Secretagogue stimulation enhances NBCe1 (electrogenic Na(+)/HCO(3)(-) cotransporter) surface expression in murine colonic crypts.
A Na(+)/HCO(3)(-) cotransporter (NBC) is located in the basolateral membrane of the gastrointestinal epithelium, where it imports HCO(3)(-) during stimulated anion secretion. Having previously demonstrated secretagogue activation of NBC in murine colonic crypts, we now asked whether vesicle traffic and exocytosis are involved in this process. Electrogenic NBCe1-B was expressed at significantly higher levels than electroneutral NBCn1 in colonic crypts as determined by QRT-PCR. In cell surface biotinylation experiments, a time-dependent increase in biotinylated NBCe1 was observed, which occurred with a peak of +54.8% after 20 min with forskolin (P < 0.05) and more rapidly with a peak of +59.8% after 10 min with carbachol (P < 0.05) and which corresponded well with the time course of secretagogue-stimulated colonic bicarbonate secretion in Ussing chamber experiments. Accordingly, in isolated colonic crypts pretreated with forskolin and carbachol for 10 min, respectively, and subjected to immunohistochemistry, the NBCe1 signal showed a markedly stronger colocalization with the E-cadherin signal, which was used as a membrane marker, compared with the untreated control. Cytochalasin D did not change the observed increase in membrane abundance, whereas colchicine alone enhanced NBCe1 membrane expression without an additional increase after carbachol or forskolin, and LY294002 had a marked inhibitory effect. Taken together, our results demonstrate a secretagogue-induced increase of NBCe1 membrane expression. Vesicle traffic and exocytosis might thus represent a novel mechanism of intestinal NBC activation by secretagogues.
Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Germany.
Haoyang
H
Yu
Brigitte
B
Riederer
Nicole
N
Stieger
Walter F
WF
Boron
Gary E
GE
Shull
Michael P
MP
Manns
Ursula E
UE
Seidler
Oliver
O
Bachmann
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism*; D019921:Biotinylation; D015820:Cadherins / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism; D018679:Cholinergic Agonists / Q000494:pharmacology; D003078:Colchicine / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D003106:Colon / Q000187:drug effects* / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D003592:Cytophotometry; D004305:Dose-Response Relationship, Drug; D020536:Enzyme Activators / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects; D005765:Gastrointestinal Agents / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D007413:Intestinal Mucosa / Q000187:drug effects* / Q000378:metabolism; D007419:Intestinal Secretions / Q000187:drug effects*; D008564:Membrane Potentials; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D016133:Polymerase Chain Reaction; D022142:Secretory Vesicles / Q000187:drug effects / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D027982:Sodium-Bicarbonate Symporters / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors
D001639:Bicarbonates; D015820:Cadherins; D018679:Cholinergic Agonists; D020536:Enzyme Activators; D005765:Gastrointestinal Agents; C498899:Slc4a4 protein, mouse; D027982:Sodium-Bicarbonate Symporters; D005576:Colforsin; D002217:Carbachol; D012964:Sodium; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D003078:Colchicine