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 In addition, when Na⁺ had been replaced by NMDG⁺, contractions were further inhibited by both nifedipine and niflumic acid (to 3.0 ± 1.8 and 24.4 ± 8.1% of control, respectively). Nifedipine also reduced contractions when Na⁺ had been replaced by Li⁺ (to 10.7 ± 3.4% to control), the niflumic acid had no effect (116.0 ± 4.5% of control).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.

     

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.     

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.     

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.     

EFFECTS OF BKCa CHANNELS ON THE REDUCTION OF CYTOSOLIC Ca2+ IN cGMP-INDUCED RELAXATION OF GUINEAPIG TRACHEA

1. In order to examine the mechanisms of cGMP-induced relaxation in airway smooth muscle, the effects of atrial natriuretic peptide (ANP) and 8-brom cGMP on muscle tone were studied by measuring isometric tension, while the effects on cytosolic Ca²⁺ concentrations were studied by measuring the spectra of fura-2 loaded in guinea-pig tracheal strips. 2. Atrial natriuretic peptide and 8-brom cGMP caused a concentration-dependent inhibition of spontaneous tone in the guinea-pig trachea. The relaxant effects of these agents on spontaneous tone were markedly suppressed in the presence of iberiotoxin (IbTX), a selective inhibitor of large-conductance Ca²⁺-activated K⁺ (BKca) channels. Iberiotoxin (30 nmol/L) markedly affected the maximal effect induced by ANP and 8-brom cGMP and augmented EC₇₀ values for ANP and EC₅₀values for 8-brom cGMP approximately 27- and 17-fold, respectively. The inhibitory effects of IbTX on relaxation induced by these agents were diminished in the presence of 1 μmol/L nifedipine, an antagonist of voltage-operated Ca²⁺channels (VOCC). 3. The inhibitory action of ANP and 8-brom cGMP on spontaneous tone was not affected by the presence of 10 μmol/L glibenclamide, an inhibitor of ATP-sensitive K⁺ channels, and 100 nmol/L apamin, an inhibitor of small-conductance Ca²+-activated K⁺ channels. When these agents were applied to tissues precontracted by high (40mmol/L) K⁺, the relaxant effects of these agents markedly diminished. 4. The extracellular Ca²⁺-dependent contraction was inhibited in the presence of 0.3 μmoI/L ANP or 0.1 mmol/L 8-brom cGMP. Concentration—response curves to extracellular Ca²⁺ (0.03—2.4 mmol/L) were markedly diminished by exposure to these agents. The maximal effect induced by extracellular Ca²⁺ was affected by these agents. 5. Atrial natriuretic peptide caused an inhibition of spontaneous tone accompanied by a reduction in the intracellular Ca²⁺ concentration. In the presence of IbTX, the elimination of both muscle tone and cytosolic Ca²⁺ by ANP was suppressed. 6. We conclude that ANP and 8-brom cGMP activate BKca channels and that the inhibition of Ca²⁺ influx through VOCC, mediated by BKca channel activation, may be involved in cGMP-dependent bronchodilation.     

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 ENDOTHELIUM AND K+ CHANNELS IN DOBUTAMINE-INDUCED RELAXATION IN RAT MESENTERIC ARTERY

