cGMP and Ca2+ regulation of ion transport across the isolated porcine distal colon epithelium

M. D. DuVall, S. M. O'Grady

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Abstract

Intact epithelium from the porcine distal colon was stripped of serosal muscle and mounted in Ussing chambers to investigate the regulation of Na, Cl, and K transport by guanosine 3',5'-cyclic monophosphate (cGMP) and elevations in intracellular [Ca2+]. Under voltage-clamped conditions cGMP (250 μM) produced an increase in tissue short-circuit current (I(sc)) that reached a maximal value within 10-20 min and remained elevated >40 min. This response was associated with an inhibition of NaCl absorption and stimulation of Cl and K secretion. In the absence of Cl the I(sc) also slowly increased but returned to baseline values within 20 min. Bicarbonate removal from both serosal and mucosal solutions or serosal bumetanide (20 μM) reduced the effect of cGMP on I(sc) by ~40%. When performed simultaneously, these conditions reduced the cGMP response by ~60%. Transepithelial Na and Cl flux measurements indicated that serosal bumetanide blocked increased Cl secretion without effecting changes in NaCl absorption. In contrast, mucosal amiloride blocked the effects of cGMP on NaCl absorption but not Cl secretion. The cGMP I(sc) response was potentiated in the presence of 1 mM, but not 10 μM, amiloride. Moreover, 1 mM amiloride inhibited I(sc) under control conditions but was ineffective in the presence of cGMP. The Ca2+ ionophore ionomycin (3 μM) produced a transient increase in the I(sc) that was also associated with a decrease in transepithelial NaCl absorption and an increase in Cl and K secretion. In contrast to cGMP, the ionomycin I(sc) response was eliminated after Cl removal from the bath. Bicarbonate replacement and bumetanide treatment reduced the ionomycin I(sc) response by 33% and 66%, respectively. Under both conditions, the ionomycin response was completely eliminated. Amiloride (1 mM) did not affect the ionomycin I(sc) response. We conclude that 1) cGMP stimulated Cl-secretion by activating a bumetanide-sensitive transport pathway; 2) ionomycin-stimulated Cl secretion was sensitive to both bumetanide and HCO3 replacement, suggesting that multiple anion secretory pathways are activated by increases in intracellular calcium; 3) the effect of ionomycin on Na absorption was not altered by amiloride, suggesting that ionomycin may regulate an amiloride-insensitive Na+ absorptive pathway; and 4) cGMP inhibited a Na+ absorption pathway that was blocked by 1 mM amiloride.

Original languageEnglish (US)
Pages (from-to)R1026-R1033
JournalAmerican Journal of Physiology - Regulatory Integrative and Comparative Physiology
Volume267
Issue number4 36-4
DOIs
StatePublished - 1994

Keywords

  • amiloride
  • bumetanide
  • secretion
  • sodium hydrogen exchange

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