Changes in extracellular calcium within the physiological range influence receptor-mediated inositol phosphate responses in brain and tracheal smooth muscle slices

Summary The effect of changes in extracellular calcium concentration ([Ca²⁺]_e) on the incorporation of myo-[2-³H]-inositol into phosphoinositides and agonist-stimulated ³H-inositol phosphates (³H-InsPs) was examined in rat cerebral cortex and bovine tracheal smooth muscle slices. In brain slices, reduction in [Ca²⁺]_e from 2.4 to 1.2 mmol/l resulted in an approximate doubling of the carbachol and noradrenaline-stimulated ³H-InsP response with no effect on the EC₅₀ values. An identical effect of varying [Ca²⁺]_e was observed for carbachol-stimulated ³H-InsP formation in tracheal smooth muscle with a further increase in ³H-InsPs evident at [Ca²⁺]_e 0.6 mmol/l. In this tissue the effect of changes in [Ca ²⁺]_e on the incorporation of myo-[2-³H]-inositol into the total phosphoinositide pool directly paralleled the changes in ³H-InsPs except in conditions of no added calcium when ³H-InsP responses were markedly impaired. Additional studies in brain slices using buffer where the added calcium varied between 0 and 2.4 mmol/l, showed that both the carbachol stimulated formation of separate inositol phosphates during short incubation periods and incorporation of myo-[2-³H]-inositol into PtdInsP and PtdInsP₂ under basal conditions was maximal at [Ca²⁺]_e 0.3 mmol/l. Omitting Ca²⁺]_e from the buffer resulted in maximal labelling of PtdIns but a decrease in PtdInsP and PtdInsP₂ labelling (compared with the level at [Ca²⁺]_e 0.3 mmol/l) and a markedly impaired inositol polyphosphate response. Alterations in [Ca²⁺]_e following ³H-inositol labelling but immediately prior to carbachol stimulation did not influence ³H-inositol polyphosphate responses. It is therefore clear that even relatively small changes in [Ca²⁺]_e markedly influence agonist-stimulated ³H-InsP responses in brain and tracheal smooth muscle slices and that these reflect changes in the labelling of substrate inositol lipids. These findings have important practical implications for studies examining ³H-InsP responses in central and peripheral tissues and the differential effect of very low [Ca²⁺]_e on PtdIns and PtdlnsP/PtdInsP₂ labelling may explain in part the severe decrease in ³H-InsPs seen under these conditions despite apparent maximal total phosphoinositide labelling. Send offprint requests to S. R. Nahorski at the above address     

Caffeine inhibits cytoplasmic Ca2+ oscillations induced by carbachol and guanosine 5′-O-(3-thiotriphosphate) in hyperpolarized pancreatic ß-cells

The effects of caffeine on cytoplasmic Ca²⁺ oscillations induced by carbachol and guanosine 5-O-(3-thiotriphosphate) (GTP--S) were studied in individual mouse pancreatic ß-cells clamped at a hyperpolarized potential. Addition of 10 mM caffeine did not affect the cytoplasmic Ca²⁺ concentration ([Ca²⁺]₁) in ß-cells exposed to 20 mM glucose and hyperpolarized with diazoxide. Under similar conditions 100 M carbachol induced a typical response with a marked [Ca²⁺]_i peak followed by a lower sustained elevation. Irrespective of whether 10 mM caffeine was present, there were [Ca²⁺]_i transients with frequencies of 1–5/min superimposed on the sustained phase in 50–60% of the cells. In previously non-exposed cells the introduction of 10 mM caffeine caused temporary lowering of the sustained phase with disappearance of the transients. Subsequent omission of caffeine in the continued presence of carbachol caused a marked [Ca²⁺]_i peak followed by reappearance of the [Ca²⁺]_i, transients. However, in cells oscillating in the presence of caffeine its omission caused disappearance of the transients. In this case reintroduction of caffeine restored the transients.In cells kept at –70 mV by a patch pipette containing 100 M GTP--S and 3 mM Mg-ATP there were [Ca²⁺]_i transients with frequencies of 0.5–2.5/min. These transients were sufficiently pronounced to activate repetitively a K⁺ current. Addition of 10 mM caffeine caused disappearance of the [Ca²⁺]_i transients or reduction of their amplitudes and frequencies.The results indicate that caffeine does not activate Ca²⁺-induced Ca²⁺ release in hyperpolarized ß-cells but inhibits the Ca²⁺-mobilizing effect of inositol 1,4,5-trisphosphate. Correspondence to: E. Gylfe at the above address     

