Dopamine-dependent cyclic AMP generating system in chick retina and its relation to melatonin biosynthesis.

Stimulation of a D4-like dopamine (DA) receptors inhibits a cAMP-dependent increase in serotonin N-acetyltransferase activity and melatonin biosynthesis in the chick retina. In order to gain more insight into the molecular mechanisms underlying this suppressive action of DA, the effects of selective stimulation of the D2-family of DA receptors (including the D4-subtype) on cAMP formation were examined in chick retina using two experimental approaches: measurements of adenylyl cyclase activity in retinal homogenates, and cAMP accumulation in eye cup preparation prelabeled with [3H]adenine. The DA-sensitive adenylyl cyclase system is well expressed in chick retina. DA increased both basal and forskolin-stimulated adenylyl cyclase activity. This effect of DA was antagonized by SCH 23390 (a blocker of D1-family of DA receptors) and not affected by sulpiride (a D20-family blocker). Incubation of retinal homogenates with quinpirole (a predominant agonist of D3/D4 DA receptor subtypes) did not produce any major changes in adenylyl cyclase activity. On the other hand, activation of D4-like DA receptor subtype by quinpirole decreased forskolin-stimulated cAMP formation in intact chick retina maintained in "eye-cup" preparations. It is suggested that D4-like DA receptors regulating melatonin biosynthesis in chick retina may be indirectly linked to the cAMP generating system.

Adrenocorticotropic hormone activation of adenylate cyclase in raphe neurons: multiple regulatory pathways control serotonergic neuronal differentiation.

The RN46A cell line was derived from embryonic day 13 rat medullary raphe cells by infection with a retrovirus encoding the temperature-sensitive mutant of SV40 large T antigen (tsT-ag). The RN46A cell line is neuronally restricted and constitutively differentiates following a shift to nonpermissive temperature. Differentiated RN46A cells express low levels of tryptophan hydroxylase (TPH) but no detectable levels of serotonin (5-HT). Treatment of cultures with the adrenocorticotropic hormone peptide ACTH4-10 up-regulates the expression of TPH immunoreactivity in differentiated RN46A cells, but 5-HT synthesis requires initial treatment with ACTH4-10, followed by partial membrane depolarizing conditions. Up-regulation of TPH by ACTH4-10 is apparently due to activation of adenylate cyclase, whereas the increased 5-HT synthesis with membrane depolarization can be blocked with the voltage-sensitive Ca(2+)-channel blockers nifedipine and omega-conotoxin. ACTH4-10 treatment also markedly up-regulates the expression of the 5-HT reuptake transporter, as do dibutyryl cyclic AMP and forskolin; chronic membrane depolarization has no effect on 5-HT reuptake. The expression of the high-affinity 5-HT1A receptor is increased threefold by ACTH4-10 treatment during differentiation and fivefold by differentiation under partial membrane depolarizing conditions. Combining ACTH4-10 treatment and membrane depolarization does not increase expression of the 5-HT1A receptor further. 5-HT release is constitutive in ACTH-treated RN46A cells and linked to spontaneous synaptic vesicle fusion in RN46A cells. Considered with previous results, these data indicate that multiple effectors, ACTH, brain-derived neurotrophic factor, and membrane depolarization, have both distinct and overlapping effects that regulate specific elements of the serotonergic neuronal phenotype during differentiation and maturation.

Studies on the neurotoxicity of 6,7-dihydroxy-1-methyl-1,2,3,4-tetrahydroisoquinoline (salsolinol) in SH-SY5Y cells.

