Intracellular calcium ion response to glucose in beta-cells of calbindin-D28k nullmutant mice and in betaHC13 cells overexpressing calbindin-D28k

This article describes studies on the glucose-induced responses of intracellular Ca²⁺ concentration ([Ca²⁺]_i), insulin release, and redistribution of calbindin-D_28k, a calcium-binding regulatory protein, in β-cells of pancreatic islets of calbindin-D_28k knockout (KO) and wild-type mice (C57BL6) as well as in βHC-13 control cells and βHC-13 CaBP40 cells (β-cell line overexpressing calbindin-D_28k). Upon increasing the glucose concentration from 2.8 to 30 mM, islets of KO mice showed a significantly greater increase in [Ca²⁺]_i (mean increase in [Ca²⁺]_i, i.e., Δ[Ca²⁺], was 296 nM) compared with wild-type mice (Δ[Ca²⁺]_i=97 nM). βHC-13 CaBP40 cells showed little change in [Ca²⁺]_i upon elevation of glucose from 5.5 to 32.7 mM, whereas βHC-13 control cells exhibited significant increases in [Ca²⁺]_i (Δ[Ca²⁺]_i=510 nM). Similarly, upon addition of 30 mM glucose, the rate of insulin release increased from 25.2 (basal rate) to 145.2 pg/mL/min in βHC-13 control cells, whereas in βHC-13 CaBP40 cells the rate of insulin release was only 27.5 pg/mL/min in high glucose. Thus, levels of calbindin-D_28k in β-cells affect both [Ca²⁺]_i and insulin secretion in response to glucose. The three-dimensional reconstruct of confocal immunofluorescent images showed that glucose caused redistribution of calbindin-D_28k resulting in co-localization in the region of L-type voltage-dependent calcium channels (VDCC). This colocalization may be an important regulatory function concerning Ca²⁺ influx via L-type VDCC and exocytosis of insulin granules.     

Calcium binding kinetics of troponin C strongly modulate cooperative activation and tension kinetics in cardiac muscle

Tension development and relaxation in cardiac muscle are regulated at the thin filament via Ca²⁺ binding to cardiac troponin C (cTnC) and strong cross-bridge binding. However, the influence of cTnC Ca²⁺-binding properties on these processes in the organized structure of cardiac sarcomeres is not well-understood and likely differs from skeletal muscle. To study this we generated single amino acid variants of cTnC with altered Ca²⁺ dissociation rates (k_off), as measured in whole troponin (cTn) complex by stopped-flow spectroscopy (I61Q cTn > WT cTn > L48Q cTn), and exchanged them into cardiac myofibrils and demembranated trabeculae. In myofibrils at saturating Ca²⁺, L48Q cTnC did not affect maximum tension (T_max), thin filament activation (k_ACT) and tension development (k_TR) rates, or the rates of relaxation, but increased duration of slow phase relaxation. In contrast, I61Q cTnC reduced T_max, k_ACT and k_TR by 40-65% with little change in relaxation. Interestingly, k_ACT was less than k_TR with I61Q cTnC, and this difference increased with addition of inorganic phosphate, suggesting that reduced cTnC Ca²⁺-affinity can limit thin filament activation kinetics. Trabeculae exchanged with I61Q cTn had reduced T_max, Ca²⁺ sensitivity of tension (pCa₅₀), and slope (n_H) of tension-pCa, while L48Q cTn increased pCa₅₀ and reduced n_H. Increased cross-bridge cycling with 2-deoxy-ATP increased pCa₅₀ with WT or L48Q cTn, but not I61Q cTn. We discuss the implications of these results for understanding the role of cTn Ca²⁺-binding properties on the magnitude and rate of tension development and relaxation in cardiac muscle.     

Effects of a high-sodium diet on renal tubule Ca<Superscript>2+</Superscript>transporter and claudin expression in Wistar-Kyoto rats

Background

Urinary Ca²⁺ excretion increases with dietary NaCl. NaCl-induced calciuria may be associated with hypertension, urinary stone formation and osteoporosis, but its mechanism and long-term effects are not fully understood. This study examined alterations in the expressions of renal Ca²⁺ transporters, channels and claudins upon salt loading to better understand the mechanism of salt-induced urinary Ca²⁺ loss.

Methods

Eight-week old Wistar-Kyoto rats were fed either 0.3% or 8% NaCl diet for 8 weeks. Renal cortical expressions of Na⁺/Ca²⁺ exchanger 1 (NCX1), Ca²⁺ pump (PCMA1b), Ca²⁺ channel (TRPV5), calbindin-D_28k, and claudins (CLDN-2, -7, -8, -16 and ?19) were analyzed by quantitative PCR, western blot and/or immunohistochemistry.

