Addition Of Cyclopiazonic Acid Research Articles

Inhibitory effect of Pyr6 (an Orai channel blocker) on agonist-induced contractions in rat uterus.

Both human and rat myometrium express stromal interaction molecule (STIM) and Orai/transient receptor potential canonical (TRPC) proteins, which are components of plasma membrane Ca2+ store-operated channels. There are reports that these proteins mediate agonist-induced Ca2+ influx in cultured myometrial cells. In this study, we aimed to determine the effects of Pyr6, an Orai channel blocker, on different agonist-induced contractions in isolated segments of rat uterus. In Ca2+ -free Tyrode's solution, Pyr6 (3μM) promoted a reduction in both the magnitude and frequency of Ca2+ (1 mM)-induced uterine contractions after the addition of carbachol (CCh, 100 μM), but not after the addition of oxytocin (OT, 150 nM). In Ca2+ (0.18 mM)-Tyrode's solution, Pyr6 completely relaxed uterine contractions induced by both CCh and cloprostenol (300 nM), but not those induced by either KCI (40-80 mM) or OT. The addition of Pyr6 abolished the oscillatory uterine contractions induced by Ca2+ after the addition of cyclopiazonic acid (CPA, 10μM). When pre-incubated (5min), Pyr6 reduced the magnitude of both CCh-induced phasic and tonic contractions. The addition of Pyr2 (3μM), an Orai and TRPC channel blocker, abolished uterine contractions induced by CCh or OT. Considering Pyr6 as an Orai channel blocker and its inhibitory effect on uterine contractions induced by CCh, CPA, and cloprostenol, we suggest that Orai channels are required for the maintenance of contractions induced by these agonists in rat uterus.

The cross-bridge dynamics is determined by two length-independent kinetics: Implications on muscle economy and Frank–Starling Law

The cellular mechanisms underlying the Frank–Starling Law of the heart and the skeletal muscle force-length relationship are not clear. This study tested the effects of sarcomere length (SL) on the average force per cross-bridge and on the rate of cross-bridge cycling in intact rat cardiac trabeculae (n=9). SL was measured by laser diffraction and controlled with a fast servomotor to produce varying initial SLs. Tetanic contractions were induced by addition of cyclopiazonic acid, to maintain a constant activation. Stress decline and redevelopment in response to identical ramp shortenings, starting at various initial SLs, was analyzed. Both stress decline and redevelopment responses revealed two distinct kinetics: a fast and a slower phase. The duration of the rapid phases (4.2±0.1msec) was SL-independent. The second slower phase depicted a linear dependence of the rate of stress change on the instantaneous stress level. Identical slopes (70.5±1.6 [1/s], p=0.33) were obtained during ramp shortening at all initial SLs, indicating that the force per cross-bridge and cross-bridge cycling kinetics are length-independent. A decrease in the slope at longer SLs was obtained during stress redevelopment, due to internal shortening. The first phase is attributed to rapid changes in the average force per cross-bridge. The second phase is ascribed to both cross-bridge cycling between its strong and weak conformations and to changes in the number of strong cross-bridges. Cross-bridge cycling kinetics and muscle economy are length-independent and the Frank–Starling Law cannot be attributed to changes in the force per cross-bridge or in the single cross-bridge cycling rates.

Arginine vasopressin, via activation of post-junctional V1 receptors, induces contractile effects in mouse distal colon

The aim of this study was to analyze whether arginine vasopressin (AVP) may be considered a modulator of intestinal motility. In this view, we evaluated, in vitro, the effects induced by exogenous administration of AVP on the contractility of mouse distal colon, the subtype(s) of receptor(s) activated and the action mechanism. Isometric recordings were performed on longitudinal and circular muscle strips of mouse distal colon. AVP (0.001nM–100nM) caused concentration-dependent contractile effects only on the longitudinal muscle, antagonized by the V1 receptor antagonist, V-1880. AVP-induced effect was not modified by tetrodotoxin, atropine and indomethacin. Contractile response to AVP was reduced in Ca2+-free solution or in the presence of nifedipine, and it was abolished by depletion of calcium intracellular stores after repetitive addition of carbachol in calcium-free medium with addition of cyclopiazonic acid. U-73122, an inhibitor of the phospholipase C, effectively antagonized AVP effects, whilst it was not affected by an adenylyl cyclase inhibitor. Oxytocin induced an excitatory effect in the longitudinal muscle of distal colon at very high concentrations, effect antagonized by V-1880. The results of this study shown that AVP, via activation of V1 receptors, is able to modulate positively contractile activity of longitudinal muscle of mouse distal colon, independently by enteric nerve activation and prostaglandin synthesis. Contractile response is achieved by increase in cytoplasmatic Ca2+ concentration via extracellular Ca2+ influx from L-type Ca2+ channels and via Ca2+ release from intracellular stores through phospholipase C pathway. No modulation has been observed on the contractility of the circular muscle.

