A Ca2+-activated Cl− conductance in interstitial cells of Cajal linked to slow wave currents and pacemaker activity

Interstitial cells of Cajal (ICC) are unique cells that generate electrical pacemaker activity in gastrointestinal (GI) muscles. Many previous studies have attempted to characterize the conductances responsible for pacemaker current and slow waves in the GI tract, but the precise mechanism of electrical rhythmicity is still debated. We used a new transgenic mouse with a bright green fluorescent protein (copGFP) constitutively expressed in ICC to facilitate study of these cells in mixed cell dispersions. We found that ICC express a specialized 'slow wave' current. Reversal of tail current analysis showed this current was due to a Cl⁻ selective conductance. ICC express ANO1, a Ca²⁺-activated Cl⁻ channel. Slow wave currents are not voltage dependent, but a secondary voltage-dependent process underlies activation of these currents. Removal of extracellular Ca²⁺, replacement of Ca²⁺ with Ba²⁺, or extracellular Ni²⁺ (30 μ m ) blocked the slow wave current. Single Ca²⁺-activated Cl⁻ channels with a unitary conductance of 7.8 pS were resolved in excised patches of ICC. These are similar in conductance to ANO1 channels (8 pS) expressed in HEK293 cells. Slow wave current was blocked in a concentration-dependent manner by niflumic acid (IC₅₀= 4.8 μ m ). Slow wave currents are associated with transient depolarizations of ICC in current clamp, and these events were blocked by niflumic acid. These findings demonstrate a role for a Ca²⁺-activated Cl⁻ conductance in slow wave current in ICC and are consistent with the idea that ANO1 participates in pacemaker activity.     

Inhibition by thromboxane antagonists of airway hyperresponsiveness to histamine induced by 13,14-dihydro-15-keto-PGF2α in guinea-pigs

Summary. We studied the effect of intravenous administration of 13,14-dihydro-15-keto-prostaglandin (PG) F_27alpha; on airway responsiveness to histamine and airway wall thickening in guinea-pigs. Guinea-pigs were killed and the lungs were fixed in formalin. Slides from paraffin-embedded sections of the lungs were stained and the airways that were cut in transverse section were measured by tracing enlarged images using a digitizer. Moreover, airway resistance (Raw) was determined by a pulmonary mechanics analyser and we calculated two indices, an index of airway wall thickening and the one of airway hyperresponsiveness to histamine, from changes of baseline-Raw and peak-Raw following intravenous administration of histamine before and after the intravenous administration of 13,14-dihydro-l5-keto-PGF_2n. Intravenous administration of 10/μg/kg 13,14-dihydro-15-keto-PGF_2α for 1 h did not induce an increase of the relative thickness of the airway wall by the histological examination. In analysis of airway function, intravenous administration of 10μg/kg 13,14-dihydro-15-keto-PGF_2α for 1 h induced airway hyperresponsiveness to histamine without airway wall thickening. Thromboxane A₂ (TXA₂) receptor antagonists ONO-NT-126 and ONO-8809 inhibited the 13,14-dihydro-15-keto-PGF_2α-induced airway hyperresponsiveness to histamine, suggesting that the effect of 13,14-dihydro-15-keto-PGF_2α on bronchial hyperresponsiveness is likely to be mediated through TXA₂.     

Aquaporin-1 and HCO3−-Cl− transporter-mediated transport of CO2 across the human erythrocyte membrane

