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1.
Ca+ sparks are rare in healthy adult mammalian skeletal muscle but may appear when adult fiber integrity is compromised, and occur in embryonic muscle but decline as the animal develops. Here we used cultured adult mouse flexor digitorum brevis muscle fibers to monitor occurrence of Ca2+ sparks during maintenance of adult fiber morphology and during eventual fiber morphological dedifferentiation after various times in culture. Fibers cultured for up to 3 days retain normal morphology and striated appearance. Ca2+ sparks were rare in these fibers. At 5–7 days in culture, many of the original muscle fibers exhibit sprouting and loss of striations, as well as the occurrence of spontaneous Ca2+ sparks. The average rate of occurrence of Ca2+ sparks is >10-fold higher after 5–7 days in culture than in days 1–3. With the use of fibers cultured for 7 days, application of the Ca2+ channel blockers Co2+ or nifedipine almost completely suppressed the occurrence of Ca2+ sparks, as previously shown in embryonic fibers, suggesting that Ca2+ sparks may be generated by similar mechanisms in dedifferentiating cultured adult fibers and in embryonic fibers before final differentiation. The sarcomeric disruption observed under transmitted light microscopy in dedifferentiating fibers was accompanied by morphological changes in the transverse (T) tubular system, as observed by fluorescence confocal imaging of both an extracellular marker dye and membrane staining dyes. Changes in T tubule morphology coincided with the appearance of Ca2+ sparks, suggesting that Ca2+ sparks may either be a signal for, or the result of, disruption of DHPR-ryanodine receptor 1 coupling. calcium ion signaling; muscle remodeling; fluo 4; calcium ion imaging  相似文献   

2.
During the cardiac action potential, Ca2+ entry through dyhidropyridine receptor L-type Ca2+ channels (DHPRs) activates ryanodine receptors (RyRs) Ca2+-release channels, resulting in massive Ca2+ mobilization from the sarcoplasmic reticulum (SR). This global Ca2+ release arises from spatiotemporal summation of many localized elementary Ca2+-release events, Ca2+ sparks. We tested whether DHPRs modulate Ca2+sparks in a Ca2+ entry-independent manner. Negative modulation by DHPR of RyRs via physical interactions is accepted in resting skeletal muscle but remains controversial in the heart. Ca2+ sparks were studied in cat cardiac myocytes permeabilized with saponin or internally perfused via a patch pipette. Bathing and pipette solutions contained low Ca2+ (100 nM). Under these conditions, Ca2+ sparks were detected with a stable frequency of 3–5 sparks·s–1·100 µm–1. The DHPR blockers nifedipine, nimodipine, FS-2, and calciseptine decreased spark frequency, whereas the DHPR agonists Bay-K8644 and FPL-64176 increased it. None of these agents altered the spatiotemporal characteristics of Ca2+ sparks. The DHPR modulators were also without effect on SR Ca2+ load (caffeine-induced Ca2+ transients) or sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity (Ca2+ loading rates of isolated SR microsomes) and did not change cardiac RyR channel gating (planar lipid bilayer experiments). In summary, DHPR modulators affected spark frequency in the absence of DHPR-mediated Ca2+ entry. This action could not be attributed to a direct action of DHPR modulators on SERCA or RyRs. Our results suggest that the activity of RyR Ca2+-release units in ventricular myocytes is modulated by Ca2+ entry-independent conformational changes in neighboring DHPRs. exitation-contraction coupling; ryanodine receptor; sarco(endo)plasmic reticulum Ca2+-ATPase; dihydropyridine receptor; sarcoplasmic reticulum  相似文献   