Direct interference of HIV protease inhibitors with pancreatic β-cell function

The aim of the present study was to evaluate whether HIV protease inhibitors directly interfere with stimulus-secretion coupling in pancreatic -cells. Insulin secretion was determined by a radioimmunoassay (RIA), cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) with the fluorescence dye fura-2 and whole-cell membrane currents with the patch-clamp technique.Glucose-induced insulin secretion was inhibited in a concentration-dependent manner by ritonavir and nelfinavir but not by indinavir. Ritonavir and nelfinavir lowered [Ca²⁺]_c in the presence of a stimulatory glucose concentration whereas indinavir again had no effect. Ritonavir and nelfinavir completely inhibited the effect of tolbutamide, which normally increases [Ca²⁺]_c by blocking K_ATP channels. This observation points to an action of both drugs on K_ATP channels or a step distal to these channels in stimulus-secretion coupling. Ritonavir was used to further evaluate the direct effects of HIV protease inhibitors on -cell ion channel currents. Unexpectedly, ritonavir inhibited neither the whole-cell K_ATP current nor the whole-cell L-type Ca²⁺ current. Tolbutamide almost completely suppressed the K_ATP current in the presence of ritonavir excluding that ritonavir alters the tolbutamide sensitivity of the K_ATP channel. Ritonavir increased the length and decreased the frequency of glucose-induced action potentials. This effect can be attributed to inhibition of voltage-dependent K⁺ currents. Intracellular stores seem not to be involved in the ritonavir-induced lowering of [Ca²⁺]_c.In conclusion, different HIV protease inhibitors surprisingly reveal distinct effects on insulin secretion. Ritonavir inhibits insulin secretion by lowering [Ca²⁺]_c but this effect is evidently independent of the opening of K_ATP channels or the closure of voltage-dependent Ca²⁺ channels, which are commonly considered to play a key role in stimulus-secretion coupling.Abbreviations [Ca²⁺]_c Cytosolic Ca²⁺ concentration - RIA Radioimmunoassay     

TRPV1 in Salivary Gland Epithelial Cells Is Not Involved in Salivary Secretion via Transcellular Pathway

Transient receptor potential vanilloid subtype 1 (TRPV1) was originally found in sensory neurons. Recently, it has been reported that TRPV1 is expressed in salivary gland epithelial cells (SGEC). However, the physiological role of TRPV1 in salivary secretion remains to be elucidated. We found that TRPV1 is expressed in mouse and human submandibular glands (SMG) and HSG cells, originated from human submandibular gland ducts at both mRNA and protein levels. However, capsaicin (CAP), TRPV1 agonist, had little effect on intracellular free calcium concentration ([Ca²⁺]_i) in these cells, although carbachol consistently increased [Ca²⁺]_i. Exposure of cells to high temperature (>43℃) or acidic bath solution (pH5.4) did not increase [Ca²⁺]_i, either. We further examined the role of TRPV1 in salivary secretion using TRPV1 knock-out mice. There was no significant difference in the pilocarpine (PILO)-induced salivary flow rate between wild-type and TRPV1 knock-out mice. Saliva flow rate also showed insignificant change in the mice treated with PILO plus CAP compared with that in mice treated with PILO alone. Taken together, our results suggest that although TRPV1 is expressed in SGEC, it appears not to play any direct roles in saliva secretion via transcellular pathway.     

Effects of the Chinese herb component phellopterin on the increase in cytosolic free calcium in PC12 cells

The present study investigated the effects of the Chinese Herb component, phellopterin on high K⁺ and glutamate‐induced extracellular calcium influx and caffeine or cyclopiazonic acid (CPA)‐induced calcium release from internal stores in attached PC12 cells. Attached cells were loaded with the calcium fluorescent indicator Fluo‐3/AM with the final concentration of 5 µM for 50 min at 37°C and cytosolic free Ca²⁺ measured as fluorescent intensity (FI) (excitation: 488 nm; emission: 535 nm). When PC12 cells were exposed to extracellular Ca²⁺([Ca²⁺]₀) 2.0 mM, the FI for resting [Ca²⁺]_i was 1,188±163, high K⁺ (75 mM) and glutamate (10 mM) induced an increase in [Ca²⁺]_i with peak values of 4,270±982 and 3,096±402, respectively. Phellopterin (0.1–100 µM) had no apparent effect on resting [Ca²⁺]_i, but inhibited high K⁺ and glutamate induced the increase in [Ca²⁺]_i in a dose‐dependent manner. When PC12 cells were exposed to Ca²⁺‐free solution, the FI for resting [Ca²⁺]_i was 804±77. Caffeine (40 mM) and CPA (30 µM) stimulated Ca²⁺ release from caffeine‐ryanodine and inositol 1,4,5‐tris‐phosphate (InsP₃₎‐sensitive internal calcium stores, inducing an increase in [Ca²⁺]_i to 2,938±362 and 1,816±291, respectively. Phellopterin (0.1–100 µmol/L) inhibited caffeine and CPA stimulated intracellular calcium release in a dose‐dependent manner. In summary, phellopterin, a novel component isolated from Changii radix, inhibited Ca²⁺ influx induced by stimulation of voltage‐gated and receptor‐dependent calcium channels with a greater inhibition of receptor‐dependent calcium channels. It also inhibited Ca²⁺ release from caffeine‐ryanodine and InsP₃‐sensitive internal stores, being more potent for caffeine stimulation. Phellopterin may be a promising candidate for the development of new classes of calcium antagonists. Drug Dev Res 68:79–83, 2007. © 2007 Wiley‐Liss, Inc.     