Recent studies have suggested that aquaporin-1 (AQP1) as well as the HCO₃⁻-Cl⁻ transporter may be involved in CO₂ transport across biological membranes, but the physiological importance of this route of gas transport remained unknown. We studied CO₂ transport in human red blood cell ghosts at physiological temperatures (37 °C). Replacement of inert with CO₂-containing gas above a stirred cell suspension caused an outside-to-inside directed CO₂ gradient and generated a rapid biphasic intracellular acidification. The gradient of the acidifying gas was kept small to favour high affinity entry of CO₂ passing the membrane. All rates of acidification except that of the approach to physicochemical equilibrium of the uncatalysed reaction were restricted to the intracellular environment. Inhibition of carbonic anhydrase (CA) demonstrated that CO₂-induced acidification required the catalytic activity of CA. Blockade of the function of either AQP1 (by HgCl₂ at 65 μM) or the HCO₃⁻-Cl⁻ transporter (by DIDS at 15 μM) completely prevented fast acidification. These data indicate that, at low chemical gradients for CO₂, nearly the entire CO₂ transport across the red cell membrane is mediated by AQP1 and the HCO₃⁻-Cl⁻ transporter. Therefore, these proteins may function as high affinity sites for CO₂ transport across the erythrocyte membrane.     

The human tumour suppressor gene SLC5A8 expresses a Na+–monocarboxylate cotransporter

The orphan cotransport protein expressed by the SLC5A8 gene has been shown to play a role in controlling the growth of colon cancers, and the silencing of this gene is a common and early event in human colon neoplasia. We expressed this protein in Xenopus laevis oocytes and have found that it transports small monocarboxylic acids. The electrogenic activity of the cotransporter, which we have named SMCT (sodium monocarboxylate transporter), was dependent on external Na⁺ and was compatible with a 3 : 1 stoichiometry between Na⁺ and monocarboxylates. A portion of the SMCT-mediated current was also Cl⁻ dependent, but Cl⁻ was not cotransported. SMCT transports a variety of monocarboxylates (similar to unrelated monocarboxylate transport proteins) and most transported monocarboxylates demonstrated K _m values near 100 μ m , apart from acetate and d -lactate, for which the protein showed less affinity. SMCT was strongly inhibited by 1 m m probenecid or ibuprofen. In the absence of external substrate, a Na⁺-independent leak current was also observed to pass through SMCT. SMCT activity was strongly inhibited after prolonged exposure to high external concentrations of monocarboxylates. The transport of monocarboxylates in anionic form was confirmed by the observation of a concomitant alkalinization of the cytosol. SMCT, being expressed in colon and kidney, represents a novel means by which Na⁺, short-chain fatty acids and other monocarboxylates are transported in these tissues. The significance of a Na⁺–monocarboxylate transporter to colon cancer presumably stems from the transport of butyrate, which is well known for having anti-proliferative and apoptosis-inducing activity in colon epithelial cells.     

Fluid flow induces mechanosensitive ATP release, calcium signalling and Cl− transport in biliary epithelial cells through a PKCζ-dependent pathway

ATP in bile is a potent secretogogue, stimulating cholangiocyte Cl⁻ and fluid secretion via binding to membrane P2 receptors, though the physiological stimuli involved in biliary ATP release are unknown. The goal of the present studies was to determine the potential role of fluid flow in biliary ATP release and secretion. In both human Mz-Cha-1 biliary cells and normal rat cholangiocyte monolayers, exposure to flow increased relative ATP release which was proportional to the shear stress. In parallel studies, shear was associated with an increase in [Ca²⁺]_i and membrane Cl⁻ permeability, which were both dependent on extracellular ATP and P2 receptor stimulation. Flow-stimulated ATP release was dependent on [Ca²⁺]_i, exhibited desensitization with repetitive stimulation, and was regulated by PKCζ. In conclusion, both human and rat biliary cells exhibit flow-stimulated, PKCζ-dependent, ATP release, increases in [Ca²⁺]_i and Cl⁻ secretion. The finding that fluid flow can regulate membrane transport suggests that mechanosensitive ATP release may be a key regulator of biliary secretion and an important target to modulate bile flow in the treatment of cholestatic liver diseases.     

