ROLE OF EXTRACELLULAR Na+, Ca2+-ACTIVATED Cl- CHANNELS AND BK CHANNELS IN THE CONTRACTION OF Ca2+ STORE-DEPLETED TRACHEAL SMOOTH MUSCLE

• 1 In the present study, we investigated the series of events involved in the contraction of tracheal smooth muscle induced by the re‐addition of Ca²⁺ in an in vitro experimental model in which Ca²⁺ stores had been depleted and their refilling had been blocked by thapsigargin.
• 2 Mean (±SEM) contraction was diminished by: (i) inhibitors of store‐operated calcium channels (SOCC), namely 100  µ mol/L SKF‐96365 and 100  µ mol/L 1‐(2‐trifluoromethylphenyl) imidazole (to 66.3 ± 4.4 and 41.3 ± 5.2% of control, respectively); (ii) inhibitors of voltage‐gated Ca²⁺ channels Ca_V1.2 channels, namely 1  µ mol/L nifedipine and 10  µ mol/L verapamil (to 86.2 ± 3.4 and 76.9 ± 5.9% of control, respectively); and (iii) 20  µ mol/L niflumic acid, a non‐selective inhibitor of Ca²⁺‐dependent Cl^? channels (to 41.1 ± 9.8% of control). In contrast, contraction was increased 2.3‐fold by 100 nmol/L iberiotoxin, a blocker of the large‐conductance Ca²⁺‐activated K⁺ (BK) channels.
• 3 Furthermore, contraction was significantly inhibited when Na⁺ in the bathing solution was replaced by N‐methyl–d ‐glucamine (NMDG⁺) to 39.9 ± 7.2% of control, but not when it was replaced by Li⁺ (114.5 ± 24.4% of control). In addition, when Na⁺ had been replaced by NMDG⁺, contractions were further inhibited by both nifedipine and niflumic acid (to 3.0 ± 1.8 and 24.4 ± 8.1% of control, respectively). Nifedipine also reduced contractions when Na⁺ had been replaced by Li⁺ (to 10.7 ± 3.4% to control), the niflumic acid had no effect (116.0 ± 4.5% of control).
• 4 In conclusion, the data of the present study demonstrate the roles of SOCC, BK channels and Ca_V1.2 channels in the contractions induced by the re‐addition of Ca²⁺ to the solution bathing guinea‐pig tracheal rings under conditions of Ca²⁺‐depleted sacroplasmic reticulum and inhibition of sarcoplasmic/endoplasmic reticulum calcium ATPase. The contractions were highly dependent on extracellular Na⁺, suggesting a role for SOCC in mediating the Na⁺ influx.

     

DYSREGULATION OF Ca2+ MOVEMENT IN PLATELETS FROM PATIENTS WITH ACUTE ISCHAEMIC STROKE

• 1 Platelets play a pivotal role during acute ischaemic stroke. An increase in cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) triggers intracellular signal transduction, leading to platelet aggregation and thrombosis. In the present study, we examined the differences between platelets from acute ischaemic stroke patients and at‐risk controls in terms of the increase in platelet [Ca²⁺]_i.
• 2 Thirty‐one patients with acute ischaemic stroke and 27 at‐risk controls were enrolled in the present study. Platelet [Ca²⁺]_i was measured using the fluorescent dye fura‐2 after stimulation with 100 µmol/L arachidonic acid (AA), 10 µmol/L ADP, 1 µmol/L platelet‐activation factor (PAF) and 0.1 U/mL thrombin.
• 3 Basal [Ca²⁺]_i was higher in the stroke group compared with at‐risk controls, irrespective of the presence or absence of extracellular Ca²⁺. In Ca²⁺‐containing medium, both PAF and ADP, but not AA and thrombin, significantly increased platelet [Ca²⁺]_i in the stroke group compared with the at‐risk controls. However, in Ca²⁺‐free medium, only PAF significantly increased platelet [Ca²⁺]_i in the stroke group compared with the at‐risk controls. Basal [Ca²⁺]_i and PAF‐induced platelet [Ca²⁺]_i increases were still higher in the stroke group at the subacute stage than in the at‐risk controls.
• 4 The results of the present study provide direct evidence that Ca²⁺ signalling in platelets from acute ischaemic stroke patients was altered in response to particular stimuli. The dysregulation of Ca²⁺ movement in platelets may persist up to the subacute stage of ischaemic stroke.

