Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells

A novel neuropeptide which stimulates adenylate cyclase in rat anterior pituitary cell cultures was isolated from ovine hypothalamic tissues. Its amino acid sequence was revealed as: His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln- Met-Ala- Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gln-Arg-Val-Lys-Asn-Lys - NH2. The N-terminal sequence shows 68% homology with vasoactive intestinal polypeptide (VIP) but its adenylate cyclase stimulating activity was at least 1000 times greater than that of VIP. It increased release of growth hormone (GH), prolactin (PRL), corticotropin (ACTH) and luteinizing hormone (LH) from superfused rat pituitary cells at as small a dose as 10(-10)M (GH, PRL, ACTH) or 10(-9)M (LH). Whether these hypophysiotropic effects are the primary actions of the peptide or what physiological action in the pituitary is linked with the stimulation of adenylate cyclase by this peptide remains to be determined.     

Processing of the luteinizing hormone-releasing hormone precursor in the preoptic area and hypothalamus of the rat

The LHRH precursor is known to contain the decapeptide and a 56 amino acid peptide termed gonadotropin-releasing hormone-associated peptide (GAP). The purpose of our study was to characterize the proLHRH and its processed products from the cell body and fiber region and from the nerve terminal region of LHRH neurons. The median eminence (ME) and a tissue block containing the preoptic area and hypothalamus (POH) were dissected separately. Tissues were homogenized and peptides were separated according to mol wt. Three different LHRH antisera bound to one immunoreactive (IR) substance which eluted at approximately 1200 mol wt. Subsequently, this material coeluted with synthetic LHRH on a reversed-phase column as a single peak. There was approximately 1.6-fold more LHRH-like IR in the ME than in the POH. The four different GAP antisera recognized multiple mol wt forms of GAP-like IR at approximately 16,000 to 14,000, 8,200, 6,500, 3,500, and 2,800 mol wt. There were more of the high mol wt materials and less of the 6500 and lower mol wt materials in the POH than in the ME. The most abundant species in both regions was the 6500 mol wt form. This IR substance coeluted with synthetic rat GAP1-56 on a reversed-phase column as a single peak. These experiments demonstrate 1) that multiple IR forms of the LHRH prohormone exist in the POH of the rat and 2) that nerve terminals of the LHRH neurons contain LHRH, GAP1-56, and some lower mol wt GAP-like substances. These results provide the first information concerning the processing scheme for the LHRH prohormone in the rat brain.     

Effects of ethanol on rat hypothalamic luteinizing hormone releasing hormone. A study utilizing radioimmunoassay

To further understand the mechanism of action by which ethanol (ETOH) decreases plasma luteinizing hormone (LH) levels, the effects of multiple i.p. injections of EOH (1.0--1.5 g/kg) or saline on hypothalamic luteinizing hormone releasing hormone (LHRH) and plasma LH concentrations were evaluated in intact and castrate male rats. After injections, animals were decapitated, brains rapidly removed, and blocks containing the hypothalamus [with median eminence (ME)] were isolated. Hypothalami were subjected to acetic acid extraction and LHRH content quantitated via radioimmunoassay (RIA). Hypothalamic LHRH was found to be inversely correlated with plasma LH. In response to castration, both saline and ETOH-treated rats showed a decrease in hypothalamic LHRH content with a concomitant increase in plasma LH; however, the ETOH-treated animals retained significantly greater concentrations of LHRH and showed significantly lower plasma LH levels when compared to saline-treated controls. Likewise, ETOH-treated intact animals showed significant increases in LHRH content, with LH levels remaining significantly lower than the saline-treated intact controls. Thus, these data from both intact and castrate rats provide evidence to support the hypothesis that alcohol-induced decreases in LH levels are due to a diminished release rate of hypothalamic LHRH.     

“Unblocking” Serum Activity <Emphasis Type="Italic">in vitro</Emphasis> in the Polyoma System may Correlate with Antitumour Effects of Antiserum <Emphasis Type="Italic">in vivo</Emphasis>

In a variety of tumour systems, individuals carrying progressively growing neoplasms have lymphoid cells with a specific cytotoxic effect on cultured tumour cells from the same individual^1–4. Since the sera of tumour-bearing individuals have been shown to prevent tumour cell destruction by immune lymphocytes in vitro^2,5–8 and since this serum blocking activity appears early in primary and transplant tumour development^5,7, it has been suggested that the appearance of this serum blocking activity might be responsible for the progressive growth of tumours in individuals having cytotoxic lymphocytes. Counteraction of this blocking activity would thus be of primary importance in facilitating the function of an already existing or bolstered cell-mediated immunity. The serum blocking activity might be inhibited in various ways, by preventing the formation of blocking antibody or by interfering with its action (“unblocking”), as demonstrated in Moloney sarcoma regressor sera⁹. This type of serum also has a therapeutic effect on Moloney sarcomas in vivo^10,11, which has been tentatively attributed to its unblocking activity^8,9 or, possibly, to a complement-dependent cytotoxicity¹⁰. Tumour growth in the Moloney sarcoma system, however, might be due in part to continuous recruitment of neoplastic cells by virus-induced transformation and so the therapeutic effect could be due to a virus-neutralizing serum activity^9,10.     

