Mechanisms of calcium transport in the giant axon of the squid and their physiological role

The giant axon of the squid has been extensively used as a model for studying Ca regulation in excitable cells. Different techniques (extrusion, injection and dialysis) have been employed to characterize Ca fluxes across the axon membrane. Since both Ca efflux and influx are markedly dependent on [Ca²⁺]_i, considerable effort has been dedicated to determine the resting value of the [Ca²⁺]_i. Results from different laboratories indicate that the [Ca²⁺]_i, in a normal fibre, range from 20–100 nM. Under dialysis conditions (internal control), with an imposed [Ca²⁺]_i of 80 nM, Ca influx is balanced by an outward Ca movement of about 40 f/CS. Ca extrusion occurs through two parallel transport systems: one having a high affinity for [Ca²⁺]_i, dependent on ATP, not affected by Na_i, Na_o, Ca_o, Mg_o and inhibited by internal vanadate (uncoupled component), the other, more prominent at relatively high [Ca²⁺]_i, does not require ATP, is inhibited by Na_i activated by Na_o and not inhibited by vanadate. (Na_o-dependent component). The existence of these two systems provide the axon with an effective way to maintain in the long term a constant low [Ca²⁺]_i in spite of short term fluctuations due to increased Ca influx during nervous activity.     

A non-invasive vibrating calcium-selective electrode measures acetylcholine-induced calcium flux across the sarcolemma of a smooth muscle

To determine possible sources of Ca²⁺ during excitation-contraction coupling in smooth muscle, a vibrating Ca²⁺-selective electrode was used to measure Ca²⁺ flux during the process of contraction. The smooth muscle model was the longitudinal muscle of the body wall of a sea cucumberSclerodactyla briareus. Because acetylcholine caused slow contractions of the muscle that were inhibited by Ca²⁺ channel blockers diltiazem and verapamil in earlier mechanical studies, we chose a vibrating Ca²⁺-selective electrode as our method to test the hypothesis that acetylcholine may be stimulating Ca²⁺ influx across the sarcolemma, providing a Ca²⁺ source during excitation-contraction coupling. Acetylcholine treatment stimulated a net Ca²⁺ efflux that was both dose and time dependent. We then tested two L-type Ca²⁺ channel blockers, diltiazem and verapamil, and two non-specific Ca²⁺ blockers, cobalt (Co²⁺) and lanthanum (La³⁺) on acetylcholine-induced Ca²⁺ flux. All four Ca²⁺ blockers tested potently inhibited Ca²⁺ efflux induced by physiological doses of acetylcholine. We propose that the acetylcholine-induced Ca²⁺ efflux was the result of, first, Ca²⁺ influx through voltage-sensitive L-type Ca²⁺ channels, then the rapid extrusion of Ca²⁺ by an outwardly directed carrier such as the Na–Ca exchanger as suggested by Li⁺ substitution experiments. The vibrating Ca²⁺ electrode has provided new insights on the active and complex role the sarcolemma plays in Ca²⁺ homeostasis and regulating Ca²⁺ redistribution during excitation-contraction coupling.Abbreviations ACh acetylcholine - E-C coupling excitation-contraction coupling - LMBW longitudinal muscle of the body wall     

Effects of elevated cytosolic calcium on ACh-induced swine tracheal smooth muscle contraction

