Intracellular chloride regulation in amphibian dorsal root ganglion neurones studied with ion-selective microelectrodes.

1. Intracellular Cl- activity (aiCl) and membrane potential (Em) were measured in frog dorsal root ganglion neurones (DRG neurones) using double-barrelled Cl- -selective microelectrodes. In standard Ringer solution buffered with HEPES (5 mM), equilibrated with air or 100% O2, the resting membrane potential was -57.7 +/- 1.0 mV and aiCl was 23.6 +/- 1.0 mM (n = 53). The value of aiCl was 2.6 times the activity expected for an equilibrium distribution and the difference between Em and ECl was 25 mV. 2. Removal of external Cl- led to a reversible fall in aiCl. Initial rates of decay and recovery of aiCl were 4.1 and 3.3 mM min-1, respectively. During the recovery of aiCl following return to standard Ringer solution, most of the movement of Cl- occurred against the driving force for a passive distribution. Changes in aiCl were not associated with changes in Em. Chloride fluxes estimated from initial rates of change in aiCl 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 Cl- activity (aoCl). The relationship between aiCl and aoCl had a sigmoidal shape with a half-maximal activation of about 50 mM-external Cl-. 4. The steady-state aiCl depended on the simultaneous presence of extracellular Na+ and K+. Similarly, the active reaccumulation of Cl- after intracellular Cl- depletion 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 aiCl observed in Cl- -free solutions was also inhibited by bumetanide. 6. Cell volume changes were calculated from the observed changes in aiCl. Cells were estimated to shrink in Cl- -free solutions to about 75% their initial volume, at an initial rate of 6% min-1. 7. The present results provide direct evidence for the active accumulation of Cl- in 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

Peripheral and Central Antinociceptive Action of Na\u3csup\u3e+\u3c/sup\u3e–K\u3csup\u3e+\u3c/sup\u3e–2Cl\u3csup\u3e−\u3c/sup\u3e Cotransporter Blockers on Formalin-Induced Nociception in Rats

The possible local peripheral and spinal (intrathecal) antinociceptive effect of Na+–K+–2Cl− cotransporter (NKCC) inhibitors was investigated in the rat formalin test. Nociceptive flinching behavior induced by formalin (1%) injection in the hind paw was assessed following administration of cotransporter inhibitors. Local peripheral pretreatment in the ipsilateral paw with bumetanide (ED30, 27.1±12.7 μg/paw), piretanide (ED30, 109.2±21.6 μg/paw) or furosemide (ED30, 34.3±5.0 μg/paw), but not vehicle (DMSO 100%), produced dose-dependent antinociception in phase 2 of the test. Local bumetanide had the greatest effect (∼70% antinociception). Bumetanide also inhibited formalin-induced flinching behavior during phase 1 (ED30, 105.6±99.1 μg/paw). Spinal intrathecal pretreatment with bumetanide (ED30, 194.6±97.9 μg), piretanide (ED30, 254.4±104.9 μg) or furosemide (ED30, 32.0±6.9 μg), but not vehicle (DMSO 100%), also produced antinociception in phase 2. In this case, only intrathecal furosemide reduced flinching behavior during phase 1 (ED30, 99.4±51.4 μg) and had the maximal antinociceptive effect in phase 2 (∼65% antinociception). The opioid receptor-antagonist naloxone (2 mg/kg, s.c.) did not reverse antinociception induced by either peripheral or spinal administration of NKCC blockers. Our data suggest that the Na+–K+–2Cl− cotransporter localized in sensory neurons at intraspinal and peripheral sites is involved in formalin-induced nociception

Peripheral and Central Antinociceptive Action of Na\u3csup\u3e+\u3c/sup\u3e–K\u3csup\u3e+\u3c/sup\u3e–2Cl\u3csup\u3e−\u3c/sup\u3e Cotransporter Blockers on Formalin-Induced Nociception in Rats

The possible local peripheral and spinal (intrathecal) antinociceptive effect of Na+–K+–2Cl− cotransporter (NKCC) inhibitors was investigated in the rat formalin test. Nociceptive flinching behavior induced by formalin (1%) injection in the hind paw was assessed following administration of cotransporter inhibitors. Local peripheral pretreatment in the ipsilateral paw with bumetanide (ED30, 27.1±12.7 μg/paw), piretanide (ED30, 109.2±21.6 μg/paw) or furosemide (ED30, 34.3±5.0 μg/paw), but not vehicle (DMSO 100%), produced dose-dependent antinociception in phase 2 of the test. Local bumetanide had the greatest effect (∼70% antinociception). Bumetanide also inhibited formalin-induced flinching behavior during phase 1 (ED30, 105.6±99.1 μg/paw). Spinal intrathecal pretreatment with bumetanide (ED30, 194.6±97.9 μg), piretanide (ED30, 254.4±104.9 μg) or furosemide (ED30, 32.0±6.9 μg), but not vehicle (DMSO 100%), also produced antinociception in phase 2. In this case, only intrathecal furosemide reduced flinching behavior during phase 1 (ED30, 99.4±51.4 μg) and had the maximal antinociceptive effect in phase 2 (∼65% antinociception). The opioid receptor-antagonist naloxone (2 mg/kg, s.c.) did not reverse antinociception induced by either peripheral or spinal administration of NKCC blockers. Our data suggest that the Na+–K+–2Cl− cotransporter localized in sensory neurons at intraspinal and peripheral sites is involved in formalin-induced nociception

Intracellular Chloride Regulation in Amphibian Dorsal Root Ganglion Neurones Studied With Ion-Selective Microelectrodes

1. Intracellular Cl- activity (aiCl) and membrane potential (Em) were measured in frog dorsal root ganglion neurones (DRG neurones) using double-barrelled Cl- -selective microelectrodes. In standard Ringer solution buffered with HEPES (5 mM), equilibrated with air or 100% O2, the resting membrane potential was -57.7 +/- 1.0 mV and aiCl was 23.6 +/- 1.0 mM (n = 53). The value of aiCl was 2.6 times the activity expected for an equilibrium distribution and the difference between Em and ECl was 25 mV. 2. Removal of external Cl- led to a reversible fall in aiCl. Initial rates of decay and recovery of aiCl were 4.1 and 3.3 mM min-1, respectively. During the recovery of aiCl following return to standard Ringer solution, most of the movement of Cl- occurred against the driving force for a passive distribution. Changes in aiCl were not associated with changes in Em. Chloride fluxes estimated from initial rates of change in aiCl 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 Cl- activity (aoCl). The relationship between aiCl and aoCl had a sigmoidal shape with a half-maximal activation of about 50 mM-external Cl-. 4. The steady-state aiCl depended on the simultaneous presence of extracellular Na+ and K+. Similarly, the active reaccumulation of Cl- after intracellular Cl- depletion 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 aiCl observed in Cl- -free solutions was also inhibited by bumetanide. 6. Cell volume changes were calculated from the observed changes in aiCl. Cells were estimated to shrink in Cl- -free solutions to about 75% their initial volume, at an initial rate of 6% min-1. 7. The present results provide direct evidence for the active accumulation of Cl- in 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