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)

The role of protein kinase A (PKA), protein kinase C (PKC), and protein phosphatases in the process of secretin stimulation of fluid and bicarbonate secretion from biliary epithelium was examined using a novel isolated bile duct unit (IBDU) model from rat liver. Sp-adenosine 3',5'-cyclic monophosphothiolate (Sp-cAMPS), 100 microM, a PKA-specific agonist, significantly increased secretion during a 30-min perfusion (+61%, P < 0.01). In contrast, preincubation and perfusion of Rp-cAMPS, 100 microM, a specific PKA inhibitor, reduced the ability of secretin to stimulate both fluid secretion (111 vs. 25%; P < 0.01) and Cl-/HCO3- exchanger activity (80 vs. 28%). Neither the PKC agonist phorbol 12-myristate 13-acetate, 10 microM, nor the PKC antagonist staurosporine showed any effect on either basal or secretin-stimulated fluid secretion or Cl-/HCO3- exchange activity in IBDU. Okadaic acid, a specific inhibitor of protein phosphatases 1 and 2A, also had no effect on basal fluid secretion or on the basal activity of the Cl-/HCO3- exchanger. However, okadaic acid resulted in persistence of secretion after removal of secretin, in contrast to the reduction in secretion observed in controls. These findings indicate that PKA but not PKC is involved in the signal transduction of secretin-stimulated fluid secretion and Cl-/HCO3- exchange activity in rat bile duct epithelium, a process inactivated by dephosphorylation by protein phosphatases 1 and/or 2A.
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PMID:Role of kinases and phosphatases in the regulation of fluid secretion and Cl-/HCO3- exchange in cholangiocytes. 927 8

There is good evidence that gallbladder epithelium is permeable to a diverse range of molecules which move into the epithelial cell from the lumen or the basement membrane. Morphological investigations have shown both secretory mucous droplets, components of the endocytosis pathway together with evidence of a system allowing passage of molecules across the basement membrane. This indicates that the gallbladder epithelium may be influenced by molecules presented via the apical and basal membranes, complicating our understanding of gallbladder function, particularly in disease. Gallbladder disease increases the proteoglycan content of the basement membrane, but the implication of this in terms of permeability remains to be defined. Indeed, it remains unknown whether this precedes disease or is a manifestation of the disease process. The removal of water from hepatic bile by gallbladder involves two counter ion transport systems. Autoradiography shows that ion transport occurs into the lateral intracellular spaces but it remains unclear whether this leads to a hypertonic solution in these spaces causing an osmotically driven water absorption or if the process involves an osmotically linked isotonic secretion. These ion pumps are reversible, for water is absorbed during the interdigestive phase but fluid is secreted into the lumen during digestion or in the presence of disease. Appropriate neural stimulation can increase or decrease fluid absorption from the lumen while vasoactive intestinal peptide or secretin promote fluid secretion, probably mediated by prostaglandins leading to raised cyclic AMP acting at the cellular level. Immediate control may depend on intracellular Ca2+ which activates a calmodulin-protein kinase, phosphorylating the counter ion transporters to downregulate their activity. Failure of this regulatory process may explain the initial increase in bile concentrating potential seen in the development of gallstones although the mechanism of such failure remains unknown. More concentrated bile increases movement of biliary compounds into gallbladder epithelial cells which alter gallbladder function in a complex manner. Secondary bile acids are raised in gallstone disease and increase permeability of the gallbladder epithelium to molecules including cholesterol. This cholesterol absorbed from the lumen may have paramount importance to gallbladder function. Raised biliary cholesterol reduces gallbladder motility, possibly by increasing the amount of cholesterol in gallbladder muscle membranes and reducing contraction in response to cholecystokinin. However, increased secondary bile acids are also associated with an alteration in phospholipid acyl groups which may alter ion transport activity and/or cholesterol solubility within the micelle/vesicle. As the acyl groups show increased arachidonate levels the production of prostaglandins could be raised, although currently it is not known if this phospholipid arachidonate enters the epithelial cells. In addition, gallbladder inflammation is associated with raised phospholipase A2 activity, leading to formation of fatty acids and lysophospholipid which causes membrane damage. The fatty acids are likely to displace cholesterol from the micelle but may also act directly on the epithelium, possibly increasing prostaglandin production and thus stimulating mucin secretion. Increased mucin secretion is seen early in gallstone disease but the evidence presently available cannot determine if this is a causative factor.
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PMID:Biochemical and morphological correlations in human gallbladder with reference to membrane permeability. 933 Mar 51

