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)

We measured cortisol and precursor steroid production in response to ACTH, cholera toxin, and forskolin by the dispersed adrenocortical cells prepared from the adrenal glands of 10 patients with different forms of Cushing's syndrome. The cells prepared from the hyperplastic adrenal glands from 4 patients with Cushing's disease responded in a dose-dependent manner to ACTH, cholera toxin, and forskolin. The adrenal cells prepared from 4 encapsulated adrenal adenomas showed no (n = 2), a lowered (n = 1), or a clear (n = 1) response of cortisol release to ACTH. The cells prepared from the adrenal glands of 1 patient with dysplastic micronodular adrenal glands showed a limited response to ACTH, while the cells from an adrenocortical carcinoma, which secreted very little cortisol per cell, were unresponsive to ACTH, cholera toxin, and forskolin. The reaction of the dispersed adrenal cells from these 10 patients to ACTH, cholera toxin, and forskolin showed a close correlation (P less than 0.001 in all instances). This suggests that the defect in autonomous glands is not located at the level of the ACTH receptor, but, rather, involves the adenylate cyclase complex as a whole or its coupling to cAMP-dependent protein kinase. The release into the medium of the cortisol precursors deoxycortisol, 17-hydroxyprogesterone, and progesterone showed that the four autonomous nodules were characterized by a significantly higher deoxycortisol/cortisol ratio in the medium (P less than 0.01), suggesting a relative blockade of 11 beta-hydroxylase in these adrenal adenomas. This was further substantiated in cells from several adrenals by a significant increase in the release of these precursors in response to ACTH in the absence of a cortisol response. We conclude the following. 1) Adrenal adenoma formation in patients with Cushing's syndrome is accompanied by a parallel decrease in the stimulation of the release of steroid hormones in response to ACTH, cholera toxin, and forskolin. This points to a defect in the adenoma cells beyond the ACTH receptor. 2) Adrenal adenoma formation in patients with Cushing's syndrome is accompanied by a relative blockade of 11 beta-hydroxylase activity. 3) By comparing the preoperative dynamic tests of the pituitary-adrenal axis, the plasma ACTH concentration, the morphology of the adrenal glands, and their in vitro responsiveness, a gradual transition from pituitary to (partial) adrenal autonomy could be recognized in several patients.
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PMID:Characterization of adrenal autonomy in Cushing's syndrome: a comparison between in vivo and in vitro responsiveness of the adrenal gland. 215 30

We examined the effects of removing extracellular Ca2+ (Ca2+e), depleting intracellular Ca2+ (Ca2+i), inhibiting cAMP-dependent calmodulin, and blocking voltage-sensitive Ca2+ channels on the secretion of ACTH by perifused dispersed rat anterior pituitary cells. The cells were stimulated with synthetic arginine vasopressin (AVP), oxytocin (OT), and angiotensin-II (AII), all of which are thought to act via the Ca2+/inositol phosphate-dependent protein kinase-C pathway, with synthetic ovine CRF, which acts via the cAMP-dependent protein kinase-A pathway, and with dioctanoylglycerol, which directly activates protein kinase-C. All three secretagogues elicited an initial spike phase ACTH secretory response that peaked within 1 or 2 min and ended within 6 min. AVP and OT also elicited a sustained plateau phase response that lasted for as long as the cells were exposed to the secretagogue, but AII did not. Removal of Ca2+e diminished the initial spike phase by 30-50%, but depletion of Ca2+i virtually abolished it. In contrast, the sustained phase of the response to AVP and OT was abolished by removal of Ca2+e. The effect of dioctanoylglycerol, which elicits a sustained progressive increase in ACTH release, but no initial spike phase, was also greatly inhibited by Ca2+e removal; no greater effect was observed when Ca2+i was depleted. Blockade of L-type voltage-sensitive Ca2+ channels with nimodipine, a dihydropyridine drug, had the same effect as Ca2+e removal on both the initial spike and sustained plateau phases of the response to AVP. Inhibiting cAMP-dependent calmodulin with penfluridol had no effect on the initial spike phase, but reduced the sustained plateau phase of the response to AVP. Removal of Ca2+e or depletion of Ca2+i did not abolish the synergistic ACTH secretory response to the combination of AVP and CRF.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Roles of intracellular and extracellular calcium in the kinetic profile of adrenocorticotropin secretion by perifused rat anterior pituitary cells. II. Arginine vasopressin, oxytocin, and angiotensin-II stimulation. 215 30

