Mechanisms of vasoinhibitory effect of cardioprotective agent, CP-060S, in rat aorta

Vasoinhibitory effects of (−)-(S)-2-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-3-[3-[N-methyl-N-[2-(3,4-methylenedioxyphenoxy)ethyl]amino]propyl]-1,3-thiazolidin-4-one hydrogen fumarate (CP-060S), a synthesized cardioprotective agent, were examined. In the rat aortic rings, the contractile responses to cumulative application of angiotensin II, [Arg⁸]-vasopressin (vasopressin), or prostaglandin F₂ were inhibited by CP-060S in a concentration-dependent manner. The Ca²⁺-induced contractions in the presence of vasopressin or prostaglandin F₂ were also inhibited by CP-060S in a concentration-dependent manner. The inhibitory effect of 10⁻⁵ M CP-060S on phenylephrine-induced contraction was as potent as that of 10⁻⁶ M nifedipine, and the combined addition of 10⁻⁶ M nifedipine and 10⁻⁵ M CP-060S showed the effect similar to that of 10⁻⁵ M CP-060S alone. In rat aorta loaded with a Ca²⁺ indicator, fura-PE3, 10⁻⁵ M CP-060S completely inhibited the high K⁺-induced increase in cytosolic Ca²⁺ level ([Ca²⁺]_i) and contraction. In contrast, 10⁻⁵ M CP-060S only partially inhibited the increase in [Ca²⁺]_i and contraction due to phenylephrine or prostaglandin F₂. In the presence of 10⁻⁶ M nifedipine, 10⁻⁵ M CP-060S did not inhibit the increase in [Ca²⁺]_i and contraction induced by prostaglandin F₂. In a Ca²⁺-free medium, the phasic increases in contraction and [Ca²⁺]_i induced by phenylephrine were not affected by 10⁻⁵ M CP-060S. These results suggest that the vasoinhibitory effect of CP-060S in rat aortic rings is due mainly to the inhibition of L-type voltage-dependent Ca²⁺-channels.     

Effects of isoquinoline derivatives, HA1077 and H-7, on cytosolic Ca level and contraction in vascular smooth muscle

The effects of isoquinoline derivatives, HA1077 (1-[5-isoquinolinesulfonyl]-homopiperazine) and H-7 (1-[5-isoquinoline-sulfonyl]-2-methylpiperazine), on cytosolic Ca²⁺ levels ([Ca²⁺]_i) and muscle tension were examined in vascular smooth muscle of rat aorta. High K⁺ (72.7 mM) and norepinephrine (1 μM) induced a sustained contraction with a sustained increase in [Ca²⁺]_i. HA1077 and H-7 (3–10 μM) inhibited the increse in muscle tension more strongly than the increase in [Ca²⁺]_i. Verapamil (10 μM) completely inhibited the increase in [Ca²⁺]_i and the contraction induced by K⁺ whereas it inhibited the increase in [Ca²⁺]_i more strongly than the contraction due to norepinephrine. The verapamil-insensitive portion of the norepinephrine-induced contraction was inhibited by HA1077 or H-7. In Ca²⁺-free solution, 0.1 μM norepinephrine induced a transient increase in [Ca²⁺]_i and muscle tension. The transient contraction was inhibited by 10 μM HA1077 or 10 μM H-7 without inhibiting the increase in [Ca²⁺]_i. 12-Deoxyphorbol 13-isobutyrate (DPB) (1 μM) caused a sustained contraction, and this contraction was inhibited by HA1077 and H-7 at similar concentrations needed to inhibit the contractions induced by high K⁺ or norepinephrine. In rabbit mesenteric artery permeabilized with Staphylococcus aureus -toxin, 100 μM HA1077 and 100 μM H-7 inhibited the contraction induced by 0.3 μM Ca²⁺. These results suggest that the inhibitory effects of isoquinoline derivatives, HA1077 and H-7, are due to a decrease in [Ca²⁺]_i and in the Ca²⁺ sensitivity of contractile elemenst in vascular smooth muscle.     