3.
To activate skeletal muscle contraction, action potentials must be sensed by dihydropyridine receptors (DHPRs) in the T tubule, which signal the Ca2+ release channels or ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) to open. We demonstrate here an inhibitory effect of the T tubule on the production of sparks of Ca2+ release. Murine primary cultures were confocally imaged for Ca2+ detection and T tubule visualization. After 72 h of differentiation, T tubules extended from the periphery for less than one-third of the myotube radius. Spontaneous Ca2+ sparks were found away from the region of cells where tubules were found. Immunostaining showed RyR1 and RyR3 isoforms in all areas, implying inhibition of both isoforms by a T tubule component. To test for a role of DHPRs in this inhibition, we imaged myotubes from dysgenic mice (mdg) that lack DHPRs. These exhibited T tubule development similar to that of normal myotubes, but produced few sparks, even in regions where tubules were absent. To increase spark frequency, a high-Ca2+ saline with 1 mM caffeine was used. Wild-type cells in this saline plus 50 µM nifedipine retained the topographic suppression pattern of sparks, but dysgenic cells in high-Ca2+ saline did not. Shifted excitation and emission ratios of indo-1 in the cytosol or mag-indo-1 in the SR were used to image [Ca2+] in these compartments. Under the conditions of interest, wild-type and mdg cells had similar levels of free [Ca2+] in cytosol and SR. These data suggest that DHPRs play a critical role in reducing the rate of spontaneous opening of Ca2+ release channels and/or their susceptibility to Ca2+-induced activation, thereby suppressing the production of Ca2+ sparks. excitation-contraction coupling; sarcoplasmic reticulum; ryanodine receptors; Ca2+ imaging  相似文献   

4.
The kinetic behavior of Ca(2+) sparks in knockout mice lacking a specific ryanodine receptor (RyR) isoform should provide molecular information on function and assembly of clusters of RyRs. We examined resting Ca(2+) sparks in RyR type 3-null intercostal myotubes from embryonic day 18 (E18) mice and compared them to Ca(2+) sparks in wild-type (wt) mice of the same age and to Ca(2+) sparks in fast-twitch muscle cells from the foot of wt adult mice. Sparks from RyR type 3-null embryonic cells (368 events) were significantly smaller, briefer, and had a faster time to peak than sparks from wt cells (280 events) of the same age. Sparks in adult cells (220 events) were infrequent, yet they were highly reproducible with population means smaller than those in embryonic RyR type 3-null cells but similar to those reported in adult amphibian skeletal muscle fibers. Three-dimensional representations of the spark peak intensity (DeltaF/Fo) vs. full width at half-maximal intensity (FWHM) vs. full duration at half-maximal intensity (FTHM) showed that wt embryonic sparks were considerably more variable in size and kinetics than sparks in adult muscle. In all cases, tetracaine (0.2 mM) abolished Ca(2+) spark activity, whereas caffeine (0.1 mM) lengthened the spark duration in wt embryonic and adult cells but not in RyR type 3-null cells. These results confirmed that sparks arose from RyRs. The low caffeine sensitivity of RyR type 3-null cells is entirely consistent with observations by other investigators. There are three conclusions from this study: i) RyR type-1 engages in Ca(2+) spark activity in the absence of other RyR isoforms in RyR type 3-null myotubes; ii) Ca(2+) sparks with parameters similar to those reported in adult amphibian skeletal muscle can be detected, albeit at a low frequency, in adult mammalian skeletal muscle cells; and iii) a major contributor to the unusually large Ca(2+) sparks observed in normal (wt) embryonic muscle is RyR type 3. To explain the reduction in the size of sparks in adult compared to embryonic skeletal muscle, we suggest that in embryonic muscle, RyR type 1 and RyR type 3 channels co-contribute to Ca(2+) release during the same spark and that Ca(2+) sparks undergo a maturation process which involves a decrease in RyR type 3.  相似文献   

5.
Spontaneous Ca2+ sparks were observed in fluo 4-loaded myocytes from guinea pig vas deferens with line-scan confocal imaging. They were abolished by ryanodine (100 µM), but the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) blockers 2-aminoethoxydiphenyl borate (2-APB; 100 µM) and intracellular heparin (5 mg/ml) increased spark frequency, rise time, duration, and spread. Very prolonged Ca2+ release events were also observed in 20% of cells treated with IP3R blockers but not under control conditions. 2-APB and heparin abolished norepinephrine (10 µM; 0 Ca2+)-evoked Ca2+ transients but increased caffeine (10 mM; 0 Ca2+) transients in fura 2-loaded myocytes. Transients evoked by ionomycin (25 µM; 0 Ca2+) were also enhanced by 2-APB. Ca2+ sparks and transients evoked by norepinephrine and caffeine were abolished by thimerosal (100 µM), which sensitizes the IP3R to IP3. In cells voltage clamped at –40 mV, spontaneous transient outward currents (STOCs) were increased in frequency, amplitude, and duration in the presence of 2-APB. These data are consistent with a model in which the Ca2+ store content in smooth muscle is limited by tonic release of Ca2+ via an IP3-dependent pathway. Blockade of IP3Rs elevates sarcoplasmic reticulum store content, promoting Ca2+ sparks and STOC activity. calcium ion release; calcium ion transients; smooth muscle  相似文献   

