Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:4.6.1.1 (adenylate cyclase)
 19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The 7-bromoacetyl-7-desacetyl (BrAcFsk) and 7-chloroacetyl-7-desacetyl (CIAcFsk) analogs of forskolin were synthesized as alkylating agents to study the high affinity binding sites for forskolin. BrAcFsk and CIAcFsk activated adenylate cyclase in human platelet membranes with EC50 values of about 20 and 12 microM, respectively. Both analogs increased cyclic AMP in human platelets; however, they were less potent that forskolin. Forskolin inhibited [3H]forskolin binding to human platelet membranes with an IC50 of 20 nM, whereas BrAcFsk and CIAcFsk inhibited [3H] forskolin binding with IC50 values of 0.1 microM. Pretreatment of intact platelets with 10 microM BrAcFsk caused a 90% irreversible loss in [3H]forskolin binding sites, whereas pretreatment with 10 microM CIAcFsk led to a loss of 55% of the binding sites. The loss of binding sites occurred within 5 min for BrAcFsk and within 30 min for CIAcFsk. The time required for the loss of binding sites produced by either alkylating agent was increased by the inclusion of 200 microM forskolin in the pretreatment buffer. The inactive bromoacetyl analog of forskolin 7-bromoacetyl-7-desacetyl-1.9-dideoxyforskolin (1,9-dideoxy-BrAcFsk) did not activate adenylate cyclase, inhibit [3H]forskolin binding, or cause an irreversible loss of [3H]forskolin binding sites. Adenylate cyclase was assayed in membranes from platelets treated with either 10 microM BrAcFsk or 10 microM 1,9-dideoxy-BrAcFsk. The stimulation of adenylate cyclase by prostaglandin E1, guanosine-5'-O-(3-thio)triphosphate, and AIF4 was inhibited by about 50% in membranes from platelets treated with BrAcFsk. However, the stimulation of adenylate cyclase by forskolin was unaffected by preincubation with BrAcFsk. Pretreatment of human platelets with 1,9-dideoxy-BrAcFsk had no effect on the stimulation of adenylate cyclase by prostaglandin E1, AIF4, or forskolin.
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PMID:Irreversible loss of [3H]forskolin binding sites in human platelets by alpha-haloacetyl analogs of forskolin. 215 11

Many hormones elicit effects on target cells by stimulating the enzyme phospholipase-C, which catalyzes the hydrolysis of phosphoinositides to the intracellular second messengers diacylglycerol and inositol phosphates. The present study examined the roles of FSH and guanine nucleotide-binding proteins (G-proteins) in regulating the hydrolysis of phosphoinositides in Sertoli cells. Sertoli cell cultures prepared from 16- to 18-day-old rats were incubated for 24 h with myo-[2-3H] inositol to label endogenous phospholipids. Treatment of cells from 0.5-20 min with preparations of ovine FSH ranging in potency from 1-60 times that of NIH FSH S1 did not affect accumulation of inositol phosphates. Levels of total [3H]inositol phosphates [[3H]inositol mono-, di-, and triphosphates (IP, IP2, and IP3)] in FSH-treated cultures was 75-120% the levels in control cultures over the various time intervals studied. Addition of testosterone and the combination of testosterone plus retinoic acid, agents that have been shown to potentiate effects of FSH in other systems, did not affect accumulation of inositol phosphates in response to FSH. In contrast to the lack of effect on accumulation of inositol phosphates, FSH stimulated 4- to 11-fold increases in estradiol secretion over 24 h of culture, indicating that Sertoli cells were viable and responsive to FSH. AIF4- has been shown to activate G-proteins involved in regulation of adenylate cyclase activity. In the present study, AIF4- induced 4- to 5-fold increases in IP, IP2, and IP3 in experiments wherein FSH had no effect. Pretreatment of Sertoli cells with pertussis toxin (100 and 1000 ng/ml) for 24 h inhibited fluoride-induced generation of IP, IP2, and IP3 by 24-51%. Similar treatment with cholera toxin had no effect on basal or fluoride-induced generation of IP2 or IP3, but increased fluoride-induced generation of IP by 20-34%. These results suggest that phospholipase-C activity in Sertoli cells is modulated by a pertussis toxin-sensitive G-protein(s), but does not appear to be affected by FSH.
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PMID:Regulation of the phosphoinositide pathway in cultured Sertoli cells from immature rats: effects of follicle-stimulating hormone and fluoride. 313 93

