Wortmannin Inhibits the Increase in Myofilament Ca2+ Sensitivity Induced by Cross-Talk of Endothelin-1 With Norepinephrine in Canine Ventricular Myocardium

Endothelin-1 (ET-1) modulates cardiac contractility by cross-talk with norepinephrine (NE) in canine ventricular myocardium. The present experiments were performed to investigate the influence of wortmannin that has inhibitory action on phosphatidylinositol 3-kinase (PI3-K) (IC₅₀ = 3 nM) and myosin light chain kinase (MLCK) (IC₅₀ = 200 nM) on Ca²⁺ signaling and the inotropic effects of ET-1 induced by cross-talk with NE. Experiments were carried out in isolated canine ventricular trabeculae and indo-1/AM–loaded single ventricular cardiomyocytes. ET-1 alone elicited a transient small negative inotropic effect (NIE). In the presence of NE at low (1 – 10 nM) and high (100 nM) concentrations, ET-1 induced a long-lasting positive inotropic effect (PIE) or a marked sustained NIE, respectively. Wortmannin up to 300 nM did not affect the contractility; and at 1 μM and higher, it decreased the basal contraction without suppressing Ca²⁺ transients. Wortmannin (1 μM) inhibited the long-lasting PIE of ET-1 without affecting the ET-1–induced increase in Ca²⁺ transients. Wortmannin at the same concentration did not affect the ET-1–induced transient and sustained NIE and the PIE mediated by β-adrenoceptor stimulation. These results imply that wortmannin exerts selective inhibitory action on the increase in myofilament Ca²⁺ sensitivity induced by cross-talk of ET-1 with NE probably through an inhibition of MLCK in canine ventricular myocardium.     

TASK1 (K(2P)3.1) K(+) channel inhibition by endothelin-1 is mediated through Rho kinase-dependent phosphorylation

BACKGROUND AND PURPOSE

TASK1 (K_2P3.1) two-pore-domain K⁺ channels contribute substantially to the resting membrane potential in human pulmonary artery smooth muscle cells (hPASMC), modulating vascular tone and diameter. The endothelin-1 (ET-1) pathway mediates vasoconstriction and is an established target of pulmonary arterial hypertension (PAH) therapy. ET-1-mediated inhibition of TASK1 currents in hPASMC is implicated in the pathophysiology of PAH. This study was designed to elucidate molecular mechanisms underlying inhibition of TASK1 channels by ET-1.

EXPERIMENTAL APPROACH

Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record TASK1 currents from hPASMC and Xenopus oocytes.

KEY RESULTS

ET-1 inhibited TASK1-mediated I_KN currents in hPASMC, an effect attenuated by Rho kinase inhibition with Y-27632. In Xenopus oocytes, TASK1 current reduction by ET-1 was mediated by endothelin receptors ET_A (IC₅₀= 0.08 nM) and ET_B (IC₅₀= 0.23 nM) via Rho kinase signalling. TASK1 channels contain two putative Rho kinase phosphorylation sites, Ser³³⁶ and Ser³⁹³. Mutation of Ser³⁹³ rendered TASK1 channels insensitive to ET_A- or ET_B-mediated current inhibition. In contrast, removal of Ser³³⁶ selectively attenuated ET_A-dependent TASK1 regulation without affecting the ET_B pathway.

CONCLUSIONS AND IMPLICATIONS

ET-1 regulated vascular TASK1 currents through ET_A and ET_B receptors mediated by downstream activation of Rho kinase and direct channel phosphorylation. The Rho kinase pathway in PASMC may provide a more specific therapeutic target in pulmonary arterial hypertension treatment.     

Modulation of cytosolic free calcium concentration by α1-adrenoceptors in rat atrial cells

Summary The effects of ₁-adrenoceptor stimulation by phenylephrine (PE) and -adrenoceptor stimulation by isoprenaline (ISO) on Ca²⁺ current (I_Ca) and free intracellular Ca²⁺ concentration ([Ca²⁺]_i) were studied in isolated atrial myocytes from rat hearts. PE did not significantly affect the magnitude of I_Ca, whereas large increases of peak I_Ca were observed in response to ISO. In electrically driven cells, PE evoked a concentration-dependent, gradual increase in diastolic [Ca²⁺]_i and, initially, an increase in the height of peak [Ca²⁺]_i transients. When the diastolic [Ca²⁺]_i was increased to a greater extent, the amplitude of [Ca²⁺]_i transients was decreased. Simultaneous measurements of [Ca²⁺]_i and membrane potential showed that the increase in diastolic [Ca²⁺]_i was associated with a depolarization of the membrane, and the greater amplitude of [Ca²⁺]_i transients with a prolongation of the action potential (AP). The PE-induced increase in diastolic [Ca²⁺]_i was eliminated when the cells were voltage-clamped at the original resting membrane potential (RP); under these conditions, an increase in [Ca²⁺]_i transients was observed in response to PE. ISO usually caused larger increases in the amplitude of [Ca²⁺]_i transients with only minor changes in diastolic [Ca²⁺]_i. These results suggest that PE and ISO increase the amplitude of [Ca²⁺]_i transients in rat atrium in different ways. The increase in [Ca²⁺]_i transients in response to -adrenoceptor stimulation is commonly thought to be mediated by a greater conductance of voltage-dependent Ca²⁺ channels causing a greater Ca²⁺ influx and a release of more Ca²⁺ from the sarcoplasmic reticulum during the AP. The increase in diastolic [Ca²⁺]_i in response to PE is probably a consequence of the depolarization of the membrane, possibly involving the voltage-dependent Na⁺-Ca²⁺ exchange mechanism. The increase in the amplitude of the [Ca²⁺]_i transients in response to PE may be ascribed both to the initial increase in diastolic [Ca²⁺]_i and the prolongation of the AP. Send offprint requests to H. Nawrath at the above address     

