Dendritic cell development: multiple pathways to nature''s adjuvants

The endothelin (ET) isoforms ET-1, ET-2 and ET-3 applied at 100 nM triggered a transient increase in [Ca2+]i in Bergmann glial cells in cerebellar slices acutely isolated from 20-25 day-old mice. The intracellular calcium concentration ([Ca2+]i) was monitored using Fura-2-based [Ca2+]i microfluorimetry. The ET-triggered [Ca2+]i transients were mimicked by ETB receptor agonist BQ-3020 and were inhibited by ETB receptor antagonist BQ-788. ET elevated [Ca2+]i in Ca(2+)-free extracellular solution and the ET-triggered [Ca2+]i elevation was blocked by 500 nM thapsigargin indicating that the [Ca2+]i was released from InsP3-sensitive intracellular pools. The ET-triggered [Ca2+]i increase in Ca(2+)-free solution was shorter in duration. Restoration of normal extracellular [Ca2+] briefly after the ET application induced a second [Ca2+]i increase indicating the presence of a secondary Ca2+ influx which prolongs the Ca2+ signal. Pre-application of 100 microM ATP or 10 microM noradrenaline blocked the ET response suggesting the involvement of a common Ca2+ depot. The expression of ETB receptor mRNAs in Bergmann glial cells was revealed by single-cell RT-PCR. The mRNA was also found in Purkinje neurones, but no Ca2+ signalling was triggered by ET. We conclude that Bergmann glial cells are endowed with functional ETB receptors which induce the generation of intracellular [Ca2+]i signals by activation of Ca2+ release from InsP3-sensitive intracellular stores followed by a secondary Ca2+ influx.     

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. 4. Pertussis toxin (IAP) markedly inhibited the extracellular Ca2+-dependent elevation of [Ca2+]j, but had no effect on the extracellular Ca2+-independent elevation of [Ca2+], caused by ET-1 (10-7M).5. Bradykinin (10-7 M) or ATP (10- 5M) elevated [Ca2+]i and these responses also consisted of extracellular Ca2+-independent and extracellular Ca2+-dependent components. IAP had no effect on either component of the [Ca2+]i elevation induced by bradykinin or ATP.6. From these findings we conclude that, in porcine endotheliel cells in situ, ET-1 elevates [Ca2+]i as are result of a Ca2+ influx component from the extracellular space and release of intracelluarly stored Ca2+ .The Ca2+ influx is regulated by an IAP-sensitive G-protein, while the release of Ca2+ from the intracellular store is not.     

The phospholipase C inhibitor U73122 increases cytosolic calcium in MDCK cells by activating calcium influx and releasing stored calcium

The effects of the phospholipase C (PLC) inhibitor U73122 on intracellular calcium levels ([Ca2+]i) were studied in MDCK cells. U73122 elevated [Ca2+]i dose-dependently. Ca2+ influx contributed to 75% of 20 microM U73122-induced Ca2+ signals. U73122 pretreatment abolished the [Ca2+]i transients evoked by ATP and bradykinin, suggesting that U73122 inhibited PLC. The Ca2+ signals among individual cells varied considerably. The internal Ca2+ source for the U73122 response was the endoplasmic reticulum (ER) since the response was abolished by thapsigargin. The depletion of the ER Ca2+ store triggered a La3+-sensitive capacitative Ca2+ entry. Independently of the internal release and capacitative Ca2 entry, U73122 directly evoked Ca2+ influx through a La3+-insensitive pathway. The U73122 response was augmented by pretreatment of carbonylcyanide m-chlorophynylhydrozone (CCCP), but not by Na+ removal, implicating that mitochondria contributed significantly in buffering the Ca2+ signal, and that efflux via Na+/Ca2+ exchange was insignificant.     

Studies on the microsomal cytochrome P450s in the livers of carcinogen-resistant and sensitive rats during long-term administration of 3''-methyl-4-dimethylaminoazobenzene

