The role of caffeine-sensitive calcium stores in the regulation of the intracellular free calcium concentration in rat sympathetic neurons in vitro

Intracellular Ca2+ stores were studied in sympathetic neurons grown in primary culture from the superior cervical ganglion of the rat. The [Ca2+]i was measured in single cells using the fluorescent Ca2+ indicator fura-2 and a sensitive microfluorimeter. Superfusion of the cells with 10 mM caffeine elicited a rapid and transient increase in [Ca2+]i in the absence of extracellular Ca2+, indicating the presence of a caffeine-sensitive intracellular Ca2+ storage site. After depletion of the store by mobilization of Ca2+ with caffeine, it could be refilled by elevating [Ca2+]i, allowing multiple caffeine-induced [Ca2+]i transients to be elicited from a single neuron. Ryanodine (1 microM), an alkaloid that promotes Ca2+ release from the sarcoplasmic reticulum, was an effective inhibitor of the caffeine-induced [Ca2+]i transients in sympathetic neurons. Exposure to ryanodine in the presence of caffeine was required to produce a subsequent inhibition of the caffeine-induced response, suggesting a "use-dependent" inhibition that may result from depletion of the Ca2+ stores. In contrast, dantrolene Na (10 microM), an agent known to interfere with Ca2+ release from the sarcoplasmic reticulum, also blocked the caffeine-induced [Ca2+]i transients, but in a time-dependent rather than a use-dependent manner. Electrophysiological measurements using the whole cell version of the patch-clamp technique were made simultaneously with [Ca2+]i microfluorimetric recordings. The magnitude of the [Ca2+]i transients elicited by step depolarizations closely paralleled the magnitude of Ca2+ influx via voltage-sensitive Ca2+ channels, regardless of whether the magnitude of the Ca2+ current was modified by varying the test pulse duration or potential. The relationship between the magnitude of Ca2+ influx and the resulting increase in [Ca2+]i saturated at large Ca2+ influxes resulting from long depolarizations, consistent with the activation of a large capacity, low affinity [Ca2+]i buffering mechanism. Caffeine (10 mM) and ryanodine (10 microM), applied singly or together, produced a small and variable decrease in the [Ca2+]i transient resulting from cell depolarization using the whole-cell patch-clamp technique. We conclude that mammalian sympathetic neurons possess intracellular Ca2+ stores with pharmacological characteristics that closely resemble those found in muscle but that these are relatively small and produce little amplification of [Ca2+]i transients resulting from Ca2+ influx through voltage-sensitive Ca2+ channels.     

Ethanol and synaptosomal calcium homeostasis

The effect of ethanol on synaptosomal calcium homeostasis was studied in the rat using the fluorescent dye, fura-2, and 45Ca uptake. The mitochondrial poison, cyanide, caused a substantial rise in intracellular free Ca2+ concentration, [Ca2+]i, over that of control synaptosomes. This rise was enhanced by ethanol. Ethanol also augmented the rise in [Ca2+]i induced by ouabain, indicating that modulation of Na(+)-Ca2+ exchange is probably not the underlying mechanism. The Ca2+ channel blockers, verapamil and La3+, also failed to inhibit the rise in [Ca2+]i caused by ethanol. Preincubation of synaptosomes with caffeine, however, caused a significant decrease in the rise of [Ca2+]i due to ethanol, suggesting that ethanol exerts effects on Ca2+ homeostasis at the level of the endoplasmic reticulum.     

A cyclic GMP-dependent housekeeping Cl- channel in rabbit gastric parietal cells activated by a vasodilator ecabapide.

