Nicardipine as a Ca2+ channel blocker in single barnacle muscle fibers

A study has been made of the efficacy of nicardipine as a Ca2+ channel blocker by determining the magnitude of its effect on the stimulatory response of the ouabain-insensitive Na+ efflux in single barnacle muscle fibers to 100 mM external K+. The results show that nicardipine (at pH 6.5) is a potent inhibitor, the minimal effective concentration being approx. 10(-7) M and the IC(50) about 5.10(-6) M. Nicardipine, however, is not as potent as verapamil (at pH 6.5) on an equimolar basis. This is explained by assuming that the number of dihydropyridine receptors in the t-tubule membranes of barnacle fibers is not high or that verapamil is able to block the sarcoplasmic reticulum Ca2+ release channel in addition to the voltage-dependent Ca2+ channels.     

Modulation of agonist-induced Ca2+ release by SR Ca2+ load: direct SR and cytosolic Ca2+ measurements in rat uterine myocytes

Release of Ca2+ from sarcoplasmic reticulum (SR) is one of the most important mechanisms of smooth muscle stimulation by a variety of physiologically active substances. Agonist-induced Ca2+ release is considered to be dependent on the Ca2+ content of the SR, although the mechanism underlying this dependence is unclear. In the present study, the effect of SR Ca2+ load on the amplitude of [Ca2+]i transients elicited by application of the purinergic agonist ATP was examined in uterine smooth muscle cells isolated from pregnant rats. Measurement of intraluminal Ca2+ level ([Ca2+]L) using a low affinity Ca indicator, mag-fluo-4, revealed that incubation of cells in a high-Ca2+ (10 mM) extracellular solution leads to a substantial increase in [Ca2+]L (SR overload). However, despite increased SR Ca2+ content this did not potentiate ATP-induced [Ca2+]i transients. Repetitive applications of ATP in the absence of extracellular Ca2+, as well as prolonged incubation in Ca2+-free solution without agonist, depleted the [Ca2+]L (SR overload). In contrast to overload, partial depletion of the SR substantially reduced the amplitude of Ca2+ release. ATP-induced [Ca2+]i transients were completely abolished when SR Ca2+ content was decreased below 80% of its normal value indicating a steep dependence of the IP3-mediated Ca2+ release on the Ca2+ load of the store. Our results suggest that in uterine smooth muscle cells decrease in the SR Ca2+ load below its normal resting level substantially reduces the IP3-mediated Ca2+ release, while Ca2+ overload of the SR has no impact on such release.     

Intracellular calcium movements during relaxation and recovery of superfast muscle fibers of the toadfish swimbladder

The mating call of the Atlantic toadfish is generated by bursts of high-frequency twitches of the superfast twitch fibers that surround the swimbladder. At 16°C, a calling period can last several hours, with individual 80–100-Hz calls lasting ∼500 ms interleaved with silent periods (intercall intervals) lasting ∼10 s. To understand the intracellular movements of Ca²⁺ during the intercall intervals, superfast fibers were microinjected with fluo-4, a high-affinity fluorescent Ca²⁺ indicator, and stimulated by trains of 40 action potentials at 83 Hz, which mimics fiber activity during calling. The fluo-4 fluorescence signal was measured during and after the stimulus trains; the signal was also simulated with a kinetic model of the underlying myoplasmic Ca²⁺ movements, including the binding and transport of Ca²⁺ by the sarcoplasmic reticulum (SR) Ca²⁺ pumps. The estimated total amount of Ca²⁺ released from the SR during a first stimulus train is ∼6.5 mM (concentration referred to the myoplasmic water volume). At 40 ms after cessation of stimulation, the myoplasmic free Ca²⁺ concentration ([Ca²⁺]) is below the threshold for force generation (∼3 µM), yet the estimated concentration of released Ca²⁺ remaining in the myoplasm (Δ[Ca_M]) is large, ∼5 mM, with ∼80% bound to parvalbumin. At 10 s after stimulation, [Ca²⁺] is ∼90 nM (three times the assumed resting level) and Δ[Ca_M] is ∼1.3 mM, with 97% bound to parvalbumin. Ca²⁺ movements during the intercall interval thus appear to be strongly influenced by (a) the accumulation of Ca²⁺ on parvalbumin and (b) the slow rate of Ca²⁺ pumping that ensues when parvalbumin lowers [Ca²⁺] near the resting level. With repetitive stimulus trains initiated at 10-s intervals, Ca²⁺ release and pumping come quickly into balance as a result of the stability (negative feedback) supplied by the increased rate of Ca²⁺ pumping at higher [Ca²⁺].     