On the mechanism of ATP-induced shape changes in the human erythrocyte membranes: the role of ATP

In the preceding paper (Sheetz, M. and S.J. Singer. 1977. J Cell Biol. 73:638-646) it was shown that erythrocyte ghosts undergo pronounced shape changes in the presence of mg-ATP. The biochemical effects of the action of ATP are herein examined. The biochemical effects of the action of ATP are herein examined. Phosphorylation by ATP of spectrin component 2 of the erythrocyte membrane is known to occur. We have shown that it is only membrane protein that is significantly phosphorylated under the conditions where the shape changes are produced. The extent of this phosphorylation rises with increasing ATP concentration, reaching nearly 1 mol phosphoryle group per mole of component 2 at 8mM ATP. Most of this phosphorylation appears to occur at a single site on the protein molecule, according to cyanogen bromide peptide cleavage experiments. The degree of phosphorylation of component 2 is apparently also regulated by a membrane-bound protein phosphatase. This activity can be demonstrated in erythrocyte ghosts prepared from intact cells prelabeled with [(32)P]phosphate. In addition to the phosphorylation of component 2, some phosphorylation of lipids, mainly of phosphatidylinositol, is also known to occur. The ghost shape changes are, however, shown to be correlated with the degree of phosphorylation of component 2. In such experiment, the incorporation of exogenous phosphatases into ghosts reversed the shape changes produced by ATP, or by the membrane-intercalating drug chlorpromazine. The results obtained in this and the preceding paper are consistent with the proposal that the erythrocyte membrane possesses kinase and phosphates activities which produce phosphorylation and dephosphorylation of a specific site on spectrin component 2 molecules; the steady-state level of this phosphorylation regulates the structural state of the spectrin complex on the cytoplasmic surface of the membrane, which in turn exerts an important control on the shape of the cell.     

A novel hypothalamic peptide, pituitary adenylate cyclase activating peptide, modulates Sertoli cell function in vitro.

Pituitary adenylate cyclase-activating peptide (PACAP), a novel hypothalamic peptide that has been shown to exist in several tissues including the testis, was examined for its effects on cultured rat Sertoli cells. PACAP stimulates cAMP accumulation in Sertoli cells cultured from 15-day-old rats in the presence or absence of methylisobutylxanthine, a phosphodiesterase inhibitor, and in the presence of pertussis toxin, a blocker of the adenylate cyclase inhibitory pathway. Maximal stimulation, which is 20-40% of that attainable with FSH, occurs at PACAP concentrations of 10 nM: the ED50 is approximately 100 pM. The ability of PACAP to stimulate Sertoli cell cAMP declines with increasing age of donor animals (15-60 days of age) in a fashion similar to the FSH effect. PACAP stimulation of Sertoli cell cAMP accumulation is additive with submaximal, but not maximal, concentrations of FSH or forskolin. PACAP also stimulates the secretion of lactate, estradiol, and inhibin in a concentration-dependent manner. The stimulation of Sertoli cell cAMP accumulation by PACAP is not altered by a vasoactive intestinal peptide antagonist, and vasoactive intestinal peptide alone does not stimulate cAMP accumulation, indicating that PACAP is not acting via vasoactive intestinal peptide receptors. Further experiments are needed to determine whether PACAP is synthesized within the testis and if so, in which cell types; however, the present data clearly demonstrate that PACAP can modulate Sertoli cell function in vitro.     

Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone

Gold salts and phenylbutazone selectively inhibit the synthesis of PGF_2α and PGE₂ respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE₂ and PGF_2α syntheses equally. It is postulated that selective inhibitors may have a different mode of action and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.     

Selective stimulation of somatostatin receptor subtypes: differential effects on Ras/MAP kinase pathway and cell proliferation in human neuroblastoma cells

In previous studies we have showed that somatostatin (SST) inhibits cell division, mitogen-activated protein (MAP) kinase and Ras activity in the human neuroblastoma cell line SY5Y. In the present study, we have assessed the role of a series of SST analogs, three of which were selective for SSTR1, SSTR2 or SSTR5, in these cellular events. All the analogs inhibited forskolin-induced cAMP accumulation. Selective stimulation of SSTR1 or SSTR2 but not of SSTR5 inhibited platelet-derived growth factor (PDGF)-induced [(3)H]thymidine incorporation. The three analogs inhibited PDGF-stimulated MAP kinase activity, at least at an early time. In contrast, none of the analogs used individually was able to inhibit PDGF-stimulated Ras activity. A combined stimulation of SSTR2 and SSTR5 was necessary to obtain a significant inhibitory effect, suggesting the possibility of receptor heterodimerization. These results indicate that SST inhibition of Ras and MAP kinase activities takes place via different pathways and that SST inhibition of PDGF-induced cell proliferation occurs via a Ras-independent pathway.     

A web server to locate periodicities in a sequence

Summary : FT is a tool written in C++, which implements the Fourier analysis method to locate periodicities in aminoacid or DNA sequences. It is provided for free public use on a WWW server with a Java interface. Availability : The server address is http://o2.db. uoa.gr/FT Contact : shamodr@atlas.uoa.gr