Anoxia-induced elevation of cytosolic Ca<Superscript>2+</Superscript> concentration depends on different Ca<Superscript>2+</Superscript> sources in rice and wheat protoplasts

The anoxia-dependent elevation of cytosolic Ca²⁺ concentration, [Ca²⁺]_cyt, was investigated in plants differing in tolerance to hypoxia. The [Ca²⁺]_cyt was measured by fluorescence microscopy in single protoplasts loaded with the calcium-fluoroprobe Fura 2-AM. Imposition of anoxia led to a fast (within 3 min) significant elevation of [Ca²⁺]_cyt in rice leaf protoplasts. A tenfold drop in the external Ca²⁺ concentration (to 0.1 mM) resulted in considerable decrease of the [Ca²⁺]_cyt shift. Rice root protoplasts reacted upon anoxia with higher amplitude. Addition of plasma membrane (verapamil, La³⁺ and EGTA) and intracellular membrane Ca²⁺-channel antagonists (Li⁺, ruthenium red and cyclosporine A) reduced the anoxic Ca²⁺-accumulation in rice. Wheat protoplasts responded to anoxia by smaller changes of [Ca²⁺]_cyt. In wheat leaf protoplasts, the amplitude of the Ca²⁺-shift little depended on the external level of Ca²⁺. Wheat root protoplasts were characterized by a small shift of [Ca²⁺]_cyt under anoxia. Plasmalemma Ca²⁺-channel blockers had little effect on the elevation of cytosolic Ca²⁺ in wheat protoplasts. Intact rice seedlings absorbed Ca²⁺ from the external medium under anoxic treatment. On the contrary, wheat seedlings were characterized by leakage of Ca²⁺. Verapamil abolished the Ca²⁺ influx in rice roots and Ca²⁺ efflux from wheat roots. Anoxia-induced [Ca²⁺]_cyt elevation was high particularly in rice, a hypoxia-tolerant species. In conclusion, both external and internal Ca²⁺ stores are important for anoxic [Ca²⁺]_cyt elevation in rice, whereas the hypoxia-intolerant wheat does not require external sources for [Ca²⁺]_cyt rise. Leaf and root protoplasts similarly responded to anoxia, independent of their organ origin.     

Focal increases in [Ca]i may account for apparent low Ca sensitivity in swine carotid artery

Estimates of [Ca²⁺]_i sensitivity in intact smooth muscle are frequently obtained by measuring [Ca²⁺]_i with indicators such as aequorin or Fura-2. We investigated whether focal in increases in [Ca²⁺]_i could impair such measures of [Ca²⁺]_i sensitivity. Stimulation of swine carotid artery with 10 μM histamine increased aequorin estimated [Ca²⁺]_i, Fura-2 estimated [Ca²⁺]_i and Ca²⁺ sensitivity without significantly altering the aequorin/Fura-2 ratio (an estimate of [Ca²⁺]_i homogeneity). Subsequent inhibition of Na⁺/Ca²⁺ exchange by replacement of Na⁺ in the PSS with choline⁺ significantly increased aequorin-estimated [Ca²⁺]_i but only minimally increased Fura-2 estimated [Ca²⁺]_i, myosin light chain (MLC) phosphorylation and force. This resulted in a large increase in the aequorin/Fura-2 ratio, suggesting an increase in [Ca²⁺] inhomogeneity. Addition of 100 μM histamine to tissues in the choline⁺ buffer initially increased both aequorin and Fura-2 estimated [Ca²⁺]_i but after 10 min exposure both of the [Ca²⁺]_i estimates declined to pre-histamine levels. Histamine addition significantly increased MLC phosphorylation and force, indicating increased Ca²⁺ sensitivity, but the aequorin/Fura-2 ratio remained elevated and uncharged from pre-histamine values. These data show that under certain conditions, aequorin and Fura-2 can yield widely differing estimates of [Ca²⁺]_i, and thus can cause misleading assessments of Ca²⁺ sensitization mechanisms. These discrepancies may arise from inhomogeneous or focal increases in [Ca²⁺]_i which can be evaluated with the aequorin/Fura-2 ratio.     