Sodium dependence of the inotropic effect of a reduction in extracellular potassium concentration

Summary The positive inotropic effect of a reduction of [K⁺]_e from 5.9 mM to 1.2 mM or less depends on the extracellular concentration of sodium. The increase in contractility is greatly reduced or prevented if [Na⁺]_e has been diminished from 140 mM to 70 mM (with the addition of sucrose).A reduction of [K⁺]_e to 1.2 mM leads to an increase in membrane resting potential (MRP) by an average of 16 mV (from –71 mV to –87 mV) and to a diminution of the overshoot by 7 mV, both changes not being influenced by a reduction of [Na⁺]_e.The increase in the rate of depolarization is relatively small (6%) indicating that the sodium-carrying system in guinea-pig ventricular muscle is almost completely activated at a MRP not far from –70 mV.The sodium-dependence of the inotropic effect caused by a reduction of [K⁺]_e points to the significance of the impairment of the sodium pump by the reduction of [K⁺]_e in the production of the inotropic effect. The mechanism of the inotropic effect is assumed to consist of an increase in calcium influx through an increased sodium/calcium exchange as a consequence of a rise in [Na⁺]_i.The duration of the AP of guinea-pig papillary muscle is dependent on the relation [Ca²⁺]_e/[Na⁺]_e ². A diminution of [Na⁺]_e to 50%, therefore, does not alter the length of the plateau phase of the AP (at 30% repolarization) provided that [Ca²⁺]_e has been reduced simultaneously to one fourth.The duration of the plateau of the AP is shortened by an average of 22 msec in response to a reduction of [K⁺]_e to 1.2 mM. This shortening of the plateau is the only alteration in the electrical behaviour of the cardiac cell which is correlated with the positive inotropic effect of the reduction of [K⁺]_e, since it occurs only with [Na⁺]_e of 140 mM but not with 70 mM. Considering the antagonism between calcium and sodium in regard to their effects upon AP duration, the shortening of the plateau is assumed to be indicative of a reduction of slow sodium inward current as a result of an increase in calcium influx.Supported by the Deutsche Forschungsgemeinschaft.     

Influence of oligopeptide aldehydes on intracellular Ca2+ concentration in rat pituitary cells

We investigated the effects of some synthetic tripeptide aldehydes, earlier shown to influence pituitary hormone secretion and ⁴⁵Ca²⁺ uptake, on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) of rat anterior pituitary cells in suspension. Boc-D-Phen- Leu-Phenylalaninal or Boc-D-Phe-Leu-Prolinal in the tested range of 1–100 or 200 μM, respectively, were ineffective in influencing basal [Ca²⁺]_i but caused a concentration-dependent inhibition in K⁺ (25 mM)-induced [Ca²⁺]_i elevation. The IC₅₀ of both effects was about 50 μM. In contrast, they did not interfere with the stimulation caused by the calcium channel agonist BAY K 8644 and were also ineffective in influencing the receptor-mediated stimulus of thyrotropin-releasing hormone on [Ca²⁺]_i. On the basis of the present and foregoing results the possible involvement of calcium channels is discussed, but different mechanisms mediating the tripeptide aldehyde inhibition are also considered. A third tripeptide aldehyde. Boc-Gln-Leu-Lysinal (Boc-GLL), showed ionophore-like properties. This nontoxic substance caused a dose-dependent rise up to 400% (at 100 μM) in [Ca²⁺]_i. Its effect is not mediated by voltage-dependent calcium channels, as it cannot be inhibited either by the classicalp calcium channel antagonists verapamil and nifedipine, or by the above-mentioned inhibitory tripeptide aldehydes. When we decreased the extracellular Ca²⁺ concentration by the addition of 4 mM EGTA, the effect was inverted and Boc-GLL caused a large fall in [Ca²⁺]_i. We suggest that Boc-GLL may open cell membrane pores through which Ca²⁺ moves along the concentration gradient. The calcium flux can be inhibited by 20 mM Mg²⁺ and 100 μM Co²⁺ but not by 500 μM La³⁺. Thus, tripeptide aldehydes. depending on their structure, may decrease or increase [Ca²⁺]_i via uncoventional mechanisms and may serve as tools for dissecting details of cell calcium homeostasis.     