We have demonstrated that anterior pituitary corticotropes can be identified cytochemically by their capacity to bind potent biotinylated analogs of CRH. In addition, 50-80% of corticotropes bind biotinylated arginine vasopressin (AVP). The percentage of CRH-bound cells is rapidly reduced after 1-h exposure to glucocorticoids. However, the rapid effects of glucocorticoids on AVP binding by corticotropes have not been tested. The first aim of this study was to examine the binding capacity of small and large corticotropes enriched to 90% by counterflow centrifugation. Biotinylated analogs of CRH or AVP were detected cytochemically on the cells by avidin-biotin-peroxidase complex protocols. At least 80% of the cells bound CRH after 1 day of culture. More large corticotropes bound AVP (93%) than small corticotropes (80%). AVP pretreatment of large corticotropes stimulated an increase in CRH-bound cells to over 90%, but it had no effect on CRH binding by small corticotropes. Corticosterone pretreatment (100 nM) for 10 min caused a 50% reduction in the percentage of cells that bound CRH and in the levels of ACTH released in response to biotinylated CRH. After 30 and 60 min of pretreatment, the percentages of CRH-bound cells were reduced by 75%, and ACTH levels remained low. No reduction in percentages of AVP-bound cells was evident at any time point after corticosterone pretreatment. These studies stimulated further tests based on previous reports that showed that AVP or its activated second messengers enhanced CRH binding. We reasoned that this potentiation might promote a recovery in CRH binding to corticosterone-inhibited cells. However, 1-h stimulation by AVP or activation of calcium channels (by Bay K 8644) or protein kinase-C by 12-O-tetradecanoyl-phorbol-13-acetate did not restore CRH binding. AVP evoked a partial recovery in ACTH release. Furthermore, Bay K and 12-O-tetradecanoyl-phorbol-13-acetate pretreatment effectively blocked the fast feedback effects of corticosterone on CRH-mediated ACTH release. Thus, these studies demonstrate that glucocorticoids rapidly inhibit CRH-receptor binding in a domain that is not affected by AVP potentiation of ACTH release. Perhaps they immobilize transport processes needed to bring unoccupied CRH receptors to the surface for binding and cytochemical detection.
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PMID:Rapid corticosterone inhibition of corticotropin-releasing hormone binding and adrenocorticotropin release by enriched populations of corticotropes: counteractions by arginine vasopressin and its second messengers. 215 74

We studied the mechanism that underlies the desensitization of calf adrenal glomerulosa cells induced by 4 h of ACTH treatment. In control cells, acute ACTH treatment provoked sizeable increases in aldosterone, cAMP, and diacylglycerol, and translocated protein kinase-C from cytosol to membrane. In desensitized cells, acute ACTH effects on aldosterone and cAMP decreased by 25-60%, and diacylglycerol levels and protein kinase-C translocation were persistently stimulated and not substantially affected by further acute ACTH treatment. After 4 h of treatment with 1 microM phorbol 12-myristate 13-acetate (PMA) there were no acute effects of ACTH on the production of aldosterone, cAMP, or diacylglycerol or on protein kinase-C, which was already strongly translocated. These results suggest that ACTH-mediated desensitization of calf adrenal glomerulosa cells may be at least partially mimicked by long term treatment with phorbol esters and could be due to ACTH-induced increases in diacylglycerol-protein kinase-C signaling.
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PMID:Treatment of primary cultures of calf adrenal glomerulosa cells with adrenocorticotropin (ACTH) and phorbol esters: a comparative study of the effects on aldosterone production and ACTH signaling system. 215 84