     

INVOLVEMENT OF PROTEIN KINASE C IN THE REGULATION OF Na+/Ca2+ EXCHANGER IN BOVINE ADRENAL CHROMAFFIN CELLS

• 1 The Na⁺/Ca²⁺ exchanger (NCX) exchanges Na+ and Ca²⁺ bidirectionally through the forward mode (Ca²⁺ extrusion) or the reverse mode (Ca²⁺ influx). The present study was undertaken to clarify the role of protein kinase C (PKC) in the regulation of NCX in bovine adrenal chromaffin cells. The Na⁺‐loaded cells were prepared by treatment with 100 µmol/L ouabain and 50 µmol/L veratridine. Incubation of Na⁺‐loaded cells with Na⁺‐free solution in the presence of the Ca²⁺ channel blockers nicardipine (3 µmol/L) and ω‐conotoxin MVIIC (0.3 µmol/L) caused Ca²⁺ uptake and catecholamine release.
• 2 The Na⁺‐dependent Ca²⁺ uptake and catecholamine release were inhibited by 2‐[4‐[(2,5‐difluorophenyl)methoxy]phenoxy]‐5‐ethoxyaniline (SEA0400; 1 µmol/L) and 2‐[2‐[4‐(4‐nitrobenzyloxy)phenyl]isothiourea (KB‐R7943; 10 µmol/L), both NCX inhibitors. These results indicate that the Na⁺‐dependent responses are mostly due to activation of the NCX working in the reverse mode.
• 3 In addition, we examined the effects of PKC inhibitors and an activator on the NCX‐mediated Ca²⁺ uptake and catecholamine release. Bisindolylmaleimide I (0.3–10 µmol/L) and chelerythrine (3–100 µmol/L), both PKC inhibitors, inhibited NCX‐mediated responses. In contrast, phorbol 12,13‐dibutyrate (0.1–10 µmol/L), a PKC activator, enhanced the responses. Bisindolylmaleimide I and chelerythrine, at effective concentrations for inhibition of Na⁺‐dependent catecholamine release, had a little or no effect on high K⁺‐induced catecholamine release in intact cells or on Ca²⁺‐induced catecholamine release in β‐escin‐permeabilized cells.
• 4 These results suggest that PKC is involved in the activation of NCX in bovine adrenal chromaffin cells.

     

ROLE OF CYCLIC NUCLEOTIDES IN THE CONTROL OF CYTOSOLIC Ca2+ LEVELS IN VASCULAR ENDOTHELIAL CELLS

• 1 Endothelial cells have a key role in the cardiovascular system. Most endothelial cell functions depend on changes in cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) to some extent and Ca²⁺ signalling acts to link external stimuli with the synthesis and release of regulatory factors in endothelial cells. The [Ca²⁺]_i is maintained by a well‐balanced Ca²⁺ flux across the endoplasmic reticulum and plasma membrane.
• 2 Cyclic nucleotides, such as cAMP and cGMP, are very important second messengers. The cyclic nucleotides can affect [Ca²⁺]_i directly or indirectly (via the actions of protein kinase (PK) A or PKG‐mediated phosphorylation) by regulating Ca²⁺ mobilization and Ca²⁺ influx. Fine‐tuning of [Ca²⁺]_i is also fundamental to protect endothelial cells against damaged caused by the excessive accumulation of Ca²⁺.
• 3 Therapeutic agents that control cAMP and cGMP levels have been used to treat various cardiovascular diseases.
• 4 The aim of the present review is to discuss: (i) the functions of endothelial cells; (ii) the importance of [Ca²⁺]_i in endothelial cells; (iii) the impact of excessive [Ca²⁺]_i in endothelial cells; and (iv) the balanced control of [Ca²⁺]_i in endothelial cells via involvement of cyclic nucleotides (cAMP and cGMP) and their general effectors.

