SUMMARY1. Intracellular Cl-activity (a',) and membrane potential (Em) were measured in frog dorsal root ganglion neurones (DRG neurones) using double-barrelled C1--selective microelectrodes. In standard Ringer solution buffered with HEPES (5 mM), equilibrated with air or 100% 02, the resting membrane potential was -57-7 + 1'0 mV and a'I was 23-6 + 10 mm (n = 53). The value of a'1 was 2-6 times the activity expected for an equilibrium distribution and the difference between Em and Ec1 was 25 mV.2. Removal of external Cl-led to a reversible fall in a'c. Initial rates of decay and recovery of a', were 4-1 and 3-3 mm min-', respectively. During the recovery of a' following return to standard Ringer solution, most of the movement of Cl-occurred against the driving force for a passive distribution. Changes in ai, were not associated with changes in Em. Chloride fluxes estimated from initial rates of change in a', when external Cl-was removed were too high to be accounted for by electrodiffusion.3. The intracellular accumulation of Cl-was dependent on the extracellular Clactivity (ao1). The relationship between at1 and aO I had a sigmoidal shape with a halfmaximal activation of about 50 mM-external Cl-.4. The steady-state a', depended on the simultaneous presence of extracellular Na+ and K+. Similarly, the active reaccumulation of Cl-after intracellular Cldepletion was abolished in the absence of either Na+ or K+ in the bathing solution.5. The reaccumulation of Cl-was inhibited by furosemide (0-5-1 x 10-3 M) or bumetanide (10-5 M). The decrease in a'i observed in Cl--free solutions was also inhibited by bumetanide.6. Cell volume changes were calculated from the observed changes in at,. Cells were estimated to shrink in Cl--free solutions to about 75 % their initial volume, at an initial rate of 6% min-'.* Address for correspondence and reprint requests.F. J. ALVAREZ-LEEFMANS AND OTHERS 7. The present results provide direct evidence for the active accumulation of Clin DRG neurones. The mechanism of Cl-transport is electrically silent, dependent on the simultaneous presence of external Cl-, Na+ and K+ and inhibited by loop diuretics. It is suggested that a Na+: K+: Cl-co-transport system mediates the active transport of Cl-across the cell membrane of DRG neurones.

No abstract

. Alterations in the balance of NKCC1 and KCC2 activity may determine the switch from hyperpolarizing to depolarizing effects of GABA, reported in the subiculum of epileptic patients with hippocampal sclerosis. We studied the expression of NKCC (putative NKCC1) and KCC2 in human normal temporal neocortex by Western blot analysis and in normal and epileptic regions of the subiculum and the hippocampus proper using immunocytochemistry. Western blot analysis revealed NKCC and KCC2 proteins in adult human neocortical membranes similar to those in rat neocortex.NKCC and KCC2 immunolabeling of pyramidal and nonpyramidal cells was found in normal and epileptic hippocampal formation. In the transition between the subiculum with sclerotic regions of CA1, known to exhibit epileptogenic activity, double immunolabeling of NKCC and KCC2 revealed that approximately 20% of the NKCC-immunoreactive neurons do not express KCC2. In these same areas some neurons were distinctly hyperinnervated by parvalbumin (PV) positive hypertrophic basket formations that innervated mostly neurons expressing NKCC (74%) and to a lesser extent NKCCimmunonegative neurons (26%). Hypertrophic basket formations also innervated KCC2-positive (76%) and -negative (24%) neurons. The data suggest that changes in the relative expression of NKCC1 and KCC2 in neurons having aberrant GABA-ergic hyperinnervation may contribute to epileptiform activity in the subicular regions adjacent to sclerotic areas of the hippocampus.

Ares GR, Caceres P, Alvarez-Leefmans FJ, Ortiz PA. cGMP decreases surface NKCC2 levels in the thick ascending limb: role of phosphodiesterase 2 (PDE2). Am J Physiol Renal Physiol 295: F877-F887, 2008. First published August 6, 2008 doi:10.1152/ajprenal.00449.2007.-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 concentrationdependent manner (basal ϭ 100%; db-cGMP 100 M ϭ 77 Ϯ 7%; 500 M ϭ 54 Ϯ 10% and 1,000 M ϭ 61 Ϯ 8%). A different cGMP analog 8-bromo-cGMP (8-Br-cGMP) also decreased surface NKCC2 levels by 25%, (basal ϭ 100%; 8-Br-cGMP ϭ 75 Ϯ 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 NaCl 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 Ϯ 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.

