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.     

Cyclic ADP-ribose induces Ca2+ release from caffeine-insensitive Ca2+ pools in canine salivary gland cells

Cyclic ADP-ribose (cADPR), a novel putative messenger of the ryanodine receptor, was examined regarding its ability to mobilize Ca2+ from intracellular Ca2+ stores in isolated cells of parotid and submandibular glands of the dog. cADPR induced a rapid and transient Ca2+ release in the digitonin-permeabilized cells of salivary glands. cADPR-induced Ca2+ release was inhibited by ryanodine receptor antagonists ruthenium red, ryanodine, benzocaine, and imperatoxin inhibitor but not by the inositol 1,4,5-trisphosphate (IP3)-receptor antagonist heparin. Thapsigargin, at a concentration of 3 to 30 microM, inhibited IP3-induced Ca2+ release, while higher concentrations were required to inhibit cADPR-induced Ca2+ release. Cross-potentiation was observed between cADPR and ryanodine or SrCl2, suggesting that cADPR sensitizes the Ca2+-induced Ca2+ release mechanism. Cyclic AMP plays a stimulatory role on cADPR- and IP3-induced Ca2+ release in digitonin-permeabilized cells. Calmodulin also potentiated cADPR-induced Ca2+ release, but inhibited IP3-induced Ca2+ release. Acetylcholine and ryanodine caused the rise in intracellular free Ca2+ concentration ([Ca2+]i) in intact submandibular and parotid cells. Caffeine did not produce any increase in Ca2+ release or [Ca2+]i rise in any preparation. ADP-ribosyl cyclase activity was found in the centrifuged particulate fractions of the salivary glands. These results suggest that cADPR serves as an endogenous modulator of Ca2+ release from Ca2+ pools through a caffeine-insensitive ryanodine receptor channel, which are different from IP3-sensitive pools in canine salivary gland cells. This system is positively regulated by cyclic AMP and calmodulin.     

Induction of hippocampal long-term depression requires release of Ca2+ from separate presynaptic and postsynaptic intracellular stores

Studies have suggested that an increase in intracellular [Ca2+] is necessary for the induction of both long-term potentiation (LTP) and long-term depression (LTD) of synaptic transmission, and that release of Ca2+ from intracellular storage pools can be necessary to induce LTP. We investigated whether release of Ca2+ from intracellular stores also is required for the induction of LTD at Schaffer collateral-CA1 synapses in hippocampal slices. Both thapsigargin (1 microM) and cyclopiazonic acid (1 microM), compounds that deplete all intracellular Ca2+ pools by blocking LTP-dependent Ca2+ uptake into intracellular compartments, blocked the induction, but not maintenance, of LTD by low-frequency stimulation (LFS) (1 Hz/15 min) without affecting baseline synaptic transmission. Washout of the reversible inhibitor cyclopiazonic acid restored the ability to induce LTD. In contrast, thapsigargin did not block depotentiation of LTP by 1 Hz LFS, suggesting that LTP causes a reduction in the threshold [Ca2+] necessary for LTD. Selective depletion of the ryanodine receptor-gated Ca2+ pool by bath application of ryanodine (10 microM) also blocked the induction of LTD, indicating a requirement for Ca(2+)-induced Ca2+ release. Impalement of CA1 pyramidal neurons with microelectrodes containing thapsigargin (500 nM to 200 microM) prevented the induction of LTD at synapses on that neuron without blocking LTD in the rest of the slice. In contrast, similar filling of CA1 pyramidal neurons with ryanodine (2 microM to 5 mM) did not block the induction of LTD. From these data, we conclude that the induction of LTD requires release of Ca2+ both from a presynaptic ryanodine-sensitive pool and from postsynaptic (presumably IP3-gated) stores.     

