Jet anesthesia and jet local anesthesia for the 21st century.

The introduction of the hollow needle and glass syringe in the middle of the 19th century was one gigantic step in the progress of medicine to the current state of art. However, the needle with the pain it causes, become the source of fear for many patients. Indeed, immunization of millions of people who feared the needle, against the deadly contagious diseases, would not have been possible without the introduction of a jet-injector, the Med-E-Jet (Peace Gun), a pain-free way of delivering immunizing medication.

Painless intravenous catheterization by intradermal jet injection of lidocaine: a randomized trial.

STUDY OBJECTIVE: To compare efficacy and cost of lidocaine cutaneous anesthesia by two jet injectors to routine needle infiltration for pain relief of intravenous (i.v.) catheterization, hypothesizing that jet injection of lidocaine is less painful than its needle infiltration. DESIGN: Randomized, prospective, controlled trial. SETTING: University hospital outpatient surgical unit. PARTICIPANTS: 75 surgical patients ASA I and II. INTERVENTIONS: Three groups of 25 patients each were given intradermal lidocaine anesthesia via conventional 25-gauge needle/syringe; by MedEJet or Biojector jet injector prior to IV catheterization with an 18-gauge Jelco catheter. MEASUREMENTS AND MAIN RESULTS: Visual analogue pain scores (VAS) (0 = no pain, 10 = intolerable pain) and subjective pain intensity scores (PIS) (0 = not painful, 4 = intolerable pain) at lidocaine application and at i.v. catheterization, were recorded. Cost assessment of each method was made. At local anesthetic application, no pain by proportion of VAS = 0 with MedEJet: 25/25 (confidence interval [CI]: 0.868, 0.999) and Biojector: 24/25 (CI 0.804, 0.991) was noted, but-22 of 25 patients experienced pain with needle administration: (with VAS = 0; 3/25 [CI: 0.044, 0.302]) (posterior probability [PP] > 0.999). The corresponding VAS scores (means +/- SD) were 0.00 +/- 0.00, 0.04 +/- 0.20, and 2.4 +/- 2.23 (p < 0.001). No pain by proportion of PIS = 0 with MedEJet: 25/25 (CI: 0.868, 0.999 and Biojector: 23/25 (0.749, 0.976) was noted, but pain in 20/25 was felt with the needle: 5/25 (CI: 0.090, 0.394) (PP > 0.999). The corresponding PIS scores were 0.00 +/- 0.00, 0.16 +/- 0.55, and 1.24 +/- 1.00 (p < 0.001). At i.v. catheterization, no pain by proportion of VAS = 0 with MedEJet: 22/25 (CI: 0.698, 0.956) or Biojector: 21/25 (CI: 0.651, 0.934) was noted; but pain in 19/25 with needle administration was experienced: 6/25 (CI: 0.116, 0.436) (PP > 0.999). The corresponding scores were 0.12 +/- 0.33, 0.44 +/- 0.20, and 1.64 +/- 1.50 (p < 0.001). No pain by proportion of PIS = 0 with MedEJet: 24/25 (CI: 0.804, 0.991) or Biojector: 24/25 (CI: 0.804, 0.991) was noted, but pain was apparent in 12/25 with needle administration: 13/25 (CI: 0.334, 0.701) (PP > 0.999). The corresponding scores were 0.00 +/- 0.00, 0.00 +/- 0.00, and 0.76 +/- 0.88 (p < 0.001). Cost per application: MedEJet = $0.13; needle/syringe = $0.50; Biojector = $0.94. CONCLUSIONS: Almost completely painless i.v. catheterization was carried out by jet injection of lidocaine, but needle infiltration produced discomfort or pain and did not significantly reduce discomfort or pain at the i.v. needle insertion.

Genetic deficiency of acylation stimulating protein (ASP(C3ades-Arg)) does not cause hyperapobetalipoproteinemia in mice.

The acylation stimulating protein (ASP) is a 76-amino acid peptide that has been proposed as a potent mediator of triglyceride synthesis and, when functionally impaired, as a major cause of hyperapobetalipoproteinemia (HyperapoB). Purification and sequence analysis of ASP from human sera have revealed that ASP is identical to the complement C3-derived activation peptide C3ades-Arg. Because C3 is the precursor for C3ades-Arg and therefore ASP, a deficiency in C3 would be predicted to result in a phenotype characteristic of HyperapoB. To test this hypothesis in vivo, the current study was undertaken in which ASP(C3ades-Arg)-deficient mice were used as a model system. No significant differences were found in the triglyceride, cholesterol, or free fatty acid concentrations in the plasma of fasted normal and ASP(C3ades-Arg)-deficient animals. In addition, plasma lipoprotein analyses indicated that the very low density lipoprotein, low density lipoprotein, and high density lipoprotein cholesterol and triglyceride concentrations as well as the apolipoprotein B-48 and B-100 levels were not significantly different in the plasma of ASP(C3ades-Arg)-deficient and wild type mice. Furthermore, when challenged with an oral fat load, the ASP(C3ades-Arg)-deficient mice showed no impaired ability to clear triglycerides and free fatty acids from their circulation when compared with their wild-type littermates. Collectively, these results indicate that ASP(C3ades-Arg) deficiency does not cause HyperapoB in mice and that the physiological importance of impaired ASP(C3ades-Arg) function as a cause of hyperapobetalipoproteinemia needs to be reevaluated.