Results

Fractional excretion of Ca²⁺ increased 6.0 fold with high-salt diet. Renal cortical claudin-2 protein decreased by approximately 20% with decreased immunological staining on tissue sections. Claudin-16 and ?19 expressions were not altered. Renal cortical TRPV5, calbindin-D_28k and NCX1 expressions increased 1.6, 1.5 and 1.2 fold, respectively.

Conclusions

Chronic high-salt diet decreased claudin-2 protein and increased renal TRPV5, calbindin-D_28k, and NCX1. Salt loading is known to reduce the proximal tubular reabsorption of both Na⁺ and Ca²⁺. The reduction in claudin-2 protein expression may be partly responsible for the reduced Ca²⁺ reabsorption in this segment. The concerted upregulation of more distal Ca²⁺-transporting molecules may be a physiological response to curtail the loss of Ca²⁺, although the magnitude of compensation does not seem adequate to bring the urinary Ca²⁺ excretion down to that of the normal-diet group.

     

Intracellular Ca- and PKC-dependent upregulation of T-type Ca channels in LPC-stimulated cardiomyocytes

Lysophosphatidylcholine (LPC) accumulation in intracellular and/or interstitial space in cardiomyocytes may underlie as a mechanism for tachycardia and various arrhythmias during cardiac ischemia, which is usually accompanied by elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). The present study was therefore designed to investigate possible mechanisms responsible for [Ca²⁺]_i elevation by LPC focusing on T-type Ca²⁺ channel current (I_Ca.T). LPC as well as phorbol 12-myristate 13-acetate (PMA) significantly accelerated the beating rates of neonatal rat cardiomyocytes. Augmentation of I_Ca.T by LPC was dependent on the intracellular Ca²⁺ concentration: an increase of I_Ca.T was significantly larger in high [Ca²⁺]_i condition (pCa = 7) than those in low [Ca²⁺]_i condition (pCa = 11). In heterologous expression system by use of human cardiac Ca_V3.1 and Ca_V3.2 channels expressed in HEK293 cells, LPC augmented Ca_V3.2 channel current (I_Cav3.2) in a concentration-dependent manner but not Ca_V3.1 channel current (I_Cav3.1). Augmentation of I_Cav3.2 by LPC was highly [Ca²⁺]_i dependent: I_Cav3.2 was unchanged when pCa was 11 but was markedly increased when [Ca²⁺]_i was higher than 10⁻¹⁰ M (pCa ≤ 10) by LPC application (10-50 μM). A specific inhibitor of protein kinase Cα (Ro-32-0432) attenuated the increase of I_Cav3.2 by LPC. LPC stimulates I_Ca.T in a [Ca²⁺]_i-dependent manner via PKCα activation, which may play a role in triggering arrhythmias in pathophysiological conditions of the heart.     

H(2)O(2)-induced left ventricular dysfunction in isolated working rat hearts is independent of calcium accumulation

Reactive oxygen species (ROS) and intracellular Ca²⁺ overload play key roles in myocardial ischemia-reperfusion (IR) injury but the relationships among ROS, Ca²⁺ overload and LV mechanical dysfunction remain unclear. We tested the hypothesis that H₂O₂ impairs LV function by causing Ca²⁺ overload by increasing late sodium current (I_Na), similar to Sea Anemone Toxin II (ATX-II). Diastolic and systolic Ca²⁺ concentrations (d[Ca²⁺]_i and s[Ca²⁺]_i) were measured by indo-1 fluorescence simultaneously with LV work in isolated working rat hearts. H₂O₂ (100 μM, 30 min) increased d[Ca²⁺]_i and s[Ca²⁺]_i. LV work increased transiently then declined to 32% of baseline before recovering to 70%. ATX-II (12 nM, 30 min) caused greater increases in d[Ca²⁺]_i and s[Ca²⁺]_i. LV work increased transiently before declining gradually to 17%. Ouabain (80 μM) exerted similar effects to ATX-II. Late I_Na inhibitors, lidocaine (10 μM) or R56865 (2 μM), reduced effects of ATX-II on [Ca²⁺]_i and LV function, but did not alter effects of H₂O₂. The antioxidant, N-(2-mercaptopropionyl)glycine (MPG, 1 mM) prevented H₂O₂-induced LV dysfunction, but did not alter [Ca²⁺]_i. Paradoxically, further increases in [Ca²⁺]_i by ATX-II or ouabain, given 10 min after H₂O₂, improved function. The failure of late I_Na inhibitors to prevent H₂O₂-induced LV dysfunction, and the ability of MPG to prevent H₂O₂-induced LV dysfunction independent of changes in [Ca²⁺]_i indicate that impaired contractility is not due to Ca²⁺ overload. The ability of further increases in [Ca²⁺]_i to reverse H₂O₂-induced LV dysfunction suggests that Ca²⁺ desensitization is the predominant mechanism of ROS-induced contractile dysfunction.     

