Release of thyroid stimulating hormone from chick anterior pituitary glands by thyrotropin releasing hormone (TRH)

Chicks two and ten days-of-age respond to a wide range of thyrotropin releasing hormone (TRH) dosages as measured by thyroid uptake of ³²P. The duration of hormone and ³²P action is important. Excellent responses were obtained with the injection of 1.0 μCi³²P at one hour and TRH either at one or four hours before autopsy in both two-day and ten-day-old birds. The ³²P uptake in the thyroid glands was increased by doses of hormone which ranged from 40 nanograms to 125,000 nanograms and was bimodal. Analysis of the data when calculated using log¹⁰ of dose was best accomplished by the use of 5th-degree polynomial equations. It is suggested that the bimodal response is a result of a dual action of TRH. First, TRH initiates the release of stored TSH from the anterior pituitary; and second, TRH stimulates the secretion of newly synthesized TSH by the anterior pituitary.     

Regulation of gonadotropin secretion in the anterior pituitary

Studies on the regulation of gonadotropin secretion in dissociated pituitary cell cultures are described. Initial studies employing a ferritin-labelled analogue of gonadotropin hormone releasing hormone (GnRH) to localize its receptor sites on the gonadotropin cell surface that while these receptor sites initially have a random monodisperse distribution, binding of the ligand causes coarse aggregation and internalization of the GnRH receptor. These events are not due to the multivalency of the ligand and probably reflect redistributive events in vivo. By using an octapeptide analogue GnRH that binds to the GnRH receptor but lacks gonadotropin releasing activity in conjunction with sequence-specific antisera it is shown that antibodies that bind the octapeptide can induce the octapeptide to release gonadotropin. These data suggest that receptor aggregation is important in GnRH stimulation. Finally immunocytochemical studies are described in which golg-protein-A-antibody complexes are used to identify gonadotropins on ultrathin frozen sections of porcine pituitary cells. These studies indicate that in porcine gonadotropin cells the majority of the secretory granules contain both luteinizing hormone and follicle-stimulating hormone.     

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.     

Distribution of angiotensinogen immunoreactivity in rat anterior pituitary glands

Angiotensin II (AII) has been previously shown to be localized in the gonadotropes of the rat anterior pituitary gland. Renin and angiotensin-converting enzyme, two enzymes that participate in the generation of AII, also have been shown to be present in gonadotropes. To determine whether angiotensinogen, the precursor to AII, is present in the same cells, we have stained rat anterior pituitary sections with an antirat angiotensinogen antiserum. Angiotensinogen staining was observed in cells that had a distinctive distribution at the periphery of the gland; the number of these cells and the intensity of the staining were increased in the pituitaries of rats that had been nephrectomized 24 hr before sacrifice. When double staining was performed, we never observed colocalization of angiotensinogen with any of the known pituitary hormones or with S100 protein. The results show that in the rat anterior pituitary gland, angiotensinogen is present, at least for the most part, in cells that are different from those containing renin, angiotensin-converting enzyme, and AII.     

Presence of ACTH-potentiating factors in rat anterior pituitary glands

High molecular weight ACTH fractions, obtained through gel filtration of boiled rat anterior pituitary extract, induced a marked increase in corticosterone production from isolated rat adrenal cells in the presence of low concentrations of ACTH-(1-24). This indicates the presence of heat-stable factors augmenting the steroidogenic action of ACTH in the rat anterior pituitary. We also noted that these factors potentiated the activity not only of ACTH-1(1-24) but also of ACTH-(1-8). The ACTH-potentiating factors in rat anterior pituitary extract are possibly present in heterogeneous forms according to their molecular weights (8,000, 10,000 and 15,000), their mobility in ion-exchange chromatography and their content in RIA-ACTH activity. Of these three forms, the former comigrated with biological ACTH activity. The remaining two forms were free of it. Since the effect of potentiating factors on modified ACTH-(1-9), shown to be less susceptible to proteolytic degradation from ACTH-(1-24), was similar to the effect on ACTH-(1-24), it is suggested that potentiation was not due to an inhibition of ACTH proteolysis.     

Regulation of glucose transporters by connective tissue activating peptide-III isoforms.

Connective tissue activating peptide-III (CTAP-III) is a component of platelet alpha-granules which elicits a series of responses in connective tissue cells referred to as activation, including increased glucose consumption and mitogenesis and increased secretion of hyaluronic acid and glycosaminoglycans. As anticipated by a requirement for glucose or glucose precursors in the activation process, an early event following CTAP-III activation of connective tissue cells is an increase in glucose transport. The present study investigates the molecular basis for this increase in glucose transport. Murine 3T3-F442A fibroblasts were found to respond to CTAP-III in a manner similar to human connective tissue cells (synovial cells, chondrocytes, skin fibroblasts). CTAP-III increases the rate of glucose transport to similar extents at 4 and 24 h, and at physiologic (micrograms/ml) concentrations of CTAP-III. A proteolytic cleavage product of recombinant CTAP-III (rCTAP-III-Leu-21 (des-1-15)), also known as neutrophil-activating peptide-2 (NAP-2), was found to be equally effective as CTAP-III, whereas NAP-1/interleukin-8, another member of the CTAP-III super-family, was ineffective in stimulating glucose transport. This contrasts with neutrophil chemotaxis, in which CTAP-III (des-1-15)/NAP-2 acts similarly to NAP-1/interleukin 8 while CTAP-III is ineffective. CTAP-III appears to elicit a different type of glucose transport response than many other growth factors in that its response is sustained (greater than or equal to 24 h) rather than transient (peak approximately 4 h) in confluent as well as in subconfluent cells. Western blot analysis using antibodies to the GLUT-1 glucose transporter revealed an increased level of GLUT-1 protein in response to CTAP-III isoforms that corresponded in magnitude (on a percentage basis) to the increased level of glucose transport. The increased levels of GLUT-1 protein in response to CTAP-III and rCTAP-III-Leu-21 (des-1-15)/NAP-2 were accompanied by an increase in levels of GLUT-1 mRNA of a magnitude sufficient to account for observed increased levels of GLUT-1. These results are consistent with CTAP-III isoforms stimulating glucose transport in connective tissue cells by increasing levels of GLUT-1 mRNA and is one of the few known instances in which increases in levels of GLUT-1 mRNA and protein are sufficient to account for observed increases in glucose transport. They also provide further evidence that CTAP-III (des-1-15)/NAP-2 binds to more than one type of receptor and that CTAP-III acts in a manner different than other well characterized growth factors (e.g. platelet-derived growth factor, transforming growth factor-beta) in that it causes a sustained (greater than or equal to 24 h) elevation in glucose transport in confluent as well as subconfluent cells.     