V. A new route, jet injection of lidocaine for skin wheal for painless intravenous catheterization.

OBJECTIVE: The objective of this study was to compare the efficacy of intradermal lidocaine anesthesia by two jet injectors to the routine needle infiltration and to the topical EMLA cream. SUBJECTS AND METHODS: In a randomized, prospective, controlled trial, 100 consenting surgicenter patients in a university hospital setting were divided into four groups (n = 25, each); intradermal lidocaine anesthesia was given either by the conventional 25 g needle/syringe or the Med-E-Jet or Biojector injector or EMLA cream was applied on the skin. Visual analogue pain scores (VAS) or verbal pain intensity scores (PIS) were reported by the patients at lidocaine application and i.v. catheterization. Cost was also assessed. RESULTS: At lidocaine application, no pain was reported, since proportions of VAS = 0 were 25/25 (CI: 0.868, 0.999) with Med-E-Jet; 24/25 (0.804, 0.991) with Biojector; 25/25 (0.868, 0.999) with EMLA; in contrast to pain, 3/25 (0.044, 0.302) with the needle (PP > 0.999). The VAS scores (mean +/- SD) were 0.00 +/- 0.00, 0.04 +/- 0.20, 0.00 +/- 0.00, and 2.4 +/- 2.2 respectively (p < 0.00 1). No pain was reported by proportions of PIS = 0 with Med-E-Jet: 25/25 (CI: 0.868, 0.999); with Biojector: 23/25 (0.749, 0.976); EMLA 25/25 (0,868, 0.999); but pain with the needle: 5/25 (0.090, 0.394) (PP > 0.999). The mean +/- SD PIS scores were 0.00 +/- 0.00, 0.16 +/- 0.55, 0.00 +/- 0.00, and 1.24 +/- 1.00, respectively (p < 0.001). At i.v. catheterization, the proportions of VAS = 0 scores were 22/25 with Med-E-Jet (0.698, 0.956); 21/25 (0.651, 0.934) with Biojector; but some pain with needle: 6/25 (0.116, 0.436) (PP > 0.999). The mean +/- SD VAS scores were: 0.12 +/- 0.33, 0.44 +/- 0.20, and 1.64 +/- 1.50, respectively (p < 0.001). No pain was reported by PIS = 0 scores in 24/25 (0.804, 0.991) with Med-E-Jet; 24/25 (0.804, 0.991) with the Biojector; but pain by zero PIS scores 13/25 (0.334, 0.703) in half of the patients in the needle group (PP > 0.999). The mean +/- SD scores were 0.00 +/- 0.00, 0.00 +/- 0.00, and 0.76 +/- 0.88, respectively (p < 0.001). The EMLA cream was not evaluated because of inadequate duration of application prior to anesthetic induction. Cost/application were: Med-E-Jet = $ 0.13; needle = $ 0.50; Biojector = $ 0.94 and EMLA = $ 3.76. CONCLUSION: Almost completely painless i.v. catheterization by jet injection of lidocaine was accomplished, while needle infiltration produced pain/discomfort and did not significantly reduce it at the i.v. needle insertion.

A critical evaluation of the putative role of C3adesArg (ASP) in lipid metabolism and hyperapobetalipoproteinemia.

The acylation stimulating protein, ASP is a small, basic serum protein capable of stimulating triglyceride synthesis in cultured fibroblasts and adipocytes. Sequence analysis of ASP has shown that ASP is identical to C3adesArg the inactive fragment of the complement anaphylatoxin peptide, C3a. It has been proposed that C3adesArg (ASP) can be generated by mature adipocytes secreting the three complement proteins: complement protein C3, factor B and factor D (adipsin). There have also been indications that adipocytes may express a specific C3adesArg (ASP)-receptor that is distinct from the recently cloned C3a-receptor. This suggests that C3adesArg (ASP) acts as an adipocyte autocrine and that it plays a central role in the metabolism of adipose tissue. Based on these observations a hypothesis for the etiology of hyperapobetalipoproteinemia (hyperapoB) has been proposed. Hyperapobetalipoproteinemia (hyperapoB), is a familial lipoprotein disorder characterized by increased hepatic secretion of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) particles. If C3adesArg (ASP) function in the adipose tissue is impaired, a reduced rate of triglyceride synthesis will follow, generating an increased flux of fatty acids to the liver. In response to an increased flow of fatty acids, the liver will increase its production of VLDL particles yielding the phenotype of hyperapoB. This review critically assesses this hypothesis and the potential role of C3adesArg (ASP) as a major determinant for triglyceride synthesis in the light of data collected in vitro and in vivo.

The role of the N-methyl-D-aspartic acid receptor in the relaxant effect of ketamine on tracheal smooth muscle.

