Diabetes Autoantibodies Mediate Neural- and Endothelial Cell- Inhibitory Effects Via 5-Hydroxytryptamine- 2 Receptor Coupled to Phospholipase C/Inositol Triphosphate/Ca2+ Pathway

Aims: To identify the G-protein coupled receptor(s) on neuroblastoma and endothelial cells which mediate neural- and endothelial cell-inhibitory effects in plasma autoantibodies from a subset of older type 2 diabetes with neurologic and vascular co-morbidity. To determine the mechanism(s) of neurite retraction induced by diabetic pathologies' auto antibodies.

Methods: Protein-A eluates from plasma of 11 diabetic patients having nephropathy, moderate-severe obesity and/or complications in which increased inflammation plays a role (depression, Parkinson's disease, atrial fibrillation, obstructive sleep apnea) were tested for neurite retraction and decreased survival in N2A neuroblastoma cells, and decreased survival in pulmonary artery endothelial cells. Specific antagonists of G protein coupled receptors belonging to the G alpha q subfamily of hetero trimetric G proteins or the phospholipase C/inositol triphosphate/Ca2+ pathway were tested for modulatory effects on diabetic pathologies' autoantibody-induced N2A neurite retraction, or cell survival.

Results: Co-incubation with specific antagonists of the 5-hydroxytryptamine- 2A receptor significantly prevented acute N2A neurite retraction induced by 50-100 nM concentrations of diabetic pathologies' autoantibodies. Protection against neurite retraction (M100907> spiperone> ketanserin) closely paralleled the antagonists' potency order at the 5-HT2-AR. Neuroblastoma or endothelial cell death (after 24 hours incubation) with 50-100 nM autoantibodies was completely or nearly completely (91%) prevented by co-incubation with 200 nM M100907, a highly selective 5-HT2-AR antagonist. Alpha-1 adrenergic, angiotensin II, metabotropic glutamate 5, or endothelin A (100 nM-10µM) receptor antagonists did not substantially inhibit autoantibody-induced cell death. The intracellular calcium chelator (BAPTA-AM, 50 µM) and inhibitors of the inositol triphosphate (IP3) receptor (2-APB, 50µM), and phospholipase C-gamma (U73144, 1µM) each significantly protected against autoantibody-induced acute N2A neurite retraction.

Conclusion: These data suggest that neural- and endothelial- inhibitory effects in autoantibodies from older adult diabetes with nephropathy and obesity/inflammation-associated complications are mediated by agonist autoantibodies directed against the 5-hydroxytryptamine 2 receptor positively coupled to the phospholipase C/inositol triphosphate/ cytosolic Ca2+ release pathway.