Mechanism of basolateral membrane H+/OH-/HCO-3 transport in the rat proximal convoluted tubule. A sodium-coupled electrogenic process

In order to examine the mechanism of basolateral membrane H+/OH-/HCO-3 transport, a method was developed for the measurement of cell pH in the vivo doubly microperfused rat proximal convoluted tubule. A pH-sensitive fluorescein derivative, (2',7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein, was loaded into cells and relative changes in fluorescence at two excitation wavelengths were followed. Calibration was accomplished using nigericin with high extracellular potassium concentrations. When luminal and peritubular fluids were pH 7.32, cell pH was 7.14 +/- 0.01. Decreasing peritubular pH from 7.32 to 6.63 caused cell pH to decrease from 7.16 +/- 0.02 to 6.90 +/- 0.03. This effect occurred at an initial rate of 2.4 +/- 0.3 pH units/min, and was inhibited by 0.5 mM SITS. Lowering the peritubular sodium concentration from 147 to 25 meq/liter caused cell pH to decrease from 7.20 +/- 0.03 to 6.99 +/- 0.01. The effect of peritubular sodium concentration on cell pH was inhibited by 0.5 mM SITS, but was unaffected by 1 mM amiloride. In addition, when peritubular pH was decreased in the total absence of luminal and peritubular sodium, the rate of cell acidification was 0.2 +/- 0.1 pH units/min, a greater than 90% decrease from that in the presence of sodium. Cell depolarization achieved by increasing the peritubular potassium concentration caused cell pH to increase, an effect that was blocked by peritubular barium or luminal and peritubular sodium removal. Lowering the peritubular chloride concentration from 128 to 0 meq/liter did not affect cell pH. These results suggest the existence of an electrogenic, sodium-coupled H+/OH-/HCO-3 transport mechanism on the basolateral membrane of the rat proximal convoluted tubule.     

Basolateral membrane Na(+)-independent Cl-/HCO3- exchange in the inner stripe of the rabbit outer medullary collecting tubule

The inner stripe of the outer medullary collecting tubule is a major distal nephron segment in urinary acidification. To examine the mechanism of basolateral membrane H+/OH-/HCO3- transport in this segment, cell pH was measured microfluorometrically in the inner stripe of the rabbit outer medullary collecting tubule perfused in vitro using the pH-sensitive fluorescent dye, (2',7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein. Decreasing peritubular pH from 7.4 to 6.8 (changing [HCO3-] from 25 to 5 mM) caused a cell acidification of 0.25 +/- 0.02 pH units, while a similar luminal change resulted in a smaller cell acidification of only 0.04 +/- 0.01 pH units. Total replacement of peritubular Cl- with gluconate caused cell pH to increase by 0.18 +/- 0.04 pH units, an effect inhibited by 100 microM peritubular DIDS and independent of Na+. Direct coupling between Cl- and base was suggested by the continued presence of peritubular Cl- removal-induced cell alkalinization under the condition of a cell voltage clamp (K(+)-valinomycin). In addition, 90% of basolateral membrane H+/OH-/HCO3- permeability was inhibited by complete removal of luminal and peritubular Cl-. Peritubular Cl(-)-induced cell pH changes were inhibited two-thirds by removal of exogenous CO2/HCO3- from the system. The apparent Km for peritubular Cl- determined in the presence of 25 mM luminal and peritubular [HCO3-] was 113.5 +/- 14.8 mM. These results demonstrate that the basolateral membrane of the inner stripe of the outer medullary collecting tubule possesses a stilbene-sensitive Cl-/HCO3- exchanger which mediates 90% of basolateral membrane H+/OH-/HCO3- permeability and may be regulated by physiologic Cl- concentrations.     