A decrease of both [Ca]e and [H]e produces cell damage in the perfused rat heart

Cell damage of the Langendorff-perfused rat heart in response to a decrease of both [Ca²⁺]_e and [H⁺]_e is described. At pH_e = 7.7, lactate dehydrogenase (LDH) release could be induced during perfusion with media of reduced [Ca²⁺]_e (0.1–0.4 mmol/I). Decreasing pH_e to normal abolished LDH release. The gap junction channel blocker heptanol (2 mmol/I) also reduced enzyme release, and polyethylene glycol (9% PEG₆₀₀₀) totally prevented cell damage. Elevation of buffer capacity of perfusion media or perfusion flow both increased LDH release. Cell damage could also be aggravated by substituting 10 mmol/I of [Na⁺]_e by foreign cations. At [Ca²⁺]_e = 0.1 mmol/I and pH_e = 7.7, [Ca²⁺]_i and [Na⁺]_i of non-lysed cells were markedly increased (in HCO₃/CO₂ buffered media to about 7.0 μmol/I and 36 mmol/I, respectively; in HEPES-buffered media, to about 5.0 μmol/I and 55 mmol/l; physiological values of [Ca²⁺]_i and [Na⁺]_i are around 0.1 μmol/I and 10 mmol/I, respectively), whereas pH_i was not appreciably elevated. In contrast to myocytes in the intact heart, [Ca²⁺]_i of isolated cardiomyocytes under similar conditions was decreased to about 75 nmol/I and LDH release was negligible; pH_i of isolated cardiomyocytes, as in intact myocardium, did not change appreciably. The results indicate that Ca²⁺ overload is produced at lowered [Ca²⁺]_e and [H⁺]_e by an influx of Ca²⁺ through gap junctional leaks.     

Effects of sustained low-flow ischemia and reperfusion on Ca2+ transients and contractility in perfused rat hearts

We investigated changes in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) and in left ventricular contractility during sustained ischemia and reperfusion in isolated beating rat hearts. Hearts from male Sprague-Dawley rats were perfused retrogradely and were loaded with 4 M fura-2. Low-flow global ischemia was induced by reducing perfusion flow to 10% and by electric pacing. The hearts were exposed to ischemia for 10 min or 30 min and then reperfused. [Ca²⁺]_i was measured by monitoring the ratio of 500 nm fluorescence excited at 340 and 380 nm while simultaneously measuring left ventricular pressure (LVP). To determine diastolic [Ca²⁺]_i, background autofluorescence was subtracted. LVP rapidly decreased from 82.3 ± 8.2 to 17.1 ± 2.9 mmHg , whereas the amplitude of the Ca²⁺ transient did not change significantly during the first 1 min of ischemia. After 10 min of ischemia, the amplitude decreased to 60.8 ± 10.6% (p < 0.05) and diastolic [Ca²⁺]_i increased by 26.3 ± 2.9% (p < 0.001) compared with the pre-ischemic value (n = 8). When the hearts were reperfused after 10 min of ischemia, the amplitude of the Ca²⁺ transient and LVP recovered to 79.0 ± 7.2% and 73.2 ± 7.5 mmHg, respectively. Whereas diastolic [Ca²⁺]_i decreased to the pre-ischemic value. In the hearts exposed to 30 min of ischemia (n = 10), diastolic [Ca²⁺]_i increased even further by 32.7 ± 5.3% at the end of ischemia and continued increasing during the 10 min of reperfusion by 42.6 ± 15.6%. Six of 10 hearts developed ventricular fibrillation (VF) and intracellular Ca²⁺ overload after reperfusion. Recovery of LVP after reperfusion was significantly smaller in the hearts exposed to 30 min of ischemia than in the hearts exposed to 10 min of ischemia (58.9 ± 11.7 vs. 97.2 ± 3.0% of pre-ischemic value, p < 0.05). Diastolic [Ca²⁺]_i also increased under hypoxic conditions (N₂ bubbling) in this model. These results suggest that increases in diastolic [Ca²⁺]_i might play an important role in myocardial contractile dysfunction and viability in ischemia-reperfusion injury.     

