T3-Agarose: A reliable tool to study nongenomic effects of thyroid hormones

Nongenomic effects are nowadays described for most hormones of the nuclear receptor superfamily, such as thyroid hormones (T3, T4). These effects are mainly membrane –located, such as calcium mobilization, glucose uptake, intracellular pH, activation of PKC, and the MAPK pathway; they show a time-course of seconds to minutes. They may be mediated by specific receptors on the plasma membrane of different cells. In these experiments it is important to use agonist hormones conjugated to non-diffusible anchor molecules, such as Agarose. The hormone binds to the membrane and cannot enter the cell: this is an easy way to study membrane effects. T3-Agarose was prepared as reported at the website: www.amershambiosciences.com. T3-Agarose is a membrane-impermeant derivative based on the use of activated CH Sepharose 4BTM, a pre-activated medium for covalent immobilization of either proteins or other ligands containing primary amino groups. The group to be coupled is the amino group (-NH2) and the matrix Agarose (4%), the coupling conditions are pH 5-10, 4-25 °C, 1-4 h. The contamination by free T3 is very low, about 1 mol free T3/10E5 mol T3-Agarose as assessed by RIA. T3-Agarose (1 nM) was able to elicit nongenomic effects of thyroid hormone, in fact it increased intracellular pH and gave rise to an increase in intracellular calcium concentration similar to that obtained with the same concentrations of T3 (1 nM) in L-6 myoblasts. Nuclear responses were not affected by T3-Agarose. The results appear promising and the same technique will be used in the near future also for T4 and analogs that mimic thyroid hormone effects.