1. In order to examine the possible involvement of the endothelium and K⁺ channel activation in the relaxation induced by dobutamine, a β-adrenoceptor agonist, in rat isolated mesenteric arteries, the effects of inhibitors of nitric oxide (NO) activity, blockers of K⁺ channels and high extracellular K⁺ were studied by measuring isometric tension in both endothelium-intact and -denuded arteries. 2. Dobutamine inhibited the phenylephrine (PE)-induced sustained tension with a pEC₅₀ of 7.40±0.08 in endothelium-intact arteries. Removal of functional endothelium attenuated the effect of dobutamine. The relaxation induced by dobutamine was inhibited by the β₁-adrenoceptor antagonist CGP20712A (3μ.mol/L) but not by the β₂-adrenoceptor antagonist ICI 118 551 (3 μmol/L) in endothelium-denuded arteries. 3. Pretreatment with N^G-nitro-l -arginine (l -NNA; 100 μmol/L) or methylene blue (3 μmol/L) induced a similar degree of inhibition of the dobutamine-induced relaxation in endothelium-intact arteries, while N^G-nitro-d -arginine (100 (μmol/L) and indomethacin (10 μmol/L) had no effect. In contrast, pretreatment with L -NNA (100 μmol/L) did not affect the relaxation induced by sodium nitroprusside (SNP) or forskolin. Methylene blue (3μmol/L) inhibited the relaxant response to SNP. 4. Charybdotoxin (CTX; 100nmol/L), iberiotoxin (IBX; 100nmol/L) and tetraethylammonium ions (TEA⁺; 3mmol/L) significantly reduced the dobutamine-induced relaxation. Tetrapentylammonium ions (TPA⁺; 5μmol/L) markedly inhibited the relaxant effect of dobutamine. The pECso values for control and in the presence of TPA⁺ in endothelium-intact arteries were 7.35±0.11 and 6.14±0.17, respectively, and 6.35±0.09 and 5.87±0.17 for control and in the presence of TPA⁺ in endothelium-denuded arteries, respectively. In contrast, glibenclamide (3 μ-mol/L) was ineffective. At 5 μmol/L, TPA⁺ also inhibited the relaxation induced by forskolin. 5. The maximal relaxation of PE-contracted arteries induced by 3μmol/L dobutamine was completely abolished in the 60mmol/L K⁺-contracted arteries with and without endothelium, while dobutamine at a concentration greater than 3 μmol/L induced inhibition of the high-K⁺ response. 6. The present results indicate that endothelium, probably NO but not prostacyclin, was involved in the dobutamine-induced relaxation in rat mesenteric arteries. Activation of CTX-, IBX- and TPA⁺-sensitive K⁺ channels contributed towards the observed relaxation. Loss of the ability to relax the 60 mmol/L K⁺-contracted arteries suggests that endothelium-derived vasoactive factors affected by concentrations of dobutamine less than 3 μmol/L may also act through K⁺ channels in our preparations. Higher concentrations of dobutamine may have a direct, endothelium-independent relaxant effect.     

CHARACTERIZATION OF BINDING SITES FOR [3H]-IDAZOXAN, [3H]-P-AMINOCLONIDINE AND [3H]-RAUWOLSCINE IN THE KIDNEY OF THE DOG

1. We characterized the binding of [³H]-rauwolscine, [³H]-p-aminoclonidine and [³H]-idazoxan in a dog kidney membrane preparation. Our aim was to determine the pharmacological nature of the α₂-adrenoceptor- and imidazoline-preferring binding sites in this organ. 2. [³H]-Rauwolscine bound to an apparent single site with an affinity (K_D) of 2.2 nmol/ L and a maximum density (B_max) of 58.5 fmol/mg protein, when 10 μmol/L idazoxan defined non-specific binding. However displacement studies demonstrated that a number of compounds, including prazosin, inhibited [³H]-rauwolscine binding in a complex manner consistent with displacement from two distinct binding sites. The majority (69%) of the [³H]-rauwolscine binding sites had a relatively low affinity for prazosin (K_I= 398 nmol/L), while the remainder had a relatively high affinity for prazosin (K_I= 7.9 nmol/ L). 3. [³H]-p-Aminoclonidine bound to an apparent single site (K_D= 5.2 nmol/L; B_max= 72.4 fmol/mg protein), when 10 μmol/L phentolamine defined non-specific binding. When 1 μmol/L of the potent and selective α₂-adrenoceptor antagonist 2-methoxyidazoxan was included in the incubate, no specific binding was detected. We therefore conclude that under the conditions of this experiment [³H]-p-aminoclonidine binds only to α₂-adrenoceptors in the dog kidney. 4. [³H]-Idazoxan bound to two sites, with a higher (K_D= 0.95 nmol/L; B_max= 43.9 fmol/mg protein) and lower (K_D= 9.1 nmol/L; B_max= 93.8 fmol/mg protein) affinity, respectively, when 1 mmol/L phentolamine defined non-specific binding. When 10 μmol/ L GTPγS was included in the incubate, the low affinity site was unaffected but the maximum binding at the higher affinity site was reduced by 79%. 2-Methoxyidazoxan displaced [³H]-idazoxan in a monophasic manner and with low potency (IG₅₀=11.5 μmol/L). Yohimbine, efaroxan, clonidine, rilmenidine, guanabenz and idazoxan itself displaced [³H]-idazoxan in a complex manner; the slope of the displacement curves being less than unity. 5. We conclude that the dog kidney contains a heterogeneous population of α₂-adrenoceptors that can be labelled either with [³H]-rauwolscine or [³H]-p-aminoclonidine. The dog kidney also contains a heterogeneous population of non-adrenoceptor imidazoline-preferring binding sites of the I₂-subtype, that can be labelled with [³H]-idazoxan. The binding site for which [³H]-idazoxan has the highest affinity appears to be coupled to a guanine nucleotide binding regulatory protein.     

