Inhalation anesthesia induced by isoflurane alters penicillin disposition in swine tissues.

Twelve healthy swine were dosed with penicillin G intramuscularly. Fluids and tissues samples were collected at the end of two periods of general anesthesia, performed 24 h apart. Tissue samples were collected by minimally invasive laparoscopy under general anesthesia at 8 and 28 h postdose. Four nonanesthetized, penicillin-treated pigs were euthanized at 8 h postdose, and a second set of four similarly treated control pigs were sacrificed 28 h postdose. Liver penicillin tissue concentrations from animals that underwent anesthesia and laparoscopic tissue collection had tissue concentrations that were higher than nonanesthetized pigs at both time points. Urine, plasma, kidney, skeletal, and cardiac muscle showed no differences between the two groups. Laparoscopic tissue collection under general anesthesia in swine induces physiological changes that cause alterations in tissue pharmacokinetics not seen in conscious animals.

Ethanol exposure decreases pituitary corticotropin-releasing factor binding, adenylate cyclase activity, proopiomelanocortin biosynthesis, and plasma beta-endorphin levels in the rat.

Animals exposed continuously for 14 days to ethanol vapor in an inhalation chamber at sufficient ethanol vapor concentration to maintain blood ethanol levels from 100-250 mg/100 ml exhibited approximately 36% lower corticotropin-releasing factor binding and 24% lower adenylate cyclase activity in anterior (AL) and neurointermediate lobe (NIL) membranes of the pituitary gland compared to controls not treated with ethanol. To determine the effect of chronic ethanol exposure on proopiomelanocortin (POMC) biosynthesis, the levels of POMC mRNA in the AL and NIL were quantified by Northern blot and slot blot techniques. Ethanol treatment for 1, 7, or 14 days produced a time-related decrease in POMC mRNA levels, relative to total RNA levels, in both the AL and NIL. Ethanol treatment caused a greater reduction in NIL POMC mRNA than in AL POMC mRNA. Exposure to ethanol vapors for 14 days decreased immunoreactive beta-endorphin in plasma by approximately 82%. The observed reduction of immunoreactive beta-endorphin in plasma after long term exposure of rats to ethanol may be related to the alcohol-mediated decrease in corticotropin-releasing factor binding and adenylate cyclase activity, which, in turn, leads to decreased intracellular POMC levels through reduced production of POMC mRNA in the AL and NIL of the rat pituitary gland.

Effect of gender on development and diurnal rhythm of prostaglandin receptors in rat aorta.

1 The prostaglandin endoperoxide H2 analogue, U-46619 (15-hydroxy-11, 9-epoxymethanoprosta-5Z, 13E-enoic acid), was used to determine the effect of gender on the isotonic contractile response of the superfused rat aorta preparation over a 24 h period and at 10 and 16 weeks of age. 2 The maximal responses of the male aortae were 20 +/- 3% and 36 +/- 4% (P less than 0.001) greater than the female at 10 and 16 weeks, respectively. Similarly, sensitivity of the male aorta to the PGH2 analogue increased with age. 3 The male but not the female exhibited a diurnal rhythm in which both the maximum contractile response and sensitivity were significantly decreased at night. 4 We conclude that these gender differences may be related to the secretion of androgen, since reported peak serum testosterone over a 44 h period and testosterone changes with maturation are coincident with the maximum response of male aortae to the PGH2 analogue.

Gender differences in prostaglandin receptors of rat aorta.

The maximal contractile response to the prostaglandin endoperoxide H2 analogue U46619, prostaglandins E2, D2 and F2 alpha and the sensitivity of the superfused aorta preparation to these drugs (except PGF2 alpha) is greater in the male than the female rat. In contrast, gender differences were not observed in the response to noradrenaline of 5-hydroxytryptamine. In previous studies, testosterone unlike oestrogen or progesterone, increased the response of both rabbit and rat aorta to U46619. We conclude that prostaglandin receptors in rat thoracic aorta may be hormonally regulated.

Lipoxygenase stimulating effects of hydroxylated docosahexaenoates produced by human platelets.

