Istaroxime stimulates SERCA2a and accelerates calcium cycling in heart failure by relieving phospholamban inhibition

Background and Purpose

Calcium handling is known to be deranged in heart failure. Interventions aimed at improving cell Ca²⁺ cycling may represent a promising approach to heart failure therapy. Istaroxime is a new luso-inotropic compound that stimulates cardiac contractility and relaxation in healthy and failing animal models and in patients with acute heart failure (AHF) syndrome. Istaroxime is a Na-K ATPase inhibitor with the unique property of increasing sarcoplasmic reticulum (SR) SERCA2a activity as shown in heart microsomes from humans and guinea pigs. The present study addressed the molecular mechanism by which istaroxime increases SERCA2a activity.

Experimental Approach

To study the effect of istaroxime on SERCA2a-phospholamban (PLB) complex, we applied different methodologies in native dog healthy and failing heart preparations and heterologous canine SERCA2a/PLB co-expressed in Spodoptera frugiperda (Sf21) insect cells.

Key Results

We showed that istaroxime enhances SERCA2a activity, Ca²⁺ uptake and the Ca²⁺-dependent charge movements into dog healthy and failing cardiac SR vesicles. Although not directly demonstrated, the most probable explanation of these activities is the displacement of PLB from SERCA2a.E2 conformation, independently from cAMP/PKA. We propose that this displacement may favour the SERCA2a conformational transition from E2 to E1, thus resulting in the acceleration of Ca²⁺ cycling.

Conclusions and Implications

Istaroxime represents the first example of a small molecule that exerts a luso-inotropic effect in the failing human heart through the stimulation of SERCA2a ATPase activity and the enhancement of Ca²⁺ uptake into the SR by relieving the PLB inhibitory effect on SERCA2a in a cAMP/PKA independent way.     

Diadenosine pentaphosphate is a potent activator of cardiac ryanodine receptors revealing a novel high-affinity binding site for adenine nucleotides

Background and purpose:

Diadenosine polyphosphates are normally present in cells at low levels, but significant increases in concentrations can occur during cellular stress. The aim of this study was to investigate the effects of diadenosine pentaphosphate (Ap5A) and an oxidized analogue, oAp5A on the gating of sheep cardiac ryanodine receptors (RyR2).

Experimental approach:

RyR2 channel function was monitored after incorporation into planar bilayers under voltage-clamp conditions.

Key results:

With10 µmol·L⁻¹ cytosolic Ca²⁺, a significant ‘hump’ or plateau at the base of the dose–response relationship to Ap5A was revealed. Open probability (Po) was significantly increased to a plateau of approximately 0.2 in the concentration range 100 pmol·L⁻¹–10 µmol·L⁻¹. High Po values were observed at >10 µmol·L⁻¹ Ap5A, and Po values close to 1 could be achieved. Nanomolar levels of ATP and adenosine also revealed a hump at the base of the dose–response relationships, although GTP did not activate at any concentration, indicating a common, high-affinity binding site on RyR2 for adenine-based compounds. The oxidized analogue, oAp5A, did not significantly activate RyR2 via the high-affinity binding site; however, it could fully open the channel with an EC₅₀ of 16 µmol·L⁻¹ (Ap5A EC₅₀ = 140 µmol·L⁻¹). Perfusion experiments suggest that oAp5A and Ap5A dissociate slowly from their binding sites on RyR2.

Conclusions and implications:

The ability of Ap5A compounds to increase Po even in the presence of ATP and their slow dissociation from the channel may enable these compounds to act as physiological regulators of RyR2, particularly under conditions of cellular stress.     

Ca2+-calmodulin can activate and inactivate cardiac ryanodine receptors

Background and purpose:

Ca²⁺-calmodulin (Ca²⁺CaM) is widely accepted as an inhibitor of cardiac ryanodine receptors (RyR2); however, the effects of physiologically relevant CaM concentrations have not been fully investigated.

Experimental approach:

We investigated the effects of low concentrations of Ca²⁺CaM (50–100 nmol·L⁻¹) on the gating of native sheep RyR2, reconstituted into bilayers. Suramin displaces CaM from RyR2 and we have used a gel-shift assay to provide evidence of the mechanism underlying this effect. Finally, using suramin to displace endogenous CaM from RyR2 in permeabilized cardiac cells, we have investigated the effects of 50 nmol·L⁻¹ CaM on sarcoplasmic reticulum (SR) Ca²⁺-release.

