scholarly journals Role of Reduced Sarco-Endoplasmic Reticulum Ca2+-ATPase Function on Sarcoplasmic Reticulum Ca2+ Alternans in the Intact Rabbit Heart

2021 ◽  
Vol 12 ◽  
Author(s):  
Lianguo Wang ◽  
Rachel C. Myles ◽  
I-Ju Lee ◽  
Donald M. Bers ◽  
Crystal M. Ripplinger
Keyword(s):  
Endoplasmic Reticulum ◽  
Sarcoplasmic Reticulum ◽  
Optical Mapping ◽  
Cyclopiazonic Acid ◽  
Rabbit Heart ◽  
Intracellular Ca ◽  
Cardiac Alternans ◽  
Intact Rabbit ◽  
Rapid Ventricular Pacing ◽  
Serca Inhibition

Sarcoplasmic reticulum (SR) Ca2+ cycling is tightly regulated by ryanodine receptor (RyR) Ca2+ release and sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) Ca2+ uptake during each excitation–contraction coupling cycle. We previously showed that RyR refractoriness plays a key role in the onset of SR Ca2+ alternans in the intact rabbit heart, which contributes to arrhythmogenic action potential duration (APD) alternans. Recent studies have also implicated impaired SERCA function, a key feature of heart failure, in cardiac alternans and arrhythmias. However, the relationship between reduced SERCA function and SR Ca2+ alternans is not well understood. Simultaneous optical mapping of transmembrane potential (Vm) and SR Ca2+ was performed in isolated rabbit hearts (n = 10) using the voltage-sensitive dye RH237 and the low-affinity Ca2+ indicator Fluo-5N-AM. Alternans was induced by rapid ventricular pacing. SERCA was inhibited with cyclopiazonic acid (CPA; 1–10 μM). SERCA inhibition (1, 5, and 10 μM of CPA) resulted in dose-dependent slowing of SR Ca2+ reuptake, with the time constant (tau) increasing from 70.8 ± 3.5 ms at baseline to 85.5 ± 6.6, 129.9 ± 20.7, and 271.3 ± 37.6 ms, respectively (p < 0.05 vs. baseline for all doses). At fast pacing frequencies, CPA significantly increased the magnitude of SR Ca2+ and APD alternans, most strongly at 10 μM (pacing cycle length = 220 ms: SR Ca2+ alternans magnitude: 57.1 ± 4.7 vs. 13.4 ± 8.9 AU; APD alternans magnitude 3.8 ± 1.9 vs. 0.2 ± 0.19 AU; p < 0.05 10 μM of CPA vs. baseline for both). SERCA inhibition also promoted the emergence of spatially discordant alternans. Notably, at all CPA doses, alternation of SR Ca2+ release occurred prior to alternation of diastolic SR Ca2+ load as pacing frequency increased. Simultaneous optical mapping of SR Ca2+ and Vm in the intact rabbit heart revealed that SERCA inhibition exacerbates pacing-induced SR Ca2+ and APD alternans magnitude, particularly at fast pacing frequencies. Importantly, SR Ca2+ release alternans always occurred before the onset of SR Ca2+ load alternans. These findings suggest that even in settings of diminished SERCA function, relative refractoriness of RyR Ca2+ release governs the onset of intracellular Ca2+ alternans.

A role for the sarcoplasmic reticulum in Ca2+ extrusion from rabbit inferior vena cava smooth muscle

1998 ◽  
Vol 274 (1) ◽  
pp. H123-H131 ◽  
Author(s):  
Mark A. Nazer ◽  
Cornelis Van Breemen
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Sarcoplasmic Reticulum ◽  
Inferior Vena Cava ◽  
Inferior Vena ◽  
Vena Cava ◽  
Cyclopiazonic Acid ◽  
Free Solution ◽  
Intracellular Ca ◽  
In Series

