Alan N. Charney

Na+-K+-activated adenosine triphosphatase and intestinal electrolyte transport. Effect of adrenal steroids.

Sodium-potassium-activated adenosine triphosphatase (Na-K-ATPase) is associated with electrolyte transport in many tissues. To help delineate its role in intestinal transport, changes in rat intestinal electrolyte and water transport induced by injecting methylprednisolone acetate 3 mg/100 g or deoxycorticosterone acetate (DOCA) 0.5 mg/100 g per day for 3 days were correlated with changes in Na-K-ATPase activity. Methylprednisolone increased sodium and water absorption, potassium secretion, transmural potential difference, and Na-K-ATPase activity in the jejunum, ileum, and colon. Examination of isolated epithelial cells demonstrated that the jejunal and ileal increase in Na-K-ATPase occurred in both the villus tip and crypermeability, Mg-ATPase, and adenylate cyclase activities were unchanged by methylprednisolone. DOCA increased sodium and water absorption, potassium secretion, transmural potential difference, and Na-K-ATPase activity in the colon alone. Colonic Mg-ATPase and adenylate cyclase activities were unaffected. Jejunal and ileal enzyme activity, electrolyte transport, and permeability were unchanged by DOCA. Methylprednisolone and DOCA were not additive in their effect on colonic Na-K-ATPase activity. Methylprednisolone and DOCA increased electrolyte and water transport and Na-K-ATPase activity concomitantly in specific segments of small intestine and colon. These data are consistent with an important role for Na-K-ATPase in intestinal electrolyte and water transport.

Effects of acid-base variables on ion transport in rat colon.

Alterations in arterial acid-base variables have important effects on colonic electrolyte transport in vivo. To confirm the relative effects of these variables and to characterize the transport processes involved, we measured unidirectional 22Na and 36Cl fluxes across short-circuited, distal colonic mucosa of Sprague-Dawley rats. Stripped tissues were studied in Hepes buffer and in Ringers solutions at HCO3 concentrations of 11, 21, and 39 mM, and CO2 tensions between 0 and 69.6 mmHg. Increases in PCO2, but not in either pH or HCO3 concentration, caused similar increases in JNanet and JClnet (net flux of sodium and chloride, respectively) from -0.2 +/- 0.3 and -1.5 +/- 0.4 mu eq/cm2 per h at PCO2 = 0 to 6.8 +/- 0.6 and 7.6 +/- 0.7 mu eq/cm2 per h, respectively, at PCO2 = 69.6 mmHg. These increases were accounted for by changes in Jms and were accompanied by small decreases in Isc. 1 mM acetazolamide decreased both JNanet and JClnet and their responses to increases in CO2. 0.75 mM luminal amiloride prevented the increase in sodium absorption, but did not affect the CO2-induced increase in chloride absorption. In the presence of amiloride, CO2 increased JR (residual flux). 0.1 mM luminal furosemide did not affect the CO2-induced increases in JNanet in the absence or presence of amiloride. Changes in HCO3 concentration did not alter JR. We conclude that ambient CO2 effects active, electroneutral sodium absorption in the rat distal colon. The process stimulated by CO2 is dependent on mucosal carbonic anhydrase activity and most likely represents Na/H and Cl/HCO3 ion exchange.

Modulation of chloride secretion in the rat colon by intracellular bicarbonate

Extracellular HCO3- stimulates colonic net Cl- absorption in part by inhibiting basal Cl- secretion. This inhibition was investigated by measuring serosal-to-mucosal Cl- flux across short-circuited colonic segments from Sprague-Dawley rats. Mucosal intracellular pH and bicarbonate were estimated using the pH-sensitive dye BCECF. When extracellular [HCO3-] ([HCO3-]e) was increased from 0 to 39 mmol/L at PCO2 33 mm Hg, mucosal intracellular [HCO3-] ([HCO3-]i) increased to 25.3 mmol/L and serosal-to-mucosal Cl- flux decreased from 13.0 to 7.1 microEq.cm-2.h-1. When PCO2 was increased to 72 mm Hg at [HCO3-]e 39 mmol/L, [HCO3-]i increased to 29.8 mmol/L and serosal-to-mucosal Cl- flux decreased to 5.9 microEq.cm-2.h-1. In Ringers solution containing 21 mmol/L HCO3- and 20 mmol/L Cl- (but not 100 mmol/L Cl-), increasing PCO2 from 21 to 70 mm Hg increased [HCO3-]i to 22.6 mmol/L and decreased serosal-to-mucosal Cl- flux from 3.0 to 1.7 microEq.cm-2.h-1. Overall, serosal-to-mucosal Cl- flux was inversely related to [HCO3-]i on either side of an [HCO3-]i plateau of 9-18 mmol/L at which flux was stable. These data suggest that [HCO3-]i is an important modulator of basal Cl- secretion in rat distal colon.

