T-channels and Na<Superscript>+</Superscript>,Ca<Superscript>2+</Superscript>-exchangers as components of the Ca<Superscript>2+</Superscript>-system of regulation of activity of the heart myocardium of the frog <Emphasis Type="Italic">Rana temporaria</Emphasis>

Earlier we have shown that regulation of rhythm and strength of the frog heart contractions, mediated by transmitters of the autonomic nervous system, is of the Ca²⁺-dependent character. In the present work, we studied chronoand inotropic effect of verapamil—an inhibitor of Ca²⁺-channels of the L-type, of nickel chloride-an inhibitor of Ca²⁺—channels of the T-type and of Na⁺,Ca²⁺exchangers as well as of adrenaline and acetylcholine (ACh) after nickel chloride. It has been found that the intracardially administered NiCh₂ at a dose of 0.01 μg/kg produced a sharp fall of amplitude of action potential (AP) and an almost twofold deceleration of heart rate (HR). The intracardiac administration of NiCh₂ (0.01 μg/kg) on the background of action of verapamil (6 mg/kg, i/m) led as soon as after 3 min to even more prominent HR deceleration and to further fall of the AP amplitude by more than 50% as compared with norm. An intracardiac administration of adrenaline (0.5 mg/kg) partly restored the cardiac activity. However, preservation of the myocardium electrical activity in such animals was brief and its duration did not exceed several minutes. Administration of Ni²⁺ on the background of acetylcholine (3.6 mg/kg) led to almost complete cessation of cardiac activity. As soon as 3 min after injection of this agent the HR decreased to 2 contractions/min. On electrograms (EG), the 10-fold fall of the AP amplitude was recorded. To elucidate role of extraand intracellular Ca²⁺ in regulation of strength of heart contractions, isometric contraction of myocardium preparations was studied in response to action of NiCl₂ (10–200 μM), verapamil (70 μM), adrenaline (5 μM), and acetylcholine (0.2 μM) after NiCl₂. It has been found that Ni²⁺ causes a dose-dependent increase of the muscle contraction amplitude. Minimal change of the contraction amplitude (on average, by 14.9% as compared with control) was recorded at a Ni²⁺ concentration of 100 μM. An increase of Ni²⁺ in the sample to 200 μM increased the cardiac contraction strength, on average, by 41%. The negative inotropic action of verapamil was essentially reduced by 100 μM Ni²⁺. Adrenaline added to the sample after Ni²⁺ produced stimulating effect on the cardiac muscle, with an almost twofold rise of the contraction amplitude. ACh (0.2 μM) decreased the cardiac contraction amplitude, on average, by 56.3%, whereas Ni²⁺ (200 μM) administered after ACh not only restored, but also stimulated partly the myocardial work. Within several parts of percent there was an increase of such isometric contraction parameters as amplitude of the effort developed by muscle, maximal rate, maximal acceleration, time of semirise and semifall. The obtained experimental results indicate that the functional activity of the frog pacemaker and contractile cardiomyocytes is regulated by Ca²⁺-dependent mechanisms. Structure of these mechanisms includes the potential-controlled Land T-channels of the plasma membrane as well as Na⁺,Ca²-exchangers characteristic exclusively of contractile cardiomyocytes. The existence of these differences seems to be due to the cardiomyocyte morphological peculiarities that appeared in evolution at the stage of the functional cell specialization.     

Regulation of the RyR channel gating by Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript>

Ryanodine receptors (RyRs) are the Ca²⁺ release channels in the sarcoplasmic reticulum in striated muscle which play an important role in excitation-contraction coupling and cardiac pacemaking. Single channel recordings have revealed a wealth of information about ligand regulation of RyRs from mammalian skeletal and cardiac muscle (RyR1 and RyR2, respectively). RyR subunit has a Ca²⁺ activation site located in the luminal and cytoplasmic domains of the RyR. These sites synergistically feed into a common gating mechanism for channel activation by luminal and cytoplasmic Ca²⁺. RyRs also possess two inhibitory sites in their cytoplasmic domains with Ca²⁺ affinities of the order of 1 μM and 1 mM. Magnesium competes with Ca²⁺ at these sites to inhibit RyRs and this plays an important role in modulating their Ca²⁺-dependent activity in muscle. This review focuses on how these sites lead to RyR modulation by Ca²⁺ and Mg²⁺ and how these mechanisms control Ca²⁺ release in excitation-contraction coupling and cardiac pacemaking.     