1. The membrane potential of rabbit gastric parietal cells is dominated by a Cl- channel with a subpicosiemens single channel conductance in the basolateral membrane. The effects of 3-[[[2-(3,4-dimethoxyphenyl)ethyl]carbamoyl]amino-N-methylbenzamide++ + (DQ-2511: ecabapide), a vasodilator, on the opening of this Cl-1 channel, the cyclic GMP content and the intracellular free Ca2+ concentration ([Ca2+]i) of parietal cells were investigated by whole-cell patch-clamp technique, enzyme immunoassay and Fura 2-fluorescence measurement. 2.Ecabapide stimulated the opening of the Cl-1 channel as determined by the reversal potential. This stimulation was concentration-dependent, and its EC50 value was 0.2 microM. Both the basal and ecabapide-induced openings of the channel were inhibited by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, 500 microM), a Cl- channel blocker. Another Cl- channel blocker, niflumic acid (500 microM) was much less effective. 3. The power spectra of the currents before and after the addition of ecabapide (10 microM) were analysed. Both spectra contained only one Lorentzian (1/f2) component. 4. 6-Anilino-5,8-quinolinedione (LY83583; 5 microM) which prevents activation of soluble guanylate cyclase, significantly inhibited both the basal and ecabapide (10 microM)-induced openings of the Cl- channel. 5. Ecabapide (0.01-100 microM) concentration-dependently elevated the cyclic GMP content in the parietal cell-rich suspension. The EC50 value was 0.2 microM. 6. In single Fura 2-loaded parietal cells, ecabapide (10-100 microM) did not increase [Ca2+]i. 7. These results indicate that ecabapide stimulates an intracellular production of cyclic GMP in the parietal cell without increasing [Ca2+]i, and leads to an activation of the housekeeping Cl- channel.     

Calcium-activated currents in cultured neurones from rat dorsal root ganglia.

1. Voltage-activated Ca2+ currents and caffeine (1 to 10 mM) were used to increase intracellular Ca2+ in rat cultured dorsal root ganglia (DRG) neurones. Elevation of intracellular Ca2+ resulted in activation of inward currents which were attenuated by increasing the Ca2+ buffering capacity of cells by raising the concentration of EGTA in the patch solution to 10 mM. Low and high voltage-activated Ca2+ currents gave rise to Cl- tail currents in cells loaded with CsCl patch solution. Outward Ca2+ channel currents activated at very depolarized potentials (Vc + 60 mV to + 100 mV) also activated Cl- tail currents, which were enhanced when extracellular Ca2+ was elevated from 2 mM to 4 mM. 2. The Ca(2+)-activated Cl- tail currents were identified by estimation of tail current reversal potential by use of a double pulse protocol and by sensitivity to the Cl- channel blocker 5-nitro 2-(3-phenyl-propylamino) benzoic acid (NPPB) applied at a concentration of 10 microM. 3. Cells loaded with Cs acetate patch solution and bathed in medium containing 4 mM Ca2+ also had prolonged Ca(2+)-dependent tail currents, however these smaller tail currents were insensitive to NPPB. Release of Ca2+ from intracellular stores by caffeine gave rise to sustained inward currents in cells loaded with Cs acetate. Both Ca(2+)-activated tail currents and caffeine-induced inward currents recorded from cells loaded with Cs acetate were attenuated by Tris based recording media, and had reversal potentials positive to 0 mV suggesting activity of Ca(2+)-activated cation channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

粉防己碱对培养乳牛基底动脉平滑肌细胞内游离钙浓度的影响

目的：研究粉防己碱对培养乳牛基底动脉平滑肌细胞游离钙浓度（[Ca^2 ]i)的影响。方法：利用AR－CM－MIC阳离子测定系统,采用Fura 2-AM为指示剂,测量单个细胞内[Ca^2 ]i。结果：粉防己碱10－100μmol/L对培养乳牛基底动脉平滑肌细胞静息[Ca^2 ]i无明显影响。在细胞外钙为1．3mmol/L,粉防己碱可浓度依赖性地抑制KC1引起[Ca^2 ]i的升高。咖啡因10mmol/L可诱导一次[Ca^2 ]i瞬间快速升高,随后自发回复到静息水平,粉防己碱10和30μmol/L对咖啡因诱导的[Ca^2 ]i瞬间升高没有作用,但高浓度（100μmol/L）粉防己碱抑制了[Ca^2 ]i瞬间升高。在细胞外钙为1.3mmol/L,苯肾上腺素10μmol/L可引起双相[Ca^2 ]i变化,包括快速升高相和持续升高相。在细胞外钙为零,苯肾上腺素仅引起[Ca^2 ]i的快速升高相。粉防己碱可浓度依赖性地抑制苯肾上腺素引起[Ca^2 ]i快速升高相。结论：在培养乳牛基底动脉平滑肌细胞,粉防己碱可能通过影响电压依赖性和苯肾上腺素受体介导的钙通道而抑制钙内流。高浓度粉防己碱也可能影响肌浆网钙释放或钙摄取。     