6.
Voltage dependence of Ca2+ sparks in intact cerebral arteries   总被引:4,自引:0,他引:4  
Ca2+ sparks have beenpreviously described in isolated smooth muscle cells. Here we presentthe first measurements of local Ca2+ transients("Ca2+ sparks") in an intactsmooth muscle preparation. Ca2+sparks appear to result from the opening of ryanodine-sensitive Ca2+ release (RyR) channels in thesarcoplasmic reticulum (SR). Intracellular Ca2+ concentration([Ca2+]i)was measured in intact cerebral arteries (40-150 µm in diameter) from rats, using the fluorescentCa2+ indicator fluo 3 and a laserscanning confocal microscope. Membrane potential depolarization byelevation of external K+ from 6 to30 mM increased Ca2+ sparkfrequency (4.3-fold) and amplitude (~2-fold) as well as globalarterial wall[Ca2+]i(~1.7-fold). The half time of decay (~50 ms) was not affected bymembrane potential depolarization. Ryanodine (10 µM), which inhibitsRyR channels and Ca2+ sparks inisolated cells, and thapsigargin (100 nM), which indirectly inhibitsRyR channels by blocking the SRCa2+-ATPase, completely inhibitedCa2+ sparks in intact cerebralarteries. Diltiazem, an inhibitor of voltage-dependentCa2+ channels, lowered global[Ca2+]iand Ca2+ spark frequency andamplitude in intact cerebral arteries in a concentration-dependentmanner. The frequency of Ca2+sparks (<1s1 · cell1),even under conditions of steady depolarization, was too low tocontribute significant amounts ofCa2+ to globalCa2+ in intact arteries. Theseresults provide direct evidence that Ca2+ sparks exist in quiescentsmooth muscle cells in intact arteries and that changes of membranepotential that would simulate physiological changes modulate bothCa2+ spark frequency and amplitudein arterial smooth muscle.

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7.
To better understand the role of the transient expression of ryanodine receptor (RyR) type 3 (RyR3) on Ca2+ homeostasis during the development of skeletal muscle, we have analyzed the effect of expression levels of RyR3 and RyR1 on the overall physiology of cultured myotubes and muscle fibers. Dyspedic myotubes were infected with RyR1 or RyR3 containing virions at 0.2, 0.4, 1.0, and 4.0 moieties of infection (MOI), and analysis of their pattern of expression, caffeine sensitivity, and resting free Ca2+ concentration ([Ca2+]r) was performed. Although increased MOI resulted in increased expression of each receptor isoform, it did not significantly affect the immunopattern of RyRs or the expression levels of calsequestrin, triadin, or FKBP-12. Interestingly, myotubes expressing RyR3 always had significantly higher [Ca2+]r and lower caffeine EC50 than did cells expressing RyR1. Although some of the increased sensitivity of RyR3 to caffeine could be attributed to the higher [Ca2+]r in RyR3-expressing cells, studies of [3H]ryanodine binding demonstrated intrinsic differences in caffeine sensitivity between RyR1 and RyR3. Tibialis anterior (TA) muscle fibers at different stages of postnatal development exhibited a transient increase in [Ca2+]r coordinately with their level of RyR3 expression. Similarly, adult soleus fibers, which also express RyR3, had higher [Ca2+]r than did adult TA fibers, which exclusively express RyR1. These data show that in skeletal muscle, RyR3 increases [Ca2+]r more than RyR1 does at any expression level. These data suggest that the coexpression of RyR1 and RyR3 at different levels may constitute a novel mechanism by which to regulate [Ca2+]r in skeletal muscle. ryanodine receptor; calcium release; ryanodine binding; muscle fibers  相似文献   

