Biomedical subjects

J X Wilson

Publications and source records attributed to J X Wilson.

At least 37 records · Page 2Linked to original sources

PubMed1995

Ascorbate concentration in osteoblastic cells is elevated by transforming growth factor-beta.

Transforming growth factor-beta modulates the proliferation, differentiation, and synthetic activity of osteoblasts, but its mechanisms of action are not fully understood. Because ascorbate also influences osteoblast differentiation and is a cofactor for collagen synthesis, the present study examined the effect of transforming growth factor-beta on the initial rate of transport and steady-state concentration of ascorbate in an osteoblastic cell line. UMR-106 rat osteosarcoma cells accumulated reduced vitamin C from culture medium. Virtually all accumulation of ascorbate was accomplished by a saturable Na(+)-dependent transport mechanism. Transforming growth factor-beta increased the initial rate of ascorbate transport, measured in either attached or suspended cells. Within 24 h, the growth factor also increased the steady-state intracellular concentration of ascorbate, without significantly changing cell volume or the DNA or protein content of cultures. These data provide evidence that Na(+)-ascorbate cotransport activity controls ascorbate concentration in osteoblasts. Furthermore, the results indicate that both the transport rate and steady-state concentration of ascorbate in these cells are regulated by transforming growth factor-beta.

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PubMed1993

Cardiac and sympathetic effects of middle cerebral artery occlusion in the spontaneously hypertensive rat.

Acute increases in sympathetic activity, plasma catecholamine concentrations and myocardial damage, occur following middle cerebral artery occlusion (MCAO) in Wistar rats. Hypertension is a major risk factor for stroke. The autonomic responses to MCAO in the spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats were therefore investigated. Arterial pressure (AP), heart rate (HR), renal sympathetic nerve discharge (SND), plasma catecholamines and ECG were measured in 16 SHR and 16 WKY male urethane-anesthetized rats, which were subjected to either MCAO or sham MCAO. Cerebral infarct size did not differ between SHR and WKY rats, as shown by tetrazolium staining. Initial AP was significantly higher in SHR (96 +/- 4 mmHg) than in WKY (70 +/- 1 mmHg; P < 0.05). No significant differences in initial HR or plasma catecholamine levels were observed between SHR and WKY. By 6 hours after MCAO, AP, SND and plasma epinephrine in SHR decreased significantly, while HR showed a significant increase. SND and plasma catecholamines in the WKY showed increases that did not reach significant levels following MCAO. The QT interval of the ECG was significantly prolonged in the WKY MCAO rats, which also had a higher frequency of cardiac myocytolysis than the other groups. Unlike the increases in autonomic variables following MCAO in Wistar rats, SHR exhibit significant decreases in SND and AP, while WKY show slight, but non-significant increases. These differences in the autonomic reaction to MCAO may reflect genetic differences in the response to cerebral ischemia.

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PubMed1992

Plasma and organ catecholamine levels following stimulation of the rat insular cortex.

The posterior insular cortex of the rat contains an area of cardiac chronotropic representation within which tachycardia sites occur rostrally to those producing bradycardia. In the current study using ketamine-anesthetized rats, the insular cortex was stimulated for 1 h using a phasic technique synchronized with the cardiac cycle. Tachycardia was associated with an increase in plasma norepinephrine concentration; epinephrine remained unchanged. This indicates a neural origin of the norepinephrine increment. The tachycardia response was completely blocked by atenolol. Plasma catecholamine levels remained unchanged during stimulation of insular bradycardia sites. Atenolol was without effect during stimulation-induced bradycardia which was completely blocked by atropine. Total cardiac norepinephrine concentration inversely correlated with change in heart rate during stimulation of tachycardia sites. No correlation between intracardiac catecholamines and heart rate variables was found for the bradycardia or control sites. These results indicate that in the ketamine-anesthetized rat, whereas insular stimulation-induced tachycardia is dependent on the sympathetic nervous system, bradycardia elicited by insular cortex stimulation is mediated by parasympathetic mechanisms. No correlation was identified between renal or skeletal muscle norepinephrine levels and any heart rate parameter. This implies that the sympathetic effects of phasic insular microstimulation may be exerted mainly on cardiac nerves, and less so in other visceral beds.

