CLC-3 chloride channels in the pulmonary vasculature.

Volume-sensitive outwardly rectifying anion channels (VSOACs) are expressed in pulmonary artery smooth muscle cells (PASMCs) and have been implicated in cell proliferation, growth, apoptosis and protection against oxidative stress. In this chapter, we review the properties of native VSOACs in PASMCs, and consider the evidence that ClC-3, a member of the ClC superfamily of voltage dependent Cl- channels, may be responsible for native VSOACs in PASMCs. Finally, we examine whether or not native VSOACs and heterologously expressed ClC-3 channels function as bona fide chloride channels or as chloride/proton antiporters.

Maladaptive homeostatic plasticity in a rodent model of central pain syndrome: thalamic hyperexcitability after spinothalamic tract lesions.

Central pain syndrome (CPS) is defined as pain associated with a lesion of the CNS and is a common consequence of spinal cord injuries. We generated a rodent model of CPS by making unilateral electrolytic or demyelinating lesions centered on the spinothalamic tract in rats. Thermal hyperalgesia and mechanical allodynia occurred in both hind paws and forepaws by 7 d postlesion and were maintained >31 d. Field potentials in the ventral posterior lateral nucleus (VPL) in thalamic brain slices from lesioned animals displayed an increased probability of burst responses. Ethosuximide, a T-type calcium channel blocker, eliminated busting in lesioned thalamic slices and attenuated lesion-induced hyperalgesia and allodynia. We conclude that CPS in this model results from an increase in the excitability of thalamic nuclei that have lost normal ascending inputs as the result of a spinal cord injury and suggest that ethosuximide will relieve human CPS by restoring normal thalamic excitability.

Cardiac-specific overexpression of the human short CLC-3 chloride channel isoform in mice.

1. ClC-3 has been proposed as a molecular candidate responsible for volume-sensitive outwardly rectifying anion channels (VSOAC) in cardiac and smooth muscle cells. To further test this hypothesis, we produced a novel line of transgenic mice with cardiac-specific overexpression of the human short ClC-3 isoform (hsClC-3). 2. Northern and western blot analyses demonstrated that mRNA and protein levels of the short isoform (sClC-3) in the heart were significantly increased in hsClC-3-overexpressing (OE) mice compared with wild-type (WT) mice. Heart weight : bodyweight ratios for OE mice were significantly smaller compared with age-matched WT mice. 3. Electrocardiogram recordings indicated no difference at rest, whereas echocardiographic recordings revealed consistent reductions in left ventricular diastolic diameter, left ventricular posterior wall thickness at end of diastole and interventricular septum thickness in diastole in OE mice. 4. The VSOAC current densities in atrial cardiomyocytes were significantly increased by ClC-3 overexpression compared with WT cells. No differences in VSOAC current properties in OE and WT atrial myocytes were observed in terms of outward rectification, anion permeability (I(-) > Cl(-) > Asp(-)) and inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid and glibenclamide. The VSOAC in atrial myocytes from both groups were totally abolished by phorbol-12,13-dibutyrate (a protein kinase C activator) and by intracellular dialysis of an N-terminal anti-ClC-3 antibody. 5. Cardiac cell volume measurements revealed a significant acceleration of the rate of regulatory volume decrease (RVD) in OE myocytes compared with WT. 6. In conclusion, enhanced VSOAC currents and acceleration of the time-course of RVD in atrial myocytes of OE mice is strong evidence supporting an essential role of sClC-3 in native VSOAC function in mouse atrial myocytes.

Pelvic nerve input mediates descending modulation of homovisceral processing in the thoracolumbar spinal cord of the rat.

