Molecular forms of human growth hormone secreted in vivo: nonspecificity of secretory stimuli

Several molecular forms of human GH (hGH) are present in blood, but their individual regulation is largely unknown. To examine the factors controlling the secretion of individual hGH variants, the relationship between the mixture of circulating hGH forms and the type of preceding secretory stimulus was studied in 18 normal subjects and 5 acromegalic patients. The stimuli employed were L-dopa, GH-releasing hormone-(1-40), exercise, spontaneous sleep-related and daytime secretory bursts, estrogens, and TRH in the acromegalic patients. Three monomeric hGH forms (22K, 20K, and acidic hGH) were identified in all samples; their mean relative proportions were 76.4%, 15.8%, and 7.9%, respectively. These proportions were similar in all subjects, regardless of stimulus, sex, or presence of acromegaly (P greater than 0.25). We conclude that the release of individual hGH forms is not stimulus specific, but, rather, that the secretory granule contents are released in toto upon somatotroph stimulation.     

Ultrastructural-immunocytochemical localization of growth hormone and prolactin in human pituitaries.

Immunocytochemical staining using the unlabeled antibody peroxidase-antiperoxidase method was undertaken to localize and characterize in ultrathin sections of human pituitaries the cells responsible for the secretion of GH and PRL. Somatotrophs in seven pituitaries stained with human (h) PRL-absorbed antiserum to hGH, were abundant, round to ovoid, densely granulated cells, whose mean (+/-SD) granule diameter was 368 +/- 60 nm. Lactotrophs immunostained with antiserum to hPRL were less numerous, angular or branching cells, with fewer round to ovoid granules, the mean diameter (+/-SD) of which was 185 +/- 35 nm in six pituitaries. The somewhat larger PRL granules (up to a mean diameter of 360 nm) seen in two of three additional pituitaries may have been related to the previous therapeutic administration of estrogen. Whereas the immunostained GH-secreting cells resemble the presumed somatotrophs identified in other studies on the basis of nonimmunological staining, the immunostained PRL-secreting cells differ considerably from the cells with large (600--1000 nm) granules designated as lactotrophs by several previous investigators. The hazards of ultrastructural identification of human pituitary cell types on purely morphological (as opposed to immunocytochemical) grounds are emphasized.     

Nitric oxide stimulates growth hormone secretion from human fetal pituitaries and cultured pituitary adenomas

Nitric oxide (NO), a highly reactive free radical, has been identified as a neurotransmitter in the central and peripheral nervous system. NO synthase (NOS) is the enzyme responsible for NO production from L-arginine and plays an important role in regulating the release of several hypothalamic peptides. In the pituitary, NO was found to increase growth hormone (GH) secretion in several in vitro and in vivomodels. However, its role in human GH regulation is unknown. The aim of this study was to investigate the regulatory effects of NO on human GH and prolactin secretion using primary cell cultures of human fetal pituitaries and cultured hormone-secreting adenomas. Incubation of the human fetal pituitaries (21-24 wk gestation) in the presence of sodium nitroprusside (SNP; 1 mM), a NO donor, for 4 h resulted in a 50-75% increase in GH secretion, similar to the stimulatory effect evoked by growth hormone-releasing hormone (GHRH) (10 nM). However, fetal PRL secretion was not affected by SNP. GH release was also stimulated (40-70% increase) by SNP in 60% of the cultured GH-secreting adenomas studied. SNP-induced GH release was inhibited in both fetal and adenomatous cells by PTI0, a NO scavenger. The addition of cGMP (0.1-1 mM), the second messenger of multiple NO actions, enhanced fetal and adenomatous GH secretion by 55-95%. Neuronal NOS (nNOS) was expressed in normal (fetal and adult) human pituitary tissues and in GH-secreting adenomas. Examination of its functional expression using L-arginine (1 microM) yielded a 35% increase in GH release from cultured GH-secreting adenoma. This response was blocked by a NOS inhibitor with high selectivity for the neuronal enzyme and by a guanylyl cyclase inhibitor. In conclusion, NO stimulates human GH in cultured fetal pituitaries and GH-secreting adenomas. Cyclic GMP is probably involved in this hormonal regulation.     

Hepatic receptors for homologous growth hormone in the eel

The specific binding of 125I-labeled eel growth hormone (eGH) to liver membranes of the eel was examined. The specific binding to the 10,000g pellet was greater than that to the 600g pellet. The specific binding was linear up to about 100 mg fresh tissue, and was saturable with increasing amounts of membrane. The specific binding was pH-, temperature-, and time-dependent, with the optimum pH at 7.4, and greater specific binding was obtained at 15 and 25 degrees than at 35 degrees. Scatchard analysis of liver binding gave an association constant of 1.1 x 10(9) M-1 and a capacity of 105 fmol/mg protein. The receptor preparation was highly specific for GHs. Natural and recombinant eel GHs as well as recombinant salmon GH competed equally with 125I-eGH for the receptor sites of the 10,000g liver membrane. Ovine GH was more potent in displacing the labeled eGH than the homologous eel hormone. Tilapia GH and ovine prolactin (PRL) were needed in greater amounts (40 times) than eGH to displace the labeled eGH. Salmon and tilapia PRLs were still less potent (500 times) than eGH. There was no displacement with eel PRL. No significant change in the specific binding was seen 1 week after hypophysectomy, whereas injection of eGH into the hypophysectomized eel caused a significant reduction after 24 hr. The binding to the membrane fractions from gills, kidney, muscle, intestine, and brain was low and exclusively nonspecific, indicating the presence of specific GH receptors predominantly in the liver.     

