Segmental differences in K-transport across rabbit proximal and distal colon in vivo and in vitro

Net K-transport was investigated in three different segments of rabbit proximal and distal colon. In vivo studies used a dialysis method, and in vitro studies an everted sac preparation. Results from both methods are in good agreement and show net K-absorption in the two segments of the proximal colon, and net K-secretion in the distal colon. K-absorption in the proximal colon seems to be an active mechanism, because K is transported against an electrical potential gradient. The present study is a comparative approach to demonstrate the heterogeneity of an organ function, and represents the first direct comparison of K-transport in various segments of rabbit colon under in vitro and in vivo conditions.     

Potassium transport by rabbit descending colon, in vitro

Studies of the bidirectional fluxes of K across segments of rabbit descending colon indicate that: a) when the tissue is short-circuited, the net flux does not differ significantly from zero under control conditions and in the presence of aldosterone; and b) the bidirectional fluxes of K conform to the Ussing flux-ratio equation over a wide range of transepithelial electrochemical potential differences. These and other findings strongly suggest that the movements of K across the epithelium are restricted to paracellular routes and are entirely passive. Studies dealing with the mechanism of homocellular K transport indicate that: a) K is actively transported into the cells across the basolateral membranes against an electrochemical potential difference of approximately 30 mV; and b) the active uptake of K may be mediated by a rheogenic Na-K exchange pump that is also responsible for transcellular Na transport. These results are entirely consistent with the model proposed by Koefoed-Johnson and Ussing for isolated frog skin.     

Effect of nitric oxide on electrolyte transport across the porcine proximal colon

The effect of nitric oxide (NO) on ion transport in the porcine proximal colon was investigated in slide-stripped epithelia mounted in Ussing chambers. The serosal addition of the NO-donors sodium nitroprusside (SNP, 0.5 mM) or S-nitroso-N-acetylpenicillamine (SNAP, 0.5 mM) induced a steep increase of short-circuit current ( I(sc)). The stimulatory effect of SNP on I(sc) could not be blocked by piroxicam or tetrodotoxin. Potassium channel inhibitors (quinidine, tetraethylammonium or barium) added serosally reduced the SNP- or SNAP-induced increases of I(sc). In chloride-free solutions, the SNP-induced increase of I(sc) was smaller than in chloride-containing solutions. Cl(- )and Na(+) flux measurements demonstrated that SNP diminished Cl(-) and Na(+) net absorption. Pre-treatment with barium was able to block the inhibitory effect of SNP on NaCl net absorption totally. NO effects on paracellular pathways were assessed by measuring flux rates of [(14)C]-D-mannitol. SNP did not change unidirectional D-mannitol flux rates. In conclusion, NO inhibits NaCl net absorption in the proximal colon of pigs by acting directly on the enterocyte. The antiabsorptive (and/or prosecretory) effect of NO depends on a functional basolateral potassium conductance.     

P-chloromercuribenzene sulfonate blocks and reverses the effect of amiloride on sodium transport across rabbit colon in vitro.

The addition of 10(-3) M p-chloromercuribenzene sulfonate (PCMBS) to the solution bathing the mucosal surface of rabbit colon has no effect on the rate of active Na transport but blocks or reverses the inhibitory action of amiloride. The tissue must be exposed to PCMBS for 20-30 min for a complete blocking effect, and removal of PCMBS from the mucosal solution after this period of exposure does not restore the sensitivity of the tissue to amiloride. The slow time-courses of the blocking and reversal effects suggest that PCMBS does not irreversibly interact with groups directly involved in the binding of amiloride.     

Segmental action of aldosterone on water and electrolyte transport across rabbit colon in vivo

Water and electrolyte transport (Na, K, Cl) and the electrical potential difference were investigated simultaneously in five segments of rabbit colon in vivo, under control conditions and under the influence of aldosterone. A dialysis method was used. The solution was designed to mimic the electrolyte concentration of the lumen, and to simulate normal in vivo conditions. Under in vivo conditions (imposed gradients), marked segmental differences in water and electrolyte transport were present. The proximal colon secreted sodium and absorbed potassium, whereas the distal colon absorbed sodium and secreted potassium. Chloride was secreted in all segments. Aldosterone stimulated water and NaCl absorption in the proximal colon by an electroneutral mechanism, whereas in the distal colon a stimulation of electrogenic Na-absorption occurred. The results demonstrate the role of aldosterone in regulating rabbit colonic electrolyte transport. A uniform hormonal stimulation causes qualitatively and quantitatively different effects in the various segments.     