Key results:

Ca²⁺CaM activated or inhibited single RyR2, but activation was much more likely at low (50–100 nmol·L⁻¹) concentrations. Also, suramin displaced CaM from a peptide of the CaM binding domain of RyR2, indicating that, like the skeletal isoform (RyR1), suramin directly competes with CaM for its binding site on the channel. Pre-treatment of rat permeabilized ventricular myocytes with suramin to displace CaM, followed by addition of 50 nmol·L⁻¹ CaM to the mock cytoplasmic solution caused an increase in the frequency of spontaneous Ca²⁺-release events. Application of caffeine demonstrated that 50 nmol·L⁻¹ CaM reduced SR Ca²⁺ content.

Conclusions and implications:

We describe for the first time how Ca²⁺CaM is capable, not only of inactivating, but also of activating RyR2 channels in bilayers in a CaM kinase II-independent manner. Similarly, in cardiac cells, CaM stimulates SR Ca²⁺-release and the use of caffeine suggests that this is a RyR2-mediated effect.     

In vitro and in vivo antineoplastic activity of a novel bromopyrrole and its potential mechanism of action

Background and purpose:

Many bromopyrrole compounds have been reported to have in vitro antineoplastic activity. In a previous study, we isolated N-(4, 5-dibromo-pyrrole-2-carbonyl)-L-amino isovaleric acid methyl ester (B6) from marine sponges. Here, we investigated the in vitro and in vivo antineoplastic activity of B6 and its potential mechanism.

Experimental approach:

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine the in vitro antineoplastic activity of B6. Flow cytometry, western blot analysis and morphological observations were used to investigate its mechanism of action. A mouse xenograft model was used to determine its in vivo activity.

Key results:

B6 inhibited the proliferation of various human cancer cells in vitro, with highest activity on LOVO and HeLa cells. B6 also exhibited significant growth inhibitory effects in vivo in a xenograft mouse model. Acute toxicity analysis suggested that B6 has low toxicity. B6-treated cells arrested in the G1 phase of the cell cycle and had an increased fraction of sub-G1 cells. In addition, the population of Annexin V-positive/propidium iodide-negative cells increased, indicating the induction of early apoptosis. Indeed, B6-treated cells exhibited morphologies typical of cells undergoing apoptosis. Western blotting showed cleaved forms of caspase-9 and caspase-3 in cells exposed to B6. Moreover, B6-promoted Ca²⁺ release and apoptosis was associated with elevated intracellular Ca²⁺concentration.

Conclusions and implications:

B6 has significant antineoplastic activity in vitro as well as in vivo. It inhibits tumour cell proliferation by arresting the cell cycle and inducing apoptosis. With its low toxicity, B6 represents a promising antineoplastic, primary compound.     

Ca2+ paradox injury mediated through TRPC channels in mouse ventricular myocytes

BACKGROUND AND PURPOSE

The Ca²⁺ paradox is an important phenomenon associated with Ca²⁺ overload-mediated cellular injury in myocardium. The present study was undertaken to elucidate molecular and cellular mechanisms for the development of the Ca²⁺ paradox.

EXPERIMENTAL APPROACH

Fluorescence imaging was performed on fluo-3 loaded quiescent mouse ventricular myocytes using confocal laser scanning microscope.

KEY RESULTS

The Ca²⁺ paradox was readily evoked by restoration of the extracellular Ca²⁺ following 10–20 min of nominally Ca²⁺-free superfusion. The Ca²⁺ paradox was significantly reduced by blockers of transient receptor potential canonical (TRPC) channels (2-aminoethoxydiphenyl borate, Gd³⁺, La³⁺) and anti-TRPC1 antibody. The sarcoplasmic reticulum (SR) Ca²⁺ content, assessed by caffeine application, gradually declined during Ca²⁺-free superfusion, which was further accelerated by metabolic inhibition. Block of SR Ca²⁺ leak by tetracaine prevented Ca²⁺ paradox. The Na⁺/Ca²⁺ exchange (NCX) blocker KB-R7943 significantly inhibited Ca²⁺ paradox when applied throughout superfusion period, but had little effect when added for a period of 3 min before and during Ca²⁺ restoration. The SR Ca²⁺ content was better preserved during Ca²⁺ depletion by KB-R7943. Immunocytochemistry confirmed the expression of TRPC1, in addition to TRPC3 and TRPC4, in mouse ventricular myocytes.