BACKGROUND & AIMS: Colonic afferents project to the lumbosacral and thoracolumbar spinal cord via the pelvic and hypogastric/lumbar colonic nerves, respectively. Both spinal regions process inflammatory colonic stimuli. The role of thoracolumbar segments in processing acute colorectal pain is questionable, however, because the lumbosacral spinal cord appears sufficient to process reflex responses to acute pain. Here, we show that activity in pelvic nerve colonic afferents actively modulates thoracolumbar dorsal horn neuron processing of the same colonic stimulus through a supraspinal loop: homovisceral descending modulation. METHODS: Dorsal horn neurons were recorded in the rat thoracolumbar spinal cord after acute or chronic pelvic neurectomy and cervical cold block. RESULTS: Acute pelvic neurectomy or lidocaine inhibition of lumbosacral dorsal roots facilitated the excitatory response of thoracolumbar dorsal horn neurons to colorectal distention (CRD) and decreased the percentage of neurons inhibited by CRD, suggesting colonic input over the pelvic nerve inhibits thoracolumbar processing of the same stimulus. Ectopic activity developed in the proximal pelvic nerve after chronic neurectomy reactivating the inhibitory circuit, inhibiting thoracolumbar neurons. Cervical cold block alleviated the inhibition in intact or chronic neurectomized rats. However, the facilitated response after acute pelvic neurectomy was inhibited by cervical cold block, exposing an underlying descending facilitation. Inhibiting pelvic nerve input after cervical cold block had minimal effect. CONCLUSIONS: These data demonstrate that input over the pelvic nerve modulates the response of thoracolumbar spinal neurons to CRD by a supraspinal loop and that increasing thoracolumbar processing increases visceral hyperalgesia.

Neonatal hind paw injury alters processing of visceral and somatic nociceptive stimuli in the adult rat.

UNLABELLED: Tissue damage during the first few weeks after birth can have profound effects on sensory processing in the adult. We have recently reported that a short-lasting inflammation of the neonatal rat hind paw produces baseline hypoalgesia and exacerbated hyperalgesia after reinflammation of that hind paw in the adult. Because the contralateral hind paw and forepaws also displayed hypoalgesia, we speculated that effects of the initial injury were not somatotopically restricted and would alter visceral sensory processing as well. In the present study we tested this hypothesis by examining the effects of neonatal hind paw injury at P3 or P14 on visceral and somatic sensitivity in the adult rat. In P3 rats, the visceromotor response evoked by colorectal distention in the absence of colonic inflammation was attenuated in carrageenan-treated neonatal rats compared to naive rats. Colonic inflammation in the adult reversed this hypoalgesia and evoked a level of visceral hyperalgesia similar to naive rats. There were no consequences of the P14 injury observed in the adult. In a second experiment, colonic inflammation in naive rats induced viscerosomatic inhibition to thermal stimulation of the forepaw and hind paw. This inhibition was reversed, and the paw withdrawal latency was slightly decreased in neonatal (P3) carrageenan-treated rats. Rats treated on P14 appeared similar to naive rats. These data support the hypothesis that neonatal hind paw injury during a critical period permanently alters sensory processing of multiple sensory modalities in the adult. Animals develop with greater inhibitory processing of somatic and visceral stimuli throughout the neuraxis. However, inflammation in the adult in previously uninjured tissue reverses the hypoalgesia and evokes development of normal hyperexcitability associated with tissue injury. PERSPECTIVE: Trauma experienced by premature infants can lead to alterations in sensory processing throughout life. This study shows that short-term somatic tissue injury to neonatal rats during a well-defined critical period alters several aspects of viscerosensory processing in the adult, demonstrating that injury to one tissue affects sensory processing throughout the body.

Colonic inflammation decreases thermal sensitivity of the forepaw and hindpaw in the rat.

Noxious stimulation at one site on the body can inhibit noxious stimulation at distal body sites. This has been extensively demonstrated for somatic stimuli, but less so for visceral stimuli. In the present study we present a model for visceral inflammatory stimuli inhibiting somatic thermal sensitivity in awake rats. Colonic inflammation induced by mustard oil increases the hindpaw and forepaw withdrawal latency from a noxious radiant heat source by 35-50% compared to baseline responses. The duration of the effect is dose-dependent. The withdrawal latency in control rats (mineral oil in colon, mustard oil on skin) was not affected. Rotarod performance was not affected by 5% mustard oil indicating that colonic inflammation did not produce a general malaise or decrease in motor performance. These data suggest that visceral inflammation in the rat decreases somatic sensitivity similar to that reported by patients with colonic hypersensitivity from irritable bowel syndrome or inflammatory bowel diseases.

