Mechanisms of bradykinin-mediated Ca(2+) signalling in canine cultured corneal epithelial cells

Experiments were designed to differentiate the mechanisms of bradykinin receptors mediating the changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) in canine cultured corneal epithelial cells (CECs). Bradykinin and Lys-bradykinin caused an initial transient peak of [Ca(2+)](i) in a concentration-dependent manner, with half-maximal stimulation (pEC(50)) obtained at 6.9 and 7.1, respectively. Pretreatment of CECs with pertussis toxin (PTX) or cholera toxin (CTX) for 24 h did not affect the bradykinin-induced [Ca(2+)](i) changes. Application of Ca(2+) channel blockers, diltiazem and Ni(2+), inhibited the bradykinin-induced Ca(2+) mobilization, indicating that Ca(2+) influx was required for the bradykinin-induced responses. Addition of thapsigargin (TG), which is known to deplete intracellular Ca(2+) stores, transiently increased [Ca(2+)](i) in Ca(2+)-free buffer, and subsequently induced Ca(2+) influx when Ca(2+) was readded to this buffer. Pretreatment of CECs with TG completely abolished bradykinin-induced initial transient [Ca(2+)](i), but had slight effect on bradykinin-induced Ca(2+) influx. Pretreatment of CECs with 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF96365) and 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122) inhibited the bradykinin-induced Ca(2+) release and Ca(2+) influx, consistent with the inhibition of receptor-gated Ca(2+) channels and phospholipase C (PLC) in CECs, respectively. These results demonstrate that bradykinin directly stimulates B(2) receptors and subsequently Ca(2+) mobilization via a PTX-insensitive G protein in canine CECs. These results suggest that bradykinin-induced Ca(2+) influx into the cells is not due to depletion of these Ca(2+) stores, as prior depletion of these pools by TG has no effect on the bradykinin-induced Ca(2+) influx that is dependent on extracellular Ca(2+) in CECs.     

Mechanism of bradykinin-induced Ca(2+) mobilization in MG63 human osteosarcoma cells.

BACKGROUND: The effect of bradykinin on intracellular free Ca(2+) levels ([Ca(2+)](i)) in MG63 human osteosarcoma cells was explored using fura-2 as a Ca(2+) dye. METHODS/RESULTS: Bradykinin (0.1 nM-1 microM) increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 0.5 nM. The [Ca(2+)](i) signal comprised an initial peak and a fast decay which returned to baseline in 2 min. Extracellular Ca(2+) removal inhibited the peak [Ca(2+)](i )signals by 35 +/- 3%. Bradykinin (1 nM) failed to increase [Ca(2+)](i) in the absence of extracellular Ca(2+ )after cells were pretreated with thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor; 1 microM). Bradykinin (1 nM)-induced intracellular Ca(2+) release was nearly abolished by inhibiting phospholipase C with 2 microM 1-(6-((17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). The [Ca(2+)](i )increase induced by 1 nM bradykinin in Ca(2+)- free medium was abolished by 1 nM HOE 140 (a B2 bradykinin receptor antagonist) but was not altered by 100 nM Des-Arg-HOE 140 (a B1 bradykinin receptor antagonist). Pretreatment with 1 pM pertussis toxin for 5 h in Ca(2+) medium inhibited 30 +/- 3% of 1 nM bradykinin-induced peak [Ca(2+)](i) increase. CONCLUSIONS: Together, this study shows that bradykinin induced [Ca(2+)](i) increases in a concentration-dependent manner, by stimulating B2 bradykinin receptors leading to mobilization of Ca(2+) from the thapsigargin-sensitive stores in a manner dependent on inositol-1,4,5-trisphosphate, and also by inducing extracellular Ca(2+) influx. The bradykinin response was partly coupled to a pertussis toxin-sensitive G protein pathway.     

