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NaCl absorption in the medullary thick ascending limb of the loop of Henle (THAL) is mediated by the apical Na/K/2Cl cotransporter (NKCC2). Hormones that increase cGMP, such as nitric oxide (NO) and natriuretic peptides, decrease NaCl absorption by the THAL. However, the mechanism by which cGMP decreases NaCl absorption in THALs is not known. We hypothesized that cGMP decreases surface NKCC2 levels in the THAL. We used surface biotinylation to measure surface NKCC2 levels in rat THAL suspensions. We tested the effect of the membrane-permeant cGMP analog dibutyryl-cGMP (db-cGMP) on surface NKCC2 levels. Incubating THALs with dbcGMP for 20 min decreased surface NKCC2 levels in a concentration-dependent manner (basal = 100%; db-cGMP 100 [micro]M = 77 [+ or -] 7%; 500 [micro]M = 54 [+ or -] 10% and 1,000 [micro]M = 61 [+ or -] 8%). A different cGMP analog 8-bromo-cGMP (8-Br-cGMP) also decreased surface NKCC2 levels by 25%, (basal = 100%; 8-Br-cGMP = 75 [+ or -] 5%). Incubation of isolated, perfused THALs with db-cGMP decreased apical surface NKCC2 labeling levels as measured by immunofluorescence and confocal microscopy, cGMP-stimulated phosphodiesterase 2 (PDE2) mediates the inhibitory effect of NO on NaC1 absorption by THALs. Thus we examined the role of PDE2 and found that PDE2 inhibitors blocked the effect of db-cGMP on surface NKCC2. Also, a nonstimulatory concentration of db-cAMP blocked the cGMP-induced decrease in surface NKCC2. Finally, db-cGMP inhibited THAL net Cl absorption by 48 [+ or -] 4%, and this effect was completely blocked by PDE2 inhibition. We conclude that cGMP decreases NKCC2 levels in the apical membrane of THALs and that this effect is mediated by PDE2. This is an important mechanism by which cGMP inhibits NaCl absorption by the THAL. Na-K-2Cl cotransporter; trafficking; apical: NO; epithelial

Choroid plexus epithelial cells (CPECs) secrete cerebrospinal fluid (CSF). They express Na+-K+-ATPase and Na+-K+-2Cl− cotransporter 1 (NKCC1) on their apical membrane, deviating from typical basolateral membrane location in secretory epithelia. Given this peculiarity, the direction of basal net ion fluxes mediated by NKCC1 in CPECs is controversial, and cotransporter function is unclear. Determining the direction of basal NKCC1-mediated fluxes is critical to understanding the function of apical NKCC1. If NKCC1 works in the net efflux mode, it may be directly involved in CSF secretion. Conversely, if NKCC1 works in the net influx mode, it would have an absorptive function, contributing to intracellular Cl− concentration ([Cl−]i) and cell water volume (CWV) maintenance needed for CSF secretion. We resolve this long-standing debate by electron microscopy (EM), live-cell-imaging microscopy (LCIM), and intracellular Na+ and Cl− measurements in single CPECs of NKCC1+/+ and NKCC1−/− mouse. NKCC1-mediated ion and associated water fluxes are tightly linked, thus their direction is inferred by measuring CWV changes. Genetic or pharmacological NKCC1 inactivation produces CPEC shrinkage. EM of NKCC1−/− CPECs in situ shows they are shrunken, forming large dilations of their basolateral extracellular spaces, yet remaining attached by tight junctions. Normarski LCIM shows in vitro CPECs from NKCC1−/− are ~17% smaller than NKCC1+/+. CWV measurements in calcein-loaded CPECs show that bumetanide (10 μM) produces ~16% decrease in CWV in NKCC1+/+ but not in NKCC1−/− CPECs. Our findings suggest that under basal conditions apical NKCC1 is continuously active and works in the net inward flux mode maintaining [Cl−]i and CWV needed for CSF secretion. [ABSTRACT FROM AUTHOR]