CONCLUSIONS AND IMPLICATIONS

These results provide evidence that (i) the Ca²⁺ paradox is primarily mediated by Ca²⁺ entry through TRPC (probably TRPC1) channels that are presumably activated by SR Ca²⁺ depletion; and (ii) reverse mode NCX contributes little to the Ca²⁺ paradox, whereas inhibition of NCX during Ca²⁺ depletion improves SR Ca²⁺ loading, and is associated with reduced incidence of Ca²⁺ paradox in mouse ventricular myocytes.     

On the mechanism of action of phenylephrine in rat atrial heart muscle

Both in rat left atrial heart and in aortic smooth muscle preparations, phenylephrine (PE) caused a concentration-dependent increase in force of contraction (F_c) in the presence of atenolol (10 mol/l), which was antagonized by phentolamine, prazosin and WB 4101 in a competitive manner. The pA₂ values of the antagonists in the cardiac tissue were 10–20fold lower than those in the rat thoracic aorta. In the spontaneously beating right atrium, PE exerted a positive chronotropic action, which was not significantly antagonized by phentolamine or prazosin. It is therefore assumed that the effects of phenylephrine in the left atrium and in the aorta are mediated by different subtypes of ₁-adrenoceptors, whereas the effects in the sino-atrial node are probably unrelated to ₁-adrenoceptors. To further elucidate the mechanisms of the positive inotropic effect of PE, action potential configuration and ⁴⁵Ca²⁺ fluxes were monitored in the rat left atrium. The increase in F_c by PE was associated with an increase in action potential duration (APD) and a reduction in resting membrane potential (RP). In the presence of (–)-devapamil (13888), the effects of PE on APD and RP persisted, whereas the increase in F_c was antagonized in a non-competitive manner. Forskolin (300 nmol/l) enhanced the positive inotropic effect of PE. PE exerted a significant increase in ⁴⁵CA²⁺ uptake in beating preparations, which was abolished in the presence of (–)13888 (1 mol/l). In addition to the PE-induced increase in ⁴⁵Ca²⁺ uptake, a decrease in ⁴⁵Ca²⁺ efflux was observed. Similarly, depolarization of the membrane by raising [K⁺]_o to 85 mmol/l revealed an increase in ⁴⁵Ca²⁺ uptake and a decrease in ⁴⁵Ca²⁺ efflux. The latter observations support the view that the membrane potential strongly determines the movement of ⁴⁵Ca²⁺ across the membrane. It is assumed that the ₁-adrenoceptor-mediated changes in APD and RP may enhance F_c, first, by increasing net Ca²⁺ entry from the extracellular space through voltage-dependent Ca²⁺ channels and, second, by decreasing Ca²⁺ efflux possibly via the Na ⁺/Ca²⁺ exchange mechanism.     

SERCA2 activity is involved in the CNP-mediated functional responses in failing rat myocardium

BACKGROUND AND PURPOSES

Myocardial C-type natriuretic peptide (CNP) levels are increased in heart failure. CNP can induce negative inotropic (NIR) and positive lusitropic responses (LR) in normal hearts, but its effects in failing hearts are not known. We studied the mechanism of CNP-induced NIR and LR in failing hearts and determined whether sarcoplasmatic reticulum Ca²⁺ ATPase2 (SERCA2) activity is essential for these responses.

EXPERIMENTAL APPROACH

Contractility, cGMP levels, Ca²⁺ transient amplitudes and protein phosphorylation were measured in left ventricular muscle strips or ventricular cardiomyocytes from failing hearts of Wistar rats 6 weeks after myocardial infarction.

KEY RESULTS

CNP increased cGMP levels, evoked a NIR and LR in muscle strips, and caused phospholamban (PLB) Ser¹⁶ and troponin I (TnI) Ser^23/24 phosphorylation in cardiomyocytes. Both the NIR and LR induced by CNP were reduced in the presence of a PKG blocker/cGMP analogue (Rp-8-Br-Pet-cGMPS) and the SERCA inhibitor thapsigargin. CNP increased the amplitude of the Ca²⁺ transient and increased SERCA2 activity in cardiomyocytes. The CNP-elicited NIR and LR were not affected by the L-type Ca²⁺ channel activator BAY-K8644, but were abolished in the presence of isoprenaline (induces maximal activation of cAMP pathway). This suggests that phosphorylation of PLB and TnI by CNP causes both a NIR and LR. The NIR to CNP in mouse heart was abolished 8 weeks after cardiomyocyte-specific inactivation of the SERCA2 gene.

