Stimulates NaCl Absorption Research Articles

Lactobacillus acidophilus counteracts enteropathogenic E. coli-induced inhibition of butyrate uptake in intestinal epithelial cells.

Butyrate, a key short-chain fatty acid metabolite of colonic luminal bacterial action on dietary fiber, serves as a primary fuel for the colonocytes, ameliorates mucosal inflammation, and stimulates NaCl absorption. Absorption of butyrate into the colonocytes is essential for these intracellular effects. Monocarboxylate transporter 1 (MCT1) plays a major role in colonic luminal butyrate absorption. Previous studies (Tan J, McKenzie C, Potamitis M, Thorburn AN, Mackay CR, Macia L. Adv Immunol 121: 91-119, 2014.) showed decreased MCT1 expression and function in intestinal inflammation. We have previously shown (Borthakur A, Gill RK, Hodges K, Ramaswamy K, Hecht G, Dudeja PK. Am J Physiol Gastrointest Liver Physiol 290: G30-G35, 2006.) impaired butyrate absorption in human intestinal epithelial Caco-2 cells due to decreased MCT1 level at the apical cell surface following enteropathogenic E. coli (EPEC) infection. Current studies, therefore, examined the potential role of probiotic Lactobacilli in stimulating MCT1-mediated butyrate uptake and counteracting EPEC inhibition of MCT1 function. Of the five species of Lactobacilli, short-term (3 h) treatment with L. acidophilus (LA) significantly increased MCT1-mediated butyrate uptake in Caco-2 cells. Heat-killed LA was ineffective, whereas the conditioned culture supernatant of LA (LA-CS) was equally effective in stimulating MCT1 function, indicating that the effects are mediated by LA-secreted soluble factor(s). Furthermore, LA-CS increased apical membrane levels of MCT1 protein via decreasing its basal endocytosis, suggesting that LA-CS stimulation of butyrate uptake could be secondary to increased levels of MCT1 on the apical cell surface. LA-CS also attenuated EPEC inhibition of butyrate uptake and EPEC-mediated endocytosis of MCT1. Our studies highlight distinct role of specific LA-secreted molecules in modulating colonic butyrate absorption.

SUPEROXIDE INCREASES SURFACE NKCC2 EXPRESSION IN RAT THICK ASCENDING LIMBS

The apical Na/K/2Cl cotransporter NKCC2 mediates NaCl reabsorption by the thick ascending limb (TAL). We found that the free radical superoxide stimulates NaCl absorption by TALs by stimulating NKCC2 activity. NKCC2 activity is a product of the number of transporters in the apical membrane plus the rate of turnover of each transporter. We hypothesized that superoxide stimulates apical surface NKCC2 expression. Apical surface NKCC2 expression was measured in rat TAL suspensions by surface biotinylation and Western blot. We found that increasing superoxide production with 1 μM xanthine oxidase and 500 μM hypoxanthine stimulated surface NKCC2 expression by 18±5% (p<0.05). This was blocked by scavenging superoxide with 50 μM tempol. However scavenging hydrogen peroxide with 100 U/mL catalase failed to block superoxide stimulated expression (22±8% of control, p<0.05). Inhibiting endogenous nitric oxide production with L-NAME enhanced the superoxide stimulated surface NKCC2 expression from 21±6 to 41±10% (p<0.05). This effect was completely blocked by scavenging superoxide with 300 U/mL superoxide dismutase. Total NKCC2 expression was not changed by any treatment. We conclude that superoxide increases apical surface NKCC2 expression and this effect is blunted by endogenous nitric oxide. Our data suggest that superoxide stimulates NaCl absorption in part by enhanced trafficking of NKCC2 which may be involved in salt-sensitive hypertension.

