Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Forskolin-mediated increase in cyclic AMP and subsequent activation of protein kinase A were evaluated in SV40-immortalized human urothelial cells. This cell line is being used to evaluate the multistep carcinogenic process. Forskolin elicited a time- and dose-dependent increase in cyclic AMP. Increases in intracellular cyclic AMP preceded media increases in cyclic nucleotide. Large increases in intracellular cyclic AMP occurred within 5 min of forskolin addition. The lowest effective concentration of forskolin was between 0.1 and 1.0 microM. Cyclic AMP increases as large as 20- to 100-fold were observed in cells and media following forskolin addition. A 60 min preincubation with 12-O-tetradecanoylphorbol-13-acetate (TPA) did not reduce the magnitude of the forskolin increase in cyclic AMP. TPA has been shown to affect cyclic AMP metabolism in many types of cells including primary and secondary cultures of urothelial cells. In the latter, preincubation with TPA reduces the magnitude of the forskolin increase. A 4.2-fold increase in protein kinase A activity was observed within 0.5 min of forskolin addition, while only small increases in cyclic AMP (1.6-fold) were detected within 1 min. Much more cyclic AMP is synthesized than is needed to maximally activate protein kinase A. While results demonstrate a forskolin-responsive cyclic AMP system, this system does not appear to be regulated by TPA. Lack of regulation of this second messenger system by TPA may be part of the immortalization process.
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PMID:Cyclic AMP response in SV40 immortalized human bladder cells. 165 Feb 92

The acrosome reaction of spermatozoa appears to be analogous to various somatic cell exocytotic events which involve cascade reactions, i.e., transmission of an external signal across the cell membrane resulting in activation of an "amplifier" enzyme and the generation of a second messenger. Using a synchronous acrosome reaction system (De Jonge et al., J. Androl., 10:232-239, '89a), it was found that analogues of the second-messenger cAMP, dibutyryl cAMP (dbcAMP) and 8-bromo cAMP, stimulated the acrosome reaction of capacitated spermatozoa. Additionally, treatment of spermatozoa with either xanthine or non-xanthine phosphodiesterase inhibitors induced a significant (P less than 0.05) increase in the percent acrosome reaction after a period of capacitation in comparison to untreated controls. These results indicate that analogues of cAMP or inhibitors which prevent cAMP hydrolysis can induce the human sperm acrosome reaction. Subsequent experiments were conducted to test whether the amplifier enzyme in the cascade reaction, adenylate cyclase, has a role in the acrosome reaction. Forskolin, an adenylate cyclase stimulator, caused a significant (P less than 0.01) increase in the percent acrosome reaction in comparison to controls. Modulators of adenylate cyclase--adenosine, 2'-0-methyladenosine, and 2',3'-dideoxyadenosine--significantly (P less than 0.01) inhibited the forskolin-induced acrosome reaction. dbcAMP was able to overcome the inhibition by adenosine. Two inhibitors of protein kinase A, the Walsh inhibitor and H-8, caused a significant (P less than 0.01) inhibition of the dbcAMP-induced acrosome reaction. Finally, in the absence of extracellular calcium, dbcAMP induced a significant (P less than 0.01) increase in the acrosome reaction in contrast to A23187. These results suggest that: 1) a molecular mechanism for the human sperm acrosome reaction involves the cAMP second-messenger system; i.e., activation of adenylate cyclase, the amplifier enzyme that produces cAMP, production of cAMP as a second messenger, and activation of cAMP-dependent kinase A; and that 2) activation of adenylate cyclase occurs after calcium influx.
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PMID:Modulation of the human sperm acrosome reaction by effectors of the adenylate cyclase/cyclic AMP second-messenger pathway. 165 65

