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PubMed1990

Cytoplasmic Ca2+ oscillations evoked by receptor stimulation, G-protein activation, internal application of inositol trisphosphate or Ca2+: simultaneous microfluorimetry and Ca2+ dependent Cl- current recording in single pancreatic acinar cells.

The effects of acetylcholine (ACh), cholecystokinin (CCK), internally applied GTP-gamma-S, inositol trisphosphate [Ins (1,4,5) P3] or Ca2+ on the cytoplasmic free Ca2+ concentration [( Ca2+]i) were assessed by simultaneous microfluorimetry (fura-2) and measurement of the Ca2(+)-dependent Cl- current (patch-clamp whole-cell recording) in single internally perfused mouse pancreatic acinar cells. ACh (0.1-0.2 microM) evoked an oscillating increase in [Ca2+]i measured in the cell as a whole (microfluorimetry) which was synchronous with oscillations in the Ca2(+)-dependent Cl- current reporting [Ca2+]i close to the cell membrane. In the same cells a lower ACh concentration (0.05 microM) evoked shorter repetitive Cl- current pulses that were not accompanied by similar spikes in the microfluorimetric recording. When cells did not respond to 0.1 microM ACh, caffeine (1 mM) added on top of the sustained ACh stimulus resulted in [Ca2+]i oscillations seen synchronously in both types of recording. CCK (10 nM) also evoked [Ca2+]i oscillations, but with much longer intervals between slightly broader Ca2+ pulses. Internal perfusion with 100 microM GTP-gamma-S evoked [Ca2+]i oscillations with a similar pattern. Ins (1,4,5) P3 (10 microM) evoked repetitive shortlasting spikes in [Ca2+]i that were only seen in the Cl- current traces, except in one small cell where these spikes were also observed synchronously in the microfluorimetric recording. Caffeine (1 mM) broadened these Ca2+ pulses. [Ca2+]i was also directly changed, bypassing the normal signalling process, by infusion of a low or high Ca2+ solution into the pipette.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1990

Effects of compound 48/80 on the Ca2+ release by reversal of the Ca2+ pump and by the Ca2+ channel of sarcoplasmic reticulum membranes.

The effect of the calmodulin antagonist, compound 48/80, on the Ca2+ release from skeletal muscle sarcoplasmic reticulum was investigated. Both the Ca2+ release by reversal of the Ca2+ pump and the Ca2+ release by the Mg2(+)-controlled Ca2+ channel were studied. It was observed that, when reversal of the pump is inoperative and Mg2+ is not present in the reaction medium, 48/80 stimulates Ca2+ release from the vesicles. In contrast, in the presence of Mg2+, which blocks the Ca2+ channel, 48/80 inhibits Ca2+ release induced by ADP and Pi. This effect is strong at low concentrations of Pi (approximately 1 mM), whereas high concentrations (approximately 15 mM) protect the system against the drug. Furthermore, it was observed that 48/80 has a maximum effect on the channel-mediated Ca2+ release at concentrations of about 20 micrograms/ml, whereas maximal inhibition of the pump-mediated Ca2+ release occurs at concentrations of about 60-80 micrograms/ml. The results indicate that both the Ca2+ channel complex and the Ca2(+)-ATPase may be target systems for the effects of 48/80 on the Ca2+ transport activity of sarcoplasmic reticulum. However, the Ca2+ channel is more sensitive to the drug, suggesting an involvement of calmodulin on this mechanism of Ca2+ release.

Adenosine Diphosphate↗
PubMed1988

(Ca2+ + Mg2+)-ATPase activity associated with the maintenance of a Ca2+ gradient by sarcoplasmic reticulum at submicromolar external [Ca2+]. The effect of hypothyroidism.

