Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Forskolin, a natural diterpene, is a novel, potent activator of a variety of adenylate cyclase systems; its mechanism and site of action are still disputed. We report here that, while forskolin activates liver adenylate cyclase up to 15-fold, it does not activate cyclase of ram sperm which comprises a pure catalytic subunit. However, forskolin activation of sperm adenylate cyclase activity could be observed after reconstitution with the regulatory component-enriched membrane from human erythrocytes. The diterpene weakly (3-5-fold) activated the cytosolic, soluble cyclase found in rat and ram tests, but this effect was lost when the cyclase was purified by gel chromatography. Our data are not compatible with the hypothesis that forskolin acts directly on the catalytic subunit, but rather suggest that it acts via another regulatory component, part of, or different from, the GTP-binding regulatory complex.
Mol Cell Endocrinol
PMID:Forskolin requires more than the catalytic unit to activate adenylate cyclase. 689 46

Incubation of SH-SY5Y neural cells with mycophenolic acid (MPA), an inhibitor of inosine monophosphate dehydrogenase (the key enzyme in purine nucleotide biosynthesis), reduced the cellular content of GTP by 94% relative to its concentration in control cells (43 nmol/mg protein) without altering the level of GDP. Although in GTP-depleted intact cells the receptor binding parameters (Kd and Bmax) of the opioid antagonist [3H]naltrexone were unchanged from those in untreated cells, the binding affinity of the mu-selective opioid agonist [3H]Tyr-D-Ala-Gly-(Me)- Phe-Gly-ol ([3H]DAMGO) was enhanced 2-fold. Furthermore, the kinetics of ligand/receptor interaction revealed that in the nucleotide-depleted cells, the dissociation rate constant for [3H]DAMGO was reduced by 44%. Initial exposure of SH-SY5Y cells to pertussis toxin reduced high-affinity ligand binding by 95% and abolished the effect of MPA treatment. Renewed incubation of the GTP-depleted cells with guanosine restored the original GTP levels and agonist binding. Neither MPA nor guanosine treatment changed the Bmax of [3H]DAMGO binding. Forskolin- and prostaglandin E1-stimulated adenylyl cyclase activities were decreased significantly in GTP-depleted cells. DAMGO and [D-Pen2,D-Pen5]enkephalin inhibitions of adenylyl cyclase were also affected with MPA treatment. Maximal inhibition of forskolin-stimulated adenylyl cyclase activity by both of the agonists was reduced, whereas MPA caused a 2-fold reduction in potency for DAMGO. The results show that reduction in endogenous GTP levels leads to noticeable changes in agonist, receptor, and G protein interactions, as measured by agonist binding, and to subsequent diminution of the signal transduction, as reflected by the cAMP levels.
Mol Pharmacol 1995 Oct
PMID:Reversible modulation of opioid receptor binding in intact neural cells by endogenous guanosine triphosphate. 747 95

We have identified and studied a posttranscriptional mechanism of lactate dehydrogenase A (LDH) subunit gene expression at the level of mRNA stability. Using the well differentiated rat C6 glioma cell line as a model system, the effects of activators of the protein kinase A and C pathways on the half-life of LDH A mRNA were measured by two independent methods: 1) by the RNA synthesis inhibitor-chase method using actinomycin D, and 2) by analysis of decay of LDH A [3H]mRNA in [3H]uridine-labeled cells. By each method, the half-life of relatively short-lived LDH A mRNA was increased 5- to 7-fold in 8- (4-chloro-phenylthio) cAMP or forskolin-treated and about 3-fold in 12-0-tetradecanoylphorbol-13- acetate (TPA) or dioctanoylglycerol-treated cells. Forskolin acted synergistically with TPA to prolong LDH A mRNA half-life from 55 min to more than 20 h. The relatively rapid basal decay rate of LDH A mRNA was also considerably slowed in the presence of the protein phosphatase inhibitor okadaic acid, suggesting a functional role for protein phosphorylation in the stabilization process. In glioma cells stably transformed with a protein kinase A catalytic subunit expression vector, overexpression of the catalytic subunit stabilized LDH mRNA to the degree seen in forskolin-treated cells. In cells transfected with a protein kinase A inhibitor-expression vector, cAMP-mediated stabilization of LDH A mRNA half-life was prevented. Furthermore, both staurosporin and 3- [1-(3-dimethylaminopropyl)-indol-3-yl]-3-(indol- 3-yl)- maleimide, inhibitors of protein kinase C, prevented the TPA-induced stabilization of LDH A mRNA. We conclude from the experimental data that the protein kinase A and C signal pathways play an active functional role in regulating LDH A mRNA stability and act cooperatively to achieve LDH A mRNA stability regulation.
Mol Endocrinol 1995 Aug
PMID:Lactate dehydrogenase A subunit messenger RNA stability is synergistically regulated via the protein kinase A and C signal transduction pathways. 747 96