UNLABELLED: Ketamine and magnesium (Mg2+), well known bronchodilators, have been used to treat patients with status asthmaticus. Both can block the N-methyl-D-aspartic acid (NMDA) receptor. NMDA receptors exist in the airway, and their activation seems to be linked to the release actions of sensory neuropeptides resulting in increased airway tone. We sought to determine whether ketamine relaxes the guinea pig trachea contracted by histamine by blocking the NMDA receptor. Female guinea pigs (250-400 g) were killed with an overdose of pentobarbital. The trachea was removed and cut spirally into strips 3 mm wide and 15 mm long. The strips were mounted in a 10-mL organ bath filled with Tyrode's solution bubbled through with 95% O2/5% CO2 at 37 degrees C. Strip contractions were measured isometrically with a force displacement transducer. We then studied the effect of NMDA receptor antagonists on histamine-induced tracheal contraction. In this protocol, we examined the effect of ketamine, Mg2+, zinc (Zn2+), or MK-801 (a noncompetitive NMDA receptor blocker) on strips contracted by 10(-5) M histamine. After full contraction was attained, ketamine (0.5-1.5 mM), MgSO4 (2-8 mM), ZnCl2(0.2-0.8 mM), or MK-801 (1.5-6 x 10(-5) M) was added, and the strip tension was measured again. We also studied the effect of NMDA on the relaxation by ketamine. After full contraction by 10(-5) M histamine, 0.5-1.5 mM KET was added alone or in combination with 0.1 mM NMDA, and the strip tension was measured again. Finally, we measured the effect of MK-801 on the relaxant effect of ketamine. After full contraction by 10(-5) M histamine, 0.5-2 mM ketamine was added alone or in combination with 0.75 or 1.5 x 10(-5) M MK-801, and the strip tension was measured again. All NMDA receptor antagonists tested reversed the tracheal contraction induced by histamine in a dose-dependent manner. However, neither the agonist NMDA nor the noncompetitive receptor blocker MK-801 affected tracheal relaxation induced by ketamine. We conclude that ketamine relaxes the tracheal smooth muscle contracted by histamine through a mechanism independent of NMDA receptors. The decreased bronchomotor tone induced by ketamine is probably due to interference with a Ca2+-requiring step necessary to maintain the contraction caused by histamine. IMPLICATIONS: Stimulation of the N-methyl-D-aspartic acid (NMDA) receptor in the airway results in airway constriction. The bronchodilator ketamine blocks the NMDA receptor. However, ketamine relaxes the guinea pig trachea contracted by histamine through a mechanism independent of the NMDA receptor.

Resistance of chylomicron and VLDL remnants to post-heparin lipolysis in ApoE-deficient mice: the role of apoE in lipoprotein lipase-mediated lipolysis in vivo and in vitro.

The interaction of lipoprotein lipase (LPL) with triglyceride-rich lipoproteins is governed by a number of factors, such as apolipoprotein (apo) C-II. The role of apoE in lipolysis is controversial. We made the unexpected observation that apoE-deficient mice were resistant to heparin-induced lipolysis; this study aims at examining the underlying mechanism for this observation. Compared to wild-type mice, apoE-deficient mice had significantly higher very low density lipoprotein (VLDL) and chylomicron remnant (VLDL/CMR) concentrations and moderately lower lipase activity (15.5 +/- 1.3 mU/ml vs. 22.9 +/- 2.5 mU/ml). Unlike in wild-type mice where the injection of heparin reduced total plasma triglycerides by 50% and VLDL/CMR triglycerides by over 95%, the injection of heparin into apoE-deficient mice did not significantly affect plasma lipids. Similarly, in vitro, purified human LPL (hLPL) almost completely hydrolyzed VLDL/CMR isolated from wild-type mice, but had no effect on VLDL/CMR from apoE-deficient mice. However, when the amount of apoE-deficient VLDL/CMR was reduced to an equivalent level as in wild-type mice, LPL hydrolyzed 94% of VLDL/CMR triglycerides. In order to increase the ratio of LPL to VLDL/CMR in vivo, we injected an adenovirus containing the human LPL cDNA into apoE-deficient mice, which produced marked liver-specific overexpression of LPL and significant reduction of VLDL/CMR (93%) and total plasma triglyceride concentrations (87%). Thus, apoE is not required for LPL activity in vivo or in vitro. Under certain pathological conditions, such as severe hyperlipidemia, the LPL pathway may be saturated and efficient lipolysis can proceed only if the ratio of substrate particles to LPL is adjusted to a more normal range.

Ketamine inhibits the tonic response to carbachol and histamine in the guinea pig trachea.

The contractile response of smooth muscles to spasmogens can be divided into two components by modifying the extracellular Ca2+ concentration. The phasic component depends on the mobilization of Ca2+ from intracellular stores whereas the tonic component depends, to a large extent, on the influx of extracellular Ca2+. The present study was designed to investigate the effect of ketamine on the tonic response to carbachol or histamine in the guinea pig trachea. Tracheal spirals from female guinea pigs were mounted in organ baths filled with aerated physiological buffer, and their isometric tension was measured. The phasic response to 10(-7) M carbachol or 10(-5) M histamine in Ca(2+)-free, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-containing buffer and the tonic response to each spasmogen after restoring [Ca2+] in the buffer was measured in the absence or presence of ketamine. In the presence of normal physiological buffer, ketamine decreased the contractions induced by carbachol or histamine in a dose-dependent fashion. No measurable phasic response to either carbachol or histamine was obtained in our preparation. Ketamine (5 x 10(-5) M-10(-3) M) reduced the tonic response to 10(-7) M carbachol to 79.5 +/- 2.7-4.3 +/- 0.7% of the response without ketamine. Similarly, ketamine (5 x 10(-4) M-2 x 10(-3) M) decreased the tonic response to 10(-5) M histamine to 80.7 +/- 3.9-23.0 +/- 3.2% of the response in the absence of ketamine. Our findings support the hypothesis that ketamine inhibits the paracrine agent-induced contractions of smooth muscles by interfering with the influx of extracellular Ca2+ or with an intracellular event(s) requiring extracellular Ca2+.

A new route, jet injection for anesthetic induction in children--III. Ketamine pharmacokinetic studies.

