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:4.6.1.1 (adenylate cyclase)
19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the previous studies we have shown that testosterone increases lipolytic responsiveness to catecholamines in rat white adipocytes, and that is associated with an up-regulation of beta-adrenergic receptor density. However, the postreceptor events involved in the testosterone induced enhancement of beta-adrenergic receptor activated lipolysis in these cells have not been adequately studied, and were therefore investigated in the present study. Male Sprague Dawley rats were divided into three groups: control, castrated, and castrated treated with testosterone. The beta-adrenergic receptor-mediated cAMP accumulation, measured with RIA after isoproterenol (a beta-adrenergic agonist) stimulation was decreased in castrated rats, and reversed by testosterone treatment, suggesting a testosterone effect at or proximal to adenylate cyclase. However, no differences between the groups were found in abundance of G alpha protein messenger RNAs (G alpha s, G alpha i-1, and G alpha i-2) as analyzed by Northern blot and a solution hybridization RNase protection assay, or in G protein mass measured with a quantitative enzyme-linked immunosorbent assay in fat cell membrane preparation. Lipolysis stimulated by N6-monobutyryl-cAMP was reduced in castrated rats and recovered by testosterone treatment, suggesting that components distal to the adenylate cyclase, i.e. protein kinase A (PKA) and/or hormone sensitive lipase (HSL) also are involved in testosterone regulation of lipolysis. In conclusion, these and previous results suggest that the testosterone-induced increase in lipolytic response to catecholamines in rat white adipocytes is mediated through several events including an increased beta-adrenergic receptor density, probably an increased adenylate cyclase activity and an increased protein kinase A/hormone sensitive lipase activity at the postreceptor level with apparent absence of effect on the expression of G-proteins.
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PMID:Postreceptor events involved in the up-regulation of beta-adrenergic receptor mediated lipolysis by testosterone in rat white adipocytes. 838 92

The presence of mRNA transcripts and/or immunoreactivity for PTH-related protein (PTHrP) in several normal mammalian tissues suggests a possible paracrine or autocrine role for this hormone. Since immunohistochemical studies of human ovary demonstrate the presence PTHrP immunoreactivity in this tissue, we wondered if ovarian follicular fluid (OVFF) might contain PTHrP. We retrospectively analyzed 28 OVFF samples obtained at ova harvest in 21 women undergoing in vitro fertilization. Fourteen samples contained significant adenylate cyclase-stimulating activity in a PTHrP-sensitive bioassay. In a subsequent prospective analysis, 41 of 45 freshly obtained OVFF samples demonstrated significant activity. This bioactivity was completely neutralized by antisera to PTHrP, but was unaffected by antisera to PTH. Fifteen OVFF samples were also analyzed in a sensitive 2-site immunoradiometric assay for PTHrP, and all 15 demonstrated significant levels of the hormone. The PTHrP levels did not correlate with the presence of an ovum in the follicle or with follicular fluid calcium. Short term (24- to 48-h) cultures of granulosa-luteal cells established from 5 OVFF samples demonstrated constitutive secretion of PTHrP using the immunoradiometric assay. Neither progesterone nor estrogen affected basal secretion. RNase protection analysis of cellular RNA prepared from cultured granulosa-luteal cells demonstrated the presence of mRNA for PTHrP in these cells. We conclude that 1) human OVFF obtained after stimulation with FSH and LH contain high concentrations of PTHrP; and 2) the granulosa-luteal cell is capable of secreting PTHrP both in vivo and in vitro.
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PMID:Human granulosa-luteal cells secrete parathyroid hormone-related protein in vivo and in vitro. 849 23

