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Query: UNIPROT:P06889 (Mol)
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The presence of gap junctions between osteoblastic cells has been previously reported. For this study we used the rat osteosarcoma cell line UMR 106, which expresses the osteoblastic phenotype, as a model to characterize further the nature, physiology, and regulation of gap junctions. Northern blot analysis identified a 3.0-kilobase RNA species corresponding to the gap junction protein connexin 43. The presence of two other connexin RNA species (26 and 32) could not be detected by this method in these cells. The identified connexin RNA was amplified by reverse transcription coupled to polymerase chain reaction; the sequence of the amplified product appears identical to the sequence of a cloned rat heart connexin 43 gene. After treatment with PTH, forskolin, and 8-Br-cAMP (a cAMP analog), the levels of connexin 43 RNA in UMR 106 cells increased. Further evidence for the role of PTH and cAMP in the physiology of gap junctions in these cells was obtained with Lucifer yellow dye transfer experiments. Gap-junctional intercellular communication increased in response to PTH and forskolin (an inducer of adenylate cyclase activity). Expression of connexin 43 RNA increased severalfold in response to PTH in a concentration- and time-dependent fashion. Connexin 43 RNA and its PTH-mediated stimulation were also observed in several other osteoblastic cell lines. The roles of PTH and forskolin in regulating the physiological state of gap junctions were confirmed in primary cultures of rat calvaria osteoblasts.
Mol Endocrinol 1992 Sep
PMID:Hormonal regulation of intercellular communication: parathyroid hormone increases connexin 43 gene expression and gap-junctional communication in osteoblastic cells. 133 76

The product of the CYP11A gene, cholesterol side chain cleavage cytochrome P450, catalyzes the initial step of steroidogenesis. A major mechanism whereby steroid hydroxylase gene transcription is regulated in the adrenal cortex requires the pituitary peptide hormone, ACTH, which acts via cAMP. We have previously identified a transcriptional enhancer in the 5'-flanking sequence [-183 to -83 base pairs (bp)] of the bovine CYP11A gene, which activates transcription of a beta-globin promoter/reporter gene in transiently transfected mouse Y1 adrenocortical tumor cells in response to the activator of adenylate cyclase, forskolin. Further deletion analysis has located the minimal cAMP-responsive sequence (CRS) to -118 to -100 bp. Analysis of DNA-protein interactions using nuclear extracts from Y1 cells revealed two protein binding sites, which were shown by competition analysis to be closely related to the two protein binding sites identified previously in the CRS of the human CYP21 gene. Namely, within the cAMP responsive fragment -118 to -100 bp, a sequence with a high degree of similarity to the consensus binding sequence for the ubiquitous transcription factor Sp1 is present, and binding of protein to this site was abolished by competition with excess GC box oligonucleotide. The second partially overlapping site is located 3' of the putative Sp1-binding site and binds to a protein identical or closely related to a putative adrenal-specific protein. Whereas the adrenal-specific protein binding site of the CYP21 CRS was previously shown to be sufficient to confer cAMP-responsive activation of transcription, the homologous site within the CYP11A CRS appears to have an attenuating effect on transcription.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1992 Oct
PMID:3',5'-cyclic adenosine monophosphate-dependent transcription of the CYP11A (cholesterol side chain cleavage cytochrome P450) gene involves a DNA response element containing a putative binding site for transcription factor Sp1. 133 53

