Bumetanide-sensitive 86Rb Influx Research Articles

Shrinkage insensitivity of NKCC1 in myosin II-depleted cytoplasts from Ehrlich ascites tumor cells

Protein phosphorylation/dephosphorylation and cytoskeletal reorganization regulate the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) during osmotic shrinkage; however, the mechanisms involved are unclear. We show that in cytoplasts, plasma membrane vesicles detached from Ehrlich ascites tumor cells (EATC) by cytochalasin treatment, NKCC1 activity evaluated as bumetanide-sensitive (86)Rb influx was increased compared with the basal level in intact cells yet could not be further increased by osmotic shrinkage. Accordingly, cytoplasts exhibited no regulatory volume increase after shrinkage. In cytoplasts, cortical F-actin organization was disrupted, and myosin II, which in shrunken EATC translocates to the cortical region, was absent. Moreover, NKCC1 activity was essentially insensitive to the myosin light chain kinase (MLCK) inhibitor ML-7, a potent blocker of shrinkage-induced NKCC1 activity in intact EATC. Cytoplast NKCC1 activity was potentiated by the Ser/Thr protein phosphatase inhibitor calyculin A, partially inhibited by the protein kinase A inhibitor H89, and blocked by the broad protein kinase inhibitor staurosporine. Cytoplasts exhibited increased protein levels of NKCC1, Ste20-related proline- and alanine-rich kinase (SPAK), and oxidative stress response kinase 1, yet they lacked the shrinkage-induced plasma membrane translocation of SPAK observed in intact cells. The basal phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was increased in cytoplasts compared with intact cells, yet in contrast to the substantial activation in shrunken intact cells, p38 MAPK could not be further activated by shrinkage of the cytoplasts. Together these findings indicate that shrinkage activation of NKCC1 in EATC is dependent on the cortical F-actin network, myosin II, and MLCK.

Insulin-independent, MAPK-dependent stimulation of NKCC activity in skeletal muscle.

Na(+)-K(+)-Cl(-) cotransporter (NKCC) activity in quiescent skeletal muscle is modest. However, ex vivo stimulation of muscle for as little as 18 contractions (1 min, 0.3 Hz) dramatically increased the activity of the cotransporter, measured as the bumetanide-sensitive (86)Rb influx, in both soleus and plantaris muscles. This activation of cotransporter activity remained relatively constant for up to 10-Hz stimulation for 1 min, falling off at higher frequencies (30-Hz stimulation for 1 min). Similarly, stimulation of skeletal muscle with adrenergic receptor agonists phenylephrine, isoproterenol, or epinephrine produced a dramatic stimulation of NKCC activity. It did not appear that stimulation of NKCC activity was a reflection of increased Na(+)-K(+)-ATPase activity because insulin treatment did not stimulate NKCC activity, despite insulin's well-known stimulation of Na(+)-K(+)-ATPase activity. Stimulation of NKCC activity could be blocked by pretreatment with inhibitors of mitogen-activated protein kinase (MAPK) kinase 1/2 (MEK1/2) activity, indicating that activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) MAPKs may be required. These data indicate a regulated NKCC activity in skeletal muscle that may provide a significant pathway for potassium transport into skeletal muscle fibers.