Age-related alterations in responses of the nucleus basalis magnocellularis neurons to frontal cortex stimulation in rats.

The present study investigated the age-related alterations in responses of the nucleus basalis magnocellularis (nbM) neurons to frontal cortex (FCX) stimulation. Single unit extracellular recording from the nbM neurons were obtained with glass micropipettes in urethane-anesthetized rats. A total of 137 units were located within the nbM in the three age groups (young, 3 months; adult, 12 months; old, 24 months). FCX stimulation elicited responses in 91% of the 137 neurons. Most of them were excited. The frequency of occurrence of excitatory responses in the nbM neurons was decreased with aging. The thresholds and latencies of excitatory responses evoked by FCX stimulation were increased in old rats. The mean peak-firing rate of exciting phase was gradually reduced with aging. These findings indicate that there might be some functional changes in the nbM neurons with aging.

Sex differences in spinal processing of transient and inflammatory colorectal stimuli in the rat.

Sex differences in the spinal processing of somatic and visceral stimuli contribute to greater female sensitivity in many pain disorders. The present study examined spinal mechanisms that contribute to sex differences in visceral sensitivity. The visceromotor response to colorectal distention (CRD) was more robust in normal female rats and after intracolonic mustard oil compared with that in male rats. No sex difference was observed in the CRD-evoked response of lumbosacral (LS) and thoracolumbar (TL) colonic afferents in normal and mustard oil-treated rats, but there was a sex difference in spontaneous activity that was exacerbated by intracolonic mustard oil. The response of visceroceptive dorsal horn neurons to CRD was greater in normal female rats in the LS and TL spinal segments. The effect of intracolonic mustard oil on the CRD-evoked response of different phenotypes of visceroceptive dorsal horn neurons was dependent on sex and segment. The NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (APV) dose-dependently attenuated the visceromotor response in normal rats with greater effect in male rats. Correspondingly, there was greater cell membrane expression of the GluN1 subunit in dorsal horn extracts in female rats. After intracolonic mustard oil, there was no longer a sex difference in the effect of APV nor GluN1 expression in LS segments, but greater female expression in TL segments. These data document a sex difference in spinal processing of nociceptive visceral stimuli from the normal and inflamed colon. Differences in dorsal horn neuronal activity and NMDA receptor expression contribute to the sex differences in the visceral sensitivity observed in awake rats.

Differential processing of noxious colonic input by thoracolumbar and lumbosacral dorsal horn neurons in the rat.

Previous studies suggest the lumbosacral (LS) spinal cord processes acute colorectal stimuli whereas the thoracolumbar (TL) and LS spinal segments process inflammatory stimuli. In this study, the effects of colorectal distention (CRD) on TL and LS dorsal horn neuronal activity were recorded in Nembutal-anesthetized male rats both with and without colonic inflammation. Both single cells (before and after inflammation) and populations (multiple cells from noninflamed or inflamed rats) were studied. CRD-responsive neurons had excitatory Abrupt (on-off with stimulus) or Sustained (prolonged after discharge) responses or were Inhibited by CRD. In noninflamed rats, a significantly greater percentage of LS neurons (63% Abrupt, 27% Sustained) were excited by CRD than TL neurons (61% Abrupt, 3% Sustained). The remaining cells were Inhibited (10% LS, 36% TL). LS Abrupt neurons had lower thresholds and greater response magnitudes to CRD compared with TL Abrupt neurons. After colonic inflammation, TL neurons became more excitable: the percentage of Inhibited neurons decreased, the response magnitude of Abrupt neurons increased, and the threshold decreased. In contrast, in single-cell recordings, the response of LS Sustained neurons increased, whereas LS Abrupt neurons decreased. These data suggest that in noninflamed rats, the net response to CRD of TL visceroceptive spinal sensory neurons is less than that of LS neurons. Colonic inflammation increases the net response of TL neurons and differentially modulates the response of LS neurons. These differences may contribute to the functional dichotomy between the TL and LS spinal segments in processing acute and inflammatory colorectal pain.

