The stimulatory effect of beta-endorphin on the plasma prolactin levels was diminished in the rats treated with 6-hydroxydopamine

Intraventicular injection of beta-endorphin (beta LPH_61^?91) in urethane anesthetized male rats led to a dose dependent increase of plasma prolactin levels. Intravenous injection of apomorphine completely abolished the stimulatory effect of beta-endorphin. Animals treated with 6-hydroxydopamine (6-OHDA) and 6-OHDA plus desmethylimipramine showed inhibition of beta-endorphin induced prolactin release. These results suggest that beta-endorphin presynaptically inhibits the activity of dopaminergic neurones, leading to the stimulation of plasma prolactin levels.     

P2-purinergic receptors are coupled to two signal transduction systems leading to inhibition of cAMP generation and to production of inositol trisphosphate in rat hepatocytes

Stimulation of P2-purinergic receptors by ATP resulted in activation of phosphorylase, which was associated with marked production of inositol trisphosphate (Ins-P3), in rat hepatocytes. ATP also inhibited forskolin-induced accumulation of cAMP in the presence of a phosphodiesterase inhibitor. On the contrary, adenosine or AMP never inhibited the cAMP accumulation, but increased hepatocyte cAMP; the stimulation was antagonized by a methylxanthine. Thus, P1-purinergic receptors are linked to adenylate cyclase in a stimulatory fashion in hepatocytes. Various kinds of purine nucleotides stimulating P2-receptors can be divided into two groups on the basis of their relative abilities to stimulate Ins-P3 production and to inhibit cAMP accumulation; the first group including adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), ADP, 5-adenylyl imidodiphosphate, GTP, and guanosine 5'-O-(3-thiotriphosphate) has an efficacy similar to that of ATP, and the second group of nucleotides including alpha, beta-methyleneadenosine 5'-triphosphate, beta, gamma-methyleneadenosine 5'-triphosphate (App(CH)2)p), and GDP exerts considerable inhibitory effects on cAMP accumulation, but only slight effects on inositol lipid metabolism. Treatment of hepatocytes with islet-activating protein, pertussis toxin, blocked the nucleotide-induced inhibition of cAMP accumulation, but exerted only a small effect on Ins-P3 production. In membranes prepared from hepatocytes, forskolin-stimulated adenylate cyclase was inhibited by GTP. This GTP-induced inhibition of the enzyme was susceptible to islet-activating protein and dependent on the concentration of ATP (or its derivatives, ATP gamma S or App(CH2)p). It is concluded that there are two types of P2-purinergic receptors: one is linked to adenylate cyclase via an inhibitory guanine nucleotide regulatory protein (Gi) and the other is linked to phospholipase C.     

Effect of calcium overload on the phosphoinositide breakdown in the rat left ventricular papillary muscle

We investigated the effect of Ca²⁺ overload on the phospholipase C-catalyzed hydrolysis of phosphoinositides in the rat left ventricular papillary muscle. Ca²⁺ overload on the papillary muscle was induced by treatment with 0.3 mM ouabain in Ca²⁺-containing medium following either Ca²⁺-containing or Ca²⁺-free superfusion. The phosphoinositide breakdown was evaluated by determining accumulations of [³H]inositol phosphates ([³H]IPs) in the tissues prelabeled with [³H]inositol. Ca²⁺ repletion following Ca²⁺-free superfusion resulted in a rapid but small increase in resting tension that was not followed by contracture, nor was it associated with a significant increase in [³H]IPs accumulations. Treatment with ouabain following Ca²⁺-containing superfusion increased resting tension after a lag period of several minutes and produced contracture associated with an increase in [³H]IPs accumulations. The ouabain induced increases in resting tension, and accumulations of [³H]IPs were significantly potentiated by prior Ca²⁺-free superfusion instead of Ca²⁺-containing superfusion. There was a significant positive correlation between increases in resting tension and the phosphoinositide breakdown. The increased resting tension and the accumulations of [³H]IPs were not antagonized by treatments with prazosin plus atropine or indomethacin, but were abolished by superfusion with Ca²⁺-free buffer solution. Although the enhanced phospholipase C-catalyzed hydrolysis of phosphoinositides appears to be a consequence rather than a cause of increased intracellular Ca²⁺, such a biochemical change may provoke a positive feedback mechanism to develop the muscle contracture through the putative intracellular messenger action of inositol triphosphate and diacylglycerol.Abbreviations [³H]IPs [³H]Inositol Phosphates - IP Inositol Phosphate - IP₂ Inositol Bisphosphate - IP₃ Inositol Trisphosphate - PI Phosphatidylinositol - PI-4-P Phosphatidylinositol-4-phosphate - PI-4,5-P₂ Phosphatidylinositol 4,5-bisphosphate - PRZ Prazosin - ATR Atropine - INDO Indomethacin - min Minutes     