8.
LocalCa2+ transients("Ca2+ sparks") caused bythe opening of one or the coordinated opening of a number of tightlyclustered ryanodine-sensitiveCa2+-release (RyR) channels in thesarcoplasmic reticulum (SR) activate nearbyCa2+-dependentK+(KCa) channels to cause anoutward current [referred to as a "spontaneous transientoutward current" (STOC)]. TheseKCa currents cause membranepotential hyperpolarization of arterial myocytes, which would lead tovasodilation through decreasingCa2+ entry throughvoltage-dependent Ca2+ channels.Therefore, modulation of Ca2+spark frequency should be a means to regulation ofKCa channel currents and hencemembrane potential. We examined the frequency modulation ofCa2+ sparks and STOCs byactivation of protein kinase C (PKC). The PKC activators, phorbol12-myristate 13-acetate (PMA; 10 nM) and 1,2-dioctanoyl-sn-glycerol (1 µM),decreased Ca2+ spark frequency by72% and 60%, respectively, and PMA reduced STOC frequency by 83%.PMA also decreased STOC amplitude by 22%, which could be explained byan observed reduction (29%) inKCa channel open probability inthe absence of Ca2+ sparks. Thereduction in STOC frequency occurred in the presence of an inorganicblocker (Cd2+) ofvoltage-dependent Ca2+ channels.The reduction in Ca2+ sparkfrequency did not result from SRCa2+ depletion, sincecaffeine-induced Ca2+ transientsdid not decrease in the presence of PMA. These results suggest thatactivators of PKC can modulate the frequency ofCa2+ sparks, through an effect onthe RyR channel, which would decrease STOC frequency (i.e.,KCa channel activity).

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9.
Phospholamban (PLB) inhibits the sarcoplasmic reticulum (SR)Ca2+-ATPase, and this inhibition is relieved bycAMP-dependent protein kinase (PKA)-mediated phosphorylation. The roleof PLB in regulating Ca2+ release throughryanodine-sensitive Ca2+ release channels, measured asCa2+ sparks, was examined using smooth muscle cells ofcerebral arteries from PLB-deficient ("knockout") mice(PLB-KO). Ca2+ sparks were monitored opticallyusing the fluorescent Ca2+ indicator fluo 3 or electricallyby measuring transient large-conductance Ca2+-activatedK+ (BK) channel currents activated by Ca2+sparks. Basal Ca2+ spark and transient BK current frequencywere elevated in cerebral artery myocytes of PLB-KO mice. Forskolin, anactivator of adenylyl cyclase, increased the frequency ofCa2+ sparks and transient BK currents in cerebral arteriesfrom control mice. However, forskolin had little effect on thefrequency of Ca2+ sparks and transient BK currents fromPLB-KO cerebral arteries. Forskolin or PLB-KO increased SRCa2+ load, as measured by caffeine-induced Ca2+transients. This study provides the first evidence that PLB is criticalfor frequency modulation of Ca2+ sparks and associated BKcurrents by PKA in smooth muscle.

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10.
FKBP12.6 and cADPR regulation of Ca2+ release in smooth muscle cells   总被引:1,自引:0,他引:1  
Intracellular Ca2+ release through ryanodine receptors (RyRs) plays important roles in smooth muscle excitation-contraction coupling, but the underlying regulatory mechanisms are poorly understood. Here we show that FK506 binding protein of 12.6 kDa (FKBP12.6) associates with and regulates type 2 RyRs (RyR2) in tracheal smooth muscle. FKBP12.6 binds to RyR2 but not other RyR or inositol 1,4,5-trisphosphate receptors, and FKBP12, known to bind to and modulate skeletal RyRs, does not associate with RyR2. When dialyzed into tracheal myocytes, cyclic ADP-ribose (cADPR) alters spontaneous Ca2+ release at lower concentrations and produces macroscopic Ca2+ release at higher concentrations; neurotransmitter-evoked Ca2+ release is also augmented by cADPR. These actions are mediated through FKBP12.6 because they are inhibited by molar excess of recombinant FKBP12.6 and are not observed in myocytes from FKBP12.6-knockout mice. We also report that force development in FKBP12.6-null mice, observed as a decrease in the concentration/tension relationship of isolated trachealis segments, is impaired. Taken together, these findings point to an important role of the FKBP12.6/RyR2 complex in stochastic (spontaneous) and receptor-mediated Ca2+ release in smooth muscle. FK506 binding protein 12.6; ryanodine receptor type 2; calcium sparks; calcium-activated chloride currents  相似文献   

