Involvement of xanthine oxidase in oxidative stress and iron release during hyperthermic rat liver perfusion.

The hepatotoxic effects of hyperthermia have been proposed to be related to lipid peroxidation as a consequence of oxidative stress. This can result from exposure of the cell to "radical oxygen" species such as the superoxide and hydrogen peroxide generated by the activity of the oxidase form (type O) of xanthine oxidase (XO), which is converted to that form by perfusion of the liver at hyperthermic temperatures. These radical species are not reactive enough in themselves to cause cell damage but require the presence of a catalyst such as low molecular weight chelated iron. In these studies, ferritin was shown to be a source of iron for the oxidative stress of hyperthermia. (a) Iron was released from ferritin in vitro by the activity of rat liver XO. The rate of iron release from ferritin in this incubation system was a function of the amount of type O XO present and the temperature. Inclusion of allopurinol or superoxide dismutase in the incubation resulted in significantly lower rates of iron release. (b) Livers from Sprague-Dawley rats were perfused at 42.5 degrees and 37 degrees C for 1 h. During the recirculating perfusion, loss of iron from the liver into the perfusate was significantly greater (P less than 0.05) at 42.5 degrees C than at 37 degrees C. Also, there was a pronounced increase in the lactate dehydrogenase and aspartate aminotransferase enzymes in the perfusate during perfusion at 42.5 degrees C. Furthermore, intrahepatic levels of low molecular weight chelated iron were significantly (P less than 0.05) increased following perfusion at 42.5 degrees C. All these responses were abrogated by the inclusion of allopurinol in the perfusate. (c) Oxidative stress, assessed by the efflux of glutathione and oxided glutathione from the liver at 42.5 degrees and 37 degrees C, was significantly (P less than 0.05) increased at the hyperthermic temperature. This oxidative stress was inhibited by iron chelation and allopurinol. These results demonstrate that there is a causal relationship between the generation of superoxide by type O XO produced by hyperthermic perfusion and mobilization of iron from ferritin to form a pool of low molecular weight chelated iron. This iron pool in combination with active oxygen species leads to oxidative stress and lipid peroxidation.

Cannabinoid CB1 receptor-mediated inhibition of prolactin release and signaling mechanisms in GH4C1 cells.

The GH4C1 cell line was used to study the cellular mechanisms of cannabinoid-mediated inhibition of PRL release. Cannabinoid CB1 receptor activation inhibited vasoactive intestinal polypeptide- and TRH-stimulated PRL release, but not its basal secretion. The cannabinoid-mediated inhibition of TRH-stimulated PRL release was reversed by the CB1 receptor-specific antagonist, SR141,716A, and was abolished by pertussis toxin pretreatment, indicating that G alpha subunits belonging to the G(i)alpha and G(o)alpha family were involved in the signaling. Photoaffinity labeling using [alpha-32P] azidoaniline GTP showed that cannabinoid receptor stimulation in cell membranes produced activation of four G alpha subunits (G(i)alpha2, G(i)alpha3, G(o)alpha1, and G(o)alpha2), which was also reversed by SR141,716A. The CB1 receptor agonists, WIN55,212-2 and CP55,940, inhibited cAMP formation and calcium currents in GH4C1 cells. The subtypes of calcium currents inhibited by WIN55,212-2 were characterized using holding potential sensitivity and calcium channel blockers. WIN55,212-2 inhibited the omega-conotoxin GVIA (Conus geographus)- and omega-agatoxin IVA (Aigelenopsis aperta)-sensitive calcium currents, but not the nisoldipine-sensitive calcium currents, suggesting the inhibition of N- and P-type, but not L-type, calcium currents. Taken together, the present findings indicate that CB1 receptors can couple through pertussis toxin-sensitive G alpha subunits to inhibit adenylyl cyclase and calcium currents and suppress PRL release from GH4C1 cells.

Lipid peroxidation caused by hyperthermic perfusion of rat liver.

The data presented support the premise that hyperthermia-induced hepatocellular injury is the end result of lipid peroxidation. Evidence for lipid peroxidation is the formation of diene conjugates and the decrease in microsomal P450 and glucose-6-phosphatase activity during hyperthermic liver perfusion.