Increased Na/H antiporter and Na/3HCO3 symporter activities in chronic hyperfiltration. A model of cell hypertrophy

The effect of chronic hyperfiltration, a model of cell hypertrophy, on H/HCO3 transporters was examined in the in vivo microperfused rat proximal tubule. Hyperfiltration was induced by uninephrectomy with subsequent increased dietary protein. After 2 wk the hyperfiltration group had a higher glomerular filtration rate (2.21 +/- 0.13 vs. 1.48 +/- 0.12 ml/min), associated with increased kidney weight (1.71 +/- 0.05 vs. 1.23 +/- 0.04 g). HCO3 absorptive rate measured in tubules perfused with an ultrafiltrate-like solution (25 mM HCO3) was higher in the hyperfiltration group (183 +/- 17 vs. 109 +/- 16 pmol/mm per min). The activities of the apical membrane Na/H antiporter and basolateral membrane Na/3HCO3 symporter were assayed using the measurement of cell pH [(2'7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein] in the doubly microperfused tubule in the absence of contact with native fluids. After 2 wk of hyperfiltration Na/H antiporter activity, assayed as the effect of luminal Na removal on cell pH, was increased 114%. Basolateral membrane Na/3HCO3 symporter activity, assayed as the effect of a decrease in peritubular [HCO3] (25 to 5 mM) or in peritubular [Na] (147 to 25 mM) in the absence of luminal and peritubular chloride, was increased 77 and 113%, respectively, in the hyperfiltration group. Steady-state cell pH, measured with physiologic, ultrafiltrate-like luminal and peritubular perfusates, was significantly higher in the hyperfiltration group (7.27 +/- 0.02 vs. 7.14 +/- 0.03). In similar studies, performed 24 h after uninephrectomy and protein feeding, kidney weight was increased 10%, Na/H antiporter activity 39%, and Na/3HCO3 symporter activity 46%. At this time cell pH was not different between the two groups. The results demonstrate that chronic hyperfiltration is associated with parallel increases in Na/H antiporter and Na/3HCO3 symporter activities. If a decrease in cell pH is the signal that triggers these adaptations, it occurs early, and the adaptations can be maintained in the absence of sustained cell acidification.     

Basolateral membrane Cl/HCO3 exchange in the rat proximal convoluted tubule. Na-dependent and -independent modes

To examine whether Cl-coupled HCO3 transport mechanisms were present on the basolateral membrane of the mammalian proximal tubule, cell pH was measured in the microperfused rat proximal convoluted tubule using the pH-sensitive, intracellularly trapped fluorescent dye (2',7')- bis(carboxyethyl)-(5,6)-carboxyfluorescein. Increasing the peritubular Cl concentration from 0 to 128.6 meq/liter caused cell pH to decrease from 7.34 +/- 0.04 to 7.21 +/- 0.04 (p less than 0.001). With more acid extracellular fluid (pH 6.62), a similar increase in the peritubular Cl concentration caused cell pH to decrease by a similar amount from 6.97 +/- 0.04 to 6.84 +/- 0.05 (p less than 0.001). This effect was blocked by 1 mM SITS. To examine the Na dependence of Cl/HCO3 exchange, the above studies were repeated in the absence of luminal and peritubular Na. In alkaline Na-free solutions, peritubular Cl addition caused cell pH to decrease from 7.57 +/- 0.06 to 7.53 +/- 0.06 (p less than 0.025); in acid Na-free solutions, peritubular Cl addition caused cell pH to decrease from 7.21 +/- 0.04 to 7.19 +/- 0.04 (p less than 0.05). The effect of Cl on cell pH was smaller in the absence of luminal and peritubular Na than in its presence. To examine whether the previously described Na/(HCO3)n greater than 1 cotransporter was coupled to or dependent on Cl, the effect of lowering the peritubular Na concentration from 147 to 25 meq/liter was examined in the absence of ambient Cl. Cell pH decreased from 7.28 +/- 0.03 to 7.08 +/- 0.03, a response similar to that observed previously in the presence of Cl. The results demonstrate that Cl/HCO3 (or Cl/OH) exchange is present on the basolateral membrane. Most of Cl/HCO3 exchange is dependent on the presence of Na and may be coupled to it. The previously described Na/(HCO3)n greater than 1 cotransporter is the major basolateral membrane pathway for the coupling of Na and HCO3 and is not coupled to Cl.     

Intestinal bicarbonate secretion by marine teleost fish--why and how?

