Localized Ca^2＋ uncaging induces Ca^2＋ release through IP3R in smooth muscle

AIM: Our previous study indicated that there are two types of Ca2+ release events seen in intact mouse bladder tissue. In this study our aim is to investigate the mechanism that underlies the phenomena of Ca2+ release in smooth muscle. METHODS: Single cells were isolated and tissue segments were prepared by cutting the detrusor into 0.1 cm x 0.5 cm strips running along the axis from the neck to the fundus. Single cells and intact tissue strips were co-loaded with the Ca2+ indicator and caged Ca2+ by incubation with 10 micromol/L Fluo-4 AM and DMNP-EDTA-AM. Fluo-4 AM fluorescence was detected by laser scanning confocal microscopy, and local uncaging of DMNP-EGTA was achieved by brief exposure to the output of a diode-pumped, Ti:sapphire laser tuned to 730 nm. RESULTS: Local uncaging of caged Ca2+ was able to trigger Ca2+ release events in both single cells and tissue strips from mouse bladder. The Ca2+ release events could not be blocked by ryanodine alone, but the property of the Ca2+ release was markedly altered. Surprisingly, in the presence of ryanodine, Xestospongin C completely inhibited the Ca2+ release events both in single cell and tissue experiments. CONCLUSION: (1) Two photon flash photolysis (TPFP) triggers Ca2+ induced Ca2+ release. This process involves release through type 2 ryanodine receptor channels; (2) TPFP results in the release of Ca2+ through inositol 1,4,5-trisphosphate receptors in the absence of phospholipase C activation.     

Ca^2＋ sparks and Ca^2＋ glows in superior cervical ganglion neurons

AIM: Ca2+ release from the endoplasmic reticulum (ER) is an integral component of neuronal Ca2+ signaling. The present study is to investigate properties of local Ca2+ release events in superior cervical ganglion (SCG) neurons. METHODS: Primary cultured SCG neurons were prepared from neonatal rats (P3-P7). Low concentration of caffeine was used to induce Ca2+ release from the ER Ca2+ store, and intracellular Ca2+ was recorded by high-resolution line scan confocal imaging and the Ca2+ indicator Fluo-4. RESULTS: Two populations of local Ca2+ release events with distinct temporal characteristics were evoked by 1.5 mmol/L caffeine near the surface membrane in the soma and the neurites of SCG neurons. Brief events similar to classic Ca2+ sparks lasted a few hundreds of milliseconds, whereas long-lasting events displayed duration up to tens of seconds. Typical somatic and neurite sparks were of 0.3- and 0.52-fold increase in local Fluo-4 fluorescence, respectively. Typical Ca2+ glows were brighter (deltaF/F0 approximately 0.6), but were highly confined in space. The half maximum of full duration of neurite sparks was much longer than those in the soma (685 vs 381 ms). CONCLUSION: Co-existence of Ca2+ sparks and Ca2+ glows in SCG neurons indicates distinctive local regulation of Ca2+ release kinetics. The local Ca2+ signals of variable, site-specific temporal length may bear important implications in encoding a 'memory' of the trigger signal.     

Regulation of TRP-like muscarinic cation current in gastrointestinal smooth muscle with special reference to PLC/InsP3/Ca^2＋ system

Acetylcholine, the main enteric excitatory neuromuscular transmitter, evokes membrane depolarization and contraction of gastrointestinal smooth muscle cells by activating G protein-coupled muscarinic receptors. Although the cholinergic excitation is generally underlined by the multiplicity of ion channel effects, the primary event appears to be the opening of cation-selective channels; among them the 60 pS channel has been recently identified as the main target for the acetylcholine action in gastrointestinal myocytes. The evoked cation current, termed mI(CAT), causes either an oscillatory or a more sustained membrane depolarization response, which in turn leads to increases of the open probability of voltage-gated Ca2+ channels, thus providing Ca2+ entry in parallel with Ca2+ release for intracellular Ca2+ concentration rise and contraction. In recent years there have been several significant developments in our understanding of the signaling processes underlying mICAT generation. They have revealed important synergistic interactions between M2 and M3 receptor subtypes, single channel mechanisms, and the involvement of TRPC-encoded proteins as essential components of native muscarinic cation channels. This review summarizes these recent findings and in particular discusses the roles of the phospholipase C/InsP3/intracellular Ca2+ release system in the mI(CAT) physiological regulation.     

