Receptor-mediated phosphoinositide metabolism in the rat cerebellar granule cell.

Cerebellar granule cells in culture have recently become a widely used neuronal preparation, because of their receptor integrity and ability to release the neurotransmitter, glutamate. The basis of this thesis was the detailed investigation of receptor-mediated phosphoinositide signalling and its role in intracellular Ca2+ in this cell type. The muscarinic activated accumulation of 3H-InsPx in this cell is the most potent reported, stimulating some 20 fold over basal and was selected as the agonist of choice for further study. Pharmacological determination of antagonist inhibition of 3H-NMS binding and 3H-InsPx production, identified the muscarinic receptor coupled to PI hydrolysis as of the M3 receptor subtype. Carbachol induced 3H-InsPx was demonstrated, at least in part, to originate from the hydrolysis of PtdInsP2, due to the capacity of the cell to produce Ins(1,4,5)P3, the intracellular Ca2+-mobilising second-messenger. The calcium mobilising properties now well established for Ins(1,4,5)P3, were confirmed in the cerebellar granule cell. 45Ca2+ release by Ins(1,4,5)P3 was stereospecific, heparin sensitive. Displacement of 32P-Ins(1,4,5)P3 confirmed the presence of Ins(1,4,5)P3 sites, that had previously gone undetected in this cell type. Fluorescent Ca2+ indicators, like fura-2, have recently been developed and enable the detailed analysis of intracellular [Ca2+] changes to be performed in intact cells, both in population and at the single cell level. Single cell studies in fura-2 loaded cerebellar granule cells, showed considerable heterogeneity in their responsiveness to carbachol. Very few cells demonstrated a classical receptor mediated Ins(1,4,5)P3-type increase in [Ca2+]i, the majority being dependent on extracellular Ca2+. Most cells exhibited a muscarinic-mediated Ca2+ entry phenomenon, that was only visible in the absence of extracellular Mg2+, suggesting some added complexity that needs further investigation. The possibility of a role of Ins(1,4,5)P3 in neurotransmitter release in this cell type is considered.