Advancing age alters rapid and spontaneous refilling of caffeine-sensitive calcium stores in sympathetic superior cervical ganglion cells.

Intracellular calcium concentration ([Ca2+]i) release from smooth endoplasmic reticulum (SER) stores plays an important role in cell signaling. These stores are rapidly refilled via influx through voltage-gated calcium channels or spontaneously via store-operated calcium channels and subsequent pumping by SER Ca2+-ATPases. We measured [Ca2+]i transients in isolated fura 2-loaded superior cervical ganglion cells from 6-, 12-, 20-, and 24-mo-old Fischer 344 rats. For rapid refilling, [Ca2+]i transients were elicited by a 1) 5-s exposure to K+, 2) caffeine to release Ca2+ from SER stores, 3) K+ to refill SER Ca2+ stores, and 4) caffeine. The percent difference between the peak and rate of rise of the first and second caffeine-evoked [Ca2+]i transient significantly declined over the age range of 12-24 mo. To estimate spontaneous refilling, cells were depolarized for 5 s with 68 mM K+ (control), followed by a 10-s exposure to 10 mM caffeine "conditioning stimulus" to deplete [Ca2+]i stores. Caffeine was then rapidly applied for 5 s at defined intervals from 60 to 300 s. Integrated caffeine-evoked [Ca2+]i transients were measured and plotted as a percentage of the K+ response vs. time. The derivative of the refilling time curves significantly declined over the age range from 12-24 mo. Overall, these data suggest that the ability of superior cervical ganglion cells to sustain release of [Ca2+]i following rapid or spontaneous refilling declines with advancing age. Compromised ability to sustain calcium signaling may possibly alter the overall function of adrenergic neurons innervating the cerebrovasculature.     

Neuronal calcium stores

Neuronal calcium stores associated with specialized intracellular organelles, such as endoplasmic reticulum and mitochondria, dynamically participate in generation of cytoplasmic calcium signals which accompany neuronal activity. They fulfil a dual role in neuronal Ca2+ homeostasis being involved in both buffering the excess of Ca2+ entering the cytoplasm through plasmalemmal channels and providing an intracellular source for Ca2+. Increase of Ca2+ content within the stores regulates the availability and magnitude of intracellular calcium release, thereby providing a mechanism which couples the neuronal activity with functional state of intracellular Ca2+ stores. Apart of 'classical' calcium stores (endoplasmic reticulum and mitochondria) other organelles (e.g. nuclear envelope and neurotransmitter vesicles) may potentially act as a functional Ca2+ storage compartments. Calcium ions released from internal stores participate in many neuronal functions, and might be primarily involved in regulation of various aspects of neuronal plasticity.     

Alkalinization-Induced Changes in Intracellular Calcium in Rat Spinal Cord Neurons

It is well-known that pH changes can influence a lot of cellular processes. In this work, we have specifically studied the influence of alkalinization, which can be developed in spinal cord neurons during hyperventilation (respiratory alkalosis) and chronic renal failure (metabolic alkalosis) on calcium homeostasis. Application of Tyrode solution with increased pH (pH = 8.8) to secondary sensory neurons isolated from rat spinal dorsal horn induced elevation of intracellular free calcium concentration in the cytosol ([Ca2+]i) if applied after membrane depolarization. Repetitive application of alkaline solution led to disappearance of such elevations. Depletion of endoplasmic reticulum (ER) calcium stores by 30 mM caffeine almost completely blocked the effect of elevated extracellular pH. If caffeine-induced [Ca2+]i transients were evoked during alkalinization, their amplitudes were decreased by 41%. Preapplication of 500 nM ionomycin resulted in disappearance of alkalinization-induced [Ca2+]i transients, whereas prolonged applications (for 20 min) of 200 nM thapsigargin, a blocker of Ca2+ ATPase of the endoplasmic reticulum, resulted in disappearance of the rapid phase of the [Ca2+]i transients induced by alkalinization. Preapplication of the mitochondrial protonophore CCCP (10 microM) also induced changes in the alkalinization-induced calcium response--it lost its peak and was transformed into an irregular wave terminating in several seconds. The data obtained indicate that alkalinization induces an increase of [Ca2+]i level in the investigated neurons via a combined action of both intracellular Ca2+-accumulating structures--the endoplasmic reticulum and mitochondria. This suggestion was supported by morphological data that both structures in these neurons are tightly connected and may interact during release of accumulated calcium ions.     