The site of action of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) in ACTH-induced stimulation of steroidogenesis was examined in rat adrenocortical fasciculata cells. Prior addition of AA861, a specific inhibitor of 5-lipoxygenase, had no significant effect on cyclic AMP-dependent protein kinase activity and cholesterol esterase activities, when stimulated by ACTH in adrenocortical cells, compared with that stimulated by ACTH alone. Cholesterol accumulation in the mitochondria of cells treated with ACTH and cycloheximide was also not altered by pretreatment with AA861. We found, however, that pregnenolone formation, stimulated by ACTH, decreased in a dose-dependent manner when cells were pretreated with AA861. The inhibition of ACTH-stimulated pregnenolone formation by treatment with AA861 was restored only by prior addition of 5-HPETE. Furthermore, addition of AA861 also did not affect the conversion of pregnenolone into corticosterone. In conclusion, 5-HPETE may act at the level of the mitochondria in ACTH-induced steroidogenesis in rat adrenal fasciculata cells.
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PMID:Possible site of action of 5-hydroperoxyeicosatetraenoic acid derived from arachidonic acid in ACTH-stimulated steroidogenesis in rat adrenal glands. 215 71

To further characterize the subcellular mechanisms by which inhibin suppresses GnRH-stimulated gonadotropin release, anterior pituitary cells from adult male Sprague-Dawley rats were treated on day 2 of culture with or without purified 31-kDa bovine inhibin (1-300 pM) for a further 3 days. On day 5, the pretreated cells were washed and incubated in the absence or presence of various secretagogues for 4 h. At the end of the stimulation, the media were saved, and cells were lysed for measurement of both extracellular and intracellular FSH and LH by specific RIAs. Released hormone was expressed as the proportion of total (released plus intracellular) hormone that was available for release in each case. This manipulation of the data corrects for the differential effect of the inhibin pretreatments to suppress intracellular FSH before the stimulation period. Pretreatment for 3 days with inhibin suppressed the proportions of FSH and LH released during 4 h in response to 1) phorbol 12-myristate 13-acetate (100 nM), an activator of protein kinase-C, by maxima of 48% and 53% with inhibin median inhibitory concentrations (IC50) of 17 and 18 pM, respectively; 2) mezerein (100 nM), another type of activator of protein kinase-C, by maxima of 49% and 50% with inhibin IC50 of 19 and 20 pM, respectively; 3) high extracellular K+ (60 mM) by 42% (P less than 0.01) and 38% (P less than 0.01), respectively, with 130 pM inhibin; 4) the calcium ionophore, A23187 (100 microM) by maxima of 54% and 56% with IC50 of 18 and 17 pM, respectively; and 5) GnRH (10 nM) by maxima of 52% and 53% with IC50 of 18 and 19 pM, respectively. However, inhibin had no effect on the proportional release of gonadotropin induced by melittin, an activator of phospholipase-A2. Finally, inhibin had no effect on ACTH release either under basal conditions or in response to CRF (10 nM), phorbol 12-myristate 13-acetate (100 nM), or A23187 (100 microM). We conclude that inhibin suppresses the stimulated release of hormones from gonadotrophs in part by a mechanism common to both gonadotropins that is independent of the previously described inhibitory effect of inhibin on the GnRH receptor. The results are consistent with an action at a site(s) beyond the GnRH receptor, such as protein kinase-C and calmodulin.
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PMID:Effect of inhibin on activators of protein kinase-C and calcium-mobilizing agents which stimulate secretion of gonadotropins in vitro: implication of a postgonadotropin-releasing hormone receptor effect of inhibin on gonadotropin release. 216 51