Characteristics of phosphate-induced Ca2+ efflux from the SR in mechanically skinned rat skeletal muscle fibers

The effects of P_i onsarcoplasmic reticulum (SR) Ca²⁺ regulation were studied inmechanically skinned rat skeletal muscle fibers. Brief application ofcaffeine was used to assess the SR Ca²⁺ content, andchanges in concentration of Ca²⁺([Ca²⁺]) within the cytosol were detected withfura 2 fluorescence. Introduction of P_i (1-40 mM)induced a concentration-dependent Ca²⁺ efflux from the SR.In solutions lacking creatine phosphate (CP), the amplitude of theP_i-induced Ca²⁺ transient approximatelydoubled. A similar potentiation of P_i-induced Ca²⁺ release occurred after inhibition of creatine kinase(CK) with 2,4-dinitrofluorobenzene. In the presence of ruthenium red or ryanodine, caffeine-induced Ca²⁺ release was almostabolished, whereas P_i-induced Ca²⁺ release wasunaffected. However, introduction of the SR Ca²⁺ ATPaseinhibitor cyclopiazonic acid effectively abolishedP_i-induced Ca²⁺ release. These data suggestthat P_i induces Ca²⁺ release from the SR byreversal of the SR Ca²⁺ pump but not via the SRCa²⁺ channel under these conditions. If this occurs inintact skeletal muscle during fatigue, activation of a Ca²⁺efflux pathway by P_i may contribute to the reporteddecrease in net Ca²⁺ uptake and increase in resting[Ca²⁺].

     

Ca2+-induced sarcoplasmic reticulum Ca2+ release in myotubularin-deficient muscle fibers

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.     

DHPR activation underlies SR Ca2+ release induced by osmotic stress in isolated rat skeletal muscle fibers

Changes in skeletal muscle volume induce localized sarcoplasmic reticulum (SR) Ca²⁺ release (LCR) events, which are sustained for many minutes, suggesting a possible signaling role in plasticity or pathology. However, the mechanism by which cell volume influences SR Ca²⁺ release is uncertain. In the present study, rat flexor digitorum brevis fibers were superfused with isoosmotic Tyrode''s solution before exposure to either hyperosmotic (404 mOsm) or hypoosmotic (254 mOsm) solutions, and the effects on cell volume, membrane potential (E_m), and intracellular Ca²⁺ ([Ca²⁺]_i) were determined. To allow comparison with previous studies, solutions were made hyperosmotic by the addition of sugars or divalent cations, or they were made hypoosmotic by reducing [NaCl]_o. All hyperosmotic solutions induced a sustained decrease in cell volume, which was accompanied by membrane depolarization (by 14–18 mV; n = 40) and SR Ca²⁺ release. However, sugar solutions caused a global increase in [Ca²⁺]_i, whereas solutions made hyperosmotic by the addition of divalent cations only induced LCR. Decreasing osmolarity induced an increase in cell volume and a negative shift in E_m (by 15.04 ± 1.85 mV; n = 8), whereas [Ca²⁺]_i was unaffected. However, on return to the isoosmotic solution, restoration of cell volume and E_m was associated with LCR. Both global and localized SR Ca²⁺ release were abolished by the dihydropyridine receptor inhibitor nifedipine by sustained depolarization of the sarcolemmal or by the addition of the ryanodine receptor 1 inhibitor tetracaine. Inhibitors of the Na-K-2Cl (NKCC) cotransporter markedly inhibited the depolarization associated with hyperosmotic shrinkage and the associated SR Ca²⁺ release. These findings suggest (1) that the depolarization that accompanies a decrease in cell volume is the primary event leading to SR Ca²⁺ release, and (2) that volume-dependent regulation of the NKCC cotransporter contributes to the observed changes in E_m. The differing effects of the osmotic agents can be explained by the screening of fixed charges by divalent ions.     

Ca2+ Modulation of sarcoplasmic reticulum Ca2+ release in rat skeletal muscle fibers

