Intracellular Ca dynamics and sarcomere length in single ventricular myocytes

The measurements of the sarcomere length in dissociated cardiac ventricular myocytes are discussed using mainly our own experimental data. The striation periodicity of these unloaded cells was found to be that which is to be expected of a myocyte free of the ultrastrucural constraints imposed upon it by the normal syncytial matrix of the ventricular wall. The sarcomere length and [Ca²⁺] relationship was consistent as expected from the intact tissue, when it was measured soon after partial rupturing the cell membrane. Miniature fluctuations of individual sarcomere length were demonstrated during rest, which was augmented by the Ca²⁺ overload. The [Ca²⁺] could be estimated from the measurements of sarcomere length during the positive staircase of contraction. The usefulness of the optical measurement of sarcomere pattern was indicated.     

Activation of Ca2+/Mg2+ ATPase in heart sarcolemma upon electrical stimulation

Electrical stimulation of the rat heart sarcolemmal membranes with a square wave current was found to increase Ca²⁺-ATPase activity. This activation of the enzyme was dependent upon the voltage of the electric current, frequency of stimulation and duration of stimulation of the sarcolemmal membranes. The increase in ca²⁺-ATPase was reversible upon terminating the electrical stimulation. The activation of sarcolemmal Ca²⁺-ATPase due to electrical stimulation was markedly depressed when the reaction was carried out at high pH (7.8 to 8.2), low pH (6.6 to 7.0), high temperatures (45 to 50°C) and low temperatures (17 to 25°C) of the incubation medium. Ca²⁺-antagonists, verapamil and D-600, unlike other types of inhibitors such as propranolol and ouabain, were found to reduce the activation of sarcolemmal Ca²⁺-ATPase by electrical stimulation. These results support the view that Ca²⁺/Mg²⁺ ATPase may be involved in the gating mechanism for opening Ca²⁺-channels in the sarcolemmal membrane upon excitation of the cardiac muscle.     

Modulation of Ca2+ mobilization by protein kinase C in the submandibular duct cell line A253

The expression of protein kinase C (PKC) isoforms and the modulation of Ca²⁺ mobilization by PKC were investigated in the human submandibular duct cell line A253. Three new PKC (nPKC) isoforms (, , and ) and one atypical PKC (aPKC) isoform () are expressed in this cell line. No classical PKC (cPKC) isoforms were present. The effects of the PKC activator phorbol 12-myristate-13-acetate (PMA) and of the PKC inhibitors calphostin C (CC) and bisindolymaleimide I (BSM) on inositol 1,4,5-trisphosphate (IP₃) and Ca²⁺ responses to ATP and to thapsigargin (TG) were investigated. Pre-exposure to PMA inhibited IP₃ formation, Ca²⁺ release and Ca²⁺ influx in response to ATP. Pre-exposure to CC or BSM slightly enhanced IP₃ formation but inhibited the Ca²⁺ release and the Ca²⁺ influx induced by ATP. In contrast, pre-exposure to PMA did not modify the Ca²⁺ release induced by TG, but reduced the influx of Ca²⁺ seen in the presence of this Ca²⁺-ATPase inhibitor. These results suggest that PKC modulates elements of the IP₃/Ca²⁺ signal transduction pathway in A253 cells by (1) inhibiting phosphatidylinositol turnover and altering the sensitivity of the Ca²⁺ channels to IP₃, (2) altering the activity, the sensitivity to inhibitors, or the distribution of the TG-sensitive Ca²⁺ ATPase, and (3) modulating Ca²⁺ entry pathways.     