The fluorescent indicator Fura-2 was used to characterize the store-operated Ca2+ entry in insulin-releasing pancreatic beta-cells. To avoid interference with voltage-dependent Ca2+ entry, the cells were hyperpolarized with 400 microM diazoxide and the channel blocker methoxyverapamil was also present in some experiments. The cytoplasmic Ca2+ concentration ([Ca2+]j) of hyperpolarized mouse beta-cells was strikingly resistant to changes in external Ca2+. In cells exposed to 20 mM glucose, stimulation with 100 microM carbachol induced an initial [Ca2+]j peak followed by a sustained increase due to store-operated influx of the cation. Store-operated influx was also induced by the intracellular Ca(2+)-ATPase inhibitor thapsigargin. In the presence of store-operated influx, [Ca2+]j became markedly sensitive to variations in external Ca2+, but this sensitivity was blocked by La3+. In beta-cells exposed to both Ca2+ and Mn2+ there was slow Mn2+ quenching of the Fura-2 fluorescence, which was accelerated upon stimulation of store-operated influx. This acceleration was reversed by glucose-stimulated filling of the internal Ca2+ stores. The store-operated Ca2+ entry increased markedly during culture of the beta-cells. Activation of protein kinase C by the phorbol ester 12-O-tetradecanoylphorbol-13 acetate, inhibition of serine/threonine phosphatase by okadaic acid and inhibition of tyrosine kinase by genistein had little effect on the store-operated influx of Ca2+. In beta-cells equilibrated in 5 mM Sr2+, carbachol exposure resulted in a pronounced cytoplasmic Sr2+ ([Sr2+]j) peak due to intracellular mobilization, but little or no sustained elevation. Moreover, after activating the store-operated pathway by exposure to thapsigargin, variations in extracellular Sr2+ between 0-2 mM had only marginal effects on [Sr2+]j. Although the store-operated influx apparently accounts for a minor fraction of the Ca2+ entry, its depolarizing influence may under certain conditions be up-regulated with resulting distortion of the beta-cell function.     

Different mechanisms for [Ca2+]i oscillations induced by carbachol and high concentrations of [Ca2+]o in the rat ventricular myocyte

1. The purpose of the present study was to explore the different mechanisms of [Ca2+]i oscillations induced by high concentrations of either carbachol (CCh) or extracellular Ca2+ ([Ca2+]o). First, we compared the oscillations induced by CCh at concentrations of 100-300 micromol/L and [Ca2+]o (5 mmol/L) in the single rat ventricular myocyte. Second, we studied CCh- and [Ca2+]o-induced [Ca2+]i oscillations following either interference with the production of inositol trisphosphate (IP3), reductions in cytosolic Ca2+ ([Ca2+]i), inhibition of Ca2+ influx and Na+-Ca2+ exchange or depletion of Ca2+ from its intracellular store. 2. The [Ca2+]i oscillations induced by CCh were frequent and were superimposed on [Ca2+]i transients in electrically stimulated cells, whereas those induced by high [Ca2+]o were occasional and occurred in quiescent cells and between [Ca2+]i transients in electrically stimulated cells. In both cases, [Ca2+]i oscillations were preceded by an increase in resting levels of [Ca2+]i. 3. Carbachol-induced [Ca2+]i oscillations were accompanied by an increase in amplitude and prolongation of the time of decline to 80% of the peak of the [Ca2+]i transient, while high [Ca2+]o-induced [Ca2+]i oscillations were the opposite. 4. A reduction of [Ca2+]o to 0.1 mmol/L and treatment with Ni2+ or ryanodine or 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid AM (BAPTA-AM) abolished the [Ca2+]i oscillations induced by both CCh and high [Ca2+]o. 5. The calcium channel blockers verapamil and nifedipine and inhibitors of phospholipase C (neomycin and U-73122) abolished the [Ca2+]i oscillations induced by CCh; Li+ accelerated the onset of the [Ca2+]i oscillations induced by CCh. 6. These observations suggest that the mechanisms responsible for the [Ca2+]i oscillations induced by CCh and high [Ca2+]o are different from each other. Other than an increase in extracellular Ca2+ influx as a mechanism common for both CCh- and high [Ca2+]o-induced [Ca2+]i oscillations, the CCh-induced [Ca2+]i oscillations involve influx of Ca2+ via L-type Ca2+ channels, Na+-Ca2+ exchange, mobilization of intracellular Ca2+ and IP3 production.     

Effect of 5-hydroxytryptamine on intracellular calcium dynamics in cultured rat vascular smooth muscle cells