Feedback mechanisms in the regulation of intracellular calcium ([Ca2+]i) in the peripheral nociceptive system: role of TRPV-1 and pain related receptors

Multimodal stimuli like heat, cold, bacterial or mechanical events are able to elicit pain, which is necessary to guarantee survival. However, the control of pain is of major clinical importance. The perception and transduction of pain is differentially modulated in the peripheral and central nervous system (CNS): while peripheral structures modulate these signals, the perception of pain occurs in the CNS. In recent years major advances have been made in the understanding of the processes which are involved in pain sensation. For the peripheral pain reception, the importance of specific pain receptors of the transition receptor pore (TRP)-family (e.g. the TRPV-1 receptor) has been analyzed. These receptors/channels are localized at the cell membrane of nociceptive neurones as well as in membranes of intracellular calcium stores like the endoplasmic reticulum. While the associated channel conducts different ions, a major proportion is calcium. Therefore, this review focuses on (1) the modulations of intracellular calcium ([Ca2+]i) initiated by the activation of pain receptors and (2) the consequences of [Ca2+]i changes for the processing of pain signals at the peripheral side. The possible interference of TRPV-1 induced [Ca2+]i modulations to the function of other membrane receptors and channels, like voltage gated calcium, sodium or potassium channels, or co-expressed CB1-receptors will be discussed. The latter interactions are of specific interest since the analgetic properties of endo- and exo-cannabinoids are mediated by CB1 receptors and their activation significantly modulates the calcium induced release of pain related transmitters. Furthermore, multiple cross links between different pain modulating intracellular pathways and their dependence on [Ca2+]i modulations will be illuminated. Overall, this review will summarize new insights resulting in the understanding of the prominent influence of [Ca2+]i for processes which are involved in pain sensation.     

Calcium release from the Golgi apparatus and the endoplasmic reticulum in HeLa cells stably expressing targeted aequorin to these compartments

Extracellular agonists mobilize Ca2+ from SERCA-comprising intracellular Ca2+ stores located in both the Golgi apparatus and the endoplasmic reticulum. Ca2+ release from both these compartments was studied in HeLa cells stably expressing the luminescent Ca2+ indicator aequorin specifically targeted to these compartments. Changes in lumenal [Ca2+] as detected by the aequorin measurements were correlated with parallel changes in total Ca2+ content of the stores. The latencies and initial rates of Ca2+ release from the Golgi apparatus and the endoplasmic reticulum were quite similar. However, maximal Ca2+ release measured with Golgi-targeted aequorin terminated faster than that from the endoplasmic reticulum. The rate and extent of Ca2+ depletion from both compartments correlated well with the peak amplitude of the cytosolic [Ca2+] rise. Time-course experiments further revealed that the peak of the cytosolic Ca2+ response occurred before the lumenal [Ca2+] reached its lowest level. We conclude that both the Golgi apparatus and the endoplasmic reticulum contribute to the rise in cytosolic [Ca2+] upon agonist stimulation, but the kinetics of the Ca2+ release are different.     

Age-related alterations in caffeine-sensitive calcium stores and mitochondrial buffering in rat basal forebrain

The properties of caffeine- and thapsigargin-sensitive endoplasmic reticulum calcium stores were compared in acutely dissociated basal forebrain neurons from young and aged F344 rats by ratiometric microfluorimetry. The ability of these stores to sequester and release calcium resembles that observed in other central neurons, with an important role of mitochondrial calcium buffering in regulating the response to caffeine. An age-related reduction in the filling state of the stores in resting cells appears to be mediated by increased rapid calcium buffering, which reduces the availability of calcium for uptake into the stores. An age-related decrease in the amplitude of maximal caffeine-induced calcium release was attributed to increased mitochondrial buffering. There were no age-related differences in the sensitivity to caffeine or in the calcium sequestration/release process at the level of the endoplasmic reticulum per se. These findings demonstrate the importance of interactions between cellular calcium buffering mechanisms and provide details regarding age-related changes in calcium homeostasis which have been thought to occur in these and other neurons associated with age-related neuronal dysfunctions.     

Bcl-2 increases emptying of endoplasmic reticulum Ca2+ stores during photodynamic therapy-induced apoptosis.