Recent evidence indicates that the N-terminal extracellular domain of receptors in the secretin-glucagon receptor family is responsible for ligand recognition. In this report, the N-terminal ectodomain of the human secretin receptor (HSR) was expressed in Escherichia coli, and the ability of this recombinant protein to interact with secretin was investigated by functional assays. The cDNA region encoding the N-terminal ectodomain of HSR linked to the polyhistidine fusion partner was expressed in E. coli. The resulting fusion protein was purified and used for competitive studies. A permanently transfected cell line with the HSR expressed was used in this study. The cell line was able to respond to secretin leading to the elevation of both intracellular cAMP and protein kinase-A activity. Using this cell line, incubation of secretin with the recombinant protein led to a dose-dependent inhibition of both cAMP production and protein kinase-A activity. These findings strongly suggested that the N-terminal ectodomain of HSR alone can act as a functional domain that provides a means to study ligand-receptor interactions of this receptor. The His-tagged recombinant HSR ectodomain may also be used for screening secretin-specific agonists and antagonists by affinity chromatography in the future.
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PMID:Functional antagonism of the human secretin receptor by a recombinant protein encoding the N-terminal ectodomain of the receptor. 944 May 1

The secretin receptor is a member of a structurally distinct class of G protein-coupled receptors designated as Class II. The molecular mechanisms of secretin receptor signal termination are unknown. Using transiently transfected HEK 293 cells expressing the secretin receptor, we investigated its mechanisms of desensitization. Binding of [125I]-secretin to plasma membranes of receptor-expressing cells was specific, with a Kd of 2 nM. Secretin evoked an increase in cellular cAMP with an EC50 of 0.4 nM. The response was maximal by 20 min and desensitized rapidly and completely. Immunoprecipitation of a functional, N-terminal epitope-tagged secretin receptor was used to demonstrate agonist-dependent receptor phosphorylation, with an EC50 of 14 nM. Pretreatment with protein kinase A or C inhibitors failed to alter secretin-stimulated cAMP accumulation. G protein-coupled receptor kinases (GRKs) are known to be involved in the desensitization of Class I G protein-coupled receptors; therefore, the effect of cotransfection of GRKs on secretin-stimulated cAMP signaling and phosphorylation was evaluated. GRKs 2 and 5 were the most potent at augmenting desensitization, causing a 40% reduction in the maximal cAMP response to secretin. GRK 5 also caused a shift in the EC50 to the right (p < 0.05). GRK 4 and GRK 6 did not alter dose-dependent signaling, and GRK 3 was intermediate in effect. Receptor phosphorylation correlated with desensitization for each GRK studied, whereas second messenger-dependent kinase phosphorylation appeared to be less important in secretin receptor signal termination. We demonstrate agonist-dependent secretin receptor phosphorylation coincident with profound receptor desensitization of the signaling function in HEK 293 cells, suggesting a role for receptor phosphorylation in this paradigm. Although GRK activity appears important in secretin receptor desensitization in HEK 293 cells, protein kinases A and C appear to play only a minor role. These results demonstrate that the GRK-arrestin system regulates Class II G protein-coupled receptors.
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PMID:A role for receptor kinases in the regulation of class II G protein-coupled receptors. Phosphorylation and desensitization of the secretin receptor. 950 76

