Effect of SH-group reagents on net water transport in frog urinary bladder

The basal rate of water reabsorption and its acceleration by oxytocin, cyclic AMP (cAMP) or serosal hypertonicity in frog urinary bladders were monitored before and after exposure of the mucosal surface to sulfhydryl (SH) reactive reagents. The following observations were made: 1. N-ethylmaleimide (NEM, 10(-5)M) did not modify the basal water flux, but did potentiate the hydrosmotic response to oxytocin. At higher NEM concentrations, an increase in the basal flux was observed, while the oxytocin-induced water flux was strongly inhibited, if not, nullified. 2. Iodoacetamide (IAM, 10(-3)M) did not modify the basal water flux but did inhibit the oxytocin-, cAMP-, and serosal hypertonicity-induced increase in water permeability. Furthermore, the time course of the hydrosmotic response to oxytocin was significantly increased. 3. 5,5' dithio-bis-(2-nitrobenzoic acid) (DTNB, 10(-3)M) modified neither the basal nor the oxytocin-induced water flux when incubated at pH 8.1, but potentiated the inhibitory effect of NEM. However, at a mucosal pH of 6.5, DTNB inhibited the response to oxytocin by 30%. These results suggest that: (1) the three SH reagents affect differently the basal and the oxytocin-induced water pathways; and that (2) each of the changes in the oxytocin-induced paths occurs at a step following the hormonally-induced increase in intracellular cAMP concentration.     

Evidence for aquaporin-mediated water transport in nematocytes of the jellyfish Pelagia noctiluca

Nematocytes, the stinging cells of Cnidarians, have a cytoplasm confined to a thin rim. The main cell body is occupied by an organoid, the nematocyst, containing the stinging tubule and venom. Exposed to hypotonic shock, nematocytes initially swell during an osmotic phase (OP) and then undergo regulatory volume decrease (RVD) driven by K(+), Cl(-) and obligatory water extrusion mechanisms. The purpose of this report is to characterize the OP. Nematocytes were isolated by the NaSCN/Ca(2+) method from tentacles of the jellyfish Pelagia noctiluca, collected in the Strait of Messina, Italy. Isolated nematocytes were subjected to hyposmotic shock in 65% artificial seawater (ASW) for 15 min. The selective aquaporin water channel inhibitor HgCl(2) (0.1-25 μM) applied prior to osmotic shock prevented the OP and thus RVD. These effects were attenuated in the presence of 1mM dithiothreitol (DTT), a mercaptide bond reducing agent. AgNO(3) (1 μM) and TEA (tetraethylammonium, 100 μM), also reported to inhibit water transport, did not alter the OP but significantly diminished RVD, suggesting different modes of action for the inhibitors tested. Based on estimates of the nematocyte surface area and volume, and OP duration, a relative water permeability of ~10(-7) cm/sec was calculated and the number of putative aquaporin molecules mediating the OP was estimated. This water permeability is 3-4 orders of magnitude lower in comparison to higher order animals and may constitute an evolutionary advantage for Cnidarian survival.     

Lithium and Protein Kinase C Modulators Regulate Swelling-Activated K-Cl Cotransport and Reveal a Complete Phosphatidylinositol Cycle in Low K Sheep Erythrocytes

K-Cl cotransport (COT), a ouabain-insensitive, Cl-dependent bidirectional K flux, is ubiquitously present in all cells, plays a major role in ion and volume homeostasis, and is activated by cell swelling and a variety of chemical interventions. Lithium modulates several cation transport pathways and inhibits phospholipid turnover in red blood cells (RBCs). Lithium also inhibits K-Cl COT by an unknown mechanism. To test the hypothesis whereby Li inhibits swelling-activated K-Cl COT by altering either its osmotic response, its regulation, or by competing with K for binding sites, low K (LK) sheep (S) RBCs were loaded with Li by Na/Li exchange or the cation ionophore nystatin. K-Cl COT was measured as the Cl-dependent, ouabain-insensitive K efflux or Rb influx. The results show that Li altered the cell morphology, and increased both cell volume and diameter. Internal (Li _i ) but not external (Li _o ) Li inhibited swelling-activated K-Cl COT by 85% with an apparent K _i of ∼7 mm. In Cl, Li _i decreased K efflux at relative cell volumes between 0.9 and 1.2, and at external pHs between 7.2 and 7.4. Li _i reduced the V _max and increased the K _m for K efflux in Cl. Furthermore, Li _i increased the production of diacylglycerol in a bimodal fashion, without significant effects on the phosphatidylinositol concentration, and revealed the presence of a complete PI cycle in LK SRBCs. Finally, phorbol ester treatment and PD89059, an inhibitor of mitogen-activated protein kinase (ERK2) kinase, caused a time-dependent inhibition of K-Cl COT. Hence, Li _i appears to inhibit K-Cl COT by acting at an allosteric site on the transporter or its putative regulators, and by modulation of the cellular phospholipid metabolism and a PKC-dependent regulatory pathway, causes an altered response of K-Cl COT to pH and volume. Received: 1 November 1999/Revised: 6 June 2000     