Photodynamic therapy (PDT) is clinically approved for the treatment of several types of cancer as well as age-related macular degeneration, the leading cause of blindness in the elderly. PDT using the photosensitizer verteporfin has been previously shown to induce rapid apoptosis via a mitochondrial-caspase activation pathway. The impact of PDT on other cellular organelles such as the endoplasmic reticulum (ER) is undefined. The effect of PDT on intracellular Ca2+ ([Ca2+]i) in control and Bcl-2-overexpressing HeLa cells was assessed. A greater [Ca2+]i transient was observed for Bcl-2 overexpressing cells in response to PDT. The PDT-induced Ca2+ release was due to the emptying of Ca2+ from ER and possibly mitochondrial stores and was not due to an influx of Ca2+ from the medium. For Bcl-2-transfected cells, the release of Ca2+ was incomplete as determined by a further [Ca2+]i transient produced by the addition of the Ca2+ ionophore ionomycin after PDT. Furthermore, extrusion of Ca2+ was not hindered while ER-mediated sequestration of Ca2+ was impaired after PDT. Impairment of ER-mediated sequestration of Ca2+ may be due to the immediate caspase-independent depletion of sarco/endoplasmic reticulum Ca2+ ATPase-2 (SERCA2) that occurred in response to PDT in birth HeLa/Neo and Bcl-2 overexpressed HeLa cells. In summary, PDT induced the rapid degradation of SERCA2 and release of ER and mitochondrial Ca2+ stores. Although overexpression of Bcl-2 did not protect against SERCA2 degradation, it may influence the release of Ca2+ from ER and mitochondrial stores in PDT-treated cells.     

Role of Ca2+ in diperoxovanadate-induced cytoskeletal remodeling and endothelial cell barrier function

Diperoxovanadate (DPV), a potent inhibitor of protein tyrosine phosphatases and activator of tyrosine kinases, alters endothelial barrier function via signaling pathways that are incompletely understood. One potential pathway is Src kinase-mediated tyrosine phosphorylation of proteins such as cortactin that regulate endothelial cell (EC) cytoskeleton assembly. As DPV modulates endothelial cell signaling via protein tyrosine phosphorylation, we determined the role of DPV-induced intracellular free calcium concentration ([Ca2+]i) in activation of Src kinase, cytoskeletal remodeling, and barrier function in bovine pulmonary artery endothelial cells (BPAECs). DPV in a dose- and time-dependent fashion increased [Ca2+]i, which was partially blocked by the calcium channel blockers nifedipine and Gd3+. Treatment of cells with thapsigargin released Ca2+ from the endoplasmic reticulum, and subsequent addition of DPV caused no further change in [Ca2+]i. These data suggest that DPV-induced [Ca2+]i includes Ca release from the endoplasmic reticulum and Ca influx through store-operated calcium entry. Furthermore, DPV induced an increase in protein tyrosine phosphorylation, phosphorylation of Src and cortactin, actin remodeling, and altered transendothelial electrical resistance in BPAECs. These DPV-mediated effects were significantly attenuated by BAPTA (25 microM), a chelator of [Ca2+]i. Immunofluorescence studies reveal that the DPV-mediated colocalization of cortactin with peripheral actin was also prevented by BAPTA. Chelation of extracellular Ca2+ by EGTA had marginal effects on DPV-induced phosphorylation of Src and cortactin; actin stress fibers formation, however, affected EC barrier function. These data suggest that DPV-induced changes in [Ca2+]i regulate endothelial barrier function using signaling pathways that involve Src and cytoskeleton remodeling.     

Xestospongin C empties the ER calcium store but does not inhibit InsP3-induced Ca2+ release in cultured dorsal root ganglia neurones

The action of Xestospongin C (XeC) on calcium concentration in the cytosol ([Ca2+]i) and within the lumen of endoplasmic reticulum (ER) ([Ca2+]L) was studied using cultured dorsal root ganglia (DRG) neurones. Application of 2.5 microM of XeC triggered a slow [Ca2+]i transient as measured by Fura-2 video-imaging. The kinetics and amplitude of XeC-induced [Ca2+]i response was similar to that triggered by 1 microM thapsigargin (TG). The [Ca2+]L was monitored in cells loaded with low-affinity Ca2+ indicator Mag-Fura-2. The cytosolic portion of Mag-Fura-2 was removed by permeabilisation of the plasmalemma with saponin. Application of XeC to these permeabilised neurones resulted in a slow depletion of the ER Ca2+ store. XeC, however, failed to inhibit inositol 1,4,5-trisphosphate (InsP3)-induced [Ca2+]L responses. We conclude that XeC is a potent inhibitor of sarco(endo)plasmic reticulum calcium ATPase, and it cannot be regarded as a specific inhibitor of InsP3 receptors in cultured DRG neurones.     