Cholecystokinin-JMV-180 and pilocarpine are potent inhibitors of cholecystokinin and carbachol actions on guinea pig pancreatic acinar cells

The release of amylase and the elevation of cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) in response to cholecystokinin-octapeptide (CCK-8), the cholecystokinin analogue JMV-180, the stable choline ester carbamylcholine (carbachol) and the muscarinic agonist pilocarpine were studied in guinea-pig pancreatic acinar cells. The maximal amylase and [Ca²⁺]_i responses to JMV-180 and pilocarpine were 12-15% of the corresponding responses to CCK-8 and carbachol. The amylase and [Ca²⁺]_i responses to maximal concentrations of CCK-8 and carbachol were inhibited in concentration-dependent manners by JMV-180 and pilocarpine, respectively. In individual acinar cells, JMV-180 and pilocarpine like low concentrations of CCK-8 and carbachol caused oscillations of [Ca²⁺]_i. The sustained [Ca²⁺]_i responses to maximal concentrations of CCK-8 and carbachol were transformed into oscillatory responses during simultaneous exposure to JMV-180 and pilocarpine, respectively. Maximal concentrations of JMV-180 and pilocarpine did not cause homologous or heterologous desensitization of the [Ca²⁺]_i responses but inhibited desensitization evoked by maximal concentrations of CCK-8 or carbachol. JMV-180 and pilocarpine acted as weak, partial agonists exhibiting effective inhibition of the acinar cell responses to full agonists. The effects appeared to be best explained by interactions with two forms of the respective receptor with JMV-180 and pilocarpine acting as partial agonists for one state of the receptor and as antagonist for the second state. Received: 19 August 1996 / Accepted: 21 January 1997     

BDE-47 and 6-OH-BDE-47 modulate calcium homeostasis in primary fetal human neural progenitor cells via ryanodine receptor-independent mechanisms

Polybrominated diphenyl ethers (PBDEs) are bioaccumulating flame retardants found in rising concentrations in human tissue. Epidemiological and animal studies have raised concern for their potential to induce developmental neurotoxicity (DNT). Considering the essential role of calcium homeostasis in neurodevelopment, PBDE-induced disturbance of intracellular calcium concentration ([Ca²⁺]_i) may underlie PBDE-induced DNT. To test this hypothesis, we investigated acute effects of BDE-47 and 6-OH-BDE-47 on [Ca²⁺]_i in human neural progenitor cells (hNPCs) and unraveled involved signaling pathways. Short-time differentiated hNPCs were exposed to BDE-47, 6-OH-BDE-47, and multiple inhibitors/stimulators of presumably involved signaling pathways to determine possible effects on [Ca²⁺]_i by single-cell microscopy with the fluorescent dye Fura-2. Initial characterization of calcium signaling pathways confirmed the early developmental stage of hNPCs. In these cells, BDE-47 (2 μM) and 6-OH-BDE-47 (0.2 μM) induce [Ca²⁺]_i transients. This increase in [Ca²⁺]_i is due to extracellular Ca²⁺ influx and intracellular release of Ca²⁺, mainly from the endoplasmic reticulum (ER). While extracellular Ca²⁺ seems to enter the cytoplasm upon 6-OH-BDE-47 by interfering with the cell membrane and independent of Ca²⁺ ion channels, ER-derived Ca²⁺ is released following activation of protein lipase C and inositol 1,4,5-trisphosphate receptor, but independently of ryanodine receptors. These findings illustrate that immature developing hNPCs respond to low concentrations of 6-OH-BDE-47 by an increase in [Ca²⁺]_i and provide new mechanistic explanations for such BDE-induced calcium disruption. Thus, these data support the possibility of a critical window of PBDE exposure, i.e., early human brain development, which has to be acknowledged in risk assessment.     