Molecular mechanisms of regulation of fast-inactivating voltage-dependent transient outward K+ current in mouse heart by cell volume changes.

The K(v)4.2/4.3 channels are the primary subunits that contribute to the fast-inactivating, voltage-dependent transient outward K(+) current (I(to,fast)) in the heart. I(to,fast) is the critical determinant of the early repolarization of the cardiac action potential and plays an important role in the adaptive remodelling of cardiac myocytes, which usually causes cell volume changes, during myocardial ischaemia, hypertrophy and heart failure. It is not known, however, whether I(to,fast) is regulated by cell volume changes. In this study we investigated the molecular mechanism for cell volume regulation of I(to,fast) in native mouse left ventricular myocytes. Hyposmotic cell swelling caused a marked increase in densities of the peak I(to,fast) and a significant shortening in phase 1 repolarization of the action potential duration. The voltage-dependent gating properties of I(to,fast) were, however, not altered by changes in cell volume. In the presence of either protein kinase C (PKC) activator (12,13-dibutyrate) or phosphatase inhibitors (calyculin A and okadaic acid), hyposmotic cell swelling failed to further up-regulate I(to,fast). When expressed in NIH/3T3 cells, both K(v)4.2 and K(v)4.3 channels were also strongly regulated by cell volume in the same voltage-independent but PKC- and phosphatase-dependent manner as seen in I(to,fast) in the native cardiac myocytes. We conclude that K(v)4.2/4.3 channels in the heart are regulated by cell volume through a phosphorylation/dephosphorylation pathway mediated by PKC and serine/threonine phosphatase(s). These findings suggest a novel role of K(v)4.2/4.3 channels in the adaptive electrical and structural remodelling of cardiac myocytes in response to myocardial hypertrophy, ischaemia and reperfusion.

P2Y purinergic receptor regulation of CFTR chloride channels in mouse cardiac myocytes.

The intracellular signalling pathways and molecular mechanisms responsible for P2-purinoceptor-mediated chloride (Cl(-)) currents (I(Cl,ATP)) were studied in mouse ventricular myocytes. In standard NaCl-containing extracellular solutions, extracellular ATP (100 microm) activated two different currents, I(Cl,ATP) with a linear I-V relationship in symmetrical Cl(-) solutions, and an inwardly rectifying cation conductance (cationic I(ATP)). Cationic I(ATP) was selectively inhibited by Gd(3+) and Zn(2+), or by replacement of extracellular NaCl by NMDG; I(Cl,ATP) was Cl(-) selective, and inhibited by replacement of extracellular Cl(-) by Asp(-); both currents were prevented by suramin or DIDS pretreatment. In GTPgammaS-loaded cells, I(Cl,ATP) was irreversibly activated by ATP, but cationic I(ATP) was still regulated reversibly. GDPbetaS prevented activation of the I(Cl,ATP,) even though pertussis toxin pretreatment did not modulate I(Cl,ATP). These results suggest that activation of I(Cl,ATP) occurs via a G-protein coupled P2Y purinergic receptor. The I(Cl,ATP) persistently activated by GTPgammaS, was inhibited by glibenclamide but not by DIDS, thus exhibiting known pharmacological properties of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels. In ventricular cells of cftr(-/-) mice, extracellular ATP activated cationic I(ATP), but failed to activate any detectable I(Cl,ATP). These results provide compelling evidence that activation of CFTR Cl(-) channels in mouse heart are coupled to G-protein coupled P2Y purinergic receptors.

