Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.48 (transcriptase)
 9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Evidence is presented that two isoforms of the voltage-dependent, dihydropyridine-sensitive calcium channel alpha 1 subunit are present in newborn and adult skeletal muscle and that expression of these isoforms is developmentally regulated. A voltage-dependent calcium channel alpha 1 cDNA from newborn muscle was cloned and found to be identical to that published from the adult, except that it was 2 kb shorter owing to an internal deletion. Nucleotide sequences, Northern blots, reverse-transcriptase PCR experiments, and sequencing of the PCR product confirmed that a segment corresponding to the inner two repeats of the structural prototype four homologous motifs is missing from the immature isoform. Immunological studies using antisera raised against synthetic peptides that correspond to sequences in the two isoforms show that the abbreviated transcript is predominant in newborn muscle, whereas the four-repeat isoform is the major species in the adult.
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PMID:A two-motif isoform of the major calcium channel subunit in skeletal muscle. 131 66

Dihydropyridine-sensitive voltage-dependent calcium channels (VDCC) play a crucial role in insulin secretion. We recently have cloned a human alpha 1-subunit of the VDCC expressed in pancreatic beta-cells, designated CACN4. In this study we have isolated complementary DNAs encoding two forms of rat CACN4 (rCACN4A and rCACN4B) from a rat insulinoma RINm5F complementary DNA library. Rat CACN4A is a protein of 2203 amino acids and is the rat homolog of human CACN4, whereas rCACN4B lacks 535 amino acids in the carboxyl-terminal region, probably due to alternative splicing. We have found two additional variations, one in the intracellular loop between repeats I and II and the other in the extracellular region between the third and fourth segments of repeat IV. Reverse transcriptase-polymerase chain reaction analysis of rat pancreatic islet messenger RNA reveals that these variants are present in pancreatic islets. In addition, whole-cell voltage-clamp recordings of Chinese hamster ovary cells stably expressing the alpha 1-subunit (rCACN4A or rCACN4B) with or without the calcium channel beta 2-subunit show that coexpression of rCACN4A with the beta 2-subunit or rCACN4B with the beta 2-subunit elicits L-type VDCC currents, whereas expression of the alpha 1-subunit alone does not, indicating that CACN4 can associate functionally with the beta 2-subunit and that the beta-subunit is essential for functional expression of CACN4. These results suggest that there are various subtypes of CACN4 expressed in pancreatic beta-cells, and that both rCACN4A and rCACN4B can function as VDCC. Furthermore, the present study suggests that the expression of the beta-subunit as well as the alpha 1-subunit may participate in the regulation of insulin secretion.
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PMID:Molecular diversity and functional characterization of voltage-dependent calcium channels (CACN4) expressed in pancreatic beta-cells. 776 Aug 45

The activation of osteoblast calcium channels by many bone regulatory factors suggests an important role for intracellular calcium signaling in the control of bone remodeling. At least six different genes for the alpha 1 subunit of voltage-gated calcium channels have been cloned including L-type (alpha 1S, alpha 1C, and alpha 1D) and non-L-type (alpha 1A, alpha 1B, and alpha 1E) isoforms. The goal of the present study was to identify which of these calcium channel isoforms are transcribed in human osteoblast-like cell lines (hFOB, MG-63, SAOS-2, TE-85, G-292) and in cultures of normal human osteoblasts. Reverse transcriptase-PCR was used to amplify sequences corresponding to each of the alpha 1 subunits using isoform specific primers. The products of the PCR reaction were cloned and sequenced to verify their identify and used to probe southern blots of the PCR reactions. The results indicate that among the different types of osteoblast-like cells examined, two calcium channel isoforms were always expressed (alpha 1C and alpha 1A), three isoforms were variably expressed (alpha 1S, alpha 1D and alpha 1B), and one isoform was not expressed in any of the osteoblast-like cells (alpha 1E) but was easily detected in human brain controls. Our results indicate that mRNAs for multiple calcium channel alpha 1 subunits are expressed in human osteoblasts, including both L-type and non-L-type isoforms. In addition, significant heterogeneity exists between the different osteoblast cell models examined in the type and mRNA abundance of the different calcium channel isoforms.
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PMID:Expression of mRNAs for the alpha 1 subunit of voltage-gated calcium channels in human osteoblast-like cell lines and in normal human osteoblasts. 1065 63