PKC signaling in CF/T43 cell line: regulation of NKCC1 by PKC-δ isotype

Cystic fibrosis (CF) airway epithelial cells have a reduced mass of ether-linked diacylglycerols which might alter protein kinase C (PKC)-regulated Cl secretion. PKC regulation of basolateral Na-K-2Cl cotransport (NKCC1) was investigated in CF nasal polyp epithelial cells and a CF/T43 cell line to ascertain whether PKC signaling was altered in CF. NKCC1 was detected as bumetanide-sensitive 86Rb influx. Methoxamine, a α 1-adrenergic agonist, increased PKC activity in cytosol and a particulate fraction for a prolonged time period, as predicted from previous studies on the generation of diglycerides induced with methoxamine. Short-term stimulation of CF/T43 cells for 40 s promoted a shift in PKC-δ and -ζ to a particulate fraction, increased activity of immune complexes of cytosolic PKC-δ and of particulate PKC-ζ and increased activity of NKCC1. Pretreatment with antisense oligonucleotide to PKC-δ blocked methoxamine-stimulated PKC-δ activity, reduced PKC-δ mass by 61.4%, and prevented methoxamine-stimulated activity of NKCC1. Sense and missense oligonucleotide to PKC-δ and antisense oligonucleotide to PKC-ζ did not alter expression of PKC-δ or the effects of methoxamine. These results demonstrate that PKC-δ-dependent activation of NKCC1 is preserved in CF cells and suggest that regulation of NKCC1 is independent of low ether-linked diglyceride mass.

Sibling resemblance of erythrocyte ion transporters in French-Canadian sibling-pairs affected with essential hypertension.

Erythrocyte Na+/Li+ countertransport and Na+,K+ cotransport are increased in some Caucasians with essential hypertension. This study examines the relative contributions of genetic and shared environmental factors to the activity of these ion carriers in French-Canadian sibling-pairs affected with essential hypertension. The activity of Na+/Li+ countertransport and Na+,K+ cotransport (rate of Na+ o-dependent Li+ efflux and bumetanide-sensitive 86Rb influx, respectively) was measured in 122 French-Canadian siblings with essential hypertension, including 36 brother/brother and 48 sister/sister pairs. Sibling/sibling correlations were estimated using the FCOR program of the S.A.G.E. package. Na+/Li+ countertransport and Na+,K+ cotransport were respectively higher by 27% (P = 0.002) and 42% (P = 0.0009) in erythrocytes from men compared with women. Intra-individual correlation analysis did not reveal a significant effect of age on the activity of these ion transporters in both males and females, and an influence of plasma lipids (triglycerides, cholesterol, low-density lipoprotein, high-density lipoprotein) in females. In males, Na+,K+ cotransport was correlated with the level of serum triglycerides only (P = 0.01). Familial correlation analysis showed that sibling resemblance of Na+/Li+ countertransport and Na+,K+ cotransport was higher in men (r = 0.26 and 0.39) than in women (r = 0.01 and 0.03, respectively). The present data indicate that different factors contribute to the regulation of monovalent ion carriers in erythrocytes from Caucasian men and women with essential hypertension. The activity of erythrocyte Na+/Li+ countertransport and Na+,K+ cotransport appears to be more strongly determined by inheritable factors in men than in women.

The role of transmembrane domain 2 in cation transport by the Na-K-Cl cotransporter.

The human and shark Na-K-Cl cotransporters (NKCC), although 74% identical in amino acid sequence, exhibit marked differences in ion transport and bumetanide binding. We have utilized shark-human chimeras of NKCC1 to search for regions that confer the kinetic differences. Two chimeras (hs3.1 and its reverse sh3.1) with a junction point located at the beginning of the third transmembrane domain were examined after stable transfection in HEK-293 cells. Each carried out bumetanide-sensitive 86Rb influx with cation affinities intermediate between shark and human cotransporters. In conjunction with the previous finding that the N and C termini are not responsible for differences in ion transport, the current observations identify the second transmembrane domain as playing an important role. Site-specific mutagenesis of two pairs of residues in this domain revealed that one pair is indeed involved in the difference in Na affinity, and a second pair is involved in the difference in Rb affinity. Substitution of the same residues with corresponding residues from NKCC2 or the Na-Cl cotransporter resulted in cation affinity changes, consistent with the hypothesis that alternative splicing of transmembrane domain 2 endows different versions of NKCC2 with unique kinetic behaviors. None of the changes in transmembrane domain 2 was found to substantially affect Km(Cl), demonstrating that the affinity difference for Cl is specified by the region beyond predicted transmembrane domain 3. Finally, unlike Cl, bumetanide binding was strongly affected by shark-human replacement of transmembrane domain 2, indicating that the bumetanide-binding site is not the same as the Cl-binding site.