Animals↗

PubMed1992

Effect of age on autonomic and cardiac responses in a rat stroke model.

The cardiovascular system and its responses change with increasing age. This has seldom been considered in experimental models of stroke, although most strokes occur in the elderly. We studied 57 male Wistar rats in three age groups: 47 to 70 days old (juvenile), 110 to 152 days old (young adult), and 186 to 245 days old (mature adult), each group being subdivided into experimental and sham operation groups. All rats underwent occlusion or sham occlusion of the left middle cerebral artery and monitoring of the mean arterial blood pressure, heart rate, sympathetic nerve activity, plasma catecholamine levels, and electrocardiogram. Eight of the 12 rats in the oldest group died within 6 hours of the middle cerebral artery occlusion; of these, the youngest was 186 days old. The mature adult rats that died before completion of the experiment showed the highest level of sympathetic nerve activity and the only significant increase in the QT interval of the electrocardiogram. Following middle cerebral artery occlusion, sympathetic nerve activity increased in the young adult rats but most strikingly in the mature adult rats that died before the end of the 6-hour experiments. Plasma norepinephrine levels were significantly elevated at 4 and 6 hours after middle cerebral artery occlusion in the oldest group and only at 6 hours in the juvenile rats. The results of this study are consistent with impaired sympathetic and cardiovascular regulation in the mature adult rat. High sympathetic activity may represent one mechanism leading to fatal cardiac arrhythmias. Age-related impairment of sympathetic regulation may contribute to the higher mortality seen among elderly patients with stroke.

Aging↗

PubMed1992

Asymmetry of sympathetic consequences of experimental stroke.

Asymmetries of sympathetic regulation at the level of the inferior cervical ganglia have long been recognized. Lateralization of autonomic representation may also occur in the brain, since inactivation of the left and right hemispheres by intracarotid amobarbital produces an increase and decrease in heart rate, respectively. However, this conclusion has remained tentative, since the differential effect of lateralized brain lesions on sympathetic activity has not been studied systematically. Forty-eight urethan-anesthetized Wistar rats were divided into three groups: a group given left middle cerebral artery occlusion, and a group given sham operation. Heart rate, mean arterial blood pressure, renal sympathetic nerve discharge, and electrocardiogram were monitored throughout the 4-hour experiments. Plasma epinephrine and norepinephrine levels were measured at baseline and 1 and 4 hours after occlusion or sham occlusion. The mean arterial pressure decreased in the group given sham operation and to lesser extent in the group given left middle cerebral artery occlusion. By contrast, mean arterial pressure did not fall in the group given right middle cerebral artery occlusion and at 4 hours was significantly higher than control values in the sham-occluded rats. Renal sympathetic nerve discharge was decreased in the sham-occluded group, increased significantly from 20 minutes to 2 hours in the group given left middle cerebral artery occlusion, and increased from about 20 minutes to the end of the experiment in the group given right middle cerebral artery occlusion. The plasma norepinephrine level was significantly elevated at 1 hour (93%) and 4 hours (44%) only in the group given right middle cerebral artery occlusion.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1992

Adaptive regulation of ascorbate transport in osteoblastic cells.

Osteoblasts possess a concentrative L-ascorbate (vitamin C) uptake mechanism involving a Na(+)-dependent ascorbate transporter located in the plasma membrane. The transporter is specific for ascorbate and stereoselective for L-ascorbate over D-isoascorbate. The present study examined the effects of ascorbate supplementation and deprivation on the activity of this transport system. L-ascorbate transport activity was determined by measuring uptake of the vitamin by ROS 17/2.8 osteosarcoma cells during 1 minute incubations with 5 microM L-[14C]ascorbate. The initial rate of L-[14C]ascorbate uptake by ROS 17/2.8 cells grown for 18 h in L-ascorbate-replete medium was 89 +/- 8 nmol/g protein per minute. Following removal of L-ascorbate from the growth medium, the initial rate of uptake increased within 6 h to 126 +/- 13 nmol/g protein per minute. Conversely, the initial rate of uptake by cells grown in ascorbate-free medium decreased following the addition of L-ascorbate, but not D-isoascorbate, to the medium. The effect of ascorbate pretreatment was specific for ascorbate transport in that preincubation of cultures with L-ascorbate did not affect uptake of 2-deoxy-D-glucose. Kinetic analysis revealed that modulation of ascorbate transport arose from changes in the apparent maximum rate of transport (Vmax) without changes in the affinity of the transport system for L-ascorbate. These experiments are the first to show that ascorbate transport by osteoblastic cells responds to vitamin C deprivation and supplementation. Adaptation of transport activity to substrate availability may play an important role in the physiological regulation of intracellular ascorbate levels.

