Sweet taste transduction in hamster: Role of protein kinases

Two different second-messenger pathways have been implicated in sweet taste transduction: sugars produce cyclic AMP (cAMP), whereas synthetic sweetene rs stimulate production of inositol 1,4,5-tris-phosphate (IP3) and diacylgl ycerol (DAG). Both sugars and sweeteners depolarize taste cells by blocking the same resting K+ conductance, but the intermediate steps in the transdu ction pathways have not been examined. In this study, the loose-patch recor ding technique was used to examine the role of protein kinases and other do wnstream regulatory proteins in the two sweet transduction pathways. Bursts of action currents were elicited from similar to 35% of fungiform taste bu d?, in response to sucrose (200 mM) or NC-00274-01 (NC-01, 200 mu M), a syn thetic sweetener. To determine whether protein kinase C (PKC) plays a role in sweet transduction, taste buds were stimulated with the PKC activator PD Bu (10 mu M). In all sweet-responsive taste buds tested (n = 11), PDBu elic ited burst of action currents. In contrast, PDBu elicited responses in only 4 of 19 sweet-unresponsive taste buds. Inhibition of PKC by bisindolylmale imide I (0.15 mu M) resulted in inhibition of the NC-01 response by similar to 75%, whereas the response to sucrose either increased or remained uncha nged. These data suggest that activation of PKC is required for the transdu ction of synthetic sweeteners. To determine whether protein kinase A (PKA) is required for the transduction of sugars, sweet responses were examined i n the presence of the membrane-permeant PKA inhibitor H-89 (10 and 19 mu M) . Surprisingly, H-89 did not decrease responses to either sucrose or NC-01. Instead, responses to both compounds were increased in the presence of the inhibitor. These data suggest that PKA is not required for the transductio n of sugars, but may play a modulatory role in both pathways, such as adapt ation of the response. We also examined whether Ca2+-calmodulin dependent c AMP phosphodiesterase (CaM-PDE) plays a role in sweet taste transduction, b y examining responses to sucrose and synthetic sweeteners in the presence o f the CaM-PDE inhibitor W-7 (100 mu M). Inhibition resulted in an increase in the response to sucrose, whereas the response to NC-01 remained unchange d. These data suggest that the pathways for sugars and sweeteners are negat ively coupled; the Ca2+ that is released from intracellular stores during s timulation with synthetic sweeteners may inhibit the response to sucrose by activation of CaM-PDE.