Effects of lidocaine and bupivacaine on isolated rabbit mesenteric capacitance veins.

BACKGROUND AND OBJECTIVES: The direct effects of circulating lidocaine and bupivacaine on splanchnic capacitance veins have not been examined previously. This article reports on the effects of clinically relevant concentrations of lidocaine and bupivacaine on adrenergic responsiveness of isolated rabbit mesenteric veins and examines the mechanism of changes. METHODS: Rings of ileal mesenteric capacitance veins were suspended in tissue baths for isometric tension measurements. Effects of lidocaine and bupivacaine on contractile responses to adrenergic nerve stimulation, exogenous norepinephrine (10(-6) M NE), and potassium chloride (80 mM KCl) were examined in endothelium-intact, L-NAME (10(-4) M) treated or denuded veins. RESULTS: Constriction in response to adrenergic nerve stimulation was attenuated by lidocaine and bupivacaine in a dose-dependent manner, with the potency of bupivacaine being higher than lidocaine. Unstimulated or potassium-constricted veins with and without endothelium were unaffected by lidocaine (0.25-100 microg/mL) and bupivacaine (0.1-100 microg/mL). In veins preconstricted by exogenously administered NE, a cumulative increase of both anesthetics produced no effect at low doses, an augmentation of constriction to NE at 5-20 microg/mL bupivacaine and 20-100 microg/mL lidocaine, and minimal effect at 50-100 microg/mL bupivacaine. These actions persisted in denuded or L-NAME treated veins. Nonincremental delivery of high concentrations of lidocaine or bupivacaine produced relaxation of NE and potassium-constricted rings in the absence and presence of L-NAME. CONCLUSIONS: Lidocaine and bupivacaine in concentrations typical during uncomplicated regional anesthesia inhibit adrenergic neurotransmission in rabbit mesenteric capacitance veins and produce modest venodilatation. Higher doses, resembling concentrations during accidental intravascular injection, result in substantial loss in vasomotor control of these capacitance vessels, which may contribute to hemodynamic effects.

Localization of mercury in the rat ovary after oral administration of mercuric chloride.

The Timm sulphide-silver method was used for histochemical localization of mercury in the ovaries of young rats fed with mercuric chloride. After 12 weeks of mercuric chloride administration test animals showed disturbances in the estrous cycle manifested by the prolongation of diestrous phase. In the ovaries of these rats the deposits of mercury were shown to occur particularly in macrophages of atretic young and mature ovarian follicles and corpora lutea, within granulosa cells of atretic follicles, and in the lutein cells of freshly formed corpora lutea. Follicular fluid and, irregularly, germinal epithelium also showed the ability to mercury accumulation. The ovaries of control animals treated according to the Timm procedure demonstrated negative histochemical reactivity.

Enzyme histochemistry of cultured ovarian cells. I. Histochemistry of porcine preovulatory granulosa cells in culture.

Histochemical activity of hydroxysteroid dehydrogenases as well as alkaline and acid phosphatase was investigated in porcine granulosa cells cultured in vitro. Granulosa cells, isolated from preovulatory procine ovarian follicles, during in vitro culture showed activity of enzymes participating in steroid biosynthesis. High activity of delta53betta0H-SDH and G6P-DH as well as the activity of alkaline phosphatase, appearing in the course of culture, could be evidence of progressive luteinization of the cells. Activity of 17beta0H-SDH was lower and exhibited strong fluctuations, similarly low was 20xOH-SDH. Gonadotropic hormones caused the increase of synthesis and accumulation of intracellular lipids. They stimulated alkaline and acid phosphatase, and also the activity of the dehydrogenases. LH had the most visible effect. Estradiol stimulated the activity of acid but not alkaline phosphatase and was not influencing, even lowering the activity of dehydrogenases.