The effects of various drugs on the myocardial inotropic response

• 1.1. The signal transduction process mediated by cyclic AMP that leads to the characteristic positive inotropic effect (PIE) in association with a positive lusitropic effect (acceleration of rate of twitch relaxation) has been well established. Relationships between accumulation of cyclic AMP, changes in intracellular Ca²⁺ transients and the PIE differ, however, depending on the mechanism of particular drugs that affect different steps in the metabolism of cyclic AMP. Selective partial agonists of β₁-adrenoceptors and inhibitors of phosphodiesterase (PDE) III cause the accumulation of less cyclic AMP for a given PIE than does isoproterenol. In addition, in aequorin-microinjected canine ventricular muscle, selective inhibitors of PDE III, OPC 18790 and Org 9731, produced smaller decreases in the responsiveness of myofilaments to Ca²⁺ ions than isoproterenol, while a partial agonist of β₁-adrenoceptors, denopamine, elicits a decrease in Ca²⁺ responsiveness of the same extent as does isoproterenol.
• 2.2. Activation of myocardial α₁-adrenoceptors, as well as stimulation of receptors for endothelin and angiotensin II, which accelerates hydrolysis of phosphoinositide (PI) to result in production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) are associated with very similar inotropic regulation: (1) the dependence on the species of animals of induction of the PIE; (2) an excellent correlation between the extent of acceleration of hydrolysis of PI and the PIE; (3) isometric contraction curves associated with a negative lusitropic effect; (4) the PIE associated with increases in myofibrillar responsiveness to Ca²⁺ ions; and (5) the selective inhibition of the PIE by an activator of protein kinase C (PKC), phorbol 12,13-dibutyrate (PDBu), with little effect on the PIE of isoproterenol and Bay k 8644.
• 3.3. A novel class of cardiotonic agents, namely, Ca²⁺ sensitizers such as EMD 53998 and Org 30029, act on the Ca²⁺-binding site of troponin C, increasing the affinity of these sites for Ca²⁺ ions, or at the actin-myosin interface to facilitate the cycling of cross-bridges. These agents produce a PIE with little change or decrease in Ca²⁺ transients and may bring about a significant breakthrough in the development of drugs for reversal of myocardial failure in the treatment of congestive heart failure.

     

Functional and morphological properties of pericytes in suburothelial venules of the mouse bladder

Background and Purpose

In suburothelial venules of rat bladder, pericytes (perivascular cells) develop spontaneous Ca²⁺ transients, which may drive the smooth muscle wall to generate spontaneous venular constrictions. We aimed to further explore the morphological and functional characteristics of pericytes in the mouse bladder.

Experimental Approach

The morphological features of pericytes were investigated by electron microscopy and fluorescence immunohistochemistry. Changes in diameters of suburothelial venules were measured using video microscopy, while intracellular Ca²⁺ dynamics were visualized using Fluo-4 fluorescence Ca²⁺ imaging.

Key Results

A network of α-smooth muscle actin immunoreactive pericytes surrounded venules in the mouse bladder suburothelium. Scanning electron microscopy revealed that this network of stellate-shaped pericytes covered the venules, while transmission electron microscopy demonstrated that the venular wall consisted of endothelium and adjacent pericytes, lacking an intermediate smooth muscle layer. Pericytes exhibited spontaneous Ca²⁺ transients, which were accompanied by phasic venular constrictions. Nicardipine (1 μM) disrupted the synchrony of spontaneous Ca²⁺ transients in pericytes and reduced their associated constrictions. Residual asynchronous Ca²⁺ transients were suppressed by cyclopiazonic acid (10 μM), 2-aminoethoxydiphenyl borate (10 μM), U-73122 (1 μM), oligomycin (1 μM) and {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 (10 μM), but unaffected by ryanodine (100 μM) or YM-244769 (1 μM), suggesting that pericyte Ca²⁺ transients rely on Ca²⁺ release from the endoplasmic reticulum via the InsP₃ receptor and also require Ca²⁺ influx through store-operated Ca²⁺ channels.

