Pharmacology of the human red cell voltage-dependent cation channel. Part II: inactivation and blocking

Pharmacological modulation of the nonselective voltage-dependent cation (NSVDC) channel from human erythrocytes was studied. Using the inorganic cations ruthenium red and La3+, as well as the organic thiol group reagents iodoacetamide (IAA) and N-ethylmaleimide (NEM), it was possible to demonstrate a concentration-dependent decrease in the voltage-activated conductance, reflecting an inhibition or inactivation of the channel. Initial voltage activation was achieved by injecting human red cells into sucrose-substituted Ringers with a low chloride concentration, which causes a strongly positive membrane potential to develop, initially determined by the equilibrium potential for Cl- ( approximately +100 mV). Due to the voltage- and time-dependent activation of the cation channel, net effluxes, minimized by addition of a chloride conductance blocker, occurred and Vm gradually decreased and stabilized at a value less positive than E(Cl), reflecting the increased cation conductance, g+, reaching 1.5-2.0 microS/cm2. In the presence of inhibitors of the NSVDC channel, both the membrane potential repolarization and the cation efflux were diminished.     

The human red cell voltage-dependent cation channel. Part III: Distribution homogeneity and pH dependence

The homogeneity of the distribution of the non-selective voltage-dependent cation channel (the NSVDC channel) in the human erythrocyte, and the pH dependence was investigated. Activation of this channel caused a uniform cellular dehydration, which was characterized by the changes in the erythrocyte osmotic resistance profiles: after 1/2 h of activation, the osmolarity at 50% hemolysis changed from 73 mM (control) to 34 mM NaCl, corresponding to 0.48% and 0.21% NaCl respectively. Unchanging standard deviations show participation of the entire erythrocyte population, which implies an even distribution of the NSVDC channel among the cells. Inactivation of the NSVDC channel with N-ethyl-maleimide (NEM) or blocking of the Cl(-) conductance with NS1652 retarded the migration of the resistance profiles towards lower osmolarities. The NSVDC channel activation was blocked by a decrease of the intracellular -- but not the extracellular -- pH. The apparent pK(A) value for the effect was estimated to be 6.5, and the specific histidine reagent 2.4'-dibromoacetophenone (DBAB) inactivated the NSVDC channel.     

Piezo1 activation augments sickling propensity and the adhesive properties of sickle red blood cells in a calcium-dependent manner

Haemoglobin S polymerization in the red blood cells (RBCs) of individuals with sickle cell anaemia (SCA) can cause RBC sickling and cellular alterations. Piezo1 is a mechanosensitive protein that modulates intracellular calcium (Ca²⁺) influx, and its activation has been associated with increased RBC surface membrane phosphatidylserine (PS) exposure. Hypothesizing that Piezo1 activation, and ensuing Gárdos channel activity, alter sickle RBC properties, RBCs from patients with SCA were incubated with the Piezo1 agonist, Yoda1 (0.1–10 μM). Oxygen-gradient ektacytometry and membrane potential measurement showed that Piezo1 activation significantly decreased sickle RBC deformability, augmented sickling propensity, and triggered pronounced membrane hyperpolarization, in association with Gárdos channel activation and Ca²⁺ influx. Yoda1 induced Ca²⁺-dependent adhesion of sickle RBCs to laminin, in microfluidic assays, mediated by increased BCAM binding affinity. Furthermore, RBCs from SCA patients that were homo−/heterozygous for the rs59446030 gain-of-function Piezo1 variant demonstrated enhanced sickling under deoxygenation and increased PS exposure. Thus, Piezo1 stimulation decreases sickle RBC deformability, and increases the propensities of these cells to sickle upon deoxygenation and adhere to laminin. Results support a role of Piezo1 in some of the RBC properties that contribute to SCA vaso-occlusion, indicating that Piezo1 may represent a potential therapeutic target molecule for this disease.     