Short-term regulation of cytosolic Ca2+, cytosolic pH and vacuolar pH under NaCl stress in the charophyte alga Nitellopsis obtusa

Isolated characean internodal cells of Nitellopsis obtusa can be stored in artificial pond water for many days, but they cannot survive in 100mol m^?3 NaCl solution unless more than several mol m^?3 Ca²⁺ is added. Short-term effects of NaCl stress on the cytosolic concentration of Ca²⁺ ([Ca²⁺]_c), cytosolic pH (pH_c) and vacuolar pH (pH_v) were studied in relation to the external concentration of Ca²⁺ ([Ca²⁺]_e). Changes in [Ca²⁺]_c were measured with light emission from a Ca²⁺-sensitive photoprotein, semisynthetic fch-aequorin which had been injected into the cytosol. Both pH_c and pH_v were measured with double-barrelled pH-sensitive microelectrodes. When internodal cells were treated with 100 mol m^?3 NaCl (0–1 mol m^?3 NaCl (0.1 mol m^?3 [Ca²⁺]_e), [Ca²⁺]_c increased and then recovered to the original level within 60 min. The time course of the transient change in [Ca²⁺]_c was not influenced by the level of [Ca²⁺]_c (0.1 and 10 mol m^?3). In some cases, the transient increase in [Ca²⁺]_c was induced only by increasing external osmotic pressure with sorbitol. In response to treatment with 100 mol m^?3 NaCl (0.1 mol m^?3 [Ca²⁺]_c), pH_c decreased by 0.1–0.2 units after 10min but recovered after 30–60 min, while pH_v increased by 0.4–0.5 units after 2–50 min and tended to recover after 60 min. The initial changes in both pH_c and pH_v were suppressed when [Ca²⁺]_e was raised from 0.1 to 10mol m^?3. These results show that the charophyte alga Nitellopsis can regulate [Ca²⁺]_c, pH_c and pH_v under NaCl stress in the short term and that the protective effect of Ca²⁺ on salinity stress is apparently unrelated to perturbation of Ca²⁺ and pH homeostasis.     

Cardiac functions and taurine's actions at different extracellular calcium concentrations in forced swimming stress-loaded rats

Modulation of the sinus rate and contractile force by taurine at different extracellular Ca²⁺ concentrations ([Ca²⁺]_o) was examined using rat right atria loaded with forced swimming stress. Serum concentration of corticosterone profoundly increased in stress-loaded rats as compared with native rats. The taurine level in serum also increased in stress-loaded rats, but was not changed in the different heart tissues and aorta. Heat-shock protein (HSP72) was detectable in cardiac muscles and in the lumen of cardiac blood vessels of stress-loaded rats using a monoclonal antibody. Increasing [Ca²⁺]_o (from 0.9 to 3.6 mM) enhanced the sinus rate and contractile force in a [Ca²⁺]_o-dependent fashion in native rats, but not in stress-loaded rats. Taurine (1–20 mM) caused a negative chronotropic and inotropic effect in a concentration-dependent manner. At 1.8 mM [Ca²⁺]_o, the negative chronotropic effect of taurine (10–20 mM) was attenuated in stress-loaded rats as compared with native rats. These results indicate that swimming stress causes a release of taurine into the serum and reduces the sensitivity to [Ca²⁺]_o. Taurine administration might, in part, exhibit the protective actions on acute stress-induced responses.     

Osmotic stress and abscisic acid reduce cytosolic calcium activities in roots of Arabidopsis thaliana*

Cytosolic Ca²⁺· ([Ca²⁺]_i, and elongation growth were measured in the roots of Arabidopsis thaliana. Exposure of plant tissues to high NaCl and abscisic acid (ABA) concentrations results in a reduction in the rate of growth, but the mechanism by which growth is inhibited is not understood. Both NaCl and ABA treatments are known to influence [Ca²⁺]_i, and in this study we measured the effects of salinity and ABA on [Ca²⁺]_i in cells from the meristematic region of Arabidopsis roots. The Ca²⁺-sensitive dye Fura-2 and ratiometric techniques were used to measure [Ca²⁺]_i in cells of the root meristem region. Resting [Ca²⁺]_i was found to be between 100 and 200 μmol m^?3 in roots of untreated plants. Resting [Ca²⁺]_i changed in response to changes in the [Ca²⁺] surrounding growing roots. An increase of external [Ca²⁺] increased [Ca²⁺]_i; conversely, a decrease of external [Ca²⁺] decreased [Ca²⁺]_i. Exposure of roots to NaCl caused a rapid reduction of [Ca²⁺]_i, a response that was proportional to the external NaCl concentration. Thus, as the NaCl concentration was increased, [Ca²⁺]_i in root meristematic cells decreased. Root elongation was also inhibited in proportion to the external NaCl concentration, with maximal inhibition occurring at 120 mol m^?3 NaCl. The [Ca²⁺]_i of root meristem cells also changed in response to ABA, and the magnitude of the effect of ABA was dependent upon ABA concentration. Treatment with 0.2 mmol m^?3 ABA caused a momentary increase in [Ca²⁺]_i followed by a decrease after 15 min, but 10 mmol m^?3 ABA caused an immediate decline in [Ca²⁺]_i. There was a strong positive correlation between [Ca²⁺]_i and root elongation rates. Experiments with the ABA-deficient Arabidopsis mutant aba-3 indicated that the reduction in [Ca²⁺]_i brought about by NaCl was unlikely to be mediated via changes in endogenous ABA. Experiments with solutes such as sorbitol, KCl and NaNO₃ indicated that the effects of NaCl could be mimicked by other solutes and was not specific for NaCl.     