The jet injector route for ketamine was used on 30 children 1-6 years of age undergoing various surgical procedures. A randomly selected dose of 2.5, 3.5, or 6.0 mg/kg of ketamine was given to induce anesthesia. Peak plasma ketamine levels did not follow a simple arithmetic increment related to dose. Dosage based on mg/m2 body surface area or mg/kg body weight provided similar blood levels of ketamine. The beta-phase t1/2 of ketamine in these children was shorter than that found in adults. Considerable individual variability was observed in both the plasma levels to a given dose of jet-injected ketamine and in the beta-phase t1/2. The ketamine beta-t1/2s were not dose related.

Ketamine relaxes airway smooth muscle contracted by endothelin.

Endothelins (ETs) are synthesized not only in vascular endothelial cells but also in airway epithelial cells. Increased ET-1 has been demonstrated in bronchial epithelium of asthmatic patients, and, in severe asthma attacks, ET-1 increases in plasma and bronchoalveolar lavage fluid. In this study, we investigated whether ketamine (KET) relaxes ET-induced tracheal contractions. Female guinea pigs were killed with an overdose of pentobarbital. The trachea was removed and cut spirally into two strips that were mounted in an organ bath filled with Krebs-bicarbonate buffer. The response of each strip to 10(-7) M carbachol was taken as 100% contraction to which the response to ET was referred. The contribution of the epithelium to the relaxant effect of KET was studied in denuded tracheae or in the presence of 5 x 10(-5) M indomethacin. ET-1 (3 x 10(-8) M) induced contractions that were 76 +/- 3% of those induced by carbachol. KET reversed the response to ET-1 in a dose-dependent fashion. Similarly, ET-2 (3 x 10(-8) M) induced contractions that were 74 +/- 5% of those induced by carbachol, and KET also reversed this response in a dose-dependent manner. In epithelium-denuded strips, ET-1 induced contractions that were 104 +/- 3% of those induced by carbachol, and KET still reversed this response. The tonic phase of the response to ET-1 was equal (100 +/- 6%) to the response to carbachol, and KET did not affect it significantly. In the presence of ryanodine, KET reduced the ET-1-induced contraction from 67 +/- 2% to 36 +/- 3.%, P < 0.01. In the presence of nicardipine, KET also inhibited the ET-1-induced contraction. We conclude that KET relaxes the tracheal smooth muscle contracted by ETs via a mechanism that is independent of the tracheal epithelium. The relaxant effect of KET on the ET-induced contraction of the trachealis muscle is not dependent upon blockade of 1) sarcolemma influx of Ca2+ through the dihydropyridine Ca2+ channel or 2) the release of intracellular Ca2+ through the ryanodine-sensitive intracellular Ca2+ channel. It is likely that the action of KET relaxing ET-induced tracheal contractions is at some point of the inositol 1,4,5-trisphosphate signaling pathway.

The relaxant effect of ketamine on guinea pig airway smooth muscle is epithelium-independent.

Airway epithelial cells and vascular endothelial cells modulate the tone of the underlying smooth muscle by releasing relaxing factors such as prostanoids and nitric oxide (NO). In the present study, we investigated whether the relaxant effect of ketamine depends on any of the epithelium-derived relaxing factors. Tracheae of female guinea pigs were cut spirally into strips (15 x 3 mm) and mounted in water-jacketed organ baths filled with Krebs-bicarbonate buffer aerated with a mixture of 95% O2 and 5% CO2 at 37 degrees C. Changes in the tension of the strips were measured isometrically with a force displacement transducer and recorded with a polygraph. In the first set of experiments, we examined the effect of ketamine on the concentration-response curves for histamine and carbachol in strips in which the epithelium was kept intact and in strips with denuded epithelium. In the second and third set of experiments, we studied the effect of indomethacin, a cyclooxygenase inhibitor, and N-omega-nitro-L-arginine methylester(L-NAME), a NO synthase inhibitor, on the relaxant activity of ketamine on tracheal strips contracted by histamine or carbachol. The following results were obtained: 1. Mechanical denudation of the tracheal epithelium shifted the concentration-response curve for histamine to the left (the 50% effective concentration [EC50] value of histamine decreased from 3.5 +/- 0.02 x 10(-6) M in the intact strips to 0.98 +/- 0.01 x 10(-6) M in denuded strips, P < 0.001). However, removal of the tracheal epithelium did not change the response to carbachol (the EC50 for carbachol was 1.1 +/- 0.02 x 10(-7) M in intact strips versus 0.88 +/- 0.01 x 10(-7) M after epithelial removal, P > 0.05). 2. Ketamine shifted to the right the concentration-response curves for histamine and carbachol in both intact and denuded tracheae. 3. Indomethacin did not alter the relaxant effect of ketamine on the tracheae contracted by either histamine (the concentration that inhibits 50% [IC50] of ketamine = 1.5 +/- 0.01 x 10(-3) M in control strips and 1.3 +/- 0.04 x 10(-3) M in strips pretreated with indomethacin, P > 0.05) or carbachol (the IC50 of ketamine was 2.5 +/- 0.02 x 10(-4) M in control strips and 2.4 +/- 0.01 x 10(-4) M in strips pretreated with indomethacin, P > 0.05). 4. L-NAME did not influence the relaxant effect of ketamine on tracheae contracted by either histamine (the IC50 of ketamine = 1.6 +/- 0.05 x 10(-3) M in control strips and 1.6 +/- 0.05 x 10(-3) M in strips pretreated with L-NAME, P > 0.05) or carbachol (the IC50 of ketamine = 2.6 +/- 0.04 x 10(-4) M in control strips and 2.3 +/- 0.01 x 10(-4) M in trips pretreated with L-NAME, P > 0.05). These results indicate that neither the mechanical removal of the tracheal epithelium nor the blockade of the release of potent mediators from tracheal epithelial cells influence the relaxant effect of ketamine on guinea pig tracheal strips contracted by histamine or carbachol. We conclude that ketamine relaxes the airway smooth muscle by an epithelium-independent mechanism.