Bradykinin stimulates phospholipase D in PC12 cells by a mechanism which is independent of increases in intracellular Ca2+

These experiments were designed to learn the role of bradykinin induced changes in intracellular Ca²⁺ in the activation of phospholipase D activity in PC12 cells. Ionomycin at a concentration of 0.1M caused an increase in intracellular Ca²⁺ comparable to bradykinin, but had no effect on phospholipase D activity. Carbachol, ATP, and thapsigargin also increased intracellular Ca²⁺ but had no effect on phospholipase D activity. Increases in intracellular Ca²⁺ may be a necessary but not a sufficient factor in the activation of phospholipase D. To investigate this issue, the bradykinin induced increase in intracellular Ca²⁺ was blocked by preincubating the cells in Ca²⁺-free media plus EGTA or in media containing the intracellular Ca²⁺ chelator BAPTA/AM. These preincubations completely blocked the bradykinin induced increase in intracellular Ca²⁺ but only attenuated the bradykinin mediated activation of phospholipase D. Physiological increases in intracellular Ca²⁺ apparently do not mediate the effect of bradykinin on phospholipase D.     

Endogenous,Ca2+-dependent cysteine-protease cleaves specifically the ryanodine receptor/Ca2+ release channel in skeletal muscle

The association of an endogenous, Ca²⁺-dependent cysteine-protease with the junctional sarcoplasmic reticulum (SR) is demonstrated. The activity of this protease is strongly stimulated by dithiothreitol (DTT), cysteine and β-mercaptoethanol, and is inhibited by iodoacetamide, mercuric chloride and leupeptin, but not by PMSF. The activity of this thiol-protease is dependent on Ca²⁺ with half-maximal activity obtained at 0.1 μm and maximal activity at 10 μm. Mg²⁺ is also an activator of this enzyme (CI₅₀=22 μm). These observations, together with the neutral pH optima and inhibition by the calpain I inhibitor, suggest that this enzyme is of calpain I type. This protease specifically cleaves the ryanodine receptor monomer (510 kD) at one site to produce two fragments with apparent molecular masses of 375 and 150 kD. The proteolytic fragments remain associated as shown by purification of the cleaved ryanodine receptor. The calpain binding site is identified as a PEST (proline, glutamic acid, serine, threonine-rich) region in the amino acid sequence GTPGGTPQPGVE, at positions 1356–1367 of the RyR and the cleavage site, the calmodulin binding site, at residues 1383–1400. The RyR cleavage by the Ca²⁺-dependent thiol-protease is prevented in the presence of ATP (1–5 mm) and by high NaCl concentrations. This cleavage of the RyR has no effect on ryanodine binding activity but stimulates Ca²⁺ efflux. A possible involvement of this specific cleavage of the RyR/Ca²⁺ release channel in the control of calpain activity is discussed.     

Role of protein kinase C in the regulation of cytosolic Ca2+ in A431 cells: Separation of growth factor and bradykinin pathways

Summary Calcium signaling systems in nonexcitable cells involve activation of Ca²⁺ entry across the plasma membrane and release from intracellular stores as well as activation of Ca²⁺ pumps and inhibition of passive Ca²⁺ pathways to ensure exact regulation of free cytosolic Ca²⁺ concentration ([Ca²⁺] _i ). A431 cells loaded with fura-2 cells were used as a model system to examine regulation of Ca²⁺ entry and intracellular release. Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-) both stimulated Ca²⁺ entry and release while bradykinin appeared only to release Ca²⁺ from intracellular stores. The possible role of protein kinase C (PKC) in modulating the [Ca²⁺] _i response to these agonists was examined by four methods. Low concentrations of TPA (2×10^–10 m) had no effect on Ca²⁺ release due to EGF, TGR- or bradykinin but resulted in a rapid return of [Ca²⁺] _i to baseline levels for EGF or TGF-. Addition of the PKC inhibitor staurosporine (1 and 10nm)_completely inhibited the action of TPA on EGF-induced [Ca²⁺] _i changes. An inhibitor of diglyceride kinase (R59022) mimicked the action of TPA. Down-regulation of PKC by overnight incubation with 0.1 or 1 m TPA produced the converse effect, namely prolonged Ca²⁺ entry following stimulation with EGF or TGF-. To show that one effect of TPA was on Ca²⁺ entry, fura-2 loaded cells were suspended in Mn²⁺ rather than Ca²⁺ buffers. Addition of EGF or TGF- resulted in Ca²⁺ release and Mn²⁺ entry. TPA but not the inactive phorbol ester, 4--phorbol-12,13-didecanoate, inhibited the Mn²⁺ influx. Thus, PKC is able to regulate Ca²⁺ entry due to EGF or TGF- in this cell type. A431 cells treated with higher concentrations of TPA (5×10^–8 m) inhibited not only Ca²⁺ entry but also Ca²⁺ release due to EGF/TGF- but had no effect on bradykinin-mediated Ca²⁺ release, suggesting differences in the regulation of the intracellular stores responsive to these two classes of agonists. Furthermore, sequential addition of EGF or TGF- gave a single transient of [Ca²⁺] _i , showing a common pool of Ca²⁺ for these agonists. In contrast, sequential addition of EGF (or TGF-) and bradykinin resulted in two [Ca²⁺] _i transients equal in size to those obtained with a single agonist. Ionomycin alone was able to fully deplete intracellular Ca²⁺ stores, whereas ionomycin following either EGF (or TGF-) or bradykinin gave an elevation of the [Ca²⁺] _i signal equal to that of the second agonist. These data indicate that there are separate pools of intracellular Ca²⁺ for EGF-mediated Ca²⁺ release which also respond differently to TPA.     