Effects of short chain fatty acids on colonic Na+ absorption and enzyme activity

Short chain fatty acids (SCFA) stimulate colonic Na+ absorption and inhibit cAMP and cGMP-mediated Cl- secretion. It is uncertain whether SCFA have equivalent effects on absorption and whether SCFA inhibition of Cl- secretion involves effects on mucosal enzymes. Unidirectional Na+ fluxes were measured across stripped colonic segments in the Ussing chamber. Enzyme activity was measured in cell fractions of scraped colonic mucosa. Mucosal 50 mM acetate, propionate, butyrate and poorly metabolized isobutyrate stimulated proximal colon Na+ absorption equally (300%). Neither 2-bromo-octanoate, an inhibitor of beta-oxidation, nor carbonic anhydrase inhibition affected this stimulation. All SCFA except acetate stimulated distal colon Na+ absorption 200%. Only one SCFA affected proximal colon cGMP phosphodiesterase (PDE) (18% inhibition by 50 mM butyrate). All SCFA at 50 mM stimulated distal colon cAMP PDE (24-43%) and decreased forskolin-stimulated mucosal cAMP content. None of the SCFA affected forskolin-stimulated adenylyl cyclase in distal colon or ST(a)-stimulated guanylyl cyclase in proximal colon. Na+-K+-ATPase in distal colon was inhibited 23-51% by the SCFA at 50 mM. We conclude that all SCFA (except acetate in distal colon) stimulate colonic Na+ absorption equally, and the mechanism does not involve mucosal SCFA metabolism or carbonic anhydrase. SCFA inhibition of cAMP-mediated secretion may involve SCFA stimulation of PDE and inhibition of Na+-K+-ATPase.

Non-catalytic role of carbonic anhydrase in rat intestinal absorption

Carbonic anhydrase (CA) inhibition reduces NaCl absorption in rat distal ileum, a pH-sensitive, low CA activity tissue, and in distal colon, a CO(2)-sensitive, high CA activity tissue. We hypothesized that CA plays a non-catalytic role in NaCl absorption in these segments. Unidirectional fluxes of Na(+) and Cl(-), and total HCO(3)(-) generation (estimated as the sum of radiolabeled HCO(3)(-) and CO(2) produced from glucose) were measured in Ussing chambers in nominally CO(2), HCO(3)(-)-free HEPES Ringer. Measurements were made in the presence and absence of 0.1 mM methazolamide, a membrane-permeant CA inhibitor. Ringer pH reduction from 7.6 to 7.1 stimulated ileal but not colonic Na(+) and Cl(-) absorption. In the ileum, methazolamide reduced J(ms)(Na) and J(ms)(Cl) and caused net Cl(-) secretion at pH 7.6, and prevented the stimulatory effect of lowering pH. In the colon, methazolamide reduced Na(+) and Cl(-) absorption at pH 7.6. Total HCO(3)(-) generation was minimal in HEPES at pH 7.6 and 7.1 in both segments, was minimally affected by methazolamide, and did not account for the changes in Cl(-) absorption caused by pH or methazolamide. We conclude that CA plays a role in ileal and colonic NaCl absorption independent of its catalytic function.

NaCl Absorption in the Rabbit Ileum

In vivo and in vitro studies suggest that acid-base variables regulate ion transport in the rabbit ileum. The relative importance of these variables on active Na’ and Cl- absorption has not been defined. Isolated, stripped ileal segments were studied under short-circuited conditions in the Ussing flux chamber. Unidirectional “Na and 38C1 fluxes were measured after changes in bathing solution pH, Pco2, and/or [HCO,J. When pH was decreased from 7.6 to 7.1, net flux of Na’ increased from 0.1 k 0.7 to 2.6 f 0.7 ~Eq/cm’ per hour and net flux of Cl- increased from -2.0 k 0.9 to 1.3 + 0.6 pEq/cm’ per hour. These changes were rapid in onset, completely reversible, and accounted for by changes in mucosal-to-serosal fluxes of these ions. They were accompanied by small decreases in short-circuit current, but there were no changes in residual flux. These pH effects were not inhibited by the presence of luminal bumet