Effects of Ca channel blockers on Ca loading induced by metabolic inhibition and hyperkalemia in cardiomyocytes

The effects of the L-type (nifedipine and verapamil) and the T-type (mibefradil) Ca²⁺ channel blockers on the increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by NaCN metabolic inhibition and hyperkalemia were examined in chicken cardiomyocytes using fluorescence imaging with Fura-2. NaCN induced a slow and sustained rise in [Ca²⁺]_i, which was not affected by pretreating the cells for 5 min with nifedipine, verapamil, or mibefradil at 100 nM or 10 μM. Pretreatment of the cells with 10 μM nifedipine, verapamil, or mibefradil for 5 min remarkably inhibited the K⁺-induced increase in [Ca²⁺]_i. These inhibitory effects diminished after 48-h pretreatment with nifedipine or verapamil but not with mibefradil. Ryanodine also induces an increase in [Ca²⁺]_i, and this effect was enhanced by 48-h pretreatment of the cells with 10 μM verapamil but not with 10 μM mibefradil. We conclude that the NaCN-induced increase in [Ca²⁺]_i is independent of the Ca²⁺ influx though the L-type or T-type Ca²⁺ channels. Chronic inhibition of the L-type Ca²⁺ channels but not T-type channels may enhance the ryanodine receptor-mediated Ca²⁺ release, which may be responsible for the development of tolerance to their inhibitory effects on K⁺-induced increase in [Ca²⁺]_i.     

Opposite effects of Bay k 8644 and nicardipine on the inhibitory effect of Ca on rat myometrium

The effect of Ca²⁺ on the oxytocin-induced, sustained contraction of rat uterine muscle in Ca-free medium after prolonged incubation with 3 mM EGTA (Ca-free contraction) was investigated. A micromolar concentration of Ca²⁺ caused phasic contraction followed by relaxation while a submicromolar concentration caused relaxation only. Cumulative addition of Ca²⁺ (10⁻⁸-3×10⁻⁶ M) caused dose-dependent relaxation (Ca reversal). This relaxation was inhibited by nicardipine and enhanced by Bay k 8644, and the effects of these two drugs were potentiated in 45.6 mM K⁺ medium. It is concluded that the inhibitory effect of Ca²⁺ on Ca-free contraction is caused by the influx of a minute amount of Ca²⁺. Thus, Ca²⁺ has dual actions in the cell: activation at concentrations higher than 10⁻⁶ M, and inhibition alone at concentrations lower than 10⁻⁷ M.     

Force-frequency relationship in intact mammalian ventricular myocardium: physiological and pathophysiological relevance

The force–frequency relationship (FFR) is an important intrinsic regulatory mechanism of cardiac contractility. The FFR in most mammalian ventricular myocardium is positive; that is, an increase in contractile force in association with an increase in the amplitude of Ca²⁺ transients is induced by elevation of the stimulation frequency, which reflects the cardiac contractile reserve. The relationship is different depending on the range of frequency and species of animal. In some species, including rat and mouse, a ‘primary-phase’ negative FFR is induced over the low-frequency range up to approximately 0.5–1 Hz (rat) and 1–2 Hz (mouse). Even in these species, the FFR over the frequency range close to the physiological heart rate is positive and qualitatively similar to that in larger mammalian species, although the positive FFR is less prominent. The integrated dynamic balance of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) is the primary cellular mechanism responsible for the FFR and is determined by sarcoplasmic reticulum (SR) Ca²⁺ load and Ca²⁺ flux through the sarcolemma via L-type Ca²⁺ channels and the Na⁺-Ca²⁺ exchanger. Intracellular Na⁺ concentration is also an important factor in [Ca²⁺]_i regulation. In isolated rabbit papillary muscle, over a lower frequency range (<0.5 Hz), an increase in duration rather than amplitude of Ca²⁺ transients appears to be responsible for the increase in contractile force, while over an intermediate frequency range (0.5–2.0 Hz), the amplitude of Ca²⁺ transients correlates well with the increase in contractile force. Over a higher frequency range (>2.5 Hz), the contractile force is dissociated from the amplitude of Ca²⁺ transients probably due to complex cellular mechanisms, including oxygen limitation in the central fibers of isolated muscle preparations, while the amplitude of Ca²⁺ transients increases further with increasing frequency (‘secondary-phase’ negative FFR). Calmodulin (CaM) may contribute to a positive FFR and the frequency-dependent acceleration of relaxation, although the role of calmodulin has not yet been established unequivocally. In failing ventricular myocardium, the positive FFR disappears or is inverted and becomes negative. The activation and overexpression of cardiac sarcoplasmic reticulum Ca²⁺ ATPase (SERCA2a) is able to reverse these abnormalities. Frequency-dependent alterations of systolic and diastolic force in association with those of Ca²⁺ transients and diastolic [Ca²⁺]_i levels are excellent indicators for analysis of cardiac excitation-contraction coupling, and for evaluating the severity of cardiac contractile dysfunction, cardiac reserve capacity and the effectiveness of therapeutic agents in congestive heart failure.     