Increased cytosolic Ca(2+) concentration in endothelial cells by calmodulin antagonists

Many functions of endothelial cells are Ca(2+)/calmodulin dependent, whereas the role of calmodulin in the regulation of cytosolic Ca(2+) ([Ca(2+)](i)) remains largely unexplained. In the present study, effects of various calmodulin antagonists on [Ca(2+)](i) were investigated in cultured aortic endothelial cells loaded with the Ca(2+)-sensitive dye fura-2/AM, and were compared with those of calmodulin-dependent protein kinase II (CaM kinase II) inhibitors. The calmodulin antagonists W-7, calmidazolium and fendiline provoked dose-dependent increases in [Ca(2+)](i). However, the CaM kinase II inhibitors KN-93 and lavendustin C had no effect on [Ca(2+)](i). In the absence of extracellular Ca(2+), pretreatment of cells with bradykinin (BK) and thapsigargin completely prevented W-7-stimulated increase in [Ca(2+)](i). Alternatively, pretreatment with W-7 also completely blocked BK- and thapsigargin-stimulated increases in [Ca(2+)](i). The time course of the Ca(2+)-response in W-7 treated cells was identical to that in thapsigargin-treated cells, but not that in BK-stimulated cells, suggesting that calmodulin antagonists could share a common signaling pathway with thapsigargin to increase [Ca(2+)](i) in endothelial cells. These findings indicate that calmodulin is involved in the regulation of [Ca(2+)](i), and may play an important role in the uptake of Ca(2+) to intracellular stores.     

Connexin 40 and ATP-dependent intercellular calcium wave in renal glomerular endothelial cells

Endothelial intracellular calcium ([Ca(2+)](i)) plays an important role in the function of the juxtaglomerular vasculature. The present studies aimed to identify the existence and molecular elements of an endothelial calcium wave in cultured glomerular endothelial cells (GENC). GENCs on glass coverslips were loaded with Fluo-4/Fura red, and ratiometric [Ca(2+)](i) imaging was performed using fluorescence confocal microscopy. Mechanical stimulation of a single GENC caused a nine-fold increase in [Ca(2+)](i), which propagated from cell to cell throughout the monolayer (7.9 +/- 0.3 microm/s) in a regenerative manner (without decrement of amplitude, kinetics, and speed) over distances >400 microm. Inhibition of voltage-dependent calcium channels with nifedipine had no effect on the above parameters, but the removal of extracellular calcium reduced Delta[Ca(2+)](i) by 50%. Importantly, the gap junction uncoupler alpha-glycyrrhetinic acid or knockdown of connexin 40 (Cx40) by transfecting GENCs with Cx40 short interfering RNA (siRNA) almost completely eliminated Delta[Ca(2+)](i) and the calcium wave. Breakdown of extracellular ATP using a scavenger cocktail (apyrase and hexokinase) or nonselective inhibition of purinergic P2 receptors with suramin, had similar blocking effects. Scraping cells off along a line eliminated physical contact between cells but did not effect calcium wave propagation. Using an ATP biosensor technique, we detected a significant elevation in extracellular ATP (Delta = 76 +/- 2 microM) during calcium wave propagation, which was abolished by Cx40 siRNA treatment (Delta = 6 +/- 1 microM). These studies suggest that connexin 40 hemichannels and extracellular ATP are key molecular elements of the glomerular endothelial calcium wave, which may serve important juxtaglomerular functions.     

C2-Ceramide increases cytoplasmic calcium concentrations in human parathyroid cells