Store operated Ca2+ entry dependent contraction of coronary artery smooth muscle: Inhibition by peroxide pretreatment

The sarco/endoplasmic reticulum (SER) Ca2+ pool is refilled by the SER Ca2+ pump (SERCA) using cytosolic Ca2+ and/or extracellular Ca2+ entering the cell. The effects of the SERCA pump inhibitor cyclopiazonic acid (CPA) were studied in pig coronary artery smooth muscle using two protocols. In protocol A, the SERCA pump was inhibited by adding CPA to cells/tissues in Ca2+-containing solution, whereas in protocol B, CPA was added to cells/tissues in Ca2+-free solution, followed by reintroduction of extracellular Ca2+. Addition of CPA increased cytosolic Ca2+ in cultured smooth muscle cells and elicited contraction in de-endothelialized coronary arteries in both protocols. Based on pharmacological experiments, the CPA-induced contraction of de-endothelialized arteries in protocol B resulted from store operated Ca2+ entry (SOCE). Reactive oxygen species such as peroxides are known to damage the SERCA pump in this tissue. Consistently, CPA-induced contractions were decreased in arteries pre-treated with hydrogen peroxide in protocol A. However, this pretreatment also decreased the force of contraction due to SOCE in protocol B, suggesting that it closed SOCE. We propose that the closure of SOCE triggered by exposure to reactive oxygen species may be a protective mechanism, so that Ca2+ entry by this pathway is disallowed when SERCA is damaged in pathologies such as ischemia–reperfusion.

Activation of P2Y receptors by ATP and by its analogue, ADPβS, triggers two calcium signal pathways in the longitudinal muscle of mouse distal colon

Our previous research showed that ATP and adenosine 5′-O-2-thiodiphosphate (ADPβS) induce contractile effects in the longitudinal muscle of mouse distal colon via activation of P2Y receptors which are not P2Y 1 or P2Y 12 subtypes. This study investigated the nature of the P2Y receptor subtype(s) and the mechanisms leading to the intracellular calcium concentration increase necessary to trigger muscular contraction. Motor responses of mouse colonic longitudinal muscle to P2Y receptor agonists were examined in vitro as changes in isometric tension. ATP or ADPβS induced muscular contraction, which was not affected by P2Y 11 or P2Y 13 selective antagonists. Calcium-free solution or the calcium channel blocker, nifedipine, failed to modify the contractile responses to ATP or ADPβS, which were virtually abolished by depletion of calcium intracellular stores after repetitive addition of carbachol in calcium-free medium with addition of cyclopiazonic acid. Neomycin or U-73122, phospholipase C inhibitors, or 2-aminoethoxy-diphenylborate (2-APB), membrane-permeant IP 3 receptor inhibitor reduced the response to ATP, whilst ryanodine or ruthenium red, inhibiting calcium release from ryanodine-sensitive stores, abolished the response to ADPβS. Responses to maximally effective concentrations of ATP and ADPβS were not fully additive. Desensitisation with ADPβS antagonized the contractile effects of ATP, as desensitisation with ATP antagonized the response to ADPβS. In the longitudinal muscle of mouse distal colon, ATP and ADPβS induce muscular contraction via a P2Y receptor, coupled to differential signal pathways leading to intracellular calcium increase.

State-Dependent Mechanisms of LTP Expression Revealed by Optical Quantal Analysis

The expression mechanism of long-term potentiation (LTP) remains controversial. Here we combine electrophysiology and Ca(2+) imaging to examine the role of silent synapses in LTP expression. Induction of LTP fails to change p(r) at these synapses but instead mediates an unmasking process that is sensitive to the inhibition of postsynaptic membrane fusion. Once unmasked, however, further potentiation of formerly silent synapses leads to an increase in p(r). The state of the synapse thus determines how LTP is expressed.

