Oleamide-mediated sleep induction does not depend on perturbation of membrane homeoviscosity

To verify whether the sleep-inducing properties of oleamide were related to its ability to perturb membrane homeoviscosity, affecting 5-HT(2A) receptors, we compared the effects of oleamide and oleic acid, the latter lacking both the sleep-inducing effect and the action on 5-HT(2A) receptors. In binding studies the two compounds did not directly interact with rat brain cortex 5-HT(2A) receptors, nor did they increase the affinity of a 5-HT(2A) agonist, either in vitro or ex vivo. They had similar fluidizing effects, in vitro at high concentrations (>/=10 microM), and ex vivo after a dose of 100 mg/kg, and they reduced locomotor activity with similar potency. There thus appears to be no causal relationship between the fluidizing effects of oleamide and its sleep-inducing properties.     

Regulated expression of the prolactin gene in rat pituitary tumor cells

Prolactin (PRL) gene expression in three strains of GH cells (rat pituitary tumor cells) has been quantitated by measurement of: (a) intracellular and extracellular PRL, (b) cytoplasmic translatable PRL-specific mRNA (mRNAPRL), and (c) molecular hybridization of cytoplasmic poly(A) RNA to cDNAPRL (DNA complementary to mRNAPRL). Three GH cell lines utilized in this investigation were a PRL-producing (PRL+) strain, GH4C1, a PRL nonproducing 5-bromo-deoxyuridine resistnat (PRL- BrdUrdr) strain, F1BGH12C1, and a new strain, 928-9b, derived by fusion of PRL+ cells with a nuclear monolayer of the PRL-, BrdUrdr GH cell strain. PRL production is a characteristic of 928-9b cells, but the level of PRL production (2-4 micrograms/mg protein/24 h) is much lower than that of the PRL+ strain, GH4C1 (15-25 micrograms/mg protein/24 h). Levels of cytoplasmic translatable mRNAPRL and cytoplasmic PRL-RNA sequences quantitated with a cDNAPRL probe were also much lower in 928-9b as compared to the PRL+ parent. PRL-RNA sequences could not be detected in the PRL- strain. Thyrotopin-releasing hormone (TRH) stimulates PRL synthesis about threefold and inhibit a growth hormone (GH) synthesis 72% in the PRL+ strain. TRH has no effect on the synthesis of either PRL or GH in the 928-9b strain, although TRH receptors could be detected in these cells. Stimulation of PRL synthesis in the PRL+ strain by TRH could be correlated with increases in levels of cytoplasmic translatable mRNAPRL and increases in cytoplasmic PRL-RNA sequences. These results demonstrate that the graded expression of the PRL gene at the basal level, and in response to TRH, is caused by the regulated production of specific mRNA, i.e., mRNAPRL in these three GH cell strains.     

Further evidence that prolactin does not affect gonadotropin release at pituitary level

In a primary monolayer cell culture of the anterior pituitary from mature male rats the effects of exogenous rPrl (rPrl exog.) and endogenously secreted rPrl (rPrl endog.) on basal and LHRH stimulated LH secretion were investigated. In pilot studies basal Prl- and LH secretion as well as influence of various LHRH concentrations (10(-1)-10(+3) ng/ml) on Prl- and LH release were observed. The influence of exogenous rPrl was studied at various concentrations (50-500 ng/ml) and with preincubation periods of 2 hrs and 6 hrs before starting LHRH stimulation. The dopamine agonist bromocriptine and the dopamine antagonist sulpirid were preferentially used to prove physiologic function of the cell system presented. Basal LH secretion started after a delay of 3 hrs, whereas basal Prl secretion began immediately showing a linear rise for 9 hrs. LHRH stimulation resulted in a non-linear dose and time dependent LH secretion. LHRH showed no influence on endogenous Prl (rPrl endog.) secretion of the mammotroph cells. Exogenous Prl (rPrl exog.) did not affect spontaneous Prl release excluding ultra short loop inhibition in this cell system. Furthermore, exogenous Prl had no effect on either basal or LHRH stimulated LH secretion even after a preincubation period of up to 6 hrs and at concentrations generally observed for prolactin secreting tumors. Bromocriptine suppressed endogenous Prl release and did not affect LH secretion. Sulpirid had no influence on either Prl or LH secretion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of VIP on prolactinemia in turkey anterior pituitary cells: role of cAMP second messenger in VIP-induced prolactin gene expression