Human platelet suspensions are capable of lipoxygenating docosahexaenoic acid (22:6n3) to an 11(S)-OH-, 14(S)-OH- or 17(S)-OH-22:6n3. The structure and stereochemical purity of these derivatives were confirmed by GC/MS and chiral phase LC analysis. The purified OH-22:6n3 positional isomers which are formed by human platelets were capable of inducing a concentration-dependent contractile response in the guinea-pig lung parenchymal strip at sub-micromolar concentrations. OH-22:6n3 may act in part through stimulation of leukotriene (LT) production as an increase in peptidyl-LT levels (LTC4, LTD4 and LTE4) occurred during the OH-22:6n3-induced contraction in this preparation. Both specific lipoxygenase inhibitors (caffeic acid, 20 uM and NDGA, 50 uM) and a LT receptor antagonist (FPL55712, 20 uM) significantly inhibited the contractile response. Moreover, the OH-22:6n3 positional isomers induced a concentration-dependent increase in LTB4 and LTC4 production in the guinea-pig chopped lung preparation. Other hydroxylated fatty acids and parent fatty acids which were tested (12-OH-20:4n6, 5-OH-20:4n6, 12-OH-20:5n3, 20:5n3 and 22:6n3) did not significantly contract this airway smooth muscle preparation or alter LT production. The hydroxylated 22:6n3 metabolites may modulate airway smooth muscle function in part through the release of peptidyl-LTs from the guinea-pig lung.

Docosahexaenoic acid (22:6n3)-induced relaxation of the rat aorta.

Dietary consumption of fish and fish oil supplements, containing eicosapentaenoic acid and docosahexaenoic acid, has been associated with favorable alterations in the cardiovascular system, such as, a reduction in blood pressure. Therefore, the effects of docosahexaenoic acid (22:6n3) on isometric tension of rat aortic smooth muscle were investigated. A concentration-dependent (1-127 microM) relaxation (6-30%) was induced by 22:6n3 in non-precontracted vessels. Docosaehexaenoic acid, concentration dependently (1-44 microM) reversed contractions of rat aortic rings induced by phenylephrine (7-43%) and by U44069 (8-52%). These results indicate that the relaxant effects produced by 22:6n3 in the rat aorta are concentration-dependent and not specific to the contractile agonist.

The effect of chronic alcohol inhalation on blood pressure and the pressor response to noradrenaline and the thromboxane-mimic U46619.

Rats were exposed to alcohol vapor for 6 days and the mean blood ethanol concentration (BEC) was obtained for each subject. Blood pressure and its reactivity to noradrenaline and a thromboxane-mimic U46619 were directly measured on day 6 via a catheter implanted in the tail artery of normal and ethanol-treated animals. The mean BEC for each subject correlated with mean arterial blood pressure (MAP); an increase in BEC was associated with a decrease in MAP (p less than 0.02). The mean MAP of subjects with BEC less than 168 mg% was 8% higher than normal (not significant), whereas, the mean MAP of subjects with BEC greater than 182 mg% decreased 27 +/- 4% (p less than 0.01). Conversely, the pressor response to U46619 was markedly enhanced (p less than 0.005) in rats with mean BEC greater than 182 mg% at all doses investigated (12.5-3200 ng per rat). Increases in the pressor response to noradrenaline in ethanol-treated rats were significant only when maximally stimulated by 400 and 800 ng doses (p less than 0.03). A 3-fold increase in sensitivity for U46619 was seen in subjects with high mean BEC, however, sensitivity for noradrenaline did not significantly change. Vasoreactivity was not effected in rats with mean BEC less than 168 mg%. These data demonstrate that a moderate mean BEC for 6 days induces a tendency towards a mild hypertension, whereas, high mean BEC induces marked hypotension which is associated with hyperreactivity. Long-term exposure to high blood ethanol concentrations may predispose the alcohol-dependent rats to hypertensive disease and vasospastic disorders, at least partially, as a result of enhanced sensitivity to prostaglandins such as thromboxane.

The structure-activity relationship of lipoxygenase products of long-chain polyunsaturated fatty acids: effects on human platelet aggregation.