Activation of the Ca(2+)-activated nonselective cation channel by diacylglycerol analogues in rat cardiomyocytes

INTRODUCTION: Cardiac hypertrophy is associated with changes in electrophysiologic properties due to ionic channel modifications and increases in protein kinase C (PKC) activity and diacylglycerol (DAG) content. These changes may contribute to an increased propensity for arrhythmia. Similar electrophysiologic modifications have been reported in adult rat cardiomyocytes undergoing dedifferentiation in primary culture. METHODS AND RESULTS: Single-channel measurements on such cells identified the appearance of a Ca(2+)-activated nonselective cation channel (NSC(Ca)) during the dedifferentiation process. The current study investigated the sensitivity of this channel to PKC and DAG analogues. In the cell-attached configuration, channel conductance was 20.2 pS under physiologic conditions. Perfusion with the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG, 0.1 mM) or the PKC activator phorbol 12-myristate 13-acetate (PMA, 0.5 microM) increased the channel normalized open probability (nPo), whereas in the presence of the PKC inhibitor calphostin C (1 microM), only OAG retained this effect. In the inside-out configuration, perfusion of both DAG analogues OAG (0.1 mM) and 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG, 10 microM) on the inside of the membrane increased nPo. These results indicate that DAG regulates the NSC(Ca) channel via both the PKC pathway and by a direct interaction. CONCLUSION: DAG content, PKC activity, and channel expression increased during hypertrophy. This indicates that the NSC(Ca) channel exhibits high activity in this condition and, therefore, is a candidate for the genesis of arrhythmias in ventricular cardiomyocytes. In addition, regulation of the channel by DAG and PKC contributes to current understanding of the physiologic role of this channel, which shares properties with the cloned TRPM4b channel.     

Measurement of lymphocyte activation by a chromatin topooptical reaction. II. Application for detecting drug allergy. A clinical and experimental study

Lymphocyte chromatin activation was measured in 18 clinical cases of suspected or proven drug allergy as well as in 4 other persons with neither history nor signs of hypersensitivity. The different drugs were tested in the reaction in such dilution that the final concentrations ranged between 50 and 10.000 nanomoles. Within this range between 100 and 1250 nanomoles a bellshaped dose response curve was found in all drug-allergic subjects, measuring the neutral-red chromatin topooptical reaction. At the peak of this curve signs of cytotoxicity could be demonstrated with appreciable chromatin "desactivation", an increase of protein-like substance in the cells' supernatant and a morphological cellular damage. The exact drug concentration at which the lymphocyte autocytotoxicity occurred was inversely proportional with the extent of drug allergy in a given patient. The average lymphocyte chromatin birefringence measured at different non-cytotoxic drug concentrations was directly proportional with the extent of drug allergy. The ratio of the above characteristics gave a score (with the dimension of cm2/microM) which corresponded to the clinical picture. The score was low (55,7 +/- 5,8 cm2/microM) in the control subjects as well as in the drug allergic ones tested with other, i.e. nonsensitizing drugs. The score was high, (309,8 +/- 54,4 cm2/microM) however, when drug allergic patients' lymphocytes were challenged by the proper drug(s). There was neither false negativity in the positive group nor false positivity in the negative group of patients. Scores above 75 cm2/microM are considered as positive, those above 80 cm2/microM are undoubtedly positive. The relation of this rapid test to the lymphocyte transformation test as well as its advantages over the latter are discussed.     

Angiotensin II activates intermediate-conductance Ca2+ -activated K+ channels in arterial smooth muscle cells

Angiostensin II (Ang II) regulates the migration and proliferation of vascular smooth muscle cells. Recent studies indicate that intermediate-conductance Ca2+ -activated K+ (IKca) channels have an important role in cell migration and proliferation. It is not known, however, whether the action of Ang II is linked to IKca channel regulation. Here, we investigated the modulation of IKca channels by Ang II in artery smooth muscle cells. Functional IKca channel expression in cultured embryonic rat aorta smooth muscle (A10) cells was studied using the patch-clamp technique. These cells predominantly express IKca channels. In contrast, large-conductance Ca2+ -activated K+ (BKca) currents were rarely observed in excised patches. Ang II increased the IKca current in a contration-dependent manner. Losartan (1.0 microM), an AT1 selective antagonist, abolished the activation of IKca channels by Ang II. Pretreatment with 100 microM myristoylated protein kinase C inhibitor peptide 20-28 or 10 microM GF109203X completely abolished the AngII-induced activation of IKca currents, whereas the action of Ang II was not prevented in the presence of 100 microM Rp-cyclic 3', 5'-hydrogen phosphotiate adenosine triethylammonium, a protein kinase A inhibitor, or 1.0 microM KT-5823, a protein kinase G inhibitor. A membrane permeant analogue of diacylglycerol 1, 2-dioctanoyl-sn-glycerol (10 microM) induced the activation of IKca currents. These data suggest that Ang II activates IKca channels through the activation of protein kinase C, and the AT1 receptor is involved in the regulation of these channels.     