Ion and Bumetanide Binding by the Na-K-Cl Cotransporter

The Na-K-Cl cotransporter (NKCC) plays a key role in electrolyte secretion and absorption across polarized epithelia. The structure of the Na-K-Cl cotransporter transport protein is not known, but from analysis of the primary amino acid sequence and biochemical studies, it has been inferred that the protein has large cytoplasmic N and C termini and a hydrophobic central domain containing 12 transmembrane helices. Both the central domain and the C-terminal domain are highly conserved within the cation-chloride cotransporter family. This paper examines the role of these three domains in interacting with the transported ions and with the inhibitor bumetanide. We have used a chimera approach, exploiting the functional differences between the structurally similar shark and human secretory Na-K-Cl cotransporters (sNKCC1 and hNKCC1). These transporters are 74% identical to one another and have similar transport and regulatory behaviors; however, sNKCC1 differs markedly from hNKCC1 with regard to apparent affinities for the cotransported ions and for bumetanide. We prepared six sNKCC1-hNKCC1 chimeras in which N and C termini were interchanged between species. When transfected in HEK-293 cells, each chimera carried out bumetanide-sensitive 86Rb influx, demonstrating transporter synthesis and cell surface delivery. Monoclonal antibodies J3 and J7 were used to detect the chimeric proteins, and the epitopes for these antibodies were localized to residues 49-196 and 1050-1168, respectively, in the shark sequence. For each of two chimeras that were examined, the time course of activation in low Cl- medium was the same as for the parent proteins; activation was found to proceed through a change in Vmax rather than Km. For the six chimeras, the apparent affinities for Na+, K+, Cl-, and bumetanide segregated exactly according to whether the large hydrophobic central domain was derived from sNKCC1 or hNKCC1. Significantly, the well-conserved C terminus does not appear to contain residues involved in the shark-human affinity differences. These results demonstrate that residues involved with ion translocation and inhibitor binding are within the large central domain that contains the 12 predicted transmembrane helices.

Increased Inward Passive Permeability in Vitro to Sodium in Uraemic Erythrocytes

1. We have reported a normal sodium (Na) pump, but decreased loop-diuretic-sensitive Na efflux in erythrocytes from patients with chronic renal failure on haemodialysis, suggesting a different mode of co-transport in uraemia. 2. The present work extends these findings and examines in vitro simultaneous unidirectional and radiolabelled Na and K fluxes through the Na/K/Cl co-transport and the Na/K pump in washed erythrocytes from seven subjects with chronic renal failure and seven controls. Erythrocyte cytosolic calcium was also examined. 3. Ouabain-sensitive 86Rb influx was similar in patients and controls (1.76 +/- 0.19 versus 1.72 +/- 0.13 mmol h-1 litre-1 of erythrocytes) as was ouabain-sensitive 22Na efflux (3.62 +/- 0.36 versus 4.04 +/- 0.39 mmol h-1 litre-1 of erythrocytes). 4. Bumetanide-sensitive 86Rb and 22Na influx and 22Na efflux were measured at three concentrations (4, 8 and 12 mmol/l) of external K. In chronic renal failure, mean bumetanide-sensitive 22Na efflux was decreased at all external K concentrations compared with controls, and at physiological concentrations (4 mmol/l) external K was lower than controls (0.14 +/- 0.01 versus 0.38 +/- 0.05 mmol h-1 litre-1 of erythrocytes, P < 0.01). Mean bumetanide-sensitive 86Rb influx was also reduced in chronic renal failure at all external K concentrations, and at 4 mmol/l external K was lower than controls (0.13 +/- 0.04 versus 0.34 +/- 0.04 mmol h-1 litre-1 of erythrocytes, P < 0.01). Conversely, bumetanide-sensitive 22Na influx was markedly increased at all external K levels in chronic renal failure, and at 4 mmol/l external K values were elevated compared with controls (0.64 +/- 0.18 versus 0.34 +/- 0.04 mmol h-1 litre-1 of erythrocytes, P < 0.001). The mean cytosolic calcium concentration was higher in erythrocytes in chronic renal failure than controls (134.4 +/- 8.6 versus 63.7 +/- 5.8 nmol/l, P < 0.001). 5. Thus, in washed erythrocytes incubated in artificial media there is a markedly increased ouabain-insensitive Na influx in subjects with chronic renal failure which might be explained in part by the higher levels of cytosolic calcium. In vivo, this cell defect combined with suppression of the Na/K pump could lead to intracellular Na accumulation and play a role in uraemic complications.

