Ca2+ release by inositol-trisphosphorothioate in isolated triads of rabbit skeletal muscle.

The effectiveness of the nonmetabolizable second messenger analogue DL-myo-inositol 1,4,5-trisphosphorothioate (IPS3) described by Cooke, A. M., R. Gigg, and B. V. L. Potter, (1987b. Jour. Chem. Soc. Chem. Commun. 1525-1526.) was examined in triads purified from rabbit skeletal muscle. A Ca2+ electrode uptake-release assay was used to determine the size and sensitivity of the IPS3-releasable pool of Ca2+ in isolated triads. Uptake was initiated by 1 mM MgATP, pCa 5.8, pH 7.5 Release was initiated when the free Ca2+ had lowered to pCa approximately 7. We found that 5-25 microM myo-inositol 1,4,5-trisphosphate (IP3), and separately IPS3, consistently released 5-20% of the Ca2+ pool actively loaded into triads. Single channel recording was used to determine if ryanodine receptor Ca2+ release channels were affected by IPS3 at the same myoplasmic Ca2+ and IPS3 concentrations. Open probability of ryanodine receptor Ca2+ release channels was monitored in triads fused to bilayers over long periods (200 s) in the absence and following addition of 30 microM IPS3 to the same channel. At myoplasmic pCa approximately 7, IPS3 had no effect in the absence of MgATP (Po = 0.0094 +/- 0.001 in control and Po = 0.01 +/- 0.006 after IPS3) and slightly increased activity in the presence of 1 mM MgATP (Po = 0.024 +/- 0.03 in control and Po = 0.05 +/- 0.03 after IPS3). Equally small effects were observed at higher myoplasmic Ca2+. The onset of channel activation by IPS3 or IP3 was slow, on the time scale 20-60 s. We suggest that in isolated triads of rabbit skeletal muscle, IP3-induced release of stored Ca2+ is probably not mediated by the opening of Ca2+ release channels.     

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.     

The effects of Mg2+ on the Ca2+-binding properties and Ca2+-induced tyrosine-fluorescence changes of calmodulin isolated from rabbit skeletal muscle

Calmodulin from phosphorylase kinase (the delta subunit) was obtained as a homogeneous protein in a spectroscopically pure form, and its interaction with Ca2+ and Mg2+ was studied. 1. Determination of the binding of Ca2+ to calmodulin in a buffer of low ionic strength (0.001 M) show that it contained six binding sites for this divalent cation. 2. Employment of a buffer of high ionic strength (0.18 M) allowed two Ca2+/Mg2+-binding sites (KdCa2+ = 4.0 microM), which showed Ca2+ - Mg2+ competition (KdMg2+ = 0.75 mM), to be distinguished from two Ca2+-specific binding sites (KdCa2+ = 40 microM). The remaining two Ca2+-binding sites are not observed under these conditions and are probably Mg2+-specific binding sites. Thus, the binding sites on calmodulin are remarkably similar to those of the homologous Ca2+-binding protein, troponin C [Potter and Gergely (1975) J. Biol. Chem. 250, 4628, 4633]. 3. The conformational states of calmodulin are defined by Ca2+, Mg2+ and salt concentrations, which can be differentiated by their Ca2+ affinity and their relative tyrosine fluorescence intensity. In a buffer of high ionic strength, Mg2+ induces a conformation which enhances the apparent affinity for Ca2+. Addition of Ca2+ leads to an enhancement of the tyrosine fluorescence intensity, which remains enhanced even upon removal of Ca2+ by chelation with EGTA. Only additional chelation of Mg2+ with EDTA reduces the tyrosine fluorescence intensity. 4. Comparison of the Ca2+-binding parameters of phosphorylase kinase, which were previously determined under identical experimental conditions [Kilimann and Heilmeyer (1977) Eur. J. Biochem. 73, 191-197], with those reported here on calmodulin isolated from this enzyme, allows the conclusion that Ca2+ binding to the holoenzyme occurs by binding to the delta subunit exclusively. 5. Ca2+ binding and Ca2+ activation of phosphorylase kinase are compared and discussed in relation to the Ca2+ and Mg2+-induced conformation changes of calmodulin.     