Activation of single-channel currents in mouse fibroblasts by platelet-derived growth factor.

A nonselective cation channel that we characterized in the mouse L-cell membrane becomes quiescent with serum deprivation (arrested cell growth) and rapidly active upon readdition of serum or, specifically, platelet-derived growth factor (PDGF). Using the patch-clamp technique, we find that the predominant channel in the LMTK- cell line is a bursting nonselective cation channel (the NS channel). In cell-attached and inside-out patches, the channel has a conductance of 28 pS; equal selectivity for Na+, K+, and Cs+; and no anion or divalent cation permeability. The channel open probability is voltage insensitive and in inside-out patches does not correlate with intracellular calcium (0.5 nM to 50 microM). When cultures are rendered quiescent by incubation in serum-free medium, channel open probability is virtually 0 as compared to 0.26 (+/- 0.17) in exponentially growing cultures. If mitogenesis is initiated by readdition of serum to quiescent cells while maintaining cell-attached recording, there is a rapid (15-30 s) activation of the channel (n = 12). The open probability of the patch increases (greater than 0.75) for 2-3 min and then decreases. We have attempted applications of several growth factors (fibroblast-derived growth factor, epidermal growth factor, insulin, bombesin, alpha-thrombin, and vasopressin, individually or in combination) but find that only PDGF (5-100 ng/ml; n = 9) produces channel activation. This activation should provide a Na+ entry pathway parallel to that of the Na/H exchanger.     

Pharmacological preconditioning in rabbit myocardium is blocked by chloride channel inhibition

OBJECTIVES: We have recently proposed that chloride (Cl(-)) channels contribute to ischemic preconditioning (IPC) in the myocardium. To further evaluate this hypothesis, we investigated the role of Cl(-) channels in pharmacological preconditioning. METHODS: Isolated rabbit cardiomyocytes and isolated buffer-perfused rabbit hearts were initially preconditioned with a 10 min exposure to either an adenosine receptor agonist [2-chloro-N(6)-cyclopentyladenosine (CCPA, 200 nM) and/or N(6)-2-(4-aminophenyl)ethyladenosine (APNEA, 1 microM)] or the PKC activator phorbol 12-myristate 13-acetate (PMA, 1 microM) followed by a 10 or 20 min washout or not preconditioned (control). Cardiomyocytes or whole hearts were then subjected to prolonged ischemic period (45 min simulated ischemia or 40 min of regional myocardial ischemia, respectively) followed by 60 min reperfusion (resuspension in oxygenated medium or release of the transient coronary occlusion, respectively). RESULTS: Indanyloxyacetic acid 94, a selective Cl(-) channel inhibitor that produced substantial inhibition of the regulatory volume decrease (RVD) when given at 10 microM concentration in cultured cardiomyocytes, was administered before ischemia to block RVD through Cl(-) channel inhibition. CCPA, APNEA and PMA significantly (P<0.01) reduced the % of dead cardiomyocytes (by trypan blue staining) after 45 min SI/60 min SR, as compared to controls, while IAA-94 abolished this protection but did not affect PKCepsilon translocation by IPC. We confirmed that IAA-94 blocked IPC-, APNEA- and PMA-induced protection against infarction in the isolated heart model. CONCLUSIONS: These findings support our contention that Cl(-) channels are downstream effectors of IPC.     