The present study elucidated the precise mechanism of 5-hydroxytryptamine (5-HT)-induced increase of intracellular Ca2+ concentration ([Ca2+]i) in cultured vascular smooth muscle cells isolated from rat aortic media. [Ca2+]i was measured using fluorescent Ca2+ indicator, fura-2. 5-HT caused a dose-dependent increase in [Ca2+]i, which was completely inhibited by ketanserin. alpha-Methyl-5-HT had an equipotent effect to 5-HT. Diltiazem at 10 microM partially suppressed the 5-HT-induced increase in [Ca2+]i. 5-HT also augmented Mn2+ influx, when monitored by Mn2+ quenching of fura-2 fluorescence. When extracellular Ca2+ (1.3 mM) was removed, a decrease in resting level and a small, transient increase in [Ca2+]i were observed. 5-HT stimulation also induced an increase in the production of inositol triphosphate. 5-HT-induced increase in [Ca2+]i was significantly, but partially inhibited by staurosporin and H-7. Phorbol 12-myristate 13-acetate induced an increase in [Ca2+]i, which was abolished by removal of extracellular Ca2+. 5-HT-induced increase in [Ca2+]i was not affected by the pretreatment with pertussis toxin (PTX), and was not accompanied by a change in cyclic AMP content. These results suggest that, in cultured rat aortic smooth muscle cells, 5-HT increases [Ca2+]i via 5-HT2 receptor subtype by inducing influx of extracellular Ca2+ partially through L-type voltage-dependent Ca2+ channel, as well as by mobilizing Ca2+ from its intracellular stores. Activation of protein kinase C may be positively involved in the regulatory mechanism of Ca2+ influx, but PTX-sensitive G protein and cyclic AMP seem to be not involved.     

Delta9-tetrahydrocannabinol activates [Ca2+]i increases partly sensitive to capacitative store refilling

Delta9-tetrahydrocannabinol induces [Ca2+]i increases in DDT1MF-2 smooth muscle cells. Both Ca2+ entry and release from intracellular Ca2+ stores were concentration dependently activated. The Ca2+ entry component contributed most to the increases in [Ca2+]i. Stimulation with delta9-tetrahydrocannabinol after functional downregulation of intracellular Ca2+ stores by longterm thapsigargin treatment, still induced a major Ca2+ entry and a minor Ca2+ release component. Thapsigargin sensitive influx and release were selectively inhibited by the cannabinoid CB1 receptor antagonist SR141716A. No effects on [Ca2+]i were obtained after stimulation with the CB2 receptor agonist palmitoylethanolamide. This study is the first demonstration of (1) Ca2+ release from thapsigargin sensitive intracellular stores and capacitative Ca2+ entry via CB1 receptor stimulation and of (2) an additional delta9-tetrahydrocannabinol induced thapsigargin insensitive component, mainly representing Ca2+ influx which is neither mediated by CB1 nor CB2 receptor stimulation.     

Dissociation of intracellular Ca2+ release and Ca2+ entry response to 5-hydroxytryptamine in cultured canine tracheal smooth muscle cells

The relationship between the agonist-sensitive Ca2+ pool and those discharged by the Ca2+ -ATPase inhibitor thapsigargin (TG) were investigated in canine tracheal smooth muscle cells (TSMCs). In fura-2-loaded TSMCs, 5-hydroxytryptamine (5-HT) stimulated a rapid increase in intracellular Ca2+ ([Ca2+]i), followed by a sustained plateau phase that was dependent on extracellular Ca2+. In such cells, TG produced a concentration-dependent increase in [Ca2+]i, which remained elevated over basal level for several minutes and was substantially attenuated in the absence of extracellular Ca2+. Application of 5-HT after TG demonstrated that the TG-sensitive compartment partly overlapped the 5-HT-sensitive stores. Pre-treatment of TSMCs with TG significantly inhibited the increase in [Ca2+]i induced by 5-HT in a time-dependent manner. Similar results were obtained with two other Ca2+ -ATPase inhibitors, cyclopiazonic acid and 2,5-di-t-butylhydroquinone. Although these inhibitors had no effect on phosphoinositide hydrolysis, Ca2+ -influx was stimulated by these agents. These results suggest that depletion of the agonist-sensitive Ca2+ stores is sufficient for activation of Ca2+ influx. Some characteristics of the Ca2+ -influx activated by depletion of internal Ca2+ stores were compared with those of the agonist-activated pathway. 5-HT-stimulated Ca2+ influx was inhibited by La3+, membrane depolarisation, and the novel Ca2+ -influx blocker 1-?beta-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl?-1H-imidazole hydrochloride (SKF96365). Likewise, activation of Ca2+ influx by TG also was blocked by La3+, membrane depolarisation, and SKF96365. These results suggest that (1) in the absence of PI hydrolysis, depletion of the agonist-sensitive internal Ca2+ stores in TSMCs is sufficient for activation of Ca2+ influx, and (2) the agonist-activated Ca2+ influx pathway and the influx pathway activated by depletion of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool are indistinguishable.     