氯通道阻断剂对H_2O_2诱导的胰岛RIN-mβ细胞凋亡的影响

目的观察氯通道阻断剂对H2O2诱导的胰岛RIN-mβ细胞凋亡的影响,探讨氯通道在RIN-mβ细胞凋亡中的作用。方法采用H2O2诱导的胰岛RIN-mβ细胞凋亡模型,观察氯通道阻断剂(DIDS、NPPB和NFA)对细胞存活率、形态学变化、凋亡的影响。结果氯通道阻断剂单用时对胰岛RIN-mβ细胞活力无明显影响,但能明显提高H2O2处理的胰岛RIN-mβ细胞的存活率。与模型对照组相比,氯通道阻断剂DIDS、NPPB和NFA对抗组细胞存活率明显增加(P<0.01),细胞凋亡率明显降低(P<0.01)。结论氯通道阻断剂对H2O2诱导的RIN-mβ细胞损伤和凋亡具有明显的保护作用。     

Caffeine-evoked, calcium-sensitive membrane currents in rabbit aortic endothelial cells.

1. Single cell photometry and whole-cell patch clamp recording were used to study caffeine-induced intracellular Ca2+ signals and membrane currents, respectively, in endothelial cells freshly dissociated from rabbit aorta. 2. Caffeine (5 mM) evoked a transient increase in [Ca2+]i in fura-2-loaded endothelial cells. Pretreatment of cells with 10 microM ryanodine did not alter resting [Ca2+]i but irreversibly inhibited the caffeine-induced rise in [Ca2+]i. The caffeine-induced increase in [Ca2+]i was not attenuated by the removal of extracellular Ca2+ and did not stimulate the rate of Mn2+ quench of fura-2 fluorescence. 3. Bath application of caffeine evoked a dose- and voltage-dependent outward current. The rate of onset and amplitude of the caffeine-evoked outward current increased with higher caffeine concentrations and membrane depolarization. The relationship between caffeine-evoked current amplitude and membrane potential was non linear, suggesting that the channels underlying the current are voltage-sensitive. 4. In the absence of extracellular Ca2+, the amplitude of the caffeine-evoked outward current was reduced by approximately 50% but the duration of the current was prolonged compared to that observed in the presence of external Ca2+. Ca(2+)-free external solutions produced an unexpected increase in both the frequency and amplitude of spontaneous transient outward currents (STOCs). 5. Inclusion of heparin (10 micrograms ml-1) in the patch pipette abolished the acetylcholine (ACh)-induced outward current but failed to inhibit either STOCs or the caffeine-evoked outward current in native endothelial cells. In the absence of extracellular Ca2+, heparin did not affect either STOCs or the caffeine-induced outward current.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cl~-通道阻断剂对5-HT和CPA引起的兔脑椎基底动脉平滑肌细胞Ca~(2+)内流的影响

目的　在培养的兔脑椎基底动脉平滑肌细胞上观察5 HT和CPA诱导的Ca2 + 内流的特性 ,电压依赖性Ca2 + 通道 (VDC)抑制药尼莫地平 ,非电压依赖性Ca2 + 通道抑制药SK&F963 65及Cl-通道阻断剂DIDS、NPPB对两种激动剂引起 [Ca2 + ]i 反应的影响 ,以探讨脑血管平滑肌细胞中 5 HT引起Ca2 + 内流的特性、Cl-通道与Ca2 + 内流的关系。方法　采用生物荧光双波长影像分析系统瞬即测定单细胞胞质[Ca2 + ]i 技术。结果　① 5 HT和CPA均能诱导平滑肌细胞[Ca2 + ]i 呈双相升高 ,并且 5 HT诱导的Ca2 + 释放是环匹阿尼酸 (CPA)敏感Ca2 + 池的一部分 ;②尼莫地平对 5 HT和CPA触发的Ca2 + 内流无明显影响 ,而SK&F963 65可阻止二者触发的Ca2 + 内流 ;③Cl-通道阻断剂DIDS、NPPB呈浓度依赖性抑制Ca2 + 内流 ,在SK&F963 65最大限度抑制Ca2 + 内流后 ,DIDS、NPPB可进一步抑制Ca2 + 内流 ;而Ca2 +内流被DIDS、NPPB分别最大抑制后 ,SK&F963 65也可进一步抑制Ca2 + 内流。结论　 5 HT引起的Ca2 + 内流是经SK&F963 65敏感的非VDC ,其中包含Ca2 + 释放引起的Ca2 + 内流 (CRAC)成分与非CRAC成分 ,并且这两部分Ca2 +内流均与DIDS、NPPB敏感的Cl-通道开放有关     

Ca2+ entry activated by emptying of intracellular Ca2+ stores in ileal smooth muscle of the rat.