Ca2+extrusion from rabbit inferior vena cava smooth muscle was studied using ratiometric fura 2 fluorimetry. Concomitant blockade of the plasma membrane Ca2+-adenosinetriphosphatase (ATPase; PCMA), Na+-Ca2+exchanger, and sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) completely prevented the decline in intracellular Ca2+ concentration ([Ca2+]i) normally observed when Ca2+ is removed from the extracellular space (ECS) after stimulated Ca2+ influx. Blockade of the Na+-Ca2+exchanger by removal of external Na+ reduced the rate of [Ca2+]idecline by 47%. Blockade of SERCA with cyclopiazonic acid reduced it by 23%, and this was not additive to the effects of Na+ removal. Exposure to nominally Ca2+-free solution prevented the sarcoplasmic reticulum (SR) from reloading only if the Na+-Ca2+exchanger was operational. Our results can be explained by an SR contribution to Ca2+ extrusion in which SERCA is arranged in series with Na+-Ca2+exchange.

Regulation of sarcoplasmic reticulum Ca2+ reuptake in porcine airway smooth muscle

2008 ◽  
Vol 294 (4) ◽  
pp. L787-L796 ◽  
Author(s):  
Venkatachalem Sathish ◽  
Figen Leblebici ◽  
Sertac N. Kip ◽  
Michael A. Thompson ◽  
Christina M. Pabelick ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Airway Smooth Muscle ◽  
Small Interfering Rna ◽  
Regulatory Protein ◽  
Plasmic Reticulum ◽  
Intracellular Ca ◽  
Subsequent Exposure ◽  
Interfering Rna ◽  
Serca Inhibition

Regulation of intracellular Ca2+ concentration ([Ca2+]i) in airway smooth muscle (ASM) during agonist stimulation involves sarcoplasmic reticulum (SR) Ca2+ release and reuptake. The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is key to replenishment of SR Ca2+ stores. We examined regulation of SERCA in porcine ASM: our hypothesis was that the regulatory protein phospholamban (PLN) and the calmodulin (CaM)-CaM kinase (CaMKII) pathway (both of which are known to regulate SERCA in cardiac muscle) play a role. In porcine ASM microsomes, we examined the expression and extent of PLN phosphorylation after pharmacological inhibition of CaM (with W-7) vs. CaMKII (with KN-62/KN-93) and found that PLN is phosphorylated by CaMKII. In parallel experiments using enzymatically dissociated single ASM cells loaded with the Ca2+ indicator fluo 3 and imaged using fluorescence microscopy, we measured the effects of PLN small interfering RNA, W-7, and KN-62 on [Ca2+]i responses to ACh and direct SR stimulation. PLN small interfering RNA slowed the rate of fall of [Ca2+]i transients to 1 μM ACh, as did W-7 and KN-62. The two inhibitors additionally slowed reuptake in the absence of PLN. In other cells, preexposure to W-7 or KN-62 did not prevent initiation of ACh-induced [Ca2+]i oscillations (which were previously shown to result from repetitive SR Ca2+ release/reuptake). However, when ACh-induced [Ca2+]i oscillations reached steady state, subsequent exposure to W7 or KN-62 decreased oscillation frequency and amplitude and slowed the fall time of [Ca2+]i transients, suggesting SERCA inhibition. Exposure to W-7 completely abolished ongoing ACh-induced [Ca2+]i oscillations in some cells. Preexposure to W-7 or KN-62 did not affect caffeine-induced SR Ca2+ release, indicating that ryanodine receptor channels were not directly inhibited. These data indicate that, in porcine ASM, the CaM-CaMKII pathway regulates SR Ca2+ reuptake, potentially through altered PLN phosphorylation.

scholarly journals Refractoriness of sarcoplasmic reticulum Ca2+ release determines Ca2+ alternans in atrial myocytes

2012 ◽  
Vol 302 (11) ◽  
pp. H2310-H2320 ◽  
Author(s):  
Vyacheslav M. Shkryl ◽  
Joshua T. Maxwell ◽  
Timothy L. Domeier ◽  
Lothar A. Blatter
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Cellular Level ◽  
Release Mechanism ◽  
Atrial Myocytes ◽  
Restitution Curve ◽  
Intracellular Ca ◽  
Cardiac Alternans ◽  
Kinetics Of ◽  
Beating Frequency

Cardiac alternans is a recognized risk factor for cardiac arrhythmia and sudden cardiac death. At the cellular level, Ca2+ alternans appears as cytosolic Ca2+ transients of alternating amplitude at regular beating frequency. Cardiac alternans is a multifactorial process but has been linked to disturbances in intracellular Ca2+ regulation. In atrial myocytes, we tested the role of voltage-gated Ca2+ current, sarcoplasmic reticulum (SR) Ca2+ load, and restitution properties of SR Ca2+ release for the occurrence of pacing-induced Ca2+ alternans. Voltage-clamp experiments revealed that peak Ca2+ current was not affected during alternans, and alternans of end-diastolic SR Ca2+ load, evaluated by application of caffeine or measured directly with an intra-SR fluorescent Ca2+ indicator (fluo-5N), were not a requirement for cytosolic Ca2+ alternans. Restitution properties and kinetics of refractoriness of Ca2+ release after activation during alternans were evaluated by four different approaches: measurements of 1) the delay (latency) of occurrence of spontaneous global Ca2+ releases and 2) Ca2+ spark frequency, both during rest after a large and small alternans Ca2+ transient; 3) the magnitude of premature action potential-induced Ca2+ transients after a large and small beat; and 4) the efficacy of a photolytically induced Ca2+ signal (Ca2+ uncaging from DM-nitrophen) to trigger additional Ca2+ release during alternans. The results showed that the latency of global spontaneous Ca2+ release was prolonged and Ca2+ spark frequency was decreased after the large Ca2+ transient during alternans. Furthermore, the restitution curve of the Ca2+ transient elicited by premature action potentials or by photolysis-induced Ca2+ release from the SR lagged behind after a large-amplitude transient during alternans compared with the small-amplitude transient. The data demonstrate that beat-to-beat alternation of the time-dependent restitution properties and refractory kinetics of the SR Ca2+ release mechanism represents a key mechanism underlying cardiac alternans.

Butyrylcholinesterase in the Adult Rabbit Heart

1971 ◽  
Vol 29 ◽  
pp. 284-285
Author(s):  
M. Hagopian ◽  
V.M. Tennyson
Keyword(s):  
Endoplasmic Reticulum ◽  
Sarcoplasmic Reticulum ◽  
Papillary Muscle ◽  
Cholinesterase Activity ◽  
Ultrastructural Level ◽  
Rabbit Heart ◽  
Adult Rabbit ◽  
Intercalated Disc ◽  
Complex Forms ◽  
T System

The papillary muscle of the adult rabbit heart was studied by a modification of the copper thiocholine technique for the localization of cholinesterase activity. At the ultrastructural level the muscle shows evidence of butyrylcholinesterase (BuChE) in view of the fact that an electron-opaque reaction product, a copper thiocholine complex, forms when butyrylthiocholine (BuThCh) is used as substrate. The deposition of the reaction product was abolished by preincubation with iso-OMPA (tetraisopropyl pyrophosphortetramide) which specifically inhibits BuChE, but not by BW284C51 (1,5-bis [4-allyldimethylammoniumphenyl] penta-3-one dibromide) which inhibits acetylcholinesterase (AChE). When acetylthiocholine (AThCh) was used as substrate, no end product was found.BuChE activity is seen primarily in the terminal sacs of the sarcoplasmic reticulum (SR) in the region adjacent to the T system (Fig. 1), within the subsarcolemmal vesicles or tubules, and in the endoplasmic reticulum (Fig. 2) of the perinuclear zone. The reaction sites are found occasionally in some areas of the longitudinal elements of the SR, on the nuclear envelope (Fig.2), and in the cisterns which are associated with the intercalated disc. The discs are always negative.

Sarcoplasmic reticulum Ca2+ uptake is not decreased in aorta from deoxycorticosterone acetate hypertensive rats: functional assessment with cyclopiazonic acid

1995 ◽  
Vol 73 (11) ◽  
pp. 1536-1545 ◽  
Author(s):  
Rita C. A. Tostes ◽  
Lusiane M. Bendhack ◽  
Oren Traub ◽  
R. Clinton Webb
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Relaxation Rate ◽  
Vascular Reactivity ◽  
Specific Inhibitor ◽  
Cyclopiazonic Acid ◽  
Hypertensive Rats ◽  
Deoxycorticosterone Acetate ◽  
Intracellular Calcium Mobilization ◽  
Intracellular Ca ◽  
And Control

Ca2+ plays a major role in vascular contraction, and a defect in intracellular Ca2+ regulation has been associated with increased vascular reactivity in hypertension. To test the hypothesis that the sarcoplasmic reticulum does not adequately buffer Ca2+ in deoxycorticosterone acetate (DOCA) hypertension, contractile experiments were performed with a specific inhibitor of the sarcoplasmic reticulum Ca2+ ATPase, cyclopiazonic acid (CPA). Contractile force in aortic strips from DOCA and control rats was measured, using standard muscle bath procedures, to evaluate (i) Ca2+ handling, assessing caffeine and serotonin (5HT) induced contractions in Ca2+-free buffer and (ii) relaxation rate after 5HT washout. Contractile responses elicited with 5HT (3 × 10−6 mol/L) and caffeine (20 mmol/L) were greater in DOCA than in control arteries. CPA (1 × 10−7 to 3 × 10−5 mol/L) reduced phasic contractions to 5HT and caffeine in DOCA and control aorta, and no differences in the IC50 values were observed. Aortae from DOCA rats contracted when placed in normal buffer, subsequent to treatment with Ca2+-free buffer, but control aortae did not. CPA potentiated these responses in DOCA aorta and only caused a modest contraction in control aorta. CPA-induced contraction did not occur in Ca2+-free buffer, and it was inhibited by nifedipine (IC50 = 4 × 10−9 mol/L). The relaxation rate, after 5HT washout (3 × 10−6 mol/L), was increased in DOCA aorta (2.6 ± 0.3 min) compared with control (1.7 ± 0.2 min), and CPA (10−5 mol/L) increased the relaxation rate in both groups. The results support the hypothesis of defective Ca2+ handling in DOCA hypertension. However, an increased Ca2+ influx, and not a decreased buffering ability of the sarcoplasmic reticulum, contributes to the enhanced vascular reactivity observed in DOCA hypertension.Key words: vascular smooth muscle, intracellular calcium mobilization, caffeine, cyclopiazonic acid, sarcoplasmic reticulum, deoxycorticosterone (DOCA) hypertension.

Intracellular Ca2+ stores in chemoreceptor cells of the rabbit carotid body: significance for chemoreception

2000 ◽  
Vol 279 (1) ◽  
pp. C51-C61 ◽  
Author(s):  
I. Vicario ◽  
A. Obeso ◽  
A. Rocher ◽  
J. R. López-Lopez ◽  
C. González
Keyword(s):  
Significant Role ◽  
Carotid Body ◽  
Time Course ◽  
Cyclopiazonic Acid ◽  
Structural Identity ◽  
High K ◽  
Intracellular Ca ◽  
Intact Rabbit ◽  
Chemoreceptor Cells

The notion that intracellular Ca2+ (Cai 2+) stores play a significant role in the chemoreception process in chemoreceptor cells of the carotid body (CB) appears in the literature in a recurrent manner. However, the structural identity of the Ca2+ stores and their real significance in the function of chemoreceptor cells are unknown. To assess the functional significance of Cai 2+ stores in chemoreceptor cells, we have monitored 1) the release of catecholamines (CA) from the cells using an in vitro preparation of intact rabbit CB and 2) the intracellular Ca2+ concentration ([Ca2+]i) using isolated chemoreceptor cells; both parameters were measured in the absence or the presence of agents interfering with the storage of Ca2+. We found that threshold [Ca2+]i for high extracellular K+ (Ke +) to elicit a release response is ≈250 nM. Caffeine (10–40 mM), ryanodine (0.5 μM), thapsigargin (0.05–1 μM), and cyclopiazonic acid (10 μM) did not alter the basal or the stimulus (hypoxia, high Ke +)-induced release of CA. The same agents produced Cai 2+transients of amplitude below secretory threshold; ryanodine (0.5 μM), thapsigargin (1 μM), and cyclopiazonic acid (10 μM) did not alter the magnitude or time course of the Cai 2+responses elicited by high Ke +. Several potential activators of the phospholipase C system (bethanechol, ATP, and bradykinin), and thereby of inositol 1,4,5-trisphosphate receptors, produced minimal or no changes in [Ca2+]i and did not affect the basal release of CA. It is concluded that, in the rabbit CB chemoreceptor cells, Cai 2+ stores do not play a significant role in the instant-to-instant chemoreception process.

scholarly journals Intracellular organelles and calcium homeostasis in rods and cones

2007 ◽  
Vol 24 (5) ◽  
pp. 733-743 ◽  
Author(s):  
TAMAS SZIKRA ◽  
DAVID KRIŽAJ
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cell Body ◽  
Cyclopiazonic Acid ◽  
Rods And Cones ◽  
Fold Reduction ◽  
Intracellular Ca ◽  
Intracellular Organelles ◽  
Spatial Buffering ◽  
Kinetics Of

The role of intracellular organelles in Ca2+homeostasis was studied in salamander rod and cone photoreceptors under conditions that simulate photoreceptor activation by darkness and light. Sustained depolarization evoked a Ca2+gradient between the cell body and ellipsoid regions of the inner segment (IS). The standing pattern of calcium fluxes was created by interactions between the plasma membrane, endoplasmic reticulum (ER), and mitochondria. Pharmacological experiments suggested that mitochondria modulate both baseline [Ca2+]i in hyperpolarized cells as well as kinetics of Ca2+entry via L type Ca2+channels in cell bodies and ellipsoids of depolarized rods and cones. Inhibition of mitochondrial Ca2+sequestration by antimycin/oligomycin caused a three-fold reduction in the amount of Ca2+accumulated into intracellular organelles in both cell bodies and ellipsoids. A further 50% decrease in intracellular Ca2+content within cell bodies, but not ellipsoids, was observed after suppression of SERCA-mediated Ca2+uptake into the ER. Inhibition of Ca2+sequestration into the endoplasmic reticulum by thapsigargin or cyclopiazonic acid decreased the magnitude and kinetics of depolarization-evoked Ca2+signals in cell bodies of rods and cones and decreased the amount of Ca2+accumulated into internal stores. These results suggest that steady-state [Ca2+]i in photoreceptors is regulated in a region-specific manner, with the ER contribution predominant in the cell body and mitochondrial buffering [Ca2+] the ellipsoid. Local [Ca2+]i levels are set by interactions between the plasma membrane Ca2+channels and transporters, ER and mitochondria. Mitochondria are likely to play an essential role in temporal and spatial buffering of photoreceptor Ca2+.

scholarly journals TRIC-A Channel Maintains Store Calcium Handling by Interacting With Type 2 Ryanodine Receptor in Cardiac Muscle

2020 ◽  
Vol 126 (4) ◽  
pp. 417-435 ◽  
Author(s):  
Xinyu Zhou ◽  
Ki Ho Park ◽  
Daiju Yamazaki ◽  
Pei-hui Lin ◽  
Miyuki Nishi ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Sarcoplasmic Reticulum ◽  
Cardiac Muscle ◽  
Ryanodine Receptor ◽  
Calcium Handling ◽  
Striated Muscles ◽  
Human Embryonic Kidney Cells ◽  
Intracellular Ca ◽  
Ion Conducting

Rationale: Trimeric intracellular cation (TRIC)-A and B are distributed to endoplasmic reticulum/sarcoplasmic reticulum intracellular Ca 2+ stores. The crystal structure of TRIC has been determined, confirming the homotrimeric structure of a potassium channel. While the pore architectures of TRIC-A and TRIC-B are conserved, the carboxyl-terminal tail (CTT) domains of TRIC-A and TRIC-B are different from each other. Aside from its recognized role as a counterion channel that participates in excitation-contraction coupling of striated muscles, the physiological function of TRIC-A in heart physiology and disease has remained largely unexplored. Objective: In cardiomyocytes, spontaneous Ca 2+ waves, triggered by store overload–induced Ca 2+ release mediated by the RyR 2 (type 2 ryanodine receptor), develop extrasystolic contractions often associated with arrhythmic events. Here, we test the hypothesis that TRIC-A is a physiological component of RyR 2 -mediated Ca 2+ release machinery that directly modulates store overload–induced Ca 2+ release activity via CTT. Methods and Results: We show that cardiomyocytes derived from the TRIC-A −/− (TRIC-A knockout) mice display dysregulated Ca 2+ movement across sarcoplasmic reticulum. Biochemical studies demonstrate a direct interaction between CTT-A and RyR 2 . Modeling and docking studies reveal potential sites on RyR 2 that show differential interactions with CTT-A and CTT-B. In HEK293 (human embryonic kidney) cells with stable expression of RyR 2 , transient expression of TRIC-A, but not TRIC-B, leads to apparent suppression of spontaneous Ca 2+ oscillations. Ca 2+ measurements using the cytosolic indicator Fura-2 and the endoplasmic reticulum luminal store indicator D1ER suggest that TRIC-A enhances Ca 2+ leak across the endoplasmic reticulum by directly targeting RyR 2 to modulate store overload–induced Ca 2+ release. Moreover, synthetic CTT-A peptide facilitates RyR 2 activity in lipid bilayer reconstitution system, enhances Ca 2+ sparks in permeabilized TRIC-A −/− cardiomyocytes, and induces intracellular Ca 2+ release after microinjection into isolated cardiomyocytes, whereas such effects were not observed with the CTT-B peptide. In response to isoproterenol stimulation, the TRIC-A −/− mice display irregular ECG and develop more fibrosis than the WT (wild type) littermates. Conclusions: In addition to the ion-conducting function, TRIC-A functions as an accessory protein of RyR 2 to modulate sarcoplasmic reticulum Ca 2+ handling in cardiac muscle.

Ca2+-Induced Ca2+ Release in Aplysia Bag Cell Neurons Requires Interaction Between Mitochondrial and Endoplasmic Reticulum Stores

2008 ◽  
Vol 100 (1) ◽  
pp. 24-37 ◽  
Author(s):  
Julia E. Geiger ◽  
Neil S. Magoski
Keyword(s):  
Endoplasmic Reticulum ◽  
Cyclopiazonic Acid ◽  
Neuroendocrine Cells ◽  
Action Potential Firing ◽  
Simultaneous Imaging ◽  
Carbonyl Cyanide ◽  
Potential Firing ◽  
Intracellular Ca ◽  
Bag Cell Neurons ◽  
Protein Kinase C Activation

Intracellular Ca2+ is influenced by both Ca2+ influx and release. We examined intracellular Ca2+ following action potential firing in the bag cell neurons of Aplysia californica. Following brief synaptic input, these neuroendocrine cells undergo an afterdischarge, resulting in elevated Ca2+ and the secretion of neuropeptides to initiate reproduction. Cultured bag cell neurons were injected with the Ca2+ indicator, fura-PE3, and subjected to simultaneous imaging and electrophysiology. Delivery of a 5-Hz, 1-min train of action potentials (mimicking the fast phase of the afterdischarge) produced a Ca2+ rise that markedly outlasted the initial influx, consistent with Ca2+-induced Ca2+ release (CICR). This response was attenuated by about half with ryanodine or depletion of the endoplasmic reticulum (ER) by cyclopiazonic acid. However, depletion of the mitochondria, with carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, essentially eliminated CICR. Dual depletion of the ER and mitochondria did not reduce CICR further than depletion of the mitochondria alone. Moreover, tetraphenylphosphonium, a blocker of mitochondrial Ca2+ release, largely prevented CICR. The Ca2+ elevation during and subsequent to a stimulus mimicking the full afterdischarge was prominent and enhanced by protein kinase C activation. Traditionally, the ER is seen as the primary Ca2+ source for CICR. However, bag cell neuron CICR represents a departure from this view in that it relies on store interaction, where Ca2+ released from the mitochondria may in turn liberate Ca2+ from the ER. This unique form of CICR may be used by both bag cell neurons, and other neurons, to initiate secretion, activate channels, or induce gene expression.

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