11.
Uridine 5'-triphosphate (UTP), a potent vasoconstrictor that activatesphospholipase C, shifted Ca2+ signaling from sparks towaves in the smooth muscle cells of rat cerebral arteries. UTPdecreased the frequency of Ca2+ sparks and transientCa2+-activated K+ (KCa) currentsand increased the frequency of Ca2+ waves. The UTP-inducedreduction in Ca2+ spark frequency did not reflect adecrease in global cytoplasmic Ca2+, Ca2+influx through voltage-dependent Ca2+ channels (VDCC), orCa2+ load of the sarcoplasmic reticulum (SR), since globalCa2+ was elevated, blocking VDCC did not prevent theeffect, and SR Ca2+ load did not decrease. However,blocking protein kinase C (PKC) with bisindolylmaleimide I did preventUTP reduction of Ca2+ sparks and transient KCacurrents. UTP decreased the effectiveness of caffeine, which increasesthe Ca2+ sensitivity of ryanodine-sensitiveCa2+ release (RyR) channels, to activate transientKCa currents. This work supports the concept thatvasoconstrictors shift Ca2+ signaling modalities fromCa2+ sparks to Ca2+ waves through the concertedactions of PKC on the Ca2+ sensitivity of RyR channels,which cause Ca2+ sparks, and of inositol trisphosphate(IP3) on IP3 receptors to generateCa2+ waves.

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12.
The regulationof intracellular Ca2+ signals in smooth muscle cells andarterial diameter by intravascular pressure was investigated in ratcerebral arteries (~150 µm) using a laser scanning confocal microscope and the fluorescent Ca2+ indicator fluo 3. Elevation of pressure from 10 to 60 mmHg increased Ca2+spark frequency 2.6-fold, Ca2+ wave frequency 1.9-fold, andglobal intracellular Ca2+ concentration([Ca2+]i) 1.4-fold in smooth muscle cells,and constricted arteries. Ryanodine (10 µM), an inhibitor ofryanodine-sensitive Ca2+ release channels, or thapsigargin(100 nM), an inhibitor of the sarcoplasmic reticulumCa2+-ATPase, abolished sparks and waves, elevated global[Ca2+]i, and constricted pressurized (60 mmHg) arteries. Diltiazem (25 µM), a voltage-dependentCa2+ channel (VDCC) blocker, significantly reduced sparks,waves, and global [Ca2+]i, and dilatedpressurized (60 mmHg) arteries. Steady membrane depolarization elevatedCa2+ signaling similar to pressure and increased transientCa2+-sensitive K+ channel current frequencye-fold for ~7 mV, and these effects were prevented by VDCCblockers. Data are consistent with the hypothesis that pressure inducesa steady membrane depolarization that activates VDCCs, leading to anelevation of spark frequency, wave frequency, and global[Ca2+]i. In addition, pressure inducescontraction via an elevation of global[Ca2+]i, whereas the net effect of sparks andwaves, which do not significantly contribute to global[Ca2+]i in arteries pressurized to between 10 and 60 mmHg, is to oppose contraction.

