hormone secretion. Experimental and clinical evidence support the presence of a dual component in the action of GABA; one mediated via the central nervous system and the other exerted directly at the anterior pituitary level. The two sites of action may be responsible for the excitatory and inhibitory effects of GABA on pituitary hormone and especially prolactin secretion. The future characterization of this system will provide a better understanding of the involvement of GABA in the physiology of anterior pituitary hormone secretion and will contribute to the development of new pharmacological agents for the therapy of neuroendocrine disorders.     

Expression of P2Y receptors in the rat anterior pituitary

In this study, the distribution patterns of P2Y₁, P2Y₂ P2Y₄, P2Y₆, P2Y₁₂, and P2Y₁₃ receptors in the anterior pituitary cells of rat were studied with double-labeling immunofluorescence and Western blot. The results showed that P2Y receptors were widely expressed in the anterior pituitary. P2Y₁ and P2Y₄ receptors were found to be expressed in the majority of gonadotrophs and thyrotrophs, P2Y₂ receptors were expressed in a small subpopulation of lactotrophs and almost all the folliculo-stellate cells, that were also stained with S100 protein immunoreactivity. P2Y₆ receptors were expressed in macrophages. P2Y₁₃ receptors were expressed in a small subpopulation of cells in the rat anterior pituitary, the identity of which needs to be clarified. P2Y₁ and P2Y₄ receptors are co-expressed in some gonadotrophs and thyrotrophs. Corticotrophs and somatotrophs were found not to express P2Y receptors in this study. FSH and TSH were shown to coexist in the same endocrine cells in rat anterior pituitary. The present data suggests that purines and/or pyrimidines could be involved in regulating the functions of gonadotrophs and thyrotrophs via P2Y₁ and P2Y₄ receptors, some lactotrophs via P2Y₂ receptors, and folliculo-stellate cells via P2Y₂ receptors in the rat anterior pituitary.     

Expression of P2X receptors in the rat anterior pituitary

Purinergic Signalling - In this study, the distribution patterns of P2X1 to P2X7 receptors in the anterior pituitary cells of rat were studied with single-, double-, and triple-labeling...     

Effects of alloxan-induced diabetes on dopaminergic receptors in rat striatum and anterior pituitary

The binding of [3H]-spiroperidol after 4 weeks of hyperglycemia was determined in the rat striatum and anterior pituitary. Alloxan-induced diabetes increased the number of dopaminergic binding sites in the striatum but not in the anterior pituitary. The interaction of metoclopramide with striatal dopaminergic receptors was slightly modified, while that of dopamine, bromocriptine and haloperidol was unaffected. These results suggest that chronic hyperglycemia exerts selective effects on nigrostriatal dopaminergic system in the rat.     

Deglycosylation and proteolysis of photolabeled D2 dopamine receptors of the porcine anterior pituitary

Dopamine D2 receptor binding subunits of the porcine anterior pituitary were visualized by autoradiography following photoaffinity labeling with [125I]N-azidophenethylspiperone and sodium dodecyl-sulfate polyacrylamide gel electrophoresis. The ligand binding subunit comprising the pituitary D2 dopamine receptor migrated as two distinct bands of apparent Mr approximately equal to 150,000 and 118,000, substantially higher than neuronal D2 receptor subunits from porcine or canine brain. The glycoprotein nature of pituitary D2 receptor binding subunits was investigated by the use of exo- and endo-glycosidase treatments and peptide mapping experiments. Photoaffinity labeled polypeptides of the anterior pituitary were susceptible to both neuraminidase and alpha-mannosidase digestion as indexed by their increased electrophoretic mobility on sodium dodecyl-sulfate polyacrylamide gels, and suggests the presence of both complex type and terminal mannose carbohydrate residues. Moreover, the additive effects of sequential treatment with these enzymes suggests that both types of carbohydrate chains are present on each receptor peptide. N-linked deglycosylation of pituitary D2 photolabeled receptors with glycopeptidase-F produced a further increase in the mobility of the labeled protein to apparent Mr approximately equal to 44,000, similar to that of deglycosylated D2 binding subunits of porcine and canine brain. Peptide mapping experiments following limited proteolysis with Staphylococcus aureus V8 proteinase and papain demonstrated that deglycosylated D2 dopamine receptors (Mr = 44,000), in different tissues and species, were homologous. Taken together, these data suggest that despite the differences in the overall molecular weight and tissue specific glycosylation pattern of pituitary D2 dopamine receptors, the primary structure of mammalian D2 receptors appears to be conserved.     

Circadian rhythms in rat brain alpha- and beta-adrenergic receptors are modified by chronic imipramine

Circadian rhythms of α- and ß-adrenergic receptor number, with different wave forms, as well as differences in timing of maximal binding, are present in rat brain. Chronic treatment with the tricyclic antidepressant drug imipramine modifies these rhythms: peak binding of both receptors occurs 4–12 hours later than in controls, the 24-hour mean is decreased by 15–30%, and the amplitude is increased by 20–30%. Delaying of the phase position of neurotransmitter receptor rhythms by a tricyclic antidepressant may be relevant to its clinical mode of action, since depressive patients appear to have abnormally phase-advanced circadian rhythms.     

