Dominant role of sarcoendoplasmic reticulum Ca2+-ATPase pump in Ca2+ homeostasis and exocytosis in rat pancreatic beta-cells

The exocytosis of insulin-containing granules from pancreatic beta-cells is tightly regulated by changes in cytosolic Ca2+ concentration ([Ca2+]i). We investigated the role of the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) pump, Na+/Ca2+ exchanger, and plasma membrane Ca2+-ATPase pump in the Ca2+ dynamics of single rat pancreatic beta-cells. When the membrane potential was voltage clamped at -70 mV (in 3 mm glucose at approximately 22 or 35 C), SERCA pump inhibition dramatically slowed (approximately 4-fold) cytosolic Ca2+ clearance and caused a sustained rise in basal [Ca2+]i via the activation of capacitative Ca2+ entry. SERCA pump inhibition increased ( approximately 1.8-fold) the amplitude of the depolarization-triggered Ca2+ transient at approximately 22 C. Inhibition of the Na+/Ca2+ exchanger or plasma membrane Ca2+-ATPase pump had only minor effects on Ca2+ dynamics. Simultaneous measurement of [Ca2+]i and exocytosis (with capacitance measurement) revealed that SERCA pump inhibition increased the magnitude of depolarization-triggered exocytosis. This enhancement in exocytosis was not due to the slowing of the cytosolic Ca2+ clearance but was closely correlated to the increase in the peak of the depolarization-triggered Ca2+ transient. When compared at similar [Ca2+]i with controls, the rise in basal [Ca2+]i during SERCA pump inhibition did not cause any enhancement in the magnitude of the ensuing depolarization-triggered exocytosis. Therefore, we conclude that in rat pancreatic beta-cells, the rapid uptake of Ca2+ by SERCA pump limits the peak amplitude of depolarization-triggered [Ca2+]i rise and thus controls the amount of insulin secretion.     

Transient receptor potential channel 6-mediated, localized cytosolic [Na+] transients drive Na+/Ca2+ exchanger-mediated Ca2+ entry in purinergically stimulated aorta smooth muscle cells

The Na+/Ca2+ exchanger (NCX) is increasingly recognized as a physiological mediator of Ca2+ influx and significantly contributes to salt-sensitive hypertension. We recently reported that Ca2+ influx by the NCX (1) is the primary mechanism of Ca2+ entry in purinergically stimulated rat aorta smooth muscle cells and (2) requires functional coupling with transient receptor potential channel 6 nonselective cation channels. Using the Na+ indicator CoroNa Green, we now directly observed and characterized the localized cytosolic [Na+] ([Na+]i) elevations that have long been hypothesized to underlie physiological NCX reversal but that have never been directly shown. Stimulation of rat aorta smooth muscle cells caused both global and monotonic [Na+]i elevations and localized [Na+]i transients (LNats) at the cell periphery. Inhibition of nonselective cation channels with SKF-96365 (50 micromol/L) and 2-amino-4-phosphonobutyrate (75 micromol/L) reduced both global and localized [Na+]i elevations in response to ATP (1 mmol/L). This effect was mimicked by expression of a dominant negative construct of transient receptor potential channel 6. Selective inhibition of NCX-mediated Ca2+ entry with KB-R7943 (10 micromol/L) enhanced the LNats, whereas the global cytosolic [Na+] signal was unaffected. Inhibition of mitochondrial Na+ uptake with CGP-37157 (10 micromol/L) increased both LNats and global cytosolic [Na+] elevations. These findings directly demonstrate NCX regulation by LNats, which are restricted to subsarcolemmal, cytoplasmic microdomains. Analysis of the LNats, which facilitate Ca2+ entry via NCX, suggests that mitochondria limit the cytosolic diffusion of LNats generated by agonist-mediated activation of transient receptor potential channel 6-containing channels.     

Elevated cytosolic Na+ decreases mitochondrial Ca2+ uptake during excitation-contraction coupling and impairs energetic adaptation in cardiac myocytes