Stimulation of apical Cl−/HCO3−(OH−) exchanger, SLC26A3 by neuropeptide Y is lipid raft dependent

Neuropeptide Y (NPY), an important proabsorptive hormone of the gastrointestinal tract has been shown to inhibit chloride secretion and stimulate NaCl absorption. However, mechanisms underlying the proabsorptive effects of NPY are not fully understood. The present studies were designed to examine the direct effects of NPY on apical Cl⁻/HCO₃⁻(OH⁻) exchange activity and the underlying mechanisms involved utilizing Caco2 cells. Our results showed that NPY (100 nM, 30 min) significantly increased Cl⁻/HCO₃⁻(OH⁻) exchange activity (∼2-fold). Selective NPY/Y1 or Y2 receptor agonists mimicked the effects of NPY. NPY-mediated stimulation of Cl⁻/HCO₃⁻(OH⁻) exchange activity involved the ERK1/2 MAP kinase-dependent pathway. Cell surface biotinylation studies showed that NPY does not alter DRA (apical Cl⁻/HCO₃⁻(OH⁻) exchanger) surface expression, ruling out the involvement of membrane trafficking events. Interestingly, DRA was found to be predominantly expressed in the detergent-insoluble (DI) and low-density fractions (LDF) of human colonic apical membrane vesicles (AMVs) representing lipid rafts. Depletion of membrane cholesterol by methyl-β-cyclodextrin (MβCD, 10 mM, 1 h) remarkably decreased DRA expression in the DI fractions. Similar results were obtained in Triton-X 100-treated Caco2 plasma membranes. DRA association with lipid rafts in the DI and LDF fractions of Caco2 cells was significantly enhanced (∼45%) by NPY compared with control. MβCD significantly decreased Cl⁻/HCO₃⁻(OH⁻) exchange activity in Caco2 cells as measured by DIDS- or niflumic acid-sensitive ³⁶Cl⁻ uptake (∼50%). Our results demonstrate that NPY modulates Cl⁻/HCO₃⁻(OH⁻) exchange activity by enhancing the association of DRA with lipid rafts, thereby resulting in an increase in Cl⁻/HCO₃⁻(OH⁻) exchange activity. Our findings suggest that the alteration in the association of DRA with lipid rafts may contribute to the proabsorptive effects of NPY in the human intestine.

Resurrecting Hope for Antioxidant Treatment of Cardiovascular Disease

Oxidative stress has been associated with cardiovascular disease and the development of hypertension in both humans and in animal models. Antioxidant therapy has been successful in ameliorating disease in animals, but the results from human studies have been largely disappointing. Despite these negative results, a wealth of evidence suggests that oxidative stress is involved in the development of cardiovascular disease both in humans and animal models. Because global attenuation of cellular reactive oxygen species (ROS) may not be advantageous, more recent therapeutic strategies target specific subcellular sources of oxidative stress. Most studies implicate plasmalemmal membrane-bound NADPH oxidase as a key mediator of ROS generation in cardiovascular disease. However, in the current issue of Circulation Research , Dikalova et al1 demonstrate that specific attenuation of mitochondrial derived oxidants prevents the development of angiotensin II–induced hypertension in the mouse. Perhaps more interesting is the mechanism: inhibition of a mitochondrial ROS-triggered activation of NADPH oxidase, interrupting a vicious cycle of ROS production. The novel approach of targeting the inciting source of ROS (mitochondria) to prevent accelerated oxidant production may hold more promise for humans by eliminating pathological but not homeostatic sources of ROS. Seminal observations by Harrison et al2 showed evidence of increased oxidative stress within the systemic vasculature in an Angiotensin II–induced rat model of hypertension, piquing interest in a possible etiologic role for ROS in this condition. In animal models, stimulating ROS production can induce hypertension,2,3 whereas treatment with a variety of antioxidants including vitamin C and E, or targeted inhibition of enzymatic oxidant production, can reduce blood pressure.3–6 Hypertensive patients demonstrate increased NAD(P)H oxidase activity7 with elevated levels of hydrogen peroxide in plasma.8 Antioxidant treatment leads to enhanced vasodilation in both forearm and coronary arteries of human hypertensive subjects,9,10 indicating a functional vasomotor …