Conclusions and Implications

The pericytes in mouse bladder can generate spontaneous Ca²⁺ transients and contractions, and thus have a fundamental role in promoting spontaneous constrictions of suburothelial venules.     

Cigarette smoke exposure up-regulates endothelin receptor B in human pulmonary artery endothelial cells: molecular and functional consequences

BACKGROUND AND PURPOSE

Pulmonary arteries from smokers and chronic obstructive pulmonary disease patients show abnormal endothelium-dependent vascular reactivity. We studied the effect of cigarette smoke extract (CSE) on endothelin receptor B (ET_B) expression in human pulmonary artery endothelial cells (HPAECs) and its role in endothelial dysfunction.

EXPERIMENTAL APPROACH

ET_B receptor expression was measured by real time RT-PCR, Western blot and immunofluorescence. Cell contraction, intracellular Ca²⁺, F/G-actin, RhoA activity, myosin light chain phosphorylation, ET, NO, thromboxane (Tx)A₂ and reactive oxygen species (ROS) were measured by traction microscopy, fluorescence microscopy, phalloidin fluorescence, colorimetric assay, Western blot, elisa and DCFDA fluorescence respectively.

KEY RESULTS

Cigarette smoke extract dose-dependently increased ET_B receptor expression in HPAECs after 24 h incubation. CSE-induced ET_B expression was attenuated by bosentan, the ET_B receptor antagonist BQ788, the Rho kinase antagonist Y27632 and the antioxidant N-acetylcysteine. A monoclonal antibody to ET-1 prevented CSE-induced ET_B receptor overexpression. Twenty-four hour exposure to ET-1 dose-dependently increased ET_B receptor expression, mimicking the effect of CSE. CSE-induced ET_B receptor overexpression caused greater cell contraction; increased intracellular Ca²⁺; increased F/G-actin and RhoA activity; increased myosin light chain phosphorylation; augmented TxA₂ and ROS production; and decreased NO after acute ET-1 (10 nM). These effects were attenuated by bosentan, BQ788, Y27632 and N-acetylcysteine.

CONCLUSIONS AND IMPLICATION

Cigarette smoke extract induced ET_B receptor overexpression by a feed forward mechanism mediated partly by ET release, promoting HPAEC dysfunction and attenuated by ET_B receptor blockade, Rho kinase and ROS inhibition. These results provide support for the use of bosentan in CS-related endothelial dysfunction.     

cAMP- and Ca²(+) /calmodulin-dependent protein kinases mediate inotropic, lusitropic and arrhythmogenic effects of urocortin 2 in mouse ventricular myocytes

BACKGROUND AND PURPOSE

Urocortin 2 is beneficial in heart failure, but the underlying cellular mechanisms are not completely understood. Here we have characterized the functional effects of urocortin 2 on mouse cardiomyocytes and elucidated the underlying signalling pathways and mechanisms.

EXPERIMENTAL APPROACH

Mouse ventricular myocytes were field-stimulated at 0.5 Hz at room temperature. Fractional shortening and [Ca²⁺]_i transients were measured by an edge detection and epifluorescence system respectively. Western blots were carried out on myocyte extracts with antibodies against total phospholamban (PLN) and PLN phosphorylated at serine-16.

KEY RESULTS

Urocortin 2 elicited time- and concentration-dependent positive inotropic and lusitropic effects (EC₅₀: 19 nM) that were abolished by antisauvagine-30 (10 nM, n = 6), a specific antagonist of corticotrophin releasing factor (CRF) CRF₂ receptors. Urocortin 2 (100 nM) increased the amplitude and decreased the time constant of decay of the underlying [Ca²⁺]_i transients. Urocortin 2 also increased PLN phosphorylation at serine-16. H89 (2 µM) or KT5720 (1 µM), two inhibitors of protein kinase A (PKA), as well as KN93 (1 µM), an inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), suppressed the urocortin 2 effects on shortening and [Ca²⁺]_i transients. In addition, urocortin 2 also elicited arrhythmogenic events consisting of extra cell shortenings and extra [Ca²⁺]_i increases in diastole. Urocortin 2-induced arrhythmogenic events were significantly reduced in cells pretreated with KT5720 or KN93.

CONCLUSIONS AND IMPLICATIONS

Urocortin 2 enhanced contractility in mouse ventricular myocytes via activation of CRF₂ receptors in a cAMP/PKA- and Ca²⁺/CaMKII-dependent manner. This enhancement was accompanied by Ca²⁺-dependent arrhythmogenic effects mediated by PKA and CaMKII.     