Increased intracellular calcium concentration ([Ca²⁺]_i) is required for smooth muscle contraction. In tracheal and other tonic smooth muscles, contraction and elevated [Ca²⁺]_i are maintained as long as an agonist is present. To evaluate the physiological role of steady-state increases in Ca²⁺ on tension maintenance, [Ca²⁺]_i was elevated using ionomycin, a Ca²⁺ ionophore or charybdotoxin, a large-conductance calcium-activated potassium channel (K_Ca) blocker prior to or during exposure of tracheal smooth muscle strips to Ach (10^–9 to 10^–4 M). Ionomycin (5 µM) in resting muscles induced increases in [Ca²⁺]_i to 500±230 nM and small increases in force of 2.6±2.3 N/cm². This tension is only 10% of the maximal tension induced by ACh. Charybdotoxin had no effect on [Ca²⁺]_i or tension in resting muscle. After pretreatment of muscle with ionomycin, the concentration-response relationship for ACh-induced changes in tension shifted to the left (EC₅₀=0.07±0.05 µM ionomycin; 0.17±0.07 µM, control, p<0.05). When applied to the muscles during steady-state responses to submaximal concentrations of ACh, both ionomycin and charybdotoxin induced further increases in tension. The same magnitude increase in tension occurs after ionomycin and charybdotoxin treatment, even though the increase in [Ca²⁺]_i induced by charybdotoxin is much smaller than that induced by ionomycin. We conclude that the resting muscle is much less sensitive to elevation of [Ca²⁺]_i when compared to muscles stimulated with ACh. Steady-state [Ca²⁺]_i limits tension development induced by submaximal concentrations of ACh. The activity of K_Ca moderates the response of the muscle to ACh at concentrations less than 1 µM.     

In vivo calcium regulation in diabetic skeletal muscle

In skeletal muscle, dysfunctional contractile activity has been linked to impaired intracellular Ca²⁺ concentration ([Ca²⁺]_i) regulation. Muscle force production is impaired and fatigability and muscle fragility deteriorate with diabetes. Use of a novel in vivo model permits investigation of [Ca²⁺]_i homeostasis in diabetic skeletal muscle. Within this in vivo environment we have shown that diabetes perturbs the Ca²⁺ regulatory system such that resting [Ca²⁺]_i homeostasis following muscle contractions is compromised and elevations of [Ca²⁺]_i are exacerbated. This review considers the impact of diabetes on the capacity of skeletal muscle to regulate [Ca²⁺]_i, following muscle contractions and, in particular, the relationship between muscle fatigue and elevated [Ca²⁺]_i in a highly ecologically relevant circulation-intact environment. Importantly, the role of mitochondria in calcium sequestration and the possibility that diabetes impacts this process is explored. Given the profound microcirculatory dysfunction in diabetes this preparation offers the unique opportunity to study the interrelationships among microvascular function, blood-myocyte oxygen flux and [Ca²⁺]_i as they relate to enhanced muscle fatigability and exercise intolerance.     

A calcium-stimulated, ouabain-inhibited ATPase in a myocardial fraction enriched with sarcolemma

An active intracellular to extracellular Ca²⁺ efflux has been proposed in heart muscle. A myocardial sarcolemmal ATPase stimulated by an intracellular pCa and serving as a Ca²⁺ pump has been postulated. A recently developed myocardial sarcolemmal preparation has not permitter a search for such an enzymatic activity. In these studies, an ATPase has been demonstrated in the sarcolemma which is activated by Mg²⁺, stimulated as the Ca²⁺ is raised to a pCa of 6, and is inhibited by ouabain. These findings suggest a mechanism by which Ca²⁺ within the myocardium may be modulated and thus how the force of contraction may be altered by cardiac glycosides.     

Altered calcium homeostasis and cell injury in silica-exposed alveolar macrophages

