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.

Photoactivation of vascular iNOS and elevation of cGMP in vivo: possible mechanism for photovasorelaxation and inhibition of restenosis in an atherosclerotic rabbit models.

Recently, intravascular low-power red laser light (LPRLL) therapy has been proposed for the prevention of postangioplasty restenosis due to the observed inhibition of experimental neointimal formation. The objective of this study was to determine the impact of endoluminal LPRLL on vascular levels of inducible nitric oxide synthase (iNOS) and cyclic guanosine monophosphate (cGMP) to help define the mechanism of this effect. Eight atherosclerotic male adult New Zealand White rabbits weighing 4-6 kg were used in these studies. The iliac arteries were treated in separate zones with: (1) balloon inflation only; (2) laser illumination only; and (3) balloon inflation + laser illumination. An uninjured zone of the iliac artery served as a control. Laser irradiation (630 nm) was delivered to the vessel wall via a Cold laser Illuminator (Cook, Inc., Bloomington, IN), with a 3 mm-diameter balloon. Experiments demonstrated that vascular cGMP levels obtained immediately following treatment in the balloon only group was the lowest (0.29 +/- 0.05 pmol/mg protein) and significantly lower compared with the uninjured controls (1.01 +/- 0.07 pmol/ mg protein) (P < 0.001). In the laser only treated group cGMP levels were significantly increased (2.87 +/- 0.12 pmol/mg protein) compared with the uninjured control (P < 0.001) and the balloon only group (P < 0.001). Vascular cGMP levels in the balloon + laser group (2.09 +/0.07 pmol/mg protein) was also increased compared to the balloon only (P < 0.001) and control (P < 0.001) groups. Qualitative analysis of Western blot demonstrated that laser illumination induces iNOS. In contrast balloon dilatation did not induce iNOS. Balloon + laser treatment, however, tended to restore the expression of iNOS. Our study demonstrated that intravascular low dose laser irradiation induces iNOS and elevates vascular cGMP in an in vivo atherosclerotic rabbit model.

The US Food and Drug Administration investigational device exemptions (IDE) and clinical investigation of cardiovascular devices: information for the investigator.

The conduct of a clinical investigation of a medical device to determine the safety and effectiveness of the device is covered by the investigational device exemptions (IDE) regulation. The purpose of IDE regulation is "to encourage, to the extent consistent with the protection of public health and safety and with ethical standards, the discovery and development of useful devices intended for human use, and to that end to maintain optimum freedom for scientific investigators in their pursuit of this purpose" (Federal Food, Drug, and Cosmetic Act). Conducting a clinical investigation may require an approved IDE application. The US Food and Drug Administration encourages early interaction with the agency through the pre-IDE process during the development of a device or technology and during the preparation of an IDE application. This facilitates approval of the IDE application and progression into the clinical investigation. This paper reviews the terminology and applicability of the IDE regulation and the type of study that requires an IDE application to the Food and Drug Administration. The pre-IDE process and the development of an IDE application for a significant risk study of a cardiovascular device are discussed.

The US Food and Drug Administration investigational device exemptions and clinical investigation of cardiovascular devices: information for the investigator.

The conduct of a clinical investigation of a medical device to determine the safety and effectiveness of the device is covered by the investigational device exemptions (IDE) regulation. The purpose of IDE regulation is "to encourage, to the extent consistent with the protection of public health and safety and with ethical standards, the discovery and development of useful devices intended for human use, and to that end to maintain optimum freedom for scientific investigators in their pursuit of this purpose." Conducting a clinical investigation may require an approved IDE application. The US Food and Drug Administration encourages early interaction with the agency through the pre-IDE approval process during the development of a device or technology and during the preparation of an IDE application. This facilitates approval of the IDE application and progression into the clinical investigation. This paper reviews the terminology and applicability of the IDE regulation and the type of study that requires an IDE application to the Food and Drug Administration. The pre-IDE approval process and the development of an IDE application for a significant risk study of a cardiovascular device are discussed.

The combined effects of chronic ethanol/desipramine treatment on beta-adrenoceptor density and coupling efficiency in rat brain.