Ascorbic Acid↗

PubMed1992

Effect of oxygen on ascorbic acid uptake and concentration in embryonic chick brain.

The effects of oxygen on ascorbic acid concentration and transport were studied in chick embryo (Gallus gallus domesticus). During normoxic incubations, plasma ascorbic acid concentration peaked on fetal day 12 and then fell, before increasing again on day 20 when pulmonary respiration began. In contrast, cerebral ascorbic acid concentration rose after day 6, was maintained at a relatively high level during days 8-18, and then fell significantly by day 20. Exposure of day 16 embryos for 48 h to 42% ambient O2 concentration decreased ascorbic acid concentration by four-fifths in plasma and by one-half in brain, compared to values in normoxic (21% O2) or hypoxic (15% O2) controls. Hyperoxic preincubation of embryos also inhibited ascorbic acid transport, as evidenced by decreased initial rates of saturable and Na(+)-dependent [14C]ascorbic acid uptake into isolated brain cells. It may be concluded that changes in ascorbic acid concentration occur in response to oxidative stress, consistent with a role for the vitamin in the detoxification of oxygen radicals in fetal tissues. However, changing O2 levels have less effect on ascorbic acid concentration in brain than in plasma, indicating regulation of the vitamin by brain cells. Furthermore, the effect of hyperoxia on cerebral vitamin C may result, in part, from inhibition of cellular ascorbic acid transport.

Animals↗

PubMed1992

Developmental profiles of antioxidant enzymes and trace metals in chick embryo.

It has been previously well documented that partial pressure of oxygen (PO2) and weight-specific rate of O2 consumption in chick embryo (Gallus gallus domesticus) transiently increase midway through the 21-day in ovo incubation period. The present study found that these oxidative changes were paralleled by the concentrations of glutathione (GSH) and Zn in liver and by the specific activity of superoxide dismutase (SOD) in brain. Levels of antioxidant enzymes and their trace metal cofactors were markedly higher in liver than in brain. Hepatic catalase activity changed in parallel with the concentration of its cofactor, Fe. However, the relative abundance of metal cofactors did not appear to be the determining influence on other antioxidant enzyme activities. Rates of extra-mitochondrial hydrogen peroxide release were also much greater in liver than in brain. Taken together, the results of this initial study of embryonic chick antioxidant systems suggest that certain antioxidants may be regulated by PO2 and rate of oxidative metabolism during fetal development.

Animals↗

PubMed1992

Transforming growth factor-beta stimulates ascorbate transport activity in osteoblastic cells.

Transforming growth factor-beta (TGF beta) modulates the proliferation and differentiation of a number of cell types, including osteoblasts. TGF beta has been shown to stimulate matrix synthesis by connective tissue cells, but its mechanism of action is poorly understood. Because ascorbate (reduced vitamin C) also influences osteoblastic differentiation and is required as a cofactor for collagen synthesis, the present study examined the effect of TGF beta on osteoblastic ascorbate uptake. Saturable Na(+)-dependent uptake of ascorbate by cultures of UMR-106 rat osteosarcoma cells proceeded linearly with time for at least 10 min at 37 C. Exposure of cultures to TGF beta 1 stimulated initial rates of saturable Na(+)-dependent ascorbate transport, but did not affect nonspecific uptake or binding of the vitamin. Cells pretreated for 24 h with either vehicle or TGF beta 1 (3 ng/ml) and then assayed for transport of L-[14C] ascorbate (10 microM) showed significantly different transport activities (vehicle, 30 +/- 2; TGF beta 1, 44 +/- 3 nmol ascorbate/g protein/min; n = 14; P less than 0.005). Kinetic studies revealed that TGF beta 1 increased the maximum velocity of ascorbate transport without changing the affinity of the transporter for the vitamin, since the apparent maximum velocity increased from 83 to 106 nmol ascorbate/g protein/min; while the apparent Km remained unchanged at 20 microM L-ascorbate. The effect of this growth factor on ascorbate transport appeared to require protein synthesis, because it was completely blocked by cycloheximide. These results are consistent with TGF beta 1 increasing the rate of synthesis of either new Na+ ascorbate cotransporters or a regulatory protein that interacts with existing transporters to increase their turnover number. Enhanced uptake of ascorbate may contribute to the increase in collagen synthesis induced by TGF beta.