     

Pharmacological changes in cellular Ca2+ homeostasis parallel initiation of atrial arrhythmogenesis in murine langendorff-perfused hearts

• 1 Intracellular Ca²⁺ overload has been associated with established atrial arrhythmogenesis. The present experiments went on to correlate acute initiation of atrial arrhythmogenesis in Langendorff‐perfused mouse hearts with changes in Ca²⁺ homeostasis in isolated atrial myocytes following pharmacological procedures that modified the storage or release of sarcoplasmic reticular (SR) Ca²⁺ or inhibited entry of extracellular Ca²⁺.
• 2 Caffeine (1 mmol/L) elicited diastolic Ca²⁺ waves in regularly stimulated atrial myocytes immediately following addition. This was followed by a decline in the amplitude of the evoked transients and the disappearance of such diastolic events, suggesting partial SR Ca²⁺ depletion.
• 3 Cyclopiazonic acid (CPA; 0.15 µmol/L) produced more gradual reductions in evoked Ca²⁺ transients and abolished diastolic Ca²⁺ events produced by the further addition of caffeine.
• 4 Nifedipine (0.5 µmol/L) produced immediate reductions in evoked Ca²⁺ transients. Further addition of caffeine produced an immediate increase followed by a decline in the amplitude of the evoked Ca²⁺ transients, without eliciting diastolic Ca²⁺ events.
• 5 These findings correlated with changes in spontaneous and provoked atrial arrhythmogenecity in mouse isolated Langendorf‐perfused hearts. Thus, caffeine was pro‐arrhythmogenic immediately following but not > 5 min after application and both CPA and nifedipine pretreatment inhibited such arrhythmogenesis.
• 6 Together, these findings relate acute atrial arrhythmogenesis in intact hearts to diastolic Ca²⁺ events in atrial myocytes that, in turn, depend upon a finite SR Ca²⁺ store and diastolic Ca²⁺ release following Ca²⁺‐induced Ca²⁺ release initiated by the entry of extracellular Ca²⁺.

     

INTERACTIONS OF Na+, H2O2 AND THE Na+-Ca2+ EXCHANGER STIMULATE Ca2+ RELEASE IN CK1.4 CELLS

1. The present study aimed to demonstrate that interactions of cations, hydrogen peroxide (H₂O₂) and the Na⁺-Ca²⁺exchanger stimulate Ca²⁺ release and oscillations of cytosolic Ca²⁺ [Ca²⁺]i in non-transfected Chinese Hamster Ovary (CHO) C1 cells and in transfected CHO (CK1.4) cells that contained an expression vector coding the Na⁺-Ca²⁺ exchanger sequence. 2. The [⁴⁵Ca²⁺] uptake assay, fura-2 fluorescence imaging and 2² and 2³ factorial orthogonal statistics provide comparative, direct, efficient, quantitative and transient methods to delineate the effects of such interactions on Ca²⁺ influx, Ca²⁺release and [Ca²⁺]i in C1 and CK1.4 cells. 3. In contrast to the control of either Na⁺-, Ca²⁺- or H₂O₂-free or CI cells, an elevated [⁴⁵Ca²⁺] uptake was induced by Ca²⁺, Na⁺ and H₂O₂ individually and in combination, intracellular Ca²⁺ release was activated by H₂O₂ and by combinations of either H₂O₂ and Na⁺, H₂O₂ and the Na⁺-Ca²⁺ exchanger, Na⁺ and the Na⁺-Ca²⁺ exchanger or by H₂O₂, Na⁺ and the Na⁺-Ca²⁺ exchanger and a rise in [Ca²⁺]i was triggered by H₂O₂, Na⁺ and a combination of Na⁺ and the Na⁺-Ca²⁺exchanger. 4. These results indicate that interactions between H₂O₂, Na⁺ and the Na⁺-Ca²⁺ exchanger stimulate intracellular Ca²⁺mobilization via Ca²⁺-induced Ca²⁺ release mechanisms, ATP-activated G-protein coupled P_2y-purinoceptor-sensitive pathways, Na⁺-Ca²⁺ exchanger-mediated Ca²⁺ influx and cation-π interaction (a strong non-covalent force between the cation and the π face of an aromatic structure in the transmembrane protein). 5. The present findings provide important clues for understanding Ca²⁺ signal transduction mechanisms from the plasma membrane to the endoplasmic reticulum.     