Background: Increased blood levels of ammonia (NH3) and ammonium (NH4+), i.e. hyperammonemia, leads to cellular brain edema in humans with acute liver failure. The pathophysiology of this edema is poorly understood. This is partly due to incomplete understanding of the osmotic effects of the pair NH3/NH4+ at the cellular and molecular levels. Cell exposure to solutions containing NH3/NH4+ elicits changes in intracellular pH (pHi), which can in turn affect cell water volume (CWV) by activating transport mechanisms that produce net gain or loss of solutes and water. The occurrence of CWV changes caused by NH3/NH4+ has long been suspected, but the mechanisms, magnitude and kinetics of these changes remain unknown. Methods: Using fluorescence imaging microscopy we measured, in real time, parallel changes in pHi and CWV caused by brief exposure to NH3/NH4+ of single cells (N1E-115 neuroblastoma or NG-108 neuroblastoma X glioma ) loaded with the fluorescent indicator BCECF. Changes in CWV were measured by exciting BCECF at its intracellular isosbestic wavelength (∼438 nm), and pHi was measured ratiometrically. Results: Brief exposure to isosmotic solutions (i.e. having the same osmolality as that of control solutions) containing NH4Cl (0.5- 30 mM) resulted in a rapid, dose-dependent swelling, followed by isosmotic regulatory volume decrease (iRVD). NH4Cl solutions in which either extracellular [NH3] or [NH4+] was kept constant while the other was changed by varying the pH of the solution, demonstrated that [NH3]o rather than [NH4+]o is the main determinant of the NH4Cl-induced swelling. The iRVD response was sensitive to the anion channel blocker NPPB, and partly dependent on external Ca2+. Upon removal of NH4Cl, cells shrank and displayed isosmotic regulatory volume increase (iRVI). Regulatory volume responses could not be activated by comparable CWV changes produced by anisosmotic solutions, suggesting that membrane stretch or contraction by themselves are not sufficient to trigger these responses. Inhibition of glutamine synthetase partially blocked the NH4Cl-induced swelling. Conclusions: A quantitative description of the osmotic changes produced by exposure to NH3/NH4+ in single neurons and glial cells shows that ∼35 to 45% of the initial cell swelling can be explained by intracellular accumulation of NH4+ due to rapid permeation and protonation of NH3. Another∼23% of the swelling can be accounted for by rapid glutamine accumulation. The results are discussed in terms of basic cell physiology and their potential relevance to the pathophysiology of hyperammonemic cellular brain edema. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

GABA depolarizes and excites central neurons during early development, becoming inhibitory and hyperpolarizing with maturation. This "developmental shift" occurs abruptly, reflecting a decrease in intracellular Cl- concentration ([Cl-]i) and a hyperpolarizing shift in Cl- equilibrium potential due to upregulation of the K+-Cl- cotransporter KCC2b, a neuron-specific Cl- extruder. In contrast, primary afferent neurons (PANs) are depolarized by GABA throughout adulthood because of expression of NKCC1, a Na+-K+-2Cl- cotransporter that accumulates Cl- above equilibrium. The GABAA-mediated depolarization of PANs determines presynaptic inhibition in the spinal cord, a key mechanism gating somatosensory information. Little is known about developmental changes in Cl- transporter expression and Cl- homeostasis in PANs. Whether NKCC1 is expressed in PANs of all phenotypes or is restricted to subpopulations (e.g., nociceptors) is debatable. Likewise, whether PANs express KCC2s is controversial. We investigated NKCC1 and K+-Cl- cotransporter expression in rat and mouse dorsal root ganglion (DRG) neurons with molecular methods. Using fluorescence imaging microscopy, we measured [Cl-]i in acutely dissociated rat DRG neurons (P0 -P21) loaded with N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide and classified with phenotypic markers. DRG neurons of all sizes express two NKCC1 mRNAs, one full-length and a shorter splice variant lacking exon 21. Immunolabeling with validated antibodies revealed ubiquitous expression of NKCC1 in DRG neurons irrespective of postnatal age and phenotype. As maturation progresses [Cl-]i decreases gradually, persisting above equilibrium in >95% mature neurons. DRG neurons express mRNAs for KCC1, KCC3s, and KCC4, but not for KCC2s. Mechanisms underlying PANs' developmental changes in Cl- homeostasis are discussed and compared with those of central neurons. [ABSTRACT FROM AUTHOR]

Choroid plexus epithelial cells (CPECs) secrete cerebrospinal fluid (CSF). They express Na + -K + -ATPase and Na + -K + -2Cl - cotransporter 1 (NKCC1) on their apical membrane, deviating from typical basolateral membrane location in secretory epithelia. Given this peculiarity, the direction of basal net ion fluxes mediated by NKCC1 in CPECs is controversial, and cotransporter function is unclear. Determining the direction of basal NKCC1-mediated fluxes is critical to understanding the function of apical NKCC1. If NKCC1 works in the net efflux mode, it may be directly involved in CSF secretion. Conversely, if NKCC1 works in the net influx mode, it would have an absorptive function, contributing to intracellular Cl - concentration ([Cl - ] i ) and cell water volume (CWV) maintenance needed for CSF secretion. We resolve this long-standing debate by electron microscopy (EM), live-cell-imaging microscopy (LCIM), and intracellular Na + and Cl - measurements in single CPECs of NKCC1+/+ and NKCC1-/- mouse. NKCC1-mediated ion and associated water fluxes are tightly linked, thus their direction is inferred by measuring CWV changes. Genetic or pharmacological NKCC1 inactivation produces CPEC shrinkage. EM of NKCC1-/- CPECs in situ shows they are shrunken, forming large dilations of their basolateral extracellular spaces, yet remaining attached by tight junctions. Normarski LCIM shows in vitro CPECs from NKCC1-/- are ~17% smaller than NKCC1+/+. CWV measurements in calcein-loaded CPECs show that bumetanide (10 μM) produces ~16% decrease in CWV in NKCC1+/+ but not in NKCC1-/- CPECs. Our findings suggest that under basal conditions apical NKCC1 is continuously active and works in the net inward flux mode maintaining [Cl - ] i and CWV needed for CSF secretion.

