Abstract
Homogenates of bovine splenic nerve and of vas deferens were subjected to differential and density gradient centrifugation to investigate their noradrenaline-storing organelles. The subcellular fractions obtained were analysed by immunoblotting in order to define the presence of various antigens in small dense-core and large dense-core vesicles. In both large granule and microsomal fractions from splenic nerve only one type of noradrenaline-storing vesicle was found, which represents the large dense-core vesicles. These organelles contained chromogranin A, chromogranin B, cytochrome b-561, carboxypeptidase H, glycoprotein II, glycoprotein III, dopamine β-hydroxylase and the monoamine carrier which are also present in adrenal chromaffin granules. The subcellular distribution of synaptin/ synaptophysin was more complex since this protein was apparently present in two organelles: in a light vesicle which did not contain significant amounts of antigens found in large dense-core vesicles (dopamine β-hydroxylase, cytochrome b-561 and the monoamine carrier) and in the dense fractions of the gradient, possibly within large dense-core vesicles. In the microsomal gradient from vas deferens several markers (catecholamines, synaptin/synaptophysin and dopamine β-hydroxylase) were found in a bimodal distribution, which is consistent with their presence in small and large dense-core vesicles. When the larger granules were removed with higher centrifugation speed a microsomal fraction containing only light vesicles was obtained. After gradient centrifugation of this fraction several components (catecholamines, dopamine β-hydroxylase, cytochrome b-561, the monoamine carrier and synaptin/synaptophysin) were concentrated in a peak at low density; apparently only small dense-core vesicles were now present. We conclude that sympathetic axons contain only one type of noradrenaline-storing vesicle, i.e. the large dense-core vesicle, which resembles in all investigated aspects adrenal chromaffin granules. On the other hand, in sympathetic nerve terminals two types of vesicles are found. The membranes of these two vesicles have at least three components in common: dopamine β-hydroxylase, the monoamine carrier and cytochrome b-561.
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