Low concentrations of caffeine raise intracellular calcium concentration only in the presence of extracellular calcium in cultured molluscan neurons

• 1.1. The effects of low concentrations of caffeine (100 and 300 μM) on the intracellular calcium concentration [Ca²⁺]_i in four cultured, identified neurons of the pond snail Lymnaea stagnalis (L) were investigated.
• 2.2. Intracellular CA²⁺ levels in these neurons were measured with the cell-permeable Ca²⁺ indicator Fura-2/AM, both in the presence and absence of extracellular Ca²(O-Ca²⁺/EGTA).
• 3.3. In the presence of Ca²⁺ in the external medium, caffeine was found to induce a substantial elevation in the free [Ca²⁺]_i in all cell types.
• 4.4. In some cases, the rise in [Ca²⁺]_i was found to be both time- and concentration-dependent.
• 5.5. Low doses of caffeine did not produce any appreciable rise in [Ca²⁺]_i in the absence of Ca²⁺ in the external medium, but calcium was still available from stores, as clinical concentrations of halothane rose [Ca²⁺]_i in the absence of extracellular calcium.
• 6.6. These results indicate that the actions of caffeine, when applied at low concentrations, are dependent on extracellular calcium.

     

Intracellular influx of calcium induced by quartz particles in alveolar macrophages

Historical studies report that cellular injury and silicosis are related to cytosolic free calcium (Ca²⁺). Moreover, reactive oxygen species (ROS) have been linked to cellular injury. However, the detail mechanism of the increase in [Ca²⁺]_i and the relationship between [Ca²⁺]_i and ROS production remains unknown. Quartz particle has been found to increase [Ca²⁺]_i and activate the generation of ROS. Our hypothesis is that [Ca²⁺]_i increase induced by quartz particle is from extracellular Ca²⁺ through the Ca²⁺ channel, and [Ca²⁺]_i increase is believed to activate ROS production. In order to examine this hypothesis, we treated rat alveolar macrophages with quartz (SiO₂) particles and used laser scanning confocal microscopy to measure [Ca²⁺]_i and the fluorescence intensity of ROS. Time- and dose-dependent increases in [Ca²⁺]_I and ROS in macrophages as well as cell viability were observed. Through chelating extracellular Ca²⁺ with ethylene glycol tetraacetic acid and releasing intracellular Ca²⁺ with thapsigargin, we found that 72.7% of the [Ca²⁺]_i increase was due to the influx of Ca²⁺ from the extracellular environment, via Ca²⁺ channels in the plasma membrane. By adding mannitol to scavenge hydroxyl radicals (OH·), and removing surface iron from the quartz particles to reduce OH· generation, we observed a reduced level of ROS generation, whereas the increase in [Ca²⁺]_i was unaffected. When using EGTA to reduce [Ca²⁺]_i, we observed a decrease in ROS production. This study suggests that the [Ca²⁺]_i influx was independent of OH· production, and the [Ca²⁺]_i increase resulted in ROS production. These results further indicate that there is a strong relationship between cytosolic free Ca²⁺ content and cellular injury as well as silica exposure.     

Cyanidin-3-glucoside Inhibits ATP-induced Intracellular Free Ca2+ Concentration,ROS Formation and Mitochondrial Depolarization in PC12 Cells

Flavonoids have an ability to suppress various ion channels. We determined whether one of flavonoids, cyanidin-3-glucoside, affects adenosine 5''-triphosphate (ATP)-induced calcium signaling using digital imaging methods for intracellular free Ca²⁺ concentration ([Ca²⁺]_i), reactive oxygen species (ROS) and mitochondrial membrane potential in PC12 cells. Treatment with ATP (100µM) for 90 sec induced [Ca²⁺]_i increases in PC12 cells. Pretreatment with cyanidin-3-glucoside (1µ g/ml to 100µg/ml) for 30 min inhibited the ATP-induced [Ca²⁺]_i increases in a concentration-dependent manner (IC₅₀=15.3µg/ml). Pretreatment with cyanidin-3-glucoside (15µg/ml) for 30 min significantly inhibited the ATP-induced [Ca²⁺]_i responses following removal of extracellular Ca²⁺ or depletion of intracellular [Ca²⁺]_i stores. Cyanidin-3-glucoside also significantly inhibited the relatively specific P2X2 receptor agonist 2-MeSATP-induced [Ca²⁺]_i responses. Cyanidin-3-glucoside significantly inhibited the thapsigargin or ATP-induced store-operated calcium entry. Cyanidin-3-glucoside significantly inhibited the ATP-induced [Ca²⁺]_i responses in the presence of nimodipine and ω-conotoxin. Cyanidin-3-glucoside also significantly inhibited KCl (50 mM)-induced [Ca²⁺]_i increases. Cyanidin-3-glucoside significantly inhibited ATP-induced mitochondrial depolarization. The intracellular Ca²⁺ chelator BAPTA-AM or the mitochondrial Ca²⁺ uniporter inhibitor RU360 blocked the ATP-induced mitochondrial depolarization in the presence of cyanidin-3-glucoside. Cyanidin-3-glucoside blocked ATP-induced formation of ROS. BAPTA-AM further decreased the formation of ROS in the presence of cyanidin-3-glucoside. All these results suggest that cyanidin-3-glucoside inhibits ATP-induced calcium signaling in PC12 cells by inhibiting multiple pathways which are the influx of extracellular Ca²⁺ through the nimodipine and ω-conotoxin-sensitive and -insensitive pathways and the release of Ca²⁺ from intracellular stores. In addition, cyanidin-3-glucoside inhibits ATP-induced formation of ROS by inhibiting Ca²⁺-induced mitochondrial depolarization.     