Using a transgenic mouse derived GnRH expressing neuronal cell line, GT1-3, we studied the effects of activation of cAMP, Ca2+ and protein kinase C pathways by forskolin, ionomycin and the phorbol ester phorbol 12-myristate 13-acetate (PMA), respectively, upon gonadotropin-releasing hormone (GnRH) secretion, cellular peptide content, mRNA and RNA primary transcript levels. Forskolin, ionomycin and phorbol ester all caused an increase in GnRH secretion in GT1-3 cells in a time and dose-dependent manner during a short-term (1 h) static incubation. Prolonged treatment with forskolin (10 microM), ionomycin (1 microM) and PMA (10 nM) for 12 or 24 h resulted in significant decreases in GnRH mRNA levels. Time-course studies showed that the increases in GnRH secretion stimulated by forskolin, ionomycin and PMA were gradually attenuated over time in parallel with the decreases in mRNA expression. In contrast, there were only small and variable changes in the GnRH cellular content. Studies using a GnRH antagonist (100 microM) suggested that the released GnRH has a negative feedback effect on its own secretion. However, co-incubation with the GnRH antagonist did not alter the inhibitory effects on GnRH mRNA levels by the secretagogues. Further studies on the transcriptional effects of forskolin, ionomycin and PMA on GnRH gene expression in GT1-3 cells revealed that all three secretagogues suppressed GnRH RNA primary transcript levels, with forskolin having a slower time course of action. Thus, the inhibition of cytoplasmic GnRH mRNA, and presumably its synthesis, after 12-24 h of secretagogue treatment may be due at least in part to a suppression of GnRH gene transcription.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Endocrinol 1994 Jun
PMID:Second messenger regulation of mouse gonadotropin-releasing hormone gene expression in immortalized mouse hypothalamic GT1-3 cells. 752 6

Aromatase cytochrome P450 mRNA and activity was strongly expressed in THP 1 myeloid leukaemia cells after treatment with phorbol-myristate-acetate (PMA) and dexamethasone, low level expression was caused by calcitriol. mRNA species of 4.0, 3.0, 2.4 and 1.1 kb size were differentially stimulated. After calcitriol-mediated differentiation (72 h, measured by CD 14 expression) mRNA expression was further enhanced by PMA (45-fold), dexamethasone (15-fold), oestradiol (3.7-fold), testosterone (2.5-fold) and androstenedione (3.5-fold). Forskolin, cAMP and follicle stimulating hormone had no stimulatory effect. Oestradiol formation from testosterone (oestradiol radioimmunoassay in culture supernatants) increased to > 2000 pg/ml/10(6) cells/24 h after PMA-stimulation, mirrored mRNA expression and was suppressed below 10% of original values in the presence of 4-OH-androstenedione. Exons I.2 and I.4 were expressed in PMA-stimulated cells only, exon I.3 in both PMA- and dexamethasone-stimulated cells. A new splicing variant was expressed after calcitriol-stimulation, which did not hybridize to an exon II-derived oligonucleotide but to an exon III-derived one. Local aromatisation of androgens into oestradiol may be important in the concerted crosstalk of cells of the monocyte/macrophage lineage with their respective tissues in inflammation and bone metabolism.
Mol Cell Endocrinol 1995 Apr 28
PMID:Expression and regulation of aromatase cytochrome P450 in THP 1 human myeloid leukaemia cells. 754 22

The role of ACTH, forskolin and 8Br-cAMP on the regulation of mRNA abundance, protein levels and enzymatic activity of cytochrome P450 21-hydroxylase (P450c21, CYP21) were investigated in guinea pig adrenal cell cultures. In untreated cells, 21-hydroxylase activity was diminished throughout a 48 h period of incubation. Although incubation with forskolin and 8Br-cAMP restored 21-hydroxylase activity to normal levels, the addition of ACTH did not prevent the decrease of 21-hydroxylase activity. Treatment of cells with RU486 for 24 h inhibited 21-hydroxylase activity by 93%; however, after removal of the drug a slight increase of enzyme activity was observed; this rise was enhanced by the addition of ACTH. Forskolin and 8Br-cAMP increased the levels of 21-hydroxylase activity to the same range as seen in untreated cells. In cells that were not pretreated with RU486, incubation with cycloheximide for 1 h had no effect on 21-hydroxylase activity and could not prevent the modest increase of 21-hydroxylase activity induced by forskolin or 8Br-cAMP after 48 h of incubation. In RU486-treated cells, cycloheximide blocks the stimulation of enzyme activity induced by ACTH, forskolin and 8Br-cAMP. Our findings indicate that 21-hydroxylase activity can be stimulated by ACTH, forskolin or 8Br-cAMP solely in the presence of reduced enzymatic activity. Western immunoblot analysis of P450c21 protein levels in untreated or RU486-treated adrenal cells indicate that P450c21 protein levels were in the same range and further incubation with ACTH caused a similar elevation of P450c21 protein levels in both the untreated and RU486-treated cells. Northern blot analysis on RNA isolated from adrenal cells showed that RU486 did not alter the basal steady state levels of P450c21 mRNA. As well, incubation with ACTH or 8Br-cAMP increased the levels of P450c21 transcript to the same extent in both untreated and RU486-treated cells. These results taken together provide additional evidence for the presence of an adrenal specific protein factor(s) modulating 21-hydroxylase activity.
J Steroid Biochem Mol Biol 1995 Jul
PMID:Regulation of guinea pig adrenal P450c21 messenger RNA, protein and activity by RU486. 763 12