Opioid receptor antagonists increase [Ca^2＋]i in rat arterial smooth muscle cells in hemorrhagic shock

INTRODUCTION Circulation hypotension induced by hemorrhagicshock is one of the main causes of death after severewounds or trauma. When the hemorrhagic shock hasdeveloped to a decompensatory stage, it is difficult toreverse the hypotension using usual vasoconstrictor,such as norepinephrine. One of the explanations ofhypotension is due to the hyposensitivity of arterialsmooth muscle cells (SMC) to vascular constrictorstimuli[1,2]. Previous studies showed that the vascularhyporesponse was r…     

Sympathetically evoked Ca^2＋ signaling in arterial smooth muscle

The sympathetic nervous system plays an essential role in the control of total peripheral vascular resistance and blood flow, by controlling the contraction of small arteries. Perivascular sympathetic nerves release ATP, norepinephrine (NE) and neuropeptide Y. This review summarizes our knowledge of the intracellular Ca2+ signals that are activated by ATP and NE, acting respectively on P2X1 and alpha1-adrenoceptors in arterial smooth muscle. Each neurotransmitter produces a unique type of post-synaptic Ca2+ signal and associated contraction. The neural release of ATP and NE is thought to vary markedly with the pattern of nerve activity, probably reflecting both pre- and post-synaptic mechanisms. Finally, we show that Ca2+ signaling during neurogenic contractions activated by trains of sympathetic nerve fiber action potentials are in fact significantly different from that elicited by simple bath application of exogenous neurotransmitters to isolated arteries (a common experimental technique), and end by identifying important questions remaining in our understanding of sympathetic neurotransmission and the physiological regulation of contraction of small arteries.     

Isoprenaline enhances local Ca^2＋ release in cardiac myocytes

AIM: Contraction of cardiac myocytes is controlled by the generation and amplification of intracellular Ca2+ signals. The key step of this process is the coupling between sarcolemma L-type Ca2+ channels (LCCs) and ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR). Beta-adrenergic stimulation is an important regulatory mechanism for this coupling process. But the details underlied the global level, which require local Ca2+ release study are still unclear. The present study is to explore the effects of beta-adrenergic stimulation on local Ca2+ release. METHODS: Using confocal microscopy combined with loose-seal patch-clamp approaches, effects of isoprenaline (1 micromol/L), a beta-adrenergic agonist, on local SR Ca2+ release triggered by Ca2+ influx through LCCs in intact rat cardiac myocytes were investigated. RESULTS: Isoprenaline increased the intensity of ensemble averaged local Ca2+ transients, the peak of which displayed a typical bell-shaped voltage-dependence over the membrane voltages ranging from approximately -40 mV to approximately +35 mV. Further analysis showed that this enhancement could be explained by the increased coupling fidelity (which refers the increased probability of RyRs activation upon depolarization), and the increased amplitude of evoked Ca2+ sparks [due to more Ca2+ releases through local RyRs]. In addition, isoprenaline decreased the first latency, which displayed a typical 'U ' shaped voltage-dependence, showing the available acceleration and synchronization of beta-adrenergic stimulation on intracellular calcium release. CONCLUSIONS: Isoprenaline enhances local Ca2+ release in cardiac myocytes. These results underscore the importance of regulation of beta-adrenergic stimulation on local intermolecular signals between LCCs and RyRs in heart cells.     

Effects of AMP579 and adenosine on L-type Ca^2＋ current in isolated rat ventricular myocytes

Aim: To compare the effects of AMP579 and adenosine on L-type Ca^2 current (ICa-L) in rat ventricular myocytes and explore the mechanism by which AMP579 acts on ICa-L.Methods: ICa-L was recorded by patch-clamp technique in whole-cell configuration. Results: Adenosine (10nmol/L to 50μmol/L) showed no effect on basal ICa-L, but it inhibited the ICa-L induced by isoproterenol 10nmol/L in a concentration-dependent manner with the IC50 of 13.06μmol/L. Similar to adenosine,AMP579 also showed an inhibitory effect on the ICa-L induced by isoproterenol.AMP579 and adenosine (both in 10μmol/L) suppressed isoproterenol-induced ICa-L by 11.1% and 5.2%, respectively. In addition, AMP579 had a direct inhibitory effect on basal ICa-L in a concentration-dependent manner with IC50(1. 17μmol/L). PD116948 (30μmol/L), an adenosine A1 receptor blocker, showed no action on the inhibitory effect of AMP579 on basal ICa-L. However, GF109203X (0.4μmol/L), a special protein kinase C (PKC) blocker, could abolish the inhibitory effect of AMP579 on basal ICa-L. So the inhibitory effect of AMP579 on basal ICa-L was induced through activating PKC, but not linked to adenosine A1 receptor. Conclusion:AMP579 shows a stronger inhibitory effect than adenosine on the ICa-L induced by isoproterenol. AMP579 also has a strong inhibitory effect on basal ICa-L in rat ventricular myocytes. Activation of PKC is involved in the inhibitory effect of AMP579 on basal ICa-L at downstream-mechanism.     

