Application of the tyrosinase assay to normal melanocytes in culture

Using a special selection technique, normal guinea-pig melanocytes were maintained in highly purified but sparse cultures (approximately 10(4) cells/25 cm2 culture vessel) which showed little proliferative activity. The applicability of the Pomerantz tyrosinase assay was tested in this in vitro model system using three different approaches, namely crude cell extracts and viable cell cultures either in situ or in suspension. The latter modifications both proved too insensitive, whereas crude cell extracts allowed accurate measurements of the basal tyrosinase activity and its stimulation by various agents. In unstimulated cultures basal tyrosinase activities ranged from 30% to 700% (mean 260%) above the blank values; intra-assay and inter-assay variability were 4.2% and 77.5%, respectively. Stimulation with alpha-MSH (10(-5) M, 10(-6) M), beta-MSH (10(-5) M), choleratoxin (10(-11) M) and cAMP (10(-4) M) plus theophylline (10(-4) M) resulted in an increase of tyrosinase activity 30-65% above basal values. Melanotropin potentiating factor (10(-8) M) enhanced the effects of alpha-MSH (10(-6) M) by 20%. This assay modification provides a sensitive tool for comparative studies of melanogenesis in normal melanocytes, malignant melanocytes and otherwise altered melanocytes.