Untersuchung zur Beteiligung von Proteinkinase A und Tyrosinkinasen an der Calcium-abhängigen Signaltransduktion von Glucagon-like Peptide 1(7-36)amid in Insulin-sezernierenden Zellen des endokrinen Pankreas

Glucagon-like peptide 1 (7?36)amide (GLP-1) is an insulinotropic intestinal peptide hormone with a potential role as antidiabetogenic therapeutic agent. It mediates a potentiation of glucose-induced insulin secretion. The hormone acts through a specific receptor coupled to a stimulatory G protein, which activates the adenylate cyclase followed by an elevation of intracellulare cAMP levels. Subsequently, the cytosolic free calcium concentration increases resulting in an elevation of the glucose-induced insulin secretion. In contrast to the cAMP elevation, the ensuing steps mostly are not known or are subject to controversial discussion. Effects of elevated cAMP are often mediated by protein kinase A. Also protein tyrosine kinases are frequently involved in signal transduction. Previous studies often investigated glucose-insensitive ß-cells or only one protein kinase inhibitor resulting in conflicting observations. For this purpose, this issue was readdress, employing both a suitable ß-cell model and adequate tools for protein kinase A and protein tyrosine kinase inhibition. As a a suitable ß-cell model, isolated mouse islets were used as well as the differentiated ß-cell line INS-1. For measurements of the intracellulare calcium concentration, both typs of ß-cells were loaded with Fura-2 and changes in the membrane potential performed using Bisoxonol. Two specific inhibitors of protein kinase A, Rp-cAMPS (up to 3 mM) and KT5720 (up to 10 mM), did not inhibit the GLP-1- and forskolin-induced calcium elevation or membrane depolarisation. Another protein kinase A inhibitor, H-89, reduced the calcium elevation and membrane depolarisation only when applied at high concentrations (10?40 mM), higher than sufficient for protein kinase A inhibition in many cell types. Furthermore, at these concentrations, H-89 also inhibited presumably protein kinase A-independent processes such as glucose-induced calcium elevation suggesting a protein kinase A-independent action of H-89. None of the three inhibitors of protein kinase A leads to an inhibition, suggesting a protein kinase A-independent signal transduction of GLP-1. The tyrosine kinase inhibitors (up to 100µM) and Herbimycin A (up to 20µM) both, Genistein more then Herbimycin A, depending on the concentration lead to a pronouced inhibition of the glucose-induced calcium elevation and calcium oscillations. The origin, wether it is a toxic reaction or a specific inhibition of tyrosine kinase is not yet known. In relation to these massive changes within the glucose reaction no further inhibition of the GLP-1- and forskolin-effects were detected. The potent but unselective protein kinase inhibitor staurosporine at proper concentration caused no inhibition of the GLP- 1- and forskolin-induced calcium elevation and membrane depolarisation. These results suggest that the GLP-1-induced elevation of intracellulare calcium concentration and membrane depolarisation is mediated independently of protein kinase A or tyrosine kinase. Probably, no protein kinase is participated. Hhe calcium dependent signal transduction of GLP-1 is possibly mediated through a nonselective cation channel activated either through cAMP directly, in combination with a hyperpolarisation or through EPAC, a cAMP regulated guanine nucleotide exchange factor. In addition, it is established that GLP-1 mediates a calcium-independent stimulation of the secretion machinery, resulting in enhanced recruitment of secretory granules to the plasma membrane.