Quinine and quinidine inhibit and reveal heterogeneity of K-Cl cotransport in low K sheep erythrocytes

Low K (LK) sheep red blood cells (SRBCs) serve as a model to study K-Cl cotransport which plays an important role in cellular dehydration in human erythrocytes homozygous for hemoglobin S. Cinchona bark derivatives, such as quinine (Q) and quinidine (QD), are effectively used in the treatment of malaria. In the present study, we investigated in LK SRBCs, the effect of various concentrations of Q and QD on Cl-dependent K efflux and Rb influx (K(Rb)-Cl flux), activated by either swelling in hyposmotic media, thiol alkylation with N-ethylmaleimide (NEM), or by cellular Mg (Mg _i ) removal through A23187 in the presence of external chelators. K efflux or Rb influx were determined in Cl and NO₃ medium and K(Rb)-Cl flux was defined as the Cl-dependent (Cl minus NO₃) component. K(Rb)-Cl flux stimulated by all three interventions was inhibited by both Q and QD in a dose-dependent manner. Maximum inhibition of K(Rb)-Cl flux occurred at Q and QD concentrations ?1 mm. The inhibitory effect of Q was manifested in Cl, but not in NO₃, whereas QD reduced K and Rb fluxes both in Cl and NO₃ media. The mean 50% inhibitory concentration (IC⁵⁰) of Q and QD to inhibit K(Rb)-Cl flux varied between 0.23 and 2.24 mm. From determinations of the percentages of inhibition of the different components of K and Rb fluxes, we found that SRBCs possess a Cl-dependent QD-sensitive and a Cl-dependent QD-insensitive K efflux and Rb influx. These two components vary in magnitude depending on the manipulation and directional flux, but in average they are about 50% of the total Cl-dependent flux. This study raises the possibility that, in SRBCs, the Cl-dependent K(Rb) fluxes are heterogeneous. This work was supported by a grant from the National Institutes of Health (NIH DK5RO1 37,160).     

Cation transport in vascular endothelial cells and aging

Summary To understand the generation and maintenance of Na and K gradients in cultured vascular endothelial cells, net Na and K movements were studied. Ouabain-sensitive (OS) net Na gain and K loss were estimated as the difference between the cation content in the presence of ouabain and that in the control. Ouabain-and furosemide-resistant (OFR) fluxes were determined in the presence of the two inhibitors. When the normal medium bicarbonate and phosphate buffers were replaced by N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid both the OS ans OFR fluxes decreased more than 50%. Ouabain-sensitive and ouabain-and furosemide-resistant fluxes decreased with increasing cellular age (passage number) an effect not observed when the cation movements were studied in the absence of bicarbonate and phosphate. These results suggest that cultured vascular endothelial cells possess bicarbonate-and phosphate-dependent Na and K pathways which account for a significant portion of their passive movements. Furthermore, the behavior of cation permeabilities with passage number suggests that these modulations may be related to the cellular aging process.     

Ion transport in a human lens epithelial cell line exposed to hyposmotic and apoptotic stress

Membrane transport changes in human lens epithelial (HLE‐B3) cells under hyposmotic and apoptotic stress were compared. Cell potassium content, K_i, uptake of the K congener rubidium, Rb_i, and water content were measured after hyposmotic stress induced by hypotonicity, and apoptotic stress by the protein‐kinase inhibitor staurosporine (STP). Cell water increased in hyposmotic (150 mOsm) as compared to isosmotic (300 mOsm) balanced salt solution (BSS) by >2‐fold at 5 min and decreased within 15 min to baseline values accompanied by a 40% K_i loss commensurate with cell swelling and subsequent cell shrinkage likely due to regulatory volume decrease (RVD). Loss of K_i, and accompanying water, and Rb_i uptake in hyposmotic BSS were prevented by clotrimazole (CTZ) suggesting water shifts associated with K and Rb flux via intermediate conductance K (IK) channels, also detected at the mRNA and protein level. In contrast, 2 h after 2 µM STP exposure, the cells lost ～40% water and ～60% K_i, respectively, consistent with apoptotic volume decrease (AVD). Indeed, water and K_i loss was at least fivefold greater after hyposmotic than after apoptotic stress. High extracellular K and 2 mM 4‐aminopyridine (4‐AP) but not CTZ significantly reduced apoptosis. Annexin labeling phosphatidylserine (PS) at 15 min suggested loss of lipid asymmetry. Quantitative PCR revealed significant IK channel expression during prolonged hyposmotic stress. Results suggest in HLE‐B3 cells, IK channels likely partook in and were down regulated after RVD, whereas pro‐apoptotic STP‐activation of 4‐AP‐sensitive voltage‐gated K channels preceded or accompanied PS externalization before subsequent apoptosis. J. Cell. Physiol. 223: 110–122, 2010. © 2009 Wiley‐Liss, Inc.     