Ca^2＋ signaling during vertebrate somitogenesis

A variety of Ca2+ signals, in the form of intercellular pulses and waves, have been reported to be associated with the various sequential stages of somitogenesis: from convergent extension and the formation of the paraxial mesoderm; during the patterning of the paraxial mesoderm to establish segmental units; throughout the formation of the morphological boundaries that delineate the segmental units, and finally from within the maturing somites as they undergo subsequent development and differentiation. Due to both the technical challenges presented in imaging intact, developing embryos, and the subtle nature of the Ca2+ transients generated, they have proved to be difficult to visualize. However, a combination of cultured cell preparations and improvements in explant and whole embryo imaging techniques has begun to reveal a new and exciting class of developmental Ca2+ signals. In this chapter, we review the small, but expanding, number of reports in the literature and attempt to identify common characteristics of the somitogenic Ca2+ transients, such as their mode of generation, as well as their spatial and temporal features. This may help to elucidate the significance and function of these intriguing Ca2+ transients and thus integrate them into the complex signaling networks that orchestrate early developmental events.     

Ca^2＋ sparks evoked by depolarization of rat ventricular myocytes involve multiple release sites

AIM:To investigate the fundamental nature of calcium release events (Ca^2 ‘sparks’) evoked in rat ventricular myocytes during excitation-contraction (E-C) coupling. METHODS: High-resolution line-scan confocal imaging with the fluorescent calcium indicator and patch-clamp techniques were used to study the spontaneous Ca^2 sparks and sparks evoked by depolarization. RESULTS: 1)Line scans oriented along the length of the cell showed that both spontaneous sparks and sparks evoked by depolarization to -35mV appeared to arise at single sites spacing about 1.80μm apart (ie, the sarcomere length), and measurements of their longitudinal spread (full-width at halfmaximal amplitude:FWHM) followed single Gaussian distributions with means of 2.6μm. 2)Different to this,transverse line scans often revealed spontaneous and evoked sparks that appeared to arise near-synchronously from paired sites. Measurements of transverse FWHM of both spontaneous and evoked sparks showed bimodal distributions, which were fit well by the sums of two Gaussian curves with means of 1.8 and 2.9μm for spontaneous sparks and ith means of 1.9 and 3.1 μm for evoked sparks. Relative areas under the two Gaussian curves were 1.73:1 and 1.85:1, respectively, for spontaneous and evoked sparks. CONCLUSIONS: Ca^2 sparks evoked by depolarization are not ′unitary′ events, but often involve multiple sites of origin along Z-lines, as previously shown for spontaneous sparks. Thus, Ca^2 released during sparks directly triggered by influx through L-type Ca^2 channels may, in turn, trigger neighboring sites. The restricted involvement of only a few transverse release sites preserves the essential feature of the ‘local control’ theory of E-C coupling.     

Inhibitory effect of Ca^2＋ on in vivo gene transfer by electroporation

AIM: To investigate the specific effects of Ca2+ on transgene expression during electroporation-mediated gene transfer in mice. METHODS: Skeletal muscle and skin were subjected to in vivo electroporation with a luciferase reporter plasmid, with or without Ca2+ and various other ions. RESULTS: For in vivo electroporation, the presence of just 10 mmol/L Ca2+ in the DNA solution drastically reduced the resulting transgene expression, to less than 5% of control values. Only Ca2+, not other ions, caused inhibition, and the effect was not tissue specific. More surprisingly, even when Ca2+ ions were delivered by electroporation before or after DNA administration, similar effects were still observed. CONCLUSION: The inhibitory effect of Ca2+ on in vivo gene transfer by electroporation is specific, ie, the inhibitory effect may be related to the cell membrane properties after electroporation and the subsequent resealing event.     