Effects of extracellular calcium ([Ca(2+)](ext)) on parathyroid cells are mainly due to the activation of a plasma membrane calcium receptor (CaR) coupled with release of intracellular calcium. In addition, high [Ca(2+)](ext) activates the sphingomyelin pathway in bovine parathyroid cells, generating ceramides and sphingosine. This study explored the direct effects of synthetic ceramides on [Ca(2+)](i) in human parathyroid cells. Cells from five parathyroid adenomas removed from patients with primary hyperparathyroidism were dispersed and maintained in primary culture. Intracellular calcium concentration ([Ca(2+)](i)) [Ca(2+)](i) was monitored using standard quantitative fluorescence microscopy in Fura-2/AM-loaded cells. Laser scanning microscopy was used to monitor the intracellular distribution of a fluorescent ceramide analogue (BODIPY-C5). After addition of 10 microM C2-ceramide (N-acetyl-d-erythro-sphingosine), [Ca(2+)](i) increased rapidly (30-60 s) to a peak three times above basal levels in 70% of cells (37/55 cells in four experiments). This effect appeared to be due to release of Ca(2+) from intracellular stores rather than Ca(2+) entry from the extracellular medium. C2-responsive cells had a smaller [Ca(2+)](i) response to subsequent stimulation with the CaR agonist-neomycin (1 mM). These responses were specific to C2 since C6-ceramide (N-hexanoyl-d-erythro-sphingosine) did not affect basal [Ca(2+)](i) nor the responses to an increase in [Ca(2+)](ext) and to neomycin. C5-BODIPY generated intense perinuclear fluorescence, suggesting targeting of the ceramides to the Golgi apparatus. These data demonstrate that endogenous generation of ceramides has the potential to modulate changes in [Ca(2+)](i) and secretion in response to [Ca(2+)](ext) in human parathyroid cells.     

Aldosterone- and testosterone-mediated intracellular calcium response in skeletal muscle cell cultures

Fast nongenomic steroid actions in several cell types seem to be mediated by second messengers such as intracellular calcium ([Ca(2+)](i)) and inositol 1,4,5-trisphosphate (IP(3)). We have shown the presence of both slow calcium transients and IP(3) receptors associated with cell nuclei in cultured skeletal muscle cells. The effect of steroids on [Ca(2+)](i) was monitored in Fluo 3-acetoxymethyl ester-loaded myotubes by either confocal microscopy or fluorescence microscopy, with the use of out-of-focus fluorescence elimination. The mass of IP(3) was determined by radioreceptor displacement assay. [Ca(2+)](i) changes after either aldosterone (10-100 nM) or testosterone (50-100 nM) were observed; a relatively fast (<2 min) calcium transient, frequently accompanied by oscillations, was evident with both hormones. A slow rise in [Ca(2+)](i) that reached its maximum after a 30-min exposure to aldosterone was also observed. Calcium responses seem to be fairly specific for aldosterone and testosterone, because several other steroid hormones do not induce detectable changes in fluorescence, even at 100-fold higher concentrations. The mass of IP(3) increased transiently to reach two- to threefold the basal level 45 s after addition of either aldosterone or testosterone, and the IP(3) transient was more rapid than the fast calcium signal. Spironolactone, an inhibitor of the intracellular aldosterone receptor, or cyproterone acetate, an inhibitor of the testosterone receptor, had no effect on the fast [Ca(2+)](i) signal or in the increase in IP(3) mass. These signals could mean that there are distinct nongenomic pathways for the action of these two steroids in skeletal muscle cells.     

Luteinizing hormone (LH) acts through PKA and PKC to modulate T-type calcium currents and intracellular calcium transients in mice Leydig cells

LH increases the intracellular Ca(2+) concentration ([Ca(2+)](i)) in mice Leydig cells, in a process triggered by calcium influx through T-type Ca(2+) channels. Here we show that LH modulates both T-type Ca(2+) currents and [Ca(2+)](i) transients through the effects of PKA and PKC. LH increases the peak calcium current (at -20mV) by 40%. A similar effect is seen with PMA. The effect of LH is completely blocked by the PKA inhibitors H89 and a synthetic inhibitory peptide (IP-20), but only partially by chelerythrine (PKC inhibitor). LH and the blockers induced only minor changes in the voltage dependence of activation, inactivation or deactivation of the currents. Staurosporine (blocker of PKA and PKC) impaired the [Ca(2+)](i) changes induced by LH. A similar effect was seen with H89. Although PMA slowly increased the [Ca(2+)](i) the subsequent addition of LH still triggered the typical transients in [Ca(2+)](i). Chelerythrine also does not avoid the Ca(2+) transients, showing that blockage of PKC is not sufficient to inhibit the LH induced [Ca(2+)](i) rise. In summary, these two kinases are not only directly involved in promoting testosterone synthesis but also act on the overall calcium dynamics in Leydig cells, mostly through the activation of PKA by LH.     