Calcium entry activated by store depletion in human umbilical vein endothelial cells

We have used the patch clamp technique combined with simultaneous measurement of intracellular Ca2+ to record ionic currents activated by depletion of intracellular Ca(2+)-stores in endothelial cells from human umbilical veins. Two protocols were used to release Ca2+ from intracellular stores, i.e. loading of the cells via the patch pipette with Ins(1,4,5)P3, and extracellular application of thapsigargin. Ins(1,4,5)P3 (10 microM) evoked a transient increase in [Ca2+]i in cells exposed to Ca(2+)-free extracellular solutions. A subsequent reapplication of extracellular Ca2+ induced an elevation of [Ca2+]i. These changes in [Ca2+]i were very reproducible. The concomitant membrane currents were neither correlated in time nor in size with the changes in [Ca2+]i. Similar changes in [Ca2+]i and membrane currents were observed if the Ca(2+)-stores were depleted with thapsigargin. Activation of these currents was prevented and holding currents at -40 mV were small if store depletion was induced in the presence of 50 microM NPPB. This identifies the large currents, which are activated as a consequence of store-depletion, as mechanically activated Cl- currents, which have been described previously [1,2]. Loading the cells with Ins(1,4,5)P3 together with 10 mM BAPTA induced only a very short lasting Ca2+ transient, which was not accompanied by activation of a detectable current, even in a 10 mM Ca(2+)-containing extracellular solution. Also thapsigargin does not activate any membrane current if the pipette solution contains 10 mM BAPTA (ruptured patches). The contribution of Ca(2+)-influx to the membrane current during reapplication of 10 mM extracellular calcium to thapsigargin-pretreated cells was estimated from the first time derivative of the corresponding Ca2+ transients at different holding potentials. These current values showed strong inward rectification, with a maximal amplitude of 1.0 +/- 0.3 pA at -80 mV (n = 8; membrane capacitance 59 +/- 9 pF).(ABSTRACT TRUNCATED AT 250 WORDS)     

From angels to handmaidens: changing constructions of nursing''s public image in post-war Britain

The molecular and ionic mechanisms responsible for the regulation of mucus exocytosis in human airway gland cells remain poorly defined. To determine whether dynamic changes of intracellular free Ca2+ concentration [Ca2+]i can promote different exocytotic responses, we monitored dynamic changes in [Ca2+]i and secretory granule (SG) exocytosis in individual human tracheal submucosal serous gland (HTG) cells. These changes were in response to exposure of the cells to three different secretagogues associated with airway inflammation and disease: human neutrophil elastase (HNE), histamine, and ATP. Dynamic changes in [Ca2+]i from single cells were determined with Indo-1/AM using quantitative UV laser microspectrofluorometry. The rate of SG exocytosis was measured in single cells by fluorescence videomicroscopy of SG degranulation and by the ELISA method. Exposure of HTG cells to a low concentration of HNE (1.0 microM) caused a high rate of SG exocytosis (52% decrease in the initial quinacrine fluorescence) during the first 8-min stimulation period compared with that observed following exposure of the cells to 100 microM histamine (10% decrease) or 100 microM ATP (6% decrease). In contrast to a rapid and transient rise in [Ca2+]i induced by histamine (1.0-100 microM) and ATP (10-100 microM), HNE (0.01-1 microM) generated asynchronous oscillations in [Ca2+]i over the first 8-min period. Depletion of internal Ca2+ stores with thapsigargin (500 nM) induced a significant reduction (P < 0.01) in the observed increases in [Ca2+]i upon addition of each of the secretagogues, but did not greatly affect the SG exocytotic responses. Interestingly, the removal of extracellular Ca2+ (+5 mM EGTA) significantly reduced (P < 0.01) both the [Ca2+]i increases and the rate of SG exocytosis following exposure to the secretagogues. We also demonstrate that the influx of extracellular Ca2+ and [Ca2+]i oscillations rather than the absolute level of [Ca2+]i regulate the rapid onset and extent of exocytotic responses to HNE in airway gland cells. Taken together, these results provide strong evidence that [Ca2+]i is a critical intracellular messenger in the regulation of exocytosis process in human airway gland cells.     