Possible mechanism of ciliary stimulation by extracellular ATP: Involvement of calcium-dependent potassium channels and exogenous Ca2+

Summary Ciliary motility was examined optically in tissue cultures from frog palate epithelium and frog's esophagus as a function of extracellular concentration of adenosine 5-triphosphate (ATP) and related compounds. The addition of micromolar concentration of ATP caused a strong enhancement of frequency and wave velocity in the direction of the effective stroke. Since adenosine 5-[, imido]-triphosphate (AMP-PNP), a nonhydrolyzable analog of ATP, produces the same effects, ATP hydrolysis is not required. The overall potency is ATP AMP-PNP>ADP adenosine>AMP. It is suggested that both the phosphate and the base moieties are involved in ATP binding.The enhancement of ciliary activity by extracellular ATP is dependent on the presence of extracellular Ca²⁺, which can be replaced by extracellular Mg²⁺. The effect of a number of potent inhibitors of the voltage-gated calcium channels on the stimulation of ciliary activity by ATP were examined. No effect was detected in the concentration range within which these agents are specific. On the other hand, quinidine, a potent inhibitor of K⁺ (calcium-dependent) channels, inhibits the effect of ATP.The following model is suggested: exogenous ATP interacts with a membrane receptor in the presence of Ca²⁺, a cascade of events occurs which mobilizes intracellular calcium, thereby increasing the cytosolic free Ca²⁺ concentration which consequently opens the calcium-activated K⁺ channels, which then leads to a change in membrane potential. The ciliary response to these changes is the enhancement of ciliary activity.This work was supported by a grant from the Fund for Basic Research administered by the Israel Academy of Science and Humanities.     

Regulation of Ca2+ mobilization by prolactin in mammary gland cells: possible role of secretory pathway Ca2+-ATPase type 2

Regulatory role of prolactin (PRL) on Ca2+ mobilization in human mammary gland cell line MCF-7 was examined. Direct addition of PRL did not affect cytoplasmic Ca2+ concentration ([Ca2+]i); however, treatment with PRL for 24h significantly decreased the peak level and duration time of [Ca2+]i elevation evoked by ATP or thapsigargin (TG). Intracellular Ca2+ release by IP3 or TG in permeablized cells was not decreased after PRL-treatment, indicating that the Ca2+ release was not impaired by PRL treatment. Extracellular Ca2+ entry evoked by ATP or TG was likely to be intact, because entry of extracellular Ba2+ was not affected by PRL treatment. Among Ca2+-ATPases expressed in MCF-7 cells, we found significant increase of secretory pathway Ca2+-ATPase type 2 (SPCA2) mRNA in PRL-treated cells by RT-PCR experiments including quantitative RT-PCR. Knockdown of SPCA2 by siRNA in PRL-treated cells showed similar Ca2+ mobilization to that in PRL-untreated cells. The present results suggest that PRL facilitates Ca2+ transport into Golgi apparatus and may contribute the supply of Ca2+ to milk.     

