Inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)R) and ryanodine receptors (RyR) were localized in cultured rodent muscle fractions by binding of radiolabeled ligands (IP(3) and ryanodine), and IP(3)R were visualized in situ by fluorescence immunocytological techniques. Also explored was the effect of K(+) depolarization on IP(3) mass and Ca(2+) transients studied using a radio-receptor displacement assay and fluorescence imaging of intracellular fluo 3. RyR were located in a microsomal fraction; IP(3)R were preferentially found in the nuclear fraction. Fluorescence associated with anti-IP(3)R antibody was found in the region of the nuclear envelope and in a striated pattern in the sarcoplasmic areas. An increase in external K(+) affected membrane potential and produced an IP(3) transient. Rat myotubes displayed a fast-propagating Ca(2+) signal, corresponding to the excitation-contraction coupling transient and a much slower Ca(2+) wave. Both signals were triggered by high external K(+) and were independent of external Ca(2+). Slow waves were associated with cell nuclei and were propagated leaving "glowing" nuclei behind. Different roles are proposed for at least two types of Ca(2+) release channels, each mediating an intracellular signal in cultured skeletal muscle.

The dihydropyridine receptor (DHPR), normally a voltage-dependent calcium channel, functions in skeletal muscle essentially as a voltage sensor, triggering intracellular calcium release for excitation-contraction coupling. In addition to this fast calcium release, via ryanodine receptor (RYR) channels, depolarization of skeletal myotubes evokes slow calcium waves, unrelated to contraction, that involve the cell nucleus (Jaimovich, E., R. Reyes, J.L. Liberona, and J.A. Powell. 2000. Am. J. Physiol. Cell Physiol. 278:C998–C1010). We tested the hypothesis that DHPR may also be the voltage sensor for these slow calcium signals. In cultures of primary rat myotubes, 10 μM nifedipine (a DHPR inhibitor) completely blocked the slow calcium (fluo-3-fluorescence) transient after 47 mM K+ depolarization and only partially reduced the fast Ca2+ signal. Dysgenic myotubes from the GLT cell line, which do not express the α1 subunit of the DHPR, did not show either type of calcium transient following depolarization. After transfection of the α1 DNA into the GLT cells, K+ depolarization induced slow calcium transients that were similar to those present in normal C2C12 and normal NLT cell lines. Slow calcium transients in transfected cells were blocked by nifedipine as well as by the G protein inhibitor, pertussis toxin, but not by ryanodine, the RYR inhibitor. Since slow Ca2+ transients appear to be mediated by IP3, we measured the increase of IP3 mass after K+ depolarization. The IP3 transient seen in control cells was inhibited by nifedipine and was absent in nontransfected dysgenic cells, but α1-transfected cells recovered the depolarization-induced IP3 transient. In normal myotubes, 10 μM nifedipine, but not ryanodine, inhibited c-jun and c-fos mRNA increase after K+ depolarization. These results suggest a role for DHPR-mediated calcium signals in regulation of early gene expression. A model of excitation-transcription coupling is presented in which both G proteins and IP3 appear as important downstream mediators after sensing of depolarization by DHPR.

] i was monitored in Fluo 3-acetoxymethyl esterloaded myotubes by either confocal microscopy or fluorescence microscopy, with the use of out-of-focus fluorescence elimination. The mass of IP 3 was determined by radioreceptor displacement assay. [Ca 2ϩ ] i changes after either aldosterone (10-100 nM) or testosterone (50-100 nM) were observed; a relatively fast (Ͻ2 min) calcium transient, frequently accompanied by oscillations, was evident with both hormones. A slow rise in [Ca 2ϩ ] i that reached its maximum after a 30-min exposure to aldosterone was also observed. Calcium responses seem to be fairly specific for aldosterone and testosterone, because several other steroid hormones do not induce detectable changes in fluorescence, even at 100-fold higher concentrations. The mass of IP 3 increased transiently to reach two-to threefold the basal level 45 s after addition of either aldosterone or testosterone, and the IP 3 transient was more rapid than the fast calcium signal. Spironolactone, an inhibitor of the intracellular aldosterone receptor, or cyproterone acetate, an inhibitor of the testosterone receptor, had no effect on the fast [Ca 2ϩ ] i signal or in the increase in IP 3 mass. These signals could mean that there are distinct nongenomic pathways for the action of these two steroids in skeletal muscle cells. steroid hormones; inositol 1,4,5-trisphosphate; calcium waves; nongenomic pathway STEROID HORMONES ARE CAPABLE of producing rapid (within 2 min) effects in several cell types (12,22,36). These rapid responses are not compatible with the classical mechanism of action proposed for these hormones, which involves binding to intracellular receptors, transcriptional processes, and protein synthesis (1, 20). Thus, for rapid steroid effects, the existence of membrane receptors for steroids has been proposed (2, 37). In skeletal muscle, steroid hormones have long been known to modulate gene expression, and hormones like testosterone have been related to both muscle hypertrophy (4, 25) and upregulation of a number of proteins (31). In particular, glucocorticoids and mineralocorticoids have been related to up-and downregulation, respectively, of sodium pumps in skeletal muscle (10), implying the presence of steroid hormone receptors in muscle cells. Because all these effects have been the result of chronic (hours to days) treatments, it would be interesting to know whether exposure to these hormones is also accompanied by fast nongenomic events.The signal transduction mechanism for the rapid action of steroids in some cellular models could be regulated by second messengers such as intracellular calcium ([Ca 2ϩ

In the heart, insulin-like growth factor-1 (IGF-1) is a pro-hypertrophic and anti-apoptotic peptide. In cultured rat cardiomyocytes, IGF-1 induced a fast and transient increase in Ca

The fluorescent calcium signal from rat myotubes in culture was monitored after field-stimulation with tetanic protocols. After the calcium signal sensitive to ryanodine and associated to the excitation-contraction coupling, a second long-lasting calcium signal refractory to ryanodine was consistently found. The onset kinetics of this slow signal were slightly modified in nominally calcium-free medium, as were both the frequency and number of pulses during tetanus. No signal was detected in the presence of tetrodotoxin. The participation of the dihydropyridine receptor (DHPR) as the voltage sensor for this signal was assessed by treatment with agonist and antagonist dihydropyridines (Bay K 8644 and nifedipine), showing an enhanced and inhibitory response, respectively. In the dysgenic GLT cell line, which lacks the alpha1(S) subunit of the DHPR, the signal was absent. Transfection of these cells with the alpha1(S) subunit restored the slow signal. In myotubes, the inositol 1,4,5-trisphosphate (IP(3)) mass increase induced by a tetanus protocol preceded in time the slow calcium signal. Both an IP(3) receptor blocker and a phospholipase C inhibitor (xestospongin C and U73122, respectively) dramatically inhibit this signal. Long-lasting, IP(3)-generated slow calcium signals appear to be a physiological response to activity-related fluctuations in membrane potential sensed by the DHPR.