Intracellular Ca2+ signaling and store-operated Ca2+ entry are required in Drosophila neurons for flight

Neuronal Ca²⁺ signals can affect excitability and neural circuit formation. Ca²⁺ signals are modified by Ca²⁺ flux from intracellular stores as well as the extracellular milieu. However, the contribution of intracellular Ca²⁺ stores and their release to neuronal processes is poorly understood. Here, we show by neuron-specific siRNA depletion that activity of the recently identified store-operated channel encoded by dOrai and the endoplasmic reticulum Ca²⁺ store sensor encoded by dSTIM are necessary for normal flight and associated patterns of rhythmic firing of the flight motoneurons of Drosophila melanogaster. Also, dOrai overexpression in flightless mutants for the Drosophila inositol 1,4,5-trisphosphate receptor (InsP₃R) can partially compensate for their loss of flight. Ca²⁺ measurements show that Orai gain-of-function contributes to the quanta of Ca²⁺-release through mutant InsP₃Rs and elevates store-operated Ca²⁺ entry in Drosophila neurons. Our data show that replenishment of intracellular store Ca²⁺ in neurons is required for Drosophila flight.     

Theoretical investigation of action potential duration dependence on extracellular Ca in human cardiomyocytes

Reduction in [Ca²⁺]_o prolongs the AP in ventricular cardiomyocytes and the QT_c interval in patients. Although this phenomenon is relevant to arrhythmogenesis in the clinical setting, its mechanisms are counterintuitive and incompletely understood. To evaluate in silico the mechanisms of APD modulation by [Ca²⁺]_o in human cardiomyocytes. We implemented the Ten Tusscher-Noble-Noble-Panfilov model of the human ventricular myocyte and modified the formulations of the rapidly and slowly activating delayed rectifier K⁺ currents (I_Kr and I_Ks) and L-type Ca²⁺ current (I_CaL) to incorporate their known sensitivity to intra- or extracellular Ca²⁺. Simulations were run with the original and modified models at variable [Ca²⁺]_o in the clinically relevant 1 to 3 mM range. The original model responds with APD shortening to decrease in [Ca²⁺]_o, i.e. opposite to the experimental observations. Incorporation of Ca²⁺ dependency of K⁺ currents cannot reproduce the inverse relation between APD and [Ca²⁺]_o. Only when I_CaL inactivation process was modified, by enhancing its dependency on Ca²⁺, simulations predict APD prolongation at lower [Ca²⁺]_o. Although Ca²⁺-dependent I_CaL inactivation is the primary mechanism, secondary changes in electrogenic Ca²⁺ transport (by Na⁺/Ca²⁺ exchanger and plasmalemmal Ca²⁺-ATPase) contribute to the reversal of APD dependency on [Ca²⁺]_o. This theoretical investigation points to Ca²⁺-dependent inactivation of I_CaL as a mechanism primarily responsible for the dependency of APD on [Ca²⁺]_o. The modifications implemented here make the model more suitable to analyze repolarization mechanisms when Ca²⁺ levels are altered.     

Relationships between dopamine-induced changes in cytosolic free calcium concentration ([Ca2+]i) and rate of prolactin secretion

This study was undertaken to investigate the relationship between dopamine (DA) induced changes in the cytosolic calcium concentration ([Ca²⁺]_i) and the rate of prolactin secretion using GH₄ZR₇, a rat pituitary cell line, which express only one subtype of D₂ receptor. GH₄ZR₇ cells were loaded with Fluo-3, a fluorescent Ca²⁺ indicator, and then perifused with two different doses of DA (10⁻⁷ mol/L and 5×10⁻⁴ mol/L). We monitored changes in [Ca²⁺]_i and rate of prolactin release simultaneously by attaching a spectrofluorometer to a dynamic perifusion system. DA has stimulatory and inhibitory effect on prolactin secretion in GH₄ZR₇ cells; 10⁻⁷ mol/L DA slightly increased [Ca²⁺]_i and stimulated prolactin release, whereas 5×10⁻⁴ mol/L DA decreased [Ca²⁺]_i and inhibited prolactin secretion. When the cells were pretreated with pertussis toxin (PTX), 10⁻⁷ mol/L DA had no significant change in [Ca²⁺]_i while stimulating prolactin release, and 5×10⁻⁴ mol/L DA reduced [Ca²⁺]_i without having any significant effect on the rate of prolactin secretion. The results of this study demonstrate that changes in [Ca²⁺]_i do not always correlate with the rate of prolactin release from lactotrophs. The dissociation between [Ca²⁺]_i and prolactin release is somewhat expected considering the diverse role of [Ca²⁺]_i and post-[Ca²⁺]_i events, which can change the rate of prolactin release.     