DDAVP increases surface NKCC2 levels via activation of PKA in rat thick ascending limbs

cAMP stimulates NaCl absorption in the thick ascending limb (THAL) by increasing surface NKCC2. Arginine vasopressin (AVP) stimulates NaCl absorption and increases cAMP in the THAL via V2 receptors (V2R). It is not known whether AVP increases surface NKCC2 by activation of cAMP‐dependent protein kinase A (PKA). We hypothesize that stimulation of V2R in the THAL increases surface NKCC2 levels via PKA. We used surface biotinylation and Western blot to measure NKCC2 in the plasma membrane of rat medullary THAL suspensions. We found that 30 minutes stimulation of V2R with dDAVP (1 μM) increased surface NKCC2 by 23 ± 9 % (p &lt;0.05). In the presence of the phosphodiesterase inhibitor IBMX (0.5 mM), dDAVP increased surface NKCC2 by 44 ± 11 % (p &lt;0.05). However, in the presence of the PKA inhibitor H‐89 (10 μM), addition of dDAVP/IBMX did not significantly change surface NKCC2 (basal: 100%, dDAVP/IBMX +H‐89: 108 ± 16%, n = 5). In control experiments, H‐89 alone did not significantly affect surface NKCC2 (basal: 100%, H‐89: 92 ± 4%, n = 6). The total NKCC2 pool was not affected by dDAVP/IBMX (basal: 100%, dDAVP/IBMX: 109 ± 11%, n = 5) or by 10 μM H‐89 (basal: 100%, H‐89: 107 ± 10%, n =5). The intracellular protein GAPDH was not detected in the surface fraction, indicating that intracellular proteins are not biotinylated. We concluded that acute V2R stimulation increases surface NKCC2 levels in the THAL via PKA activation.

Superoxide Stimulates NaCl Absorption in the Thick Ascending Limb Via Activation of Protein Kinase C

Abnormal production of superoxide (O(2)(-)) contributes to hypertension, in part because of its effects on the kidney. The thick ascending limb absorbs 20% to 30% of the filtered load of NaCl. O(2)(-) stimulates NaCl absorption by the thick ascending limb by enhancing Na(+)/K(+)/2Cl(-) cotransporter activity; however, the signaling mechanism is unknown. We hypothesized that O(2)(-) stimulates NaCl absorption by activating protein kinase C (PKC). To test this, we measured the effect of O(2)(-) on: (1) Cl(-) absorption in the presence and absence of PKC inhibitors, (2) total PKC activity, and (3) activation of specific PKC isoforms. Isolated perfused medullary thick ascending limbs were exposed to O(2)(-) generated by xanthine oxidase (1 mU/mL) and hypoxanthine (0.5 mmol/L). O(2)(-) increased Cl(-) absorption by 42% (from 76.2+/-3.6 to 108.2+/-11.9 pmol/min per millimeter; n=5; P<0.05). After treatment with the general PKC inhibitor staurosporine (10 nmol/L), O(2)(-) did not stimulate Cl(-) absorption (Delta-5.7+/-8.6%; n=6). In thick ascending limb suspensions, O(2)(-) increased total PKC activity by 33% (from 66+/-11 to 88+/-12 mU/mg protein; n=5; P<0.05) and increased PKC-alpha and PKC-delta activity by 1.75- and 0.37-fold, respectively. The PKC-alpha/beta-selective inhibitor Gö976 (100 nmol/L) blocked the ability of O(2)(-) to stimulate Cl(-) absorption by isolated perfused medullary thick ascending limbs (Delta4.5+/-15.0%; n=5). The role of PKC-delta could not be studied because of cell necrosis caused by the selective inhibitor rottlerin. We conclude that PKC-alpha is required for O(2)(-)-stimulated NaCl absorption in the thick ascending limb.