Outpatient general anesthesia: a comparison of a combination of midazolam plus propofol and propofol alone.

STUDY OBJECTIVE: To compare the hemodynamics, efficacy, safety, and postoperative recovery of patients following the use of either midazolam plus propofol or placebo plus propofol for induction and maintenance of general anesthesia for outpatient surgical procedures of less than two hours' duration. DESIGN: Prospective, parallel, randomized, double-blind, placebo-controlled, multicenter study. SETTING: Ten outpatient surgery centers. PATIENTS: 203 ASA physical status I, II, and III patients undergoing various outpatient surgical procedures. INTERVENTIONS: Patients were randomly assigned to one of the two treatment groups. For induction of anesthesia, Group 1 received midazolam (0.077 +/- 0.0021 mg/kg) via slow intravenous (IV) push plus continuous infusion propofol (provided in a concentration of 5 mg/ml), and Group 2 received placebo plus full-concentration (10 mg/ml) propofol. Thereafter, Group 1 received half-concentration propofol and Group 2 received full-concentration propofol via continuous infusion for maintenance of anesthesia. Investigators administered doses of study medication in a blinded fashion as required to achieve the desired clinical effect. Drugs used to maintain anesthesia were restricted to study drug, short-acting opioids, and nitrous oxide. Succinylcholine chloride or vecuronium were used to facilitate intubation of study patients. MEASUREMENTS AND MAIN RESULTS: There were no statistically significant differences between the midazolam/propofol and placebo/propofol groups with respect to the mean (SE) decrease in mean arterial pressure from pre-dose to time of intubation or from time of intubation to initiation of surgery; the mean (SE) time required from initiation of study medication to completion of intubation [6.7 (0.23) minutes vs. 7.0 (0.26) minutes, respectively]; or the mean (SE) amount of propofol required to induce and maintain anesthesia [6.03 (0.329) mg/kg vs. 9.71 (0.489) mg/kg, respectively]. There was no significant difference between the two treatment groups in the time to recovery following the completion of surgery (as assessed by Aldrete Post Anesthesia Recovery Score). Most patients (approximately 79%) in both groups rated the quality of the anesthetic regimen as excellent; however, as assessed by patient questionnaires, fewer patients in the midazolam/ propofol group were able to recall the events surrounding their surgical procedure as compared with patients in the placebo/ propofol group (89.2% vs. 77.9%; p = 0.022). There were no differences between the two groups with respect to the frequency or severity of adverse events. CONCLUSIONS: Concomitantly administered midazolam and reduction-concentration propofol did not exacerbate the well-described hypotensive effects of full-strength propofol during induction of anesthesia. The time to intubation was equivalent with the combination of midazolam/propofol as compared with propofol alone. Recovery from the two regimens was not significantly different. However, reduced recall of perioperative events was observed more often in the midazolam/propofol regimen compared with propofol alone.

Adenovirus-mediated gene transfer of human lipoprotein lipase ameliorates the hyperlipidemias associated with apolipoprotein E and LDL receptor deficiencies in mice.

Lipoprotein lipase (LPL) is the rate-limiting enzyme for the hydrolysis of triglyceride-rich lipoproteins. We tested the efficacy of adenovirus-mediated gene transfer of LPL as treatment of experimental hyperlipidemias associated with apolipoprotein (apoE) deficiency (apoE-/-) and low-density lipoprotein receptor (LDLr) deficiency (LDLr-/-) in mice. Replication-defective adenovirus containing the human LPL cDNA driven by a cytomegalovirus promoter (Ad.hLPL) efficiently transduced CHO-ldlA7 cells in vitro, inducing in these cells the production of bioactive LPL (73 mU/ml). Intravenous injection of Ad.hLPL (2 x 10(9) pfu) led to high-level expression of hLPL mRNA and LPL activity in the liver (88.3 mU/ml) and in post-heparin plasma (116.1 mU/ml). Overexpression of LPL resulted in marked reductions in total plasma cholesterol (TC; 48%, 43%, 25%) and triglycerides (TTg; 63%, 40%, 70%, p < 0.01) in apoE-/-, LDLr-/-, and wild-type (WT) mice, respectively. Fast protein liquid chromatography (FPLC) fractionation of plasma lipoproteins showed a marked decrease in very-low-density lipoprotein (VLDL)/chylomicron remnant cholesterol (V/CR-C) in apoE-/- (83%), LDLr-/- (84%), and WT mice (58%, p < 0.01). VLDL/chylomicron remnant triglycerides (V/CR-Tg) were virtually eliminated in apoE-/- (92%), LDLr-/- (86%), and WT mice (84%, p < 0.05). No significant changes were detected in LPL activities, plasma lipids, or lipoproteins of mice injected with a control virus, Ad.Luc, containing the luciferase instead of the LPL cDNA. In summary, infusion of Ad.hLPL leads to increased liver and post-heparin plasma LPL activities, significantly reduced TC, TTg, V/CR-C, and V/CR-Tg in WT mice, as well as in mice with apoE and LDLr deficiencies. Adenovirus-mediated LPL gene transfer to the liver is an effective means of reversing many of the lipoprotein abnormalities in apoE- and LDLr-deficient mice.

Macrophage-mediated 15-lipoxygenase expression protects against atherosclerosis development.