The effect of hydroperoxy and hydroxy derivatives of various fatty acids on human platelet aggregation was determined to delineate potencies and structure-activity function. In this regard, the 22-carbon n-3 fatty acids are the most potent inhibitors in comparison to the n-6 lipoxygenase derivatives. Submicromolar levels of the docosapentaenoic (22:5) and especially docosahexaenoic (22:6) n-3 hydroperoxy and hydroxy derivatives specifically antagonize the platelet aggregating effect to arachidonic acid (AA, 20:4n-6) but not that of ADP or collagen. Chain length (22-C > 20-C), double-bond position (n-3 > n-6), and double-bond number (6 > 5 > 4) influence the degree of inhibition of AA-induced aggregation of human platelets. Moreover, significant differences in potency were associated with specific structural aspects of 22:6n-3 lipoxygenase derivatives of 22:6n-3 as follows: functional group (OOH > OH) and positional isomer (14-OOH, 14-OH, 20-OOH > 11-OOH, 17-OOH > 10-OOH > 11-OH, 8-OOH, 7-OOH > 4-OOH).

RO 15-4513 induces seizures in DBA/2 mice undergoing alcohol withdrawal.

RO 15-4513, an imidazodiazepine that has been reported to reverse some of the behavioral effects of ethanol, was given to DBA/2 mice. Although no animals treated with a 6 mg/kg dose of this drug had seizures, 20% of animals given 20 mg/kg of this drug had tonic seizures. Ethanol withdrawal was induced in DBA/2 mice treated with 4-methyl pyrazole using an inhalation paradigm. Mice were more likely to have a seizure during ethanol withdrawal if treated with RO 15-4513 (6 mg/kg) than if they received the vehicle. These data suggest administering RO 15-4513 as an alcohol antagonist to alcoholic subjects may increase the incidence of seizures.

Inhibition of platelet aggregation in whole blood after exposure of rats to alcohol by inhalation.

The dose-effect relationship between blood alcohol concentration (BAC) and altered platelet function was examined in whole blood in a rat model of alcohol exposure by inhalation, using the impedance method of ex vivo whole blood platelet aggregation. With rates of alcohol addition to the chamber air inflow from 29 to 56 mg ethanol/l air/min, BAC was dependent on duration of exposure and concentration of alcohol in the air. Next, 3, 6, and 9 h exposures to the highest delivery rate were used, and platelet aggregability was tested. After 9 h, BAC reached 453 +/- 16 mg% and aggregation responses to three doses of collagen were significantly lower than in control blood (p < 0.01). Less consistent inhibition was observed with arachidonic acid and ADP, and also when exposure duration was reduced. However, some significant inhibition of collagen-induced aggregation (p < 0.05) was observed with BAC as low as 127 +/- 15 mg%. These experiments demonstrate that in vivo alcohol exposure inhibits, in a concentration-dependent manner, ex vivo rat whole blood platelet aggregation, at BACs readily attained in humans by ingestion.

Polyunsaturated fatty acids and ethanol.

Ethanol exposure leads to a loss in membrane polyunsaturated fatty acids (PUFA). It is proposed that polyunsaturated species of phospholipids are not randomly distributed, but are concentrated in the cytosolic leaflets of the plasma membrane and are preferentially associated with membrane proteins. These lipids affect the physical state of environments surrounding membrane proteins and thereby serve to regulate many cellular functions. Disruption of these environments may occur even when a small percentage of total polyunsaturates is lost due to ethanol exposure. One possible mechanism of ethanol-induced polyunsaturate loss may be activation of a phospholipase A2 enzyme which is selective for these species of phospholipids. Fatty acids released would stimulate the production of prostaglandins and/or leukotrienes. Similarly, the released docosahexaenoate can be metabolized by rat brain to leukotriene-like compounds which are biologically active in smooth muscle systems. This metabolism is stimulated by ethanol in human platelets, in vitro.

The effect of alcohol inhalation on the cardiovascular state of the rat.

