Jimmy D. Neill

Cloning, sequencing, and expression of human gonadotropin releasing hormone (GnRH) receptor☆

Gonadotropin releasing hormone is a hypothalamic decapeptide that stimulates the release of gonadotropic hormones from the anterior pituitary gland. Therapeutically, the human pituitary GnRH receptor is the target of agonists used in the suppression of prostate cancer. Here we report the isolation of a cDNA representing this receptor. It encodes a protein with a transmembrane topology similar with that of other G protein-coupled, 7-transmembrane receptors. Binding studies of the cloned receptor demonstrate high affinity and pharmacological properties similar with the native human pituitary GnRH receptor. Northern blot and reverse transcriptase/PCR analysis revealed that its mRNA is expressed in pituitary, ovary, testis, breast, and prostate but not in liver and spleen. Availability of a human GnRH receptor cDNA should permit the design of improved analogs for therapeutic applications.

Angiotensin II type-1 receptor subtype cDNAs: differential tissue expression and hormonal regulation.

A rat angiotensin, type 1A (AT1A) receptor cDNA was cloned recently and shown to be a member of the 7-transmembrane, G-protein coupled family of receptors. Here, we report the cloning, sequencing, and expression of a previously unsuspected second form of the type 1 receptor (AT1B) in the rat which exhibits high similarity with the AT1A receptor relative to amino acid sequence (95% identity), binding of angiotensin II analogs, and utilization of Ca+2 as its intracellular second messenger. The adrenal and pituitary gland express primarily AT1B mRNA whereas vascular smooth muscle and lung express primarily AT1A mRNA. Estrogen treatment suppressed AT1B but not AT1A mRNA levels in the pituitary gland. Thus, the unexpected existence of two putative AT1 receptor genes appears to be related to the differential regulation of their expression rather than to different functional properties of the encoded receptor proteins.

Minireview: GnRH and GnRH Receptor Genes in the Human Genome

Four different GnRHs and one GnRH receptor are reported to be expressed in various mammals, whereas 13 GnRHs and numerous GnRH receptors have been identified in various nonmammalian vertebrates. The nucleotide sequencing of the human genome provided the opportunity to determine which of these peptides and receptors might be expressed in primates. Of the four GnRHs reportedly expressed in mammals, only GnRH I (mammalian GnRH) and GnRH II (chicken GnRH II) genes were identified in the human genome. Three GnRH receptor or receptor-like genes were identified: 1) the well-established GnRH I receptor gene located on chromosome 4; 2) an apparent GnRH II receptor gene located on chromosome 1, and; 3) a sterile GnRH II receptor-like homolog gene on chromosome 14. A cDNA cloned from monkey RNA that was 96% identical with the putative human GnRH receptor type II gene encoded a 379-amino acid G protein-coupled/7-transmembrane receptor having a C-terminal cytoplasmic tail. The experimentally expressed GnRH II receptor was functional with and specific for GnRH II, and, unlike the GnRH I receptor, desensitized to continuous GnRH treatment. GnRH II receptor mRNA is expressed ubiquitously in human tissues. Significant questions remain about the potential functions of the primate GnRH II receptor such as regulation of gonadotropin secretion, female sexual behavior, and tumor cell growth; also, about whether it is expressed as a full-length, functional gene transcript in humans.

Differential expression of angiotensin II receptor subtype mRNAs (AT-1A and AT-1B) in the brain.

Two highly similar rat angiotensin II, type 1 receptor cDNAs (AT1) have been described that probably are encoded by separate genes. AT1A subtype mRNA was expressed in vascular smooth muscle whereas AT1B mRNA was expressed in adrenal and pituitary. Here we measured the two AT1 subtype mRNAs in brain using reverse transcriptase/polymerase chain reactions. AT1B mRNA was predominant in subfornical organ (SFO) and organum vasculosum of the lamina terminalis (OVLT), the two regions that mediate angiotensin II-induced drinking behavior, and also in cerebellum. AT1A mRNA was predominant in the hypothalamus. Thus, the two AT1 receptor subtypes established to reside in peripheral tissues also are found in the central nervous system where the AT1B subtype may mediate drinking behavior.

Detection and measurement of secretion from individual neuroendocrine cells using a reverse hemolytic plaque assay.