TRPC4 Knockdown Suppresses Epidermal Growth Factor-induced Store-operated Channel Activation and Growth in Human Corneal Epithelial Cells

Epidermal growth factor (EGF) in corneal epithelial cells stimulates proliferation by inducing capacitative calcium entry (CCE). However, neither the identity nor the mechanism of activation of the plasma membrane influx pathway that mediates CCE is known. Accordingly, we determined, in human corneal epithelial cells, whether or not (i) CCE is dependent upon stimulation of storeoperated channel (SOC) activity, (ii) the canonical transient receptor potential (TRP) protein isoform TRPC4 is a component of such channels, and (iii) suppression of TRPC4 protein expression decreases EGF-induced stimulation of SOC activity and proliferation. The whole cell patch-clamp technique was used to monitor TRPC4-mediated stimulation of SOC activity following intracellular calcium store depletion and induction of CCE. TRPC4 small interfering RNA transfection suppressed TRPC4 protein expression. Reverse transcription-PCR and Western blot analysis were used to assess knockdown efficiency of mRNA and protein expression. [(3)H]Thymidine incorporation was used to evaluate EGF-in-duced mitogenesis. Ca(2+) transients were measured by single-cell fluorescence imaging. TRPC4 knockdown decreased mRNA and protein expression by 89 and 87%, respectively. In these cells, EGF-induced SOC activation elicited by intracellular calcium store depletion was obviated; 2) EGF-induced CCE fell by 76%; 3) EGF-induced stimulation of SOC activity was eliminated; and 4) EGF-induced increases in proliferation fell by 54%. Thus, TRPC4 is a component of SOC in human corneal epithelial cells whose activation by EGF is requisite for an optimum mitogenic response to this growth factor.

Cyclopiazonic acid reduces the coupling factor of the Ca 2+-ATPase acting on Ca 2+ binding

The mycotoxin cyclopiazonic acid (CPA) is a potent inhibitor of the sarcoplasmic reticulum Ca 2+-ATPase. The compound decreases the affinity of the Ca 2+-ATPase for Ca 2+ and reduces the maximum specific activity of the enzyme. Furthermore, CPA abolishes the cooperativity of Ca 2+ transport, showing a Ca 2+/ATP ratio ∼1 at any extent of Ca 2+ saturation. There is also an effect on the Ca 2+-binding mechanism, where the addition of CPA results in binding of only half-maximal amount of Ca 2+ observed in its absence. The experimental data suggest that in the presence of CPA, only a single Ca 2+ ion binds to the Ca 2+-ATPase.

Calcium buffering of resting, voltage-dependent Ca2+ influx by sarcoplasmic reticulum in femoral arteries from spontaneously hypertensive rats at prehypertensive stage.

We examined the Ca2+-buffering function of the sarcoplasmic reticulum (SR) in the resting state of arteries from spontaneously hypertensive rats (SHR) at a prehypertensive stage. Differences in the effects of cyclopiazonic acid (CPA) and thapsigargin, agents that inhibit SR Ca2+-ATPase, and of ryanodine, which depletes SR Ca2+, on tension and cellular Ca2+ level were assessed in endothelium-denuded strips of femoral arteries from 4-week-old SHR and normotensive Wistar-Kyoto rats (WKY). Addition of CPA, thapsigargin or ryanodine to the resting state of the strips caused an elevation of cytosolic Ca2+ level and a contraction in both WKY and SHR. These responses were larger in SHR than in WKY. The contractions were inhibited strongly by 100 nM nifedipine or 3 microM verapamil and were abolished by Ca2+-free solution. Nifedipine, verapamil or Ca2+-free solution itself caused a relaxation from the resting state of SHR strips, but not from that of WKY strips. The resting Ca2+ influx in arteries measured by a 5-min incubation with 45Ca was significantly larger in SHR than in WKY. This influx was decreased by 10 microM CPA or 10 microM ryanodine in both WKY and SHR. These results suggest that in the resting state of the femoral artery from 4-week-old SHR, the greater part of the increased Ca2+ influx via L-type Ca2+ channels is buffered by Ca2+ uptake into the SR, while some Ca2+ reaches the myofilaments, resulting in the maintenance of resting tone.

Reactive oxygen metabolites increase mitochondrial calcium in endothelial cells: implication of the Ca2+/Na+ exchanger.