Comparative study of Y<Superscript>3+</Superscript> effect on calcium-dependent processes in frog cardiac muscle and mitochondria of rat cardiomyocytes

Inotropic effects of yttrium acetate (Y³⁺) on contractions of myocardium preparations of the frog Rana ridibunda, as well as on respiration and the inner membrane potential (ΔΨ_mito) of isolated rat heart mitochondria were studied. 2 mM yttrium in Ringer solution was found to significantly reduce the amplitude of myocardium contractions, evoked by electric stimulation, and increase the half-relaxation time (n = 5). In experiments with Ca²⁺, Y³⁺ decreased the Ca²⁺-dependent basal respiration rate in rat heart mitochondria, energized by glutamate and malate, impeded the reduction in respiration of these mitochondria operating in state 3 after Chance or uncoupled by 2,4-dinitrophenol, and inhibited a Ca²⁺-induced reduction in their inner membrane potential. The data obtained are important for better understanding the mechanism underlying Y³⁺ effects on the myocardial Ca²⁺-dependent processes. Possible mechanisms of the negative inotropic effect of Y³⁺ on myocardium and its influence on the Ca²⁺-dependent processes in rat mitochondria are discussed.     

Peculiarities of Ca<Superscript>2+</Superscript>-regulation of functional activity of myocardium of frog <Emphasis Type="Italic">Rana temporaria</Emphasis>

To elucidate role of intra- and extracellular Ca²⁺ in regulation of rhythm and strength of frog heart contractions, there were studied ECC and isometric contraction of myocardium preparations in response to verapamil, adrenaline, and blockers of α- and β-adrenoreceptors. It has been shown that after an intramuscular injection of verapamil (6 mg/kg), bradycardia develops, the heart rate (HR) decreasing by 50–70%. Further, the cardiac arrest occurred; however, administration to the animals of adrenaline (100 mg/kg) restored the cardiac rhythm for a short while. After an intramuscular injection of adrenaline at doses of 0.1–10 mg/kg, no essential changes were observed in the potential action amplitude and HR; an increase of the administered adrenalin concentration to 100 mg/kg was not accompanied by the cardiac rhythm stimulation, as this takes place in homoiothermal animals and human; on the contrary, an essential HR deceleration was revealed. Phentolamine (5 mg/kg) gradually decelerated HR rhythm by 32–45%. The potential amplitude changed insignificantly. A subsequent intracardiac injection of adrenaline (100 mg/kg) on the background of block of α-adrenoreceptors produced acceleration of the rhythm (by 15–21%) and fall of the electrogram amplitude. These results can indicate that in the frog heart phentolamine interacts predominantly with α 1-adrenoreceptors. An intracardial administration of propranolol (1 mg/kg) to frogs promoted inhibition of β-adrenergic receptors and produced a gradual cardiac rhythm deceleration. In experiments on assessment of verapamil effect on the character of contractions this preparation at a concentration of 150 μM was established to produce a significant dose-dependent decrease of the contraction strength. A rise of verapamil concentration in the sample to 200 μM led to a decrease of the amplitude, on average, by 68–70% and in individual preparations—by 80–85%; however, administration into the sample of adrenaline (10 μM) restored the cardiac contraction strength. Adrenaline (1 nM–100 μM) increased markedly the contraction amplitude. Phentolamine (10 μM) did not inhibit transmission of contractile signal to cardiomyocytes; this was manifested in that the contraction amplitude after addition of adrenaline (10 μM) into the sample was approximately the same as in the sample containing no phentolamine. Propranolol (10 μM) eliminated the stimulatory action of adrenaline (10 μM). The results of these experiments indicate that in the frog ventricular cardiomyocytes the main adrenaline acceptors are β-adrenoreceptors.     

