Noradrenergic neurons release both noradrenaline and neuropeptide Y from a single pool: the large dense cored vesicles.

In peripheral adrenergic nerve endings, noradrenaline is stored in two different types of vesicles, the large and the small dense cored vesicles. A systematic study was undertaken to examine the release of noradrenaline and neuropeptide Y from dog spleen and rat vas deferens under various conditions of stimulation, particularly those which previously have demonstrated a differential regulation of exocytosis of the different types of storage vesicles. Here we present evidence that noradrenaline is released by exocytosis exclusively from the large dense cored vesicles, in which it is stored together with neuropeptide Y. Upon a single stimulation (at frequencies varying from 2-20 Hz), the release of noradrenaline and neuropeptide Y from the dog splenic nerve increased with the frequency of stimulation, but the ratio of noradrenaline to neuropeptide Y remained constant. After repeated stimulation of the splenic nerve, both substances' overflow decreased gradually and in parallel to values of 12.5% and 11.1% of the first stimulation for noradrenaline and neuropeptide Y, respectively. Similarly, repeated stimulation of the rat vas deferens (of which only 2-10% is large dense cored vesicles, whereas in the dog splenic nerve the large dense cored vesicles make up 30-40% of the total vesicle population) with 120 mM K+, in the presence of phentolamine, caused a gradual and parallel decline in the release of noradrenaline and neuropeptide Y (31.6% and 34.0%, respectively). Moreover, omega-conotoxin (10(-8) M to 10(-5) M) had a similar inhibitory effect on the release of both substances, alpha-latrotoxin (10(-9) M) evoked a parallel release of both noradrenaline and neuropeptide Y. The results indicate that noradrenaline in peripheral noradrenergic nerves is released exclusively from large dense cored vesicles by an exocytotic mechanism.

Two polypeptide toxins with opposite effects on calcium uptake in bovine chromaffin cells: isolation from the venom of the marine snail Conus distans.

Two polypeptide toxins which modulate the uptake of 45Ca2+ in bovine chromaffin cells were isolated from the venom of the marine snail Conus distans. The molecular weights were estimated by gel electrophoresis and gel filtration to be 25.5 and 24 kDa, respectively. The purified proteins were electrophoretically homogeneous. The 25.5 kDa-component caused a concentration-dependent increase of the initial rate of 45Ca2+ uptake, but it had no effect on the stimulation evoked uptake. The 24 kDa-component produced the opposite effects; it caused a concentration-dependent inhibition of the stimulation evoked 45Ca2+ uptake, but it did not affect the initial rate.

Phenotype plasticity and immunocytochemical evidence for ChAT and D beta H co-localization in fetal pig superior cervical ganglion cells.

The early expression of the cholinergic phenotype in sympathetic neurons was already studied in superior cervical ganglion cells derived from rat, quail and chicken embryo. In the present work, we set up a neuron culture derived from the superior cervical ganglia of fetal pigs. The yield is 1000 times of that of a neonatal rat [17], 100 times of a 10- to 13-day-old chick embryo [26] and 20 times of a 10-day-old quail embryo [3]. This high yield will greatly facilitate further biochemical studies concerning neuronal differentiation. Using these cells as a model, the phenotype plasticity was studied by both biochemical and immunocytochemical methods in normal physiological medium, in a high KCl (30 mM) medium and in a splenocyte co-culture. The phenotype shift occurs in the normal physiological medium and in the splenocyte co-culture, but not in the high KCl medium. Taking into account the species difference, the fetal pig superior cervical ganglion neurons behave in a comparable manner as reported in earlier studies for other animal models. Moreover, for the first time, using immunocytochemical methods, direct evidence for a co-localization of choline-acetyl-transferase and dopamine-beta-hydroxylase in mammalian fetal sympathetic neurons, at least during a certain period, is given.

Vasostatins, N-terminal products of chromogranin A, are released from the stimulated calf spleen in vitro.