There is evidence to suggest that cell injury induced in alveolar macrophages (AM) following phagocytic activation by silica particles may be mediated through changes in intracellular free calcium [Ca²⁺]_i. However, the mechanism of silica- induced cytotoxicity relative to [Ca²⁺]_i overloading is not yet clear. To provide a better insight into this mechanism, isolated rat AMs were exposed to varying concentrations of crystalline silica (particle size < 5 μm in diameter) and the fluctuation in their [Ca²⁺]_i and cell integrity were quantitatively monitored with the fluorescent calcium probe, Fura-2 AM, and the membrane integrity indicator, propidium iodide (PI). Results from this study indicate that silica can rapidly increase [Ca²⁺]_i in a dose-dependent manner with a characteristic transient calcium rise at low doses (<0.1 mg/ml) and an elevated and sustained rise at high doses (>0.1 mg/ml). Depletion of extracellular calcium [Ca²⁺]_o markedly inhibited the [Ca²⁺]_i rise (≈90%), suggesting that Ca²⁺ influx from extracellular source is a major mechanism for silica-induced [Ca²⁺]_i rise. When used at low doses but sufficient to cause a transient [Ca²⁺]_i rise, silica did not cause significant increase in cellular PI uptake during the time of study, suggesting the presevation of membrane integrity of AMs under these conditions. At high doses of silica, however, a marked increase in PI nuclear fluorescence was observed. Depletion of [Ca²⁺]_o greatly inhibited cellular PI uptake, induced by 0.1 mg/ml or higher doses of silica. This suggests that Ca²⁺ influx, as a result of silica activation, is associated with cell injury. Indeed, our results further demonstrated that the low dose effect of silica on Ca²⁺ influx is inhibited by the Ca²⁺ channel blocker nifedipine. At high doses of silica (>0.1 mg/ml), cell injury was not prevented by nifedipine or extracellular Ca²⁺ depletion, suggesting that other cytotoxic mechanisms, i.e., nonspecific membrane damage due to lipid peroxidation, are also responsible for the silica-induced cell injury. Silica had no significant effect on cellular ATP content during the time course of the study, indicating that the observed silica-induced [Ca²⁺]_i rise was not due to the impairment of Ca²⁺-pumps, which restricts Ca²⁺ efflux. Pretreatment of the cells with cytochalasin B to block phagocytosis failed to prevent the effect of silica on [Ca²⁺]_i rise. Taken together, these results suggest that the elevation of [Ca²⁺]_i caused by silica is due mainly to Ca²⁺ influx through plasma membrane Ca²⁺ channels and nonspecific membrane damage (at high doses). Neither ATP depletion nor Ca²⁺ leakage during phagocytosis was attributed to the silica-induced [Ca²⁺]_i rise. © 1993 Wiley-Liss, Inc.     

Ca2+ release and contraction induced by IP3 and contractile agonists in mammalian gastric smooth muscle

The source, time course and stoichiometry of cytosolic free Ca²⁺ ([Ca²⁺]_i) during contraction were examined in smooth muscle cells isolated from the guinea pig and human stomach. Contraction by receptor-linked agonists (eg, acetylcholine, cholecystokinin octapeptide and Met-enkephalin) was preceded by stoichiometric increases in [Ca²⁺]_i and net ⁴⁵Ca²⁺ efflux that were maintained in the absence of extracellular Ca²⁺ or in the presence of a Ca²⁺ channel blocker (13600). The intracellular Ca²⁺ store could be depleted by repeated stimulation with all agonists in Ca²⁺-free medium or in the presence of 13600 resulting in loss of contractile response; response was restored by re-exposure of the cells to Ca²⁺.The source of intracellular Ca²⁺ an the signal for its release were examined in saponin-permeabilized muscle cells. The cells retained their ability to contract in response to receptor-linked agonists and developed an ability to contract in response to inositol trisphosphate (IP₃). The cells accumulated Ca²⁺ to the same extent as intact muscle cells, but only in the presence of ATP. IP₃ caused a prompt, concentration-dependent increase in contraction, [Ca²⁺]_i and net ⁴⁵Ca²⁺ efflux. These effects were maximally similar to those produced by CCK-8 alone or in combination with IP₃: Depletion of the Ca²⁺ store by repeated stimulation of single muscle cells in Ca²⁺-free medium with IP₃, acetylcholine or CCK-8 separately resulted in loss of contractile response to all three agents; the response was restored by re-exposure of the muscle cell to a cytosol-like perfusate (Ca²⁺ 180 nM).The studies demonstrate that a product of membrane phosphoinositide hydrolysis is capable of mobilizing Ca²⁺ from a depletable, non-mitochondrial intracellular store that is utilized by receptor-linked agonists. The magnitude of IP₃-induced Ca²⁺ release is correlated with contraction.     