Ca²⁺ transients and the rate of Ca²⁺ release (dCa_REL/dt) from the sarcoplasmic reticulum (SR) in voltage-clamped, fast-twitch skeletal muscle fibers from the rat were studied with the double Vaseline gap technique and using mag-fura-2 and fura-2 as Ca²⁺ indicators. Single pulse experiments with different returning potentials showed that Ca²⁺ removal from the myoplasm is voltage independent. Thus, the myoplasmic Ca²⁺ removal (dCa_REM/dt) was studied by fitting the decaying phase of the Ca²⁺ transient (Melzer, Ríos & Schneider, 1986) and dCa_REL/dt was calculated as the difference between dCa/dt and dCa_REM/dt. The fast Ca²⁺ release decayed as a consequence of Ca²⁺ inactivation of Ca²⁺ release. Double pulse experiments showed inactivation of the fast Ca²⁺ release depending on the prepulse duration. At constant interpulse interval, long prepulses (200 msec) induced greater inactivation of the fast Ca²⁺ release than shorter depolarizations (20 msec). The correlation (r) between the myoplasmic [Ca²⁺]_i and the inhibited amount of Ca²⁺ release was 0.98. The [Ca²⁺]_i for 50% inactivation of dCa_REL/dt was 0.25 m, and the minimum number of sites occupied by Ca²⁺ to inactivate the Ca²⁺ release channel was 3.0. These data support Ca²⁺ binding and inactivation of SR Ca²⁺ release.This work was supported by Grant-in-Aid from the American Heart Association (National) and Muscular Dystrophy Association (USA). Part of this work was developed in Dr. Stefani's laboratory at Baylor College of Medicine.     

Ca2+ dependence of stimulated 45Ca efflux in skinned muscle fibers

Stimulation of sarcoplasmic reticulum Ca release by Mg reduction of caffeine was studied in situ, to characterize further the Ca2+ dependence observed previously with stimulation by Cl ion. 45Ca efflux and isometric force were measured simultaneously at 19 degrees C in frog skeletal muscle fibers skinned by microdissection; EGTA was added to chelate myofilament space Ca either before or after the stimulus. Both Mg2+ reduction (20 or 110 microM to 4 microM) and caffeine (5 mM) induced large force responses and 45Ca release, which were inhibited by pretreatment with 5 mM EGTA. In the case of Mg reduction, residual efflux stimulation was undetectable, and 45Ca efflux in EGTA at 4 microM Mg2+ was not significantly increased. Residual caffeine stimulation at 20 microM Mg2+ was substantial and was reduced further in increased EGTA (10 mM); at 600 microM Mg2+, residual stimulation in 5 mM EGTA was undetectable. Caffeine appears to initiate a small Ca2+-insensitive efflux that produces a large Ca2+-dependent efflux. Additional experiments suggested that caffeine also inhibited influx. The results suggest that stimulated efflux is mediated mainly or entirely by a channel controlled by an intrinsic Ca2+ receptor, which responds to local [Ca2+] in or near the channel. Receptor affinity for Ca2+ probably is influenced by Mg2+, but inhibition is weak unless local [Ca2+] is very low.     

Differential expression of calcineurin and SR Ca2+ handling proteins in equine muscle fibers during early postnatal growth.

During early postnatal development, the myosin heavy chain (MyHC) expression pattern in equine gluteus medius muscle shows adaptation to movement and load,resulting in a decrease in the number of fast MyHC fibers and an increase in the number of slow MyHC fibers. In the present study we correlated the expression of MyHC isoforms to the expression of sarcoplasmic(endo)reticulum Ca2+-ATPase 1 and 2a (SERCA), phospholamban (PLB), calcineurin A (CnA), and calcineurin B (CnB). Gluteus medius muscle biopsies were taken at 0, 2, 4, and 48 weeks and analyzed using immunofluorescence. Both SERCA isoforms and PLB were expressed in almost all fiber types at birth. From 4 weeks of age onward, SERCA1 was exclusively expressed in fast MyHC fibers and SERCA2a and PLB in slow MyHC fibers. At all time points, CnA and CnB proteins were expressed at a basal level in all fibers, but with a higher expression level in MyHC type 1 fibers. From 4 weeks onward, expression of only CnA was also higher in MyHC type 2a and 2ad fibers. We propose a double function of calcineurin in calcium homeostasis and maintenance of slow MyHC fiber type identity. Although equine muscle is already functional at birth, expression patterns of the monitored proteins still show adaptation, depending on the MyHC fiber type.     

Laser Raman study of internally perfused muscle fibers. Effect of Mg2+, ATP and Ca2+

Raman spectra of an intact muscle fiber and of internally perfused fibers in capillary tubes have been obtained. The use of internal perfusion has insured a good control of the concentration of Ca2+, Mg2+ and ATP. The comparison of the spectra obtained with the two types of fibers shows that the muscle structure is well preserved in capillary tubes. In addition, it appears that the sarcomere length has no significant effect on the Raman spectrum of muscle fibers. Our results on perfused fibers demonstrate that a fiber can be kept in the relaxed state for several hours, then displaying an intact fiber spectrum, when the concentration of ATP, Mg2+ and Ca2+ is maintained at 5, 2 and 0 mM, respectively. Therefore ATP and Mg2+ do not affect the Raman spectrum of muscle fibers. When one of these components is removed, or when Ca2+ is added, contraction occurs and causes major spectral changes. These results are interpreted as being due to strong electrostatic interactions between basic and acidic residues during contraction, and to a change of the alpha-helical content, or of the orientation, of some of the contractile proteins.     