The thapsigargin-evoked increase in [Ca2+]i involves an InsP3-dependent Ca2+ release process in pancreatic acinar cells

In pancreatic acinar cells, as in many other cell types, the tumour promoter thapsigargin (TG) evokes a significant increase of intracellular free Ca2+ ([Ca2+]i). The increases of [Ca2+]i evoked by TG was associated with significant changes of plasma membrane Ca2+ permeability, with [Ca2+]i values following changes in extracellular [Ca2+]. Plasma membrane Ca2+ extrusion is activated rapidly as a consequence of the rise in [Ca2+]i evoked by TG and the rate of extrusion is linearly dependent on [Ca2+]i up to 1 microM Ca2+. In contrast, the activation of the Ca2+ entry pathway is delayed and the apparent rate of Ca2+ entry is independent of [Ca2+]i. In the presence of 20 mM caffeine, which reduces the resting levels of inositol trisphosphate (InsP3), the increase of [Ca2+]i evoked by TG was significantly reduced. The reduction was manifest both as a decrease of the amplitude of the [Ca2+]i peak (30% reduction) and, more importantly, as a reduction of the apparent maximal rate of [Ca2+]i increase (from 12.3 +/- 1.0 to 6.1 +/- 0.6 nM Ca2+/s). The inhibition evoked by caffeine was reversible and the removal of caffeine in the continuous presence of TG evoked a further increase of [Ca2+]i. The amplitude of the [Ca2+]i increase upon caffeine removal was reduced as a function of the time of TG exposure. Addition of TG in the presence of 1 mM La3+, which is known to inhibit the plasma membrane Ca(2+)-activated adenosine triphosphatase, induced a much higher peak of [Ca2+]i. This increase was associated with an augmentation of the apparent rate of [Ca2+]i increase (from 12.3 +/- 1.2 to 16.1 +/- 1.9 nM Ca2+/s).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Region-specific activity of the plasma membrane Ca2+ pump and delayed activation of Ca2+ entry characterize the polarized, agonist-evoked Ca2+ signals in exocrine cells

The initial release of Ca2+ from the intracellular Ca2+ stores is followed by a second phase during which the agonist-dependent Ca2+ response becomes sensitive to the extracellular Ca2+, indicating the involvement of the plasma membrane (PM) Ca2+ transport systems. The time course of activation of these transport systems, which consist of both Ca2+ extrusion and Ca2+ entry pathways, is not well established. To investigate the participation of these processes during the agonist-evoked Ca2+ response, isolated pancreatic acinar cells were exposed to maximal concentrations of an inositol 1,4,5-trisphosphate-mobilizing agonist (acetylcholine, 10 microM) in different experimental conditions. Following the increase of [Ca2+]i, there was an almost immediate activation of the PM Ca2+ extrusion system, and maximal activity was reached within less than 2s. The rate of Ca2+ extrusion was dependent on the level of [Ca2+]i, with a steep activation at values just above the resting [Ca2+]i and reached a plateau value at 700 nM Ca2+. In contrast, the PM Ca2+ entry pathway was activated with a much slower time course. There was also a delay of 3-4 s between the maximal effective depletion of the intracellular Ca2+ stores and the activation of this entry pathway. By use of digital imaging data, the PM Ca2+ transport systems were also analyzed independently in two regions of the cells, the lumenal and the basal poles. With respect to the activation of the Ca2+ entry pathways, no significant difference existed between these two regions. In contrast, the PM Ca2+ pump displayed a different pattern of activity in these regions. In the basal pole, the pump activity was more sensitive to changes of [Ca2+]i and had a higher maximal activity. Also, in the lumenal pole, the pump became saturated at values of [Ca2+]i around 700 nM, whereas at the basal pole [Ca2+]i had a biphasic effect on the pump activity, and higher [Ca2+]i inhibited the pump. It is argued that these differences in sensitivity to the levels of [Ca2+]i and the different relationship between [Ca2+]i and the rate of extrusion at the two functional poles of the pancreatic acinar cells indicate that the plasma membrane Ca2+ ATPase might play an important role in the polarization of the Ca2+ response.     