Animals↗

PubMed1991

Insular cortex stimulation produces lethal cardiac arrhythmias: a mechanism of sudden death?

The rat posterior insular cortex has recently been shown to possess cardiac chronotropic organization and therefore may be involved in cortical mechanisms of sudden death. In order to assess the potential of this region for cardiac arrhythmogenicity, phasic microstimulation of tachycardia zones was undertaken in the urethane-anesthetized rat. The insular stimulus was triggered by the R wave of the electrocardiogram (ECG) and delayed so that resultant putative cardiac sympathetic nerve activity would be synchronous with the T wave of the ECG. This resulted in increasing degrees of heart block leading to escape rhythms, ventricular ectopics and ultimately death in asystole. Heart block was associated with elevated plasma norepinephrine levels and myocardial damage. Such effects have not been previously demonstrated for a cortical site. These data suggest that pathophysiological activation of the insular cortex by stroke, epileptic seizure, or under conditions of severe emotional stress could predispose to ECG changes, cardiac arrhythmias and sudden death.

Animals↗

PubMed1991

Expression of gap junction genes in astrocytes and C6 glioma cells.

The expression of the gap junction genes coding for the liver-type connexin32 and the heart-type connexin43 was examined in primary cultures of astrocytes and in cultures of C6 glioma cells. In both cell types, only connexin43 mRNA was detectable. However, the level of this mRNA was greatly reduced in C6 glioma cells compared to astrocytes. This was consistent with the further observation that astrocytes in primary culture were extensively dye-coupled, whereas such coupling was very restricted in cultures of C6 glioma cells. Connexin43 was immunocytochemically localized in astrocytes, but was not readily detected in C6 cells.

Animals↗

PubMed1991

Ascorbate uptake by ROS 17/2.8 osteoblast-like cells: substrate specificity and sensitivity to transport inhibitors.

Ascorbate (reduced vitamin C) is required for bone formation. We have shown previously that both the osteoblast-like cell line ROS 17/2.8 and primary cultures of rat calvarial cells possess a saturable, Na(+)-dependent uptake system for L-ascorbate (J Membr Biol 111:83-91, 1989). The purpose of the present study was to investigate the specificity of this transport system for organic anions and its sensitivity to transport inhibitors. Initial rates of ascorbate uptake were measured by incubating ROS 17/2.8 cells with [L-14C]ascorbate at 37 degrees C. Uptake of [L-14C]ascorbate (5 microM) was inhibited 98 +/- 1% by coincubation with unlabeled L-ascorbate (3 mM) and 48 +/- 4% by salicylate (3 mM), but it was not affected by 3 mM formate, lactate, pyruvate, gluconate, oxalate, malonate, or succinate. Uptake of the radiolabeled vitamin also was not affected by acute (1 minute) exposure of the cells to the Na+ transport inhibitors amiloride and ouabain or the glucose transport inhibitor cytochalasin B. In contrast, anion transport inhibitors rapidly (less than 1 minute) and reversibly blocked [L-14C]ascorbate uptake. In order of potency, these drugs were 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) approximately equal to sulfinpyrazone greater than furosemide approximately equal to 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS). These findings indicate that the ascorbate transporter is relatively specific for the ascorbate anion, since other organic anions (with the exception of salicylate) did not compete with ascorbate for uptake. Rapid and reversible inhibition by the impermeant antagonists DIDS and SITS suggests that they interact directly with the ascorbate transporter, consistent with location of the transport system in the plasma membrane.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1991

Evidence for electrogenic sodium-dependent ascorbate transport in rat astroglia.