CONCLUSIONS AND IMPLICATIONS

We conclude that CNP-induced PLB and TnI phosphorylation by PKG in concert mediate both a predictable LR as well as the less expected NIR in failing hearts.     

Mechanisms underlying the slow onset of action of a new dihydropyridine,NZ-105, on a cultured smooth muscle cell line

Summary The inhibitory effect of a new dihydropyridine derivative, (±)-2-[benzyl(phenyl)amino]ethyl-1,4-dihydro-2,6-dimethyl-5-(5,5-dimethyl-2-oxo-1,3,2-dioxaphosphorinan-2-yl)-4-(3-nitrophenyl)-3-pyridinecarboxylate hydrochloride (NZ-105), on whole cell Ca²⁺ current (I_Ca) in cultured vascular smooth muscle cells was investigated with the patch clamp technique. NZ-105 blocked I_Ca in a concentration-dependent manner when the command pulse ranged from +10 mV to –50 mV. The inhibitory effect of NZ-105 appeared at concentrations higher than 10 mol/l and it blocked I_Ca completely at a concentration of 1 nmol/l. The concentration which produced the half-maximal inhibitory effect was estimated to be around 20 mol/l. NZ-105 (500 pmol/l) completely blocked I_Ca elicited by depolarization to + 10 mV at a holding potential of –40 mV, whereas it blocked I_Ca by only 67% at a holding potential of –90 mV. NZ-105 (100 mol/l) shifted the steady-state inactivation curve by 40 mV to more negative potentials without affecting its slope factor. The blocking time constant of 500 mol/l NZ-105 was 57.6 + 9.9 s at a holding potential of –70 mV. These results indicate that NZ-105 has characteristics typical of dihydropyridines and binds to Ca²⁺ channels of vascular smooth muscle cells with a high affinity. They also suggested that the slow onset of its action is due to the slow binding of the drug to Ca²⁺ channels. Send offprint requests to S. Kokubun at the above address     

阿托伐他汀的肝毒性机制研究

目的 研究阿托伐他汀肝毒性损伤作用及机制。方法 将24只Wistar han雄鼠分为对照组和阿托伐他汀低（68.5mg/kg）、高剂量组（205.5 mg/kg）,按照10 mL/kg的药液体积给药,溶媒对照组ig等体积5% CMC-Na,连续ig 28 d。检测血清中天门冬氨酸氨基转移酶（AST）、丙氨酸氨基转移酶（ALT）、碱性磷酸酶（ALP）、尿素氮（BUN）和血肌酐（CRE）的含量,HE染色观察肝组织病理。在体外,HepG2细胞经传代培养后,给予阿托伐他汀干预24 h,检测细胞存活率,丙二醛（MDA）水平、Na⁺-K⁺-ATP酶和Ca²⁺-Mg²⁺-ATP酶活性及线粒体膜电位。结果 与对照组比较,阿托伐他汀高剂量组大鼠肝细胞弥漫性肿胀,核分裂多见,部分肝细胞极性消失,排列紊乱（P<0.05）。与对照组比较,阿托伐他汀高剂量组给药后血清中ALT和AST显著升高（P<0.05、0.01）。在体外,与对照组比较,阿托伐他汀125、250、500 μmol/L能明显抑制细胞存活率（P<0.05、0.001）。与对照组比较,阿托伐他汀500 μmol/L HepG2细胞MDA含量明显升高（P<0.01）。与对照组比较,阿托伐他汀125 μmol/L能使Na⁺-K⁺-ATP酶活性增强,500 μmol/L使Na⁺-K⁺-ATP酶活性降低（P<0.001）。与对照组比较,阿托伐他汀125、250、500 μmol/L均能使能使Ca²⁺-Mg²⁺-ATP酶活性降低（P<0.01,0.001）。与对照组比较,阿托伐他汀125、250、500 μmol/L均能降低线粒体膜电位（P<0.001）。结论 阿托伐他汀高剂量可导致肝组织损伤,其毒性作用通过破坏细胞的线粒体膜电位,抑制Na⁺-K⁺-ATP酶、Ca²⁺-Mg²⁺-ATP酶活性,细胞膜脂质过氧化,从而破坏细胞内微环境的平衡,导致细胞凋亡和坏死。     