Mitochondrial Ca2+ ([Ca2+]m) regulates oxidative phosphorylation and thus contributes to energy supply and demand matching in cardiac myocytes. Mitochondria take up Ca2+ via the Ca2+ uniporter (MCU) and extrude it through the mitochondrial Na+/Ca2+ exchanger (mNCE). It is controversial whether mitochondria take up Ca2+ rapidly, on a beat-to-beat basis, or slowly, by temporally integrating cytosolic Ca2+ ([Ca2+]c) transients. Furthermore, although mitochondrial Ca2+ efflux is governed by mNCE, it is unknown whether elevated intracellular Na+ ([Na+]i) affects mitochondrial Ca2+ uptake and bioenergetics. To monitor [Ca2+]m, mitochondria of guinea pig cardiac myocytes were loaded with rhod-2-acetoxymethyl ester (rhod-2 AM), and [Ca2+]c was monitored with indo-1 after dialyzing rhod-2 out of the cytoplasm. [Ca2+]c transients, elicited by voltage-clamp depolarizations, were accompanied by fast [Ca2+]m transients, whose amplitude (delta) correlated linearly with delta[Ca2+]c. Under beta-adrenergic stimulation, [Ca2+]m decay was approximately 2.5-fold slower than that of [Ca2+]c, leading to diastolic accumulation of [Ca2+]m when amplitude or frequency of delta[Ca2+]c increased. The MCU blocker Ru360 reduced delta[Ca2+]m and increased delta[Ca2+]c, whereas the mNCE inhibitor CGP-37157 potentiated diastolic [Ca2+]m accumulation. Elevating [Na+]i from 5 to 15 mmol/L accelerated mitochondrial Ca2+ decay, thus decreasing systolic and diastolic [Ca2+]m. In response to gradual or abrupt changes of workload, reduced nicotinamide-adenine dinucleotide (NADH) levels were maintained at 5 mmol/L [Na+]i, but at 15 mmol/L, the NADH pool was partially oxidized. The results indicate that (1) mitochondria take up Ca2+ rapidly and contribute to fast buffering during a [Ca2+]c transient; and (2) elevated [Na+]i impairs mitochondrial Ca2+ uptake, with consequent effects on energy supply and demand matching. The latter effect may have implications for cardiac diseases with elevated [Na+]i.     

Mitochondrial Ca2+ uptake during simulated ischemia does not affect permeability transition pore opening upon simulated reperfusion

OBJECTIVE: Reenergization of ischemic cardiomyocytes may be associated with acute necrotic cell death due in part to cytosolic Ca2+ overload and opening of a permeability transition pore (PTP) in mitochondria. It has been suggested that Ca2+ overload during ischemia primes mitochondria for PTP opening during reperfusion. We investigated the ability of mitochondria to uptake Ca2+ during simulated ischemia (SI) and whether this uptake determines PTP opening and cell death upon simulated reperfusion (SR). METHODS: Rat heart mitochondria were submitted to either hypoxia (anoxic chamber) or to SI (respiratory inhibition, substrate depletion and acidosis) and subsequent SR. Mitochondrial Ca2+ uptake was monitored using Ca2+ microelectrodes after exposure to different [Ca2+] up to 25 microM during SI, and PTP opening was assessed by quantification of mitochondrial swelling (changes in absorbance rate at 540 nm) and calcein release. Mitochondrial Ca2+ uptake (Rhod-2 fluorescence) and cytosolic Ca2+ rise (Fura-2 ratio fluorescence) were further investigated in HL-1 cardiac myocytes submitted to SI/SR, and the effect of reducing mitochondrial Ca2+ load (with 25 microM ruthenium red) or blocking PTP opening (with 0.5 microM cyclosporin A) on the rate of cell death was investigated in adult cardiomyocytes exposed to SI/SR. RESULTS: SI induced a progressive dissipation of mitochondrial membrane potential (TMRE fluorescence); however, prior to the completion of depolarization, high levels of Ca2+ uptake were observed in mitochondria. SR induced PTP opening but this phenomenon was not influenced by the magnitude of mitochondrial Ca2+ uptake during previous SI. Blockade of the mitochondrial Ca2+ uniporter during SI in cardiomyocytes attenuated mitochondrial Ca2+ uptake but increased cytosolic Ca2+ overload and cell death upon subsequent SR. CONCLUSION: Mitochondrial Ca2+ uptake during SI buffers cytosolic Ca2+ overload but its magnitude appears not to be an important determinant of PTP opening upon subsequent SR.     

