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- Garcia-Guzman, M, et al.
(author)
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Characterization of recombinant human P2X4 receptor reveals pharmacological differences to the rat homologue.
- 1997
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In: Molecular Pharmacology. - 0026-895X .- 1521-0111. ; 51:1, s. 109-18
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Journal article (peer-reviewed)abstract
- We isolated a cDNA from human brain encoding a purinergic receptor that shows a high degree of homology to the rat P2X4 receptor (87% identity). By fluorescence in situ hybridization, the human P2X4 gene has been mapped to region q24.32 of chromosome 12. Tissue distribution analysis of human P2X4 transcripts demonstrates a broad expression pattern in that the mRNA was detected not only in brain but also in all tissues tested. Heterologous expression of the human P2X4 receptor in Xenopus laevis oocytes and human embryonic kidney 293 cells evoked an ATP-activated channel. Simultaneous whole-cell current and Fura-2 fluorescence measurements in human embronic kidney 293 cells transfected with human P2X4 cDNA allowed us to determine the fraction of the current carried by Ca2: this was approximately 8%, demonstrating a high Ca2+ permeability. Low extracellular Zn2+ concentrations (5-10 microM) increase the apparent gating efficiency of human P2X4 by ATP without affecting the maximal response. However, raising the concentration of the divalent cation (> 100 microM) inhibits the ATP-evoked current in a non-voltage-dependent manner. The human P2X4 receptor displays a very similar agonist potency profile to that of rat P2X4 (ATP > > 2-methylthio-ATP > or = CTP > alpha, beta-methylene-ATP > dATP) but has a notably higher sensitivity for the antagonists suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid, and bromphenol blue. Chimeric constructs between human and rat isoforms as well as single-point mutations were engineered to map the regions responsible for the different sensitivity to suramin and pyridoxal-phosphate-6-azophenyl-2'4'-disulfonic acid.
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- Nilsson, B O, et al.
(author)
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Differential actions of exogenous and intracellular spermine on contractile activity in smooth muscle of rat portal vein
- 1995
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In: Acta Physiologica Scandinavica. - : Wiley. - 0001-6772 .- 1365-201X. ; 154:3, s. 65-355
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Journal article (peer-reviewed)abstract
- Effects of the naturally occurring polyamine spermine on electrical and contractile properties of the rat portal vein were studied. 1 mM spermine nearly abolished spike activity and spontaneous contractions and decreased the intracellular Ca2+ concentration ([Ca2+]i). The phasic force responses to 0.1 and 1 microM phenylephrine were partially inhibited, but not the sustain plateau contraction caused by 5 microM phenylephrine. The Ca(2+)-force relation in high-K+ (128 mM)-depolarized veins was shifted to the right, EC50 for Ca2+ increasing from 0.50 +/- 0.03 mM (control, n = 8) to 0.65 +/- 0.06 and to 0.94 +/- 0.03 at 1 (n = 4) and 10 (n = 3) mM spermine, respectively. However, at a Ca2+ concentration of 2.5 mM, giving maximal force, there was no effect of spermine (1 mM) on either force or [Ca2+]i. Whereas extracellular spermine thus reduced contractile activity at moderate levels of stimulation, increased intracellular concentration of spermine potentiated the force response to Ca2+. Intracellular loading of spermine by reversible permeabilization increased its concentration by 2-3 times. The spontaneous activity and response to phenylephrine were unchanged. However, the Ca(2+)-force relation of depolarized veins was shifted to the left, EC50 decreasing from 0.51 +/- 0.04 mM in controls (n = 7) to 0.36 +/- 0.02 mM in the loaded veins (n = 9). Spermine increased Ca(2+)-activated force in portal veins permeabilized with beta-escin. The degree of potentiation was consistent with observed effects in spermine-loaded intact veins. The results suggest that spermine at physiological intracellular concentration may contribute to the determination of Ca2+ sensitivity in vascular smooth muscle cells.
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