1. The effects of depletion of intracellular Ca2+ stores on muscle tension and the intracellular Ca2+ concentration ([Ca2+])i were studied in fura-2 loaded longitudinal smooth muscle cells of the rat ileum. 2. After exposure to a Ca(2+)-free solution, application of Ca2+ caused a small contraction and a rise in [Ca2+]i, both of which were potentiated when the muscle was challenged with carbachol or caffeine before the addition of Ca2+. 3. Cyclopiazonic acid (CPA), a specific inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase, dose-dependently decreased tension development and the rises in [Ca2+]i induced by carbachol and caffeine in the Ca(2+)-free solution, but conversely increased the Ca(2+)-induced responses even in the presence of the voltage-dependent Ca2+ channel blockers, methoxyverapamil and nifedipine. 4. The contraction and rise in [Ca2+]i evoked by Ca2+ gradually declined with time after removal of CPA, while the reverse was the case for the responses to carbachol and caffeine. 5. The Ca(2+)-induced contraction and rise in [Ca2+]i in the presence of CPA were inhibited by the replacement of Na+ with K+ or Cs+, and by the addition of Cd2+, Ba2+, Ni2+ or La3+. 6. The influx of Mn2+ was much greater in extent in the presence of CPA than in its absence. 7. These results suggest that the emptying of intracellular Ca2+ stores may activate Ca2+ influx not associated with voltage-dependent Ca2+ channels in the rat ileal smooth muscle.     

Effect of nortriptyline on intracellular Ca2+ handling and proliferation in human osteosarcoma cells

The effect of the antidepressant nortriptyline, on bone cells is unknown. In human osteosarcoma MG63 cells, the effect of nortriptyline on intracellular Ca2+ concentration ([Ca2+]i) and proliferation was measured by using fura-2 and tetrazolium, respectively. Nortriptyline (> or = 10 microM) caused a [Ca2+]i rise in a concentration-dependent manner (EC50 = 200 microM). Nortriptyline-induced [Ca2+]i rise was prevented by 60% by removal of extracellular Ca2+ but was not altered by voltage-gated Ca2+ channel blockers. In Ca2+ -free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ -ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of nortriptyline on [Ca2+]i was abolished; also, pretreatment with nortriptyline abolished thapsigargin-induced [Ca2+]i increase. U73122, an inhibitor of phospholipase C, did not affect nortriptyline-induced [Ca2+]i rise; however, activation of protein kinase C decrease nortriptyline-induced [Ca2+]i rise by 32%. Overnight incubation with 50 and 100 microM nortriptyline killed 78% and 97% of cells, respectively; while 10 microM nortriptyline had no effect. These data suggest that nortriptyline rapidly increases [Ca2+]i in human osteosarcoma cells by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release, and is cytotoxic at high concentrations.     

Effect of intracellular pH on cytosolic free [Ca2+] in human epidermoid A-431 cells.

