Are NPY and enkephalins costored in the same noradrenergic neurons and vesicles?

Separate studies show that NPY and enkephalins are widely distributed in peripheral noradrenergic neurons. In the present study, the subcellular costorage and release in response to intense sympathetic stimulation and reserpine at near therapeutic doses (0.05 mg/kg every other day) were examined. In young pig arteries and vas deferens, enkephalin and D beta H immunofluorescence show consistent but not total overlap. Also NPY is colocalized with D beta H in many fibers but with VIP (nonnoradrenergic) in others. Ultrastructural immunogold labeling indicates that individual terminals contain large dense cored vesicles (LDVs) which store either NPY or enkephalins, even though costorage of both peptides occurs. Some LDVs costore NPY and VIP, especially in the middle cerebral artery and in the lamina propria of vas deferens. Acute CNS ischemia depletes enkephalins and norepinephrine in all tissues analyzed without parallel loss of NPY. Reserpine depletes norepinephrine 70-85% but does not deplete NPY or enkephalins. The latter is in contrast to commonly used high doses known to produce nonspecific, detergent-like effects. In fact, low doses of reserpine induce a time-dependent new synthesis and processing of NPY precursor peptides in vas deferns. Contrasting effects of reserpine on NPY and enkephalin contents, new synthesis and apparent processing, and a differential response to acute CNS ischemia were found in every tissue studied. Activation of precursor neuropeptide processing occurred immediately upon intense sympathetic stimulation in most tissues. Dual localization of NPY in noradrenergic and nonnoradrenergic fibers and differences in subcellular LDV storage help explain why enkephalin correlates better than NPY with norepinephrine loss in response to acute CNS ischemia. Furthermore, the costorage of NPY and enkephalins in distinct subpopulations of noradrenergic fibers, which varies according to tissue, is likely to be under separate CNS control.

Enkephalin contents reflect noradrenergic large dense cored vesicle populations in vasa deferentia.

Opioid peptides including met- and leu-enkephalins are co-stored with catecholamines at similar concentrations in highly purified chromaffin granule fractions from bovine adrenal medulla and large dense cored vesicle (LDV) fractions from bovine splenic nerve. An initial attempt was made to test the universality of the co-storage of enkephalins in LDVs of sympathetic nerves. Based on a number of practical considerations, vasa deferentia were chosen from seven species. Leu-and met-enkephalin contents were quantitated by radio-immunoassays and norepinephrine by HPLC with electrochemical detection. Norepinephrine contents varied 11-fold and generally paralleled the density of sympathetic innervation among the species as reported in the literature. Leu-enkephalin contents varied 26-fold, generally paralleling the percentage composition of LDVs in the respective terminal varicosities among the species and animal size (axonal length). Met-enkephalin contents varied 20-fold, generally paralleling the density of sympathetic innervation, but not the percentage LDVs. Various amounts of met-enkephalin were also likely stored in cells other than sympathetic nerves, including chromaffin-like cells, the incidence of which varied according to species. Thus, the met- to leu-enkephalin ratios varied from 1.2:1 in dog; 1.7-1.9:1 in rabbit, cat and bull; 2.9:1 in man; 8.2:1 in rat; and essentially only met-enkephalin in guinea pig. The data imply differences in the processing of preproenkephalins in the same tissue of different species as well as in different cells of that tissue.

Chromogranin A: localization and stoichiometry in large dense core catecholamine storage vesicles from sympathetic nerve.

Chromogranin A is present in both adrenal medullary chromaffin granules and sympathetic nerve large dense core catecholamine storage vesicles (LDVs), yet selective stimulation of sympathetic axons provokes only minor changes in chromogranin A in the circulation. We therefore examined the stoichiometry of chromogranin A storage in purified LDVs as compared to chromaffin granules. Chromogranin A was found in LDVs on immunocytochemical sections of sympathetic axons. Sedimentation of sympathetic axon homogenates on sucrose-D2O gradients localized chromogranin A, norepinephrine, enkephalins and dopamine-beta-hydroxylase to the same gradient particulate fractions, suggesting that they inhabit a particle of the same buoyant density, the LDV. Chromogranin A was identified in LDV by radioimmunoassay, immunoblotting and immunocytochemistry. Purified LDVs contained 17.8 +/- 4.8% of cell total chromogranin A, at 27.9 +/- 3.5-fold enrichment over the original axon homogenate. When LDVs were lysed, all of the chromogranin A immunoreactivity originated from the soluble vesicle core rather than the LDV membrane. Although chromogranin A/catecholamine ratios were similar in LDVs and adrenal chromaffin granules, chromogranin A was a quantitatively minor protein in LDVs, accounting for only 0.16 +/- 0.015% of total LDV protein, as compared to 35.2 +/- 1.4% of total chromaffin granule protein. Each LDV particle contained approximately 0.94 +/- 0.09 chromogranin A molecules. Immunocytochemical data suggested that chromogranin A is costored in large dense core noradrenergic vesicles in subpopulations of sympathetic axons, analogous to enkephalins and neuropeptide Y. Thus, only profound changes in exocytotic catecholamine release from sympathetic axon LDVs would be expected to perturb circulating chromogranin A concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum dopamine beta-hydroxylase activity following subarachnoid hemorrhage in man.

Serum dopamine beta-hydroxylase activity was followed in seven patients who had experienced primary subarachnoid hemorrhage resulting from a ruptured intracranial aneurysm. This preliminary study suggested that dopamine beta-hydroxylase levels were initially elevated following subarachnoid hemorrhage, after which they progressively declined during the first week post hemorrhage. After a minimum was reached, the activity rose again to a stable level two to three weeks post hemorrhage.

Dopamine beta-hydroxylase distribution in density gradients: physiological and artefactual implications.

Knowledge of the vesicular origin of circulating dopamine beta-hydroxylase (DbetaH) is indispensable for any attempts to explain the parallelism or lack of it between circulating enzyme and catecholamines as they may relate to physiological stress, forms of hypertension, neurological disorders, and the response to pharmacological agents. The present study represents an effort to evaluate and to place in proper perspective data based on the DbetaH activity found in the region of the light vesicle peak of noradrenaline (NA), which is used as a quantitative measure of a population of small terminal vesicles. Distributions of vesicles and subvesicular components are compared with DbetaH and NA in sucrose-D2O density gradients used to prepare relatively pure fractions of large dense cored vesicles (LDV) from bovine splenic nerve. Although NA in sedimentable particles of the light vesicle peak is likely to be a valid measure of a small vesicle population, the following is demonstrated: (1) A substantial fraction (25%-37%) of the total sedimentable DbetaH activity can be proven to distribute in the region of the light vesicle peak from a tissue with an insignificant small vesicle population. Based on studies of vesicles from sequential nerve segments, this enzyme activity probably corresponds to a population of "immature" LDV which are undergoing axoplasmic transport and have not synthesized their full complement of transmitter. (2) Physical lysis which depletes the matrix of LDV causes redistribution of DbetaH activity from the heavy vesicle peak into the region of the light vesicle peak. Analogously, DbetaH associated with exocytosed LDV and retrograde transport particles is also likely to contaminate the region of the light vesicle peak. (3) Based on available data, it can be calculated that each small dense cored vesicle could contain only 0.1-0.5 molecules of DbetaH and that a contamination of only 0.016% LDV can account for all of the DbetaH reported to occur in the light vesicle peak of normal rat vas deferens preparations.