Background: Increased blood levels of ammonia (NH3) and ammonium (NH4(+)), i.e. hyperammonemia, leads to cellular brain edema in humans with acute liver failure. The pathophysiology of this edema is poorly understood. This is partly due to incomplete understanding of the osmotic effects of the pair NH3/NH4(+) at the cellular and molecular levels. Cell exposure to solutions containing NH3/NH4(+) elicits changes in intracellular pH (pHi), which can in turn affect cell water volume (CWV) by activating transport mechanisms that produce net gain or loss of solutes and water. The occurrence of CWV changes caused by NH3/NH4(+) has long been suspected, but the mechanisms, magnitude and kinetics of these changes remain unknown., Methods: Using fluorescence imaging microscopy we measured, in real time, parallel changes in pHi and CWV caused by brief exposure to NH3/NH4(+) of single cells (N1E-115 neuroblastoma or NG-108 neuroblastoma X glioma ) loaded with the fluorescent indicator BCECF. Changes in CWV were measured by exciting BCECF at its intracellular isosbestic wavelength (∼438 nm), and pHi was measured ratiometrically., Results: Brief exposure to isosmotic solutions (i.e. having the same osmolality as that of control solutions) containing NH4Cl (0.5- 30 mM) resulted in a rapid, dose-dependent swelling, followed by isosmotic regulatory volume decrease (iRVD). NH4Cl solutions in which either extracellular [NH3] or [NH4(+)] was kept constant while the other was changed by varying the pH of the solution, demonstrated that [NH3]o rather than [NH4(+)]o is the main determinant of the NH4Cl-induced swelling. The iRVD response was sensitive to the anion channel blocker NPPB, and partly dependent on external Ca(2+). Upon removal of NH4Cl, cells shrank and displayed isosmotic regulatory volume increase (iRVI). Regulatory volume responses could not be activated by comparable CWV changes produced by anisosmotic solutions, suggesting that membrane stretch or contraction by themselves are not sufficient to trigger these responses. Inhibition of glutamine synthetase partially blocked the NH4Cl-induced swelling., Conclusions: A quantitative description of the osmotic changes produced by exposure to NH3/NH4(+) in single neurons and glial cells shows that ∼35 to 45% of the initial cell swelling can be explained by intracellular accumulation of NH4(+) due to rapid permeation and protonation of NH3. Another∼23% of the swelling can be accounted for by rapid glutamine accumulation. The results are discussed in terms of basic cell physiology and their potential relevance to the pathophysiology of hyperammonemic cellular brain edema., (© 2014 S. Karger AG, Basel.)

The solute carrier protein family 12 group A, member one (Slc12a1) and two (Slc12a2) encode several splice variants of the kidney-specific and the ubiquitous isoforms, respectively, of the bumetanide (BTD)-sensitive Na-dependent K2Cl co-transporter. The Slc12a2 co-transporter is involved in the maintenance of a high intracellular chloride concentration [Cl(-)](i) in β-cells and its inhibition with BTD blocks glucose-induced insulin secretion. In β-cells, [Cl(-)](i) plays an important role in glucose-induced depolarization and insulin secretion. Glucose promotes electrogenic efflux of Cl(-) contributing to β-cell's electrical and secretory activity. To identify the expression pattern of Slc12a1 and Slc12a2 genes in β-cells we have used RT-PCR, Western blotting and immunolocalization studies in mouse pancreatic islets, β-cell lines and rat tissues. Our results demonstrate expression of specific splice variants of Slc12a1 and Slc12a2 transcripts in β-cells i.e., variants 1 of Slc12a1 (NKCC2A) and Slc12a2 (NKCC1a). Molecular cloning and characterization of Slc12a1 variant 1 transcripts from β-cells revealed an alternative splicing event involving the 5'-UTR region. NKCC2A expression at the protein level in islets and β-cells was confirmed by immunoblotting and immunolocalization. Further, NKCC2A, NKCC1a and pro-insulin co-localized in β-cells but not in the exocrine pancreas. Therefore, our results provide for the first time evidence of NKCC2A expression in pancreatic β-cells where it may play a role in insulin secretion., (Copyright © 2012 S. Karger AG, Basel.)