The formation and maintenance of Ca2+-filling levels by sarcoplasmic reticulum vesicles from euthyroid (control) and hypothyroid skeletal muscle were investigated using the Ca2+-indicator quin-2, at [Ca2+] in the medium [( Cao2+]) of 0.05-0.3 microM. Rapid ATP-dependent Ca2+ uptake resulted in a steady-state Ca2+-filling level, Cai2+, within one minute. This Ca2+ gradient was maintained for at least three minutes, during which less than 20% of the ATP was consumed. Cai2+ was maximal (120 nmol/mg) for [Cao2+] greater than 0.3 microM and decreased to 40 nmol/mg at [Cao2+] of 0.05 microM. Preparations from both experimental groups showed qualitatively and quantitatively the same relationship between Cai2+ and [Cao2+] at steady state, despite a significantly lower Ca2+-pump content of hypothyroid sarcoplasmic reticulum, which resulted in a 25% lower maximal (Ca2+ + Mg2+)-ATPase activity. Maintenance of the steady state, at all levels of Cai2+, was associated with net ATP consumption by the Ca2+ pump and cycling of Ca2+, which processes were 30% slower in the hypothyroid group as compared to the control group. Determination of the passive efflux of Ca2+, as well as the fraction of leaky or unsealed sarcoplasmic reticulum fragments, excluded either of these possibilities as an explanation for the relatively high (Ca2+ + Mg2+)-ATPase rates at steady state. On the basis of these and previously reported results, it is concluded that the maintenance of a Ca2+ gradient by sarcoplasmic reticulum under physiological conditions with respect to external [Ca2+] and the concentrations of ATP, ADP and Pi, is associated with the cycling of Ca2+ coupled to net ATP hydrolysis. Using the obtained data it is calculated that the sarcoplasmic reticulum may account for 20% of the resting metabolic rate in skeletal muscle. Consequently, together with the previously reported lower sarcoplasmic reticulum content of skeletal muscle in hypothyroidism, we calculate that about one third of the decrease in basal metabolic rate in this thyroid state can be related to the alterations of the sarcoplasmic reticulum.

Adenosine Triphosphate↗
PubMed1989

Contribution of external and internal Ca2+ to changes in intracellular free Ca2+ produced by mitogens in Swiss 3T3 fibroblasts: the role of dihydropyridine sensitive Ca2+ channels.

Changes in intracellular free Ca2+ concentration [( Ca2+]i) produced by growth factors and mitogens have been studied using aequorin-loaded Swiss 3T3 cells. Decreasing free Ca2+ in the external medium by using EGTA had no significant effect on the increase in [Ca2+]i produced by vasopressin, bradykinin, bombesin or prostaglandin E2, but reduced the increase in [Ca2+]i produced by platelet derived growth factor (PDGF) by 58%, by prostaglandin E1 44% and by prostaglandin F2 alpha 47%. The dihydropyridine Ca2+-channel antagonist nifedipine at 10 microM inhibited the [Ca2+]i response to PDGF by 41% in both the presence of and in the absence of external Ca2+. Methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl) pyridine-5-carboxylate (BAY K8644), a Ca2+-channel agonist, at 10 microM produced an increase in [Ca2+]i and decreased the [Ca2+]i response to PDGF by 39%. Nifedipine did not block 45Ca2+ uptake or release by inositol 1,4,5-trisphosphate in saponin-permeabilized Swiss 3T3 fibroblasts but BAY K8644 inhibited 45Ca2+ release by inositol 1,4,5-trisphosphate. The results suggest that the increase in [Ca2+]i caused by PDGF in Swiss 3T3 fibroblasts is due to the influx of external Ca2+ through dihydropyridine sensitive Ca2+ channels, as well as release of internal Ca2+.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1990

Receptor-activated cytoplasmic Ca2+ spiking mediated by inositol trisphosphate is due to Ca2(+)-induced Ca2+ release.