Inhibition of store-operated Ca(2+) channels prevent ethanol-induced intracellular Ca(2+) increase and cell injury in a human hepatoma cell line

Elevated intracellular Ca²⁺ content is implicated in ethanol-induced hepatocyte apoptosis and necrosis. Extracellular Ca²⁺ influx has been suggested to play a role in this process. However, the exact Ca²⁺-permeable channel involved in the plasma membrane is still unclear. This study investigated the role of store-operated calcium entry (SOCE) in ethanol-induced cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) increase and hepatotoxicity. Ethanol (25-800 mM) dose-dependently increased [Ca²⁺]_i content and hepatocyte damage in HepG2 cells. 2-aminoethoxydiphenyl borate (2-APB), the proved efficient antagonist of SOCs, dose-dependently suppressed the ethanol (200 nM)-increased [Ca²⁺]_i content and protected against ethanol-induced viability loss and transaminase leakage. Exposure to 200 mM ethanol for 24 h significantly upregulated the mRNA and protein expression of calcium release-activated calcium channel protein 1 (CRACM1, Orai1) and stromal interaction molecule 1 (STIM1), the two main molecular constituents of SOCs, which was sustained for at least 72 h. In addition, small interfering RNA knockdown of STIM1 attenuated the ethanol-increased [Ca²⁺]_i content and hepatotoxicity. Taken together, these data indicate that the Ca²⁺ channel of SOCE may be involved in the pathogenesis of ethanol-induced intracellular Ca²⁺ elevation and consequent hepatocyte damage.     

Discovery and structural optimization of 1-phenyl-3-(1-phenylethyl)urea derivatives as novel inhibitors of CRAC channel

Aim:

Ca²⁺-release-activated Ca²⁺ (CRAC) channel, a subfamily of store-operated channels, is formed by calcium release-activated calcium modulator 1 (ORAI1), and gated by stromal interaction molecule 1 (STIM1). CRAC channel may be a novel target for the treatment of immune disorders and allergy. The aim of this study was to identify novel small molecule CRAC channel inhibitors.

Methods:

HEK293 cells stably co-expressing both ORAI1 and STIM1 were used for high-throughput screening. A hit, 1-phenyl-3-(1-phenylethyl)urea, was identified that inhibited CRAC channels by targeting ORAI1. Five series of its derivatives were designed and synthesized, and their primary structure-activity relationships (SARs) were analyzed. All derivatives were assessed for their effects on Ca²⁺ influx through CRAC channels on HEK293 cells, cytotoxicity in Jurkat cells, and IL-2 production in Jurkat cells expressing ORAI1-SS-eGFP.

Results:

A total of 19 hits were discovered in libraries containing 32 000 compounds using the high-throughput screening. 1-Phenyl-3-(1-phenylethyl)urea inhibited Ca²⁺ influx with IC₅₀ of 3.25±0.17 μmol/L. SAR study on its derivatives showed that the alkyl substituent on the α-position of the left-side benzylic amine (R1) was essential for Ca²⁺ influx inhibition and that the S-configuration was better than the R-configuration. The derivatives in which the right-side R3 was substituted by an electron-donating group showed more potent inhibitory activity than those that were substituted by electron-withdrawing groups. Furthermore, the free N–H of urea was not necessary to maintain the high potency of Ca²⁺ influx inhibition. The N,N′-disubstituted or N′-substituted derivatives showed relatively low cytotoxicity but maintained the ability to inhibit IL-2 production. Among them, compound 5b showed an improved inhibition of IL-2 production and low cytotoxicity.

Conclusion:

1-Phenyl-3-(1-phenylethyl)urea is a novel CRAC channel inhibitor that specifically targets ORAI1. This study provides a new chemical scaffold for design and development of CRAC channel inhibitors with improved Ca²⁺ influx inhibition, immune inhibition and low cytotoxicity.     

