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Cystic fibrosis (CF) is a hereditary disease typically characterized by infection-associated chronic lung inflammation. The persistent activation of toll-like receptor (TLR) signals is considered one of the mechanisms for the CF hyperinflammatory phenotype; however, how negative regulatory signals of TLRs associate with CF inflammation is still elusive. Here, we showed that the cell surface expression of a single immunoglobulin interleukin-1 receptor (IL-1R)-related molecule (SIGIRR), a membrane protein essential for suppressing TLRs- and IL-1R-dependent signals, was remarkably decreased in CF airway epithelial cells compared to non-CF cells. Notably, CF airway epithelial cells specifically and highly expressed a unique, alternative splice isoform of the SIGIRR that lacks exon 8 (Δ8-SIGIRR), which results in the production of a C-terminal truncated form of the SIGIRR. Δ8-SIGIRR was expressed intracellularly, and its over-expression abolished the cell surface expression and function of the full-length SIGIRR (WT-SIGIRR), indicating its dominant-negative effect leading to the deficiency of anti-inflammatory activity in CF cells. Consistently, IL-37, a ligand for the SIGIRR, failed to suppress viral dsRNA analogue poly(I:C)-dependent JNK activation and IL-8 production, confirming the reduction in the functional WT-SIGIRR expression in the CF cells. Together, our studies reveal that SIGIRR-dependent anti-inflammatory activity is defective in CF airway epithelial cells due to the unique splicing switch of the SIGIRR gene and provides the first evidence of IL-37-SIGIRR signaling as a target of CF airway inflammation.

Background Pharbitidis Semen (the seeds of Pharbitis nil), traditionally used as a purgative in Japan, China and Korea, contains a resin glycoside fraction named pharbitin, which is known as a purgative ingredient. Due to the complex nature of pharbitin, little is known about either the action on intestinal tension caused by resin glycoside itself or by its components. Methods In this study, we investigated the effects of pharbitin, the glycosidic acid fraction (pharbitic acid) and the aglycone fraction (phar-genin) generated from pharbitin on peristalsis of colon and ileum isolated from mice with the Magnus method. Results We demonstrated that pharbitin (3–30 μg/mL) concentration-dependently increased tonus of mice colon via acetylcholine receptors, its components phar-genin (1.27–12.7 μg/mL) and pharbitic acid (10–1000 μg/mL) also had the increment on colon tonus. On the other hand, ileum tension decreased in the presence of pharbitin. Conclusions The effects of resin glycoside of Pharbitidis Semen on colon tonus are different with those on ileum tonus isolated from mice. In the next step it is necessary to investigate details of its pharmacological mechanism.

Single immunoglobulin interleukin-1 receptor-related molecule (SIGIRR) is one of the immunoglobulin-like membrane proteins that is crucial for negative regulation of toll-like receptor 4 (TLR4) and interleukin-1 receptor. Despite the importance of understanding its expression and function, knowledge is limited on the regulatory mechanism in the epithelial tissues, such as the liver, lung, and gut, where its predominant expression is originally described. Here, we found expression of SIGIRR in non-epithelial innate immune cells, including primary peripheral blood monocytes, polymorphonuclear neutrophils, monocytic RAW264 cells, and neutrophilic-differentiated HL-60 cells. Consistent with previous findings in epithelial tissues, SIGIRR gene and protein expression were also down-regulated by LPS treatment in a time-dependent manner in primary blood monocytes and polymorphonuclear neutrophils. A reduction was also observed in RAW264 and differentiated HL-60 cells. Notably, exogenous introduction of the dominant negative form of TLR4 and siRNA of p38 resulted in inhibition of LPS-induced SIGIRR down-regulation, whereas treatment with p38 activator anisomycin showed a dose-dependent decrease in SIGIRR expression, suggesting TLR4-p38 signal as a critical pathway for LPS-induced SIGIRR down-regulation. Finally, reporter gene and chromatin immunoprecipitation assays demonstrated that Sp1 is a key factor that directly binds to the proximal promoter of SIGIRR gene and consequently regulates basal SIGIRR expression, which is negatively regulated by the LPS-dependent TLR4-p38 pathway. In summary, the data precisely demonstrate how LPS down-regulates SIGIRR expression and provide a role of LPS signal that counteracts Sp1-dependent basal promoter activation of SIGIRR gene via TLR4-p38 pathway in non-epithelial innate immune cells.