Activation of muscarinic receptors elicits inotropic responses in ventricular muscle from rats with heart failure through myosin light chain phosphorylation

Background and purpose:

Muscarinic stimulation increases myofilament Ca²⁺ sensitivity with no apparent inotropic response in normal rat myocardium. Increased myofilament Ca²⁺ sensitivity is a molecular mechanism promoting increased contractility in failing cardiac tissue. Thus, muscarinic receptor activation could elicit inotropic responses in ventricular myocardium from rats with heart failure, through increasing phosphorylation of myosin light chain (MLC).

Experimental approach:

Contractile force was measured in left ventricular papillary muscles from male Wistar rats, 6 weeks after left coronary artery ligation or sham surgery. Muscles were also frozen, and MLC-2 phosphorylation level was quantified.

Key results:

Carbachol (10 µmol·L⁻¹) evoked a positive inotropic response only in muscles from rats with heart failure approximating 36% of that elicited by 1 µmol·L⁻¹ isoproterenol (20 ± 1.5% and 56 ± 6.1% above basal respectively). Carbachol-evoked inotropic responses did not correlate with infarction size but did correlate with increased left ventricular end diastolic pressure, heart weight/body weight ratio and lung weight, primary indicators of the severity of heart failure. Only muscarinic receptor antagonists selective for M₂ receptors antagonized carbachol-mediated inotropic effects with the expected potency. Carbachol-evoked inotropic responses and increase in phosphorylated MLC-2 were attenuated by MLC kinase (ML-9) and Rho-kinase inhibition (Y-27632), and inotropic responses were abolished by Pertussis toxin pretreatment.

Conclusion and implications:

In failing ventricular muscle, muscarinic receptor activation, most likely via M₂ receptors, provides inotropic support by increasing MLC phosphorylation and consequently, myofilament Ca²⁺ sensitivity. Enhancement of myofilament Ca²⁺ sensitivity, representing a less energy-demanding mechanism of inotropic support may be particularly advantageous in failing hearts.     

Contribution of Rho-kinase to membrane excitability of murine colonic smooth muscle

BACKGROUND AND PURPOSE

The Rho-kinase pathway regulates agonist-induced contractions in several smooth muscles, including the intestine, urinary bladder and uterus, via dynamic changes in the Ca²⁺ sensitivity of the contractile apparatus. However, there is evidence that Rho-kinase also modulates other cellular effectors such as ion channels.

EXPERIMENTAL APPROACH

We examined the regulation of colonic smooth muscle excitability by Rho-kinase using conventional microelectrode recording, isometric force measurements and patch-clamp techniques.

KEY RESULTS

The Rho-kinase inhibitors, Y-27632 and H-1152, decreased nerve-evoked on- and off-contractions elicited at a range of frequencies and durations. The Rho-kinase inhibitors decreased the spontaneous contractions and the responses to carbachol and substance P independently of neuronal inputs, suggesting Y-27632 acts directly on smooth muscle. The Rho-kinase inhibitors significantly reduced the depolarization in response to carbachol, an effect that cannot be due to regulation of Ca²⁺ sensitization. Patch-clamp experiments showed that Rho-kinase inhibitors reduce GTPγS-activated non-selective cation currents.

CONCLUSIONS AND IMPLICATIONS

The Rho-kinase inhibitors decreased contractions evoked by nerve stimulation, carbachol and substance P. These effects were not solely due to inhibition of the Ca²⁺ sensitization pathway, as the Rho-kinase inhibitors also inhibited the non-selective cation conductances activated by excitatory transmitters. Thus, Rho-kinase may regulate smooth muscle excitability mechanisms by regulating non-selective cation channels as well as changing the Ca²⁺ sensitivity of the contractile apparatus.     

Distinct effects of CGRP on typical and atypical smooth muscle cells involved in generating spontaneous contractions in the mouse renal pelvis

Background and purpose:

We investigated the cellular mechanisms underlying spontaneous contractions in the mouse renal pelvis, regulated by calcitonin gene-related peptide (CGRP).

Experimental approach:

Spontaneous contractions, action potentials and Ca²⁺ transients in typical and atypical smooth muscle cells (TSMCs and ATSMCs) within the renal pelvis wall were recorded separately using tension and intracellular microelectrode recording techniques and Fluo-4 Ca²⁺ imaging. Immunohistochemical and electron microscopic studies were also carried out.

Key results:

Bundles of CGRP containing transient receptor potential cation channel, subfamily V, member 1-positive sensory nerves were situated near both TSMCs and ATSMCs. Nerve stimulation reduced the frequency but augmented the amplitude and duration of spontaneous phasic contractions, action potentials and Ca²⁺ transients in TSMCs. CGRP and agents increasing internal cyclic adenosine monophosphate (cAMP) mimicked the nerve-mediated modulation of TSMC activity and suppressed ATSMCs Ca²⁺ transients. Membrane hyperpolarization induced by CGRP or cAMP stimulators was blocked by glibenclamide, while their negative chronotropic effects were less affected. Glibenclamide enhanced TSMC Ca²⁺ transients but inhibited ATSMC Ca²⁺ transients, while both 5-hydroxydecanoate and diazoxide, a blocker and opener of mitochondrial ATP-sensitive K⁺ channels, respectively, reduced the Ca²⁺ transient frequency in both TSMCs and ATSMCs. Inhibition of mitochondrial function blocked ATSMCs Ca²⁺ transients and inhibited spontaneous excitation of TSMCs.