In endothelial cells, a bolus of hydrogen peroxide (H2O2) or oxygen metabolites generated by hypoxanthine-xanthine oxidase (HX-XO) increased the mitochondrial calcium concentration [Ca2+]m. Both agents caused a biphasic increase in [Ca2+]m which was preceded by a rise in cytosolic free calcium concentration [Ca2+]c (18 and 6 seconds for H2O2 and HX-XO, respectively). The peak and plateau elevations of [Ca2+] were consistently higher in the mitochondrial matrix than in the cytosol. In Ca2+-free/EGTA medium, the plateau phase of elevated [Ca2+] evoked by H2O2 due to capacitative Ca2+ influx was abolished in the cytosol, but was maintained in the mitochondria. In contrast to H2O2 and HX-XO, ATP which binds the P2Y purinoceptors induced an increase in [Ca2+]m that was similar to that of [Ca2+]c. When cells were first stimulated with inositol 1,4, 5-trisphosphate-generating agonists or the Ca2+-ATPase inhibitor cyclopiazonic acid (CPA), subsequent addition of H2O2 did not affect [Ca2+]c, but still caused an elevation of [Ca2+]m. Moreover, the specific inhibitor of the mitochondrial Ca2+/Na+ exchanger, 7-chloro-3,5-dihydro-5-phenyl-1H-4.1-benzothiazepine-2-on (CGP37157), did not potentiate the effects of H2O2 and HX-XO on [Ca2+]m, while causing a marked increase in the peak [Ca2+]m and a significant attenuation of the rate of [Ca2+]m efflux upon addition of histamine or CPA. In permeabilized cells, H2O2 mimicked the effects of CGP37157 causing an increase in the basal level of matrix free Ca2+ and decreased efflux. Dissipation of the electrochemical proton gradient by carbonylcyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), and blocade of the Ca2+ uptake by ruthenium red prevented [Ca2+]m increases evoked by H2O2. These results demonstrate that the H2O2-induced elevation in [Ca2+]m results from a transfer of Ca2+ secondary to increased [Ca2+]c, and an inhibition of the Ca2+/Na+ electroneutral exchanger of the mitochondria.

Possible mechanism underlying the potent vasoconstrictor actions of cyclopiazonic acid on dog cerebral arteries

A sustained Ca 2+ influx via L-type Ca 2+ channels has been shown in the resting state of dog cerebral arteries. Sarcoplasmic reticulum is now recognized to serve as a buffer barrier to Ca 2+ entry in vascular smooth muscle cells. To clarify whether sarcoplasmic reticulum of the cerebral arteries can buffer the sustained Ca 2+ influx, effects of cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic reticulum Ca 2+-ATPase, were determined in endothelium-denuded strips of the cerebral (basilar, posterior communicating, middle cerebral), mesenteric and coronary arteries of the dog. The addition of CPA (0.1–10 μM) during the resting state of the strips caused a concentration-dependent contraction in the three cerebral arteries. The CPA-induced contraction was extremely small in the mesenteric or coronary artery. The CPA-induced contractions in the cerebral arteries were inhibited concentration-dependently by nifedipine (1–100 nM). Nifedipine itself induced relaxation from the resting state of cerebral arteries, suggesting a maintenance of basal tone. The CPA-induced potent contraction seen in the cerebral arteries could be mimicked in the mesenteric artery by elevating the extracellular K + concentration (14.9 mM) or adding Bay k 8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate] (100 nM) to produce an increase in Ca 2+ influx via L-type Ca 2+ channels. We conclude that, in the resting state of dog cerebral arteries, (1) the greater part of the sustained Ca 2+ influx is buffered by Ca 2+ uptake into the sarcoplasmic reticulum, (2) therefore, the inhibition of sarcoplasmic reticulum Ca 2+-ATPase by CPA causes a potent contraction, and (3) the maintenance of basal tone suggests that some Ca 2+ that entered via L-type Ca 2+ channels always reaches the myofilaments in the resting state.

Effects of cyclopiazonic acid on Ca2+ regulation by the sarcoplasmic reticulum in saponin-permeabilized skeletal muscle fibres.