We have studied the hypothesis that 6,7-dihydroxy-1-methyl-1,2,3,4-tetrahydroisoquinoline (salsolinol) is neurotoxic. Salsolinol induced a significant time and dose related inhibition of 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazoyl blue (MTT) reduction, and increased lactate dehydrogenase release (LDH) release from human SH-SY5Y neuroblastoma cells, at concentrations within the range of 1-methyl-4-phenylpyridinium (MPP+) cytotoxicity, in vitro. Cytotoxicity was not inhibited by the addition of antioxidants, monoamine oxidase inhibitors or imipramine. In confluent monolayers, salsolinol stimulated catecholamine uptake with EC50 values of 17 muM and 11 muM, for noradrenaline and dopamine, respectively. Conversely, at concentrations above 100 muM, salsolinol inhibited the uptake of noradrenaline and dopamine, with IC50 values of 411 muM and 379 muM, respectively. The inhibition of catecholamine uptake corresponded to the increase displacement of [3H]nisoxetine from the uptake 1 site by salsolinol, as the Ki (353 muM) for displacement was similar to the IC50 (411 and 379 muM) for uptake. Salsolinol stimulated catecholamine uptake does not involve the uptake recognition site, or elevation of cAMP, cGMP, or inhibition of protein kinase C. Salsolinol also inhibited both carbachol (1 mM) and K+ (100 mM, Na+ adjusted) evoked released of noradrenaline from SH-SY5Y cells, with IC50 values of 500 muM and 120 muM, respectively. In conclusion, salsolinol appears to be cytotoxic to SH-SY5Y cells, via a mechanism that does not require uptake 1, bioactivation by monoamine oxidase, or membrane based free radical damage. The effects of salsolinol on catecholamine uptake, and the mechanism of toxicity require further investigation.

Tubulin stimulates adenylyl cyclase activity in rat striatal membranes via transfer of guanine nucleotide to Gs protein.

Previous studies of rat cerebral cortex and rat C6 glioma cells have demonstrated that dimeric tubulin is capable of activating the G proteins Gs and Gil via transfer of guanine nucleotide from tubulin to Gs alpha and Gil alpha. To provide further information regarding cytoskeletal modulation of adenylyl cyclase, the present study examined effects of tubulin on the activation of the enzyme in rat striatal membranes. Tubulin, prepared from rat brain by polymerization with the hydrolysis-resistant GTP analog 5'-guanylylimidodiphosphate (GppNHp) caused significant activation of adenylyl cyclase by approximately 130%. Furthermore, tubulin-GppNHp activated SKF 38393-sensitive adenylyl cyclase and potentiated forskolin-stimulated activity of the enzyme. When tubulin, polymerized with the hydrolysis-resistant photoaffinity GTP analog [32p]p3 (4-azidoanilido)-p1-5'-GTP ([32P]AAGTP), was incubated with striatal membranes, AAGTP was transferred from tubulin to Gs alpha as well as Gi alpha with the extents of nucleotide transfers being 7.6 +/- 0.8% and 17.8 +/- 1.4% of AAGTP originally bound to tubulin, respectively. These results indicate that, in rat striatum, the tubulin dimer participates in the stimulatory regulation of adenylyl cyclase by transferring guanine nucleotide to Gs alpha, supporting the hypothesis that tubulin contributes to the regulation of neuronal signal transduction.

Experimental studies of the effect of Forskolin on the lowering of intraocular pressure.

PURPOSE: To determine the effect of domestic Forskolin on lowering of intraocular pressure (IOP) in rabbits. METHODS: Measure the normal IOP and ocular hypertension by water load in rabbits using Perkins tonometer. Measure the adenylate cyclase (AC) activity by Ho & Sutherland's method. RESULTS: The results show that Forskolin significantly lowered the normal IOP of rabbits and blocked the ocular hypertension induced by water load in rabbits (P < 0.01). The maximum decrease value of 2%, 1% and 0.5% of the Forskolin was 0.59, 0.36 and 0.19 kPa (1kPa = 7.5 mmHg), which showed the noticeable dose-effect relationship. Topical ocular application of Forskolin lowered IOP in half an hour, reached to a peak in 2-3 hours and remained significantly for 10 hours. The pupillary diameter did not change when IOP was reduced. The Forskolin had potent stimulative properties to AC. The greater the ability of the Forskolin to stimulate AC, the stronger the effect of IOP lowering. CONCLUSIONS: The Forskolin had the effect on lowering the IOP of rabbits. The mechanism of IOP reduction by Forskolin is related to its AC activation.