Primary Structure, Functional Expression, and Chromosomal Localization of the Bumetanide-sensitive Na-K-Cl Cotransporter in Human Colon

By moving chloride into epithelial cells, the Na-K-Cl cotransporter aids transcellular movement of chloride across both secretory and absorptive epithelia. Using cDNA probes from the recently identified elasmobranch secretory Na-K-Cl cotransporter (sNKCC1) (Xu, J. C., Lytle, C. Zhu, T. T., Payne, J. A., Benz, E., and Forbush, B., III (1994) Proc. Natl. Acad. Sci. 91, 2201-2205), we have identified the human homologue. By screening cDNA libraries of a human colonic carcinoma line, T84 cell, we identified a sequence of 4115 bases from overlapping clones. The deduced protein is 1212 amino acids in length, and analysis of the primary structure indicates 12 transmembrane segments. The primary structure is 74% identical to sNKCC1, 91% identical to a mouse Na-K-Cl cotransporter (mNKCC1), 58% identical to rabbit and rat renal Na-K-Cl cotransporters (NKCC2), and 43% identical to the thiazide-sensitive Na-Cl cotransporters from flounder urinary bladder and rat kidney. Similar to sNKCC1 and mNKCC1, the 5'-end of the human colonic cotransporter is rich in G + C content. Interestingly, a triple repeat (GCG)7 occurs within the 5'-coding region and contributes to a large alanine repeat (Ala15). Two sites for N-linked glycosylation are predicted on an extracellular loop between putative transmembrane segments 7 and 8. A single potential site for phosphorylation by protein kinase A is present in the predicted cytoplasmic C-terminal domain. Northern blot analysis revealed a 7.4-7.5-kilobase transcript in T84 cells and shark rectal gland and a approximately 7.2-kilobase transcript in mammalian colon, kidney, lung, and stomach. Metaphase spreads from lymphocytes were probed with biotin-labeled cDNA and avidin fluorescein (the cotransporter gene was localized to human chromosome 5 at position 5q23.3). Human embryonic kidney cells stably transfected with the full-length cDNA expressed a approximately 170-kDa protein recognized by anti-cotransporter antibodies. Following treatment with N-glycosidase F, the molecular mass of the expressed protein was similar to that predicted for the core protein from the cDNA sequence (132-kDa) and identical to that of deglycosylated T84 cotransporter (approximately 135-kDa). The stably transfected cells exhibited a approximately 15-fold greater bumetanide-sensitive 86Rb influx than control cells, and this flux required external sodium and chloride. Flux kinetics were consistent with an electroneutral cotransport of 1Na:1K:2Cl. Preincubation in chloride-free media was necessary to activate fully the expressed cotransporter, suggesting a [Cl]-dependent regulatory mechanism.

Choroid Plexus Potassium Cotransport: Modulation by Osmotic Stress and External Potassium

