Regulation of ion concentration by Drosophila hydei Na+, K+, Ca2+, and Cl-

The ionic composition of the haemolymph of osmotically unencumbered larvae of Drosophila hydei shows a pattern that is typical (Bone, 1944) for highly developed phytophagous insect larvae: 36 mval/l. Cl^?; 56 mval/l. Na⁺; 31 mval/l. K⁺; approximately 18 mval/l. Ca²⁺ at an osmolality of 299 mOsmol/l.The larvae are able to maintain their most favourable ionic concentrations in the haemolymph after experimental osmotic stress in hypertonic as well as in hypotonic media. The reactions are most distinct with an increase or decrease of Cl^? concentration of the external medium. Characteristic regulating processes begin, and the Cl^? concentration of the haemolymph adjusts to the ‘standard’ again. The principal lapse shows the functional representation of an intensely suppressed oscillation. There seems to be a two-point regulation which requires the existence of a Cl^? ion depot and the existence of Cl^?-sensitive receptors.     

Na+, K+, and Cl- transport in resting pancreatic acinar cells

To understand the role of Na+, K+, and Cl- transporters in fluid and electrolyte secretion by pancreatic acinar cells, we studied the relationship between them in resting and stimulated cells. Measurements of [Cl-]i in resting cells showed that in HCO3(-)-buffered medium [Cl- ]i and Cl- fluxes are dominated by the Cl-/HCO3- exchanger. In the absence of HCO3-, [Cl-]i is regulated by NaCl and NaK2Cl cotransport systems. Measurements of [Na+]i showed that the Na(+)-coupled Cl- transporters contributed to the regulation of [Na+]i, but the major Na+ influx pathway in resting pancreatic acinar cells is the Na+/H+ exchanger. 86Rb influx measurements revealed that > 95% of K+ influx is mediated by the Na+ pump and the NaK2Cl cotransporter. In resting cells, the two transporters appear to be coupled through [K+]i in that inhibition of either transporter had small effect on 86Rb uptake, but inhibition of both transporters largely prevented 86Rb uptake. Another form of coupling occurs between the Na+ influx transporters and the Na+ pump. Thus, inhibition of NaK2Cl cotransport increased Na+ influx by the Na+/H+ exchanger to fuel the Na+ pump. Similarly, inhibition of Na+/H+ exchange increased the activity of the NaK2Cl cotransporter. The combined measurements of [Na+]i and 86Rb influx indicate that the Na+/H+ exchanger contributes twice more than the NaK2Cl cotransporter and three times more than the NaCl cotransporter and a tetraethylammonium-sensitive channel to Na+ influx in resting cells. These findings were used to develop a model for the relationship between the transporters in resting pancreatic acinar cells.     

Potassium transport in nonpigmented epithelial cells of ocular ciliary body: inhibition of a Na+, K+, Cl- cotransporter by protein kinase C.

The mechanisms by which 86Rb+ (used as a tracer for K+) enters human nonpigmented ciliary epithelial cells were investigated. Ouabain-inhibitable bumetanide-insensitive 86Rb+ transport accounted for approximately 70-80% of total, whereas bumetanide-inhibitable ouabain-insensitive uptake accounted for 15-25% of total. K+ channel blockers such as BaCl2 reduced uptake by approximately 5%. Bumetanide inhibited 86Rb+ uptake with an IC50 of 0.5 microM, while furosemide inhibited with an IC50 of about 20 microM. Bumetanide-inhibitable 86Rb+ uptake was reduced in Na(+)-free or Cl(-)-free media, suggesting that Na+ and Cl- were required for optimal uptake via this mechanism. These characteristics are consistent with a Na+, K+, Cl- cotransporter in NPE cells. Treatment of NPE cells for 15 min with phorbol 12-myristate, 13-acetate (PMA), an activator of protein kinase C, caused a 50-70% decrease in 86Rb+ uptake via the Na+, K+, Cl- cotransporter. Other 86Rb+ uptake mechanisms were not affected. 86Rb+ uptake via the Na+, K+, Cl- cotransporter could be inhibited by other phorbol esters and by dioctanoylglycerol, an analog of diacylglycerol, but not by 4 alpha phorbol didecanoate, an ineffective activator of protein kinase C. Staurosporine, a protein kinase C inhibitor, blocked phorbol ester inhibition of 86Rb+ uptake. These data suggest that a Na+, K+, Cl- cotransporter in NPE cells is inhibited by activation of protein kinase C.     