Superoxide enhances Na-K-2Cl cotransporter activity in the thick ascending limb

Superoxide (O2-) enhances Na reabsorption by the thick ascending limb (THAL). Na absorption in this segment involves the Na-K-2Cl cotransporter, K channel, and Na-K-ATPase. We hypothesized that O2- stimulates NaCl absorption primarily by enhancing Na-K-2Cl cotransport. First, we measured steady-state intracellular Na (Nai) and chloride (Cli). Xanthine oxidase (XO; 0.75 mU/ml) and hypoxanthine (HX; 0.125 mM) were added to the bath to increase O2-. During the control period, Nai was 12.2 +/- 1.9 mM. After treatment with O2-, it rose to 20.9 +/- 3.3 mM, a 71% increase (P < 0.01). Cli also increased (P < 0.01). Neither XO nor HX alone had a significant effect on Nai or Cli. Next, we tested cotransport activity by measuring the initial rate of increase in Nai caused by changing luminal Na-Cl-K from 50/0/0 to 140/134/4 mM. During the control period, the initial rate of increase was 0.13 +/- 0.02 arbitrary units (AU)/min. After treatment with O2-, it was 0.22 +/- 0.04 AU/min (P < 0.025), a 69% increase. Neither XO nor HX alone had a significant effect. Furosemide completely blocked the increase in intracellular Na in the control and O2- treatment periods. Next, we studied K channel activity by measuring the depolarization caused by increasing luminal K from 1 to 25 mM using a voltage-sensitive dye. During the control period, maximum depolarization was 7.31 +/- 0.77 AU. After O2- treatment, it was 6.18 +/- 0.90 AU (P < 0.05), a 15% decrease. Finally, we assessed the effects of O2- on Na-K-ATPase activity in THAL suspensions by measuring ATP hydrolysis. Vmax and K1/2 for Na were not affected by O2-. We concluded that O2- stimulates THAL NaCl absorption primarily by enhancing Na entry via Na-K-2Cl cotransport.

The role of reactive oxygen species in the regulation of tubular function

The phrase reactive oxygen species covers a number of molecules and atoms, including the quintessential member of the group, O2-; singlet oxygen; H2O2; organic peroxides; and OONO-. While nitric oxide (NO) is also technically a member of the reactive oxygen species family, it is generally considered with a different class of compounds and will not be considered here. To our knowledge, there are currently no published data reporting the effects of reactive oxygen species on net transepithelial flux in the proximal nephron. However, there is evidence that OONO- regulates Na+/K+ adenosine triphosphatase (ATPase) activity as well as paracellular permeability. While it is easy to speculate that such an effect on the pump would decrease net transepithelial solute and water reabsorption, one cannot do so without knowing how other transporters are affected. O2- stimulates NaCl absorption by the thick ascending limb by activating protein kinase C and blunting the effects of NO. The effects of O2- on thick ascending limb NaCl absorption may be important for the initiation of salt-sensitive hypertension. To our knowledge, there are no published data concerning the role of reactive oxygen species in the regulation of solute absorption in either the distal convoluted tubule or the collecting duct. However, OONO- inhibits basolateral K+ channels in the cortical collecting duct, although the net effect of such inhibition is unknown. While the regulation of tubular transport by reactive oxygen species is important to overall salt and water balance, we know very little about where and how these regulators act along the nephron.

Superoxide stimulates NaCl absorption by the thick ascending limb.