Astrocytes, a subtype of glial cells, have been demonstrated to have an abundant number of receptors for pituitary adenylate cyclase activating polypeptide (PACAP), a neuropeptide of the VIP/secretin family which stimulates cAMP accumulation 1000 times more potent than VIP in astrocytes. PACAP is reported to stimulate the proliferation of astrocytes at low concentrations at which it does not yet stimulate the cAMP accumulation. In the present study, we examined the effect of PACAP on the activation of mitogen-activated protein kinase (MAPK), one of the important intracellular signals for the proliferation, and compared it with that of epidermal growth factor (EGF). To investigate the activation of MAPK, we focused on ERK2, one of MAPK, in cultured rat astrocytes. The activation of ERK2 was determined by immunoblotting and measurement of the activity in terms of the phosphorylating activity of immunoprecipitates with MAPK antibody on myelin basic protein. One pM of PACAP38 temporarily activated ERK2 at 10 min. In contrast, EGF activated ERK2 from 10 min to 60 min continuously. As for the dose-response effect, PACAP stimulated ERK2 at as low a concentration as 10-14 M and peaked at 10-12 M. Thereafter, its activating effect gradually decreased at 10-10 M and returned to the basal level at 10-8 M, forming a bell-shaped dose-dependency. Neither an inhibitor of PKA (H89) nor inhibitors of PKC (staurosporine and calphostin C) had any effect on the ERK2 activation induced by 1 pM PACAP38. Dibutyryl cAMP suppressed ERK2 activity in a dose-dependent manner. These data clearly demonstrated that PACAP stimulates MAPK in both a PKA- and a PKC-independent manner in cultured rat astrocytes.
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PMID:Pituitary adenylate cyclase activating polypeptide (PACAP) stimulates mitogen-activated protein kinase (MAPK) in cultured rat astrocytes. 962 27

Bicarbonate excretion in bile is a major function of the biliary epithelium. It is driven by the apically located Cl-/HCO3- exchanger which is functionally coupled with a cAMP-dependent Cl- channel (CFTR). A number of hormones and/or neuropeptides with different mechanisms and at different intracellular levels regulate, in concert, the processes underlying bicarbonate excretion in the biliary epithelium. Secretin induces a bicarbonate rich choleresis by stimulating the activity of the Cl-/HCO3- exchanger by cAMP and protein kinase A mediated phosphorylation of CFTR regulatory domain. Protein phosphatase 1/2A are involved in the run-down of secretory stimulus after secretin removal. Acetylcholine potentiates secretin-choleresis by inducing a Ca(++)-calcineurin mediated "sensitization" of adenyl cyclase to secretin. Bombesin and vasoactive intestinal peptide also enhance the Cl-/HCO3- exchanger activity, but the intracellular signal transduction pathway has not yet been defined. Somatostatin and gastrin inhibit basal and/or secretin-stimulated bicarbonate excretion by down-regulating the secretin receptor and decreasing cAMP intracellular levels induced by secretin.
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PMID:Hormonal regulation of bicarbonate secretion in the biliary epithelium. 962 62