Distinct Na+/K+/2Cl- cotransporter localization in kidneys and gills of two euryhaline species, rainbow trout and killifish

The kidney is an organ playing an important role in ion regulation in both freshwater (FW) and seawater (SW) fish. The mechanisms of ion regulation in the fish kidney are less well studied than that of their gills, especially at the level of transporter proteins. We have found striking differences in the pattern of Na⁺/K⁺/2Cl^- cotransporter (NKCC) expression between species. In the killifish kidney, NKCC is apically localized in the distal and collecting tubules and basolaterally localized in the proximal tubules. However, in the SW killifish gill, NKCC is basolaterally co-localized with Na⁺/K⁺-ATPase, whereas in FW, NKCC immunoreactivity is primarily apical, although still colocalized within the same mitochondria-rich cell with basolateral Na⁺/K⁺-ATPase. Rainbow trout kidney has NKCC only in the apical membrane of the distal and collecting tubules in both environments, with no signal being detected in the proximal tubule. On the other hand, in the trout gill, NKCC is found basolaterally in both FW and SW environments. An important observation is that, in the gills of rainbow trout, the trailing edge of the filament possesses mostly Na⁺/K⁺-ATPase-positive but NKCC-negative mitochondria-rich cells, whereas in the region between and at the roots of the gill lamellae, most mitochondria-rich cells exhibit both Na⁺/K⁺-ATPase- and NKCC-positive immunoreactivity. These results suggest that the differential localization of transporters between the two species represents differences in function between these two euryhaline fishes with different life histories and strategies. Funding for this research was provided by NSERC Discovery Grants to G.G.G. and W.S.M., an Alberta Ingenuity Fund PDF, and a fellowship from the NSERC Research Capacity Development Grant to F.K.     

Mitogen activated protein kinase 14-1 regulates serum glucocorticoid kinase 1 during seawater acclimation in Atlantic killifish, Fundulus heteroclitus

The Atlantic killifish (Fundulus heteroclitus) is an environmental sentinel organism used extensively for studies of environmental toxicants and osmoregulation. Previous research in our laboratory has shown that acute acclimation to seawater is mediated by an increase in SGK1. SGK1 promotes the trafficking of CFTR chloride channels from intracellular vesicles to the plasma membrane of the gill within the first hour in seawater resulting in increased chloride secretion. Although we have shown that the increase in gill SGK1 does not require activation of the glucocorticoid receptor, the mechanisms that mediate the rise SGK1 during acute acclimation is unknown. To test the hypothesis that mitogen activated protein kinase (MAPK14) is responsible for the rise in SGK1 we identified the coding sequence of killifish MAPK14-1 and designed a translational blocking vivo-morpholino targeting MAPK14-1. Injection of the MAPK14-1 vivo-morpholino resulted in a 30% reduction of MAPK14-1 and a 45% reduction in phosphorylated-MAPK14-1 protein in the gill of killifish transitioned from freshwater to seawater. Knock down of phosphorlyated-MAPK14-1 completely blocked the rise in SGK1 mRNA and protein in the killifish gill, providing the first direct and in vivo evidence that MAPK14-1 is necessary for acute seawater acclimation.     

Activation of the bumetanide-sensitive Na+,K+,2Cl- cotransporter (NKCC2) is facilitated by Tamm-Horsfall protein in a chloride-sensitive manner

Active transport of NaCl across thick ascending limb (TAL) epithelium is accomplished by Na(+),K(+),2Cl(-) cotransporter (NKCC2). The activity of NKCC2 is determined by vasopressin (AVP) or intracellular chloride concentration and includes its amino-terminal phosphorylation. Co-expressed Tamm-Horsfall protein (THP) has been proposed to interact with NKCC2. We hypothesized that THP modulates NKCC2 activity in TAL. THP-deficient mice (THP(-/-)) showed an increased abundance of intracellular NKCC2 located in subapical vesicles (+47% compared with wild type (WT) mice), whereas base-line phosphorylation of NKCC2 was significantly decreased (-49% compared with WT mice), suggesting reduced activity of the transporter in the absence of THP. Cultured TAL cells with low endogenous THP levels and low base-line phosphorylation of NKCC2 displayed sharp increases in NKCC2 phosphorylation (+38%) along with a significant change of intracellular chloride concentration upon transfection with THP. In NKCC2-expressing frog oocytes, co-injection with THP cRNA significantly enhanced the activation of NKCC2 under low chloride hypotonic stress (+112% versus +235%). Short term (30 min) stimulation of the vasopressin V2 receptor pathway by V2 receptor agonist (deamino-cis-D-Arg vasopressin) resulted in enhanced NKCC2 phosphorylation in WT mice and cultured TAL cells transfected with THP, whereas in the absence of THP, NKCC2 phosphorylation upon deamino-cis-D-Arg vasopressin was blunted in both systems. Attenuated effects of furosemide along with functional and structural adaptation of the distal convoluted tubule in THP(-/-) mice supported the notion that NaCl reabsorption was impaired in TAL lacking THP. In summary, these results are compatible with a permissive role for THP in the modulation of NKCC2-dependent TAL salt reabsorptive function.     

