The NO synthase inhibitor L-NNA depresses neurohypophysial vasopressin but not its precursor amidating enzymes in salt-loaded rats.

The arginine vasopressin (AVP) producing hypothalamo-neurohypophysial system also has high activities of NO-synthase. Vasopressin production and secretion is drastically upregulated during salt intake and the NO-producing enzyme may be involved. We have studied the influence of the NO-synthase inhibitor NG-nitro-L-arginine (L-NNA) on neurohypophysial and hypothalamic AVP and its amidating enzymes in salt-loaded and control rats as well as on stimulated AVP release in vitro in such rats. Rats were given 2% NaCl solution as the only fluid for 4 days and then returned to tap water. The specific amount of AVP (microgram (mg protein)-1) and the activities of peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), components of its amidating processing enzyme in the supraoptic (SON) and paraventricular nuclear (PVN) regions, did not change during the salt-loading or the following recovery period. In contrast, the AVP and PHM and PAL in the neurohypophysis fell drastically during the salt loading. After that, PHM and PAL increased even more rapidly than AVP, the latter reaching control levels in about 10 days. Salt loading did not change the protein content of the neurohypophysis. When salt loading was performed after administration of L-NNA, the neurohypophysial AVP at the end of the salt loading and 3 days later was lower than in rats not receiving L-NNA, whereas PHM and PAL were not affected. Fractional AVP release from isolated neurohypophyses of salt loaded rats treated with L-NNA and stimulated with K+ was similar to that found in non-treated rats. It is suggested that L-NNA may affect translation or precursor processing of AVP.

Effect of hypoosmolality on the abundance, poly(A) tail length and axonal targeting of arginine vasopressin and oxytocin mRNAs in rat hypothalamic magnocellular neurons.

Arginine vasopressin (AVP) and oxytocin (OT) mRNAs are targeted to the axonal compartment of rat hypothalamic magnocellular neurons. Salt-loading results in a considerable rise in hypothalamic and axonal AVP mRNA but only a moderate increase for axonal OT mRNA. Here we report that hypoosmolality gives rise to a rapid decrease of axonal AVP encoding transcripts to undetectable levels after 2 weeks. The levels of OT mRNA in the axonal compartment did not change significantly. In the hypothalamus the mRNA for AVP also decreased. The size of the poly(A) tract of AVP encoding transcripts appeared to be strictly correlated with plasma osmolality. In contrast, the amount and size of OT encoding mRNAs were only moderately or not influenced by hypoosmolar stimuli.

Transport of BC1 RNA in hypothalamo-neurohypophyseal axons.

Ample evidence indicates that in nerve cells, several individual proteins are locally synthesized in postsynaptic domains in dendrites. By contrast, axonal terminals, at least in mammals, are generally thought to lack protein synthetic capacity. However, axonal nerve endings of the hypothalamo-neurohypophyseal tract have recently been shown to contain mRNAs encoding vasopressin, oxytocin, dynorphin, and neurofilament. In this report, we identify BC1 RNA, a small RNA polymerase III transcript that is specifically expressed in neurons, in hypothalamo-neurohypophyseal axons. BC1 RNA has previously been shown to be located in somatic and dendritic domains of various types of neurons in the rat nervous system. Here we present evidence to show that BC 1 RNA, like several neuropeptide mRNAs, is axonally transported from magnocellular hypothalamic neurons to neurosecretory nerve endings in the posterior pituitary. BC1 RNA, which has been reported to be a component of a ribonucleoprotein particle, is thus colocalized with dendritic mRNAs in dendritic domains and with axonal mRNAs in axonal domains, respectively. Such colocalization is indicative of functional interactions of BC1 RNA with those mRNAs that are targeted to extrasomatic domains of nerve cells.

Amidating processing enzyme complex for bioactive peptides (PAM) shows differences in specific activity and form in secretory granules isolated from the proximal and distal parts of the hypothalamo-neurohypophyseal tract in rats.

In rats the PAM specific activity in hypothalamic and neurohypophyseal extracts was 0.58 +/- 0.8, respectively 1.78 +/- 0.6 nmol.mg prot.-1 x h-1 (n = 5). PHM specific activity in the soluble part of the granules was higher in the neurohypophyseal than in the hypothalamic granules, and the fraction of total PHM and PAL present in the soluble part increased with the distance from the hypothalamus from some 45% to approx. 85%. Western blots of membrane and soluble granule fractions showed prevalence of higher mol. wt. forms in hypothalamic granules. It would appear that higher mol. wt. forms of PAM are processed by proteolytic enzymes during transport in the neuron and that non-neural cells in the neurohypophysis have a considerable PAM activity.

Polyamines in nerve terminals and secretory granules isolated from neurohypophyses.

