DIABETIC NEUROPATHY: RECEPTORS AND SIGNALING MECHANISM

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Diabetic neuropathy, one of the major complications of chronic diabetes is characterized by several biochemical alterations including a reduction in myo-inositol levels, decrease in Na+K+-ATPase activity and abnormalities in phosphoinositide (PPI) metabolism and adenylyl cyclase activity. The current hypothesis regarding the pathophysiology of diabetic neuropathy assign a central role to the decrease in myo-inositol uptake-dependent phosphoinositide turnover and hence availability of second messengers, like diacylglycerol which modulate Na+K+-ATPase activity through activation of protein kinase C. While it is generally accepted that the reduction in Na+K+-ATPase plays a key role in the development of neuropathy, its precise regulation is far from clear. Recent evidence from our and other labs suggests that peripheral nerve possesses muscarinic, bradikynin, purinergic and beta-adrenergic receptors which seem to be linked to stimulation of the breakdown of PPI or adenylyl cyclase. We have recently identified several types of guanine-nucleotide binding (G) proteins, that are known to be involved in other tissues in coupling between the agonist-receptor complexes and the adenylyl cyclase or phospholipase C systems. In nerve and myelin membranes from experimental diabetic animals we demonstrated that some of these signalling pathways are altered. With this evidence, we propose to: 1) characterize the relative levels of G-proteins in total and subcellular fractions of sciatic nerve from diabetic rats by ADP- ribosylation with bacterial toxins and immunoblotting; 2) investigate the receptor-linked signalling pathways that modulate the activity of phospholipase C and adenylyl cyclase and their influence on Na+K+-ATPase activity in sciatic nerve slices or membranes by: a)activation of phospholipase C through muscarinic, purinergic and bradikynin receptors b) modulation of adenylyl cyclase activity through activation of beta- adrenergic and muscarinic receptors: 3) evaluate how the alterations in Na+K+-ATPase in nerves from diabetic animals correlate with changes in the different components of the above mentioned signalling pathway and 4) determine if in cultured Schwann cells the above mentioned agonists are linked to cAMP and phospholipase C and if these pathways are altered under conditions that mimic diabetes like high glucose and myo-inositol free culture conditions.