Vasoactive intestinal peptide (VIP) is the avian prolactin (PRL)-releasing factor. In the turkey, hypothalamic VIP immunoreactivity and mRNA content, as well as VIP levels in hypophyseal portal blood, are closely related to the state of prolactinemia and the reproductive stage. The present study investigated the role of VIP on prolactinemia in turkey anterior pituitary (AP) cells through PRL gene expression and the role of a cAMP second messenger system on VIP-induced PRL expression. In primary AP cells harvested from hens in different prolactinemic states, steady state promoter activities were positively correlated with secreted PRL levels. VIP increased PRL promoter activities in AP cells from hens with intermediate PRL levels (laying), but not in AP cells from hypoprolactinemic hens (nonphotostimulated reproductively quiescent). However, in AP cells from hyperprolactinemic hens (incubating), PRL promoter activity was down-regulated by VIP. PRL mRNA steady state levels were significantly decreased by the cAMP analogue, 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), and PRL secretion was down-regulated by the phosphodiesterase blocker, 3-isobutyl-1-methylxanthine (IBMX) in a dose-dependent manner, suggesting that the cAMP second messenger system might be involved in the inhibitory action of dopamine upon VIP-stimulated PRL secretion and gene expression at the pituitary level. In a study of VIP immediate and long-term effects on c-fos expression in relation to PRL expression, VIP dramatically induced c-fos mRNA expression within 5 min, suggesting that VIP-induced c-fos expression might be involved in VIP-stimulated PRL secretion and gene expression. These results provide additional evidence of the functional significance of VIP in PRL gene expression and suggest that changes in PRL promoter activity by VIP may be one of the important inductive mechanisms leading to prolactinemia.     

Action of diphtheria toxin does not depend on the induction of large,stable pores across biological membranes

Summary Vero cells exposed to diphtheria toxin at pH 4.5 leak monovalent cations but not amino acids or phosphorylated metabolites; affected cells do not take up trypan blue. Monovalent cation leakage is inhibited by 1mmCd²⁺, but not by 1mmZn²⁺ or Ca²⁺. Cd²⁺ blocks calcein leakage from liposomes and closes diphtheria toxin-induced channels in lipid bilayers. It is concluded that translocation of the A fragment of diphtheria toxin across biological membranes does not depend on the formation of large stable pores, but that small Cd²⁺-sensitive pores may play a role.     

Vacuolar ethylene formation does not depend on membrane potential

Enzymatic synthesis of ethylene in the vacuole is assumed to require membrane integrity. The possibility that this reflects dependence on the vacuolar membrane potential was investigated. Vacuoles were released from protoplasts isolated from leaves of Vicia faba L. cv. Cyprus. The dependence of the ethylene-forming activity on tonoplast integrity was re-examined by immobilization of the vacuoles in a cross-linked polymeric matrix and subsequent permeabilization of the tonoplast with toluene, a pore-forming reagent. The relationship between the vacuolar ethylene formation and the membrane potential of free vacuoles was investigated by following the uptake of thiocyanate using permeabilized, depolarized and hyperpolarized vacuoles. Toluene and the proton conductor carbonyl cyanide m -chlorophenylhydrazone (CCCP) caused loss of ethylene-forming activity and depolarized the vacuolar membrane potential. However, depolarization of the membrane potential with choline chloride and hyperpolarization by ATP did not affect ethylene biosynthesis. These conflicting results lead to the conclusion that vacuolar ethylene biosynthesis is not dependent on the vacuolar membrane potential. The possibility that the inhibition of ethylene biosynthesis by toluene and CCCP may result from direct hydrophobic interactions between these compounds and hydrophobic components of the ethylene-forming enzyme is discussed.     