Effects of capsaicin on Ca(2+) release from the intracellular Ca(2+) stores in the dorsal root ganglion cells of adult rats

We have investigated the effect of capsaicin on Ca(2+) release from the intracellular calcium stores. Intracellular calcium concentration ([Ca(2+)](i)) was measured in rat dorsal root ganglion (DRG) neurons using microfluorimetry with fura-2 indicator. Brief application of capsaicin (1 microM) elevated [Ca(2+)](i) in Ca(2+)-free solution. Capsaicin-induced [Ca(2+)](i) transient in Ca(2+)-free solution was evoked in a dose-dependent manner. Resiniferatoxin, an analogue of capsaicin, also raised [Ca(2+)](i) in Ca(2+)-free solution. Capsazepine, an antagonist of capsaicin receptor, completely blocked the capsaicin-induced [Ca(2+)](i) transient. Caffeine completely abolished capsaicin-induced [Ca(2+)](i) transient. Dantrolene sodium and ruthenium red, antagonists of the ryanodine receptor, blocked the effect of capsaicin on [Ca(2+)](i). However, capsaicin-induced [Ca(2+)](i) transient was not affected by 2-APB, a membrane-permeable IP(3) receptor antagonist. Furthermore, depletion of IP(3)-sensitive Ca(2+) stores by bradykinin and phospholipase C inhibitors, neomycin, and U-73122, did not block capsaicin-induced [Ca(2+)](i) transient. In conclusion, capsaicin increases [Ca(2+)](i) through Ca(2+) release from ryanodine-sensitive Ca(2+) stores, but not from IP(3)-sensitive Ca(2+) stores in addition to Ca(2+) entry through capsaicin-activated nonselective cation channel in rat DRG neurons.     

Carmustine augments the effects of tert-butyl hydroperoxide on calcium signaling in cultured pulmonary artery endothelial cells

The effects of oxidant stress and inhibition of glutathione reductase on the bradykinin-stimulated changes in cytosolic free Ca2+ concentration ([Ca2+]i) of calf pulmonary artery endothelial cells were determined using the intracellular fluorescent probe, fura-2. Changes in [Ca2+]i upon stimulation with bradykinin were measured after incubation of cells with the chemical oxidant tert-butyl hydroperoxide (0.4 mM) for various times. After 60 min, bradykinin-stimulated Ca2+ influx was significantly decreased. With more prolonged incubations with the peroxide, bradykinin had little effect on cytosolic calcium concentration. Preincubation of cells with the glutathione reductase inhibitor, carmustine, led to elevated basal [Ca2+]i, yet the cells remained responsive to bradykinin. However, incubation of carmustine-treated cells with tert-butyl hydroperoxide for 30 min dramatically reduced both bradykinin-stimulated release of Ca2+ from internal stores and influx of Ca2+ from the extracellular space. These results suggest that inhibition of glutathione reductase alters cytosolic Ca2+ homeostasis and enhances the effects of oxidative stress on signal transduction in vascular endothelial cells.     

Acute, nongenomic effect of thyroid hormones in preventing calcium overload in newborn rat cardiocytes

