Desensitization of glucagon-like peptide 1 receptors in insulin-secreting βTC3 cells: Role of PKA-independent mechanisms

Abstract

1. The cellular processes involved in the desensitization of the glucagon-like peptide 1 receptors were investigated by measurements of the glucagon-like peptide 1(7-36)amide (GLP-1(7-36)amide)-induced increases in intracellular free Ca2+ concentration ([Ca2+](i)) in insulin-secreting βTC3 cells. 2. In the presence of 11.2 mM glucose, stimulation with GLP-1(7-36)amide led to a small membrane depolarization (< 10 mV), induction of electrical activity and a rapid increase in [Ca2+](i). The increase in [Ca2+](i) was not observed in the presence of the L-type Ca2+-channel antagonist nifedipine. However, nifedipine was ineffective when applied after addition of GLP-1(7-36)amide. 3. The increase in [Ca2+](i) evoked by GLP-1-(7-36)amide was transient and even in the continued presence of the agonist, [Ca2+](i) returned to the basal value within 4-5 min. The latter process was slowed, but not prevented, by inhibition of protein kinase C (PKC) by staurosporine and Ro31-8220. 4. Short pretreatment of the cells with the phorbol ester, 4-β-phorbol-12-β-myristate-13-α-acetate (PMA), an activator of PKC, reduced the GLP-1(7-36)amide-evoked increase in [Ca2+](i) by 75%. This effect of PMA was fully reversed by staurosporine and Ro31-8220. 5. The ability of GLP-1(7-36)amide to increase [Ca2+](i) disappeared upon pre-exposure of the cells to the hormone (desensitization). This process was maximal within 5 min of exposure to the agonist. Following removal of the agonist from the medium, the ability to respond to subsequent stimulation by GLP-1(7-36)amide recovered gradually with time; half and complete recovery requiring > 20 min and 60 min, respectively. The desensitizing action of GLP-1(7-36)amide persisted in the presence of either staurosporine or forskolin and did not require an elevation of [Ca2+](i). 6. Our data suggest that the GLP-1(7-36)amide-evoked increase in [Ca2+](i) is initiated by Ca2+-influx though voltage-dependent and nifedipine-sensitive L-type Ca2+ channels but depends principally on Ca2+ mobilization from internal stores for its maintenance. The desensitization of the GLP-1 receptors that occurs in the continued presence of the agonist does not result from the activation of protein kinase A or Ca2+-dependent kinases/phosphatases. Our data indicate that activation of PKC may contribute to the desensitization of the GLP-1 receptors but that other (PKC-independent) mechanisms also participate in this process.