Jayne L. Hart

Attenuation of endothelium-mediated vasodilation by halothane.

To determine whether halothane alters endothelium-mediated vasodilation of vascular smooth muscle, isolated ring preparations of rabbit aorta and canine femoral and carotid arteries were suspended for isometric tension recordings in Krebs-Ringer bicarbonate solution at 37°C. Acetylcholine and bradykinin have been shown to relax these norepinephrine contracted arteries via an endothelium-dependent process. In this study, these relaxations were reversibly and significantly attenuated by 2% halothane. However, halothane did not affect relaxations caused by nitroglycerin, which, in these vessels, acts by an endothelium independent mechanism. These results suggest that halothane is not interfering with cyclic guanylate-monophosphate mediated relaxation of vascular smooth muscle, but may interfere with the synthesis, release, or transport of the endothelium-derived relaxing factor. In addition, during contractions evoked by norepinephrine, halothane caused significant decreases in tension in both the canine carotid and rabbit aortic preparations, but increased tension in the femoral artery rings. These effects were not altered by mechanical removal of the endothelium. These results suggest a direct action of halothane on the vascular smooth muscle, which can result in either an increase or decrease in tension, depending on the specific vessel. In addition to its direct vascular effect, this study suggests a new action of halothane; it interferes with endothelium-derived relaxing factor-mediated relaxation of vascular smooth muscle. This action may contribute in part to the vascular alterations seen clinically during administration of halothane.

Effects of halothane and isoflurane on carbon monoxide-induced relaxations in the rat aorta

Background Halothane and isoflurane previously were reported to attenuate endothelium‐derived relaxing factor/nitric oxide‐mediated vasodilation and cyclic guanosine monophosphate (cGMP) formation in isolated rat aortic rings. Carbon monoxide has many chemical and physiologic similarities to nitric oxide. This study was designed to investigate the effects of halothane and isoflurane on carbon monoxide‐induced relaxations and cGMP formation in the isolated rat aorta. Methods isometric tension was recorded continuously from endothelium denuded rat aortic rings suspended in Krebs‐filled organ baths. Rings precontracted with submaximal concentrations of norepinephrine were exposed to cumulative concentrations of carbon monoxide (26–176 micro Meter). This procedure was repeated three times, with anesthetics delivered 10 min before the second procedure. Carbon monoxide responses of rings contracted with the same concentration of norepinephrine (10‐8 M and 2 x 10‐8 M) used in the anesthetic‐exposed preparations also were examined. The concentrations of cGMP were determined in denuded rings using radioimmunoassay. The rings were treated with carbon monoxide (176 micro Meter, 30 s) alone, or carbon monoxide after a 10‐min incubation with halothane (0.34 mM or 0.72 mM). To determine whether the sequence of anesthetic delivery influenced results, vascular rings pretreated with halothane were compared with non‐pretreated rings. Results Carbon monoxide (26–176 micro Meter) caused a dose‐dependent reduction of norepinephrine‐induced tension, with a maximal relaxation of 1.51+/‐0.07 g (85+/‐7% of norepinephrine‐induced contraction). Halothane (0.34 mM and 0.72 mM) significantly attenuated the carbon monoxide‐induced relaxations, but only the highest concentration of isoflurane (0.53 mM) significantly attenuated the carbon monoxide‐induced relaxations. Carbon monoxide (176 micro Meter) significantly increased cGMP content (+88.1+/‐7.1%) and preincubation of the aortic rings with halothane (0.34 mM and 0.72 mM) inhibited this increase (‐70.7 +/‐6.8% and ‐108.1+/‐10.6%, respectively). When aortic rings and carbon monoxide were added simultaneously to Krebs solution equilibrated with halothane (0.72 mM), no inhibition of cGMP formation occurred. Conclusion Carbon monoxide‐induced endothelium‐independent relaxations of rat aortic rings were decreased by clinically relevant concentrations of halothane and isoflurane. The carbon monoxide‐induced elevations of cGMP were attenuated by halothane only when the anesthetic was incubated with aortic rings before carbon monoxide treatment. The possible clinical significance of the actions of the anesthetics on this endogenous vasodilator is yet to be determined.