The effects of the sarcoplasmic reticulum (SR) Ca2+ pump inhibitor cyclopiazonic acid (CPA) were studied in saponin-permeabilized frog skeletal muscle fibres. Release of Ca2+ from the SR was triggered by brief (2 s) applications of 40 mM caffeine at 2-min intervals. Changes in [Ca2+] within the fibre were monitored continuously using Fura-2 fluorescence. At a bathing [Ca2+] of 100 nM, introduction of 20 microM CPA induced a slow release of Ca2+ from the SR. The following one to two caffeine-induced Ca2+ transients were markedly increased in amplitude and duration. Thereafter, the caffeine-induced Ca2+ transients decreased progressively and were barely detectable 6-7 min after introduction of CPA. However, increasing the bathing [Ca2+] or increasing the Ca2+ loading period resulted in a partial recovery of the caffeine-induced Ca2+ transients, suggesting that pump inhibition is incomplete, even in the presence of 100 microM CPA. The slow Ca2+ efflux induced by CPA was insensitive to ryanodine, but absent following abolition of SR Ca2+ pump activity by ATP withdrawal. These results suggest that the caffeine-induced Ca2+ transient reflects a balance between efflux via the SR Ca2+ channel and reuptake by the Ca pump. Ca2+ release upon addition of CPA may result from inhibition of SR Ca2+ uptake, which reveals a tonic Ca2+ efflux that is independent of the Ca2+ release channels.

Changes in intracellular Ca2+ concentration induced by L-type Ca2+ channel current in guinea pig gastric myocytes.

We investigated the relationship between voltage-operated Ca2+ channel current and the corresponding intracellular Ca2+ concentration ([Ca2+]i) change (Ca2+ transient) in guinea pig gastric myocytes. Fluorescence microspectroscopy was combined with conventional whole cell patch-clamp technique, and fura 2 (80 microM) was added to CsCl-rich pipette solution. Step depolarization to 0 mV induced inward Ca2+ current (ICa) and concomitantly raised [Ca2+]i. Both responses were suppressed by nicardipine, an L-type Ca2+ channel blocker, and the voltage dependence of Ca2+ transient was similar to the current-voltage relation of ICa. When pulse duration was increased by up to 900 ms, peak Ca2+ transient increased and reached a steady state when stimulation was for longer. The calculated fast Ca2+ buffering capacity (B value), determined as the ratio of the time integral of ICa divided by the amplitude of Ca2+ transient, was not significantly increased after depletion of Ca2+ stores by the cyclic application of caffeine (10 mM) in the presence of ryanodine (4 microM). The addition of cyclopiazonic acid (CPA, 10 microM), a sarco(endo)plasmic reticulum Ca(2+)-ATPase inhibitor, decreased B value by approximately 20% in a reversible manner. When KCl pipette solution was used, Ca(2+)-activated K+ current [IK(Ca)] was also recorded during step depolarization. CPA sensitively suppressed the initial peak and oscillations of IK(Ca) with irregular effects on Ca2+ transients. The above results suggest that, in guinea pig gastric myocyte, Ca2+ transient is tightly coupled to ICa during depolarization, and global [Ca2+]i is not significantly affected by Ca(2+)-induced Ca2+ release from sarcoplasmic reticulum during depolarization.

Potent vasoconstrictor actions of cyclopiazonic acid and thapsigargin on femoral arteries from spontaneously hypertensive rats.

1. Ca2+ buffering function of sarcoplasmic reticulum (SR) in the resting state of arteries from spontaneously hypertensive rats (SHR) was examined. Differences in the effects of cyclopiazonic acid (CPA) and thapsigargin, agents which inhibit the Ca(2+)-ATPase of SR, on tension and cellular Ca2+ level were assessed in endothelium-denuded strips of femoral arteries from 13-week-old SHR and normotensive Wistar-Kyoto rats (WKY). 2. In resting strips preloaded with fura-PE3, the addition of CPA (10 microM) or thapsigargin (100 nM) caused an elevation of cytosolic Ca2+ level ([Ca2+]i) and a contraction. These responses were significantly greater in SHR than in WKY. 3. The additional of verapamil (3 microM) to the resting strips caused a decrease in resting [Ca2+]i, which was significantly greater in SHR than in WKY. In SHR, but not in WKY, this decrease was accompanied by a relaxation from the resting tone, suggesting the maintenance of myogenic tone in the SHR artery. 4. Verapamil (3 microM) abolished differences between SHR and WKY. The effects of verapamil were much greater on the contraction than on the [Ca2+]i. 5. The resting of Ca2+ influx in arteries measured after a 5 min incubation of the artery with 45Ca was not increased by CPA or thapsigargin in either SHR or WKY. The net Ca2+ entry measured after a 30 min incubation of the artery with 45Ca was decreased by CPA or thapsigargin in both SHR and WKY. The resting Ca2+ influx was significantly higher in SHR than in WKY, and was decreased by nifedipine (100 nM) in the SHR artery, but was unchanged in the WKY artery. 6. The resting 45Ca efflux from the artery was increased during the addition of CPA (10 microM). This increase was less in SHR than in WKY. The resting 45Ca efflux was the same in SHR and WKY. 7. These results suggest that (1) the Ca2+ influx via L-type voltage-dependent Ca2+ channels (VDCCs) was increased in the resting state of the SHR femoral artery, (2) the greater part of the increased Ca2+ influx was buffered by Ca2+ uptake into the SR and some Ca2+ reached the myofilaments resulting in the maintenance of the myogenic tone, and (3) therefore the functional elimination of SR by CPA or thapsigargin caused a large elevation of [Ca2+]i and a potent contraction in this artery. During this process, the contraction was mainly due to the basal Ca2+ influx via L-type VDCCs. The present study also showed the existence of a relatively large compartment of [Ca2+]i which does not contribute to the contraction during the addition of CPA or thapsigargin.