In isolated nerve terminals from ox neurohypophyses the following concentrations of polyamines [pmol (microgram protein)-1 (mean +/- SEM)] were found: spermine: 2.07 +/- 0.14 (n = 3), spermidine: 0.22 +/- 0.01 (n = 4), putrescine: 0.20 +/- 0.01 (n = 4). In secretory granules isolated from the same tissue, the concentrations were: spermine: 0.57 +/- 0.02 (n = 3), spermidine: 0.07 +/- 0.04 (n = 3), putrescine: 0.13 +/- 0.04 (n = 3). After incubation of isolated nerve terminals with the polyamines, they were taken up as a function of time and concentration, approaching saturation at high concentrations. The kinetic parameters of their synthesizing enzyme, ornithine decarboxylase, in ox neurohypophyseal nerve terminals (apparent Km 0.75 mM and Vmax 22.5 pmol mg protein-1 h-1) were comparable to those previously found in cerebral cortex of rats. When isolated, hemilobes from rat neurohypophyses were incubated in a medium which contained spermidine (5 mM), and were stimulated by 56 mM K+, release of vasopressin was smaller than in control experiments. However, after removal of spermidine and after restimulation, 50 min after initial stimulation, the release was significantly elevated. It is suggested that polyamines may take part in modulation of vasopressin release.

High ascorbic acid content in the rat endocrine pancreas.

The peptidyl alpha-amidation of biologically active peptides (a number of which are found in the endocrine pancreas) requires several co-factors, including ascorbic acid. In the present study, tissue contents and developmental changes of ascorbic acid in the rat endocrine pancreas were measured using a highly sensitive HPLC system. High concentrations were found in neonatal rats, with the highest value, 42.5 nmol/mg protein, in 2-day-old rats. The concentration decreased gradually with age to 19.4 nmol/mg in 5-week-old rats. The exocrine pancreas had a lower concentration, but a peak was also observed in 2-day-old rats. In freshly isolated islet cells, an intracellular concentration of 7.5 mmol/l was estimated in 5-7-day-old rats. Secretory granules isolated from 4-6-day-old rat islets contained 10.6 nmol/mg protein. Culturing islets or cells in ascorbic acid free medium resulted in a marked decrease in their contents. Ascorbic acid in secretory granules from such islets decreased at a relatively lower rate. Addition of ascorbic acid to cultured cells or islets reduced the loss markedly. Increasing the glucose concentration in islet culture medium in the presence of 100 mumols/l ascorbic acid increased the islet ascorbic acid concentration.

Transport of ascorbic acid and dehydroascorbic acid by pancreatic islet cells from neonatal rats.

Several amidated biologically active peptides such as pancreastatin, thyrotropin-releasing hormone, pancreatic polypeptide and amylin are produced in endocrine pancreatic tissue which contains the enzyme necessary for their final processing, i.e. peptidylglycine alpha-amidating mono-oxygenase (EC 1.14.17.3). The enzyme needs ascorbic acid for activity as well as copper and molecular oxygen. The present work shows that pancreatic islet cells prepared from overnight cultures of isolated islets from 5-7-day-old rats accumulate 14C-labelled ascorbic acid by a Na(+)-dependent active transport mechanism which involves a saturable process (estimated Km 17.6 microM). Transport was inhibited by ouabain, phloridzin, cytochalasin B, amiloride and probenecid. Glucose inhibited or stimulated uptake, depending on the length of incubation time of the cells. The uptake of dehydroascorbic acid was linearly dependent on concentration. Dehydroascorbic acid was converted to ascorbic acid by an unknown mechanism after uptake. The uptake of both ascorbic acid and dehydroascorbic acid was inhibited by tri-iodothyronine, and uptake of ascorbic acid, but not of dehydroascorbic acid, was inhibited by glucocorticoids. Isolated secretory granules contained a fairly low concentration of iron but a high concentration of copper.

Parallel expression of synaptophysin and evoked neurotransmitter release during development of cultured neurons.

Primary cultures of GABAergic cerebral cortex neurons and glutamatergic cerebellar granule cells were used to study the expression of synaptophysin, a synaptic vesicle marker protein, along with the ability of each cell type to release neurotransmitter upon stimulation. The synaptophysin expression and neurotransmitter release were measured in each of the culture types as a function of development for up to 8 days in vitro, using the same batch of cells for both sets of measurements to obtain optimal comparisons. The content and the distribution of synaptophysin in the developing cells were assessed by quantitative immunoblotting and light microscope immunocytochemistry, respectively. In both cell types, a close parallelism was found between the temporal pattern of development in synaptophysin expression and neurotransmitter release. This temporal pattern differed between the two types of neurons. The cerebral cortex neurons showed a biphasic time course of increase in synaptophysin content, paralleled by a biphasic pattern of development in their ability to release [3H]GABA in response to depolarization by glutamate or elevated K+ concentrations. In contrast, a monophasic, approximately linear increase in the synaptophysin content and stimulated [3H]D-aspartate release was found in the cerebellar granule cells. These results, particularly regarding the GABAergic neurons, offer correlative evidence in support of the notion that a vesicular pool of these amino acid neurotransmitters may be intimately involved in their release, subsequent to depolarization stimuli.

Characteristics of ascorbic acid uptake by isolated ox neurohypophyseal nerve terminals and the influence of glucocorticoid and tri-iodothyronine on uptake.