Regulation of electrolyte transport across cultured endometrial epithelial cells by prolactin

The effect of prolactin (PRL) on ion transport across the porcine glandular endometrial epithelial cells was studied in primary cell culture using the short-circuit current technique. Addition of 1 microg/ml PRL either to the apical solution or to the basolateral solution produced a peak followed by a sustained increase in Isc, but with a lesser response when PRL was added apically. Basolateral addition of PRL increased the Isc in a concentration-dependent manner with a maximum effect at 1 microg/ml and an effective concentration value of 120 ng/ml. The PRL-stimulated Isc was significantly reduced by pretreatment with an apical addition of 5-nitro-2-(3-phenylpropylamino) benzoic acid (200 microM), diphenylamine-2-carboxylic acid (1 mM) or 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (200 microM), Cl(-) channel blockers, but not by amiloride (10 microM), a Na(+) channel blocker. In addition, pretreatment with bumetanide (200 microM), a Na(+)-K(+)-2Cl(-) cotransporter inhibitor, in the basolateral solution significantly reduced the PRL-stimulated Isc. Replacement of Cl(-) or in the bathing solutions also decreased the Isc response to PRL. Pretreatment of the monolayer with AG490 (50 microM), an inhibitor of JAK2 activity significantly inhibited the PRL-induced increase in Isc. Western blot analysis of the porcine endometrial epithelial cells revealed the presence of short isoform of PRL receptor (PRLR-S) that could be regulated by 17beta-estradiol. The results of this investigation showed that PRL acutely stimulated anion secretion across the porcine endometrial epithelial cells possibly through PRLR-S present in both apical and basolateral membranes. The PRL response appeared to be mediated by the JAK2-dependent pathway.     

Gating kinetics of the cyclic-GMP-activated channel of retinal rods: flash photolysis and voltage-jump studies.

The gating kinetics of the cGMP-activated cation channel of salamander retinal rods have been studied in excised membrane patches. Relaxations in patch current were observed after two kinds of perturbation: (i) fast jumps of cGMP concentration, generated by laser flash photolysis of a cGMP ester ("caged" cGMP), and (ii) membrane voltage jumps, which perturb activation of the channel by cGMP. In both methods the speed of activation increased with the final cGMP concentration. The results are explained by a simple kinetic model in which activation involves three sequential cGMP binding steps with bimolecular rate constants close to the diffusion-controlled limit; fully liganded channels undergo rapid open-closed transitions. Voltage perturbs activation by changing the rate constant for channel closing, which increases with hyperpolarization. Intramolecular transitions of the fully liganded channel limit the kinetics of activation at high cGMP concentrations (greater than 50 microM), whereas at physiological cGMP concentrations (less than 5 microM), the kinetics of activation are limited by the third cGMP binding step. The channel appears to be optimized for rapid responses to changes in cytoplasmic cGMP concentration.     

Design of a potent and selective inhibitor of the intermediate-conductance Ca2+-activated K+ channel, IKCa1: a potential immunosuppressant

The antimycotic clotrimazole, a potent inhibitor of the intermediate-conductance calcium-activated K(+) channel, IKCa1, is in clinical trials for the treatment of sickle cell disease and diarrhea and is effective in ameliorating the symptoms of rheumatoid arthritis. However, inhibition of cytochrome P450 enzymes by clotrimazole limits its therapeutic value. We have used a rational design strategy to develop a clotrimazole analog that selectively inhibits IKCa1 without blocking cytochrome P450 enzymes. A screen of 83 triarylmethanes revealed the pharmacophore for channel block to be different from that required for cytochrome P450 inhibition. The "IKCa1-pharmacophore" consists of a (2-halogenophenyl)diphenylmethane moiety substituted by an unsubstituted polar pi-electron-rich heterocycle (pyrazole or tetrazole) or a -CN group, whereas cytochrome P450 inhibition absolutely requires the imidazole ring. A series of pyrazoles, acetonitriles, and tetrazoles were synthesized and found to selectively block IKCa1. TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole) inhibits the cloned and the native IKCa1 channel in human T lymphocytes with a K(d) of 20-25 nM and is 200- to 1,500-fold selective over other ion channels. Using TRAM-34, we show that blocking IKCa1 in human lymphocytes, in the absence of P450-inhibition, results in suppression of mitogen-stimulated [(3)H]thymidine incorporation of preactivated lymphocytes with EC(50)-values of 100 nM-1 microM depending on the donor. Combinations of TRAM-34 and cyclosporin A are more effective in suppressing lymphocyte mitogenesis than either compound alone. Our studies suggest that TRAM-34 and related compounds may hold therapeutic promise as immunosuppressants.     