The dependence of ascorbate uptake on external cations was studied in primary cultures of rat cerebral astrocytes. Initial rates of ascorbate uptake were diminished by lowering the external concentrations of either Ca2+ or Na+. The Na(+)-dependence of astroglial ascorbate uptake gave Hill coefficients of approximately 2, consistent with a Na(+)-ascorbate cotransport system having stoichiometry of 2 Na+:1 ascorbate anion. Raising external K+ concentration incrementally from 5.4 to 100 mM, so as to depolarize the plasma membrane, decreased the initial rate of ascorbate uptake, with the degree of inhibition depending on the level of K+. The depolarizing ionophores gramicidin and nystatin slowed ascorbate uptake by astrocytes incubated in 5.4 mM K+; whereas, the nondepolarizing ionophore valinomycin did not. Qualitatively similar results were obtained whether or not astrocytes were pretreated with dibutyryl cyclic AMP (0.25 mM for 2 weeks) to induce stellation. These data are consistent with the existence of an electrogenic Na(+)-ascorbate cotransport system through which the rate of ascorbate uptake is modulated by endogenous agents, such as K+, that alter astroglial membrane potential.

Animals↗

PubMed1991

Accumulation of noradrenaline and its oxidation products by cultured rodent astrocytes.

The accumulation of [3H]noradrenaline ([3H]NA) and its oxidation products was studied in primary cultures of cerebral astrocytes. Astroglial accumulation of radiolabeled catecholamine ([3H] NA and oxidation products) was enhanced by manganese or iron, but it was inhibited by unlabeled NA, dopamine or ascorbate. Tissue: medium ratios of radioactivity increased as extracellular [3H]NA was oxidized. When extracellular oxidation was prevented by ascorbate, as confirmed by high performance liquid chromatography with electrochemical detection, either ouabain pretreatment or nominally Na(+)-free incubation medium inhibited approximately one-half of specific [3H]NA accumulation by rat (but not mouse) astrocytes. These observations suggest that neurological responses to trace metals and ascorbate may arise from the effects of these agents on the clearance of extracellular catecholamines. Astrocytes can accumulate oxidation products of NA more rapidly than they take up NA itself, but ascorbate at physiological concentrations prevents the oxidation process in extracellular fluid. Furthermore, in the presence of ascorbate, Na(+)-dependent transport mediates a significant component of NA accumulation in rat astrocytes.

Animals↗

PubMed1990

Substrate regulation of ascorbate transport activity in astrocytes.

Astrocytes possess a concentrative L-ascorbate (vitamin C) uptake mechanism involving a Na(+)-dependent L-ascorbate transporter located in the plasma membrane. The present experiments examined the effects of deprivation and supplementation of extracellular L-ascorbate on the activity of this transport system. Initial rates of L-ascorbate uptake were measured by incubating primary cultures of rat astrocytes with L-[14C]ascorbate for 1 min at 37 degrees C. We observed that the apparent maximal rate of uptake (Vmax) increased rapidly (less than 1 h) when cultured cells were deprived of L-ascorbate. In contrast, there was no change in the apparent affinity of the transport system for L-[14C]ascorbate. The increase in Vmax was reversed by addition of L-ascorbate, but not D-isoascorbate, to the medium. The effects of external ascorbate on ascorbate transport activity were specific in that preincubation of cultures with L-ascorbate did not affect uptake of 2-deoxy-D-[3H(G)]glucose. We conclude that the astroglial ascorbate transport system is modulated by changes in substrate availability. Regulation of transport activity may play a role in intracellular ascorbate homeostasis by compensating for regional differences and temporal fluctuations in external ascorbate levels.

Animals↗

PubMed1990

Regulation of ascorbic acid concentration in embryonic chick brain.

The relationship between ascorbic acid concentration and cellular transport mechanisms was studied in chicken embryos (Gallus gallus domesticus). Unincubated (Day 0) fertile eggs did not contain detectable levels of ascorbic acid as assayed by high performance liquid chromatography with electrochemical detection. However, ascorbic acid concentration in brain increased to 5.6 nmol/mg tissue by Day 10 in ovo and then gradually declined 32% before birth. These levels were an order of magnitude greater than in skeletal muscle, where ascorbic acid concentration decreased sixfold between Days 8-20. Uptake of ascorbic acid was measured in brain cells that were either freshly isolated or grown in primary culture. Saturable, temperature- and Na(+)-dependent ascorbic acid transport was evident in freshly isolated cells as early as Day 6 and persisted throughout the period of ontogenic development. Primary cultures of embryonic chick brain cells were observed to take up ascorbic acid through a high-affinity (apparent Km = 37 microM, Vmax = 106 nmol ascorbic acid/g protein/min) mechanism. This transport system may maintain the high concentrations of ascorbic acid observed in the central nervous system during the ontogenic period when the levels of ascorbic acid in peripheral tissues change drastically.