Intestinal fluids of most marine teleosts are alkaline (pH 8.4-9.0) and contain high levels of HCO(3)(-) equivalents (40-130 mM) which are excreted at a significant rate (>100 microEq kg(-1) h(-1)). Recent research reveals the following about this substantial HCO(3)(-) secretion: (1) It is not involved in acid-base regulation or neutralisation of stomach acid, but increases in parallel with drinking rate at elevated ambient salinities suggesting a role in osmoregulation; (2) In species examined so far, all sections of the intestine can secrete bicarbonate; (3) The secretion is dependent on mucosal Cl(-), sensitive to mucosal DIDS, and immuno-histochemistry indicates involvement of an apical Cl(-)/HCO(3)(-) exchanger. In addition, hydration of CO(2) via carbonic anhydrase in combination with proton extrusion appears to be essential for bicarbonate secretion. The mode of proton extrusion is currently unknown but potential mechanisms are discussed. One consequence of the luminal alkalinity and high bicarbonate concentrations is precipitation of calcium and magnesium as carbonate complexes. This precipitation is hypothesised to reduce the osmolality of intestinal fluids and thus play a potential role in water absorption and osmoregulation. The present studies on European flounder reveal that elevated luminal calcium (but not magnesium) concentrations stimulate intestinal bicarbonate secretion both acutely and chronically, in vitro and in vivo. At the whole animal level, the result of this elevated bicarbonate secretion was increased calcium precipitation with an associated reduction in the osmolality of rectal fluids and plasma. These observations suggest direct functional links between intestinal bicarbonate secretion, divalent cation precipitation and osmoregulation in marine teleost fish.     

Cell pH and luminal acidification inNecturus proximal tubule

Summary Cellular potential and pH measurements (pH _i ) were carried out in the perfused kidney ofNecturus on proximal tubules with standard and recessed-tip glass microelectrodes under control conditions and after stimulation of tubular bicarbonate reabsorption. Luminal pH and net bicarbonate reabsorption were measured in parallel experiments with recessed-tip glass or antimony electrodes, both during stationary microperfusions as well as under conditions of isosmotic fluid transport. A mean cell pH of 7.15 was obtained in control conditions. When the luminal bicarbonate concentration was raised to 25 and 50mm, pH _i rose to 7.44 and 7.56, respectively. These changes in pH _i were fully reversible. Under all conditions intracellular H⁺ was below electrochemical equilibrium. Thus the maintenance of intracellular pH requires active H⁺ extrusion across one or both of the cell membranes. The observed rise in pH _i and the peritubular depolarization after stimulation of bicarbonate reabsorption are consistent with enhanced luminal hydrogen ion secretion and augmentation of peritubular bicarbonate exit via an anion-conductive transport pathway.     

Basolateral membrane Na/base cotransport is dependent on CO2/HCO3 in the proximal convoluted tubule

The mechanism of basolateral membrane base transport was examined in the in vitro microperfused rabbit proximal convoluted tubule (PCT) in the absence and presence of ambient CO2/HCO3- by means of the microfluorometric measurement of cell pH. The buffer capacity of the cells measured using rapid NH3 washout was 42.8 +/- 5.6 mmol.liter-1.pH unit-1 in the absence and 84.6 +/- 7.3 mmol.liter-1.pH unit-1 in the presence of CO2/HCO3-. In the presence of CO2/HCO3-, lowering peritubular pH from 7.4 to 6.8 acidified the cell by 0.30 pH units and lowering peritubular Na from 147 to 0 mM acidified the cell by 0.25 pH units. Both effects were inhibited by peritubular 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS). In the absence of exogenous CO2/HCO3-, lowering peritubular pH from 7.4 to 6.8 acidified the cell by 0.25 pH units and lowering peritubular Na from 147 to 0 mM decreased cell pH by 0.20 pH units. Lowering bath pH from 7.4 to 6.8 induced a proton flux of 643 +/- 51 pmol.mm-1.min-1 in the presence of exogenous CO2/HCO3- and 223 +/- 27 pmol.mm-1.min-1 in its absence. Lowering bath Na from 147 to 0 mM induced proton fluxes of 596 +/- 77 pmol.mm-1.min-1 in its absence. The cell acidification induced by lowering bath pH or bath Na in the absence of CO2/HCO3- was inhibited by peritubular SITS or by acetazolamide, whereas peritubular amiloride had no effect. In the absence of exogenous CO2/HCO3-, cyanide blocked the cell acidification induced by bath Na removal, but was without effect in the presence of exogenous CO2/HCO3-. We reached the following conclusions. (a) The basolateral Na/base n greater than 1 cotransporter in the rabbit PCT has an absolute requirement for CO2/HCO3-. (b) In spite of this CO2 dependence, in the absence of exogenous CO2/HCO3-, metabolically produced CO2/HCO3- is sufficient to keep the transporter running at 30% of its control rate in the presence of ambient CO2/HCO3-. (c) There is no apparent amiloride-sensitive Na/H antiporter on the basolateral membrane of the rabbit PCT.     