PIP: A histochemical evaluation of hydroxysteroid dehydrogenase (OH-SDH) activity in porcine granulosa cells isolated from preovulatory ovarian follicles and cultured in cell culture is presented. Granulosa cells during in vitro culture showed activity of enzymes participating in steroid biosynthesis. High activity of delta-5,3beta OH-SDH and glucose-6-phosphate dehydrogenase as well as the activity of alkaline phosphatase, appearing in the course of culture, could be evidence of progressive luteinization of the cells. 17beta OH-SDH activity was lower and exhibited strong fluctuations. 20alpha OH-SDH was similarly low. Gonadotropic hormones caused the increase of synthesis and accumulation of intracellular lipids, and stimulated alkaline and acid phosphatase and dehydrogenase activity. Luteinizing hormone had the most visible effect.

Effects of epidural anesthesia on splanchnic capacitance.

Splanchnic veins play an important role in the active control of total body circulatory capacitance. The effects of epidural anesthesia on splanchnic venous capacitance have not previously been examined. A rabbit model using direct measures of mesenteric vein diameter and sympathetic efferent nerve activity was used to test the response to epidural lidocaine at three different doses and to intramuscular lidocaine at two doses. Epidural anesthesia produced hypotension, mesenteric venodilatation, and interruption of sympathetic activity. Maximal changes of these parameters were comparable in the three epidural dosage groups but were more prolonged with increasing dose. High-dose systemic lidocaine caused smaller changes in arterial pressure and sympathetic activity. Further experiments were done to investigate the mechanism of splanchnic venodilatation. Passive vein distension and effects of circulating lidocaine or catecholamines are not likely contributing factors. Blocks limited to thoracic segments, but including the origin of splanchnic preganglionic fibers, produce comparable mesenteric venodilatation and sympathetic interruption as extensive thoracolumbar blocks. Blocks limited to lumbar segments, however, showed mesenteric venoconstriction and increased splanchnic sympathetic activity. The variable responses in splanchnic capacitance with the onset of epidural anesthesia are the result of the competing influences of increased sympathetic activity from decreasing blood pressure and blockade of sympathetic fibers to the splanchnic veins.

Effect of mercury nitrate on the histochemical activity of some dehydrogenases in primary cultures of rat kidney tubular cells.

Direct effect of sublethal and lethal doses of mercury nitrate on histochemical activity of G6PDH, LDH and SDH was investigated in primary cultures of rat tubular cells. Enzyme activities were studied histochemically after administration of mercury nitrate for periods extending from 5 min. to 24 hrs and also after 2,3,4 and 5 following days. The sublethal doses of mercury nitrate containing 1 microgram and 5 microgram of mercury were found to decrease histochemical activity of the studied dehydrogenases as early as 10 and 15 minutes. Their inhibitory effect was much stronger after 24 h and depended on the dose of mercury compound. The lethal doses of mercury nitrate containing 15 microgram and 20 microgram of mercury depressed the activity of dehydrogenases within 5 min. after administration. The loss of enzyme activities usually preceded the appearance of necrotic signs in the cultured cells. It was also found that the cultured cells of kidney tubules treated with sublethal doses of mercury nitrate were usually able to regain the normal level of the enzymic activity within 2-5 days.

Enzyme histochemistry of cultured ovarian cells. III. Histochemical characteristics of bovine granulosa and theca interna cells grown separately in cell culture.

Granulosa and theca interna cells were isolated from bovine preovulatory ovarian follicles. They were cultured separately but in the same conditions of cell culture. Both cell types, grown as monolayers, were investigated histochemically with special regard to the activity of several hydroxysteroid dehydrogenases: delta53betaOH-SDH, 17betaOH-SDH, 20alphaOH-SDH and G6P-DH. Bovine granulosa and theca interna cells during in vitro culture showed high activity of delta53betaOH-SDH and G6P-DH, the enzymes essential to progesterone biosynthesis. Enzyme pattern of cultured cells indicated continuation in vitro of luteinization, which in the normal preovulatory follicle of the bovine ovary begins prior to ovulation. There was investigated as well the influence of single doses of gonadotrophic hormones and estradiol on growth, lipid contents and enzymic activity of cultured in vitro bovine granulosa and theca interna cells.

Enzyme histochemistry of cultured ovarian cells. II. Histochemistry of porcine theca interna cells in tissue culture.