Animals↗

PubMed1990

Ascorbic acid potentiates the inhibitory effect of dopamine on prolactin release: a putative supplementary agent for PIF.

Dopamine has a catechol group which can be easily oxidized by mild oxidizing agents. Ascorbic acid has been routinely added to a dopamine solution in order to protect it from oxidation. We have examined the effect of ascorbic acid on dopaminergic inhibition of prolactin release. Male rat pituitary cells were dispersed using trypsin and cultured for 5-7 days before experiments. Ascorbic acid did not stimulate nor inhibit prolactin release in both static monolayer culture and dynamic perifusion systems, but potentiated by approximately 100 times the inhibitory effect of dopamine on prolactin release. In order to differentiate chemical protection from potentiation, we tested the potentiation effect of isoascorbic acid which is an epimer of biologically active L-ascorbic acid but is biologically less active. Our results indicated that isoascorbic acid caused less potentiation of the dopaminergic effect on prolactin release than did ascorbic acid. In a perifusion system, a high concentration of dopamine (100 nmol/l) was unable to inhibit prolactin release for a 1 h experimental period, but a low concentration of dopamine (10 nmol/l) plus ascorbic acid (10 mumol/l) inhibited prolactin release for the entire 1 h perifusion period. There is a strong possibility that ascorbic acid may be a physiological supplementary agent for the prolactin-release inhibiting factor (PIF) since the blood concentration of ascorbic acid is rather high (23-85 mumol/l).

Animals↗

PubMed1989

Autonomic and myocardial changes in middle cerebral artery occlusion: stroke models in the rat.

Stroke models in larger animals such as the cat, dog and monkey are becoming increasingly more expensive and less readily available. However, the rat is an excellent model for focal cerebral ischemia. Rats are readily available, inexpensive and their neuroanatomy and brain function have been studied extensively. Increases in plasma catecholamines and myocardial damage have been observed in clinical stroke. We examined autonomic and myocardial changes in two rat stroke models. In one model only the middle cerebral artery was occluded (MCAO) while the other model involved occlusion of both the MCA and the common carotid artery (MCAO/CCAO). Arterial blood pressure and heart rate were monitored continuously in 25 male rats (326-430 g) that underwent one of the following procedures: (1) MCAO only; (2) MCAO/CCAO; (3) CCAO only; and (4) sham occlusions (SHAM). Arterial blood samples (0.5 ml) for radioenzymatic assay of norepinephrine (NE) and epinephrine (E) were taken twice before the occlusions and at 90 and 180 min after the occlusions. The animals were perfused at the end of the experiment and the heart removed and examined histologically. Tetrazolium salts were reacted with oxidative enzymes to delineate the region of inadequate perfusion. The mean blood pressure and pulse pressure of the SHAM, MCAO/CCAO and CCAO groups significantly declined from initial values (from an average of 78 to 53 mm Hg) during the course of the experiment. However, the mean blood pressure and pulse pressure of the MCAO rats did not change during the experiment, so that the final mean blood pressure and pulse pressure were significantly higher than in the other 3 groups. The levels of both NE and E increased significantly (NE, 1443 +/- 285.9 to 4095 +/- 929 pg/ml; E, 2402 +/- 623 to 3741 +/- 1166 pg/ml) following occlusion in the MCAO group only while the other 3 groups did not change. Four of 6 hearts in the MCAO group were abnormal, showing evidence of subendocardial hemorrhage, ischemic damage or subendocardial congestion. MCAO also resulted in a consistent region of the brain with inadequate perfusion including the insular cortex. These autonomic and myocardial changes appear to mimic some of the changes seen clinically in stroke patients and provide the first acute stroke model for studying autonomic dysfunction in the rat.

Animals↗