8-isoprostaglandin E(2) activates Ca(2+)-dependent K(+) current via cyclic AMP signaling pathway in murine renal artery

The inhibitory pathway of 8-isoprostaglandin E₂ was investigated in murine renal arterial smooth muscle. K⁺ current was augmented in a concentration-dependent fashion, with an average increase of 123 ± 28% (n = 6) following application of 10^− 5 M 8-isoPGE₂. This augmentation was observed in the presence of 4-aminopyridine (4-AP, 10^− 3 M) but not that of charybdotoxin (ChTx, 10^− 7 M). Fluorimetric recordings showed marked concentration-dependent increase of cytosolic Ca²⁺ levels by 8-isoPGE₂, while an enzyme-linked immunosorbent assay (ELISA)-based cyclic AMP assay showed increased cAMP levels by 10^− 7 M 8-isoPGE₂ challenge. The isoprostane-induced augmentation was prevented by the ryanodine receptor blocker ruthenium red (10^− 5 M) or the adenylate cyclase blocker SQ 22536 (10^− 4 M). The protein kinase A (PKA) inhibitor H89 (10^− 5 M) inhibited resting K⁺ currents (78 ± 5%, n = 5) but did not prevent 8-isoPGE₂ from augmenting the remaining K⁺ current. We conclude that 8-isoPGE₂ enhances Ca²⁺-dependent K⁺ currents in murine renal artery through a cAMP-dependent pathway which may involve internally sequestered Ca²⁺.     

CGP37157 modulates mitochondrial Ca homeostasis in cultured rat dorsal root ganglion neurons

The effects of 7-chloro-3,5-dihydro-5-phenyl-1H-4,1-benzothiazepine-2-on (CGP37157), an inhibitor of mitochondrial Na⁺/Ca²⁺ exchange, on depolarization-induced intracellular free Ca²⁺ concentration ([Ca²⁺]_i) transients were studied in cultured rat dorsal root ganglion neurons with indo-1-based microfluorimetry. A characteristic plateau in the recovery phase of the [Ca²⁺]_i transient resulted from mitochondrion-mediated [Ca²⁺]_i buffering. It was blocked by metabolic poisons and was not dependent on extracellular Ca²⁺. CGP37157 produced a concentration-dependent decrease in the amplitude of the mitochondrion-mediated plateau phase (IC₅₀=4±1 μM). This decrease in [Ca²⁺]_i was followed by an increase in [Ca²⁺]_i upon removal of the drug, suggesting that Ca²⁺ trapped in the matrix was released when the CGP37157 was removed from the bath. CGP37157 also inhibited depolarization-induced Ca²⁺ influx at the concentrations required to see effects on [Ca²⁺]_i buffering. Thus, CGP37157 inhibits mitochondrial Na⁺/Ca²⁺ exchange and directly inhibits voltage-gated Ca²⁺ channels, suggesting caution in its use to study [Ca²⁺]_i regulation in intact cells.     

Mechanisms of pinacidil-induced vasodilatation

The mechanism of the vasodilator effect of pinacidil was examined. Pinacidil (0.1–100 μM) inhibited the increases in cytosolic Ca²⁺ ([Ca²⁺]_i) and muscle tension due to norepinephrine in rat aorta. In contrast, a Ca²⁺ channel blocker, verapamil, inhibited the norepinephrine-stimulated [Ca²⁺]_i more strongly than the contraction. Higher concentrations of pinacidil (3–100 μM) inhibited the verapamil-insensitive portion of the contraction and [Ca²⁺]_i. An inhibitor of ATP-sensitive K⁺ channels, glibenclamide, antagonized the inhibitory effect of low concentrations ( 10 pM) of pinacidol. Pinacidil did not change the contraction induced by Ca²⁺ in vascular smooth muscle permeabilized with Staphylococcus aureus -toxin. Norepinephrine (in the presence of GTP), 12-deoxyphorbol 13-isobutyrate (in the absence of GTP), and treatment with GTP_γS potentiated the contraction of permeabilized smooth muscle induced by the addition of Ca²⁺. Pinacidil (100 μM) inhibited the potentiation due to GTP_γS or noepinephrine but not to phorbol ester. These results suggest that pinacidil has dual effects on vascular smooth muscle contraction. At lower concentrations (>0.1 μM), it decreases [Ca²⁺]_i, possibly by activating ATP-sensitive K+ channels. At higher concentrations (> 3 μM), it may additionally inhibit the receptor-mediated, GTP-binding protein-coupled phosphatidyl inositol turnover.     