By means of histochemical technique the activities of delta53beta-, 17beta-, 20alpha-hydroxysteroid dehydrogenases and glucose-6-phosphate dehydrogenase, as well as alkaline and acid phosphatase were investigated in monolayer cultures of theca interna cells, isolated from preovulatory porcine ovarian follicles. It was found that theca interna cells exhibited high and constant activity of delta53beta-hydroxysteroid dehydrogenase and G6P-DH, whereas activities of both 17beta- and 20alpha-hydroxysteroid dehydrogenase were lower and showed some fluctuations during in vitro culture. Addition of LH to the medium brought about the increase of all studied dehydrogenases. FSH was less effective. Estradiol showed quite and inhibiting effect. All the hormones mentioned above caused the increase of alkaline and acid phosphatase activity in cultured porcine theca interna cells.

Cytochemistry and ultrastructure of the prenatal porcine adrenal medulla.

The ultrastructure and cytochemistry of fetal porcine adrenal medullae have been studied at 60, 80, and 100 days of gestation. Adrenal medullae from fetuses at 60 days of pregnancy consisted of norepinephrine cells only. Some cells containing chromaffin granules were seen in the process of mitosis. A few epinephrine cells were present in the outer medullary zone at 80 days at pregnancy, their number increasing by the 100 day of pregnancy. Chromaffin cells containing both norepinephrine and epinephrine storing granules were also present at 80 and 100 days of gestation. Norepinephrine and epinephrine specific granular vesicles in the fetal adrenal medullary cells were smaller than those reported for the adult pig. The general ultrastructural characteristics of the porcine fetal adrenal medulla were similar to those reported for prenatal adrenal medulla of other species.

Hyperthermic liver toxicity: a role for oxidative stress.

Rat livers were perfused at 37 degrees C, 41 degrees C, 42 degrees C, 42.5 degrees C, and 43 degrees C for 2 hr. Among perfusate constituents analyzed were urea, total amino acids, N-acetyl-beta-glucosaminidase (NAG), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), malonaldehyde (MDA), glutathione (GSH), oxidized glutathione (GSSG), allantoin, potassium, phosphate, and glucose. After perfusion, livers were homogenized and analyzed for xanthine oxidase (XO) activity, GSH content, and lysosomal lability. Perfusate AST, LDH, NAG, potassium, glucose, and phosphate increased significantly with time, and there were significant differences in the final values between 37 degrees C and 42 degrees C, 42.5 degrees C and 43 degrees C (P less than .05). GSH levels increased significantly at all temperatures after 90 and 120 min, whereas GSSG levels differed significantly at 60, 90, and 120 min for 37 degrees C vs. 42 degrees C, 42.5 degrees C, and 43 degrees C (P less than .05). Mean MDA levels at 37 degrees C differed from those at 41 degrees C and 43 degrees C (P less than .05) at each temperature. Allantoin levels increased significantly with time of perfusion; mean levels at 37 degrees C were significantly different from mean levels at each temperature at 60, 90, and 120 min. GSH liver tissue levels decreased with perfusion at hyperthermic temperatures; mean values at 41 degrees C, 42 degrees C, and 42.5 degrees C, and 43 degrees C differed from 37 degrees C mean values (P less than .01). Type O XO increased after 120 min perfusion from 6.4% +/- 2.0% at 37 degrees C to 55% +/- 30%, 43% +/- 27%, and 63% +/- 29% at 42 degrees C, 42.5 degrees C, and 43 degrees C, respectively. Lysosomal lability increased after perfusion at 42.5 degrees C. There was a significant increase in nonsedimentable NAG activity at 42.5 degrees C (P less than .05). These data support the premise that hyperthermic toxicity to the liver may be a consequence of oxidative stress brought about by enhanced adenosine triphosphate (ATP) consumption and conversion of XO to type O. Such conversion results in superoxide formation and subsequent depletion of cellular GSH, labilization of the lysosomes, and plasma membrane damage.

Effects of hyperthermia on xanthine oxidase activity and glutathione levels in the perfused rat liver.