Long-term activation of capacitative Ca2+ entry in mouse microglial cells

The cytoplasmic free calcium concentration ([Ca2+]i) was measured in cultured microglial cells with the Ca2+-sensitive fluorescent dye Fura-2 using a digital imaging system. Stimulation of P2 purinergic receptors by ATP or UTP always evoked a [Ca2+]i elevation. The ATP-induced Ca2+ response involved both Ca2+ influx through ionotropic receptors and Ca2+ release from intracellular pools, whereas UTP selectively stimulated intracellular Ca2+ release. When intracellular Ca2+ release was stimulated in the absence of extracellular Ca2+, the readmission of extracellular Ca2+ caused a large rebound [Ca2+]i increase. Following this rebound, [Ca2+]i did not return to the initial resting level, but remained for long periods of time (up to 20 min), at a new, higher steady-state level. Both the amplitude of the rebound Ca2+ transient and the new plateau level strongly correlated with the degree of intracellular Ca2+ depletion, indicating the activation of a store-operated Ca2+ entry pathway. The elevated steady-state [Ca2+]i level was associated with a significant increase in the plasma membrane permeability to Ca2+, as changes in extracellular Ca2+ were reflected in almost immediate changes of [Ca2+]i. Similarly, blocking plasma-lemmal Ca2+ channels with the non-specific agonist La3+ (50 microM) caused a decrease in [Ca2+]i, despite the continuous presence of Ca2+ ions in the extracellular medium. After the establishment of the new, elevated steady-state [Ca2+]i level, stimulation of P2U metabotropic purinoreceptors did not induce a [Ca2+]i response. In addition, application of either thapsigargin (1 microM) or carbonyl cyanide chlorophenyl hydrazone (10 microM) failed to affect [Ca2+]i. We conclude that the maximal depletion of intracellular Ca2+ stores in mouse brain microglia determines the long-term activation of a plasma membrane Ca2+ entry pathway. This activation appears to be associated with a significant decrease in the capability of the intracellular Ca2+ stores to take up cytosolic Ca2+ once they have been maximally depleted.     

Mechanical stimulation initiates intercellular Ca2+ signaling in intact tracheal epithelium maintained under normal gravity and simulated microgravity

We investigated mechanically induced cell-to-cell Ca2+ signaling in a preparation of rabbit tracheal epithelium close to its in vivo condition. We used confocal microscopy to analyze changes in intracellular free calcium concentration ([Ca2+]i) in intact ciliated tracheal mucosal explants loaded with the Ca2+-indicator dye, fluo-3. When a single cell in the epithelium was transiently stimulated with a microprobe, [Ca2+]i increased in the stimulated cell and then increased in surrounding cells. In the absence of extracellular Ca2+, the [Ca2+]i increases had a smaller amplitude and spread to fewer cells. Treatment of the cells with thapsigargin, in the presence of extracellular Ca2+, more markedly reduced the spread of elevated [Ca2+]i. These results suggest that the propagated [Ca2+]i increases are due to mobilization of Ca2+ from intracellular stores and, possibly, the influx of extracellular Ca2+. The mechanically stimulated [Ca2+]i increases were accompanied by propagated increases in ciliary beat frequency. Since microgravity has been shown to alter signal transduction, we investigated whether simulated microgravity affects the mechanically stimulated cell-to-cell Ca2+ signaling observed in tracheal epithelium. Tissues were maintained for 3-8 d in a rotating wall vessel which simulates microgravity conditions. Cells maintained in simulated microgravity exhibited mechanically induced [Ca2+]i increases not significantly different in magnitude, in speed of propagation, or in the number of cells involved, from tissue maintained at unit gravity. Our results suggest that intercellular Ca2+ signaling coordinates cellular activity, including ciliary beating, within the tracheal epithelium in vivo and that this function is not compromised in microgravity.     

Inhibition of bradykinin-induced calcium increase by phosphatase inhibitors in neuroblastoma x glioma hybrid NG108-15 cells

Prior treatment of NG108-15 cells with phosphatase inhibitors including okadaic acid and calyculin A inhibited the elevation of cytosolic Ca2+ concentration ([Ca2+]i) induced by bradykinin by approximately 63%. This inhibition was dependent on the concentration of okadaic acid with an IC50 of 0.15 nM. Okadaic acid treatment only lowered the maximal response of [Ca2+]i increase and had no effect on the EC50 value for bradykinin regardless of the presence of extracellular Ca2+. Neither the capacity of 45Ca2+ accumulation within intracellular nonmitochondrial Ca2+ stores nor the magnitude of [Ca2+]i increase induced by thapsigargin was reduced by the treatment of okadaic acid. In contrast, the same phosphatase inhibitor treatment inhibited the bradykinin-evoked inositol 1,4,5-trisphosphate (IP3) generation, the Mn2+ influx, and the capacity of mitochondrial Ca2+ accumulation. Furthermore, the sensitivity of IP3 in the Ca2+ release was suppressed by okadaic acid pretreatment. Our results suggest that the reduction of bradykinin-induced [Ca2+]i rise by the promotion of protein phosphorylation was attributed to the reduced activity of phospholipase C, the decreased sensitivity to IP3, and the slowed rate of Ca2+ influx. Thus, phosphorylation plays a role in bradykinin-sensitive Ca2+ signaling cascade in NG108-15 cells.     