The effect of trimethyltin on acetylcholine release in the guinea-pig trachea

The purpose of the present work was to characterise the effects of trimethyltin on the release of acetylcholine from parasympathetic nerves and its effect on the postjunctional cholinergic stimulation of a smooth muscle. The guinea-pig trachea has been used as a model. Prejunctionally, trimethyltin (3.0 × 10⁻³ M) significantly enhanced in a reversible manner the high K⁺ (75 mM) evoked release of endogenous acetylcholine and [³H]acetylcholine. The evoked release of endogenous acetylcholine and [³H]acetylcholine was released from a pool of acetylcholine being independent of extraneuronal Ca²⁺ in the presence, but not in the absence of trimethyltin. The effect of trimethyltin on the release was not inhibited by low Ca²⁺ (0 mM and 1.0 × 10⁻⁴ M) or by Ca²⁺ channel blockers (verapamil, 1.0 × 10⁻⁴ M, flunarizine, 1.0 × 10⁻⁴ M, ω-conotoxin GVIA, 2.0 × 10⁻⁷ M and ω-agatoxin, 2.0 × 10⁻⁷ M). The present results also demonstrate that trimethyltin induce emptying of a non-vesicular, probably a cytoplasmic storage pool of acetylcholine, since AH5183 (2.0 × 10⁻⁵ M), an inhibitor of the translocation of acetylcholine into synaptic vesicles, and -latrotoxin (1.0 × 10⁻⁸ M), a toxin from black widow spider venom inducing vesicle depletion, had no inhibitory effects on the release of [³H]acetylcholine evoked by trimethyltin (3.0 × 10⁻³ M). The release of [³H]acetylcholine was moreover enhanced by trimethyltin when the vesicular uptake of [³H]acetylcholine was inhibited by AH5183, probably as a result of a higher cytoplasmic concentration of [³H]acetylcholine. Trimethyltin also reduced the neuronal uptake of [³H]choline and this was probably due to a depolarising effect of trimethyltin on the cholinergic nerve terminals. A similar depolarisation induced by trimethyltin was observed during patch clamping of GH₄ C₁ neuronal cells. Postjunctionally, trimethyltin had no effect by itself or on the carbachol-induced smooth muscle contraction, indicating that trimethyltin did not have a general depolarising effect on smooth muscle cells or an effect on muscarinic receptors. Furthermore, the reduced electrical field-induced contraction and the subsequent increase in the basal smooth muscle tension that was observed by addition of trimethyltin was activity-dependent, and was most probably due to emptying of a nervous non-vesicular storage pool of acetylcholine, followed by rapid hydrolysis of acetylcholine by acetyl- and pseudocholinesterases.     

Homologous and heterologous desensitisation of somatostatin-induced increases in intracellular Ca and inositol 1,4,5-trisphosphate in CHO-K1 cells expressing human recombinant somatostatin sst5 receptors