Recent advances in technology have dramatically increased the resolution with which we may examine many features of biological systems. Intracellular recording and tracer injection techniques allow one to study the function of individual neurons and later characterize the same cells morphologically. In situ hybridization techniques can give us information about messenger RNA levels in single cells. More established techniques such as immunocytochemistry and electron microscopy also provide information at the cellular and even subcellular level. With each of these technological advances we have learned more about the mechanisms underlying cell function. We are also beginning to appreciate the role of heterogeneity among cells in relation to the function of the whole organism. Application of the reverse hemolytic plaque assay to the study of hormone or neurotransmitter secretion should help clarify this role. This technique permits accurate quantitation of hormone secreted from a large number of cells. Thus while cells can be studied individually they can also be categorized into functional subpopulations. As discussed in this chapter, many other techniques may be applied on cells which have already been functionally defined with the plaque assay. This should result in a clearer understanding of the roles of secretagogue binding and internalization, activation of second messenger systems, protein synthesis, and the cytoskeleton in hormone secretion. In the plaque assays described in this chapter individual pituitary cells are isolated in culture free from possible interactive effects coming from other cells. While these interactions are no doubt critical to the understanding of the function of the organism as a whole they can result in totally uninterpretable results. In fact, when we have gained some understanding into the functioning of individual cells it should be possible using the plaque assay to study the interactions among cells in a controlled fashion.

Detection and measurement of hormone secretion from individual pituitary cells.

Publisher Summary The most enduring debate in neuroendocrinology is whether feedback control by peripheral hormones occurs indirectly at the hypothalamus or directly on the pituitary gland. Of all the hormones of the pituitary gland, the secretion of luteinizing hormone (LH) has provoked the greatest controversy probably because of the complexity of its regulation. Dual inhibitory and stimulatory effects of ovarian estradiol are observed depending on whether its concentrations are low or high, respectively. This chapter explains about immunecytochemistry on plaque-forming cells, growth hormone/prolactin secretion from the same pituitary cells, regulation of secretion among the different pituitary cell types, and measurement of gonadotropin-releasing hormone (GnRH) binding and LH release by individual gonadotropes. The preovulatory surge of LH secretion is evoked by estrogen acting partly at the hypothalamus to increase GnRH secretion and partly at the pituitary gland to increase its responsiveness to the releasing actions of GnRH. This increased responsiveness is characterized by an elevation in the number of GnRH receptors and an increase in the fraction of the LH content that maximal doses of GnRH can release. The reverse hemolytic plaque assay for measurement of LH secretion by individual gonadotropes has been used to determine if subpopulations of cells exist and, if so, whether they contribute to such physiologic phenomena observed in vivo.

Rat gonadotropin-releasing hormone (GnRH) receptor : tissue expression and hormonal regulation of its mRNA

The binding of gonadotropin-releasing hormone (GnRH) to its receptor in the anterior pituitary gland is the key molecular interaction regulating the reproductive process of mammals. Here, we report the isolation of a cDNA representing this receptor from rat anterior pituitary and the regulation of expression of its mRNA. The rat GnRH receptor cDNA was composed of 2909 nucleotides and encoded a protein containing 327 amino acids having a seven transmembrane topology. Northern blot analysis on RNA from rat pituitary, ovary and testis showed four different transcripts (5.0, 4.5, 2.5 and 1.3 kb) of which the 5.0 kb form was most abundant. The levels of expression of the transcripts were found to be highest in the pituitary followed by the ovary and the testis (about 40% and 5% compared to pituitary, respectively). Using the more sensitive reverse transcriptase/PCR technique, we also detected GnRH receptor mRNA in the adrenal and the hypothalamus. Measurement of pituitary GnRH receptor mRNA levels (the 5.0 kb form) during the estrous cycle showed the lowest levels at estrus (1.0-fold), a 2.2 +/- 0.57 (mean +/- SEM) -fold increase at diestrus I, a 3.5 +/- 0.41-fold increase at diestrus II, a 2.6 +/- 0.34-fold increase on the morning of proestrus, and a 1.9 +/- 0.25-fold on the afternoon of proestrus. Removal of the ovaries led to a 2.7 +/- 0.29-fold increase in GnRH receptor mRNA levels in the pituitary gland; treatment of ovariectomized rats with estrogen resulted in a significant decrease in GnRH receptor mRNA levels. Our studies demonstrate ovarian regulation of GnRH receptor mRNA expression in the anterior pituitary gland.