Effect of halothane on Ca2+-induced Ca2+ release from sarcoplasmic reticulum vesicles isolated from rat skeletal muscle

Halothane induces the release of Ca2+ from a subpopulation of sarcoplasmic reticulum vesicles that are derived from the terminal cisternae of rat skeletal muscle. Halothane-induced Ca2+ release appears to be an enhancement of Ca2+-induced Ca2+ release. The low-density sarcoplasmic reticulum vesicles which are believed to be derived from nonjunctional sarcoplasmic reticulum lack the capability of both Ca2+-induced and halothane-induced Ca2+ release. Ca2+ release from terminal cisternae vesicles induced by halothane is inhibited by Ruthenium red and Mg2+, and require ATP (or an ATP analogue), KCl (or similar salt) and extravesicular Ca2+. Ca2+-induced Ca2+ release has similar characteristics.     

Ca2+ regulation in detrusor smooth muscle from developing fetal sheep bladders

Sheep fetus is a useful model to study in utero bladder outflow obstruction but little is known about cell physiology of fetal bladders. To remedy this defect we have characterised intracellular Ca(2+) regulation in fetal sheep myocytes of different developmental ages. Fetal detrusor myocytes had a similar resting [Ca(2+)](i) to adult cells and exhibited transient [Ca(2+)](i) increases in response to carbachol, ATP, high-K, caffeine and low-Na. The carbachol transients were abolished by atropine and caffeine; the ATP response was blocked by alpha,beta-methylene ATP; high-K-evoked [Ca(2+)](i) rises were antagonised by verapamil. The maximal responses to carbachol, high-K, caffeine and low-Na in fetal cells were similar to those of adult counterparts, whilst the ATP response was smaller (p < 0.05). These variables were largely similar between the three gestational groups with the exception of ATP-induced response between early fetal and adult bladders (p < 0.05). Dose-response curves to carbachol demonstrated an increase of potency between mid-gestation and early adulthood (p < 0.05). These data show that muscarinic receptors coupled to intracellular Ca(2+) release, P2X receptor-linked Ca(2+) entry, depolarisation-induced Ca(2+) rise via L-type Ca(2+) channels, Na(+)/Ca(2+) exchange and functional intracellular Ca(2+) stores are all operational in fetal bladder myocytes. Whilst most of Ca(2+) regulators are substantially developed and occur at an early fetal age, a further functional maturation for cholinergic sensitivity and purinergic efficacy continues throughout to adulthood.     

Inhibition of the veratridine-induced increase in cytosolic Ca2+ and respiration by Ca2+ antagonists in isolated cardiac myocytes.

1. We studied the effect of verapamil, nitrendipine, 3',4'-dichlorobenzamil (DCB) and Cd2+ on the increase in cytosolic free Ca2+ ([Ca2+]c) and the rate of O2-uptake induced by depolarization of isolated rat cardiac myocytes with veratridine. 2. The degree of inhibition by the several drugs tested on the increase in [Ca2+]c and respiration was dependent on extracellular Ca2+, pH and Na+. 3. Low verapamil and nitrendipine concentrations (2.5 microM) were fully effective in Ca2+ channel blockade, as indicated from experiments with isoproterenol and in a low-Na+ medium. 4. A complete inhibition of veratridine-induced increase in [Ca2+]c and O2-uptake was attained with higher Ca2+ blocker concentrations (25-30 microM), implying that these processes depend to a major extent on some other Ca2+ transport system, probably Na+/Ca2+ exchange.     