Purification of CMP-N-acetylneuraminic acid synthetase from bovine anterior pituitary glands

CMP-beta-N-acetylneuraminic acid (CMP-neuNAc) is the substrate for the sialylation of glycoconjugates by sialyltransferases in microbes and higher eukaryotes. CMP-neuNAc synthetase catalyzes the formation of this substrate, CMP-neuNAc, from CTP and neuNAc. In this report we describe the purification of CMP-neuNAc synthetase from bovine anterior pituitary glands. The enzyme was purified by ion exchange, gel filtration, and affinity chromatography. The protein was homogeneous on SDS-PAGE with a molecular weight of 52 kDa, a subunit size similar to that of the E.coli K1 (48.6 kDa). The identity of the 52 kDa protein band was confirmed by native gel electrophoresis in that the position of the enzyme activity in gel slices coincided with the position of major bands in the stained gel. Photoaffinity labeling with 125I-ASA-CDP ethanolamine resulted in the modification of a 52 kDa polypeptide that was partially protected against modification by the substrate CTP. Enzyme activity in crude fractions could be adsorbed onto an immunoadsorbent prepared from antibody against the purified 52 kDa protein. Taken together these data suggest that the 52 kDa polypeptide purified by this procedure described in this report is indeed CMP-neuNAc synthetase. The active enzyme chromatographed on a gel filtration column at 158 kDa suggesting it exists in its native form as an oligomer.     

On the innervation of mammalian endocrine glands (anterior pituitary and parathyroids)

Summary Electron microscopic studies have been carried out on the innervation of the mammalian anterior pituitary and parathyroids. The total area of grid squares (2.25·10^–2mm²) examined was 2000 per gland and species. In the pituitary pars distalis and in the parenchyma of the parathyroid gland we did not observe a single axon profile. According to the equation proposed by Hennig (1963) we have calculated that there might be—if any—0.133 mm of nerves per 1 mm³ tissue in those two endocrine glands (level of significance 0.95). Comparing these results to the degree of innervation in brown adipose tissue containing more than 160 mm nerve per 1 mm³ tissue we can not imagine that such a small degree of innervation is of any biological importance.In the pituitary pars tuberalis two types of axon terminals have been found both inside and outside the basement membrane surrounding the epithelial complexes. One type contains synaptic and two populations of smaller dense-cored vesicles, the other one contains a population of larger granules which have some properties of the classical elementary granules. Further investigations have to clarify the functional significance of those nerve endings.This investigation was supported by the Deutsche Forschungsgemeinschaft.     

Characterization of the mannosyl and fucosyl transferases in the ovine anterior pituitary glands.

Ovine anterior pituitary glands contain mannosyl- and fucosyl-transferases localized in the microsomes and able to incorporate mannose or fucose as such from GDP-mannose or GDP-fucose into endogenous glycoproteins. The requirements and conditions necessary for maximum activity were investigated. The value of the Km is very similar for the two enzyme systems, 3 X 10(-7) M in the case of mannosyl-transferases and 5 X 10(-7) M in the case of fucosyl-transferases.     

Expression of retinaldehyde dehydrogenase 1 in the anterior pituitary glands of adult rats

Retinoic acid (RA) plays a critical role in cell growth and tissue development and is also a regulatory factor of pituitary function. However, whether RA is generated in the pituitary gland and plays a role as a paracrine and/or autocrine factor is generally unknown. RA is synthesized from retinoids through oxidation processes. Dehydrogenases that catalyze the oxidation of retinal to RA are members of the retinaldehyde dehydrogenase (RALDH) family. Recently, we demonstrated that RALDH2 and RALDH3, but not RALDH1, were expressed in the developing anterior pituitary gland of rats, but the expression of RALDHs in the adult pituitary gland was not determined. Therefore, we have now examined the expression of RALDH1, RALDH2, and RALDH3 mRNAs in the pituitary gland of adult rats. Analysis by quantitative real-time polymerase chain reaction of adult pituitary glands has revealed a high level of RALDH1 mRNA but not of RALDH2 mRNA or RALDH3 mRNA. We have also detected mRNA expression for RALDH1 in the anterior pituitary gland by in situ hybridization with digoxigenin-labeled cRNA probes. Double-staining for RALDH1 mRNA and pituitary hormones or S-100 protein, a marker of folliculo-stellate cells (FS-cells), has revealed RALDH1 mRNA expression in a portion of prolactin-producing cells, marginal layer cells, and FS-cells. Our results suggest that RA is generated in the adult anterior pituitary gland, and that it may act locally on pituitary cells. This work was supported by a Grant-in-Aid for Young Scientists (B) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (18790149) and by the Foundation of Growth Science.