Na+/H+ exchanger (NHE) activity is regulated by several types of receptors directly coupled to distinct classes (i.e. Gs, Gi, Gq, and G12) of heterotrimeric (alpha beta gamma) GTP-binding proteins (G proteins), which, upon activation, modulate production of various second messengers (e.g. cAMP, cGMP, diacylglycerol, inositol trisphosphate, and Ca2+). Recently, four isoforms of the rat Na+/H+ exchanger were identified by molecular cloning. To examine their intrinsic responsiveness to G protein and second messenger stimulation, three of these isoforms, NHE-1, -2, and -3, were stably expressed in mutant Chinese hamster ovary cells devoid of endogenous NHE activity (AP-1 cells). Incubation of cells with either AIF4-, a general agonist of G proteins, or cholera toxin, a selective activator of G alpha s that stimulates adenylate cyclase, accelerated the rates of amiloride-inhibitable 22Na+ influx mediated by NHE-1 and -2, whereas they inhibited that by NHE-3. Similarly, short term treatment with phorbol 12-myristate 13-acetate, which mimics diacylglycerol activation of protein kinase C (PKC), or with agents (i.e. forskolin, 8-(4-chlorophenylthio)-cAMP, and isobutylmethylxanthine) that lead to activation of cAMP-dependent protein kinase (PKA) also stimulated transport by NHE-1 and NHE-2 but depressed that by NHE-3. The effects of phorbol 12-myristate 13-acetate were blocked by depleting cells of PKC or by inhibiting PKC using chelerythrine chloride, confirming a role for PKC in modulating NHE isoform activities. Likewise, the PKA antagonist, H-89, attenuated the effects of elevated cAMPi on NHE-1, -2, and -3, further demonstrating the regulation by PKA. Unlike cAMPi, elevation of cGMPi by treatment with dibutyryl-cGMP or 8-bromo-cGMP had no influence on NHE isoform activities, thereby excluding the possibility of a role for cGMP-dependent protein kinase in these cells. These data support the concept that the NHE isoforms are differentially responsive to agonists of the PKA and PKC pathways.
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PMID:Plasma membrane Na+/H+ exchanger isoforms (NHE-1, -2, and -3) are differentially responsive to second messenger agonists of the protein kinase A and C pathways. 749 49

D1 and D5 dopamine receptor genes, stably expressed in GH4C1 rat somatomammotrophic cells, display identical binding values and stimulate adenylate cyclase. Approximately 60% of D1 receptors were in the agonist high-affinity state and were converted to the low-affinity state by 100 microM guanyl-5'-ylimidodiphosphate [Gpp(NH)p]. Of the 48% of D5 receptors in the high-affinity state, only half were modulated by 100 microM Gpp(NH)p; in the presence of the G protein activator, AIF4-, the high-affinity sites of D5 receptors were abolished by Gpp(NH)p, suggesting tight coupling between D5 receptors and G proteins. The high-affinity sites of D1, but not D5, receptors were reduced after pertussis toxin treatment of cells. Thus, whereas D1 receptors in GH4C1 cells couple to both Gs, the G stimulatory protein, and a pertussis toxin-sensitive G protein, D5 receptors couple to Gs and a pertussis toxin-insensitive G protein. Neither D1 nor D5 receptors were able to stimulate phosphoinositide metabolism in these cells. The ability of D5, but not D1, receptors to couple to novel G proteins may be significant in assigning a functional role for these receptors.
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PMID:Differential coupling of D1 and D5 dopamine receptors to guanine nucleotide binding proteins in transfected GH4C1 rat somatomammotrophic cells. 772 95

The effects of increases in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) on carbachol-induced generation of inositol phosphates (IPs) and increases in intracellular Ca2+ ([Ca2+]i) were investigated in canine cultured tracheal smooth muscle cells (TSMCs). The cAMP elevating agents, cholera toxin (CTX) and forskolin, induced concentration- and time-dependent cAMP formation with half-maximal effects (-logEC50) at concentrations of 7.6 +/- 1.3 g/ml and 4.8 +/- 0.9 M, respectively. Forskolin caused a concentration-dependent inhibition of carbachol-induced increase in [Ca2+]i with half-maximal inhibition (-logEC50) at 5.2 +/- 0.7 M. Pretreatment of TSMCs with either CTX (10 micrograms/ml, 4 h), forskolin (10-100 microM, 30 min), or dibutyryl cAMP (1 mM, 30 min) inhibited carbachol-stimulated Ca2+ mobilization and IPs accumulation. The inhibitory effects of these agents produced both depression of the maximal response and a shift to the right of the concentration-response curve of carbachol without changing the EC50 values. After treatment with forskolin for 24 h, carbachol-induced IPs accumulation and Ca2+ mobilization were close to those of control group. SQ-22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine, 10 microM], an inhibitor of adenylate cyclase, and HA-1004 [N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride, 50 microM], an inhibitor of cAMP-dependent protein kinase (PKA), attenuated the ability of forskolin to inhibit carbachol-induced IPs accumulation. Moreover, the inactive analogue of forskolin, 1,9-dideoxy forskolin, did not inhibit these responses evoked by carbachol, suggesting that activation of cAMP/PKA was involved in these inhibitory effects of forskolin. The KD and Bmax values of the muscarinic receptor (mAChR) for [3H]-N-methyl scopolamine binding were not significantly changed by forskolin treatment for 30 min and 24 h, suggesting that the inhibitory effect of forskolin is distal to the mAChR. The locus of this inhibition was further investigated by examining the effect of forskolin treatment on AIF4(-)-stimulated IPs accumulation in canine TSMCs. The AIF4(-)-induced response was inhibited by forskolin, supporting the notion that G protein(s) are directly activated by AIF4- and uncoupled to phospholipase C by forskolin treatment. We conclude that cAMP elevating agents inhibit carbachol-stimulated generation of IPs and Ca2+ mobilization in canine cultured TSMCs. Since generation of IPs and increases in [Ca2+]i are very early events in the activation of mAChRs, attenuation of these events by cAMP elevating agents might well contribute to the inhibitory effect of cAMP on tracheal smooth muscle formation.
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PMID:Effect of cAMP elevating agents on carbachol-induced phosphoinositide hydrolysis and calcium mobilization in cultured canine tracheal smooth muscle cells. 873 64