Ca<Superscript>2+</Superscript> Buffering Capacity of Mitochondria After Oxygen-Glucose Deprivation in Hippocampal Neurons

In an earlier study, we showed that mitochondria hyperpolarized after short periods of oxygen-glucose deprivation (OGD), and this response appeared to be associated with subsequent apoptosis or survival. Here, we demonstrated that hyperpolarization following short periods of OGD (30 min; 30OGD group) increased the cytosolic Ca²⁺ ([Ca²⁺]_c) buffering capacity in mitochondria. After graded OGD (0 min (control), 30 min, 120 min), rat cultured hippocampal neurons were exposed to glutamate, evoking Ca²⁺influx. The [Ca²⁺]_c level increased sharply, followed by a rapid increase in mitochondrial Ca²⁺ [Ca²⁺]_m. The increase in the [Ca²⁺]_m level accompanied a reduction in the [Ca²⁺]_c level. After reaching a peak, the [Ca²⁺]_c level decreased more rapidly in the 30OGD group than in the control group. This buffering reaction was pronounced in the 30OGD group, but not in the 120OGD group. The enhanced buffering capacity of the mitochondria may be linked to preconditioning after short-term ischemic episodes.     

Phosphodiesterase inhibition and Ca2+ sensitization

Inhibitors of phosphodiesterase type III (PDE III) enhance cardiac contractile force by elevating the intracellular calcium concentration [Ca²⁺]_i by impairing cAMP degradation thus increasing cAMP levels. The drugs are more effective in healthy than in failing hearts since basal cAMP production is diminished in the latter. However, long term treatment with PDE-III inhibitors does not appear to be beneficial due to increased risk of potentially lethal arrhythmias caused by augmentation of [Ca²⁺]_i[1). This risk should be absent in Ca²⁺ sensitizers. Recently, thiadiazinone derivatives have been synthetized in which the potency for Ca²⁺ sensitization is many-fold larger than the potency for PDE-III inhibition. The Ca²⁺-sensitizing action resides in the [+]-enantiomers, while the [–]-enantiomers show weak PDE-III inhibition. In the enantiomer pair [+]-EMD 60263 and [–]-EMD 60264, only the former concentration-dependently increased force of contraction in isolated cardiac preparations and myocytes. In the Langendorff-perfused guinea-pig heart, force was reversibly increased, whereas [–]-EMD 60264 even produced a negative inotropic response despite of its PDE inhibitory activity. Heart rate, however, was reduced by both enantiomers. Perfusion pressure remained unaffected. The effects were fully reversible upon wash-out of the enantiomers. [+]-EMD 60263 also enhanced cell shortening of human myocytes from both normal and failing hearts. In contrast to the opposite effects on contractility, both enantiomers prolong the action potential duration by blocking the rapidly activating component of the delayed rectifier K⁺ current. Thus they also possess class III antiarrhythmic activity. The therapeutic potential of these agents has yet to be assessed in clinical studies.     

Neuron-derived factors negatively modulate ryanodine receptor-mediated calcium release in cultured mouse astrocytes

Changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) produced by ryanodine receptor (RyR) agonist, caffeine (caf), and ionotropic agonists: N-methyl-d-aspartate (NMDA) receptor (NMDAR) agonist, NMDA and P2X7 receptor (P2X7R) agonist, 3′-O-(4-benzoyl)benzoyl adenosine 5′-triphosphate (BzATP) were measured in cultured mouse cortical astrocytes loaded with the fluorescent calcium indicator Fluo3-AM in a confocal laser scanning microscope. In mouse astrocytes cultured in standard medium (SM), treatment with caf increased [Ca²⁺]_i, with a peak response occurring about 10 min after stimulus application. Peak responses to NMDA or BzATP were observed about <1 min and 4.5 min post stimulus, respectively. Co-treatment with NMDA or BzATP did not alter the peak response to caf in astrocytes cultured in SM, the absence of the effects being most likely due to asynchrony between the response to caf, NMDA and BzATP. Incubation of astrocytes with neuron-condition medium (NCM) for 24 h totally abolished the caf-evoked [Ca²⁺]_i increase. In NCM-treated astrocytes, peak of [Ca²⁺]_i rise evoked by NMDA was delayed to about 3.5 min, and that induced by BzATP occurred about three minutes earlier than in SM. The results show that neurons secrete factors that negatively modulate RyR-mediated Ca²⁺-induced Ca²⁺ release (CICR) in astrocytes and alter the time course of Ca²⁺ responses to ionotropic stimuli.     

Inotropic effects of adrenaline on the anoxic or hypercapnic myocardium of rainbow trout and eel

Summary The effects of adrenaline on the development of force under anoxia and hypercapnic acidosis (13% CO₂ in 30 mM HCO ₃ ^– ) were examined in isolated, electrically stimulated cardiac ventricle strips of rainbow trout and eel.During anoxia or acidosis applied 15 min in advance, the adrenaline concentration of the bathing solution was increased in 5 steps from 0 to 10^–4 M with 5 min at each step. Before levelling off the contractile tension increased by 145±42% (mean±SE) in the anoxic, 80±14% in the acidotic and 152±41% in the control trout cardiac strips. Except for the acidotic strips the corresponding values tended to be lower for the eel strips being 46±9%, 57±17% and 57±9%, respectively. The inotropic affinity for adrenaline was lower in the trout than in the eel myocardium. For the trout myocardium it remained unchanged, while it decreased somewhat for the eel myocardium under anoxia or acidosis.Adding to the muscle bath 10^–5 M adrenaline resulted in an increase in force development by about 90% for the trout myocardium and 50% for the eel myocardium. 5 min later anoxia or hypercapnic acidosis was applied for 30 min followed by 30 min at control conditions. Relative to the force values recorded just before anoxia or acidosis was applied, the changes in contractile force during these periods were the same with and without adrenaline. Thus adrenaline appears to have a persistent positive inotropic effect in the fish myocardium during and after oxygen lack or acidosis.     

Different calcium sensitivity in osteoclasts on glass and on bone and maintenance of cytoskeletal structures on bone in the presence of high extracellular calcium

The sensitivity of rat osteoclasts to increased extracellular calcium concentrations ([Ca²⁺]_e) was investigated by single cell measurements of free cytosolic calcium concentrations ([Ca²⁺]_i), by changes in microfilament organization of resorbing osteoclasts, and by in vitro bone resorption assays. Osteoclasts cultured on glass and on bone showed clear differences in their responses, as in 44% and 52% of osteoclasts on glass but in only 21% and 25% of osteoclasts on bone [Ca²⁺]_i increased when [Ca²⁺]_e was increased from 2 mM to 6 or 10 mM via perfusion, respectively. Bone resorption was inhibited without changes in the osteoclast numbers only by 10 mM [Ca²⁺]_e in 2 day cultures. Furthermore, there were no changes in the organization of microfilament structures in resorbing osteoclasts after increased [Ca²⁺]_e (up to 20 mM [Ca²⁺]_e, 30 min incubation). These results suggest that the sensitivity of osteoclasts to increased [Ca²⁺]_e is dependent on their activation phase (resting/migrating vs. resorbing) and that resorbing osteoclasts are not sensitive to increased [Ca²⁺]_e or that the sensing system cannot be reached in polarized resorbing osteoclasts. In contrast, increasing [Ca²⁺]_i through the use of calcium ionophores dispersed specific microfilament structures at the sealing zone transiently in a few minutes. This shows that [Ca²⁺]_i is used as a signaling mechanism to inactivate osteoclasts, with a similar end result on microfilament structures at the sealing zone as caused by increased concentration of cAMP and activation of protein kinase C. © 1996 Wiley-Liss, Inc.     

