Blockade Of Chloride Channels Research Articles

Clues and new evidences in arterial hypertension: unmasking the role of the chloride anion.

The present review will focus on the role of chloride anion in cardiovascular disease, with special emphasis in the development of hypertensive disease and vascular inflammation. It is known that acute and chronic overload of sodium chloride increase blood pressure and have pro-inflammatory and pro-fibrotic effects on different target organs, but it is unknown how chloride may influence these processes. Chloride anion is the predominant anion in the extracellular fluid and its intracellular concentration is dynamically regulated. As the queen of the electrolytes, it is of crucial importance to understand the physiological mechanisms that regulate the cellular handling of this anion including the different transporters and cellular chloride channels, which exert a variety of functions, such as regulation of cellular proliferation, differentiation, migration, apoptosis, intracellular pH and cellular redox state. In this article, we will also review the relationship between dietary, serum and intracellular chloride and how these different sources of chloride in the organism are affected in hypertension and their impact on cardiovascular disease. Additionally, we will discuss the approach of potential strategies that affect chloride handling and its potential effect on cardiovascular system, including pharmacological blockade of chloride channels and non-pharmacological interventions by replacing chloride by another anion.

Decreased intracellular chloride promotes ADP induced platelet activation through inhibition of cAMP/PKA instead of activation of Lyn/PI3K/Akt pathway

Decrease of chloride concentration contributes to cardiovascular diseases, however, whether decrease of chloride concentration is involved in platelet activation remains elusive. In the present study, we found that ACI patients had lower serum chloride which would be rescued after Aspirin administration. ADP induced chloride concentration reduction in platelets. Blockade of chloride channel prevented ADP-induced platelet adhesion, activation and aggregation, however, decreasing the extracellular chloride concentration promoted ADP-induced platelet adhesion and activation. Decrease of the extracellular chloride concentration facilitated the inactivation of Src family kinase Lyn, which was not involved in PI3K/Akt phosphorylation. Nevertheless, low chloride concentration promoted the production of platelet cytosol Gαi2 subunit. This subunit prevents AC from converting ATP into cAMP, which therefore, inhibited the phosphorylation of PKA to promote platelet activation. In conclusion, decreased intracellular chloride promotes ADP induced platelet activation through the Gαi2/cAMP/PKA pathway instead of the Lyn/PI3K/Akt signal pathway.

Potential role of potassium and chloride channels in regulation of silymarin-induced apoptosis in Candida albicans.

Silymarin, which is derived from the seeds of Silybum marianum, has been widely used to prevent and treat liver diseases. In our previous study, we reported that at concentrations above the minimal inhibitory concentration (MIC), silymarin exhibited antifungal activity against Candida albicans by targeting its plasma membrane. However, the antifungal mechanism at concentration below the MIC remains unknown. Therefore, we aimed to determine the underlying mechanism of antifungal effects of silymarin at concentration below the MIC. To evaluate the inhibitory effects on the ion channels, C. albicans cells were separately pretreated with potassium and chloride channel blockers. The antifungal activity of silymarin at sub-MIC was affected by the ion channel blockers. Potassium channel blockade inhibited the antifungal effects, whereas chloride channel blockade slightly enhanced these effects. Subsequently, we found that silymarin induced disturbances in calcium homeostasis via the cytosolic and mitochondrial accumulation of calcium. Furthermore, apoptotic responses, such as phosphatidylserine exposure, loss of mitochondrial membrane potential (MMP), DNA damage, and caspase activation were induced in response to silymarin treatment. The increases in intracellular calcium level and pro-apoptotic changes were prevented when potassium ion channels were blocked. In contrast, these changes were enhanced upon chloride channels blockade; however, this did not affect the intracellular calcium levels and MMP loss. Thus, we showed that silymarin treatment at concentration below the MIC induced apoptosis in C. albicans; additionally, ion channels contributed these effects. © 2018 IUBMB Life, 70(3):197-206, 2018.