Relaxation of the trabecular smooth muscle, which is necessary for penile erection, is controlled locally by neurotransmitters and vasoactive agents. The goal of this study was to identify and characterize muscarinic acetylcholine receptor (mAChR) subtypes expressed in cultured human corpus cavernosum smooth muscle cells (HCC SMC). Binding analysis with L-[benzilic-4,4'-3H(N)]quinuclidinyl benzilate ([3H]QNB) demonstrated the expression of specific muscarinic receptor binding sites in HCC SMC. Analysis of total RNA isolated from whole corpus cavernosum tissue and smooth muscle cells, by RNase protection assays, demonstrated the expression of mRNA transcripts for m1, m2, m3, and m4 mAChR subtypes in whole tissue and m2 and m4 subtypes in cultured cells. In situ hybridization with specific m2 and m4 probes further confirmed the expression of m2 and m4 mRNA transcripts in cultured cells. Carbachol (CCh), a nonselective cholinergic agonist, inhibited cAMP synthesis at low concentrations (0.1-1 microM) and stimulated cAMP synthesis at high concentrations (100 microM), in cultured HCC SMC. CCh (100 microM) further augmented forskolin (FSK), isoproterenol (ISO), and prostaglandin E1 (PGE1)-induced cAMP synthesis. These observations suggest that, in vivo, in HCC, ACh may activate m3 mAChR subtypes on endothelial cells or m2 and m4 subtypes on the SMC. Although m2 and m4 are thought to inhibit adenylate cyclase (AC), the augmentation of cAMP synthesis by high concentrations of CCh in SMC suggests an alternative mechanism of coupling to G-proteins that stimulates AC activity. These studies show that HCC tissue expresses different subtypes of mAChR (m1, m2, m3, and m4), whereas cultured HCC SMC express m2 and m4 subtypes. It is suggested that m2 and m4 receptor subtypes may play an important role in maintaining trabecular smooth muscle tone in vivo. The augmentation of FSK-, ISO, and PGE1-induced cAMP synthesis by CCh suggests possible development of a multidrug therapeutic approach to treatment of erectile dysfunction.
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PMID:Expression of functional muscarinic acetylcholine receptor subtypes in human corpus cavernosum and in cultured smooth muscle cells. 872 95

ARPP-21 (cAMP-regulated phosphoprotein, Mr = 21,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis) is a phosphoprotein highly enriched in concentration in the neurons of the limbic striatum. It is likely a third messenger in the intracellular cascade of events following neuronal stimulation by first-messenger activators of the adenylate cyclase system, including dopamine via the D1 receptor. ARPP-21 expression is restricted to telencephalic post-mitotic, post-migrational neurons, and its precise pattern of temporal and spatial expression makes it an attractive candidate for the study of transcriptional regulation of neuronal maturation. To define genomic regions likely to contain functional promoter elements, we isolated the murine ARPP-21 gene. Primer extension and T2 RNase protection analyses identified multiple transcription start sites, but 1.3 kb of 5'-flanking DNA revealed few consensus transcription factor binding sequences. A series of transient transfection assays in clonal cell lines which do not express ARPP-21 identified a basal promoter active in both neuronal and non-neuronal lines. Expression in all lines was decreased by the inclusion of regions further upstream, and extinguished by the inclusion of the first intron. Further analyses are likely to reveal cell specific regulatory sequences.
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PMID:ARPP-21: murine gene structure and promoter identification of a neuronal phosphoprotein enriched in the limbic striatum. 886 51

Perlecan, the basement membrane heparan sulfate proteoglycan (HSPG), has been fully cloned from mouse and human tissues. When a cRNA probe of murine perlecan cDNA was employed in RNase protection assay to test whether rat glomerular epithelial cells (GEC) constitutively express perlecan, several bands of hybridization were seen, suggesting that sequences between rat and murine perlecan may not be identical. Using primers based on published cDNA sequences of murine and human perlecan and poly A+ RNA of rat GEC, we synthesized a 497 bp product (RPD-I) by RT-PCR. The deduced aminoacid sequence showed an 85% and 88% homology with domain I of murine and human perlecan, respectively. The three putative sites containing the consensus sequence SGD for attachment of heparan sulfate chains were fully conserved in the rat perlecan as was a site (NFT) for attachment of N-linked oligosaccharide. RPD-I detected a > 9.5 kb transcript of perlecan in RNA of GEC, similar in size to that present in rat glomeruli. Employing a riboprobe synthesized from RPD-I in RNase protection assay we examined whether dbcAMP regulated perlecan expression in the GEC. At 1, 6, 24 and 48 h of incubation, 1 mM dbcAMP caused 43%, 32%, 47% and 40% reduction in mRNA abundance of perlecan, respectively. Immunoprecipitation showed a corresponding reduction of 61%, 70% and 65% in the synthesis of 35SO4 labeled basement membrane HSPG by the GEC following 12, 24 and 48 h of incubation with dbcAMP. Following incubation for 1 and 24 h prostaglandins, PGE1 and PGE2 (1 uM), known activators of glomerular adenylate cyclase, reduced perlecan mRNA abundance to a similar extent as dbcAMP on northern analysis. Our results show that glomerular basement membrane HSPG synthesized by the GEC belongs to the perlecan family. Decrease of GEC perlecan gene expression and synthesis by cAMP and prostaglandins may be of relevance to proteinuric states characterized by activation of these mediators.
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PMID:Cyclic AMP regulates basement membrane heparan sulfate proteoglycan, perlecan, metabolism in rat glomerular epithelial cells. 890 27