Mexiletine block of wild-type and inactivation-deficient human skeletal muscle hNav1.4 Na+ channels

Mexiletine is a class 1b antiarrhythmic drug used for ventricular arrhythmias but is also found to be effective for paramyotonia congenita, potassium-aggravated myotonia, long QT–3 syndrome, and neuropathic pain. This drug elicits tonic block of Na⁺ channels when cells are stimulated infrequently and produces additional use-dependent block during repetitive pulses. We examined the state-dependent block by mexiletine in human skeletal muscle hNav1.4 wild-type and inactivation-deficient mutant Na⁺ channels (hNav1.4-L443C/A444W) expressed in HEK293t cells with a β1 subunit. The 50% inhibitory concentrations (IC₅₀) for the inactivated-state block and the resting-state block of wild-type Na⁺ channels by mexiletine were measured as 67.8 ± 7.0 μ m and 431.2 ± 9.4 μ m , respectively ( n = 5). In contrast, the IC₅₀ for the block of open inactivation-deficient mutant channels at +30 mV by mexiletine was 3.3 ± 0.1 μ m ( n = 5), which was within the therapeutic plasma concentration range (2.8–11 μ m ). Estimated on- and off-rates for the open-state block by mexiletine at +30 mV were 10.4 μ m ⁻¹ s⁻¹ and 54.4 s⁻¹, respectively. Use-dependent block by mexiletine was greater in inactivation-deficient mutant channels than in wild-type channels during repetitive pulses. Furthermore, the IC₅₀ values for the block of persistent late hNav1.4 currents in chloramine-T-pretreated cells by mexiletine was 7.5 ± 0.8 μ m ( n = 5) at +30 mV. Our results together support the hypothesis that the in vivo efficacy of mexiletine is primarily due to the open-channel block of persistent late Na⁺ currents, which may arise during various pathological conditions.     

Subunit composition and role of Na+,K+-ATPases in adrenal chromaffin cells

Adrenal medullary (AM) cells are exposed to high concentrations of cortical hormones, one of which is a ouabain-like substance. Thus, the effects of ouabain on catecholamine secretion and distribution of Na⁺,K⁺-ATPase α and β subunits in rat and guinea-pig AM cells were examined using amperometry and immunological techniques. While exposure to 1 μ m ouabain did not have a marked effect on resting secretion, it induced an increase in secretion due to mobilization of Ca²⁺ ions that were stored during a 4 min interval between muscarine applications. Immunocytochemistry revealed that Na⁺,K⁺-ATPase α1 subunit-like and β3 subunit-like immunoreactive (IR) materials were distributed ubiquitously at the cell periphery, whereas α2- and β2-like IR materials were present in restricted parts of the cell periphery. The α1 and α2 subunits were mainly immunoprecipitated from AM preparations by anti-β3 and anti-β2 antisera, respectively. Peripheral BODIPY-FL-InsP₃ binding sites were localized below membrane domains with α2- and β2-like IR materials. The results indicate that in AM cells, α1β3 isozymes of Na⁺,K⁺-ATPase were present ubiquitously in the plasma membrane, while α2β2 isozymes were in the membrane domain closely associated with peripheral Ca²⁺ store sites. This close association of the α2β2 isozyme with peripheral Ca²⁺ store sites may account for the facilitation of mobilization-dependent secretion in the presence of 1 μ m ouabain.     

Adenosine inhibition via A1 receptor of N-type Ca2+ current and peptide release from isolated neurohypophysial terminals of the rat

Effects of adenosine on voltage-gated Ca²⁺ channel currents and on arginine vasopressin (AVP) and oxytocin (OT) release from isolated neurohypophysial (NH) terminals of the rat were investigated using perforated-patch clamp recordings and hormone-specific radioimmunoassays. Adenosine, but not adenosine 5'-triphosphate (ATP), dose-dependently and reversibly inhibited the transient component of the whole-terminal Ba²⁺ currents, with an IC₅₀ of 0.875 μ m. Adenosine strongly inhibited, in a dose-dependent manner (IC₅₀= 2.67 μ m ), depolarization-triggered AVP and OT release from isolated NH terminals. Adenosine and the N-type Ca²⁺ channel blocker ω-conotoxin GVIA, but not other Ca²⁺ channel-type antagonists, inhibited the same transient component of the Ba²⁺ current. Other components such as the L-, Q- and R-type channels, however, were insensitive to adenosine. Similarly, only adenosine and ω-conotoxin GVIA were able to inhibit the same component of AVP release. A₁ receptor agonists, but not other purinoceptor-type agonists, inhibited the same transient component of the Ba²⁺ current as adenosine. Furthermore, the A₁ receptor antagonist 8-cyclopentyltheophylline (CPT), but not the A₂ receptor antagonist 3, 7-dimethyl-1-propargylxanthine (DMPGX), reversed inhibition of this current component by adenosine. The inhibition of AVP and OT release also appeared to be via the A₁ receptor, since it was reversed by CPT. We therefore conclude that adenosine, acting via A₁ receptors, specifically blocks the terminal N-type Ca²⁺ channel thus leading to inhibition of the release of both AVP and OT.     