This study characterizes the correlation between intracellular pH (pHi) and the cytosolic free Ca2+ concentration ([Ca2+]i) in suspended and adherent human epidermoid A-431 cells. Using the fluorescent dyes 2,7-bis(carboxyethyl)carboxyfluorescein acetoxymethyl ester (BCECF) and fura-2, the resting pHi and [Ca2+]i in suspended cells were 7.23 +/- 0.03 and 209 +/- 30 nM; those in adherent cells were 7.28 +/- 0.02 and 87 +/- 5 nM. Removal of external Ca2+ did not change the resting pHi but reduced the resting [Ca2+]i, indicating the resting level of [Ca2+]i is in part maintained by an influx of Ca2+ from the external medium. When both suspended and adherent cells were acidified or alkalinized, resting [Ca2+]i was altered. An intracellular acidification induced a fall in [Ca2+]i, and a rise in pHi induced a rise in [Ca2+]i. These changes in [Ca2+]i were correlated with an uptake of 45Ca2+ from the external medium, whereas no Ca2+ efflux occurred. The alteration in [Ca2+]i induced by modification of pHi was abolished in the absence of external Ca2+ or by adding 2 mM CoCl2, LaCl3, and attenuated by the addition of 2 mM MnCl2 to the bathing medium. It was insensitive to the voltage-gated Ca2+ channel blockers nifedipine or verapamil (1 mM). CoCl2, LaCl3, and MnCl2 each induced changes in pHi and [Ca2+]i but verapamil and nifedipine did not. Because CoCl2, LaCl3, and MnCl2 are also known to block Na+/Ca2+ exchange, intracellular Na+ ([Na+]i) was measured by flame photometry in acidified or alkalinized cells. In either case no change in [Na+]i was observed. Furthermore, treatment with amiloride (100 microM), a blocker of the Na+/Ca2+ exchanger, did not inhibit the pH-induced changes in [Ca2+]i. 1,2-bis(o-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (100 microM), a Ca2+ chelator, induced a decrease in pHi as well as a reduction of [Ca2+]i, also supporting the direct relation between pHi and [Ca2+]i. 3,4,5-Trimethoxybenzoic acid 8-(diethylamino)ocytl ester HCl (TMB-8) (100 microM), a known blocker of intracellular Ca2+ mobilization, did not change the resting pHi and [Ca2+]i in normal cells or cells acidified or alkalinized. This observation, taken together with data from cells incubated in the absence of external Ca2+, suggests intracellular Ca2+ pools are not involved in changes in [Ca2+]i that result from a modification of pHi. Resting pHi and [Ca2+]i in cells treated with either 8-bromo-dibutyryl cAMP (1 mM) or forskolin (150 microM) are not changed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of voltage-independent Ca2+ channels activated by endothelin-1

To clarify Ca2+ entry channels involved in the endothelin-1 (ET-1)-induced increase in the intracellular concentration ([Ca2+]i), we performed whole-cell recordings of patch-clamp techniques and monitoring of [Ca2+]i with Ca2+ indicators fura-2 and fluo-3 in A7r5 cells (a cell line derived from rat thoracic aortic smooth muscle cells). With whole-cell recordings, lower concentrations (< or = 1 nM) of ET-1 activated a Ca(2+)-permeable nonselective cation channel (designated NSCC-1). In contrast, higher concentrations (> or = 1 nM) of ET-1 activated two types of Ca(2+)-permeable nonselective cation channel (designated NSCC-1 and NSCC-2) and store-operated Ca2+ channel (SOCC). Importantly, we found that these Ca2+ channels can be pharmacologically discriminated using blockers of the so-called receptor operated Ca2+ influx such as SK&F 96365 and LOE 908. That is, NSCC-1 is resistant to SK&F 96365 but sensitive to LOE 908; NSCC-2 is sensitive to both SK&F 96365 and LOE 908; SOCC is sensitive to SK&F 96365 but resistant to LOE 908. Using these blockers, we analyzed the ET-1-induced increase in [Ca2+]i. The increase in [Ca2+]i induced by lower concentrations of ET-1 was resistant to SK&F 96365 but sensitive to LOE 908. In contrast, the increase in [Ca2+]i induced by higher concentrations of ET-1 was partially suppressed to approximately 30% of controls by either SK&F 96365 or LOE 908 alone, and it was abolished by their combination. These results show that the increase in [Ca2+]i induced by lower concentrations (< or = 1 nM) of ET-1 results from Ca2+ influx through NSCC-1, whereas the increase in [Ca2+]i induced by higher concentrations (> or = 10 nM) of ET-1 results from Ca2+ influx through NSCC-1, NSCC-2 and SOCC.     