Functional characterization of thapsigargin and agonist-insensitive acidic Ca2+ stores in Drosophila melanogaster S2 cell lines

The role of acidic intracellular calcium stores in calcium homeostasis was investigated in the Drosophila Schneider cell line 2 (S2) by means of free cytosolic calcium ([Ca2+]i) and intracellular pH (pHi) imaging together with measurements of total calcium concentrations within intracellular compartments. Both a weak base (NH4Cl, 15 mM) and a Na+/H+ ionophore (monensin, 10 microM) evoked cytosolic alkalinization followed by Ca2+ release from acidic intracellular Ca2+ stores. Pretreatment of S2 cells with either thapsigargin (1 microM), an inhibitor of endoplasmic reticulum Ca(2+)-ATPases, or with the Ca2+ ionophore ionomycin (10 microM) was without effect on the amplitude of Ca2+ release evoked by alkalinization. Application of the cholinergic agonist carbamylcholine (100 microM) to transfected S2-DM1 cells expressing a Drosophila muscarinic acetylcholine receptor (DM1) emptied the InsP3-sensitive Ca2+ store but failed to affect the amplitude of alkalinization-evoked Ca2+ release. Glycyl-L-phenylalanine-beta-naphthylamide (200 microM), a weak hydrophobic base known to permeabilize lysosomes by osmotic swelling, triggered Ca2+ release from internal stores, while application of brefeldin A (10 microM), an antibiotic which disperses the Golgi complex, resulted in a smaller increase in [Ca2+]i. These results suggest that the alkali-evoked calcium release is largely attributable to lysosomes, a conclusion that was confirmed by direct measurements of total calcium content of S2 organelles. Lysosomes and endoplasmic reticulum were the only organelles found to have concentrations of total calcium significantly higher than the cytosol. However, NH4Cl (15 mM) reduced the level of total calcium only in lysosomes. Depletion of acidic Ca2+ stores did not elicit depletion-operated Ca2+ entry. They were refilled upon re-exposure of cells to normal saline ([Ca2+]o = 2 mM), but not by thapsigargin-induced [Ca2+]i elevation in Ca(2+)-free saline.     

Effect of gossypol on intracellular Ca2+ regulation in human hepatoma cells

Gossypol is a natural toxicant present in cottonseeds, and is hepatotoxic to animals and human. The effect of gossypol on cytosolic free Ca2+ levels ([Ca2+]i) in HA22/VGH human hepatocytes was explored using fura-2 as a fluorescent Ca2+ indicator. Gossypol increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 2 microM. The Ca2+ signal was reduced by removing extracellular Ca2+ or by 10 microM La3+, but was not affected by nifedipine, verapamil or diltiazem. Pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+ partly reduced 10 microM gossypol-induced Ca2+ release; and conversely pretreatment with gossypol abolished thapsigargin-induced Ca2+ release. The Ca2+ release induced by 10 microM gossypol was not changed by inhibiting phospholipase C with 2 microM U73122 or by depleting ryanodine-sensitive Ca2+ stores with 50 microM ryanodine. Together, the results suggest that in human hepatocytes, gossypol induced a [Ca2+]i increase by causing store Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and by inducing Ca2+ influx.     

A localized elevation of cytosolic free calcium is associated with cytokinesis in the zebrafish embryo

Cytokinesis, a key step in cell division, is known to be precisely regulated both in its timing and location. At present, the regulatory mechanism of cytokinesis is not well understood, although it has been suggested that calcium signaling may play an important role in this process. To test this notion, we introduced a sensitive fluorescent Ca2+ indicator into the zebrafish embryo and used confocal microscopy to measure the spatiotemporal variation of intracellular free Ca2+ concentration ([Ca2+]i) during cell cleavage. It was evident that a localized elevation of [Ca2+]i is closely associated with cytokinesis. First, we found that during cytokinesis, the level of free Ca2+ was elevated locally precisely at the cleavage site. Second, the rise of free Ca2+ was very rapid and occurred just preceding the initiation of furrow contraction. These observations strongly suggest that cytokinesis may be triggered by a calcium signal. In addition, we found that this cytokinesis-associated calcium signal arose mainly from internal stores of Ca2+ rather than from external free Ca2+; it could be blocked by the antagonist of inositol trisphosphate (InsP3) receptors. These findings suggest that the localized elevation of [Ca2+]i is caused by the release of free Ca2+ from the endoplasmic reticulum through the InsP3-regulated calcium channels.     