X-linked adrenoleukodystrophy is a severe and progressive neurodegenerative disease caused by the peroxisomal transporter ATP-binding cassette, subfamily D, member 1 gene mutations. The defect of this gene product results in accumulation of very-long-chain fatty acids in organs and serum, central demyelination, and peripheral axonopathy. Although there are different magnetic resonance (MR) findings which reflect various phenotypes in adrenoleukodystrophy, some cases present with specific symmetrical occipital white-matter lesions. We describe a patient with adult-onset X-linked adrenoleukodystrophy with topographic disorientation, whose brain MR images revealed T2-signal hyperintensity along the occipito-pontine tract and lateral lemnisci, but not in the cortico-spinal tract in the brainstem. The occipito-pontine tract and lateral lemnisci were clearly detected using diffusion-tensor fiber tracking, suggesting that the topographic disorientation of this patient might be related to the occipito-pontine tract. MR tractography can effectively identify the occipito-pontine tract and may help to localize the fibers associated with clinical symptoms.

Alpha(1)-acid glycoprotein (AGP) is an acute phase protein. Whereas the expression of AGP in an inflammatory state is enhanced by inflammatory cytokines including interleukin-1, 6 (IL-1 and IL-6), and tumor necrosis factor-alpha (TNF-alpha), the biological significance of AGP remains unclear. In the current study, the anti-inflammatory effect of AGP on the acute inflammatory state was examined in vivo and in vitro. AGP suppressed carrageenan-, dextran- and kaolin-induced paw edema and vascular permeability in rat. These results suggest that both initial inflammatory mediators (serotonin and histamine) and later inflammatory mediators (prostaglandin and bradykinin) are involved in the anti-inflammatory effects of AGP. In fact, prostaglandin E(2) (PGE(2)) generation in plasma was significantly inhibited by AGP. Moreover, AGP inhibited the migration of neutrophils treated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) through membrane filter. In addition, AGP significantly suppressed superoxide generation from neutrophils that has been treated with fMLP or phorbol 12-myristate 13-acetate. These results imply that the anti-inflammatory effect of AGP may involve the inhibition of neutrophils migration. The data obtained in this study support a scenario in which an increase in AGP concentration in pathological conditions suppresses inflammation reactions induced by autacoids and neutrophils activities and that AGP plays an important role in the maintenance in the body.

Drug-induced contraction of gastrointestinal tracts seems to depend upon the extent of their rhythmic contraction that is driven by the activity of gastrointestinal pacemaker cells. In BALB/c mice chronically administrated with a neutralizing anti-c-Kit monoclonal antibody (ACK2), rhythmic contraction of the gastrointestinal tract was impaired and contractile responses to drugs, including acetylcholine, prostaglandin F(2alpha), and bradykinin, were anomalously augmented. Histochemical analysis of the c-kit-positive cells in the gastrointestinal tract revealed the decreased number of c-kit-positive cells in the ACK2-treated animals, which lead to the impaired rhythmic contraction. Since the intestinal c-kit-positive cells in primary culture developed Ca(2+)-dependent rhythmic Cl(-) current, the rhythmic current is supposed to be an origin of gastrointestinal pacemakers. The extent of anomaly in drug-induced contraction correlated with the extent of impairment in rhythmic contraction. The drug-induced anomalous contraction in the preparation from ACK2-treated animals, which is accompanied by the impaired rhythmic contraction, was mimicked when the gastrointestinal segments from control animals were superfused with a low temperature organ bath solution at 25 degrees C. These results suggest that rhythmic discharge of excitation of smooth muscle cells, which is triggered by rhythmic excitatory input from c-kit cells, regulates the extent of drug-induced contraction.

We examined the effects of human alpha(1)-acid glycoprotein on isometric tension of mouse aortic rings. alpha(1)-Acid glycoprotein (7.5-75 microM) produced a transient, concentration-dependent relaxation of the phenylephrine-precontracted preparation. Although N(G)-nitro-L-arginine methyl ester or removal of endothelium rarely affected the alpha(1)-acid glycoprotein-induced relaxation, extracellular heparin inhibited the alpha(1)-acid glycoprotein-induced relaxation. In 10 mM Ca(2+)-containing external solutions, the alpha(1)-acid glycoprotein-induced relaxation was significantly potentiated. In the 60 mM KCl-precontracted preparation, alpha(1)-acid glycoprotein produced weaker relaxation than in the phenylephrine-precontracted preparation. These results suggest that the vasorelaxant effect of alpha(1)-acid glycoprotein is mainly achieved by block of Ca(2+) entry in the vascular smooth muscle cells. The interaction between alpha(1)-acid glycoprotein molecules and plasmalemmal Ca(2+) entry channels may be modified by extracellular Ca(2+) and heparin.