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates catecholamine release and biosynthesis in sympathetic postganglionic cells. Moreover, PACAP receptor activation in cultured adrenal chromaffin and superior cervical ganglion cells has been reported to increase the expression of the gene coding for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. However, the relative contribution of transcriptional and posttranscriptional mechanisms to the effects of PACAP on TH gene expression has not been evaluated. Therefore, in this study we compared the temporal effects of PACAP on TH gene transcription with the duration of its effects on TH mRNA levels. We had previously shown that vasoactive intestinal polypeptide, peptide histidine isoleucine, and secretin, peptides closely related to PACAP, induce TH gene expression through a cyclic AMP (cAMP)-dependent pathway. Therefore, using a mutant PC12 cell line deficient in cAMP-dependent protein kinase II (PKA), we also evaluated the role of the cAMP pathway in the effect of PACAP on TH gene expression. Continuous treatment of wild-type PC12 cells with PACAP (1 nM) increased TH mRNA levels maximally by 12 h and maintained TH mRNA at near maximal levels for at least 2 days. In contrast, the rate of TH gene transcription, as measured by a nuclear run-on assay, was maximal by 1 h and returned to basal levels by 3 h. The fact that a new steady-state level of TH mRNA was achieved and maintained for days in the absence of a sustained increase in TH gene transcription supports the involvement of posttranscriptional mechanisms. Removal of PACAP after 12 h, a time at which TH gene transcription was at basal levels, resulted in a subsequent return of TH mRNA to unstimulated levels within 36 h. Thus, continuous PACAP stimulation is required to maintain sustained increases in TH mRNA levels in the absence of a sustained elevation of transcription. To examine the role of the cAMP pathway in these effects, we compared the effects of PACAP in wild-type PC12 cells and in a mutant PC12 cell line (A126-1B2) that is deficient in PKA. PACAP failed to stimulate either TH mRNA levels or TH gene transcription in the mutant cells. In contrast to the effects of PACAP, dexamethasone increased TH mRNA levels by the same magnitude in both cell lines. It is noteworthy that stimulation of the PKA-deficient mutant cells with a combination of PACAP and dexamethasone (1 microM) produced a synergistic increase in TH mRNA levels, which was nearly twice that induced by dexamethasone stimulation alone. This synergistic effect was not transcriptionally mediated. The effect of the combined treatment on TH gene transcription was identical to the effect of dexamethasone alone. Taken together, these data indicate that PACAP regulates TH gene expression through a transcriptional mechanism requiring an intact cAMP pathway and through posttranscriptional mechanisms under the control of a cAMP-independent pathway(s).
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PMID:Transcriptional and posttranscriptional control of tyrosine hydroxylase gene expression during persistent stimulation of pituitary adenylate cyclase-activating polypeptide receptors on PC12 cells: regulation by protein kinase A-dependent and protein kinase A-independent pathways. 968 37

Nerve fibers containing bombesin (BB)/gastrin-releasing polypeptide (GRP), pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal polypeptide (VIP), or galanin are known to innervate the mucosa of the upper small intestine. Both BB/GRP and PACAP have been shown to elicit secretin secretion in vivo. We studied whether the above-mentioned neuropeptides can act directly on secretin-producing cells, including the murine neuroendocrine cell line STC-1 and a secretin cell-enriched preparation isolated from rat upper small intestinal mucosa. Secretin release from both cell types was stimulated by various agents known to elicit secretin release and by the neuropeptides BB, GRP, and PACAP, suggesting a comparable response between the two cell preparations. The effects of neuropeptides were further studied in STC-1 cells. BB, GRP, and PACAP stimulated secretin release time and concentration dependently. VIP also stimulated secretin release concentration dependently. Stimulation by BB/GRP or PACAP was accompanied by elevation of inositol-1,4,5-trisphosphate (IP3) or cAMP, respectively. The stimulatory effect of PACAP on secretin release was synergistically enhanced by BB without any synergistic increase in IP3 or cAMP production, suggesting cross talk between different signal transduction pathways downstream of the production of these two second messengers. The L-type Ca2+ channel blocker diltiazem (10 microM) and the Ca2+ chelator EGTA (1 mM) significantly inhibited BB-stimulated secretin release by 64% and 59%, respectively, and inhibited PACAP-stimulated release by 75% and 55%, respectively. The protein kinase A-specific inhibitor Rp-cAMPS (100 microM) also inhibited both BB- and PACAP-stimulated secretin release by 30% and 62%, respectively. Galanin inhibited BB- and PACAP-stimulated secretin release and production of second messengers in a concentration-dependent and pertussis toxin-sensitive manner. These results suggested that the neuropeptides BB/GRP, PACAP, VIP, and galanin can modulate secretin release in secretin-producing cells and that STC-1 cells can serve as a useful model for studying the cellular mechanism of secretin secretion elicited by luminal secretagogues and neuropeptides.
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PMID:Modulation of secretin release by neuropeptides in secretin-producing cells. 968 45