Contribution of intracellular calcium stores to an increase in cytosolic calcium concentration induced by Mannheimia haemolytica leukotoxin

The contribution of intracellular calcium stores to Mannheimia haemolytica leukotoxin (LKT)-induced increase in cytosolic calcium concentration was studied by pharmacologically inhibiting transport of calcium across the plasma and endoplasmic reticulum membranes of bovine neutrophils exposed to LKT. Active intracellular storage of calcium by sarcoplasmic/endoplasmic reticulum calcium ATPase, influx of extracellular calcium across the plasma membrane, and release of stored calcium via inositol triphosphate receptors and ryanodine-sensitive calcium channels were inhibited using thapsigargin, lanthanum chloride, xestospongin C, and magnesium chloride, respectively. Pre-incubation with thapsigargin attenuated the increase in cytosolic calcium concentration produced by LKT, thus confirming the involvement of intracellular calcium stores. Inhibitory effects of lanthanum chloride, xestospongin C, and magnesium chloride indicated that the increase in cytosolic calcium concentration induced by LKT resulted from both influx of calcium across the plasma membrane and release of calcium from intracellular stores.     

Tetracaine stimulates insulin secretion from the pancreatic beta-cell by release of intracellular calcium

The role of intracellular calcium stores in stimulus-secretion coupling in the pancreatic beta-cell is largely unknown. We report here that tetracaine stimulates insulin secretion from collagenase-isolated mouse islets of Langerhans in the absence of glucose or extracellular calcium. We also found that the anesthetic evokes a dose-dependent rise of the intracellular free-calcium concentration ([Ca2+]i) in cultured rat and mouse beta-cells. The tetracaine-specific [Ca2+]i rise also occurs in the absence of glucose, or in beta-cells depolarized by exposure to a Ca(2+)-deficient medium (< 1 microM) or elevated [K+]o. Furthermore, tetracaine (> or = 300 microM) depolarized the beta-cell membrane in mouse pancreatic islets, but inhibited Ca2+ entry through voltage-gated Ca2+ channels in HIT cells, an insulin-secreting cell line. From these data we conclude that tetracaine-enhancement of insulin release occurs by mechanisms that are independent of Ca2+ entry across the cell membrane. The tetracaine-induced [Ca2+]i rise in cultured rat beta-cells and insulin secretion from mouse islets is insensitive to dantrolene (20 microM), a drug that inhibits Ca2+ release evoked by cholinergic agonists in the pancreatic beta-cell, and thapsigargin (3 microM), a blocker of the endoplasmic reticulum (ER) Ca2+ pump. We conclude that the Ca2+ required for tetracaine-potentiated insulin secretion is released from intracellular Ca2+ stores other than the ER. Furthermore, tetracaine-induced Ca2+ release was unaffected by the mitochondrial electron transfer inhibitors NaN3 and rotenone. Taken together, these data show that a calcium source other than the ER and mitochondria can affect beta-cell insulin secretion.     

Dynamic regulation of intracellular calcium signals through calcium release channels

After the seminal work of Ebashi and coworkers which established the essential role of the intracellular Ca2+ concentration ([Ca2+]i) in the regulation of skeletal muscle contraction, we have witnessed an explosive elongation of the list of cell functions that are controlled by the [Ca2+]i. In numerous instances, release of intracellular Ca2+ stores plays important roles in Ca2+ signalling which displays significant variation in spatio-temporal pattern. There are two families of Ca2+ release channels, ryanodine receptors and inositol 1,4,5-trisphosphate (IP3) receptors. These Ca2+ release channels are structurally and functionally similar. In particular, the activity of both types of channels is regulated by the [Ca2+]i. The [Ca2+]i dependence of the Ca2+ release channel activity provides both types of channels with properties of a Ca2+ signal amplifier. This function of the ryanodine receptor is important in striated muscle excitation-contraction coupling, whereas that of the IP3 receptor seems to be the basis of the generation of Ca2+ waves. Thus the wide variety of Ca2+ signalling patterns seem to be critically dependent on the [Ca2+]i dependence of the Ca2+ release channels.     