CIC-3 chloride channel blockade protects mouse photoreceptor-derived 661W cells against ischemia-reperfusion-induced injury in vitro

Exposure to ischemia/reperfusion leads to the development and progression of retinal degenerative diseases. However, the exact mechanisms are not fully understood. In this article, the role of CIC-3 chloride channel in OGD-R (oxygen-glucose deprivation followed by reperfusion)-induced retinal damage was examined. Mouse photoreceptor-derived 661W cells were treated with the CIC-3 antisense oligonucleotide before exposure to OGD-R. Cell viability, mitochondrial membrane potential, cytochrome-c level, DNA fragmentation, caspase activity and protein expression were detected. Pretreatment of 661W cells with CIC-3 antisense oligonucleotide significantly decreased OGD-R-mediated toxicity. In addition, apoptosis-related biochemical indicators showed that pre-incubation of CIC-3 antisense oligonucleotide would elevate the mitochondrial membrane potential, decrease the release of cytochrome-c as well as formation of DNA fragmentation, and inhibit activities of caspase-3 and caspase- 9 in exogenous OGD-R-treated 661W cells. Moreover, treatment with CIC-3 antisense oligonucleotide changed the expression of apoptosis-related protein. These results suggest that CIC-3 chloride channel mediates OGD-R-induced apoptosis, at least partially through mitochondrial membrane potential pathway and increasing the levels of proapoptotic molecules in 661W cells. CIC-3 chloride channel blockade may provide a new therapeutic approach for preventing ischemia/reperfusion-induced retinal neural damage.

Chloride channel blockade relaxes airway smooth muscle and potentiates relaxation by β-agonists.

Severe bronchospasm refractory to β-agonists continues to cause significant morbidity and mortality in asthmatic patients. We questioned whether chloride channels/transporters are novel targets for the relaxation of airway smooth muscle (ASM). We have screened a library of compounds, derivatives of anthranilic and indanyloxyacetic acid, that were originally developed to antagonize chloride channels in the kidney. We hypothesized that members of this library would be novel calcium-activated chloride channel blockers for the airway. The initial screen of this compound library identified 4 of 20 compounds that relaxed a tetraethylammonium chloride-induced contraction in guinea pig tracheal rings. The two most effective compounds, compounds 1 and 13, were further studied for their potential to either prevent the initiation of or relax the maintenance phase of an acetylcholine (ACh)-induced contraction or to potentiate β-agonist-mediated relaxation. Both relaxed an established ACh-induced contraction in human and guinea pig ex vivo ASM. In contrast, the prevention of an ACh-induced contraction required copretreatment with the sodium-potassium-chloride cotransporter blocker bumetanide. The combination of compound 13 and bumetanide also potentiated relaxation by the β-agonist isoproterenol in guinea pig tracheal rings. Compounds 1 and 13 hyperpolarized the plasma cell membrane of human ASM cells and blocked spontaneous transient inward currents, a measure of chloride currents in these cells. These functional and electrophysiological data suggest that modulating ASM chloride flux is a novel therapeutic target in asthma and other bronchoconstrictive diseases.

Superoxide anion radicals activate hepatic stellate cells after entry through chloride channels: A new target in liver fibrosis

It is generally accepted that reactive oxygen species (ROS) play an important role in the pathogenesis of liver fibrosis. ROS, however, constitute a group of species with varying properties making it likely that their contribution to the pathological mechanism varies. LX-2 hepatic stellate cells (HSCs) were exposed to superoxide anion radicals (O2−) generated by xanthine and xanthine oxidase. To rule out that the activation of HSCs is due to hydrogen peroxide derived from O2−, control incubations with copper, zinc-superoxide dismutase and tempol were studied as well. Influx of O2− activated HSCs, evidenced by the expression of α-smooth muscle actin and the secretion of transforming growth factor β1 and collagen. We further found that blockade of chloride channels with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB) or indanyloxyacetic acid (IAA-94) prevented the increase of intracellular O2− levels as well as the activation of HSCs. These findings suggest that O2− is involved in the development of liver fibrosis and that entry of O2−, through chloride channels, in stellate cells is critical for their activation. This study provides new insight into the mechanism by which ROS induce liver fibrosis. Furthermore, our data suggest that chloride channels constitute a potential target for new anti-fibrotic drugs.

