Bradykinin induces elevations of cytosolic calcium through mobilisation of intracellular and extracellular pools in bovine aortic endothelial cells.

1. In the presence of 1.8 mM extracellular calcium, bradykinin (0.3 nM-100 nM) induced a biphasic elevation of intracellular calcium ([Ca2+]i) in bovine aortic endothelial cells, consisting of an initial, large transient component followed by a lower sustained component. 2. When endothelial cells were bathed in nominally calcium-free solution containing 0.5 mM EGTA, bradykinin induced only a transient elevation of [Ca2+]i: the magnitude of this was significantly smaller than that obtained in the presence of extracellular calcium and the sustained phase was abolished. In the continued presence of bradykinin, re-addition of extracellular calcium to achieve a level of around 1.8 mM resulted in the induction of a biphasic elevation of [Ca2+]i consisting of a large initial component followed by a lower sustained component. 3. In the presence of 1.8 mM extracellular calcium, caffeine (5 mM) induced a small elevation of [Ca2+]i. When endothelial cells were bathed in nominally calcium-free solution containing 0.5 mM EGTA, the caffeine-induced elevation of [Ca2+]i was almost completely abolished. 4. In the presence of 1.8 mM extracellular calcium, treatment of endothelial cells with the calcium influx blocker, nickel chloride (4 mM), had no effect on resting [Ca2+]i or on the magnitude of the bradykinin-induced initial transient elevation of [Ca2+]i but abolished the sustained component. 5. In the presence of 1 mM extracellular calcium, treatment with the calcium chelator EGTA (2 mM; 1 min) had no effect on resting [Ca2+]i but the magnitude of the bradykinin-induced initial transient elevation of [Ca2+]i was significantly reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of diltiazem on calcium concentrations in the cytosol and on force of contractions in porcine coronary arterial strips.

1. Using front-surface fluorometry with fura-2-loaded porcine coronary arterial strips, we simultaneously measured effects of a Ca2+ antagonist, diltiazem, on cytosolic Ca2+ concentrations [( Ca2+]i) and on tension development. 2. In the presence of extracellular Ca2+ (1.25 mM), histamine concentration-dependently induced abrupt (the first component) and then sustained (the second component) elevations of [Ca2+]i. In the absence of extracellular Ca2+, histamine induced transient elevations of [Ca2+]i, and the time course was similar to that of the first component observed in the presence of extracellular Ca2+. Histamine caused a greater contraction for a given change in [Ca2+]i than did potassium, at [Ca2+]i over 300 nM. 3. Diltiazem, 10(-8)M to 10(-5)M, concentration-dependently inhibited the second component of [Ca2+]i elevation and tension development induced by histamine (10(-5) M). Only at higher concentrations (over 10(-5) M) did diltiazem inhibit the first component of increases in [Ca2+]i and tension development induced by histamine, both in the presence and absence of extracellular Ca2+. 4. Diltiazem (10(-6) M) inhibited increases in [Ca2+]i and tension development induced by cumulative applications of extracellular Ca2+ during K(+)-depolarization. The curve of [Ca2+]i against tension of these Ca2(+)-induced contractions obtained in diltiazem-treated strips overlapped with that obtained in untreated strips. This suggests that diltiazem has no direct effects on contractile elements. 5. In contrast, the histamine-induced Ca2(+)-tension curve (second component) was shifted in parallel to the left by diltiazem.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sensitivity of G-protein involved in endothelin-1-induced Ca2+ influx to pertussis toxin in porcine endothelial cells in situ.

1. We designed a new method to determine quantitatively the intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells in situ, using front-surface fluorometry and fura-2-loaded porcine aortic valvular strips. Using this method, we investigated the characteristics of the G-protein involved in endothelin-1 (ET-1)-induced changes in [Ca2+]i of endothelial cells in situ. 2. Endothelial cells were identified by specific uptake of acetylated-low density lipoprotein labelled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL). Double staining with DiI-Ac-LDL and fura-2 showed that the valvular strip was covered with a monolayer of endothelial cells and that the cellular component which contributed to the fura-2 fluorescence, [Ca2+]i signal, was exclusively endothelial cells. 3. ET-1 (10(-7) M) induced an elevation of [Ca2+]i consisting of two components: the first was a rapid and transient elevation to reach a peak, followed by a second, sustained elevation (the second phase). The first phase was composed of extracellular Ca(2+)-independent and -dependent components, while the second phase was exclusively extracellular Ca(2+)-dependent. The extracellular Ca(2+)-independent component of the first phase was due to the release of Ca2+ from intracellular storage sites. The second phase and part of the first phase of [Ca2+]i elevation were attributed to the influx of extracellular Ca2+. The Ca2+ influx component was completely inhibited by 10(-3) M Ni2+ but was not affected by 10(-5) M diltiazem.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-desethylamiodarone modulates intracellular calcium concentration in endothelial cells