1. The characteristics have been examined of the high threshold calcium channel current in cultured rat dorsal root ganglion (DRG) neurones recorded in the presence of guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S; 200 microM in the patch pipette). This current, termed IBa, GTP gamma S, was slowly activating and showed little inactivation over 100 ms. 2. External application of forskolin (10 microM) to elevate internal cyclic AMP levels increased the amplitude of IBa, GTP gamma S whereas it had no effect on the control IBa. This cyclic AMP-dependent protein kinase (PKI; 25 microM). 3. The cyclic AMP-dependent phosphorylation induced enhancement of IBa, GTP gamma S was voltage dependent and either did not occur or was observed only transiently at a holding potential (VH) of -30 mV. The forskolin-stimulated enhancement seen at VH -80 mV was lost with a t1/2 of about 1 min when VH was depolarized to -30 mV. Cholera toxin pre-treatment also increased the amplitude of IBa, GTP gamma S at VH -80 mV but not at VH -30 mV. 4. The calcium channel antagonist (-)-202-791 (5 microM) increased the amplitude of IBa, GTP gamma S when applied at VH -80 mV, but either not, or only transiently, at VH -30 mV, as previously observed. This 'agonist' effect of (-)-202-791 was prevented by PKI and was occluded by prior enhancement of IBa, GTP gamma S with forskolin. (-)-202-791 did not increase cyclic AMP levels in DRG neurones. 5. The 'agonist' response of IBa, GTP gamma S to D600 (10 microM) was also occluded by application of forskolin (10 microM) in the patch pipette. Forskolin alone, applied in this manner, increased IBa, GTP gamma S to a similar extent to D600 applied alone. 6. The agonist effect of (+)-202-791 (5 microM) on IBa, GTP gamma S was not prevented by prior enhancement with forskolin, nor was it prevented by PKI. 7. In conclusion, internal GTP gamma S activates G proteins which may interact directly with calcium channels to influence the kinetics of activation and to reduce steady-state inactivation of the channels. There is also an indirect effect on the generation of second messengers such as cyclic AMP. It is likely that forskolin enhances IBa, GTP gamma S by increasing activated Gs coupling to adenylyl cyclase and increasing cyclic AMP generation. The mechanism of action of (-)-202-791 to enhance IBa, GTP gamma S also involves cyclic AMP-dependent phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Ca2+ channel currents in rat sensory neurones: interaction between guanine nucleotides, cyclic AMP and Ca2+ channel ligands. 165 19

1. Calcium currents (ICa) were measured in frog ventricular myocytes using the whole-cell patch clamp technique and a perfused pipette. The effect of internal perfusion with the hydrolysis-resistant GTP analogue, GppNHp (5'guanylylimidodiphosphate), on basal ICa and ICa stimulated with forskolin or isoprenaline was examined to gain insight into the role of G proteins in ICa regulation. 2. Without added guanine nucleotides, isoprenaline stimulated ICa approximately 14-fold with an EC50 of 0.09 microM. Forskolin stimulated ICa approximately 10-fold with an EC50 of 0.30 microM. 3. Internal 30 microM-GppNHp produced an approximately 80% decrease in ICa elevated by 0.3 microM-isoprenaline or 3 microM-forskolin. The inhibition of isoprenaline stimulation was due to a decrease in the maximal stimulation from approximately 14-fold to approximately 14-fold without a significant change in the EC50. In contrast, the reduction in forskolin stimulation was due to a 22-fold increase in the EC50 to 11.4 microM, with little change in maximal stimulation. 4. The inhibition of stimulated ICa by GppNHp is likely to be mediated by a G protein, because the effects of GppNHp are irreversible, and are blocked by excess GTP. ICa is affected similarly by GppNHp and by ACh. This suggests that GppNHp activates the same G protein that is normally activated by ACh, but activation by GppNHp occurs in the absence of agonist occupation of the muscarinic receptor. 5. The increase in the EC50 for forskolin produced by internal GppNHp was reversed by exposure to isoprenaline, which itself did not affect ICa amplitude. On average, exposure to isoprenaline in the presence of GppNHp caused an irreversible 81-fold decrease in the EC50 for forskolin to 0.14 microM. Stimulation of ICa by forskolin after internal GppNHp and exposure to isoprenaline was completely blocked by the protein kinase A inhibitor PKI(5-22). 6. These effects do not involve the phospholipase C system, because they are not mimicked by phorbol esters or internal inositol 1,4,5-trisphosphate (IP3) and are not blocked by bromophenacyl bromide or neomycin. 7. Direct effects of G proteins on ICa were not evident, because internal perfusion with PKI(5-22) completely inhibited isoprenaline- or forskolin-stimulated increases in ICa, and neither ACh nor internal GppNHp (30-500 microM) affected basal ICa or ICa elevated by internally perfused cyclic AMP. 8. These results suggest that the predominant site of action of the inhibitory G protein activated by either GppNHp or ACh is adenylyl cyclase. Furthermore, the internally perfused frog cardiomyocytes may provide a useful approach for probing the detailed interactions of G proteins, forskolin, and adenylyl cyclase in an intact cell.
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PMID:Regulation of Ca2+ current in frog ventricular cardiomyocytes by 5'-guanylylimidodiphosphate and acetylcholine. 165 25