GABA UPTAKE BY GAT1 MODULATES LONG-TERM OPTICAL CHANGES FOLLOWING ELECTRICAL STIMULATION OF THE PITUITARY GLAND NEUROINTERMEDIATE LOBE

Intrinsic optical changes that follow infundibular stalk stimulation of the neurointermediate lobe of the mouse pituitary gland exhibit three different phases that reflect three distinct physiological events. The first (E-wave) is the rapid light-scattering increase that is associated with a nerve terminal volume increase (mechanical spike), and that accompanies excitation of the neurohypophysial terminals by the invading action potential; the second (S-wave) is the slower light-scattering decrease that is tightly correlated with the secretion of the peptide hormones oxytocin and arginine vasopressin, and the third is the long-duration response (R-wave) that reflects cell volume changes in the pars intermedia. We have studied the E-wave and the S-wave in earlier publications. The R-wave, considered here, is sensitive to chloride replacement as well as to blockade of chloride channels. By blocking GABAAreceptors (which are ligand-gated chloride channels) with pharmacological agents, and by applying GABA directly into the bathing solution, or evoking its release from GABAergic inputs, we have demonstrated that this long-duration optical response is sensitive to chloride movements and reflects GABA-induced changes in the intrinsic optical properties of the pars intermedia. The full time-course of this optical response takes minutes and, therefore, has to embody some other process (or processes) related to the restoration of resting physiological chloride concentrations, following the opening and closing of GABAA-receptor channels. Here we demonstrate that the shape of the R-wave, the long-lasting light-scattering signal, is indeed affected by the activity of GAT1, one of the sodium- and chloride-dependent GABA transporters.

GABA Uptake by GAT-1 Modulates Long-Term Optical Changes following Secretion in the Mouse Neurohypophysis

Intrinsic optical changes in the mouse pituitary gland associated with secretion exhibit three phases (waves) that reflect three distinct physiological events. The first (E-wave) is the rapid light scattering change that is associated with a nerve terminal volume increase (mechanical spike) that accompanies excitation of the neurohypophysial terminals by the invading action potential (Kim et al., Biophys. J. 92(9) 3122-3129 (2007)); the second (S-wave) is the slower, opposite change in light scattering that reflects the secretion of the peptide hormones, oxytocin and arginine vasopressin, and the third is the long duration response (recovery phase or R-wave) that reflects cell volume changes in the pars intermedia. This recovery phase is sensitive to chloride replacement and to blockade of chloride channels. Experiments that employ pharmacological blockers of GABAA receptors (chloride channels), as well as direct bath application of GABA, alter the R-wave in a manner that reflects GABA-induced changes in the intrinsic optical properties of the pars intermedia (Kosterin et al., Neuroendocrinology 92, 158-167 (2010)). Because the full time-course of this optical response takes minutes, we suggested that, in addition to chloride-channel opening / closing, some additional processes related to the restoration of resting physiological chloride concentrations may occur. Indeed, we demonstrate here that the time-course of this long term light scattering signal is also affected by the turnover of GAT-1, the sodium- and chloride-dependent GABA-transporter.Supported by RO1 040966.

Calcium-Activated Chloride Currents Prolongs the Duration of Contractions in Pregnant Rat Myometrial Tissue

We investigated the importance of pharmacologically blocking calcium-activated chloride (I(Cl(Ca))) and L-type calcium currents on isometric contractions of strips of D21 pregnant rat myometrial tissue, while simultaneously measuring the electrical activity of the tissue strips with extracellular contact electrodes. When measured with contact electrodes, the duration of the spiking activity directly reflects the duration of the tissue-level plateau potential. We correlated the number of spikes, durations of spiking activity, and the spiking frequencies with changes of the area under the force curves as a function of exposure to low doses of anthracene-9-carboxylate (9-AC, a non-specific Cl channel blocker), chlorotoxin (a specific I(Cl(Ca)) blocker) and nifedipine (an L-type calcium channel blocker). The area under the force curve was measured only during spiking electrical activity, thereby separating pharmacological effects on tissue relaxation from those that modulate force production. Blocking chloride channels reduced impulse, shortened the duration of spiking activity, and reduced the number of spikes generated in each contraction. This was observed without a change in the frequency of spike production or a reduction of peak force. Nifedipine reduced impulse, shortened the duration of spiking activity, and reduced the number of spikes. In contrast to chloride channel blockade, nifedipine reduced maximum spike frequency and peak force. Taken together, our data suggest that blocking L-type calcium channels reduces impulse directly by reducing peak force, and indirectly by reducing activation of I(Cl(Ca)) , which shortens the duration of the contraction.