1. Under voltage-clamped conditions, gastric smooth muscle cells of BALB/c mice developed spontaneous (STOCs) and caffeine- (I(CAF)) and carbachol-induced (I(CCh)) transient outward currents. 2. In fura-2 microscopic measurements of intracellular Ca(2+) concentration ([Ca(2+)](i)), caffeine and carbachol (CCh) provoked similar transient [Ca(2+)](i) elevations. 3. Both I(CCh) and CCh-induced [Ca(2+)](i) elevation of single smooth muscle cells occurred in an 'all-or-nothing' fashion in contrast to the reproducible caffeine responses. 4. On the basis of the suppression of STOCs and I(CAF) by nicardipine, tetraethylammonium and iberiotoxin, but not by charybdotoxin nor apamin, it was suggested that both currents were generated by large conductance type Ca(2+)-activated K(+) channels. 5. In measurements of isometric tension, caffeine produced relaxation of gastric smooth muscle strips in a concentration-dependent manner (0.1 -- 3 mM). The concentration-dependent relaxation with caffeine was mimicked by dibutyryl cyclic AMP which produced potentiation of contraction triggered by 50 mM KCL. 6. At caffeine concentrations >3 mM, a transient contraction followed by relaxation was provoked as the quasi maximal response to caffeine. In the quasi maximal response, caffeine acted as a potent relaxant in smooth muscle strips precontracted with 50 mM KCl or 3 microM CCh. 7. The relaxation with caffeine was significantly accelerated in those strips precontracted with KCl or CCh. All these results suggest that ryanodine-sensitive Ca(2+) release, which is triggered by caffeine, is an important modifier of Ca(2+) homeostasis in the cytoplasm and the contractility of gastric smooth muscle cells of mice.

Adrenoceptor-mediated modulation of a caffeine (CAF)-induced [Ca(2+)](i) elevation and resulting Ca(2+)-activated K(+) current (I(CAF)) in bovine ciliary muscle (CM) cells were investigated.The nystatin-perforated patch clamp technique for the measurement of membrane currents and a microscope based fura-2 fluorescence imaging of [Ca(2+)](i) were applied to CM cells freshly dissociated with collagenase and identified with smooth muscle-specific alpha-isoactin.Under voltage-clamped conditions, noradrenaline (NA) potentiated I(CAF) in a NA concentration-dependent manner without producing current responses to NA when NA was applied alone. NA-induced potentiation of I(CAF) occurred within 20 sec after the application of NA, while the NA-potentiated I(CAF) gradually recovered to the control level within 30 min after removal of NA. Despite the little current response to NA applied alone, NA elicited a [Ca(2+)](i) elevation in a manner similar to that induced by CAF although the NA-induced [Ca(2+ )](i) elevation was smaller than the CAF-induced [Ca(2+ )](i) elevation. In contrast to the significant potentiation of I(CAF) with NA, NA produced little potentiation of the CAF-induced [Ca(2+)](i) elevation. The NA-induced potentiation of I( CAF) was antagonized by an alpha(1) adrenoceptor antagonist, prazosin. Neither clonidine nor isoproterenol had an effect on I(CAF), suggesting that alpha(2) and beta adrenoceptor are not involved in the response to NA.These results suggest that NA potentiates I( CAF) via alpha(1) adrenoceptor activation and that the NA-induced potentiation occurs at Ca(2+)-activated K(+) channels but not CAF-induced Ca(2+) releasing sites.

We have recently found that mouse megakaryocytes responded to extracellular alkalinization to pH > 8.0, generating a K+ current under voltage-clamped conditions with the whole cell recording mode of the patch-clamp technique. The purpose of this study was to physiologically and pharmacologically characterize the alkaline-dependent K+ conductance of the megakaryocyte membrane. The alkalinization-induced K+ current (I(ALK)) did not seem to be Ca2+-dependent since I(ALK) was allowed to be generated under intracellularly Ca2+-buffered conditions with 10 mM EGTA, which completely prevented the generation of caffeine-induced Ca2+-activated currents of mouse megakaryocytes; and no [Ca2+]i elevation was evoked by the alkalinization protocol in contrast to a significant increase in [Ca2+]i in response to caffeine when [Ca2+]i was measured with a fura 2 ratiometry. I(ALK) was strongly suppressed with tetraethylammonium (TEA), 4-aminopyridine (4-AP) and streptomycin (SM), but was completely resistant to quinidine (QND). The values of IC50 for the suppression of I(ALK) with TEA, 4-AP and SM were 5.6, 0.47 and 1.5 mM, respectively. Voltage-gated K+ currents (I(K)) of the same megakaryocyte preparation were weakly suppressed with TEA and 4-AP, while they were significantly suppressed with either SM or QND. These results suggest that mouse megakaryocytes possess K+ conductance that was activated by extracellular alkalinization and that probably differs from conventional K+ conductance in its pharmacological properties.