Conclusions and implications:

The negative chronotropic effects of CGRP result primarily from suppression of ATSMC Ca²⁺ transients rather than opening of plasmalemmal ATP-sensitive K⁺ channels in TSMCs. The positive inotropic effects of CGRP may derive from activation of TSMC L-type Ca²⁺ channels. Mitochondrial Ca²⁺ handling in ATSMCs also plays a critical role in generating Ca²⁺ transients.     

Role of perinuclear mitochondria in the spatiotemporal dynamics of spontaneous Ca2+ waves in interstitial cells of Cajal-like cells of the rabbit urethra

BACKGROUND AND PURPOSE

Although spontaneous Ca²⁺ waves in interstitial cells of Cajal (ICC)-like cells (ICC-LCs) primarily arise from endoplasmic reticulum (ER) Ca²⁺ release, the interactions among mitochondrial Ca²⁺ buffering, cellular energetics and ER Ca²⁺ release in determining the spatiotemporal dynamics of intracellular Ca²⁺ remain to be elucidated.

EXPERIMENTAL APPROACH

Spontaneous Ca²⁺ transients in freshly isolated ICC-LCs of the rabbit urethra were visualized using fluo-4 Ca²⁺ imaging, while the intracellular distribution of mitochondria was viewed with MitoTracker Red.

KEY RESULTS

Spontaneous Ca²⁺ waves invariably originated from the perinuclear region where clusters of mitochondria surround the nucleus. Perinuclear Ca²⁺ dynamics were characterized by a gradual rise in basal Ca²⁺ that preceded each regenerative Ca²⁺ transient. Caffeine evoked oscillatory Ca²⁺ waves originating from anywhere within ICC-LCs. Ryanodine or cyclopiazonic acid prevented Ca²⁺ wave generation with a rise in basal Ca²⁺, and subsequent caffeine evoked a single rudimentary Ca²⁺ transient. Inhibition of glycolysis with 2-deoxy-glucose or carbonyl cyanide 3-chlorophenylhydrazone, a mitochondrial protonophore, increased basal Ca²⁺ and abolished Ca²⁺ waves. However, caffeine still induced oscillatory Ca²⁺ transients. Mitochondrial Ca²⁺ uptake inhibition with RU360 attenuated Ca²⁺ wave amplitudes, while mitochondrial Ca²⁺ efflux inhibition with {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 suppressed the initial Ca²⁺ rise to reduce Ca²⁺ wave frequency.

CONCLUSIONS AND IMPLICATIONS

Perinuclear mitochondria in ICC-LCs play a dominant role in the spatial regulation of Ca²⁺ wave generation and may regulate ER Ca²⁺ release frequency by buffering Ca²⁺ within microdomains between both organelles. Glycolysis inhibition reduced mitochondrial Ca²⁺ buffering without critically disrupting ER function. Perinuclear mitochondria may function as sensors of intracellular metabolites.     

Oxidative species increase arginase activity in endothelial cells through the RhoA/Rho kinase pathway

BACKGROUND AND PURPOSE

NO produced by endothelial NOS is needed for normal vascular function. During diabetes, aging and hypertension, elevated levels of arginase can compete with NOS for available l-arginine, reducing NO and increasing superoxide (O₂^.−) production via NOS uncoupling. Elevated O₂^.− combines with NO to form peroxynitrite (ONOO^-), further reducing NO. Oxidative species increase arginase activity, but the mechanism(s) involved are not known. Our study determined the mechanism involved in peroxynitrite and hydrogen peroxide-induced enhancement in endothelial arginase activity. We hypothesized that oxidative species increase arginase activity through PKC-activated RhoA/Rho kinase (ROCK) pathway.

EXPERIMENTAL APPROACH

Arginase activity/expression was analysed in bovine aortic endothelial cells (BAEC) treated with an ONOO^- generator (SIN-1) or H₂O₂. Pretreatment with inhibitors of Rho kinase (Y-27632) or PKC (Gö6976) was used to investigate the mechanism involved in arginase activation.

KEY RESULTS

Exposure to SIN-1 (25 µM, 24 h) or H₂O₂ (25 µM, 8 h) increased arginase I expression and arginase activity (35% and 50%, respectively), which was prevented by ROCK inhibitor, Y-27632, PKC inhibitor, Gö6976 or siRNA to p115-Rho GEF. There was an early activation of p115-Rho GEF (SIN-1, 2 h; H₂O₂, 1 h) and Rho A (SIN-1, 4 h; H₂O₂, 1 h) that was prevented by using the PKC inhibitor. Exposure to SIN-1 and H₂O₂ also reduced NOS activity, which was blocked by pretreatment with p115-RhoGEF siRNA.

