Seon H. Shin

Oxidative stress induced by ascorbate causes neuronal damage in an in vitro system

Of particular physiological interest, ascorbate, the ionized form of ascorbic acid, possesses strong reducing properties. However, it has been shown to induce oxidative stress and lead to apoptosis under certain experimental conditions. Ascorbate in the brain is released during hypoxia, including stroke, and is subsequently oxidized in plasma. The oxidized product (dehydroascorbate) is transported into neurons via a glucose transporter (GLUT) during a reperfusion period. The dehydroascorbate taken up by cells is reduced to ascorbate by both enzymatic and non-enzymatic processes, and the ascorbate is stored in cells. This reduction process causes an oxidative stress, due to coupling of redox reactions, which can induce cellular damage and trigger apoptosis. Ascorbate treatment decreased cellular glutathione (GSH) content, and increased the rates of lipid peroxide production in rat cortical slices. Wortmannin, a specific inhibitor of phosphatidylinositol (PI)-3-kinase (a key enzyme in GLUT translocation), prevented the ascorbate induced-decrease of GSH content, and suppressed ascorbate-induced lipid peroxide production. However, wortmannin was ineffective in reducing hydrogen peroxide (H(2)O(2))-induced oxidative stress. The oxidative stress caused ceramide accumulation, which was proportionally changed with lipid peroxides when the cortical slices were treated with ascorbate. These differential effects support the hypothesis that GLUT efficiently transports the dehydroascorbate into neurons, causing oxidative stress.

Glutathione protects PC12 cells from ascorbate- and dopamine-induced apoptosis.

Abstract The role of reduced glutathione (GSH) on ascorbate- and dopamine-induced apoptosis in PC12 cells was investigated. Ascorbate is a potent reducing agent and is thus expected to protect against dopamine-induced apoptosis. However, we found that both ascorbate and dopamine killed PC12 cells and ascorbate enhanced dopamine-induced toxicity. The EC50 of cell toxicity induced by ascorbate, dopamine and dopamine plus 0.1 mM ascorbate during 24-h treatment were 0.93±0.15 mM, 0.18±0.05 mM and 0.13±0.04 mM, respectively. When the medium contained 10 mM GSH, the EC50 increased approximately three- and sevenfold for ascorbate and dopamine, respectively. With increased treatment duration, no further toxic effects of ascorbate or dopamine were observed. The GSH synthesis inhibitor, dl-buthionine-(S,R)-sulfoximine (BSO), induced cell toxicity and potentiated the toxic effects of ascorbate and dopamine, suggesting that endogenous GSH participates in protecting against basal oxidative stress. We conclude that both ascorbate and dopamine induce apoptosis in PC12 cells and further that GSH protects them from apoptosis. This study indicates that the toxic effects of ascorbate are potentially due to an oxidative mechanism, similar to that induced by dopamine.

Prolactin secretion in acute stress is controlled by prolactin releasing factor.

Abstract Experiments were carried out to demonstrate that the surge of prolactin release with ether stress is due to the release of a prolactin releasing factor rather than an inhibition of release of prolactin inhibiting factor (PIF). When the normal male rat was exposed to ether dopamine (30 ng/10 μl/min), a putative PIF, was infused through the right carotid artery, the prolactin surge still occurred. The elevated circulating prolactin level induced by estradiol implantation was lowered by the infusion of dopamine (30 ng/10 μl/min), indicating that the infused dopamine was reaching the adenohypophysis. The lowered prolactin concentration caused by the infusion of dopamine was elevated by ether stress. The hypothesis that the prolactin surge following ether stress is due to the inhibition of PIF is unlikely since the surge subsequent to ether stress occurred during a constant infusion of the putative PIF, dopamine. We concluded that the prolactin surge is due to the stimulation of PRF secretion rather than an inhibition of PIF secretion.