Receptor-mediated inositol 1,4,5-trisphosphate (Ins-(1,4,5)P3) generation evokes fluctuations in the cytoplasmic Ca2+ concentration ([Ca2+]i). Intracellular Ca2+ infusion into single mouse pancreatic acinar cells mimicks the effect of external acetylcholine (ACh) or internal Ins(1,4,5)P3 application by evoking repetitive Ca2+ release monitored by Ca2(+)-activated Cl- current. Intracellular infusion of the Ins(1,4,5)P3 receptor antagonist heparin fails to inhibit Ca2+ spiking caused by Ca2+ infusion, but blocks ACh- and Ins(1,4,5)P3-evoked Ca2+ oscillations. Caffeine (1 mM), a potentiator of Ca2(+)-induced Ca2+ release, evokes Ca2+ spiking during subthreshold intracellular Ca2+ infusion. These results indicate that ACh-evoked Ca2+ oscillations are due to pulses of Ca2+ release through a caffeine-sensitive channel triggered by a small steady Ins(1,4,5)P3-evoked Ca2+ flow.

Acetylcholine↗
PubMed1994

Interaction between Ca2+ release from inositol trisphosphate sensitive stores and Ca2+ entry through neuronal Ca2+ channels expressed in Xenopus oocyte.

Rat cerebellar RNA injected into Xenopus oocytes leads to the expression of putative P-type voltage-dependent Ca2+ channels (VDCCs). The monitoring of intracellular Ca2+ variations by recording the Ca2+ dependent chloride current in voltage clamped oocytes indicates that activation of these Ca2+ channels by depolarization gives rise to two distinct components of cytosolic Ca2+ elevation. If the early component (T1) can be directly attributed to the Ca2+ entry through VDCCs, the second delayed one (T2) is related to a Ca2+ release from InsP3 sensitive stores activated following Ca2+ entry. Modifications of cytosolic Ca2+ by direct injection of Ca2+ into oocytes or by increasing the Ca2+ influx through VDCCs suggest that the Ca2+ release from intracellular InsP3 sensitive stores can be modulated in a differential manner. Namely, discrete elevations of cytosolic Ca2+ switch on the Ca2+ release whereas higher Ca2+ concentrations dampen the release. These results suggest a functional coupling between P-type VDCCs and InsP3 receptors.

Animals↗
PubMed1988

Imaging of cytosolic Ca2+ transients arising from Ca2+ stores and Ca2+ channels in sympathetic neurons.

Changes in cytosolic free Ca2+ concentration [( Ca2+]i) due to Ca2+ entry or Ca2+ release from internal stores were spatially resolved by digital imaging with the Ca2+ indicator fura-2 in frog sympathetic neurons. Electrical stimulation evoked a rise in [Ca2+]i spreading radially from the periphery to the center of the soma. Elevated [K+]o also increased [Ca2+]i, but only in the presence of external Ca2+, indicating that Ca2+ influx through Ca2+ channels is the primary event in the depolarization response. Ca2+ release or uptake from caffeine-sensitive internal stores was able to amplify or attenuate the effects of Ca2+ influx, to generate continued oscillations in [Ca2+]i, and to persistently elevate [Ca2+]i above basal levels after the stores had been Ca2(+)-loaded.

Animals↗
PubMed1997

Inhibition of M3 muscarinic acetylcholine receptor-mediated Ca2+ influx and intracellular Ca2+ mobilization in neuroblastoma cells by the Ca2+/calmodulin-dependent protein kinase inhibitor 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-trosyl]-4-phenylpiperazin e (KN-62).