Inositol 1,4,5-trisphosphate-induced Ca2+ release is regulated by cytosolic Ca2+ in intact skeletal muscle

Microinjection of inositol 1,4,5-trisphosphate (InsP3) into intact skeletal muscle fibers isolated from frogs (Rana temporaria) increased resting cytosolic Ca2+ concentration ([Ca2+]i) as measured by double-barreled Ca2+-selective microelectrodes. In contrast, microinjection of inositol 1-phosphate, inositol 1,4-biphosphate, and inositol 1,4,5,6-tetrakisphosphate did not induce changes in [Ca2+]i. Incubation in low-Ca2+ solution, or in the presence of L-type Ca2+ channel blockers did not affect InsP3-induced release of cytosolic Ca2+. Neither ruthenium red, a blocker of ryanodine receptor Ca2+-release channels, nor cytosolic Mg2+, a known inhibitor of the Ca2+-induced Ca2+-release process, modified the InsP3-induced release of cytosolic Ca2+. However, heparin, a blocker of InsP3 receptors, inhibited InsP3-induced release of cytosolic Ca2+. Also, pretreatment with dantrolene or azumulene, two inhibitors of cytosolic Ca2+ release, reduced [Ca2+]i, and prevented InsP3 from inducing release of cytosolic Ca2+. Incubation in caffeine or lengthening of the muscle increased [Ca2+]i and enhanced the ability of InsP3 to induce release of cytosolic Ca2+. These results indicate that InsP3, at physiological concentrations, induces Ca2+ release in intact muscle fibers, and suggest that the InsP3-induced Ca2+ release is regulated by [Ca2+]i. A Ca2+-dependent effect of InsP3 on cytosolic Ca2+ release could be of importance under physiological or pathophysiological conditions associated with alterations in cytosolic Ca2+ homeostasis.     

Transient inhibition by chemotactic peptide of a store-operated Ca2+ entry pathway in human neutrophils

Emptying the intracellular calcium stores of fura-2-loaded human neutrophils by treatment with the endomembrane ATPase inhibitor thapsigargin leads to a maintained increase of [Ca2+]i by Ca2+ entry through a store-operated Ca2+ entry pathway. Under these conditions, [Ca2+]i was reduced transiently by N-formyl-methionyl-leucyl-phenylalanine (fMLP) and permanently by phorbol 12,13-dibutyrate (PDB). Platelet-activating factor (PAF) had no effect. The fMLP- and PDB-induced [Ca2+]i decreases were not due to stimulated Ca2+ efflux but to inhibition of store-operated Ca2+ entry pathway. PDB and fMLP, but not PAF, inhibited the entry of Ca2+, Mn2+, and Ba2+ in thapsigargin-treated cells. This inhibition was dependent on [Ca2+]i, barely detectable at [Ca2+]i of 50 nM and increasingly strong and fast to appear at 170 and 630 nM. Inhibition of entry by fMLP was complete within 5-10s, disappeared within 2-3 min, and was partially prevented by staurosporin (100 nM). Inhibition by PDB was equally fast, but no recovery was detected within 5 min, and it was fully prevented by staurosporin. The inhibitory effect of fMLP had similar characteristics when PAF was used instead of thapsigargin to induce the entry of Ca2+ or Mn2+. We conclude that fMLP, but not PAF, is able to produce a transient inhibition of store-operated Ca2+ entry pathway, probably mediated by protein kinase C. This action could be part of a general homeostatic mechanism designed to moderate [Ca2+]i increases induced by some agonists.     

Ca2+-induced Ca2+ release in chromaffin cells seen from inside the ER with targeted aequorin

The presence and physiological role of Ca2+-induced Ca2+ release (CICR) in nonmuscle excitable cells has been investigated only indirectly through measurements of cytosolic [Ca2+] ([Ca2+]c). Using targeted aequorin, we have directly monitored [Ca2+] changes inside the ER ([Ca2+]ER) in bovine adrenal chromaffin cells. Ca2+ entry induced by cell depolarization triggered a transient Ca2+ release from the ER that was highly dependent on [Ca2+]ER and sensitized by low concentrations of caffeine. Caffeine-induced Ca2+ release was quantal in nature due to modulation by [Ca2+]ER. Whereas caffeine released essentially all the Ca2+ from the ER, inositol 1,4, 5-trisphosphate (InsP3)- producing agonists released only 60-80%. Both InsP3 and caffeine emptied completely the ER in digitonin-permeabilized cells whereas cyclic ADP-ribose had no effect. Ryanodine induced permanent emptying of the Ca2+ stores in a use-dependent manner after activation by caffeine. Fast confocal [Ca2+]c measurements showed that the wave of [Ca2+]c induced by 100-ms depolarizing pulses in voltage-clamped cells was delayed and reduced in intensity in ryanodine-treated cells. Our results indicate that the ER of chromaffin cells behaves mostly as a single homogeneous thapsigargin-sensitive Ca2+ pool that can release Ca2+ both via InsP3 receptors or CICR.     