Fate of intraluminal glutamine in the perfused renal tubule

To determine the fate of intraluminal glutamine and specifically the role of brush border gamma glutamyltransferase in its hydrolysis and reabsorption, proximal convoluted tubules of rabbits were isolated and perfused with an artificial ultrafiltrate containing 1 mM 14C-glutamine and 3H-PEG as a volume absorption marker. The tubules, average length 0.80 +/- 0.09 mm, were bathed in perfusate containing albumin, 6.5 percent but no glutamine. Aliquots of collectate and bathing media were monitored for total 14C counts while the distribution of radioactive 14C between glutamine and glutamate in the collectate was determined by separation on a Dowex X8 formate form ion-exchange column. After 3 ten minute control periods the perfusate was switched to one containing 1 mM AT-125 in addition to glutamine and after equilibration an additional 3 collections were obtained. Control period glutamine load averaged 16.1 +/- 2.4 pmole/min of which 35 percent was absorbed and 38 and 27 percent excreted as glutamine and glutamate respectively; of the absorbed glutamine 25 percent was metabolized. During AT-125 administration, glutamine delivery averaged 15.0 +/- 2.1 pmole/min of which 57 percent was absorbed; increased absorption occurred at the expence of intraluminal glutamate formation which fell to less than 10 percent. Thus luminal transport and gamma glutamyltransferase mediated hydrolysis appear to compete for available glutamine. Significantly, reducing intraluminal glutamine hydrolysis doubles the cellular metabolism of absorbed glutamine suggesting that extracellular conversion of glutamine to glutamate alters the metabolic fate of filtered glutamine.     

Role of intracellular buffering power on the mitochondria-cytosol pH gradient in the rat liver perfused at 4 degrees C

The factors regulating the amplitude and the pH gradient between cytosol and mitochondria (DeltapHmito-cyt) were investigated in the isolated rat liver perfused at 4 degrees C. Liver ATP content, pH, and buffering power of cytosolic and mitochondrial compartments were evaluated in situ using phosphorus-31 nuclear magnetic resonance spectroscopy. No DeltapHmito-cyt was detected in the liver perfused without bicarbonate. Permeant weak acid in the perfusate (H2CO3, 25 mM, or isobutyric acid, 25, 50, or 100 mM) acidified both cytosol and mitochondria and revealed a DeltapHmito-cyt from 0.06 to 0.31 pH unit. Nevertheless, the manipulations of the DeltapHmito-cyt were more effective under bicarbonate-free conditions, due to the absence of buffering by H2CO3/HCO-3. In the absence of bicarbonate, the intracellular buffering power was threefold higher in the mitochondria (110 mmol/pH unit at pHmito 7.16) than in the cytosol (44 mmol/pH unit at pHcyt 7.30) and dependent on the matrix and cytosol pH, respectively. These buffering powers were almost double in the presence of bicarbonate. In the bicarbonate-free perfused liver, the respiratory activity was 0.08 +/- 0.02 micromol O2/min. g liver wet weight and the ATP turnover was only 40 +/- 7 nmol/min. g liver wet weight, indicating the weak activity of liver mitochondria when DeltapHmito-cyt was <0.05 pH unit. The ATP turnover during a 50 mM isobutyric acid load was 35 +/- 4 nmol/min. g liver wet weight whereas DeltapHmito-cyt rose to 0.26 +/- 0.02 pH unit and pHmito remained alkaline. Hence, although DeltapHmito-cyt was increased the ATP turnover remained unchanged. This work is the first evaluation of the mitochondrial buffering power in the isolated liver. The DeltapHmito-cyt observed within various acid loads reflected the differential titration of cytosol and mitochondria containing proteins and H2CO3/HCO-3 buffering systems. Moreover, no direct relationship between DeltapHmito-cyt and ATP turnover could be shown.     

