Serotonin inhibits Na+/H+ exchange activity via 5-HT4 receptors and activation of PKC alpha in human intestinal epithelial cells.
OBJECTIVE
Increased serotonin levels have been implicated in the pathophysiology of diarrhea associated with celiac and inflammatory diseases. However, the effects of serotonin on Na+ /H+ exchange (NHE) activity in the human intestine have not been investigated fully. The present studies examined the acute effects of 5-hydroxytryptamine (5-HT) on NHE activity using Caco-2 cells as an in vitro model.


METHODS
Caco-2 cells were treated with 5-HT (.1 micromol/L, 1 h) and NHE activity was measured as ethyl-isopropyl-amiloride (EIPA)-sensitive 22Na uptake. The effect of 5-HT receptor-specific agonists and antagonists was examined. The role of signaling intermediates in 5-HT-mediated effects on NHE activity was elucidated using pharmacologic inhibitors and immunoblotting.


RESULTS
NHE activity was inhibited significantly (approximately 50%-75%, P < .05) by .1 micromol/L 5-HT via inhibition of maximal velocity (Vmax) without any changes in apparent affinity (Km) for the substrate Na+ . NHE inhibition involved a decrease of both NHE2 and NHE3 activities. Studies using specific inhibitors and agonists showed that the effects of 5-HT were mediated by 5-HT4 receptors. 5-HT-mediated inhibition of NHE activity was dependent on phosphorylation of phospholipase C gamma 1 (PLC gamma 1) via activation of src-kinases. Signaling pathways downstream of PLC gamma 1 involved increase of intracellular Ca 2+ levels and subsequent activation of protein kinase C alpha (PKC alpha). The effects of 5-HT on NHE activity were not cell-line specific because T84 cells also showed NHE inhibition.


CONCLUSIONS
A better understanding of the regulation of Na+ absorption by 5-HT offers the potential for providing insights into molecular and cellular mechanisms involved in various diarrheal and inflammatory disorders.
Gill
Ravinder K
RK
Section of Digestive Diseases and Nutrition, University of Illinois at Chicago, Jesse Brown VA Medical Center, IL 60612, USA.
Saksena
Seema
S
Tyagi
Sangeeta
S
Alrefai
Waddah A
WA
Malakooti
Jaleh
J
Sarwar
Zaheer
Z
Turner
Jerrold R
JR
Ramaswamy
Krishnamurthy
K
Dudeja
Pradeep K
PK
D018938:Caco-2 Cells; D020013:Calcium Signaling / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology / Q000378:metabolism*; D051966:Phospholipase C gamma; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D044422:Receptors, Serotonin, 5-HT4 / Q000378:metabolism*; D012701:Serotonin / Q000494:pharmacology*; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors*; D010738:Type C Phospholipases / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism
C081517:SLC9A2 protein, human; C000613385:SLC9A3 protein, human; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D044422:Receptors, Serotonin, 5-HT4; D012701:Serotonin; D011505:Protein-Tyrosine Kinases; D019061:src-Family Kinases; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D010738:Type C Phospholipases; D051966:Phospholipase C gamma