Abstract
The objective of this study was to determine if zinc, when added in combination with ractopamine hydrochloride (RH), would stabilize the interaction of RH with the β-adrenergic receptor, as indicated by altered cAMP concentrations, mRNA quantity, or protein abundance. Cultured bovine skeletal muscle cells were established and treated after 120 h for 6, 24, and 96 h with differentiation media of specific treatments. Treatments were applied in a factorial arrangement with two levels of zinc (0 μM or 1 μM) and two levels of RH (0 μM or 10 μM) in differentiation media. cAMP levels were measured at 6, 24, and 96 h, while mRNA and protein were measured at 24 and 96 h. At 6 h, no differences (P > 0.05) were detected in cAMP levels between the treatments. However, at 24 h the 10 μM RH, 1 μM zinc treatment had the greatest concentrations of cAMP (P P = 0.05) compared to the control. No differences were detected in mRNA (β1-adrenergic receptor, β2-adenergic receptor, AMPKα, myosin heavy chain I, myosin heavy chain IIA, and myosin heavy chain IIX) concentrations between treatments. Protein quantity of the β1-adrenergic receptor and β2-adrenergic receptor did not differ between treatments. These results indicate that zinc, in combination with RH, may help sustain the RH response during prolonged exposure as indicated by increased cAMP concentrations.
Highlights
At 6 h of treatment cAMP production was nearly identical for the two treatments, at 24 h cells that were treated with Zn in combination produced more (P < 0.05) cAMP than those treated with only ractopamine hydrochloride (RH)
Reports by Swaminath et al [11] showed that Zn binds to the β2-adrenergic receptor (β2AR) and provided evidence that Zn binds to the receptor, but that it causes an increase in agonist affinity, resulting in greater production of the secondary messenger cAMP
At 6 h of treatment, results were inconclusive on whether cAMP production was affected by Zn administration with RH; at 24 h of treatment, an increase (P < 0.05) in receptor activity was shown in those cells provided a combination of both RH and Zn
Summary
Pippig et al [8] showed that in cell culture studies, desensitization leads to a decrease in cAMP production as a result of down-regulation of adenylyl cyclase activity Another form of desensitization has been reported that causes the receptor to sequester within an intracellular vesicle, losing its functionality to bind extracellular agents and couple with intracellular mechanisms necessary for normal activity [9] [10]. It has been postulated that zinc binding to the receptor causes a conformational change that affects receptor functionality It is not known how the combination of RH and Zn would influence the β-adrenergic receptors ability to produce cAMP, and its regulation of downstream mRNA and protein synthesis. Our objective was to determine if Zn, when administered in combination with RH, resulted in a prolonged, extended production of cAMP that could be verified by downstream altered mRNA and protein expression
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