The thick ascending limb of the loop of Henle (THAL) plays an important role in the regulation of NaCl and water reabsorption. In vivo studies have shown that the free radical superoxide (O) stimulates Na and water reabsorption by the kidney. However, it is not known whether O regulates transport along the nephron in general or in the THAL specifically. We hypothesized that O stimulates THAL NaCl reabsorption. Cl absorption was measured in isolated, perfused THALs from Sprague-Dawley rats. First, we tested whether extracellular O stimulates Cl absorption. Addition of the O-generating system xanthine oxidase/hypoxanthine increased Cl absorption from 112.7 +/- 12.0 to 146.2 +/- 13.9 pmol. mm(-1). min(-1), a 33% increase (P < 0.03). When superoxide dismutase (300 U/ml) was present in the bath, addition of xanthine oxidase/hypoxanthine did not significantly increase Cl absorption (116.9 +/- 13.8 vs. 102.5 +/- 8.5 pmol. mm(-1). min(-1)). Furthermore, adding 200 nM H(2)O(2) to the bath did not significantly affect Cl absorption (from 130.3 +/- 13.7 to 125.3 +/- 19.6 pmol. mm(-1). min(-1)). Because extracellular O stimulated Cl absorption, we next tested whether endogenously produced O could stimulate transport. Under basal conditions, THALs produced detectable amounts of O, as measured by lucigenin-enhanced chemiluminescence. Adding the O scavenger tempol to the bath decreased Cl absorption from 198.1 +/- 35.4 to 132.4 +/- 23.5 pmol. mm(-1). min(-1), a 31% decrease (P < 0.02). To make sure tempol was not exerting cytotoxic effects, we tested whether its effect was reversible. With tempol in the bath, Cl absorption was 117.2 +/- 9.3 pmol. mm(-1). min(-1). Sixty minutes after tempol was removed from the bath, Cl absorption had increased to 149.2 +/- 9.1 pmol. mm(-1). min(-1) (P < 0.05). We concluded that both exogenous and endogenous O stimulate THAL NaCl absorption. To our knowledge, these are the first data showing a direct effect of O on nephron transport.

Ion exchangers mediating NaCl transport in the renal proximal tubule.

We have studied the mechanisms of NaCl transport in the mammalian proximal tubule. Studies of isolated brush-border membrane vesicles confirmed the presence of Na+-H+ exchange and identified Cl(-)-formate and Cl(-)-oxalate exchangers as possible mechanisms of uphill Cl- entry. We found that formate and oxalate each stimulate NaCl absorption in microperfused proximal tubules. Stimulation of NaCl absorption by formate was blocked by the Na+-H+-exchange inhibitor EIPA, whereas stimulation by oxalate was blocked by omission of sulfate from the perfusion solutions. These observations were consistent with recycling of formate from lumen to cell by H+-coupled formate transport in parallel with Na+-H+ exchange and recycling of oxalate by oxalate-sulfate exchange in parallel with Na+-sulfate cotransport. Using isoform-specific antibodies, we found that NHE1 is present on the basolateral membrane of all nephron segments, whereas NHE3 is present on the apical membrane of cells in the proximal tubule and the loop of Henle. The inhibitor sensitivity of Na+-H+ exchange in renal brush-border vesicles and of HCO3- absorption in microperfused tubules suggested that NHE3 is responsible for most, if not all, apical membrane Na+-H+ exchange in the proximal tubule. The role of NHE3 in mediating proximal tubule HCO3- absorption and formate-dependent Cl- absorption was confirmed by studies in NHE3 null mice. Finally, we cloned and functionally expressed CFEX, an anion transporter expressed on the apical surface of proximal tubule cells and capable of mediating Cl(-)-formate exchange.

Essential role of NHE3 in facilitating formate-dependent NaCl absorption in the proximal tubule.

The absorption of NaCl in the proximal tubule is markedly stimulated by formate. This stimulation of NaCl transport is consistent with a cell model involving Cl(-)-formate exchange in parallel with pH-coupled formate recycling due to nonionic diffusion of formic acid or H(+)-formate cotransport. The formate recycling process requires H(+) secretion. Although Na(+)-H(+) exchanger isoform NHE3 accounts for the largest component of H(+) secretion in the proximal tubule, 40-50% of the rates of HCO absorption or cellular H(+) extrusion persist in NHE3 null mice. The purpose of the present investigation is to use NHE3 null mice to directly test the role of apical membrane NHE3 in mediating NaCl absorption stimulated by formate. We demonstrate that formate stimulates NaCl absorption in the mouse proximal tubule microperfused in vivo, but the component of NaCl absorption stimulated by formate is absent in NHE3 null mice. In contrast, stimulation of NaCl absorption by oxalate is preserved in NHE3 null mice, indicating that oxalate-stimulated NaCl absorption is independent of Na(+)-H(+) exchange. The virtually complete dependence of formate-induced NaCl absorption on NHE3 activity raises the possibility that NHE3 and the formate transporters are functionally coupled in the brush border membrane.