Oxidative modification of LDL increases its atherogenicity, and 15-lipoxygenase (15-LO) has been implicated in the process. To address this issue, we generated transgenic rabbits that expressed 15-LO in a macrophage-specific manner and studied their susceptibility to atherosclerosis development when they were fed a high-fat, high-cholesterol (HFHC) diet (Teklad 0533 rabbit diet 7009 with 10% corn oil and 0.25% cholesterol) for 13.5 wk. Transgenic and nontransgenic rabbits developed similar degrees of hypercholesterolemia and had similar levels of triglyceride, VLDL, LDL, and HDL. Quantitative morphometric analysis of the aortic atherosclerosis indicated that the transgenic animals (n = 19) had significantly smaller lesion areas (9.8+/-6.5%, mean+/-SD) than their littermate controls (n = 14, 17.8+/-15.0%) (P < 0.05). In a subgroup (n = 9) of transgenic rabbits that received the HFHC diet plus the antioxidant N',N '-diphenyl-phenylenediamine (1%), the extent of lesion involvement (9.8+/-7.5%) did not differ from the subgroup (n = 10) that received the regular HFHC diet (9.7+/-5.9%). Since the results were unexpected, we repeated the experiments. Again, we found that the nontransgenic littermates (n = 12) had more extensive lesions (11.6+/-10.6%) than the transgenic rabbits (n = 13; 9.5+/-7.8%), although the difference was not significant. In a third set of experiments, we crossed 15-LO transgenic rabbits with Watanabe heritable hyperlipidemic (WHHL) rabbits and found that the lesion area in the 15-LO transgenic/heterozygous WHHL rabbits (n = 14) was only about one third (7.7+/-5.7%) that found in nontransgenic heterozygous WHHL littermate controls (n = 11, 20.7+/-19.4%) (P < 0.05). These data suggest that overexpression of 15-LO in monocytes/macrophages protects against lipid deposition in the vessel wall during early atherogenesis in these rabbit models of atherosclerosis.

Complete phenotypic characterization of apobec-1 knockout mice with a wild-type genetic background and a human apolipoprotein B transgenic background, and restoration of apolipoprotein B mRNA editing by somatic gene transfer of Apobec-1.

We have produced gene knockout mice by targeted disruption of the apobec-1 gene. As recently reported by Hirano et al. (Hirano, K.-I., Young, S. G., Farese, R. V., Jr., Ng, J., Sande, E., Warburton, C., Powell-Braxton, L. M., and Davidson, N. O. (1996) J. Biol. Chem. 271, 9887-9890), these animals do not edit apolipoprotein (apo) B mRNA or produce apoB-48. In this study we have performed a detailed analysis of the lipoprotein phenotypic effects of apobec-1 gene disruption that were not examined in the previous study. We first analyzed the plasma lipoproteins in knockout animals with a wild-type genetic background. Although there was no difference in plasma cholesterol between apobec-1(-/-), +/-, or +/+ mice, there was a marked (176%) increase in plasma apoB-100, from 1.8 +/- 1.2 mg/dl in apobec-1(+/+) mice to 2.7 +/- 0.6 mg/dl in apobec-1(+/-) and 5.0 +/- 1.4 mg/dl in apobec-1(-/-) mice. Plasma apoE was similar in these animals. By fast protein liquid chromatography (FPLC) analysis, there was a significant decrease in plasma high density lipoprotein (HDL) cholesterol in apobec-1(-/-) mice. We further fractionated the plasma lipoproteins into d < 1.006, 1.006-1.02, 1.02-1.05, 1.05-1.08, 1.08-1.10, and 1.10-1.21 g/ml classes, and found a marked (30-40%) reduction in the cholesterol and protein content in the (d 1.08-1.10 and 1.10-1.21) HDL fractions, corroborating the FPLC data. SDS-gel analysis revealed an absence of apoB-48, an increase in apoB-100 in the very low density lipoprotein (VLDL) and low density lipoprotein (LDL) fractions, and a small decrease in apoA-I in the HDL fractions in the apobec-1(-/-) samples. We next raised the basal plasma apoB levels in the apobec-1(-/-) animals by cross-breeding them with human apoB transgenic (TgB) mice. The plasma apoB-100 was 3-fold higher in apobec-1(-/-)/TgB+/- mice (26.6 +/- 18.3 mg/dl) than in apobec-1(+/+)/TgB+/- mice (9.8 +/- 3.9 mg/dl, p < 0.05). The apobec-1(-/-)/TgB+/- mice had a plasma cholesterol levels of 170 +/- 28 mg/dl and triglyceride levels of 106 +/- 31 mg/dl, which are 80% and 58% higher, respectively, than the corresponding values of 94 +/- 21 mg/dl and 67 +/- 11 mg/dl in apobec+/+/TgB+/- mice. By FPLC, the apobec-1(-/-)/TgB+/- animals developed markedly elevated plasma LDL cholesterol (518.5 +/- 329.5 microg/ml) that is 373% that of apobec1(+/+)/TgB+/- mice (139.0 +/- 87.0 microg/ml) (p < 0.05). The elevated plasma triglyceride was accounted for mainly by a 97% increase in VLDL triglyceride in the apobec1(-/-)/TgB+/- mice. We conclude that apobec-1(-/-) animals have a distinctive lipoprotein phenotype characterized by significant hyperapoB-100 and HDL deficiency in mice with a wild-type genetic background. Furthermore, the abolition of apoB mRNA editing elevates plasma total cholesterol and LDL cholesterol in apobec-1(-/-) animals with a TgB background. Finally, to exclude the possibility that absence of apoB mRNA editing was a secondary effect of chronic Apobec-1 deficiency, we treated apobec-1(-/-) mice with a replication-defective mouse Apobec-1 adenoviral vector and found that we could acutely restore apoB mRNA editing in the liver. These experiments indicate that Apobec-1 is an essential component of the apoB mRNA editing machinery and absence of editing in the knockout animals is a direct consequence of the absence of functional Apobec-1.