Fish growth hormone receptor: molecular characterization of two membrane-anchored forms.

A RT-PCR approach was used to clone and sequence the full-length growth hormone receptor (GHR) of a teleost fish, the turbot (Scophthalmus maximus). Total liver RNA was amplified by RT-PCR with degenerate primers designed in extracellular and cytoplasmic regions, and a single DNA fragment of 1100 bp was obtained. The entire coding region was obtained by 5' and 3' RACE assays, and comprises an open-reading frame of 633 amino acids. This sequence shows the characteristic motifs of the class I cytokine receptor superfamily, and its amino acid identity with mammalian, avian, reptilian and amphibian GHRs is 32-36%. The 3' RACE also revealed the occurrence of an alternate messenger encoding a membrane-anchored truncated receptor, which could facilitate the production of GH-binding protein in fish species. This report represents the first data on fish GHR sequence, and it provides evidence for the conservation of this receptor throughout vertebrate evolution.     

Existence of multiple forms of follicle-stimulating hormone within the anterior pituitaries of cynomolgus monkeys

Ovariectomized cynomolgus monkeys were treated with physiological levels of estradiol and progesterone. A reduction in serum levels of FSH was observed after steroid exposure. Anterior pituitary homogenates were prepared from monkeys after 0, 12, 24, or 36 h of exposure to estradiol and progesterone and quantitated for FSH activity by radioreceptor assay (RRA) and RIA. Pituitary FSH activity (expressed as RRA/RIA) increased with duration of exposure to steroids. Forms of FSH within these pituitaries were separated by the column isoelectric focusing technique, chromatofocusing. All pituitary homogenates tested contained FSH isohormones that eluted at similar isoelectric points. Each FSH isohormone exhibited a mol wt similar to that of a purified FSH standard, but differed in ability to displace labeled FSH from a biological receptor preparation. FSH forms with basic isoelectric points exhibited greater RRA/RIA values than forms with more acidic isoelectric points. The relative proportion of the more basic FSH forms increased within pituitary tissue with duration of exposure to steroids. All FSH forms were secreted by pituitary cells in culture. The biochemical basis for the microheterogeneity appears to be the degree of sialic acid incorporation into the FSH molecule. The results of these studies demonstrate that the cynomolgus monkey pituitary responds to the surrounding hormonal milieu by altering the relative proportions of FSH forms present within that gland.     

Specificity of amphibian and reptilian pituitaries for various forms of gonadotropin-releasing hormones in vitro

In vitro perifusion was employed to compare the potencies of mammalian, avian, salmon, and lamprey gonadotropin-releasing hormones (GnRHs) on the release of luteinizing hormone (LH) from the pituitaries of an amphibian (Rana pipiens) and a reptile (Chrysemys picta). The chicken-I and salmon GnRH variants were equipotent with mammalian GnRH in both the frog and the turtle glands. By contrast, the lamprey GnRH was inactive (less than 1% as potent as the others). Lamprey GnRH also failed to stimulate LH release or to induce GnRH priming when administered chronically to the frog gland. These results support the hypothesis that the GnRH receptors on nonmammalian pituitary cells are much less specific than those of the mammal with regard to the amino acid at position 8 of the GnRH molecule. These data suggest that the native GnRH variant or the one most like that found in the brain of a species is not necessarily the most potent biologically in that species. However, the nonmammalian pituitary does show some specificity with regard to the structure of natural GnRHs in that none of the tetrapod species studied is responsive to lamprey GnRH.     

Effect of pulsatile gonadotropin-releasing hormone on the release of luteinizing hormone and follicle-stimulating hormone in vitro by anterior pituitaries from lactating and cycling rats