Calcium pools in Ehrlich carcinoma cells. A major, high affinity Ca2+ pool is sensitive to both inositol 1,4,5-trisphosphate and thapsigargin

To investigate the presence and the size of different non-mitochondrial Ca2+ pools of Ehrlich ascites tumor cells (EATCs), digitonin-permeabilized cells were allowed to accumulate Ca2+ in the presence of mitochondrial inhibitors and treated with the reticular Ca(2+)-ATPase inhibitor thapsigargin, IP3 and the Ca2+ ionophore A23187. Emptying of thapsigargin-sensitive Ca2+ stores prevented any Ca2+ release by IP3, and, after IP3 addition, little or no Ca2+ was released by thapsigargin. In both instances, a further Ca2+ release was accomplished by A23187. The IP3-thapsigargin-sensitive pool and the residual A23187-sensitive one corresponded to approximately 60 and 37% of non-mitochondrial stored Ca2+, respectively. In intact EATCs, IP3-dependent agonists and thapsigargin discharged Ca2+ pools almost completely overlapping, and A32187 released a minor residual Ca2+ pool. The IP3-insensitive pool appeared to have a relatively low affinity for Ca2+ (below 600 nM). The high affinity, IP3-sensitive Ca2+ pool was discharged in a 'quantal' manner following step additions of sub maximal [IP3], and the IP3-induced fractional Ca2+ release was more marked at higher concentrations of stored (luminal) Ca2+, The IP3-sensitive Ca2+ pool appeared to be devoid of the Ca(2+)-activated Ca2+ release channel since caffeine did not released any Ca2+ in intact and permeabilized EATCs, and Western blot analyses of EATC microsomal membranes failed to detect any known ryanodine receptor isoform.     

Release-activated Ca2+ transport in neurons of frog sympathetic ganglia

Frog sympathetic ganglion neurons exhibit a novel Ca2+ uptake mechanism, release-activated calcium transport or RACT, which is manifest in both cytosolic and store [Ca2+] signals as greatly accelerated Ca2+ uptake after Ca2+ release from internal stores. RACT is activated by Ca2+ release but not by Ca2+ entry and serves to selectively refill Ca2+ stores after release. RACT lowers cytosolic [Ca2+] with a rate constant about 1.6 times that of the SERCA pump with empty ER. RACT is thapsigargin-insensitive, was eliminated by ryanodine, but was not affected by blocking mitochondrial or plasma membrane Ca2+ transport. A Ca2+ flux model with RACT in the ER membrane reproduced the cytosolic and store [Ca2+] responses to all stimuli.     

Histamine causes Ca2+ entry via both a store-operated and a store-independent pathway in bovine adrenal chromaffin cells

The characteristics and properties of the increase in cytosolic [Ca2+] that occurs in bovine adrenal medullary chromaffin cells on exposure to histamine have been investigated. Specifically, these experiments were conducted to determine how much external Ca2+ enters the cell through a (capacitative) Ca2+ entry pathway activated as a consequence of intracellular Ca2+ store mobilization, relative to that which enters independently of store depletion via other channels activated by histamine. In Fura-2 loaded cells continued exposure to histamine (10 microM) caused a rapid but transient increase in cytosolic [Ca2+] followed by a lower plateau that was sustained as long as external Ca2+ was present. In the absence of external Ca2+, only the initial brief transient was observed. In cells previously treated with thapsigargin (100 nM) in Ca(2+)-free medium to deplete the internal Ca2+ stores, histamine caused no increase in cytosolic [Ca2+] when external Ca2+ was absent. Re-introduction of external Ca2+ to thapsigargin-treated store-depleted cells caused a sustained increase in cytosolic [Ca2+] that was further increased (P < 0.0002) upon exposure to histamine. The histamine-evoked increase was prevented by the H1-receptor antagonist, mepyramine (2 microM). A comparison was made between store-dependent Ca2+ entry consequent upon store mobilization with histamine in Ca(2+)-free medium and plateau phase Ca2+ entry resulting from stimulation with histamine in Ca(2+)-containing medium. The latter was found to be approximately 3 times greater in magnitude than the former (P < 0.0001) at the same concentration of histamine (10 microM). It is concluded that histamine causes Ca2+ entry not only via a capacitative entry pathway secondary to internal store mobilization, but also causes substantial Ca2+ entry through other pathways.     