Tyrosine kinase, phosphatidylinositol 3-kinase, and protein kinase C regulate insulin-stimulated NaCl absorption in the thick ascending limb

We have previously shown a direct stimulatory effect of insulin on NaCl absorption in the medullary thick ascending limb of Henle's loop (mTAL). To further investigate the signal transduction involved, we determined whether tyrosine kinase, phosphatidylinositol 3-kinase (PI3-kinase), and/or protein kinase C (PKC) regulate insulin-stimulated NaCl absorption in the mTAL by in vitro microperfusion methods. In control experiments, insulin increased transepithelial voltage (V(te)) and net lumen-to-bath Cl- flux (J(Cl)). Genistein and methyl 2,5-dihydroxycinnamate, two specific tyrosine kinase inhibitors, abolished the effects of insulin. Wortmannin, a specific PI3-kinase inhibitor, inhibited the action of insulin. The effects of insulin also were inhibited by staurosporin and calphostin C, which are dissimilar inhibitors of PKC. These results indicate that insulin stimulates NaCl absorption in the mTAL through tyrosine kinase, PI3-kinase, and PKC-mediated mechanisms. Moreover, because we have reported previously that insulin causes no detectable change in cytosolic free Ca2+ in the mTAL cells, the present results also suggest that insulin-induced PKC activation is not related to inositol 1,4,5-triphosphate (IP3) production.

Brush Border Phosphatidylinositol 3-Kinase Mediates Epidermal Growth Factor Stimulation of Intestinal NaCl Absorption and Na+/H+ Exchange

In terminally differentiated ileal villus Na+-absorptive cells, epidermal growth factor (EGF) stimulates NaCl absorption and its component brush border Na+/H+ exchanger, acting via basolateral membrane receptors, and as we confirm here, a brush border tyrosine kinase. In the present study we show that brush border phosphatidylinositol 3-kinase (PI 3-kinase) is involved in EGF stimulation of NaCl absorption and brush border Na+/H+ exchange. In rabbit ileum studied with the Ussing chamber-voltage clamp technique, EGF stimulation of active NaCl absorption is inhibited by the selective PI 3-kinase inhibitor wortmannin. PI 3-kinase, a largely cytosolic enzyme, translocates specifically to the brush border of ileal absorptive cells following EGF treatment. This translocation occurs as early as 1 min after EGF treatment and remains increased at the brush border for at least 15 min. EGF also causes a rapid (1 min) and large (4-5-fold) increase in brush border PI 3-kinase activity. Involvement of PI 3-kinase activity in intestinal Na+ absorption is established further by studies done in the human colon cancer cell line, Caco-2, stably transfected with the intestinal brush border isoform of the Na+/H+ exchanger, NHE3 (Caco-2/NHE3 cells). Brush border Na+/H+ exchange activity was measured using the pH-sensitive fluorescent dye 2'7'-bis(carboxyethyl)5-(6)-carboxyfluorescein. EGF added to the basolateral surface but not apical surface of Caco-2/NHE3 cells increased brush border Na+/H+ exchange activity. The EGF-induced increase in brush border Na+/H+ exchange activity was completely abolished in cells pretreated with wortmannin. EGF treatment caused increased tyrosine phosphorylation of PI 3-kinase in both ileal brush border membranes and Caco-2/NHE3 cells, suggesting that a tyrosine kinase upstream of the PI 3-kinase is involved in the EGF effects on Na+ absorption. In conclusion, the present study provides evidence in two separate intestinal models, the ileum and a human colon cancer cell line, that PI 3-kinase is an intermediate in EGF stimulation of intestinal Na+ absorption.

Brush-border tyrosine phosphorylation stimulates ileal neutral NaCl absorption and brush-border Na(+)-H+ exchange.