Characterization of the Ca2+- or Mg2+-ATPase of transverse tubule membranes isolated from rabbit skeletal muscle

Transverse tubule membranes isolated from rabbit skeletal muscle have high levels of a Ca2+- or Mg2+-ATPase with Km values for Ca-ATP or Mg-ATP in the 0.2 mM range, but do not display detectable levels of ATPase activity activated by micromolar [Ca2+]. The transverse tubule enzyme is less temperature or pH dependent than the Ca2+-ATPase of sarcoplasmic reticulum and hydrolyzes equally well ATP, ITP, UTP, CTP, and GTP. Of several ionic, non-ionic, and zwitterionic detergents tested, only lysolecithin solubilizes the transverse tubule membrane while preserving ATPase activity. After extraction of about 50% of the transverse tubule proteins by solubilization with lysolecithin most of the ATPase activity remains membrane bound, indicating that the Ca2+- or Mg2+-ATPase is an intrinsic membrane enzyme. A second extraction of the remaining transverse tubule proteins with lysolecithin results in solubilization and partial purification of the enzyme. Sedimentation of the Ca2+- or Mg2+-ATPase, partially purified by lysolecithin solubilization, through a continuous sucrose gradient devoid of detergent leads to additional purification, with an overall 3- to 5-fold purification factor. The purified enzyme preparation contains two main protein components of molecular weights 107,000 and 30,000. Cholesterol, which is highly enriched in the transverse tubule membrane, copurifies with the enzyme. Transverse tubule membrane vesicles also display ATP-dependent calcium transport which is not affected by phosphate or oxalate. The possibility that the Ca2+- or Mg2+-ATPase is the enzyme responsible for the Ca2+ transport displayed by isolated transverse tubules is discussed.     

Effect of extracellular Ca2+ on plasma membrane Ca2+ inflow and cytoplasmic free Ca2+ in isolated hepatocytes

An initial rapid phase and a subsequent slow phase of 45Ca2+ uptake were observed following the addition of 45Ca2+ to Ca2+-deprived hepatocytes. The magnitude of the rapid phase increased 15-fold over the range 0.1-11 mM extracellular Ca2+ (Ca2+o) and was a linear function of [Ca2+]o. The increases in the rate of 45Ca2+ uptake were accompanied by only small increases in the intracellular free Ca2+ concentration. In cells made permeable to Ca2+ by treatment with saponin, the rate of 45Ca2+ uptake (measured at free Ca2+ concentrations equal to those in the cytoplasm of intact cells) increased as the concentration of saponin increased from 1.4 to 2.5 micrograms per mg wet weight cells. Rates of 45Ca2+ uptake by cells permeabilized with an optimal concentration of saponin were comparable with those of intact cells incubated at physiological [Ca2+o], but were substantially lower than those for intact cells incubated at high [Ca2+o]. It is concluded that Ca2+ which enters the hepatocyte across the plasma membrane is rapidly removed by binding and transport to intracellular sites and by the plasma membrane (Ca2+ + Mg2+)-ATPase and the plasma membrane Ca2+ inflow transporter is not readily saturated with Ca2+o.     

Large-conductance voltage- and Ca2+-activated K+ channels regulate human detrusor smooth muscle function

The large-conductance voltage- and Ca(2+)-activated K(+) (BK) channel is expressed in many smooth muscle types, but its role in human detrusor smooth muscle (DSM) is unclear. With a multidisciplinary approach spanning channel molecules, single-channel activity, freshly isolated human DSM cells, intact DSM preparations, and the BK channel specific inhibitor iberiotoxin, we elucidated human DSM BK channel function and regulation. Native human DSM tissues were obtained during open surgeries from patients with no preoperative history of overactive bladder. RT-PCR experiments on single human DSM cells showed mRNA expression of BK channel α-, β(1)-, and β(4)-subunits. Western blot and immunocytochemistry confirmed BK channel α, β(1), and β(4) protein expression. Native human BK channel properties were described using the perforated whole cell configuration of the patch-clamp technique. In freshly isolated human DSM cells, BK channel blockade with iberiotoxin inhibited a significant portion of the total voltage step-induced whole cell K(+) current. From single BK channel recordings, human BK channel conductance was calculated to be 136 pS. Voltage-dependent iberiotoxin- and ryanodine-sensitive transient BK currents were identified in human DSM cells. In current-clamp mode, iberiotoxin inhibited the hyperpolarizing membrane potential transients and depolarized the cell resting membrane potential. Isometric DSM tension recordings revealed that BK channels principally control the contractions of isolated human DSM strips. Collectively, our results indicate that BK channels are fundamental regulators of DSM excitability and contractility and may represent new targets for pharmacological or genetic control of urinary bladder function in humans.     