Ion transport by rabbit colon

Summary Descending rabbit colon, stripped ofmuscularis externa, absorbs Na and Cl under short-circuit conditions and exhibits a residual ion flux, consistent with HCO₃ secretion, whose magnitude is approximately equal to the rate of active Cl absorption. Net K transport was not observed under short-circuit conditions. The results of ion replacement studies and of treatment with ouabain or amiloride suggest that the short-circuit currentI _sc is determined solely by the rate of active Na transport and that the net movements of Cl and HCO₃ are mediated by a Na-independent, electrically-neutral, anion exchange process. Cyclic AMP stimulates an electrogenic Cl secretion, abolishes HCO₃ secretion but does not affect the rate of Na absorption under short-circuit conditions. Studies of the effect of transepithelial potential difference on the serosa-to-mucosa fluxesJ _sm ⁱ of Na, K and Cl suggest thatJ _sm ^Na ,J _sm ^K and one-third ofJ _sm ^Cl may be attributed to ionic diffusion. The permeabilities of the passive conductance pathway(s) are such thatP _KP _NaP _Cl=1.00.070.11. Electrolyte transport byin vitro rabbit colon closely resembles that reported fromin vivo studies of mammalian colon and thus may serve as a useful model for the further study of colonic ion transport mechanisms.     

Effect of anions on amiloride-sensitive,active sodium transport across rabbit colon,in Vitro

Summary replacement of Cl in the solutions bathing partial mucosal strips of rabbit descending colon with sulfate, isethionate, hydroxypropane-sulfonate and, to a lesser degree, ethanesulfonate stimulates active Na absorption (J _net ^Na ) when the baso-lateral pump mechanism is not saturated. These effects are rapid in onset and are readily reversible. Our findings indicate that these stimulatory anions decrease the resistance of the amiloridesensitive Na entry step at the mucosal membrane (R _Na ^m ). However, when the active Na pump mechanism at the baso-lateral membrane is saturated these stimulatory anions do not decrease the resistance of the Na entry process. These findings suggest the presence of a negative feedback between the activity of the pump mechanism and the resistance of the Na entry step which may be mediated by the size of the intracellular Na transport pool. In other words, it seems that when the baso-lateral pump is operating at its maximal rate the resistance to Na entry across the mucosal membrane through the amiloride-sensitive pathway is at a minimum and cannot be further decreased.     

Transtubular transport of proteins in rabbit proximal tubules

The purpose of the present experiments was to study possible different pathways of intracellular transport of proteins after luminal and basolateral uptake in isolated rabbit proximal tubules. Tubules were exposed to cationized ferritin (CF) in the perfusion fluid and horseradish peroxidase (HRP) in the bath simultaneously or to HRP in the bath alone for 30 min. The peritubular fluid (bath) and perfusion fluid were then exchanged and the tubules either fixed immediately or allowed to function during chase-periods for 10, 20, 30, or 60 min before fixation to follow the migration of the proteins through the cells. The proteins were to a large extent found separated in different vacuoles and lysosomes at all time periods studied, indicating separate pathways after uptake via the luminal and basolateral membranes respectively. About 0.5% of the CF taken up by the cells was transported through the cells and became located in the intercellular spaces. HRP was transported from the peritubular fluid to the apical cytoplasm of the tubules indicated by a gradual accumulation of small HRP-containing vesicles, first in the basal part of the cells and then in the apical cytoplasm. In tubules perfused with both CF and HRP in the perfusate, the CF and HRP were found together in apical vacuoles and lysosomes. After perfusion with HRP alone, this tracer was found in similar large vacuoles and lysosomes in the apical cytoplasm, in contrast to the small HRP-filled vacuoles seen after uptake from the bath.     

Electrolyte transport across the basolateral membrane of the parietal cells

The ion-transport properties of the basal lateral membranes of intact isolated parietal cells were studied at the cellular and subcellular level. The presence of an amiloride-sensitive Na+:H+ exchange was demonstrated in cells by proton gradient-driven Na+ uptake and by changes in cell pH as monitored by dimethylcarboxylfluorescein fluorescence both in a fluorimeter and on single isolated cells using a fluorescence microscope and an attached intensified photodiode array spectrophotometer. The presence of the Na+:H+ antiport in vesicles was shown both by intravesicular acidification monitored by acridine orange fluorescent quenching and by proton gradient-dependent Na+ uptake. The presence of Cl-:HCO-3 exchange was determined in intact cells by monitoring changes in cell pH due to Cl- uptake and was shown to be 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid- and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid-sensitive. In vesicles, Cl-:HCO-3 exchange was demonstrated by Cl- flux measurement. The apparent affinities for both Cl- and HCO-3 on either side of the membrane were determined to be Km Cli = 20 mM, Km Clout = 17.5 mM, Km HCO-3in = 2.5 mM, and Km HCO-3out = 7.5 mM. A K+ conductance in cells and vesicles was demonstrated by monitoring K+ gradient-dependent 86Rb uptake. No evidence was found for the presence of a Cl- conductance in either cells or vesicles but a H+ conductance was found to be present in vesicles but not in intact cells. In the latter, by determining the effect of either Na+ or Cl- gradients on cell pH and by flux calculations it was concluded that the Cl-:HCO-3 exchange was the major passive flux mechanism for pH regulation in this cell type.