Blockade of insulin sensitive steady-state R-type Ca2+ channel by PN 200-110 in heart and vascular smooth muscle

The effect of high K^– concentration, insulin and the L-type Ca^2– channel blocker PN 200-110 on cytosolic intracellular free calcium ([Ca²⁺]_i) was studied in single ventricular myocytes of 10-day-old embryonic chick heart, 20-week-old human fetus and rabbit aorta (VSM) single cells using the Ca²⁺-sensitive fluorescent dye, Fura-2 microfluorometry and digital imaging technique. Depolarization of the cell membrane of both heart and VSM cells with continuous superfusion of 30 mM [K⁺]_o induced a rapid transient increase of [Ca²⁺]_i that was followed by a sustained component. The early transient increase of [Ca²⁺]_i by high [⁺]_o was blocked by the L-type calcium channel antagonist nifedipine. However, the sustained component was found to be insensitive to this drug. PN 200-110 another L-type Ca²⁺ blocker was found to decrease both the early transient and the sustained increase of [Ca²⁺]_i induced by depolarization of the cell membrane with high [K⁺]_o. Insulin at a concentration of 40 to 80 U/ml only produced a sustained increase of [Ca²⁺]_i that was blocked by PN 200-110 or by lowering the extracellular Ca²⁺ concentration with EGTA. The sustained increase of [Ca²⁺], induced by high [K⁺]_o or insulin was insensitive to metabolic inhibitors such as KCN and ouabain as well to the fast Na⁺ channel blocker, tetrodotoxin and to the increase of intracellular concentrations of cyclic nucleotides. Using the patch clamp technique, insulin did not affect the L-type Ca²⁺ current and the delayed outward K⁺ current. These results suggest that the early increase of (Ca²⁺]_i during depolarization of the cell membrane of heart and VSM cells with high [K⁺]_o is due to the opening and decay of an L-type Ca ²⁺ channel. However, the sustained increase of [Ca²⁺]_i during a sustained depolarization is due to the activation of a resting (R) Ca ²⁺ channel that is insensitive to lowering [ATP]_i and sensitive to insulin.     

Post-conditioning protects cardiomyocytes from apoptosis via PKCε-interacting with calcium-sensing receptors to inhibit endo(sarco)plasmic reticulum–mitochondria crosstalk

The intracellular Ca²⁺ concentration ([Ca²⁺]_i) is increased during cardiac ischemia/reperfusion injury (IRI), leading to endo(sarco)plasmic reticulum (ER) stress. Persistent ER stress, such as with the accumulation of [Ca²⁺]_i, results in apoptosis. Ischemic post-conditioning (PC) can protect cardiomyocytes from IRI by reducing the [Ca²⁺]_i via protein kinase C (PKC). The calcium-sensing receptor (CaR), a G protein-coupled receptor, causes the production of inositol phosphate (IP₃) to increase the release of intracellular Ca²⁺ from the ER. This process can be negatively regulated by PKC through the phosphorylation of Thr-888 of the CaR. This study tested the hypothesis that PC prevents cardiomyocyte apoptosis by reducing the [Ca²⁺]_i through an interaction of PKC with CaR to alleviate [Ca²⁺]_ER depletion and [Ca²⁺]_m elevation by the ER-mitochondrial associated membrane (MAM). Cardiomyocytes were post-conditioned after 3 h of ischemia by three cycles of 5 min of reperfusion and 5 min of re-ischemia before 6 h of reperfusion. During PC, PKC_ε translocated to the cell membrane and interacted with CaR. While PC led to a significant decrease in [Ca²⁺]_i, the [Ca²⁺]_ER was not reduced and [Ca²⁺]_m was not increased in the PC and GdCl₃–PC groups. Furthermore, there was no evident ?ψ_m collapse during PC compared with ischemia/reperfusion (I/R) or PKC inhibitor groups, as evaluated by laser confocal scanning microscopy. The apoptotic rates detected by TUNEL and Hoechst33342 were lower in PC and GdCl₃–PC groups than those in I/R and PKC inhibitor groups. Apoptotic proteins, including m-calpain, BAP31, and caspase-12, were significantly increased in the I/R and PKC inhibitor groups. These results suggested that PKC_ε interacting with CaR protected post-conditioned cardiomyocytes from programmed cell death by inhibiting disruption of the mitochondria by the ER as well as preventing calcium-induced signaling of the apoptotic pathway.     