A prostaglandin D2 receptor antagonist modifies experimental asthma in sheep

Background Prostaglandin (PG) D₂ is the major cylooxygenase metabolite released by mast cells upon allergen stimulation, and elicits responses through either the prostanoid DP1 receptor and/or the chemoattractant receptor homologous molecule expressed on T-helper type 2 (Th2) cells (CRTH2/DP2). Experimental evidence suggests that stimulation of one or both these receptors contributes to asthma pathophysiology.
Objective The aim of this study was to test the hypothesis that the prostanoid DP1 receptor contributes to asthma pathophysiology by determining the efficacy of an orally active antagonist for this receptor, S-5751, on allergen-induced bronchoconstriction, airway hyperresponsiveness (AHR) and cellular inflammation in the sheep model of asthma.
Methods PGD₂-induced cyclic adenosine monophosphate (cAMP) production in platelet-rich plasma was used to establish the in vitro efficacy of S-5751. In vivo , sheep naturally allergic to Ascaris suum were challenged with an aerosolized antigen with and without S-5751 treatment (given 4 days before and for 6 days after the challenge).
Results S-5751 inhibited PGD₂-induced cAMP production in platelet-rich plasma with an IC₅₀ value of 0.12 μ m . S-5751 at 30 mg/kg, but not at 3 mg/kg, reduced the early bronchoconstriction and inhibited the late bronchoconstriction. AHR and inflammatory cell infiltration in bronchoalveolar lavage fluid at days 1 and 7 were also inhibited with the 30 mg/kg dose. The responses observed with S-5751 at 30 mg/kg were comparable with those with montelukast treatment (0.15 mg/kg, twice a day, intravenous); however, S-5751 did not block inhaled leukotrieneD₄-induced broncoconstriction.
Conclusion Prostanoid DP1 receptor inhibition may represent an alternative target for asthma therapy.     

Role of extracellular Ca2+ in gating of CaV1.2 channels

We examined changes in ionic and gating currents in Ca_V1.2 channels when extracellular Ca²⁺ was reduced from 10 m m to 0.1 μ m . Saturating gating currents decreased by two-thirds ( K _D≈ 40 μ m ) and ionic currents increased 5-fold ( K _D≈ 0.5 μ m ) due to increasing Na⁺ conductance. A biphasic time dependence for the activation of ionic currents was observed at low [Ca²⁺], which appeared to reflect the rapid activation of channels that were not blocked by Ca²⁺ and a slower reversal of Ca²⁺ blockade of the remaining channels. Removal of Ca²⁺ following inactivation of Ca²⁺ currents showed that Na⁺ currents were not affected by Ca²⁺-dependent inactivation. Ca²⁺-dependent inactivation also induced a negative shift of the reversal potential for ionic currents suggesting that inactivation alters channel selectivity. Our findings suggest that activation of Ca²⁺ conductance and Ca²⁺-dependent inactivation depend on extracellular Ca²⁺ and are linked to changes in selectivity.     