Properties of calcium channels in cardiac muscle and vascular smooth muscle

The voltage-dependent slow channels in the myocardial cell membrane are the major pathway by which Ca²⁺ ions enter the cell during excitation for initiation and regulation of the force of contraction of cardiac muscle. The slow channels have some special properties, including functional dependence on metabolic energy, selective blockade by acidosis, and regulation by the intracellular cyclic nucleotide levels. Because of these special properties of the slow channels, Ca²⁺ influx into the myocardial cell can be controlled by extrinsic factors (such as autonomic nerve stimulation or circulating hormones) and by intrinsic factors (such as cellular pH or ATP level). The slow Ca²⁺ channels of the heart are regulated by cAMP in a stimulatory fashion. Elevation of cAMP produces a very rapid increase in number of slow channels available for voltage activation during excitation. The probability of a slow channel opening and the mean open time of the channel are increased. Therefore, any agent that increases the cAMP level of the myocardial cell will tend to potentiate I_si, Ca²⁺ influx, and contraction. The myocardial slow Ca²⁺ channels are also regulated by cGMP, in a manner that is opposite to that of CAMP. The effect of cGMP is presumably mediated by means of phosphorylation of a protein, as for example, a regulatory protein (inhibitory-type) associated with the slow channel. Preliminary data suggest that calmodulin also may play a role in regulation of the myocardial slow Ca²⁺ channels, possibly mediated by the Ca²⁺-calmodulin-protein kinase and phosphorylation of some regulatory-type of protein. Thus, it appears that the slow Ca²⁺ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of extrinsic and intrinsic factors.VSM cells contain two types of Ca²⁺ channels: slow (L-type) Ca²⁺ channels and fast (T-type) Ca²⁺ channels. Although regulation of voltage-dependent Ca²⁺ slow channels of VSM cells have not been fully clarified yet, we have made some progress towards answering this question. Slow (L-type, high-threshold) Ca²⁺ channels may be modified by phosphorylation of the channel protein or an associated regulatory protein. In contrast to cardiac muscle where cAMP and cGMP have antagonistic effects on Ca²⁺ slow channel activity, in VSM, cAMP and cGMP have similar effects, namely inhibition of the Ca²⁺ slow channels. Thus, any agent that elevates cAMP or cGMP will inhibit Ca²⁺ influx, and thereby act to produce vasodilation. The Ca²⁺ slow channels require ATP for activity, with a K_0.5 of about 0.3 mM. C-kinase may stimulate the Ca²⁺ slow channels by phosphorylation. G-protein may have a direct action on the Ca²⁺ channels, and may mediate the effects of activation of some receptors. These mechanisms of Ca²⁺ channel regulation may be invoked during exposure to agonists or drugs, which change second messenger levels, thereby controlling vascular tone.     

Ketamine inhibition of cytoplasmic calcium signalling in rat pheochromocytoma (PC-12) cells

This study examines the mechanism of action of ketamine, a dissociative anesthetic, with a specific focus on its ability to inhibit changes in the concentration of intracellular free calcium, [Ca²⁺]_i, in PC-12 cells. The resting [Ca²⁺]_i as measured with the fluorescent probe Fura-2 AM in control cells is 184.8±8.6 nM (mean±SEM, n = 15). Changes in [Ca²⁺]_i via influx through voltage-gated calcium channels after membrane depolarization with potassium chloride were monitored in the absence and presence of various concentrations of ketamine. Potassium-depolarization caused a dose-dependent rapid increase in [Ca²⁺]_i, averaging 62±5%, 33±2% and 18±3% (n = 10 each) above control levels for 70 mM, 50 mM and 35 mM KCl, respectively. Ketamine, in the dosage range studied (5 – 500 μM), inhibited the increase in [Ca²⁺]_i stimulated by potassium-depolarization in a dose-dependent manner. The computer-fitted dose-response curve of the pooled data yielded a half maximal suppression concentration, ED₅₀, of 33 μM. In conclusion, this study demonstrates that ketamine inhibits Ca²⁺ influx through voltage-gated Ca²⁺ channels in PC-12 cells at clinically relevant doses, and may play a role in ketamine's action as a general anesthetic agent.     