Rats were exposed to alcohol vapors by an inhalation technique and blood pressure and its reactivity and platelet aggregation were measured. Acute exposure to alcohol levels which produced moderate blood alcohol concentration (BAC) was associated with increased vascular production of prostacyclin (PGI2) and plasma catecholamines, whereas platelet production of thromboxane (TXA2) decreased. Blood pressure is elevated in these animals, however, the platelet aggregating and pressor effects of noradrenaline (NE) were decreased. Chronic exposure to high BAC is associated with a dramatic reduction in vascular and platelet prostaglandin (PG) production and a marked increase in plasma catecholamine levels. Platelet aggregation decreased in these animals, however, the pressor effect of TXA2 and NE was significantly increased. The fatty acid precursors to PG were reduced by 50% in the lipid extracts of these preparations. These findings suggest that alterations in fatty acid metabolism may lead to a functional deficiency in PG production from dependent rats. Qualitative differences may exist between acute and chronic exposure with respect to the cardiovascular state. Locally produced PG and circulating catecholamines may mediate alcohol-induced alterations in vascular smooth muscle tone and platelet aggregation.

Effects of acute and chronic ethanol exposure on the response of rat aorta to a thromboxane mimic, U46619.

The spasmogenic properties of a stable thromboxane mimic, U46619, were determined in the isolated rat aorta after 1, 4, 8, or 12 hr of acute ethanol exposure in vitro or after 12 days of chronic exposure by inhalation. Acute ethanol exposure (87-822 mM) increased the baseline tension of aortic rings in a concentration-dependent manner. Moderate concentrations of ethanol (11 and 43 mM) decreased the maximum tensile response of rat aortic rings to U46619 after 8 and 4 hr of exposure, respectively. In contrast, higher ethanol concentrations (87 and 411 mM) did not significantly effect the maximum tensile response to U46619. Ethanol at 822 mM completely inhibited the response to U46619 and this inhibition could be 85% reversed after removal of ethanol. The inhibitory effect of 1.64 M ethanol was irreversible. Sensitivity to U46619 was inversely related to the ethanol concentration (greater than or equal to 43 mM) and incubation time. Similarly, chronic exposure to moderate blood ethanol levels (92-198 mg/100 ml) for 12 days decreased the maximum tensile response whereas high levels did not effect aortic contractility in vitro. Sensitivity of aorta from rats with mean blood ethanol levels greater than or equal to 92 mg/100 ml decreased at least one order of magnitude. The results suggest that the effect of physiologically tolerable ethanol concentrations on the amplitude of the tensile response to U46619 is biphasic both during acute exposure in vitro or following a chronic exposure period in vivo and the inhibitory effect on sensitivity for U46619 is both concentration- and time-dependent in the rat aorta preparation.

Effect of gender and sex steroids on the contractile response of canine coronary and renal blood vessels.

The effect of gender, gonadal steroids, and antiandrogen/antiestrogen-treatment on the isotonic response of isolated preparations of the left anterior descending coronary artery (LAD), left circumflex coronary artery, and renal artery and vein of sexually mature dogs was investigated. The maximum isotonic response of the coronary and renal vasculature to the thromboxane A2 (TXA2)-mimetic U46619 was significantly greater, and the EC50 value was significantly lower in males as compared with females. Moreover, similar gender differences in the contractile response of the coronary vasculature to norepinephrine were observed. Pretreatment of male dogs with the antiandrogens flutamide or cyproterone acetate reduced the maximum contractile response of the LAD to the TXA2-mimetic. Pretreatment of female dogs with testosterone resulted in an increase in both the maximum contractile response and EC50 value to U46619. Antiestrogen treatment of female dogs with tamoxifen was associated with an increase in the maximum contractile response of the LAD to U46619. Estrogen pretreatment of male dogs decreased both the maximum contractile response and the EC50 value to U46619. Therefore, there is a sex difference in LAD and LCX contractile responses to both U46619 and norepinephrine. These results suggest that smooth muscle reactivity of dog coronary artery to the TXA2-mimetic U46619 may be susceptible to regulation by both androgens and estrogens. The observed gender differences in the catecholamine response may be similarly altered by changes in the hormonal milieu.