Intracellular and luminal pH measurements of Malpighian tubules of the mosquito Aedes aegypti: the effects of cAMP

In order to understand the critical role that hydrogen ions play in fluid secretion in Malpighian tubules, intracellular and luminal pH and K+ measurements were performed in isolated Malpighian tubules of the yellow fever mosquito (Aedes aegypti). The intracellular pH was 7.03+/-0.05 (n=15 Malpighian tubules (MT)) and the luminal pH was 7.19+/-0.09 (n=99 MT) when bathed in saline at a pH of 7.0. The lumen potential is positive, thus net proton secretion into the lumen is active. The intracellular and the luminal K+ concentrations were 75+/- 9 mM (n=15) and 102+/-13 mM (n=9 MT) respectively. Cyclic AMP analogues accelerated fluid secretion and at the same time acidified the cell without affecting the luminal pH. Both effects were abolished by an isomer of adenosine-3',5' cyclic monophosphothioate (cAMPS), the Rp-cAMPS, known to inhibit protein kinase A. The results suggest that in the presence of cAMP the properties of the cation/H+ exchanger are affected and that this may be a result of phosphorylation of a Na+/2H+ antiporter located on the apical membrane.     

Study of acute pharmacologic effects of prolactin on calcium and water transport in the rat colon by an in vivo perfusion technique

We investigated the acute effect of intraperitoneally administered prolactin on calcium and water transport in colon of sexually mature female Wistar rats using an in vivo perfusion technique. Test solution containing (in mM) NaCl, 100; KCl, 4.7; MgSO4, 1.2; CaCl2, 20; D-glucose, 11; sodium ferrocyanide (Na4Fe(CN)6), an index of net water transport, 20; and 0.7 (microCi 45CaCl2 (1 Ci = 37 GBq) was perfused througth the 8-cm colonic loop for 60 min at perfusion rates of 0.5 or 1.0 mL x min(-1). Calcium and water transport was also studied under a no flow condition to stimulate the condition often found in the colon by in vivo ligated colonic loop for 30 min. Control results showed no correlation between calcium transport and water flux. Flow of luminal solution at 0.5 and 1.0 mL x min(-1) was found to reverse net calcium absorption from 0.04+/-0.01 nmol x g(-1) dry weight x h(-1) to net calcium secretion of 0.04+/-0.04 and 0.9+/-0.02 nmol x g(-1) dry weight x h(-1), respectively. Neither 0.4, 0.6, nor 1.0 mg x kg(-1) prolactin had any effect on calcium fluxes in the colon. On the other hand, at a perfusion rate of 1 mL x min(-1), 0.4 mg x kg(-1) prolactin significantly decreased net water absorption from 3.86+/-0.90 to 0.88+/-0.64 mL x g(-1) dry weight x h(-1) (P < 0.001), and the higher doses of 0.6 and 1.0 mg x kg(-1) prolactin reversed net water absorption to net water secretion of 2.20+/-0.63 and 2.33+/-0.89 mL x g(-1) dry weight x h(-1), respectively (P < 0.001). The stimulatory effect of prolactin on water transport was completely abolished by reducing the perfusion rate from 1.0 mL x min(-1) to zero. The stimulatory effect of prolactin on water secretion at perfusion rate of 1.0 mL x min(-1) was also abolished when luminal [Na+] was reduced from 180 to 80 mM. We concluded that, unlike in the small intestine, calcium fluxes in the colon are not related to water transport and did not respond at all to prolactin. Water transport, on the other hand, was reversed from net absorption to secretion by prolactin. We propose that this prolactin-induced water secretion is probably mediated by recycling of luminal sodium in the vicinity of tight junctions.     

Effects of luminal pH and luminal HCO3 on CI-HCO3 exchange across turtle urinary bladder

The Cl-HCO3 exchange rate across turtle urinary bladder was studied by measuring the net flux of 36Cl in paired tissues. Serosal bicarbonate concentration and pH were held constant (10 mM and 7.6, respectively) while luminal composition was altered. The rate of net chloride absorption was the same at a luminal pH of 5 or 7 and at luminal HCO-3 concentrations of 0 and 10 mM. These results indicate that the affinity of the exchanger for luminal Cl- is much higher than that for luminal HCO-3 or OH-. Vanadate, an inhibitor of a number of ATPases, had no significant effect on chloride absorption.     