Isolated nerve endings (neurosecretosomes) from ox neurohypophyses took up L-[14C]ascorbic acid by a process or processes which showed energy dependence and which could be inhibited by unlabelled ascorbic acid in micromolar concentrations and by isoascorbic acid in millimolar concentrations, whereas dehydroascorbic acid only inhibited in concentrations of about 100 mM. The uptake showed saturation with increasing concentration of ascorbic acid and a Km value of 97 microM. Uptake was inhibited by increasing glucose concentration in the medium or by adding cytochalasin B, phloridzin, ethanol or probenecid to the medium. The uptake was inhibited by lowering the sodium concentration and by lack of calcium. These facts suggest the presence of both a glucose-dependent uptake and a sodium-dependent uptake. Cortisol and tri-iodothyronine inhibited uptake. This effect of cortisol, but not of tri-iodothyronine, was dependent on the presence of sodium in the medium. For both hormones it was still present when phloridzin or probenecid was added to the medium.

Chronic osmotic stimulation reduces vasopressin but not synaptophysin content in rat neurohypophysis.

The content of synaptophysin, a vesicular integral membrane protein of neurons and endocrine cells, and that of vasopressin was measured in neurohypophyses of rats during chronic osmotic stimulation. The animals received 2% NaCl in their drinking water for up to 4 days. Synaptophysin content of neurohypophyses was determined using quantitative immunoblotting, vasopressin content was measured by radioimmunoassay. Salt loading caused a decrease in the content of vasopressin to about 15% of that of control animals, whether expressed per neurohypophysis or relative to the total tissue protein. In contrast, no change was found in the synaptophysin content. Taken together with published evidence of changes in the relative numbers of the hormone-containing neurosecretory granules (NSGs) and the microvesicles (MVs) under the conditions of chronic osmotic stimulation, these results strongly indicate the surface density of synaptophysin on NSGs to be significantly lower than its surface density on MVs.

Evidence for presence of peptide alpha-amidating activity in pancreatic islets from newborn rats.

The peptide alpha-amidating activity of a homogenate of pancreatic islets from 5-7-day-old rats was investigated, using as substrate a glycine-extended tripeptide (D-Tyr-Val-Gly). The islet homogenates had a marked amidating activity, with a Km of 57 microM, a Vmax. of 185 pmol/h per mg and a pH optimum of 7.0. This activity was dependent on the presence of ascorbic acid (in the reduced form) and Cu2+, the optimum concentrations being 4 mM and 40 microM respectively. On fractionation of the homogenate, the highest specific activity was found in the soluble fraction. Exocrine pancreatic tissue showed very low levels of amidating activity.

Rats with physically disconnected hypothalamo-pituitary tracts no longer contain vasopressin-oxytocin gene transcripts in the posterior pituitary lobe.

In rats, vasopressin- and oxytocin-encoding mRNAs are present in the posterior but absent in the anterior lobe of the pituitary gland. RNase protection experiments indicate that in the posterior pituitary and hypothalamus identical transcriptional start points are used. Furthermore, the two transcripts from posterior pituitary and hypothalamus show identical nucleotide sequences. Animals operated by paired electrical lesions in such a way that connections between the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus and the posterior pituitary lobe are destroyed continue to express the vasopressin and oxytocin gene in the hypothalamus but not in the posterior pituitary. Operated animals subjected to chronic intermittent salt loading for 6 days similarly contain vasopressin and oxytocin encoding transcripts in the hypothalamus but not in the posterior pituitary.

Uptake of ascorbic acid by freshly isolated cells and secretory granules from the intermediate lobe of ox hypophyses.

Mechanically isolated cells from the intermediate lobe of ox hypophyses contained 40.6 +/- 3.7 nmol mg-1 protein (mean +/- SE, n = 5) of ascorbic acid. They accumulated radioactivity time dependently, on incubation with L-[14C]ascorbic acid in ionic medium dominated by NaCl. No definite saturation of uptake occurred when mechanically isolated cells were incubated with increasing ascorbic acid concentrations up to 0.6 mM. But if such cells were purified on a Percoll gradient, a clear saturation of uptake could be observed. Acetylsalicylic acid reduced the uptake markedly. When cells loaded with L-[14C]ascorbic acid were homogenized and placed on a Percoll gradient, the radioactivity was recovered in several subcellular fractions. Decrease of the Na+ concentration or presence of ouabain in the medium did not cause noticeable changes in uptake by non-purified cells, whereas uptake by purified cells was clearly sodium-dependent. Phloridzin inhibited uptake. Secretory granules from pars intermedia contained 40.0 +/- 3.8 nmol mg-1 protein of ascorbic acid (mean +/- SE, n = 3) and could accumulate L-[14C]ascorbic acid rapidly in a KCl-dominated medium. The uptake was not saturable with ascorbic acid concentration and was not influenced by the presence of I mM ATP + I mM Mg2+ in the medium. The concentration of copper and iron in isolated cells was comparable to that in isolated neurohypophysial nerve terminals, whereas the concentration of zinc was considerably higher in the pars intermedia cells. The concentration of Cu, Zn, Fe and Co in secretory granules from pars intermedia was higher than in secretory granules from neurohypophyses.