The main in-vivo metabolite of amiodarone, N-desethylamiodarone (DEAM), possesses clinically relevant class-II antiarrhythmic and vasodilator activities. Vasodilation by DEAM is endothelium dependent and involves a sustained and biphasic increase in cytosolic free Ca2+ concentration ([Ca2+]i). The aims of this study were to explore the mechanisms mediating the DEAM-induced increase in [Ca2+]i in endothelial cells and to determine whether this increase in [Ca2+]i was associated with altered cell proliferation. Cultured bovine aortic endothelial cells were loaded with the Ca2+-sensitive fluorescent dye Fura-2/AM, and [Ca2+]i measured spectrofluorimetrically. DEAM increased [Ca2+]i concentration dependently (EC50 approximately 6 microM) both in the presence and absence of extracellular Ca2+. In the presence of extracellular Ca2+, the response of [Ca2+]i to DEAM (10 microM) consisted of an initial rise to a plateau followed by a second increase to micromolar levels. The initial plateau was reduced by the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (200 nM) and by the antioxidant ascorbic acid (100 microM). The initial rate of rise in [Ca2+]i was decreased by blocking mitochondrial Ca2+ release with cyclosporine A (1 microM). Under Ca2+-free conditions, the response of [Ca2+]i to DEAM (10 microM) was also biphasic, consisting of an initial transient peak and a second slow increase. When extracellular Ca2+ was restored, [Ca2+]i rose to micromolar concentrations. The initial peak was abolished by thapsigargin, but not altered by ascorbic acid or cyclosporine A. Both the second [Ca2+]i increase and that due to restoring extracellular Ca2+ were reduced by ascorbic acid but not affected by thapsigargin or cyclosporine A. The DEAM-induced generation of free radicals and sustained increase in [Ca2+]i might alter cell proliferation and endothelial cell proliferation was indeed concentration-dependently inhibited by DEAM (IC50 approximately 2.5 microM). In conclusion, the DEAM-induced [Ca2+]i increase in endothelial cells is due to Ca2+ influx from the extracellular space and to Ca2+ release from endoplasmic reticulum and mitochondria and involves enhanced generation of free radicals.     

Effects of dilazep on cytoplasmic calcium ion concentrations and arachidonic acid metabolism in activated human platelets

The effects of dilazep (tetrahydro-1H-1,4-diazepine-1,4(5H)-dipropanol bis(3,4,5-trimethoxybenzoate)-di-hydrochloride monohydrate, Comelian), a coronary and cerebral vasodilator and an antiplatelet drug, on the cytoplasmic Ca2+ concentration ([Ca2+]i) and arachidonic acid (AA) metabolism in activated human platelets were investigated. [Ca2+]i (free calcium ion concentration) of aequorin-loaded platelets was estimated by using the platelet ionized calcium aggregometer. AA metabolism was studied by the determination of AA metabolites, hydroxyheptadecatrienoic acid and 12-hydroxyeicosatetraenoic acid, using reversed-phase high performance liquid chromatography. When platelets were preincubated with dilazep (0-0.5 mmol/l), the drug inhibited both platelet aggregation and [Ca2+]i elevation induced by thrombin, AA and collagen in a concentration dependent manner, while only aggregation was inhibited after stimulation with the Ca ionophore A23187 (calcimycin). Both influx and release of Ca2+ into platelet cytoplasm induced by thrombin or AA were inhibited by dilazep, while neither of them was affected when induced by A23187. Oral ingestion of dilazep as a 100-mg capsule significantly depressed the [Ca2+]i elevation induced by thrombin, AA and collagen after 3 h. Dilazep inhibited endogenous AA metabolism by platelets induced by thrombin, although it enhanced exogenous one. Thus, dilazep inhibited platelet aggregation induced by any agonists including A23187, while [Ca2+]i elevation was inhibited by the drug only when the receptor-mediated agonist was used. Furthermore, it is suggested that dilazep inhibited AA liberation from platelet membrane phospholipids, leading to reduced production of all endogenous AA metabolites after platelet activation although metabolites of exogenous AA could be increased.     

