Adenosine Receptors Machinery and Purinergic Receptors in Rat Primary Skeletal Muscle Cells
Mansour HaddadDepartment of Clinical Sciences, Faculty of Pharmacy, Philadelphia University, P.O Box 3341, Irbid, P.C 211-10.Jordan.
Abstract: GPCRs are the largest family of proteins in the human genome. One family of GPCRs is adenosine receptors, which are divided into A1, A2A, A2B and A3 adenosine receptors based on pharmacology and coupling to cAMP production. Another family of receptors is P2Y receptors, which are divided into P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14. Indeed, GPCRs are targets for huge numbers of therapeutic drugs, although the role of skeletal muscle in the action of these drugs is unclear. The purpose of this research was to identify genes encoding adenosine and purinergic receptors highly expressed in skeletal muscle and in cultured preparations thereof. Skeletal muscle cells were cultured from vastus lateralis obtained from male Wistar (180-200 g) rats as Blau and Webster method with slight modification (Blau et al., 1981). mRNA expression, cAMP and calcium measurement were examined by conventional methods (Akaneya et al., 2006; Hudson et al.; Millns et al., 2001). Data were reported as means of triplicate or quadruplicate wells generated from two animals. Statistical analysis was conducted using one or two-way ANOVA with Bonferroni’s multiple comparisons test. mRNA encoding Gs- (adora2a, adora2b), Gi- (adora1), and Gq-coupled (p2ry1, p2ry2 and p2ry6) receptors were detected using gene microarray (Agilent, all ranked <24000 out of 41090). Treatment of myotubes with NECA (5'-N-ethylcarboxamidoadenosine) (10-7 and 10-5 M) for 10 minutes elicited a significant increase in cAMP (18 ± 0.33 compared to basal levels of 4.3 ± 0.15 pmol/well), which were significantly inhibited in the presence of the A2B antagonist PSB603 (Fredholm et al.) (10-5 M, 5.3 ± 0.33 pmol/well). Treatment of myotubes with the A2A-selective agonist CGS21680 (10-7 M) failed to evoke a significant cAMP elevation. Neither basal nor forskolin (1 µM)–evoked elevation of cAMP was altered in the presence of the A1-selective agonist S-ENBA (10-7 M). All 748 myoblast cells (imaged from four animals; 150-250 cells/animal) showed an elevation in intracellular calcium levels in response to 1 mM ATP, with an increase in fluorescence ratio (the max of response (peak) obtained from drug minus the baseline (DFR); Fura-2 340/380 nm excitation) of 0.33 ± 0.06. 575 cells responded to 10-5 M UTP (0.18 ± 0.08 DFR). Responses to ATP were insensitive to the P2Y1 antagonist MRS2179 (10-7 M). In summary, we observe expression and functional responses to example members of the adenosine and purinergic GPCR families in rat skeletal muscle preparations.
Keywords: GPCRs; cAMP; calcium and myotubes; adenosine receptors; purinergic receptors Back to TOC