Pregnancy-specific modulatory role of mitochondria on adenosine 5'-triphosphate-induced cytosolic [Ca2+] signaling in uterine artery endothelial cells

Vascular endothelial cells respond to extracellular ATP by inositol 1,4,5-trisphosphate-mediated Ca2+ release from the endoplasmic reticulum followed by Ca2+ influx and subsequent synthesis of vasodilators. In this study, the contribution of mitochondria in shaping the ATP-induced Ca2+ increase was examined in ovine uterine artery endothelial cells from nonpregnant and pregnant (late gestation) ewes (NP- and P-UAEC, passage 4). The mitochondrial protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) induced a rapid mitochondrial depolarization. CCCP also slowly increased cytosolic [Ca2+] ([Ca2+]c), which then gradually declined to 10-20 nM above resting level. Pretreatment with CCCP for 30 min significantly inhibited both ATP and thapsigargin-induced [Ca2+]c, with inhibition in NP-UAEC more effective than in P-UAEC. Pretreatment of mitochondrial permeability transition pore inhibitor cyclosporine A did not affect CCCP-induced mitochondrial depolarization, but delayed CCCP-induced [Ca2+]c for about 12-15 min (we termed this the "window of time"). During the cyclosporine A-delayed window of time of CCCP-induced [Ca2+]c, ATP induced a normal Ca2+ response, but after this window of time, ATP-induced [Ca2+]c was significantly inhibited. Pretreatment of oligomycin B to prevent intracellular ATP depletion by F0F1-ATPase did not reduce the inhibition of ATP-induced [Ca2+]c by CCCP. Ruthenium red, a mitochondrial Ca2+ uptake blocker, did not mimic the inhibition of Ca2+ signaling by CCCP. In conclusion, our data show that mitochondrial Ca2+ depletion after dissipation of mitochondrial membrane potential with CCCP inhibits ATP-induced [Ca2+]c, mediated at the level of Ca2+ release from the endoplasmic reticulum. Moreover, our data revealed that P-UAEC is more resistant to the inhibitory effect of CCCP on [Ca2+]c than NP-UAEC.     

Evolution of metabolic and functional derangements of pancreatic islets in phosphate depletion.

Phosphate depletion (PD) causes a rise in basal level of cytosolic calcium ([Ca2+]i) of pancreatic islets, a decrease in their basal and stimulated ATP content, a reduction in the maximum velocity (Vmax) of Ca2+ adenosine triphosphatase (ATPase) and Na(+)-K+ ATPase, impaired glucose-induced calcium signal and decreased glucose-induced insulin secretion. The sequence of events that lead to these derangements during the evolution of PD are not defined. The present study examined this issue by measuring the metabolic and functional profile of pancreatic islets weekly during the evolution of PD over a period of 6 weeks, and whether phosphate repletion reverses these abnormalities. The results show that initial abnormalities are a rise in Vmax of Ca2+ ATPase and modest rise in basal [Ca2+]i. This was followed by a fall in basal and stimulated ATP content. With the fall in ATP content, the Vmax of Ca2+ ATPase and Na(+)-K+ ATPase decreases and the rise in [Ca2+]i becomes more pronounced. A decrease in glucose-induced insulin secretion becomes evident with the fall in ATP, the decrease in glucose-induced calcium signal, and/or delta[Ca2+]i/basal[Ca2+]i. All functional and metabolic derangements of the pancreatic islets returned to normal after phosphate repletion. Taken together, our data are consistent with the notion that PD is associated with an initial increase in calcium influx into the islets. This is followed by modest but significant rise in [Ca2+]i which, in turn, would inhibit mitochondrial oxidation and ATP generation leading to a decrease in ATP content. The latter compromises the activity of Ca2+ ATPase and Na(+)-K+ ATPase which are involved, directly or indirectly, in calcium extrusion out of the islets. The increased influx of calcium combined with decreased calcium extrusion is followed by a further rise in basal levels of [Ca2+]i. This sequence of events continues until a steady state is reached and is characterized by reduced basal and stimulated ATP content, reduced Vmax of Ca2+ ATPase and Na(+)-K+ ATPase and elevated basal level of [Ca2+]i. Phosphate repletion reverses all these abnormalities.     

Human platelet Ca2+-ATPases: new markers of cell differentiation as illustrated in idiopathic scoliosis

The aetiology of adolescent idiopathic scoliosis (AIS), the most common form of scoliosis, is unclear. Previous studies showed controversial platelet abnormalities including intracellular calcium. Platelet Ca2+ homeostasis is controlled by a multi-Ca2+-ATPase system including SERCA (sarco/endoplasmic reticulum Ca2+-ATPase) and PMCA (plasma membrane Ca2+-ATPase) isoforms. Here, we first investigated the expression of PMCA4b, SERCA3a and SERCA2b isoforms in platelets of 17 patients with AIS. Patients presenting thoracic curves were found to present a higher PMCA4b expression coupled to a lower SERCA3a one in agreement with an abnormality in platelet maturation. Indeed, using PMA-treated MEG 01 cells, an in vitro model of megakaryocytopoiesis, we found an increase in SERCA3a expression, associated to a caspase-3 mediated C terminal proteolysis of PMCA4b. To look whether platelets reflect a basic defect in cell differentiation, we next identified osteoblast Ca2+-ATPases and studied their expressions in AIS. Major expressions of PMCA4b and SERCA2b were found in normal osteoblasts. Comparing platelets and osteoblasts in two additional patients with AIS, we found opposite and concerted regulations of the expressions of PMCA4b and caspase-3 substrate, PARP in both cell types. A systemic defect in cell differentiation involving caspase-3 can be proposed as a novel mechanism in the etiopathogenesis of the most frequent type of AIS. *R. Bredoux and E. Corvazier contributed equally to this work.     