Ca 2+ effluxes from the sarcoplasmic reticulum vesicles of frog muscle: effects of cyclopiazonic acid and thapsigargin

Call efflux from frog muscle sarcoplasmic reticulum (SR) vesicles was studied by measuring external free [Ca 2+] using Fluo-3 fluorescence. Light SR vesicles were preloaded with Ca 2+ in the presence of ATP and inorganic phosphate (P i). Calcium pump reversal was activated by either depletion of the medium ATP by apyrase in the presence of 20 mM P i, or resuspending preloaded vesicles in an ATP-free solution containing 1 mM ADP and 20 mM P i. Cyclopiazonic acid (CPA) and thapsigargin (TG), at concentrations of 2.5 μM, which completely inhibit Ca 2+ uptake, both inhibited the pump reversal efflux almost completely. When active Ca 2+ uptake was stopped by either ATP-depletion or addition of CPA, a leak efflux of 6–7 nmole/mg/min was recorded. TG (2.5 μM) reduced this leak by over 50%, suggesting that TG, but not CPA, can slow the passage of calcium ions through the Ca 2+-ATPase passive channel.

Rhythmic relaxations of active tension in the rabbit large arteries induced by a combination of cyclopiazonic acid and Bay K 8644.

1. We previously demonstrated that cyclopiazonic acid (CPA), an inhibitor of Ca(2+)-ATPase in the sarcoplasmic reticulum, induced rhythmic relaxations of active tension in the endothelium-denuded small arteries of the mesentery and the ear of the rabbit, but that this agent failed to induce rhythmic responses in the endothelium-denuded rabbit femoral artery. 2. In the present study, an attempt was made to induce rhythmic relaxations of active tension in the endothelium-denuded rabbit femoral artery and the thoracic aorta, both of which were suspended in organ chambers for isometric tension recordings, by using CPA plus Bay K 8644, an L-type Ca2+ channel agonist, to induce an excessive increase in cytosolic Ca2+. 3. CPA or Bay K 8644 alone failed to produce rhythmic relaxations in the femoral artery that had been contracted with phenylephrine. In contrast, rhythmic responses were induced by the sequential treatment of the femoral artery with CPA and Bay K 8644. 4. The rhythmic relaxations of active tension in the femoral artery induced by CPA plus Bay K 8644 were inhibited by charybdotoxin and by iberiotoxin, both of which are antagonists of the Ca(2+)-activated K+ channel, but not by glibenclamide, a blocker of the ATP-sensitive K+ channel. 5. The endothelium-denuded rabbit aorta also exhibited rhythmic responses by the sequential addition of CPA and Bay K 8644. These responses were sensitive to charybdotoxin. 6. These findings indicate that, like small arteries, the large femoral and aortic arteries of the rabbit are also capable of displaying rhythmic relaxations of active tension; these relaxations may be in part attributed to the activation of the Ca(2+)-activated K+ channel as a result of the Ca2+ overload caused by CPA and Bay K 8644.

