Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of the protein kinase C activator, phorbol myristate acetate (PMA), on cytosolic calcium levels and adrenocorticotropin (ACTH) release from the mouse anterior pituitary tumor cell line, AtT-20, were compared to those induced by the hormone, corticotropin-releasing factor (CRF), a stimulant of cAMP-dependent protein kinase activity. Cytosolic calcium levels were measured using the fluorescence probe Quin 2. PMA induced a time- and concentration-dependent rise in cytosolic calcium levels and ACTH release from AtT-20 cells that was blocked by verapamil and nifedipine, antagonists of voltage-regulated calcium channels, and tetraethylammonium (TEA), a K+ channel antagonist. The inactive phorbol ester, 4-phorbol 12,13-didecanoate, did not alter cytosolic calcium levels or ACTH release. Several minutes after the initial stimulation of calcium influx by PMA, cytosolic calcium levels returned to basal levels despite the continued presence of the phorbol ester. A short pretreatment (2-4 min) of AtT-20 cells with PMA abolished the ability of K+, CRF, and forskolin to raise intracellular calcium levels. These findings indicate that phorbol esters induce a secondary inhibition of calcium influx after an initial stimulation. In contrast to the effects of PMA, CRF induced a sustained rise in cytosolic calcium levels and did not reduce the subsequent stimulation of calcium influx by K+ or PMA. CRF-stimulated calcium influx was blocked by verapamil but not TEA. The ability of CRF to elevate cytosolic calcium levels was mediated by cAMP-dependent protein kinase because the insertion of a synthetic peptide inhibitor of cAMP-dependent protein kinase activity into AtT-20 cells attenuated the ability of CRF and forskolin but not PMA to raise cytosolic calcium levels. The results suggest that activators of protein kinase C and cAMP-dependent protein kinase regulate intracellular calcium levels in AtT-20 cells through different mechanisms.
 ...
PMID:Activators of protein kinase C and cyclic AMP-dependent protein kinase regulate intracellular calcium levels through distinct mechanisms in mouse anterior pituitary tumor cells. 282 94

Dispersed chick adrenocortical cells were incubated with avian parathyroid hormone (aPTH) or ACTH. Accumulation of cyclic AMP (cAMP), activity of cAMP-dependent protein kinase and the secretion of corticosterone and aldosterone, in response to these hormones, were measured. Accumulation of cAMP and activity of cAMP-dependent protein kinase were stimulated by both aPTH and ACTH as well as by cholera toxin. Cyclic AMP production followed a similar time-course when stimulated by either peptide hormone. Stimulation of steroid hormone secretion was detectable after 20 min of incubation with ACTH, but only after 40 min with aPTH. The maximal steroid hormone secretion by adrenocortical cells was similar when induced by either peptide hormone. The aPTH concentrations needed for half-maximal response of corticosterone and aldosterone secretion were higher than those for ACTH (2.5- and 2-fold respectively), but still within the physiological range. The 11 beta-hydroxylase inhibitor metyrapone inhibited the secretion of both corticosterone and aldosterone when induced by either aPTH or ACTH. The results suggest that aPTH is almost as potent as ACTH in stimulating the secretion of corticosterone and aldosterone from chick adrenocortical cells and utilizes a cAMP-dependent pathway similar to that of ACTH.
 ...
PMID:Stimulation of chick adrenal steroidogenesis by avian parathyroid hormone. 282 8