Intracellular Na+ regulation in cardiac myocytes

Intracellular [Na+] ([Na+]i) is regulated in cardiac myocytes by a balance of Na+ influx and efflux mechanisms. In the normal cell there is a large steady state electrochemical gradient favoring Na+ influx. This potential energy is used by numerous transport mechanisms, including Na+ channels and transporters which couple Na+ influx to either co- or counter-transport of other ions and solutes. Six sarcolemmal Na+ influx pathways are discussed in relatively quantitative terms: Na+ channels, Na+/Ca2+ exchange, Na+/H+ exchange, Na+/Mg2+ exchange, Na+/HCO3- cotransport and Na+/K+/2Cl- cotransport. Under normal conditions Na+/Ca2+ exchange and Na+ channels are the dominant Na+ influx pathways, but other transporters may become increasingly important during altered conditions (e.g. acidosis or cell volume stress). Mitochondria also exhibit Na+/Ca2+ antiporter and Na+/H+ exchange activity that are important in mitochondrial function. These coupled fluxes of Na+ with Ca2+, H+ and HCO3- make the detailed understanding of [Na+]i regulation pivotal to the understanding of both cardiac excitation-contraction coupling and pH regulation. The Na+/K+-ATPase is the main route for Na+ extrusion from cells and [Na+]i is a primary regulator under physiological conditions. [Na+]i is higher in rat than rabbit ventricular myocytes and the reason appears to be higher Na+ influx in rat with a consequent rise in Na+/K+-ATPase activity (rather than lower Na+/K+-ATPase function in rat). This has direct functional consequences. There may also be subcellular [Na+]i gradients locally in ventricular myocytes and this may also have important functional implications. Thus, the balance of Na+ fluxes in heart cells may be complex, but myocyte Na+ regulation is functionally important and merits focused attention as in this issue.     

Parallel adaptive feedback enhances reliability of the Ca2+ signaling system

Despite large cell-to-cell variations in the concentrations of individual signaling proteins, cells transmit signals correctly. This phenomenon raises the question of what signaling systems do to prevent a predicted high failure rate. Here we combine quantitative modeling, RNA interference, and targeted selective reaction monitoring (SRM) mass spectrometry, and we show for the ubiquitous and fundamental calcium signaling system that cells monitor cytosolic and endoplasmic reticulum (ER) Ca(2+) levels and adjust in parallel the concentrations of the store-operated Ca(2+) influx mediator stromal interaction molecule (STIM), the plasma membrane Ca(2+) pump plasma membrane Ca-ATPase (PMCA), and the ER Ca(2+) pump sarco/ER Ca(2+)-ATPase (SERCA). Model calculations show that this combined parallel regulation in protein expression levels effectively stabilizes basal cytosolic and ER Ca(2+) levels and preserves receptor signaling. Our results demonstrate that, rather than directly controlling the relative level of signaling proteins in a forward regulation strategy, cells prevent transmission failure by sensing the state of the signaling pathway and using multiple parallel adaptive feedbacks.     

Regulation of arachidonic acid release by calcium influx in human endothelial cells.

In response to stimuli, endothelial cells release arachidonic acid, a lipid precursor of various vasoactive substances. We have investigated the relationships between cytosolic Ca2+ movements and arachidonic acid release in human umbilical vein endothelial cells. Histamine, a receptor-dependent agonist, and thapsigargin, a specific inhibitor of sarco-/endoplasmic Ca2+ pumps, time- and dose-dependently increased the release of [1-14C]-arachidonic acid. This release was inhibited by AACOCF3, a selective inhibitor of cytosolic phospholipase A2 (PLA2). In the absence of Ca2+ influx, arachidonic acid release was suppressed in both histamine- and thapsigargin-stimulated cells, despite marked elevations of cytosolic Ca2+ concentration ([Ca2+]i). In the presence of Ca2+ influx, arachidonic acid release was reduced in cells treated with BAPTA, an intracellular Ca2+ buffer, or with SK&F 96365, a receptor-operated Ca2+ channel blocker. Arachidonic acid release was analyzed as a function of the two successive phases of Ca2+ response to stimulation: Ca2+ peak and plateau phase, reflecting Ca2+ mobilization from internal stores and Ca2+ influx, respectively. The amount of arachidonic acid released was directly related to [Ca2+]i values measured at the influx phase with a 80 nM [Ca2+]i threshold, similar to that reported for PLA2 translocation. This suggests that Ca2+ entry from the extracellular space is essential for activating cytosolic PLA2 in human endothelial cells.     