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide which was first isolated from ovine hypothalamic tissue by screening for pituitary adenylate cyclase stimulating activity. Our previous data showed that radioimmunoassayable PACAP and PACAP-binding sites were detected in the whole rat brain as early as embryonic day 14(E14). In order to understand more precisely the developmental pattern of the synthesis of PACAP and its receptors in the brain, we studied the expression of PACAP and its receptor genes in the prenatal and postnatal mouse brain using RNase protection assay. The mRNAs for both PACAP and its receptor were detected as early as 9.5 days of gestation (E9.5) in the whole head of mouse embryos. The levels of PACAP mRNA in the brain increased during the prenatal period peaking at postnatal day 0 (P0). On the other hand, the levels of PACAP receptor mRNA gradually increased after E9.5. The levels sharply increased at P6 (479.0 +/- 82.5% of P0 levels), and then fell to the P3 levels at P10. These data together with our previous study on the ontogeny of PACAP immunoreactivity and its binding sites in the rat brain support the view that PACAP plays an important regulatory role in the development of brain.
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PMID:Ontogeny of pituitary adenylate cyclase activating polypeptide and its receptor mRNA in the mouse brain. 895 77

We previously reported that ES20-receptor binding activates phosphoinositide (PI) turnover, resulting in an increase in inositol-1,4,5-trisphosphate, which in turn mobilizes intracellularly stored calcium in the vomeronasal (VN) sensory epithelium of garter snakes. We also found that the activity of adenylate cyclase (AC) in the VN organ is very sensitive to Ca2+ but insensitive to calmodulin regulation. A 250-bp fragment of adenylate cyclase type VI (AC-VI) was obtained from brain cDNA of garter snake by RT-PCR with degenerate primers. The 250-bp fragments were amplified, cloned, and sequenced. Both Northern blot and RNase protection assays revealed that the vomeronasal organ (VNO) and brain contained more abundance of AC type VI than the main olfactory epithelium. A 3.8-kb cDNA was then cloned from the vomeronasal cDNA library of garter snakes and sequenced. The 5' cDNA was obtained by means of 5' RACE PCR and sequenced. We have successfully cloned a 5200-nucleotide cDNA from VNO of garter snakes containing an open reading frame++ encoding 1150 amino acids of AC-VI protein. The vomeronasal AC is termed AC(VN) . AC(VN) shows a high degree of homology with type VI AC of rat, mouse, or human. In situ hybridization with digoxigenin-labeled cRNA demonstrated that AC(VN) mRNA was abundant in the sensory epithelium but not in the nonsensory epithelium of the mushroom body of the vomeronasal organ of garter snakes.
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PMID:Chemosignal transduction in the vomeronasal organ of garter snakes: cloning of a gene encoding adenylate cyclase from the vomeronasal organ of garter snakes. 984 34

Among the five members of the melanocortin receptor (MC-R) family, MC2 and MC5 are expressed in peripheral tissues. The receptor MC2 (ACTH receptor) almost exclusively expressed in the adrenal cortex whereas MC5-R is expressed in several organs including the adrenal cortex. Both receptors bind ACTH and activate adenylate cyclase. The aim of this work was to study the spatial distribution of MC5-R among the different zones of the bovine adrenal cortex and to analyze the regulation of its expression by its own ligands, ACTH and alpha-MSH and by angiotensin II (AII). Using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and RNase protection assay, MC5-R was detected only in the glomerulosa zone whereas MC2-R was present in both glomerulosa and fasciculata zones of adult adrenal cortex. Treatments by ACTH, alpha-MSH, or AII increased the MC5-R mRNA level in glomerulosa cells by factors 7, 5, and 4.5, respectively. However, although potentially regulated by hormones, MC5-R is expressed at a level at least 100 times less than MC2-R, suggesting that MC5-R expression might only be at trace levels in grown adults, but could be much higher during embryogenesis.
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PMID:Expression and regulation of melanocortin receptor-5 (MC5-R) in the bovine adrenal cortex. 1068 56