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。     

Modulation of agonist-induced calcium mobilisation in bovine aortic endothelial cells by phorbol myristate acetate and cyclic AMP but not cyclic GMP.

1. In bovine aortic endothelial cells (BAEC), thrombin (1 mu ml-1), bradykinin (1-10 nM) and adenosine triphosphate (ATP) (0.3 microM-100 microM) each induced a biphasic elevation of cytosolic calcium ([Ca2+]i), consisting of an initial transient followed by a sustained plateau phase. 2. Pretreatment of BAEC with 4 beta-phorbol 12-myristate 13-acetate (PMA; 100 nM) reduced the magnitude of the initial transient elevation of [Ca2+]i, induced by thrombin (1 mu ml-1), low concentrations of bradykinin (1 nM) or ATP (0.3 microM, 3 microM), but not by higher concentrations of the latter two agonists. Addition of PMA (100 nM) during the plateau phase of the increase in [Ca2+]i induced by thrombin (1 mu ml-1), bradykinin (10 nM) or ATP (30 microM) resulted in a fall in [Ca2+]i. 3. The inhibitory effects of PMA (100 nM) were inhibited by staurosporine (100 nM) but not mimicked by the inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD; 100 nM). Furthermore, staurosporine (100 nM) increased [Ca2+]i when added during the plateau phase of the increase in [Ca2+]i induced by thrombin or bradykinin. In contrast, staurosporine (100 nM) reduced [Ca2+]i when added during the plateau phase of the increase in [Ca2+]i induced by ATP (30 microM). 4. Pretreatment with forskolin (10 microM) had no effect on the magnitude of the initial transient elevation of [Ca2+]i induced by thrombin (1 mu ml-1), bradykinin (1 nM and 10 nM) or ATP (30 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of endothelin receptor subtypes mediating Ca2+ mobilization and contractile response in rabbit iris dilator muscle.

1. We investigated the characteristics of endothelin (ET)-induced contraction and changes in intracellular Ca2+ concentration ([Ca2+]i) using the fura-2-loaded and non-loaded rabbit iris dilator. ET-1 and ET-2 (3-100 nM) and ET-3 (30-100 nM) caused contraction in a concentration-dependent fashion. 2. The selective ETB-receptor agonists, IRL1620 and sarafotoxin S6c produced only a small contraction or no contraction at a concentration of 1 microM. The rank order of potencies for the contraction (pD2 value) was ET-1 = ET-2 > ET-3 >> sarafotoxin S6c = IRL1620. 3. The contractile response to ET-3 was antagonized by pretreatment with BQ-123 (10 nM), a selective ETA receptor antagonist. The contractile responses to ET-1 and ET-2 were antagonized by pretreatment with BQ-123 (10 microM), but not at a concentration of 10 nM. 4. ETs increased [Ca2+]i and sustained muscle contraction. ET-1 (100 nM), ET-2 (100 nM), and ET-3 (1 microM) induced an elevation of [Ca2+]i consisting of two components: first a rapid and transient elevation to reach a peak, followed by a second, sustained elevation; a sustained contraction was produced without a transient contraction. The ETB receptor-selective agonist, IRL1620 (1 microM) and sarafotoxin S6c (1 microM) also induced a rapid and transient elevation of [Ca2+]i to reach a peak and a sustained elevation, together with only a small contraction or no contraction. 5. ET-1 (100 nM) induced a transient increase in [Ca2+]i in a Ca(2+)-free, 2 mM EGTA-containing physiological saline solution (Ca(2+)-free PSS), and a small sustained contraction which was significantly different from that induced by ET-1 (100 nM) in normal PSS. The ET-1-induced increase in [Ca2+]i and sustained contraction were not affected by the voltage-dependent Ca2+ channel blocker, nicardipine (10 microM). The ET-1-induced transient increase in [Ca2+]i was significantly reduced by the sarcoplasmic reticulum (SR) Ca(2+)-ATPase inhibitor, cyclopiazonic acid (30 microM); however, the ET-1-induced sustained contraction was not affected by this agent. 6. The selective ETA receptor antagonist, BQ-123 (100 nM) reduced the ET-3 (100 nM)-induced contraction, but did not affect the transient increase or elevation of the second phase of [Ca2+]i. However, this antagonist at 1 microM did not affect the ET-1 (100 nM)- and ET-2 (100 nM)-induced elevation of [Ca2+]i and contractile response, or the IRL1620-induced elevation of [Ca2+]i. 7. The selective ETB receptor antagonist, BQ-788 (1 microM) reduced the transient increase in [Ca2+]i induced by ET-1 (30 nM), ET-2 (30 nM), ET-3 (100 nM) and IRL1620 (1 microM), but did not affect the sustained elevation of [Ca2+]i and contractile responses produced by ET-1, ET-2 and ET-3. 8. Pretreatment with IRL1620 (1 microM) reduced the increase in [Ca2+]i induced by IRL1620 (1 microM) and sarafotoxin S6c (1 microM), as well as the ET-1 (100 nM)-, ET-2 (100 nM)- and ET-3 (1 microM)-induced elevation of [Ca2+]i, whereas in the presence of IRL1620, ET-1-, ET-2- and ET-3-induced contractions were unaltered. 9. These results suggest that ETA and ETB receptor subtypes exist in the rabbit iris dilator muscle, and that the ETA receptor is divided into: (1) BQ-123-sensitive ETA subtypes activated by ET-1, ET-2 and ET-3, and (2) BQ-123-insensitive ETA subtypes activated by ET-1 and ET-2, which cause the sustained increase of [Ca2+]i and contraction; in contrast, ETB receptor subtypes are activated by ET-1, ET-2, ET-3, IRL1620 and sarafotoxin S6c and cause the transient and sustained increase in [Ca2+]i which is not able to contract the smooth muscle.     

