Uptake of dehydroascorbic acid and ascorbic acid to isolated nerve terminals and secretory granules from ox neurohypophyses

When uptake of L-[14C]ascorbic acid ([14C]AA) to various organs in guinea-pigs was studied after intracardiac injection, the adenohypophysis, pars intermedia, and the neurohypophysis had an uptake per milligramme protein which was about half of the uptake to the adrenals. Adrenal uptake was 20 +/- 2.8 pmol mg-1 protein microCi-1 injected. The uptake to the different parts of the hypophysis was considerably higher than the uptake to pancreas, liver, kidney, spleen and other organs. When isolated nerve endings (neurosecretosomes) from ox neurohypophyses were incubated with a medium containing labelled dehydroascorbic acid ([14C]DHA), the uptake was much slower than when the medium contained labelled ascorbic acid. The uptake of [14C]DHA showed a linear dependence on concentration, and was not influenced by addition of Mg2+ and ATP. Addition of Mg2+ + ATP, omission of Ca2+ and Mg2+ or exchange of Na+ in the medium with K+ had no effect on the uptake of ascorbic acid. When isolated secretory granules from ox neurohypophyses were incubated with a medium containing [14C]DHA, uptake was considerably faster than the uptake when they were incubated in a medium containing [14C]AA. The uptake of dehydroascorbic acid was linear with the concentration in the medium and was not changed by addition of Mg2+ ATP. Addition of 10 mM NH4Cl or exchange of 120 mM K+ in the incubation medium with Na+ did not change the uptake of dehydroascorbic acid. The contents of copper, zinc, iron and cobalt were determined in isolated nerve endings (A) and membranes (B) as well as in lysate (C) from isolated neurosecretory granules. The results (in nmol mg-1 protein) were for Cu: (A): 0.25 +/- 0.01 (SEM), (B): 0.67 +/- 0.16, (C): 0.22 +/- 0.06; for Zn: (A): 0.53 +/- 0.13, (B): 6.97 +/- 0.75, (C): 1.8 +/- 0.53; and for Fe: (A): 15.6 +/- 1.9, (B): 6.92 +/- 0.32, (C): 3.15 +/- 0.43. In all preparations the cobalt content was below the detection limit (less than 5 pmol mg-1 protein).     

Ascorbic acid uptake to isolated nerve terminals and secretory granules from ox neurohypophyses

Isolated nerve terminals (neurosecretosomes) from cow neurohypophyses accumulated radioactivity when they were incubated with L[14C]-ascorbic acid in an ionic medium dominated by NaCl. Uptake of radioactivity was saturable with ascorbic acid concentration. Replacement of Na+ with Li+ in the incubation medium or presence of ouabain inhibited the accumulation. Isolated, purified cow neurosecretory granules contained 14 +/- 2 nmol ascorbate (n = 10) per mg of protein. When such granules were incubated with L[14C]-ascorbic acid in a KCl dominated medium, they took up radioactivity slowly. The accumulation was not saturable with ascorbic acid concentration and was not influenced by the presence of Mg2+ATP.     

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.     

Polyamines in nerve terminals and secretory granules isolated from neurohypophyses

In isolated nerve terminals from ox neurohypophyses the following concentrations of polyamines [pmol (μg 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 K_m 0.75 mm and V_max 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 mrs 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.     

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.     

The uptake of ascorbic acid and dehydroascorbic acid by chromaffin granules of the adrenal medulla.

Adrenal chromaffin granules incubated with [¹⁴C]ascorbate or [¹⁴C]dehydroascorbate accumulated radioactively labeled compounds. Dehydroascorbate was generated from ascorbate by including oxidized cytochrome c in the incubation mixture but thin layer chromatography revealed that dehydroascorbate was present even in the absence of cytochrome c. This observation coupled with the fact that radioactively labeled compound was accumulated much faster in the presence of cytochromec than in its absence indicates that dehydroascorbate but not ascorbate is taken up by the chromaffin granules. Uptake of radioactivity was not saturable with ascorbate or dehydroascorbate concentration, and granules which had taken up the radioactively labeled compound lost most of the material in 12 h. In addition 1 mm adenosine 5′-triphosphate--Mg²⁺ + had no effect on either ascorbate or dehydroascorbate uptake. These data indicate that uptake proceeds via an energy independent mechanism. The rate of uptake of dehydroascorbate is 50-fold lower than the rate of catecholamine uptake.Ascorbate but not dehydroascorbate acts as a cofactor for the enzyme dopamine β-hydroxylase in the conversion of dopamine to norepinephrine. Dehydroascorbate taken up by the granules must therefore be reduced in order to be used by dopamine i3-hydroxylase. We suggest that the chromaffin granule electron transport system may be involved in this reduction.     