1. Our objective was to evaluate the mechanism of cyclic AMP-dependent arterial smooth muscle relaxation. Cyclic AMP-dependent relaxation has been proposed to result from either (a) a decrease in intracellular [Ca2+] or (b) a decrease in [Ca2+] sensitivity of myosin light chain kinase by protein kinase A-dependent phosphorylation of myosin kinase. 2. We evaluated these proposed mechanisms by examining forskolin-induced changes in aequorin-estimated myoplasmic [Ca2+], [cyclic AMP], myosin phosphorylation and stress generation in agonist-stimulated or KCl-depolarized swine common carotid media tissues. 3. Forskolin, an activator of adenylyl cyclase, increased [cyclic AMP] and reduced [Ca2+], myosin phosphorylation and stress in tissues pre-contracted with phenylephrine or histamine. This relaxation was not associated with an alteration of the [Ca2+] sensitivity of phosphorylation, nor the dependence of stress on phosphorylation. 4. Forskolin pre-treatment attenuated, but did not abolish, agonist-induced increases in [Ca2+] and stress. 5. These results suggest that cyclic AMP-induced relaxation of the agonist-stimulated swine carotid media is primarily caused by cyclic AMP-mediated decreases in myoplasmic [Ca2+].
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PMID:Cyclic AMP relaxes swine arterial smooth muscle predominantly by decreasing cell Ca2+ concentration. 165 11

Ethanol inhibits adenosine uptake, thereby increasing the concentration of extracellular adenosine. Elevation of extracellular adenosine increases intracellular cAMP concentration via activation of adenosine A2 receptors. Extracellular adenosine is also required for the subsequent development of ethanol-induced heterologous desensitization. Here we report that activation of cAMP-dependent protein kinase is necessary for inhibition of adenosine uptake by ethanol and for the consequent accumulation of extracellular adenosine. Ethanol does not inhibit adenosine uptake in mutants of the S49 cell line that lack receptor-stimulated cAMP production (unc cells) or cAMP-dependent protein kinase activity (kin- cells). Forskolin, which bypasses the receptor-coupling defect in unc cells to increase cAMP levels, restores inhibition of adenosine uptake by ethanol. In contrast, in kin- cells forskolin did not restore inhibition of adenosine uptake by ethanol, despite similar increases in cAMP levels. Taken together, these results suggest that cAMP-dependent protein kinase phosphorylates a component of the nucleoside transporter, thereby regulating the sensitivity of adenosine transport to ethanol.
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PMID:cAMP-dependent protein kinase regulates inhibition of adenosine transport by ethanol. 165 11

