Specific Binding Sites for ¹²⁵I-Iodo Insulin in the Rat Submandibular Gland: In Vivo and In Vitro Studies

Summary

The presence of specific binding sites for ¹²⁵I-iodo insulin in microsomes from rat submandibular gland was demonstrated. Binding of ¹²⁵I-iodo insulin was a time and temperature-dependent process that reached a plateau after 4 h at 4°C. At 25°C, specific binding reached a maximum at 20 min and then showed a decline within the first hour. At this temperature the specific binding of ¹²⁵I-iodo insulin was 2.9 times enhanced by increasing the ionic strength of the incubation medium to 2M NaCl.

Equilibrium binding data analysis gave rise to a non-linear Scatchard plot, whose high affinity component presented a K_D of 2.5 ± 0.1 nM with a binding capacity of 254 ± 9 fmol/mg microsomal protein. A K_D value of 55±1 nM and a binding capacity of 658 ± 34 fmol/mg microsomal protein were obtained for the low affinity component.

Kinetics studies showed enhancement dissociation of the ¹²⁵I-iodo insulin bound to the microsomes, in the presence of an excess of unlabelled insulin.

In vivo studies proved that the specific uptake of radioactivity reached a maximum at 2-5 min after the administration of ¹²⁵I-iodo insulin. A significant reduction of the uptake was observed when the labelled insulin was injected together with increasing amounts of unlabelled hormone. This reduction was dose dependent and the concentration of unlabelled insulin that prevented 50% of the glandular uptake of ¹²⁵I-iodo insulin was close to 1.5 μg/100g b.w.

These observations are consistent with the existence of insulin receptors in the submandibular gland of the rat which are similar in their characteristics to those present in other tissues.

Key-Words:

Insulin Receptors - Submandibular Gland - Insulin Binding - Submaxillary Gland