Uncoupling secretion and tip growth in lily pollen tubes: evidence for the role of calcium in exocytosis

Cytoplasmic calcium concentration ([Ca²⁺]_i) and extracellular calcium (Ca²⁺_o) influx has been studied in pollen tubes of Lilium longliflorum in which the processes of cell elongation and exocytosis have been uncoupled by use of Yariv phenylglycoside ((β-D-Glc)₃). Growing pollen tubes were pressure injected with the ratio dye fura-2 dextran and imaged after application of (β-D-Glc)₃, which binds arabinogalactan proteins (AGPs). Application of (β-D-Glc)₃ inhibited growth but not secretion. Ratiometric imaging of [Ca²⁺]_i revealed an initial spread in the locus of the apical [Ca²⁺]_i gradient and substantial elevations in basal [Ca²⁺]_i followed by the establishment of new regions of elevated [Ca²⁺]_i on the flanks of the tip region. Areas of elevated [Ca²⁺]_i corresponded to sites of pronounced exocytosis, as evidenced by the formation of wall ingrowths adjacent to the plasma membrane. Ca²⁺_o influx at the tip of (β-D-Glc)₃-treated pollen tubes was not significantly different to that of control tubes. Taken together these data indicate that regions of elevated [Ca²⁺]_i, probably resulting from Ca²⁺_o influx across the plasma membrane, stimulate exocytosis in pollen tubes independent of cell elongation.     

The role of TRPP2 in agonist-induced gallbladder smooth muscle contraction

TRPP2 channel protein belongs to the superfamily of transient receptor potential(TRP) channels and is widely expressed in various tissues, including smooth muscle in digestive gut. Accumulating evidence has demonstrated that TRPP2 can mediate Ca~(2+) release from Ca~(2+) stores. However, the functional role of TRPP2 in gallbladder smooth muscle contraction still remains unclear. In this study, we used Ca~(2+) imaging and tension measurements to test agonist-induced intracellular Ca~(2+) concentration increase and smooth muscle contraction of guinea pig gallbladder, respectively. When TRPP2 protein was knocked down in gallbladder muscle strips from guinea pig, carbachol(CCh)-evoked Ca~(2+) release and extracellular Ca~(2+) influx were reduced significantly, and gallbladder contractions induced by endothelin 1 and cholecystokinin were suppressed markedly as well. CCh-induced gallbladder contraction was markedly suppressed by pretreatment with U73122, which inhibits phospholipase C to terminate inositol 1,4,5-trisphosphate receptor(IP3) production, and 2-aminoethoxydiphenyl borate(2APB), which inhibits IP3 recepor(IP3R) to abolish IP3R-mediated Ca~(2+) release. To confirm the role of Ca~(2+) release in CCh-induced gallbladder contraction, we used thapsigargin(TG)-to deplete Ca~(2+) stores via inhibiting sarco/endoplasmic reticulum Ca~(2+)-ATPase and eliminate the role of store-operated Ca~(2+) entry on the CCh-induced gallbladder contraction. Preincubation with 2 μmol L~(-1) TG significantly decreased the CCh-induced gallbladder contraction. In addition, pretreatments with U73122, 2APB or TG abolished the difference of the CCh-induced gallbladder contraction between TRPP2 knockdown and control groups. We conclude that TRPP2 mediates Ca~(2+) release from intracellular Ca~(2+) stores, and has an essential role in agonist-induced gallbladder muscle contraction.     

Simplified calcium transport and storage pathways

The control of calcium concentration in the cytoplasm of most cells involves both the influx and efflux of Ca⁺⁺ from extracellular fluid and the release and uptake of Ca⁺⁺ from two separate, but interacting intracellular membrane-bound Ca⁺⁺ stores: (1) the ryanodine receptor-activated calcium store (RyR) and (2) the inositol-trisphosphate (IP₃) receptor calcium store (Golovina and Blaustein, 1997, Spatially and functionally distinct Ca²⁺ stores in sarcoplasmic and endoplasmic reticulum. Science 275, 1643–1648). A more complete understanding of calcium pathways may lead to the development of new strategies to reduce the pathophysiology induced by severe hyperthermia, exercise, hypoxia, and other stresses. This review discusses the fundamental mechanisms involved in the control of Ca_i, the main regulator of biochemical processes, and ultimately, of physiological responses to moderate and severe physical exercise and stress.     