Relation of exocytotic release of gamma-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the gamma-aminobutyric acid (GABA) carrier, NNC-711, (1-[(2-diphenylmethylene)amino]oxyethyl)- 1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride, blocks the Ca(2+)-independent release of [3H]GABA from rat brain synaptosomes induced by 50 mM K+ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca(2+)-dependent release of [3H]GABA in the total absence of carrier-mediated release. Reversal of the Na+/Ca2+ exchanger was used to increase the intracellular free Ca2+ concentration ([Ca2+]i) to test whether an increase in [Ca2+]i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca2+]i may rise to values above 400 nM, as a result of Na+/Ca2+ exchange, without inducing release of [3H]GABA, but subsequent K+ depolarization immediately induced [3H]GABA release. Thus, a rise of only a few nanomolar Ca2+ in the cytoplasm induced by 50 mM K+ depolarization, after loading the synaptosomes with Ca2+ by Na+/Ca2+ exchange, induced exocytotic [3H]GABA release, whereas the rise in cytoplasmic [Ca2+] caused by reversal of the Na+/Ca2+ exchanger was insufficient to induce exocytosis, although the value for [Ca2+]i attained was higher than that required for exocytosis induced by K+ depolarization. The voltage-dependent Ca2+ entry due to K+ depolarization, after maximal Ca2+ loading of the synaptosomes by Na+/Ca2+ exchange, and the consequent [3H]GABA release could be blocked by 50 microM verapamil. Although preloading the synaptosomes with Ca2+ by Na+/Ca2+ exchange did not cause [3H]GABA release under any conditions studied, the rise in cytoplasmic [Ca2+] due to Na+/Ca2+ exchange increased the sensitivity to external Ca2+ of the exocytotic release of [3H]GABA induced by subsequent K+ depolarization. Thus, our results show that the vesicular release of [3H]GABA is rather insensitive to bulk cytoplasmic [Ca2+] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca2+ entry through Ca2+ channels near the active zones.     

Molecular cloning and functional characterization of a novel receptor-activated TRP Ca2+ channel from mouse brain

Characterization of mammalian homologues of Drosophila TRP proteins, which induce light-activated Ca2+ conductance in photoreceptors, has been an important clue to understand molecular mechanisms underlying receptor-activated Ca2+ influx in vertebrate cells. We have here isolated cDNA that encodes a novel TRP homologue, TRP5, predominantly expressed in the brain. Recombinant expression of the TRP5 cDNA in human embryonic kidney cells dramatically potentiated extracellular Ca2+-dependent rises of intracellular Ca2+ concentration ([Ca2+]i) evoked by ATP. These [Ca2+]i transients were inhibited by SK&F96365, a blocker of receptor-activated Ca2+ entry, and by La3+. Expression of the TRP5 cDNA, however, did not significantly affect [Ca2+]i transients induced by thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPases. ATP stimulation of TRP5-transfected cells pretreated with thapsigargin to deplete internal Ca2+ stores caused intact extracellular Ca2+-dependent [Ca2+]i transients, whereas ATP suppressed [Ca2+]i in thapsigargin-pretreated control cells. Furthermore, in ATP-stimulated, TRP5-expressing cells, there was no significant correlation between Ca2+ release from the internal Ca2+ store and influx of extracellular Ca2+. Whole-cell mode of patch-clamp recording from TRP5-expressing cells demonstrated that ATP application induced a large inward current in the presence of extracellular Ca2+. Omission of Ca2+ from intrapipette solution abolished the current in TRP5-expressing cells, whereas 10 nM intrapipette Ca2+ was sufficient to support TRP5 activity triggered by ATP receptor stimulation. Permeability ratios estimated from the zero-current potentials of this current were PCa:PNa:PCs = 14.3:1. 5:1. Our findings suggest that TRP5 directs the formation of a Ca2+-selective ion channel activated by receptor stimulation through a pathway that involves Ca2+ but not depletion of Ca2+ store in mammalian cells.     