Inhibition of cell proliferation and arrest of cell cycle progression by blocking chloride channels in human laryngeal cancer cell line Hep-2

Chloride channel (ClC) is involved in normal physiological processes and pathology of various diseases. Although it is recognized that blockade of ClC inhibits the cell proliferation, it is not well understood the potential function of ClC in laryngeal cancer. In this study, we investigated the effect of the ClC inhibitor on cell proliferation, cell cycle progression in human laryngeal cancer cell line Hep-2, as well as the effect on the phosphorylation levels of ERK1/2 and AKT1. In this study crystal violet method was used to study the effect of the ClC inhibitor, 5-nitro-2-(3-phenylpropylamino) benzoic acid, NPPB, on Hep-2 cell proliferation. The impaction of the inhibitor on the cell cycle distribution was investigated by the flow cytometry (FCM). Western blot was performed to measure the phosphorylation levels of ERK1/2 and AKT1. Our data indicated ClC played an important role in Hep-2 cell proliferation and cell cycle. NPPB inhibited Hep-2 cell proliferation when compared with the controls. Blockade of ClC arrested cell cycle progression and suppressed the phosphorylation of ERK1/2 and AKT1 in Hep-2 cells by inhibition of cell proliferation by CIC inhibitor (NPPB) could be through arresting cell cycle progression, which is probably by suppressing phosphorylation of ERK1/2 and AKT1.

Pharmacologic blockade of chloride channel synergistically enhances apoptosis of chemotherapeutic drug-resistant cancer stem cells

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is the most commonly used chemotherapeutic agent in the treatment of human glioblastoma multiforme (GBM). However, BCNU chemotherapy fails due to subpopulations of intrinsic resistant-cells within the tumor mass. In our previous study, we dissociated BCNU-resistant cancer stem cells (CSCs) and observed the over-expression of multiple ion channel genes related to drug efflux. In the present study, we identified chloride intracellular channel 1 (CLIC1) in dissociated-BCNU-resistant CSCs using 2-DE and MALDI-TOF/MS analysis. To develop the specific target therapy of BCNU-resistant CSCs, we evaluated the drug-sensitivity of these CSCs using the chloride channel blocker, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). When combined with BCNU, DIDS synergistically increased the apoptotic events of BCNU-resistant CSCs in vitro and augmented BCNU sensitivity ex vivo. These findings suggest that CLIC1 is involved in the resistance of BCNU-resistant CSCs and BCNU/DIDS combined-therapy can provide valuable insight for promoting apoptosis or sensitizing glioblastomas to BCNU chemotherapy.

Blockade of chloride channels suppresses engulfment of microspheres in the microglial cell line, BV-2

Microglial cells, monocyte-derived cells in the brain, phagocytose apoptotic and necrotic cells during neurodegenerative processes. As uptake of particles increases cellular volume and swelling-activated chloride channels participate in volume regulation, we investigated the phagocytotic capacity of microglial cells exposed to blockers of swelling-activated chloride channels. We visualized engulfment of hydrophobic polystyrenic microspheres (4 microm in diameter) in the microglial cell line, BV-2, using scanning electron microscopy and confocal laser microscopy. Microspheres instead of apoptotic cells were used because in scanning electron micrographic images, engulfed particles were clearly discriminated from attached ones. Exposure of BV-2 cells to the chloride channel blocker, flufenamic acid (200 microM) or NPPB (200 microM), eliminated uptake of microspheres almost completely. SITS (1 mM), which blocks chloride channels and to some extend K-Cl cotransporters (KCC), had only a moderate inhibiting impact on particle uptake. DIOA, a compound that inhibits KCC as well as chloride channels, did not inhibit particle uptake in BV-2 cells. Osmotic challenge by hypoosmotic saline (60% saline) elicited a swelling-activated chloride channel sensitive to SITS (1 mM) and flufenamic acid (200 microM). Because uptake of particles requires formation of engulfment pseudopodia, we hypothesize that engulfment pseudopodia are initially nothing but local swellings and that activation of swelling-activated chloride channels participates in the formation of these swellings.