Sarcoballs: direct access to sarcoplasmic reticulum Ca2+-channels in skinned frog muscle fibers.

In skeletal muscle, twitch contraction is caused by the rapid release of Ca2+ from the sarcoplasmic reticulum (SR) (Endo, M. 1977. Physiol. Rev. 57:71-108) via Ca2+ conducting channels in the SR membrane (Smith, J. S., R. Coronado, and G. Meissner, 1985. Nature (Lond.). 316:446-449; Suarez-Isla, B. A., G. Orozco, P. F. Heller, and J. P. Froehlich. 1986. Proc. Natl. Acad. Sci. USA. 83:7741-7745). To facilitate study of these and other intracellular channels, we have developed a method which allows direct patch-clamp recording of currents through SR channels in native membrane. The Ca2+-release channel studied using this method exhibits two predominant conductance levels (80-100 pS and 120-160 pS), conducts Ca2+ preferentially over K+ (PCa/Pk = 6.5), is highly voltage sensitive, blocked on one side by ruthenium red (1 microM), and displays enhanced activity in the presence of caffeine (5 mM). Studied in skinned fibers, this channel appears fundamentally similar to homologous channels from isolated rabbit SR incorporated into bilayers, with some distinct differences.     

Direct measurement of Ca2+ concentration in the SR of living cardiac myocytes

Although abnormal sarcoplasmic reticulum (SR) Ca(2+) handling may cause heart failure, there has been no method to directly measure Ca(2+) concentration in SR ([Ca(2+)](SR)) of living cardiomyocytes. We have measured [Ca(2+)](SR) by expressing novel fluorescent Ca(2+) indicators yellow cameleon (YC) 2.1, YC3er, and YC4er in cultured neonatal rat cardiomyocytes. The distribution of YC2.1 was uniform in the cytoplasm, while that of YC3er/YC4er, containing the signal sequence which recruits them to SR, showed reticular pattern and was co-localized with SERCA2a. The treatment with caffeine reversibly decreased the emission ratio (R) in YC3er/YC4er-expressing myocytes, and the treatment with ryanodine and thapsigargin decreased R irreversibly. During the contraction-relaxation cycle, R was changed periodically in the YC2.1- and YC3er-expressing myocytes, but its direction of the change was opposite. These results suggest that YC3er/YC4er were specifically localized and functioned in SR as a [Ca(2+)](SR) indicator. This technique would be useful to understand the function of SR in failing myocardium.     

Enhancement of Ca2+-induced Ca2+ release in calpain treated rabbit skinned muscle fibers.

Calpain treatment of rabbit skinned muscle fibers resulted in proteolysis of junctional foot protein or Ca2+ release channel of the sarcoplasmic reticulum. Electrophoretic and immunoblot analyses indicate that calpain cleaves off approximately 130 kDa peptide from the N-terminus. After such treatment, Ca2+ capacity of the sarcoplasmic reticulum remained normal and both Ca2+ and adenine nucleotide dependence of Ca2+-induced Ca2+ release mechanism were retained. However, the Ca2+-activated Ca2+ release rate was increased by two fold after the proteolysis. The results suggest the presence of functional domains in the junctional foot protein, and the N-terminus domain controls the activity of the Ca2+ channel without changing Ca2+ and nucleotide sensitivities.     

Regulation of SERCA Ca2+ pump expression by cytoplasmic Ca2+ in vascular smooth muscle cells

Vascular smooth muscle cells (VSMC) express three isoforms of the sarcoplasmic or endoplasmic reticulum Ca2+-ATPase (SERCA) pump; SERCA2b predominates (91%), whereas SERCA2a (6%) and SERCA3 (3%) are present in much smaller amounts. Treatment with thapsigargin (Tg) or A-23187 increased the level of mRNA encoding SERCA2b four- to fivefold; SERCA3 increased about 10-fold, whereas SERCA2a was unchanged. Ca2+ chelation prevented the Tg-induced SERCA2b increase, whereas Ca2+ elevation itself increased SERCA2b expression. These responses were discordant with those of 78-kDa glucose-regulated protein/immunoglobulin-binding protein (grp78/BiP), an endoplasmic reticulum stress-response protein. SERCA2b mRNA elevation was much larger than could be accounted for by the observed increase in message stability. The induction of SERCA2b by Tg did not require protein synthesis, nor was it affected by inhibitors of calcineurin, protein kinase C, Ca2+/calmodulin-dependent protein kinase, or tyrosine protein kinases. Treatment with the nonselective protein kinase inhibitor H-7 prevented Tg-induced SERCA2b expression from occurring, whereas another nonselective inhibitor, staurosporine, was without effect. We conclude that changes in cytosolic Ca2+ control the expression of SERCA2b in VSMC via a mechanism involving a currently uncharacterized, H-7-sensitive but staurosporine-insensitive, protein kinase.     