The mechanisms responsible for somatostatin (SRIF)-induced increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and subsequent desensitisation were studied in CHO-K1 cells expressing human sst₅ receptors (CHOsst₅ cells). To study the nature of the desensitisation, interactions with uridine triphosphate (UTP) were examined. SRIF (pEC₅₀ 7.10) and UTP (pEC₅₀ 5.14) caused concentration-dependent increases in [Ca²⁺]_i but the SRIF maximum was about 40% of that to UTP. SRIF-, but not UTP-, induced increases in [Ca²⁺]_i were transient and abolished by pertussis toxin. SRIF and UTP caused sustained increases in Ins(1,4,5)P₃ but the SRIF maximum was about 30% of that to UTP. Removal of [Ca²⁺ ]_e attenuated the SRIF-induced peak rise in [Ca²⁺]_i but had no effect on the peak increases in Ins(1,4,5)P₃. UTP-induced increases in [Ca²⁺]_i and Ins(1,4,5)P₃ were attenuated in the absence of [Ca²⁺]_e. Following pre-exposure to SRIF (1 μM) or UTP (100 μM) for 5 min, subsequent SRIF responses were desensitised. Similar results were obtained in the absence of [Ca²⁺]_e. Pre-exposure to SRIF had no effect on subsequent responses to UTP but in the absence of [Ca²⁺]_e, responses to UTP were attenuated. The results suggest that SRIF but not UTP-induced increases in [Ca²⁺]_i in CHOsst₅ cells are mediated by pertussis toxin sensitive G proteins and are caused by an entry of extracellular Ca²⁺ and release from an Ins(1,4,5)P₃ sensitive Ca²⁺ store. Homologous or heterologous desensitisation of agonist-induced increases in [Ca²⁺]_i could be demonstrated in the presence or absence of extracellular Ca²⁺ respectively, and the latter appeared to involve depletion of a common intracellular Ca²⁺ store.     

A novel aspect of lindane testicular toxicity: in vitro effects on peritubular myoid cells

The in vitro effects of the insecticide lindane have been investigated in rat testis peritubular myoid cells (PMCs). Upon PMC exposure to lindane, polarity increase and decrease of dipole dynamics were seen at the membrane level (EC50 20 μM), leading to a partial dissipation of the membrane intrinsic dipole potential. The initial membrane depolarization was increased by Cl⁻ efflux and limited by Ca²⁺-activated repolarizing currents. Concomitantly, lindane produced an increase in [Ca²⁺]_i (EC50 125 μM) resulting from both Ca²⁺ release from an inositol 1,4,5-trisphosphate-sensitive intracellular store and a voltage-dependent Ca²⁺ influx from the extracellular medium. Of particular interest from a toxicologic point of view, insecticide concentrations well below those effective in altering ion homeostasis potently inhibited both [Ca²⁺]_i increase and contraction induced by the natural agonists vasopressin and endothelin-1 (IC50s < 10 μM). These data demonstrate that PMCs are highly susceptible to lindane and suggest that the insecticide may exert testicular toxicity by interfering with hormone-regulated PMC function.     

Enhancement by captopril of bradykinin-induced calcium transients in cultured endothelial cells of the bovine aorta

Using microscopic fluorometry and fura-2-loaded cultured bovine aortic endothelial cells, we determined the effects of captopril, an angiotensin converting enzyme (ACE) inhibitor, on bradykinin-induced Ca²⁺ transients in endothelial cells. In the presence of extracellular Ca²⁺, 10⁻⁹ M bradykinin induced an early rise in the transients followed by sustained elevations of cytosolic calcium concentration ([Ca²⁺]_i). Bradykinin concentration-dependently increased [Ca²⁺]_i (EC₅₀ 6.7 × 10⁻⁹ M). Captopril, 10⁻⁵ M, enhanced and prolonged the bradykinin-induced Ca²⁺ transients and shifted the concentration-response curve to the left (EC₅₀ 8.5 × 10⁻¹⁰ M). In porcine coronary aterial strips with intact endothelium, cumulative applications of bradykinin induced an endothelium-dependent relaxation during prostaglandin F_2α-induced contraction (EC₅₀ = 2.0 × 10⁻⁹ M). Treatment with 10⁻⁵ M captopril enhanced the bradykinin-induced relaxation and shifted the concentration-response curve to the left (EC₅₀ = 7.6 × 10⁻¹⁰ M). Thus, captopril enhances the bradykinin-induced relaxation by mechanisms mainly dependent on the endothelium, namely the inhibition of ACE.     

Effects of [Na] and [Ca] on the responses to milrinone in rat cardiac preparations