Agonist-stimulated calcium entry in primary cultures of human cerebral microvascular endothelial cells.

Primary cultures of human cerebral microvascular endothelial cells (HCMEC) were loaded with fura-2. The intracellular free Ca2+ concentration ([Ca2+]i) was measured by digital imaging microscopy. Agonists ATP (100 micro), thrombin (10 units/ml), and histamine (25 microM) induced a transient [Ca2+]i increase. Histamine (100 microM) induced a biphasic [Ca2+]i increase with an initial [Ca2+]i peak followed by a [Ca2+]i plateau. The [Ca2+]i plateau was blocked by the receptor-operated Ca2+ channel (ROC) blockers SK&F 96365 and NCDC, indicating a contribution by Ca2+ influx through ROC to the [Ca2+]i plateau. However, this [Ca2+]i plateau was not blocked by the voltage-gated Ca2+ channel (VGC) blocker diltiazem (DTZ). Depolarization with 80K+ or application of the VGC agonist BAY K 8644 did not alter the resting [Ca2+]i; but 80K+ reduced the histamine (100 microM) induced [Ca2+]i plateau. These results show that HCMEC are devoid of functional VGC. Thus the membrane potential (Em) regulates Ca2+ entry mainly by enhancing the electrochemical Ca2+ gradient, such that hyperpolarization increases while depolarization decreases [Ca2+]i. Blockade of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) by CPA increased [Ca2+]i. This effect was dependent on extracellular Ca2+ and reduced by iberiotoxin (IBTX) blockade of Ca2+-activated K+ channels (Kca), suggesting a role for Kca in regulating Ca2+ influx. Ca2+ is the principal activator of endothelial nitric oxide synthase (eNOS), which stimulates cyclic GMP production. The final result that the eNOS inhibitor L-NAME enhanced the histamine (100 microM) induced [Ca2+]i plateau suggests a negative feedback loop (via cGMP) of endothelial NO on its own synthesis in the regulation of endothelial [Ca2+]i signal.     

Plasma membrane calcium ATPase overexpression in arterial smooth muscle increases vasomotor responsiveness and blood pressure

In vascular smooth muscle cells (SMCs), several mechanisms act in concert to regulate the intracellular calcium concentration [Ca2+]i, which may in turn affect vascular tone. One such mechanism is the extrusion of Ca2+ by the plasma membrane calcium ATPase (PMCA). To address, in particular, the role of the neuronal nitric oxide synthase (nNOS)-associating isoform PMCA4b in regulating vascular tone, a doxycycline-responsive transgene for human PMCA4b was overexpressed in arterial SMCs of mice. Overexpression of hPMCA4b resulted in a 2-fold increase in total aortic PMCA4 protein expression and significant real-time RT-PCR-documented differences in the levels of endogenous mouse PMCA1, PMCA4, SERCA2, and IP3R1 gene expression in arterial SMCs. Whereas no significant difference in basal [Ca2+]i or Ca2+ sensitivity was observed in vascular SMCs or mesenteric arteries, respectively, from hPMCA4b-overexpressing versus control mice, hPMCA4b-overexpressing mice revealed a reduced set-point and increased extent of myogenic response and heightened sensitivity to vasoconstrictors. Treatment of arteries with an nNOS inhibitor resulted in a reduced set-point and increased extent of the myogenic response in control but not hPMCA4b-overexpressing mice. Moreover, aortic SMCs from hPMCA4b-overexpressing mice exhibited reduced levels of cGMP under both basal and phenylephrine-stimulated conditions. These changes were associated with significant doxycycline-reversible elevations in blood pressure. Taken together, these data show that overexpression of hPMCA4b in arterial SMCs increases vascular reactivity and blood pressure, an effect that may be mediated in part by negative regulation of nNOS.     

