Volume-dependent regulation of sodium and potassium fluxes in cultured vascular smooth muscle cells: dependence on medium osmolality and regulation by signalling systems.
To identify ion transport systems involved in the maintenance of vascular smooth muscle cell volume the effects of incubation medium osmolality and ion transport inhibitors on the volume and 86Rb and 22Na transport in cultured smooth muscle cells from rat aorta (VSMC) have been studied. A decrease of medium osmolality from 605 to 180 mosm increased intracellular water volume from 0.6 to 1.3 microliters per 10(6) cells. Under isosmotic conditions, cell volume was decreased by ouabain (by 10%, P less than 0.005) but was not influenced by bumetanide, furosemide, EIPA and quinidine. These latter compounds were also ineffective in cell volume regulation under hypotonic buffer conditions. Under hyperosmotic conditions, cell volume was decreased by bumetanide (by approximately 7%, P less than 0.05) and by ethylisopropyl amiloride (by approximately 13%, P less than 0.005). Ouabain-sensitive 86Rb influx was decreased by 30-40% under hypoosmotic conditions. An increase in medium osmolality from 275 to 410 mosm resulted in an approximately eightfold increase in bumetanide-inhibited 86Rb influx and 86Rb efflux. The (ouabain and bumetanide)-insensitive component of 86Rb influx was not dependent on the osmolality of the incubation medium. However (ouabain and bumetanide)-insensitive 86Rb efflux was increased by approximately 1.5-2 fold in VSMC incubated in hypotonic medium. Ethylisopropyl amiloride-inhibited 22Na influx was increased by approximately sixfold following osmotic-shrinkage of VSMC. The data show that both Na+/H+ exchange and Na+/K+/2Cl- cotransport may play a major role in the regulatory volume increase in VSMC. Basal and shrinkage-induced activities of Na+/K+/2Cl- cotransport in VSMC were similarly sensitive to inhibition by either staurosporin, forskolin, R24571 or 2-nitro-4-carboxyphenyl N,N-diphenylcarbomate (NCDC). In contrast basal and shrinkage-induced Na+/K+/2Cl- cotransport were differentially inhibited by NaF (by 30 and 65%, respectively), suggesting an involvement of guanine nucleotide binding proteins in the volume-sensitive activity of this carrier. Neither staurosporin, forskolin, R24571 nor NCDC influenced shrinkage-induced Na+/H+ exchange activity. NaF increased Na+/H+ exchanger activity under both isosmotic and hyperosmotic conditions. These data demonstrate that different intracellular signalling mechanisms are involved in the volume-dependent activation of the Na+/K+/2Cl- cotransporter and the Na+/H+ exchanger.
Orlov
S N
SN
Department of Research, University Hospitals, Basel, Switzerland.
Resink
T J
TJ
Bernhardt
J
J
Buhler
F R
FR
D000470:Alkaloids / Q000494:pharmacology; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002034:Bumetanide / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002352:Carrier Proteins / Q000502:physiology; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019204:GTP-Binding Proteins / Q000502:physiology; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000502:physiology*; D009994:Osmolar Concentration; D010042:Ouabain / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000502:physiology; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D051381:Rats; D011921:Rats, Inbred WKY; D012414:Rubidium Radioisotopes; D015290:Second Messenger Systems / Q000187:drug effects / Q000502:physiology*; D015222:Sodium Channels / Q000187:drug effects / Q000502:physiology*; D012969:Sodium Fluoride / Q000494:pharmacology; D012979:Sodium Radioisotopes; D017923:Sodium-Hydrogen Exchangers; D028021:Sodium-Potassium-Chloride Symporters; D019311:Staurosporine
D000470:Alkaloids; D002147:Calmodulin; D002352:Carrier Proteins; D010716:Phosphatidylinositols; D015221:Potassium Channels; D012414:Rubidium Radioisotopes; D015222:Sodium Channels; D012979:Sodium Radioisotopes; D017923:Sodium-Hydrogen Exchangers; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide; D005576:Colforsin; D010042:Ouabain; D000584:Amiloride; D012969:Sodium Fluoride; D000242:Cyclic AMP; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019311:Staurosporine