Prooxidant effects of ascorbate in rat brain slices

Ascorbate is a well‐known reducing agent, but it can generate oxidative potential under appropriate condition. In rat cerebral cortex homogenate, 1 mM ascorbate decreased thiobarbituric acid‐reactive substances (TBARS) content to 86% ± 4% of control values, confirming that ascorbate is a reducing agent. However, ascorbate increased TBARS, in a dose‐related manner, in slices prepared from cerebral cortex. Ferrous ion (Fe2+) had little effect on ascorbate‐induced lipid oxidation in cortical slices, and EDTA did not have an influence on the ascorbate‐induced oxidative action. Conversely, ascorbate plus Fe2+ elevated TBARS content to more than threefold over ascorbate alone in tissue homogenates. In summary, ascorbate is a reducing agent in the brain tissue homogenate but has an oxidizing effect in brain slices. A hypothesis is proposed to explain the oxidative effects of ascorbate in cortical slices, wherein extracellular ascorbate is oxidized to dehydroascorbate, which is rapidly carried into the cells via a glucose transporter (GLUT). The dehydroascorbate in cytosol is then reduced back to ascorbate, and, during the reduction process, cellular components are oxidized. J. Neurosci. Res. 58:328–336, 1999.

Adenohypophysis Has an Inherent Property for Pulsatile Prolactin Secretion

Hypophysectomized male rats with adenohypophyses grafted under the kidney capsule (HG rat) for 3 weeks with or without estradiol implantation, were used to examine prolactin (PRL) secretion independent of hypothalamic influence. Changes in the circulating PRL concentration were monitored by taking blood samples every 2 min via an indwelling atrial cannula. The circulating PRL concentration did not remain at a constant level but fluctuated with time. When estradiol (100 microgram/kg) was administered to HG rats through the indwelling cannula the size of the PRL pulses was magnified. The ability of the grafted adenohypophyses to generate pulsatile changes in the plasma PRL indicates that the adenohypophysis possesses an inherent interlactotroph communication system to permit synchronized PRL secretion without the direct involvement of hypothalamic PRL-releasing factors or inhibiting factors.

A possible phagocytic role for folliculo-stellate cells of anterior pituitary following estrogen withdrawal from primed male rats

SummaryUltrastructural changes suggesting a phagocytic role for the nongranular folliculo-stellate cells of the anterior pituitary are investigated in estrogen-primed male rats after withdrawal of estrogen. Morphological changes in mammotropes following the removal of a subcutaneous estradiol-containing Silastic implant include the formation of intracellular lipid bodies. These lipid bodies appear to be associated with enhanced estrogen-dependent prolactin secretion in mammotropes. Seven and 24 h after estrogen withdrawal intracellular lipid within mammotropes seems to be released into the intercellular space. Seventy-two h after estrogen withdrawal, lipid droplets are almost entirely cleared from mammotropes while folliculo-stellate cells become packed with lipid globules. Folliculo-stellate cells also undergo dramatic hypertrophy 7 and 24 h after the removal of E2-containing implants. Extensive intercellular junctions including zonulae adhaerentes, desmosomes, and putative gap junctions are formed. Intercellular junctions delineate extravascular channels into which numerous microvilli project. Folliculo-stellate cells appear capable of accumulating many lipid droplets, presumably related to mammotrope metabolism. What appear to be large secondary lysosomes as well as the lipid droplets are observed within folliculostellate cells; lipid, therefore, may be degraded through a lysosomal pathway in folliculo-stellate cells.