Single calcium channels in native sarcoplasmic reticulum membranes from skeletal muscle.

Electrical properties of native sarcoplasmic reticulum membranes from rabbit skeletal muscle were investigated using the patch-clamp technique. Bilayers were assembled at the tip of patch pipettes from monolayers formed at the air-water interface of sarcoplasmic reticulum membrane suspensions. The membranes were found to contain a spontaneously active cation channel of small conductance (5 pS in 200 mM CaCl2, symmetrical solutions) that was selective for Ca2+ and Ba2+. Between 50 and 200 mM CaCl2 (symmetrical) the increase in conductance as a function of [Ca2+] fit a hyperbola (K0.5, 83 mM, and gamma max, 7.9 pS) that extrapolated to a single-channel conductance of 0.5 pS at physiological Ca2+ levels. The channel opened in bursts followed by long silent periods of up to a minute. During a burst the channel fluctuated very rapidly with time constants in the millisecond range. The mean burst duration was voltage dependent, increasing from 1.8 s at a pipette voltage of +60 mV to 4.1 s at +80 mV. Over this range, burst frequency decreased with increasing voltage such that the fraction of time spent in the open state (fb) remained constant. Application of 1.6 mM caffeine resulted in activation of the channel that appeared as an increase in mean burst duration. In contrast, 50 microM dantrolene significantly decreased burst frequency, whereas 10 microM nitrendipine had no effect. The functional and pharmacological properties of this Ca2+ channel suggest that it may be important in mediating Ca2+ release from the sarcoplasmic reticulum during excitation-contraction coupling.     

Inhibition of T cell proliferation by selective block of Ca2+-activated K+ channels

T lymphocytes express a plethora of distinct ion channels that participate in the control of calcium homeostasis and signal transduction. Potassium channels play a critical role in the modulation of T cell calcium signaling, and the significance of the voltage-dependent K channel, Kv1.3, is well established. The recent cloning of the Ca(2+)-activated, intermediate-conductance K(+) channel (IK channel) has enabled a detailed investigation of the role of this highly Ca(2+)-sensitive K(+) channel in the calcium signaling and subsequent regulation of T cell proliferation. The role IK channels play in T cell activation and proliferation has been investigated by using various blockers of IK channels. The Ca(2+)-activated K(+) current in human T cells is shown by the whole-cell voltage-clamp technique to be highly sensitive to clotrimazole, charybdotoxin, and nitrendipine, but not to ketoconazole. Clotrimazole, nitrendipine, and charybdotoxin block T cell activation induced by signals that elicit a rise in intracellular Ca(2+)-e.g., phytohemagglutinin, Con A, and antigens such as Candida albicans and tetanus toxin in a dose-dependent manner. The release of IFN-gamma from activated T cells is also inhibited after block of IK channels by clotrimazole. Clotrimazole and cyclosporin A act synergistically to inhibit T cell proliferation, which confirms that block of IK channels affects the process downstream from T cell receptor activation. We suggest that IK channels constitute another target for immune suppression.     