Crosstalk between insulin receptor and G protein-coupled receptor signaling systems leads to Ca oscillations in pancreatic cancer PANC-1 cells

We examined crosstalk between the insulin receptor and G protein-coupled receptor (GPCR) signaling pathways in individual human pancreatic cancer PANC-1 cells. Treatment of cells with insulin (10 ng/ml) for 5 min markedly enhanced the proportion of cells that display an increase in intracellular [Ca²⁺] induced by picomolar concentrations of the GPCR agonist neurotensin. Interestingly, insulin increased the proportion of a subpopulation of cells that exhibit intracellular [Ca²⁺] oscillations in response to neurotensin at concentrations as low as 50-200 pM. Insulin enhanced GPCR-induced Ca²⁺ signaling in a time- and dose-dependent manner; a marked potentiation was obtained after an exposure to a concentration of 10 ng/ml for 5 min. Treatment with the mTORC1 inhibitor rapamycin abrogated the increase in GPCR-induced [Ca²⁺]_i oscillations produced by insulin. Our results identify a novel aspect in the crosstalk between insulin receptor and GPCR signaling systems in pancreatic cancer cells, namely that insulin increases the number of [Ca²⁺]_i oscillating cells induced by physiological concentrations of GPCR agonists through an mTORC1-dependent pathway.     

Eicosapentaenoic Acid Enhances Nitric Oxide Production by Cultured Human Endothelial Cells

It is unclear whether the abnormal relaxation seen in diabetes is due to decreased levels of nitric oxide (NO) and how eicosapentaenoic acid (EPA, C20:5ω3) affects the endothelial production of NO. We investigated the effects of EPA ethyl ester (EPA-E) and elevated glucose on NO production by human endothelial cells (HUE). EPA-E (0.3 mM) significantly enhanced [NO₂⁻] production and the intracellular concentration of free Ca²⁺within 3 min after EPA-E was added to the cultures. High levels of glucose (27.5 mM) significantly increased endothelial glucose, sorbitol and fructose, and inhibited [NO₂⁻] production. However, EPA-E (0.3 mM) prevented the inhibition of [NO₂⁻] production due to the activation of the Ca²⁺-calmodulin system of NO synthase. EPA-E decreased the glucose-mediated inhibition of NO production by HUE. These results suggest this agent might ameliorate endothelial dysfunction associated with diabetes.     

In situ visualization of the intracellular Ca2+ dynamics at the border of the acute myocardial infarct

Ischemic insult to the heart produces myocyte Ca²⁺ ([Ca²⁺]_i) overload. However, little is known about spatiotemporal changes in [Ca²⁺]_i within the ischemic heart in situ at the cellular level. Using real-time confocal microscopy, we successfully visualized [Ca²⁺]_i dynamics at the border zone on the subepicardial myocardium of the heart 2 h after coronary ligations followed by loading with fluo 3/AM. Three distinct regions were identified in the acute infarcted heart. In intact regions, the myocytes showed spatially uniform Ca²⁺ transients synchronously to QRS complex in the electrocardiogram. The myocytes at the infarcted regions showed no fluorescence intensity (FI). At the border zones between the intact and infarcted regions, Ca²⁺ waves emerged sporadically and randomly, instead of Ca²⁺ transients, at a mean frequency of 11.5 ± 8.5 min/cell with a propagation velocity of 151.0 ± 35.7 m/sec along the longitudinal axis of the individual myocytes. In addition, some myocytes within the border zone exhibited homogeneously high static FI, indicating severe Ca²⁺ overload. In summary, we provided the first direct evidence of abnormal [Ca²⁺]_i dynamics in acute infarcted hearts at the cellular level. The observed diversity in spatiotemporal [Ca²⁺]_i dynamics at the border zone may contribute to the arrhythmias or contractile failure in acute myocardial infarction.     

Cytosolic calcium regulation in rat afferent vagal neurons during anoxia

Sensory neurons are able to detect tissue ischaemia and both transmit information to the brainstem as well as release local vasoactive mediators. Their ability to sense tissue ischaemia is assumed to be primarily mediated through proton sensing ion channels, lack of oxygen however may also affect sensory neuron function. In this study we investigated the effects of anoxia on isolated capsaicin sensitive neurons from rat nodose ganglion. Acute anoxia triggered a reversible increase in [Ca²⁺]_i that was mainly due to Ca²⁺-efflux from FCCP sensitive stores and from caffeine and CPA sensitive ER stores. Prolonged anoxia resulted in complete depletion of ER Ca²⁺-stores. Mitochondria were partially depolarised by acute anoxia but mitochondrial Ca²⁺-uptake/buffering during voltage-gated Ca²⁺-influx was unaffected. The process of Ca²⁺-release from mitochondria and cytosolic Ca²⁺-clearance following Ca²⁺ influx was however significantly slowed. Anoxia was also found to inhibit SERCA activity and, to a lesser extent, PMCA activity. Hence, anoxia has multiple influences on [Ca²⁺]_i homeostasis in vagal afferent neurons, including depression of ATP-driven Ca²⁺-pumps, modulation of the kinetics of mitochondrial Ca²⁺ buffering/release and Ca²⁺-release from, and depletion of, internal Ca²⁺-stores. These effects are likely to influence sensory neuronal function during ischaemia.     