Silver ions induce Ca2+ release from the SR in vitro by acting on the Ca2+ release channel and the Ca2+ pump.

Silver nitrate (AgNO3) is a sulfhydryl oxidizing agent that induces a biphasic Ca2+ release from isolated sarcoplasmic reticulum (SR) vesicles by presumably oxidizing critical sulfhydryl groups in the Ca2+ release channel (CRC), causing the channel to open. To further examine the effects of AgNO3 on the CRC and the Ca2+-ATPase, Ca2+ release was measured in muscle homogenates prepared from rat hindlimb muscle using indo 1. Cyclopiazonic acid (CPA) and ruthenium red (RR) were used to inhibit the Ca2+-ATPase and block the CRC, respectively, before inducing Ca2+ release with both AgNO3 and 4-chloro-m-cresol (4-CMC), a releasing agent specific for the CRC. With AgNO3 and CPA, the early rapid rate of release (phase 1) was increased (P < 0.05) by 42% (314 +/- 5 vs. 446 +/- 39 micromol x g protein(-1) x min(-1)), whereas the slower, more prolonged rate of release (phase 2) was decreased (P < 0.05) by 72% (267 +/- 39 vs. 74 +/- 7.7 micromol x g protein(-1) x min(-1)). RR, in combination with AgNO3, had no effect on phase 1 (P > 0.05) (314 +/- 51 vs. 334 +/- 43 micromol x g protein(-1) x min(-1)) and decreased phase 2 (P < 0.05) by 65% (245 +/- 34 vs. 105 +/- 8.2 micromol x g protein(-1) x min(-1)). With 4-CMC, CPA had no effect (P > 0.05) on either phase 1 or 2. With addition of RR, phase 1 was reduced (P < 0.05) by 59% (2,468 +/- 279 vs. 1,004 +/- 87 micromol x g protein(-1) x min(-1)), and RR completely blocked phase 2. Both AgNO3 and 4-CMC fully inhibited Ca2+-ATPase activity measured in homogenates. These findings indicate that AgNO3, but not 4-CMC, induces Ca2+ release by acting on both the CRC and the Ca2+-ATPase.     

ATP-energized Ca2+ pump in isolated transverse tubules of skeletal muscle

A modified protocol for isolation of transverse tubules incorporated an extra stage of purification. The existence of an ATP-energized Ca2+ pump in transverse tubules isolated from rabbit skeletal muscle has been demonstrated. Isolated transverse tubules had a Ca-ATPase activity of 0.78 mu mol/min . mg; this was 300% in excess of that activity attributable to sarcoplasmic reticulum contamination. The distribution of part of the CaATPase activity and ATP-energized Ca2+ uptake coincided with the distribution of transverse tubules in isopycnic sucrose gradients loaded with mechanically disrupted triad junctions. Transverse tubules accumulated over 70 nmol of Ca2+/mg of protein; this uptake was abolished by the Ca2+ ionophore A23187. Neither digitoxin nor monensin inhibited Ca2+ uptake, indicating that Ca2+ accumulation did not occur through a sodium/calcium exchange. Conditions for half-maximal Ca2+ uptake were 5 micro M free Ca2+ and 10 micro M ATP. The Ca2+ pump of isolated transverse tubules was distinguished from the Ca2+ pump of sarcoplasmic reticulum and sarcolemma in that the transverse tubule Ca2+ pump: 1) was not enhanced by oxalate; 2) was not energized by acetyl phosphate, p-nitrophenyl phosphate, or 3-O-methylfluorescein phosphate; and 3) did not hydrolyze p-nitrophenyl phosphate or 3-O-methyl-fluorescein phosphate. Using Ca2+-dependent 3-O-methylfluorescein phosphatase as a marker for sarcoplasmic reticulum, the contamination of the transverse tubule preparation was calculated to be 6%. This agreed with a contamination level of 5% estimated by freeze-fracture electron microscopy.