Cyclic AMP‐Independent Inhibition of Voltage‐Sensitive Calcium Channels by Forskolin in PC12 Cells
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Abstract: Forskolin has been used to stimulate adenylyl cyclase. However, we found that forskolin inhibited voltage‐sensitive Ca 2+ channels (VSCCs) in a cyclic AMP (cAMP)‐independent manner in PC12 cells. Ca 2+ influx induced by membrane depolarization with 70 m M K + was inhibited when cells were preincubated with 10 µ M forskolin. Almost maximum inhibitory effect on Ca 2+ influx without any significant increase in cellular cAMP level was observed in PC12 cells exposed to forskolin for 1 min. In addition, the forskolin effect on Ca 2+ influx was not affected by the presence of 2′,5′‐dideoxyadenosine, an inhibitor of adenylyl cyclase that reduces dramatically forskolin‐induced cAMP production. 1,9‐Dideoxyforskolin, an inactive analogue of forskolin, also inhibited ∼80% of Ca 2+ influx induced by 70 m M K + without any increase in cAMP. The data suggest that forskolin and its analogue inhibit VSCCs in PC12 cells and that the inhibition is independent of cAMP generation.Keywords:
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An improved affinity support for the purification of adenylyl cyclase was prepared from 7-desacetyl-7-aminoethylaminocarbonyl forskolin. This analog allows convenient synthesis of an affinity matrix that is chemically stable, with-standing repeated use for up to two years, and efficient, yielding purifications of adenylyl cyclase from solubilized bovine brain membranes of 2,000-6,000 fold in a single step. Immunoblotting data suggest that the majority of the enzyme purified in this fashion differs from forms described previously. Since the specific activity of this preparation is substantially higher than that described in previous reports, it is possible that the purification described here selects, presumably on the basis of affinity for forskolin, for a form of adenylyl cyclase with higher specific activity than any described previously.
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In previous studies it has been shown that forskolin and noradrenaline stimulate cAMP production in in vitro preparations of rat brain tissue, and that baclofen can inhibit the forskolin stimulation, and augment the noradrenaline stimulation. The purpose of the present study was to determine the pharmacological profile of this GABAb receptor mediated response, and investigate its physiological relevance by using in vivo microdialysis in awake freely moving rats, and by determining the class of cells in which the response occurs. Thus, in a cross-chopped rat cortical slice preparation, it was confirmed that (-)Baclofen inhibited forskolin stimulated adenylyl cyclase activity and augmented noradrenaline stimulated adenylyl cyclase activity. The potency of five further agonists was investigated (SKF97541, CGP47656, CG44533, 3-APA and CGP44532). Of these agonists two compounds were significantly more potent as inhibitors of forskolin stimulated adenylyl cyclase than as augmenters of noradrenaline stimulated adenylyl cyclase activity, these were (-)baclofen (pEC50 of 6.07±0.29 and 5.04±0.17 respectively (p<0.01)), and CGP47656 (pEC50=6.44±0.05 and 4.08±0.26 respectively (p<0.01)). The specificity of (-)baclofen and CGP47656 may be due to the low intrinsic activity of these compounds, and a relatively high receptor reserve for the GABAB receptor mediated inhibition of forskolin stimulated adenylyl cyclase activity. Six antagonists (CGP49311A, CGP46381, CGP45024, CGP35348, CGP45397, CGP36742) were also tested for their ability to antagonize 30 μM (-)baclofen. These ranged in potency in the forskolin assay from CGP49311A (pEC50 5.4±0.30) to CGP36742 (pEC50 3.87±0.16). Each compound had similar potency in the two assays. In vivo microdialysis was used to investigate the ability of forskolin, noradrenaline and (-)baclofen to modulate adenylyl cyclase activity in the frontal cortex of rats. Forskolin (100 μM) administered through the dialysis probe (flow rate 2 μl.min-l, 30 min fractions), caused a significant (p<0.01) elevation in cAMP in dialysates. A two pulse protocol gave a control S2/S1 ratio of 7.3±0.25. (-)Baclofen (10 μM, 100 μM, 1 mM and 10 mM) was administered through the probe during the second forskolin pulse, however, the S2/S1 ratios in the presence of (-)baclofen did not differ significantly from control (1.38±0.37, 1.27±0.52, 1.14±0.13 and 3.66±1,92 respectively). Using different conditions (flow rate 1.2 μ1 min-1, 10 min fractions) noradrenaline administered through the probe also caused a significant increase in cAMP concentration in dialysates (p<0.05), however a second pulse was ineffective. The inclusion of (-)baclofen in the perfusion medium during the first pulse increased the concentration of cAMP in dialysates, but the difference was not significant. Experiments were also conducted to determine the relative contribution made to forskolin and noradrenline stimulated adenylyl cyclase activity by glial cells and neurons. Flourocitrate, a glial cell specific metabolic inhibitor, reduced forskolin stimulated adenylyl cyclase activity by 78% and eliminated the stimulation caused by noradrenaline in cross chopped cortical slices. In cultured glial cells both forskolin and noradrenaline caused large increases in cAMP production (306 fold and 93 fold respectively). However, these responses were not modulated by 100 ?M (-)baclofen. These data suggest that most noradrenaline, and a proportion of forskolin and noradrenaline stimulated adenylyl cyclase activity may be associated with glial cells. In addition, the long term effects of GABAB receptor activation and antagonism were investigated. Thus rats were dosed with (-)baclofen and the GABAB antagonists CGP46381 and CGP36742 for three weeks ((-)baclofen, 10 mg.kg-1day-1; antagonists 100 mg.kg-1day-1, IP). No regulation of GABAB receptor function, as assayed by modulation of adenylyl cyclase activity in frontal cortex, was detected. Autoradiography detected an up regulation of radioligand receptor binding in the lateral thalamic nucleus (115% increase) in response to CGP46381, and CGP36742 caused an up regulation in the lateral dorsal thalamic nucleus (103 %), the substantia nigra (156%) and the CA3 pyramidal cell layer of the hippocampus (68%). (-)Baclofen caused a complex spectrum of up and down regulation in different brain regions which requires confirmation in a more detailed study. Genetic Absence Epilepsy Rats from Strasbourg are a strain of rats susceptible to absence seizures. An autoradiographic study of the brains of these animals revealed no differences in the levels of radioligand binding to GABAA and GABAB receptors between these animals and controls. This suggests that an alteration in these receptor populations might not underlie this genetic condition.
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Adenylyl cyclase activity can be reconstituted by simple mixture of the two cytosolic domains of the enzyme after their independent synthesis in Escherichia coli. We have synthesized and purified the C1a domain of type I adenylyl cyclase and the C2 domain of the type II enzyme to assess their interactions with each other and with the activators Gsalpha and forskolin. In the absence of an activator, the fragments associate with low affinity and display low catalytic activity. This basal activity can be stimulated more than 100-fold by either forskolin or activated Gsalpha. Further, the addition of these activators increases the apparent affinity of the fragments for each other. Stimulation of catalysis by Gsalpha and forskolin is synergistic. These data suggest a model wherein either Gsalpha or forskolin enhances association of the other activator with adenylyl cyclase, as well as facilitating the interaction between the C1 and C2 domains of the enzyme.
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Although the number of beta-adrenoceptors is unchanged with age in rat heart, both beta-adrenoceptor and postreceptor activation of adenylyl cyclase decreases with age. Pharmacologic data suggest that it is the amount of adenylyl cyclase enzyme units that limit activation of adenylyl cyclase with senescence, but direct quantitation of either G protein or adenylyl cyclase in rat heart with age is lacking. To quantitate the amount of adenylyl cyclase and G proteins with age directly, we assessed forskolin-stimulated adenylyl cyclase activity, the number of [3H] forskolin binding sites, and stimulatory G protein (Gs alpha) and inhibitory G protein (Gi alpha) immunoreactivity in the ventricles from 6- and 24-month-old F-344 rats. The amount of Gs alpha and Gi alpha was unchanged with age in both crude membranes and partially purified membranes from ventricles. In contrast, there was a 32% decrease in the ability of forskolin to stimulate adenylyl cyclase maximally and a 41% decrease in the number of forskolin binding sites with age. Sensitivity for forskolin activation was unchanged with age, but there was a slight increase in affinity for [3H]forskolin binding. The decrease in the amount of adenylyl cyclase with age correlates with the diminished capacity to activate adenylyl cyclase with age and may account for the reduced beta-adrenergic signal transduction observed in senescent rat heart.