Mechanisms of U46619‐ and 5‐HT‐induced contraction of bovine pulmonary arteries: role of chloride ions

Thromboxane A(2) and 5-hydroxytryptamine (5-HT) are implicated in pulmonary hypertension. The involvement of chloride, voltage-operated calcium channels (VOCCs), store-operated calcium channels (SOCCs) and the Rho kinase in the contractile response of bovine pulmonary arteries (BPA) to the thromboxane A(2) mimetic U46619 and 5-HT was investigated. Endothelium-intact ring segments of BPA were mounted in Krebs/Henseleit buffer (37 degrees C) under a tension of 2g and gassed with 95%O(2)/5%CO(2). Depletion or removal of extracellular chloride, inhibition of chloride and SOCC, Na:K:2Cl, Cl/HCO(3), Rho kinase inhibited contractions to U46619. Combining Rho kinase inhibition and chloride channel blockade (with NPPB) almost abolished the contractions to U46619. In contrast 5-HT-induced contraction was inhibited by verapamil and mibefradil. Depletion of stored calcium with caffeine almost abolished the response to U46619 but not 5-HT. The contraction by the sarco(endo)plasmic reticulum Ca(2+)-ATPase inhibitor CPA was abolished by SOCC and chloride channel blockade (with NPPB) and by chloride depletion. This study suggests that the contractile response of BPA to U46619 involves Rho kinase together with a chloride-sensitive mechanism, which does not involve VOCC but may have a role in calcium release and calcium entry via SOCC. In contrast contraction of the BPA by 5-HT appears to involve verapamil- and mibefradil-sensitive VOCC. This study may indicate that the use of calcium channel blockers in the management of pulmonary hypertension may not always be effective and that Rho kinase and chloride channels may be targets for the development of new therapies.

Calcium and chloride channel activation by angiotensin II-AT1receptors in preglomerular vascular smooth muscle cells

The pathways responsible for the rapid and sustained increases in [Ca(2+)](i) following activation of ANG II receptors (AT(1)) in renal vascular smooth muscle cells were evaluated using fluorescence microscopy. Resting intracellular calcium concentration [Ca(2+)](i) averaged 75 +/- 9 nM. The response to ANG II (100 nM) was characterized by a rapid initial increase of [Ca(2+)](i) by 74 +/- 6 nM (n = 35) followed by a decrease to a sustained level of 12 +/- 2 nM above baseline. The average time from peak to 50% reduction from the peak value (50% time point) was 32 +/- 4 s. AT(1) receptor blockade with 1 microM candesartan (n = 5) prevented the responses to ANG II. In nominally calcium-free conditions (n = 8), the peak increase in [Ca(2+)](i) averaged 42 +/- 7 nM but the sustained phase was absent and the 50% time point was reduced to 11 +/- 4 s. L-type calcium channel blockade with diltiazem reduced the peak [Ca(2+)](i) to 24 +/- 8 nM and the sustained level to 4 +/- 2 nM (n = 10). In cells preincubated in low Cl(-) (3.0 mM), the peak response to ANG II was suppressed as was the sustained response. Blockade of chloride channels with DIDS eliminated both the peak and sustained responses (n = 11); chloride channel blockade with DPC (n = 17) suppressed the peak increase in [Ca(2+)](i) to 18 +/- 5 and also prevented the sustained response. IP3 receptor blockade by 10 microM TMB-8 (n = 6) reduced the peak to 22 +/- 8 and prevented the sustained response. Exposure to 10 microM TMB-8 in the presence of Ca(2+)-free medium prevented the ANG II response (n = 9). In the presence of 100 microM DPC and 10 microM TMB-8 (n = 7), the ANG II response was also prevented. Thus the rapid initial increase in [Ca(2+)](i) is due not only to release from intracellular stores, but also to Ca(2+) influx from the extracellular fluid. Although Ca(2+) entry via L-type calcium channels is responsible for the major portion of the sustained response, other entry pathways participate. The finding that chloride channel blockers markedly attenuate both rapid and sustained responses indicates that chloride channel activation contributes to, rather than being the consequence of, the initial rapid response.

