Regulation of NHE3 by nitric oxide in Caco-2 cells.
The effect of nitric oxide (NO) on Na+/H+ exchange (NHE) activity was investigated utilizing Caco-2 cells as an experimental model. Incubation of Caco-2 cells with 10(-3) M S-nitroso-N-acetylpenicillamine (SNAP), a conventional donor of NO, for 20 min resulted in a approximately 45% dose-dependent decrease in NHE activity, as determined by assay of ethylisopropylamiloride-sensitive 22Na uptake. A similar decrease in NHE activity was observed utilizing another NO-specific donor, sodium nitroprusside. SNAP-mediated inhibition of NHE activity was not secondary to a loss of cell viability. NHE3 activity was significantly reduced by SNAP (P < 0.05), whereas NHE2 activity was essentially unaltered. The effects of SNAP were mediated by the cGMP-dependent signal transduction pathway as follows: 1) LY-83583 and 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), specific inhibitors of soluble guanylate cyclase, blocked the inhibitory effect of SNAP on NHE; 2) 8-bromo-cGMP mimicked the effects of SNAP on NHE activity; 3) the SNAP-induced decrease in NHE activity was counteracted by a specific protein kinase G inhibitor, KT-5823 (1 microM); 4) chelerythrine chloride (2 microM) or calphostin C (200 nM), specific protein kinase C inhibitors, did not affect inhibition of NHE activity by SNAP; 5) there was no cross activation by the protein kinase A-dependent pathway, as the inhibitory effects of SNAP were not blocked by Rp-cAMPS (25 microM), a specific protein kinase A inhibitor. These data provide novel evidence that NO inhibits NHE3 activity via activation of soluble guanylate cyclase, resulting in an increase in intracellular cGMP levels and activation of protein kinase G.
Section of Digestive and Liver Diseases, Department of Medicine, University of Illinois at Chicago and West Side Veterans Affairs Medical Center, Chicago, Illinois 60612, USA.
Ravinder K
RK
Gill
Seema
S
Saksena
Irfan Ali
IA
Syed
Sangeeta
S
Tyagi
Waddah A
WA
Alrefai
Jaleh
J
Malakooti
Krishnamurthy
K
Ramaswamy
Pradeep K
PK
Dudeja
D018938:Caco-2 Cells; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006152:Cyclic GMP / Q000502:physiology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000502:physiology; D004789:Enzyme Activation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D009569:Nitric Oxide / Q000494:pharmacology / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009573:Nitrites / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000502:physiology
D004791:Enzyme Inhibitors; D020030:Nitric Oxide Donors; D009573:Nitrites; C081517:SLC9A2 protein, human; C000613385:SLC9A3 protein, human; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D009569:Nitric Oxide; D026423:S-Nitroso-N-Acetylpenicillamine; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D011493:Protein Kinase C; D000254:Sodium-Potassium-Exchanging ATPase; D006152:Cyclic GMP