Expression of the human beta 2-adrenoceptor in NCB20 cells results in agonist activation of adenylyl cyclase and agonist-mediated selective down-regulation of Gs alpha.

Murine neuroblastoma x embryonic Chinese hamster brain NCB20 cells were transfected with a construct containing the human beta 2-adrenoceptor under the control of a beta-actin promoter. Two clones were selected for detailed analysis: D1, which expressed some 12.7 pmol/mg of membrane protein, and L9, which expressed 1.2 pmol/mg of membrane protein of the receptor. Incubation with the beta-adrenoceptor agonist isoprenaline resulted in stimulation of adenylyl cyclase activity in both of the clones, whereas no such activation was observed in wild-type NCB20 cells. The EC50 for isoprenaline stimulation of adenylyl cyclase activity in membranes of clone D1 (0.8 nM) was significantly lower, however, than in membranes of clone L9 (10.4 nM). Although the maximal adenylyl cyclase stimulation by isoprenaline was similar in both clones, D1 had a higher basal activity. Immunoblotting studies with specific antipeptide antisera directed against various G protein alpha subunits showed that treatment of the cells with isoprenaline resulted in a 35% reduction in the membrane-associated levels of Gs alpha in membranes of clone L9 cells and a 50% reduction in Gs alpha levels in membranes prepared from clone D1. Isoprenaline treatment had no effect on the levels of Gs alpha in wild-type NCB20 cells, and such treatment had no effect on the levels of other G protein alpha subunits such as Gq/G11 and Gi2 in any of the cell lines investigated. Time course analysis revealed that half-maximal loss of Gs alpha in clone D1 was achieved within 1-2 h of addition of agonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Diazepam potentiates the positive inotropic effect of isoprenaline in rat ventricle strips: role of cyclic AMP.

The responses of the electrically driven right ventricle strip of the rat heart to isoprenaline and other cyclic AMP-related inotropic agents were recorded in the absence and in the presence of diazepam. Isoprenaline, in concentrations ranging from 10 nM to 1 microM, significantly increased, in a concentration-dependent manner, the contractile force in this preparation. Diazepam (10 microM) produced a leftward shift in the isoprenaline concentration-response curve and significantly reduced its EC50. Higher concentrations of diazepam (100 microM) produced no further shift, but reduced the maximum of the concentration-response curve of isoprenaline. Forskolin (0.5-10 microM), which directly stimulates adenyl cyclase, also produced a concentration-dependent increase in cardiac contractility. Diazepam (10 microM) displaced to the left the concentration-response curve for forskolin and reduced its EC50. The cyclic AMP analogous dibutyryl cyclic AMP (0.1-1 mM) produced concentration-dependent positive inotropic effects which were not significantly modified in the presence of diazepam (10 microM). Diazepam (10 microM) significantly enhanced the cyclic AMP production induced by isoprenaline (0.1 microM) and forskolin (10 microM) by about 136% and 35% respectively. These results indicate that diazepam potentiates the positive inotropic effect induced by beta-adrenoceptor agonists, probably by increasing cyclic AMP production induced by these agents.

Rat fibroblasts synthesize T-kininogen in response to cyclic-AMP, prostaglandin E2 and cytokines.