The hepatotoxic effects of hyperthermic liver perfusion were investigated in male Fischer 344 rat livers. Perfusions were carried out at 37, 41, 42, 42.5, and 43 degrees C for 2 hr. During the 2 hr, the perfusate was analyzed for activity of aspartate aminotransferase (AST), lactate dehydrogenase (LDH), N-acetyl-beta-glucosaminidase (NAG), and glutathione (GSH), oxidized glutathione (GSSG), allantoin, and potassium. After perfusion, each liver was homogenized and analyzed for total xanthine oxidase (XO) activity, percentage type-D and type-O XO, and total GSH content. Perfusate AST, LDH, NAG, and potassium levels were increased significantly with time and were significantly different in all hyperthermic perfusions from the 37 degrees C perfusion values by the end of the perfusion. Perfusate GSH + GSSG levels were increased significantly in all hyperthermic perfusions after 60 min. Liver GSH levels were significantly lowered following perfusion at hyperthermic temperatures. There was a temperature-dependent increase in the percentage of XO in the type-O form following perfusion at hyperthermic temperatures, which was strongly and positively correlated with the loss of hepatic GSH. These data support the hypothesis that hyperthermic toxicity to the liver is the result of oxidative stress brought about by conversion of XO to the type-O form.

Effects of epidural and systemic lidocaine on sympathetic activity and mesenteric circulation in rabbits.

BACKGROUND: The mechanisms producing hemodynamic changes during epidural anesthesia are incompletely understood. This study examines the sympathetic block and splanchnic venodilatation that result from extensive thoracolumbar epidural anesthesia in rabbits using direct measurements of sympathetic efferent nerve activity (SENA) and mesenteric vein diameter (VD). METHODS: Epidural catheters were inserted in rabbits anesthetized with alpha-chloralose, paralyzed with vecuronium, and receiving mechanical ventilation. Arterial pressure was monitored with a femoral cannula, heart rate was determined from the pressure signal, SENA was measured from a postganglionic splanchnic nerve, and VD was measured from segments of ileum externalized in situ. Epidural anesthesia was induced with 0.4 ml/kg lidocaine, using concentrations of either 0.5, 1, or 1.5%. Control animals received intramuscular lidocaine in a dose of either 6 or 15 mg/kg. After recovery from epidural anesthesia, complete sympathetic blockade was induced by systemic administration of the ganglionic blocker hexamethonium (HX). Individual groups included from five to eight animals. RESULTS: A mild decrease in arterial pressure and SENA followed the larger dose of intramuscular lidocaine, but no changes occurred in VD in the control animals exposed to systemic lidocaine at levels comparable to that in the epidural groups (0.96-3.58 micrograms/ml). Epidural injectate extended from T2 to L5. All concentrations of epidural lidocaine produced comparable degrees of hypotension (-53.5 to -61.4%), decreased SENA (-82.6 to -95.5%), and increased VD (7.5 to 10.2%). The duration of the changes was greater with more concentrated lidocaine. Hexamethonium produced changes in arterial pressure and VD comparable to those evoked by epidural anesthesia. CONCLUSIONS: Epidural anesthesia increases splanchnic venous capacitance by markedly decreasing splanchnic sympathetic nerve activity.

Effects of sevoflurane on inward rectifier K+ current in guinea pig ventricular cardiomyocytes.

The effects of sevoflurane on the inward rectifier potassium current (IKIR) were examined in guinea pig ventricular cardiomyocytes using the whole cell patch-clamp methodology. Sevoflurane had a unique dual effect on the steady-state current amplitude, producing a reversible, concentration- and voltage-dependent block of the inward current at potentials negative to the potassium equilibrium potential (EK) but enhancing the outward current positive to EK. Accordingly, the steady-state conductance negative to EK was reduced by sevoflurane, but conductance positive to EK was increased. The chord conductance-voltage relationship showed depolarizing shifts at 0.7, 1.3, and 1.6 mM sevoflurane. When the myocytes were dialyzed with 10 mM Mg2+, but not with 1.0 mM Mg2+, sevoflurane further slowed current activation kinetics. With 10 mM intracellular Mg2+, the outward current enhancement by sevoflurane and the associated shifts in half-activation potential were abolished. Polyamines abolished all effects of sevoflurane on IKIR. With the use of the Woodhull model for voltage-dependent block, we determined the sevoflurane interaction site with the inward rectifier potassium channel to be at an electrical distance of 0.2 from the extracellular side.