The role of Ca2+/calmodulin-dependent protein kinase (CaM kinase; EC 2.7.1.123) in the generation of Ca2+ signals by muscarinic acetylcholine receptors (mAChR) was studied. Changes in intracellular Ca2+ concentrations ([Ca2+]i) induced by mAChR activation were monitored in SK-N-SH human neuroblastoma cells using the dye Fura-2. SK-N-SH cells express M3 mAChR, as well as CaM kinase types II and IV, which are specifically inhibited by the CaM kinase antagonist KN-62 (1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazi ne). Carbamylcholine (100 microM) elicited an initial transient peak in [Ca2+]i due to mobilization of Ca2+ from internal stores, followed by a sustained elevation in [Ca2+]i that depended on the influx of extracellular Ca2+ and which was inhibited by EGTA and Ni2+. These mAChR-induced Ca2+ signals were diminished to an equal extent by preincubating the cells with 0.01 to 100 microM KN-62. KN-62 inhibited mAChR-induced Ca2+ influx and mobilization from internal stores by about 25-30%, producing a half-maximal effect at approximately 1 microM. In contrast, KN-62 (25 microM) almost completely abolished carbamylcholine-stimulated entry of divalent cations through Mn2+-permeant channels, as revealed by Mn2+ quenching of Fura-2 fluorescence. KN-62 also almost completely abolished Ca2+ influx induced by depolarization of the cells with 25 mM K+ (IC50 = 3 microM). These results suggest that CaM kinases regulate both the mobilization of intracellular Ca2+ and the stimulation of Ca2+ influx that are induced by mAChR activation, and indicate that the mAChR-induced influx of Ca2+ occurs through Ca2+ channels other than, or in addition to, the voltage-gated calcium channels or Mn2+-permeant channels which are inhibited by KN-62.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1998

In situ characterization of the Ca2+ sensitivity of large conductance Ca2+-activated K+ channels: implications for their use as near-membrane Ca2+ indicators in smooth muscle cells.

The Ca2+ sensitivity of large conductance Ca2+- and voltage-activated K+ channels (BKV,Ca) has been determined in situ in freshly isolated myocytes from the guinea pig urinary bladder. In this study, in situ denotes that BKV,Ca channel activity was recorded without removing the channels from the cell. By combining patch clamp recording in the cell-attached configuration and microfluorometry of fura-2, we were able to correlate BKV,Ca channel activity with changes in cytoplasmic intracellular [Ca2+] ([Ca2+]i). The latter were induced by ionomycin, an electroneutral Ca2+ ionophore. At 0 mV, the Hill coefficient (nH) and the [Ca2+]i to attain half of the maximal BKV,Ca channel activity (Ca50) were 8 and 1 microM, respectively. The data suggest that this large Hill number was not a consequence of the difference between the near-membrane [Ca2+] ([Ca2+]s) and the bulk [Ca2+]i, indicated by fura-2. High Hill numbers in the activation by Ca2+ of BKV,Ca channels have been seen by different groups (e.g., filled squares in Fig. 4 of Silberberg, S. D., A. Lagrutta, J. P. Adelman, and K. L. Magleby. 1996. Biophys. J. 70:2640-2651). However, such high nH has always been considered a peculiarity rather than the rule. This work shows that a high Ca2+ cooperativity is the normal situation for BKV,Ca channels in myocytes from guinea pig urinary bladder. Furthermore, the Ca50 did not display any significant variation among different channels or cells. It was also evident that BKV,Ca channel activity could decrease in elevated [Ca2+]i, either partially or completely. This work implies that the complete activation of BKV,Ca channels occurs with a smaller increment in [Ca2+]s than previously expected from in vitro characterization of the Ca2+ sensitivity of these channels. Additionally, it appears that the activity of BKV,Ca channels in situ does not strictly follow changes in near-membrane [Ca2+].

Animals↗
PubMed1998

Solution structures of the Ca2+-free and Ca2+-bound C2A domain of synaptotagmin I: does Ca2+ induce a conformational change?

C2 domains are widespread Ca2+-binding modules that are particularly abundant in proteins involved in membrane traffic and signal transduction. The C2A domain of synaptotagmin I is believed to play a key role in neurotransmitter release through its Ca2+-dependent interactions with syntaxin and phospholipids. Elucidating the structural consequences of Ca2+ binding to the C2A domain is critical for understanding its mechanism of action and for models of the functions of other C2 domains. We have determined the solution structure of the Ca2+-free and Ca2+-bound forms of the C2A domain of synaptotagmin I by NMR spectroscopy. Our data represent the first structure determination of a C2 domain in its Ca2+-free and Ca2+-bound forms. Three Ca2+ ions were included in the Ca2+-bound structure, yielding a Ca2+-binding motif that involves five aspartate side chains and one serine side chain. Ca2+ immobilizes the structure of the C2A domain but does not produce a significant conformational change from a well-defined conformation to another. Thus, the mechanism of action of the C2A domain of synaptotagmin I is different from that used by Ca2+-binding proteins of the EF-hand family. The main effect of Ca2+ binding on the C2A domain is to change its electrostatic potential rather than its structure. These results support a model whereby the C2A domain functions as an electrostatic switch in neurotransmitter release. The similarity between the structures of the synaptotagmin I C2A domain and the PLC-delta1 C2 domain suggests that the latter binds four Ca2+ ions and acts by a similar mechanism. This mechanism may also be valid for other C2 domains that share the unusual ability to bind multiple Ca2+ ions in a tight cluster at the tip of the domain.