CONCLUSIONS AND IMPLICATIONS

Our data indicate that the oxidative species ONOO^- and H₂O₂ increase arginase activity/expression through PKC-mediated activation of RhoA/Rho kinase pathway.     

Potentiation by endothelin-1 of Ca2+ sensitivity of contractile elements depends on Ca2+ influx through L-type Ca2+ channels in the canine cerebral artery

• 1.1. Endothelin-1 (ET-1) contracted canine cerebral artery in a concentration-dependent manner with an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), and at higher concentrations it produced a greater contraction with a smaller increase in [Ca²⁺]_i.
• 2.2. Ca²⁺ channel antagonist such as d-cis-diltiazem inhibited the tension more effectively than the [Ca²⁺]_i increased by ET-1.
• 3.3. In Ca²⁺-free solution containing 0.2 mM EGTA, ET-1 elicited a transient increase in [Ca²⁺]_i and tension.
• 4.4. In the Staphylococcus aureus α-toxin-permeabilized artery, ET-1 shifted the pCa-tension relationship leftwards in the presence of GTP.
• 5.5. These findings suggest that ET-I contracts the canine cerebral artery by increasing not only the Ca²⁺ influx through L-type Ca²⁺ channels, but also Ca²⁺ release from the intracellular storage sites, and also Ca²⁺ sensitivity of contractile elements. The degree of Ca²⁺ sensitivity is strongly affected by [Ca²⁺]_i which is increased by the Ca²⁺ influx through L-type Ca²⁺ channels.

     

Independent [Ca2+]i increases and cell proliferation induced by the carcinogen safrole in human oral cancer cells

The effect of the carcinogen safrole on intracellular Ca²⁺ movement and cell proliferation has not been explored previously. The present study examined whether safrole could alter Ca²⁺ handling and growth in human oral cancer OC2 cells. Cytosolic free Ca²⁺ levels ([Ca²⁺]_i) in populations of cells were measured using fura-2 as a fluorescent Ca²⁺ probe. Safrole at a concentration of 325 M started to increase [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced by 40% by removing extracellular Ca²⁺, and was decreased by 39% by nifedipine but not by verapamil or diltiazem. In Ca²⁺-free medium, after pretreatment with 650 M safrole, 1 M thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor) barely induced a [Ca²⁺]_i rise; in contrast, addition of safrole after thapsigargin treatment induced a small [Ca²⁺]_i rise. Neither inhibition of phospholipase C with 2 M U73122 nor modulation of protein kinase C activity affected safrole-induced Ca²⁺ release. Overnight incubation with 1 M safrole did not alter cell proliferation, but incubation with 10–1000 M safrole increased cell proliferation by 60±10%. This increase was not reversed by pre-chelating Ca²⁺ with 10 M of the Ca²⁺ chelator BAPTA. Collectively, the data suggest that in human oral cancer cells, safrole induced a [Ca²⁺]_i rise by causing release of stored Ca²⁺ from the endoplasmic reticulum in a phospholipase C- and protein kinase C-independent fashion and by inducing Ca²⁺ influx via nifedipine-sensitive Ca²⁺ entry. Furthermore, safrole can enhance cell growth in a Ca²⁺-independent manner.     

Epigallocatechin-3-gallate increases intracellular [Ca<Superscript>2+</Superscript>] in U87 cells mainly by influx of extracellular Ca<Superscript>2+</Superscript> and partly by release of intracellular stores

Green tea has been receiving considerable attention as a possible preventive agent against cancer and cardiovascular disease. Epigallocatechin-3-gallate (EGCG) is a major polyphenol component of green tea. Using digital calcium imaging and an assay for [³H]-inositol phosphates, we determined whether EGCG increases intracellular [Ca²⁺] ([Ca²⁺]_i) in non-excitable human astrocytoma U87 cells. EGCG induced concentration-dependent increases in [Ca²⁺]_i. The EGCG-induced [Ca²⁺]_i increases were reduced to 20.9% of control by removal of extracellular Ca²⁺. The increases were also inhibited markedly by treatment with the non-specific Ca²⁺ channel inhibitors cobalt (3 mM) for 3 min and lanthanum (1 mM) for 5 min. The increases were not significantly inhibited by treatment for 10 min with the L-type Ca²⁺ channel blocker nifedipine (100 nM). Treatment with the inhibitor of endoplasmic reticulum Ca²⁺-ATPase thapsigargin (1 µM) also significantly inhibited the EGCG-induced [Ca²⁺]_i increases. Treatment for 15 min with the phospholipase C (PLC) inhibitor neomycin (300 µM) attenuated the increases significantly, while the tyrosine kinase inhibitor genistein (30 µM) had no effect. EGCG increased [³H]-inositol phosphates formation via PLC activation. Treatment for 10 min with mefenamic acid (100 µM) and flufenamic acid (100 µM), derivatives of diphenylamine-2-carboxylate, blocked the EGCG-induced [Ca²⁺]_i increase in non-treated and thapsigargin-treated cells but indomethacin (100 µM) did not affect the increases. Collectively, these data suggest that EGCG increases [Ca²⁺]_i in non-excitable U87 cells mainly by eliciting influx of extracellular Ca²⁺ and partly by mobilizing intracellular Ca²⁺ stores by PLC activation. The EGCG-induced [Ca²⁺]_i influx is mediated mainly through channels sensitive to diphenylamine-2-carboxylate derivatives.     

