Differential Regulation of Intracellular Signaling Systems by Sodium Fluoride in Rat Glioma Cells

Abstract: We investigated the rapid and slow effects of NaF on intracellular signaling systems such as Ca²⁺ homeostasis and cyclic GMP (cGMP) generation in rat glioma C6 cells, using the Ca²⁺-sensitive dye fura-2 and cGMP enzyme immunoassay. We found that the following: (a) NaF enhanced cGMP generation in a concentration-dependent manner. This enhancement was abolished by pretreatment with 100 µ M BAPTA tetraacetoxymethyl ester or in the presence of W-7 in a concentration-dependent manner. N ^G-Monomethyl- l -arginine (NMMA), a competitive inhibitor of nitric oxide synthase (NOS), also inhibited the NaF-induced generation of cGMP. These results suggest that NaF-induced cGMP generation occurs via a calcium/calmodulin- and NOS-dependent pathway. (b) The basal intracellular Ca²⁺ concentration ([Ca²⁺]_i) was transiently greater at 1 and 3 h after pretreatment with NaF. W-7 and W-13 antagonized the increase in [Ca²⁺]_i, whereas NMMA had little effect. This suggests that the NaF-induced change in basal [Ca²⁺]_i was mediated by a calmodulin-dependent pathway but was independent of a NOS-sensitive pathway. (c) The serotonin (5-HT)-induced intracellular mobilization of Ca²⁺ was reduced by pretreating the cells with NaF. The reduction in Ca²⁺ mobilization was antagonized by genistein, a tyrosine kinase inhibitor. W-7, W-5, and H-8 had no effect. Results suggest that NaF differentially regulates the cGMP generation, basal [Ca²⁺]_i, and 5-HT_2A receptor function in C6 glioma cells.     

Chronic Exposure to Adenosine Receptor Agonists and Antagonists Reciprocally Regulates the A1 Adenosine Receptor-Adenylyl Cyclase System in Cerebellar Granule Cells

Abstract: Chronic treatment with the adenosine receptor antagonist caffeine evokes an up-regulation of A₁ adenosine receptors and increased coupling of the receptor to G proteins in rat brain membranes. However, chronic agonist exposure has not been explored. Primary cultures of cerebellar granule cells were exposed chronically to A₁ adenosine receptor agonists and antagonists. Exposure to the A₁ adenosine receptor agonist N ⁶-cyclopentyladenosine resulted in (1) a time- and concentration-dependent reduction in the density of receptors labeled by 1,3-[³H]dipropyl-8-cyclopentylxanthine, (2) an enhanced ability of guanyl nucleotides to decrease the fraction of A₁ adenosine receptor sites displaying high affinity for 2-chloroadenosine, and (3) a functional uncoupling of receptors from adenylyl cyclase (EC 4.6.1.1). The adenosine antagonists caffeine and 8- p -sulfophenyltheophylline produced alterations in A₁ adenosine receptor homeostasis that were antipodal to those associated with agonist treatment. Antagonist exposure (1) increased the density of A₁ adenosine receptors in cerebellar granule cell membranes, (2) blunted the effect of guanyl nucleotides on receptor coupling to G proteins, and (3) increased the functional coupling of receptors to adenylyl cyclase inhibition. Forskolin treatment of cerebellar granule cells did not affect receptor density, suggesting that cyclic AMP is not involved in the regulation of A₁ adenosine receptor expression.     

EVOLUTIONARY POTENTIAL AND LOCAL GENETIC DIFFERENTIATION IN A PHENOTYPICALLY PLASTIC TRAIT OF A CYCLICAL PARTHENOGEN,DAPHNIA MAGNA

The frequent use of neutral markers to quantify genetic variation in natural populations emphasizes the role of stochastic events in explaining genetic differentiation between populations. Complementary studies on ecologically relevant traits are needed to assess the role of natural selection acting on adaptive variation in the development of local genetic differentiation. To test the hypothesis of local adaptation in the cyclical parthenogenetic species Daphnia magna, the phototactic behavior in the absence and presence of fish kairomone was assayed for 30 clones derived from resting eggs isolated from three habitats characterized by a different predation pressure by fish. Clones derived from populations in which fish are present were, on average, more negatively phototactic in and more responsive to the presence of fish kairomone than clones derived from a fishless habitat. In addition, the results show a high genetic diversity for the traits studied in all three gene pools investigated, indicating a high potential for microevolutionary changes in behavior of these Daphnia populations in the face of changes in predation pressure. The results of the present study indicate that working with large samples at the expense of having less precise estimates of genotypic values for specific genotypes may result in a loss of information with regard to the evolutionary potential of local populations.     