Alkaline pH- and cAMP-induced V-ATPase membrane accumulation is mediated by protein kinase A in epididymal clear cells

In the epididymis, low luminal bicarbonate and acidic pH maintain sperm quiescent during maturation and storage. The vacuolar H(+)-ATPase (V-ATPase) in epididymal clear cells plays a major role in luminal acidification. We have shown previously that cAMP, luminal alkaline pH, and activation of the bicarbonate-regulated soluble adenylyl cyclase (sAC) induce V-ATPase apical accumulation in these cells, thereby stimulating proton secretion into the epididymal lumen. Here we examined whether protein kinase A (PKA) is involved in this response. Confocal immunofluorescence labeling on rat epididymis perfused in vivo showed that at luminal acidic pH (6.5), V-ATPase was distributed between short apical microvilli and subapical endosomes. The specific PKA activator N(6)-monobutyryl-3'-5'-cyclic monophosphate (6-MB-cAMP, 1 mM) induced elongation of apical microvilli and accumulation of V-ATPase in these structures. The PKA inhibitor myristoylated-PKI (mPKI, 10 microM) inhibited the apical accumulation of V-ATPase induced by 6-MB-cAMP. Perfusion at pH 6.5 with 8-(4-chlorophenylthio)-2-O-methyl-cAMP (8CPT-2-O-Me-cAMP; 10 microM), an activator of the exchange protein activated by cAMP (Epac), did not induce V-ATPase apical accumulation. When applied at a higher concentration (100 microM), 8CPT-2-O-Me-cAMP induced V-ATPase apical accumulation, but this effect was completely inhibited by mPKI, suggesting crossover effects on the PKA pathway with this compound at high concentrations. Importantly, the physiologically relevant alkaline pH-induced apical V-ATPase accumulation was completely inhibited by pretreatment with mPKI. We conclude that direct stimulation of PKA activity by cAMP is necessary and sufficient for the alkaline pH-induced accumulation of V-ATPase in clear cell apical microvilli.     

Effect of indomethacin on bombesin-like immunoreactivity, somatostatin and gastrin secretion from rat stomach

In the present study the effect of indomethacin-induced prostaglandin deficiency was examined on the release of bombesin-like immunoreactivity (BLI), a putative peptidergic neurotransmitter, from the isolated perfused rat stomach. In addition, gastrin and somatostatin (SLI) secretion was determined. Pretreatment of rats with indomethacin (2 mg/kg X h) resulted in a 3-fold increase of basal BLI secretion. In response to acetylcholine (2 X 10(-6) M) BLI rose from 2,000 to 4,000 pg/min, whereas in controls BLI increased from 400 to 1,400 pg/min. While absolute values for BLI secretion were higher in indomethacin-treated stomachs the relative increase above baseline was lower (100 vs. 250%). In control rats the increase in BLI secretion in response to acetylcholine was abolished when the acidity in the gastric lumen was increased from pH 7 to pH 2. After indomethacin, however, the stimulatory effect of acetylcholine during luminal pH 7 and pH 2 was identical. The decrease of SLI by acetylcholine at luminal pH 7 was abolished in indomethacin-treated stomachs in response to 10(-6) M acetylcholine, and 2 X 10(-6) M had even a stimulatory effect on SLI secretion. Indomethacin pretreatment reduced gastrin secretion at luminal pH 7. These data demonstrate that endogenous prostaglandins exert an inhibitory tone on basal and stimulated BLI and stimulated SLI secretion in the rat stomach. It is suggested that endogenous prostaglandins also inhibit the release of a peptidergic neurotransmitter, similar to their effect on the classical neurotransmitters acetylcholine and norepinephrine.     

Intestinal water absorption from select carbohydrate solutions in humans.

