Function of cannabinoid CB1 receptors in a cell culture model of diabetic neuropathy

Fan Zhang, Vicki Stone, Paula JW Smith. Napier University, Edinburgh, United Kingdom.

Two recent studies demonstrated the anti-nociceptive action of a mixed cannabinoid CB1/CB2 receptor agonist is preserved in diabetic mice (Doğrul et al., 2004) and rats (Ulugol et al., 2004). In the present study, we examined the function of CB1 receptors in an in vitro model of diabetic neuropathy, whereby neurite outgrowth of rat pheochromocytoma (PC12) cells was induced by nerve growth factor (50 ng/ml), in the presence of normal and high concentrations of glucose (5.5 and 50mM) to simulate normal and hyperglycaemic conditions in vivo. CB1 receptor stimulation suppresses vanilloid TRPV1 receptor-mediated calcium influx and neurotransmitter release (Millns et al., 2001). We therefore assessed CB1 receptor function by measuring the inhibitory effect of the CB1 receptor agonist HU210 on capsaicin-induced calcium influx in PC12 cells cultured in physiological and high glucose conditions.

We found a reduction in total neurite length induced by nerve growth factor in PC12 cells cultured in 50 mM glucose on day 6 (P < 0.01 versus 5.5 mM; n=70-79 from 6 independent cultures). This effect was due to raised glucose levels, used to mimic diabetic conditions, rather than any hyperosmolality effects since mannitol (50 mM) gave similar results to that of the physiological 5.5mM glucose control (P = 0.79). In fluo-4-loaded PC12 cells, capsaicin evoked an increase in fluo-4 fluorescence (≈[Ca2+]i) in a concentration-dependent manner. The proportion of cells that responded to capsaicin out of the total potassium chloride-responsive cells did not differ between the different glucose treatments (5.5 mM: 86%, n = 32/37; 50 mM: 85%, n = 29/34), but the magnitude of the response tended to be higher in cells cultured in 50 mM glucose compared to control 5.5 mM (190.8 ± 36.4 versus 121.0 ± 24.3 fluorescence intensity as % KCl response; P = 0.16). Application of the CB1 agonist HU210 (30 μM) significantly inhibited capsaicin-induced calcium influx (P < 0.01), and the inhibitory effect of HU210 was reversed by co-application of the CB1 antagonist AM251 (30 μM). This CB1-mediated inhibition was of a similar magnitude in cells cultured in high glucose (77%) versus control (80%) (n=29-42, from four independent cultures).

The results of our studies show that the function of the CB1 receptor is preserved when stimulated by an exogenous agonist under conditions mimicking hyperglycaemia. Given the neuroprotective effect of cannabinoids, CB1 receptors may be an appropriate therapeutic target in preventing the neurodegenerative process in diabetes.

Doğrul A, et al. (2004) Neurosci Lett 368:82-86

Millns PJ, et al. (2001) Br J Pharmacol 132:969-971

Ulugol A, et al. (2004) Neurosci Lett 371:167-170