Hydroxylated 22-carbon fatty acids in platelet and vascular smooth muscle function: interference with TXA2/PGH2 receptors.

Sub-micromolar levels of the lipoxygenase products of n-3 fatty acids specifically antagonize both the contractile effects of thromboxane (U46619) and its platelet aggregating effect. In addition, OH-22:6n3 inhibits thromboxane-induced decreases in cerebral blood flow of the rat. Analysis of binding parameters indicates these derivatives induce a marked decrease in the affinity of the TXA2/PGH2 receptor for thromboxane with a mild change in the number of receptor sites. The 22-carbon n-3 hydroxy fatty acids are the most potent biological antagonists of thromboxane in comparison to the n-6 hydroxy fatty acids and their parent fatty acids. Dietary permutations modify the hydroxy fatty acid profile and correlate with changes in thromboxane-mediated responses.

Formation of 15-lipoxygenase product from docosahexaenoic acid (22:6w3) by human platelets.

The metabolism of docosahexaenoic acid (22:6w3) by 15-lipoxygenase activity of washed human platelets was investigated. Platelets produced 17-hydroxydocosahexaenoic acid (HDHE) when incubated with 22:6w3. Similarly, 15-hydroxyeicosatetraenoic acid (HETE) and 13- and 9-hydroxyoctadecadienoic acids (HODD) were produced when incubated with 20:4w6 and 18:2w6, respectively. However, these products were observed only as minor components in the platelet incubation mixture. Control studies with carefully purified platelets and mononuclear cells indicated that these products were formed by the platelets. Chiral phase HPLC analysis indicated that these compounds were mainly in the S configuration with the exception of the 9-HODD, thus, confirming that a lipoxygenase is responsible for their production. The 9-HODD produced by platelets was a racemic mixture.

Ethanol inhalation and dietary n-6, n-3, and n-9 fatty acids in the rat: effect on platelet and aortic fatty acid composition.

The effects of 18-carbon n-6, n-3, and n-9 fatty acid diets and ethanol exposure on the fatty acyl composition of platelets and vascular tissue were examined. An experimental design was devised to control the dietary content of 18-carbon fatty acids. The levels of 18:3n6, 18:3n3 and 18:1n9 were varied by a formulation of dietary oils which contained similar proportions of 18:2n6. Male Sprague-Dawley rats were fed a purified diet containing 11% by weight of either borage oil (BOR) rich in 18:3n6, linseed/safflower oil (LSO) rich in 18:3n3, or sesame oil (SES) rich in 18:1n9 for 7 weeks and exposed to ethanol vapors by means of inhalation for the final 6 days of the dietary regimen. Moderate blood ethanol levels of 118 +/- 6.6 mg/dl were obtained. Total lipids were extracted from platelets and aortae, and the fatty acid distributions were analyzed by gas chromatography. BOR feeding resulted in increases in the proportion of n-6 fatty acids (18:3n6, 20:3n6, 20:4n6) in platelets and aorta. Animals fed the LSO diet had increased levels of n-3 fatty acids (18:3n3, 20:5n3, 22:6n3). The SES-based diet resulted in an increase in 18:1n9 in both aorta and platelets. Following ethanol exposure alone, the most marked change in the fatty acid profile was a decrease in 20:4n6 in the platelet. This effect was not observed in rats supplemented with BOR. No significant changes were observed in the aortic fatty acid content at this level of ethanol exposure. The results suggested that, in the rat, a diet enriched with BOR effectively prevented ethanol-induced alterations in platelet fatty acid composition.

Effect of ethanol on prostacyclin and thromboxane A2 synthesis in rat aortic rings in vitro.