P2-purinoceptor-mediated formation of inositol phosphates and intracellular Ca2+ transients in human coronary artery smooth muscle cells

1. The effects of extracellular adenosine 5'-triphosphate (ATP) on smooth muscles are mediated by a variety of purinoceptors. In this study we addressed the identity of the purinoceptors on smooth muscle cells (SMC) cultured from human large coronary arteries. Purinoceptor-mediated increases in [Ca2+]i were measured in single fura-2 loaded cells by applying a digital imaging technique, and the formation of inositol phosphate compounds was quantified after separation on an anion exchange column. 2. Stimulation of the human coronary artery SMC (HCASMC) with extracellular ATP at concentrations of 0.1-100 microM induced a transient increase in [Ca2+]i from a resting level of 49 +/- 21 nM to a maximum of 436 +/- 19 nM. The effect was dose-dependent with an EC50 value for ATP of 2.2 microM. 3. The rise in [Ca2+]i was independent of the presence of external Ca2+, but was abolished after depletion of intracellular stores by incubation with 100 nM thapsigargin. 4. [Ca2+]i was measured upon stimulation of the cells with 0.1-100 microM of the more specific P2-purinoceptor agonists alpha, beta-methyleneadenosine 5'-triphosphate (alpha,beta-MeATP), 2-methylthioadenosine 5'-triphosphate (2MeSATP) and uridine 5'-triphosphate (UTP). alpha, beta-MeATP was without effect, whereas 2MeSATP and UTP induced release of Ca2+ from internal stores with 2MeSATP being the most potent agonist (EC50 = 0.17 microM), and UTP having a potency similar to ATP. The P1 purinoceptor agonist adenosine (100 microM) did not induce any changes in [Ca2+]i. 5. Stimulation with a submaximal concentration of UTP (10 microM) abolished a subsequent ATP-induced increase in [Ca2+]i, whereas an increase was induced by ATP after stimulation with 10 microM 2MeSATP. 6. The phospholipase C (PLC) inhibitor U73122 (5 microM) abolished the purinoceptor-activated rise in [Ca2+]i, whereas pretreatment with the Gi protein inhibitor pertussis toxin (PTX, 500 ng ml-1) was without effect on ATP-evoked [Ca2+]i increases. 7. Receptor activation with UTP and ATP resulted in formation of inositol phosphates with peak levels of inositol 1, 4, 5-trisphosphate (Ins(1, 4, 5)P3) observed 5-20 s after stimulation. 8. These findings show, that cultured HCASMC express G protein-coupled purinoceptors, which upon stimulation activate PLC to induce enhanced Ins(1, 4, 5)P3 production causing release of Ca2+ from internal stores. Since a release of Ca2+ was induced by 2MeSATP as well as by UTP, the data indicate that P2y- as well as P2U-purinoceptors are expressed by the HCASMC.     

Co-existence of P2Y-and PPADS-insensitive P2U-purinoceptors in endothelial cells from adrenal medulla