Role of vasopressin in rat distal colon function

The specific role of vasopressin in colonic crypt function and its possible synergistic action with aldosterone were studied. Sprague-Dawley rats fed a high-Na⁺ (HS; 150 m m NaCl) or a low-Na⁺ (LS; 150 μ m NaCl) diet were deprived of water or infused with vasopressin, and some animals were treated with specific vasopressin receptor subtype V₁ and V₂ antagonists. The expression of the epithelial Na⁺ channel (ENaC), α-smooth muscle actin (α-SMA) and aquaporin-2 (AQP-2) were determined by immunolocalization in distal colonic mucosa. The pericryptal Na⁺ concentration was determined by confocal microscopy, using a low-affinity Na⁺-sensitive fluorescent dye (sodium red) and crypt permeability was measured by the rate of escape of fluorescein isothiocyanate-labelled dextran (10 kDa) from the crypt lumen into the pericryptal space in isolated rat distal colonic mucosa. A high plasma concentration of vasopressin raised α-SMA expression in the pericryptal sheath ( P < 0.05), increased the pericryptal Na⁺ accumulation in this space ( P < 0.01) and caused a reduction of crypt wall permeability ( P < 0.01). All these effects were reversed by selective blockade of V₁ and V₂ receptors. No synergistic effects with aldosterone were observed. Dehydration and vasopressin infusion increased AQP-2 expression in distal colonic mucosa ( P < 0.05). This action of vasopressin was prevented by tolvaptan, a specific V₂ receptor antagonist ( P < 0.05). It is concluded that vasopressin has trophic effects in the rat distal colon, increasing pericryptal myofibroblast growth which affects crypt absorption, and these effects are independent of the presence of aldosterone.     

Regenerative component of slow waves in the guinea-pig gastric antrum involves a delayed increase in [Ca2+]i and Cl− channels

Regenerative potentials were initiated by depolarizing short segments of single bundles of circular muscle isolated from the gastric antrum of guinea-pigs. When changes in [Ca²⁺]_i and membrane potential were recorded simultaneously, regenerative potentials were found to be associated with an increase in [Ca²⁺]_i, with the increase starting after a minimum latency of about 1 s. Although the increase in [Ca²⁺]_i was reduced by nifedipine, the amplitudes of the regenerative responses were little changed. Regenerative responses and associated changes in [Ca²⁺]_i were abolished by loading the preparations with the Ca²⁺ chelator MAPTA-AM. Regenerative potentials were abolished by 2-aminoethoxydiphenyl borate (2APB), an inhibitor of IP₃ induced Ca²⁺ release, by N -ethylamaleimide (NEM), an alkylating agent which blocks activation of G-proteins and were reduced in amplitude by two agents which block chloride (Cl⁻)-selective channels in many tissues. The observations suggest that membrane depolarization triggers IP₃ formation. This causes Ca²⁺ release from intracellular stores which activates Ca²⁺-dependent Cl⁻ channels.     

Properties of spontaneous Ca2+ transients recorded from interstitial cells of Cajal-like cells of the rabbit urethra in situ

Interstitial cells of Cajal-like cells (ICC-LCs) in the urethra may act as electrical pacemakers of spontaneous contractions. However, their properties in situ and their interaction with neighbouring urethral smooth muscle cells (USMCs) remain to be elucidated. To further explore the physiological role of ICC-LCs, spontaneous changes in [Ca²⁺]_i (Ca²⁺ transients) were visualized in fluo-4 loaded preparations of rabbit urethral smooth muscle. ICC-LCs were sparsely distributed, rather than forming an extensive network. Ca²⁺ transients in ICC-LCs had a lower frequency and a longer half-width than those of USMCs. ICC-LCs often exhibited Ca²⁺ transients synchronously with each other, but did not often show a close temporal relationship with Ca²⁺ transients in USMCs. Nicardipine (1 μ m ) suppressed Ca²⁺ transients in USMCs but not in ICC-LCs. Ca²⁺ transients in ICC-LCs were abolished by cyclopiazonic acid (10 μ m ), ryanodine (50 μ m ) and caffeine (10 m m ) or by removing extracellular Ca²⁺, and inhibited by 2-aminoethoxydiphenyl borate (50 μ m ) and 3-morpholino-sydnonimine (SIN-1; 10 μ m ), but facilitated by increasing extracellular Ca²⁺ or phenylephrine (1–10 μ m ). These results indicated that Ca²⁺ transients in urethral ICC-LCs in situ rely on both Ca²⁺ release from intracellular Ca²⁺ stores and Ca²⁺ influx through non-L-type Ca²⁺ channel pathways. ICC-LCs may not act as a coordinated pacemaker electrical network as do ICC in the gastrointestinal (GI) tract. Rather they may randomly increase excitability of USMCs to maintain the tone of urethral smooth muscles.     

