This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via Google Scholar Google Scholar Articles by SEGAL, J. Articles by INGBAR, S. H. Search for Related Content PubMed PubMed Citation Articles by SEGAL, J. Articles by INGBAR, S. H.

The Effect of Trypsinization on the Plasma Membrane Binding and Action of 3,5,3'-Triiodothyronine in Rat Thymocytes^*

The Charles A. Dana Research Institute and the Harvard-Thorndike Laboratories of Beth Israel Hospital, Department of Medicine, Beth Israel Hospital and Harvard Medical School Boston, Massachusetts 02215

Address requests for reprints to: Joseph Segal, Ph.D., Department of Medicine, Beth Israel Hospital, 330 Brookline Avenue, Boston, Massachusetts 02215.

Abstract

We have previously demonstrated that in the rat thymocyte T₃ produces a prompt, extranuclear effect to increase cellular cAMP concentration and 2-deoxyglucose (2-DG) uptake. We have also demonstrated the presence of specific receptors for T₃ in the rat thymocyte plasma membrane. From these and other observations we have suggested that T₃ initiates these actions by binding to its receptors on the surface of this cell. To test this hypothesis further we have now examined whether a decrease in the binding of T₃ to the thymocyte plasma membrane, induced by mild trypsinization, attenuates the effect of T₃ on thymocyte cAMP concentration and 2-DG uptake.

Mild trypsin treatment of rat thymocytes in cell suspension did not cause appreciable nonspecific damage to the cells, since it did not change the ability of the cells to exclude either trypan blue or mannitol, a marker of extracellular water. Further, treatment with trypsin did not alter the ability of thymocytes to bind concanavalin A. It did produce, however, a dose-related decrease in the binding of T₃ to its plasma membrane binding sites owing to a decrease in their number, but not their affinity. This was associated with a progressive decrease in the T₃-induced increase in both 2-DG uptake and cAMP concentration. Thus, low concentrations of trypsin had no significant effect on 2-DG uptake in the absence of T₃, but decreased significantly the increase in sugar uptake induced by T₃, whereas a higher concentration of trypsin (5 mg/ml) decreased basal 2-DG uptake by only 23%, but abolished the response to T₃ altogether. T₃, epinephrine, and trypsin alone induced dose-related increases in cellular cAMP concentration, the response to trypsin exceeding that to T₃ or epinephrine. Trypsinization also resulted in progressive decrease in the response of cellular cAMP to both hormones, so that at a trypsin concentration of 5 mg/ml no further increase was produced by either hormone. The possibility that a maximum activation of adenylate cyclase by trypsin could explain these results was rendered unlikely by experiments with prostaglandin E₁ (PGE₁). The increase in cellular cAMP concentration induced by this agent was vastly greater than that produced by trypsin alone. Although the response to PGEi was greatly inhibited by trypsin, cellular cAMP concentration in thymocytes incubated with trypsin (5 mg/ml) plus PGE₁ was much higher than that in cells treated with trypsin alone. This suggests that adenylate cyclase was not maximally activated by trypsin, and that the inhibition of the response to T₃ and epinephrine that trypsin induced cannot be ascribed to this mechanism.

These findings indicate that trypsin-sensitive proteins are functionally important components of the T₃ receptor in the thymocyte plasma membrane, and they support the suggestion that binding of T₃ to such receptors is a prerequisite of its effect to increase cAMP concentration and 2-DG uptake in these cells. (Endocrinology 118; 1863–1868, 1986)

Footnotes

^* Supported in part by Grant No. AM-18416 from the NIADDK, National Institutes of Health, Bethesda, Maryland.

Received November 6, 1985.