The histamine H1 receptor in GT1-7 neuronal cells is regulated by calcium influx and KN-62, a putative inhibitor of calcium/calmodulin protein kinase II.

1. In GT1-7 cells, histamine stimulated the initial [Ca2+]i transient in a dose-dependent manner with a best-fit EC50 value of 4.2 +/- 4.2 microM (mean +/- s.e.mean, n = 4) and a best-fit maximal effect of 138 +/- 56 nM (n = 4) increase above basal calcium levels. 2. Pretreatment of cells with 30 microM histamine for 30 min desensitized the population mean peak calcium signal by 53% to 75 +/- 9 nM, (n = 3, P < 0.04). Analysis of the individual cells revealed that 39 +/- 7% (n = 94 cells from 8 experiments) of pretreated cells exhibited desensitized histamine-stimulated [Ca2+]i transients of < or = 1 standard deviation below the control cells mean calcium transient level. 3. The desensitization induced by histamine was prevented (P < 0.01) by KN-62 (10 microM), a putative inhibitor of the calcium/calmodulin-dependent protein kinase II (CaMKII). KN-62 (10 microM) alone did not induce [Ca2+]i mobilization, nor did it antagonize the histamine-stimulated [Ca2+]i signal. In addition, KN-62 did not appear to have its effect by hastening the rate of recovery from desensitization. 4. Histamine pretreatment in nominal (zero calcium + 0.2 mM EGTA) or in low (0.3 mM) extracellular calcium did not induce histamine receptor desensitization, supporting a role for extracellular calcium in the homologous H1 receptor desensitization process. 5. Histamine (30 microM) stimulated at least four different types of [Ca2+]i signals in GT1-7 cells. The majority (61%) were of single spikes with the remaining cells showing some form of calcium oscillatory behaviour. The proportion of GT1-7 cells showing histamine-induced calcium oscillations was histamine concentration-dependent and significantly reduced after acute desensitization. KN-62, when present during histamine pretreatment, prevented this fall in calcium oscillation. Under the conditions of nominal or 0.3 mM extracellular calcium the proportion of cells exhibiting histamine-stimulated calcium oscillations was not significantly different from the controls. 6. Bradykinin stimulated a [Ca2+]i transient in GT1-7 cells with a population mean peak response of 147 +/- 8 nM (n = 5) over basal levels. The bradykinin-induced [Ca2+]i signal was without any calcium oscillatory activity. Histamine pretreatment caused the heterologous desensitization of the bradykinin [Ca2+]i signal (44% reduction, P < 0.007), which was unaffected by KN-62. 7. The results presented here suggest that the histamine-mediated homologous H1 receptor desensitization process involves extracellular calcium and can be blocked by KN-62, a putative inhibitor of CaMKII. In contrast, KN-62 does not appear to prevent the histamine-mediated heterologous desensitization cascade. These findings suggest fundamental differences in the mechanisms underlying homologous and heterologous H1 receptor desensitization pathways in GT1-7 neuronal cells.     