The drug genistein, a tyrosine (Tyr) kinase inhibitor, was used to define a role for Tyr phosphorylation in regulation of basal and stimulated neutral NaCl absorption in rabbit ileum. Brush-border vesicles contain Tyr-phosphorylated peptides. Genistein freeze-thawed into the vesicles caused a concentration-dependent inhibition of at least three peptides with M(r) 111,000, 83,000, and 80,000. Studied with the Ussing chamber-voltage clamp technique, genistein added to the ileal mucosal surface inhibited neutral NaCl absorption. Direct addition of genistein to brush-border vesicles made from ileal villus cells inhibited brush-border Na(+)-H+ exchange but not D-glucose-stimulated Na+ uptake. These effects were not duplicated by genistin, a drug with similar structure to genistein but lacking Tyr kinase inhibiting properties. Serosal but not mucosal epidermal growth factor (EGF) stimulated NaCl absorption. Mucosal genistein but not genistin also altered second-messenger regulation of neutral NaCl absorption, inhibiting the effect of Ca2+ ionophore A-23187 and of serosal EGF but not affecting the transport changes caused by 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP). In contrast, the Cl secretory effects indicated by the increase in short-circuit current for all three agents, A-23187, EGF, and 8-BrcAMP, were inhibited by mucosal genistein. These results suggest that 1) a Tyr kinase is involved in basally stimulating ileal neutral NaCl absorption and brush-border Na(+)-H+ exchange; 2) EGF stimulates NaCl absorption by an effect exerted from the serosal surface, but the effect also involves a brush-border Tyr kinase; 3) brush-border Tyr kinase is involved in the ability of Ca2+ ionophore A-23187 to inhibit neutral NaCl absorption but is not involved in the transport effects of cAMP. This study suggests that Tyr kinase(s) acting over short time periods is involved in stimulation of neutral NaCl absorption and brush-border Na(+)-H+ exchange and also in Ca(2+)-induced inhibition of NaCl absorption. These studies represent the first example of a brush-border Tyr kinase being involved in short-term signal transduction in intestinal epithelial cells.

L-glutamine with D-glucose stimulates oxidative metabolism and NaCl absorption in piglet jejunum.

To explore the relationship between intestinal fluid absorption and oxidative metabolism, we measured the effects of amino acids and glucose on piglet jejunal ion transport and oxygen consumption (QO2) in vitro. Jejunal QO2 was stimulated by L-glutamine and D-glucose but not by the nonmetabolizable organic solutes methyl beta-D-glucoside or L-phenylalanine. QO2 was maximally enhanced by the combination of D-glucose and L-glutamine (5 mM). Even though 5 mM L-glutamine was previously found to be insufficient to stimulate NaCl absorption, 5 mM L-glutamine enhanced jejunal NaCl flux when combined with equimolar mucosal D-glucose. Either D-glucose or methyl beta-D-glucoside caused an increase in short-circuit current (Isc), an increase in Na+ absorption in excess of Isc, and a decrease in Cl- secretion, when L-glutamine was substituted for D-glucose (10 mM) on the serosal side. This relationship suggests that mucosal sugars, if combined with L-glutamine, enhance neutral NaCl absorption as well as electrogenic Na+ flow. (Aminooxy)acetate, an inhibitor of alanine aminotransferase, abolished the stimulation of QO2 and the NaCl-absorptive response to L-glutamine. We conclude that the oxidative metabolism fueled by L-glutamine is linked to a NaCl-absorptive mechanism in the intestine. We propose that the CO2 produced by glutamine metabolism yields carbonic acid, which dissociates to H+ and HCO3-, which may stimulate parallel antiports in the apical membrane.

Ca2+ channel blockers interact with alpha 2-adrenergic receptors in rabbit ileum.