Steady-state [Ca2+]i-force relationship in intact twitching cardiac muscle: direct evidence for modulation by isoproterenol and EMD 53998

We have developed a novel method for measuring steady-state force-[Ca2+]i relations in isolated, membrane-intact rat trabeculae that are microinjected with Fura-2 salt. Twitches are markedly slowed after inhibition of phasic Ca2+ release and uptake from the sarcoplasmic reticulum by addition of cyclopiazonic acid and ryanodine. During relaxation of slowed twitches, force and [Ca2+]i trace a common trajectory in plots of force versus [Ca2+]i, despite very different histories of contraction. The common trajectory thereby provides a high resolution determination of the steady-state relation between force and [Ca2+]i. Using this method, we show that 1 microM isoproterenol, a beta-adrenergic agonist, causes a rightward shift (Hill function K1/2 increased from 0.39 +/- 0.07 microM to 0.82 +/- 0.23 microM, p < 0.02, n = 6) and a decreased slope (nH decreased from 5.4 +/- 1.1 to 4.0 +/- 1.4, p < 0.02) of the steady-state force-[Ca2+]i curve, with no change in maximal force (Fmax = 99.2 +/- 2.2% of control). In contrast, 2 microM EMD 53998, a racemic thiadiazinone derivative, causes a leftward shift (K1/2 decreased from 0.42 +/- 0.02 microM to 0.30 +/- 0.06 microM, p < 0.02, n = 4) with no change in slope of the steady-state force-[Ca2+]i curve, accompanied by a modest increase in maximal force (Fmax = 107.1 +/- 4.6% of control, p < 0.02). To gain mechanistic insight into these modulatory events, we developed a simple model of cooperative thin filament activation that predicts steady-state force-[Ca2+]i relationships. Model analysis suggests that isoproterenol decreases cooperativity arising from nearest-neighbor interactions between regulatory units on the thin filament, without change in the equilibrium constant for Ca2+ binding. In contrast, the effects of EMD 53998 are consistent with an increase in the affinity of strong-binding cross-bridges, without change in either the affinity of troponin C for Ca2+ or cooperative interactions.

Measurement of Sarcoplasmic Reticulum Function in Mammalian Skeletal Muscle: Technical Aspects

Two recently developed methods for measuring the maximal rate of Ca2+ uptake and Ca2+ ATPase activity (EC 3.1.6.38) in vitro use muscle homogenate rather than isolated sarcoplasmic reticulum (SR). In this study we investigated technical aspects of these assays, including specificity and variability of the assays, the effect of different freezing treatments on maximal Ca2+ uptake and Ca2+ ATPase activity of human and rat muscle homogenate, stability of the homogenate, and the modification of the Ca2+ uptake assay to measure Ag+-induced Ca2+ release. Addition of cyclopiazonic acid (20 μm) blocked Ca2+ uptake, demonstrating specificity of the assay. Using frozen muscle homogenate, the inter- and intraassay variation for both assays was less than 9%. Whereas homogenates were stable to freezing for both Ca2+ uptake and Ca2+ ATPase activity, there was a significant (P < 0.05) decrease in activity when muscle was freeze-dried or quickly frozen in small pieces. After 1 h on ice, rat muscle homogenate Ca2+ uptake and Ca2+ ATPase activity had decreased by 6.0% (ns) and 3.6% (P < 0.05), respectively; after 3 h, activity had decreased by 15.3 and 14.7%, respectively (P < 0.01). The Ca2+ uptake assay was modified to allow measurement of Ag+-induced Ca2+ release. Following homogenate addition, after the SR vesicles were loaded with Ca2+ and [Ca2+] had declined to a plateau, AgNO3 (141 μm) was added, initiating release of Ca2+ into the assay solution. Addition of dithiothreitol (4 mM) blocked the Ag+-induced Ca2+ release, demonstrating specificity of the assay.

Differential Effects of Endoplasmic Reticulum Ca&lt;sup&gt;2+&lt;/sup&gt;-ATPase Inhibitors on Madin-Darby Canine Kidney Cells