Expression and roles of Cav1.3 (α1D) L-type Ca²+ channel in atrioventricular node automaticity

Atrioventricular node (AV node) is the hub where electrical input from the atria is propagated and conveyed to the ventricles. Despite its strategic position and role in governing impulse conduction between atria and ventricles, there is paucity of data regarding the contribution of specific ion channels to the function of the AV node. Here, we examined the roles of Ca_v1.3 L-type Ca²⁺ channel in AV node by taking advantage of a mouse model with null mutation of Ca_v1.3 (Ca_v1.3^−/−). Ca_v1.3 null mutant mice show evidence of AV node dysfunction with AV block, suggesting the tissue-specific function of the Ca_v1.3 channel. In keeping with this assertion, we demonstrate that Ca_v1.3 isoform is highly expressed in the isolated AV node cells. Furthermore, AV node isolated from Ca_v1.3 null mutant mice show a significant decrease in the firing frequency of spontaneous action potentials suggesting that Ca_v1.3 L-type Ca²⁺ channel plays significant roles in the automaticity of the AV node. Because of the distinct voltage-dependence of Ca_v1.2 and Ca_v1.3 Ca²⁺ channels, Ca_v1.2 alone does not suffice to maintain normal AV node function. Ca_v1.3 currents activate at more hyperpolarizing voltage compared to Ca_v1.2 currents. Consequently, Ca_v1.2 Ca²⁺ channel cannot functionally substitute for Ca_v1.3 isoform in the AV node of Ca_v1.3 null mutant mice. Thus, our study demonstrates that the distinct biophysical properties of Ca_v1.3 Ca²⁺ channel play critical roles in the firing frequency of AV node tissues.     

Activation of Connexin-43 Hemichannels Can Elevate [Ca]iand [Na]iin Rabbit Ventricular Myocytes During Metabolic Inhibition

ATP depletion due to ischemia or metabolic inhibition (MI) causes Na⁺and Ca²⁺accumulation in myocytes, which may be in part due to opening of connexin-43 hemichannels. Halothane (H) has been shown to reduce conductance of connexin-43 hemichannels and to protect the heart against ischemic injury. We therefore investigated the effect of halothane on [Ca²⁺]_iand [Na⁺]_iin myocytes during MI. Isolated rabbit left ventricular myocytes were loaded with 4μ m fluo-3 AM for 30 min, or with 5 μ m sodium green AM for 60 min at 37°C. After washing, the myocytes were exposed to: (1) Normal HEPES solution; (2) MI solution (2 m NaCN, 20 m 2-deoxy- -glucose and 0-glucose); or (3) MI+H (0.95 m , 4.7 m ) for 60 min. Propidium iodide (PI, 25 μ m) was added to all samples before data acquisition. The fluorescence intensity was measured by flow cytometry with 488 nm excitation and 530 nm emission for fluo-3 or sodium green, and 670 nm for PI. The [Ca²⁺]_iand [Na⁺]_iwere then calculated by calibration. In some experiments, the effect of 10 μ m tetrodotoxin (TTX) and 20 μ m nifedipine (NIF) were studied. Metabolic inhibition for 60 min caused a significant increase in [Ca²⁺]_iand [Na⁺]_iin myocytes when compared to controls, which was significantly reduced by halothane in a dose-dependent fashion. In the presence of TTX and NIF, halothane also significantly reduced the rise in the [Ca²⁺]_iand [Na⁺]_iin myocytes subjected to MI. 1-heptanol, another gap junction blocker, had similar effects. Thus, halothane reduced [Ca²⁺]_iand [Na⁺]_ioverload produced by MI in myocytes. This effect is not solely due to block of voltage-gated Na⁺and Ca²⁺channels, and is likely mediated by inhibiting the opening of connexin-43 hemichannels.     

Regulation of insulin secretion: a matter of phase control and amplitude modulation

The consensus model of stimulus–secretion coupling in beta cells attributes glucose-induced insulin secretion to a sequence of events involving acceleration of metabolism, closure of ATP-sensitive K⁺ channels, depolarisation, influx of Ca²⁺ and a rise in cytosolic free Ca²⁺ concentration ([Ca²⁺]_c). This triggering pathway is essential, but would not be very efficient if glucose did not also activate a metabolic amplifying pathway that does not raise [Ca²⁺]_c further but augments the action of triggering Ca²⁺ on exocytosis. This review discusses how both pathways interact to achieve temporal control and amplitude modulation of biphasic insulin secretion. First-phase insulin secretion is triggered by the rise in [Ca²⁺]_c that occurs synchronously in all beta cells of every islet in response to a sudden increase in the glucose concentration. Its time course and duration are shaped by those of the Ca²⁺ signal, and its amplitude is modulated by the magnitude of the [Ca²⁺]_c rise and, substantially, by amplifying mechanisms. During the second phase, synchronous [Ca²⁺]_c oscillations in all beta cells of an individual islet induce pulsatile insulin secretion, but these features of the signal and response are dampened in groups of intrinsically asynchronous islets. Glucose has hardly any influence on the amplitude of [Ca²⁺]_c oscillations and mainly controls the time course of triggering signal. Amplitude modulation of insulin secretion pulses largely depends on the amplifying pathway. There are more similarities than differences between the two phases of glucose-induced insulin secretion. Both are subject to the same dual, hierarchical control over time and amplitude by triggering and amplifying pathways, suggesting that the second phase is a sequence of iterations of the first phase. Adapted from the A. E. Renold Lecture 2008.     

Ischemic preconditioning and diazoxide limit mitochondrial Ca2+ overload during ischemia/reperfusion: Role of reactive oxygen species

BACKGROUND:

Generation of reactive oxygen species (ROS) is associated with cardioprotection imparted by ischemic preconditioning (IPC) and pharmacological PC (PPC). The authors have previously shown that IPC or PPC, using the mitochondrial ATP-sensitive K⁺ channel opener diazoxide (DZ), reduce mitochondrial Ca²⁺ ([Ca²⁺]_m) during ischemia and reperfusion.

OBJECTIVES:

To test the hypothesis that both IPC and PPC (using DZ) lead to reduced [Ca²⁺]_m and improved functional recovery via a ROS-dependent mechanism.

METHODS:

Intracellular Ca²⁺ ([Ca²⁺]_i) and [Ca²⁺]_m were measured in isolated perfused rat hearts loaded with the fluorescent indicator indo-1 acetoxymethyl ester. [Ca²⁺]_m was determined by quenching the cytosolic indo-1 signal using manganese before ischemia (25 min). IPC and DZ (100 μM) group hearts were studied with and without the ROS scavenger N-2-mercaptopropionyl glycine (400 μM) (2-MPG).

RESULTS:

Both IPC and DZ significantly reduced [Ca²⁺]_i and [Ca²⁺]_m on reperfusion compared with the control. Administration of 2-MPG with washout before ischemia significantly attenuated the reduction in [Ca²⁺]_m observed on reperfusion in both the IPC and DZ groups. Additionally, the myocardial functional protection imparted by IPC or DZ was lost with the administration of 2-MPG.

CONCLUSIONS:

The [Ca²⁺]_m-reducing effect of IPC and DZ was attenuated with the administration of 2-MPG, resulting in decreased myocardial functional performance and increased release of creatine kinase, a marker of cellular injury. It can be concluded that IPC and DZ impart their protective effect via a mechanism involving ROS generation before the ischemic episode.     

Hepatocyte growth factor disrupts cell contact and stimulates an increase in type 3 inositol triphosphate receptor expression, intracellular calcium levels, and apoptosis of rat ovarian surface epithelial cells

The present studies revealed that hepatocyte growth factor (HGF) disrupts cell contact, increases both type 3 IP₃ receptor and intracellular calcium ([Ca²⁺) levels and induces apoptosis of rat ovarian surface epithelial cells (ROSE-179 cells). Type 3 IP₃ receptor was only increased in cells that lost cell contact. Disrupting cell contact by depleting extracellular calcium (Ca²⁺) also resulted in an increase in [Ca²⁺]_i levels and an increase in apoptosis. These responses were prevented by the addition of 0.7 mM Ca²⁺. Actinomycin D and cyclo-heximide prevented apoptosis that resulted from Ca²⁺ removal. In situ hybridization studies revealed that type 3 IP₃ receptor was expressed at relatively low levels by ROSE-179 cells cultured with Ca²⁺ but at high levels in the absence of Ca²⁺. ROSE-179 cells cultured in Ca²⁺-free medium with type 3 IP₃ receptor antisense oligonucleotide lost cell contact but did not show an increase in either type 3 IP₃ receptor protein, [Ca²⁺]_i, or apoptosis. The nonsense oligonucleotide did not alter these responses to Ca²⁺ removal. Thus, the disruption of cell contact by either HGF or Ca²⁺ depletion increases the expression of type 3 IP₃ receptor, which causes an increase in [Ca²⁺]_i and the apoptotic death of ROSE-179 cells.     

Mitochondrial Ca2+ flux and respiratory enzyme activity decline are early events in cardiomyocyte response to H2O2

