L163,255, a Synthetic Growth Hormone Secretagogue, Raises [Ca2+]i by Promoting Intracellular Calcium Stores Depletion in Intact Fast-Twitch Fibers of Rat Skeletal Muscle

Abstract

The synthetic growth hormone secretagogues, GHS, and the endogenous ghrelin are small molecules proposed as pharmacological tools for the treatment of GH deficiency conditions in view of their ability to stimulate the GH release. Other than in pituitary gland, GHS receptor binding sites are documented in peripheral tissues accounting for a series of GHS pleiotropic activities. Accordingly, a direct action of GHS on skeletal muscle has been proposed, as they reduced resting chloride and potassium conductances in muscle fibres, probably through the activation of a GHS-receptor linked to PLC/PKC/Ca2+ signalling (Pierno et al. 2003. Br. J. Pharmacol. 139:575–584). By using fura-2 fluorescent measurements, we evaluated the effect of L163,255, a non peptidic GHS, on calcium homeostasis of rat Extensor Digitorum Longus (EDL) fibers mechanically isolated. In vitro application of L163,255 increased cytosolic calcium concentration, [Ca2+]i, in a dose-dependent manner with an IC50 of ∼ 300 μM. Particularly, application of 200μM L163,255 led to [Ca2+]i increase from 26±2 nM to 164±34 nM after 10 min of incubation.Removal of external Ca2+ in the bath solution did not abolish L163,255 effects. On the contrary, pre-incubation with the Ca2+-ATPase inhibitor thapsigargin or the mitochondrial permability transition pore inhibitor Cyclosporin A partially and strongly reduced L163,255-induced Ca2+ transient respectively, suggesting the involvement of thapsigargin-sensitive calcium stores and mitochondria in the drug action. These data support the presence of GHS receptors on skeletal muscle and highlight that GHS, proposed as therapeutic drugs, by altering calcium homeostasis could interfere with skeletal muscle functionality.