The steroid 21-hydroxylase (21-OHase) gene is selectively expressed at high levels in cells of the adrenal cortex and is transcriptionally regulated by corticotropin (ACTH). In this study, we examined the contribution of cis-acting nucleotide sequences to the regulated expression of the mouse 21-OHase gene. The 5'-flanking sequences of the mouse 21-OHase gene, extending 330 bp upstream from the transcription initiation site, were placed in front of the human growth hormone (hGH) reporter gene, and expression of the fusion gene was measured following transient transfection in Y1 mouse adrenocortical tumor cells. The 330 bp of 21-OHase flanking sequence directed both basal and ACTH-stimulated expression of hGH in Y1 adrenocortical cells but did not direct hGH expression in I-10 mouse testicular Leydig cells or in mouse fibroblast L cells. The 21-OHase/hGH fusion gene was poorly expressed in Y1 mutants defective in cAMP-dependent protein kinase activity. These results indicate that sequences necessary for adrenal cell-selective and ACTH-regulated expression of the 21-OHase gene reside within the first 330 bp of 5'-flanking DNA and that constitutive expression of the gene requires the integrity of cAMP-dependent protein kinase. The constitutive expression of hGH in Y1 cells was decreased dramatically (40-fold) when the 21-OHase flanking sequences in front of hGH were shortened to 156 bp from the transcription initiation site and was restored when the upstream sequences of the 21-OHase gene, from -330 to -150, were added back; the sequences from -330 to -150 were equally effective in either the correct or reverse orientation. From these observations, we conclude that an enhancer element is contained within the sequences from -330 to -150 bp upstream of the 21-OHase transcription initiation site.
 ...
PMID:An enhancer element and a functional cyclic AMP-dependent protein kinase are required for expression of adrenocortical 21-hydroxylase. 284 6

The tumor-promoting agent 12-0-tetradecanoyl-phorbol-13-acetate (TPA) caused a time- and dose-dependent morphological change in Y-1 adrenocortical tumor cells. The morphological alteration was apparent 2 hr following addition of 1 microgram/ml TPA to cell cultures and became more striking with longer treatment times. Smaller doses of TPA took a longer time to produce an effect. Cultures grown in the presence of TPA exhibited more rounding and piling up of cells than similar cultures maintained in medium lacking TPA. These TPA-stimulated morphological changes were reversible, and after 24 hr in TPA-free media, the cultured cells began to flatten. After 96 hr in TPA-free media they resembled the control cultures. The reversibility of the morphological change was also dose dependent: cells treated with 1 microgram/ml TPA took a longer time to resume the typical control morphology than did cultures treated with 0.01 microgram/ml TPA. In addition, TPA treatment resulted in a decrease in cell growth rate, an increase in steroid production, and an increase in the localization of free catalytic units of cAMP-dependent protein kinase in the cytoplasm. The steroidogenic effect of ACTH on the cell population was inhibited in cultures maintained in TPA. The results of this study indicate that TPA induces morphological changes in the Y-1 adrenocortical tumor cell population while increasing steroidogenesis and the activation of cAMP-dependent protein kinase and decreasing cell growth rate.
 ...
PMID:Characterization of the morphological, growth, and steroidogenic effect of TPA on mouse Y-1 adrenal cortical tumor cells in culture. 284 97

Release of adrenal catecholamine by carbachol has been shown to be coincident with an increase in intracellular cAMP levels. Bovine adrenal medullary (BAM) cells were prepared and maintained in culture and used to examine the role of cAMP in stimulus-secretion coupling. The addition of ACTH to these cells caused a 10- to 50-fold increase in cellular cAMP without an effect on catecholamine secretion, suggesting cortical cell contamination. Percoll density separation of both BAM cells and adrenal cortical cells revealed that the greatest cAMP responses to ACTH corresponded to the catecholamine-containing cell fractions and not to those density layers where cortical cells sedimented. BAM cells isolated on Percoll did not metabolize [14C]cholesterol to steroids as would be expected were the ACTH-stimulated cAMP accumulations due to cortical cell contamination of the cultures. ACTH stimulated protein phosphorylation in 32P-labeled BAM cells in a manner indistinguishable from that induced by carbachol and forskolin. The major soluble phosphoprotein to be affected by these agents had a relative mol wt of 55-57 kdaltons on sodium dodecyl sulfate-gels and corresponded to tyrosine hydroxylase, which is a specific marker enzyme in the adrenal for chromaffin cells. We propose that bovine adrenal chromaffin cells express ACTH receptors which are coupled to adenylate cyclase. While no acute effect of ACTH was found on catecholamine secretion, ACTH may play a direct role in the regulation of catecholamine synthesis by stimulating the phosphorylation of tyrosine hydroxylase by cAMP-dependent protein kinase.
 ...
PMID:Direct effects of adrenocorticotropic hormone on bovine adrenomedullary cells: adenosine 3',5'-monophosphate-dependent phosphorylation of tyrosine hydroxylase. 286 12