Oxidative stress is involved in mitochondrial apoptosis, and plays a critical role in ischemic heart disease and cardiac failure. Exposure of cardiomyocytes to H₂O₂ leads to oxidative stress and mitochondrial dysfunction. In this study, we investigated the temporal order of mitochondrial-related events in the neonatal rat cardiomyocyte response to H₂O₂ treatment. At times ranging from 10 to 90 min after H₂O₂ treatment, levels were determined for respiratory complexes I, II, IV and V, and citrate synthase activities, mitochondrial Ca²⁺ flux, intracellular oxidation, mitochondrial membrane potential and apoptotic progression. Complexes II and IV activity levels were significantly reduced within 20 min of H₂O₂ exposure while complexes I and V, and citrate synthase were unaffected. Mitochondrial membrane potential declined after 20 and 60 min of H₂O₂ exposure while intracellular oxidation, declining complex I activity and apoptotic progression were detectable only after 60 min. Measurement of mitochondrial Ca²⁺ ([Ca²⁺]_m) using rhodamine 2 detected an early accumulation of [Ca²⁺]_m occurring between 5 and 10 min. Pretreatment of cardiomyocytes with either ruthenium red or cyclosporin A abrogated the H₂O₂-induced decline in complexes II and IV activities, indicating that [Ca²⁺]_m flux and onset of mitochondrial permeability transition pore opening likely precede the observed early enzymatic decline. Our findings suggest that [Ca²⁺]_m flux represents an early pivotal event in H₂O₂-induced cardiomyocyte damage, preceding and presumably leading to reduced mitochondrial respiratory activity levels followed by accumulation of intracellular oxidation, mitochondrial membrane depolarization and apoptotic progression concomitant with declining complex I activity.     

Hyperpolarization induced by K+ channel openers inhibits Ca2+ influx and Ca2+ release in coronary artery

The vasodilating mechanisms of the K⁺ channel openers—cromakalim, pinacidil, nicorandil, KRN2391, and Ki4032—were examined by measurement of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) using the fura-2 method in canine or porcine coronary arterial smooth muscle. The five K⁺ channel openers all produced a reduction of [Ca²⁺]_i in 5 and 30 mM KCl physiological salt solution (PSS), the effects of which were antagonized by tetrabutylammonium (TBA) or glibenclamide, but failed to affect [Ca²⁺]_i in 45 and 90 mM MCl-PSS. Cromakalim and Ki4032 only partially inhibited the 30 mM KCl-induced contractures, whereas pinacidil, nicorandil, and KRN2391 nearly abolished contractions produced by high KCl-PSS. The increased [Ca²⁺]_i and force produced by a thromboxane A₂ analogue, U46619, were inhibited by K⁺ channel openers and verapamil. In the absence of extracellular Ca²⁺, U46619 induced a transient increase in [Ca²⁺]_i with a contraction, which is effectively inhibited by cromakalim and Ki4032. Their inhibitory effects were blocked by TBA and counteracted by 20 mM KCl-induced depolarization. Cromakalim and Ki4032 did not affect caffeine-induced Ca²⁺ release. Cromakalim reduced U46619-induced IP₃ production and TBA blocked this inhibitory effect. Thus, cromakalim and Ki4032 are more specific K⁺ channel openers than pinacidil, nicorandil, and KRN2391. The vasodilation related with a reduction of [Ca²⁺]_i produced by K⁺ channel openers is due to the hyperpolarization of the plasma membrane resulting in not only the closure of voltage-dependent Ca²⁺ channels but also inhibition of the production of IP₃ and Ca²⁺ release from intracellular stores related to stimulation of the thromboxane A₂ receptor.     

Orai-STIM–mediated Ca2+ release from secretory granules revealed by a targeted Ca2+ and pH probe