Physiological Evidence for the Existence of Prolactin Releasing Factor: Stress-Induced Prolactin Secretion Is not Linked to Dopaminergic Receptors

Experiments were undertaken to demonstrate the existence of a physiological role of prolactin releasing factor (PRF). Hypothalamic control of prolactin secretion is achieved by secretion of prolactin inhibiting factor (PIF) and/or PRF. Since the putative PIF is dopamine, complete blockage of the dopaminergic PIF receptors should permit demonstration of PRF activity. The changes in prolactin concentration were monitored by taking a blood sample every 2 min through an indwelling atrial cannula. An excessive amount of pimozide (3 mg/kg) was used to block dopaminergic receptors and prolactin concentration was elevated as a result. This higher concentration of circulating prolactin was maintained for more than 30 h after a bolus injection of pimozide. In this situation, lacking functional dopaminergic PIF receptors, there were fluctuations in the circulating prolactin concentration throughout the experiment. If, after pimozide administration, rats were exposed to an acute (ether) stress, the stress consistently elevated the circulating prolactin concentration. Since there are no functional dopaminergic PIF receptors available under these conditions, a dopaminergic PIF cannot be involved in producing the prolactin surge induced by the acute stress. Therefore, these results suggest that a physiological PRF is involved in the generation of this prolactin surge, through the evidence is not direct.

Effect of Testosterone on Hypothalamic LH-RH Content

Hypothalamic LH-RH content in male rats is lowered after castration. The s.c. implantation of testosterone or testosterone propionate-packed Silastic tubing (from 0.5 to 6 cm in length) in a range which encompassed the normal circulating plasma testosterone concentration, prevented this lowered LH-RH content 21 days following castration and simultaneous implantation. The temporal response to implanted testosterone was then studied: rats were killed 1,2,4,8,14 and 21 days after castration and simultaneous implantation of 3 cm testosterone-packed Silastic tubing. The hypothalamic LH-RH content began to decrease in the castrated group after 4 days and fell progressively thereafter. However, the hypothalamic LH-RH content of the castrated group maintained with constant levels of testosterone showed no such reduction at any time following castration. These experiments indicate that circulating testosterone in physiological concentrations can maintain a normal hypothalamic LH-RH content and demonstrate an action of testosterone, in physiological concentrations, in the feedback regulation of LH-RH secretion.

Zinc and Copper inhibit nerve growth factor-mediated protection from oxidative stress-induced apoptosis

We have previously provided evidence that two transition metal cations, Zn2+ and Cu2+, can alter the conformation of nerve growth factor (NGF), rendering it unable to bind to its receptors or to activate signal transduction pathways. In the present study, we have assessed the influence of Zn2+ and Cu2+ on NGF-mediated protection from an oxidative insult. Exposure of rat pheochromocytoma (PC12) cells to hydrogen peroxide resulted in an increase in cell death via apoptosis, which was inhibited by NGF. Zn2+ and Cu2+, when added to cultures at a concentration of 100 microM, prevented NGF-mediated survival-promoting effects. Neither of these ions had an effect on basal cell viability (in the absence of NGF) after an oxidative insult. These results demonstrate that Zn2+ and Cu2+ can selectively inhibit NGF-mediated resistance to an oxidative stress, and have significant implications for neuronal function under both physiological and pathological (e.g. cerebral ischemia) conditions.

Vasopressin Has a Direct Effect on Prolactin Release in Male Rats

Prolactin-releasing (PRF) activity was found in Pitressin (a commercial extract from posterior pituitary for vasopressin). Injection of Pitressin into conscious free-moving rats implanted with a permanent atrial indwelling cannula, produced a transient increase in prolactin concentration in the circulation. In order to find out whether the PRF activity was due to vasopressin or to an unidentified component in the Pitressin, we tested synthetic lysine vasopressin and demonstrated that vasopressin (1 U/kg) elevated plasma prolactin concentration about threefold. In contrast, oxytocin (1 U/kg) did not alter the prolactin concentration. In order to find out whether the effect of vasopressin is a direct or indirect action, we tested the vasopressin effect on hypophysectomized rats which had previously been implanted with 2 adenohypophyses under the kidney capsule. Again this dose (2 U/kg) of vasopressin elevated circulating plasma prolactin. These experiments indicate that vasopressin can elevate circulating prolactin concentration in nonestrogen-primed normal male rats and that vasopressin also stimulates prolactin secretion from transplanted glands dissociated from direct hypothalamic control.