Ca<Superscript>2+</Superscript>-Dependent Phosphorylation of RyR2 Can Uncouple Channel Gating from Direct Cytosolic Ca<Superscript>2+</Superscript> Regulation

Phosphorylation of the cardiac ryanodine receptor (RyR2) is thought to be important not only for normal cardiac excitation-contraction coupling but also in exacerbating abnormalities in Ca²⁺ homeostasis in heart failure. Linking phosphorylation to specific changes in the single-channel function of RyR2 has proved very difficult, yielding much controversy within the field. We therefore investigated the mechanistic changes that take place at the single-channel level after phosphorylating RyR2 and, in particular, the idea that PKA-dependent phosphorylation increases RyR2 sensitivity to cytosolic Ca²⁺. We show that hyperphosphorylation by exogenous PKA increases open probability (P _o) but, crucially, RyR2 becomes uncoupled from the influence of cytosolic Ca²⁺; lowering [Ca²⁺] to subactivating levels no longer closes the channels. Phosphatase (PP1) treatment reverses these gating changes, returning the channels to a Ca²⁺-sensitive mode of gating. We additionally found that cytosolic incubation with Mg²⁺/ATP in the absence of exogenously added kinase could phosphorylate RyR2 in approximately 50% of channels, thereby indicating that an endogenous kinase incorporates into the bilayer together with RyR2. Channels activated by the endogenous kinase exhibited identical changes in gating behavior to those activated by exogenous PKA, including uncoupling from the influence of cytosolic Ca²⁺. We show that the endogenous kinase is both Ca²⁺-dependent and sensitive to inhibitors of PKC. Moreover, the Ca²⁺-dependent, endogenous kinase–induced changes in RyR2 gating do not appear to be related to phosphorylation of serine-2809. Further work is required to investigate the identity and physiological role of this Ca²⁺-dependent endogenous kinase that can uncouple RyR2 gating from direct cytosolic Ca²⁺ regulation.     

A comparative study of the effects of Pr<Superscript>3+</Superscript> and La<Superscript>3+</Superscript> ions on calcium dependent processes in frog cardiac muscle and rat heart mitochondria

The inotropic effect of Pr³⁺ and La³⁺ ions on the heart muscle of frog Rana ridibunda, as well as the influence of the ions on respiration, swelling, and the potential (ΔΨ_mito) on the inner membrane of Ca²⁺- loaded rat heart mitochondria, energized by glutamate and malate or succinate in the presence of rotenone were studied. It was found that 2 mM Pr³⁺ in Ringer’s solution reduces the force of spontaneous contractions and those induced by electrical stimulation in the heart; it had a negative chronotropic effect, decreasing the frequency of spontaneous contractions. Pr³⁺ and La³⁺ prevented a decrease in the 2,4-dinitrophenol (DNP)- uncoupled respiration of energized rat heart mitochondria, swelling of these organelles in salt media, and a reduction in ΔΨ_mito on the inner mitochondrial membrane that were induced by Ca²⁺ ions. Retardation by Pr³⁺ and La³⁺ ions of these calcium-induced effects may suggest that in the inner mitochondrial membrane these metals inhibit the opening of the mitochondrial permeability transition pore caused by Ca²⁺ overload of mitochondria. The data we obtained are important for a better understanding of the mechanisms of the damaging action of rare-earth elements on Ca²⁺-dependent processes in the vertebrate myocardium.     

Nicotine-induced Ca<Superscript>2+</Superscript>-myristoyl Switch of Neuronal Ca<Superscript>2+</Superscript> Sensor VILIP-1 in Hippocampal Neurons: A Possible Crosstalk Mechanism for Nicotinic Receptors

Visinin-like protein (VILIP-1) belongs to the neuronal Ca²⁺ sensor family of EF-hand Ca²⁺-binding proteins that regulate a variety of Ca²⁺-dependent signal transduction processes in neurons. It is an interaction partner of α4β2 nicotinic acetylcholine receptor (nAChR) and increases surface expression level and agonist sensitivity of the receptor in oocytes. Nicotine stimulation of nicotinic receptors has been reported to lead to an increase in intracellular Ca²⁺ concentration by Ca²⁺-permeable nAChRs, which in turn might lead to activation of VILIP-1, by a mechanism described as the Ca²⁺-myristoyl switch. It has been postulated that this will lead to co-localization of the proteins at cell membranes, where VILIP-1 can influence functional activity of α4-containing nAChRs. In order to test this hypothesis we have investigated whether a nicotine-induced and reversible Ca²⁺-myristoyl switch of VILIP-1 exists in primary hippocampal neurons and whether pharmacological agents, such as antagonist specific for distinct nAChRs, can interfere with the Ca²⁺-dependent membrane localization of VILIP-1. Here we report, that only α7- but not α4-containing nAChRs are able to elicit a Ca²⁺-dependent and reversible membrane-translocation of VILIP-1 in interneurons as revealed by employing the specific receptor antagonists dihydro-beta-erythroidine and methylallylaconitine. The nAChRs are associated with processes of synaptic plasticity in hippocampal neurons and they have been implicated in the pathology of CNS disorders, including Alzheimer’s disease and schizophrenia. VILIP-1 might provide a novel functional crosstalk between α4- and α7-containing nAChRs.     

Evolution of mechanisms of Ca<Superscript>2+</Superscript>-signaling. Role of Ca<Superscript>2+</Superscript> in regulation of fundamental cell functions

The review considers mechanisms of Ca²⁺-dependent regulation of cell growth, differentiation, and apoptosis in cells of the higher eukaryotes by modulation of the signal Ras-MAPK pathway.     

Modulation of the reaction cycle of the Na<Superscript>+</Superscript>:Ca<Superscript>2+</Superscript>, K<Superscript>+</Superscript> exchanger

Ca²⁺ concentration in retinal photoreceptor rod outer segment (OS) strongly affects the generator potential kinetics and the receptor light adaptation. The response to intense light stimuli delivered in the dark produce potential changes exceeding 40 mV: since the Ca²⁺ extrusion in the OS is entirely controlled by the Na⁺:Ca²⁺, K⁺ exchanger, it is important to assess how the exchanger ion transport rate is affected by the voltage and, in general, by intracellular factors. It is indeed known that the cardiac Na⁺:Ca²⁺ exchanger is regulated by Mg-ATP via a still unknown metabolic pathway. In the present work, the Na⁺:Ca²⁺, K⁺ exchanger regulation was investigated in isolated OS, recorded in whole-cell configuration, using ionic conditions that activated maximally the exchanger in both forward and reverse mode. In all species examined (amphibia: Rana esculenta and Ambystoma mexicanum; reptilia: Gecko gecko), the forward (reverse) exchange current increased about linearly for negative (positive) voltages and exhibited outward (inward) rectification for positive (negative) voltages. Since hyperpolarisation increases Ca²⁺ extrusion rate, the recovery of the dark level of Ca²⁺ (and, in turn, of the generator potential) after intense light stimuli results accelerated. Mg-ATP increased the size of forward and reverse exchange current by a factor of ∼2.3 and ∼2.6, respectively, without modifying their voltage dependence. This indicates that Mg-ATP regulates the number of active exchanger sites and/or the exchanger turnover number, although via an unknown mechanism. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.     

Muscarinic acetylcholine receptor compounds alter net Ca<Superscript>2+</Superscript> flux and contractility in an invertebrate smooth muscle

Responses of a holothurian smooth muscle to a range of muscarinic (M₁ to M₅) acetylcholine receptor (mAChR) agonists and antagonists were surveyed using calcium (Ca²⁺)-selective electrodes and a mechanical recording technique. Most of the mAChR agonists and antagonists tested increased both contractility and net Ca²⁺ efflux, with M₁-specific agents like oxotremorine M being the most potent in their action. To investigate the possible sources of Ca²⁺ used during mAChR activation, agents that disrupt intracellular Ca²⁺ ion sequestration [cyclopiazonic acid (CPA), caffeine, ryanodine], the phosphoinositide signaling pathway [lithium chloride (LiCl)], and L-type Ca²⁺ channels (diltiazem and verapamil) were used to challenge contractions induced by oxotremorine M. These contractions were blocked by treatment with CPA, caffeine, LiCl, and by channel blockers, diltiazem and verapamil, but were unaltered by ryanodine. Our data suggest that this smooth muscle had an M_1,3,5-like receptor that was associated with the phosphoinositide signaling pathway that relied on intracellular Ca²⁺ stores, but secondarily used extracellular Ca²⁺ via the opening of L-type channels.     

The significance of extracellular Ca<Superscript>2+</Superscript> in contractile responses of chick amnion

Neurotransmitter receptors are formed during chick embryo development in the amnion, an avascular extraembryonic membrane devoid of innervation. Carbachol induces phasic and tonic contractions mediated by M₃ cholinoceptors in an amniotic membrane strip isolated from 11–14-day-old chick embryo. The carbachol effect on the amnion contractile activity was studied in normal physiological salt solution, during depolarization by K⁺, exposure to nifedipine, and in calcium-free medium. Voltage-dependent and receptor-operated Ca²⁺ channels as well as calcium from intracellular stores are involved in the contractile response to carbachol. Phasic contractions of the amnion are mainly induced by calcium ions entering through voltage-dependent calcium channels, while tonic contractions are also maintained by receptor-operated channels. Ca²⁺-activated potassium channels can serve as a negative feedback factor in regulation of the amnion contractile responses.     

Ca<Superscript>2+</Superscript> signalling in cardiovascular disease: the role of the plasma membrane calcium pumps

The plasma membrane calcium ATPases (PMCA) are a family of genes which extrude Ca²⁺ from the cell and are involved in the maintenance of intracellular free calcium levels and/or with Ca²⁺ signalling, depending on the cell type. In the cardiovascular system, Ca²⁺ is not only essential for contraction and relaxation but also has a vital role as a second messenger in signal transduction pathways. A complex array of mechanisms regulate intracellular free calcium levels in the heart and vasculature and a failure in these systems to maintain normal Ca²⁺ homeostasis has been linked to both heart failure and hypertension. This article focuses on the functions of PMCA, in particular isoform 4 (PMCA4), in the heart and vasculature and the reported links between PMCAs and contractile function, cardiac hypertrophy, cardiac rhythm and sudden cardiac death, and blood pressure control and hypertension. It is becoming clear that this family of calcium extrusion pumps have essential roles in both cardiovascular health and disease.     

Life after the birth of the mitochondrial Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchanger,NCLX

Powered by the mitochondrial membrane potential, Ca²⁺ permeates the mitochondria via a Ca²⁺ channel termed Ca²⁺ uniporter and is pumped out by a Na⁺/Ca²⁺ exchanger, both of which are located on the inner mitochondrial membrane. Mitochondrial Ca²⁺ transients are critical for metabolic activity and regulating global Ca²⁺ responses. On the other hand, failure to control mitochondrial Ca²⁺ is a hallmark of ischemic and neurodegenerative diseases. Despite their importance, identifying the uniporter and exchanger remains elusive and their inhibitors are non-specific. This review will focus on the mitochondrial exchanger, initially describing how it was molecularly identified and linked to a novel member of the Na⁺/Ca²⁺ exchanger superfamily termed NCLX. Molecular control of NCLX expression provides a selective tool to determine its physiological role in a variety of cell types. In lymphocytes, NCLX is essential for refilling the endoplasmic reticulum Ca²⁺ stores required for antigendependent signaling. Communication of NCLX with the store-operated channel in astroglia controls Ca²⁺ influx and thereby neuro-transmitter release and cell proliferation. The refilling of the Ca²⁺ stores in the sarcoplasmic reticulum, which is controlled by NCLX, determines the frequency of action potential and Ca²⁺ transients in cardiomyocytes. NCLX is emerging as a hub for integrating glucose-dependent Na⁺ and Ca²⁺ signaling in pancreatic β cells, and the specific molecular control of NCLX expression resolved the controversy regarding its role in neurons and β cells. Future studies on an NCLX knockdown mouse model and identification of human NCLX mutations are expected to determine the role of mitochondrial Ca²⁺ efflux in organ activity and whether NCLX inactivation is linked to ischemic and/or neurodegenerative syndromes. Structure-function analysis and protein analysis will identify the NCLX mode of regulation and its partners in the inner membrane of the mitochondria.     

Tachyphylaxis to the inhibitory effect of L-type channel blockers on ACh-induced [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> oscillations in porcine tracheal myocytes

Summary Discrepancies about the role of L-type voltage-gated calcium channels (VGCC) in acetylcholine (ACh)-induced [Ca²⁺]_i oscillations in tracheal smooth muscle cells (TSMCs) have been seen in recent reports. We demonstrate here that ACh-induced [Ca²⁺]_i oscillations in TMCS were reversibly inhibited by three VGCC blockers, nicardipine, nifedipine and verapamil. Prolonged (several minutes) application of VGCC blockers, led to tachyphylaxis; that is, [Ca²⁺]_i oscillations resumed, but at a lower frequency. Brief (15–30 s) removal of VGCC blockers re-sensitized [Ca²⁺]_i oscillations to inhibition by the agents. Calcium oscillations tolerant to VGCC blockers were abolished by KB-R7943, an inhibitor of the reverse mode of Na⁺/Ca²⁺ exchanger (NCX). KB-R7943 alone also abolished ACh-induced [Ca²⁺]_i oscillations. Enhancement of the reverse mode of NCX via removing extracellular Na⁺ reversed inhibition of ACh-induced [Ca²⁺]_i oscillations by VGCC blockers. Inhibition of non-selective cation channels using Gd³⁺ slightly reduced the frequency of ACh-induced [Ca²⁺]_i oscillations, but did not prevent the occurrence of tachyphylaxis. Altogether, these results suggest that VGCC and the reverse mode of NCX are two primary Ca²⁺ entry pathways for maintaining ACh-induced [Ca²⁺]_i oscillations in TSMCs. The two pathways complement each other, and may account for tachyphylaxis of ACh-induced [Ca²⁺]_i oscillations to VGCC blockers.     

Convergence of Ca<Superscript>2+</Superscript> signaling pathways in adipocytes. The role of <Emphasis Type="Italic">L</Emphasis>-arginine and protein kinase G in generation of transient and periodic Ca<Superscript>2+</Superscript> signals

Experiments on cultured mouse adipocytes (9 days in vitro) using fluorescent microscopy have shown that activation of α₁- and α₂-adrenoceptors by norepinephrine (NE) or α₂-adrenoreceptors by L-arginine evokes transient Ca²⁺ signals, while activation of m₃-cholinoreceptors by acetylcholine (ACh) or betaine causes sustained or damped Ca²⁺ oscillations. The presence in the incubation medium of L-arginine at a low concentration (100–200 μM) is necessary for a vigorous manifestation of these effects, apparently due to transition of protein kinase G (PKG) and phosphodiesterase V into an active state. In the presence of 1–10 mM L-arginine, the amplitude of the Ca²⁺ transient response to NE increases and signal duration decreases. ACh and NE upon a sequential addition mutually potentiate their effects. Using an inhibitory analysis we show that the observed modes are related to the operation of a signaling pathway with the participation of phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB), endothelial NO synthase (eNOS), cytoplasmic guanylate cyclase (sGC), protein kinase G (PKG), ADP-ribosyl cyclase (CD38), and the ryanodine receptor (RyR). The formation of several loops of positive feedbacks (PF) and negative feedbacks (NF) in the signaling system is possible: (i) short PF loops due to Ca²⁺-induced Ca²⁺ release (CICR) from internal stores through the inositol trisphosphate receptor (IP₃R) and RyR participating in the transient signal formation; (ii) long PF loop Ca²⁺ → eNOS → sGC → PKG → CD38 → RyR → Ca²⁺, which can provide necessary conditions for calcium oscillations arising from short PF loops (CICR); (iii) several NF loops based on PKG-mediated inhibition of IP₃R and activation of Ca²⁺-ATPases of sarco(endo)plasmic reticulum and of the plasma membrane providing a shutdown of signaling by the pathway phospholipase C → IP₃R → Ca²⁺ and limiting Ca²⁺ rise caused by the pathway PI3K → PKB → eNOS → sGC → PKG → CD38 → RyR → Ca²⁺. Convergence of signaling pathways that involve α₁-, α₂-, and m₃-receptors and then Gβγ-subunits of G_q and G_q proteins acting on PI3Kγ can provide activation of cytoplasmic PKG, which plays a key role in producing transient responses, in activation of Ca²⁺ removal and generation of [Ca²⁺]_i oscillations. PKG inhibition (implemented here by KT5823 application) in the presence of any agonist results in rupture of NF loops controlling Ca²⁺ transporting systems activity that leads to uncontrolled [Ca²⁺]_i rise and cell death.     

Minocycline chelates Ca<Superscript>2+</Superscript>, binds to membranes,and depolarizes mitochondria by formation of Ca<Superscript>2+</Superscript>-dependent ion channels

Minocycline (an anti-inflammatory drug approved by the FDA) has been reported to be effective in mouse models of amyotrophic lateral sclerosis and Huntington disease. It has been suggested that the beneficial effects of minocycline are related to its ability to influence mitochondrial functioning. We tested the hypothesis that minocycline directly inhibits the Ca²⁺-induced permeability transition in rat liver mitochondria. Our data show that minocycline does not directly inhibit the mitochondrial permeability transition. However, minocycline has multiple effects on mitochondrial functioning. First, this drug chelates Ca²⁺ ions. Secondly, minocycline, in a Ca²⁺-dependent manner, binds to mitochondrial membranes. Thirdly, minocycline decreases the proton-motive force by forming ion channels in the inner mitochondrial membrane. Channel formation was confirmed with two bilayer lipid membrane models. We show that minocycline, in the presence of Ca²⁺, induces selective permeability for small ions. We suggest that the beneficial action of minocycline is related to the Ca²⁺-dependent partial uncoupling of mitochondria, which indirectly prevents induction of the mitochondrial permeability transition.     

Klotho attenuates isoproterenol-induced hypertrophic response in H9C2 cells by activating Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase and inhibiting the reverse mode of Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript>-exchanger

Cardiac hypertrophy plays a major role in heart failure and is related to patient morbidity and mortality. Calcium overloading is a main risk for cardiac hypertrophy, and Na⁺/K⁺-ATPase (NKA) has been found that it could not only regulate intracellular Na⁺ levels but also control the intracellular Ca²⁺ ([Ca²⁺]_i) level through Na⁺/Ca²⁺-exchanger (NCX). Recent studies have reported that klotho could affect [Ca²⁺]_i level. In this study, we aimed at exploring the role of klotho in improving isoproterenol-induced hypertrophic response of H9C2 cells. The H9C2 cells were randomly divided into control and isoproterenol (ISO) (10 μM) groups. Klotho protein (10 μg/ml) or NKAα2 siRNA was used to determine the changes in isoproterenol-induced hypertrophic response. The alterations of [Ca²⁺]_i level were measured by spectrofluorometry. Our results showed that H9C2 cells which were treated with isoproterenol presented a higher level of [Ca²⁺]_i and hypertrophic gene expression at 24 and 48 h compared with the control group. Moreover, the expressions of NKAα1 and NKAα2 were both increased in control and ISO groups after treating with klotho protein; meanwhile, the NKA activity was increased and NCX activity was decreased after treatment. Consistently, the [Ca²⁺]_i level and hypertrophic gene expression were decreased in ISO group after klotho protein treatment. However, these effects were both prevented by transfecting with NKAα2 siRNA. In conclusion, these findings demonstrated that klotho inhibits isoproterenol-induced hypertrophic response in H9C2 cells by activating NKA and inhibiting the reverse mode of NCX and this effect may be associated with the upregulation of NKAα2 expression.     