The effects of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a potent anion transport blocker, on transmembrane action potentials (APs) and the sodium current (I[Na]) of guinea pig ventricular myocytes were examined by using conventional microelectrode and whole-cell patch-clamp recording techniques. In papillary muscle preparations, DIDS (> or =0.1 mM) suppressed the maximal upstroke velocity (.v[max]) of the AP without significant changes in other AP parameters. Extracellular application of DIDS on single cardiomyocytes isolated from the guinea pig ventricle markedly reduced the peak amplitude of the tetrodotoxin (TTX)-sensitive and voltage-activated sodium current. The concentration-dependent block of DIDS could be expressed by the Hill equation with a Hill coefficient of 0.97 and a dissociation constant of 0.15 mM at a holding potential of (VH) -120 mV. DIDS (0.1 mM) shifted the steady-state inactivation curve for I(Na) toward more negative potentials by 6.0 +/- 0.5 mV and the activation curve to more positive potentials by 5.0 +/- 1.0 mV, although the slope factors were unaffected. With repetitive depolarizing pulses from -120 mV, DIDS produced a use-dependent block on the I(Na). Recovery of I(Na) from inactivation was slowed (time constant = 245 ms, compared with 10 ms of control) in the presence of 0.1 mM DIDS. In the two-pulse experiments, DIDS produced two distinct phases of development of I(Na) block, the rapid phase (tau = 5 ms) caused by an open channel block, and the slower phase (tau = 382 ms) induced by an inactivated channel block. These results suggest that the Cl- transport blocker DIDS has a direct inhibitory effect on the cardiac sodium channel. DIDS-induced use dependence of I(Na) block may result from the interaction of the drug with sodium channels in both the open and inactivated channel states.

The dependence of plasmalemma Ca2+ influx and Ca2+ release from intracellular stores on Ca2+ activated K+ channels of bovine ciliary muscle (CM) cells were examined.The nystatin-perforated patch clamp technique for the measurement of membrane currents and a microscope based fura-2 fluorescence imaging of [Ca2+]i were applied to CM cells freshly dissociated with collagenase and identified with smooth muscle-specific alpha-isoactin.At holding voltages (VH) of-60 mV, CM cells showed spontaneous transient outward currents (STOCs) and caffeine (10(-4) M) induced large transient outward currents (ICAF). Both STOCs and ICAF were abolished by tetraethylammonium chloride (10(-3) M) and charybdotoxin (10(-7) M), but not by apamin (10(-6) M), suggesting that both currents are mediated by Ca(2+)-activated K+ channels similar to those with medium (MK) or large (BK-type) conductance. Both STOCs and ICAF were gradually abolished in the nominally Ca(2+)-free and Co(2+)-containing solutions but were resistant to L-type Ca2+ channel blockers, including nicardipine, verapamil and diltiazem and a N-type channel blocker, omega-contoxin. The [Ca2+]i-elevation during high K+ (100 mM)-depolarization was prevented by Ca(2+)-free and Co(2+)-containing solutions but not by nicardipine.These results suggest that CM cells possess MK or BK type-like Ca(2+)-activated K+ channels and that L-type Ca2+ channels play minor roles for the maintenance of Ca(2+)-dependent responses in contrast to other types of smooth muscle cells.

Reduction of voltage-activated Ca2+ currents by intracellular application of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) through ultraviolet (UV) photolysis of the caged compound, is followed by a re-augmentation to control levels within 10 min, independently of the divalent cation used. The Ca2+ current inhibition by the gamma-aminobutyric acid type B (GABAB) receptor agonist baclofen, which is also thought to be mediated by a GTP-binding protein (G-protein), is potentiated when GTP gamma S is uncaged during agonist superfusion. The authors suggest that GTP gamma S activates G-protein-dependent pathways that are not activated by the baclofen receptor.

The effects of plant diterpenes, horminone (HMN) and taxodione (TXN), on the GABAA receptor-operated Cl-current (IGABA) were investigated in voltage-clamped and internally perfused neurones dissociated from frog dorsal root ganglia. Both diterpenes depressed IGABA in a concentration-dependent manner similar to that of picrotoxin. Concentrations required to elicit 50% depression of the IGABA were 10(-6) M for picrotoxin, 10(-5) M for HMN and 10(-4) M for TXN. Blocking and restoration kinetics of the IGABA with HMN were also similar to those of picrotoxin. Time constants for both the blockade and restoration of the IGABA with TXN were more than five times greater than those with HMN.