In this study, we examined the acute effects of thyroid hormones (TH) T(3) and T(4), leading to improvement of myocardial function through activation of Ca(2+) extrusion mechanisms and, consequently, prevention of intracellular calcium overload. Extracellular calcium elevation from 1.8 to 3.8 mM caused immediate increase in intracellular calcium level ([Ca(2+)](i)) in newborn cardiomyocyte cultures. Administration of 10 or 100 nM T(3) or T(4) rapidly (within 10 sec) decreased [Ca(2+)](i) to its control level. Similar results were obtained when [Ca(2+)](i) was elevated by decreasing extracellular Na(+) concentration, causing backward influx of Ca(2+) through Na(+)/Ca(2+) exchanger, or by administration of caffeine, releasing Ca(2+) from the sarcoplasmic reticulum (SR). Under these conditions, T(3) or T(4) decreased [Ca(2+)](i). T(3) and T(4) also exhibited protective effects during ischemia. T(3) or T(4) presence during hypoxia for 120 min in culture medium restricted the increase of [Ca(2+)](i) and prevented the pathological effects of its overload. An inhibitor of SR Ca(2+)-ATPase (SERCA2a), thapsigargin, increases [Ca(2+)](i) and in its presence neither T(3) nor T(4) had any effect on the [Ca(2+)](i) level. The reduction of [Ca(2+)](i) level by T(3) and T(4) was also blocked in the presence of H-89 (a PKA inhibitor), and by calmodulin inhibitors. The effect of TH on the reduction of [Ca(2+)](i) was prevented by propranolol, indicating that the hormones exert their effect through interaction with adrenergic receptors. These results support our hypothesis that TH prevent calcium overload in newborn rat cardiomyocytes, most likely by a direct, acute, and nongenomic effect on Ca(2+) transport into the SR.     

ANG II AT2 receptor modulates AT1 receptor-mediated descending vasa recta endothelial Ca2+ signaling

We tested whether the respective angiotensin type 1 (AT(1)) and 2 (AT(2)) receptor subtype antagonists losartan and PD-123319 could block the descending vasa recta (DVR) endothelial intracellular calcium concentration ([Ca(2+)](i)) suppression induced by ANG II. ANG II partially reversed the increase in [Ca(2+)](i) generated by cyclopiazonic acid (CPA; 10(-5) M), acetylcholine (ACh; 10(-5) M), or bradykinin (BK; 10(-7) M). Losartan (10(-5) M) blocked that effect. When vessels were treated with ANG II before stimulation with BK and ACh, concomitant AT(2) receptor blockade with PD-123319 (10(-8) M) augmented the suppression of endothelial [Ca(2+)](i) responses. Similarly, preactivation with the AT(2) receptor agonist CGP-42112A (10(-8) M) prevented AT(1) receptor stimulation with ANG II + PD-123319 from suppressing endothelial [Ca(2+)](i). In contrast to endothelial [Ca(2+)](i) suppression by ANG II, pericyte [Ca(2+)](i) exhibited typical peak and plateau [Ca(2+)](i) responses that were blocked by losartan but not PD-123319. DVR vasoconstriction by ANG II was augmented when AT(2) receptors were blocked with PD-123319. Similarly, AT(2) receptor stimulation with CGP-42112A delayed the onset of ANG II-induced constriction. PD-123319 alone (10(-5) M) showed no AT(1)-like action to constrict microperfused DVR or increase pericyte [Ca(2+)](i). We conclude that ANG II suppression of endothelial [Ca(2+)](i) and stimulation of pericyte [Ca(2+)](i) is mediated by AT(1) or AT(1)-like receptors. Furthermore, AT(2) receptor activation opposes ANG II-induced endothelial [Ca(2+)](i) suppression and abrogates ANG II-induced DVR vasoconstriction.     

Pressure-induced endothelial Ca(2+) oscillations in lung capillaries

Endothelial second messenger responses may contribute to the pathology of high vascular pressure but remain poorly understood because of the lack of direct in situ quantification. In lung venular capillaries, we determined endothelial cytosolic Ca(2+) concentration [Ca(2+)](i) by the fura 2 ratioing method. Pressure elevation increased mean endothelial [Ca(2+)](i) by Ca(2+) influx through gadolinium-inhibitable channels and amplified [Ca(2+)](i) oscillations by Ca(2+) release from intracellular stores. Endothelial [Ca(2+)](i) transients were induced by pressure elevations of as little as 5 cmH(2)O and increased linearly with higher pressures. Heptanol inhibition of [Ca(2+)](i) oscillations in a subset of endothelial cells indicated that oscillations originated from pacemaker endothelial cells and were propagated to adjacent nonpacemaker cells by gap junctional communication. Our findings indicate the presence of a sensitive, active endothelial response to pressure challenge in lung venular capillaries that may be relevant in the pathogenesis of pressure-induced lung microvascular injury.     