Niflumic acid inhibits ATP-stimulated exocytosis in a mucin-secreting epithelial cell line.

ATP is an efficacious secretagogue for mucin and chloride in the epithelial cell line HT29-Cl.16E. Mucin release has been measured as [3H]glucosamine-labeled product in extracellular medium and as single-cell membrane capacitance increases indicative of exocytosis-related increases in membrane area. The calcium-activated chloride channel blocker niflumic acid, also reported to modulate secretion, was used to probe for divergence in the purinergic signaling of mucin exocytosis and channel activation. With the use of whole cell patch clamping, ATP stimulated a transient capacitance increase of 15 +/- 4%. Inclusion of niflumic acid significantly reduced the ATP-stimulated capacitance change to 3 +/- 1%, although normalized peak currents were not significantly different. Ratiometric imaging was used to assess intracellular calcium (Cai2+) dynamics during stimulation. In the presence of niflumic acid, the ATP-stimulated peak change in Cai2+ was unaffected, but the initial response and overall time to Cai2+ peak were significantly affected. Excluding external calcium before ATP stimulation or including the capacitative calcium entry blocker LaCl3 during stimulation muted the initial calcium transient similar to that observed with niflumic acid and significantly reduced peak capacitance change, suggesting that a substantial portion of the ATP-stimulated mucin exocytosis in HT29-Cl.16E depends on a rapid, brief calcium influx through the plasma membrane. Niflumic acid interferes with this influx independent of a chloride channel blockade effect.

Ion channels in cultured microglia.

Inward and, depending on activation state, outward potassium currents are the dominant ion channels in microglial cells in culture. During transition between resting and activated phases, there is also an upregulated expression of stretch/swelling-activated chloride currents. Pharmacological blockade of the specific potassium channels does not prevent the transition, whereas blockade of chloride channels does, suggesting that this current may be involved in phase changes. Interestingly, this chloride current is far less studied than the potassium currents with regard to the different microglial phases. One puzzling finding when studying microglial state is that despite changes in current densities and membrane oscillations during transition, there is no evidence of an accompanying change in membrane potential. In other cells of the immune system, membrane oscillations and alterations in membrane potential are correlated with transitions in cellular phases. This discrepancy in microglia may be a result of the fact that almost all ion channel and membrane potential studies in culture are undertaken with concomitant dialysis of cytoplasm with pipette solution. Further complicating matters is that the few studies that use microglia in situ, find fundamental differences in ion channel current patterns of "resting" microglia as well as different temporal changes to pathological events or stimuli.

Blockade of chloride channels reveals relaxations of rat small mesenteric arteries to raised potassium.

1. Raised extracellular K(+) relaxes some arteries, and has been proposed as Endothelium-Derived Hyperpolarizing Factor (EDHF). However, relaxation of rat small mesenteric arteries to K(+) is highly variable. We have investigated the mechanism of K(+)-induced dilatation and relaxation of pressurized arteries and arteries mounted for measurement of isometric force. 2. Raising [K(+)](o) from 5.88 - 10.58 mM did not dilate or relax pressurized or isometric arteries. Relaxation to raised [K(+)](o) was revealed in the presence of 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB); this effect of NPPB was concentration-dependent (IC(50): 1.16 microM). 3. Relaxations to raised [K(+)](o) in the presence of NPPB, were abolished by 30 microM Ba(2+) or endothelial-denudation. Acetycholine (10 microM) relaxed endothelium-intact arteries in presence of raised [K(+)](o) NPPB and Ba(2+). 4. Relaxations to raised [K(+)](o) were revealed in hyperosmotic superfusate (+60 mM sucrose). These relaxations were abolished by 30 microM Ba(2+). In the presence of raised [K(+)](o), 60 mM sucrose and 30 microM Ba(2+), 10 microM acetycholine still relaxed all arteries. 5. Fifty microM 18 alpha-glycyrrhetinic acid (18 alpha-GA), a gap junction inhibitor, depressed relaxations to both 10 microM acetylcholine and raised [K(+)](o), in the presence of 10 microM NPPB. 6. In summary, blockade of a volume-sensitive Cl(-) conductance in small rat mesenteric arteries, using NPPB or hyperosmotic superfusion, reveals a endothelium-dependent, Ba(2+) sensitive dilatation or relaxation of rat mesenteric arteries to raised [K(+)](o). We conclude that inwardly rectifying potassium channels on the endothelium underlie relaxations to raised [K(+)](o) in rat small mesenteric arteries.