G02 AND Gi3 PROTEINS MEDIATE THE ACTION OF SOMATOSTATIN ON MEMBRANE Ca2+ AND K+ CURRENTS IN OVINE PITUITARY SOMATOTROPHS

1. Growth hormone (GH) secretion from the anterior pituitary gland is mainly regulated by hypothalamic GH-releasing hormone (GHRH) and somatostatin (SRIF). Somatostatin reduces both spontaneous and GHRH-stimulated GH secretion. 2. Exocytosis of GH is mainly determined by the intracellular free Ca²⁺ concentration ([Ca²⁺]_i), which is regulated by the influx of Ca²⁺ via membrane Ca²⁺ channels. Somatostatin reduces the influx of Ca²⁺ through two separate mechanisms, namely a direct action on Ca²⁺ channels and an indirect action on membrane potentials through the activation of K⁺ channels. 3. In the present experiments, somatotroph-enriched cells were obtained from the ovine pituitary gland by means of collagenase dissociation and Percoll-gradient centrifugation. Further identification was based on the effect of SRIF (10 nmol/L) on Ca²⁺ or K⁺ currents. 4. A significant reduction in Ca²⁺ currents and an increase in K⁺ currents was obtained in response to local application of SRIF (10 nmol/L), but vehicle application had no effect. The responses of Ca²⁺ and K⁺ currents to SRIF were reversible after removal of SRIF. 5. Dialysis of GTP-λ-s (200 μmol/L) abolished the recovery phase of K⁺ current response to SRIF after its removal, whereas GDP-β-s (200 μmol/L) totally blocked the response. Pretreatment of the cells with pertussis toxin (100 nmol/L) overnight abolished the Ca²⁺ current response to SRIF. 6. Intracellular dialysis of antibodies to αo, α_{1_3}, ai_1-2 and ai₃summits of the G-proteins into cells via whole-cell patch-clamp pipettes was confirmed by immunofluorescent staining of the antibodies. 7. Dialysis of anti-ai_1-3 or anti-@aLi₃ antibodies significantly attenuated the increase in the K⁺ current in response to 10 nmol/L SRIF, whereas neither anti-αo nor anti-αi_2 antibodies diminished the effect of SRIF on the K⁺ current. 8. Dialysis of anti-αo antibodies significantly attenuated the reduction in the Ca²⁺ current that was obtained upon application of 10 nmol/L SRIF. Neither anti-αi-2 nor anti-αi3 antibody dialysis diminished the effect of SRIF on the Ca²⁺ current. 9. Dialysis of the ao common antisense oligonucleotides (ASm) but not the αi₃ AS significantly diminished the inhibitory effect of SRIF on the Ca²⁺ current. This effect of ao ASm dialysis occurred at 12 h incubation after dialysis, reaching a maximal level at 48 h and partially recovering at 72 h incubation. Antisense oligonucleotides specific for αo₁ (αo₁ AS) or αo₂(α0₂ AS) were dialysed into somatotrophs and only αo₂ AS significantly attenuated the inhibition of SRIF on the Ca²⁺ current. 10. It is concluded that the G_i3 protein mediates the effect of SRIF on the K⁺ current and that the G02 protein mediates the effect of SRIF on the Ca²⁺ current in primary cultured ovine somatotrophs.     

MECHANISMS FOR REGULATION OF ARTERIAL TONE BY Ca2+- DEPENDENT K+ CHANNELS IN HYPERTENSION

1. The membrane potential and reactivity of arterial smooth muscle cells is regulated by a variety of K⁺ channels, which are highly expressed in vascular smooth muscle meuscle membrances. 2. Of these K⁺ channel types, the high-conductance, Ca²⁺-ependent K⁺ channel appears to be up-regulated in arterial smooth muscle membrances from hypertensive animals. 3. Patch-clamp studies show that whole-cell membrances and membrane patches of arterial smooth muscle obtained from rats with genetic or renal hypertension show an increased macroscopic and single-channel Ca²⁺-activated K⁺ current. Pharmacological block of this K⁺ current profoundly constricts aortic, renal, mesenteric and femoral arteries obtained from the same hypertensive animals, suggesting that Ca²⁺-dependent K⁺ current is a critical determinant of resting membrane potential in arterial muscle exposed to elevated blood pressure. 4. Thus, K⁺ efflux through Ca²⁺-dependent K⁺ channels appears to constitute an important homeostatic mechanism for buffering increases in arterial reactivity in hypertension.     

Pharmacology of ORAI channels as a tool to understand their physiological functions

Store-operated Ca²⁺ entry is a major Ca²⁺ entry mechanism that is present in most cell types. In immune cells, store-operated Ca²⁺ entry is almost exclusively mediated by Ca²⁺ release-activated Ca²⁺ (CRAC) channels. Ca²⁺ entry through these channels and the corresponding cytosolic Ca²⁺ signals are required for many immune cell functions, including all aspects of T-cell activation. ORAI proteins are the molecular correlates for the CRAC channels. The three human members, ORAI1, ORAI2 and ORAI3, are activated through the stromal interaction molecules (STIM)1 and 2 following depletion of endoplasmic reticulum Ca²⁺ stores. Different combinations of STIM and ORAI can form different CRAC channels with distinct biophysical properties. In this article, we review and discuss mechanistic and functional implications of two important CRAC/ORAI inhibitors, 2-APB and BTP2, and the antibiotic G418 that has also been reported to interfere with ORAI channel function. The use of pharmacological tools should help to assign distinct physiological and pathophysiological functions to different STIM–ORAI protein complexes.     