The stimulation of steroidogenesis by antimitotic drugs has been studied in wild-type (Y-1) and cAMP-dependent protein kinase-deficient (kin-8) mouse adrenal tumor cell lines. Unlike some other cells, Y-1 cells do not increase their cAMP output upon exposure to antimitotic drugs such as colchicine, vinblastine or podophyllotoxin, which readily increase steroidogenesis. Moreover, no increase in cAMP can be detected over an extended time span. Stabilization of tubulin polymers by taxol or high concentrations of vinblastine blocks ACTH-, cholera toxin- or colchicine-stimulated steroidogenesis without major effects on cAMP levels. Colchicine and podophyllotoxin stimulate steroidogenesis in the cAMP-dependent protein kinase-deficient mutant to the same degree as in the wild-type Y-1 cells, although absolute steroid yields are lower in the mutant cells. We suggest that the antimitotic agents stimulate adrenal steroidogenesis by a cAMP-independent pathway that may involve facilitation of cholesterol access to the mitochondrion.
 ...
PMID:Cyclic AMP-independent stimulation of steroidogenesis in Y-1 adrenal tumor cells by antimitotic agents. 287 35

To define the role of cAMP in the actions of ACTH, the dissociation of cAMP-dependent protein kinase and the subsequent intracellular location of its free catalytic units were monitored after exposure of Y-1 cells to ACTH, FSH, or cyclic nucleotide analog. To accomplish this, a fluorescinated cytochemical probe was used that complexes specifically with free catalytic units from cAMP-dependent protein kinase. Also, the effects of hormone or nucleotide on secretion of fluorogenic steroids and DNA synthesis were examined. Y-1 cells dissociated protein kinase in a dose-dependent fashion when exposed to ACTH or cAMP analog, but did not respond to FSH, which was one of the control agents used. After 30 min of treatment with 1.5 X 10(-10) M ACTH, free catalytic units were observed only in the cytoplasm of Y-1 cells, whereas a similar time of exposure to 3 X 10(-10) M ACTH led to the appearance of catalytic units in nucleolus as well as in cytoplasm. ACTH (6 X 10(-10) M) caused a rise in cytoplasmic and nucleolar protein kinase dissociation proportionally greater than that seen in cultures exposed to 3 X 10(-10) M ACTH. Upon treatment with 6 X 10(-10) M ACTH, the amount of free catalytic units in cytoplasm and nucleolus was detectably greater than that in controls within 1 min of stimulation and continued to rise with increasing time of exposure to hormone. The nuclear, mostly nucleolar, content of free catalytic unit appeared to peak after 15 min of stimulation, while cytoplasmic enzyme levels continued to rise up to 60 min. Exposure of Y-1 cells to nucleotide analog caused cAMP-dependent protein kinase dissociation with temporal kinetics and a subcellular distribution similar to that seen after ACTH stimulation. We conclude that actions of ACTH are mediated by cAMP-dependent protein kinases. Further, there appear to be two intracellular pools of protein kinase, one nucleolar, the other cytoplasmic, and these may be independently regulated, with the nucleolar enzyme requiring higher concentrations of ACTH for dissociation than those needed for cytoplasm protein kinase. These observations may be relevant to the fact that more ACTH is required to inhibit DNA synthesis than is necessary to enhance steroid production.
 ...
PMID:Intracellular kinetics of free catalytic units dissociated from adenosine 3',5'-monophosphate-dependent protein kinase in adrenocortical tumor cells (Y-1). 298 Oct 71