Eight men positioned a triple-lumen tube in the duodenojejunum. By use of segmental perfusion, 2, 4, 6, or 8% solutions of glucose (111-444 mM), sucrose (55-233 mM), a maltodextrin [17-67 mM, avg. chain length = 7 glucose units (7G)], or a corn syrup solid [40-160 mM, avg. chain length = 3 glucose units (3G)] were perfused at 15 ml/min for 70 min after a 30-min equilibration period. All solutions were made isotonic with NaCl, except 6 and 8% glucose solutions, which were hypertonic. An isotonic NaCl solution was perfused as control. Water absorption (range: 9-15 ml.h-1.cm-1) did not differ for the 2, 4, and 6% CHO solutions but was greater (P < 0.05) than absorption from control (3.0 +/- 2.2 ml.h-1.cm-1). The 8% glucose and 3G solutions reduced (P < 0.05) net water flux compared with their 2, 4, and 6% solutions, but 8% sucrose and 8% 7G solutions promoted water absorption equivalent to lower CHO concentrations. Water absorption was independent of [Na+] in the original solution. In the test segment, 1) Na+ flux correlated with net water flux (r = 0.72, P < 0.01), K+ (r = 0.78, P < 0.01), and [Na+] (r = 0.68, P < 0.001); 2) Na+ absorption occurred at luminal [Na+] as low as 50 mM; 3) glucose transport increased linearly over the luminal concentration range of 40-180 mM; and 4) net water flux was similar over a range of glucose-to-Na+ concentration ratios of 0.4:1 to 3.5:1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of chloride on pH microclimate and electrogenic Na+ absorption across the rumen epithelium of goat and sheep

Active Na+ absorption across rumen epithelium comprises Na+/H+ exchange and a nonselective cation conductance (NSCC). Luminal chloride is able to stimulate Na+ absorption, which has been attributed to an interaction between Cl-/HCO3- and Na+/H+ exchangers. However, isolated rumen epithelial cells also express a Cl- conductance. We investigated whether Cl- has an additional effect on electrogenic Na+ absorption via NSCC. NSCC was estimated from short-circuit current (Isc) across epithelia of goat and sheep rumen in Ussing chambers. Epithelial surface pH (pHs) was measured with 5-N-hexadecanoyl-aminofluorescence. Membrane potentials were measured with microelelectrodes. Luminal, but not serosal, Cl- stimulated the Ca2+ and Mg2+ sensitive Isc. This effect was independent of the replacing anion (gluconate or acetate) and of the presence of bicarbonate. The mean pHs of rumen epithelium amounted to 7.47 +/- 0.03 in a low-Cl- solution. It was increased by 0.21 pH units when luminal Cl- was increased from 10 to 68 mM. Increasing mucosal pH from 7.5 to 8.0 also increased the Ca2+ and Mg2+ sensitive Isc and transepithelial conductance and reduced the fractional resistance of the apical membrane. Luminal Cl- depolarized the apical membrane of rumen epithelium. 5-Nitro-2-(3-phenylpropylamino)-benzoate reduced the divalent cation sensitive Isc, but only in low-Cl- solutions. The results show that luminal Cl- can increase the microclimate pH via apical Cl-/HCO3- or Cl-/OH- exchangers. Electrogenic Na+ absorption via NSCC increases with pH, explaining part of the Cl- effects on Na+ absorption. The data further show that the Cl- conductance of rumen epithelium must be located at the basolateral membrane.     

Release of bombesin-like immunoreactivity from the isolated perfused rat stomach

In the present study the release of bombesin-like immunoreactivity (BLI), somatostatin and gastrin was determined form the isolated perfused rat stomach. Gastric inhibitory polypeptide (GIP, 2 X 10(-9) M) had no effect on BLI while stimulating somatostatin and gastrin release. In these experiments the luminal pH of the stomach was kept at pH 7. Reduction of the luminal pH to 2 resulted in an inhibition of BLI secretion by GIP while gastrin release was abolished and somatostatin remained unaffected compared to luminal pH 7. Acetylcholine (10(-6) and 2 X 10(-6) M) elicited a dose-dependent stimulation of BLI secretion while gastrin was stimulated and somatostatin secretion suppressed independent of the administered dose. The present data demonstrate that release of bombesin-like immunoreactivity can be modulated by intestinal hormones and neurotransmitters and is integrated into the complex system of gastrointestinal neuroendocrine regulation.     

pH-dependent electrical properties and buffer permeability of the Necturus renal proximal tubule cell