Arginine vasopressin (AVP) stimulates biphasic release of ACTH from anterior pituitary corticotrophs. The response consists of an initial transient spike phase lasting less than 3 min and a subsequent sustained plateau phase that persists for as long as AVP is present. AVP also acts synergistically with CRF on ACTH release. We have previously shown that the initial spike phase of the response mainly requires release of intracellular Ca2+ and is independent of calmodulin, whereas the sustained plateau phase, like the monophasic sustained response elicited by CRF, involves the influx of extracellular Ca2+ via L-type voltage-sensitive Ca2+ channels and activation of calmodulin. We have also shown that the synergism between AVP and CRF does not require extracellular Ca2+ influx. In this study we examined the role of Ca2+/phospholipid-dependent protein kinase-C (PKC) in the two phases of the response to AVP and in the synergism between AVP and CRF. We exploited the observation that prolonged exposure to phorbol esters down-regulates PKC. Dispersed adult male rat anterior pituitary cells were incubated in static suspension culture for 4-5 days, 0.5 microM phorbol 12-myristate 13-acetate (PMA) or 0.0005% dimethylsulfoxide vehicle alone was added, and the incubation was continued for 24 h. The cells were preperifused with PMA-free perifusion medium for 3 h and then perifused with various agents for 10-20 min. Effluent fractions were collected every 30 sec or 1 min and subjected to ACTH RIA. Pretreatment with PMA inhibited the subsequent response to 100 nM PMA and 100 microM dioctanoylglycerol, but not to 5 microM forskolin or to depolarization with 56 mM KCl, demonstrating specific down-regulation of PKC. PMA pretreatment had no effect on the initial spike phase of the response to AVP, but inhibited the sustained plateau phase by 57% (P less than 0.005) and, consequently, the integrated total response by 33% (P less than 0.05). Pretreatment had no effect on the response to CRF. However, pretreatment with PMA completely blocked both phases of the synergistic response to the combination of AVP and CRF. These results indicate that activation of PKC is required for the sustained phase of the response to AVP and both phases of its synergistic interaction with the protein kinase-A pathway, but is not involved in the initial spike phase of the response to AVP, which presumably is mediated by inositol 1,4,5-trisphosphate-stimulated mobilization of intracellular Ca2+, or in the independent activation of the protein kinase-A pathway by CRF.
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PMID:Role of protein kinase-C in the adrenocorticotropin secretory response to arginine vasopressin (AVP) and the synergistic response to AVP and corticotropin-releasing factor by perifused rat anterior pituitary cells. 216 16

This study was undertaken to define the roles of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) in the regulation of adrenocorticotropin (ACTH) release and biosynthesis in cultured ovine anterior pituitary cells and to define the intracellular mechanisms responsible for their action. At 4 h, CRF and AVP increased both ACTH release and total ACTH content, with AVP clearly the more potent agonist (maximal ACTH release: AVP, 22.8-fold; CRF, 7.6-fold; maximal increment in total ACTH content: AVP, 1.9-fold; CRF, 1.1-fold; EC50 for ACTH release: AVP, 2.3 +/- 0.5 nM; CRF, 9.2 +/- 5.0 nM). The increase in total ACTH content was interpreted to reflect an augmentation of ACTH biosynthesis since it was abolished by 10 microM cycloheximide. Exposure of the anterior pituitary cells to increasing concentrations of forskolin or 8-bromo-cAMP elicited increases in ACTH release and total ACTH content that were similar to those caused by CRF. A 30-min incubation with phorbol 12-myristate 13-acetate (PMA) caused a dose-related translocation of protein kinase C from the cytosol to the cell membrane; after 4 h, the increases in ACTH release and total ACTH content in response to increasing concentrations of PMA were similar to those caused by AVP. Chronic (24 h) exposure to 150 nM PMA caused an almost total depletion of both cytosolic and membrane-bound protein kinase C activities. When protein kinase C-depleted cells were subsequently exposed to AVP, the increases in ACTH release and total ACTH content were markedly attenuated, but the responses to CRF were preserved. Finally, the combination of CRF and AVP, CRF and PMA, or AVP and 8-bromo-cAMP increased ACTH release and total ACTH content in a synergistic manner. We conclude that: 1) in ovine anterior pituitary cells, AVP is the predominant regulator of ACTH secretion and biosynthesis; 2) the action of AVP is predominantly mediated by activation of protein kinase C, whereas the action of CRF is likely to be mediated by activation of the cAMP-dependent protein kinase (protein kinase A); and 3) the ability of CRF and AVP to increase total ACTH content and secretion in a synergistic manner provides a demonstration in normal pituitary cells that protein kinases C and A may interact in a unidirectional manner to regulate ACTH biosynthesis in addition to ACTH release. This interaction may take place within, or between, individual corticotropes.
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PMID:The biosynthesis and secretion of adrenocorticotropin by the ovine anterior pituitary is predominantly regulated by arginine vasopressin (AVP). Evidence that protein kinase C mediates the action of AVP. 216 7