Ischemia-reperfusion induces myocardial infarction through mitochondrial Ca²⁺ overload

Both mitochondria and the sarcoplasmic reticulum (SR) are essential for myocardial homeostasis and control of cardiac function. Uptake of Ca(2+) from the cytosol into SR is mediated by the Ca(2+)-dependent ATPase SERCA2a, which is reversibly inhibited by phospholamban (PLN). We previously showed that removal of PLN inhibition of SERCA2a with an antibody to (anti-) PLN reduces cytosolic Ca(2+) overload, thereby attenuating the spread of contraction bands and fodrin proteolysis, during reperfusion after cardiac ischemia. We have now examined the effects of anti-PLN injection into the heart on the development of myocardial infarction (MI) after ischemia-reperfusion in rats. Whereas anti-PLN injection attenuated cytosolic Ca(2+) overload, it did not affect MI size 6h after the onset of reperfusion and actually increased it at 30 min. The antibody also increased the release of apoptosis-inducing factor (AIF) from mitochondria into the cytosol, indicative of enhanced opening of the mitochondrial permeability transition pore (mPTP). Administration of an mPTP blocker at the time of reperfusion or of a blocker of the mitochondrial Ca(2+) uniporter significantly suppressed the release of AIF and the development of MI. These results indicate that the enhancement of SR Ca(2+) loading by anti-PLN injection facilitated Ca(2+) uniporter-dependent mitochondrial Ca(2+) uptake and thereby induced mPTP opening and MI development during early reperfusion. The enhancement of SR Ca(2+) loading thus aggravates MI in a manner independent of cytosolic Ca(2+) overload. Given that cytosolic Ca(2+) overload induces contraction bands, our findings are inconsistent with a causal relation between contraction bands and MI.     

Ca2+ uptake by the sarcoplasmic reticulum in ventricular myocytes of the SERCA2b/b mouse is impaired at higher Ca2+ loads only

SERCA2a is the cardiac-specific isoform of Ca2+-ATPase of the sarcoplasmic reticulum (SR). A reduction of SERCA2a has been implicated in the contractile dysfunction of heart failure, and partial knockout of the SERCA2 gene (Atp2a2+/- mice) reiterated many of the features of heart failure. Yet, mice with a mutation of Atp2a2, resulting in full suppression of the SERCA2a isoform and expression of the SERCA2b isoform only (SERCA2b/b), showed only moderate functional impairment, despite a reduction by 40% of the SERCA2 protein levels. We examined in more detail the Ca2+ handling in isolated cardiac myocytes from SERCA2b/b. At 0.25 Hz stimulation, the amplitude of the [Ca2+]i transients, SR Ca2+ content, diastolic [Ca2+]i, and density of ICaL were comparable between WT and SERCA2b/b. However, the decline of [Ca2+]i was slower (t1/2 154+/-7 versus 131+/-5 ms; P<0.05). Reducing the amplitude of the [Ca2+]i transient (eg, SR depletion), removed the differences in [Ca2+]i decline. In contrast, increasing the Ca2+ load revealed pronounced reduction of SR Ca2+ uptake at high [Ca2+]i. There was no increase in Na+-Ca2+ exchange protein or function. Theoretical modeling indicated that in the SERCA2b/b mouse, the higher Ca2+ affinity of SERCA2b partially compensates for the 40% reduction of SERCA expression. The lack of SR depletion in the SERCA2b/b may also be related to the absence of upregulation of Na+-Ca2+ exchange. We conclude that for SERCA isoforms with increased affinity for Ca2+, a reduced expression level is better tolerated as Ca2+ uptake and storage are impaired only at higher Ca2+ loads.     

Evidence for mechanistic alterations of Ca2+ homeostasis in Type 2 diabetes mellitus

Altered cytosolic Ca2+ is implicated in the aetiology of many diseases including diabetes but there are few studies on the mechanism(s) of the altered Ca2+ regulation. Using human lymphocytes, we studied cytosolic calcium (Cai) and various Ca2+ transport mechanisms in subjects with Type 2 diabetes mellitus and control subjects. Ca2+-specific fluorescent probes (Fura-2 and Fluo-3) were used to monitor the Ca2+ signals. Thapsigargin, a potent and specific inhibitor of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), was used to study Ca2+-store dependent Ca2+ fluxes. Significant (P<0.05) elevation of basal Ca, levels was observed in lymphocytes from diabetic subjects. Cai levels were positively correlated with fasting plasma glucose and HbA1c. There was also a significant (P<0.05) reduction in plasma membrane calcium (PMCA) ATPase activity in diabetic subjects compared to controls. Cells from Type 2 diabetics exhibited an increased Ca2+ influx (as measured both by Fluo-3 fluorescence and 45Ca assays) as a consequence of thapsigargin-mediated Ca2+ store depletion. Upon addition of Mn2+ (a surrogate of Ca2+), the fura-2 fluorescence decayed in an exponential fashion and the rate and extent of this decline was steeper and greater in cells from type 2 diabetic patients. There was also a significant (P<0.05) difference in the Na+/Ca2+ exchange activity in Type 2 diabetic patients, both under resting conditions and after challenging the cells with thapsigargin, when the internal store Ca2+ sequestration was circumvented. Pharmacological activation of protein kinase C (PKC) in cells from patients resulted in only partial inhibition of Ca2+ entry. We conclude that cellular Ca2+ accumulation in cells from Type 2 diabetes results from (a) reduction in PMCA ATPase activity, (b) modulation of Na+/Ca2+ exchange and (3) increased Ca2+ influx across the plasma membrane.     