Rhythmic Fluctuations in the Concentration of Intracellular Mg2+ in Association with Spontaneous Rhythmic Contraction in Cultured Cardiac Myocytes

Magnesium ions (Mg²⁺) play a fundamental role in cellular function, but the cellular dynamic changes of intracellular Mg²⁺ remain poorly delineated. The present study aims to clarify whether the concentration of intracellular Mg²⁺ possibly changes cyclically in association with rhythmic contraction and intracellular Ca²⁺ oscillation in cultured cardiac myocytes from neonatal rats. To do this, we performed a noise analysis of fluctuations in the concentration of intracellular Mg²⁺ in cardiac myocytes. The concentration was estimated by loading cells with either Mg‐fluo4/AM or KMG‐20/AM. Results revealed that the intensity of Mg‐fluo‐4 or KMG‐20 fluorescence fluctuated cyclically in association with the rhythmic contraction of cardiac myocytes. In addition, the simultaneous measurement of Fura2 and Mg‐fluo‐4 fluorescence revealed phase differences between the dynamics of the two signals, suggesting that the cyclic changes in the Mg‐fluo‐4 or KMG‐20 fluorescent intensity actually reflected the changes in intracellular Mg²⁺. The complete termination of spontaneous rhythmic contractions did not abolish Mg²⁺ oscillations, suggesting that the rhythmic fluctuations in intracellular Mg²⁺ did not result from mechanical movements. We suggest that the concentration of intracellular Mg²⁺ changes cyclically in association with spontaneous, cyclic changes in the concentration of intracellular Ca²⁺ of cardiac myocytes. A noise analysis of the fluctuation of subtle changes in fluorescence intensity could contribute to the elucidation of novel functional roles of Mg²⁺ in cells.     

Voltage-dependent Ca2+ fluxes in skeletal myotubes determined using a removal model analysis

The purpose of this study was to quantify the Ca2+ fluxes underlying Ca2+ transients and their voltage dependence in myotubes by using the "removal model fit" approach. Myotubes obtained from the mouse C2C12 muscle cell line were voltage-clamped and loaded with a solution containing the fluorescent indicator dye fura-2 (200 microM) and a high concentration of EGTA (15 mM). Ca2+ inward currents and intracellular ratiometric fluorescence transients were recorded in parallel. The decaying phases of Ca2+-dependent fluorescence signals after repolarization were fitted by theoretical curves obtained from a model that included the indicator dye, a slow Ca2+ buffer (to represent EGTA), and a sequestration mechanism as Ca2+ removal components. For each cell, the rate constants of slow buffer and transport and the off rate constant of fura-2 were determined in the fit. The resulting characterization of the removal properties was used to extract the Ca2+ input fluxes from the measured Ca2+ transients during depolarizing pulses. In most experiments, intracellular Ca2+ release dominated the Ca2+ input flux. In these experiments, the Ca2+ flux was characterized by an initial peak followed by a lower tonic phase. The voltage dependence of peak and tonic phase could be described by sigmoidal curves that reached half-maximal activation at -16 and -20 mV, respectively, compared with -2 mV for the activation of Ca2+ conductance. The ratio of the peak to tonic phase (flux ratio) showed a gradual increase with voltage as in rat muscle fibers indicating the similarity to EC coupling in mature mammalian muscle. In a subgroup of myotubes exhibiting small fluorescence signals and in cells treated with 30 microM of the SERCA pump inhibitor cyclopiazonic acid (CPA) and 10 mM caffeine, the calculated Ca2+ input flux closely resembled the L-type Ca2+ current, consistent with the absence of SR Ca2+ release under these conditions and in support of a valid determination of the time course of myoplasmic Ca2+ input flux based on the optical indicator measurements.     