Focal adhesion kinase and Src mediate integrin regulation of insulin receptor phosphorylation.

We show here that phosphorylation of the insulin receptor and insulin receptor substrate-1 is increased when suspended cells are replated on fibronectin. This is not due to decreased numbers of cell surface receptors, alteration of insulin binding, or stimulation of a phosphatase activity in non-adherent cells. Expression of Src together with focal adhesion kinase (FAK) in suspended cells restores insulin-induced receptor autophosphorylation to levels observed in fibronectin-attached cells. Conversely, expression of dominant-negative mutants of either Src or FAK abolishes potentiation of insulin receptor phosphorylation by cell adhesion. The results suggest that both Src and FAK participate in integrin-mediated regulation of insulin receptor signal.     

Intracellular Na+, K+ and Cl- activities in Ehrlich ascites tumor cells

Ehrlich ascites tumor cell membrane potential (Vm) and intracellular Na+, K+ and Cl- activities were measured under steady-state conditions in normal saline medium (Na+ = 154, K+ = 6, Cl- 150 mequiv./l). Membrane potential was estimated to be -23.3 +/- 0.8 mV using glass microelectrodes. Intracellular ion activities were estimated with similar glass electrodes rendered ion-selective by incorporation of ion-specific ionophores. Measurements of Vm and ion-activity differences were made in the same populations of cells. Under these conditions the intracellular Na+, K+ and Cl- activities are 4.6 +/- 0.5; 68.3 +/- 8.0; and 43.6 +/- 2.1 mequiv./l, respectively. The apparent activity coefficients for Na+ and K+ are 0.18 +/- 0.02 and 0.41 +/- 0.05 respectively. These are significantly lower than the activity coefficients expected for the ions in physiological salt solutions (0.71 and 0.73, respectively). The activity coefficient for intracellular Cl- (0.67 +/- 0.03), however, is close to that of the medium (0.73), and the transmembrane electrochemical potential difference for Cl- is not different from zero. The results establish that the energy available from the Na+ electrochemical gradient is much greater than previously estimated from chemical measurements.     

Interleukin-1beta upregulates Na+-K+-2Cl- cotransporter in human middle ear epithelia

Disruption of periciliary fluid homeostasis is the main pathogenesis of otitis media with effusion (OME), one of the most common childhood diseases. Although the underlying molecular mechanisms are unclear, it has been suggested that the altered functions of ion channels and transporters are involved in the fluid collection of middle ear cavity of OME patients. In the present study, we analyzed the effects of a major cytokine interleukin (IL)-1beta, which was known to be involved in the pathogenesis of OME, on Na(+)-K(+)-2Cl(-) cotransporter (NKCC) in human middle ear cells. Intracellular pH (pH(i)) was measured in primary cultures of normal human middle ear epithelial (NHMEE) cells using a double perfusion chamber, which enabled us to analyze the membrane-specific transporter activities. NKCC activities were estimated by the pH(i) reduction due to bumetanide-sensitive intracellular uptake of NH(4) (+). In NHMEE cells, NKCC activities were observed only in the basolateral membrane, and immunoblotting using specific antibodies revealed the expression of NKCC1. Interestingly, IL-1beta treatments augmented the basolateral NKCC activities and increased NKCC1 expression. In addition, IL-1beta treatments stimulated bumetanide-sensitive fluid transport across the NHMEE cell monolayers. Furthermore, an elevated NKCC1 expression was observed in middle ear cells from OME patients when compared to those from control individuals. The above results provide in vitro and in vivo evidence that the inflammatory cytokine IL-1beta upregulates NKCC1 in middle ear epithelial cells, which would be one of the important underlying mechanisms of excess fluid collection in OME patients.     

