Abstract
Basal calcium leak into smooth muscle was identified 30 years ago yet remains poorly understood. We characterized this leak measuring 45Ca2+ uptake into cultured rat aortic smooth muscle cells. Wash solution (0 degrees C) containing lanthanum (3 mM) removed extracellular tracer and increased cellular 45Ca2+ retention more effectively than EGTA (0.2 mM). Basal Ca2+ entry was 1.45 x 10(9) Ca2+ x cell(-1) x min(-1). This translated to approximately 250 micromol(-1) x min(-1) given cell volumes of 4-15 pl as determined by 3-D image reconstruction. Gadolinium (100 microM) blocked 80% of the leak and exhibited a biphasic concentration-response relation (IC50s=1 microM and 2 mM). Organic ion channel blockers also inhibited approximately 80% of the leak; 45% by nifedipine (10 microM), 7% was exclusively blocked by SKF 96365 (1-[b-[3-(4-Methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole) (50 microM) and 23% was exclusively sensitive to 2-aminoethoxy-diphenylborate (2-APB, 75 microM). Reverse transcriptase polymerase chain reaction revealed TrpC1, 4 and 6 mRNA, and we propose that 2-APB may selectively block TrpC4-containing channels. We conclude that basal Ca2+ entry is mainly due to a basal open probability of excitable Ca2+ -channels.
Publication types
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Comparative Study
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Boron Compounds / pharmacology
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Calcium / metabolism*
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Calcium / pharmacokinetics
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Calcium Channel Blockers / pharmacology
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Calcium Channels / genetics
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Calcium Radioisotopes
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Dose-Response Relationship, Drug
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Egtazic Acid / pharmacology
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Gadolinium / pharmacology
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism
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Imidazoles / pharmacology
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Lanthanum / pharmacology
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Male
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Membrane Proteins / genetics
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Microscopy, Confocal
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Muscle, Smooth, Vascular / cytology
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Muscle, Smooth, Vascular / drug effects
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Muscle, Smooth, Vascular / metabolism*
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Nerve Tissue Proteins / genetics
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Nifedipine / pharmacology
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RNA, Messenger / genetics
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RNA, Messenger / metabolism
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Rats
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Rats, Inbred WKY
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Reverse Transcriptase Polymerase Chain Reaction
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Synaptosomal-Associated Protein 25
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TRPC Cation Channels
Substances
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Boron Compounds
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Calcium Channel Blockers
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Calcium Channels
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Calcium Radioisotopes
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Imidazoles
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Membrane Proteins
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Nerve Tissue Proteins
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RNA, Messenger
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Recombinant Fusion Proteins
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Snap25 protein, rat
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Synaptosomal-Associated Protein 25
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TRPC Cation Channels
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transient receptor potential cation channel, subfamily C, member 1
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Green Fluorescent Proteins
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Egtazic Acid
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Lanthanum
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Gadolinium
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2-aminoethoxydiphenyl borate
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1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole
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Nifedipine
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Calcium