Inhibitory effects of (1R,9S)-β-hydrastine on calcium transport in PC12 cells

(1R,9S)-beta-Hydrastine (BHS), at 100 microM, has been shown to mainly reduce the K+-induced dopamine release and Ca2+ influx by blocking the L-type Ca2+ channel and inhibit the caffeine activated store-operated Ca2+ channels, but not those activated by thapsigargin, in PC12 cells. In this study, the effects of BHS on Ca2+ transport from Ca2+ stores in the absence of external Ca2+ were investigated in PC12 cells. BHS decreased the basal intracellular Ca2+ concentration ([Ca2+]i) in the absence of external Ca2+ in PC12 cells. In the absence of external Ca2+, pretreating PC12 cells with 100 microM BHS reduced the rapid increase in the [Ca2+]i elicited by 20 mM caffeine, but not that by 1 microM thapsigargin. In addition, BHS inhibited the increase in the [Ca2+]i elicited by restoration of 2 mM CaCl2 after the Ca2+ stores had been depleted by 20 mM caffeine, but not those depleted by 1 microM thapsigargin, in the absence of external Ca2+. These results suggested that BHS mainly inhibited Ca2+ leakage from the Ca2+ stores and the caffeine-stimulated release of Ca2+ from the caffeine-sensitive Ca2+ stores in PC12 cells.     

Effects of Cl channel blockers on Ca-activated chloride and potassium currents in smooth muscle cells from rabbit portal vein.

1. The effects of some chloride channel antagonists were studied on the calcium-activated chloride current (ICl(Ca)) in smooth muscle cells from the rabbit portal vein with the perforated patch technique. 2. 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) and 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS) reduced the amplitude of spontaneous transient inward currents (STICs, calcium-activated chloride currents) in a concentration-dependent manner. The concentrations required to reduce the amplitude by 50% (IC50) of STICs were 2.1 x 10(-4) M and 6.4 x 10(-4) M for DIDS and SITS, respectively. This effect was not voltage-dependent. 3. The time constant of decay of STICs (tau), which is voltage-dependent, was increased by about 30% by SITS and decreased by about 20% by DIDS. The effect of DIDS and SITS on tau was similar at holding potentials of -50 and +50 mV. 4. These compounds did not modify the characteristics of spontaneous transient outward currents (STOCs, calcium-activated potassium currents). 5. DIDS and SITS decreased the amplitude of ICl(Ca) evoked by noradrenaline and caffeine less potently than STICs with IC50 values of 7.5 x 10(-4) M and 1.8 x 10(-3) M, respectively. 6. DIDS and SITS increased the calcium-activated potassium current (IK(Ca) evoked by noradrenaline and caffeine by 3-6 fold. 7. Anthracene-9-carboxylic acid (A-9-C) inhibited STICs in a voltage-dependent fashion and was about 3 fold more active at +50 mV than at -50 mV. A-9-C increased STIC tau and this effect was enhanced by depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of magnesium and chloride ions on tone and contractility of vascular muscle.

Replacement of extracellular chloride ions ([Cl-]o) by other anions, on contractility and the effects of extracellular magnesium ions ([Mg2+]o) on spontaneous mechanical activity, as well as on agonist-induced responses of rat aorta and portal vein, were studied. Replacement of [Cl-]o with acetate (Ac-) or isethionate (Ise-) ions resulted in an increase and decrease, respectively, of the spontaneous mechanical activity frequency in portal vein; the amplitudes of the spontaneous mechanical activity were attenuated by Ac- and Ise- substitution. Withdrawal of [Mg2+]o in Cl(-)-containing media resulted in elevation of tension development in rat aortas, whereas a similar maneuver in media with Ac- or Ise-, substituted for [Cl-]o, resulted in abrogation of this tension development. Use of disulfonic stilbene anion-channel blockers, DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonate, 400-600 microM) and SITS (4,4'-acetamido-4'-isothiocyano-2,2'-stilbene disulfonic acid, 400-600 microM), failed to influence either spontaneous mechanical activity or basal tone of rat portal portal vein or aortas. Incubation of DIDS or SITS in Mg(2+)-free media also failed to influence mechanical responses to withdrawal of [Mg2+]o. Use of the Cl- cation transport inhibitor bumetanide (30-80 microM) also failed to alter spontaneous mechanical activity or basal tone in either the presence or absence of [Mg2+]o. Ac- and Ise- substitution attenuated norepinephrine- and K(+)-induced contractile responses in portal vein and aorta, Caffeine-induced contractions of aortas were potentiated by withdrawal of [Mg2+]o in Cl(-)-containing media but inhibited in Ac(-)- or Ise(-)-substituted solutions. In the presence of [Mg2+]o, substitution of foreign anions resulted in alterations in the agonist contractile dose-response curves; EC50s were increased whereas maximum tensions were depressed. Withdrawal of [Mg2+]o amplified these effects. Substitution of Ac- or Ise- for [Cl-]o in the presence or absence of [Mg2+]o depressed contractions induced by Ca acetate in aortas and portal vein. These results suggest that: (1) Cl- plays an important role in regulating spontaneous mechanical activity, basal tone, and contractility in rat aorta and portal vein; and (2) Cl- probably physiologically mediates some of the effects and actions of [Mg2+]o on intracellular release of and influx of Ca2+ in these smooth muscles.     