T-Kininogen is a plasma protein characterized as a kinin-precursor, a cysteine protease inhibitor and an acute phase protein in the rat. Rat fibroblasts prepared from meninges or embryos and 3Y1-B clone 1-6 cells, a rat fibroblast cell line, secreted T-kininogen. Incubating these cells with 1 mM Bt2cAMP or a combination with 1 microM dexamethasone resulted in a marked increase in T-kininogen secretion, as well as in the incorporation of radioactive methionine into newly synthesized T-kininogen. Secretion of T-kininogen by meningeal fibroblasts was stimulated by forskolin, prostaglandin E2, bradykinin and cytokines, such as tumor necrosis factor alpha, interleukin-1 alpha (IL-1) and IL-6. Expression of T-kininogen mRNA was demonstrated in meningeal fibroblasts by Northern blot hybridization using T-kininogen cDNA as a probe, and the expression was stimulated by Bt2cAMP, prostaglandin E2, and the cytokines described above. In contrast, expression of T-kininogen mRNA in rat hepatocytes was not altered by Bt2cAMP, prostaglandin E2, tumor necrosis factor and IL-1, whereas it was greatly stimulated by IL-6, suggesting the differential regulation of T-kininogen gene expression in fibroblasts and hepatocytes. These results demonstrated for the first time, that rat fibroblasts express the T-kininogen gene, and that the expression is regulated by inflammatory mediators and cytokines.

Effects of melanocyte-stimulating hormone on wild-type and white axolotl neural crest cells.

The goals of the current research were twofold: to study the effects of melanocyte-stimulating hormone (MSH) on undifferentiated axolotl (Ambystoma mexicanum) neural crest cells and to determine whether wild-type or white mutant axolotl neural crest cells respond differently to MSH or to either of two agonists of the MSH signal transduction pathway (cholera toxin or N6,O2-dibutyryl adenosine 3',5'-monophosphate (dbcAMP). We found that MSH induces melanophore differentiation in axolotl neural crest cells in a dose-dependent manner; however, white, but not wild-type, neural crest cells are inhibited by the highest concentration of MSH (6 x 10(-6) M) tested. The effects of cholera toxin and dbcAMP indicate that the differentiation of melanophores from white neural crest cells may be more cAMP sensitive than wild-type neural crest cells. These results suggest that MSH is likely to play a regulatory role in the initial pigment pattern formation of wild-type and white axolotls.

Increased concentrations of proteins Gi1 and Gi2 in adipocytes from aged rats alter the sensitivity of adenylyl cyclase to inhibitory and stimulatory agonists.

We used a series of antipeptide antisera to estimate the relative amounts of G proteins in adipocytes from young lean versus aged obese Sprague-Dawley rats. Western blots were analyzed using antisera that recognize (1) the alpha-subunits of Gi1 and Gi2 (serum SG1), (2) the alpha-subunit of Gi3 (serum I3B) (3) two forms of the alpha-subunit of Gs (serum CS1), and (4) forms of the beta-subunits common to all the G proteins (serum BN2). Adipocyte membranes from aged rats contained approximately fivefold to sixfold more alpha i1 and alpha i2 than those from young rats, but almost equal amounts of alpha i3. Membranes from aged rats had a modestly higher (50%) amount of a 43-kd and normal amounts of a 47-kd form of alpha s. Membranes from old rats also had approximately a threefold higher amount of beta-subunits, consistent with increased concentrations of some of the G proteins but not others. Finally, the functional consequences of these differences in G proteins was investigated by measuring the effect of N6-phenylisopropyl adenosine ([PIA] an A1-adenosine receptor agonist) and isoproterenol on adenylyl cyclase activity. Adenylyl cyclase was more sensitive to inhibition by PIA in membranes from old rats than from young rats, but was less sensitive to stimulation by isoproterenol, suggesting that the differences we observed are functionally active G proteins. These findings may account for the altered sensitivity of adipocytes from old rats to antilipolytic and lipolytic hormones.

Determinants of potency and temperature-dependent function in the Aplysia bag cell peptides.

Structure-activity relationships were determined for the natural bag cell peptides (BCPs) and for a series of synthetic analogues in terms of their ability to stimulate (at 30 degrees C) and to inhibit (at 15 degrees C) bag cell adenyl cyclase. We found that the core RLRF motif shared by all these peptides is active in this assay, and is stimulatory. The histidine residue C-terminal to this motif in beta-BCP is superfluous in this respect. An electronegative residue C-terminal to RLRF is sufficient to induce temperature-dependent function. The Ala-Pro pair that is N-terminal to this motif in alpha-BCP increases potency, but does not alter function.