Differential effect of diltiazem and TA-3090 on calcium homeostasis of neutrophils.

The effects of diltiazem and TA-3090, an 8-chloro analog of diltiazem, on cellular responses and calcium homeostasis of human neutrophils were investigated. TA-3090, at 10 to 20 microM, enhanced lysozyme release and superoxide generation induced in neutrophils by n-formyl-methionyl-leucyl-phenylalanine (FMLP). Higher concentrations of TA-3090 inhibited responses at IC50s between 70 and 85 microM. Diltiazem by comparison inhibited responses at an IC50 of about 200 microM. The two drugs had little or no effect on early signaling events: inositol 1,4,5-trisphosphate formation triggered by FMLP was not affected. Moreover, 500 microM TA-3090 or diltiazem did not significantly affect FMLP-triggered Ca2+ transients. (Cytoplasmic free Ca2+ levels ([Ca2+]i) were monitored in fura-2-loaded neutrophils.) Diltiazem alone caused a limited influx of extracellular Ca2+ which increased basal [Ca2+]i by twofold. Internal Ca2+ stores were not released. TA-3090, in contrast, induced a biphasic rise in [Ca2+]i--an initial mobilization of intracellular Ca2+ stores was followed after 10-15 min by a persistent influx of extracellular Ca2+ which increased [Ca2+]i to 1.3 +/- 0.7 (SD) microM. Complementary studies with semipermeabilized neutrophils showed that TA-3090 but not diltiazem directly released Ca2+ from intracellular stores. In TA-3090-treated cells, lactate dehydrogenase release was correlated with delayed influx of extracellular Ca2+. The chelation of extracellular Ca2+ by EGTA prevented LDH release. Present results show that TA-3090 and diltiazem initially blocked cell signaling at steps subsequent to phospholipase C activity. With TA-3090-treated cells, elevated [Ca2+]i ensuing from prolonged incubations likely activated inappropriate reactions leading to cell lysis and death.     

Long-lasting activation of cation current by low concentration of endothelin-1 in mouse fibroblasts and smooth muscle cells of rabbit aorta.

1. Recombinant human ETA receptors were expressed in a mouse fibroblast cell line (Ltk- cell) and functional coupling of the receptors with Ca2+ permeable channels at low concentrations of endothelin-1 (ET-1) was investigated using whole-cell recordings and monitoring the changes in intracellular free Ca2+ concentrations ([Ca2+]i) with a Ca2+ indicator, fluo-3. A similar type of coupling was investigated in freshly dispersed vascular smooth muscle cells (VSMCs) of rabbit thoracic aorta by use of whole-cell recordings. 2. In Ltk- cells expressing recombinant human ETA receptors, concentrations of ET-1 (10(-8) M, 10(-9) M) evoked an initial transient peak and a subsequent sustained elevation in [Ca2+]i whereas a lower concentration of ET-1 (10(-10) M) evoked only a sustained elevation of [Ca2+]i. After removal of extracellular Ca2+, ET-1 evoked only an initial peak without a sustained elevation of [Ca2+]i. The sustained elevation induced by 10(-10) M ET-1 was blocked by 300 microM mefenamic acid (a cation channel blocker) but not by 10 microM nifedipine (a blocker of voltage-operated Ca2+ channel). 3. In whole-cell recordings with Ltk- cells, a brief (3-5 min) application of ET-1 (10(-10) M) induced a sustained inward current at a holding potential of -60 mV. The current-voltage relationship revealed that the reversal potential of the ET-1-induced current was close to 0 mV (1.9 mV) and was not altered by reducing the concentration of Cl- in the bath solution, indicating that the current is carried by cations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between cytosolic calcium concentration and force in the papaverine-induced relaxation of medial strips of pig coronary artery.