Identification of calmodulin-binding proteins on membranes of secretory granules isolated from bovine neurohypophyses

Membrane proteins from isolated, purified ox neurohypophyseal secretory granules were separated by sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis (PAGE). Using a gel overlay technique, after renaturation procedures, it was demonstrated that ¹²⁵J calmodulin bound in a Ca²⁺-dependent way to two protein bands with molecular weights (MW) of 58,000 and 52,000. Binding of small amounts of calmodulin to other protein bands was independent of calcium. No calmodulin binding to granule content proteins could be detected. Treatment of the granules with trypsin prior to separation of membrane proteins removed the Ca²⁺-dependent binding proteins from the granule membrane. On incubation of granules with [y-³²P]ATP, protein bands with MW of 52,000 and 45,000 showed a marked phosphorylation activity. The 52,000 band had the same electrophoretic mobility as one of the calmodulin-binding bands. However, no effect of calmodulin on phosphorylation of this band could be demonstrated.     

Electrophysiological study of single Leydig cells freshly isolated from rat testis

Changes in the membrane potential of isolated Leydig cells produced by modified ionic solutions were investigated in vitro either by a total change of the bathing medium (procedure P1) or by a flush of the solution around the impaled cell (procedure P2). In standard Earle's solution, the impalement of 198 cells by a glass microelectrode was accompanied by an hyperpolarization (MP1 = -37.6 +/- 0.7 mV) (means +/- S.E.M.) followed by a gradual depolarization to a steady state potential (MP2 = -25.1 +/- 0.6 mV) (Joffre et al. 1984). Experiments with K modified media (P1) showed that MP2, and to a greater extend MP1, were dependent on the external K. A tenfold increase of K decreased MP2 by 16 mV and MP1 by 25 mV. When the extracellular Cl was reduced (P1) by substituting Cl with a less permeant anion, MP2 was unchanged and MP1 was significantly decreased. A transient depolarization of MP2 occurred when a low Cl medium was flushed (P2). An equimolar Na replacement by choline chloride (P1) did not change MP1 or MP2, during the first 10 min. It suppressed MP1 and decreased MP2 after a 15 min exposure. MP1 reappeared and MP2 increased after the restoration of the normal Na solution (P1 and P2). The modification of external Ca from 0 to 3.6 mM (P1) increased both MP1 and MP2. MP1 was never cancelled in 0 mM Ca. In 18 mM Ca, MP1 was suppressed and MP2 decreased. Restoration of 1.8 mM Ca was rapidly accompanied by the MP1 reappearance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophysiological study of single Leydig cells freshly isolated from rat testis

Single Leydig cells were isolated from rat testis by a collagenase digestion procedure and purified through a 21,000g self generated densities gradient of 35% Percoll. A method including collagen and fibronectin was proposed to attach freshly prepared Leydig cells to the bottom of plastic Petri dishes. Four hours after the isolation of the cells, it was simultaneously possible to determine their membrane potential by a standard electrophysiological technique using intracellular microelectrodes and to judge cellular integrity by direct microscopic observations. In standard Earle's solution, changes of membrane potentials appeared to be biphasic. On 198 impaled cells, 18±1 s after the impalement was effective, the membrane potential reached a most negative value (MP₁) (−37.6±0.7 mV), followed by a gradual depolarization to a steady state (MP₂) (−25.1±0.6 mV) which remained constant for a few minutes. In standard Earle's solution, the membrane resistance was low or decreasing towards the most negative potential, then it increased towards the steady potential. At this state, the average value of the cell input resistance was 65.9±6.0 MΩ (n=16). No action potential was observed either in standard Earle's solution or under a depolarizing current state. It was concluded that the electrophysiological characteristics of the Leydig cell are similar to those of fibroblasts and macrophages, three types of cells with the same mesenchymal origin, present in the interstitial tissue of the rat testis. But the resting potential of the Leydig cell is higher and this secreting cell does not elicit hyperpolarizing oscillations at the steady state, under mechanical or electrical stimuli.     