The choroid plexuses are involved in CSF secretion and CSF K homeostasis. This study examines the potential role of K cotransport in these two processes using isolated rat lateral ventricle choroid plexuses. Bumetanide-sensitive 86Rb influx and efflux were measured to assess the response of K cotransport to changes in media osmolality and K concentration. Alterations in osmolality had no effect on K uptake (in the presence or absence of bumetanide). However, the efflux rate constant for K was 0.29 +/- 0.02, 0.44 +/- 0.04, and 0.84 +/- 0.06 min-1 in 240, 300, and 424 mOsm/kg solutions, respectively (p < 0.001). This increase in efflux with osmolality, an opposite effect to that found in many cells, was solely due to enhanced K cotransport. The increased cotransport may be involved in limiting brain shrinkage during hyperosmotic stress if the cotransporter is present on the apical membrane. The rate of bumetanide-sensitive efflux was unaffected by changes in external [K]. However, the rate of K uptake (measured on return to normal [K] media) was reduced gradually by exposure to low [K]. It was 21 +/- 1, 19 +/- 3, 13 +/- 2, and 6 +/- 1 nmol/mg/min after 0, 10, 30, and 60-min exposure to 1 mM K. Sixty minutes of exposure to 1 mM [K] abolished the bumetanide-sensitive K uptake present in plexuses exposed continually to normal media. This modulation of K cotransport by external [K] may be important in CSF K homeostasis by limiting K loss from the CSF if CSF [K] is low.

Genetic control of erythrocyte volume regulation: effect of a single gene (rol) on cation metabolism.

In laboratory mice we previously defined a gene, rol (resistance to osmotic lysis), based on its effect on erythrocyte osmotic fragility. Here we report a physiological characterization of rol gene action utilizing congenic strains developed for the purpose; these two strains have a common genetic background and differ only by the two alleles of rol, susceptible (rols) or resistant (rolr). In comparison to rols/s erythrocytes, rolr/r cells have a reduced mean cell volume, a higher mean corpuscular hemoglobin concentration and hemolytic volume, and respond differently to swelling induced by ion influx. Rolr/r erythrocytes also have reduced cell water and K, which are associated with a threefold higher activity of the Na-K-Cl cotransporter (measured as ouabain-resistant, bumetanide-sensitive 86Rb influx) and 30% higher Na pump activity. Apart from differences in ion transport and water content, the content of 2,3-diphosphoglycerate (2,3-DPG) in rolr/r cells is 15% lower than in rols/s ones. Analyses of membrane structural components revealed no rol-associated differences in their phospholipid or fatty acid content, nor were strain differences evident among the membrane and cytoskeletal proteins and their posttranslational modifications (phosphorylation and fatty acylation). Rol is not the structural gene for either the alpha- or the beta-chain of hemoglobin and has no effect on erythrocyte production or destruction. The concerted effect of rol variation on erythrocyte volume, water and cation content, cation cotransport, and 2,3-DPG levels is similar in many ways to the variation observed among individual humans for the same characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell growth and Na-K-Cl cotransport responses of vascular smooth muscle cells of Milan rats.

The present study examines the role of serum growth factors in the proliferative response and Na-K-Cl cotransport activity of vascular smooth muscle cells from Milan normotensive (MNS) and hypertensive (MHS) rats. Cells from thoracic aorta of both strains were cultured in 10% serum medium and made quiescent by 72 hours in 0.3% serum medium. MHS cells grown with 10% serum had a shorter population doubling time than MNS cells between passages 8 and 12 (13.8 +/- 1.7 versus 20.1 +/- 1.6 hours, P < .01, n = 4). MHS cells also exhibited a higher response of thymidine incorporation into nucleic acid to serum, epidermal, and platelet-derived growth factor BB. In MHS cells epidermal (100 ng/mL) and platelet (50 ng/mL) growth factors increased thymidine incorporation 2- and 10-fold, respectively. In MNS cells epidermal factor did not induce a significant response, and that of platelet factor was twofold lower than in MHS cells. Binding curves revealed a higher number of receptors for platelet than epidermal growth factor in both strains and a similar number of both receptors in MHS and MNS cells. Quantitative immunoblots of these receptor proteins confirmed the observation that the greater proliferation of MHS cells could not be related to a higher number of growth factor receptors. Cotransport activity (bumetanide-sensitive 86Rb influx in nanomoles per milligram protein per 5 minutes) was found to be significantly higher in MHS cells (16 +/- 3, n = 18) than MNS cells (8 +/- 3, n = 15) at confluence as well as in the log phase of serum-stimulated growth.(ABSTRACT TRUNCATED AT 250 WORDS)

The regulation of Na/K/2Cl cotransport and bumetanide binding in avian erythrocytes by protein phosphorylation and dephosphorylation. Effects of kinase inhibitors and okadaic acid.