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13.
We studied whether acetaldehyde, which is produced by alcohol consumption, impacts ryanodine receptor (RyR) activity and muscle force. Exposure to 50–200 µM acetaldehyde enhanced channel activity of frog RyR and rabbit RyR1 incorporated into lipid bilayers. An increase in acetaldehyde to 1 mM modified channel activity in a time-dependent manner, with a brief activation and then inhibition. Application of 200 µM acetaldehyde to frog fibers increased twitch tension. The maximum rate of rise of tetanus tension was accelerated to 1.5 and 1.74 times the control rate on exposure of fibers to 50 and 200 µM acetaldehyde, respectively. Fluorescence monitoring with fluo 3 demonstrated that 200–400 µM acetaldehyde induced Ca2+ release from the sarcoplasmic reticulum (SR) in frog muscles. Acetaldehyde at 1 mM inhibited twitch tension by 12%, with an increased relaxation time after a small, transient twitch potentiation. These results suggest that moderate concentrations of acetaldehyde can elicit Ca2+ release from the SR by increasing the open probability of the RyR channel, resulting in increased tension. However, the effects of acetaldehyde at clinical doses (1–30 µM) are unlikely to mediate alcohol-induced acute muscle dysfunction. ryanodine receptor; single-channel current; fluo 3 fluorescence; calcium ion release; calcium ion uptake  相似文献   

14.
We hypothesized that activation of heat shock protein 70 (HSP70) by preconditioning, which is known to confer delayed cardioprotection, attenuates the impaired handling of Ca2+ at multiple sites. To test the hypothesis, we determined how the ryanodine receptor (RyR), sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), and Na+/Ca2+ exchanger (NCX) handled Ca2+ in rat ventricular myocytes preconditioned with a -opioid receptor agonist, U50488H (UP), followed by blockade of HSP70 with a selective antisense oligonucleotide and subsequently subjected to simulated ischemia. We determined the following: 1) the Ca2+ transients induced by electrical stimulation and caffeine, which provide the overall picture of Ca2+ homeostasis; 2) expression of RyR, SERCA, and NCX; and 3) Ca2+ fluxes via NCX by the use of 45Ca2+ in the rat ventricular myocyte. We found that UP increased the activity of RyR, SERCA, and NCX and the expression of RyR and SERCA. These effects led to increases in the release of Ca2+ from the sarcoplasmic reticulum via RyR and in the removal of Ca2+ from the cytoplasm by reuptake of Ca2+ to the SR via SERCA and by extrusion of Ca2+ out of the cell via NCX. UP also reduced mitochondrial Ca2+ accumulation. All of the effects of UP were either abolished or significantly attenuated by blockade of HSP70 synthesis with a selective antisense oligonucleotide. The results are evidence that activation of HSP70 by preconditioning improves the ischemia-impaired Ca2+ homeostasis at multiple sites in the heart, which may be responsible, at least partly, for attenuated Ca2+ overload, improved recovery in contractile function, and cardioprotection. intracellular Ca2+, -opioid receptor; Na+/Ca2+ exchanger; ryanodine receptor; sarco(endo)plasmic reticulum Ca2+-ATPase  相似文献   

15.
Forskolin, which elevates cAMP levels, and sodium nitroprusside(SNP) and nicorandil, which elevate cGMP levels, increased, by two- tothreefold, the frequency of subcellularCa2+ release("Ca2+ sparks") throughryanodine-sensitive Ca2+ release(RyR) channels in the sarcoplasmic reticulum (SR) of myocytes isolatedfrom cerebral and coronary arteries of rats. Forskolin, SNP,nicorandil, dibutyryl-cAMP, and adenosine increased the frequency ofCa2+-sensitiveK+(KCa) currents["spontaneous transient outward currents" (STOCs)] bytwo- to threefold, consistent withCa2+ sparks activating STOCs.These agents also increased the mean amplitude of STOCs by 1.3-fold, aneffect that could be explained by activation ofKCa channels, independent ofeffects on Ca2+ sparks. To testthe hypothesis that cAMP could act to dilate arteries throughactivation of the Ca2+sparkKCa channel pathway,the effects of blockers of KCachannels (iberiotoxin) and of Ca2+sparks (ryanodine) on forskolin-induced dilations of pressurized cerebral arteries were examined. Forskolin-induced dilations were partially inhibited by iberiotoxin and ryanodine (with no additive effects) and were entirely prevented by elevating externalK+. Forskolin lowered averageCa2+ in pressurized arteries whileincreasing ryanodine-sensitive, caffeine-inducedCa2+ transients. These experimentssuggest a new mechanism for cyclic nucleotide-mediated dilationsthrough an increase in Ca2+ sparkfrequency, caused by effects on SRCa2+ load and possibly on the RyRchannel, which leads to increased STOC frequency, membrane potentialhyperpolarization, closure of voltage-dependentCa2+ channels, decrease inarterial wall Ca2+, and,ultimately, vasodilation.