Ca2+ sparks promote myogenic tone in retinal arterioles

Background and Purpose

Ca²⁺ imaging reveals subcellular Ca²⁺ sparks and global Ca²⁺ waves/oscillations in vascular smooth muscle. It is well established that Ca²⁺ sparks can relax arteries, but we have previously reported that sparks can summate to generate Ca²⁺ waves/oscillations in unpressurized retinal arterioles, leading to constriction. We have extended these studies to test the functional significance of Ca²⁺ sparks in the generation of myogenic tone in pressurized arterioles.

Experimental Approach

Isolated retinal arterioles (25–40 μm external diameter) were pressurized to 70 mmHg, leading to active constriction. Ca²⁺ signals were imaged from arteriolar smooth muscle in the same vessels using Fluo4 and confocal laser microscopy.

Key Results

Tone development was associated with an increased frequency of Ca²⁺ sparks and oscillations. Vasomotion was observed in 40% of arterioles and was associated with synchronization of Ca²⁺ oscillations, quantifiable as an increased cross-correlation coefficient. Inhibition of Ca²⁺ sparks with ryanodine, tetracaine, cyclopiazonic acid or nimodipine, or following removal of extracellular Ca²⁺, resulted in arteriolar relaxation. Cyclopiazonic acid-induced dilatation was associated with decreased Ca²⁺ sparks and oscillations but with a sustained rise in the mean global cytoplasmic [Ca²⁺] ([Ca²⁺]_c), as measured using Fura2 and microfluorimetry.

Conclusions and Implications

This study provides direct evidence that Ca²⁺ sparks can play an excitatory role in pressurized arterioles, promoting myogenic tone. This contrasts with the generally accepted model in which sparks promote relaxation of vascular smooth muscle. Changes in vessel tone in the presence of cyclopiazonic acid correlated more closely with changes in spark and oscillation frequency than global [Ca²⁺]_c, underlining the importance of frequency-modulated signalling in vascular smooth muscle.     

Evidence for a distinct Ca2+ antagonist receptor for the novel benzothiazinone compound HOE 166

Summary The pharmacological and binding properties of the novel enantiomerically pure benzothiazinone (R)-(+)-3, 4-dihydro-2-isopropyl-4-methyl-2-[2-[4-[4-[2-(3,4,5-trimethoxyphenyl)-ethyl]-piperazinyl]-butoxyl-phenyl]-2H-1, 4-benzothiazine-3-one dihydrochloride (HOE 166), are described. HOE 166 stereoselectively inhibited KCl^– but not noradrenaline-induced contractions of guinea-pig pulmonary arteries, rabbit aorta, rat mesenteric artery preparations and k-strophantin-induced enhancement of guinea-pig papillary muscle contraction in a dose-dependent manner. KCl-induced smooth muscle contraction was inhibited by HOE 166 with IC₅₀-values of 70 nM (5–11 times less potent than nifedipine, 2–16 times more potent than verapamil), the respective S-(–)-enantiomer being 10-fold less potent. HOE 166 decreased the upstroke velocity of the slow action potential in partially depolarized guinea-pig papillary muscle at similar concentrations than nifedipine. To investigate possible interactions with the calcium channel, HOE 166 and its S-(–)-enantiomer were characterized by radioligand binding studies in heart, brain and skeletal muscle transverse-tubule membranes. HOE 166 was a 4–15 times more potent inhibitor of reversible (+)-[³H]PN200-110, (–)-[³H]desmethoxyverapamil and d-cis [³H]diltiazem binding compared to its pharmacologically less active (S)-(–)-enantiomer, with IC₅₀ values in the low nanomolar range. Extensive equilibrium and kinetic studies suggest that HOE 166 exerts its Ca²⁺-antagonistic effect by binding to a Ca²⁺-channel-associated drug receptor which is distinct from the 1,4-dihydropyridine, phenylalkylamine or benzothiazepine-selective domain. This HOE 166-selective site is, however, allosterically linked to the other sites of the Ca²⁺ antagonist receptor complex. We conclude that HOE 166 is a novel calcium antagonist.Send offprint requests to H. Glossmann     