1. The mechanisms of vasorelaxation induced by papaverine were investigated using front-surface fluorometry and fura-2-loaded medial strips of the pig coronary artery. 2. In the presence of extracellular Ca2+ (1.25 x 10(-3) M), histamine (10(-4) M) induced abrupt elevations of cytosolic calcium concentration, [Ca2+]i reaching a peak within 12 s (the first phase); after making a slight shoulder, [Ca2+]i declined gradually to reach sustained levels (the second phase). Force rapidly rose to reach maximum levels in 3 min, then gradually declined. Papaverine (10(-7)-10(-5) M) inhibited both the first and the second phases of [Ca2+]i elevation and the development of force induced by histamine, in a concentration-dependent manner. 3. In the absence of extracellular Ca2+, histamine (10(-4) M) induced a transient increase in [Ca2+]i and force, both of which were inhibited in a concentration-dependent manner by papaverine (10(-7)-10(-5) M). When papaverine was washed out, a second application of 10(-4) M histamine also induced transient increases in [Ca2+]i and force. The smaller the first response, the greater was the second response. The total amount of [Ca2+]i released from intracellular stores by the first and second application of histamine in the presence of papaverine was smaller than in its absence, thereby indicating a reduction of Ca2+ in the histamine-sensitive store. However, while papaverine (10(-5) M) did not affect the transient increase in [Ca2+]i induced by 2 x 10(-2) M caffeine, contractions were inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel effect of N-palmitoyl-L-serine phosphoric acid on cytosolic Ca2+ levels in human osteoblasts

The effect of N-palmitoyl-L-serine phosphoric acid (L-NASPA), which has been used as an inhibitor of lysophosphatidic acid receptors, on intracellular Ca2+ concentration ([Ca2+]i) in human osteosarcoma MG63 cells was measured by using fura-2. L-NASPA (0.1-10 microM) caused a rapid and transient plateau [Ca2+]i rise in a concentration-dependent manner (EC50=0.5 microM). The L-NASPA-induced [Ca2+]i rise was partly reduced by removal of extracellular Ca2+ but was not altered by L-type voltage-gated Ca2+ channel blockers. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, induced a [Ca2+]i rise, after which the increasing effect of L-NASPA on [Ca2+]i was completely inhibited; also, pretreatment with L-NASPA partly reduced thapsigargin-induced [Ca2+]i rise. U73122, an inhibitor of phospholipase C, abolished histamine (but not L-NASPA)-induced [Ca2+]i rise. Overnight incubation with 1 microM L-NASPA did not affect cell proliferation, but 10-20 microM L-NASPA exerted 4% and 15% inhibition, respectively. Collectively, L-NASPA rapidly increased [Ca2+]i in MG63 cells by evoking both extracellular Ca2+ influx and intracellular Ca2+ release, and is cytotoxic at higher concentrations.     

Effect of riluzole on Ca2+ movement and cytotoxicity in Madin-Darby canine kidney cells

Riluzole is a drug used in the treatment of amyotrophic lateral sclerosis; however, its in vitro action is unclear. In this study, the effect of riluzole on intracellular Ca2+ concentration ([Ca2+]i) in Madin-Darby canine kidney (MDCK) cells was investigated using the Ca2+ -sensitive fluorescent dye, fura-2. Riluzole (100-500 microM) caused a rapid and sustained increase of [Ca2+]i in a concentration-dependent manner (EC50 = 150 microM). Some 40 and 50% of this [Ca2+]i increase was prevented by the removal of extracellular Ca2+ and the addition of La3+, respectively, but was unchanged by dihydropyridines, verapamil and diltiazem. In Ca2+ -free medium, thapsigargin - an inhibitor of the endoplasmic reticulum (ER) Caz+ -ATPase--caused a monophasic [Ca2+]i increase, after which the increasing effect of riluzole on [Ca2+]i was attenuated by 70%; in addition, pre-treatment with riluzole abolished thapsigargin-induced [Ca2+]i increases. U73122, an inhibitor of phospholipase C (PLC), abolished ATP (but not riluzole)-induced [Ca2+]i increases. At concentrations of 250 and 500 microM, riluzole killed 40 and 95% cells, respectively. The cytotoxic effect of riluzole (250 microM) was unaltered by pre-chelating cytosolic Ca2+ with BAPTA. Collectively, in MDCK cells, riluzole rapidly increased [Ca2+]i by stimulating extracellular Ca2+ influx via an La3+ -sensitive pathway and intracellular Ca2+ release from the ER via, as yet, unidentified mechanisms. Furthermore, riluzole caused Ca2+ -unrelated cytotoxicity in a concentration-dependent manner.     

