Roles of Ca2+ and protein tyrosine kinase in insulin action on cell volume via Na+ and K+ channels and Na+/K+/2Cl- cotransporter in fetal rat alveolar type II pneumocyte.
The aim of the present study was to investigate the roles of Ca2+ and protein tyrosine kinase (PTK) in the insulin action on cell volume in fetal rat (20-day gestational age) type II pneumocytes. Insulin (100 nm) increased cell volume in the presence of extracellular Ca2+ (1 mm), while cell shrinkage was induced by insulin in the absence of extracellular Ca2+ (<1 nm). This insulin action in a Ca2+-containing solution was completely blocked by co-application of bumetanide (50 microm, an inhibitor of Na+/K+/2Cl- cotransporter) and amiloride (10 microm, an inhibitor of epithelial Na+ channel), but not by the individual application of either bumetanide or amiloride. On the other hand, the insulin action on cell volume in a Ca2+-free solution was completely blocked by quinine (1 mm, a blocker of Ca2+-activated K+ channel), but not by bumetanide and/or amiloride. These observations suggest that insulin activates an amiloride-sensitive Na+ channel and a bumetanide-sensitive Na+/K+/2Cl- cotransporter in the presence of 1 mm extracellular Ca2+, that the stimulatory action of insulin on an amiloride-sensitive Na+ channel and a bumetanide-sensitive Na+/K+/2Cl- cotransporter requires Ca2+, and that in a Ca2+-free solution insulin activates a quinine-sensitive K+ channel but not in the presence of 1 mm Ca2+. The insulin action on cell volume in a Ca2+-free solution was almost completely blocked by treatment with BAPTA (10 microm) or thapsigargin (1 microM, an inhibitor of Ca2+-ATPase which depletes the intracellular Ca2+ pool). Further, lavendustin A (10 microm, an inhibitor of receptor type PTK) blocked the insulin action in a Ca2+-free solution. These observations suggest that the stimulatory action of insulin on a quinine-sensitive K+ channel is mediated through PTK activity in a cytosolic Ca2+-dependent manner. Lavendustin A, further, completely blocked the activity of the Na+/K+/2Cl- cotransporter in a Ca2+-free solution, but only partially blocked the activity of the Na+/K+/2Cl- cotransporter in the presence of 1 mm Ca2+. This observation suggests that the activity of the Na+/K+/2Cl- cotransporter is maintained through two different pathways; one is a PTK-dependent, Ca2+-independent pathway and the other is a PTK-independent, Ca2+-dependent pathway. Further, we observed that removal of extracellular Ca2+ caused cell shrinkage by diminishing the activity of the amiloride-sensitive Na+ channel and the bumetanide-sensitive Na+/K+/2Cl- cotransporter, and that removal of extracellular Ca2+ abolished the activity of the quinine-sensitive K+ channel. We conclude that the cell shrinkage induced by removal of extracellular Ca2+ results from diverse effects on the cotransporter and Na+ and K+ channels.
Lung Biology and MRC Group in Lung Development, Hospital for Sick Children Research Institute and Department of Pediatrics, University of Toronto Faculty of Medicine, Toronto, Ontario, Canada M5G 1X8.
Y
Y
Marunaka
N
N
Niisato
H
H
O'Brodovich
M
M
Post
A K
AK
Tanswell
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002034:Bumetanide / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002352:Carrier Proteins / Q000502:physiology*; D048429:Cell Size / Q000187:drug effects; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D007231:Infant, Newborn; D007328:Insulin / Q000494:pharmacology*; D010636:Phenols / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D015221:Potassium Channels / Q000187:drug effects* / Q000502:physiology; D011247:Pregnancy; D011254:Pregnancy in Diabetics; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D011650:Pulmonary Alveoli / Q000166:cytology / Q000187:drug effects* / Q000196:embryology / Q000378:metabolism; D011803:Quinine / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D011972:Receptor, Insulin / Q000187:drug effects; D012127:Respiratory Distress Syndrome, Newborn / Q000503:physiopathology; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D015222:Sodium Channels / Q000187:drug effects* / Q000502:physiology; D028021:Sodium-Potassium-Chloride Symporters; D019284:Thapsigargin / Q000494:pharmacology
D002352:Carrier Proteins; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D007328:Insulin; D010636:Phenols; D015221:Potassium Channels; D015222:Sodium Channels; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide; C062898:lavendustin A; D004533:Egtazic Acid; D019284:Thapsigargin; D000584:Amiloride; D011803:Quinine; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D000252:Calcium-Transporting ATPases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium