Occurrence Of Ca2 Research Articles

Interaction of background Ca2+ influx, sarcoplasmic reticulum threshold and heart failure in determining propensity for Ca2+ waves in sheep heart.

Ventricular arrhythmias can cause death in heart failure (HF). A trigger is the occurrence of Ca2+ waves which activate a Na+‐Ca2+ exchange (NCX) current, leading to delayed after‐depolarisations and triggered action potentials. Waves arise when sarcoplasmic reticulum (SR) Ca2+ content reaches a threshold and are commonly induced experimentally by raising external Ca2+, although the mechanism by which this causes waves is unclear and was the focus of this study. Intracellular Ca2+ was measured in voltage‐clamped ventricular myocytes from both control sheep and those subjected to rapid pacing to produce HF. Threshold SR Ca2+ content was determined by applying caffeine (10 mM) following a wave and integrating wave and caffeine‐induced NCX currents. Raising external Ca2+ induced waves in a greater proportion of HF cells than control. The associated increase of SR Ca2+ content was smaller in HF due to a lower threshold. Raising external Ca2+ had no effect on total influx via the L‐type Ca2+ current, I Ca‐L, and increased efflux on NCX. Analysis of sarcolemmal fluxes revealed substantial background Ca2+ entry which sustains Ca2+ efflux during waves in the steady state. Wave frequency and background Ca2+ entry were decreased by Gd3+ or the TRPC6 inhibitor BI 749327. These agents also blocked Mn2+ entry. Inhibiting connexin hemi‐channels, TRPC1/4/5, L‐type channels or NCX had no effect on background entry. In conclusion, raising external Ca2+ induces waves via a background Ca2+ influx through TRPC6 channels. The greater propensity to waves in HF results from increased background entry and decreased threshold SR content.Key points Heart failure is a pro‐arrhythmic state and arrhythmias are a major cause of death.At the cellular level, Ca2+ waves resulting in delayed after‐depolarisations are a key trigger of arrhythmias. Ca2+ waves arise when the sarcoplasmic reticulum (SR) becomes overloaded with Ca2+.We investigate the mechanism by which raising external Ca2+ causes waves, and how this is modified in heart failure.We demonstrate that a novel sarcolemmal background Ca2+ influx via the TRPC6 channel is responsible for SR Ca2+ overload and Ca2+ waves.The increased propensity for Ca2+ waves in heart failure results from an increase of background influx, and a lower threshold SR content.The results of the present study highlight a novel mechanism by which Ca2+ waves may arise in heart failure, providing a basis for future work and novel therapeutic targets.

Al-Substituted Tobermorites: An Effective Cation Exchanger Synthesized from "End-of-Waste" Materials.

The policies to meet the “zero waste” regime and transition to sustainable circular economy can no longer ignore the use of wastes in place of natural resources, and these daunting and vital societal challenges are nowadays being faced by several nations. The main objective of this work was to search waste materials suitable for a quick and environmentally friendly production of a nanoporous geomaterial able to trap toxic metals at the solid/liquid interface. More specifically, the end-of-waste from the thermal inertization of cement–asbestos and glass powder from domestic glass containers have been employed as sources for the hydrothermal synthesis of a tobermorite-rich material (TRM) successfully tested for the selective removal of Pb2+, Zn2+, Cd2+, and Ni2+ from aqueous solutions. The synthesis was carried out in alkaline solution under mild hydrothermal conditions (120 °C) within 24 h. The quantitative phase analyses of the TRM carried out by applying the Rietveld method showed the occurrence of a large amount of well-crystallized 11 Å Al-substituted tobermorites and an amorphous phase and a lower content of aragonite and calcite. Chemical analyses and thermogravimetric measurements coupled with simultaneous evolved gas mass spectrometry highlighted that Al3+ for Si4+ substitutions in the wollastonite-like tetrahedral chains of tobermorites are balanced by the occurrence of Ca2+, Na+, and K+ cations in the interlayer rather than by (OH)− for O2– substitutions in the CaO polyhedra. Time-dependent removal tests clearly indicated that metal cations are selectively adsorbed depending on their concentration in solution. Moreover, the kinetic curves showed that the removal of metals from solution is fast and the equilibrium is almost reached in the first 8 h.

Identification of the Causative Genes of Calcium Deficiency Disorders in Horticulture Crops: A Systematic Review

The occurrence of calcium (Ca2+) deficiency disorders is a severe problem in the production of horticulture crops. Recently, several studies have investigated the role of gene expression in Ca2+ deficiency disorders and/or Ca2+ accumulation, providing an indication of the mechanism of Ca2+ deficiency disorders at the genetic level. To determine the relation between gene expression and the occurrence of Ca2+ deficiency disorders, we conducted a systematic review of the literature using the Preferred Reporting Items for Systematic Reviews and Meta-analyses protocol. In our initial search, we extracted studies investigating the relationships between Ca2+ deficiency disorders (tipburn and blossom-end rot) and gene expression. In our second search, we extracted studies involving functional analyses of the genes associated with Ca2+ deficiency and/or Ca2+ accumulation in plant organs. Thirty-seven articles were extracted from both searches. Studies on Ca2+ movement-related genes (Ca2+ antiporters, calreticulin, Ca2+ pumps, Ca2+ channels, and pectin methylesterases) accounted for the majority of these articles. Particularly, the effects of the expression of CAXs (Ca2+/H+ antiporters) and CRT (calreticulin) on the occurrence of Ca2+ deficiency disorders were demonstrated in studies extracted from both searches. Further research focusing on these genes may reveal the causative genes for Ca deficiency disorders in different horticulture crops. We hope that the knowledge synthesized in this systematic review will contribute to the accumulation of further knowledge and elucidation of the causes of Ca2+ deficiency disorders.

The relationship between anion distribution in process water and flotation properties of iron oxides

It is inevitable for the presence of dissolved ions, especially inorganic cations or anions, in the process water of an industrial flotation system. They could alter the chemistry of a flotation system and interfere with the interaction between minerals and reagents. The purpose of this study was to identify the relationship between anion distribution in the process water and the flotation performance of a typical industrial iron ore concentrator when the process conditions were stable. An effort was also made to understand how the anions affect the flotation properties of the iron ore through a series of water chemical analyses by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES), ore sample analysis by X-ray Diffraction (XRD), bench flotation tests by using the industrial iron ore, micro-flotation tests by using pure quartz, and zeta potential measurement. The data from water chemical analyses indicated that sulfate ion concentrations in the process water, especially from the roughing stage, are more relevant to the SiO2 content and Fe recovery in concentrate than chloride ion. This can be explained by the results from flotation tests, suggesting that the anions at higher valence tend to have more significant influences on lowering the SiO2 content. But it substantially depends on anion concentration, pulp pH, and type of metal ions. At 8.5–9.2, SO42− ions have promotive effects on the recovery of the quartz pre-modified by FeCl2, or FeCl3 in the water. This is probably due to a shift towards acidic direction on the zeta potential of quartz surfaces in the presence of sulfate ions. These anions, however, have negative effects on the quartz recovery with the occurrence of Ca2+ ions. The precipitation of CaSO4 on the mineral surfaces could be attributed to it.

Cardiotoxicity of sorafenib is mediated through elevation of ROS level and CaMKII activity and dysregulation of calcium homoeostasis.

Sorafenib, a multi-kinase inhibitor, is recommended as a new standard therapy for advanced hepatocellular carcinoma (HCC); however, it also exhibits severe cardiotoxicity and the toxicity mechanisms are not completely elucidated. Recent studies suggested that sorafenib-enhanced ROS may partially contribute to its anti-HCC effect, which implies that redox mechanism might also be involved in sorafenib's cardiotoxicity. In this study, we aimed to investigate if sorafenib is able to induce oxidative stress and how this may impair cellular functions in cardiomyocyte, ultimately accounting for its cardiotoxicity. Our results showed that in isolated rat hearts, sorafenib caused ventricular arrhythmias and left ventricular dysfunction, which were alleviated by the antioxidant N-(2-mercaptopropionyl)-glycine (MPG). In isolated ventricular myocytes, sorafenib increased diastolic intracellular Ca2+ levels, decreased Ca transients and the occurrence of Ca2+ waves. These changes were eliminated by MPG, CaMKII inhibitor KN-93 and the mitochondrial permeability transition pore (mPTP)inhibitor cyclosporin A (CsA). Moreover, the levels of oxidized and phosphorylated CaMKII were significantly increased. Sorafenib elevated ROS levels, which was reversed by CsA and MPG; additionally, sorafenib reduced the activity of mitochondrial complex III and augmented mitochondrial ROS production. In vivo rats treated with sorafenib exhibited a reduction of antioxidant defence and abnormal histological alterations including hypertrophy, increased fibrosis, disordered myofibrils and damaged mitochondria, which were protected by MPG. We conclude that sorafenib induces the disruption of Ca2+ homoeostasis and cardiac injury via enhanced ROS potentially through inhibiting mitochondrial complex III, the opening of mPTP and overactivating CaMKII. These results provide a potential strategy for preventing or reducing cardiotoxicity of sorafenib.

1178Unsuspected role of the cardiac PKA type I in excitation-contraction coupling and in heart failure development

Abstract The cAMP-dependent protein kinase (PKA) consists of two regulatory (R) and two catalytic (C) subunits and comprises two subtypes, PKAI and PKAII, defined by the nature of their regulatory subunits, RIα and RIIα respectively. Whereas PKAII is thought to play a key role in β-adrenergic (β-AR) regulation of cardiac contractility, the function of PKAI is unclear. To address this question, we generated mice with cardiomyocyte-specific and conditional invalidation of the RIα subunit of PKA. Tamoxifen injection in 8 weeks-old mice resulted in a >70% decrease in RIα protein without modification of other PKA subunits, which was associated with ∼2-fold increased basal PKA activity in RIα-KO mice (p<0.05, N=6/group). This translated into enhanced cardiac contraction and relaxation, as observed in vivo by increased fractional shortening and E-wave velocity (p<0.05, N=10/group) and ex vivo by increased LV pressure and maximal rate of contraction and relaxation (p<0.05, N=9/group). L-type Ca2+ current density was increased in ventricular myocytes from RIα-KO, and β-AR stimulation was decreased by ∼50% (p<0.05, n=38 cells for WT, and, n=40 for RIα-KO). Consistently, Ca2+ transients amplitude and relaxation kinetics were increased, along with increased occurrence of Ca2+ sparks and waves (p<0.05, n=44 cells for WT, and, n=50 for RIα KO). Phosphorylation of Ca2+ channels (CaV1.2), PLB, RyR2 and cMyBP-C at PKA sites was increased >2-fold (p<0.05, N=6/group) in RIα KO without modification of total protein expression. With age, these mice developed a congestive heart failure (HF) phenotype with massive hypertrophy and fibrosis which eventually led to death in 50% of RIα-KO mice at 50 weeks (versus 0% in WT, p<0.01). These results reveal a previously unsuspected role of PKA type I in cardiac excitation-contraction coupling and HF.

Molecular mechanisms underlying the α-tomatine-directed apoptosis in human malignant glioblastoma cell lines A172 and U-118 MG.

In the present study, the molecular mechanisms involved in the α-tomatine-induced apoptosis in human glioblastoma cell lines A172 and U-118 MG were investigated. Wright staining and ApopTag assays were conducted to confirm the apoptosis induced by α-tomatine treatment. Fura-2 assay determined an enhancement in free Ca2+ intracellularly, indicating the occurrence of Ca2+-dependent apoptosis induction. Western blot experiments were also performed to predict the apoptosis by measuring the changes in the Bax:Bcl-2 ratio. Increase of calpain activity triggered caspase-12 expression, which in turn further activated caspase-9. In addition, an increase in the ratio of Bax:Bcl-2 accounted for the mitochondrial release of cytochrome c into the cytosol for caspase-3 and caspase-9 activation. Elevated activity of calpain and caspase-3 yielded spectrin breakdown products with 145 and 120 kDa, respectively. Caspase-3 activation further cleaved the inhibitor of caspase activated DNase, while the apoptosis-inducing factor detected in the cytosol suggested that apoptosis was independent of caspase. The apoptosis induction was further supported by decreased expression levels of nuclear factor-κB and increased expression of the inhibitor of nuclear factor, IκBα. In conclusion, the presented experimental results revealed the stimulation of different molecular mechanisms for α-tomatine-mediated apoptosis in A172 and U-118 MG human glioblastoma cell lines.

Ca2+ Microdomains in T-Lymphocytes

Early Ca2+ signaling is characterized by occurrence of Ca2+ microdomains formed by opening of single or clusters of Ca2+ channels, thereby initiating first signaling and subsequently activating global Ca2+ signaling mechanisms. However, only few data are available focusing on the first seconds and minutes of Ca2+ microdomain formation and related signaling pathways in activated T-lymphocytes. In this review, we condense current knowledge on Ca2+ microdomain formation in T-lymphocytes and early Ca2+ signaling, function of Ca2+ microdomains, and microdomain organization. Interestingly, considering the first seconds of T cell activation, a triphasic Ca2+ signal is becoming apparent: (i) initial Ca2+ microdomains occurring in the first second of T cell activation, (ii) amplification of Ca2+ microdomains by recruitment of further channels in the next 5–10 s, and (iii) a transition to global Ca2+ increase. Apparently, the second messenger nicotinic acid adenine dinucleotide phosphate is the first second messenger involved in initiation of Ca2+ microdomains. Ryanodine receptors type 1 act as initial Ca2+ release channels in CD4+ T-lymphocytes. Regarding the temporal correlation of Ca2+ microdomains with other molecular events of T cell activation, T cell receptor-dependent microdomain organization of signaling molecules Grb2 and Src homology [SH2] domain-containing leukocyte protein of 65 kDa was observed within the first 20 s. In addition, fast cytoskeletal changes are initiated. Furthermore, the involvement of additional Ca2+ channels and organelles, such as the Ca2+ buffering mitochondria, is discussed. Future research developments will comprise analysis of the causal relation between these temporally coordinated signaling events. Taken together, high-resolution Ca2+ imaging techniques applied to T cell activation in the past years paved the way to detailed molecular understanding of initial Ca2+ signaling mechanisms in non-excitable cells.

Evidence that NO/cGMP/PKG signalling cascade mediates endothelium dependent inhibition of IP3R mediated Ca2+ oscillations in myocytes and pericytes of ureteric microvascular network in situ

In ureteric microvessels the antagonistic relationship between Ca2+ signalling in endothelium and Ca2+ oscillations in myocytes and pericytes of arterioles and venules involves nitric oxide (NO), but the underlying mechanisms are not well understood. In the present study we investigated the effects of carbachol and NO donor SNAP on Ca2+ signalling and vasomotor responses of arterioles and venules in intact urteric microvascular network in situ using confocal microscopy. Vasomotor responses of arterioles and venules induced by AVP correlated with the occurrence of Ca2+ oscillations in the myocytes and pericytes and were not abolished by the removal of Ca2+ from extracellular fluid. Carbachol-induced rise of intracellular Ca2+ in endothelium was accompanied by the termination of the Ca2+ oscillations in myocytes and pericytes. This carbachol-induced inhibitory effect on Ca2+ oscillations in myocytes and pericytes was reversed by ODQ, an inhibitor of soluble guanylyl cyclase (sGC) and by Rp-8-pCPT-cGMPS, an inhibitor of protein kinase G (PKG). Ca2+ oscillations in myocytes and pericytes were also effectively blocked by NO donor SNAP. An Inhibitory effect of SNAP was markedly enhanced by zaprinast, a selective inhibitor of cGMP-specific phosphodiesterase-5, and reversed by sGC inhibitor, ODQ and PKG inhibitor, Rp-8-pCPT-cGMPS. The cGMP analogue and selective PKG activator 8pCPT-cGMP also induced inhibition of the AVP-induced Ca2+ oscillations in myocytes and pericytes. SNAP had no effects on Ca2+ oscillations induced by caffeine in distributing arcade arterioles. Consequently, we conclude that NO- mediated inhibition of Ca2+ oscillations in myocytes and pericytes predominantly recruits the cGMP/PKG dependent pathway. The inhibitory effect of NO/cGMP/PKG cascade is associated with suppressed Ca2+ release from the SR of myocytes and pericytes selectively via the inositol triphosphate receptor (IP3R) channels.

Serca Stimulation Increases Intra-Sr Ca2+Threshold for Ca2+ Waves in Cadiomyocytes

In cardiac muscle, stimulation of β-adrenergic receptors leads to activation of PKA via the second messenger cAMP. PKA-dependent phosphorylation of several targets subsequently amplifies the L-type Ca2+ current, relieves the SERCA inhibition by PLB and is proposed to increase the Ca2+ sensitivity of the RyRs (by phosphorylation at serine 2808). Together, these mechanisms account for positive inotropy, but can also be arrhythmogenic via facilitation of spontaneous Ca2+ waves. Unexpectedly, the level of Ca2+ inside the SR ([Ca2+]SR) needed to initiate such waves has been reported to increase upon β-adrenergic stimulation, an observation which cannot be easily reconciled with elevated Ca2+ sensitivity of the RyRs. We tested the hypothesis that this change of Ca2+ wave threshold could occur indirectly, subsequent to SERCA disinhibition. Cytosolic and intra-SR Ca2+ waves were simultaneously recorded with confocal line-scans in permeabilized mouse cardiomyocytes with rhod-2 and fluo-5-N, respectively. We analyzed changes of SR Ca2+ content and several Ca2+ wave parameters. Ochratoxin A (OTA) was used to specifically stimulate the SERCA and the findings were compared to those in the presence of cAMP, which besides SERCA stimulation also leads to RyR phosphorylation. While cAMP resulted in a modest increase of Ca2+ wave thresholds (F/F0 9%±3.5%), OTA lead to a substantial increase (30±5.1%). Both compounds resulted in an acceleration of SR refilling (OTA earlier than cAMP), confirming SERCA stimulation. While cAMP provoked in the occurrence of Ca2+ sparks and mini waves, possibly accounting for the less pronounced SR loading, this was not observed in OTA. Together these results suggest that SERCA stimulation alone can elevate the intra-SR threshold for the generation of Ca2+ waves, independently of RyR phosphorylation. Supported by SNF.

Mechanisms by which Cytoplasmic Calcium Wave Propagation and Alternans Are Generated in Cardiac Atrial Myocytes Lacking T-Tubules—Insights from a Simulation Study

This study investigated the mechanisms underlying the propagation of cytoplasmic calcium waves and the genesis of systolic Ca2+ alternans in cardiac myocytes lacking transverse tubules (t-tubules). These correspond to atrial cells of either small mammals or large mammals that have lost their t-tubules due to disease-induced structural remodeling (e.g., atrial fibrillation). A mathematical model was developed for a cluster of ryanodine receptors distributed on the cross section of a cell that was divided into 13 elements with a spatial resolution of 2 μm. Due to the absence of t-tubules, L-type Ca2+ channels were only located in the peripheral elements close to the cell-membrane surface and produced Ca2+ signals that propagated toward central elements by triggering successive Ca2+-induced Ca2+ release (CICR) via Ca2+ diffusion between adjacent elements. Under control conditions, the Ca2+ signals did not fully propagate to the central region of the cell. However, with modulation of several factors responsible for Ca2+ handling, such as the L-type Ca2+ channels (Ca2+ influx), SERCA pumps (sarcoplasmic reticulum (SR) Ca2+ uptake), and ryanodine receptors (SR Ca2+ release), Ca2+ wave propagation to the center of the cell could occur. These simulation results are consistent with previous experimental data from atrial cells of small mammals. The model further reveals that spatially functional heterogeneity in Ca2+ diffusion within the cell produced a steep relationship between the SR Ca2+ content and the cytoplasmic Ca2+ concentration. This played an important role in the genesis of Ca2+ alternans that were more obvious in central than in peripheral elements. Possible association between the occurrence of Ca2+ alternans and the model parameters of Ca2+ handling was comprehensively explored in a wide range of one- and two-parameter spaces. In addition, the model revealed a spontaneous second Ca2+ release in response to a single voltage stimulus pulse with SR Ca2+ overloading and augmented Ca2+ influx. This study provides what to our knowledge are new insights into the genesis of Ca2+ alternans and spontaneous second Ca2+ release in cardiac myocytes that lack t-tubules.

Nitric oxide induces airway smooth muscle cell relaxation by decreasing the frequency of agonist-induced Ca2+ oscillations

Nitric oxide (NO) induces airway smooth muscle cell (SMC) relaxation, but the underlying mechanism is not well understood. Consequently, we investigated the effects of NO on airway SMC contraction, Ca2+ signaling, and Ca2+ sensitivity in mouse lung slices with phase-contrast and confocal microscopy. Airways that were contracted in response to the agonist 5-hydroxytryptamine (5-HT) transiently relaxed in response to the NO donor, NOC-5. This NO-induced relaxation was enhanced by zaprinast or vardenafil, two selective inhibitors of cGMP-specific phosphodiesterase-5, but blocked by ODQ, an inhibitor of soluble guanylyl cyclase, and by Rp-8-pCPT-cGMPS, an inhibitor of protein kinase G (PKG). Simultaneous measurements of airway caliber and SMC [Ca2+]i revealed that airway contraction induced by 5-HT correlated with the occurrence of Ca2+ oscillations in the airway SMCs. Airway relaxation induced by NOC-5 was accompanied by a decrease in the frequency of these Ca2+ oscillations. The cGMP analogues and selective PKG activators 8Br-cGMP and 8pCPT-cGMP also induced airway relaxation and decreased the frequency of the Ca2+ oscillations. NOC-5 inhibited the increase of [Ca2+]i and contraction induced by the photolytic release of inositol 1,4,5-trisphosphate (IP3) in airway SMCs. The effect of NO on the Ca2+ sensitivity of the airway SMCs was examined in lung slices permeabilized to Ca2+ by treatment with caffeine and ryanodine. Neither NOC-5 nor 8pCPT-cGMP induced relaxation in agonist-contracted Ca2+-permeabilized airways. Consequently, we conclude that NO, acting via the cGMP–PKG pathway, induced airway SMC relaxation by predominately inhibiting the release of Ca2+ via the IP3 receptor to decrease the frequency of agonist-induced Ca2+ oscillations.

Differential Hypertrophic Remodeling Of Cardiomyocytes Determines Distinct Types Of Arrhythmias In The Ischemic Failing Heart: Key Role Of The Ryanodine Receptor

A better understanding of the mechanisms responsible for sudden cardiac death (SCD) in heart failure (HF) may influence treatment strategies. We investigated whether early and delayed afterdepolarizations (EADs, DADs) coexist in HF, and how they are initiated during disease progression. Cells were isolated from the left ventricle of rats 8 weeks after myocardial infarction (PMI) and from age-matched sham-operated animals, and studied using the whole-cell patch-clamp technique. Cellular arrhythmias were triggered exclusively in PMI cells (40 %) using trains of 5 stimulations at 2.0 Hz. EADs and DADs occurred in distinct cell populations. Cell membrane capacitance measurements showed that EADs occurred in normal-sized PMI cells ( 200 pF). All cells exhibited prolonged action potentials (AP) due to decreased Ito currents. However, additional modifications in Ca2+-dependent ionic currents were observed in hypertrophic cells: a decrease in the inward rectifier K+ current IK1 and a slowing of L-type Ca2+ current (ICaL) inactivation, responsible for the poor adaptation of both ICaL and AP to abrupt changes in the pacing rate. The occurrence of Ca2+ sparks, reflecting ryanodine receptor (RyR2) activity, also increased with hypertrophy. Fluorescence measurements using Fluo-4 AM revealed that the amplitudes of [Ca2+]i transients, Ca2+ load of the sarcoplasmic reticulum (SR) and Ca2+ spark amplitude were inversely correlated with cell size.The trophic status of cardiomyocytes determines the type of arrhythmia triggered in PMI rats, based on differential electrophysiological remodeling reflecting early-mild and late-severe modifications in the function of the ryanodine receptor RyR2.

Effects of perfusion pressure on Ca2+ sparks in rat renal afferent arterioles

Mechanical force can be transduced into freshly isolated renal vascular smooth muscle cells (VSMCs) to trigger Ca2+ sparks. Sparks can trigger Spontaneous Transient Inward Currents (STICs), and regulate myogenic tone via membrane depolarization. Electrophysiological studies indicate the presence of STICs in isolated renal VSMCs. Hence, we sought to determine whether the occurrence of Ca2+ sparks is increased during pressure induced myogenic constriction. Line scan images were collected from perfused rat afferent arterioles at 500 Hz using laser scanning confocal microscopy. We detected spontaneous Ca2+ sparks in 40% of the VSMCs at a perfusion pressure of 80 mm Hg. These sparks were suppressed by 50 μM ryanodine and were augmented by 0.5 mM caffeine. The mean spontaneous Ca2+ spark frequencies were 0.14±0.04 sparks/s (n=23 cells) and 0.24±0.11 sparks/s (n=13 cells) at 80 mm Hg and 120 mm Hg, respectively. These suggest that the increase in frequency of sparks is probably related to pressure induced myogenic constriction. Intriguingly, the frequency of Ca2+ sparks observed in SHR afferent arteriole was double compared to that of normotensive rats at the same perfusion pressure. These observations implicate that the enhanced Ca2+ sparks frequency may contribute to the increase of renal vascular resistance in SHR.

Miniature Synaptic Events Elicited by Presynaptic Ca2+ Rise Are Selectively Suppressed by Cannabinoid Receptor Activation in Cerebellar Purkinje Cells

Activation of cannabinoid receptors suppresses neurotransmitter release in various brain regions. In cerebellar Purkinje cells (PCs), cannabinoid agonists suppress both EPSC and IPSC evoked by stimulating the corresponding inputs. However, cannabinoid agonists suppress miniature IPSC (mIPSC) but not miniature EPSC (mEPSC) at normal external Ca2+ concentration ([Ca2+]o). Therefore, cannabinoid agonists are thought to suppress release machinery for IPSCs but not that for EPSCs. Here we investigated the possible cause of this difference and found that cannabinoid agonists selectively suppressed Ca2+-enhanced miniature events. A cannabinoid agonist, WIN55,212-2 (5 microM), did not affect mEPSC frequency with 2 mM extracellular Ca2+ (Ca2+(o)). However, WIN55,212-2 became effective when mEPSC frequency was enhanced by elevation of presynaptic Ca2+ level by perfusion with 5 mM Ca2+(o) or bath application of A23187, a Ca2+ ionophore. In contrast, WIN55,212-2 suppressed mIPSC frequency with 2 mM Ca2+(o), but it became ineffective when the presynaptic Ca2+ level was lowered by perfusion with a Ca2+-free solution containing BAPTA-AM. Experiments with systematic [Ca2+]o changes revealed that mIPSC but not mEPSC regularly involved events elicited by presynaptic Ca2+ rise with 2 mM Ca2+(o). Importantly, Ca2+-enhancement of mEPSC and mIPSC was not attributable to activation of voltage-dependent Ca2+ channels. Activation of GABAB receptor or group III metabotropic glutamate receptor, which couple to G(i/o)-protein, also preferentially suppressed Ca2+-enhanced miniature events in PCs. These results suggest that the occurrence of Ca2+-enhanced miniature events at normal [Ca2+]o determines the sensitivity to the presynaptic depression mediated by cannabinoid receptors and other G(i/o)-coupled receptors in PCs.

Modeling of Molecular and Cellular Mechanisms Involved in Ca<sup>2+</sup> Signal Encoding in Airway Myocytes

In airway myocytes signal transduction via cytosolic calcium plays an important role. In relation with experimental results we review models of basic molecular and cellular mechanisms involved in the signal transduction from the myocyte stimulation to the activation of the contractile apparatus. We concentrate on mechanisms for encoding of input signals into Ca2+ signals and the mechanisms for their decoding. The mechanisms are arranged into a general scheme of cellular signaling, the so-called bow-tie architecture of signaling, in which calcium plays the role of a common media for cellular signals and links the encoding and decoding part. The encoding of calcium signals in airway myocytes is better known and is presented in more detail. In particular, we focus on three recent models taking into account the intracellular calcium handling and ion fluxes through the plasma membrane. The model of membrane conductances was originally proposed for predicting membrane depolarization and voltage-dependent Ca2+ influx triggered by initial cytosolic Ca2+ increase as observed on cholinergic stimulation. Cellular models of intracellular Ca2+ handling were developed to investigate the role of a mixed population of InsP3 receptor isoforms and the cellular environment in the occurrence of Ca2+ oscillations, and the respective role of the sarcoplasmic reticulum, mitochondria, and cytosolic Ca2+-binding proteins in cytosolic Ca2+ clearance. Modeling the mechanisms responsible for the decoding of calcium signals is developed in a lesser extent; however, the most recent theoretical studies are briefly presented in relation with the known experimental results.

Fusion pore expansion is a slow, discontinuous, and Ca2+-dependent process regulating secretion from alveolar type II cells.

In alveolar type II cells, the release of surfactant is considerably delayed after the formation of exocytotic fusion pores, suggesting that content dispersal may be limited by fusion pore diameter and subject to regulation at a postfusion level. To address this issue, we used confocal FRAP and N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl) pyridinium dibromide (FM 1-43), a dye yielding intense localized fluorescence of surfactant when entering the vesicle lumen through the fusion pore (Haller, T., J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. 1998. Proc. Natl. Acad. Sci. USA. 95:1579–1584). Thus, we have been able to monitor the dynamics of individual fusion pores up to hours in intact cells, and to calculate pore diameters using a diffusion model derived from Fick's law. After formation, fusion pores were arrested in a state impeding the release of vesicle contents, and expanded at irregular times thereafter. The expansion rate of initial pores and the probability of late expansions were increased by elevation of the cytoplasmic Ca2+ concentration. Consistently, content release correlated with the occurrence of Ca2+ oscillations in ATP-treated cells, and expanded fusion pores were detectable by EM. This study supports a new concept in exocytosis, implicating fusion pores in the regulation of content release for extended periods after initial formation.

Abnormalities in gap junctions and Ca2+ dynamics in cardiomyocytes at the border zone of myocardial infarcts.

Abnormalities in gap junction function and Ca2+ dynamics are believed to be important factors in arrhythmogenesis after myocardial infarction. To elucidate the relationship between changes in Ca2+ dynamics and gap junctions, we analyzed by real-time in situ Ca2+ imaging of fluo-3 loaded whole hearts the spatiotemporal occurrence of Ca2+ waves and the localization of connexin43 (Cx43) at the border zone of myocardial infarcts induced in the rat by coronary ligation. At early time points (2-4 hours postligation), different regions of the left ventricle showed distinct changes in cytosolic free Ca2+ concentrations [Ca2+]i. While some cardiomyocytes of infarcted regions exhibited high levels of resting fluo-3 fluorescence, at border zones frequent Ca2+ waves were observed. Some of the waves were abolished by spontaneous Ca2+ transients and others were not. Intact myocardium apart from infarcted regions exhibited homogenous Ca2+ transients. Confocal imaging of Cx43 and actin filaments in the rat heart fixed 2 hours after coronary ligation revealed that Cx43 was markedly decreased in the area of myocyte necrosis with contraction bands and in the neighboring myocardium. These results suggest that abnormal expression and function of gap junctions could be associated with Ca2+ waves at the border zone of myocardial infarcts, possibly through Ca2+ overload.