Sevoflurane and bradykinin-induced calcium mobilization in pulmonary arterial valvular endothelial cells in situ: sevoflurane stimulates plasmalemmal calcium influx into endothelial cells

Kinins locally synthesized in the cardiovascular tissue are believed to contribute to the regulation of cardiovascular homeostasis by stimulating the endothelial cells to release nitric oxide, prostacyclin, or a hyperpolarizing factor via autocrine-paracrine mechanisms. This study was designed to investigate the action of sevoflurane on bradykinin-induced Ca2+ mobilization in endothelial cells in situ. Utilizing fura-2-loaded rat pulmonary arterial valve leaflets, the effects of sevoflurane were examined on bradykinin-induced increases in intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells in situ. In the presence of extracellular Ca2+ (1.5 mM), bradykinin (3-30 microM) produced an initial phasic and a subsequent tonic increase in [Ca2+]i in a concentration-dependent manner. However, it produced only the phasic increase in [Ca2+]i in the absence of extracellular Ca2+. Sevoflurane (5%, 0.67 mM) inhibited both the phasic and tonic responses to bradykinin. In these experiments, sevoflurane (3-5%) generated sustained increases (approximately 20-40% of the bradykinin-induced maximal increase in [Ca2+]i) in the resting [Ca2+]i level. Sevoflurane still increased [Ca2+]i after depletion of the intracellular Ca stores with ionomycin (0.1 microM ). However, the sevoflurane-induced increase in [Ca2+]i was eliminated by removal of the extracellular Ca and attenuated by NiCl (1-3 mM). In conclusion, in the pulmonary arterial valvular endothelial cells, sevoflurane inhibits both bradykinin-induced Ca2+ release from the intracellular stores and bradykinin-induced plasmalemmal Ca2+ influx. In addition, sevoflurane appears to stimulate the plasmalemmal Ca2+ influx and thereby increase the endothelial [Ca2+]i level. Sevoflurane might influence the pulmonary vascular tone through its direct action on the pulmonary arterial valvular endothelial cells.     

Glutamate-induced increase in intracellular Ca2+ concentration in isolated hippocampal neurones.

A system for real-time quantitative monitoring of intracellular free calcium ion concentration ([Ca2+]i) on a single cell basis was developed by the combination of a fluorescent Ca2+ indicator fura-2, a fluorescence microscope, a video-camera and photometrical devices. It was applied to rat individual hippocampal neurones under culture for detection of L-glutamate-induced alterations in the [Ca2+]i level. L-Glutamate (0.01-100 microM) induced a dose-dependent elevation of the [Ca2+]i. The [Ca2+]i in the rat hippocampal neurone was found to be around 30 nM in the resting state, and was increased up to 500 nM by the application of 100 microM L-glutamate. N-methyl-D-aspartate, kainate and quisqualate in a concentration of 10 microM also increased the [Ca2+]i level in the same single neurone, but their efficacy varied between individual cells. The L-glutamate-induced [Ca2+]i elevation was abolished after removal of extracellular Ca2+ and was much reduced by Mg2+ (3 mM). The increase was, however, still observed in a Na+-free medium. The L-glutamate-induced [Ca2+]i elevation was not affected substantially after treatment with nitrendipine (10 microM) which blocked the increase in [Ca2+]i induced by an isotonic high KCl-medium (50 mM). The present results suggest that the L-glutamate-induced [Ca2+]i elevation in the hippocampal neurone is due to an influx of Ca2+ through both L-glutamate receptor-coupled and voltage-sensitive ionic channels.     

The effects of ATP and alpha,beta-methylene-ATP on cytosolic Ca2+ level and force in rat isolated aorta.