Calcium↗
PubMed1986

Effects of vasopressin and La3+ on plasma-membrane Ca2+ inflow and Ca2+ disposition in isolated hepatocytes. Evidence that vasopressin inhibits Ca2+ disposition.

Vasopressin caused a 40% inhibition of 45Ca uptake after the addition of 0.1 mM-45Ca2+ to Ca2+-deprived hepatocytes. At 1.3 mM-45Ca2+, vasopressin and ionophore A23187 each caused a 10% inhibition of 45Ca2+ uptake, whereas La3+ increased the rate of 45Ca2+ uptake by Ca2+-deprived cells. Under steady-state conditions at 1.3 mM extracellular Ca2+ (Ca2+o), vasopressin and La3+ each increased the rate of 45Ca2+ exchange. The concentrations of vasopressin that gave half-maximal stimulation of 45Ca2+ exchange and glycogen phosphorylase activity were similar. At 0.1 mM-Ca2+o, La3+ increased, but vasopressin did not alter, the rate of 45Ca2+ exchange. The results of experiments performed with EGTA or A23187 or by subcellular fractionation indicate that the Ca2+ taken up by hepatocytes in the presence of La3+ is located within the cell. The addition of 1.3 mM-Ca2+o to Ca2+-deprived cells caused increases of approx. 50% in the concentration of free Ca2+ in the cytoplasm [( Ca2+]i) and in glycogen phosphorylase activity. Much larger increases in these parameters were observed in the presence of vasopressin or ionophore A23187. In contrast with vasopressin, La3+ did not cause a detectable increase in glycogen phosphorylase activity or in [Ca2+]i. It is concluded that an increase in plasma membrane Ca2+ inflow does not by itself increase [Ca2+]i, and hence that the ability of vasopressin to maintain increased [Ca2+]i over a period of time is dependent on inhibition of the intracellular removal of Ca2+.

Animals↗
PubMed1993

Receptor-operated Ca2+ channels in gastric parietal cells: gastrin and carbachol induce Ca2+ influx in depleting intracellular Ca2+ stores.

The mechanism whereby gastrin-type receptor and muscarinic M3-type receptor regulate free intracellular Ca2+ concentration ([Ca2+]i) was studied in rabbit gastric parietal cells stimulated by either gastrin or carbachol. Both agonists induced a biphasic [Ca2+]i response: a transient [Ca2+]i rise, followed by a sustained steady state depending on extracellular Ca2+. Gastrin and carbachol also caused a rapid and transient increase in Mn2+ influx (a tracer for bivalent-cation entry). Pre-stimulation of cells with one agonist drastically decreased both [Ca2+]i increase and Mn2+ influx induced by the other. Neither diltiazem nor pertussistoxin treatment had any effect on agonist-stimulated Mn2+ entry. Thapsigargin, a Ca(2+)-pump inhibitor, induced a biphasic [Ca2+]i increase, and enhanced the rate of Mn2+ entry. Preincubation of cells with thapsigargin inhibits the [Ca2+]i increase as well as Mn2+ entry stimulated by gastrin or by carbachol. Thapsigargin induced a weak but significant increase in Ins(1,4,5)P3 content, but this agent had no effect on the agonist-evoked Ins(1,4,5)P3 response. In permeabilized parietal cells, Ins(1,4,5)P3 and caffeine caused an immediate Ca2+ release from intracellular pools, followed by a reloading of Ca2+ pools which can be prevented in the presence of thapsigargin. We conclude that (i) gastrin and carbachol mobilize common Ca2+ intracellular stores, (ii) Ca2+ permeability secondary to receptor activation involves neither a voltage-sensitive Ca2+ channel nor a GTP-binding protein from the G1 family, and (iii) agonists regulate common Ca2+ channels in depleting intracellular Ca2+ stores.