Endoplasmic reticulum (ER) Ca2+-ATPases regulate intracellular free calcium ([Ca2+]in) in renal epithelial cells. Interestingly, treatment of certain cell types with ER Ca2+-ATPase inhibitors such as thapsigargin (TG), cyclopiazonic acid (CPA), and 2,5-di-tert-butyl-l,4-benzohydroquinone (BHQ) may result in distinct effects on [Ca2+]m homeostasis. In this study, we investigated whether these distinct effects are due to Ca2+ entry after Ca2+ depletion of the ER. Therefore, we coloaded Madin-Darby canine kidney cells with SNARF-1 and Fura-2 (see text) to simultaneously measure intracellular pH (pHin) and [Ca2+]in, respectively. Subsequently, Madin-Darby canine kidney cells were treated both in the presence and absence of extracellular Ca2+ ([Ca2+]ex) with ER-Ca2+-ATPase inhibitors (TG, CPA, and BHQ). In the presence of [Ca2+]ex, both TG and BHQ caused a sustained increase in [Ca2+]in and a decrease in pHιn. In contrast, CPA induced a transient increase in [Ca2+]m in the presence of [Ca2+]ex. Acute [Ca2+]ex removal resulted in a decrease in [Ca2+]in. In the absence of [Ca2+]ex, the [Ca2+]in increase induced by either TG, CPA, or BHQ was suppressed. These data suggest that the TG-, BHQ-, and CPA-sensitive-Ca2+ pool is readily depleted by removal of [Ca2+]ex. Readdition of [Ca2+]ex increased [Ca2+]ιn transiently in the absence of drugs. In the presence of CPA, the readdition of [Ca2+]ex increased [Ca2+]in also transiently. In contrast, readdition of [Ca2+]ex resulted in a sustained increase in [Ca2+]in when either TG or BHQ was present. This latter effect was reversed by subsequent addition of CPA (in the presence of [Ca2+]ex), or when CPA was added prior to the readdition of [Ca2+]ex. These data suggest that CPA, unlike TG and BHQ, also decreases Ca2+ entry across the plasma membrane. Altogether, our data indicate that ER Ca2+-ATPase inhibitors exhibit differential effects on [Ca2+]in homeostasis in Madin-Darby canine kidney cells.

Effect of cyclopiazonic acid, an inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase, on the frequency-dependence of the contraction-relaxation cycle of the guinea-pig isolated atrium.

1. The relevance of a functional sarcoplasmic reticulum (SR) membrane system to the contraction-relaxation cycle and to the force-frequency relationship of guinea-pig atrial tissue was investigated. Cyclopiazonic acid (CPA) was used to inhibit selectively the activity of the SR Ca(2+)-ATPase. IC50 values of 0.2 microM or 1.0 microM were measured in guinea-pig isolated SR membranes in the absence or presence of millimolar ATP, respectively. CPA (0.3-30 microM) did not inhibit the activity of the sarcolemmal Na(+)-Ca(2+)-exchanger as measured in isolated cardiac cell membrane preparations. 2. In guinea-pig isolated left atrium paced at 2.5 Hz (30 degrees C), CPA (1-100 microM) produced a concentration-dependent reduction in developed tension and a fall in the maximum rate of tension increase (+dT/dtmax) and decrease (-dT/dtmax). The twitch duration was markedly increased due to a prolongation of the time to peak tension, and in particular, the relaxation phase. 3. The contraction-relaxation cycle of the left atrium showed a marked dependence on the frequency of stimulation. The developed tension and +dT/dtmax showed a progressive increase from 0.5 Hz, reaching peak values at a stimulation rate of 1.5-2.5 Hz, the positive staircase phenomenon. Higher frequencies of stimulation caused a fall in these parameters. Resting tension was unaffected. The time-course of the contraction-relaxation cycle was also frequency-dependent, with both time to peak tension and relaxation time showing a progressive fall from 2.0-3.5 Hz. 4. The addition of CPA (30 microM) caused marked alterations in the frequency-dependence of the contraction-relaxation cycle. The frequency-dependence of developed tension, + dr/dtmax and dT/dt max was shifted downwards, particularly at higher frequencies, and the frequency at which peak values of+ dT/dtmax and - dT/dtmax were reached was shifted leftwards. The resting tension of the tissues in the presence of 30 micro M CPA was increased markedly at frequencies greater than 2 Hz. The time-course of the contraction-relaxation cycle was markedly prolonged between 1.0 and 3.5 Hz, due to an effect on both time to peak tension and relaxation time.5. In conclusion, these results show that CPA is a highly selective inhibitor of the cardiac SR Ca2+-ATPase, without effect on the sarcolemmal Na+-Ca2+-exchanger, and suggest that a functional SR Ca2+-ATPase is necessary for the normal contraction-relaxation cycle of guinea-pig cardiac tissue.Additionally, the results suggest an increasing dependence of tension development on SR Ca2+-ATPase with increasing frequency, which may reflect either a frequency-dependent activation of this enzyme or the diminished contribution of the Na+-Ca2+ exchanger. These results also provide novel support for the mechanism of the depressed force-frequency relation found in cardiac tissue of heart failure patients, in which there is a reduced expression of Ca2+-ATPase.