Structure-function relationship of lipoprotein lipase-mediated enhancement of very low density lipoprotein binding and catabolism by the low density lipoprotein receptor. Functional importance of a properly folded surface loop covering the catalytic center.

We examined the structure-function relationship of human lipoprotein lipase (hLPL) in its ability to enhance the binding and catabolism of very low density lipoproteins (VLDL) in COS cells. Untransfected COS cells did not bind to or catabolize normal VLDL. Expression of wild-type hLPL by transient transfection enhanced binding, uptake, and degradation of the VLDL (a property of LPL that we call bridge function). Heparin pretreatment and a monoclonal antibody ID7 that blocks LDL receptor-binding domain of apoE both inhibited binding, and apoE2/E2 VLDL from a Type III hyperlipidemic subject did not bind. However, LDL did not reduce 125I-VLDL binding to the hLPL-expressing cells, whereas rabbit beta-VLDL was an effective competitor. By contrast, LDL reduced uptake and degradation of 125I-VLDL to the same extent as excess unlabeled VLDL or beta-VLDL. These data suggest that binding occurs by direct interaction of VLDL with LPL but the subsequent catabolism of the VLDL is mediated by the LDL receptor. Mutant hLPLs that were catalytically inactive, S132A, S132D, as well as the partially active mutant, S251T, and S172G, gave normal enhancement of VLDL binding and catabolism, whereas the partially active mutant S172D had markedly impaired capacity for the process; thus, there is no correlation between bridge function and lipolytic activity. A naturally occurring genetic variant hLPL, S447-->Ter, has normal bridge function. The catalytic center of LPL is covered by a 21-amino acid loop that must be repositioned before a lipid substrate can gain access to the active site for catalysis. We studied three hLPL loop mutants (LPL-cH, an enzymatically active mutant with the loop replaced by a hepatic lipase loop; LPL-cP, an enzymatically inactive mutant with the loop replaced by a pancreatic lipase loop; and C216S/C239S, an enzymatically inactive mutant with the pair of Cys residues delimiting the loop substituted by Ser residues) and a control double Cys mutant, C418S/C438S. Two of the loop mutants (LPL-cH and LPL-cP) and the control double Cys mutant C418S/C438S gave normal enhancement of VLDL binding and catabolism, whereas the third loop mutant, C216S/C239S, was completely inactive. We conclude that although catalytic activity and the actual primary sequence of the loop of LPL are relatively unimportant (wild-type LPL loop and pancreatic lipase loops have little sequence similarity), the intact folding of the loop, flanked by disulfide bonds, must be maintained for LPL to express its bridge function.

Droperidol inhibits tracheal contraction induced by serotonin, histamine or carbachol in guinea pigs.

PURPOSE: Droperidol (D) is effective in the treatment of patients with status asthmaticus. It has been reported that D inhibits the bronchoconstriction induced by serotonin (5-HT) but not that by histamine (H) or acetylcholine. However, haloperidol, another butyrophenone, is known to interact with and inhibit calmodulin, an intracellular Ca(++)-binding protein which is important in the contraction of smooth muscles. The present study was designed to investigate the effects of D on tracheal contractions induced by 5-HT, H or carbachol (C) and to determine the contribution of alpha-adrenoceptors to the relaxant effect of D in vitro. METHODS: Tracheas of female guinea pigs were cut spirally into strips and mounted in water-jacketed organ baths in Tyrode's solution, aerated with a mixture of 95% O2 and 5% CO2 at 37 degrees C. The changes in isometric tension induced by each spasmogen in the strips were measured with a transducer and a polygraph. RESULTS: We found that D inhibited the tracheal contractions induced by 5-HT, H or C in a concentration-dependent manner. At 1.25 x 10(-6) M D blocked the effect of 10(-4) M 5-HT by 44.1 +/- 4.3% and at 2.5 x 10(-6) M by 63.8 +/- 3.8%. Similarly, at 5.0 x 10(-6) M concentration, D blocked the effect of 10(-5) M H by 27.7 +/- 5.3% and at 10(-5) M by 56.2 +/- 2.6%. Furthermore, 5 x 10(-6) M of D reduced the contractions produced by 10(-7) M C by 37.1 +/- 3.0% and 10(-5) M of D by 76.1 +/- 3.2%. The inhibiting effect of D was strongest on contractions induced by 5-HT. Prazosin (10(-6) M) affected neither 5-HT-induced contractions nor the inhibition by D. CONCLUSION: Our data indicate that D partially blocks the contractile responses not only to 5-HT, an effect which would be mediated through a blockade of the 5-HT receptors, but also to H or C, probably through inhibition of calmodulin. Our data support previous reports indicating that droperidol may be an important therapeutic agent in the treatment of patients with hyperreactive airways.

Relaxant effect of ketamine and its isomers on histamine-induced contraction of tracheal smooth muscle.

