Biological activity, binding, and metabolic fate of Ac‐[Nle4, D‐Phe7]α‐MSH4–11NH2 with the F1 variant of B16 melanoma cells

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Abstract
The α-MSH (α-melanocyte-stimulating hormone) agonist, Ac-[Nle4, D-Phe7]α-MSH4–11NH2 (hereafter called ND4–11α-MSH), is at least 10-fold more potent than α-MSH as a stimulus of tyrosinase activity in F1 variant cells of B16 melanoma. The binding to these cells during an incubation with 5 nM (3H)ND4–11α-MSH at 37°C is maximal at 0–30 min, 22 fmol/106 cells, but declines to 40% of this value at 4 hr. In the presence of 5 nM (3H)ND4–11α-MSH at 37°C, the acid soluble (cell surface) radioactivity decreased rapidly from 11.4 fmol/106 cells at 5 min to 4.6 fmol/106 cells at 4 hr. Chromatographic analysis of media and cellular samples revealed that there was no evidence of degradation of (3H)ND4–11α-MSH in the medium but there was evidence of intracellular degradation of (3H)ND4–11α-MSH. Ammonium chloride (10mM) resulted in an increase in acid resistant radioactivity (internalized hormone) at 4 hr. The binding to F1 variant cells during an incubation with 0.155 nM or 5 nM (3H)ND4–11α-MSH at 4°C was constant from 4 hr to 24 hr. Under these conditions, there was no time-dependent change in the acid soluble radioactivity from 4 to 24 hr. Scatchard analysis of (3H)ND4–11α-MSH binding to F1 variant cells at 4°C demonstrated that there were approximately 4500 receptors per cetl and an association constant of 17.1 nM−l. These results are consistent with a process of (3H)ND4–1α-MSH binding to its receptor followed by internalization of the receptor-hormone complex and then intracellular degradation of the hormone.

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