A pertussis toxin-sensitive GTP-binding protein plays a role in the G0-G1 transition of rat hepatocytes following establishment in primary culture

Acute spontaneous c-myc gene expression and sustained increase of a GTP-binding protein(s) (G-protein) which is sensitive to islet-activating protein (IAP), pertussis toxin, occurred early during primary culture of adult rat hepatocytes. Following these earlier events, DNA synthesis was demonstrated in response to EGF and insulin. Addition of IAP immediately after plating of primary cultures inhibited c-myc expression and the hormone-induced DNA synthesis. Addition at 24 h or later following cell inoculation, however, produced only weak effects on DNA synthesis, even though the IAP-sensitive G-proteins were completely inactivated. We conclude that the IAP-sensitive G-protein(s) plays a role in the earlier process(es) of the G0-G1 transition, which is essential for the initiation of growth factor-dependent DNA synthesis.     

Effect of taurine on growth hormone and prolactin secretion in rats: possible interaction with opioid peptidergic system

The effect of taurine on growth hormone (GH) and prolactin (PRL) secretion was investigated in the urethane-alpha-chloralose anesthetized rats, considering the interaction with endogenous opioid peptidergic system. Intraventricular injection of taurine (0.25 and 1.0 mumol) stimulated GH and PRL secretion in a dose-dependent manner. However, 4.0 mumol taurine failed to show these effect. The intravenous infusion of naloxone (4 mg/kg b.w.) completely inhibited both the GH and PRL secretion induced by taurine (1.0 mumol). The combined treatment of taurine (1.0 mumol) and FK33-824 (Met-enkephalin derivative, 100 micrograms/kg b.w., i.v.) significantly increased GH and PRL responses induced by taurine or FK33-824 alone. These results indicate that taurine is an effective stimulator of GH and PRL secretion in rats, and that the mechanism of this action involves the opioid peptidergic system in the hypothalamus.     

Kinetic analysis of displacement photocurrents elicited in two types of bacteriorhodopsin model membranes.

Fast displacement photocurrents have been reported in bacteriorhodopsin model membranes by several groups of investigators since 1977. A fast component (B1) is associated with positive charge displacement in the direction opposite to that of a physiological proton translocation. A slower component (B2) of opposite polarity is associated with positive charge displacement in the same direction as the proton translocation. Using two slightly different methods for model membrane formation, we observed photosignals with or without a significant B2 component under appropriate conditions. By means of the tunable voltage clamp method of measurement (Hong, F.T., and D. Mauzerall, 1974, Proc. Natl. Acad. Sci. USA, 71:1564-1568) we demonstrated that the time course of the B1 signal is completely predictable by an equivalent circuit containing a chemical capacitance. From the equivalent circuit analysis, we obtained a first-order relaxation time constant of 12.3 +/- 0.7 microseconds at room temperature. We also found a slight temperature dependence of the B1 relaxation with an activation energy of 2.54 +/- 0.24 kcal/mol. We found no pH dependence of the B1 component in the range of 0 to 11, whereas the B2 component is diminishing in a graded manner when the pH is varied from 0 to 10. These results are diametrically different from what reported previously (Drachev, L.A., A.D. Kaulen, L.V. Khitrina, and V.P. Skulachev, 1981, Eur. J. Biochem., 117:461-470). Our results support the interpretation that the B1 component is generated by an intramolecular charge displacement accompanying the light-induced reactions of bacteriorhodopsin and that the B2 component is generated by a process of proton uptake from the intracellular aqueous phase and subsequent release into the same aqueous phase. The impact of the present results on the conventional practice of identifying photointermediates of bacteriorhodopsin by spectroscopic means is discussed.     