We have recently reported that His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP-6) synergizes with GH-releasing factor (GRF) to increase GH release and cAMP accumulation in rat pituitary cells in vitro. This study was undertaken to further investigate the mechanism of action of GHRP-6 on GH release, particularly the involvement of protein kinase-C. Forskolin (10(-5) M), A23187 (10(-6) M), and phorbol 12-myristate 13-acetate (PMA; 10(-7) M) all stimulated GH release. However, only PMA can mimic the synergistic effects of GHRP-6 on GRF-stimulated GH release and intracellular cAMP accumulation. 4 alpha-Phorbol 12,13-didecanoate, an inactive phorbol ester, was unable to stimulate GH release or potentiate the effect of GRF. Extracellularly added phospholipase-C not only stimulated GH release in a dose-dependent manner, but also potentiated GRF-induced GH release. Phloretin, a protein kinase-C inhibitor, in a concentration range of 10-250 microM had very little or no effect on basal and GRF-stimulated GH release, but markedly inhibited the stimulatory effects induced by either PMA or GHRP-6. Incubation of rat pituitary cells with 10(-6) M PMA for 24 h completely down-regulated protein kinase-C, since such PMA-pretreated cells did not release GH in response to a second dose of PMA. The protein kinase-C-depleted cells had an attenuated GHRP-6 response, but they responded normally to GRF. Moreover, the synergistic effects of GHRP-6 and GRF on GH release and cAMP accumulation were also greatly inhibited by protein kinase-C down-regulation. These data suggest that the effects of GHRP-6 on GH release, either alone or together with GRF, are at least partially mediated via the activation of protein kinase-C.
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PMID:Evidence for a role of protein kinase-C in His-D-Trp-Ala-Trp-D-Phe-Lys-NH2-induced growth hormone release from rat primary pituitary cells. 165 29

The role of signal transduction systems was examined in the secretion of GH-releasing hormone (GHRH) and somatostatin (SS) from perifused rat hypothalamic fragments. Forskolin, an adenylate cyclase activator, stimulated the release of GHRH and SS in a concentration-dependent manner (10-100 microM) with greatest stimulation for GHRH at 100 microM (mean +/- SE, 249 +/- 14%) and for SS at 30 microM (172 +/- 18%). (Bu)2cAMP also augmented GHRH and SS release. The protein kinase-C activator phorbol 12-myristate 13-acetate did not significantly stimulate basal GHRH or SS release at concentrations of 10 nM to 1 microM. The calcium ionophore A23187 enhanced the release of GHRH and SS in a concentration-dependent manner (2-20 microM), with the greatest responses of 282 +/- 50% at 10 microM and 189 +/- 24% at 20 microM, respectively. Potentiation by phorbol 12-myristate 13-acetate of forskolin-stimulated GHRH and SS release was observed. A23187 at 10 microM did not enhance forskolin-stimulated GHRH release, but did potentiate forskolin-stimulated SS release in a more than additive response. We conclude that there is 1) cAMP stimulation of hypothalamic GHRH and SS release, 2) a modulating role of protein kinase-C on cAMP-stimulated release of GHRH and SS, 3) a stimulatory role of the calcium messenger system for GHRH and SS release, 4) interaction of the signal pathways with differences in net GHRH and SS responses, and 5) a modulatory effect of protein kinase-C in perifused hypothalamic fragments which differs from the stimulation of basal GHRH and SS release reported in fetal-derived hypothalamic cell cultures. Our observations suggest an important regulatory role of interacting signal transduction systems in the hypothalamic secretion of GHRH and SS.
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PMID:Signal transduction systems in growth hormone-releasing hormone and somatostatin release from perifused rat hypothalamic fragments. 167 98