Effects of lindane (gamma-benzenehexachloride; gamma-BHC) on GABA-evoked Cl- current (IGABA) in freshly dissociated frog sensory (dorsal root ganglion) neurons were studied and compared with those of tert-butylbicycloortho benzoate (TBOB) and picrotoxin by the use of the suction-pipette method [13]. Drugs were applied with a rapid drug-application method, "Concentration-clamp" technique. At concentration of GABA of > 3 x 10(-6) M, at least two components of the IGABA were recognized distinct degree of desensitization. Those were defined as the peak and plateau components in the text. At low concentration (3 x 10(-7) M) of gamma-BHC, only the plateau component of IGABA at 10(-5) M were depressed without changing the peak amplitude. While gamma-BHC at high concentration (3 x 10(-5) M) depressed both the peak and plateau current components. The gamma-BHC-induced depression of IGABA seemed to be IGABA-component-dependent. A detailed analysis of the gamma-BHC action in the concentration-response relationship for GABA revealed that the IGABA with strong desensitization was preferentially blocked by gamma-BHC (3 x 10(-5) M). The rate of recovery of the IGABA from gamma-BHC-induced block depended on the concentration of GABA. The lower the concentration of GABA, the slower the recovery. The GABAA receptor Cl- channels were proposed to be classified into two types of the gamma-BHC-sensitive and -resistant ones.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of glycine receptor-ionophore complex by internally perfused cyclic AMP was investigated and compared to that of GABA in the acutely dissociated ventromedial hypothalamic neurons using whole-cell and outside-out patch-clamp techniques. Cyclic AMP significantly reduced both GABA- and glycine-gated chloride currents. The reduction in glycine-induced chloride current was specific in that only the fast-desensitizing one gated by high concentrations of glycine (30-100 microM) was affected. Cyclic AMP did not modulate the non-desensitizing current induced by lower concentrations (6-10 microM). Addition of N-[-2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride, a protein kinase A inhibitor, did not have a significant effect on its own but prevented the attenuation of fast desensitizing glycine current induced by cyclic AMP. Both the reversal potential and inactivation kinetics of glycine current were not affected by the activation of protein kinase A, suggesting that cyclic AMP-mediated attenuation is not due to an enhancement of desensitization. In outside-out patch studies intracellular perfusion of cyclic AMP reduced the open probability of the 100 microM glycine-activated channels without affecting that of the 6 microM glycine-activated channels. In conclusion, cyclic AMP selectively modulates the channel open frequency of the glycine receptor when activated at higher concentrations through a protein kinase A-mediated phosphorylation.

1. Effects of penicillin G (PCN) on glycine (Gly)-evoked Cl- current (IGly) were investigated in acutely dissociated rat ventro-medial hypothalamic (VMH) neurones by the whole cell mode of patch clamp technique. 2. When PCN was applied simultaneously with Gly, PCN depressed IGly like a Cl- channel blocker. 3. The PCN-induced blocking action was clearly observed at a low PCN concentration (30 u), while the maximal blockade was achieved by 600 u (units per 10 ml) PCN. 4. When tested solution containing both PCN and Gly was quickly substituted with one containing Gly only, a new rebound-like transient current (I(T)) which also passed through Cl- channel, was elicited. 5. The peak amplitude of I(T) induced by PCN at concentrations higher than 100 u was greater than that induced by glycine alone. We termed this phenomenon PCN-induced potentiation of IGly. In all cells tested, PCN potentiated IGly. 6. At a lower PCN concentration below 30 u, I(T) generation was not clear in the presence of 10(-5) M gamma-aminobutyric acid. With PCN a higher concentration than 300 u, I(T) amplitude was greater than that of the original peak IGly. This was observed in 18 neurones out of 21. The maximal amplitude of the I(T) was achieved with 600 u PCN.

The origin of rhythmicity in gastrointestinal motility was long thought to involve the activity of interstitial cells of Cajal (ICC) that locate in close association with enteric neurons and smooth muscle cells. We have demonstrated that significant decrease in the number of cells immunopositive to c-Kit, a type of tyrosine kinase receptor, in the gastrointestinal tract of mice mutated at the W/c-kit locus and BALB/c mice administered with neutralizing c-Kit antibody leads to the impaired autonomic motility of the gastrointestinal tract. It is also demonstrated that ICC express c-kit which plays important roles in development and maintenance of the ICC network in the gastrointestinal tract. ICC, derived from mesenchymal cells, are classified into smooth muscle type and fibroblast type by their morphology and tissue location. The ligand for c-Kit, Sl factor (SLF), has shown to be expressed in enteric neurons and gastrointestinal smooth muscle cells. Studies with mutant mice and transgenic mice have suggested that functional c-Kit/SLF is required for the differentiation and proliferation of ICC as pacemakers and mediators of neural regulation in gastrointestinal motility. Here we review the genetic basis of autonomic gastrointestinal motility and the pathophysiological models.