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。     

赛庚啶对离体犬心肌肌质网Ca2+,Mg2+—ATP酶活性和45Ca2+摄取功能的影响

当赛庚啶浓度在8×10^-6mol/L～2×10^-4mol/L之间时,该药对正常犬心肌肌质网Ca²⁺,Mg²⁺—ATP酶活性几乎没有影响,仅在10^-3mol/L时对该酶活性才有一定的抑制作用(抑制率为39.85%,P<0.01)。正常犬心肌肌质网的⁴⁵Ca²⁺摄取过程有明显的时间依赖性,至第30 min,其⁴⁵Ca²⁺摄取量可达312.79±22.25 nmol/mg protein.赛庚啶对心肌肌质网的~(45)Ca²⁺摄取有一定的抑制作用,其IC₅₀为1.94×10^-4mol/L。     

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.     

STORE-OPERATED Ca2+ CHANNELS AND MICRODOMAINS OF Ca2+ IN LIVER CELLS

• 1 Oscillatory increases in the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt) play essential roles in the hormonal regulation of liver cells. Increases in [Ca²⁺]_cyt require Ca²⁺ release from the endoplasmic reticulum (ER) and Ca²⁺ entry across the plasma membrane.
• 2 Store‐operated Ca²⁺ channels (SOCs), activated by a decrease in Ca²⁺ in the ER lumen, are responsible for maintaining adequate ER Ca²⁺. Experiments using patch‐clamp recording and the fluorescent Ca²⁺ reporter fura‐2 indicate there is only one type of SOC in rat liver cells. These SOCs have a high selectivity for Ca²⁺ and properties essentially indistinguishable from those of Ca²⁺ release‐activated Ca²⁺ (CRAC) channels.
• 3 Although Orai1, a CRAC channel pore protein, and stromal interaction molecule 1 (STIM1), a CRAC channel Ca²⁺ sensor, are components of liver cell SOCs, the mechanism of activation of SOCs, and in particular the role of subregions of the ER, are not well understood.
• 4 Recent experiments have used the transient receptor potential vanilloid 1 (TRPV1) non‐selective cation channel, ectopically expressed in liver cells, and a choleretic bile acid to deplete Ca²⁺ from different ER subregions. The results of these studies have provided evidence that only a small component of the ER is required for STIM1 redistribution and the activation of SOCs.
• 5 It is concluded that different Ca²⁺ microdomains in the ER and cytoplasmic space are important in both the activation of SOCs and in the signalling actions of Ca²⁺ in liver cells. Future experiments will investigate the nature of these microdomains further.

     

K+ release from rat parotid cells: An α1-adrenergic mediated event

To characterize α-adrenergic receptors in rat parotid gland tissue, 9,10-[9,10-³H(N)]-dihydro-α-ergocryptine ([³h]dhe) and [³H]prazosin binding to membranes and stimulated K⁺ release from parotid cell aggregates were examined. Prazosin (selective α₁-adrenergic antagonist), displacement of [³H]DHE binding from parotid membranes was biphasic, indicating the presence of both α₁- and α₂-adrenergic receptors. The numbers of α₁- and α₂-receptors were about equal. α₁-Adrenergic receptors were further studied by [³H]prazosin binding. [³H]Prazosin binding was a rapid, reversible, saturable and stereospecific process, with high affinity (K_D = 0.38 nM) and low capacity (B_max = 380 fmoles ligand bound/g tissue, 10.1 fmoles/mg protein) as determined by Scatchard analysis. The characteristics of [³H]prazosin binding were in good agreement with those of catecholamine-stimulated K⁺ release, suggesting that K⁺ release from rat parotid gland cells is an α₁-adrenergic mediated effect.     