Vasostatins are the N-terminal chromogranin A peptides 7 approximately 22 kDa. They have been shown to be present in several endocrine tissues and exhibit vasoinhibitory activity in vitro. In a first series of experiments, we investigated the presence and subcellular localization of vasostatins in the bovine splenic nerve. Experimental results, obtained using gradient centrifugation, showed that noradrenaline was enriched 25-fold in the large dense core vesicle fraction, compared with the original homogenate. In the latter fraction, the 7 and 18 kDa peptides were observed following immunodetection with antiserum to chromogranin A1-40 and laser densitometric scanning revealed these two fragments as the major N-terminal fragments. Subsequently, we examined the release of the 7 and 18 kDa peptides from perfused calf spleen during veratridine (20 microM) or 1,1-dimethyl-4-phenylpiperazinium iodide (20 microM) stimulation. In the prestimulation samples, we were not able to detect these peptides, however, following stimulation, the 7 and 18 kDa chromogranin A fragments became apparent. The vasostatin-immunoreactivity, in both bovine chromaffin granule lysate and calf spleen perfusate, elutes at the same retention time on reversed-phase high performance liquid chromatography. The present study demonstrated that vasostatins are present in the large dense core vesicles of sympathetic axons and are released from the nerve terminals in response to stimulation. The release of vasostatins from sympathetic nerves in the spleen suggest an in vivo function for N-terminal chromogranin A products of neuronal origin.

Evidence against differential release of noradrenaline, neuropeptide Y, and dopamine-beta-hydroxylase from adrenergic nerves in the isolated perfused sheep spleen.

The subcellular storage and release of noradrenaline (NA), dopamine-beta-hydroxylase (D beta H), and neuropeptide Y (NPY) was studied in the isolated perfused sheep spleen. Subcellular distribution studies showed a bimodal distribution for NA which was well reflected by D beta H and indicated the occurrence of two types of NA storage vesicles. The most dense, presumably large dense-cored vesicles (LDV), contain both membrane-bound and soluble D beta H; the less dense presumably corresponds to small dense-cored vesicles (SDV) and at least does not contain soluble D beta H. The distribution of NPY is extended but shows a peak only at the position of LDV, indicating that LDV contain NPY. Continuous electrical stimulation of the splenic nerve at 2 Hz, 5 Hz, 10 Hz, and 20 Hz or at 20 hz with bursts induced the release of NA, NPY, and D beta H. The ratio among these components was constant. The fractional release of D beta H and NA was comparable at all frequencies used; that of NPY was 10-20 times lower, suggesting the occurrence of a large nonreleasable NPY pool. The present data argue against a high frequency stimulation or intermittent stimulation-induced preferential release of NPY from adrenergic neurons and question the concept of frequency-dependent chemical coding of sympathetic transmission in general. The simplest interpretation of our data is that NA and NPY are released at all frequencies from a single pool. The present finding might signify that only large dense-cored vesicles are involved in the sympathetic stimulation-evoked secretion of catecholamines from adrenergic nerve terminals of the isolated sheep spleen.

Inhibition of evoked neurotransmitter release from rat hippocampus by a polypeptide toxin isolated from the marine snail Conus distans.

The active fraction, isolated and partially purified from the crude venom of the marine snail Conus distans, with a molecular mass of about 25 kDa, inhibits neurotransmitter release in rat hippocampus. This toxin (distans Toxin) inhibits the electrically evoked tritium labelled noradrenaline release from rat hippocampal slices in a dose and time dependent manner. The neurotransmitter release is mainly regulated by N-type of voltage sensitive Ca(2+)-channels. The distans toxin behaves as a partial antagonist of calcium in the buffer, possibly by competing with calcium for this type of voltage sensitive Ca(2+)-channels.

Relationship between external calcium concentration and noradrenaline- and neuropeptide Y-evoked release from perfused dog spleen.