Freshly dissociated bladder smooth muscle cells of mice developed spontaneous, caffeine- (ICAF) and carbachol-induced (ICCh) currents under voltage-clamped conditions. Spontaneous currents, ICAF and ICCh were blocked with tetraethylammonium at 3 x 10(-4)-10(-2) M but were resistant to both charybdotoxin (10(-7)-10(-6) M) and iberiotoxin (10(-7)-10(-6) M). The reversal potential for each current indicated that K+ channels play a major role in the generation of each current. Both spontaneous currents and ICAF but not ICCh were abolished in nominally Ca2+-free and nicardipine (10(-6) M)-containing media. These results suggest that the activity of L-type voltage-sensitive Ca2+ channels is important in the generation and maintenance of spontaneous currents and ICAF but not ICCh. Ryanodine (10(-6) M) prevented spontaneous currents, ICAF and caffeine-induced [Ca2+]i elevation but not ICCh and carbachol-induced [Ca2+]i elevation, suggesting that the response of bladder smooth muscle cells to carbachol may involve a Ca2+ store distinct from that for caffeine. Pretreatment with carbachol suppressed ICAF to 22 +/- 7% (n = 7) and the caffeine-induced [Ca2+]i elevation to 25 + 3% (n = 6). Similarly, caffeine suppressed ICCh to 23 +/- 4% (n = 9) and the carbachol-induced [Ca2+]i elevation to 24 +/- 6% (n = 6).

Chronic injection of an anti-c-KIT receptor tyrosine kinase monoclonal antibody (ACK2) results in the disruption of the normal motility patterns of young BALB/c mice intestine. This effect is accompanied by a drastic decrease in the number of intestinal c-kit-expressing (c-kit +) cells when studied immuno-histochemically with the fluorescence-labelled antibody. In order to clarify the mechanism underlying the ACK2 action and the physiological roles of intestinal c -kit + cells, we studied the excitability of intestinal c -kit + cells in primary culture by use of the nystatin perforated-patch-clamp technique. Under voltageclamp at −40 mV, the majority of c -kif +cells tested (59/70) elicited rhythmic current waves with an amplitude and frequency of 263±24 pA and 2.30±0.25 cycles/min (mean±SEM), respectively. Intracellular perfusion of the c -kit + cells with ethylenebis (okonitrilo) tetraacetate (EGTA) as well as a nominally Ca2+-free external solution or low holding voltage (

Effects of bradykinin (BK) on the membrane conductance and level of cytoplasmic free Ca 2+ in undifferentiated and differentiated neuroblastoma-glioma hybrid (NG108-15) cells were studied using the nystatin-perforated patch-clamp technique and fura-2 fluorometry. Under voltage clamp at −20 mV, undifferentiated cells responded to BK at > 10 −9 M, producing a biphasic current composed of an apamin-sensitive Ca 2+ -activated K + outward current and non-specific cationic inward current. Both current components corresponding to a biphasic elevation of [Ca 2+ ] i were completely prevented by an intracellular perfusion with EGTA (1 mM) under conventional whole cell recording condition. Undifferentiated cells revealed almost no voltage sensitive Ca 2+ current. In NG108-15 cells differentiated with 8-Br-cyclic AMP (1 mM) or rolipram (1 mM), an inhibitor of type IV phosphodiesterase, BK concentration required for the non-specific cationic current with amplitude of > 100 pA was much greater than that of undifferentiated cells. This suggests that the differentiated cells decreased BK-sensitivity in induction of the non-specific cationic current. The non-specific cationic channel is suggested to play roles as a source of Ca 2+ entry in undifferentiated NG108-15 cells.

Effects of penicillin-G (PCN) on GABA-evoked Cl- current (IGABA) were investigated in freshly dissociated frog sensory neurons by the use of the concentration-clamp technique combined with the suction-pipette method. Under conditions where the internal and external solutions allowed only Cl- permeability, PCN elicited triphasic modulation on IGABA, consisting of two modes of blockade on IGABA and a following rebound (rebound-like transient IGABA). Simultaneously applied PCN and GABA depressed IGABA immediately (phasic blockade), with the depressed IGABA slightly recovering in amplitude to achieve a stable level of blockade (tonic blockade). When a solution containing a mixture or PCN and GABA was quickly replaced by one containing GABA alone, a rebound-like transient Cl- current (IR) was evoked. Each component of the PCN actions on IGABA was PCN- and GABA-concentration-dependent. The reversal potential for each component of the PCN actions on IGABA was close to the chloride equilibrium potential (ECl) calculated using the Nernst equation. The current-voltage (I-V) relations for both the phasic and tonic blockade revealed inward rectification, while I-V curves for the control IGABA and the IR were outwardly rectified. The degree of IGABA-desensitization and the amplitude of the IR correlated well. The data suggest that partial removal of the GABAA receptor-desensitization may result in generation of the IR.