Mechanisms of calcium elevation in the micromeres of sea urchin embryos

The micromeres, the first cells to be specified in sea urchin embryos, are generated by unequal cleavage at the fourth cell division. The micromeres differentiate autonomously to form spicules and dispatch signals to induce endomesoderm in the neighbouring macromeres cells in the embryo. Using a calcium indicator Fura-2/AM and a mixture of dextran conjugated Oregon green-BAPTA 488 and Rhodamine red, the intracellular calcium ion concentration ([Ca2+]i) was studied in embryos at the 16-cell stage. [Ca2+]i was characteristically elevated in the micromeres during furrowing at the 4th cleavage. Subsequently, Ca2+ oscillated for about 10 min in the micromeres, resulting in episodic high levels of [Ca2+]i. High [Ca2+]i regions were associated with regional localizations of the endoplasmic reticulum (ER), though not with ER accumulated at the vegetal pole of the micromeres during the 4th division. Pharmacological studies, using a blocker of IP3-mediated Ca2+ release (Xestospongin), a store-operated Ca2+ entry inhibitor (2 aminoethoxydiphenyl borate (2-APB)) and an inhibitor of stretch-dependent ion channels (gadolinium), suggest that the high [Ca2+]i and oscillations in the micromeres are triggered by calcium influx caused by the activation of stretch-dependent calcium channels, followed by the release of calcium ions from the endoplasmic reticulum. On the basis of these new findings, a possible mechanism for autonomous formation of the micromeres is discussed.     

SERCA pump optimizes Ca2+ release by a mechanism independent of store filling in smooth muscle cells

Thapsigargin-sensitive sarco/endoplasmic reticulum Ca(2+) pumps (SERCAs) are involved in maintaining and replenishing agonist-sensitive internal stores. Although it has been assumed that release channels act independently of SERCA pumps, there are data suggesting the opposite. Our aim was to study the relationship between SERCA pumps and the release channels in smooth muscle cells. To this end, we have rapidly blocked SERCA pumps with thapsigargin, to avoid depletion of the internal Ca(2+) stores, and induced Ca(2+) release with either caffeine, to open ryanodine receptors, or acetylcholine, to open inositol 1,4,5-trisphosphate receptors. Blocking SERCA pumps produced smaller and slower agonist-induced [Ca(2+)](i) responses. We determined the Ca(2+) level of the internal stores both indirectly, measuring the frequency of spontaneous transient outward currents, and directly, using Mag-Fura-2, and demonstrated that the inhibition of SERCA pumps did not produce a reduction of the sarco/endoplasmic reticulum Ca(2+) levels to explain the decrease in the agonist-induced Ca(2+) responses. It appears that SERCA pumps are involved in sustaining agonist-induced Ca(2+) release by a mechanism that involves the modulation of Ca(2+) availability in the lumen of the internal stores.     

Cell calcium and its regulation in smooth muscle

Two novel methods used to study smooth muscles-electron probe X-ray microanalysis and Ca2+-sensitive indicators (which are used for resolving, respectively, the spatial distribution and temporal distribution of calcium)-are briefly reviewed and the major findings obtained are summarized. In smooth muscle the sarcoplasmic reticulum is the major intracellular source of Ca2+; mitochondria do not play a significant role in the physiological regulation of [Ca2+]i. Under pathological conditions mitochondria can reversibly accumulate large amounts of calcium. Resting [Ca2+]i generally ranges from 80 to 200 nM, and is lower in phasic than in tonic smooth muscles. Removal of extracellular Ca2+ and Ca2+ entry blockers can reduce [Ca2+]i, but the effects of beta-adrenergic agents are variable. Increases in [Ca2+]i are triggered by electrical stimulation, depolarization with high K+, and excitatory agonists. Stretch, after a delay of several seconds, can cause an increase in [Ca2+]i in some smooth muscles. There is also a delay of approximately 200-400 ms between the initiation of the rise of Ca2+ and contraction that follows spontaneous action potentials or electrical stimulation. Agonist-induced Ca2+ release, a major mechanism of pharmacomechanical coupling, has been demonstrated in smooth muscles depolarized with high K; evidence suggests that it is mediated by G proteins that couple receptors to phospholipase C. Ca2+ release can be triggered directly in permeabilized smooth muscle with inositol 1,4,5-trisphosphate. Even though Ca2+ is the major physiological regulator of contraction, Ca2+ sensitivity of the regulatory-contractile apparatus differs in different (phasic and tonic) smooth muscles, and can be modulated in a given smooth muscle. The force [Ca2+]i ratio is higher during agonist-stimulated than during high K+-induced contractions, owing to agonist-induced increases in Ca2+ sensitivity mediated by G proteins. In some phasic smooth muscles (guinea pig ileum), the time course of the initial myosin light chain phosphorylation is extremely rapid and returns to basal levels while force remains elevated. In these smooth muscles there is also a marked decrease in the Ca2+ sensitivity of the regulatory-contractile apparatus during maintained depolarization in Ca2+-free or low Ca2+ solutions. It has been suggested that regulation of myosin light chain phosphatase plays a major role in the modulation of the Ca2+ sensitivity manifested as either potentiation or desensitization to [Ca2+]i.     