Effects of salinity and turgor on calcium influx in Chara

Measurements were made of the influx of ⁴⁵Ca into internodal cells of Chara corallina in solutions containing high concentrations of NaCl. Increasing salinity in the range 4–100mol m^?3 NaCl resulted in a doubling of Ca²⁺ influx at the plasmalemma. A time-course of Ca²⁺ influx in 50 mol m^?3 NaCl, 0.5mol m^?3 CaCl₂ showed that while influx at the plasmalemma increased only 1.5-fold, influx to the vacuole increased by up to 15-fold. This was interpreted as being due to inhibition of active Ca²⁺ efflux from the cell. The stimulation of Ca²⁺ influx by increasing salinity appeared to be principally a response to reduced turgor since similar stimulations were obtained when turgor was reduced by NaCl, Na₂SO₄ or mannitol. When cells were plasmolysed Ca²⁺ influx increased by 10–20-fold. The increased permeability was relatively specific for Ca²⁺ and was inhibitable by La³⁺. Survival of cells in high salt conditions was increased by 30 mmol m^?3 La³⁺, which inhibited Ca²⁺ influx. Paradoxically, survival can also be extended by increasing external Ca²⁺ which leads to a higher influx. Therefore, it seems unlikely that the ameliorative effect of Ca²⁺ on the sensitivity of plants to high NaCl is mediated by Ca²⁺ entry across the plasmalemma. It seems more likely that the principal role of Ca²⁺ under these conditions is exerted externally through the control of membrane voltage and permeability.     

Involvement of Ca<Superscript>2+</Superscript> signaling in tachykinin-mediated contractile responses in swine trachea

Summary Neuropeptide tachykinins, present within sensory nerves, have been implicated as neurotransmitters involved in nonadrenergic and noncholinergic airway muscle contraction. The signal transduction pathways of tachykinins on muscle contraction and Ca²⁺ mobilization were investigated in swine trachea. Tachykinins, substance P (SP) and neurokinin A (NKA), concentration (1 nM to 1 μM)-dependently induced contractile responses with removal of epithelium, whereas neurokinin B (NKB) did not alter the muscle tension. The SP- and NKA-evoked muscle contractions were inhibited by NK1-R antagonist L732138, but not by either NK2-R antagonist MDL29913 or NK3-R antagonist SB218795. Consistently, SP-elicited increase in [Ca²⁺]_i was abolished by NK1-R antagonist, neither by NK2-R nor NK3-R antagonists. The SP-induced muscular responses were significantly inhibited by L-type Ca²⁺ channel blocker verapamil and withdrawal of external Ca²⁺. Caffeine (10 mM) or ryanodine (50 μM) also partly suppressed the SP-induced muscle responses. Inhibition of inositol 1,4,5-trisphosphate (InsP₃) receptor with 2-APB (75 μM) potently attenuated SP-evoked Ca²⁺ mobilization and muscle contraction, which was further inhibited by 2-APB under Ca²⁺-free external solution, but not completely. Unexpectedly, simultaneous blockade of InsP₃ receptor and ryanodine receptor (RyR) by 2-APB and ryanodine enhanced SP-evoked muscle contraction and Ca²⁺ mobilization. This potentiation was virtually abolished by removal of external Ca²⁺, suggesting native Ca²⁺ channels may contribute to this phenomenon. These results demonstrate that tachykinins produce a potent muscle contraction associated with Ca²⁺ mobilization via tachykinin NK1- R-dependent activation of multiple signal transduction pathways involving Ca²⁺ influx and release of Ca²⁺ from InsP₃- and ryanodine-sensitive Ca²⁺ stores. Blockade of both InsP₃ receptor and RyR enhances the Ca²⁺ influx through native Ca²⁺ channels in plasma membrane, which is crucial to Ca²⁺ signaling in response to NK1 receptor activation.     

