Activation of the Na+-K+(NH4+)-2Cl(-)- cotransporter from rat submandibular glands in response to VIP.
A cellular suspension from rat submandibular glands was prepared with collagenase. The intracellular pH (pHi) was estimated with 2',7'-bis-(2-carboxy-ethyl)-5(6)-carboxyfluorescein (BCECF). After exposure to NH4Cl, the pHi transiently increased (diffusion of NH3) and then dropped (influx of NH4+). Isoproterenol increased 2.5-fold the rate of NH4+ influx; bumetanide, an inhibitor of the Na+-K+-2Cl(-)-cotransporter blocked the response to isoproterenol, confirming that the beta-adrenergic agonist stimulated the cotransporter. Forskolin (1 micromol/L) mimicked the response to isoproterenol. VIP (1 nmol/L(-1) micromol/L) also increased the activity of the cotransporter. Cyclic AMP rather than calcium was the mediator of this activation since 1) carbachol which increased the [Ca2+]i fivefold increased the uptake of NH4+ by only 50%; 2) only high concentrations of VIP significantly increased the [Ca2+]i; 3) incubation in the presence of EGTA had no effect on the response to VIP; 4) low concentrations (nmol/L) of the neuropeptide increased the intracellular level of cAMP; and 5) the stimulation of the cotransporter by VIP, forskolin, and isoproterenol was inhibited by H8, an inhibitor of cAMP-dependent protein kinase. It is concluded that the Na+-K+-2Cl(-)-cotransporter of rat submandibular glands is activated by isoproterenol, forskolin, and neuropeptides of the VIP family by a mechanism involving cAMP-dependent processes. The activation of the cotransporter by VIP could partly explain the potentiating effect of VIP on the response to sialagogues like substance P or muscarinic agonists.
Chaïb
N
N
Laboratoire de Biochimie générale et humaine, Institut de Pharmacie, Université libre de Bruxelles, Brussels, Belgium.
Kabré
E
E
Métioui
M
M
Alzola
E
E
Amsallem
H
H
Marino
A
A
Moran
A
A
Dehaye
J P
JP
D000643:Ammonium Chloride / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002034:Bumetanide / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000502:physiology; D002217:Carbachol / Q000494:pharmacology; D002352:Carrier Proteins / Q000819:agonists / Q000378:metabolism* / Q000502:physiology; D018679:Cholinergic Agonists / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid / Q000737:chemistry; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D028021:Sodium-Potassium-Chloride Symporters; D013363:Submandibular Gland / Q000187:drug effects* / Q000201:enzymology*; D014660:Vasoactive Intestinal Peptide / Q000008:administration & dosage / Q000494:pharmacology*
D002352:Carrier Proteins; D018679:Cholinergic Agonists; D005452:Fluoresceins; D005456:Fluorescent Dyes; D028021:Sodium-Potassium-Chloride Symporters; D000643:Ammonium Chloride; D002034:Bumetanide; D014660:Vasoactive Intestinal Peptide; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D002217:Carbachol; D000242:Cyclic AMP; D007545:Isoproterenol; D002118:Calcium