Invited review: effects of flow on vascular endothelial intracellular calcium signaling of rat aortas ex vivo.

To study the effects of flow on in situ endothelial intracellular calcium concentration ([Ca(2+)](i)) signaling, rat aortic rings were loaded with fura 2, mounted on a tissue flow chamber, and divided into control and flow-pretreated groups. The latter was perfused with buffer at a shear stress of 50 dyns/cm(2) for 1 h. Endothelial [Ca(2+)](i) responses to ACh or shear stresses were determined by ratio image analysis. Moreover, ACh-induced [Ca(2+)](i) elevation responses were measured in a calcium-free buffer, or in the presence of SKF-96365, to elucidate the role of calcium influx in the flow effects. Our results showed that 1) ACh increased endothelial [Ca(2+)](i) in a dose-dependent manner, and these responses were incremented by flow-pretreatment; 2) the differences in ACh-induced [Ca(2+)](i) elevation between control and flow-pretreated groups were abolished by SKF-96365 or by Ca(2+)-free buffer; and 3) in the presence of 10(-5) M ATP, shear stress induced dose-dependent [Ca(2+)](i) elevation responses that were not altered by flow-pretreatment. In conclusion, flow-pretreatment augments the ACh-induced endothelial calcium influx in rat aortas ex vivo.     

Activation of muscarinic acetylcholine receptors induces Ca(2+) mobilization in FRT cells

The effect of the muscarinic receptors agonist carbachol (Cch) on intracellular calcium concentration ([Ca(2+)](i)) and cAMP level was studied in polarized Fischer rat thyroid (FRT) epithelial cells. Cch provoked a transient increase in [Ca(2+)](i), followed by a lower sustained phase. Thapsigargin, a specific microsomal Ca(2+)-ATPase inhibitor, caused a rapid rise in [Ca(2+)](i) and subsequent addition of Cch was without effect. Removal of extracellular Ca(2+) reduced the initial transient response and completely abolished the plateau phase. Ryanodine, an agent that depletes intracellular Ca(2+) stores through stimulation of ryanodine receptors (RyRs), had no effect on [Ca(2+)](i). However, the transitory activation of [Ca(2+)](i) was dose-dependently attenuated in cells pretreated with U73122, a specific inhibitor of phospholipase C (PLC). These data suggest that the Cch-stimulated increment of [Ca(2+)](i) required IP(3) formation and binding to its specific receptors in Ca(2+) stores. Further studies were performed to investigate whether the effect of Cch on Ca(2+) entry into FRT cells was via L-type voltage-dependent Ca(2+) channels (L-VDCCs). Nicardipine, a nonspecific L-type Ca(2+) channel blocker, decreased Cch-induced increase on [Ca(2+)](i), while Bay K-8644, an L-type Ca(2+) channel agonist, slightly increased [Ca(2+)](i) in FRT cells. These data indicate that Ca(2+) entry into these nondifferentiated thyroid cells occurs through an L-VDCC, and probably through another mechanism such as a capacitative pathway. Cch did not affect the intracellular cAMP levels, but its effects on [Ca(2+)](i) were significantly reduced when cells were pretreated with forskolin, suggesting the existence of an intracellular cross-talk between PLC and cAMP mechanisms in the regulation of intracellular Ca(2+) mobilization in neoplastic FRT cells.     