Histamine acts on airway contractile elements through at least two different receptor subtypes: H1, which mediates Ca(2+)-dependent contraction, and H2, which stimulates cyclic adenosine monophosphate (cAMP) synthesis and possibly relaxation. The aim of this study was to determine the relative contribution of the different receptor subtypes to histamine-stimulated cAMP production by guinea pig tracheal smooth muscle (GPTSM) cells in primary culture. Histamine and N-alpha-methylhistamine induced concentration-dependent cAMP synthesis; these effects were entirely blocked by 10(-4) M cimetidine, an H2-receptor antagonist, whereas 10(-6) M thioperamide, a selective H3 blocker, was ineffective. The H3 agonist, R-(alpha)-methylhistamine, did not stimulate cAMP synthesis. Triprolidine, an H1 antagonist, did not modify histamine (10(-5) M)-stimulated cAMP synthesis. Histamine (10(-5) M) doubled [Ca2+]i in GPTSM. A 24-h pretreatment of GPTSM cells with 10(-6) M dexamethasone enhanced cAMP synthesized in response to 10(-5) M histamine and to 5 x 10(-6) M forskolin but did not significantly alter either the affinity or the binding capacity for [3H]-tiotidine, an H2-receptor antagonist. These results indicate that GPTSM cells in culture express H2 but not H3 receptors, which are linked to adenylate cyclase; their functional expression does not seem to be modulated by the concurrent activation of H1 receptors, whose presence in GPTSM is evidenced by a histamine-stimulated increase in [Ca2+]i. The most likely site of action of dexamethasone in enhancing histamine-stimulated cAMP synthesis is at the level of adenylate cyclase since the steroid had no effect on the H2 receptor itself.
Am J Respir Cell Mol Biol 1992 Dec
PMID:Identification of adenylate cyclase-coupled histamine H2 receptors in guinea pig tracheal smooth muscle cells in culture and the effect of dexamethasone. 133 44

In mammalian brain, physiological signals carried by cyclic AMP (cAMP) seem to be targeted to effector sites via the tethering of cAMP-dependent protein kinase II beta (PKAII beta) to intracellular structures. Recently characterized A kinase anchor proteins (AKAPs) are probable mediators of the sequestration of PKAII beta because they contain a high-affinity binding site for the regulatory subunit (RII beta) of the kinase and a distinct intracellular targeting domain. To establish a cellular basis for this targeting mechanism, we have employed immunocytochemistry to 1) identify the types of neurons that are enriched in AKAPs, 2) determine the primary intracellular location of the anchor protein, and 3) demonstrate that an AKAP and RII beta are coenriched and colocalized in neurons that utilize the adenylate cyclase-cyclic AMP-dependent protein kinase (PKA) signaling pathway. Antibodies directed against rat brain AKAP 150 were used to elucidate the regional, cellular and intracellular distribution of a prototypic anchor protein in the CNS. AKAP 150 is abundant in Purkinje cells and in neurons of the olfactory bulb, basal ganglia, cerebral cortex, and other forebrain regions. In contrast, little AKAP 150 is detected in neurons of the thalamus, hypothalamus, midbrain, and hindbrain. A high proportion of total AKAP 150 is concentrated in primary branches of dendrites, where it is associated with microtubules. We also discovered that the patterns of accumulation and localization of RII beta (and PKAII beta) in brain are similar to those of AKAP 150. The results suggest that bifunctional AKAP 150 tethers PKAII beta to the dendritic cytoskeleton, thereby creating a discrete target site for the reception and propagation of signals carried by cAMP.
Mol Biol Cell 1992 Nov
PMID:cAMP signaling in neurons: patterns of neuronal expression and intracellular localization for a novel protein, AKAP 150, that anchors the regulatory subunit of cAMP-dependent protein kinase II beta. 133 41

Interactions between beta-adrenergic and ADP purinergic receptors in C6 glioma cell membrane preparations were investigated under steady state and then pre-steady state conditions of adenylyl cyclase (EC 4.6.1.1) activity, in order to determine how fast the second receptor antagonizes the transduction mechanism of the first. Cell membranes were washed to deplete them as thoroughly as possible of low molecular weight compounds, especially ATP and ADP, and to ensure better control of both substrate and agonist nucleotide concentrations. ATP concentrations were kept constant with the use of an ATP-regenerating system; the C6 cell line exhibited very active ectonucleotidases. The purinergic agonist ADP was replaced by its nonhydrolyzable congener adenosine 5'-O-(2-thio)diphosphate (ADP beta S), which was demonstrated, like ADP, to inhibit isoproterenol-stimulated adenylyl cyclase activity in intact cells (IC50 for ADP, 0.5 +/- 0.1 microM; IC50 for ADP beta S, 25 +/- 2 microM) and in membrane preparations (IC50 for ADP beta S, 79 +/- 20 microM). In the case of membrane preparations, ADP beta S did not compete with ATP, the substrate of the cyclase-catalyzed reaction, and behaved apparently as a non-competitive inhibitor of the enzyme. The pre-steady state kinetics of isoproterenol-stimulated adenylyl cyclase activity measured with a pulsed quenched-flow apparatus have previously been shown to include two steps, the first very rapid (taking place within 1-2 sec) and giving rise to a burst of cAMP synthesis and the second much slower and corresponding to the steady state reaction. ADP beta S inhibited the occurrence of both steps with comparable IC50 values (mean value, 55 +/- 20 microM). In the presence of increasing concentrations of the purinergic receptor agonist, the time constant of the exponential burst reaction was not affected, but its amplitude progressively decreased to zero. These results showed that the extinction of the beta receptor cAMP response by the purinergic ADP receptor occurred within the dead-time of the pulsed quenched-flow apparatus, which was 50 msec. Such a rapid inhibition of cAMP production excluded modulation of isoproterenol-stimulated adenylyl cyclase activity by the ADP receptor by a pathway other than its direct negative coupling to the cyclase via a Gi protein. In this respect, the P2 purinergic ADP receptor of the C6 glioma cell line appears comparable to the P2t receptor of platelets.
Mol Pharmacol 1992 Dec
PMID:Pre-steady state study of beta-adrenergic and purinergic receptor interaction in C6 cell membranes: undelayed balance between positive and negative coupling to adenylyl cyclase. 133 11