Lead-induced ER calcium release and inhibitory effects of methionine choline in cultured rat hippocampal neurons

Lead, a ubiquitous neurotoxicant, can result in learning and memory dysfunction. Long term potentiation in the hippocampus, a potential neural substrate for learning and memory, is thought to be linked to calcium-triggered intracellular events. In this study, laser scanning confocal microscopy was used to examine the effects of Pb²⁺ on intracellular and endoplasmic reticulum free calcium concentration ([Ca²⁺]_i and [Ca²⁺]_ER) in cultured neonatal rat hippocampal neurons and their possible antagonism by methionine choline; understanding these effects would help explain the lead-induced cognitive and learning dysfunction and explore efficient safety and relief strategies. The results showed that Pb²⁺ increased [Ca²⁺]_i and decreased [Ca²⁺]_ER linearly in a time- and concentration-dependant manner, and Pb²⁺ addition after the applying of a ryanodine receptor (RyR) antagonist and an inositol-1,4,5-triphosphate receptor (IP₃R) antagonist did not increase [Ca²⁺]_i. The addition of 10, 20, or 40 mmol/L methionine choline simultaneously with addition of 10 μmol/L Pb²⁺ decreased [Ca²⁺]_i in Ca²⁺-free culture medium by 39.0%, 66.0%, and 61.6%, respectively, in a concentration-dependant manner in a certain dose range. Our results suggest that Pb²⁺ induces ER calcium release to increase the resting [Ca²⁺]_i; and methionine choline inhibit this increase in [Ca²⁺]_i.     

Enhancement by captopril of bradykinin-induced calcium transients in cultured endothelial cells of the bovine aorta

Using microscopic fluorometry and fura-2-loaded cultured bovine aortic endothelial cells, we determined the effects of captopril, an angiotensin converting enzyme (ACE) inhibitor, on bradykinin-induced Ca²⁺ transients in endothelial cells. In the presence of extracellular Ca²⁺, 10⁻⁹ M bradykinin induced an early rise in the transients followed by sustained elevations of cytosolic calcium concentration ([Ca²⁺]_i). Bradykinin concentration-dependently increased [Ca²⁺]_i (EC₅₀ 6.7 × 10⁻⁹ M). Captopril, 10⁻⁵ M, enhanced and prolonged the bradykinin-induced Ca²⁺ transients and shifted the concentration-response curve to the left (EC₅₀ 8.5 × 10⁻¹⁰ M). In porcine coronary aterial strips with intact endothelium, cumulative applications of bradykinin induced an endothelium-dependent relaxation during prostaglandin F_2α-induced contraction (EC₅₀ = 2.0 × 10⁻⁹ M). Treatment with 10⁻⁵ M captopril enhanced the bradykinin-induced relaxation and shifted the concentration-response curve to the left (EC₅₀ = 7.6 × 10⁻¹⁰ M). Thus, captopril enhances the bradykinin-induced relaxation by mechanisms mainly dependent on the endothelium, namely the inhibition of ACE.     

Differential calcium signalling by m2 and m3 muscarinic acetylcholine receptors in a single cell type