Inhibitory effect of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) in vascular smooth muscle

The inhibitory effects of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) on vascular smooth muscle contraction and cytosolic Ca2+ level ([Ca2+]i) were examined using isolated rabbit aorta loaded with a fluorescent Ca2+ indicator, fura-2. TMB-8 (100 microM) decreased the high K(+)-induced increase in muscle tension, and [Ca2+]i and 45Ca2+ influx to their respective resting levels. TMB-8 (100 microM) almost completely inhibited the increase in [Ca2+]i and 45Ca2+ influx due to norepinephrine although muscle tension was only partially decreased. A higher concentration of TMB-8 (300 microM) inhibited the remaining portion of the contraction without additional decrease in [Ca2+]i. The inhibitory effect of TMB-8 on high K(+)-induced contraction, but not on the norepinephrine-induced contraction, was antagonized by the increase in external Ca2+ concentrations or by the Ca2+ channel activators, CGP 28,392 and by Bay K8644. In Ca(2+)-free solution, norepinephrine-induced transient increases in [Ca2+]i and muscle tension and 100 microM TMB-8 inhibited these changes. The caffeine-induced transient increases in [Ca2+]i and muscle tension were also inhibited by TMB-8 at concentrations higher than those needed to inhibit the norepinephrine-induced transient changes. In permeabilized smooth muscle, TMB-8 (300 microM) did not inhibit the Ca(2+)-induced contraction. These results suggest that TMB-8 inhibits vascular smooth muscle contractility by inhibiting Ca2+ influx, Ca2+ release and Ca2+ sensitization of contractile elements.     

FK506 inhibits Cl- secretion in airway epithelium via calcineurin-independent mechanism.

FK506 (tacrolimus)-binding protein (FKBP) is associated with intracellular Ca2+ release channel and modulates its function. To elucidate the effect of FK506 on Ca2+ dynamics and Ca2+-mediated Cl- secretion in airway epithelium, we studied intracellular Ca2+ ([Ca2+]i) concentration and Cl(-)-dependent short-circuit current (Isc), in cultured bovine tracheal epithelial cells. Addition of ATP induced an increase in [Ca2+]i, and this response was dose dependently inhibited by FK506. Rapamycin, which binds FKBP with high affinity, likewise inhibited the [Ca2+]i rise, but cyclosporin A, a specific calcineurin inhibitor, did not. In Cl- secretion studies using Ussing chamber, ATP increased Ca2+-mediated Isc in amiloride-treated cells, an effect that was inhibited by FK506 and rapamycin but not by cyclosporin A. Therefore, FK506 inhibits Ca2+ mobilization in airway epithelium via FKBP but not calcineurin-dependent mechanism, which may result in the suppression of Ca2+-activated Cl- secretion.     

氯离子参与心肌细胞缺氧复氧损伤机制的研究

目的研究氯离子参加心肌细胞缺氧/复氧(A/R)损伤的机制。方法采用原代培养新生大鼠的心肌细胞A/R损伤模型,去除细胞外的氯离子(Cl--free),或分别给予Na+-K+-2Cl-共同转运体阻断剂bumetanide,Cl-/HCO3-离子交换系统抑制剂SITS和氯通道阻断剂9-AC,观察细胞存活率、MDA含量、LDH、SOD、GSH-Px酶活性变化及细胞内的钙含量、NF-κB活性变化。结果A/R组与对照组比,MDA含量、LDH活性、细胞内的钙含量、NF-κB活性升高,细胞存活率、SOD、GSH-Px活性降低;bumetanide及9-AC组与A/R组比,各项指标无统计学意义。Cl--free、SITS处理后较A/R组,MDA含量、LDH活性、细胞内的钙含量、NF-κB活性降低,而细胞存活率、SOD、GSH-Px明显升高。结论Cl-/HCO3-离子交换系统在心肌细胞A/R损伤中起了重要作用,Cl-参与心肌A/R损伤的机制与钙超载及NF-κB活性升高有关。     