RELATIVE CONTRIBUTIONS OF EXTRACELLULAR Ca2+ AND Ca2+ STORES TO SMOOTH MUSCLE CONTRACTION IN ARTERIES AND ARTERIOLES OF RAT, GUINEA-PIG DOG AND RABBIT

1. These studies describe the functional effects of modulation of the sarcoplasmic reticulum (SR) Ca²⁺ stores at three levels of the vasculature: (i) large arteries (rat and guinea-pig aorta); (ii) small resistance arteries (rat tail artery, rabbit mesenteric artery, dog mesenteric artery); and (iii) arterioles (guinea-pig submucosal arterioles of the small intestine). 2. All tissues responded to phenylephrine (PE; 10 μmol/L) with a transient contraction in Ca²⁺-free Krebs', reflecting Ca²⁺ release from PE-sensitive Ca²⁺ stores. After pretreatment with cyclopiazonic acid (CPA; 30 μmol/L) or thapsigargin (TSG; 1 μmol/L), putative SR Ca²⁺ pump inhibitors, the PE-induced contraction in a Ca²⁺-free medium was significantly inhibited in arterial tissues at all levels of the vasculature. Similarly, ryanodine (RYA; 30 μmol/L), an agonist that enhances Ca²⁺ release from the SR, also reduced the PE contraction in a Ca²⁺-free solution. 3. CPA or TSG alone in the presence of extracellular Ca²⁺, caused marked and sustained contraction in the rat and guinea-pig aorta and marked but transient or no contraction in the resistance arteries. In the rat and guinea-pig aorta, RYA caused a slowly developing tension. Little increase in basal tension was produced by RYA in resistance arteries and arterioles. 4. The findings show that an agonist-releasable Ca²⁺ pool is present at all levels of the vasculature that is independent of the size of the vessels and suggest that under normal physiological conditions there is an intimate balance between the roles of the plasma membrane and of the SR in the maintenance of vascular contractility. It appears that the role of the SR diminishes as the arteries become smaller, while Ca²⁺ fluxes across the plasma membrane predominates.     

FORCE AND INTRACELLULAR Ca2+ DURING NANC-MEDIATED RELAXATION OF RAT ANOCOCCYGEUS MUSCLE AND THE EFFECTS OF CYCLOPIAZONIC ACID

1. Simultaneous recordings of tension and [Ca²⁺]_i during NANC-mediated relaxation were made in the rat anococcygeus muscle under various conditions. 2. In muscles precontracted with guanethidine, nitrergic stimulations at 2 Hz produced a rapid decrease in both the tension and [Ca²⁺]_i. 3. The nitric oxide synthase inhibitor, N^G-nitro-L-Arginine (NOLA, 100 μmol/L) completely abolished the decreases in the [Ca²⁺]_i and force response of the NANC-mediated relaxation. 4. Noradrenergic-mediated contractions elicited by electrical field stimulation were potentiated by the addition of NOLA. In the absence of NOLA, the motor responses were larger in magnitude at 10 Hz stimulation than at 2 Hz. After NOLA, both the force response and the associated rise in [Ca²⁺]_i were substantially increased in comparison to the control stimulations. Proportionately the potentiation of the 2 Hz response was of a far greater magnitude than that of the 10 Hz response. 5. The guanylate cyclase inhibitor methylene blue (10 μmol/ L), partially inhibited the force and [Ca²⁺]_i response of the NANC relaxation. 6. Following exposure of the muscles to the sarcoplasmic reticulum Ca²⁺-ATPase inhibitor, cyclopiazonic acid, (10 μmol/ L) the responses to NANC stimulation were inhibited. The attenuated relaxation response displayed a bi-phasic timecourse and the Ca²⁺ change in comparison to that of the control was markedly smaller. In some cases, a relaxation was observed with no detectable change in the [Ca²⁺]_i. 7. The results suggest that part of the relaxation response observed with NANC-mediated relaxation in the rat anococcygeus is dependent on Ca²⁺ sequestration into the sarcoplasmic reticulum. However, other Ca²⁺ lowering mechanisms and possible Ca²⁺ independent mechanisms may also contribute to the NANC relaxation response.     