The basic organization of the human vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor (VPAC) 1 promoter was investigated after cloning the 5'-flanking region (1.4 kb) of the VPAC1 gene from a human genomic library. Subsequent functional analysis of various deletions of the 5'-flanking sequence, subcloned upstream of a luciferase reporter gene, was carried out in HT-29 cells. The minimal promoter region identified encompasses the -205/+76 sequence and contains a crucial CCAAT box (-182/-178) and a GC-rich sequence. Moreover a region (-1348/-933) containing a silencer element was identified. We previously showed that the expression of the VPAC1 receptor binding site is strictly dependent upon the enterocytic differentiation of human colon cancer Caco-2 cells [Laburthe, Rousset, Rouyer-Fessard, Couvineau, Chantret, Chevalier and Zweibaum (1987) J. Biol. Chem. 262, 10180-10184]. In the present study we show that VPAC1 mRNA increases dramatically when Caco-2Cl.20 cells differentiate, as measured by RNase protection assays and reverse transcriptase-PCR. A single transcript species of 3 kb is detected in differentiated cells by Northern-blot analysis. Accumulation of VPAC1 receptor mRNA is due to a 5-fold increase of transcription rate (run-on assay) without a change in mRNA half-life (9 h). Stable transfections of various constructs in Caco-2Cl.20 cells and subsequent analysis of reporter gene expression, during the enterocytic differentiation process over 25 days of culture, further indicated that the -254/+76 5'-flanking sequence is endowed with the regulatory element(s) necessary for transcriptional regulation of VPAC1 during differentiation. Altogether, these observations provide the first characterization of the basic organization of the human VPAC1 gene promoter and unravel the crucial role of a short promoter sequence in the strict transcriptional control of VPAC1 expression during differentiation of human colon cancer Caco-2 cells.
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PMID:The human vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor 1 (VPAC1) promoter: characterization and role in receptor expression during enterocytic differentiation of the colon cancer cell line Caco-2Cl.20. 1076 64

In Scrobicularia plana testis, a nuclear acid phosphatase (ACPase) activity was detected in mid and late spermatids with the improved Gomori-chloride procedure. Lead deposits were first observed in mid spermatids at focal points over condensed chromatin strands, increasing in density as chromatin further condensated. In late spermiogenesis, lead deposits became concentrated between chromatin aggregates, and after total DNA compaction were transfered to the nuclear periphery and then shed into the cytoplasm. The specificity of the nuclear ACPase was tested against different pH values (3.9, 7.2, 7.8, 9.0), substrates (TPP, IDP, TMP, p-NCS, ATP, GTP, AMP, ADP, AMP-PNP) and inhibitors (NaF, levamisole, Zn, vanadate, theophylline). To further specify the nature of this nuclear ACPase, other enzymes were comparatively studied at their optimal pH values and at pH 5.0: nucleoside-diphosphatase, thiamin-pyrophosphatase, inorganic trimetaphosphatase, lysosomal arylsulfatases A and B, ATPase, GTPase, 5'-nucleotidase, adenylate kinase, and adenylate cyclase. Several other controls were introduced to exclude artefactual deposits induced by lead ions and tissue molecules. The results showed that the enzyme has an optimal pH at 5.0, a high specific affinity for beta-GP, and is inhibited by NaF, which suggests that it behaves as a type B-ACPase, and all controls demonstrated the specificity of the enzymic activity. Because lead deposits were specifically and temporally associated with spermatid chromatin condensation, when DNA and RNA synthesis, histones, phosphoproteins and RNA molecules strongly decrease, it is possible to suggest that the nuclear ACPase could be associated with DNA processing during chromatin compaction or involved in the hydrolysis of 2' and 3' nucleotides resulting from nuclear RNase action during RNA degradation.
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PMID:Chromatin condensation during Scrobicularia plana spermiogenesis: a controlled and comparative enzymatic ultracytochemical study. 1079 22


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