Disruption of energy transduction in sarcoplasmic reticulum by trypsin cleavage of (Ca2++Mg2+)-ATPase

Summary Proteolytic digestion of sarcoplasmic reticulum vesicles with trypsin has been used as a structural modification with which to examine the interaction between the ATP hydrolysis site and calcium transport sites of the (Ca²⁺+Mg²⁺)-ATPase. The kinetics of trypsin fragmentation were examined and the time course of fragment production compared with ATP hydrolytic and calcium uptake activities of the digested vesicles. The initial cleavage (TD 1) of the native ATPase to A and B peptides has no effect on the functional integrity of the enzyme, hydrolytic and transport activities remaining at the levels of the undigested control. Concomitant with the second tryptic cleavage (TD 2) of the A peptide to A₁ and A₂ fragments, calcium transport is inhibited. Kinetic analysis demonstrates that the rate constant for inhibition of calcium uptake is correlated with the rate constant of a fragment disappearance. Both Ca²⁺-dependent and total ATPase activities are unaffected by this second cleavage. Passive loading of vesicles with calcium and subsequent efflux measurements show that transport inhibition is not due to increased permeability of the membrane to calcium even at substantial extents of digestion. Steady-state levels of acidstable phosphoenzyme are unaffected by either TD 1 or TD 2, indicating that uncoupling of the hydrolytic and transport functions does not increase the turnover rate of the enzyme and that TD 2 does not change the essential characteristics of the ATP hydrolysis site. Sarcoplasmic reticulum (SR) vesicles were examined for the presence of tightly bound nucleotides and are shown to contain 2.8–3.0 nmol ATP and 2.6–2.7 nmol ADP per mg SR protein. The ADP content of SR remains essentially unchanged with TD 1 cleavage of the ATPase enzyme to A and B peptides, but declines upon TD 2 in parallel with the digestion of the A fragment and the loss of calcium uptake activity of the vesicles. The ATP content is essentially constant throughout the course of trypsin digestion. The results are discussed in terms of current models of the SR calcium pump and the molecular mechanism of energy transduction.     

Fluctuations in the Concentration of Extracellular ATP Synchronized with Intracellular Ca2+ Oscillatory Rhythm in Cultured Cardiac Myocytes

Isolated and cultured neonatal cardiac myocytes contract spontaneously and cyclically. The intracellular concentration of free Ca²⁺ also changes rhythmically in association with the rhythmic contraction of myocytes (Ca²⁺ oscillation). Both the contraction and Ca²⁺ oscillatory rhythms are synchronized among myocytes, and intercellular communication via gap junctions has been considered primarily responsible for the synchronization. However, a recent study has demonstrated that intercellular communication via extracellular ATP‐purinoceptor signaling is also involved in the intercellular synchronization of intracellular Ca²⁺ oscillation. In this study, we aim to elucidate whether the concentration of extracellular ATP changes cyclically and contributes to the intercellular synchronization of Ca²⁺ oscillation among myocytes. In almost all the cultured cardiac myocytes at four days in vitro (4 DIV), intracellular Ca²⁺ oscillations were synchronized with each other. The simultaneous measurement of the concentration of extracellular ATP and intracellular Ca²⁺ revealed the extracellular concentration of ATP actually oscillated concurrently with the intracellular Ca²⁺ oscillation. In addition, power spectrum and cross‐correlation analyses suggested that the treatment of cultured cardiac myocytes with suramin, a blocker of P2 purinoceptors, resulted in the asynchronization of Ca²⁺ oscillatory rhythms among cardiac myocytes. Treatment with suramin also resulted in a significant decrease in the amplitudes of the cyclic changes in both intracellular Ca²⁺ and extracellular ATP. Taken together, the present study demonstrated the possibility that the concentration of extracellular ATP changes cyclically in association with intracellular Ca²⁺, contributing to the intercellular synchronization of Ca²⁺ oscillation among cultured cardiac myocytes.     