The Ca2+ antagonists binding cytosolic protein has properties of the Ca2+ channel.

In our previous work (Krizanová et al. 1989) we have described a protein from rabbit skeletal muscle cytosolic fraction, which is able to bind dihydropyridines and phenothiazines. In the present work conclusive evidence is provided for the ability of the phospholipid-reconstituted cytosolic protein to transport calcium. The calcium transport was stimulated by BAY K 8644 and inhibited in the presence of PN 200-110. Our observations were confirmed also by electrophysiological measurements on planar lipid bilayers. The possibility that the cytosolic fraction was contaminated with membranes could be definitely ruled out. Nevertheless, the nature of the protein under study is still in the frame of guess.     

Ca 2+ -specific removal of Z lines from rabbit skeletal muscle

Removal of rabbit psoas strips immediately after death and incubation in a saline solution containing 1 mM Ca²⁺ and 5 nM Mg²⁺ for 9 hr at 37°C and pH 7.1 causes complete Z-line removal but has no ultrastructurally detectable effect on other parts of the myofibril. Z lines remain ultrastructurally intact if 1 mM 1,2-bis-(2-dicarboxymethylaminoethoxy)-ethane (EGTA) is substituted for 1 mM Ca²⁺ and the other conditions remain unchanged. Z lines are broadened and amorphous but are still present after incubation for 9 hr at 37°C if 1 mM ethylenediaminetetraacetate (EDTA) is substituted for 1 mM Ca²⁺ and 5 mM Mg²⁺ in the saline solution. A protein fraction that causes Z-line removal from myofibrils in the presence of Ca²⁺ at pH 7.0 can be isolated by extraction of ground muscle with 4 mM EDTA at pH 7.0–7.6 followed by isoelectric precipitation and fractionation between 0 and 40% ammonium sulfate saturation. Z-line removal by this protein fraction requires Ca²⁺ levels higher than 0.1 mM, but Z lines are removed without causing any other ultrastructurally detectable degradation of the myofibril. This is the first report of a protein endogenous to muscle that is able to catalyze degradation of the myofibril. The very low level of unbound Ca²⁺ in muscle cells in vivo may regulate activity of this protein fraction, or alternatively, this protein fraction may be localized in lysosomes.     

Kinetics of Ca2+-activated K+ channels from rabbit muscle incorporated into planar bilayers. Evidence for a Ca2+ and Ba2+ blockade

The interaction of Ca2+ and Ba2+ with a Ca2+-activated K+ channel from rabbit skeletal muscle membranes is studied in planar lipid bilayers. At [Ca2+] greater than or equal to 100 microM in the cis side (the side to which the vesicles are added) and at positive voltages, the channel kinetics consisted of bursts of activity interrupted by long periods of quiescence. We found that the reciprocal of the mean burst time increases linearly with [Ca2+], whereas the mean time for the quiescent (closed) periods is independent of [Ca2+]. The number of quiescent periods is reduced by increasing [K+]. Micromolar amounts of cis Ba2+ do not activate the channel, but induce similar "slow" closings. Also, in this case, the mean burst time is inversely proportional to the [Ba2+] and the mean closed time is independent of [Ba2+]. Raising [K+] either symmetrically or only in the trans side relieved the Ba2+ effect. trans Ba2+ also induces changes in the slow kinetics, but in millimolar amounts. These results suggest that the quiescent periods correspond to a channel blocked by a Ba ion. The voltage dependence of the cis blockade indicates that the Ba2+ binding site is past the middle of the membrane field. The similarities in the slow kinetics induced by Ca2+ and Ba2+ suggest that Ca2+ blocks the channel by binding to the same site. However, binding of Ca2+ to the site is 10(5)- fold weaker.     