Characterization of receptors for thyrotropin-releasing hormone-potentiating peptide on rat anterior pituitary membranes.

Previous studies (Bulant, M., Delfour, A., Vaudry, H., and Nicolas, P. (1988) J. Biol. Chem. 263, 17189-17196; Bulant, M., Roussel, J. P., Astier, H., Nicolas, P., and Vaudry, H. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4439-4443) have shown that post-translational processing of rat thyrotropin-releasing hormone prohormone (pro-TRH) generates, besides thyrotropin-releasing hormone (TRH), a connecting decapeptide corresponding to prepro-TRH-(160-169), i.e. Ser-Phe-Pro-Trp-Met-Glu-Ser-Asp-Val-Thr. This peptide, which is named TRH-potentiating peptide (Ps4), is co-localized with TRH in the median eminence nerve endings and is involved in potentiation of the action of TRH on thyrotropin hormone release by pituitary in vitro and in vivo. To characterize the receptor(s) for TRH-potentiating peptide in the pituitary, a highly potent and metabolically stable derivative of Ps4, [I-Tyr0]Ps4, was radioiodinated. Binding of [125I-Tyr-0]Ps4 to rat pituitary membrane homogenates was specific, saturable, reversible, and linear with membrane protein concentration. Equilibrium measurements performed over a large range of concentrations revealed a single homogeneous population of high affinity binding sites (Kd = 0.22 nM; Bmax = 517 fmol/mg of membrane proteins). Several naturally occurring neuropeptides and hormones, including TRH, did not compete with [125I-Tyr0]Ps4 in the binding, which suggests the binding sites are specific to Ps4. Using C-terminal deletion analogs of [Tyr0]Ps4, we further showed the critical role the C-terminal residues Thr10, Val9, and Asp8 play in conferring high binding affinity and selectivity. Binding site tissue distribution and cross-reactivity binding studies suggest that the action of TRH-potentiating peptide is mediated through interaction with a specific pituitary cell-surface receptor which differ from those for TRH. [I-Tyr0]Ps4 reported in this paper, through its high binding affinity and specificity, its very low nonspecific binding, its high resistance to enzymatic degradation, and its high potentiating action in vitro should allow further progress in understanding the in vivo physiological function of Ps4.     

Multiple transduction mechanisms of dopamine, somatostatin and angiotensin II receptors in anterior pituitary cells

The concept of multifactorial pituitary control is now well established. As in other cell systems, integration of complex messages involves dynamic interactions of receptors and coupling mechanisms. Regulation of adenohypophyseal secretions has been shown to involve cyclic AMP production, the modulation of phosphatidylinositol phosphate breakdown and Ca2+ mobilization. Dopamine, somatostatin and angiotensin II receptors are negatively coupled to adenylate cyclase in anterior pituitary cells. In the case of angiotensin, this effect on adenylate cyclase appears paradoxical since the peptide markedly stimulates prolactin secretion. In fact, angiotensin II also markedly stimulates inositol phosphate production and this effect could account for the stimulated hormone secretion. In addition, dopamine could inhibit inositol phosphate production stimulated by angiotensin II and thyrotropin-releasing hormone. Dopamine and somatostatin also directly modulate voltage-dependent calcium channels, perhaps through a direct coupling with potassium channels. On the other hand, steroids modulate the sensitivity of adenohypophyseal cells to neurohormones by different mechanisms. In the case of somatostatin, it increases the number of specific binding sites, while in the case of dopamine estradiol affects the transduction mechanisms of D2 dopamine receptors. In conclusion, dopamine and somatostatin receptors appear coupled to various transduction mechanisms through pertussis-sensitive G proteins in anterior pituitary cells.     

Structural analysis of purified beta-adrenergic receptors

We have characterized the structure of purified beta-adrenergic receptors by a combination of photoaffinity labeling, high performance liquid chromatography (HPLC)-tryptic mapping, CNBr fragmentation, target size analysis, and electron microscopy of purified receptor molecules. Guinea pig lung beta-adrenergic receptors purified by affinity chromatography, ion exchange chromatography, and HPLC size exclusion chromatography or photoaffinity labeled with [125]-iodocyanopindolol diazirine displayed mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that corresponded to Mr = 68,000. Purified, radioiodinated guinea pig lung beta-receptors were subjected to complete trypsin digestion and subsequent reverse-phase HPLC analysis, which revealed nine peptides. Active site labeling and tryptic digestion of partially purified hamster lung beta-receptors produced one peptide, whereas CNBr digestion of the same material produced two labeled fragments, yielding information about the location of the active site within the primary sequence. Purified guinea pig lung receptors were examined with transmission electron microscopy. Electron micrographs revealed slightly asymmetric, rod-shaped structures with an average length of 13 nm and width of 3.4 nm. Many receptors were arranged as apparent dimeric structures. These findings confirm data obtained from target size analysis of guinea pig lung beta-receptors in situ which suggest that receptors may exist as oligomeric arrays in the native membrane. Taken together, these data provide information about putative functional domains of the beta-adrenergic receptor and its quaternary structure.