High molecular weight kininogen activates B2 receptor signaling pathway in human vascular endothelial cells

The nonenzymatic cofactor high molecular weight kininogen (HK) is a precursor of bradykinin (BK). The production of BK from HK by plasma kallikrein has been implicated in the pathogenesis of inflammation and vascular injury. However, the functional role of HK in the absence of prekallikrein (PK), the proenzyme of plasma kallikrein, on vascular endothelial cells is not fully defined. In addition, no clinical abnormality is seen in PK-deficient patients. Therefore, an investigation into the effect of HK, in the absence of PK, on human pulmonary artery endothelial cell (HPAEC) function was performed. HK caused a marked and dose-dependent increase in the intracellular calcium [Ca(2+)](i) level in HPAEC. Gd(3+) and verapamil potentiated the HK-induced increase in [Ca(2+)](i). HK-induced Ca(2+) increase stimulated endothelial nitric oxide (NO) and prostacyclin (PGI(2)) production. The inhibitors of B(2) receptor-dependent signaling pathway impaired HK-mediated signal transduction in HPAEC. HK had no effect on endothelial permeability at physiological concentration. This study demonstrated that HK regulates endothelial cell function. HK could play an important role in maintaining normal endothelial function and blood flow and serve as a cardioprotective peptide.     

Mobilization of intracellular calcium by peroxynitrite in arteriolar smooth muscle cells from rats

The present study was designed to investigate the possible effects of peroxynitrite (ONOO(-)) on the intracellular calcium concentration ([Ca(2+)](i)) of mesenteric arteriolar smooth muscle cells (ASMCs), and to reveal the underlying mechanisms by using fluorescence imaging analysis. The results showed that ONOO(-) could exert a concentration- and time-dependent but also a dual effect on [Ca(2+)](i). Bolus administration with a low concentration of ONOO(-) (25 microM) decreased [Ca(2+)](i), whereas higher concentrations (50 or 100 microM) increased [Ca(2+)](i) persistently. Further experiments demonstrated that pretreatment of ASMCs with calcium-free medium completely abolished [Ca(2+)](i) increase by 100 microM ONOO(-). Additionally, nifedipine, an antagonist of selective L-type voltage-gated calcium channels (VGCCs), delayed the [Ca(2+)](i) response to ONOO(-), and ryanodine, an inhibitor of intracellular calcium release from the sarcoplasmic reticulum, effectively antagonized [Ca(2+)](i) increase during the late stage of ONOO(-) exposure. Furthermore, [Ca(2+)](i) alteration by ONOO(-) appeared to be intimately associated with the subsequent membrane potential changes. Although the mechanisms by which ONOO(-) alters [Ca(2+)](i) are complex, we conclude that a series of variables such as external calcium influx, activation of VGCCs, intracellular calcium release, and membrane potential changes are involved. The decrease of [Ca(2+)](i) in ASMCs by a low concentration of ONOO(-) may participate in the pathogenesis of low vasoreactivity in shock, and the increase of [Ca(2+)](i) by high concentrations of ONOO(-) may lead to calcium overload with cellular injury.     

P2Y-receptor regulates size of endothelial cells in an intracellular Ca2+ dependent manner

The effects of P2 receptor agonists on cell size and intracellular calcium levels, [Ca(2+)](i), was investigated using cultured endothelial cells isolated from the caudal artery of male Wistar rats. Cell size and [Ca(2+)](i) were measured using a phase-contrast and fluorescent confocal microscopic image analyzer and a Calcium Green fluorescence probe. P2Y receptor agonists, 2-methylthio ATP (2meS-ATP), ADP, UTP and ATP decreased the cell size and increased [Ca(2+)](i) in endothelial cells from rat caudal artery. However, alpha,beta-methylene ATP, a P2X receptor agonist, did not induce these responses. The decrease in size and the increase in [Ca(2+)](i), by 2meS-ATP were blocked by PPADS (P2-antagonist), suramin (P2-antagonist), thapsigargin (Ca(2+) pump inhibitor) and U-73122 (phospholipase C inhibitor). The present results show that activation of P2Y receptors, not P2X receptors, induces a decrease in cell size and an increase in [Ca(2+)](i), and the pharmacological properties of these two responses are the same. We concluded that the size of endothelial cells is regulated by P2Y receptors via intracelluar Ca(2+) derived from Ca(2+) stores.     