Quercetin stimulation of calcium release from rabbit skeletal muscle sarcoplasmic reticulum

To elucidate the mechnism by which quercetin enhances the rate of tension development in skinned muscle fibers, effects on calcium release from longitudinal tubule-derived SR (LSR) after phosphate-supported calcium uptake were examined. In all studies, 100 μM quercetin (which inhibits initial calcium uptake velocity 85%) was added at or shortly after the time calcium content reached a maximum at various extravesicular Ca²⁺ concentrations (Ca_o). At moderate Ca_o (0.2–1.0 μM). where spontaneous calcium release rate depended on Ca_o, quercetin caused a marked stimulation of calcium release. This was accompanied by a 60% reduction in calcium influx and a 30-fold increase in calcium efflux. Thus, the previously reported quercetin-induced increase in the rate of tension development by skinned muscle fibers may result, at least in part, from sensitization of Ca²⁺-triggered calcium release to lower Ca_o.     

Reduction of blood pressure by store‐operated calcium channel blockers

The voltage‐operated Ca²⁺ channels (VOCC), which allow Ca²⁺ influx from the extracellular space, are inhibited by anti‐hypertensive agents such as verapamil and nifedipine. The Ca²⁺ entering from outside into the cell triggers Ca²⁺ release from the sarcoplasmic reticulum (SR) stores. To refill the depleted Ca²⁺ stores in the SR, another type of Ca²⁺ channels in the cell membrane, known as store‐operated Ca²⁺ channels (SOCC), are activated. These SOCCs are verapamil and nifedipine resistant, but are SKF 96465 (SK) and gadolinium (Gd³⁺) sensitive. Both SK and Gd³⁺ have been shown to reduce [Ca²⁺]_i in the smooth muscle, but their effects on blood pressure have not been reported. Our results demonstrated that both SK and Gd³⁺ produced a dose‐dependent reduction in blood pressure in rat. The combination of SK and verapamil produced an additive action in lowering the blood pressure. Furthermore, SK, but not Gd³⁺ suppressed proliferation of vascular smooth muscle cells in the absence or presence of lysophosphatidic acid (LPA). SK decreased the elevation of [Ca²⁺]_i induced by LPA, endothelin‐1 (ET‐1) and angiotensin II (Ang II), but did not affect the norepinephrine (NE)‐evoked increase in [Ca²⁺]_i. On the other hand, Gd³⁺ inhibited the LPA and Ang II induced change in [Ca²⁺]_i, but had no effect on the ET‐1 and NE induced increase in [Ca²⁺]_i. The combination of verapamil and SK abolished the LPA‐ or adenosine‐5′‐triphosphate (ATP)‐induced [Ca²⁺]_i augmentation. These results suggest that SOCC inhibitors, like VOCC blocker, may serve as promising drugs for the treatment of hypertension.     

Bergmann glial cells in situ express endothelinB receptors linked to cytoplasmic calcium signals

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 ET, receptor agonist BO-3020 and were inhibited by ETB receptor antagonist BQ-788. ET elevated [Ca²⁺]_i in Ca2+-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 Ca²⁺-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 μM ATP or 10 μM 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 ET_B 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.     

Myocardial mechanical dysfunction and calcium overload following rewarming from experimental hypothermia in vivo

Rewarming patients from accidental hypothermia are regularly complicated with cardiovascular instability ranging from minor depression of cardiac output to fatal circulatory collapse also termed “rewarming shock”. Since altered Ca²⁺ handling may play a role in hypothermia-induced heart failure, we studied changes in Ca²⁺ homeostasis in in situ hearts following hypothermia and rewarming. A rat model designed for studies of the intact heart in a non-arrested state during hypothermia and rewarming was used. Rats were core cooled to 15 °C, maintained at 15 °C for 4 h and thereafter rewarmed. As time-matched controls, one group of animals was kept at 37 °C for 5 h. Total intracellular myocardial Ca²⁺ content ([Ca²⁺]_i) was measured using ⁴⁵Ca²⁺. Following rewarming we found a significant reduction of stroke volume and cardiac output compared to prehypothermic control values as well as to time-matched controls. Likewise, we found that hypothermia and rewarming resulted in a more than six-fold increase in [Ca²⁺]_i to 3.01 ± 0.43 μmol/g dry weight compared to 0.44 ± 0.05 μmol/g dry weight in normothemia control. These findings indicate that hypothermia-induced alterations in the Ca²⁺-handling result in Ca²⁺ overload during hypothermia, which may contribute to myocardial failure during and after rewarming.