In the present investigation we have studied the influence of changing the [Ca²⁺] and [Na⁺] on the cardiac responses to milrinone in various preparations of rat heart. Milrinone (5 × 10⁻⁵ to 8 × 10⁻⁴ M) produced a dose-dependent positive chronotropic effect on right atrium and a positive inotropic effect on left atrium and papillary muscle of the rat. A decrease in [Ca²⁺] (from 2.2 to 1.1 mM) or an increase in [Na⁺] (from 120 to 60 mM) increased the milrinone-induced inotropic effect in left atrium and papillary muscle. However, in right atrium the chronotropic effect of milrinone was significantly decreased under these conditions. Opposite changes to milrinone-induced responses were observed when [Ca²⁺] was increased (to 3.3 mM) or when the [Na⁺] was decreased to 60 mM. Nifedipine (3 × 10⁻³ M), a selective Ca²⁺ channel blocker, significantly inhibited the chronotropic response to milrinone in right atrium. However, the inotropic response to milrinone was found to be significantly greater in the presence of nifedipine. A veratridine-induced positive inotropic effect in the left atrium was also significantly increased in the presence of nifedipine. Tetrodotoxin (TTX, 1 × 10⁻⁶ M), a fast sodium channel blocker, significantly reduced the inotropic response to milrinone in left atrium and papillary muscle. A milrinone-induced dose-dependent increase in the baseline tension was observed in the right atrium which was abolished in low [Ca²⁺] and significantly increased in high [Ca²⁺]. Our data suggest the possibility that milrinone increases Ca²⁺ influx in the right atrium to cause the chronotropic effect. Milrinone also may possess an action like veratridine, involving an increased influx of Na⁺ through fast Na⁺ channels in left atrium and papillary muscle, and this action is possibly involved in the positive inotropic effect.     

The Protective Action of Tetrodotoxin and (±)-Kavain on Anaerobic Glycolysis, ATP Content and Intracellular Na and Ca of Anoxic Brain Vesicles

Because recent reports point to Na⁺ channel blockers as protective agents directed against anoxia-induced neuronal damage including protection of anaerobic glycolysis, the influences of tetrodotoxin (TTX) and (±)-kavain on anoxic rat brain vesicles were investigated with respect to lactate synthesis, vesicular ATP content and cytosolic free Na⁺ and Ca²⁺ ([Na⁺]_i, [Ca²⁺]_i), both of the latter determined fluorometrically employing SBFI and FURA-2, respectively. After anoxia, basal lactate production was increased from 2.9 to 9.8 nmol lactate/min/mg protein. Although lactate synthesis seemed to be stable for at least 45 min of anoxia, as deduced from the linearity of lactate production, the ATP content declined continuously with a half life (τ ) af 14.5 min, indicating that anaerobic glycolysis was insufficient to cover the energy demand of anoxic vesicles. Correspondingly, [Na⁺]_i and [Ca²⁺]_i increased persistently after anoxia by 22.1 mmol/l Na⁺ and 274.9 nmol/l Ca²⁺, determined 6.3 min after onset. An additional stimulation of vesicles with veratridine accelerated the drop of ATP (τ = 5.1 min) and provoked a massive Na⁺ overload, which levelled off to 119 mmol/l Na⁺ within a few minutes. Concomitantly, [Ca²⁺]_i increased linearly with a rate of 355 nmol Ca²⁺/l/min. Despite the massive perturbation of ion homeostasis, lactate production was unaffected during the first 8 min of veratridine stimulation. However, complete inhibition of lactate synthesis took place 30 min after veratridine was added. The Na⁺ channel blockers TTX and (±)-kavain, if applied before anoxia, preserved vesicular ATP content, diminished anoxia-induced increases in [Na⁺]_i and [Ca²⁺]_i and prevented both the veratridine-induced increases of [Na⁺]_i and [Ca²⁺]_i and the inhibition of lactate production. The data indicate a considerable Na⁺ influx via voltage-dependent Na⁺ channels during anoxia, which speeds up the decline in ATP and provokes an increase in [Ca²⁺]_i. A massive Na⁺ and Ca²⁺ overload induced by veratridine failed to influence lactate synthesis directly, but initiated its inhibition. © 1997 Elsevier Science Ltd. All rights reserved.     