1. The effects of a non-selective P2-receptor agonist ATP and a selective P2x-receptor agonist alpha,beta-methylene-ATP on intracellular free Ca2+ level ([Ca2+]i) and force were examined in rat isolated aorta without endothelium. 2. Both ATP (1-1000 microM) and alpha,beta-methylene-ATP (0.1-100 microM) induced transient increase followed by small sustained increase in [Ca2+]i in a concentration-dependent manner. Compared with the force induced by a high concentration of KCl, the force induced by alpha,beta-methylene-ATP was smaller and that induced by ATP was much smaller at a given [Ca2+]i. 3. An L-type Ca2+ channel blocker, verapamil (10 microM), completely inhibited the high K(+)-stimulated [Ca2+]i and force. Verapamil partially inhibited the transient and sustained increases in [Ca2+]i induced by 10 microM alpha,beta-methylene-ATP and the sustained increase but not the transient increase induced by 1 mM ATP. 4. In the absence of extracellular Ca2+ (with 0.5 mM EGTA) 1 mM ATP caused transient increase in [Ca2+]i while 10 microM alpha,beta-methylene-ATP was ineffective 5. ATP, but not alpha,beta-methylene-ATP, increased the tissue adenosine 3':5'-cyclic monophosphate (cyclic AMP) level. 6. These data suggest that ATP and alpha,beta-methylene-ATP increase [Ca2+]i by an activation of both L-type and non-L-type Ca2+ channels. In addition, ATP, but not alpha,beta-methylene-ATP, increases [Ca2+]i by a release of Ca2+ from an intracellular Ca2+ store. Possible reasons are discussed as to why the increase in [Ca2+]i due to ATP and alpha,beta-methylene-ATP resulted in only a small contraction.     

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.     

Vasorelaxation and inhibition of the voltage-operated Ca2+ channels by FK506 in the porcine coronary artery

Using fura-2 fluorometry, the effects of FK506, an immunosuppressant, on changes in cytosolic Ca2+ concentrations ([Ca2+]i) and tension were investigated in porcine coronary arterial strips. The effects of FK506 on the activity of voltage-operated Ca2+ channels were examined by applying a whole cell patch clamp to the isolated smooth muscle cells of porcine coronary artery. FK506 inhibited the sustained increases in both [Ca2+]i and tension induced by 118 mM K+ depolarization and 100 nM U46619 in a concentration-dependent manner (1-30 microM). The extent of inhibition of the K+-induced contraction was greater than that of the U46619-induced contraction. The increases in [Ca2+]i and tension induced by histamine and endothelin- in the presence of extracellular Ca2+ were also inhibited by 10 microM FK506. FK506 (10 microM) had no effect on Ca2+ release induced by caffeine or by histamine in the Ca2+-free solution. FK506 (10 microM) had no effect on the [Ca2+]i-tension relationships of the contractions induced by cumulative increases of extracellular Ca2+ during K+ depolarization or stimulation with U46619. In the patch clamp experiments, FK506 (30 microM) partially inhibited the inward current induced by depolarization pulse from -80 mV to 0 mV. In conclusion, FK506 induces arterial relaxation by decreasing [Ca2+]i mainly due to the inhibition of the L-type Ca2+ channels, with no effect on the Ca2+ sensitivity of the contractile apparatus.     

Mechanism of endothelium-dependent relaxation induced by substance P in the coronary artery of the pig.

1. Using front-surface fluorometry of fura-2-loaded porcine coronary arterial strips with the endothelium intact, we investigated the mechanisms of vasorelaxation induced by substance P (SP). Fura-2 fluorescence signals which indicated the cytosolic Ca2+-concentration ([Ca2+]i), were observed to arise exclusively from teh smooth muscle cells in these strips. 2. During the contractions induced by U46619 (100 nM), a thromboxane A2 analogue, an SP-induced endothelium-dependent, biphasic vasorelaxation was observed, which consisted of an initial rapid relaxation phase followed by a sustained phase, with a transient decrease in [Ca2+]i. Pretreatment with indomethacin (Ind) had no effect on the SP-induced relaxation; however, pretreatment with NG-nitro-L-arginine (L-NOARG) partially, but significantly inhibited the decrease in both the [Ca2+]i and tension abolished. Thus, part of the relaxation was considered to be mediated by L-NOARG-sensitive relaxing factor (endothelium-derived relaxing factor: EDRF). 3. During the 40 mM K+-depolarization-induced contraction which may eliminate the effects of endothelium-derived hyperpolarizing factor (EDRF), the vasorelaxation reduced by SP was completely inhibited by L-NOARG. 4. During the vasorelaxation induced SP, the [Ca2+]i-tension relationships shifted to the right of the contractions induced by either U46619 or high K+-depolarization. 5. Using front-surface fluorometry of fura-2 loaded porcine aortic valvular strips, we examined the effects of SP on [Ca2+]i in endothelial cells in situ. SP induced a rapid increase in [Ca2+]i of endothelial cells in situ followed by a small sustained phase in normal PSS (5.9 mM K+). The increase in extracellular K+ had no apparent effect on the SP-induced [Ca2+]i elevation of endothelial cells.