Role of sarcoplasmic reticulum Ca<Superscript>2+</Superscript> content in Ca<Superscript>2+</Superscript> entry of bovine airway smooth muscle cells

Depletion of intracellular Ca²⁺ stores induces the opening of an unknown Ca²⁺ entry pathway to the cell. We measured the intracellular free-Ca²⁺ concentration ([Ca²⁺]i) at different sarcoplasmic reticulum (SR) Ca²⁺ content in fura-2-loaded smooth muscle cells isolated from bovine tracheas. The absence of Ca²⁺ in the extracellular medium generated a time-dependent decrement in [Ca²⁺]i which was proportional to the reduction in the SR-Ca²⁺ content. This SR-Ca²⁺ level was indirectly determined by measuring the amount of Ca²⁺ released by caffeine. Ca²⁺ restoration at different times after Ca²⁺-free incubation (2, 4, 6 and 10 min) induced an increment of [Ca²⁺]i. This increase in [Ca²⁺]i was considered as Ca²⁺ entry to the cell. The rate of this entry was slow (~0.3 nM/s) when SR-Ca²⁺ content was higher than 50% (2 and 4 min in Ca²⁺-free medium), and significantly (p<0.01) accelerated (>1.0 nM/s) when SR-Ca²⁺ content was lower than 50% (6 and 10 min in Ca²⁺-free medium). Thapsigargin significantly induced a higher rate of this Ca²⁺ entry (p<0.01). Variations in Ca²⁺ influx after SR-Ca²⁺ depletion were estimated more directly by a Mn²⁺ quench approach. Ca²⁺ restoration to the medium 4 min after Ca²⁺ removal did not modify the Mn²⁺ influx. However, when Ca²⁺ was added after 10 min in Ca²⁺-free medium, an increment of Mn²⁺ influx was observed, corroborating an increase in Ca²⁺ entry. The fast Ca²⁺ influx was Ni²⁺ sensitive but was not affected by other known capacitative Ca²⁺ entry blockers such as La³⁺, Mg²⁺, SKF 96365 and 2-APB. It was also not affected by the blockage of L-type Ca2⁺ channels with methoxyverapamil or by the sustained K⁺-induced depolarisation. The slow Ca²⁺ influx was only sensitive to SKF 96365. In conclusion, our results indicate that in bovine airway smooth muscle cells Ca²⁺ influx after SR-Ca²⁺ depletion has two rates: A) The slow Ca²⁺ influx, which occurred in cells with more than 50% of their SR-Ca²⁺ content, is sensitive to SKF 96365 and appears to be a non-capacitative Ca²⁺ entry; and B) The fast Ca²⁺ influx, observed in cells with less than 50% of their SR-Ca²⁺ content, is probably a capacitative Ca²⁺ entry and was only Ni²⁺-sensitive.     

精氨酸加压素对失血性休克大鼠心肌收缩力的影响及与Rho激酶的关系

目的 探讨精氨酸加压素(AVP)对失血性休克大鼠心肌收缩力的影响及与Rho激酶的关系。方法 失血性休克大鼠的离体乳头肌分别用AVP 0.1 μmol·L^-1 和 Y-27632 10 μmol·L^-1预孵育,或Y-27632+AVP合用时,先用Y-27632 孵育10 min后,再加入AVP作用10 min。平衡30 min后依次用含异丙肾上腺素（Iso）1和10 μmol·L^-1, 0.1, 1, 10 mmol·L^-1的H-K液灌流乳头肌,观察加入Iso前后收缩力变化。制备离体心脏,稳定后,进行相应再灌流30 min,检测左心室收缩压（LVSP）和左心室压力上升或下降的最大速率（±dp/dt_max）。结果 失血性休克2 h,经Iso 0.1,1 及10 mmol·L^-1灌流后,离体乳头肌收缩力显著低于假手术组（P<0.05）,AVP 0.1 μmol·L^-1预孵育可明显升高乳头肌对心肌的收缩力;与休克模型组相比,在Iso 1和10 mmol·L^-1灌流后,乳头肌收缩力明显升高(P<0.05);Y-27632 10 μmol·L^-1离体乳头肌收缩力无明显影响,但Y-27632+AVP可明显降低由AVP引起的乳头肌收缩力的增加。失血性休克2 h后,离体心脏血流动力学指标明显降低,AVP可明显改善休克模型大鼠离体心脏的血流动力学指标,同时Y-27632可明显拮抗由AVP引起休克大鼠离体心脏血流动力学指标的增加。结论 AVP可改善失血性休克所致的心肌收缩力的降低,其机制与其激活Rho 激酶有关。     