The mitochondria in cells that synthesize steroid hormones not only have enzymes not present in mitochondria of non-steroidogenic cells but also have unique mechanisms for regulating the steroid substrate availability for certain of these enzymes. We have considered in detail the cytochrome P-450scc system that is located in the inner mitochondrial membrane and that catalyzes the initial and rate-determining step in the steroid hormone biosynthetic pathway. The flux through this pathway is regulated both by the levels of these catalysts themselves and by the availability of the substrate cholesterol for conversion to pregnenolone. These two levels of regulation occur in different time frames but are both controlled externally by the action of tissue-specific peptide hormone. We have used the adrenal cortex fasciculata cells as our paradigmatic cell type. The overall picture seems closely similar for mitochondria in other such steroidogenic cells when analogous data are available. Thus, in adrenal cortex fasciculata cells ACTH triggers several long-term (trophic) and short-term (acute) effects upon and within mitochondria that influence the initial and rate-determining step in the steroid hormone biosynthetic pathway. The only second messenger for both effects characterized thus far is cAMP. An increase in membrane-associated cAMP rapidly activates cAMP-dependent protein kinase, which in turn phosphorylates several cellular proteins, e.g., cholesterol ester hydrolase (vide supra). The trophic action, i.e., that produced by exposure of the cells to increased levels of ACTH or cAMP for a prolonged period (minutes to hours), increases the amounts of the steroid hormone synthesizing proteins in the mitochondria by increasing the transcription of the relevant nuclear genes. This latter process is not needed for the acute increase in the rate of steroid hormone biosynthesis. Whether induction of steroidogenic enzymes requires activation of a kinase has not been determined. However, the postulated SHIP proteins provide a mechanism by which cAMP levels and protein synthesis itself may regulate this induction. Mitochondria in steroidogenic tissues exert control over this process by their ability to recognize, import and process correctly the nuclear encoded precursors of the steroidogenic enzymes. Whether control at this level is ultimately dictated by nuclear or mitochondrial gene products or by an interplay between them is still unknown.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Distinctive properties of adrenal cortex mitochondria. 217 62

Corticotropin (ACTH)-releasing factor, vasoactive intestinal peptide, and catecholamines--hormones that stimulate ACTH secretion and cAMP generation--increased cytosolic calcium in AtT-20 cells. The increase in intracellular calcium is presumably a consequence of the stimulated cAMP synthesis, since forskolin, an activator of the catalytic unit of adenylate cyclase, and the cAMP analog 8-bromoadenosine 3',5'-cyclic monophosphate (8Br-cAMP) also increased the cytosolic levels of this ion. Pretreatment with somatostatin, a neuropeptide that inhibits stimulation of the adenylate cyclase system and the secretion of ACTH blocked the increase of cytosolic calcium. The effect of 8Br-cAMP, which bypasses the cyclase, was not inhibited by somatostatin pretreatment. The source of the increased calcium appears to be mainly extracellular. This is indicated by the inability of the secretagogues to increase cytosolic calcium in a medium deprived of this ion or in the presence of blockers of voltage-gated calcium channels. The involvement of calcium channels in the calcium rise evoked by the secretagogues was supported by experiments using the whole-cell patch-clamp technique. In these experiments 8Br-cAMP increased voltage-dependent calcium currents. These results suggest the following chain of events in the receptor-mediated elevation of cytosolic calcium and the concomitant release of ACTH from AtT-20 cells: hormone-receptor binding----cAMP synthesis----protein kinase activation----calcium channel activation----increase in cytosolic calcium----many steps----ACTH release. Phorbol myristate acetate, a compound which does not stimulate cAMP generation but enhances the release of ACTH in AtT-20 cells, decreased the cytosolic calcium level.
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PMID:Hormone secretagogues increase cytosolic calcium by increasing cAMP in corticotropin-secreting cells. 241 78


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