Functional characterization of a Ca2+-activated non-selective cation channel in human atrial cardiomyocytes

Cardiac arrhythmias, which occur in a wide variety of conditions where intracellular calcium is increased, have been attributed to the activation of a transient inward current ( I _ti). I _ti is the result of three different [Ca]_i-sensitive currents: the Na⁺–Ca²⁺ exchange current, a Ca²⁺-activated chloride current and a Ca²⁺-activated non-selective cationic current. Using the cell-free configuration of the patch-clamp technique, we have characterized the properties of a Ca²⁺-activated non-selective cation channel (NSC_Ca) in freshly dissociated human atrial cardiomyocytes. In excised inside-out patches, the channel presented a linear I–V relationship with a conductance of 19 ± 0.4 pS. It discriminated poorly among monovalent cations (Na⁺ and K⁺) and was slightly permeable to Ca²⁺ ions. The channel's open probability was increased by depolarization and a rise in internal calcium, for which the K _d for [Ca²⁺]_i was 20.8 μ m . Channel activity was reduced in the presence of 0.5 m m ATP or 10 μ m glibenclamide on the cytoplasmic side to 22.1 ± 16.8 and 28.5 ± 8.6%, respectively, of control. It was also inhibited by 0.1 m m flufenamic acid. The channel shares several properties with TRPM4b and TRPM5, two members of the 'TRP melastatin' subfamily. In conclusion, the NSC_Ca channel is a serious candidate to support the delayed after-depolarizations observed in [Ca²⁺] overload and thus may be implicated in the genesis of arrhythmias.     

Palmitate increases L-type Ca2+ currents and the size of the readily releasable granule pool in mouse pancreatic β-cells

We have investigated the in vitro effects of the saturated free fatty acid palmitate on mouse pancreatic β-cells by a combination of electrophysiological recordings, intracellular Ca²⁺ ([Ca²⁺]_i) microfluorimetry and insulin release measurements. Addition of palmitate (1 m m , bound to fatty acid-free albumin) to intact islets exposed to 15 m m glucose increased the [Ca²⁺]_i by ∼30% and insulin secretion 2-fold. Palmitate remained capable of increasing [Ca²⁺]_i and insulin release in the presence of tolbutamide and in islets depolarized by high K⁺ in combination with diazoxide, indicating that the stimulation occurs independently of closure of ATP-regulated K⁺ channels (K_ATP channels). Palmitate (0.5 m m ) augmented exocytosis (measured as an increase in cell capacitance) in single β-cells and increased the size of the readily releasable pool (RRP) of granules 2-fold. Whole-cell peak Ca²⁺ currents rose by ∼25% following addition of 0.5 m m palmitate, an effect that was abolished in the presence of 10 μ m isradipine indicating that the free fatty acid specifically acts on L-type Ca²⁺ channels. The actions of palmitate on exocytosis and Ca²⁺ currents were not mimicked by intracellular application of palmitoyl-CoA. We conclude that palmitate increases insulin secretion by a K_ATP channel-independent mechanism exerted at the level of exocytosis and that involves both augmentation of L-type Ca²⁺ currents and an increased size of the RRP.     

