Stimulation-associated redistribution of H+-K+-ATPase activity in isolated gastric glands.
The objective of this work is to establish a procedure to study the stimulation-dependent membrane redistribution and properties of H+-K+-ATPase in an in vitro model system, rabbit isolated gastric glands. Stimulated (10(-4) M histamine plus 10(-5) M forskolin) and resting (10(-4) M metiamide) glands were homogenized and fractionated into PO (40 g, 5 min), P1 (400 g, 10 min), P2 (14,500 g, 10 min), P3 (48,200 g, 90 min), and supernatant, S3. Significant changes occurred in the distribution of our marker for H+-K+-ATPase (K+-p-nitrophenyl phosphatase) activity: a reduction in activity of P3 and a compensatory increment in P1. P3 showed valinomycin (Val)-dependent vesicular H+ uptake, while H+ uptake in P1 was Val independent. Direct measurements of ATPase revealed that H+-K+-ATPase activity of P3 was Val dependent and decreased by stimulation; H+-K+-ATPase activity of P1 was Val independent and increased by stimulation. Further density gradient purification of P1 showed that membranes lighter than 17% Ficoll contained higher specific H+-K+-ATPase activity, and the observed increase in H+-K+-ATPase associated with stimulation was more pronounced. Also, the lighter fractions from stimulated P1 had much latent H+-K+-ATPase activity that was unmasked by n-octylglucoside. The properties of membrane fractions from isolated glands were consistent with results obtained in vivo: high H+-K+-ATPase activity of P3 from resting glands corresponds to cytoplasmic tubulovesicles lacking KCl transport pathways; high activity of P1 from stimulated glands corresponds to apical plasma membrane vesicles containing KCl transport in addition to the H+-K+-ATPase, and full competency for the generation of HCl.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002451:Cell Compartmentation; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology; D005260:Female; D005750:Gastric Juice / Q000378:metabolism*; D005753:Gastric Mucosa / Q000378:metabolism*; D005960:Glucosides / Q000494:pharmacology; D017506:H(+)-K(+)-Exchanging ATPase; D006632:Histamine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008297:Male; D008785:Metiamide / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology; D009840:Oligomycins / Q000494:pharmacology; D011188:Potassium / Q000502:physiology; D011817:Rabbits; D013997:Time Factors
D005960:Glucosides; D009840:Oligomycins; D005576:Colforsin; C018619:octyl-beta-D-glucoside; D008785:Metiamide; D006632:Histamine; D002217:Carbachol; D000242:Cyclic AMP; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase; D011188:Potassium