Receptor subtype involved in α1-adrenergic receptor-mediated Ca^2＋ signaling in cardiomyocytes

Aim： The enhancement of intracellular Ca^＋signaling in response to α1-adrenergic receptor （α1-AR） stimulation is an essential signal transduction event in the regulation of cardiac functions, such as cardiac growth, cardiac contraction, and cardiac adaptation to various situations. The present study was intended to determine the role（s） of the α1-AR subtype（s） in mediating this response. Methods： We evaluated the effects of subtype-specific agonists and antagonists of the α1- AR on the intracellular Ca^2＋ signaling of neonatal rat ventricular myocytes using a confocal microscope. Results： After being cultured for 48 h, the myocytes exhibited spontaneous local Ca^2＋ release, sparks, and global Ca^2＋ transients. The activation of the α1-AR with phenylephrine, a selective agonist of the α1-AR, dose-dependently increased the frequency of Ca^2＋ transients with an EC5o value of 2.3 larnol/L. Blocking the α1A-AR subtype with 5-methylurapidil （5-Mu） inhi- bited the stimulatory effect of phenylephrine with an IC50 value of 6.7 nmol/L. In contrast, blockade of the α1B-AR and α1D-AR subtypes with chloroethylclonidine and BMY 7378, respectively, did not affect the phenylephrine effect. Similarly, the local Ca^2＋ spark numbers were also increased by the activation of the α1-AR, and this effect could be abolished selectively by 5-Mu. More importantly, A61603, a novel selective α1A-AR agonist, mimicked the effects of phenylephrine, but with more potency （EC50 value =6.9 nmol/L） in the potentiation of Ca^2＋ transients, and blockade of the α1A-AR by 5-Mu caused abolishment of its effects. Conclusion： These results indicate that α1-adrenergic stimulation of intracellular Ca^2＋ activity is mediated selectively by the α1-AR.     

Interaction of heavy metals with membrane Ca^2＋ channels

The objective of our study was to determine if specific types of high voltage-activated Ca^2 channels,typically found in neurons were affected differentially by MeHg,Hg^2 and Pb^2 .Expression cDNA clones of α1C,α1B or α1E subunits coding for neuronal L-,N- and R- subtypes respectively,were combined with α2b δ and β3 Ca^2 channel subunits of human neuronal origin to transfect HEK293 cells.Current was measured using whole cell voltage clamp recording techniques.It the present studies,we conclude: (1)neurotoxic heavy metals such as MeHg,Hg^2 and Pb impair the function of voltage-gated Ca^2 channels at low μmolar to sub-μmolar concentrations-concentrations in the range of which are pathologically and environmentally relevant; (2)a particular metal,i.e.Pb^2 ,may inhibit function of phenotypically distince Ca^2 channels with variable potency; (3)different metals have differing “orders of potency” at inhibiting defined populations of Ca^2 channels; (4)for “susceptible populations” of patients with either underlying diseases or genetic alter ations of Ca^2 channel function,these metals may have heightened effectiveness.As such,for these populations,environmental toxic metals could produce a more dominant neurotoxicity.     

Characteristics of Ca^2＋-exocytosis coupling in isolated mouse pancreatic β cells

AIM: To characterize Ca2+-stimulated exocytosis in isolated mouse pancreatic beta cells. METHODS: An improved method was described for isolation of mouse pancreatic beta cells by collagenase P. The Ca2+ channel current and the membrane capacitance were examined by using the whole-cell patch clamp recording technique. RESULTS: Using depolarization and flash photolysis of caged Ca2+ to induce Ca2+-dependent exocytosis in beta cell from KM mouse, we have explored the characteristics of the Ca2+ channel current and the relationship between Ca2+ signals and exocytosis. The averaged peak Ca2+ current measured at +20 mV was -60+/-6 pA (n=13). CONCLUSION: We characterized three kinetically different pools of vesicles in mouse pancreatic beta cells, namely an immediately releasable pool, a readily releasable pool, and a reserve pool.     