ATP-activated P2X2 current in mouse spermatozoa

Sperm cells acquire hyperactivated motility as they ascend the female reproductive tract, which enables them to overcome barriers and penetrate the cumulus and zona pellucida surrounding the egg. This enhanced motility requires Ca(2+) entry via cation channel of sperm (CatSper) Ca(2+)-selective ion channels in the sperm tail. Ca(2+) entry via CatSper is enhanced by the membrane hyperpolarization mediated by Slo3, a K(+) channel also present in the sperm tail. To date, no transmitter-mediated currents have been reported in sperm and no currents have been detected in the head or midpiece of mature spermatozoa. We screened a number of neurotransmitters and biomolecules to examine their ability to induce ion channel currents in the whole spermatozoa. Surprisingly, we find that none of the previously reported neurotransmitter receptors detected by antibodies alone are functional in mouse spermatozoa. Instead, we find that mouse spermatozoa have a cation-nonselective current in the midpiece of spermatozoa that is activated by external ATP, consistent with an ATP-mediated increase in intracellular Ca(2+) as previously reported. The ATP-dependent current is not detected in mice lacking the P2X2 receptor gene (P2rx2(-/-)). Furthermore, the slowly desensitizing and strongly outwardly rectifying ATP-gated current has the biophysical and pharmacological properties that mimic heterologously expressed mouse P2X2. We conclude that the ATP-induced current on mouse spermatozoa is mediated by the P2X2 purinergic receptor/channel. Despite the loss of ATP-gated current, P2rx2(-/-) spermatozoa have normal progressive motility, hyperactivated motility, and acrosome reactions. However, fertility of P2rx2(-/-) males declines with frequent mating over days, suggesting that P2X2 receptor adds a selection advantage under these conditions.     

Inhibition of phenylephrine induced hypertrophy in rat neonatal cardiomyocytes by the mitochondrial KATP channel opener diazoxide

The effect of the putative mitochondrial K(ATP) channel opener diazoxide (100 microM) was studied in terms of its ability to modulate the hypertrophic effect of 24 h treatment with the alpha(1) adrenoceptor agonist phenylephrine (PE; 10 microM) in cultured neonatal rat ventricular myocytes. PE on its own significantly increased cell size by 40%, (3)H leucine incorporation by 37% and produced more than a threefold elevation in both atrial natriuretic peptide and myosin light chain-2 expression. These effects were nearly completely prevented by diazoxide although the inhibitory effect of this agent was generally mitigated by the mitochondrial K(ATP) channel antagonists 5-hydroxydecanoic acid (100 microM) and glibenclamide (50 microM). Although PE produced an early threefold elevation in MAP kinase activation this was generally unaffected by diazoxide. PE also produced a greater than threefold increase in Na-H exchanger isoform 1 (NHE-1) expression which, was prevented by diazoxide treatment. Our study therefore, demonstrates a potential antihypertrophic influence of mitochondrial K(ATP) channel activation which, is related to diminished NHE-1 expression. Mitochondrial K(ATP) channel activation could represent an effective approach to minimize the myocardial hypertrophic process.     

Guanine nucleotide modulation of high affinity gonadotropin-releasing hormone receptors in rat mammary tumors.

We previously suggested that gonadotropin-releasing-hormone (GnRH) analogues activate the phosphoinositide pathway in rat mammary tumor membranes. In the present study we analyzed the binding of GnRH analogues to these membranes and assessed its modulation by guanine nucleotides. [125I]Buserelin (a GnRH superagonist) binding is specific because it is displaced only by GnRH analogues. Scatchard plot analysis reveals high affinity binding sites (Kd = 2.5 +/- 0.8 nM, Bmax = 250 +/- 120 fmol/mg membrane protein) and low affinity binding sites (Kd 1.1 +/- 0.3 microM, Bmax = 200 +/- 105 pmol/mg membrane protein). Guanine nucleotides increased the ED50 of [125I]buserelin displacement, and almost completely eliminated the high affinity binding. Similar results were obtained with [125I]D-Trp6-GnRH--another GnRH superagonist. The inhibition of buserelin binding by guanine nucleotides was specific for nucleotides that interact with G-binding proteins and was dose-dependent with a maximal effect at 10 microM GTP gamma S. Kinetic analysis of buserelin binding revealed that the dissociation rate increased at least 4-fold in the presence of 10 microM GTP gamma S. These results support the hypothesis that GnRH analogues interact directly with mammary tumors and activate a G-protein-dependent transducing mechanism.     