Effects of pH and Cyclic Adenosine Monophosphate on Ileal Electrolyte Transport in the Rat and Rabbit

Alterations in extracellular pH cause reciprocal changes in NaCl absorption in the rat and rabbit ileum. The presence of cholera toxin-induced secretion does not affect pH action measured by in vivo perfusion of the rat ileum. We examined the interaction of pH and cyclic adenosine monophosphate-induced secretion in the rabbit ileum. We found that alterations in arterial pH did not affect ileal absorption in the rabbit in the presence of cholera toxin-induced secretion. This was true whether transport was studied during in vivo ileal perfusion of anesthetized rabbits or by measuring Na+ and Cl- fluxes across isolated, short-circuited tissues in the Ussing chamber. The effects of pH also were blocked when normal rabbit ileum was exposed to 1 mmol/L dibutyryl cyclic adenosine monophosphate in vitro. By contrast, alterations in bathing solution pH affected ileal absorption in the rat in the presence and absence of cyclic adenosine monophosphate. Similarly, exposure to cyclic adenosine monophosphate did not affect the response of the rat colon to PCO2. These findings suggest that the apparently independent effects of pH and cyclic adenosine monophosphate in the rat ileum are not universal. In tissues such as the rabbit ileum, the mechanisms of pH and cyclic adenosine monophosphate action may have biochemical or physiological pathways in common.

Effect of Systemic pH on Models of Altered Ileal Transport in the Rat

Decreases in arterial pH markedly increase sodium, chloride, and water absorption in the normal ileum and can reverse ongoing cholera toxin-induced secretion. In the current study we examined whether these effects of pH are evident in other models of ileal secretion, and in a model of increased absorption. Rats were anesthetized and transport was measured in ileal loops during respiratory acidosis and alkalosis. Decreases in arterial pH increased absorption equally in control loops and in adjacent loops perfused with a Ringers solution containing ST toxin (cyclic guanosine monophosphate-mediated secretion), hypertonic mannitol (passive, osmotically mediated secretion), or glucose. Decreases in arterial pH increased absorption in a similar way in loops exposed to cholera toxin (cyclic adenosine monophosphate-mediated secretion) that were then perfused with glucose-Ringers solution. Alterations in arterial and luminal pH did not affect glucose absorption. These results suggest that the effect of arterial pH on ileal absorption occurs by a mechanism that is independent of these various means of altering transport.

Mechanism of bicarbonate secretion in rat (Rattus rattus) colon

1. Colonic HCO3 secretion was measured as the residual flux in male Sprague-Dawley rats (Rattus rattus). 2. Basal HCO3 secretion was increased by 1 mM dibutyryl cyclic AMP (dbcAMP) and was reduced to baseline by 0.1 mM methazolamide (Mtz) but not by SITS (1 mM), DIDS (1 mM) or amiloride (1 mM). 3. In vivo, intravenous vasoactive intestinal peptide increased HCO3 secretion and prior perfusion with 1 mM Mtz prevented this increase. 4. These results suggest that the source of basal and cAMP-stimulated HCO3 secretion is, in part, intracellular and requires the action of carbonic anhydrase.

Effect of Lithium Ingestion on Water and Electrolyte Transport in Rat Intestine

The effect of lithium ingestion on intestinal electrolyte and water transport was studied in adult Sprague-Dawley rats. We fed animals a lithium-supplemented diet for 1, 2, 4, or 16 wk before in vivo perfusion of the jejunum and colon. Lithium feeding did not alter jejunal transport of water, electrolytes, or glucose, However, at 4 and 16 wk (16-wk data given) the colon increased net water (168%), sodium (160%), and chloride (140%) absorptions, and the transmural potential difference (396%) as compared with control animals. In addition, the colon absorbed both bicarbonate and potassium against an unfavorable electrochemical gradient. The increased colonic sodium absorption was not associated with an increase in mucosal Na+, K+-ATPase activity. Furthermore, in lithium-fed animals deoxycorticosterone acetate stimulated mucosal Na+, K+-ATPase activity, but it did not further increase net sodium absorption. Neither jejunal nor colonic electrolyte transport was affected 24 h after being gavage-fed lithium. These results suggest that chronic lithium ingestion has a unique mechanism of action as other means of chronically increasing sodium absorption are associated with increased mucosal Na+, K+-ATPase activity.