Blockade of chloride channels by DIDS stimulates renin release and inhibits contraction of afferent arterioles.

Calcium-activated chloride channels have been proposed to control renin release from juxtaglomerular cells and to be involved in the excitation-contraction coupling of the renal afferent arteriole. The hypothesis was tested on renin release from rat glomeruli and in microperfused rabbit afferent arterioles with the chloride channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Renin secretion was equally enhanced by omission of extracellular calcium and by addition of 0.5 mM DIDS. The inhibitory effect of calcium was blocked by DIDS. The stimulatory effects of low calcium [with or without ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] and DIDS were not additive. In the absence of chloride, basal renin release was suppressed and the stimulatory effect of DIDS was abolished. The DIDS-induced enhancement of renin release was not dependent on bicarbonate. Norepinephrine (5 x 10(-7)-1 x 10(-6) M) and angiotensin II (1 x 10(-8)-10(-6) M) evoked reversible and dose-dependent contractions of microperfused rabbit afferent arterioles. DIDS (0.5 mM) did not affect the basal diameter of the arterioles but strongly inhibited the response to angiotensin II and attenuated the duration of the contractile response to norepinephrine. The results support the hypothesis that DIDS-sensitive calcium-activated chloride channels are involved in regulation of renin release and in the afferent arteriolar contraction after angiotensin II but do not play a pivotal role in the response to norepinephrine.

Segment-specific effect of chloride channel blockade on rat renal arteriolar contractile responses to angiotensin II

Experiments were performed to determine the effect of a chloride channel blocker (indanyloxyacetic acid, IAA-94) on renal arteriolar vasoconstrictor responses to angiotensin II (AngII). The in vitro blood-perfused juxtamedullary nephron technique was exploited to provide access to the renal microvasculature of enalaprilat-treated rats. Under control conditions, 1 to 100 nmol/L AngII evoked concentration-dependent afferent arteriolar vasoconstriction. Baseline diameter of afferent arterioles was not altered by 30 mumol/L IAA-94; however, AngII responsiveness was markedly attenuated. The afferent response to K-induced depolarization was sustained in the presence of IAA-94. In efferent arterioles, neither baseline diameter nor AngII responsiveness was altered by IAA-94. These results suggest that full expression AngII-induced afferent (but not efferent) arteriolar vasoconstriction requires participation of chloride channels, which likely engender depolarization and subsequent opening of voltage-gated calcium channels.

Participation of a Gaba-Ergic System in the Processes of Neuroimmunomodulation

Participation of a GABA-ergic system in neuroimmunomodulation was established through the use of a large number of chemical compounds which selectively modulate the activity of the GABA-BD-receptor-ionophore complex. Activation of the GABA-receptors with muscimol or activation of the BD-receptors with diazepam or tazepam had stimulatory effects upon immunogenesis. A decrease in the GABA-BD-receptor-ionophore complex activity led to a suppression of the immune response. The effect was achieved with: a blockade of the complex with bicuculline--a competitive inhibitor of the GABA-receptors: administration of a specific antagonist of the BD-receptors flumazenil or Ro 15-3505: or with blockade of chloride channels with picrotoxin. Activation of the GABA-ergic system causes an increase in bone marrow content of T-helper cells marked by L3T4. The immunomodulatory action of the GABA-ergic system is of central origin and can occur only when the hypothalamo-pituitary system is intact. Section of the pituitary stalk prevents accumulation of the T-helper cells in the bone marrow. The result show that the influence of GABA-ergic system on immunogenesis requires participation of both dopaminergic and serotoninergic systems.