Inheritance of dermatoglyphic configurations in the rat

Inheritance of dermatoglyphic configurations was studied on the palmar III interdigital pad of the rat (Rattus norvegicus). Four rats from each of the WKS and ACI/N inbred strains were mated with each other, and 54 F1 and 88 F2 hybrids were obtained. In addition, 52 offspring were produced by backcross mating between F1 hybrids and WKS and ACI/N parents. Whorls were the predominant pattern in the WKS strain and triradial patterns characterized the ACI/N strains. The F1 hybrids showed a wide range of pattern types, consisting of whorls, loops, cusps, arches, and triradial patterns. These patterns were intermediate in size between the two inbred strains. The F2 hybrids presented a distribution of patterns with a similar range as the F1, but the frequencies of some types were different. Patterns in the offspring of each backcross demonstrated a slight shift towards the characteristic pattern of the parental inbred strain. No sex difference was observed. Generally, the bilateral differences were striking, with a radial direction predominating on the left palm, and an ulnar one on the right palm, respectively. This study suggests that the dermal patterns are genetically determined, but also are influenced by environmental factors, especially in the hybrids.     

Coupling of the guanine nucleotide regulatory protein to chemotactic peptide receptors in neutrophil membranes and its uncoupling by islet-activating protein, pertussis toxin. A possible role of the toxin substrate in Ca2+-mobilizing receptor-mediated signal transduction

A chemotactic peptide stimulated the high-affinity GTPase activity in membrane preparations from guinea pig neutrophils. The enzyme stimulation was inhibited by prior exposure of the membrane-donor cells to islet-activating protein (IAP), pertussis toxin, or by direct incubation of the membrane preparations with its A-protomer (the active peptide) in the presence of NAD. The affinity for the chemotactic peptide binding to its receptors was lowered by guanyl-5'-yl beta, gamma-imidodiphosphate (Gpp(NH)p) reflecting its coupling to the guanine nucleotide regulatory protein in neutrophils. The affinity in the absence of Gpp(NH)p was lower, but the affinity in its presence was not, in the A-protomer-treated membranes than in nontreated membranes. The inhibitory guanine nucleotide regulatory protein of adenylate cyclase (Ni) was purified from rat brain, and reconstituted into the membranes from IAP-treated cells. The reconstitution was very effective in increasing formyl-Met-Leu-Phe-dependent GTPase activity and increasing the chemotactic peptide binding to membranes due to affinity increase. The half-maximal concentration of IAP to inhibit GTPase activity was comparable to that of the toxin to inhibit the cellular arachidonate-releasing response which was well correlated with ADP-ribosylation of a membrane Mr = 41,000 protein (Okajima, F., and Ui, M. (1984) J. Biol. Chem. 259, 13863-13871). It is proposed that the IAP substrate, Ni, couples to the chemotactic peptide receptor and mediates arachidonate-releasing responses in neutrophils, as it mediates adenylate cyclase inhibition in many other cell types.     

Growth of the malignant and nonmalignant human squamous cells in a protein-free defined medium

Summary A novel protein-free synthetic medium has been developed for the culture of human squamous cell carcinoma cells. This medium, designated PF86-1, supports the serial subcultivation of six out of nine human squamous cell carcinoma cell lines in a protein-free, chemically defined condition without the adapting culture from serum-containing conditions. These cell lines growing in PF86-1 exhibited nearly equal potency to grow in massive culture without noticeable changes in morphology but presented a significantly decreased level of colony forming efficiency when compared with the cells cultured in serum-containing media, suggesting the implication of some autocrine mechanism. Interestingly, this medium supported the growth of normal human squamous cells of oral mucosa and skin for more than 2 mo. in the primary explant culture in spite of high levels of calcium ion concentration, where the overgrowth of fibroblasts as contaminant was not observed. These results suggest that PF86-1 supports the growth of cells derived from epidermal tissues selectively and provides the same defined condition for growth of malignant and nonmalignant human squamous cells. It seems, therefore, that PF86-1 allows investigations on the products of squamous cell carcinoma cells or on the differences of growth mechanisms between normal and neoplastic human squamous cells.     

Subunit composition of RNA polymerase II from the fission yeast Schizosaccharomyces pombe

The subunit composition of RNA polymerase II (polII) was compared between the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. For this purpose, we partially purified the enzyme from S. pombe. Judging from the co-elution profiles in column chromatographies of both the RNA polymerase activity and the two large subunit polypeptides (subunit 1 (prokaryotic β' homologue) and subunit 2 (β homologue)), the minimum number of S. pombe polII-associated polypeptides was estimated to be ten, less than the proposed subunit number of the S. cerevisiae enzyme. These ten putative subunits of S. pombe polII correspond to subunits 1, 2, 3, 5, 6, 7, 8, 10, 11 and 12 of the S. cerevisiae counterparts