Effects of Halogenated and Non-Halogenated Anesthetics on Diaspirin Cross-Linked Hemoglobin™-Induced Contractions of Porcine Pulmonary Veins

Diaspirin crosslinked hemoglobin (DCLHb) is a resuscitative fluid presently undergoing clinical trials. Administration of DCLHb is associated with an elevation of mean arterial pressure in vivo and contraction of isolated blood vessels in vitro. The mechanisms for the vascular actions are unknown but may be due to inhibition of nitric oxide (NO). Halothane has been reported to inhibit NO induced relaxation. We examined the effect of anesthetics on DCLHb induced contraction of blood vessels. Porcine pulmonary veins were excised, cut into rings and placed in organ chambers filled with 25 ml Krebs-Ringer solution (37 degrees C). Following equilibration at their optimal length the rings were exposed to increasing concentrations of serotonin(10(-8)M-10(-5)M). Endothelial activity was confirmed by relaxation greater than 80% with ACh (10(-6)M). DCLHb (1.5 x 10(-8)M to 6 x 10(-7)M) contracted porcine pulmonary veins (1.04 +/- 0.17g to 3.45 +/- 0.22g), and halothane (0.5% and 1%) significantly inhibited these DCLHb induced contractions in a dose-related manner (-41.6 +/- 8.1% and -73.3 +/- 8.2%, respectively). At equi-molar concentrations, isoflurane had no inhibitory activity. The relative effect of these volatile anesthetics is consistent with their inhibitory actions on other heme containing proteins. Propofol (10(-5)M) only has inhibitory effects on lower concentrations of DCLHb. Fentanyl did not have inhibitory effects. These results suggest that halogenated anesthetics may interact with the heme iron of DCLHb and inhibit its binding with NO.

Effect of halothane on phenylephrine-induced vascular smooth muscle contractions in endotoxin-exposed rat aortic rings.

OBJECTIVES a) To determine the response of endotoxin-exposed vascular smooth muscle to exogenous vasoconstrictors during concomitant exposure to an inhaled anesthetic (halothane); and b) to determine if excess nitric oxide production is responsible for any altered response. DESIGN In vitro, prospective, repeated-measures, dose-response study. SETTING University/medical school experimental physiology laboratory. SUBJECTS Adult male Sprague-Dawley rats, whose aortae were studied in an in vitro preparation. INTERVENTIONS Thoracic aortae were excised from anesthetized animals and cut into 3-mm rings. After incubation in aerated organ baths containing a modified essential medium with or without Escherichia coli lipopolysaccharide (100 micrograms/mL) at 37 degrees C for 5 hrs, the rings were removed and suspended in separate baths for isometric tension recording. Phenylephrine dose-response data (10(-10) to 10(-5) M) were determined for lipopolysaccharide- and nonlipopolysaccharide-treated rings. After washout and equilibration, two vessels (one each lipopolysaccharide- and nonlipopolysaccharide-treated) were additionally exposed to 2% halothane and phenylephrine dose-response determinations were repeated for all vessels. This procedure was repeated for 1% halothane in a separate experiment. In some experiments, the nitric oxide synthase inhibitor, N omega-nitro-L-arginine (3 x 10(-4) M), was added to the bath after the washout from the second phenylephrine dose-response determination. Then, a third phenylephrine dose-response determination was performed, with and without 2% halothane. MEASUREMENTS AND MAIN RESULTS Dose-response curves were evaluated using a logistic regression analysis. In addition, absolute and percentage changes in tension were compared between the first and second contractions. Exposure to lipopolysaccharide resulted in a decrease in the maximum tension from 2.07 +/- 0.03 (controls) to 1.24 +/- 0.04 g/mg of vessel dry weight and an increase in the dose at which the contraction is 50% of maximum (ED50) from 3.78 x 10(-8) to 2.05 x 10(-7) M (p < .05). Exposure to 2% halothane produced significant reductions in the maximum tensions in both groups. The lipopolysaccharide-treated vessels showed not only a proportionately larger decrease (-51 +/- 5% vs. -18 +/- 2% in the control plus halothane group), but also a significantly greater absolute decrease (0.59 +/- 0.09 vs. 0.34 +/- 0.04 g/mg in the control plus halothane group). The addition of 1% halothane produced less pronounced decreases in tension, with only an additive effect in the lipopolysaccharide-treated vessels. The addition of N omega-nitro-L-arginine resulted in a reversal of the lipopolysaccharide-induced decrease in tension. However, 2% halothane still had a significantly greater effect on the lipopolysaccharide-exposed rings. CONCLUSIONS Exposure of rat aortic rings to lipopolysaccharide in vitro decreased the contractile response to phenylephrine. The addition of 2% halothane resulted in a more than additive decrease in tension in the lipopolysaccharide-treated vessels. Patients in septic or endotoxic shock are sensitive to most anesthetic regimens, and some of this sensitivity may be due to an altered vasoconstrictive response induced by lipopolysaccharide exposure. The inability of nitric oxide synthase inhibition to reverse this response completely suggests that induction of nitric oxide synthase and increased production of nitric oxide are not solely responsible for this finding.