Several neuropeptides have been demonstrated to coexist with classical neurotransmitters in the central and peripheral nervous systems and have been proposed as neurotransmitter or neuromodulator candidates. In this report, we investigated the relationship between the external calcium concentration and the electrically induced overflow of noradrenaline (NA) and neuropeptide Y (NPY) in dog perfused spleen. Perfusion solutions with calcium concentrations ranging from 0 to 10 mM were applied. The splenic nerves were electrically stimulated at 16 Hz. For NA analysis high-pressure liquid chromatography with electrochemical detection was used. NPY was determined by radioimmunoassay. The perfusion pressure and the overflow of NA and NPY increased in a calcium concentration dependent manner. The calcium concentration dependency of the overflow of NA and NPY was comparable, indicating a co-release of the two substances. The molar ratio of NA/NPY remained unchanged over the calcium concentration range applied and the half maximal saturation values for release of NA (5.55 mM) and NPY (6.66 mM) were similar. These results indicate that the preferential release of NPY at high frequency stimulation as previously shown in the pig spleen, if present in the dog, is not the result of a difference in calcium dependency of the evoked release of NA and NPY.

Evidence for the co-storage and co-release of neuropeptide Y and noradrenaline from large dense cored vesicles in sympathetic nerves of the bovine vas deferens.

The subcellular localization and the secretion of neuropeptide Y were studied in sympathetic nerve endings of bovine vas deferens. Immunostaining revealed a parallel distribution for neuropeptide Y and dopamine-beta-hydroxylase immunoreactivity in the network of varicose nerve fibers in the smooth muscle layers of the vas deferens. Following differential centrifugation and sucrose density gradient centrifugation, neuropeptide Y was found to coincide with noradrenaline in the more dense region of the gradient, where the large dense cored vesicles are found. Superfusion experiments demonstrated the release of neuropeptide Y and noradrenaline upon electrical stimulation. Furthermore, the neuropeptide Y secretion was shown to be Ca2+-dependent. We conclude that in the bovine vas deferens neuropeptide Y is only present in large dense cored vesicles of adrenergic neurons and that the peptide and noradrenaline are co-released from these vesicles in a calcium dependent manner.

Evidence for the coexistence and co-release of [Met]enkephalin and noradrenaline from sympathetic nerves of the bovine vas deferens.

The localization and neurosecretion of methionine-enkephalin was studied in sympathetic nerves of the bovine vas deferens. Immunostaining showed methionine-enkephalin-like immunoreactivity in a network of varicose nerve fibres in the smooth muscle layers of the vas deferens. When vas deferens homogenates were subjected to differential and sucrose density gradient centrifugation, methionine-enkephalin was found to parallel the distribution of noradrenaline in the more dense region of the gradient, where "heavy" or large dense-cored vesicles are present. Electron microscopic immunochemistry confirmed this finding and showed methionine-enkephalin-like immunoreactivity in large dense-cored vesicles. The release of methionine-enkephalin upon electrical stimulation was studied in superfusion experiments. The methionine-enkephalin secretion was shown to be Ca2+-dependent and was inhibited by adding the adrenergic neuron blocking drug guanethidine to the superfusion medium. We conclude that in the bovine vas deferens methionine-enkephalin is only present in large dense-cored vesicles of adrenergic neurons and that the peptide is released from these vesicles together with noradrenaline by a Ca2+-dependent mechanism.

Presynaptic serotonin receptors regulate the release of 3H-serotonin in hypothalamic slices of the rabbit.

Hypothalamic slices of the rabbit brain were incubated with 10(-7) M of 3H-serotonin (3H-5HT). After the incubation and an initial washout period, a nearly constant basal efflux of tritium was detected. This basal efflux was not significantly altered by Ca2+-free solution or by the 5HT-antagonist metitepin (10(-5) M), but was augmented by chlorimipramine (10(-5) M) and by unlabelled 5HT (10(-6) M); the acceleration caused by unlabelled 5HT was absent in presence of chlorimipramine (10(-5) M). Both electrical stimulation (4 Hz, 50 mA, 2 min) and high K+ (50 mM) induced an overflow of 3H. This overflow was nearly abolished in Ca2+-free solution. In presence of chlorimipramine (10(-5) M) both the tritium overflow evoked by electrical stimulation and that evoked by high K+ were augmented by metitepin (10(-5) M) and decreased in a concentration dependent manner by unlabelled serotonin (10(-8) - 10(-6) M); the latter effect was antagonized by metitepin (10(-6) M and 10(-5) M). These experiments suggest that in rabbit hypothalamic slices, the release of 3H-5HT is controlled by a negative feedback mechanism acting via presynaptic serotonin receptors.