Effect of ryanodine on sarcoplasmic reticulum Ca2+ accumulation in nonfailing and failing human myocardium

BACKGROUND: The purpose of this study was to determine whether abnormal Ca2+ release through ryanodine-sensitive Ca2+ channels in the sarcoplasmic reticulum might contribute to the abnormal [Ca2+]i homeostasis that has been described in failing human myocardium. METHODS AND RESULTS: Occupancy of low-affinity ryanodine binding sites on ryanodine-sensitive Ca2+ channels stimulates oxalate-supported, ATP-dependent Ca2+ accumulation in sarcoplasmic reticulum-derived microsomes by inhibiting concurrent Ca2+ efflux through these channels. We examined the effects of 0.5 mmol/L ryanodine on 45Ca2+ accumulation in microsomes prepared from nonfailing (n = 8) and failing (n = 10) human left ventricular myocardium. In the absence of ryanodine, 45Ca2+ accumulation reached similar levels in microsomes from nonfailing and failing hearts. Incubation with 0.5 mmol/L ryanodine caused a 52.2 +/- 6.5% increase in peak 45Ca2+ accumulation in microsomes from nonfailing hearts and a 24.3 +/- 4.1% increase in microsomes from failing hearts. The density of high-affinity ryanodine binding sites and the inhibition of [3H]ryanodine dissociation from these sites by 0.1 mmol/L ryanodine were similar in microsomes from nonfailing and failing hearts. CONCLUSIONS: These results, which demonstrate a diminished stimulation of Ca2+ accumulation by ryanodine in sarcoplasmic reticulum-derived microsomes from failing human myocardium that could be explained by an uncoupling of the occupancy of low-affinity ryanodine binding sites from the reduction in the open probability of these channels or by concurrent Ca2+ efflux through a ryanodine-insensitive mechanism, are evidence that increased efflux of Ca2+ from the sarcoplasmic reticulum may contribute to the abnormal [Ca2+]i homeostasis described in failing human myocardium.     

Differential effects of extracellular Mg2+ on thrombin-induced and capacitative Ca2+ entry in human coronary arterial endothelial cells

Receptor-mediated and capacitative Ca2+ entry are the primary Ca2+ entry pathways in endothelial cells (ECs). The mechanisms for Ca2+ entry via these pathways have not been fully elucidated. In this study, the effect of low and high external Mg2+ concentrations on these Ca2+ entry pathways was examined in human coronary arterial ECs. External Mg2+ concentration did not affect cytosolic free Mg2+ concentration. After exposure to thrombin in Ca(2+)-free medium, addition of Ca2+ to the medium caused a rise in cytosolic free Ca2+ concentration ([Ca2+]i), indicating thrombin-induced Ca2+ influx. Thrombin-induced Ca2+ influx was inhibited by not only low but also high external Mg2+ concentrations. After depletion of endoplasmic Ca2+ stores by thapsigargin, addition of Ca2+ to the medium induced an increase in [Ca2+]i, indicating capacitative Ca2+ entry. Capacitative entry was found to be accelerated by low external Mg2+ and inhibited by high external Mg2+ concentration. Results suggest that receptor-mediated Ca2+ influx requires external Mg2+ but is inhibited by increased external Mg2+ concentrations and that capacitative Ca2+ entry is reduced by external Mg2+ in human coronary arterial ECs.     

Pharmacokinetics and effects of intravesical oxybutynin on the paediatric neurogenic bladder