The Na+, K+, 2Cl- cotransporter of estuarine pufferfishes (Sphoeroides testudineus and S. greeleyi) in hypo- and hyper-regulation of plasma osmolality

The pufferfishes Sphoeroides testudineus and Sphoeroides greeleyi are estuarine species that osmoregulate efficiently, but S. testudineus tolerates seawater dilution to a much higher degree than S. greeleyi. This study aimed at testing whether NKCC is involved with their differential tolerance of seawater dilution, through the analysis of in vivo furosemide (NKCC inhibitor) injection both on hypo-regulation (in 35 per thousand salinity) and hyper-regulation (in 5 per thousand salinity). After exposure for 6 h or 5 days to both salinities, blood samples were obtained for determination of plasma osmolality, chloride, sodium and hematocrit, and muscle samples for determination of water content. Furosemide injection led to increased plasma osmolality and sodium in 35 per thousand and decreased osmolality and chloride in 5 per thousand, when compared to saline-injected controls. Furosemide injection led to hematocrit reduction in both salinities, and muscle water content increase in 5 per thousand and decrease in 35 per thousand in S. testudineus. The results are compatible with NKCC working in branchial NaCl secretion in 35 per thousand, in both species, and a higher role in cell volume regulation in blood and muscle cells of S. testudineus, in both salinities, which could partially explain the stronger capacity of S. testudineus to tolerate seawater dilution during low tide.     

Na+,K+ pump and Na+-coupled ion carriers in isolated mammalian kidney epithelial cells: regulation by protein kinase C.

This review updates our current knowledge on the regulation of Na+/H+ exchanger, Na+,K+,Cl- cotransporter, Na+,Pi cotransporter, and Na+,K+ pump in isolated epithelial cells from mammalian kidney by protein kinase C (PKC). In cells derived from different tubule segments, an activator of PKC, 4beta-phorbol 12-myristate 13-acetate (PMA), inhibits apical Na+/H+ exchanger (NHE3), Na+,Pi cotransport, and basolateral Na+,K+ cotransport (NKCCl) and augments Na+,K+ pump. In PMA-treated proximal tubules, activation of Na+,K+ pump probably plays a major role in increased reabsorption of salt and osmotically obliged water. In Madin-Darby canine kidney (MDCK) cells, which are highly abundant with intercalated cells from the collecting duct, PMA completely blocks Na+,K+,Cl- cotransport and decreases the activity of Na+,Pi cotransport by 30-40%. In these cells, agonists of P2 purinoceptors inhibit Na+,K+,Cl- and Na+,Pi cotransport by 50-70% via a PKC-independent pathway. In contrast with MDCK cells, in epithelial cells derived from proximal and distal tubules of the rabbit kidney, Na+,K+,Cl- cotransport is inhibited by PMA but is insensitive to P2 receptor activation. In proximal tubules, PKC-induced inhibition of NHE3 and Na+,Pi cotransporter can be triggered by parathyroid hormone. Both PKC and cAMP signaling contribute to dopaminergic inhibition of NHE3 and Na+,K+ pump. The receptors triggering PKC-mediated activation of Na+,K+ pump remain unknown. Recent data suggest that the PKC signaling system is involved in abnormalities of dopaminergic regulation of renal ion transport in hypertension and in the development of diabetic complications. The physiological and pathophysiological implications of PKC-independent regulation of renal ion transporters by P2 purinoceptors has not yet been examined.     

Na+/K+/Cl- cotransporter activates MAP-kinase cascade downstream to protein kinase C, and upstream to MEK