To explore the role of calmodulin (CaM) in lipolysis, studies were carried out on effects of CaM inhibitors on hormone-stimulated lipolysis, the activity of cAMP-dependent protein kinase, and phosphorylation of endogenous substrate proteins. When adipocytes were incubated with trifluoperazine (TFP) and W-7 but not with W-5, stimulation of lipolysis by epinephrine was blunted. W-7 also inhibited lipolysis induced by ACTH, 1-methyl-3-isobutylxanthine (MIX) or (Bu)2 cAMP. The binding of 3H-cAMP to its receptor protein (the regulatory subunit of protein kinase) as well as the activity of cAMP-dependent protein kinase was suppressed by W-7, and the anti-CaM antibody, but not by W-5. The CaM-dependence of the protein kinase was also proved by the fact that the protein kinase activity that was markedly reduced in CaM-depleted cell extracts, was significantly restored by addition of exogenous CaM to them. Furthermore, W-7 decreased cAMP-stimulated phosphorylation of endogenous substrate proteins (mol wt 230k, 200k, 130k, 85k, 75k, and 50kdalton), among which the one of 85kdalton is most likely to be the hormone-sensitive lipase. These findings suggest that CaM is involved in the mechanism of hormone-induced lipolysis by exerting stimulatory effects on the activation of cAMP-dependent protein kinase in cell extracts capable of phosphorylating substrate proteins including hormone-sensitive lipase.
 ...
PMID:The role of calmodulin in hormone-stimulated lipolysis. 298 17

In microsomes of bovine fasciculata reticularis cells incubated with or without 10(-8) M ACTH during 20 min, we measured covalent and non covalent cAMP binding under exchange or non-exchange conditions and cAMP-kinase activity. ACTH induced a decrease in cAMP-kinase activity and in the number of free cAMP binding sites. These results indicate an activation by ACTH of a part of microsomal cAMP-dependent protein kinase. Photoaffinity labeling of microsomal protein with 8-azido-cAMP revealed the presence of both cAMP-kinase isoenzyme I and II in this cellular fraction. Using this method, it was demonstrated that ACTH1-24 caused a preferential and nearly complete activation of microsomal protein kinase I.
 ...
PMID:Activation of microsomal cAMP-dependent protein kinase isoenzyme I by ACTH1-24 in bovine adrenal cells. 299 46

Corticotropin-releasing factor (CRF) is the most potent and effective natural stimulant of corticotropin (ACTH) secretion. In a tumor cell line of the mouse anterior pituitary (AtT-20/D16-16) consisting of a homogeneous population of corticotrophs, CRF is known to increase adenylate cyclase and cAMP-dependent protein kinase activities as well as to release ACTH. To determine whether activation of cAMP-dependent protein kinase is essential for CRF to evoke the secretion of ACTH, an inhibitor (PKI) of this kinase was inserted into AtT-20 cells. This was accomplished by first encapsulating PKI into liposomes and then covalently coupling them to protein A for binding to antibodies directed against an AtT-20 cell surface antigen, N-CAM (neural cell adhesion molecule). The binding of the liposomes to the anti-N-CAM antibodies led to the internalization of the PKI into the tumor cells. The PKI treatment greatly attenuated CRF-stimulated ACTH release as well as the secretory response to beta-adrenergic agonists. However, ACTH release in response to caerulein, an agonist of cholecystokinin 8 receptors, was not altered by the PKI treatment. CRF treatment also increased the levels of mRNA for proopiomelanocortin (POMC), the precursor for ACTH in AtT-20 cells. Application of liposomes containing PKI to AtT-20 cells blocked the ability of CRF and 8-bromo-cAMP, but not phorbol ester, to increase POMC mRNA levels. The results revealed an essential role for cAMP in mediating the effect of CRF on ACTH release and POMC gene expression.
 ...
PMID:Corticotropin-releasing factor-induced adrenocorticotropin hormone release and synthesis is blocked by incorporation of the inhibitor of cyclic AMP-dependent protein kinase into anterior pituitary tumor cells by liposomes. 299 99


<< Previous 1 2 3 4 5 6 7 8 9 Next >>