Ca2+-dependent membrane currents in vascular smooth muscle cells of the rabbit.

The membrane potential in vascular smooth muscle cells contributes to the regulation of cytosolic [Ca2+], which in turn regulates membrane potential by means of Ca2+i-dependent ionic currents. We investigated the characteristics of Ca2+i-dependent currents in rabbit coronary and pulmonary arterial smooth muscle cells. Ca2+i-dependent currents were recorded using the whole-cell patch-clamp technique while cytosolic [Ca2+] was increased by caffeine. The reversal potentials of caffeine-induced currents were between -80 and -10 mV under normal ionic conditions, whereas they were about 0 mV when K+-free NaCl solutions were used both in pipette and bath. The total substitution of extracellular Na+ with membrane-impermeable cation N-Methyl-D-glucamine did not affect caffeine-induced currents, implying no significant contribution of Na+ as a permeant ion to the currents. The substitution of extracellular NaCl with sucrose reduced outward component of the currents and shifted the reversal potentials according to the change in Cl- equilibrium potential. Upon application of the niflumic acid under K+-free conditions, most of the current induced by caffeine was inhibited. Taken together, the results of the present study indicate that K+ and Cl- currents are major components of Ca2+i-dependent currents in vascular smooth muscles isolated from coronary and pulmonary arteries of the rabbit, and the relative contribution of each type of current to total currents are not different between the two arteries.     

Effect of endurance exercise on the Ca2+ pumps from transverse tubule and sarcoplasmic reticulum of rabbit skeletal muscle.

The sarcoplasmic reticulum (SR) Ca(2+) pump is the main homeostatic regulatory mechanism in fast skeletal muscle that maintains intracellular Ca(2+) concentration ([Ca(2+)](i)) at the nanomolar level at rest. The transverse tubule (TT) Ca(2+) pump transports cytosolic Ca(2+) to the extracellular space. During prolonged muscular activity, [Ca(2+)](i) may increase. TT and SR isolated microsomal vesicles were highly purified, and the purity was checked by immunoblotting. The present study shows the effects of endurance exercise on the activities and structures of the TT and SR Ca(2+) pumps of fast skeletal muscle from rabbit at rest. The Ca(2+) pump activity increased manifolds in TT but did not change in SR. The protein denaturalization profiles obtained by differential scanning calorimetry showed 1) a shift in the transition temperature and an increase in the enthalpy of the TT Ca(2+) pump and 2) a significant change in the transition temperature of the SR Ca(2+) pump Ca(2+)-binding domain. We conclude that the TT Ca(2+) pump activity was upgraded in association with structural changes to handle the changes in [Ca(2+)](i) and TT lumen Ca(2+) concentration that occur during endurance exercise.     

Ca2+-induced Ca2+ release in skinned single smooth muscle cells isolated from guinea-pig taenia caeci

The Ca2+ release from intracellular Ca2+ storage sites of skinned single smooth muscle cells isolated from guinea-pig taenia caeci was studied. The Ca2+ release from intracellular Ca2+ storage sites of the skinned single cells was enhanced by the presence of submicromolar concentrations of Ca2+ in the solution. The Ca2+ release was enhanced by caffeine and adenine, and suppressed by Mg2+ and procaine. These results suggest that the Ca2+-induced Ca2+ release mechanism may play an important role in the release of Ca2+ from intracellular storage sites of guinea-pig taenia caeci smooth muscle cells.     

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.