Human secretin receptor is a G protein-coupled receptor that is functionally linked to the cAMP second messenger system by stimulation of adenylate cyclase. To functionally characterize the receptor and evaluate its signal transduction pathway, the full-length human secretin receptor cDNA was subcloned into the mammalian expression vector pRc/CMV and expressed in cultured CHO cells. Intracellular cAMP accumulation of the stably transfected cells was measured by a radioimmunoassay (RIA), while the extracellular acidification rate was measured by the Cytosensor microphysiometer. Human secretin and biotinylated human secretin were equipotent in both assays in a dose-dependent manner. The EC50 values of stimulating the intracellular cAMP accumulation and the extracellular acidification rate were 0.2-0.5 nM and 0.1 nM, respectively, indicating that microphysiometry is more sensitive than the cAMP assay in monitoring ligand stimulation of the human secretin receptor. The secretin-stimulated response could be mimicked by forskolin and augmented by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, indicating that the extracellular acidification response is positively correlated with intracellular cAMP level. The response could be abolished by the protein kinase A inhibitor H-89, suggesting that protein kinase A plays an essential role in the intracellular signaling of the receptor. Upon repeated stimulation by the ligand, the peak acidification responses did not change significantly at both physiological (0.03 nM and 3 nM) and pharmacological (0.3 microM) concentrations of human secretin, suggesting that the human secretin receptor did not exhibit robust homologous desensitization.
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PMID:Real-time evaluation of human secretin receptor activity using cytosensor microphysiometry. 1002 11

Gastric inhibitory polypeptide (GIP) is a 42-amino acid peptide, belonging to the VIP-secretin-glucagon superfamily, some members of this group are able to regulate adrenocortical function. GIP-receptor mRNA has been detected in the rat adrenal cortex, but investigations on the effect of GIP on steroid-hormone secretion in this species are lacking. Hence, we have investigated the distribution of GIP binding sites in the rat adrenal gland and the effect of their activation in vivo and in vitro. Autoradiography evidenced abundant [125I]GIP binding sites exclusively in the inner adrenocortical layers, and the computer-assisted densitometric analysis of autoradiograms demonstrated that binding was displaced by cold GIP, but not by either ACTH or the selective ACTH-receptor antagonist corticotropin-inhibiting peptide (CIP). The intraperitoneal (IP) injection of GIP dose-dependently raised corticosterone, but not aldosterone plasma concentration: the maximal effective dose (10 nmol/rat) elicited a twofold increase. GIP did not affect aldosterone and cyclic-AMP release by dispersed zona glomerulosa cells. In contrast, GIP enhanced basal corticosterone secretion and cyclic-AMP release by dispersed inner adrenocortical cells in a concentration-dependent manner, and the maximal effective concentration (10(-7) M) evoked 1.5- and 2.4-fold rises in corticosterone and cyclic-AMP production, respectively. GIP (10(-7) M) did not display any additive or potentiating effect on corticosterone and cyclic-AMP responses to submaximal or maximal effective concentrations of ACTH. The corticosterone secretagogue action of 10(-7) M GIP was abolished by the protein kinase A (PKA) inhibitor H-89 (10(-5)M), and unaffected by CIP (10(-6)M). Collectively, these findings indicate that GIP exerts a moderate but statistically significant stimulatory effect on basal glucocorticoid secretion in rats, acting through specific receptors coupled with the adenylate cyclase/PKA-dependent signaling pathway.
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PMID:Gastric inhibitory polypeptide stimulates glucocorticoid secretion in rats, acting through specific receptors coupled with the adenylate cyclase-dependent signaling pathway. 1046 10


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