A role of reactive oxygen species in apoptotic activation of volume-sensitive Cl(-) channel

Apoptotic volume decrease is a pivotal event triggering a cell to undergo apoptosis and is induced by ionic effluxes resulting mainly from increased K(+) and Cl(-) conductances. Here, we demonstrate that in human epithelia HeLa cells both mitochondrion- and death receptor-mediated apoptosis inducers [staurosporine and Fas ligand or tumor necrosis factor (TNF)-alpha] rapidly activate Cl(-) currents that show properties phenotypical of volume-sensitive outwardly rectifying Cl(-) channel currents, including outward rectification, voltage-dependent inactivation gating at large positive potentials, inhibition by osmotic shrinkage, sensitivity to classic Cl(-) channel blockers, and dependence on cytosolic ATP. Staurosporine, but not Fas ligand or TNF-alpha, rapidly (within 30 min) increased the intracellular level of reactive oxygen species (ROS). A ROS scavenger and an NAD(P)H oxidase inhibitor blocked the current activation by staurosporine but not by Fas ligand or TNF-alpha. A ROS scavenger also inhibited apoptotic volume decrease, caspase-3 activation, and apoptotic cell death induced by staurosporine. Thus, it is concluded that an apoptosis-triggering anion conductance is carried by the volume-sensitive outwardly rectifying Cl(-) channel and that the channel activation on apoptotic stimulation with staurosporine, but not with Fas ligand or TNF-alpha, is mediated by ROS.     

Stoichiometric requirements for trapping and gating of Ca2+ release-activated Ca2+ (CRAC) channels by stromal interaction molecule 1 (STIM1)

Store-operated Ca(2+) entry depends critically on physical interactions of the endoplasmic reticulum (ER) Ca(2+) sensor stromal interaction molecule 1 (STIM1) and the Ca(2+) release-activated Ca(2+) (CRAC) channel protein Orai1. Recent studies support a diffusion-trap mechanism in which ER Ca(2+) depletion causes STIM1 to accumulate at ER-plasma membrane (PM) junctions, where it binds to Orai1, trapping and activating mobile CRAC channels in the overlying PM. To determine the stoichiometric requirements for CRAC channel trapping and activation, we expressed mCherry-STIM1 and Orai1-GFP at varying ratios in HEK cells and quantified CRAC current (I(CRAC)) activation and the STIM1:Orai1 ratio at ER-PM junctions after store depletion. By competing for a limited amount of STIM1, high levels of Orai1 reduced the junctional STIM1:Orai1 ratio to a lower limit of 0.3-0.6, indicating that binding of one to two STIM1s is sufficient to immobilize the tetrameric CRAC channel at ER-PM junctions. In cells expressing a constant amount of STIM1, CRAC current was a highly nonlinear bell-shaped function of Orai1 expression and the minimum stoichiometry for channel trapping failed to evoke significant activation. Peak current occurred at a ratio of ～2 STIM1:Orai1, suggesting that maximal CRAC channel activity requires binding of eight STIM1s to each channel. Further increases in Orai1 caused channel activity and fast Ca(2+)-dependent inactivation to decline in parallel. The data are well described by a model in which STIM1 binds to Orai1 with negative cooperativity and channels open with positive cooperativity as a result of stabilization of the open state by STIM1.     

Pharmacological action of 5'-methylthioadenosine on isolated rabbit aorta strips

The effects of 5'-methylthioadenosine (MTA) and adenosine analogues on isolated rabbit thoracic aorta strips were studied in vitro. High concentrations (500-1,000 microM) of adenosine analogues produced dose-dependent relaxation in isolated rabbit thoracic aorta strips. The relative potencies of relaxant effect were MTA greater than N6-phenylisopropyladenosine greater than 2-chloroadenosine on a molar basis. MTA (50-1,000 microM) suppressed the contraction induced by norepinephrine in isolated rabbit thoracic aorta strips in a concentration-dependent manner. Nucleoside uptake inhibitor dipyridamole did not impair the MTA actions. Pretreatment of the aorta strips with theophylline, an adenosine receptor antagonist, blocked the actions of MTA. MTA showed a relaxant effect in KCl-contracted aorta suggesting that MTA did not affect the metabolism or reuptake of norepinephrine. The present experiments suggest that MTA has a pharmacological action on the arterial smooth muscle cells mediated through adenosine receptors.