Animals↗
PubMed2001

Plasma membrane Ca2+ release-activated Ca2+ channels with a high selectivity for Ca2+ identified by patch-clamp recording in rat liver cells.

Repetitive waves of increased cytoplasmic Ca2+ concentration play a central role in the process by which hormones regulate liver function. Maintenance of these Ca2+ waves requires Ca2+ inflow through store-operated Ca2+ channels. The properties and mechanism(s) of activation of these channels are not well understood. Store-operated Ca2+ channels (SOCs) in the H4-IIE rat liver cell line were studied by whole-cell patch clamping. Depletion of Ca2+ in intracellular stores by intracellular perfusion with either inositol 1,4,5-trisphosphate (InsP(3)) or thapsigargin in the presence of 10 mmol/L ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid (EGTA), or with 10 mmol/L EGTA alone, activated an inward current that reversed at a membrane potential above +40 mV. In physiologic extracellular medium, this inward current was carried exclusively by Ca2+ and was blocked by a variety of di- and trivalent cations. In the absence of extracellular Ca2+ and Mg2+, the inward current was carried by monovalent cations. This current was 10 to 30 times larger than that observed in the presence of extracellular Ca2+, and permitted the detection of single-channel events that corresponded to a single-channel conductance of about 40 pS. Both the Ca2+ and Na+ inward currents were blocked by the calmodulin antagonist, N-(6-amino hexyl)-5-chloro-1-naphthalenesulphonamide (W7), but not by calmidazolium or calmodulin-dependent protein kinase II fragment 290-309. It is concluded that liver cells possess plasma membrane Ca2+ channels that have a high selectivity for Ca2+, are activated by a decrease in the concentration of Ca2+ in intracellular stores through a mechanism that is unlikely to involve calmodulin, and are involved in re-filling intracellular Ca2+ stores during Ca2+ signaling.

Animals↗
PubMed1998

Co-expression of a Ca2+-inhibitable adenylyl cyclase and of a Ca2+-sensing receptor in the cortical thick ascending limb cell of the rat kidney. Inhibition of hormone-dependent cAMP accumulation by extracellular Ca2+.

The Ca2+-sensing receptor protein and the Ca2+-inhibitable type 6 adenylyl cyclase mRNA are present in a defined segment of the rat renal tubule leading to the hypothesis of their possible functional co-expression in a same cell and thus to a possible inhibition of cAMP content by extracellular Ca2+. By using microdissected segments, we compared the properties of regulation of extracellular Ca2+-mediated activation of Ca2+ receptor to those elicited by prostaglandin E2 and angiotensin II. The three agents inhibited a common pool of hormone-stimulated cAMP content by different mechanisms as follows. (i) Extracellular Ca2+, coupled to phospholipase C activation via a pertussis toxin-insensitive G protein, induced a dose-dependent inhibition of cAMP content (1.25 mM Ca2+ eliciting 50% inhibition) resulting from both stimulation of cAMP hydrolysis and inhibition of cAMP synthesis; this latter effect was mediated by capacitive Ca2+ influx as well as release of intracellular Ca2+. (ii) Angiotensin II, coupled to the same transduction pathway, also decreased cAMP content; however, its inhibitory effect on cAMP was mainly accounted for by an increase of cAMP hydrolysis, although angiotensin II and extracellular Ca2+ can induce comparable release of intracellular Ca2+. (iii) Prostaglandin E2, coupled to pertussis toxin-sensitive G protein, inhibited the same pool of adenylyl cyclase units as extracellular Ca2+ but by a different mechanism. The functional properties of the adenylyl cyclase were similar to those described for type 6. The results establish that the co-expression of a Ca2+-inhibitable adenylyl cyclase and of a Ca2+-sensing receptor in a same cell allows an inhibition of cAMP accumulation by physiological concentrations of extracellular Ca2+.