Both ethanol and desipramine influence beta-adrenoceptor regulation. We reported previously that ethanol partially counteracted desipramine's effects on beta-adrenoceptor. Previous studies utilized beta-adrenoceptor radioligands that also bind to 5-HT1B receptors, thus, changes in 5-HT1B receptors could have confounded the results. The effects of chronic ethanol, desipramine and ethanol/desipramine treatment on beta-adrenoceptor coupling efficiency to Gs protein in rat brain were examined using 125I-iodocyanopindolol after blocking binding to 5-HT1B receptors. In the frontal cortex, ethanol uncoupled beta-adrenoceptor from GS. Desipramine decreased beta-adrenoceptor density, particularly in the high-conformational state, with no effect on coupling. In combined treatment, desipramine prevented ethanol-induced uncoupling. In the hippocampus, desipramine enhanced beta-adrenoceptor coupling, but ethanol had no effect. In combination with desipramine, ethanol enhanced desipramine-induced decrease in beta-adrenoceptor density in the high-conformational state, but uncoupled beta-adrenoceptors, an effect not observed with ethanol alone. These results suggest a complex interplay between ethanol and antidepressants in modulating beta-adrenoceptor function.

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.

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.

Losses of arachidonic acid in rat liver after alcohol inhalation.

This paper presents an animal model of alcoholism in which rats were exposed to alcohol by inhalation and were fed a diet that simulated the poor diet of some alcoholics. It is hypothesized that some of the pathophysiological effects of alcohol are related to its effects on essential fatty acid metabolism and composition of vital organs. A diet that contains no 20- and 22-carbon essential fatty acids and has low levels of 18-carbon essential fatty acids was used as a dietary challenge. Addition of a second metabolic challenge, i.e., alcohol, led to loss of tissue polyunsaturates, particularly liver arachidonate. A method of cycling alcohol inhalation for 12 h/d was also presented, which was also shown to lower liver arachidonic acid content.

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).

Time- and voltage-dependent block of delayed rectifier potassium channels by docosahexaenoic acid.

Docosahexaenoic acid (22:6n3) acts at an extracellular site to produce a voltage- and time-dependent block of the delayed rectifier current (IK) similar to that classically described for intracellularly applied quaternary ammonia compounds. In dissociated cells from the pineal gland, some long-chain polyunsaturated fatty acids reduced both late sustained (IK) (for 22:6n3, IC50 = 2.5 +/- 0.3 microM) and early transient (IA) (IC50 = 2.0 +/- 0.1 microM) components of potassium current when applied extracellularly, whereas the monounsaturate oleic acid had minimal efficacy. From comparisons of other related fatty acids, it was determined that there is a structural requirement for polyunsaturation to block IK. In contrast, chain-elongated 22-carbon polyunsaturates acted similarly to their precursor 20-carbon fatty acids (arachidonic acid and eicosapentanoic acid). Block of IK by 22:6n3 was accompanied by a dose-dependent acceleration of the current decay in both whole-cell and outside-out membrane patches, and 22:6n3 increased the macroscopic inactivation rate of IA. The combined "eicosanoid" inhibitor eicosatetraenoic acid, when included in the patch pipette, did not antagonize the action of 22:6n3. Instead, eicosatetraenoic acid produced a direct block of IK when applied extracellularly at high concentrations (25 microM). Analyses of voltage- and time-dependent block by 22:6n3 support the hypothesis that certain fatty acids directly interact with and preferentially block the open state of some potassium channels. We also describe an interaction between fatty acid block and zinc; 22:6n3 failed to block either IA or IK in the presence of zinc or cadmium, whereas extracellular calcium did not affect the response. These studies suggest a possible biological function for 22:6n3 in the nervous system, which may underlie its essential role during neural development.

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.

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.

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.

Mapping rat brain structures activated during ethanol withdrawal: role of glutamate and NMDA receptors.