Brain ischemia induces serine phosphorylation of neuronal nitric oxide synthase by Ca^2＋ ／ calmodulin-dependent protein kinase II in rat hippocampus

INTRODUCTIONConstitutively expressed nitric oxide synthase(NOS) has three isoforms, including neuronal NOS(nNOS), inducible NOS (iNOS), and endothelial NOS(eNOS), which has been isolated and cloned[1]. NOScatalyzed L-arginine to generate L-citruline and nitricoxide (NO), a gaseous messenger molecule, the over-production of which plays a critical role in glutamate-induced neurotoxicity after ischemia[2]. In mutant micedeficient in nNOS and subsequent NO production, inf-arct s…     

Toosendanin increases free-Ca^2＋ concentration in NG108-15 cells via L-type Ca^2＋ channels

To examine if toosendanin (TSN) affects intracellular free-Ca2 concentration ([Ca2~]i) in neuroblastomaxglioma hybrid cells (NG108-15 cells). METHODS: The [Ca2~]i was determined by laser-scanning confocal microscopic imaging technique in which Fluo-3 was used as Ca2~ indicator. RESULTS: TSN induced an increase in resting[Ca2 ]i and in high K~-evoked Ca2~ transient in differentiated NG108-15 cells. The TSN-induced increase in [Ca2 ]i was dose-dependent and disappeared in CdC12-, nifedipine-containing or Ca2~-free solution, and appeared after washing out the Ca2~ channel blockers or adding Ca2~. CONCLUSION: TSN increased [Ca2~]i in differentiated NG108-15 cells. The [Ca2 ]i enhancement was due to the influx of extracellular Ca2 and related to L-type Ca2 channels.     

MEK inhibitor PD98059 acutely inhibits synchronized spontaneous Ca^2＋ oscillations in cultured hippocampal networks

AIM: To investigate the changes in synchronized spontaneous Ca2+ oscillations induced by mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 at different concentrations in cultured hippocampal network. METHODS: Hippocampal neurons in culture for 1-2 weeks were used for this study. Spontaneous synaptic activities of these hippocampal neurons were examined by Ca2+ imaging using calcium-sensitive dye. MEK inhibitor PD98059 (10, 30, and 60 micromol/L) and SB202474 (10 and 60 micromol/L), a negative control for mitogen-activated protein kinase (MAPK) cascade study, were applied to the cells under the microscope while imaging was taking place. RESULTS: PD98059 at a lower concentration of 10 micromol/L had little effect on the Ca2+ oscillation. At the higher concentration of 30 micromol/L, 5 min after application of PD98059, the spike frequency was decreased to 25.38% +/-7.40% (mean+/-SEM, n=16, P<0.01 vs medium control) of that of the control period. At an even higher concentration of 60 micromol/L, 5 min after application of PD98059, the spike frequency was decreased to 14.53%+/-5.34% (mean+/-SEM, n=16, P< 0.01 vs medium control) of that of the control period. The spike amplitude underwent a corresponding decrease. However, the negative control SB202474 at concentrations of 10 and 60 micromol/L had little inhibition effect on the Ca2+ oscillation. CONCLUSION: These results indicate that PD98059 inhibits synchronized spontaneous Ca2+ oscillation through inhibition of MEK, which hints that the MAPK cascade is required to maintain synchronized spontaneous Ca2+ oscillation.     

Tetrandrine inhibits Ca^2＋-activated chloride channel in cultured human umbilical vein endothelial cells

INTRODUCTION Tetrandrine (Tet, 6,6',7,12-tetramethoxy-2,2'- dim-ethyl-berbaman) is a purified bis-benzylisoquinoline al-kaloid derivedfrom the root ofa Chinese herb (Stephaniatetrandra S Moore)[1,2]. It was first shown as an anti-hypertensive agent in both normal and hypertensivesubjects in 1950s[3,4]. The primary anti-hypertensiveaction of Tet is presumably due to its vasodilatoryproperty, which was confirmed both in vivo (15 mg/kg in conscious rats) and in vitro (1-100 μmol/L,effecti…     

Residue Phe266 in S5-S6 loop is not critical for Charybdotoxin binding to Ca^2＋-activated K^＋ （mSlo1） channels

AIM: To gain insight into the interaction between the Charybdotoxin (ChTX) and BK channels. METHODS: Site-directed mutagenesis was used to make two mutants: mSlo1-F266L and mSlo1-F266A. The two mutants were then expressed in Xenopus oocytes and their effects were tested on ChTX by electrophysiology experiments. RESULTS: We demonstrate an equilibrium dissociation constant Kd=3.1-4.2 nmol/L for both the mutants mSlo1-F266L and mSlo1-F266A similar to that of the wild-type mSlo1 Kd=3.9 nmol/L. CONCLUSION: The residue Phe266 does not play a crucial role in binding to ChTX, which is opposed to the result arising from the simulation of peptide-channel interaction.