1. We have studied the effects of purinoceptor stimulation on Ca2+ signals in bovine adrenomedullary endothelial cells. [Ca2+]i was determined with the fluorescent probe fura-2 both in population samples and in single, isolated, endothelial cells in primary culture and after subculturing. 2. In endothelial cells, maintained in culture for more than one passage, several purinoceptor agonists elicited clear [Ca2+]i transient peaks that remained in the absence of extracellular Ca2+. Adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) were equipotently active, with EC50 values of 8.5 +/- 0.9 microM and 6.9 +/- 1.5 microM, respectively, whereas 2-methylthioadenosine 5'-triphosphate (2MeSATP), adenosine 5'-(alpha, beta-methylene)triphosphate (alpha, beta-MeATP) and adenosine(5')tetraphospho(5')adenosine (Ap4A) were basically inactive. Adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) was a weak agonist. The apparent potency order was UTP = ATP > ADP beta S > 2MeSATP > alpha, beta-MeATP. 3. Cross-desensitization experiments revealed that UTP or ATP, added sequentially at concentrations of maximal effect, could completely abolish the [Ca2+]i response to the second agonist. ADP beta S exerted only a partial desensitization of the response to maximal ATP, in accordance with its lower potency in raising [Ca2+]i. 4. The effect on [Ca2+]i of 100 microM ATP in subcultured cells was reduced by only 25% with 100 microM suramin pretreatment and was negligibly affected by exposure to 10 microM pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (PPADS). The concentration-effect curve for ATP was not significantly affected by PPADS, but was displaced to the right by a factor of 6.5 by 100 microM suramin. 5. In primary cultures, clear [Ca2+]i responses were elicited by 2MeSATP. Suramin totally and selectively blocked 2MeSATP responses, whereas UTP-evoked [Ca2+]i transients were mainly unaffected by suramin or PPADS. Over 80% of cells tested showed responses to both 2MeSATP and UTP. The [Ca2+]i response to UTP was not desensitized in the presence of 2MeSATP. 6. ATP and UTP stimulated the release of preloaded [3H]-arachidonic acid ([3H]-AA), both in the presence and in the absence of extracellular Ca2+, by approximately 135% with respect to basal levels. Suramin and PPADS enhanced, rather than inhibited, the [3H]-AA releasing effect of ATP by 2.5 times. Suramin also potentiated the effect of the calcium ionophore A23187. 7. These results indicate that endothelial cells from adrenomedullary capillaries co-express both P2Y- and P2U-purinoceptors. P2Y-purinoceptors are lost in culture with the first passage of the cells. The P2U-purinoceptor subtype present in these cells is insensitive to PPADS and thus similar to that found in aortic endothelial cells.     

Role of intracellular calcium in fast and slow desensitization of P2-receptors in PC12 cells

1. Combined whole-cell patch clamp recording and confocal laser scanning microscopy of [Ca2+]i transients were performed on single PC12 cells to study any correlation between membrane currents induced by ATP and elevation in [Ca2+]i. ATP was applied by pressure from micropipettes near the recorded PC12 cells continuously superfused at a fast rate. 2. Brief (20 ms) pulses of ATP elicited monophasic inward currents and [Ca2+]i increases. Long applications (2 s) of ATP (5 mM) evoked peak currents which rapidly faded during the pulse and were followed by a large rebound current, interpreted as due to rapid desensitization and recovery of P2-receptors. The associated [Ca2+]i increase grew monotonically to a peak reached only after the occurrence of the current rebound, indicating that it is unlikely this cation has a role in fast desensitization. 3. Both membrane currents and [Ca2+]i transients were linearly dependent on holding membrane potential, suggesting that Ca2+ influx is the predominant cause of [Ca2+]i elevation. This view was supported by experiments carried out in Ca(2+)-free solution. 4. Brief pulses of ATP applied after a desensitizing pulse (2 s) of the same elicited smaller inward currents and [Ca2+]i rises indicating a role for [Ca2+]i in controlling slow desensitization of P2-receptors. 5. This notion was confirmed in experiments with various [Ca2+]i chelators which differentially affected slow desensitization in relation to their buffering capacity, while sparing fast receptor desensitization. 6. These results suggest a role for [Ca2+]i in slow rather than fast desensitization of P2-receptors, thus proposing this divalent cation as an intracellular factor able to provide an efficient and reversible control over receptor activity induced by ATP.     

Simultaneous automated determination of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities in whole blood

We have shown previously that the non-steroidal anti-inflammatory drug flufenamate (FFA) causes a maintained increase in [Ca2+]i and transient increases in a Ca(2+)-activated nonselective cation current (ICAN) and a Ca(2+)-activated slow, outward Cl- current (lo-slow) in molluscan neurons [Shaw T., Lee R.J., Partridge L.D. Action of diphenylamine carboxylate derivatives, a family of non-steroidal anti-inflammatory drugs, on [Ca2+]i and Ca(2+)-activated channels in neurons. Neurosci Lett 1995; 190:121-124]. Here we demonstrate that pretreatment of neurons with 10 microM thapsigargin eliminates the FFA-induced increase in [Ca2+]i and substantially reduces both ICAN and Io-slow supporting the hypothesis that the FFA-induced increase in [Ca2+]i results primarily from Ca2+ release from a thapsigargin-sensitive intracellular store. The [Ca2+]i response appears to be sustained, not by influx of extracellular Ca2+, but by inhibitory effects of FFA on Ca2+ removal from the cytosol. Inhibition of Ca2+ efflux may be an important component of the FFA-induced activation of both ICAN and Io-slow, as Ca2+ release by thapsigargin alone is not sufficient to activate either current. Our data also demonstrate that the effects of FFA on [Ca2+]i, ICAN and Io-slow are reversible and suggest that protein phosphorylation as well as an increase in [Ca2+]i are involved in the FFA-induced activation of Io-slow. Effects on neuronal Ca2+ handling as well as activation of ICAN or Io-slow may partially explain the analgesic effects of FFA.     

