cyclic-gmp and 1-9-dideoxyforskolin

The effects of neuropeptide Y on the intracellular level of Ca2+ ([Ca2+]i) were studied in cultured rat adrenal chromaffin cells loaded with fura-2. A proportion (16%) of cells exhibited spontaneous rhythmic [Ca2+]i oscillations. In silent cells, oscillations could be induced by forskolin and 1,9-dideoxyforskolin. This action of forskolin was not modified by H-89, an inhibitor of protein kinase A. Spontaneous [Ca2+]i fluctuations and [Ca2+]i fluctuations induced by forskolin- and 1,9-dideoxyforskolin were inhibited by neuropeptide Y. Increases in [Ca2+]i induced by 10 and 20 mM KCI but not by 50 mM KCI were diminished by neuropeptide Y. However, neuropeptide Y had no effect on [Ca2+]i increases evoked by (-)BAY K8644 and the inhibitory effect of neuropeptide Y on responses induced by 20 mM KCI was not modified by omega-conotoxin GVIA, consistent with neither L- nor N-type voltage-sensitive Ca2+ channels being affected by neuropeptide Y. Rises in [Ca2+]i provoked by 10 mM tetraethylammonium were not decreased by neuropeptide Y, suggesting that K+ channel blockade reduces the effect of neuropeptide Y. However, [Ca2+]i transients induced by 1 mM tetraethylammonium and charybdotoxin were still inhibited by neuropeptide Y, as were those to 20 mM KCI in the presence of apamin. The actions of neuropeptide Y on [Ca2+]i transients provoked by 20 and 50 mM KCI, 1 mM tetraethylammonium, (-)BAY K8644 and charybdotoxin were mimicked by 8-bromo-cGMP. In contrast, 8-bromo-cAMP did not modify responses to 20 mM KCI or 1 mM tetraethylammonium. The inhibitory effects of neuropeptide Y and 8-bromo-cGMP on increases in [Ca2+]i induced by 1 mM tetraethylammonium were abolished by the Rp-8-pCPT-cGMPS, an inhibitor of protein kinase G, but not by H-89. A rapid, transient increase in cGMP level was found in rat adrenal medullary tissues stimulated with 1 microM neuropeptide Y. Rises in [Ca2+]i produced by DMPP, a nicotinic agonist, but not by muscarine, were decreased by neuropeptide Y. Our data suggest that neuropeptide Y activates a K+ conductance via a protein kinase G-dependent pathway, thereby opposing the depolarizing action of K+ channel blocking agents and the associated rise in [Ca2+]i.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adrenal Glands; Animals; Calcium Channel Agonists; Calcium Channel Blockers; Calcium Channels; Cells, Cultured; Cholinergic Agonists; Chromaffin Cells; Colforsin; Cyclic AMP; Cyclic GMP; Electrophysiology; In Vitro Techniques; Ion Channel Gating; Male; Neuropeptide Y; Potassium; Potassium Channels; Rats; Rats, Wistar

In this study, the interaction between 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) in [3H]adenine- or [3H]-guanine-prelabelled adult guinea-pig cerebellar slices was investigated. Basal levels of [3H]cGMP were enhanced by forskolin, although no plateau was reached over the concentration range tested (0.1-100 microM). However, forskolin elicited a concentration-dependent, saturable potentiation of sodium nitroprusside (SNP)-stimulated [3H]cGMP accumulation (forskolin EC50 value of 0.98 +/- 0.23 microM; 10 microM forskolin produced a 1.8 +/- 0.3-fold potentiation of the SNP response at 2.5 min). The forskolin potentiation was observed at all concentrations of SNP tested (0.001-10 mM). Forskolin also elicited a large stimulation of [3H]-cAMP in [3H]adenine-prelabelled guinea-pig cerebellar slices; however, 1,9-dideoxyforskolin failed to elicit either a [3H]cAMP response or a potentiation of the SNP-induced [3H]cGMP response at concentrations up to 100 microM. Pretreatment with oxyhaemoglobin (50 microM) inhibited the response to SNP (1 mM) and forskolin (10 microM), as well as the response evoked by the combination of SNP and forskolin. NG-Nitro-L-arginine (100 microM) inhibited the response to forskolin alone, but did not change the response to SNP or the potentiation induced by forskolin on SNP-induced [3H]cGMP levels. The protein kinase inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7; 100 microM), staurosporine (10 microM), polymyxin B (100 microM), and Ro 31-8220 (10 microM) had no effect on the [3H]cGMP response to either SNP or the combination of SNP plus forskolin. N6,2'-Dibutyryl cAMP, at concentrations up to 10 mM, was also without effect on [3H]cGMP levels induced by SNP.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 1-Methyl-3-isobutylxanthine; Animals; Bucladesine; Cerebellum; Colforsin; Cyclic AMP; Cyclic GMP; Ferrocyanides; Guinea Pigs; In Vitro Techniques; Nitric Oxide; Nitroprusside

Forskolin, a naturally occurring activator of adenylate cyclase, inhibits total and high-affinity cyclic AMP phosphodiesterase activity in soluble and particulate fractions of cultured LLC-PK1 renal epithelial cells. The naturally occurring forskolin analogue 1,9-dideoxyforskolin, which does not stimulate adenylate cyclase activity, is a more potent inhibitor of cyclic AMP phosphodiesterase activity than forskolin. To clarify the structural feature of the forskolin molecule responsible for inhibition of cyclic AMP phosphodiesterase activity, the effects of two agents which share structural identity with portions of the forskolin ring were tested. The steroid 5-pregnenolone, but not the hexose alpha-D-galactose, inhibited cyclic AMP phosphodiesterase activity in LLC-PK1 cells. Forskolin and 1,9-dideoxyforskolin both stimulate protein kinase C activity in LLC-PK1 cells. The effect of 1,9-dideoxyforskolin in stimulating LLC-PK1 protein kinase C activity can be attenuated by staurosporine. Both 5-pregnenolone and alpha-D-galactose also stimulate protein kinase C activity in LLC-PK1 cells. 5-Pregnenolone and the phorbol ester phorbol 12-myristate 13-acetate cause translocation of protein kinase C from a soluble to a particulate fraction, while both 1,9-dideoxyforskolin and alpha-D-galactose increase protein kinase C activity in both soluble and particulate fractions. Our results demonstrate that forskolin exerts diverse enzymic effects in cultured LLC-PK1 cells.

Topics: 1-Methyl-3-isobutylxanthine; 3',5'-Cyclic-AMP Phosphodiesterases; Alkaloids; Animals; Cells, Cultured; Colforsin; Cyclic GMP; Galactose; Macaca mulatta; Pregnenolone; Protein Kinase C; Staurosporine; Tetradecanoylphorbol Acetate