Involvement of Ca in globular adiponectin-induced reactive oxygen species

Globular adiponectin (gAd) induces the generation of reactive oxygen species (ROS) and nitric oxide (NO) in the murine macrophage cell line RAW 264. We investigated the role of Ca²⁺ in gAd-induced ROS and NO generation. Pretreatment with BAPTA-AM, a selective chelator of intracellular Ca²⁺ ([Ca²⁺]_i), partially reduced gAd-induced generation of ROS and NO in gAd-treated RAW 264 cells. The lowest [Ca²⁺]_i occurred 30 min after gAd treatment, after which [Ca²⁺]_i increased continually and exceeded the initial level. The mitochondrial Ca²⁺ ([Ca²⁺]_m) detected by Rhod-2 fluorescence started to increase at 6 h after gAd treatment. Pretreatment with a NAD(P)H oxidase inhibitor, diphenyleneiodonium, prevented the reduction of [Ca²⁺]_i in the early phase after gAd treatment. Calcium depletion by BAPTA-AM had no effect on the gAd-induced [Ca²⁺]_m oscillation. The administration of a specific calmodulin inhibitor, calmidazolium, significantly suppressed gAd-induced ROS and NO generation and NOS activity.     

?-Blocker Timolol Prevents Arrhythmogenic Ca2+ Release and Normalizes Ca2+ and Zn2+ Dyshomeostasis in Hyperglycemic Rat Heart

Defective cardiac mechanical activity in diabetes results from alterations in intracellular Ca²⁺ handling, in part, due to increased oxidative stress. Beta-blockers demonstrate marked beneficial effects in heart dysfunction with scavenging free radicals and/or acting as an antioxidant. The aim of this study was to address how β-blocker timolol-treatment of diabetic rats exerts cardioprotection. Timolol-treatment (12-week), one-week following diabetes induction, prevented diabetes-induced depressed left ventricular basal contractile activity, prolonged cellular electrical activity, and attenuated the increase in isolated-cardiomyocyte size without hyperglycemic effect. Both in vivo and in vitro timolol-treatment of diabetic cardiomyocytes prevented the altered kinetic parameters of Ca²⁺ transients and reduced Ca²⁺ loading of sarcoplasmic reticulum (SR), basal intracellular free Ca²⁺ and Zn²⁺ ([Ca²⁺]_i and [Zn²⁺]_i), and spatio-temporal properties of the Ca²⁺ sparks, significantly. Timolol also antagonized hyperphosphorylation of cardiac ryanodine receptor (RyR2), and significantly restored depleted protein levels of both RyR2 and calstabin2. Western blot analysis demonstrated that timolol-treatment also significantly normalized depressed levels of some [Ca²⁺]_i-handling regulators, such as Na⁺/Ca²⁺ exchanger (NCX) and phospho-phospholamban (pPLN) to PLN ratio. Incubation of diabetic cardiomyocytes with 4-mM glutathione exerted similar beneficial effects on RyR2-macromolecular complex and basal levels of both [Ca²⁺]_i and [Zn²⁺]_i, increased intracellular Zn²⁺ hyperphosphorylated RyR2 in a concentration-dependent manner. Timolol also led to a balanced oxidant/antioxidant level in both heart and circulation and prevented altered cellular redox state of the heart. We thus report, for the first time, that the preventing effect of timolol, directly targeting heart, seems to be associated with a normalization of macromolecular complex of RyR2 and some Ca²⁺ handling regulators, and prevention of Ca²⁺ leak, and thereby normalization of both [Ca²⁺]_i and [Zn²⁺]_i homeostasis in diabetic rat heart, at least in part by controlling the cellular redox status of hyperglycemic cardiomyocytes.