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Abstract: Long‐term (48‐h) forskolin treatment of rat astroglial cells led to a slight decrease (30–40%) in the response to isoproterenol, vasoactive‐intestinal peptide, guanyl 5′‐(βγ‐imido)diphosphate, guanosine 5′‐ O ‐(3‐thiotriphosphate) [GTP(S)], and AIF 4 − in crude membrane fractions. In contrast, the acute stimulatory effect of forskolin was increased by 1.25–1.5‐fold. These two opposite effects of forskolin were mediated by a cyclic AMP‐dependent mechanism. No changes in G s α, G i α, or Gβ protein levels could be determined by immunoblotting using specific antisera. No significant differences were observed in the ability of G proteins extracted from control and forskolin‐treated cells to reconstitute a full adenylyl cyclase activity in membranes from S49 cyc − cells, lacking G s α protein. G s α proteins were detected in two pools of membranes, one in the heavy sucrose fractions and the other in light sucrose fractions. Forskolin treatment of the cells shifted G s α protein toward the light‐density membranes. We did not find any significant change in the distribution of adenylyl cyclase. In contrast to the decreased stimulation of adenylyl cyclase activity by agonists acting via G s α, observed in the crude membrane fraction, the responses of adenylyl cyclase to forskolin as well as to GTP(S) were increased in the purified plasma membrane fractions. These results may indicate that sensitization of the catalyst appears to be the dominant component in the astroglial cell response to long‐term treatment by forskolin.
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Forskolin (Fsk) has been demonstrated to interact directly with the enzyme adenylyl cyclase (EC 4.6.1.1) in diverse tissues. However, the ability of Fsk to bind to and activate adenylyl cyclase varies depending on the tissue being studied. Different adenylyl cyclase subtypes have been cloned and expressed in a recombinant Sf9 expression system. This provides an opportunity to study the effects of chemically reactive derivatives of Fsk on individual adenylyl cyclase subtypes in the absence of Gs alpha. Reaction of type I adenylyl cyclase with an isothiocyanate derivative of Fsk (6-[[N-(2-isothiocyanatoethyl)amino]carbonyl]forskolin) causes irreversible inhibition of Fsk binding with an IC50 of 300 nM and irreversible inhibition of Fsk activation with an IC50 of 10 microM, suggesting that there are two sites of 6-[[N-(2-isothiocyanatoethyl)amino]carbonyl]forskolin interaction. These studies establish the usefulness of the isothiocyanate derivative of Fsk in localizing the site(s) of Fsk interaction with type I adenylyl cyclase.
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Adenylyl cyclase is a membrane-bound enzyme that catalyzes the conversion of ATP to cAMP upon various hormonal stimulations. Isoform-selectivity among forskolin derivatives that forskolin and its derivatives are a direct activator of adenylyl cyclase, can be predicted mostly by the distribution of the negative electrostatic potential of each derivative. Keywords: Adenylyl cyclase, Isoform-selectivity, cAMP, Forskolin, Electrostatic potential, First principles calculations
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Forskolin potently activates all cloned mammalian adenylyl cyclases except type IX by interacting with two homologous cytoplasmic domains (C1 and C2) that form the catalytic core. A mutational analysis of the IIC2 protein (C2domain from type II adenylyl cyclase) and forskolin analogs suggests that Ser942 interacts with the 7-acetyl group of forskolin. The C1/C2 complex has only one forskolin, one ATP, and one binding site for the α subunit of the G protein that stimulates adenylyl cyclase (Gsα) and its structure may be modeled using the three-dimensional structure of (IIC2/forskolin)2. The Ser942 mutation defines which forskolin in the (IIC2/forskolin)2structure exists in C1/C2 complex. Thus, the forskolin-binding site is close to the Gsα-binding site but distal (15–20Å) from the catalytic site. Mutation from Leu912 of IIC2 protein to tyrosine or alanine severely reduces Gsα activation and completely prevents forskolin activation. The corresponding residue of Leu912 is Tyr1082 at type IX isoform of adenylyl cyclase. Similar to recombinant type IX enzyme, soluble adenylyl cyclase derived from mouse-type IX adenylyl cyclase is sensitive to Gsα activation but not to forskolin. Changing Tyr1082 to leucine makes soluble type IX adenylyl cyclase forskolin-responsive.
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