Diclofenac, a Non-steroidal Anti-inflammatory Drug, Inhibits L-type Ca2+ Channels in Neonatal Rat Ventricular Cardiomyocytes

A non-steroidal anti-inflammatory drug (NSAID) has many adverse effects including cardiovascular (CV) risk. Diclofenac among the nonselective NSAIDs has the highest CV risk such as congestive heart failure, which resulted commonly from the impaired cardiac pumping due to a disrupted excitation-contraction (E-C) coupling. We investigated the effects of diclofenac on the L-type calcium channels which are essential to the E-C coupling at the level of single ventricular myocytes isolated from neonatal rat heart, using the whole-cell voltage-clamp technique. Only diclofenac of three NSAIDs, including naproxen and ibuprofen, significantly reduced inward whole cell currents. At concentrations higher than 3 µM, diclofenac inhibited reversibly the Na⁺ current and did irreversibly the L-type Ca²⁺ channels-mediated inward current (IC₅₀=12.89±0.43 µM) in a dose-dependent manner. However, nifedipine, a well-known L-type channel blocker, effectively inhibited the L-type Ca²⁺ currents but not the Na⁺ current. Our finding may explain that diclofenac causes the CV risk by the inhibition of L-type Ca²⁺ channel, leading to the impairment of E-C coupling in cardiac myocytes.     

Endothelin-1 induced contraction of rat aorta: contributions made by Ca2+ influx and activation of contractile apparatus associated with no change in cytoplasmic Ca2+ level

Summary The modes by which Endothelin-1 (ET) induces Ca²⁺-influx and the relative functional importance of the different sources of Ca²⁺ for ET-induced contraction were studied using fura 2-loaded and unloaded rat aortic strips. ET caused an increase in the cytosolic free Ca²⁺ level ([Ca²⁺]_i) followed by a tonic contraction in Ca²⁺-containing solution, and produced a transient elevation of [Ca²⁺]_i followed by a small sustained contraction in Ca²⁺-free medium. ET also stimulated ⁴⁵Ca influx into La²⁺-inaccessible fraction significantly. With the same change of [Ca²⁺]_i, ET caused a larger tension than that induced by high K. ET-induced contraction and [Ca²⁺]_i elevation were not significantly inhibited by 0.1–0.3 M nicardipine which nearly abolished the contraction and [Ca⁺]_i elevation produced by high K. During treatment of the strips with high K, addition of ET induced further increases in [Ca²⁺]_i and muscle tension, and vice versa. In Ca²⁺-free medium, ET-induced contraction was influenced neither by ryanodine-treatment nor by high K-treatment, although the former attenuated and the latter potentiated the [Ca²⁺]_i transient induced by ET. Further, the ET-induced sustained contraction under Ca²⁺-free conditions began to develop after the [Ca²⁺]_i level returned to the baseline. Thus, it seems that the Ca²⁺ released from the ryanodine-sensitive and -insensitive Ca²⁺ stores by ET may provide only a minor or indirect contribution, if any, to the tension development. ET might cause a contraction mainly by stimulating Ca²⁺-influx through Ca²⁺ channel(s) other than voltage-dependent Ca²⁺ channels in character, and by increasing the sensitivity of the contractile filaments to Ca²⁺ or activating them Ca²⁺-independently.Visiting from Zun Yi Medical College, China Send offprint requests to I. Takayanagi at the above address     

赛庚啶对离体犬心肌肌质网Ca2+,Mg2+—ATP酶活性和45Ca2+摄取功能的影响

当赛庚啶浓度在8×10^-6mol/L～2×10^-4mol/L之间时,该药对正常犬心肌肌质网Ca²⁺,Mg²⁺—ATP酶活性几乎没有影响,仅在10^-3mol/L时对该酶活性才有一定的抑制作用(抑制率为39.85%,P<0.01)。正常犬心肌肌质网的⁴⁵Ca²⁺摄取过程有明显的时间依赖性,至第30 min,其⁴⁵Ca²⁺摄取量可达312.79±22.25 nmol/mg protein.赛庚啶对心肌肌质网的~(45)Ca²⁺摄取有一定的抑制作用,其IC₅₀为1.94×10^-4mol/L。     