Histamine-induced Ca2+ release in bovine adrenal chromaffin cells

The histamine-induced biphasic increase of the intracellular free [Ca2+] ([Ca2+]i) was studied in bovine adrenal chromaffin cells using fura-2 microfluorimetry and the whole-cell patch-clamp technique. Both the rapid, transient Ca2+ rise and the sustained plateau component of elevated [Ca2+]i were independent of extracellular Ca2+. Incubation with the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) blocker thapsigargin diminished histamine-induced changes in [Ca2+]i. When Ca2+ release was either stimulated by IP3 or blocked with the competitive inhibitor heparin, histamine was unable to elicit the typical Ca2+ rise. Ryanodine, tetracaine and ruthenium red, all blockers of Ca2+ release from caffeine-sensitive stores, had only minor effects on the agonist-induced Ca2+ changes. The contribution of mitochondria in shaping the histamine-induced Ca2+ increase was studied using ruthenium red and the two proton ionophores carbonylcyanide m-chlorophenylhydrazone (CCCP) and carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP). Both mitochondrial uncouplers reversibly increased [Ca2+]i and induced an inward current leading to cell membrane depolarisation. In summary, these results indicate that Ca2+ from IP3-sensitive stores is essential for the generation of both the transient increase and secondary elevation in [Ca2+]i.     

The effect of L-type calcium channel modulators on the mobilization of intracellular calcium stores in guinea-pig intestinal smooth muscle.

1. The action of Ca2+ channel modulators has been examined on the intracellular Ca2+ signal in the longitudinal smooth muscle cells of the guinea-pig intestine after exposure to histamine and to agents known to affect intracellular Ca2+ stores. Isometric contraction has been measured simultaneously with front-surface fluorometry of fura 2-loaded preparations. 2. Histamine (10 microM) evoked a phasic and tonic increase in [Ca2+]i and contraction which were both sensitive to the Ca2+ channel blockers, nimodipine and D600. 3. Caffeine (10 mM) evoked in rapid increase in [Ca2+]i which was sustained as long as the preparation was exposed to the drug, whereas the contractile response was only phasic. In the presence of nimodipine 1 microM, the phasic contraction was absent although the fura 2-Ca2+ signal amounted to 32% of the control. 4. Ryanodine (10 microM) evoked a slow increase in [Ca2+]i and a contraction, both of which were reversed after exposure to nimodipine (1 microM) or D600 (10 microM). In the presence of diazoxide (500 microM), a hyperpolarizing agent, the ryanodine-evoked increase in [Ca2+]i and in muscle tone were inhibited. 5. Thapsigargin (1 microM) also produced an increase in [Ca2+]i and a contraction both of which were blocked by nimodipine (1 microM). 6. In Ca2+-free solution, histamine 10 microM evoked non-reproducible phasic Ca2+ signal and contraction. This response was recovered after refilling in Ca2+ containing solution. The recovery was blocked by nimodipine, D600 or diazoxide and was facilitated by the Ca2+ channel activator, Bay K 8644. When the refilling medium was supplemented with thapsigargin, the recovered response was significantly reduced, but Bay K 8644 still had some action. 7. The present results show that blockage of L-type Ca2+ channels inhibited changes in [Ca2+]i evoked by histamine, caffeine and ryanodine which are generally attributed to Ca2+ mobilization from intracellular stores. They also show that when the tissue was exposed to nimodipine, D600 and diazoxide during the procedure of refilling after depletion of intracellular stores, the action of histamine on [Ca2+]i and contraction was blocked. Bay K 8644 had an opposite effect even when the Ca2+ pumping activity of the sarcoplasmic reticulum was reduced by thapsigargin. This indicates that refilling of intracellular Ca2+ stores depleted by histamine in guinea-pig intestine mainly occurred through L-type Ca2+ channels.