In an effort to identify the signal transduction mechanism associated with the inhibition of juvenile hormone (JH) biosynthesis by the neuropeptides allatostatins, levels of the cyclic nucleotides cAMP and cGMP were measured in corpora allata (CA) of virgin and mated Diploptera punctata females using radioimmunoassays. Treatment of isolated CA with varying concentrations of synthetic allatostatins 1, 2, 3 or 4 did not elicit significant changes in the levels of either cAMP or cGMP in any of the test glands, suggesting that these compounds do not act as second messengers for the four allatostatins tested. Simultaneous treatment of CA with allatostatin 4 and the adenylate cyclase activator forskolin did not increase the degree of inhibition of juvenile hormone biosynthesis relative to that obtained with forskolin (5 or 50 microM) alone. We interpret these results as lending further support to the suggestion that cyclic nucleotides do not play a role in the signal transduction of allatostatins 1-4 in cockroach CA.
Mol Cell Endocrinol 1992 Nov
PMID:Assessment of the role of cyclic nucleotides in allatostatin-induced inhibition of juvenile hormone biosynthesis in Diploptera punctata. 133 20

Amiloride, a potent blocker of the sodium channel in airway epithelium, has been administered by aerosol as a therapeutic agent for cystic fibrosis. Because amiloride in high concentration has been reported to interfere with cell functions, including adrenergic responses, we tested the ability of amiloride to inhibit beta-adrenergic responses in human tracheal epithelial cells. Amiloride (10(-4) M), applied from the basolateral surface of a cell monolayer, inhibited the changes in transepithelial potential and short circuit current to isoproterenol (10(-6) M). The stimulation of cyclic adenosine monophosphate (cAMP) synthesis by isoproterenol was inhibited in dose-dependent fashion by amiloride (P = 0.007 by multivariate ANOVA with multiple samples correction). Amiloride did not affect baseline transepithelial potential, short circuit current, basal cAMP levels, cAMP response to prostaglandin E2, or basal adenylate cyclase activity measured directly in membrane preparations. Therefore, it is unlikely that amiloride exerts a nonspecific toxic effect on adenylate cyclase, receptor-cyclase coupling, or substrate or cofactor supply. The binding of [125I]iodocyanopindolol (ICYP), a beta-adrenergic receptor antagonist, to membranes from human tracheal epithelial cells could be displaced by amiloride with IC50 = 410 microM; displacement was 70% at 10(-3) M amiloride. These data are most consistent with the hypothesis that amiloride inhibits beta-adrenergic responses in airway epithelial cells by occupying beta-adrenergic receptor sites. Therapeutic administration of amiloride should take into account its affinity for adrenergic receptors.
Am J Respir Cell Mol Biol 1992 Feb
PMID:Amiloride antagonizes beta-adrenergic stimulation of cAMP synthesis and Cl- secretion in human tracheal epithelial cells. 134 24