We have recently shown that the Ca2+ response in endothelial cells evoked by readdition of Ca2+ to the medium after store depletion caused by a submaximal concentration of agonist can involve Ca2+ release from Ca2+ stores sensitive to both inositol 1,4, 5-trisphosphate and ryanodine. The present experiments were performed to determine whether this mechanism might also exist in other types of cell. For this purpose, we used the human carcinoma cell line A431, which has a varied resting [Ca2+]i. We found that the amplitude of the Ca2+ response evoked by Ca2+ readdition did not correlate with the amplitude of the preceding UTP-evoked Ca2+ release, but did positively correlate with the initial [Ca2+]i. An inspection of the two patterns of response seen in this study (the large biphasic and small plateau-shaped Ca2+ responses) revealed that there is an accelerating rise in [Ca2+]i during the biphasic response. Application of ryanodine during the plateau-shaped Ca2+ response reversibly transformed it into the biphasic type. Unlike ryanodine, caffeine did not itself evoke Ca2+ release, but it caused a further [Ca2+]i rise when [Ca2+]i had already been elevated by thapsigargin. These data suggest that in A431 cells, as in endothelial cells, the readdition of Ca2+ after agonist-evoked store depletion can evoke Ca2+-induced Ca2+ release. This indicates that Ca2+ entry may be overestimated by this widely used protocol.     

Modulation of pancreatic acinar cell to cell coupling during ACh-evoked changes in cytosolic Ca2+

The temporal changes in cytosolic free Ca2+ ([Ca2+]i), Ca2+-dependent membrane currents (Im), and gap junctional current (Ij) elicited by acetylcholine (ACh) were measured in rat pancreatic acinar cells using digital imaging and dual perforated patch-clamp recording. ACh (50 nM-5 microM) increased [Ca2+]i and evoked Im currents without altering Ij in 19 of 37 acinar cell pairs. Although [Ca2+]i rose asynchronously in cells comprising a cluster, the delay of the [Ca2+]i responses decreased with increasing ACh concentrations. Perfusion of inositol 1,4,5-trisphosphate (IP3) into one cell of a cluster resulted in [Ca2+]i responses in neighboring cells that were not necessarily in direct contact with the stimulated one. This suggests that extensive coupling between acinar cells provides a pathway for cell-to-cell diffusion of Ca2+-releasing signals. Strikingly, maximal (1-5 microM) ACh concentrations reduced Ij by 69 +/- 15% (n = 9) in 25% of the cell pairs subjected to dual patch-clamping. This decrease occurred shortly after the Im peak and was prevented by incubating acinar cells in a Ca2+-free medium, suggesting that uncoupling was subsequent to the initiation of the Ca2+-mobilizing responses. Depletion of Ca2+-sequestering stores by thapsigargin resulted in a reduction of intercellular communication similar to that observed with ACh. In addition, ACh-induced uncoupling was prevented by blocking nitric oxide production with L-nitro-arginine and restored by exposing acinar cells to dibutyryl cGMP. The results suggest that ACh-induced uncoupling and capacitative Ca2+ entry are regulated concurrently. Closure of gap junction channels may occur to functionally isolate nearby cells differing in their intrinsic sensitivity to ACh and thereby to allow for sustained activity of groups of secreting cells.     

The delta-opioid receptor regulates activity of ryanodine receptors in the human neuroblastoma cell line SK-N-BE

Recent studies have demonstrated that opioid agonists affect the cytosolic Ca2+ concentration ([Ca2+]i) either by regulating plasma membrane Ca(2+)-channel activity or by mobilizing intracellular Ca2+ stores. The present report documents the [Ca2+]i increase induced by opioid agonists in a human neuroblastoma cell line, SK-N-BE, expressing delta-opioid receptors. In the presence, as well as in the absence, of extracellular Ca2+, opioid agonists enhanced significantly [Ca2+]i, whereas carbachol, known to mobilize specifically inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores, acted only in the presence of extracellular Ca2+. The opioid-induced increase in [Ca2+]i was not affected by treatments modifying the trimeric Gl, Go, and Gs protein transduction mechanisms or the activity of adenylyl cyclase. The Ca(2+)-ATPase pump-inhibiting sesquiterpene lactone, thapsigargin, did not modify the opioid-induced [Ca2+]i response, whereas it abolished the effects of carbachol. The Ryana speciosa alkaloid, ryanodine, at concentrations known to block endoplasmic reticulum ryanodine receptors, decreased significantly the response to opioids without affecting the effects of carbachol. Thus, our results suggest that, in SK-N-BE cells, delta-opioid receptors mobilize Ca2+ from intracellular ryanodine-sensitive stores and the mechanism involved is independent of Gl/Go Gs proteins and protein kinase A activation.