Evidence for a direct interaction of thapsigargin with voltage-dependent Ca2+ channel

The effect of thapsigargin, an inhibitor of the sarco-endoplasmic reticulum Ca²⁺-ATPase, on voltage-dependent Ca²⁺ channels has been investigated in the A7r5 cell line and in membrane preparations from rat aorta, heart and brain. Patch-clamp technique showed that, at micromolar concentrations, thapsigargin inhibited the L-type Ca²⁺ channel current in A7r5 cells. It depressed the current at all voltages without change in the steady state inactivation curve. The rates of inactivation of the Ca²⁺ current were highly variable among the cells suggesting that more than one component of L-type Ca²⁺ current coexist in A7r5 cells, differing in the kinetics of inactivation. Thapsigargin appeared to be more potent on the slower-inactivating Ca²⁺ current than on the faster-inactivating one. In the same range of concentrations, thapsigargin inhibited the specific binding of ³H(+)-isradipine in intact cells while ⁴⁵Ca²⁺ uptake in intracellular stores of skinned cells was inhibited at nanomolar concentrations. The equilibrium dissociation constant of ³H(+)-isradipine was increased in the presence of thapsigargin as a result of an increase of the dissociation rate constant indicating that the inhibitory effect of the antagonist cannot be attributed to a simple competitive interaction with the dihydropyridine binding site. Maximum binding capacity was unaffected. A similar pattern of inhibition of ³H(+)-isradipine binding was observed in membrane preparations from rat aorta, heart and brain.Those results indicate that, at micromolar concentrations, thapsigargin inhibits the voltage-dependent Ca²⁺ current by a direct interaction with the L-type Ca²⁺ channels.On leave from the A. A. Bogomoletz Institute of Physiology, Kiev, Ukraine     

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.     

The involvement of a novel mechanism distinct from the thrombin receptor in the vasocontraction induced by trypsin

1. The vasocontracting effect of a serine protease trypsin and its mechanisms were investigated by monitoring the isometric tension in endothelium-denuded rings of rabbit thoracic aortae and its effects on intracellular free Ca²⁺ concentrations ([Ca²⁺]_i) in dispersed rabbit vascular smooth muscle cells with a Ca²⁺ indicator fura-2. The actions of trypsin were compared with those of thrombin.
2. Both thrombin and trypsin reversibly contracted aortic rings without endothelium in a concentration-dependent manner. The vasocontraction induced by trypsin was well correlated with the protease activity of trypsin actually added to the tissue baths containing the aortic rings and was completely blocked by soybean trypsin inhibitor and phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor.
3. The trypsin-induced contractions of the aortic rings were not the result of irreversible damage to vascular smooth muscle cells, since the contractile responses induced by noradrenaline or 30 mM KCl were unaffected by pretreatment with trypsin.
4. The contractions induced by either thrombin or trypsin were reduced to about 30% of control responses after removal of extracellular Ca²⁺, indicating that most of the contraction is dependent on extracellular Ca²⁺. By contrast, the contractions induced by either of the proteases were reduced by an antagonist of L-type voltage-operated Ca²⁺ channels, nifedipine, to about 70% of control responses, indicating that both nifedipine-sensitive and -resistant Ca²⁺ channels are involved in these contractions.
5. In the aortic rings precontracted by a maximally effective concentration of thrombin, the second application of thrombin virtually failed to induce contractions but trypsin could still induce contractions amounting to 10% of control values by it''s protease activity.
6. After the first application of a maximal concentration of thrombin, the second application of thrombin could not induce an increase in [Ca²⁺]_i, but an application of trypsin could still induce an increase in [Ca²⁺]_i in dispersed rabbit vascular smooth muscle cells.
7. These data suggest that in addition to activation of a thrombin receptor, trypsin can contract rabbit aortae by a proteinase-activated receptor 2 or a novel mechanism.

     

Ca2+ signalling by P2Y receptors in cultured rat aortic smooth muscle cells

Background and purpose:

P2Y receptors evoke Ca²⁺ signals in vascular smooth muscle cells and regulate contraction and proliferation, but the roles of the different P2Y receptor subtypes are incompletely resolved.

