Expression of both alpha 1- and alpha 2-adrenoceptors in an insulin-secreting cell line. Parallel studies of cytosolic free Ca2+ and insulin release.

Changes in intracellular free Ca2+, [Ca2+]i, and immunoreactive insulin release in response to alpha-adrenergic agents were measured in RINm5F cell suspensions. Cells were loaded with the fluorescent indicator quin 2 for monitoring [Ca2+]i. Epinephrine (1 microM), which inhibited alanine-stimulated insulin release by 73%, evoked a transient rise in [Ca2+]i. This rise is in part due to Ca2+ mobilization, since it is still present in the absence of extracellular Ca2+. The alpha 2-adrenergic agonist clonidine (10 microM) mimicked the epinephrine effect on insulin release without any change in [Ca2+]i. In contrast, the alpha 1-adrenergic agonist phenylephrine (10 and 100 microM) raised [Ca2+]i, albeit to a lesser extent than epinephrine. Phenylephrine enhanced basal, but had no effect on alanine-stimulated insulin release. To examine further the nature of the effect of epinephrine, specific alpha-adrenergic blocking agents were employed. The epinephrine-induced increase in [Ca2+]i could be inhibited by the alpha 1-adrenergic antagonists BE2254 (0.1 microM) and prazosin (0.01 microM). In the presence of these blockers, epinephrine was still able to inhibit insulin release. When alpha 2-adrenergic receptors were blocked by the addition of idazoxan (0.1 and 1 microM), epinephrine still raised [Ca2+]i. At the higher concentration of idazoxan, the epinephrine inhibition of insulin release was completely overcome. The alpha-adrenergic agonists did not attenuate the alanine-induced rise in [Ca2+]i. This study shows that both subtypes of alpha-adrenergic receptors are present in the insulin-secreting cell line RINm5F. The activation of alpha 1-adrenergic receptors leads to an increase in [Ca2+]i. In contrast, the inhibition of insulin release due to epinephrine is mediated through alpha 2-adrenergic receptors. The alpha 2-adrenergic mechanism does not involve changes in [Ca2+]i, but is rather exerted at a later step in the secretory process.