The Na/K/2Cl cotransport system in the avian erythrocyte can be activated by agents that raise intracellular cAMP suggesting the involvement of cAMP-dependent protein kinase (cAMP-PK) in its regulation. Another group of stimuli including fluoride and hypertonicity stimulate cotransport via cAMP-independent means. To further investigate the role of phosphorylation in these processes, we examined the effects of protein kinase inhibitors of 8 (p-Cl-phenylthio)-cAMP (cpt-cAMP), fluoride and hypertonic activation of cotransport in duck red cells, and [3H]bumetanide binding to isolated membranes. Preincubation of cells with the kinase inhibitors K-252a (Ki approximately 1.6 microM) and H-9 (Ki approximately 100 microM) blocked cpt-cAMP activation of bumetanide-sensitive 86Rb influx and bumetanide binding. These inhibitors also led to a rapid deactivation of cotransport and decrease in bumetanide binding when added to cells maximally stimulated by cpt-cAMP. K-252a and H-9 inhibited cotransport activation by cAMP-independent stimuli, but 10-fold higher concentrations were required, implying the involvement of a cAMP-independent phosphorylation process in the mechanism of action of these agents. Removal of stimuli that elevate cAMP leads to a rapid reversal of cotransport indicating the presence of active protein phosphatases in these cells. The protein phosphatase inhibitor okadaic acid (OA, EC50: 630 nM) stimulated both Na/K/2Cl cotransport and bumetanide binding to membranes. As with fluoride and hypertonic stimulation, the OA effect was inhibited only at relatively high concentrations of K-252a. Phosphorylation of the membrane skeletal protein goblin (Mr 230,000) at specific cAMP-dependent sites was used as an in situ marker for the state of activation of cAMP-PK. Goblin phosphorylation at these sites was increased by norepinephrine and cpt-cAMP and rapidly reversed by K-252a and H-9, confirming that both inhibitors do block cAMP-PK activity. While OA markedly increased overall phosphorylation of many erythrocyte membrane proteins, including goblin, it did not affect goblin phosphorylation at specific cAMP-dependent sites. These results implicate a cAMP-independent protein kinase in the mediation of the OA effect on cotransport and bumetanide binding. The bumetanide-binding component of the avian erythrocyte cotransporter, an Mr approximately 150,000 protein that can be photolabeled with the bumetanide analog [3H]4-benzoyl-5-sulfamoyl-3-(3-thenyloxy)-benzoic acid was found to be a phosphoprotein. These results strongly support the hypothesis that phosphorylation and dephosphorylation, possibly of the Na/K/2Cl cotransporter itself, regulates the activity of

Expression of the Na(+)-K(+)-2Cl- cotransporter in Xenopus oocytes

The Na(+)-K(+)-2Cl- cotransporter (NKCCT) is important in mediating net salt transport in a variety of cells. As a first step in obtaining structural information on this transport system, we attempted to express it in Xenopus laevis oocytes by injection of poly(A)+ mRNA from rat kidney. Four days after injection, batches of oocytes were tested for 86Rb uptake in presence and absence of 10 microM bumetanide to distinguish that fraction of influx mediated by NKCCT. In the absence of bumetanide, the oocytes formed a bimodal distribution with respect to 86Rb uptake, with some oocytes accumulating significantly more 86Rb than others. In the presence of bumetanide, or when Na was replaced with choline, that group of oocytes taking up more 86Rb did not appear. Sham-injected oocytes did not accumulate sufficient 86Rb to be distinguishable above background. Taken together, these data suggest that some of the mRNA-injected oocytes expressed the rat renal NKCCT. Crude poly(A)+ mRNA was separated by acid-urea agarose gel electrophoresis, and fractions were injected into oocytes. One fraction corresponding to messages of approximately 7 kilobases in length induced a bumetanide-sensitive 86Rb influx resembling that seen with total mRNA. Poly(A)+ mRNA fractionated on sucrose density gradients gave similar results. It is concluded that the rat kidney NKCCT has been expressed in Xenopus oocytes from a high molecular weight fraction of poly(A)+ mRNA.