The increase in intracellular free calcium associated with IgG gamma 2b/gamma 1 Fc receptor-ligand interactions: role in phagocytosis.

The concentration of cytosolic free calcium, [Ca2+]i, was measured in J774 mouse macrophages by use of the fluorescent indicator quin-2. Resting [Ca2+]i was 87 nM. Addition of a number of specific ligands to the immunoglobulin gamma 2b/gamma 1 Fc receptor resulted in a transient increase in [Ca2+]i, the magnitude of which depended on the extent of receptor aggregation. Monovalent ligands gave only a small Ca2+ signal but blocked cell response to subsequent addition of multivalent ligands. Incubation with antibody-coated erythrocytes raised macrophage [Ca2+]i to micromolar levels. [Ca2+]i changes were only partially inhibited by the absence of external Ca2+, suggesting the release of Ca2+ from internal stores in addition to an influx of external Ca2+. These internal stores were not limited to mitochondria. An optimal range of [Ca2+]i was required for phagocytosis. Buffering [Ca2+]i with quin-2 and treating cells with quinine in the absence of external Ca2+ resulted in inhibition of phagocytosis. Increasing [Ca2+]i to micromolar levels with the calcium ionophore A23187 also resulted in similar inhibitory effects. We suggest the involvement of localized cytosolic Ca2+ gradients in generating the signals necessary for phagocytosis.     

Calcium and the liver

The concentration of cytosolic free calcium ([Ca2+]i) in living cells is far below than that of plasma and must be extruded against a large electrochemical gradient. Cytosolic Ca2+ is also concentrated by two organelles, the high-affinity, low-capacity endoplasmic reticulum and the low-affinity, high-capacity mitochondria. In liver, different hormones and neurotransmitters mobilize cell glucose by using Ca2+ as an amplifier signal. These agonists promote a fast and large increase in [Ca2+]i as detected by intracellular fluorescent indicators resulting from permeabilization of the endoplasmic reticulum and stimulation of the Ca2+ influx. The hormone action is mediated by rapid hydrolysis of inositol lipids with formation of two messagers inositol [1,4,5]triphosphate and diacylglycerol. Certain bile acids also promote a pronounced release of Ca2+ from the endoplasmic reticulum in liver but independently of the hormone messengers. Mobilization of cell Ca2+ and/or H+ by these cell messengers is involved in a wide variety of other cell functions.     

pH homeostasis in pituitary GH4C1 cells: basal intracellular pH is regulated by cytosolic free Ca2+ concentration.

In GH4C1 cells, membrane depolarization induces a rapid and sustained increase in the cytosolic free calcium concentration ([Ca2+]i). In the present study we have investigated the role of [Ca2+]i in the regulation of basal intracellular pH (pHi). Depolarizing GH4C1 cells in buffer containing 0.4 mM extracellular Ca2+ decreased basal pHi from 7.02 +/- 0.04 to 6.85 +/- 0.03 (P less than 0.05). If the depolarization-induced influx of Ca2+ was inhibited by chelating extracellular Ca2+ or blocking influx through voltage-operated Ca2+ channels with nimodipine, no acidification was observed. Addition of TRH induced a rapid activation of Na+/H+ exchange in acidified cells, increasing pHi by 0.14 +/- 0.03 U. The action of TRH was blunted if extracellular Ca2+ was chelated; however, if influx of Ca2+ via voltage-operated channels was blocked by nimodipine, TRH still increased pHi. To deplete ATP, we incubated cells with 2-deoxy-D-glucose for 15-20 min and observed a decrease in basal pHi to 6.75 +/- 0.03 (P less than 0.05). No additional acidification was obtained when 2-deoxy-D-glucose-treated cells were depolarized, and no TRH-induced activation of Na+/H+ exchange was observed. Addition of ionomycin or 12-O-tetradecanoyl-phorbol-13-acetate separately to acidified cells had only modest effects on pHi; however, addition of 12-O-tetradecanoyl-phorbol-13-acetate and ionomycin together increased pHi markedly. We conclude that in GH4C1 cells, increasing [Ca2+]i reduces basal pHi through a mechanism dependent on influx of extracellular Ca2+ and independent of Na+/H+ exchange. In addition, elevation of [Ca2+]i and activation of protein kinase C act synergistically to enhance Na+/H+ exchange and increase pHi in acidified cells. Finally, normal cellular ATP is necessary for the activation of Na+/H+ exchange.     

