Biochemical evidence for the presence of small and large noradrenergic storage vesicles isolated from cat ovary in isoosmotic conditions. Distribution of dopamine-β-hydroxylase activity

The cat ovary presents unusually high levels of noradrenaline that change according to the endocrine status of the animal. Their functional meaning remains unknown. The cat ovary innervation, unlike that of other organs receiving noradrenergic innervation, has been poorly characterized on biochemical grounds. We present here a biochemical characterization of the neurotransmitter storage. By using hyperosmotic and isoosmotic gradients evidence is presented that noradrenaline is associated to two different populations of vesicles. In hyperosmomolarity conditions (sucrose gradients) “light” vesicles (density 1.12 g/ml) and “heavy” vesicles (density 1.17 g/ml) appeared. In both vesicles, noradrenaline and dopamine-β-hydroxylase were found. In isoosmotic Percoll gradients distribution of the markers also suggested the presence of two vesicle populations. Light vesicles (density 1.033 g/ml) with high dopamine-β-hydroxylase activity but very low levels of noradrenaline and adenosine triphosphate; ³H]noradrenaline, used as a specific exogenous vesicle marker, was feebly incorporated in this fraction. Heavy vesicles (density 1.041 g/ml) containing high levels of noradrenaline, adenosine triphosphate, low levels of dopamine-β-hydroxylase activity are able to incorporate high amounts of ³H]noradrenaline. In these gradients, Mg²⁺ activated ATPase activity was present in both vesicle fractions.

Sedimentation analysis by analytical differential centrifugation also disclosed two types of vesicles: large vesicles with a sedimentation coefficient between 348 and 308 and small vesicles with a sedimentation coefficient of 96 . Large vesicles were associated with noradrenaline-β-hydroxylase activity, while small vesicles were associated only with noradrenaline.

In isoosmotic conditions the use of other microsomal markers allowed us to define the degree of contamination of the vesicle fractions. It was found that the noradrenergic heavy vesicles fraction presented under 11% of 5′-nucleotidase activity of the total activity present in the gradient and less than 5% of acid phosphatase, NADH-cytochrome c reductase and monoaminooxidase of the total activities in the gradients.

In isoosmotic conditions the physical properties of presumed vesicles were apparently undisturbed supporting the current morphometric observations. Our results then suggest prevailing roles for each type of vesicle: synthesis for light vesicles, and storage and/or release for heavy ones.