A Reverse Hemolytic Plaque Assay for Microscopic Visualization of Growth Hormone Release from Individual Cells: Evidence for Somatotrope Heterogeneity

Growth hormone (GH) secreting cells direct complement-mediated plaque formation (clear zones of hemoly-sis surrounding the somatotropes) in mixed pituitary cell cultures incubated as a monolayer withGrowth hormone (GH) secreting cells direct complement-mediated plaque formation (clear zones of hemolysis surrounding the somatotropes) in mixed pituitary cell cultures incubated as a monolayer with protein-A coupled ovine erythrocytes (oRBC) in the presence of GH antiserum. Plaques were maximal in number after 4 h; growth hormone-releasing hormone (GHRH) and somatostatin increased and decreased, respectively, the rate of formation of plaques and their final sizes. Approximately 30% of all pituitary cells formed GH plaques and a similar fraction stained for GH using peroxidase-antiperoxidase immunocytochemistry (ICC). The plaque areas of individual somatotropes (reflecting the amount of GH released) covered a 20-fold range from the smallest to the largest in the 3 treatment groups. Somatostatin-treated and untreated cells formed frequency distributions of plaque sizes that were unimodal. In contrast, GHRH produced a bimodal frequency distribution suggestive of a sub-population of somatotropes preferentially responsive to this secretagogue. This new assay coupled with other morphological and biochemical techniques that can be applied to single cells will permit further analysis of these sub-populations of somatotropes.

The glycopeptide moiety of vasopressin-neurophysin precursor is neurohypophysial prolactin releasing factor

All of the classically-described hypothalamic, hypophysiotropic factors that regulate anterior pituitary hormone secretion have now been isolated and identified except for prolactin releasing factor. We report here that the 39-amino acid glycopeptide comprising the carboxyterminus of the neurohypophysial vasopressin-neurophysin precursor stimulates prolactin release from cultured pituitary cells as potently as does thyrotropin releasing hormone but has no effect on the secretion of other pituitary hormones. Furthermore, antisera to the glycopeptide administered to lactating rats attenuated suckling-induced prolactin secretion. Thus, this glycopeptide appears to be the neurohypophysial prolactin releasing factor.

Stimulation of Prolactin Secretion in Rhesus Monkeys by Vasoactive Intestinal Polypeptide

Vasoactive intestinal polypeptide (VIP) is a potent stimulus for prolactin (PRL) release in rats. The purpose of this study was to test the effect of VIP on PRL secretion in rhesus monkeys and to identify its site of action. Three experimental models were used: (1) intact monkeys during the follicular phase of the menstrual cycle; (2) female, hypophyseal stalk-transected, ovariectomized monkeys (St-OVX), and 93) monkey pituitary tissue perifused in vitro. Initial serum concentrations of immunoreactive PRL were more than 10-fold higher for ST-OVX monkeys (52.4 +/- 10.4 ng/ml, X +/- SEM; n = 6) than for intact monkeys (4. 79 +/- 1.0 ng/ml; n = 10). Intravenous administration of VIP (20 micrograms/kg body weight) induced an elevation of circulating PRL in each of the intact and ST-OVX monkeys tested. On the average, VIP treatment evoked more than a 9-fold rise in serum PRL in intact monkeys (p less than 0.01) and more than a 2-fold increase in ST-OVX monkeys (p less than 0.01), while injection of vehicle alone did not affect PRL levels in either experimental group. Addition of VIP (5 x 10(-9) - 5 x 10 (-6) M) to medium perifusing monkey pituitary tissue in vitro stimulated PRL secretion both in the presence and in the absence of dopamine (2.5 x 10(-6) M). Furthermore, the in vitro potency of VIP was comparable, on a molar basis, with that of thyrotropin releasing hormone. Collectively, these results indicate that VIP is a potent stimulus for PRL secretion in monkeys which exerts its effect, at least in part, by a direct action at the pituitary level. Therefore, VIP should now be considered as a possible PRL releasing factor in primates.