Ca2+-dependent inactivation of a store-operated Ca2+ current in human submandibular gland cells. Role of a staurosporine-sensitive protein kinase and the intracellular Ca2+ pump

Stimulation of human submandibular gland cells with carbachol, inositol trisphosphate (IP3), thapsigargin, or tert-butylhydroxyquinone induced an inward current that was sensitive to external Ca2+ concentration ([Ca2+]e) and was also carried by external Na+ or Ba2+ (in a Ca2+-free medium) with amplitudes in the order Ca2+ > Ba2+ > Na+. All cation currents were blocked by La3+ and Gd3+ but not by Zn2+. The IP3-stimulated current with 10 microM 3-deoxy-3-fluoro-D-myo-inositol 1,4,5-triphosphate and 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in the pipette solution, showed 50% inactivation in <5 min and >5 min with 10 and 1 mM [Ca2+]e, respectively. The Na+ current was not inactivated, whereas the Ba2+ current inactivated at a slower rate. The protein kinase inhibitor, staurosporine, delayed the inactivation and increased the amplitude of the current, whereas the protein Ser/Thr phosphatase inhibitor, calyculin A, reduced the current. Thapsigargin- and tert-butylhydroxyquinone-stimulated Ca2+ currents inactivated faster. Importantly, these agents accelerated the inactivation of the IP3-stimulated current. The data demonstrate that internal Ca2+ store depletion-activated Ca2+ current (ISOC) in this salivary cell line is regulated by a Ca2+-dependent feedback mechanism involving a staurosporine-sensitive protein kinase and the intracellular Ca2+ pump. We suggest that the Ca2+ pump modulates ISOC by regulating [Ca2+]i in the region of Ca2+ influx.     

Reduced expression of E-cadherin in oral squamous cell carcinoma: relationship with DNA methylation of 5'' CpG island

The caffeine-evoked effects on the intracellular Ca2+ concentration ([Ca2+]i) and on the release of dopamine by PC12 cells were investigated. Stimulation by caffeine resulted in a transient Ca2+ release which was followed by a sustained phase of Ca2+ entry through a non-voltage dependent pathway. Treatment with cyclopiazonic acid (CPA) or thapsigargin, inhibitors of the Ca2+ATPase pump of the endoplasmic reticulum, resulted in only a sustained rise in [Ca2+]i in the presence of extracellular Ca2+. Pretreatment of cells with CPA or thapsigargin abolished the subsequent Ca2+ responses to caffeine. Caffeine also evoked the release of dopamine from the cells only in the presence of extracellular Ca2+, which was mimicked by CPA. These results suggest that store-dependent Ca2+ entry evoked by caffeine has an indispensable role in the secretory response in an excitable cell line, PC12 cells.     

DNA damage induced by m-phenylenediamine and its derivative in the presence of copper ion

We have previously reported that long-term priming of human polymorphonuclear neutrophilic granulocytes (PMN) with interferon-gamma (IFN-gamma) increased the fMLP-stimulated calcium influx. We now show that also after short-term incubation with IFN-gamma, PMN calcium metabolism is modulated. Single adherent cells in three different calcium-containing buffers (high, normal, and low [Ca2+]) were stimulated with the bacterial peptide fMLP or the Ca-ATPase inhibitor thapsigargin (Tg) after about 5 min preincubation with IFN-gamma. The results of this protocol indicated that IFN-gamma increases both calcium influx and calcium sequestration. Store dependent Ca2+ influx, directly measured on readdition of calcium to Tg-treated cells incubated in EGTA buffer, was significantly enhanced in IFN-gamma-treated cells. This effect of IFN-gamma was enhanced by the tyrosine kinase inhibitor herbimycin A. Strikingly, in low extracellular calcium concentrations, IFN-gamma induced calcium transients in 20%-60% of the cells. The proportion of PMN responding with Ca2+ transients increased with decreasing extracellular calcium concentration. Average lagtime from addition of IFN-gamma to a response that could be measured was 7.3 sec, and average increase in [Ca2+] above the basal level was 790 nM. These IFN-gamma-induced transients could not be depressed by herbimycin A. Thus, IFN-gamma can increase capacitative calcium influx, induce calcium transients, and possibly affect calcium sequestration in human PMN.