Adenylate Cyclase Toxin↗
PubMed1992

Transmembrane-mediated changes in [Ca2+] are involved in the signaling pathway leading to macrophage cytocidal differentiation: implications of localized changes in intracellular [Ca2+] and of interferon priming on Ca2+ utilization.

Macrophage cytocidal activation requires the sequential impingement on the macrophage of a priming stimulus (interferon [IFN] alpha, beta, or gamma) and a triggering stimulus (such as polyinosinic acid:polycytidylic acid [poly [I:C]] or bacterial lipopolysaccharide). The mechanism of progression from the IFN-primed state to the cytocidal state is poorly understood. By quantifying the level of expression of a gene product (complement component factor B [Bf]) associated with cytocidal activation and through the use of phenotypically distinct populations of macrophages (unprimed and IFN-primed), we have investigated the functional necessity of changes in intracellular concentration of free calcium ions ([Ca2+]i) in signaling the transition from the primed to the cytocidal state. Elevating the [Ca2+]i by incubation of unprimed macrophages with the calcium ionophore, ionomycin, failed to induce the expression of Bf. By contrast, Bf was expressed at high levels when IFN-primed macrophages were exposed to ionomycin, suggesting that priming induced within the macrophages the capacity to respond to a nonspecific change in [Ca2+]i. Quantification of the [Ca2+]i in response to exposure to ionomycin revealed an initial transient elevation, followed by a secondary sustained component. No differences in these changes were observed between unprimed and IFN-primed macrophages. We therefore questioned if changes in [Ca2+]i were also implicated in the transition between the primed and the cytocidal state using the ligand, poly [I:C]. In contrast to ionomycin, incubation of IFN-primed macrophages with poly [I:C] did not sustain measurable increases in [Ca2+]i, yet fully stimulated the transition from the IFN primed to the cytocidal state. However, incubation of IFN-primed macrophages with poly [I:C] in the presence of 1) a Ca2+/ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffer calculated to clamp the extracellular concentration of free calcium ions to a value approximately equal to the resting [Ca2+]i; 2) the calcium channel blocker verapamil; or 3) the intracellular Ca2+ antagonists (W-7, W-13, and TMB-8) substantially inhibited the induction of Bf. Collectively, these data support the following conclusions. First, that changes in [Ca2+]i comprise an important element in the induction of progression from the IFN-primed to the cytocidal state. Second, the failure to detect global changes in [Ca2+]i in response to the ligand, poly [I:C], suggests that changes in [Ca2+]i or Ca2+ movement may occur in either a spatially restricted or in an asynchronous cyclical fashion and are not detected by population fluorescence measurements. Third, the source of the relevant Ca2+ is extracellular. Fourth, our findings suggest that priming influences macrophage functional responses at a locus that is distal to the changes in [Ca2+]i, thereby potentially allowing signaling processes to be utilized to initiate different cellular responses.

Animals↗
PubMed2006

Flubendiamide, a novel Ca2+ channel modulator, reveals evidence for functional cooperation between Ca2+ pumps and Ca2+ release.