Effect of diethylstilbestrol on Ca2+ handling and cell viability in human breast cancer cells

In human breast cancer cells, the effect of the widely prescribed estrogen diethylstilbestrol (DES) on intracellular Ca2+ concentrations ([Ca2+]i) and cell viability was explored by using fura-2 and trypan blue exclusion, respectively. DES caused a rise in [Ca2+]i in a concentration-dependent manner (EC50 = 15 microM). DES-induced [Ca2+]i rise was reduced by 80 % by removal of extracellular Ca2+. DES-induced Mn(2+)-associated quench of intracellular fura-2 fluorescence also suggests that DES induced extracellular Ca2+ influx. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of DES on [Ca2+]i was greatly inhibited. Conversely, pretreatment with DES to deplete intracellular Ca2+ stores totally prevented thapsigargin from releasing more Ca2+, whereas ionomycin added afterward still released some Ca2+. These findings suggest that in human breast cancer cells, DES increases [Ca2+]i by stimulating extracellular Ca2+ influx and also by causing intracellular Ca2+ release from the endoplasmic reticulum. Acute trypan blue exclusion studies suggest that 10-20 NM DES killed cells in a time-dependent manner.     

Epileptogenesis induces long-term alterations in intracellular calcium release and sequestration mechanisms in the hippocampal neuronal culture model of epilepsy

Calcium and calcium-dependent processes have been hypothesized to be involved in the induction of epilepsy. It has been shown that epileptic neurons have altered calcium homeostatic mechanisms following epileptogenesis in the hippocampal neuronal culture (HNC) and pilocarpine models of epilepsy. To investigate the mechanisms causing these alterations in [Ca2+]i homeostatic processes following epileptogenesis, we utilized the HNC model of in vitro 'epilepsy' which produces spontaneous recurrent epileptiform discharges (SREDs). Using [Ca2+]i imaging, studies were initiated to evaluate the mechanisms mediating these changes in [Ca2+]i homeostasis. 'Epileptic' neurons required much longer to restore a glutamate induced [Ca2+]i load to baseline levels than control neurons. Inhibition of Ca2+ entry through voltage and receptor gated Ca2+ channels and stretch activated Ca2+ channels had no effect on the prolonged glutamate induced increase in [Ca2+]i in epileptic neurons. Employing thapsigargin, an inhibitor of the sarco/endoplasmic reticulum calcium ATPase (SERCA), it was shown that thapsigargin inhibited sequestration of [Ca2+]i by SERCA was significantly decreased in 'epileptic' neurons. Using Ca2+ induced Ca2+ release (CICR) cell permeable inhibitors for the ryanodine receptor (dantrolene) and the IP3 receptor (2-amino-ethoxydiphenylborate, 2APB) mediated CICR, we demonstrated that CICR was significantly augmented in the 'epileptic' neurons, and determined that the IP3 receptor mediated CICR was the major release mechanism altered in epileptogenesis. These data indicate that both inhibition of SERCA and augmentation of CICR activity contribute to the alterations accounting for the impaired calcium homeostatic processes observed in 'epileptic' neurons. The results suggest that persistent changes in [Ca2+]i levels following epileptogenesis may contribute to the long-term plasticity changes manifested in epilepsy and that understanding the basic mechanisms mediating these changes may provide an insight into the development of novel therapeutic approaches to treat epilepsy and prevent or reverse epileptogenesis.