Brain structures activated during ethanol withdrawal have been mapped by visualizing c-fos mRNA expression. The regional distribution of c-fos mRNA in brain during ethanol withdrawal can be mimicked by acute injection of N-methyl-D-aspartic acid (NMDA) and is stereospecifically blocked by the NMDA receptor antagonist, MK-801. The findings reveal that the dentate gyrus and piriform cortex are selectively activated during ethanol withdrawal and suggest that this may be mediated by glutamate activation of NMDA receptors.

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.

Prolonged ethanol inhalation decreases gamma-aminobutyric acidA receptor alpha subunit mRNAs in the rat cerebral cortex.

Ethanol administration to rats by ethanol vapor inhalation (14 days) results in a 40-50% reduction in the level of gamma-aminobutyric acidA (GABAA) receptor alpha 1 subunit mRNAs [4.4 and 4.8 kilobases (kb)] in the cerebral cortex. The level of alpha 2 subunit mRNA (8.0 kb) was also reduced by 29%, whereas there was no effect of prolonged ethanol exposure on the level of alpha 3 subunit mRNA (3.1 kb). Ethanol exposure did not alter the steady state levels of cerebral cortical glutamic acid decarboxylase or beta-actin mRNAs. Moreover, no alterations in the levels of total RNA, poly(A)+ RNA, or rRNA were observed, suggesting that the ethanol-induced reductions in GABAA receptor alpha 1 and alpha 2 subunit mRNAs were not the result of a generalized effect of ethanol administration on transcription or mRNA turnover. These ethanol-induced reductions in GABAA receptor alpha subunit mRNAs may underlie alterations in GABAA receptor function or number observed following prolonged ethanol exposure in rats.

Stereochemical analysis of hydroxylated docosahexaenoates produced by human platelets and rat brain homogenate.

The stereochemical configuration of hydroxylated products of docosahexaenoic acid (22:6w3) formed by human platelets and rat brain homogenate were characterized for the first time. Chiral phase HPLC was employed along with autooxidized 22:6w3 as reference material. The 14- and 11-hydroxy 22:6w3 (HDHE) products produced by human platelets were in the S configuration. Rat brain homogenate produced all of the ten possible positional isomers when incubated with 22:6w3. Their retention behavior on the reversed and chiral phase HPLC columns and GC/MS/EI analysis indicated that they were 20-, 17-, 16-, 14-, 13-, 11-, 10-, 8-, 7- and 4-HDHE. However, stereochemical analysis revealed that each positional isomer was a racemic mixture, suggesting that these were not formed by lipoxygenation but mainly by peroxidation process.

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.

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.

Effects of chronic exposure to ethanol alone and in combination with desipramine on beta-adrenoceptors of rat brain.

The effect of chronic ethanol exposure alone or in combination with desipramine on agonist and antagonist binding to beta-adrenoceptors was studied in membrane preparations from rat frontal cortex and hippocampus. Ten day exposure of animals to ethanol vapor (25 mg/l) in inhalation chambers had no effect on binding properties of antagonist iodocyanopindolol (ICYP) in either brain region. However, ethanol in combination with chronic desipramine treatment prevented the reduction of beta-adrenoceptor density in frontal cortex produced by desipramine administration. Similar to its effects on antagonist binding, chronic ethanol exposure did not change the agonist isoproterenol binding characteristics measured in membranes from either rat frontal cortex or hippocampus. However, the combination of ethanol plus desipramine reduced the dissociation constant of the low affinity state of the receptor (KL) in frontal cortex from 23.1 +/- 3.7 microM in controls to 11.2 +/- 1.7 microM. Moreover, ethanol plus desipramine produced a greater decrease in the percentage of cortical receptors in the high affinity state for agonist (%RH) than did desipramine alone. This suggests that ethanol enhances desipramine-induced desensitization of beta-adrenoceptors in frontal cortex in spite of the prevention of reduction in density of the receptors. In hippocampal membranes, ethanol together with desipramine prevented desipramine-induced changes in agonist binding characteristics, i.e. the decrease in KH (dissociation constant from high affinity state of the receptor) and the consequent enhancement in KL/KH ratio. Thus, chronic exposure to relatively low concentrations of ethanol partially prevents effects of desipramine on beta-adrenoceptors.