Spontaneous 6-keto-PGF1 alpha and TXB2 levels in isolated rat aortic rings increased in a concentration dependent manner after a 0.5 hour incubation with moderate or high ethanol concentrations (11 mM to 218 mM). After a 1 hour incubation with moderate concentrations of ethanol less than or equal to 22 mM) spontaneous prostaglandin (PG) production did not increase although high concentrations (87 mM and 218 mM) increased both 6-keto-PGF1 alpha and TXB2 levels. Similarly, in the presence of 40 microM Na-arachidonate, high ethanol concentrations increased PG production after 0.5 and 1 hour incubation. In addition, either a 4 or an 8 hour exposure to high ethanol concentrations increased spontaneous PG production. A moderate concentration of ethanol (22 mM) increased the 6-keto-PGF1 alpha/TXB2 ratio whereas high levels (greater than or equal to 87 mM) depressed the ratio after 0.5 and 1 hour exposure. This effect was short-lived since after 4 or 8 hours incubation with high ethanol concentrations the 6-keto-PGF1 alpha/TXB2 ratio was markedly increased. The alcohol-induced changes in both spontaneous and arachidonate-stimulated PG levels were concentration dependent and related to the incubation time. Furthermore, these data suggest that there may be unbalanced production of PGI2 and thromboxane A2 in vascular tissue exposed to alcohol.

Chronic ethanol treatment decreases specific nonopiate beta-endorphin binding to hepatic and kidney membranes and lowers plasma beta-endorphin in the rat.

In a previous study we reported the presence of specific nonopiate beta-endorphin (BE) binding sites in peripheral tissues of the rat (Dave JR, Rubinstein N, Eskay RL: Evidence that beta-endorphin binds to specific receptors in rat peripheral tissue and stimulates the adenylate cyclase-adenosine 3',5'-monophosphate system. Endocrinology 117:1389-1396, 1985). The objective of this study was to determine the effect of chronic ethanol administration in vivo on specific nonopiate binding of BE in hepatic and kidney membranes. Experimental animals were exposed continuously for 14 days to ethanol vapor in an inhalation chamber at vapor levels sufficient to maintain blood ethanol levels greater than 120 mg/100 ml, whereas control animals were maintained in ethanol-free chambers. Chronic ethanol treatment decreased [125I]BE binding to hepatic and kidney membranes by approximately 35%, which was due to a decrease in the number of BE binding sites. Chronic ethanol treatment decreased immunoreactive BE in plasma by greater than 80%. In vitro exposure of hepatic and kidney membranes from control animals to ethanol resulted in a dose-related enhancement of BE binding with maximal enhancement of 50-65% being observed at 0.2% ethanol concentration. In contrast, the addition of ethanol in vitro to hepatic and kidney membranes derived from rats chronically exposed to ethanol in vivo did not affect BE binding. Our findings demonstrate that chronic alcohol exposure lowers immunoreactive BE in plasma and reduces BE binding in hepatic and kidney membranes. The observed reduction of BE binding may be due to ethanol-induced changes in membrane composition.

Inhibitory effects of n-6 and n-3 hydroxy fatty acids on thromboxane (U46619)-induced smooth muscle contraction.

Mammalian platelets are capable of enzymatically producing a number of n-6 and n-3 hydroxy fatty acids. Human platelet suspensions produce two major docosahexaenoic acid (22:6n3) metabolites, namely, 11-OH and 14-OH-22:6n3. The hydroxy fatty acids which were formed by human platelets and purified by high performance liquid chromatography specifically antagonize the contractile effects of a thromboxane mimetic, U46619, in airway, visceral and, especially, in the vascular smooth muscle preparations studied. The efficacy of OH-22:6n3 (IC25 = 1.1 microM) was compared to other n-6 and n-3 hydroxy fatty acids in the rat aortic ring preparation. The OH-22:6n3 was significantly more potent with the exception of OH-22:5n3. The rank order of their potency was 14-OH-22:5n3 > or = 14-OH-22:6n3 > 17-OH-22:6n3 > or = 11-OH-22:6n3 > or = 11-OH-22:5n3 > 12-OH-20:5n3 > or = 12-OH-20:4n6 > or = 14-OH-22:5n6 > 13-OH-18:2n6 > 14-OH-22:5n5. Antagonism of thromboxane effects may be an important aspect of the biological function of 22-carbon n-3 hydroxylated fatty acids in platelet-vascular smooth muscle cell interactions.