四肽FMRFa对大鼠心室肌Na+/Ca2+交换的抑制

目的　研究四肽FMRFa对大鼠单个心室肌细胞Na⁺/Ca²⁺交换的作用。方法　用膜片钳全细胞记录法测定成年大鼠心室肌细胞Na⁺/Ca²⁺交换电流(I_Na⁺/Ca²⁺)和其他离子通道电流。结果　FMRFa对大鼠心室肌细胞I_Na⁺/Ca²⁺呈浓度依赖性抑制,100μmol·L^-1浓度时抑制内向和外向I_Na⁺/Ca²⁺密度分别达60.1%和56.5%,对内向电流及外向电流的IC₅₀分别为20μmol·L^-1和34μmol·L^-1。FMRFa5μmol·L^-1抑制I_Na⁺/Ca²⁺内向和外向电流密度分别为38.7%和34.9%,但FMRFa5μmol·L^-1及20μmol·L^-1对L型钙电流、钠电流、瞬时外向电流和内向整流钾电流均无显著抑制作用。结论　FMRFa对大鼠心室肌细胞是一个特异性Na⁺/Ca²⁺交换抑制剂。     

Increase in force of contraction by activation of the Na+'ox/Ca2+'ox-exchangerin human myocardium

Aims The aim of the present study was to investigate whether agents which enhance force of contraction via increasing intracellular Na⁺, i.e. cAMP-independently, remain effective in failing human myocardium.Methods Cumulative concentration-response curves with (±)BDF 9148 (0.01–10 μmol l⁻¹ ), a Na⁺-channel activator, and ouabain (0.01–0.1 μmol l⁻¹ ), a Na⁺/K⁺-ATPase inhibitor, were performed on electrically driven left ventricular human papillary muscle strips (1 Hz, 37° C; dilative cardiomyopathy, NYHA IV, heart transplantation, n=16; nonfailing, donor hearts, n=5). The β-adrenoceptor agonist isoprenaline (0.001–1 μmol l⁻¹ ) and Ca²⁺ (1.8–15 mmol l⁻¹ ) were studied for control. In addition, Ca²⁺ response curves were obtained on skinned fibre preparations from left ventricular myocardium (NYHA IV, n=7) in the presence of BDF 9148 (1 μmol l⁻¹ ) or a high Na⁺ concentration (50 mmol l⁻¹ ) to investigate a possible direct or indirect interaction of (±)BDF 9148 with the myofilaments.Results While isoprenaline was significantly less effective in increasing force of contraction in failing human myocardium than in nonfailing myocardium (P<0.01), in NYHA IV, (±)BDF 9148 and ouabain were as effective as in nonfailing human tissue. In failing and nonfailing myocardium, (±)BDF 9148 and ouabain exerted positive inotropic effects similar to those of Ca²⁺. However, the potency for (±)BDF 9148 to increase force of contraction was higher in NYHA IV than in nonfailing human myocardium (P<0.05). Neither (±)BDF 9148 (1 μmol l⁻¹ ) nor an increased concentration of Na⁺ (50 mmol l⁻¹ ) altered the Ca²⁺ sensitivity or maximal developed tension of the contractile apparatus in experiments on chemically skinned left ventricular fibres.Conclusions The enhanced sensitivity of the failing human myocardium towards Na⁺-channel modulation is not due to a direct or indirect interaction of (±)BDF 9148 with cardiac myofilaments but may be due to an altered Na⁺-homeostasis in human heart failure.     

NEUROPROTECTION IN VITRO AND IN VIVO BY CELL MEMBRANE-PERMEANT Ca2+ CHELATORS

1. A method of attenuating excitotoxic and hypoxic/ ischaemic neurodegeneration in vitro and in vivo using cell-permeant Ca²⁺-chelating agents is described. 2. The mechanism of neuroprotection may depend on both pre- and post-synaptic effects on intracellular Ca²⁺ dynamics. 3. This method is unique, because it targets Ca²⁺ ions, the presumed triggers of neurodegeneration, rather than a specific cellular receptor.     