Secretory granules (SGs) sequester significant calcium. Understanding roles for this calcium and potential mechanisms of release is hampered by the difficulty of measuring SG calcium directly in living cells. We adapted the Förster resonance energy transfer-based D1-endoplasmic reticulum (ER) probe to develop a unique probe (D1-SG) to measure calcium and pH in secretory granules. It significantly localizes to SGs and reports resting free Ca²⁺ of 69 ± 15 μM and a pH of 5.8. Application of extracellular ATP to activate P2Y receptors resulted in a slow monotonic decrease in SG Ca²⁺ temporally correlated with the occurrence of store-operated calcium entry (SOCE). Further investigation revealed a unique receptor-mediated mechanism of calcium release from SGs that involves SG store-operated Orai channels activated by their regulator stromal interaction molecule 1 (STIM1) on the ER. SG Ca²⁺ release is completely antagonized by a SOCE antagonist, by switching to Ca²⁺-free medium, and by overexpression of a dominant-negative Orai1(E106A). Overexpression of the CRAC activation domain (CAD) of STIM1 resulted in a decrease of resting SG Ca²⁺ by ∼75% and completely abolished the ATP-mediated release of Ca²⁺ from SGs. Overexpression of a dominant-negative CAD construct (CAD-A376K) induced no significant changes in SG Ca²⁺. Colocalization analysis suggests that, like the plasma membrane, SG membranes also possess Orai1 channels and that during SG Ca²⁺ release, colocalization between SGs and STIM1 increases. We propose Orai channel opening on SG membranes as a potential mode of calcium release from SGs that may serve to raise local cytoplasmic calcium concentrations and aid in refilling intracellular calcium stores of the ER and exocytosis.Here, we consider the Ca²⁺ dynamics of secretory compartments and describe a unique mechanism of Ca²⁺ release from them. Cells maintain cytoplasmic Ca²⁺ at nanomolar concentrations but elevate it locally in response to many signals. Elevated cytoplasmic Ca²⁺ modulates the functions of signaling cascades and enzymes, regulating many cellular responses (1). Two major sources feed most known cytoplasmic Ca²⁺ elevations: the extracellular space and the endoplasmic reticulum (ER). Here, we are interested in an additional intracellular source of Ca²⁺.Like the ER, secretory granules (SGs) contain a lot of Ca²⁺. They bud from the trans-Golgi network, and during subsequent maturation, they acquire an intraluminal pH of 5.5–5.9 (2), electron-dense cores (3), and matrices that bind Ca²⁺ and other ions (4). Their cargo is destined for release by regulated exocytosis, often triggered by Ca²⁺ signals. Only a small fraction of SGs undergo exocytosis after a given stimulus and are then typically recovered by endocytosis (5–7). Total Ca²⁺ in SGs is remarkably high, at 30–40 mM (8, 9). The free Ca²⁺ is significantly lower, with estimates ranging from 10 to 80 μM (10–14), which is still well above that in the cytoplasm. Roles for granular Ca²⁺ remain to be understood.There is no clear picture of SG Ca²⁺ dynamics. Ca²⁺ may be captured while SG Ca²⁺ buffers transit through the secretory pathway starting with the ER, and Ca²⁺could be imported continually into mature SGs. Several mechanisms for Ca²⁺ import have been proposed, including Ca²⁺-transporting ATPases, such as “secretory pathway Ca²⁺ ATPase,” cotransport using the proton gradient across the SG, and Na⁺/Ca²⁺ exchange secondarily driven by H⁺/Na⁺ exchange (11, 15, 16). Contributing to the uncertainties in this field has been the difficulty in measuring SG Ca²⁺ directly in living cells. Low-affinity chemical Ca²⁺ probes, such as mag-fura, can measure free Ca²⁺ at granular levels and at acidic pH (10) but are limited by the difficulty in ensuring that the signals originate from the compartment of interest. Genetically targeted granular Ca²⁺ probes based on the photoprotein aequorin have been developed that have the proper Ca²⁺ affinity, pH insensitivity, and subcellular localization (11, 12), but they are consumed after releasing one photon, such that thousands of cells must be measured in suspension to have sufficient signal and imaging is precluded.Store-operated calcium entry (SOCE) is a process in which depletion of calcium stores in the ER induces calcium influx from the extracellular space (17). Stimulation of plasma membrane receptors coupled to G_q activates phospholipase C, generating inositol trisphosphate (IP₃); IP₃ releases Ca²⁺ from the ER and raises cytoplasmic Ca²⁺. Eventually, stromal interaction molecules (STIMs) in the ER membrane sense ER Ca²⁺ depletion, oligomerize, and become active. They aggregate in the ER membrane just under the plasma membrane, promoting clustering and activation of Orai1 channels on the plasma membrane, initiating SOCE in membrane patches (18–20). Local Ca²⁺ entry there raises the submembrane Ca²⁺ concentration, promoting an adaptive partial inactivation of SOCE (21) and loosening the STIM–Orai interaction (22, 23). Eventual refilling of the ER gradually reverses the activation of STIM1.We now describe the development and use of a cameleon probe for monitoring Ca²⁺ and pH in SGs. Like the aequorin-based probes, it is genetically targeted to SGs, and like other cameleons, it is based on Förster resonance energy transfer (FRET) and is usable for subcellular imaging with single cells. With this tool, we discovered a unique mechanism of Ca²⁺ release from SGs apparently involving Orai channels in the SG membrane. This mode of calcium release from SGs might elevate local Ca²⁺ concentrations and aid in the refilling of other cytoplasmic Ca²⁺ stores.     

Oscillations in cytoplasmic free calcium concentration in human pancreatic islets from subjects with normal and impaired glucose tolerance