We have compared muscarinic acetylcholine receptor (mAChR) coupling to phospholipase C (PLC) and increases in cytoplasmic Ca²⁺ concentration [Ca²⁺]_i in human embryonic kidney (HEK) cells, stably expressing either the human m3 or m2 receptor subtype. In m3 mAChR-expressing cells, carbachol stimulated inositol phosphate (InsP) formation and increased [Ca²⁺]_i with EC₅₀ values of about 2 M and 30 nM, respectively. Maximal inositol 1,4,5-trisphosphate (InsP₃) production (about fourfold) was rapid (15 s) and stable for 2 min. Maximal increases in [Ca²⁺]_i were 300–350 nM and mainly, almost 90%, due to influx of extracellular Ca²⁺. The efficacy of pilocarpine for stimulating InsP andCa²⁺ responses was not significantly different from that of carbachol. All m3 mAChR-mediated responses were pertussis toxin (PTX)-insensitive. In m2 mAChR-expressing cells, carbachol stimulated InsP formation and increased [Ca²⁺]_i with EC₅₀ values of about 20 M and 7 M, respectively. Maximal InsP formation was only 10–15% of that observed in m3 mAChR-expressing cells, whereas maximal elevations of [Ca²⁺]_i were similar in both cell types. Formation of InsP₃ was rapid (15 s to 2 min) and about twofold above basal. In contrast to m3 mAChR activation, [Ca²⁺]_i increases induced by m2 mAChR activation were exclusively due to Ca²⁺ mobilization from intracellular stores.The efficacy of pilocarpine for stimulating InsP and Ca²⁺ responses was 50% and 20% of the efficacy of carbachol, respectively. PTX treatment did not affect m2 mAChR-induced PLC stimulation, but reduced the m2 mAChR-mediated increases in [Ca²⁺]_i to 50%. In conclusion, m3 and m2 mAChRs stably expressed in HEK cells can induce similar cellular responses; however, they do so by activating apparently distinct signalling pathways. While coupling of m2 mAChR to PLC occurs in a PTX-insensitive manner, coupling to mobilization of Ca²⁺ from intracellular stores is partly PTX-sensitive and this may occur at least partly independent of PLC activation.     

DA-6034 Induces [Ca2+]i Increase in Epithelial Cells

DA-6034, a eupatilin derivative of flavonoid, has shown potent effects on the protection of gastric mucosa and induced the increases in fluid and glycoprotein secretion in human and rat corneal and conjunctival cells, suggesting that it might be considered as a drug for the treatment of dry eye. However, whether DA-6034 induces Ca²⁺ signaling and its underlying mechanism in epithelial cells are not known. In the present study, we investigated the mechanism for actions of DA-6034 in Ca²⁺ signaling pathways of the epithelial cells (conjunctival and corneal cells) from human donor eyes and mouse salivary gland epithelial cells. DA-6034 activated Ca²⁺-activated Cl^- channels (CaCCs) and increased intracellular calcium concentrations ([Ca²⁺]_i) in primary cultured human conjunctival cells. DA-6034 also increased [Ca²⁺]_i in mouse salivary gland cells and human corneal epithelial cells. [Ca²⁺]_i increase of DA-6034 was dependent on the Ca²⁺ entry from extracellular and Ca²⁺ release from internal Ca²⁺ stores. Interestingly, these effects of DA-6034 were related to ryanodine receptors (RyRs) but not phospholipase C/inositol 1,4,5-triphosphate (IP₃) pathway and lysosomal Ca²⁺ stores. These results suggest that DA-6034 induces Ca²⁺ signaling via extracellular Ca²⁺ entry and RyRs-sensitive Ca²⁺ release from internal Ca²⁺ stores in epithelial cells.     

神经肽DGAVP和Org2766对神经细胞内游离Ca2+的影响

运用Ca²⁺指示剂Fura-2作为细胞内钙离子的荧光探针,利用AR—CM—MIC阳离子测定系统,检测了分离的神经细胞内游离钙及其变化,并观测了DGAVP和Org2766对蛋白质合成抑制剂茴香霉素(ANI)引起细胞内钙离子浓度([Ca²⁺]_i)变化的影响。结果表明茴香霉素可使[Ca²⁺]_i显著升高,且有量效关系;DGAVP本身并不引起[Ca²⁺]_i发生显著变化,但适当剂量的DGAVP可显著对抗一定剂量范围内ANI升高[Ca²⁺]_i的作用,提示DGAVP对抗ANI的蛋白质合成抑制效应可能是通过拮抗ANI升高[Ca²⁺]_i这一途径实现的,另一神经肽Org2766则可能不是通过这一机制发生作用。从细胞内Ca²⁺的角度看,这两种肽的作用机理显然是不同的。     

Effects of ω-conotoxin GVIA on electrical field stimulation- and agonist-induced changes in cytosolic Ca2+ and tension in isolated rat anococcygeus muscle