Protein Kinases Are Involved in Prolonged Acetylcholine Release from Rat Hippocampus Induced by Thyrotropin-Releasing Hormone Analogue NS-3

Abstract: The effects of various protein kinase inhibitors on acetylcholine release from the rat hippocampus induced by the local application of NS-3 (montirelin hydrate, CG-3703), a thyrotropin-releasing hormone analogue, into the medial septum-diagonal band were examined using in vivo microdialysis. Perfusion of NS-3 (1 µ M ) into the medial septum-diagonal band for 20 min produced a pronounced and prolonged increase in the hippocampal acetylcholine efflux. Pretreatment of the medial septum-diagonal band with either K-252a, a nonselective protein kinase inhibitor, or selective protein kinase A inhibitor H-89 almost completely blocked the acetylcholine efflux evoked by NS-3, and selective protein kinase C inhibitor calphostin C inhibited the action of NS-3. On the other hand, NS-3 (0.1–10 µ M ) or TRH (1–100 µ M ) increased the cyclic AMP efflux from the medial septum-diagonal band in a concentration-dependent manner, as measured by microdialysis. These findings suggest that protein kinases A and C in the neurons of the medial septum-diagonal band are involved in the mechanism of the prolonged stimulation of acetylcholine release from the hippocampus induced by thyrotropin-releasing hormone and its analogue, NS-3.     

cAMP-dependent protein phosphorylation in mitochondria of bovine heart

A study is presented of the cAMP-dependent phosphorylation in bovine heart mitochondria of three proteins of 42, 16 and 6.5 kDa associated to the inner membrane. These proteins are also phosphorylated by the cytosolic cAMP-dependent protein kinase and by the purified catalytic subunit of this enzyme. In the cytosol, proteins of 16 and 6.5 kDa are phosphorylated by the cAMP-dependent kinase. It is possible that cytosolic and mitochondrial cAMP-dependent kinases phosphorylate the same proteins in the two compartments.     

Serotonin 5-HT1A Receptor Activation Increases Cyclic AMP Formation in the Rat Hippocampus In Vivo

Abstract: In vivo microdialysis was used to examine the efflux of cyclic AMP (cAMP) into the extracellular fluid of the ventral hippocampus in the freely moving rat. The changes in extracellular cAMP concentration were monitored in response to forskolin and the serotonin 5-HT_1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The basal level of hippocampal extracellular cAMP was 2.3 ± 0.2 pmol/ml (n = 6), after a 3-h postsur- gery stabilisation period. Perfusion of forskolin (100 μM) through the probe for 30 min significantly increased the efflux of cAMP, which returned to baseline levels within 90 min. 8-OH-DPAT (0.3 mg/kg s.c.) also significantly increased cAMP efflux, whereas a similar volume of saline had no effect. Desensitisation of the 8-OH-DPAT-induced increase in cAMP efflux was observed following a second administration of 8-OH-DPAT after a 4-h interval. Administration of 8-OH-DPAT did not alter the efflux of cAMP when forskolin was perfused through the probe. Pretreatment with WAY 100135 [N-tert-butyl 3–4-(2-methoxyphenyl)piperazine-1 -yl-2-phenylpropanamide dihydrochloride] (5 mg/kg s.c.), a specific 5-HT_1A receptor antagonist, prevented the 8-OH-DPAT-induced increase in cAMP efflux. The data indicate that the 8-OH-DPAT-induced increase in cAMP efflux in vivo is mediated by a 5-HT_1A receptor.     

Stimulatory Effect of Histamine on Cyclic AMP Formation in Chick Pineal Gland

Abstract: Histamine (HA) potently stimulated cyclic AMP accumulation in intact pineal glands taken from light-exposed chicks. The action of HA was stronger in the presence of forskolin and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). The effect of HA was mimicked by HA H₁- and H₂-receptor-selective agonists in the following order of potency: HA > 4-methylhistamine (H₂) > 2-methylhistamine (H₁) > 2-thiazolylethylamine (H₁) ≫ dimaprit (H₂). The HA H₃-receptor-selective agonist (R)α-methylhistamine was poorly active. The effect of HA was antagonized by selective H₂-receptor blockers (tiotidine > oxmetidine > cimetidine = ranitidine) and was not significantly affected by the selective H₁- and H₃-receptor blockers mepyramine and thioperamide. A detailed analysis of an antagonistic action of ranitidine (versus HA) revealed a noncompetitive mode of action of the H₂ blocker. The stimulatory action of the H₁ agonist 2-thiazolylethylamine (both under basal conditions and in the presence of forskolin or IBMX) was not significantly influenced by three H₁-receptor-selective blockers (mepyramine, triprolidine, and diphenhydramine), but it was totally counteracted by ranitidine. Using accepted selective agonists and antagonists of the HA H₁, H₂, and H₃ receptor we were unable to identify clearly the receptor subtype mediating the HA action on the cyclic AMP-generating system of the chick pineal. It is suggested that the receptor under consideration may represent either an H₂-like (in terms of mammalian criteria) or avian-specific HA receptor. The data suggest that HA may be considered a modulator of the pineal activity in chicks.