A suppressant effect of intracelullar free Ca 2+ on the γ-aminobutyric acid (GABA)-induced chloride inward current ( I Cl(GABA) ) was studied in isolated frog sensory neurones under whole cell voltage clamp. Voltage-dependent Ca 2+ influx elicited during the steady state of I Cl(GABA) -induced a fast, slowly recovering current relaxation in the outward direction, the amplitude of which was dependent on total Ca 2+ influx. This suppressant effect showed specificity for different divalent actions, suggesting action at a specific intracellular effector. Single channel recording revealed a Ca 2+ -dependent decrease in the duration of the open time of the GABA-gated Cl − channel without change in single channel conductance.

1. The contribution of Cl- redistribution to the decay phase of the GABA (gamma-aminobutyric acid) response was investigated in isolated frog sensory neurones, using a suction-pipette technique which allows for internal perfusion under conditions of voltage clamp. 2. In neurones perfused with 120 mM [Cl-]i and [Cl-]o at driving forces (delta VH) of less than 15 mV, no shift of GABA equilibrium potential (EGABA) occurred during a continuous application of GABA, at various concentrations. However, increases of delta VH towards negative or positive potentials over 15 mV induced EGABA shifts. 3. The degree of EGABA shift was governed by the total amount of Cl- flux across the soma membrane, an event which depends upon delta VH, GABA concentration and drug application time. 4. The time-dependent EGABA shift due to Cl- redistribution during GABA application induced a current run-down resulting from a decreased Cl- gradient and a diminished Cl- conductance (gCl), the latter brought about by a drop in the intracellular ionic density of Cl-. 5. The EGABA shift during a continuous GABA application was also affected by [Cl-]i; e.g. the shift more readily occurred at lower [Cl-]i. 6. In neurones perfused with internal and external solutions containing 120 mM-Cl- at a delta VH of less than 10 mV, the change of gCl occurred with no shift of EGABA during the continuous application of GABA at concentrations over 6 x 10(-5) M, thereby indicating a 'real' GABA receptor desensitization. The desensitization depended solely upon the agonist concentrations but not upon the amount of ICl. Under these conditions, the time course of recovery from GABA desensitization was estimated. The decrease of gCl at the desensitization phase was a single exponential. 7. At a delta VH greater than 15 mV, therefore, the decay of ICl induced by GABA concentrations over 6 x 10(-6) M consists of the sum of both the 'real' GABA receptor desensitization and the current run-down brought about by Cl- shifts. The gCl at the current decay phase consisted of a double exponential. In the present experiments we chose experimental conditions with which Cl- shift become negligible. 8. The 'pure' GABA receptor desensitization during a continuous application of GABA developed rapidly at GABA concentrations over 10(-5) M. The speed of desensitization was facilitated by increasing the magnitude of desensitization.

1. The kinetic properties of the activation and inactivation (desensitization) phases of pentobarbitone (PB)-induced inward Cl- current (ICl) were studied in isolated frog sensory neurones, following suppression of Na+, K+ and Ca2+ currents, using the concentration jump technique which combines the internal perfusion and the rapid exchange of the external solutions surrounding a neurone with time constants of 2-3 ms. The results were compared with those of the gamma-aminobutyric acid (GABA)-gated ICl. 2. The PB dose-response curve was bell-shaped and the maximum peak value was less than the current induced by 1.7 X 1.5(-5) M-GABA, the concentration at which GABA evoked a half-maximum response. 3. The activation and inactivation phases of PB-induced ICl consisted of double-exponential, fast and slow components, respectively. The time constant of the fast component (tau af) of the activation was relatively stable in a concentration range between 3 X 10(-4) and 6 X 10(-3) M. The time constant of the slow component (tau as) of the activation decreased with increasing PB concentrations. Both the fast and slow components (tau if and tau is) of the inactivation decreased with increasing PB concentrations. 4. Over a wide range of concentrations the tau af and tau as values of the PB-induced ICl were 10-30 times greater than the respective values of GABA-induced ICl. 5. At concentrations below 10(-3) M the PB-induced ICl was voltage dependent at more negative potentials than -20 mV. 6. The PB-induced ICl was blocked by bicuculline and by picrotoxin, but in a different manner. Bicuculline increased the time constants of the activation and inactivation. Picrotoxin had little effect on the activation phase but markedly facilitated the inactivation phase. 7. High concentrations of PB (over 10(-3) M) led to a decline in both the peak and plateau currents of the PB-induced ICl. A transient 'hump' current appeared with wash-out of the external solutions containing high concentrations of PB. This hump current was blocked by bicuculline in a dose-dependent manner. 8. The results suggest the possibilities that the PB receptor-ionophore complexes consist of at least two different components having different affinities and kinetics and that the PB and GABA binding sites are closely located.