The intravenous calcium injection test has been reported to be useful for the diagnosis of gastrinoma. However, the mechanism underlying calcium-evoked gastrin release is not fully understood. We investigated the mechanism of calcium-stimulated gastrin release from gastrinoma cells in vitro with a particular focus on the calcium-sensing receptor (CaR). Human gastrinoma cells were taken from mechanically minced gastrinoma tissues obtained at surgery. In the perifusion system, high [Ca2+]o induced gastrin release from gastrinoma cells. As [Ca2+]o increased, [Ca2+]i rapidly increased, as monitored by fluorometry. The response was not inhibited by nifedipine, a blocker of the voltage-dependent calcium channel. Reverse transcriptase-polymerase chain reaction and subsequent Southern blot hybridization revealed the presence of the CaR gene in human gastrinoma tissues. Moreover, the expression of CaR in gastrinoma tissues was confirmed by immunohistochemistry. Our results demonstrated that CaR was expressed in human gastrinoma cells and could be involved in the mechanism of calcium-evoked gastrin release.
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PMID:Human gastrinoma cells express calcium-sensing receptor. 1178 38

Neuroendocrine differentiation of prostate epithelial cells is usually associated with an increased aggressivity and invasiveness of prostate tumors and a poor prognosis. However, the molecular mechanisms involved in this process remain poorly understood. We have investigated the possible expression of voltage-gated calcium channels in human prostate cancer epithelial LNCaP cells and their modulation during neuroendocrine differentiation. A small proportion of undifferentiated LNCaP cells displayed a voltage-dependent calcium current. This proportion and the calcium current density were significantly increased during neuroendocrine differentiation induced by long-term treatments with cyclic AMP permeant analogs or with a steroid-reduced culture medium. Biophysical and pharmacological properties of this calcium current suggest that it is carried by low-voltage activated T-type calcium channels. Reverse transcriptase-PCR experiments demonstrated that only a single type of LVA calcium channel mRNA, an alpha(1H) calcium channel mRNA, is expressed in LNCaP cells. Quantitative real-time reverse transcriptase-PCR revealed that alpha(1H) mRNA was overexpressed during neuroendocrine differentiation. Finally, we show that this calcium channel promotes basal calcium entry at resting membrane potential and may facilitate neurite lengthening. This voltage-dependent calcium channel could be involved in the stimulation of mitogenic factor secretion and could therefore be a target for future therapeutic strategies.
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PMID:Overexpression of an alpha 1H (Cav3.2) T-type calcium channel during neuroendocrine differentiation of human prostate cancer cells. 1179 14

In the search for P2-receptors modulating the stimulation-evoked entry of calcium at processes of PC12 cells differentiated in the presence of nerve growth factor and neurotrophin-3, electrically evoked increases in free calcium were assessed by fura-2 microfluorimetry. Omission of calcium and addition of cadmium (100 microM) or the N-type calcium channel blocker omega-conotoxin GVIA (0.5 microM) abolished or markedly reduced the evoked responses. The P2Y-receptor agonists 2-methylthio adenosine 5'-diphosphate (2-methylthio-ADP), ADP, and adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS) inhibited the electrically evoked entry of calcium without any changes in basal calcium concentrations. 2-Methylthio-ADP was the most potent agonist. Adenosine, P(1),P(4)-di(adenosine-5')-tetraphosphate (Ap4A), UDP, and UTP (30 microM each) had no effect. The effect of ADPbetaS (30 microM) was abolished by the P2-antagonists reactive blue 2 (3 microM), suramin (100 microM), 2-methylthio-AMP (10 microM), p-chloromercuriphenyl sulfonic acid (1 microM), and AR-C 69931MX [N(6)-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene adenosine 5'-triphosphate] (300 nM). In contrast, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (10 microM), the selective P2Y1-receptor antagonist MRS 2179 (N(6)-methyl-2'-deoxyadenosine 3',5'-bisphosphate; 10 microM), as well as the adenosine A(1)-receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine; 100 nM), caused no change. Pretreatment with pertussis toxin abolished the effect of ADPbetaS. Reverse transcriptase-polymerase chain reaction revealed the presence of mRNA for P2Y12-receptors in nondifferentiated and differentiated PC12 cells. The results indicate that processes of differentiated PC12 cells possess P2Y12-receptors coupling to pertussis toxin-sensitive G-proteins and mediating an inhibition of the stimulation-evoked entry of calcium through omega-conotoxin GVIA-sensitive calcium channels. This suggests a role of P2Y12-receptors in neuromodulation in addition to their involvement in platelet aggregation.
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PMID:P2Y-receptors mediating an inhibition of the evoked entry of calcium through N-type calcium channels at neuronal processes. 1238 31