Irreversible platelet aggregation does not depend on lipoxygenase metabolites

Previous investigations in our laboratory demonstrated the existence of an intrinsic mechanism, termed membrane modulation, capable of restoring sensitivity to aspirin treated platelets, resulting in irreversible aggregation in response to arachidonic acid (AA). The mechanism underlying correction of aspirin induced inhibition of platelet function, however, was not clear. In the present study we have evaluated the role of lipoxygenase (LO) metabolites of AA in securing irreversible aggregation of drug induced cyclooxygenase (CO) deficient platelets. Platelets treated with aspirin or Ibuprofen did not convert radiolabeled AA to thromboxane, but generated significant quantities of hydroxy acids via the LO pathway. However, drug exposed platelets, when stirred with epinephrine first and then challenged with AA, aggregated irreversibly. Eicosatetraynoic acid (ETYA 1, U53119) inhibited AA conversion by the LO pathway, whereas 5,8,11,14-eicosatetraynoic acid (ETYA 2) inhibited AA conversion by both CO and LO enzymes. Yet, at the inhibitory concentration these fatty acids failed to prevent AA induced irreversible aggregation of CO deficient, alpha adrenergic receptor stimulated platelets. Results of four studies show that the generation of LO metabolites of AA are not essential for securing irreversible aggregation of platelets.     

Hyperglycemic effect of prolactin in anterior pituitary transplanted rats

The role of prolactin as a diabetogenic hormone was based on studies describing the ability of administered prolactin to raise the level of serum glucose. In the present study, female rats were made hyperprolactinemic by the transplantation of two anterior pituitary glands under the renal capsule, obtained from littermate donors. Chronic hyperprolactinemia increased blood glucose and impaired the glycolytic pathway of hepatic carbohydrate metabolism. However, basal serum insulin values were not different from those of controls. These data suggest that chronic hyperprolactinemia has an hyperglycemic effect and has a significant influence on glucose handling by the liver, through reduced glycolysis.     

Prolactin receptor antagonism in mouse anterior pituitary: effects on cell turnover and prolactin receptor expression

Since anterior pituitary expresses prolactin receptors, prolactin secreted by lactotropes could exert autocrine or paracrine actions on anterior pituitary cells. In fact, it has been observed that prolactin inhibits its own expression by lactotropes. Our hypothesis is that prolactin participates in the control of anterior pituitary cell turnover. In the present study, we explored the action of prolactin on proliferation and apoptosis of anterior pituitary cells and its effect on the expression of the prolactin receptor. To determine the activity of endogenous prolactin, we evaluated the effect of the competitive prolactin receptor antagonist Δ1-9-G129R-hPRL in vivo, using transgenic mice that constitutively and systemically express this antagonist. The weight of the pituitary gland and the anterior pituitary proliferation index, determined by BrdU incorporation, were higher in transgenic mice expressing the antagonist than in wild-type littermates. In addition, blockade of prolactin receptor in vitro by Δ1-9-G129R-hPRL increased proliferation and inhibited apoptosis of somatolactotrope GH3 cells and of primary cultures of male rat anterior pituitary cells, including lactotropes. These results suggest that prolactin acts as an autocrine/paracrine antiproliferative and proapoptotic factor in the anterior pituitary gland. In addition, anterior pituitary expression of the long isoform of the prolactin receptor, measured by real-time PCR, increased about 10-fold in transgenic mice expressing the prolactin receptor antagonist, whereas only a modest increase in the S3 short-isoform expression was observed. These results suggest that endogenous prolactin may regulate its own biological actions in the anterior pituitary by inhibiting the expression of the long isoform of the prolactin receptor. In conclusion, our observations suggest that prolactin is involved in the maintenance of physiological cell renewal in the anterior pituitary. Alterations in this physiological role of prolactin could contribute to pituitary tumor development.     

