Release of dopamine, noradrenaline and dopamine B-hydroxylase from mouse neuroblastoma

The murine C1300 neuroblastoma tumor was found to secrete dopamine, noradrenaline and dopamine B-hydroxylase into the circulation of tumor-bearing A/J mice. The plasma levels of dopamine, noradrenaline and dopamine B-hydroxylase increased with the size of the tumor, and the increase in noradrenaline paralleled the increase in dopamine B-hydroxylase (r = 0.86). The vesicular storage of dopamine and noradrenaline in the tumor was evidenced by a decrease of the tissue content of dopamine and noradrenaline 24 hours after the administration of reserpine (5 micrograms/g) respectively to 17.6% and 7.8% of control values. A similar observation could be made for the levels of dopamine and noradrenaline in the plasma of reserpinized C1300 mice. The total activity of dopamine B-hydroxylase in the tumor and in plasma was unaffected by the reserpine treatment. Chronic administration of 6-hydroxydopamine (100 micrograms/g for 8 days) had no effect on the tissue contents of dopamine, noradrenaline or dopamine B-hydroxylase. The release of catecholamines and dopamine B-hydroxylase from the C1300 neuroblastoma was studied in vitro on superfused tumor slices. Stimulation of these slices with 56 mM KC1 or with 5.10(-5) M tyramine failed to induce the release of endogenous dopamine, noradrenaline or dopamine B-hydroxylase above the basal outflow levels. These results are suggestive for a non-exocytotic release of catecholamines and dopamine B-hydroxylase from the neuroblastoma tumor.     

Immunohistochemical evidence for a plasma membrane localization of dopamine-beta-hydroxylase in the mouse neuroblastoma

Dopamine-beta-hydroxylase (D beta H), a glycoprotein enzyme which converts dopamine into noradrenaline, was purified from C1300 mouse neuroblastoma and used to raise antibodies in rabbits. Using an indirect immunofluorescence technique the cellular localization of D beta H in C1300 mouse neuroblastoma was compared with that of the superior cervical ganglion. C1300 neuroblastoma D beta H was found to be predominantly localized in the plasma membrane, in contrast to its intracellular localization in the superior cervical ganglion of A/J mice. At least part of the enzyme was found to be associated with the external side of the plasma membrane.     

Subcellular fractionation of splenic nerve: ATP, chromogranin A and dopamine beta-hydroxylase in noradrenergic vesicles

The subcellular particles in axons of the splenic nerve have been studied by centifrugation techniques. By differential centifrugation, five different types of particle could be distinguished and partly separated: noradrenaline-containing particles (noradrenergic vesicles), large and small lysosomes, mitochondria, and microsomal particles. In density gradient centrifugation, only one type of noradrenergic vesicle could he demonstrated. The noradrenergic vesicles and the mitochondria contain ATP. Two proteins (chromogranin A and dopamine beta-hydroxylase) are present in the noradrenergic vesicles.     

Release of dopamine beta-hydroxylase and chromogranin A upon stimulation of the splenic nerve

Two proteins present in noradrenergic vesicles of the splenic nerve (dopamine beta-hydroxylase and chromogranin A) are released into the perfusate from the spleen when the splenic nerve is stimulated. Experiments in which drugs were added to the perfusion fluid showed that the proteins were released from terminals of the splenic nerve. There was a correlation between the amounts of the proteins released and the quantity of noradrenaline released; and the release process was dependent upon calcium. It is suggested that the proteins are released from the large dense-cored vesicles present in the terminals of the splenic nerve, and that secretion from these vesicles occurs by exocytosis.