Regulatory volume increase in mammalian jejunal villus cells is due to bumetanide-sensitive NaKCl2 cotransport

We assessed ion transport mechanisms operative during regulatory volume increase (RVI) in villus enterocytes isolated in suspension from guinea pig jejunum and examined with electronic cell sizing and 86Rb influx. After validation of the electronic-sizing technique with direct measurements of cell water, the response of cell volume to hypertonic media was evaluated in detail. When shrunk by exposure to hyperosmotic media (455 mosmol/kg medium) cells demonstrated a RVI that was complete in 20 min. RVI required extracellular Na+, K+, and Cl-; this cell swelling showed the following ion sensitivity; Na+ greater than Li+ greater than choline, K+ = Rb+, and Cl- greater than or equal to Br- greater than NO3- = acetate = gluconate. Bumetanide inhibition of villus cell swelling was concentration dependent from 10(-10) to 10(-5) M (7.0 +/- 4.5% vs. 87.8 +/- 0.3%); furosemide (10(-3)M) inhibited RVI (74.1 +/- 9.5%), but amiloride (10(-4) M) had little effect on cell swelling. Disulfonic stilbenes, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (10(-4) M), generated the same inhibition of RVI in either nominally HCO3(-)-free phosphate-buffered saline (PBS) or HCO3(-)-buffered PBS, suggesting anion exchange was not involved. Ouabain (10(-4) M) stimulated cell swelling. Hypertonic shrinkage increased the initial rate of bumetanide-sensitive 86Rb influx (80 +/- 38 vs. 1,011 +/- 241 pmol.mg protein-1.min-1; P less than 0.005) and required extracellular Na+ and Cl- (11 +/- 16 vs. 28 +/- 61 pmol.mg protein-1.min-1). RVI was prevented in low-K+ media (0.2 mM), but the addition of KCl initiated cell swelling. Our data strongly suggest that RVI in jejunal villus enterocytes occurs because of the hypertonic activation of NaKCl2 cotransport.

Bradykinin and vasopressin stimulate Na+-K+-Cl- cotransport in cultured endothelial cells.

We have characterized a Na+-K+-Cl- cotransporter in vascular endothelial cells (EC) cultured from different blood vessels and species that is inhibited by the diuretics furosemide and bumetanide (50% inhibitory concentration for 86Rb influx approximately 20 microM and 0.5 microM, respectively). Inward 86Rb influx mediated via this pathway is greater than 86Rb influx transported by the Na+-K+ pump in cultured EC from bovine and pig aorta, bovine vena cava, and baboon cephalic vein but not in human umbilical or saphenous vein EC. External Na+ or Cl- -stimulated, ouabain-insensitive 86Rb influx is equal to furosemide or bumetanide-sensitive 86Rb influx. Ouabain-insensitive 22Na influx is also partially inhibited by these drugs and stimulated by increasing external K+ or Cl-. Net Na+ extrusion occurs via the Na+-K+-Cl- cotransporter in the absence of external K+, whereas net Na+ influx occurs at higher external K+ (greater than 1 mM). Maximal concentrations (100 nM) of bradykinin and vasopressin increase the initial rate of bumetanide-sensitive 86Rb influx by approximately 60 and 70% (50% effective concentration approximately 1 and 0.6 nM, respectively). Addition of either ethyleneglycol-bis(beta-aminotethylether)-N,N'-tetraacetic acid or LaCl3 (to block calcium influx) prevents bradykinin-stimulated 86Rb influx. When intracellular calcium is elevated using ionomycin (100 nM), a Ca2+ ionophore, bumetanide-sensitive 86Rb influx increases approximately twofold. In contrast, isoproterenol (100 microM) and forskolin (50 microM), adenylate cyclase stimulators, decrease furosemide-sensitive 86Rb influx.(ABSTRACT TRUNCATED AT 250 WORDS)