The effects of the voltage-dependent Ca2+ inward current (ICa) on gamma-aminobutyric acid (GABA)-induced Cl- current (ICl) in isolated frog sensory neurones were examined using the "concentration clamp" technique, an approach which combines internal perfusion by a suction pipette with a rapid external solution change method. A preceding ICa suppressed the GABA-activated ICl. While the inhibition was not influenced by changes of membrane potential between -40 and -100 mV, it was reduced when the internal EGTA concentration was increased. A preceding ICa shifted the GABA dose-response curve to the right without affecting the maximum current or the Hill coefficient (n = 2). Inhibition of the GABA-activated ICl by ICa showed a recovery in a single exponential manner, the time constant of which depended upon the extent of Ca2+ influx. At a low temperature (10 degrees C), however, the recovery from Ca2+-mediated inhibition was only slight. We conclude that, as a result of the voltage-dependent Ca2+ influx, a subsequent increase of intracellular Ca2+ concentration modulates the GABA-gated ICl in the soma membrane. Possible mechanisms are discussed.

The action of lidocaine on the Ca2+ current (ICa) was studied on single isolated neurons of frog dorsal root ganglia using a 'concentration-clamp' technique which combines intracellular perfusion with a rapid external solution change. Lidocaine decreased the peak amplitude of ICa at a threshold concentration of 10 microM. Higher concentrations gave a dose-dependent increase in inhibition of ICa. Lidocaine also depressed the Na+ current (INa) at a threshold concentration 10 times lower than that for decreasing the amplitude of ICa of neurons isolated from same ganglia. The rate of inhibitory action on ICa was slowed at more negative holding potentials (VH). Lidocaine appears to block both closed and open Ca2+ channels, but acts more profoundly on open channels.

The open channel blocking action of the Ca2+ antagonists, D-600 and Cd2+, was investigated in single neurons isolated by enzymatic treatment from dorsal root ganglia of frog. Using a 'concentration clamp' (jump) technique Ca2+ antagonists were applied to the preparation just before (less than 100 ms) or during a depolarizing step which induced the maximal peak amplitude of Ca2+ current (ICa). The inhibitory action of D-600 was more pronounced when applied during a depolarizing step than when applied just before a depolarizing step. In contrast, Cd2+ was equally inhibitory, whether applied before or during the depolarization. Thus, D-600 affects open channels more quickly or more profoundly than closed ones, while Cd2+ acts equally on both open and closed Ca2+ channels.

The effect of diazepam (DZP) on the GABA-induced macroscopic and microscopic Cl- current was investigated in isolated frog sensory neurons using both 'concentration-clamp' and patch-clamp techniques. At concentration range between 10(-9) and 10(-4) M, DZP itself evoked no response but potentiated time- and dose-dependently the subthreshold GABA responses, though at high DZP concentrations beyond 10(-5) M the potentiation ratio decreased. The potentiation effect was long-lasting and desensitized slowly over the course of several 10 minutes after washing-out of DZP. DZP potentiated GABA response without shifting the GABA reversal potential. The entire GABA dose-response curve was shifted in a parallel manner to the left by adding DZP without changing cooperatively: the Hill slope was 2.0. The potentiation of GABA response by DZP did not depend on either inward or outward direction of the Cl- current but slightly on the membrane potential. The time constants of activation of desensitization of GABA-gated Cl- current consisted of fast and slow components, respectively. The slow components were concentration-dependent, and significantly changed in the presence of DZP, while DZP had little effects on fast components. In the 'inside-out' configuration, the addition of DZP activated GABA-receptor ionophore complexes under subthreshold without changing the single Cl- channel conductance. It is concluded that DZP may act at a site to modulate GABA binding, in which DZP increases GABA binding affinity and also affects the kinetics of GABA-gated Cl- channels, indicating that DZP has dual action on the GABA-induced responses.