Altered intracellular calcium homeostasis in cerebellar granule cells of prion protein-deficient mice

Previous studies have indicated that recombinant cellular prion protein (PrP(C)), as well as a synthetic peptide of PrP(C), affects intracellular calcium homeostasis. To analyze whether calcium homeostasis in neurons is also affected by a loss of PrP(C), we performed microfluorometric calcium measurements on cultured cerebellar granule cells derived from prion protein-deficient (Prnp(0/0)) mice. The resting concentration of intracellular free calcium [Ca(2+)](i) was found to be slightly, but significantly, reduced in Prnp(0/0) mouse granule cell neurites. Moreover, we observed a highly significant reduction in the [Ca(2+)](i) increase after high potassium depolarization. Pharmacological studies further revealed that the L-type specific blocker nifedipine, which reduces the depolarization-induced [Ca(2+)](i) increase by 66% in wild-type granule cell somas, has no effect on [Ca(2+)](i) in Prnp(0/0) mouse granule cells. Patch-clamp measurements, however, did not reveal a reduced calcium influx through voltage-gated calcium channels in Prnp(0/0) mice. These data clearly indicate that loss of PrP(C) alters the intracellular calcium homeostasis of cultured cerebellar granule cells. There is no evidence, though, that this change is due to a direct alteration of voltage-gated calcium channels.     

低浓度双氢哇巴因对豚鼠心室肌细胞内游离钙浓度的影响

用激光共聚焦显微镜检查研究低浓度双氢哇巴因（DHO）对豚鼠心室肌细胞内钙浓度（[Ca^2 ]i)的影响。DHO 1fmol/L-1 mmol/L可增加心室肌细胞的[Ca^2 ]i,尤其以10pmol/L DHO为显著,Nisoldipine,EGTA或TTX可分别部分抑制10pmol/L DHO的作用,去除胞外K^ 和Na^ 后,上述作用仍存在,以上结果表明,低浓度DHO中通过激活钙通道和TTX敏感的钠通道,或许还可直接促进胞内钙释放来增加[Ca^2 ]i,并有不依赖Na^ /K^ 泵而升高[Ca^2 ]i的作用。     

Diabetes alters intracellular calcium transients in cardiac endothelial cells

Diabetic cardiomyopathy (DCM) is a diabetic complication, which results in myocardial dysfunction independent of other etiological factors. Abnormal intracellular calcium ([Ca(2+)](i)) homeostasis has been implicated in DCM and may precede clinical manifestation. Studies in cardiomyocytes have shown that diabetes results in impaired [Ca(2+)](i) homeostasis due to altered sarcoplasmic reticulum Ca(2+) ATPase (SERCA) and sodium-calcium exchanger (NCX) activity. Importantly, altered calcium homeostasis may also be involved in diabetes-associated endothelial dysfunction, including impaired endothelium-dependent relaxation and a diminished capacity to generate nitric oxide (NO), elevated cell adhesion molecules, and decreased angiogenic growth factors. However, the effect of diabetes on Ca(2+) regulatory mechanisms in cardiac endothelial cells (CECs) remains unknown. The objective of this study was to determine the effect of diabetes on [Ca(2+)](i) homeostasis in CECs in the rat model (streptozotocin-induced) of DCM. DCM-associated cardiac fibrosis was confirmed using picrosirius red staining of the myocardium. CECs isolated from the myocardium of diabetic and wild-type rats were loaded with Fura-2, and UTP-evoked [Ca(2+)](i) transients were compared under various combinations of SERCA, sarcoplasmic reticulum Ca(2+) ATPase (PMCA) and NCX inhibitors. Diabetes resulted in significant alterations in SERCA and NCX activities in CECs during [Ca(2+)](i) sequestration and efflux, respectively, while no difference in PMCA activity between diabetic and wild-type cells was observed. These results improve our understanding of how diabetes affects calcium regulation in CECs, and may contribute to the development of new therapies for DCM treatment.