Effects of halothane and isoflurane on fast and slow inactivation of human heart hH1a sodium channels.

BACKGROUND: Cloning and heterologous expression of ion channels allow biophysical and molecular studies of the mechanisms of volatile anesthetic interactions with human heart sodium channels. Volatile anesthetics may influence the development of arrhythmias arising from cardiac sodium channel dysfunction. For that reason, understanding the mechanisms of interactions between these anesthetics and cardiac sodium channels is important. This study evaluated the mechanisms of volatile anesthetic actions on the cloned human cardiac sodium channel (hH1a) alpha subunit. METHODS: Inward sodium currents were recorded from human embryonic kidney (HEK293) cells stably expressing hH1a channels. The effects of halothane and isoflurane on current and channel properties were evaluated using the whole cell voltage-clamp technique. RESULTS: Halothane at 0.47 and 1.1 mM and isoflurane at 0.54 and 1.13 mM suppressed the sodium current in a dose- and voltage-dependent manner. Steady state activation was not affected, but current decay was accelerated. The voltage dependence of steady state fast and slow inactivations was shifted toward more hyperpolarized potentials. The slope factor of slow but not fast inactivation curves was reduced significantly. Halothane increased the time constant of recovery from fast inactivation. The recovery from slow inactivation was not affected significantly by either anesthetic. CONCLUSIONS: In a heterologous expression system, halothane and isoflurane interact with the hH1a channels and suppress the sodium current. The mechanisms involve acceleration of the transition from the open to the inactivated state, stabilization of the fast and slow inactivated states, and prolongation of the inactivated state by delayed recovery from the fast inactivated to the resting state.

Mechanism of mesenteric venodilatation after epidural lidocaine in rabbits.

BACKGROUND: Increased splanchnic venous capacitance has been observed during extensive thoracolumbar epidural anesthesia in rabbits, but the mechanism is not clear. The present study examines the contributions of intravascular pressure changes, catecholamine levels, neural input, and direct effects of lidocaine to mesenteric venodilatation. METHODS: Epidural catheters were inserted in rabbits anesthetized with alpha-chloralose. Vein diameter was measured by videomicrography from segments of ileum externalized in situ. Plasma epinephrine and norepinephrine levels were measured in animals receiving epidural blockade (0.4 ml/kg lidocaine 1.5%, n = 5) and in control animals given intramuscular lidocaine 15 mg/kg (n = 5). Intraluminal pressure was monitored during the onset of epidural anesthesia (0.4 ml/kg lidocaine 1.0%, n = 9) by a servo-null micropressure technique. The effect of inhibiting norepinephrine release from sympathetic nerves in the mesenteric veins was determined by using topical tetrodotoxin (n = 8) and by assessing the effect of topical lidocaine (10 and 100 micrograms/ml, n = 5) administered in the solution bathing the mesentery. RESULTS: Epidural injectate extended from T2 to L5. Plasma epinephrine decreased 68.3 +/- 4.4% (mean +/- SEM) with epidural anesthesia, and norepinephrine was lower after epidural block than after intramuscular lidocaine (1,868 +/- 290 pg/ml vs. 3,049 +/- 712 pg/ml). Mesenteric vein pressure decreased 35.3 +/- 3.5% and vein diameter increased 10.2 +/- 3.3% during epidural blockade. Tetrodotoxin caused mesenteric venodilatation (7.6 +/- 2.0%) and prevented venodilatation by subsequent epidural lidocaine. Topical lidocaine 10 micrograms/kg produced no change in vein diameter, but lidocaine 100 micrograms/ml increased it 3.5 +/- 1.3%. CONCLUSIONS: Splanchnic venodilatation during epidural anesthesia is an active process: a decrease in intravenous pressure concurrent with dilatation indicates that vein wall tension diminished. Significant dilatation with tetrodotoxin and lack of dilatation with subsequent epidural block point to a minor role for changes in circulating catecholamines. A direct effect of lidocaine does not contribute to splanchnic venodilatation except when circulating lidocaine concentrations reach very high levels.