Voltage-gated calcium (Ca2+) channels play a key role in the control of heart contraction and are essential for normal heart development. The Cav1.2 L-type calcium channel is the predominant isoform in cardiomyocytes and is essential for excitation-contraction coupling. Although the inactivation of the Cav1.2 gene caused embryonic lethality before embryonic day E14.5, hearts were contracting before E14 depending on a dihydropyridine-sensitive calcium influx. We analyzed the consequences of the deletion of the Cav1.2 channel on the expression level of other voltage-gated calcium channels in the embryonic mouse heart and isolated cardiomyocytes. A strong compensatory up-regulation of the Cav1.3 calcium channel was observed on the mRNA as well as on the protein level. Reverse transcriptase PCR indicated that the recently identified new Cav1.3(1b) isoform was strongly up-regulated, whereas a more moderate increase was found for the Cav1.3(1a) variant. Heterologous expression of Cav1.3(1b) in HEK293 cells induced Ba2+ currents with properties similar to those found in Cav1.2 (-/-) cardiomyocytes, suggesting that this isoform constitutes a major component of the residual L-type calcium current in Cav1.2 (-/-) cardiomyocytes. In summary, our results imply that calcium channel expression is dynamically regulated during heart development and that the Cav1.3 channel may substitute for Cav1.2 during early embryogenesis.
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PMID:Enhanced expression of L-type Cav1.3 calcium channels in murine embryonic hearts from Cav1.2-deficient mice. 1290 Apr

Calcium influx activates biosynthesis of the endogenous cannabinoids 2-arachidonyl glycerol (2-AG) and anandamide (AEA). The calcium channel involved with endocannabinoid synthesis and release in neurons is still unknown. The canonical TRP (TRPC) channels are calcium-permeable channels that are a homology-based subdivision of the broader class of TRP channels. TRPC3, 6, and 7 are G-protein-gated non-selective cation channels that have been localized to lipid rafts and shown to colocalize with caveolin 1. Because endocannabinoid synthesis has been found to occur "on demand" in a calcium-dependent manner and has been linked to lipid rafts, we explored the potential role of transient receptor potential (TRP) channels in this process. Previously, we observed that after metabolism AEA and arachidonic acid (ArA) can be recycled into new endocannabinoid molecules. Consistent with these previous findings, we found that Cath.a differentiated (CAD) cells pretreated with radiolabeled ArA exhibited a robust increase in 2-AG release in response to TRPC stimulation with the diacylglycerol (DAG) analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG). Furthermore, cells pretreated with [(3)H]AEA produced a significant amount of AEA and 2-AG upon stimulation of TRPC channels. This process was not mediated through protein kinase C activation. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that only TRPC6 was present in the CAD cells. siRNA-induced knockdown of TRPC6 in the CAD cells abolished OAG-stimulated production of the endocannabionids. This evidence suggests that TRPC6 may be capable of promoting endocannabinoid synthesis in neuronal cells.
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PMID:Activation of TRPC6 channels promotes endocannabinoid biosynthesis in neuronal CAD cells. 2046 28

It has been demonstrated that neuronal cells cultured on traditional flat surfaces may exhibit exaggerated voltage gated calcium channel (VGCC) functionality. To gain a better understanding of this phenomenon, primary neuronal cells harvested from mice superior cervical ganglion (SCG) were cultured on two dimensional (2D) flat surfaces and in three dimensional (3D) synthetic poly-L-lactic acid (PLLA) and polystyrene (PS) polymer scaffolds. These 2D- and 3D-cultured cells were compared to cells in freshly dissected SCG tissues, with respect to intracellular calcium increase in response to high K(+) depolarization. The calcium increases were identical for 3D-cultured and freshly dissected, but significantly higher for 2D-cultured cells. This finding established the physiological relevance of 3D-cultured cells. To shed light on the mechanism behind the exaggerated 2D-cultured cells' functionality, transcriptase expression and related membrane protein distributions (caveolin-1) were obtained. Our results support the view that exaggerated VGCC functionality from 2D cultured SCG cells is possibly due to differences in membrane architecture, characterized by uniquely organized caveolar lipid rafts. The practical implication of use of 3D-cultured cells in preclinical drug discovery studies is that such platforms would be more effective in eliminating false positive hits and as such improve the overall yield from screening campaigns.
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PMID:Three dimensional neuronal cell cultures more accurately model voltage gated calcium channel functionality in freshly dissected nerve tissue. 2304 67