Chloroplast assemblage by mechanical stimulation and its intercellular transmission in diatom cells

Summary By touching a cell ofPleurosira laevis, a centric diatom, the chloroplasts in the cortical cytoplasm immediately migrate to the nuclear cytoplasm which is located at the center of the cell. The time required for migration, is about 10 s and that for restoration is about 15 min. The touch could be replaced by an electric stimulation. A 7 V pulse for 1 ms was required for the induction of chloroplast assemblage. To obtain chloroplast assemblage in 50% of the cells, the amount of time and voltage required were inversely proportional. Higher concentrations of KCl also induced chloroplast assemblage. Neither contact nor electric stimulation in Ca²⁺ free medium induced chloroplast assemblage. Verapamil, a Ca²⁺ channel blocker, in medium suppressed the chloroplast assemblage. Ca²⁺ ionophore caused the chloroplast assemblage. These results suggested that the depolarization of the plasma membrane was a trigger of the induction of chloroplast assemblage and the influx of Ca²⁺ to the cytoplasm through the channel was essential for this. The chloroplast assemblage was transmitted successively to the other cells of the filamentous colony, though these cells were linked only by a mucilaginous substance. Furthermore, the transmission occurred in cells which were placed separately. The maximum distance possible for this jumping transmission was 200 m in the culture medium. To search for the release of any chemical transmitter, contact stimulation was applied to one cell at the middle of a filamentous colony which was placed in water streaming along the axis of the filament. Chloroplast assemblage was induced more in downstream cells than in upstream ones. This result suggested the existence of some substance which mediated the transmission of the stimulation.     

Modulation of Na+-Ca2+ exchange in cardiac sarcolemmal vesicles by Ca2+ antagonists

Summary The purpose of this study was to examine the effect of three classes of Ca²⁺ antagonists, diltiazem, verapamil and nifedipine on Na⁺-Ca²⁺ exchange mechanism in the sarcolemmal vesicles isolated from canine heart. Na⁺-Ca²⁺ exchange and Ca²⁺ pump (ATP-dependent Ca²⁺ uptake) activities were assessed using the Millipore filtration technique. sarcolemmal vesicles used in this study are estimated to consist of several subpopulations wherein 23% are inside-out and 55% are right side-out sealed vesicles in orientation. The affect of each Ca²⁺ antagonist on the Na⁺-dependent Ca²⁺ uptake was studied in the total population of sarcolemmal vesicles, in which none of the agents depressed the initial rate of Ca²⁺ uptake until concentrations of 10 M were incubated in the incubation medium. However, when sarcolemmal vesicles were preloaded with Ca²⁺ via ATP-dependent Ca²⁺ uptake, cellular Ca²⁺ influx was depressed only by verapamil (28%) at 1 M in the efflux medium with 8 mM Na⁺. Furthermore, inhibition of Ca²⁺ efflux by verapamil was more pronounced in the presence of 16 mM Na⁺ in the efflux medium. The order of inhibition was; verapamil > diltiazem > nifedipine. These results indicate that same forms of Ca²⁺-antagonist drugs may affect the Na⁺-Ca²⁺ exchange mechanism in the cardiac sarcolemmal vesicles and therefore we suggest this site of action may contribute to their effects on the myocardium.     

Purinergic stimulation of K+-dependent Na+/Ca2+ exchanger isoform 4 requires dual activation by PKC and CaMKII