Role of cyclic nucleotide phosphodiesterase isoforms in cAMP compartmentation following β2-adrenergic stimulation of ICa,L in frog ventricular myocytes

The role of cyclic nucleotide phosphodiesterase (PDE) isoforms in the β₂-adrenergic stimulation of the L-type Ca²⁺ current ( I _Ca,L) was investigated in frog ventricular myocytes using double patch-clamp and double-barrelled microperfusion techniques. Isoprenaline (ISO, 1 nM to 10 μM) was applied on one half of the cell, either alone or in the presence of PDE inhibitors, and the local and distant responses of I _Ca,L were used to determine the gradient of local vs. distant cAMP concentration (α). IBMX (100 μM), a non-selective PDE inhibitor, reduced α from 40 to 4.4 indicating a 9-fold reduction in intracellular cAMP compartmentation when all PDE activity was blocked. While PDE1 and PDE2 inhibition had no effect, PDE3 inhibition by milrinone (3 μM) or PDE4 inhibition by Ro 20-1724 (3 μM) reduced α by 6- and 4-fold, respectively. A simultaneous application of milrinone and Ro 20-1724 produced a similar effect to IBMX, showing that PDE3 and PDE4 were the major PDEs accounting for cAMP compartmentation. Okadaic acid (3 μM), a non-selective phosphatase inhibitor, or H89 (1 μM), an inhibitor of cAMP-dependent protein kinase (PKA), had no effect on the distant response of I _Ca,L to ISO indicating that PDE activation by PKA played a minor role in cAMP compartmentation. Our results demonstrate that PDE activity determines the degree of cAMP compartmentation in frog ventricular cells upon β₂-adrenergic stimulation. PDE3 and PDE4 subtypes play a major role in this process, and contribute equally to ensure a functional coupling of β₂-adrenergic receptors with nearby Ca²⁺ channels via local elevations of cAMP.     

Tyrosine kinase and phosphatase regulation of slow delayed-rectifier K+ current in guinea-pig ventricular myocytes

The objective of this study was to investigate the involvement of tyrosine phosphorylation in the regulation of the cardiac slowly activating delayed-rectifier K⁺ current ( I _Ks) that is important for action potential repolarization. Constitutive I _Ks recorded from guinea-pig ventricular myocytes was suppressed by broad-spectrum tyrosine kinase (TK) inhibitors tyrphostin A23 (IC₅₀, 4.1 ± 0.6 μ m ), tyrphostin A25 (IC₅₀, 12.1 ± 2.1 μ m ) and genistein (IC₅₀, 64 ± 4 μ m ), but was relatively insensitive to the inactive analogues tyrphostin A1, tyrphostin A63, daidzein and genistin. I _Ks was unaffected by AG1478 (10 μ m ), an inhibitor of epidermal growth factor receptor TK, and was strongly suppressed by the Src TK inhibitor PP2 (10 μ m ) but not by the inactive analogue PP3 (10 μ m ). The results of experiments with forskolin, H89 and bisindolylmaleimide I indicate that the suppression of I _Ks by TK inhibitors was not mediated via inhibition of ( I _Ks-stimulatory) protein kinases A and C. To evaluate whether the suppression was related to lowered tyrosine phosphorylation, myocytes were pretreated with TK inhibitors and then exposed to the phosphotyrosyl phosphatase inhibitor orthovanadate (1 m m ). Orthovanadate almost completely reversed the suppression of I _Ks induced by broad-spectrum TK inhibitors at concentrations around their IC₅₀ values. We conclude that basal I _Ks is strongly dependent on tyrosine phosphorylation of Ks channel (or channel-regulatory) protein.     

A novel O2-sensing mechanism in rat glossopharyngeal neurones mediated by a halothane-inhibitable background K+ conductance