Uptake of K+ by frog gastric mucosa from submucosal side and acid secretory rate.

The K+ content in frog gastric mucosa (K+) was measured as a function of the submucosal K+ concentration ([K+sm]) in the absence of K+ on the mucosal side. The (K+) in HCO3- buffer with 95% O2-5% CO2 gas showed that the removal of external K+ induced a 27% K+ loss from the control value of 5 mM K+sm, that there was no linear relation between (K+) and [K+sm, and that the change in the (K+) versus the [K+sm] was hyperbolic, indicating that there are two different types of K+ in the mucosa: bound and free K+.     

Uptake of adenosine triphosphate by isolated adrenal chromaffin granules: A carrier-mediated transport

Partly purified chromaffin granules from bovine adrenal medulla were incubated in vitro with [³H]adenosine triphosphate (ATP). After several washes the particles were subjected to density gradient centrifugation. It was demonstrated that chromaffin granules had taken up radioactive material. Thin-layer chromatography revealed that more than 60% of the radioactivity was confined to [³H]ATP. Incubation experiments with a mixture of [³H]ATP and [³²P]ATP indicated that ATP was taken up by chromaffin granules as an intact molecule. The uptake of [³H]ATP was temperature dependent and linear up to 10 min. The rate of uptake depended upon the ATP concentration and levelled off at ATP concentrations above 2 mM. At this ATP concentration the rate was 0.268 nmol ATP/mg protein/min. The uptake of ATP was compared with that of catecholamines. Both processes were activated by Mg²⁺, but inhibited by N-ethylmaleimide and an uncoupler of oxidative phosphorylation. Atractyloside inhibited only the ATP-uptake, whereas reserpine only interfered with that of the catecholamines.It is suggested that this uptake mechanism for ATP is carrier-mediated and that it enables newly formed chromaffin granules to accumulate and to maintain a high nucleotide concentration, which is required for the formation of a storage complex with catecholamines.     

Oxytocin release is inhibited by opiates from the neural lobe, not those from the intermediate lobe

Previous experiments have shown that the amount of oxytocin released by electrical stimulation from an isolated rat neurointermediate lobe is about doubled in the presence of the opiate antagonist naloxone. To test whether the opiates from the intermediate lobe inhibit the release of oxytocin, we performed similar experiments on the isolated neural lobe, after almost total removal of intermediate lobe tissue. The potentiating effect of naloxone upon oxytocin release was undiminished, suggesting that the endogenous opiate responsible for inhibiting oxytocin secretion comes from the neural lobe itself.     

Exocytosis of secretory granules in the juxtaglomerular granular cells of kidneys

Background: There is little agreement as to the secretory process of renin granules in juxtaglomerular granular cells (JG cells) of kidneys, although a large number of studies of the regulation of renin secretion have been reported. Methods: The structural correlation between the stimuli and the secretory process was examined in mouse JG cells on renal cortical slice incubated with the beta-adrenergic agonist, isoproterenol; the loop diuretic, furocemide; the Ca²⁺ chelator, EGTA; and the actin filament-disrupting agent, cytochalasin B. Results and Conclusions: Treatment with isoproterenol (10⁻⁵−10⁻³ M) or furocemide (10⁻³M) in Ca²⁺-containing medium did not significantly affect the ultrastructure of JG cells. In slices incubated with isoproterenol or furocemide in the Ca²⁺-free medium, JG cells occasionally contained a few electron-lucent granules at the cell periphery in addition to the electrondense mature granules observed in the control slices. On rare occasions, the JG cells displayed omega-shaped cavities with electron-lucent matrices, a feature similar to the contents of electron-lucent granules. Cytochalasin B markedly promoted the effects of these stimulants in Ca²⁺-free medium. These findings suggest the participation of actin filament disassembly in the exocytotic process of the mature granules in JG cells. © 1995 Wiley-Liss, Inc.     