Experimental approach:

Quantitative PCR was used to define expression of mRNA encoding P2Y receptor subtypes in freshly isolated and cultured rat aortic smooth muscle cells (ASMC). Fluorescent indicators in combination with selective ligands were used to measure the changes in cytosolic free [Ca²⁺] in cultured ASMC evoked by each P2Y receptor subtype.

Key results:

The mRNA for all rat P2Y receptor subtypes are expressed at various levels in cultured ASMC. Four P2Y receptor subtypes (P2Y1, P2Y2, P2Y4 and P2Y6) evoke Ca²⁺ signals that require activation of phospholipase C and comprise both release of Ca²⁺ from stores and Ca²⁺ entry across the plasma membrane.

Conclusions and implications:

Combining analysis of P2Y receptor expression with functional analyses using selective agonists and antagonists, we isolated the Ca²⁺ signals evoked in ASMC by activation of P2Y1, P2Y2, P2Y4 and P2Y6 receptors.     

ML-9, a myosin light chain kinase inhibitor, reduces intracellular Ca2+ concentration in guinea pig trachealis

We investigated the effects of ML-9 [1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine], a myosin light chain kinase (MLCK) inhibitor, on intracellular Ca2+ concentration ([Ca2+]i), contraction induced by high K+ and an agonist, and capacitative Ca2+ entry in fura-2-loaded guinea pig tracheal smooth muscle. ML-9 inhibited both the increase in [Ca2+]i and the contraction induced by 60 mM K+, 1 microM methacholine or 1 microM thapsigargin, an inhibitor of the sarcoplasmic reticulum Ca2+-ATPase. However, another MLCK inhibitor, wortmannin (3 microM), inhibited the contraction elicited by these stimuli without affecting [Ca2+]i. Under the condition that the thapsigargin-induced contraction was fully suppressed by 3 microM wortmannin, 30 microM ML-9 caused a further decrease in [Ca2+]i. The inhibitory effects of ML-9 on [Ca2+]i and the contraction elicited by methacholine were similar to those of SKF-96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride), a Ca2+ channel blocker. These results indicate that ML-9 acts as a potent inhibitor of Ca2+-permeable channels independently of MLCK inhibition in tracheal smooth muscle.     

Functional, morphological and molecular characterization of bladder dysfunction in streptozotocin-induced diabetic mice: evidence of a role for L-type voltage-operated Ca2+ channels

BACKGROUND AND PURPOSE

Diabetic cystopathy is one of the most common and incapacitating complications of diabetes mellitus. This study aimed to evaluate the functional, structural and molecular alterations of detrusor smooth muscle (DSM) in streptozotocin-induced diabetic mice, focusing on the contribution of Ca²⁺ influx through L-type voltage-operated Ca²⁺ channels (L-VOCC).

EXPERIMENTAL APPROACH

Male C57BL/6 mice were injected with streptozotocin (125 mg·kg⁻¹). Four weeks later, contractile responses to carbachol, α,β-methylene ATP, KCl, extracellular Ca²⁺ and electrical-field stimulation were measured in urothelium-intact DSM strips. Cystometry and histomorphometry were performed, and mRNA expression for muscarinic M₂/M₃ receptors, purine P2X1 receptors and L-VOCC in the bladder was determined.

KEY RESULTS

Diabetic mice exhibited higher bladder capacity, frequency, non-void contractions and post-void pressure. Increased bladder weight, wall thickness, bladder volume and neural tissue were observed in diabetic bladders. Carbachol, α,β-methylene ATP, KCl, extracellular Ca²⁺ and electrical-field stimulation all produced greater DSM contractions in diabetic mice. The L-VOCC blocker nifedipine almost completely reversed the enhanced DSM contractions in bladders from diabetic animals. The Rho-kinase inhibitor Y27632 had no effect on the enhanced carbachol contractions in the diabetic group. Expression of mRNA for muscarinic M₃ receptors and L-VOCC were greater in the bladders of diabetic mice, whereas levels of M₂ and P2X1 receptors remained unchanged.

CONCLUSIONS AND IMPLICATIONS

Diabetic mice exhibit features of urinary bladder dysfunction, as characterized by overactive DSM and decreased voiding efficiency. Functional and molecular data suggest that overactive DSM in diabetes is the result of enhanced extracellular Ca²⁺ influx through L-VOCC.