Sarcoplasmic reticulum Ca2+ release channel ryanodine receptor (RyR2) plays a crucial role in aconitine-induced arrhythmias

The present study established a model of RyR₂ knockdown cardiomyocytes and elucidated the role of RyR₂ in aconitine-induced arrhythmia. Cardiomyocytes were obtained from hearts of neonatal Sprague–Dawlay rats. siRNAs were used to down-regulate RyR₂ expression. Reduction of RyR₂ expression was documented by RT-PCR, western blot, and immunofluorescence. Ca²⁺ signals were investigated by measuring the relative intracellular Ca²⁺ concentration, spontaneous Ca²⁺ oscillations, caffeine-induced Ca²⁺ release, and L-type Ca²⁺ currents. In normal cardiomyocytes, steady and periodic spontaneous Ca²⁺ oscillations were observed, and the baseline [Ca²⁺]_i remained at the low level. Exposure to 3 μM aconitine increased the frequency and decreased the amplitude of Ca²⁺ oscillations; the baseline [Ca²⁺]_i and the level of caffeine-induced Ca²⁺ release were increased but the L-type Ca²⁺ currents were inhibited after application of 3 μM aconitine for 5 min. In RyR₂ knockdown cardiomyocytes, the steady and periodic spontaneous Ca²⁺ oscillations almost disappeared, but were re-induced by aconitine without affecting the baseline [Ca²⁺]_i level; the level of caffeine-induced Ca²⁺ release was increased but L-type Ca²⁺ currents were inhibited. Alterations of RyR₂ are important consequences of aconitine-stimulation and activation of RyR₂ appear to have a direct relationship with aconitine-induced arrhythmias. The present study demonstrates a potential method for preventing aconitine-induced arrhythmias by inhibiting Ca²⁺ leakage through the sarcoplasmic reticulum RyR₂ channel.     

Fluorescent estimation on cytotoxicity of methylmercury in dissociated rat cerebellar neurons: its comparison with ionomycin

To study the cellular basis of the neurotoxicity of methylmercury, the effects of methylmercury on dissociated rat cerebellar neurons were examined using a flow cytometer, a confocal laser microscope and three fluorescent dyes, fluo-3 for monitoring the changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and for detecting live neurons, ethidium for assessing the neurons that are dead or have compromised membranes, and 5-chloromethylfluorescein (CMF) for estimating the cellular content of nonprotein thiols. Methylmercury at concentrations of 1 μM or greater increased the [Ca²⁺]_i of almost all neurons. Prolonged exposure to methylmercury (3 and 10 μM) produced a further increase in [Ca²⁺]_i, in association with compromising membranes in some neurons. Thereafter, methylmercury induced blebs on membranes of some neurons with increased [Ca²⁺]_i. Methylmercury at concentrations of 0.3 μM or greater dose-dependently decreased the cellular content of nonprotein thiols. Results suggest that methylmercury may induce the loss of membrane integrity through destabilized Ca²⁺ homeostasis and oxidative stress in mammalian brain neurons.     

Effect of a phospholipase A2 with cardiotoxin-like properties, from Bungarus fasciatus snake venom, on calcium-modulated potassium currents

, , , and . Effect of a phospholipase A₂ with cardiotoxin-like properties, from Bungarus fasciatus snake venom, on calcium-modulated potassium currents. Toxicon 27, 1339–1349, 1989.—The action of a 16,300mol. wt phospholipase A₂ with cardiotoxin-like properties from Bungarus fasciatus venom on membrane electrical properties of two human cell types was examined in vitro by using tight-seal whole-cell recording methods. Epithelial cells exhibited a voltage-and Ca²⁺-activated K⁺current; the sensitivity for voltage activation of the K⁺ current was enhanced by increasing free Ca²⁺ in the recording pipette from 10⁻⁸ M to 2 × 10⁻⁶ M. In contrast, peripheral blood lymphocytes possessed voltage-activated K⁺ currents that were inhibited by increasing intracellular Ca²⁺.

Exposure of either preparation to B.fasciatus toxin (0.2–5 × 10⁻⁶ M) for up to 30 min in the bath did not alter membrane leakage current, as judged by the maintenance of low pre-treatment values over the range of − 140mV to − 40mV. However, the sensitivity for voltage activation of the K⁺ current was enhanced in the epithelial cells even at the lowest concentrations tested. In contrast to the results with epithelial cells, toxin exposure inhibited the activation of voltage-activated K⁺ currents in human lymphocytes, suggesting a specific increase in intracellular Ca²⁺ levels in both cell types.

The fluorescent probe indo-1/AM was used to monitor cytoplasmic Ca²⁺ levels. Exposure of either lymphocytes or epithelial cells to toxin (10⁻⁶ M) resulted in a transient increase in Ca²⁺. However, while the Ca²⁺ response to toxin was transient, K-channel modulation by the toxin appeared to be irreversible over the experimental time course. The longer-lasting modulation of Ca²⁺-regulated K⁺ channels may reflect an irreversible action of the B.fasciatus phospholipase A₂ on a Ca²⁺-dependent regulatory process.     