Tau is a neuron-specific, microtubule-associated protein that forms paired helical filaments (PHFs) of Alzheimer's disease when aberrantly phosphorylated. We have attempted to elucidate the protein kinases and phosphatases that regulate tau phosphorylation. Incubation of rat, human, and rhesus monkey temporal neocortex slices with the phosphatase inhibitor okadaic acid induced epitopes of tau similar to those found in PHFs. Okadaic acid (1-20 microM) induced variant forms of tau at 60-68 kDa, which were recognized by the monoclonal antibodies Alz-50 (in humans only) and 5E2 and two polyclonal antipeptide antisera, OK-1 and OK-2. The phosphorylation-sensitive monoclonal antibody Tau-1 failed to recognize the slowest mobility forms of tau after okadaic acid treatment. FK-520 (1-10 microM), a potent inhibitor of calcineurin activity, was tested in brain slices and found not to alter tau mobility. However, combinations of FK-520 (5 microM) and okadaic acid (100 nM) caused tau mobility shifts similar to those seen after 10 microM okadaic acid treatment; similar results were seen using the calcineurin-selective inhibitor cypermethrin. Treatment of human slices with 10 microM okadaic acid decreased both protein phosphatase 2A and calcineurin activity; FK-520 inhibited only protein phosphatase 2B activity. A proposed tau-directed kinase, 42-kDa mitogen-activated protein kinase (p42mapk), was activated by okadaic acid (> 100 nM) but not FK-520 (5 microM). Nerve growth factor (100 ng/ml) activated p42mapk, particularly when used in combination with 100 nM okadaic acid; changes in tau mobility were seen when this kinase was activated. Forskolin (2 microM) antagonized the effects of nerve growth factor on both p42mapk activity and tau phosphorylation; forskolin alone had little effect on PHF-like tau formation induced by phosphatase inhibitors. These results outline complex interactions between tau-directed protein kinases and protein phosphatases and suggest potential sites for therapeutic intervention.
Mol Pharmacol 1995 Apr
PMID:Tau phosphorylation in brain slices: pharmacological evidence for convergent effects of protein phosphatases on tau and mitogen-activated protein kinase. 772 35

beta-Adrenoceptor (beta AR) responsiveness, receptor density, receptor-G protein coupling, and the possible role of membrane fluidity in receptor-G protein coupling were investigated in the rat aorta with age. The beta AR agonist isoproterenol (ISO) produced relaxation of KCl-induced aortic contractions by 97%, 21%, and 0% in aortae from 1- 6-, and 24-month-old Fischer 344 rats, respectively. Forskolin completely relaxed the contractions at all ages. beta AR density was determined in aortic membranes by saturation binding of 125I-cyanopindolol (125I-CYP). beta AR density was 76, 52, and 47 fmol/mg of protein in 1-, 6-, and 24-month-old rats, respectively. To investigate beta AR coupling to G proteins, displacement by ISO of 125I-CYP binding was determined in aortic membranes in the presence and absence of the GTP analog guanosine-5'-(beta gamma-imido)triphosphate [Gpp(NH)p] (0.1 mM). The effect of Gpp(NH)p on the ISO displacement curve for 125I-CYP binding was greatest in 1-month-old rats and decreased markedly with age. In 1-month-old aorta, in the absence of Gpp(NH)p the ISO displacement curve was biphasic and two affinity constants were determined (KH - 0.061 microM and KL = 2.4 microM). In the presence of Gpp(NH)p the ISO displacement curve was monophasic (Kd - 0.72 microM). In 6-month-old aorta, whereas an effect of Gpp(NH)p on the ISO displacement curve could still be observed [in the absence of Gpp(NH)p, KH = 0.2 microM and KL = 3.5 microM; in the presence of Gpp(NH)p, Kd - 0.83 microM], the affinity constant for high affinity agonist binding and the percentage of receptors with high affinity for agonist were decreased significantly. In 24-month-old aorta there was no effect of Gpp(NH)p on the ISO displacement curve and a single affinity constant was detected [0.7 microM and 0.8 microM in the presence and absence of Gpp(NH)p, respectively]. The presence of two affinity constants for ISO in 1- and 6-month-old aorta in the absence of Gpp(NH)p and single affinity constants in the presence of Gpp(NH)p presumably represent the G protein-coupled and uncoupled states of the beta ARs, which are not observed in 24-month-old aorta. The ability of the beta AR to form the high affinity nucleotide-sensitive complex with the agonist was restored by treatment of the membranes with cis-vaccenic acid, which increases the fluidity of the membrane.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1995 Apr
PMID:Beta-adrenoceptor-G alpha S coupling decreases with age in rat aorta. 772 38

