Ward C.R., Dise D.G., Russell K.N., et al.
Conference Proceedings, (2005). American College of Veterinary Internal Medicine, Baltimore:
The pathogenesis of feline hyperthyroidism is not understood at the cellular or molecular level. Previous studies from our laboratory have identified a specific decrease in expression of the inhibitory G proteins (Gi) in hyperthyroid cells (Hammer et al, AJVR 61, 874-879) and have further determined that Gi2 is the specific subset of Gi proteins that is decreased (Ward et al, JVIM 15, 298). Moreover, stimulatory G proteins (Gs) were determined to be present in normal levels in hyperthyroid cells. We postulate that this decrease in expression of Gi2 is part of the pathogenesis of feline hyperthyroidism. Although the expression level of Gi2 is decreased, the activity of Gi and Gs proteins has not been investigated. The purpose of this study was to determine whether G protein activity is altered in feline hyperthyroid cells.
Thyroids were harvested from hyperthyroid and age-matched euthyroid cats. They were snap frozen in liquid nitrogen. Membranes were isolated from thawed, homogenized thyroid tissue. G protein activation in the membranes was investigated by measuring specific GTPγ35S binding to the membranes in response to thyroid stimulating hormone (TSH), a natural agonist of the thyroid cell. GTPγ35S binding measures the activation phase of the G protein activation/deactivation cycle. Pertussis toxin (PTX), a specific inhibitor of Gi proteins and cholera toxin (CTX), a specific activator of Gs proteins were used to isolate the activities of Gi and Gs, respectively.
Concentration-response curves were performed to determine the optimal TSH concentration for G protein activation in the thyroid membranes. Normal thyroid (NT) membranes and hyperthyroid (HYPERTHYROID) membranes were incubated with concentrations of TSH from 0.1-100 mU/ml. Optimal GTPγ35S binding was found at 1mU/ml TSH for both NT and HT membranes. Basal GTPγ35S binding was 48% lower in the HT membranes (1.69 +/- 0.25 fmoles GTPγ35S binding/mcg protein) than the NT membranes (3.55 +/- 0.86 fmoles GTPγ35S binding/mcg protein). Upon stimulation with TSH, both NT and HYPERTHYROID membranes showed a significant (p<0.05) increase in GTPγ35S binding over background of 57 +/- 10% and 49 +/- 12%, respectively. PTX treatment of membranes, to inhibit Gi activity and highlight Gs activity, resulted in a significant (p<0.05) decrease in TSH- stimulation of NT and HT membranes to 34 +/- 5% and 26 +/- 3%, respectively. This decrease was not significant between the NT and HT groups (p>0.05). In membranes treated with CTX, TSH stimulation of GTPγ35S binding was similar (p>0.05) between NT and HT cells at 45 +/- 5% and 51 +/-7%, respectively. This stimulation was not significantly different than stimulation seen in untreated membranes (p>0.05).
We conclude that G protein activity is not altered in HT cells and that a decreased expression of Gi protein and not an alteration in activity is part of the pathogenesis of feline hyperthyroidism.