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16.
Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle triggered in susceptible individuals by inhalation anesthetics and depolarizing skeletal muscle relaxants. This syndrome has been linked to a missense mutation in the type 1 ryanodine receptor (RyR1) in more than 50% of cases studied to date. Using double-barreled Ca2+ microelectrodes in myotubes expressing wild-type RyR1 (WTRyR1) or RyR1 with one of four common MH mutations (MHRyR1), we measured resting intracellular Ca2+ concentration ([Ca2+]i). Changes in resting [Ca2+]i produced by several drugs known to modulate the RyR1 channel complex were investigated. We found that myotubes expressing any of the MHRyR1s had a 2.0- to 3.7-fold higher resting [Ca2+]i than those expressing WTRyR1. Exposure of myotubes expressing MHRyR1s to ryanodine (500 µM) or (2,6-dichloro-4-aminophenyl)isopropylamine (FLA 365; 20 µM) had no effects on their resting [Ca2+]i. However, when myotubes were exposed to bastadin 5 alone or to a combination of ryanodine and bastadin 5, the resting [Ca2+]i was significantly reduced (P < 0.01). Interestingly, the percent decrease in resting [Ca2+]i in myotubes expressing MHRyR1s was significantly greater than that for WTRyR1. From these data, we propose that the high resting myoplasmic [Ca2+]i in MHRyR1 expressing myotubes is due in part to a related structural conformation of MHRyR1s that favors "passive" calcium leak from the sarcoplasmic reticulum. ryanodine; FLA 365; bastadin 5; resting intracellular calcium concentration; sarcoplasmic reticulum  相似文献   

17.
Skeletal muscle deficiency in the 3-phosphoinositide (PtdInsP) phosphatase myotubularin (MTM1) causes myotubular myopathy which is associated with severe depression of voltage-activated sarcoplasmic reticulum Ca2+ release through ryanodine receptors. In the present study we aimed at further understanding how Ca2+ release is altered in MTM1-deficient muscle fibers, at rest and during activation. While in wild-type muscle fibers, SR Ca2+ release exhibits fast stereotyped kinetics of activation and decay throughout the voltage range of activation, Ca2+ release in MTM1-deficient muscle fibers exhibits slow and unconventional kinetics at intermediate voltages, suggestive of partial loss of the normal control of ryanodine receptor Ca2+ channel activity. In addition, the diseased muscle fibers at rest exhibit spontaneous elementary Ca2+ release events at a frequency 30 times greater than that of control fibers. Eighty percent of the events have spatiotemporal properties of archetypal Ca2+ sparks while the rest take either the form of lower amplitude, longer duration Ca2+ release events or of a combination thereof. The events occur at preferred locations in the fibers, indicating spatially uneven distribution of the parameters determining spontaneous ryanodine receptor 1 opening. Spatially large Ca2+ release sources were obviously involved in some of these events, suggesting that opening of ryanodine receptors in one cluster can activate opening of ryanodine receptors in a neighboring one. Overall results demonstrate that opening of Ca2+-activated ryanodine receptors is promoted both at rest and during excitation-contraction coupling in MTM1-deficient muscle fibers. Because access to this activation mode is denied to ryanodine receptors in healthy skeletal muscle, this may play an important role in the associated disease situation.  相似文献   