Subcellular imaging of intramitochondrial Ca2+ with recombinant targeted aequorin: significance for the regulation of pyruvate dehydrogenase activity.

Specific targeting of the recombinant, Ca2+ -sensitive photoprotein, aequorin to intracellular organelles has provided new insights into the mechanisms of intracellular Ca2+ homeostasis. When applied to small mammalian cells, a major limitation of this technique has been the need to average the signal over a large number of cells. This prevents the identification of inter- or intracellular heterogeneities. Here we describe the imaging in single mammalian cells (CHO.T) of [Ca2+] with recombinant chimeric aequorin targeted to mitochondria. This was achieved by optimizing expression of the protein through intranuclear injection of cDNA and through the use of a charge-coupled device camera fitted with a dual microchannel plate intensifier. This approach allows accurate quantitation of the kinetics and extent of the large changes in mitochondrial matrix [Ca2+] ([Ca2+](m)) that follow receptor stimulation and reveal different behaviors of mitochondrial populations within individual cells. The technique is compared with measurements of [Ca2+](m) using the fluorescent indicator, rhod2. Comparison of [Ca2+](m) with the activity of the Ca2+ -sensitive matrix enzyme, pyruvate dehydrogenase (PDH), reveals that this enzyme is a target of the matrix [Ca2+] changes. Peak [Ca2+](m) values following receptor stimulation are in excess of those necessary for full activation of PDH in situ, but may be necessary for the activation of other mitochondrial dehydrogenases. Finally, the data suggest that the complex regulation of PDH activity by a phosphorylation-dephosphorylation cycle may provide a means by which changes in the frequency of cytosolic (and hence mitochondrial) [Ca2+] oscillations can be decoded by mitochondria.     

Glucose controls cytosolic Ca2+ and insulin secretion in mouse islets lacking adenosine triphosphate-sensitive K+ channels owing to a knockout of the pore-forming subunit Kir6.2

Glucose-induced insulin secretion is classically attributed to the cooperation of an ATP-sensitive potassium (K ATP) channel-dependent Ca2+ influx with a subsequent increase of the cytosolic free Ca2+ concentration ([Ca2+]c) (triggering pathway) and a K ATP channel-independent augmentation of secretion without further increase of [Ca2+]c (amplifying pathway). Here, we characterized the effects of glucose in beta-cells lacking K ATP channels because of a knockout (KO) of the pore-forming subunit Kir6.2. Islets from 1-yr and 2-wk-old Kir6.2KO mice were used freshly after isolation and after 18 h culture to measure glucose effects on [Ca2+]c and insulin secretion. Kir6.2KO islets were insensitive to diazoxide and tolbutamide. In fresh adult Kir6.2KO islets, basal [Ca2+]c and insulin secretion were marginally elevated, and high glucose increased [Ca2+]c only transiently, so that the secretory response was minimal (10% of controls) despite a functioning amplifying pathway (evidenced in 30 mm KCl). Culture in 10 mm glucose increased basal secretion and considerably improved glucose-induced insulin secretion (200% of controls), unexpectedly because of an increase in [Ca2+]c with modulation of [Ca2+]c oscillations. Similar results were obtained in 2-wk-old Kir6.2KO islets. Under selected conditions, high glucose evoked biphasic increases in [Ca2+]c and insulin secretion, by inducing K ATP channel-independent depolarization and Ca2+ influx via voltage-dependent Ca2+ channels. In conclusion, Kir6.2KO beta-cells down-regulate insulin secretion by maintaining low [Ca2+]c, but culture reveals a glucose-responsive phenotype mainly by increasing [Ca2+]c. The results support models implicating a K ATP channel-independent amplifying pathway in glucose-induced insulin secretion, and show that K ATP channels are not the only possible transducers of metabolic effects on the triggering Ca2+ signal.