We investigated the involvement of second messenger systems in the control by pituitary cytotropic factor (CTF) of tyrosine hydroxylase (TH) expression in primary cultures of hypothalamic cells. Forskolin, an activator of adenylyl cyclase, as well as Sp-cAMP[S] [(Sp)-cyclic adenosine 3',5'-monophosphothioate], a cAMP agonist, and theophylline, an inhibitor of phosphodiesterase activity, stimulate the secretion of dihydroxyphenylalanine (DOPA) and dopamine (DA), suggesting a role for cAMP-dependent protein kinase in the secretion of catecholamines by hypothalamic dopaminergic cells. When cells were cultured with either CTF or forskolin for 14 days, a progressive increase in the secretion of DOPA and DA was observed throughout the period of incubation. At the end of the 2-week culture period, the amount of TH in the cells, determined by immunoblot analysis, was appreciably increased compared to controls. When the cells were analyzed immunocytochemically for TH, the TH-positive cells that had been incubated with CTF or forskolin for 2 weeks were found to have neurites that appeared larger than those of TH-positive cells in the controls. The diameters of the perikarya of TH-positive cells in cultures incubated with CTF also appeared larger than the controls. After incubation of hypothalamic cells with CTF for 96 h, the amount of TH mRNA in the cultures was significantly increased. When membranes isolated from PC12 cells were incubated for 10 min with 50 microM forskolin, the specific activity of adenylyl cyclase was increased 20-fold; CTF had no effect on adenylyl cyclase activity of PC12 cell membranes. Yet, CTF significantly (P less than 0.001) stimulated the secretion of DOPA and DA by PC12 cells. When hypothalamic cells were incubated with both forskolin and CTF, using doses of each that stimulated maximal secretion, the secretion of DOPA and DA was equal to sum of the secretions with each stimulant alone. These additive actions of forskolin and CTF and the failure of CTF to activate adenylyl cyclase in membranes of PC12 cells suggest that forskolin and CTF stimulate catecholamine secretion by hypothalamic dopaminergic cells through different mechanisms, perhaps through different protein kinases. When hypothalamic cells were incubated with CTF and W-7 [N-(6-aminohexyl)5-chloro-1-naphthalenesulfonamide], an inhibitor of calmodulin, the secretion of DOPA was significantly (P less than 0.001) less than that in cultures that were not incubated with W-7. The findings of this study suggest that TH expression in hypothalamic dopaminergic cells is controlled by redundant protein kinases, including cAMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase.
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PMID:Tyrosine hydroxylase expression in hypothalamic cells: analysis of the roles of adenosine 3',5'-monophosphate- and Ca2+/calmodulin-dependent protein kinases in the action of pituitary cytotropic factor. 168 36

At the initial phase of cell differentiation in mouse neuroblastoma (N18) induced by dibutyrylcyclic AMP (dbcAMP), an additional site of histone H1 was extensively phosphorylated. Forskolin and various phosphodiesterase inhibitors also induced both cell differentiation and H1 phosphorylation at the identical site. The phosphorylation preferentially occurred in a single H1 subtype (H1c) among the five (H1a-e) fractionated by high performance liquid chromatography. The three H1 subtypes of N18 (H1c, H1d, and H1e) were phosphorylated in vitro, and their amino acid sequences of the phosphopeptides were identical to the known sequence of rabbit H1 peptides containing a serine 37 residue. However, the amount of H1a and H1b phosphorylations was negligible. The serine residue was replaced by threonine residue in H1a, and H1b did not have a homologous peptide. The tryptic phosphopeptides of H1 in N18 were identical to that in rat liver H1 induced by glucagon (Langan, T.A. (1969) Proc. Natl. Acad. Sci. USA 64, 1276-1283). The results indicate that 1) the response of H1 subtypes to cAMP-dependent protein kinase in vivo and in vitro is H1 subtype-specific, and 2) the H1c phosphorylation may play an important role in the restrictive area of chromatin in both cell differentiation and hormonal stimulation mediated by cAMP.
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PMID:Subtype-specific cyclic AMP-dependent histone H1 phosphorylation at the differentiation of mouse neuroblastoma cells. 169 Jul 30


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