Flubendiamide, developed by Nihon Nohyaku Co., Ltd. (Tokyo, Japan), is a novel activator of ryanodine-sensitive calcium release channels (ryanodine receptors; RyRs), and is known to stabilize insect RyRs in an open state in a species-specific manner and to desensitize the calcium dependence of channel activity. In this study, using flubendiamide as an experimental tool, we examined an impact of functional modulation of RyR on Ca2+ pump. Strikingly, flubendiamide induced a 4-fold stimulation of the Ca2+ pump activity (EC50=11 nM) of an insect that resequesters Ca2+ to intracellular stores, a greater increase than with the classical RyR modulators ryanodine and caffeine. This prominent stimulation, which implies tight functional coupling of Ca2+ release with Ca2+ pump, resulted in a marginal net increase in the extravesicular calcium concentration despite robust Ca2+ release from the intracellular stores by flubendiamide. Further analysis suggested that luminal Ca2+ is an important mediator for the functional coordination of RyRs and Ca2+ pumps. However, kinetic factors for Ca2+ pumps, including ATP and cytoplasmic Ca2+, failed to affect the Ca2+ pump stimulation by flubendiamide. We therefore conclude that the stimulation of Ca2+ pump by flubendiamide is mediated by the decrease in luminal calcium, which may induce calcium dissociation from the luminal Ca2+ binding site on the Ca2+ pump. This mechanism should play an essential role in precise control of intracellular Ca2+ homeostasis.

Animals↗
PubMed1986

Intracellular free Ca2+ in the course of the Ca2+ paradox and during poisoning. Ca2+-selective microelectrode measurements in the perfused rat heart.

The free intracellular Ca2+ concentration of perfused rat hearts was measured using Ca2+-selective microelectrodes. In Krebs-Ringer bicarbonate buffer + 0.1 mmol/l Ca2+ (controls) the intracellular Ca2+ concentration was 0.87 +/- 0.07 mumol/l and the membrane potential was -51.5 +/- 0.3 mV. Without glucose the membrane potential approached zero after ca. 60 min, whereas the Ca2+ concentration during 110 min increased only slowly to 10.0 mumol/l. During Ca2+-free perfusion (5 min) both parameters did not change significantly. With reintroduction of 2.0 mmol/l Ca2+ the membrane potential rapidly collapsed and the intracellular Ca2+ concentration was elevated above 0.1 mmol/l within two min. Reperfusion with only 0.1 mmol/l Ca2+ decelerated both changes. Poisoning by carbonyl cyanide-p-trifluoromethoxyphenylhydrazone or antimycin A in Ca2+-free Krebs-Ringer bicarbonate buffer increased the intracellular Ca2+ concentration to 30.0 and 25.0 mumol/l, and the membrane potential was collapsed after 16 and 10 min, respectively. In antimycin A- and Ca2+-containing sucrose medium the intracellular Ca2+ during 16 min increased above 1.0 mmol/l, and the membrane potential began to increase only after 10 min. The results are consistent with the postulate of a hypothetical mechanism of cell injury, in which noxious membrane-cytoskeleton interactions are induced by an elevated intracellular Ca2+ concentration. It is concluded that Ca2+ entry via Na/Ca exchange is not fundamentally involved with induction of injury.

Animals↗
PubMed1992

Intraluminal Ca2+ dependence of Ca2+ and ryanodine-mediated regulation of skeletal muscle sarcoplasmic reticulum Ca2+ release.

The action of ryanodine upon sarcoplasmic reticulum (SR) Ca2+ handling is controversial with evidence for both activation and inhibition of SR Ca2+ release. In this study, the role of the intraluminal SR Ca2+ load was probed as a potential regulator of ryanodine-mediated effects upon SR Ca2+ release. Through dual-wavelength spectroscopy of Ca2+:antipyrylazo III difference absorbance, the intraluminal Ca2+ dependence of ryanodine and Ca(2+)-induced Ca2+ release (CICR) from skeletal SR vesicles was examined. Ryanodine addition after initiation of Ca2+ uptake (a) increased the intraluminal Ca2+ sensitivity of CICR and (b) stimulated spontaneous Ca2+ release with a delayed onset. These ryanodine effects were inversely proportional to the intraluminal Ca2+ load. Ryanodine also inhibited subsequent CICR after reaccumulation of Ca2+ released from the initial CICR. These results provide evidence that ryanodine inhibits transitions between low and high affinity Ca2+ binding states of an intraluminal Ca2+ compartment, possibly calsequestrin. Conformational transitions of calsequestrin may be reciprocally coupled to transitions between open and closed states of the Ca2+ release channel.

Adenosine Triphosphate↗