We studied the effect of IGF-I and insulin on intracellular Ca(2+) in primary cultured myotubes. IGF-I induced a fast and transient Ca(2+) increase, measured as fluo-3 fluorescence. This response was blocked by both genistein and AG538. IGF-I induced a fast inositol-1,4,5-trisphosphate (IP(3)) increase, kinetically similar to the Ca(2+) rise. The Ca(2+) signal was blocked by inhibitors of the IP(3) pathway. On the other hand, insulin produced a fast (<1 s) and transient Ca(2+) increase. Insulin-induced Ca(2+) increase was blocked in Ca(2+)-free medium and by either nifedipine or ryanodine. In the normal muscle NLT cell line, the Ca(2+ )signals induced by both hormones resemble those of primary myotubes. GLT cells, lacking the alpha1-subunit of dihydropyridine receptor (DHPR), responded to IGF-I but not to insulin, while GLT cells transfected with the alpha1-subunit of DHPR reacted to both hormones. Moreover, dyspedic muscle cells, lacking ryanodine receptors, responded to IGF-I as NLT cells, however they show no insulin-induced calcium increase. Moreover, G-protein inhibitors, pertussis toxin (PTX) and GDPbetaS, blocked the insulin-induced Ca(2+) increase without major modification of the response to IGF-I. The different intracellular Ca(2+) patterns produced by IGF-I and insulin may improve our understanding of the early action mechanisms for these hormones in skeletal muscle cells.
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A Espinosa, M Estrada, and E Jaimovich
M Estrada, A Espinosa, C J Gibson, P Uhlen, and E Jaimovich
Ca2+ oscillations are one of the most important signals within the cell. The mechanism for generation of Ca2+ oscillations is still not yet fully elucidated. We studied the role of capacitative Ca2+ entry (CCE) on intracellular Ca2+ oscillations induced by testosterone at the single-cell level in primary myotubes. Testosterone (100 nM) rapidly induced an intracellular Ca2+ rise, accompanied by Ca2+ oscillations in a majority of myotubes. Spectral analysis of the Ca2+ oscillations revealed a periodicity of 20.3 ± 1.8 s (frequency of 49.3 ± 4.4 mHz). In Ca2+-free medium, an increase in intracellular Ca2+ was still observed, but no oscillations. Neither nifedipine nor ryanodine affected the testosterone-induced Ca2+ response. This intracellular Ca2+ release was previously shown in myotubes to be dependent on inositol-1,4,5-trisphosphate (IP3). Intracellular Ca2+ store depletion in Ca2+-free medium, using a sarcoplasmic/endoplasmic reticulum calcium ATPase-pump inhibitor, followed by re-addition of extracellular Ca2+, gave a fast rise in intracellular Ca2+, indicating that CCE was present in these myotubes. Application of either testosterone or albumin-bound testosterone induced Ca2+ release and led to CCE after re-addition of Ca2+ to Ca2+-free extracellular medium. The CCE blockers 2-aminoethyl diphenylborate and La3+, as well as perturbation of the cytoskeleton by cytochalasin D, inhibited testosterone-induced Ca2+ oscillations and CCE. The steady increase in Ca2+ induced by testosterone was not, however, affected by either La3+ or cytochalasin D. These results demonstrate testosterone-induced Ca2+ oscillations in myotubes, mediated by the interplay of IP3-sensitive Ca2+ stores and Ca2+ influx through CCE.
J A García-Sáinz, M Martínez-Alfaro, M T Romero-Avila, and C González-Espinosa
In guinea pig hepatocytes angiotensin II induced phosphorylase a activation. This effect was mimicked by other angiotensins with the potency order: angiotensin II (EC50 ≈1 nm)>angiotensin III (EC50 ≈30 nm)>angiotensin I (EC50 ≈300 nm). The effect of 10 nm angiotensin II was blocked by the angiotensin II receptor AT1-selective antagonists irbesartan and losartan (K i values of ≈1 nm and ≈10 nm for irbesartan and losartan respectively) but not by the AT2-selective antagonist PD123177.
Similar data were obtained when the production of [3H]IP3 from [3H]myo-inositol-labeled cells was studied. Angiotensin II induced a dose-dependent increase in [3H]IP3 production; the maximal effect (≈3-fold) was observed at a concentration of 10 μm. This effect of angiotensin II was completely blocked by the AT1-selective antagonists irbesartan and losartan, but only in a very limited fashion by PD123177. [125I][Sar1-Ile8]angiotensin II bound with high affinity (≈3·8 nm) to a moderately abundant number of sites (≈660 fmol/mg protein) in guinea pig liver membranes. Binding competition experiments indicate the following orders of potency for agonists: angiotensin II (≈1·5 nm)>angiotensin III (≈7 nm)>angiotensin I (≈176 nm), and for antagonists: irbesartan (≈0·5 nm)>losartan (≈36 nm)>> PD123177 (>> 10 000 nm).
The functional and binding data strongly indicate that the effects of angiotensin II were mediated through AT1 receptors. Expression of the mRNA for these receptors was confirmed by RT-PCR and hybridization of the reaction product with a radiolabeled rat AT1 receptor cDNA probe.
Journal of Endocrinology (1997) 154, 133–138