PROTON AND POTASSIUM TRANSPORT BY H+/K+-ATPases

1. H⁺/K⁺-ATPases are members of the P-type ATPase multigene family. The prototypical H⁺/K⁺-ATPase is the protein that acidifies gastric luminal contents. The physiological and pharmacological significance of this pump has led to a detailed investigation of its biochemistry and molecular and cell biology. 2. Recently, a number of closely related H⁺/K⁺-ATPase isoforms have been discovered. These isoforms are present in organs other than the stomach, including the colon and kidney, where they contribute to acid—base and potassium homeostasis. The structure, expression and physiological roles of the gastric H⁺/K⁺-ATPase and other isoforms are reviewed.     

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.     

小檗碱对培养大鼠神经细胞内游离Ca2+的影响

以Fura-2/AM为细胞内钙离子的荧光指示剂,用AR-CM-MIC阳离子测定系统,直接测定了体外培养的新生大鼠神经细胞内游离钙([Ca²⁺]i)值,并观察了小檗碱(Ber)的影响。结果表明,Ber对神经细胞静息[Ca²⁺]i无明显影响,Ber1～100μmol·L^-1能剂量依赖地抑制去甲肾上腺素和H₂O₂引起的[Ca²⁺]i升高,其IC₅₀分别为39.9和17.9μmol·L^-1。高剂量Ber(10～100μmol·L^-1)能抑制高K+引起的[Ca²⁺]i升高。姐果提示,Ber对去甲肾上腺素,高K⁺及H₂O₂引起的[Ca²⁺]i升高的抑制作用可能是其抗脑缺血作用机制之一。     

BRL 38227 (levcromakalim)-induced hyperpolarization reduces the sensitivity to Ca2+ of contractile elements in canine coronary artery

Summary Potassium (K⁺) channel openers decrease intracellular free Ca²⁺ concentrations ([Ca²⁺]_i) by hyperpolarizing the membrane and deactivating the Ca²⁺-channels. To examine whether the hyperpolarization produced by K⁺-channel openers has other effects on the mechanical activity of vascular smooth muscle, we investigated the effects of levcromakalim (BRL 38227) on membrane potential, [Ca²⁺]_i, as measured with fura-2, and force of contraction induced by 30 mmol/l KCl-physiological salt solution (PSS), in canine coronary arteries. BRL 38227 hyperpolarized the membrane and reduced increases in [Ca²⁺]_i and in contractile force induced by 30 mmol/l KCl-PSS. The [Ca²⁺]_i-contractile force curve, determined in the presence of BRL 38227, was located to the right of the control curve determined by decreasing extracellular Ca²⁺ concentrations ([Ca²⁺]_o) in 30 mmol/l KCl-PSS. The [Ca²⁺ _i-contractile force curve, determined by decreasing extracellular K⁺ concentrations ([K⁺]_o), was also located to the right of that determined by decreasing [Ca²⁺]_o in 30 mmol/l KCl-PSS. The effect of BRL 38227, a reduction in the Ca ²⁺-sensitivity of contractile elements, was antagonized by the ATP-sensitive K⁺-channel blocker, glibenclamide (10^–6 or 10^–5 mol/1). These results suggest that the membrane hyperpolarization induced by BRL 38227, or the repolarization caused by reducing ([K⁺]_o), decreases the Ca²⁺-sensitivity of contractile elements of vascular smooth muscle.Correspondence to T. Yanagisawa at the above address     

间硝苯地平对老龄肾性高血压大鼠心肌和脑微粒体Na+,K+-ATP酶,Ca2+-ATP酶和Mg2+-ATP酶活性的影响

间硝苯地平(m-Nif,ig20mg·kg^-1·d^-1持续给药9周)可显著降低老龄肾性高血压大鼠(RVHR)血压和左室重量(P<0.01),增高心、脑微粒体Na⁺,K⁺-ATP酶和Ca²⁺-ATP酶活性(P<0.01),降低Mg²⁺-ATP酶活性,体外量效关系研究发现,m-Nif在较高剂量(10～1000μmol·L^-1)时可增高RVHR心脑微粒体Na⁺,K⁺-ATP酶和Ca²⁺-ATP酶活性,且随剂量增加而增高。上述结果表明,m-Nif可改善老龄RVHR心脑微粒体Na⁺,K⁺泵和Ca²⁺泵功能。     

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.

     

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.