In this study, we demonstrated that the specific inhibitors of the Na+/K+/Cl- cotransporter (NKCC1), bumetanide and furosemide, inhibited extracellular regulated kinase (ERK) phosphorylation in Balb/c 3T3 fibroblasts, stimulated with a variety of mitogens. In addition to fibroblast growth factor (FGF) shown before, the various mitogens tested in the present study (endothelial growth factor (EGF), platelet-derived growth factor (PDGF), insulin, thrombin, and the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA)). Enter, the Ras/Raf/MEK/ERK cascade via different growth factors receptors and through one of the two main routes. The results of the present study provide evidence that have led us to conclude that the target protein which is controlled by the Na+/K+/Cl- cotransporter, is downstream of tyrosine kinase receptors, as well as of the G-protein-coupled receptor (GPCR). Several additional lines of evidence supported the above conclusion: (i) furosemide inhibits phosphorylation of MAPK kinase (MEK) induced by receptor tyrosine kinase (RTK) ligands, such as PDGF, FGF, and EGF. (ii) Furosemide also inhibited ERK phosphorylation, induced by thrombin, a GPCR. (iii) Furosemide inhibited MEK and ERK phosphorylation even when ERK phosphorylation was induced by direct activation of protein kinase C (PKC) by TPA, which bypasses early steps of the mitogenic cascade. In addition, we found that furosemide did not affect PKC phosphorylation induced directly by TPA. Taken together, the results of the present study indicate that the signal transduction protein, controlled by the Na+/K+/Cl- cotransporter, must be downstream of the PKC, and at/or upstream to MEK in the Ras/Raf/MEK/ERK cascade.     

The Na+,K+,2Cl- -cotransport system in HeLa cells and HeLa cell mutants exhibiting an altered efflux pathway

We have investigated the characteristics of a transport system in HeLa cells, which turned out to be very similar to a previously described Na+, K+, 2Cl- -cotransport system. For further understanding about the physiological role of the cotransporter, we have mutagenized HeLa cells and selected progeny cells for growth in low potassium (0.2 mM) medium. The selected HeLa cells (LK1) exhibited alterations in the Na+,K+,2Cl- -cotransport system. LK1 cells showed a remarkable reduction of 86Rb+ efflux via the cotransporter when compared to the parental HeLa cells. In contrast, bumetanide-sensitive potassium influx, measured by 86Rb+ uptake, was increased in the LK1 cells (increase in Vmax). Km values of the cotransporter in HeLa cells and LK1 mutants revealed similar properties for 86Rb+ and 22Na+ uptake. In addition, (3H)-bumetanide binding studies were carried out on intact HeLa cells; 1.7 pmol/mg protein (3H)-bumetanide was specifically bound to HeLa parental cells, which could be calculated to a number of 103,000 binding sites/cell. LK1 cells present, 1.44 pmol/mg protein, specifically bound (3H)-bumetanide and, respectively, 137,000 binding sites/cell. The LK1 cells also exhibited an increase in the number of (3H)-ouabain binding sites as well as an increase in the activity of the Na+,K+-ATPase, expressed as a function of ouabain-sensitive 86Rb+ uptake. Furthermore, LK1 cells were different in the concentrations of intracellular Na+ (increases) and K+ (decreases) when compared to the HeLa parental cells. When grown in low K+ medium (0.2 mM K+), protein content and cell volume were increased in the LK1 cells, while the DNA content was not significantly different between both cell lines.     

Elevated [Cl-]i, and [Na+]i inhibit Na+, K+, Cl- cotransport by different mechanisms in squid giant axons

Bumetanide-sensitive (BS) unidirectional fluxes of (36)Cl- or (22)Na+ were measured in internally dialyzed squid giant axons while varying the intra- or extracellular concentrations of Na+ and/or Cl-. Raising either [Cl-]i or [Na+]i resulted in a concentration-dependent reduction of the BS influx of both (36)Cl- and (22)Na+. Raising [Cl-]i above 200 mM completely blocked BS influxes. However, raising [Na+]i to 290 mM resulted in saturable but incomplete inhibition of both BS Na+ influx and BS Cl- influx. The consequences of varying intracellular Cl- on cotransporter effluxes were complex. At lower [Cl-]i values (below 100 mM) intracellular Cl- activated cotransporter effluxes. Surprisingly, however, raising [Cl-]i levels > 125 mM resulted in a [Cl-]i-dependent inhibition of BS effluxes of both Na+ and Cl-. On the other hand, raising [Na+]i resulted only in the activation of the BS Na+ efflux; intracellular Na+ did not inhibit BS efflux even at 290 mM. The inhibitory effects of intracellular Na+ on cotransporter-mediated influxes, and lack of inhibitory effects on BS effluxes, are consistent with the trans-side inhibition expected for an ordered binding/release model of cotransporter operation. However, the inhibitory effects of intracellular Cl- on both influxes and effluxes are not explained by such a model. These data suggest that Cl may interact with an intracellular site (or sites), which does not mediate Cl transport, but does modulate the transport activity of the Na+, K+, Cl- cotransporter.