We have studied the postnatal changes in the levels of isoforms of stimulatory (Gs) and inhibitory (Gi) G-proteins, cAMP and cGMP in washed particulate membranes (WPM) from whole ventricles as well as from isolated ventricular myocytes and have also measured adenylyl cyclase (AC) activity in WPM prepared from isolated myocytes of adult (AD) and newborn (NB) rabbit heart. Immunoblot analysis for the levels of Gi alpha 1, G alpha 2, Gi alpha 3 and Gs alpha subunits showed that Gi alpha 2 and Gi alpha 3 were higher in WPM from whole ventricles of NB compared to AD. This ratio was much higher in WPM from isolated ventricular myocytes since Gi alpha 2 and Gi alpha 3 were either absent or present in extremely low immunodetectable levels in WPM from AD ventricular myocytes. Gi alpha 1 levels were not different for AD compared to NB WPM, whether prepared from whole ventricle or from isolated myocytes. Two forms of Gs alpha, a small form (Gs alpha-S) and a large form (Gs alpha-L), were immunodetected at 43 and 48 kDa, respectively. The Gs alpha-S form was higher in AD WPM and the Gs alpha-L form was higher in NB WPM while the total Gs alpha(L+S) was not different. The Gs alpha results for WPM from isolated myocytes were not different from the results for WPM from whole ventricles. Basal levels of cAMP were 80% higher in NB compared to AD whole ventricles and were 200% higher in NB compared to AD isolated myocytes. Levels of cGMP were 4-5 fold higher in NB than in AD myocytes and ventricular tissue. Basal AC activity was higher in NB than in AD WPM from isolated myocytes and was enhanced by Gpp(NH)p pretreatment in AD but not in NB WPM. The isoproterenol-induced increase in AC activity was higher in AD compared to NB WPM and was completely abolished by Gpp(NH)p pretreatment in NB but not in AD WPM. Forskolin caused a greater increase in AC activity in NB than in AD WPM. The post-natal decrease in the levels of Gi alpha 2 and Gi alpha 3, particularly in isolated ventricular myocytes, may help to explain the smaller effects of isoproterenol and greater muscarinic influence on ICa, as we previously showed, and the smaller effect of isoproterenol on AC activity in NB compared to AD WPM.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Cell Cardiol 1994 Dec
PMID:Postnatal changes in the G-proteins, cyclic nucleotides and adenylyl cyclase activity in rabbit heart cells. 773 Oct 49

An in vitro perifusion system was used to ascertain the role of cAMP in the genesis of the self-priming effect of gonadotrophin-releasing hormone (GnRH) in rat pituitaries. Ten-minute pulses of 20 nmol/l GnRH administered 150 min apart resulted in the manifestation of the self-priming effect, an effect which was inhibited by 5 mumol/l cycloheximide. Forskolin (1 mumol/l) which does not stimulate luteinizing hormone (LH) secretion or affect the initial LH response to GnRH significantly potentiated the second response through protein synthesis-dependent mechanisms. Additionally, an initial 10-min pulse of forskolin alone was sufficient to prime the pituitary to a subsequent pulse of GnRH 150 min later. Interestingly, similar amounts of LH were secreted in response to forskolin + GnRH or GnRH administered 150 min after forskolin. Flufenamate, an inhibitor of GnRH-stimulated increases in cAMP production prevented the manifestation of the self-priming effect of GnRH. Forskolin which bypasses the inhibitory effects of flufenamate on cAMP production reversed the flufenamate-induced inhibition of the self-priming effect of GnRH through protein synthesis-dependent processes. These results suggest that cAMP does not mediate the LH response to an initial exposure of GnRH, but does play a pivotal role in the genesis of the self-priming effect of GnRH through the stimulation of de novo protein synthesis. Once the newly synthesized proteins are available, the nucleotide is not required for the manifestation of the phenomenon.
Mol Cell Endocrinol 1995 Jan
PMID:Adenosine 3',5'-cyclic monophosphate and the self-priming effect of gonadotrophin-releasing hormone. 779 28


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