18.
To clarify whether activity of the ryanodine receptor type 2 (RyR2) is reduced in the sarcoplasmic reticulum (SR) of cardiac muscle, as is the case with the ryanodine receptor type 1 (RyR1), Ca2+-dependent [3H]ryanodine binding, a biochemical measure of Ca2+-induced Ca2+ release (CICR), was determined using SR vesicle fractions isolated from rabbit and rat cardiac muscles. In the absence of an adenine nucleotide or caffeine, the rat SR showed a complicated Ca2+ dependence, instead of the well-documented biphasic dependence of the rabbit SR. In the rat SR, [3H]ryanodine binding initially increased as [Ca2+] increased, with a plateau in the range of 10–100 µM Ca2+, and thereafter further increased to an apparent peak around 1 mM Ca2+, followed by a decrease. In the presence of these modulators, this complicated dependence prevailed, irrespective of the source. Addition of 0.3–1 mM Mg2+ unexpectedly increased the binding two- to threefold and enhanced the affinity for [3H]ryanodine at 10–100 µM Ca2+, resulting in the well-known biphasic dependence. In other words, the partial suppression of RyR2 is relieved by Mg2+. Ca2+ could be a substitute for Mg2+. Mg2+ also amplifies the responses of RyR2 to inhibitory and stimulatory modulators. This stimulating effect of Mg2+ on RyR2 is entirely new, and is referred to as the third effect, in addition to the well-known dual inhibitory effects. This effect is critical to describe the role of RyR2 in excitation-contraction coupling of cardiac muscle, in view of the intracellular Mg2+ concentration. [3H]ryanodine binding; CICR; stimulation by physiological Mg2+, excitation-contraction coupling in the heart  相似文献   

19.
Ryanodine receptor (RyR) type 1 (RyR1) exhibits a markedly lower gain of Ca2+-induced Ca2+ release (CICR) activity than RyR type 3 (RyR3) in the sarcoplasmic reticulum (SR) of mammalian skeletal muscle (selective stabilization of the RyR1 channel), and this reduction in the gain is largely eliminated using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). We have investigated whether the hypothesized interdomain interactions within RyR1 are involved in the selective stabilization of the channel using [3H]ryanodine binding, single-channel recordings, and Ca2+ release from the SR vesicles. Like CHAPS, domain peptide 4 (DP4, a synthetic peptide corresponding to the Leu2442-Pro2477 region of RyR1), which seems to destabilize the interdomain interactions, markedly stimulated RyR1 but not RyR3. Their activating effects were saturable and nonadditive. Dantrolene, a potent inhibitor of RyR1 used to treat malignant hyperthermia, reversed the effects of DP4 or CHAPS in an identical manner. These findings indicate that RyR1 is activated by DP4 and CHAPS through a common mechanism that is probably mediated by the interdomain interactions. DP4 greatly increased [3H]ryanodine binding to RyR1 with only minor alterations in the sensitivity to endogenous CICR modulators (Ca2+, Mg2+, and adenine nucleotide). However, DP4 sensitized RyR1 four- to six-fold to caffeine in the caffeine-induced Ca2+ release. Thus the gain of CICR activity critically determines the magnitude and threshold of Ca2+ release by drugs such as caffeine. These findings suggest that the low CICR gain of RyR1 is important in normal Ca2+ handling in skeletal muscle and that perturbation of this state may result in muscle diseases such as malignant hyperthermia. malignant hyperthermia; 3-[(3-cholamidopropyl)dimethylammonio]propane sulfonic acid; domain peptide 4  相似文献   

20.
The goal of the present study was to testthe hypothesis that local Ca2+ release events(Ca2+ sparks) deliver high local Ca2+concentration to activate nearby Ca2+-sensitiveK+ (BK) channels in the cell membrane of arterial smoothmuscle cells. Ca2+ sparks and BK channels were examined inisolated myocytes from rat cerebral arteries with laser scanningconfocal microscopy and patch-clamp techniques. BK channels had anapparent dissociation constant for Ca2+ of 19 µM and aHill coefficient of 2.9 at 40 mV. At near-physiological intracellularCa2+ concentration ([Ca2+]i; 100 nM) and membrane potential (40 mV), the open probability of a singleBK channel was low (1.2 × 106). A Ca2+spark increased BK channel activity to 18. Assuming that 1-100% of the BK channels are activated by a single Ca2+ spark, BKchannel activity increases 6 × 105-fold to 6 × 103-fold, which corresponds to ~30 µM to 4 µM sparkCa2+ concentration.1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acidacetoxymethyl ester caused the disappearance of all Ca2+sparks while leaving the transient BK currents unchanged. Our resultssupport the idea that Ca2+ spark sites are in closeproximity to the BK channels and that local[Ca2+]i reaches micromolar levels to activateBK channels.

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