Water transport by the Na + -K + -2Cl − cotransporter (NKCC1) was studied in confluent cultures of pigmented epithelial (PE) cells from the ciliary body of the fetal human eye. Interdependence among water, Na + and Cl − fluxes mediated by NKCC1 was inferred from changes in cell water volume, monitored by intracellular self-quenching of the fluorescent dye calcein. Isosmotic removal of external Cl − or Na + caused a rapid efflux of water from the cells, which was inhibited by bumetanide (10 μm). When returned to the control solution there was a rapid water influx that required the simultaneous presence of external Na + and Cl − . The water influx could proceed uphill, against a transmembrane osmotic gradient, suggesting that energy contained in the ion fluxes can be transferred to the water flux. The influx of water induced by changes in external [Cl − ] saturated in a sigmoidal fashion with a K m of 60 mm, while that induced by changes in external [Na + ] followed first order kinetics with a K m of about 40 mm. These parameters are consistent with ion transport mediated by NKCC1. Our findings support a previous investigation, in which we showed water transport by NKCC1 to be a result of a balance between ionic and osmotic gradients. The coupling between salt and water transport in NKCC1 represents a novel aspect of cellular water homeostasis where cells can change their volume independently of the direction of an osmotic gradient across the membrane. This has relevance for both epithelial and symmetrical cells.

Rocha-González HI, Mao S, Alvarez-Leefmans FJ. Na ϩ ,K ϩ ,2Cl Ϫ cotransport and intracellular chloride regulation in rat primary sensory neurons: thermodynamic and kinetic aspects.

Cell volume is frequently down‐regulated by the activation of anion channels. The role of cell swelling‐activated chloride channels in cell volume regulation has been studied using the patch‐clamp technique and a non‐invasive microspectrofluorimetric assay for changes in cell volume. The rate of activation of these chloride channels was shown to limit the rate of regulatory volume decrease (RVD) in response to hyposmotic solutions. Expression of the human MDR1 or mouse mdr1a genes, but not the mouse mdr1b gene, encoding the multidrug resistance P‐glycoprotein (P‐gp), increased the rate of channel activation and the rate of RVD. In addition, P‐gp decreased the magnitude of hyposmotic shock required to activate the channels and to elicit RVD. Tamoxifen selectively inhibited both chloride channel activity and RVD. No effect on potassium channel activity was elicited by expression of P‐gp. The data show that, in these cell types, swelling‐activated chloride channels have a central role in RVD. Moreover, they clarify the role of P‐gp in channel activation and provide direct evidence that P‐gp, through its effect on chloride channel activation, enhances the ability of cells to down‐regulate their volume.

GABA, a major inhibitory neurotransmitter, depolarizes hippocampal pyramidal neurons during the first postnatal week. These depolarizations result from an efflux of Cl- through GABAA-gated anion channels. The outward Cl- gradient that provides the driving force for Cl- efflux might be generated and maintained by the Na+, K+, 2Cl- cotransporter (NKCC) that keeps intracellular Cl- concentration above electrochemical equilibrium. The developmental pattern of expression of the cotransporter in the hippocampus is not known. We studied the postnatal distribution pattern of NKCC in the hippocampus using a monoclonal antibody (T4) against a conserved epitope in the C-terminus of the cotransporter molecule. We also examined the temporal relationships between the developmental pattern of NKCC expression and the formation of perisomatic GABAergic synapses. This study was aimed at determining, with antivesicular inhibitory amino acid transporter (VIAAT) antibodies, whether perisomatic GABAergic synapses are formed preferentially at the time when GABA is depolarizing. During the first postnatal week, NKCC immunolabelling was restricted to cell bodies in the pyramidal cell layer and in the strata oriens and radiatum. In contrast, at postnatal day 21 (P21) and in adult animals little or no labelling occurred in cell bodies; instead, a prominent dendritic labelling appeared in both pyramidal and nonpyramidal neurons. The ultrastructural immunogold study in P21 rat hippocampi corroborated the light-microscopy results. In addition, this study revealed that a portion of the silver-intensified colloidal gold particles were located on neuronal plasmalemma, as expected for a functional cotransporter. The formation of inhibitory synapses on perikarya of the pyramidal cell layer was a late process. The density of VIAAT-immunoreactive puncta in the stratum pyramidale at P21 reached four times the P7 value in CA3, and six times the P7 value in CA1. Electron microscopy revealed that the number of synapses per neuronal perikaryal profile in the stratum pyramidale of the CA3 area at P21 was three times higher than at P7, even if a concomitant 20% increase in the area of these neuronal perikaryal profiles occurred. It is concluded that, in hippocampal pyramidal cells, there is a developmental shift in the NKCC localization from a predominantly somatic to a predominantly dendritic location. The presence of NKCC during the first postnatal week is consistent with the hypothesis that this transporter might be involved in the depolarizing effects of GABA. The depolarizing effects of GABA may not be required for the establishment of the majority of GABAergic synapses in the stratum pyramidale, because their number increases after the first postnatal week, when GABA action becomes hyperpolarizing.