The ability of pituitaries from lactating animals to secrete LH and FSH in response to gonadotropin-releasing hormone (GnRH) was studied in vitro using a pituitary incubation system. Hemipituitaries were exposed to GnRH for 6 min during each hour of incubation. LH release by anterior pituitaries (APs) from day 5 postpartum rats nursing eight pups, in response to pulsatile exposure to GnRH, was significantly less than that released by APs from diestrous cycling females. Even though the amount of LH released by APs increased as lactation progressed, LH release by APs from day 15 postpartum rats nursing eight pups was still less than LH release by APs from diestrous females. In contrast pituitaries from lactating females nursing two pups released amounts of LH similar to that released by pituitaries from diestrous females, whereas females deprived of their litters for 48 h showed a greater response than diestrous females. Generally, there was a good quantitative relationship between the amount of LH released in vitro and plasma LH concentrations for all the intact groups studied. The ability of lactation to suppress the postcastration rise in serum LH also was demonstrated in vitro as pituitaries from ovariectomized or intact females nursing eight pups released similar amounts of LH on days 5 and 10 postpartum. However, by day 15 postpartum, even though serum LH concentrations were still very low, pituitaries from ovariectomized lactating females released LH in vitro at a rate similar to pituitaries from nonlactating rats. Serum FSH concentrations were not suppressed but similar in intact and cycling females. Also, the total amount of FSH released in vitro in response to GnRH by pituitaries from lactating and cycling females did not differ significantly, even though LH release differed greatly among these groups of animals. However, the patterns of GnRH-stimulating FSH secretion differed among intact lactating, ovariectomized lactating, and nonlactating females. Pituitary LH concentrations were similar on day 5 postpartum and diestrus and on day 15 postpartum and proestrus. Pituitary FSH concentrations on day 5 postpartum were similar to those during diestrus and proestrus and had increased 2-3 times by day 15 postpartum. Generally, there was no correlation between the amount of LH or FSH released by pituitaries in response to GnRH and pituitary gonadotropin content. In summary, the inability of pituitaries from lactating rats to respond adequately to large doses of GnRH in vitro suggests that the suckling stimulus indirectly suppresses pituitary responsiveness to GnRH. This suppression differentially affects basal LH secretion, but not basal FSH secretion, and may be the direct result of inadequate GnRH stimulation in vivo.     

Isolation and characterization of follicle-stimulating hormone and luteinizing hormone and its subunits from snapping turtle (Chelydra serpentina) pituitaries.

Highly purified luteinizing hormone and follicle-stimulating hormone have been isolated from extracts of snapping turtle (Chelydra serpentina) pituitaries. Both hormones are potent in non-mammalian gonadotropin bioassays (1.8 X NIH-LH-S1 and 30 X NIH-FSH-S1). The materials have been characterized by polyacrylamide gel electrophoresis, amino terminal group analysis, amino acid and carbohydrate content, and, in the case of turtle luteinizing hormone, ultracentrifugation. The luteinizing hormone was shown to dissociate and subunits were prepared by the countercurrent distribution technique and characterized. Biological activity of the hormone could be regenerated by recombination of the subunits. In addition, it was shown that the snapping turtle luteinizing hormone subunits could be combined with subunits from ovine luteinizing hormone with generation of significant biological activity. Comparisons in properties of the turtle gonadotropins have been made with ovine gonadotropins, showing, in many cases, similarities in properties, suggesting structural features which have been conserved during evolution.     

Purification of liver feminizing factor from rat pituitaries and demonstration of its identity with growth hormone

The identity of the pituitary factor responsible for the maintenance of a female pattern of hepatic steroid metabolism and a female level of PRL receptors has been established. Fractionation of pituitary extracts revealed that only the GH fraction had the capacity to feminize liver metabolism of androstenedione (i.e. increase 5 alpha-reductase activity and decrease 16 alpha-hydroxylase activity) and to induce PRL receptors to a female level in hypophysectomized animals. The purification of pituitary GH was performed by chromatofocusing followed by gel filtration on Sephadex G-75. GH obtained from male or female pituitary glands showed an identical chromatographic behavior and both preparations had a mol wt of 22,000 and an isoelectric point of 6.1 when analyzed by analytical sodium dodecyl sulfate-gel electrophoresis and isoelectric focusing, respectively. The degree of homogeneity of GH varied between 93% and 97% as judged from sodium dodecyl sulfate-gel electrophoresis. Purified male and female GH were equally efficient in feminizing the liver metabolism. Since degradation of the native mol wt 22,000 form reduced the feminizing capacity, we believe that the intact hormone is needed for the feminization.     

Cloning of a bullfrog growth hormone cDNA: expression of growth hormone mRNA in larval and adult bullfrog pituitaries.

A GH cDNA was specifically amplified from cDNAs constructed from total RNA of bullfrog (Rana catesbeiana) adenohypophyses employing the DNA polymerase chain reaction. Sequencing analysis revealed that the cDNA clone thus obtained was 654 bp in length, and included an open reading frame encoding the entire sequence of mature GH, with its signal peptide. Slight discrepancies were noted between the deduced amino acid sequence and that determined by direct protein sequencing of purified bullfrog GH or that deduced from the nucleotide sequence reported previously. The length of the bullfrog GH mRNA was estimated to be about 1.2 kb by Northern blot analysis. Homologies of nucleotide and amino acid sequences between GH and prolactin of bullfrog origin were 48% and 26% respectively. Using the cDNA as a probe, the content of GH mRNA in the pituitary of larval and adult bullfrogs was measured. GH mRNA levels were relatively low at the preclimax stage, and rose markedly during climax. In juvenile frogs, GH mRNA levels in the pituitary were extremely high and declined towards adulthood. This finding suggests that the increase in plasma and pituitary GH levels reported previously accompanies the increase in GH synthesis.