K⁺-dependent Na⁺/Ca²⁺-exchanger isoform 4 (NCXK4) is one of the most broadly expressed members of the NCKX (K⁺-dependent Na⁺/Ca²⁺-exchanger) family. Recent data indicate that NCKX4 plays a critical role in controlling normal Ca²⁺ signal dynamics in olfactory and other neurons. Synaptic Ca²⁺ dynamics are modulated by purinergic regulation, mediated by ATP released from synaptic vesicles or from neighbouring glial cells. Previous studies have focused on modulation of Ca²⁺ entry pathways that initiate signalling. Here we have investigated purinergic regulation of NCKX4, a powerful extrusion pathway that assists in terminating Ca²⁺ signals. NCKX4 activity was stimulated by ATP through activation of the P2Y receptor signalling pathway. Stimulation required dual activation of PKC (protein kinase C) and CaMKII (Ca²⁺/calmodulin-dependent protein kinase II). Mutating T312, a putative PKC phosphorylation site on NCKX4, partially prevented purinergic stimulation. These data illustrate how purinergic regulation can shape the dynamics of Ca²⁺ signalling by activating a signal damping and termination pathway.     

Failure of short term stimulation to reduce sarcoplasmic reticulum Ca2+-ATPase function in homogenates of rat gastrocnemius

To examine the effect of short term intense activity on sarcoplasmic reticulum (SR) Ca²⁺ sequestering function, the gastrocnemius (G) muscles of 11 anaesthetized male rats (weight, 411±8 g,X±SE) were activated using supramaximal, intermittent stimulation (one train of 0.2 msec impulses per sec of 100 msec at 100 Hz). Homogenates were obtained from stimulated white (WG-S) and red (RG-S) tissues, assayed for Ca²⁺ uptake and maximal Ca²⁺ ATPase activity and compared to contralateral controls (WG-C, RG-C). Calcium uptake (nmoles/mg protein/min) determined using Indo-l and at [Ca²⁺]_f concentrations between 300–400 nM was unaffected (p>0.05) by activity in both WG (6.14+0.43 vs 5.37+0.43) and RG (3.21+0.18 vs 3.07+0.20). Similarly, no effect (p>0.05) of contractile activity was found for maximal Ca²⁺ ATPase activity (mole/mg protein/min) determined spectrophotometrically in RG (0.276+0.03 vs 0.278+0.02). In WG, Ca²⁺ ATPase activity was 15% higher in WG-S compared to WG-C (0.412+0.03 vs 0.385+0.04). Repetitive stimulation resulted in a reduction in tetanic tension of 74% (p<0.05) by 2 min in the G muscle. By the end of the stimulation period, ATP concentration was reduced (p<0.05) by 57% in the WG and by 47% in the RG. These results indicate that the repeated generation of maximal tetanic force, at least for short term periods, need not adversely affectin vitro homogenate determination of Ca²⁺ sequestering function in spite of severe alterations in energy potential and that some other mechanism must be involved to explain the depression in Ca²⁺ uptake and Ca²⁺ ATPase activity previously noted with short term intense exercise.     

N(6)-Methyldeoxyadenosine, a nucleoside commonly found in prokaryotes, induces C2C12 myogenic differentiation

N(6)-methyl-2(')-deoxyadenosine (MedAdo) is a nucleoside naturally found in prokaryotic DNA. Interestingly, the N(6)-methylation of adenine in DNA seems to have been counter-selected during the course of evolution since MedAdo has not been detected in mammalian DNA until now. We show here that treatment with MedAdo induces myogenesis in C2C12 myoblasts. The presence of MedAdo in C2C12 DNA was investigated using a method based on HPLC coupled to electrospray ionization tandem mass spectrometry which is several thousand fold more sensitive than assays used previously. By this procedure, MedAdo is detected in the DNA from MedAdo-treated cells but remains undetectable in the DNA from control cells. Furthermore, MedAdo regulates the expression of p21, myogenin, mTOR, and MHC. Interestingly, in the pluripotent C2C12 cell line, MedAdo drives the differentiation towards myogenesis only. Thus, the biological effect of MedAdo is suppressed in the presence of BMP-2 which transdifferentiates C2C12 from myogenic into osteogenic lineage cells. Taken together these results point to MedAdo as a novel inducer of myogenesis and further extends the differentiation potentialities of this methylated nucleoside. Furthermore, these data raise the intriguing possibility that the biological effects of MedAdo on cell differentiation may have led to its counter-selection in eukaryotes.