Functional effects of novel anti-ClC-3 antibodies on native volume-sensitive osmolyte and anion channels in cardiac and smooth muscle cells.

Whether ClC-3 encodes volume-sensitive organic osmolyte and anion channels (VSOACs) remains controversial. We have shown previously that native VSOACs in some cardiac and vascular myocytes were blocked by a commercial anti-ClC-3 carboxy terminal antibody (Alm C592-661 antibody), although recent studies have raised questions related to the specificity of Alm C592-661 antibody. Therefore, we have developed three new anti-ClC-3 antibodies and investigated their functional effects on native VSOACs in freshly isolated canine pulmonary artery smooth muscle cells (PASMCs) and guinea pig cardiac myocytes. These new antibodies produced a common prominent immunoreactive band with an apparent molecular mass of 90-92 kDa in the guinea pig heart and PASMCs, and a similar molecular mass immunoreactive band was observed in the brain from homozygous Clcn3+/+ mice but not from homozygous Clcn3-/- mice. VSOACs elicited by hypotonic cell swelling in PASMCs and guinea pig atrial myocytes were nearly completely abolished by intracellular dialysis with two new anti-ClC-3 antibodies specifically targeting the ClC-3 carboxy (C670-687 antibody) and amino terminus (A1-14 antibody). This inhibition of native VSOACs can be attributed to a specific interaction with endogenous ClC-3, because 1) preabsorption of the antibodies with corresponding antigens prevented the inhibitory effects, 2) extracellular application of a new antibody raised against an extracellular epitope (Ex133-148) of ClC-3 failed to inhibit native VSOACs in PASMCs, 3) intracellular dialysis with an antibody targeting Kv1.1 potassium channels failed to inhibit native VSOACs in guinea pig atrial myocytes, and 4) anti-ClC-3 C670-687 antibody had no effects on swelling-induced augmentation of the slow component of the delayed rectifying potassium current in guinea pig ventricular myocytes, although VSOACs in the same cells were inhibited by the antibody. These results confirm that endogenous ClC-3 is an essential molecular entity responsible for native VSOACs in PASMCs and guinea pig cardiac myocytes.

Hypotonic activation of volume-sensitive outwardly rectifying chloride channels in cultured PASMCs is modulated by SGK.

The serum- and glucocorticoid-inducible kinase (SGK) is a serine/threonine protein kinase (PK) transcriptionally regulated by corticoids, serum, and cell volume. SGK regulates cell volume of various cells by effects on Na(+) and K(+) transport through membrane channels. We hypothesized a role for SGK in the activation of volume-sensitive osmolyte and anion channels (VSOACs) in cultured canine pulmonary artery smooth muscle cells (PASMCs). Intracellular dialysis through the patch electrode of recombinant active SGK, but not kinase-dead Delta60-SGK-K127M, heat-inactivated SGK, or active Akt1, partially activated VSOACs under isotonic conditions. Dialysis of active SGK before cell exposure to hypotonic medium significantly accelerated the activation kinetics and increased the maximal density of VSOAC current. Exposure of PASMCs to hypotonic medium (230 mosM) activated phosphatidylinositol 3-kinases (PI3Ks) and their downstream targets Akt/PKB and SGK but not PKC-epsilon. Inhibition of PI3Ks with wortmannin reduced the activation rate and maximal amplitude of VSOACs. Immunoprecipitated ClC-3 channels were phosphorylated by PKC-epsilon but not by SGK in vitro, suggesting that SGK may activate VSOACs indirectly. These data indicate that the PI3K-SGK cascade is activated on hypotonic swelling of PASMCs and, in turn, affects downstream signaling molecules linked to activation of VSOACs.

Regulation of volume-sensitive outwardly rectifying anion channels in pulmonary arterial smooth muscle cells by PKC.