Modulation of K⁺ channels by hypoxia is a common O₂-sensing mechanism in specialised cells. More recently, acid-sensitive TASK-like background K⁺ channels, which play a key role in setting the resting membrane potential, have been implicated in O₂-sensing in certain cell types. Here, we report a novel O₂ sensitivity mediated by a weakly pH-sensitive background K⁺ conductance in nitric oxide synthase (NOS)-positive neurones of the glossopharyngeal nerve (GPN). This conductance was insensitive to 30 m m TEA, 5 m m 4-aminopyridine (4-AP) and 200 μ m Cd²⁺, but was reversibly inhibited by hypoxia (O₂ tension ( P _O2) = 15 mmHg), 2–5 m m halothane, 10 m m barium and 1 m m quinidine. Notably, the presence of halothane occluded the inhibitory effect of hypoxia. Under current clamp, these agents depolarised GPN neurones. In contrast, arachidonic acid (5–10 μ m ) caused membrane hyperpolarisation and potentiation of the background K⁺ current. This pharmacological profile suggests the O₂-sensitive conductance in GPN neurones is mediated by a class of background K⁺ channels different from the TASK family; it appears more closely related to the THIK (tandem pore domain halothane-inhibited K⁺) subfamily, or may represent a new member of the background K⁺ family. Since GPN neurones are thought to provide NO-mediated efferent inhibition of the carotid body (CB), these channels may contribute to the regulation of breathing during hypoxia via negative feedback control of CB function, as well as to the inhibitory effect of volatile anaesthetics (e.g. halothane) on respiration.     

Descending vasa recta pericytes express voltage operated Na+ conductance in the rat

We studied the properties of a voltage-operated Na⁺ conductance in descending vasa recta (DVR) pericytes isolated from the renal outer medulla. Whole-cell patch-clamp recordings revealed a depolarization-induced, rapidly activating and rapidly inactivating inward current that was abolished by removal of Na⁺ but not Ca⁺ from the extracellular buffer. The Na⁺ current ( I _Na) is highly sensitive to tetrodotoxin  (TTX, K _d= 2.2 n m )  . At high concentrations, mibefradil (10 μ m ) and Ni⁺ (1 m m ) blocked I _Na. I _Na was insensitive to nifedipine (10 μ m ). The L-type Ca⁺ channel activator FPL-64176 induced a slowly activating/inactivating inward current that was abolished by nifedipine. Depolarization to membrane potentials between 0 and 30 mV induced inactivation with a time constant of ∼1 ms. Repolarization to membrane potentials between −90 and −120 mV induced recovery from inactivation with a time constant of ∼11 ms. Half-maximal activation and inactivation occurred at −23.9 and −66.1 mV, respectively, with slope factors of 4.8 and 9.5 mV, respectively. The Na⁺ channel activator, veratridine (100 μ m ), reduced peak inward I _Na and prevented inactivation. We conclude that a TTX-sensitive voltage-operated Na⁺ conductance, with properties similar to that in other smooth muscle cells, is expressed by DVR pericytes.     

GABA Mediates Autoreceptor Feedback Inhibition in the Rat Carotid Body Via Presynaptic GABAB Receptors and TASK-1

Background K⁺ channels exert control over neuronal excitability by regulating resting potential and input resistance. Here, we show that GABA_B receptor-mediated activation of a background K⁺ conductance modulates transmission at rat carotid body chemosensory synapses in vitro . Carotid body chemoreceptor (type I) cells expressed GABA_B(1) and GABA_B(2) subunits as well as endogenous GABA. The GABA_B receptor agonist baclofen activated an anandamide- and Ba²⁺-sensitive TASK-1-like background K⁺ conductance in chemoreceptor cell clusters, but was without effect on voltage-gated Ca²⁺ channels. Hydroxysaclofen (50 μ m ), 5-aminovaleric acid (100 μ m ) and CGP 55845 (100 n m ), selective GABA_B receptor blockers, potentiated the hypoxia-induced receptor potential; this effect was abolished by pre-treatment with pertussis toxin (PTX; 500 ng ml^-1), an inhibitor of G_i, or by H-89 (50 μ m ), a selective inhibitor of protein kinase A. The protein kinase C inhibitor chelerythrine chloride (100 μ m ) was without effect on this potentiation. GABA_B receptor blockers also caused depolarisation of type I cells in clusters, and enhanced spike discharge in spontaneously firing cells. In functional co-cultures of type I clusters and petrosal sensory neurones, GABA_B receptor blockers potentiated hypoxia-induced postsynaptic chemosensory responses mediated by the fast-acting transmitters ACh and ATP. Thus GABA_B receptor-mediated activation of TASK-1 or a related channel provides a presynaptic autoregulatory feedback mechanism that modulates fast synaptic transmission in the rat carotid body.