Chemoattractant-stimulated calcium influx in Dictyostelium discoideum does not depend on cGMP

Chemoattractant stimulation of Dictyostelium cells leads to the opening of calcium channels in the plasma membrane, causing extracellular calcium to flux into the cell. The genetically uncharacterised mutants stmF and KI8 show strongly altered chemoattractant-stimulated cGMP responses. The aberrant calcium influx in these strains has provided evidence that the chemoattractant-stimulated calcium influx is potentiated by cGMP. We have tested this hypothesis in genetically defined mutants by measuring the calcium influx in a strain that lacks intracellular cGMP due to the disruption of two guanylyl cyclases, and in a strain with increased cGMP levels caused by the disruption of two cGMP-degrading phosphodiesterases. The results reveal that the calcium influx stimulated by cAMP or folic acid is essentially identical in these strains. We conclude that cGMP is not involved in chemoattractant-stimulated calcium influx.     

Genistein-induced pituitary prolactin gene expression and prolactin release in ovariectomized ewes following a series of intracerebroventricular infusions

The aim of the study was to evaluate whether genistein, a phytoestrogen commonly present in feed plants, affects prolactin release and its gene expression in the pituitary gland. In the experimental model, genistein was infused into the third ventricle (IIIv) of the brain in ewes during the short-daylight period (November-December), when the physiological plasma level of prolactin is low. Animals were ovariectomized six weeks before the experiment, to remove the main source of endogenous estrogens, and three weeks later a stainless steel guide cannula was implanted into IIIv. Genistein (10 ng/100 microl/h, n=5) or vehicle (control, n=5) were infused in a series of four one-hour infusions at 30-min intervals (from 16:30 to 22:00). Plasma samples were collected at 15-min intervals from 14:00 to 22:00 through a catheter inserted into the jugular vein and after the experiment ewes were slaughtered. Northern blot analysis revealed that pituitary prolactin mRNA content increased significantly in response to genistein, compared to the vehicle-infused ewes (p<0.05). Prolactin concentration in plasma rose significantly during the periods of genistein infusion, as compared to the values found before infusion (p<0.05-p<0.01) as well as to the values of the concomitant periods in vehicle-infused ewes (p<0.001). Our results show an effective estrogenic action of genistein on prolactin synthesis and release in ovariectomized ewes that might in part be exerted at the central nervous system level.     

Evidence for a role of calmodulin in the regulation of prolactin gene expression

Epidermal growth factor (EGF) stimulates prolactin (PRL) gene expression in GH3 cells in a Ca2+-dependent manner (White, B. A., and Bancroft, F. C. (1983) J. Biol. Chem. 258, 4618-4622). The present report shows that the phenothiazine, calmidazolium (compound R 24571), blocks the ability of EGF plus Ca2+ to increase levels of PRL mRNA. Calmidazolium inhibition of this response is dose dependent in the range of 0.05-1.00 microM. Total inhibition of the response was consistently obtained at a level of calmidazolium (0.5 microM) that had no effect on total cytoplasmic RNA synthesis, total cytoplasmic protein synthesis, cell viability, or extent of EGF plus Ca2+-induced cell aggregation. The drug inhibited the increase in PRL mRNA when given immediately before or 48 h after treatment with EGF plus Ca2+. Another calmodulin inhibitor, W13, similarly blocked the ability of EGF plus Ca2+ to stimulate PRL mRNA, whereas the less active analog, W12, had little effect. These results implicate Ca2+-binding proteins such as calmodulin in the mechanism of action of EGF in GH3 cells, and, therefore, provide further evidence for a role of intracellular Ca2+ in the regulation of the expression of a specific eukaryotic gene, the PRL gene.