Necturus kidneys were perfused with Tris-buffered solutions at three different pH values, i.e. 7.5, 6.0 and 9.0. A significant drop in fluid absorption occurred at pH 6.0, whereas pH 9.0 did not increase volume flow significantly. When acute unilateral, i.e. either in the lumen or the peritubular capillaries, and bilateral pH changes were elicited in both directions from 7.5 to 9.0 at a constant Tris-butyrate buffer concentration, both peritubular membrane potential difference V1 and transepithelial potential difference V3 hyperpolarized, independently of the side where the change in pH was brought about. Acid perfusions at pH 6.0 caused a similar response but of opposite sign. Analysis of the potential changes shows that pH influences not only the electromotive force and resistance of the homolateral membrane, but also the electrical properties of the paracellular path. Interference of pH with Na, Cl or K conductance was assessed. Any appreciable role for sodium or chloride was excluded, whereas the potassium transference number (tK) of the peritubular membrane increased 16% in alkaline pH. However, this increase accounts only for 19 to 36% of the observed hyperpolarization. Since changes in Tris-butyrate buffer concentration at constant pH do not affect V1 or V3 considerably, the hyperpolarization in pH 9 cannot be explained by an elevation in internal pH only, or by a Tris-H+ ion diffusion potential only. The role of the permeability of the buffers: bicarbonate, butyrate and phosphate, in determining electrical membrane parameters was evaluated. Transport numbers of the buffer anions ranked as follows: tHCO3 greater than tbutyrate greater than tphosphate. It is concluded that modulation of membrane potential by extracellular pH is mediated primarily by a change in peritubular cell membrane tK and additionally by membrane currents carried by buffer anions.     

Proton nuclear magnetic resonance measurement of diffusional water permeability in suspended renal proximal tubules.

Diffusional water permeability was measured in renal proximal tubule cell membranes by pulsed nuclear magnetic resonance using proton spin-lattice relaxation times (T1). A suspension of viable proximal tubules was prepared from rabbit renal cortex by Dounce homogenization and differential sieving. T1 measured in a tubule suspension (22% of exchangeable water in the intracellular compartment) containing 20 mM extracellular MnCl2 was biexponential with time constants 1.8 +/- 0.1 ms and 8.3 +/- 0.2 ms (mean +/- SD, n = 8, 37 degrees C, 10 MHz). The slower time constant, representing diffusional exchange of water between intracellular and extracellular compartments, increased to 11.6 +/- 0.6 ms (n = 6) after incubation of tubules with 5 mM parachloromercuribenzene sulfonate (pCMBS) for 60 min at 4 degrees C and was temperature dependent with activation energy Ea = 2.9 +/- 0.4 kcal/mol. To relate T1 data to cell membrane diffusional water permeabilities (Pd), a three-compartment exchange model was developed that included intrinsic decay of proton magnetization in each compartment and apical and basolateral membrane water transport. The model predicted that the slow T1 was relatively insensitive to apical membrane Pd because of low luminal/cell volume ratio. Based on this analysis, basolateral Pd (corrected for basolateral membrane surface convolutions) is 2.0 X 10(-3) cm/s, much lower than corresponding values for basolateral Pf (10-30 X 10(-3) cm/s) measured in the intact tubule and in isolated basolateral membrane vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of VIP-induced electrolyte secretion at three levels in rat small intestine.

Duodenal, jejunal and ileal loops were prepared and an iso-osmotic test solution injected, containing 80 mM Na+, 5-mM K+, 1.2 mM Ca2+, 77 mM Cl-, 10 mM HCO3- and 136 mM mannitol. 14CPEG 4000 was used as a non-absorbable marker and 36Cl was added to measure the bidirectional fluxes. During the 60-min in vivo incubation time, the duodenum actively secreted bicarbonate, a virtually zero flux in the jejunum was observed, whereas the ileum absorbed water and chloride and secreted bicarbonate. The response to the perfused doses of 0.15 to 2.4 nmol.100 g-1.h-1 of VIP (vasoactive intestinal peptide) differed qualitatively and quantitatively in the 3 segments: VIP increased bicarbonate secretion and induced chloride secretion in the duodenum, induced chloride secretion in the jejunum without changing bicarbonate minimal influx, induced bicarbonate secretion and suppressed chloride absorption in the ileum. The minimal dose required was lower in the duodenum (0.3 nmol.100 g-1.h-1) than in the jejunum and ileum (1.2 nmol.100 g-1.h-1). The functional heterogeneity of the small intestine was clearly demonstrated after VIP stimulation.