S100 protein expression in subpopulations of neurons of rat brain.

Available data are conflicting as regards the occurrence of Ca2+ and Zn2+ binding S100 proteins in neurons of mammalian brain. Here the localization and expression of S100 was re-investigated using several different antibodies and in situ hybridization. A map is provided for the distribution of two classes of S100-positive neuron populations in the adult rat CNS. "Persistently S100-positive" neurons had large size, were strongly immunoreactive and were mainly distributed in the nuclei of the lower brainstem and cerebellum. "Variably S100-positive" neurons were preferentially found in the forebrain of rats older than 90 days and were especially numerous in limbic regions. The S100-immunoreactivity in these neurons was moderately intense, occurred with high interindividual variation and appeared related to function as suggested by variations due to anesthesia. The expression of S100 mRNA in neurons was re-investigated at high spatial resolution with non-radioactive in situ hybridization using an oligonucleotide specific for S100 beta-mRNA. Expression of S100 was demonstrated in astrocytes and in those neuron populations which were also strongly S100-immunoreactive. No expression of S100 beta message was seen in weakly immunoreactive neurons, b but this may be due to low sensitivity of the techniques used. The data suggest that the S100 proteins are synthesized in all astrocytes and in distinct subpopulations of neurons in rat brain. These neurons show a characteristic topography and vary in S100 expression probably due to their function and maturation.

Properties of the caffeine-sensitive intracellular calcium stores in mammalian neurons

Using indo-1- and fura-2-based microfluorometry for measuring the cytoplasmic free calcium concentration ([Ca2+]in), the properties of caffeine-induced Ca2+ release from internal stores were studied in rat cultured central and peripheral neurons, including dorsal root ganglion (DRG) neurons, neurons from then. cuneatus, CA1 and CA3 hippocampal regions, and pyramidal neocortical neurons. Under resting conditions, the Ca2+ content of internal stores in DRG neurons was high enough to produce caffeine-triggered [Ca2+]in transients. Prolonged exposure of caffeine depleted the caffeine-sensitive stores of releasable Ca2+; the degree of this depletion depended on caffeine concentration. The depletion of the caffeine-sensitive internal stores to some extent was linked to calcium extrusion via La3+-sensitive plasmalemmal Ca2+-ATPases. Caffeine-induced Ca2+ release deprived internal stores in DRG neurons, but they refilled themselves spontaneously within 10 min. Pharmacological manipulation with caffeine-sensitive stores interferred with the depolarization-induced [Ca2+]in transients. In the presence of low caffeine concentration (0.5–1.0 mM) in the extracellular solution, the rate of rise of the depolarization-triggered [Ca2+]in transients significantly increased (by a factor of 2.15 ± 0.29) suggesting the occurrence of Ca2+-induced Ca2+ release. When the caffeine-sensitive stores were emptied by prolonged application of caffeine, the amplitude and rate of rise of the depolarization-induced [Ca2+]in transients decreased. These findings suggest the involvement of internal caffeine-sensitive calcium stores in generation of calcium signal in sensory neurons. In contrast, in all types of central neurons tested the resting Ca2+ content of internal stores was low, but the stores could be charged by transmembrane Ca2+ entry through voltage-operated calcium channels. After charging, the stores in central neurons spontaneously lost releasable calcium content and within 10 min they became completely empty again. We suggest that internal Ca2+ stores in peripheral and central neurons, although having similar pharmacological characteristics, handle Ca2+ ions in a different manner. Calcium stores in sensory neurons are continuously filled by releasable calcium and after discharging they can be spontaneously refilled, whereas in central neurons internal calcium stores can be charged by releasable calcium only transiently. Caffeine-evoked [Ca2+]in transients in all types of neurons were effectively blocked by 10 mM ryanodine, 5 mM procaine, 10 mM dantrolene, or 0.5 mM Ba2+, thus sharing the basic properties of the Ca2+-induced Ca2+ release from endoplasmic reticulum.