The mechanism by which racemic (R(+/-)) ketamine relaxes airway smooth muscle is unclear and there is no information on the differential effects of ketamine and its isomers. In this study, we have examined the spasmolytic effect of R(+/-) ketamine and its isomers S(+) and R(-) ketamine and the role of intracellular calcium and opioid receptors in R(+/-) ketamine-induced relaxation. The tension of isolated guinea pig tracheal strips was measured isometrically with a force displacement transducer and contraction elicited with histamine 10(-5) mol litre-1. In histamine-preconstricted strips, the two ketamine isomers (4.5-18.0 x 10(-4) mol litre-1) produced equipotent relaxation. A subthreshold dose of each isomer of ketamine (10(-4) mol litre-1) which alone did not relax histamine-induced contraction (S(+), P < 0.01; R(+/-), P < 0.01; R(-), P < 0.05) significantly potentiated adrenaline 1.25-5.0 x 10(-9) mol litre-1-induced relaxation (potency: S(+) > R(+/-) > R(-)). Increase in extracellular Ca2+ (1.8-14.4 x 10(-3) mol litre-1) significantly reduced R(+/-) ketamine-induced relaxation. S(-) Bay K 8644, at concentrations up to 2.0 x 10(-6) mol litre-1, partially antagonized R(+/-) ketamine-induced relaxation whereas at 10(-5) mol litre-1 or higher it potentiated the response. Naloxone 1.5-6.0 x 10(-6) mol litre-1 did not affect the relaxation caused by R(+/-) ketamine. We conclude that although both ketamine isomers produced equipotent spasmolytic effects on airway smooth muscle precontracted with histamine, they differed in their ability to potentiate the relaxing effect of adrenaline. S(+) ketamine produced the greatest potentiation. Changes in intracellular Ca2+ level secondary to a reduction in the L-type Ca2+ current may partially mediate the spasmolytic effect of R(+/-) ketamine.

A new route, jet-injection for anesthetic induction in children - II. ketamine dose-range finding studies.

Ketamine (K) i.m. has been widely used for anesthetic induction in small children in the last decades, if mask induction has failed. In many instances, however, physical restraint was required. In order to eliminate the pain of i.m. injection and to prevent the psychological and physical trauma associated with restraint, we evaluated the utility of jet-injection (j.i.) of K for anesthetic induction in a dose-range finding study. Thirty children (age 1-6 years), whose parents gave a valid consent approved by the IRB and were scheduled for minor surgeries, were randomized into 3 equal groups: A/ketamine 6.0 mg/kg j.i., B/ketamine 3.5 mg/kg j.i., C/ketamine 2.5 mg/kg j.i. As a drying agent atropine 20 microg/kg was also given i.v. The onset of full amnesic/sedative effect of K, the scoring of sedation and emotional state, the ease of placement of the i.v. catheter, the speed of recovery by Aldrete scores, and the time for safe discharge were evaluated. Although no demographic differences were observed among the groups the duration of surgery and anesthesia were longer in the B group (41 and 49 min) than in the A or C groups. The onset of sedation was significantly (p < 0.05) faster in group A (174 sec) than in group B (312 sec) or C (303 sec). However, no significant difference was observed in the onset of complete sedation among the groups. The sedation index was the lowest representing the best sedation in group A (4.2) while in group B and C were somewhat higher (4.6 and 4.4). There were no differences in the ease of i.v. cannulation among the groups. Recovery from anesthesia was the slowest in group A, although the differences among the 3 groups did not reach statistical significance. The mean discharge times ranged from 10-13 min with no differences among the groups. Laryngospasm occurred in 4:10 in group A and 1:10 in groups B and C. Evidently the high dose of K, 6.0 mg/kg caused a proneness to laryngospasm. Since no additional benefit was derived from this high dose, the lower doses (3.0 mg/kg) of K may be sufficient for routine use. None of the children experienced unpleasant recall or pain for the injection or the whole procedure. This new route of anesthetic induction with the jet-injector utilizing K may provide pain-free and stress-free induction as compared to its i.m. injection. This technique also prevents transmission of infection and is [correction of and cost] cost effective since simultaneous and/or sequential injection can be given from a single vial of K.

The bispectral index during induction of anesthesia with midazolam and propofol.

This study evaluated the bispectral index as an indicator of anesthetic depth in relation to the cardiovascular response to intubation. Two treatments were compared: group 1 (n = 8) received propofol for induction of anesthesia (2 mg/kg bolus followed by an infusion of 0.20 mg/kg-1/min-1, group 2 (n = 8) was given 90 micrograms/kg midazolam 2 min before, followed by anesthesia with half-strength propofol (1 mg/kg bolus with infusion of 0.10 mg/kg-1/min-1). The bispectral index of the electroencephalogram, blood pressure, and heart rate were measured under unanesthetized conditions, during anesthetic induction, intubation, and a 15-min period after intubation. The duration of anesthesia and the total propofol requirement were recorded. Midazolam pretreatment produced transient decreases in blood pressure and the bispectral index. During anesthetic induction with propofol, blood pressure decreased 20% in both groups, and the bispectral index decreased to lower levels in group 1 (29 +/- 9) than in group 2 (47 +/- 22). Intubation increased blood pressure more in group 2 (50 +/- 10 mm Hg) than in group 1 (30 +/- 12 mm Hg). Throughout the rest of the surgery, more propofol was used in group 1 (77 +/- 14 micrograms/kg-1/min-1) than in group 2 (42 +/- 14 micrograms/kg-1/min-1). These results show that the decrease in bispectral index provides an indication of the blood pressure increase to intubation during propofol anesthesia. Midazolam pretreatment did not attenuate the cardiovascular response to intubation but did decrease propofol use during surgery.