PDGF activates K-Cl cotransport through phosphoinositide 3-kinase and protein phosphatase-1 in primary cultures of vascular smooth muscle cells

K-Cl cotransport (K-Cl COT, KCC) is an electroneutrally coupled movement of K and Cl present in most cells. In this work, we studied the pathways of regulation of K-Cl COT by platelet-derived growth factor (PDGF) in primary cultures of vascular smooth muscle cells (VSMCs). Wortmannin and LY 294002 blocked the PDGF-induced K-Cl COT activation, indicating that the phosphoinositide 3-kinase (PI 3-K) pathway is involved. However, PD 98059 had no effect on K-Cl COT activation by PDGF, suggesting that the mitogen-activated protein kinase pathway is not involved under the experimental conditions tested. Involvement of phosphatases was also examined. Sodium orthovanadate, cyclosporin A and okadaic acid had no effect on PDGF-stimulated K-Cl COT. Calyculin A blocked the PDGF-stimulated K-Cl COT by 60%, suggesting that protein phosphatase-1 (PP-1) is a mediator in the PDGF signaling pathway/s. In conclusion, our results indicate that the PDGF-mediated pathways of K-Cl COT regulation involve the signaling molecules PI 3-K and PP-1.     

K-Cl co-transport: immunocytochemical and functional evidence for more than one KCC isoform in high K and low K sheep erythrocytes

K-Cl co-transport (COT) is significantly higher in low K (LK), L-antigen (L) positive, than in high K (HK), M-antigen (M) positive, sheep red blood cells (SRBCs) and is inhibited by sheep allo-anti-L1 antibody. To answer the question of whether this difference in K-Cl co-transport activity resides at the level of the transporter or its regulation, a combined immunocytochemical and functional approach was taken. At least four isoforms of K-Cl COT encoded by different KCC genes are known, with 12 transmembrane domains and cytoplasmic C- and N-terminal domains (Ctd and Ntd, respectively). Polyclonal anti-rat (rt)KCC1 antibodies against a fusion peptide with 77 amino acids from the Ctd of rtKCC1 and anti-human (h)KCC3 against an 18-aa peptide from the Ntd of hKCC3, were prepared in rabbits (rb). Two distinctly separate protein bands of 180 and 145 kDa molecular mass were detected in hemoglobin-free ghosts from RBCs of two LK (one homozygous LL and one heterozygous LM) and one HK (homozygous MM) sheep by Western blots with rb anti-rtKCC1 and rb anti-hKCC3. Confocal microscopy showed specific immunostaining of KCC1 with rb anti-rtKCC1, and of KCC3 with rb anti-hKCC3, in white ghosts from both LK and HK SRBCs. To test the functional heterogeneity of K-Cl COT, the effect of the anti-L1 antibody was assessed on K-Cl COT activated by the kinase inhibitor staurosporine. Incubation of LK SRBCs with anti-L1 serum inhibited by 30% staurosporine-stimulated K-Cl COT suggesting that approximately two-thirds of the transport activity is independent of the L1 antigen. That staurosporine altered the L1 antigen/antibody reaction is unlikely since the action of another antibody, anti-Lp, stimulating the Na/K pump flux, was not modified. The present results, in conjunction with earlier work, lead to the hypothesis that the partial anti-L1 inhibition of K-Cl COT may be related to the molecular KCC dimorphism, seen in these cells with anti-KCC1 and anti-KCC3 antibodies.     

Changes in cation composition of aortic endothelial cells in culture

As a first step in the study of membrane transport characteristics of aortic endothelial cells the content of the two main cations, Na and K, was determined in cultured cells from bovine and porcine origins. The Na and K contents of cultured endothelial cells, dissociated by scraping or trypsin and collagenase treatment and subsequently separated through oil (25% dodecyl-, 75% dibutyl-phthalate), were more than 20-fold higher and five-fold lower, respectively, than those of undissociated cells. Based on daily determination of cell Na, K, and protein contents, the following findings were made. (1) Steady-state levels of Na and K were not reached in subconfluent, confluent or post-confluent monolayers. Instead, intracellular K content varied by up to two-fold, and intracellular Na by more than six-fold with marked 'peaks' after confluency. (2) Increasing the number of passages decreased cellular Na but not K content. (3) In cells cultured with 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) the protein content was decreased by five-fold. (4) The K/Na ratio was dependent on the number of passages and buffers used and varied daily. (5) Cell Na decreased and K increased exponentially with the seeding density. These data not only reveal significant changes of ion transport parameters during manipulations of endothelial cell cultures, but moreover suggest unsynchronized development of ion transport systems and/or their intermittent activation and deactivation as reflected in the variations observed in cellular cation composition.