1 It has been reported that ω -conotoxin GVIA (ω-CgTx) blocks L- and N-type voltage-sensitive Ca²⁺ channels (VSCCs) in neurones and inhibits neurotransmitter release in various tissues. The present study investigates the effects of ω-CgTx on electrical field stimulation (EFS)- and agonist-induced changes in free cytosolic Ca²⁺ ([Ca²⁺]_cyt) levels and tension in isolated fura-2 loaded rat anococcygeus muscle. 2 EFS produced frequency-dependent increases in [Ca⁺]_cyt levels and contractions. Phentolamine (1 μM) and ω-CgTx (0.1 μM) significantly inhibited EFS-induced responses and shifted the frequency-response curves to the right. 3 α-adrenoceptor agonists (noradrenaline and clonidine) and carbachol (in the presence of phentolamine) produced concentration-dependent increases in [Ca²⁺]_cyt levels and contractions. Though ω-CgTx (0.1 μM) significantly inhibited the increases in [Ca²⁺]_cyt levels induced by low doses of noradrenaline, the overall concentration–response curves of [Ca²⁺]_cyt and contractions for noradrenaline, clonidine, and carbachol were not affected by ω-CgTx. 4 When the tone of rat anococcygeus muscle was raised with either clonidine (0.1 μM) or carbachol (30 μM, in the presence of 3 μM phentolamine), EFS (2 Hz) produced reproducible decreases in [Ca²⁺]_cyt levels and relaxations. These responses were significantly inhibited by ω-CgTx when the tissue was precontracted with clonidine, but not when it was precontracted with carbachol. 5 The results of the present study suggest that in rat anococcygeus muscle, ω-CgTx inhibits the EFS-induced release of both excitatory and inhibitory neurotransmitters, probably by blocking Ca²⁺ channels on nerve terminals. Furthermore, the Ca²⁺ channels present on the smooth muscle cell membrane, which are involved in the agonist-induced Ca²⁺ influx and contractions, may not be sensitive to ω-CgTx.     

Evidence for adenosine receptor-mediated isoprenaline-antagonistic effects of the adenosine analogs PIA and NECA on force of contraction in guinea-pig atrial and ventricular cardiac preparations

Summary The effects of the adenosine agonists (–)-N⁶-phenylisopropyladenosine (PIA) and 5-N-ethylcarboxamideadenosine (NECA) on force of contraction, adenylate cyclase activity and normal as well as slow action potentials were studied in guinea-pig isolatedatrial (left auricles) andventricular preparations (papillary muscles).Inauricles PIA and NECA exerted concentration-dependent negative inotropic effects with similar potenticies (mean EC₅₀:0.05 mol l^–1 for PIA and 0.03 mol l^–1 for NECA). Similar results were obtained in the presence of isoprenaline.Inpapillary muscles PIA and NECA alone had no effect on force of contraction but produced negative inotropic effects in the presence of isoprenaline (mean EC₅₀:0.19 mol l^–1 for PIA and 0.10 mol l^–1 for NECA).In both preparations, the negative inotropic effects of PIA and NECA in the presence of isoprenaline were antagonized by the adenosine receptor antagonist 8-phenyltheophylline.In both preparations, PIA and NECA did not affect adenylate cyclase activity, both in the absence and presence of isoprenaline.Inauricles the negative inotropic effects of both nucleosides were accompanied by shortening of the action potential. This effect was also observed in the presence of isoprenaline. Inpapillary muscles the adenosine analogs did not detectably alter the shape of the normal action potential. Ca²⁺-dependent slow action potentials elicited in potassium-depolarized preparations also remained unaltered in the presence of PIA or NECA alone. However, the isoprenaline-induced enhancement of the maximal rate of depolarization of slow action potentials was attenuated by PIA or NECA.It is concluded that in guinea-pig atrial and ventricular cardiac preparations the adenosine analogs PIA and NECA exert isoprenaline-antagonistic effects on force of contraction via adenosine receptors the existence of which can thus be shown in a functional way. These receptors are not detectably coupled to the adenylate cyclase. The negative inotropic effect in theauricle is most likely due to a shortening of the action potential resulting from an activation of potassium channels, which in turn indirectly reduces the Ca²⁺ influx during the action potential. In theventricle the adenosine receptor is either not linked to these potassium channels or adenosine-sensitive potassium channels do not exist in the ventricle. Instead the activation of the receptor causes a decrease of the slow Ca²⁺ inward current but this effect is observed only when the slow Ca²⁺ inward current had previously been enhanced by a cyclic AMP-dependent mechanism.     

BAY K 8644 stimulates glucose-dependent rise of cytoplasmic Ca2+ in hyperpolarized pancreatic β-cells

Summary The effect of BAY K 8644 on the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) was studied in pancreatic -cells hyperpolarized by the K⁺ channel-activating agent diazoxide. After 50–60 min preexposure to 0–20 mM glucose in the presence of 400 M diazoxide [Ca²⁺]_i was close to the level in unstimulated -cells. The addition of 5 M BAY K 8644 then triggereed a rise of [Ca²⁺]_i dependent on Ca²⁺ influx. The magnitude of the BAY K 8644 effect increased with the glucose concentration and was almost 10-fold higher in 20 mM than in the absence of the sugar. It is concluded that glucose can modulate Ca²⁺ entry through the voltage-dependent channels by a mechanism additional to depolarization. This action may help to explain why previous exposure to the sugar results in an augmented insulin response to a second challenge.