The secretory granules of the acinar cells of the mouse submaxillary gland

Absence of membranes from the secretion granules of the acinar cells of the submaxillary gland of the mouse had led to speculation concerning mechanisms of secretion of these cells. By means of rapid perfusion fixation, smooth membranes have been identified around the secretion granules, and the mode of secretion proves to be similar to that of the other exocrine glands. The evidence suggests that potent membranolytic agents of unknown nature, capable of rapidly destroying the membranes are present in these secretory granules.     

Phenotypical and functional characterization of freshly isolated adipose tissue-derived stem cells

Adipose tissue contains a stromal vascular fraction (SVF) that is a rich source of adipose tissue-derived stem cells (ASCs). ASCs are multipotent and in vitro-expanded ASCs have the capacity to differentiate, into amongst others, adipocytes, chondrocytes, osteoblasts, and myocytes. For tissue engineering purposes, however, it would be advantageous to use the whole SVF, which can be transplanted without further in vitro selection or expansion steps. Because little is known about the freshly isolated ASCs in the SVF, we phenotypically characterized human freshly isolated ASCs, using flow cytometry. In addition, we investigated whether freshly isolated ASCs have functional properties comparable to cultured ASCs. For this, the differentiation potential of both freshly isolated ASCs and cultured ASCs into the osteogenic pathway was analyzed. Freshly isolated ASCs slightly differed in immunophenotype from cultured ASCs. Contrary to cultured ASCs, freshly isolated ASCs were shown to be highly positive for CD34, and positive for CD117 and HLA-DR. On the other hand, expression of CD105 and especially CD166 on the freshly isolated ASCs was relatively low. After osteogenic stimulation of freshly isolated ASCs, both Runx-2 and CollaI gene expression were significantly increased (p < 0.05). However, there was a difference in the kinetics of gene expression between freshly isolated and cultured ASCs and also between the different SVF isolates tested. There was no difference in alkaline phosphatase activity between freshly isolated ASCs and cultured ASCs. In addition, freshly isolated ASCs stained positive for osteonectin and showed matrix mineralization. We conclude that although there are minor differences in phenotype and kinetics of differentiation between freshly isolated ASCs and cultured ASCs, the use of freshly isolated ASCs for tissue engineering purposes involving bone repair is potentially applicable.     

The bat pituitary cleft: phasic production and release of secretory granules by microvillous cells

The ultrastructure of the two types of cells, ciliated and microvillous, lining the wall of the bat pituitary cleft was examined at different periods during the annual life cycle. Ciliated lining cells remained unchanged in structure throughout the yearly activity cycle. In contrast, microvillous lining cells exhibited striking cyclic morphological changes. Throughout the active phase of the life cycle and during most of the hibernating period, the structure of microvillous cells is unchanged. However, toward the end of hibernation, the cells produce and store large numbers of round to ovoid dense secretory granules. Just prior to arousal, the microvillous cells are packed with secretory granules. At arousal, the cells undergo marked degranulation; the granules appear to be discharged into the lumen of the cleft. Degranulation is followed by a period of proliferation of rough endoplasmic reticulum, the cavity of which is often filled with large paracrystalline inclusions. Within 1 month, microvillous cells return to a resting state and are devoid of secretory granules.     

Rectifying properties of the membrane of single freshly isolated smooth muscle cells

Single smooth muscle cells freshly isolated from the stomach muscularis of the toad Bufo marinus were studied under direct microscopic observation using intracellular microelectrodes. The deviation of the membrane potential from rest was recorded when steps of current were injected into the cell. Outward-going rectification was consistently observed both in the presence of 1.8 mM and higher external concentrations of Ca2+. There was no indication of inward-going rectification even under conditions favoring its demonstration, i.e., when the external concentration of K+ was high (108 mM) and Cl-, low (39.6 mM). In the presence of tetraethylammonium chloride (TEA), there was a marked decrease in the rectification normally observed with depolarizing currents, suggesting that a K+ conductance contributes to the outward-going rectification. This K+ conductance increased by almost two orders of magnitude over the range from -20 to 0 mV, and displayed an e-fold increase with a depolarization as small as 4-7 mV. In response to hyperpolarizing currents, the membrane potential did not always reach a plateau but at times continued to become more negative. The feasibility of the depletion of ions from the caveolae as an explanation for this observation is discussed.