Fura-2 measurement of cytosolic free Ca2+ concentration in corpus allatum cells of larval Manduca sexta

Abstract

Cytosolic free Ca2+ has been implicated in the regulation of the larval corpus allatum (CA) of the tobacco hornworm Manduca sexta since agents presumed to cause changes in intracellular Ca2+ concentrations affect both basal and neuropeptide-modulated synthesis/release of the juvenile hormones. To determine whether differences in cytosolic free [Ca2+] in CA cells were associated with different levels of gland activity, methods were developed for dissociating CA cells with the retention of biosynthetic activity and for uptake by the dissociated cells of the fluorescent Ca2+ indicator Fura-2. Digitized fluorescence microscopy of the Fura-2-loaded cells enabled measurement of intracellular Ca2+ levels in individual cells. Intracellular free Ca2+ levels were measured in CA cells from selected days during the fifth larval stadium and were found to be highest when the CA were biosynthetically active. Treatment of CA cells from day 6 with the Ca2+ ionophore ionomycin elevated the intracellular Ca2+ level, corroborating the involvement of elevated intracellular [Ca2+] in ionophore effects on juvenile hormone (JH) and JH acid synthesis. The results of the study are considered in relation to our knowledge of the role of Ca2+ in the function of endocrine gland cells.