Swelling-Induced Ca<Superscript>2+</Superscript> Influx and K<Superscript>+</Superscript> Efflux in American Alligator Erythrocytes

The American alligator can hibernate during winter, which may lead to osmotic imbalance because of reduced kidney function and lack of food consumption during this period. Accordingly, we hypothesized that their red blood cells would have a well-developed regulatory volume decrease (RVD) to cope with the homeostatic challenges associated with torpor. Osmotic fragility was determined optically, mean cell volume was measured by electronic sizing, and changes in intracellular Ca²⁺ concentration were visualized using fluorescence microscopy and fluo-4-AM. Osmotic fragility increased and the ability to regulate volume was inhibited when extracellular Na⁺ was replaced with K⁺, or when cells were exposed to the K⁺ channel inhibitor quinine, indicating a requirement of K⁺ efflux for RVD. Addition of the ionophore gramicidin to the extracellular medium decreased osmotic fragility and also potentiated volume recovery, even in the presence of quinine. In addition, hypotonic shock (0.5× Ringer) caused an increase in cytosolic Ca²⁺, which resulted from Ca²⁺ influx because it was not observed when extracellular Ca²⁺ was chelated with EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid). Furthermore, cells loaded with BAPTA-AM (1,2-bis(2-aminophenoxymethyl)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl) ester) or exposed to a low Ca²⁺-EGTA hypotonic Ringer had a greater osmotic fragility and also failed to recover from cell swelling, indicating that extracellular Ca²⁺ was needed for RVD. Gramicidin reversed the inhibitory effect of low extracellular Ca²⁺. Finally, and surprisingly, the Ca²⁺ ionophore A23187 increased osmotic fragility and inhibited volume recovery. Taken together, our results show that cell swelling activated a K⁺ permeable pathway via a Ca²⁺-dependent mechanism, and this process mediated K⁺ loss during RVD.     

A domain peptide of the cardiac ryanodine receptor regulates channel sensitivity to luminal Ca<Superscript>2+</Superscript> via cytoplasmic Ca<Superscript>2+</Superscript> sites

The clustering of cardiac RyR mutations, linked to sudden cardiac death (SCD), into several regions in the amino acid sequence underlies the hypothesis that these mutations interfere with stabilising interactions between different domains of the RyR2. SCD mutations cause increased channel sensitivity to cytoplasmic and luminal Ca²⁺. A synthetic peptide corresponding to part of the central domain (DPc10:²⁴⁶⁰G–P²⁴⁹⁵) was designed to destabilise the interaction of the N-terminal and central domains of wild-type RyR2 and mimic the effects of SCD mutations. With Ca²⁺ as the sole regulating ion, DPc10 caused increased channel activity which could be reversed by removal of the peptide whereas in the presence of ATP DPc10 caused no activation. In support of the domain destablising hypothesis, the corresponding peptide (DPc10-mut) containing the CPVT mutation R2474S did not affect channel activity under any circumstances. DPc10-induced activation was due to a small increase in RyR2 sensitivity to cytoplasmic Ca²⁺ and a large increase in the magnitude of luminal Ca²⁺ activation. The increase in the luminal Ca²⁺ response appeared reliant on the luminal-to-cytoplasmic Ca²⁺ flux in the channel, indicating that luminal Ca²⁺ was activating the RyR2 via its cytoplasmic Ca²⁺ sites. DPc10 had no significant effect on the RyR2 gating associated with luminal Ca²⁺ sensing sites. The results were fitted by the luminal-triggered Ca²⁺ feed-through model and the effects of DPc10 were explained entirely by perturbations in cytoplasmic Ca²⁺-activation mechanism.