Role of the cytoskeleton in communication between L‐type Ca2+ channels and mitochondria

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EFFECTS OF INTRACELLULAR Ca2+ CHELATING ON NORADRENALINE RELEASE IN NORMOXIC AND ANOXIC HEARTS

1. Ischaemia and anoxia induce excessive noradrenaline (NA) release in the heart by a mechanism independent of both nerve activity and extracellular Ca²⁺. The present study was designed to examine the potential role of intracellular Ca²⁺ mobilization in anoxic NA release in the heart by chelating intracellular free Ca²⁺. 2. In normoxic hearts, preloading with an intracellular fre. Ca²⁺ chelator (BAPTA) reduced neuronal NA release by 65%, confirming the effectiveness of the loading protocol. Release of NA independent of nerve activity occurred in hearts subjected to a 40 min period of anoxic, substrate-free and nominal Ca²⁺-free perfusion. Loading hearts with BAPTA prior to anoxia failed to reduce NA overflow (1561 ± 147 vs 1496 ± 206 pmol/g over 40 min). Infusion with BAPTA (20 μmol/L) during the first 25 min of the anoxic period reduced the quantity of anoxic NA release by approximately 25% from 2013 ± 124 to 1476 ± 207 pmol/g (P < 0.05). 3. Our results confirm that anoxic NA release is predominantly. Ca²⁺-independent process with Ca²⁺ mobilization from endogenous storage playing only a minor contributing role.     

TRPC1 is involved in Ca influx and cytotoxicity following Pb exposure in human embryonic kidney cells

Lead (Pb²⁺) is a divalent heavy metal ion which causes severe damage to almost all life forms and is therefore considered a notorious toxicant. Exposure to Pb²⁺ is associated with poor cognitive development in children at relatively low levels that previously were thought to be safe. The mechanism through which Pb²⁺ enters cells, however, is unclear. Previous studies have showed that Ca²⁺ release-activated Ca²⁺ protein 1 (Orai1), a component of store-operated Ca²⁺ channels (SOCs), contributes to Pb²⁺ cellular entry. Canonical transient receptor potential (TRPC1) channel 1 is a transient receptor potential (TRP) channel which is sometimes referred to as a SOC. The present study was designed to investigate the role of TRPC1 in Pb²⁺ entry and toxicity in human embryonic kidney cells (HEK293). Additionally, changes in intracellular Ca²⁺ concentration were determined through Fluo-4 and Mag-fluo-4 fluorescent Ca²⁺ imaging. Following Pb²⁺ exposure, there was a dose-dependent decrease in cell viability. Overexpression of TRPC1 increased Pb²⁺-induced cell death, while knockdown of this channel attenuated cell death. There was increased entry of Pb²⁺, as measured by inductively coupled plasma mass spectrometry (ICP-MS), following overexpression of TRPC1. Conversely, knockdown of TRPC1 led to a decrease in Pb²⁺ influx. Down-regulation of STIM1 by RNA interference attenuated the Pb²⁺ influx, and transfection with a mutant STIM1, which could not gate TRPC1, had a similar effect. Co-transfection of mutant STIM1 and mutant TRPC1, which restore the electrostatic interaction between STIM1 and TRPC1, resumed Pb²⁺ entry in HEK293 cells. Down-regulation of TRPC1 by RNA interference decreased Ca²⁺ influx whilst its overexpression increased Ca²⁺ entry in HEK293 cells. These results suggest that TRPC1 is involved in the cytotoxicity and entry of Pb²⁺ through molecular interactions with STIM1 and subsequent Ca²⁺ influx in HEK293 cells.     

MOLECULAR AND FUNCTIONAL DIVERSITY OF K+ CHANNELS

1. Amino acid sequences encoding K⁺ channels belong to several subfamilies of the voltage-gated ion channel superfamily which includes Na²⁺-, Ca²⁺-, and cyclic nucleotide gated channels. The Kv family is the largest group, and encodes delayed rectifier, A-type, and large conductance Ca²⁺ activated K⁺ channels. 2. The α-subunits of Kv channels form as tetramers of four independent subunits. Each subunit has six membrane spanning regions and a pore forming ‘P’ region. Subunits belong to subfamilies (Kv1–4, BK, Eag) comprising multiple members, each of which has distinct properties resembling each of the major types of native Kv channel when expressed as homomultimers in heterologous systems. 3. Enormous diversity of Kv channel function arises from the multiplicity of subunits, the formation of heteromultimers within subfamilies and from association with intracellular β-subunit proteins. 4. In the absence of direct structural information, mutational analyses have provided considerable insights into the structure of the voltage-sensor, pore-forming region and the sites of action of drugs, toxins and associated proteins. 5. Another subfamily, the inwardly rectifying, or K_IR, family, appears to have arisen from a deletion of the first four membrane spanning regions of ancient Kv channels, changing gating properties from outward to inward rectification. These include the G-protein gated inward rectifiers and K_atp channels.