Summary Plasma insulin levels in healthy subjects oscillate and non-insulin-dependent diabetic patients display an irregular pattern of such oscillations. Since an increase in cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) in the pancreatic beta cell is the major stimulus for insulin release, this study was undertaken to investigate the dynamics of electrical activity, [Ca²⁺]_i-changes and insulin release, in stimulated islets from subjects of varying glucose tolerance. In four patients it was possible to investigate more than one of these three parameters. Stimulation of pancreatic islets with glucose and tolbutamide sometimes resulted in the appearance of oscillations in [Ca²⁺]_i, lasting 2–3 min. Such oscillations were observed even in some islets from patients with impaired glucose tolerance. In one islet from a diabetic patient there was no response to glucose, whereas that islet displayed [Ca²⁺]_i-oscillations in response to tolbutamide, suggesting that sulphonylurea treatment can mimic the complex pattern of glucose-induced [Ca²⁺]_i-oscillations. We also, for the first time, made patch-clamp recordings of membrane currents in beta-cells in situ in the islet. Stimulation with glucose and tolbutamide resulted in depolarization and appearance of action potentials. The islet preparations responded to stimulation with a number of different secretagogues with release of insulin. The present study shows that human islets can respond to stimulation with glucose and sulphonylurea with oscillations in [Ca²⁺]_i, which is the signal probably underlying the oscillations in plasma insulin levels observed in healthy subjects. Interestingly, even subjects with impaired glucose tolerance had islets that responded with oscillations in [Ca²⁺]_i upon glucose stimulation, although it is not known to what extent the response of these islets was representative of most islets in these patients.Abbreviations [Ca²⁺]_i Cytoplasmic free Ca²⁺ - NIDDM non-insulin-dependent diabetes mellitus - DMSO dimethylsulphoxide - PC pancreatic cancer     

Diastolic transient inward current in long QT syndrome type 3 is caused by Ca overload and inhibited by ranolazine

Long QT syndrome variant 3 (LQT-3) is a channelopathy in which mutations in SCN5A, the gene coding for the primary heart Na⁺ channel alpha subunit, disrupt inactivation to elevate the risk of mutation carriers for arrhythmias that are thought to be calcium (Ca²⁺)-dependent. Spontaneous arrhythmogenic diastolic activity has been reported in myocytes isolated from mice harboring the well-characterized ΔKPQ LQT-3 mutation but the link to altered Ca²⁺ cycling related to mutant Na⁺ channel activity has not previously been demonstrated. Here we have investigated the relationship between elevated sarcoplasmic reticulum (SR) Ca²⁺ load and induction of spontaneous diastolic inward current (I_TI) in myocytes expressing ΔKPQ Na⁺ channels, and tested the sensitivity of both to the antianginal compound ranolazine. We combined whole-cell patch clamp measurements, imaging of intracellular Ca²⁺, and measurement of SR Ca²⁺ content using a caffeine dump methodology. We compared the Ca²⁺ content of ΔKPQ^+/− myocytes displaying I_TI to those without spontaneous diastolic activity and found that I_TI induction correlates with higher sarcoplasmic reticulum (SR) Ca²⁺. Both spontaneous diastolic I_TI and underlying Ca²⁺ waves are inhibited by ranolazine at concentrations that preferentially target I_NaL during prolonged depolarization. Furthermore, ranolazine I_TI inhibition is accompanied by a small but significant decrease in SR Ca²⁺ content. Our results provide the first direct evidence that induction of diastolic transient inward current (I_TI) in ΔKPQ^+/− myocytes occurs under conditions of elevated SR Ca²⁺ load.     

Effects of nitric oxide donors on vascular smooth muscles depend on a type of vascular smooth-muscle preactivation

The abilities of such therapeutic nitrovasodilators as sodium nitroprusside (SNP) and glyceryl trinitrate (GTN) to dilate vascular smooth muscles (VSM) and affect intracellular calcium concentration level ([Ca²⁺]_i) in a rat tail artery were tested under different types of preactivation. To shed light on mechanisms underlying possible differences in the action of these two nitric oxide (NO) donors, simultaneous measurements of [Ca²⁺]_i and contractile force were done. All vascular rings were precontracted either using a high-K⁺-Krebs solution or phenylephrine (PE). It was shown that the effect of both NO donors strongly depended on a type of VSM preactivation. The EC₅₀ for GTN under K⁺ stimulation of VSM comprised (2.48±1.6)×10⁻⁵ M, whereas the mean EC₅₀ under PE stimulation was (3.05±2.3)×10⁻⁴ M (p<0.05, n=9). The EC₅₀ for SNP under K⁺ stimulation of VSM comprised (1.09±0.47)×10⁻⁷ M, whereas the EC₅₀ under PE stimulation was (8.01±2.4)×10⁻⁶ M (p<0.05, n=9). GTN demonstrated a significant discrepancy in the magnitude of changes in [Ca²⁺]_i and related VSM relaxant responses, indicating the ability of GTN to relax VSM in the absence of a proportional decrease in [Ca²⁺]_i. The main peculiarity of SNP action under K⁺ stimulation as compared to PE stimulation was the transient decrease in [Ca²⁺]_i while relaxation was sustained. Therefore, both NO donors demonstrated their ability to produce vasorelaxation as a result of an alteration in myofilament calcium sensitivity. These data clearly indicate that the sensitivity of VSM to NO donors is higher under K⁺ depolarization than that seen under PE stimulation, indicating that Ca²⁺ entry through voltage-operated calcium channels is more sensitive to NO as compared to calcium mobilization by means of Ca²⁺ entry through receptor-operated calcium channels or intracellular Ca²⁺ release, or both.