Inhibition of aftercontractions and phasic calcium release by yohimbine in ferret papillary muscle

Phasic release of calcium from the sarcoplasmic reticulum occurs in all mammalian cardiac preparations when the intracellular calcium concentration is sufficiently high. The phasic calcium release is often sufficient to trigger electrophysiological responses and aftercontractions. These can be detrimental to normal cardiac function. We induced phasic calcium release in ferret papillary muscles loaded with the calcium indicator aequorin. Development of phasic calcium release was associated with an increase in resting and peak [Ca²⁺]_i. Inhibiting sodium channels with yohimbine reduced resting [Ca²⁺]_i and prevented phasic calcium release. We propose a mechasism where by reduced [Na⁺]_i, and the subsequent increased efflux of calcium via sodium/calcium exchange reduced [Ca²⁺]_i.     

Delta(9)-Tetrahydrocannabinol-induced desensitization of cannabinoid-mediated inhibition of synaptic transmission between hippocampal neurons in culture

Prolonged exposure to cannabinoids results in desensitization of cannabinoid receptors. Here, we compared the desensitization produced by the partial agonist, Δ⁹-tetrahydrocannabinol (THC) to that produced by the full agonist Win55,212-2 on cannabinoid-mediated inhibition of glutamatergic synaptic transmission. Synaptic activity between rat hippocampal neurons was determined from network-driven increases in the intracellular Ca²⁺ concentration ([Ca²⁺]_i spikes). To assess the effects of prolonged treatment, cultures were incubated with cannabinoids, washed in 0.5% fatty-acid-free bovine serum albumin to ensure the removal of the lipophilic drug and then tested for inhibition of [Ca²⁺]_i spiking by Win55,212-2. In control experiments, 0.1 μM Win55,212-2 inhibited [Ca²⁺]_i spiking by 93 ± 5%. Win55,212-2 produced significantly less inhibition of [Ca²⁺]_i spiking following 18–24 h treatment with 1 μM THC (48 ± 5%) or treatment with 1 μM Win55,212-2 (29 ± 6%). Thus, THC produced significantly less functional desensitization than Win55,212-2. The desensitization produced by THC was maximal at 0.3 μM, remained stable between 1 and 7 days of preincubation and shifted the EC₅₀ of acute inhibition by Win55,212-2 from 27 to 251 nM. Differences in the long-term effects of cannabinoid receptor agonists on synaptic transmission may prove important for evaluating their therapeutic and abuse potential.     

Action of the Na ionophore monensin on vascular smooth muscle of guinea-pig aorta

The effects of the Na⁺ ionophore monensin on contractile responses were investigated in guinea-pig aorta in normal and high K⁺ solutions. In normal K⁺ (5.4 mM) solution, monensin (2 × 10⁻⁵ M) produced a rapid increase in tension followed by slow relaxation. This contraction was markedly inhibited by phentolamine (10⁻⁵ M) or prazosin (10⁻⁶ M) and was accompanied by an increase in tritium efflux from tissue preloaded with [³H]norepinephrine. In the presence of phentolamine, monensin (1–2 × 10⁻⁵ M) or ouabain (1−2 × 10⁻⁵ M) caused only a small and slowly developing contraction. Simultaneous application of these agents caused a more rapid and greater contraction. Either monensin or ouabain gradually increased cellular Na⁺ and decreased cellular K⁺ content. When monensin was applied simultaneously with ouabain, there was a rapid increase in cellular Na⁺ and loss of cellular K⁺. In high K⁺ (65.4 mM) solution, monensin (10⁻⁶ M) slightly reduced the increased tension level but when external glucose was omitted monensin markedly inhibited the contraction. A significant decrease in tissue ATP content was observed only when monensin was applied in glucose-free solution. Similarly, hypoxia (N₂ bubbling) markedly inhibited the high K⁺ contraction and decreased the tissue ATP content only in the absence of glucose. These results suggest that monensin produces a neurogenic contraction due to the release of endogenous catecholamines and also produces a myogenic contraction by a decrease in transmembrane Na⁺ and K⁺ gradients when the Na⁺ and -K⁺ pump is inhibited by ouabain, and that monensin inhibits aerobic energy metabolism of vascular smooth muscle.