Evidence for the existence of P2Y<Subscript>1,2,4</Subscript> receptor subtypes in HEK-293 cells: reactivation of P2Y<Subscript>1</Subscript> receptors after repetitive agonist application

ATP, ADPS and UTP induced a comparable rise in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in HEK-293 cells using fura-2 microfluorimetry. The responses persisted in Ca²⁺-free medium, but were abolished following depletion of intracellular Ca²⁺ stores by cyclopiazonic acid. Cross-desensitisation experiments demonstrated that exposure to ADPS has no marked effect on UTP-induced [Ca²⁺]_i transients and vice versa. Whereas the P2Y₁ receptor-selective antagonist 2-deoxy-N⁶-methyladenosine 3,5-diphosphate (MRS 2179) abolished the responses to ADPS, it decreased and did not alter the responses to ATP and UTP respectively. Although the P2Y₁/P2Y₄ receptor-preferential antagonist pyridoxalphosphate-6-azophenyl-2,4-disulphonic acid (PPADS) abolished the responses to ADPS, and decreased those to ATP, it also depressed the UTP-induced [Ca²⁺]_i transients. Suramin, an antagonist with preference for P2Y₂ receptors decreased both the ATP- and UTP-induced [Ca²⁺]_i reactions. After numerous splittings, HEK-293 cells failed to react to ADPS; however, repeated superfusion with this P2Y₁ receptor agonist restored the [Ca²⁺]_i signals. In agreement with the functional data, real-time polymerase chain reaction and immunocytochemical studies indicated the presence of P2Y₁, P2Y₂ and P2Y₄ receptors. Our findings raise doubt with respect to the reliability of HEK-293 cells as expression systems for recombinant P2X receptors, because of a possible functional interaction with endogenous P2Y receptors.     

The negative inotropic action of canrenone is mediated by L-type calcium current blockade and reduced intracellular calcium transients

Background and purpose:

Adding spironolactone to standard therapy in heart failure reduces morbidity and mortality, but the underlying mechanisms are not fully understood. We analysed the effect of canrenone, the major active metabolite of spironolactone, on myocardial contractility and intracellular calcium homeostasis.

Experimental approach:

Left ventricular papillary muscles and cardiomyocytes were isolated from male Wistar rats. Contractility of papillary muscles was assessed with force transducers, Ca²⁺ transients by fluorescence and Ca²⁺ fluxes by electrophysiological techniques.

Key results:

Canrenone (300–600 µmol·L⁻¹) reduced developed tension, maximum rate of tension increase and maximum rate of tension decay of papillary muscles. In cardiomyocytes, canrenone (50 µmol·L⁻¹) reduced cell shortening and L-type Ca²⁺ channel current, whereas steady-state activation and inactivation, and reactivation curves were unchanged. Canrenone also decreased the Ca²⁺ content of the sarcoplasmic reticulum, intracellular Ca²⁺ transient amplitude and intracellular diastolic Ca²⁺ concentration. However, the time course of [Ca²⁺]_i decline during transients evoked by caffeine was not affected by canrenone.

Conclusion and implications:

Canrenone reduced L-type Ca²⁺ channel current, amplitude of intracellular Ca²⁺ transients and Ca²⁺ content of sarcoplasmic reticulum in cardiomyocytes. These changes are likely to underlie the negative inotropic effect of canrenone.     

PKC-Independent Stimulation of Cardiac Na+/Ca2+ Exchanger by Staurosporine

[Ca²⁺]_i transients by reverse mode of cardiac Na⁺/Ca²⁺ exchanger (NCX1) were recorded in fura-2 loaded BHK cells with stable expression of NCX1. Repeated stimulation of reverse NCX1 produced a long-lasting decrease of Ca²⁺ transients (''rundown''). Rundown of NCX1 was independent of membrane PIP₂ depletion. Although the activation of protein kinase C (PKC) was observed during the Ca²⁺ transients, neither a selective PKC inhibitor (calphostin C) nor a PKC activator (PMA) changed the degrees of rundown. By comparison, a non-specific PKC inhibitor, staurosporine (STS), reversed rundown in a dose-dependent and reversible manner. The action of STS was unaffected by pretreatment of the cells with calphostin C, PMA, or forskolin. Taken together, the results suggest that the stimulation of reverse NCX1 by STS is independent of PKC and/or PKA inhibition.