An interaction between Ca2+ channel blockers and alpha 2-adrenergic receptors has been demonstrated in rabbit ileum by studying the effect of clonidine on active electrolyte transport, under short-circuited conditions, in the presence and absence of several Ca2+ channel blocking agents. Clonidine, verapamil, diltiazem, cadmium, and nitrendipine all decrease short-circuit current and stimulate NaCl absorption to different extents with clonidine having the largest effect. Exposure to verapamil, diltiazem, and cadmium inhibited the effects of clonidine on transport, whereas nitrendipine had no such effect. Verapamil, diltiazem, and cadmium, but not nitrendipine, also decreased the specific binding of [3H]alpha 2-adrenergic agents to a preparation of ileal basolateral membranes explaining the observed decrease in the transport effects of clonidine. The effective concentrations of the Ca2+ channel blockers that inhibited the effects of clonidine on transport were fairly similar to the concentrations needed to inhibit its specific binding. The displacement of clonidine by calcium channel blockers is ascribed to a nonspecific effect of these agents, although the possibility that their effects are exerted via their binding to the calcium channels is not excluded.

Na+-K+-Cl- co-transport in the intestine of a marine teleost.

A key element in the absorption of salt and water by various epithelia is the presence, in the apical or brush border membrane, of a mechanism for directly coupling the inward movements of Na+ and Cl− (ref. 1). In this way the downhill gradient for Na+ from lumen to cell, which is maintained by the cell's Na+/K+ pump, can provide the energy for cellular accumulation and transepithelial transport of Cl−. A requirement for K+ at the serosal surface of the epithelium has long been accepted as essential to the operation of the Na+/K+ pump and therefore to salt absorption. Until very recently, however, the effect of luminal K+ on NaCl absorption has not been explored. We show here that luminal K+ stimulates NaCl absorption in the intestine of a marine teleost, the winter flounder, Pseudopleuronectes americanus. Furthermore, K+ uptake across the brush border membrane depends on the presence of both Na+ and Cl− in the bathing medium and is inhibited by furosemide, which also inhibits the coupled uptake of Na+ and Cl− (ref. 2). Thus, flounder intestine seems to have a Na+ −K+ −Cl− co-transport system similar to that demonstrated in erythrocytes3–5, cultured tumour cells6, cultured kidney cells7 and squid giant axon8. Electrophysiological studies of the thick ascending limb of Henle's loop in mammalian kidney9 and the diluting segment in amphibian kidney10 also indicate that luminal K+ is required for salt absorption. Co-transport of Na+, K+ and Cl− may therefore be characteristic of a variety of cells and tissues, including salt-absorbing epithelia.

Alpha 2-Adrenergic receptor regulation of ion transport in rabbit ileum.

Catecholamines are known to decrease short-circuit current (Isc), stimulate NaCl absorption, and inhibit HCO3 secretion in rabbit ileal mucosa in vitro. These effects appear to be mediated by alpha-adrenergic receptors because they are partially blocked by phentolamine and not by propranolol. To further characterize this receptor system, we determined the interactions of epinephrine (Epi) with alpha-subtype-selective antagonists. Prazosin (PZ), a specific alpha 1-antagonist, did not alter the Epi dose-response curve at concentrations up to 10(-5) M. Yohimbine (YO), a specific alpha 2-antagonist, completely inhibited the Epi effect on Isc. At 10(-5) M, YO increased by 70-fold the concentration of Epi required to produce a half-maximal effect (ED50; from 1.4 X 10(-7) M to 10(-5) M). YO and PZ by themselves had no significant effect on Isc in concentrations up to 10(-5) M. Clonidine, a specific alpha 2-agonist, decreased Isc with an ED50 similar to that of Epi; its effect was blocked by YO but not by PZ. Two alpha 1-selective agonists, methoxamine and phenylephrine, only caused a decrease in Isc in doses greater than 10(-5) M. This effect was reversed by YO but not by PZ. The effects of YO and PZ on Epi-modified Cl fluxes were also determined. YO completely aborted the effects of Epi on net Cl flux. No significant effects were seen with PZ. We conclude that the effects of Epi on ileal ion transport are mediated by a specific alpha 2-adrenergic receptor present in ileal mucosa and that no physiologic alpha 1-receptor function can be demonstrated.