Adenylate and guanylate cyclases, having different but related substrates, are a paradigm for the study of substrate discrimination. A prokaryotic adenylate cyclase gene, phylogenetically related to eukaryotic counterparts, was screened for mutants remodelling the enzyme's specificity. In a first step, a mutant was selected displaying a significant level of guanylate cyclase activity. This was due to a point mutation destroying most of the adenylate cyclase activity. A second selection step restored most of the original activity. This resulted from an additional mutation in the same region, thus permitting the first identification of a functional domain in adenylate and guanylate cyclases.
J Mol Biol 1992 Jun 20
PMID:From adenylate cyclase to guanylate cyclase. Mutational analysis of a change in substrate specificity. 135 50

The 98 amino acid (a.a.) N-terminus of the 126 a.a. atrial natriuretic factor (ANF) prohormone contains three peptides consisting of a.a. 1-30 (proANF 1-30), a.a. 31-67 (proANF 31-67) and a.a. 79-98 (proANF 79-98) with blood pressure lowering, sodium and/or potassium excreting properties similar to atrial natriuretic factor (a.a. 99-126, C-terminus of prohormone). ProANF 1-30 and proANF 31-67 have separate and distinct receptors from ANF in both vasculature and in the kidney to help mediate the above effects. At the cellular level proANFs 1-30, 31-67, and 79-98 as well as ANF's effects are mediated by enhancement of the guanylate cyclase (EC 4.6.1.2)-cyclic GMP system in vasculature and in the kidney. These peptides from the N-terminus of the ANF prohormone circulate normally in man and in all animal species tested. The object of the present investigation was to determine if these peptides have the ability to enhance either guanylate cyclase and/or adenylate cyclase in a variety of other tissues in addition to kidney and vasculature. ProANF 1-30, proANF 31-67, proANF 79-98, and ANF all increased rat lung, liver, heart and testes, but not spleen, particulate guanylate cyclase 2- to 3-fold at their 100 nM concentrations. Dose response curves revealed that maximal stimulation of particulate guanylate cyclase activity by these newly discovered peptides was at their 1 microM concentrations, with no further increase in activity above their 1 microM concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Biochem 1992 Jan 15
PMID:Peptides from the N-terminus of the atrial natriuretic factor prohormone enhance guanylate cyclase activity and increase cyclic GMP levels in a wide variety of tissues. 135 37

Using rat hepatocytes we confirmed our previous results that glucagon and beta-adrenergic agonists increased the enzyme activity of alanine aminotransferase (AAT) and propranolol abolished their effects. Only the enzyme activity was measured and other parameters like quantity of the enzyme or activation due to modification were not looked for. As in perfusion experiment phenylephrine and phenoxybenzamine (alpha-agonist and alpha-antagonist respectively) also alpha-antagonist respectively) also increased the AAT activity in isolated rat hepatocytes and propranolol reversed these effects. The additive effect of glucagon and phenoxybenzamine on AAT was also persistent in hepatocyte system. Fructose-1:6-bisphosphatase (Fru-P2-ase), another key enzyme in gluconeogenic pathway, was elevated by glucagon and other beta-adrenergic agonists both in liver perfusion and isolated hepatocyte experiments and was brought back to the normal level by propranolol. In this case also only the enzyme activity was measured and no other parameters were looked for. Unlike AAT this enzyme was not stimulated by phenylephrine or phenoxybenzamine. But AAT and Fru-P2-ase activities were increased significantly by adenylate cyclase activators like fluoride or forskolin. Thus, it appears that the regulation of fru-P2-ase by glucagon is purely a b-receptor mediated process whereas AAT activation shows a mixed type of regulation where some well known alpha-agonist and antagonists are behaving as beta-agonists. Results further indicate the presence of phosphodiesterase in hepatocyte membrane which was stimulated by glucagon and brought back to the normal level by propranolol. The different adrenergic compounds stated above, not only modified the activity of the above two enzymes but also stimulated glucose production by hepatocytes from alanine which was in turn abolished by propranolol as well as amino oxyacetate (AOA), a highly specified inhibitor of AAT. This confirm the participation of AAT in gluconeogenesis from alanine in liver. Forskolin and fluoride also increased the glucose production from alanine and showed additive effects with glucagon, phenylephrine and phenoxybenzamine.
Mol Cell Biochem 1992 Aug 18
PMID:Effect of adrenergic agonists and antagonists on alanine amino transferase, fructose-1:6-bisphosphatase and glucose production in hepatocytes. 135 93


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