We tested the possible role of endogenous protein kinase C (PKC) in the regulation of native volume-sensitive organic osmolyte and anion channels (VSOACs) in acutely dispersed canine pulmonary artery smooth muscle cells (PASMC). Hypotonic cell swelling activated native volume-regulated Cl(-) currents (I(Cl.vol)) which could be reversed by exposure to phorbol 12,13-dibutyrate (0.1 microM) or by hypertonic cell shrinkage. Under isotonic conditions, calphostin C (0.1 microM) or Ro-31-8425 (0.1 microM), inhibitors of both conventional and novel PKC isozymes, significantly activated I(Cl.vol) and prevented further modulation by subsequent hypotonic cell swelling. Bisindolylmaleimide (0.1 microM), a selective conventional PKC inhibitor, was without effect. Dialyzing acutely dispersed and cultured PASMC with epsilon V1-2 (10 microM), a translocation inhibitory peptide derived from the V1 region of epsilon PKC, activated I(Cl.vol) under isotonic conditions and prevented further modulation by cell volume changes. Dialyzing PASMC with beta C2-2 (10 microM), a translocation inhibitory peptide derived from the C2 region of beta PKC, had no detectable effect. Immunohistochemistry in cultured canine PASMC verified that hypotonic cell swelling is accompanied by translocation of epsilon PKC from the vicinity of the membrane to cytoplasmic and perinuclear locations. These data suggest that membrane-bound epsilon PKC controls the activation state of native VSOACs in canine PASMC under isotonic and anisotonic conditions.

Altered properties of volume-sensitive osmolyte and anion channels (VSOACs) and membrane protein expression in cardiac and smooth muscle myocytes from Clcn3-/- mice.

ClC-3, a member of the large superfamily of ClC voltage-dependent Cl(-) channels, has been proposed as a molecular candidate responsible for volume-sensitive osmolyte and anion channels (VSOACs) in some cells, including heart and vascular smooth muscle. However, the reported presence of native VSOACs in at least two cell types from transgenic ClC-3 disrupted (Clcn3(-/-)) mice casts considerable doubt on this proposed role for ClC-3. We compared several properties of native VSOACs and examined mRNA transcripts and membrane protein expression profiles in cardiac and pulmonary arterial smooth muscle cells from Clcn3(+/+) and Clcn3(-/-) mice to: (1) test the hypothesis that native VSOACs are unaltered in cells from Clcn3(-/-) mice, and (2) test the possibility that targeted inactivation of the Clcn3 gene using a conventional murine global knock-out approach may result in compensatory changes in expression of other membrane proteins. Our experiments demonstrate that VSOAC currents in myocytes from Clcn3(+/+) and Clcn3(-/-) mice are remarkably similar in terms of activation and inactivation kinetics, steady-state current densities, rectification, anion selectivity (I(-) > Cl(-)>> Asp(-)) and sensitivity to block by glibenclamide, niflumic acid, DIDS and extracellular ATP. However, additional experiments revealed several significant differences in other fundamental properties of native VSOACs recorded from atrial and smooth muscle cells from Clcn3(-/-) mice, including: differences in regulation by endogenous protein kinase C, differential sensitivity to block by anti-ClC-3 antibodies, and differential sensitivities to [ATP](i) and free [Mg(2+)](i). These results suggest that in response to Clcn3 gene deletion, there may be compensatory changes in expression of other proteins that alter VSOAC channel subunit composition or associated regulatory subunits that give rise to VSOACs with different properties. Consistent with this hypothesis, in atria from Clcn3(-/-) mice compared to Clcn3(+/+) mice, quantitative analysis of ClC mRNA expression levels revealed significant increases in transcripts for ClC-1, ClC-2, and ClC-3, and protein expression profiles obtained using two-dimensional polyacrylamide gel electrophoresis revealed complex changes in at least 35 different unidentified membrane proteins in cells from Clcn3(-/-) mice. These findings emphasize that caution needs to be exercised in simple attempts to interpret the phenotypic consequences of conventional global Clcn3 gene inactivation.