Vascular Interactions of Calcium and Reactive Oxygen Intermediates Produced Following Photoradiation

This study was designed to exarmine vascular smooth muscle contractile properties following cnhanced proudction of reactive oxygen intermediates (ROIs), which were produced by pretreating rat caudal arteries and aortas with a photosensitizer, hematoporphyrin deravative. and then illuminating them with red laser light. This treatment produced a long-lasting contraction that was dependent on the presence of extracellular calcium. Reduction in extracellular calcium relaxed the smooth muscle and replacement of calcium 30 min later increased the tension. Oxygen radical scavengers did not block the contractile effect postillumination when calcium was returned to the hathing solution: however, verapamil (5.5 μM) and nifedipine (10 μM) attenuated this contraction. The contractions were dependent on oxygen in the aerating gas mixture. Production of ROIs by isolated blood vessels was supported biochemically by a significant increase in both bath and tissue levels of oxidation products. reactive with thiobarbituric acid. and by a reduction in the tissue stain, nitroblue tetrazolium. These ROIinduced contractions were observed in vitro on large conduit arteries and also in vivo on small ear arteries. The vascular response following this acute production of ROIs may be similar to vascular abnormalities in certain pathologicl conditions where ROI production is reported to be elevated. Therefore. these results could contribute to a further understanding of mechanisms involved in these ROI-dependent vascular changes.

Endothelin plays a role in contractions of isolated pig pulmonary vessels induced by diaspirin cross-linked hemoglobin.

The current studies were undertaken to investigate the role of endothelin-1 (ET-1) and its receptors in contractions of isolated pulmonary vessels of the pig induced by diaspirin cross-linked hemoglobin (DCLHb). Second-order pulmonary arteries (PAs) and veins (PVs) were isolated from pigs, cut into rings (4 to 5 mm), and mounted at optimal passive tension in 37 degrees C Krebs-filled tissue baths bubbled with 95% O2/5% CO2. Isometric tension was recorded continuously. In paired rings, concentration responses to ET-1 (10(-10) to 10(-7) mol/L), DCLHb (10(-9) to 3x10(-6) mol/L), and N-nitro-L-arginine (LNA) (10(-6) to 5x10(-5) mol/L) in the presence and absence of the ET(A) receptor antagonist BQ123 (3x10(-5) mol/L) were determined. PVs and PAs with intact endothelium and rings from which the endothelium was removed (denuded) were pretreated with the ET(B) receptor antagonist BQ788 to determine the contribution of ET(B) receptors to ET-1, DCLHb, and LNA responses. ET-1, DCLHb, and LNA caused concentration-dependent increases in tension in all vessels. In the presence of BQ123, the 50% effective concentration (EC50) of ET-1 was significantly increased (by 5-fold to 10-fold) in all vessels. DCLHb concentration responses were significantly attenuated-in the PVs by 45% and in the PAs by 79%-during treatment with BQ123. BQ123 attenuated LNA responses in PVs by 35% and in PAs by 87%. Treatment with BQ788 had no effect on endothelium-intact PVs or PAs but significantly increased ET-1 EC50 (log of the molar concentration) from -9.0+/-0.22 to -7.8+/-0.05 in denuded PAs. The ET-1 EC50 was significantly decreased in denuded PAs (-9.0+/-0.22) as compared with responses in endothelium-intact PAs (-8.1+/-0.18). DCLHb concentration responses were attenuated by 71% and LNA responses by 80% during antagonism with BQ788 in the intact PAs only. These data demonstrate that ET-1 plays a role in DCLHb-induced contractions in the PA and PV. The contributions of ET are mediated by both ET(A) and ET(B) receptors in the PA but only by ET(A) receptors in the PV. These results suggest that the vasoconstrictor actions of DCLHb, which have previously been shown to depend on its interference with endothelium-generated NO, may also involve ET. This may reflect the importance of the interaction of these two endothelium-generated physiologic antagonists in the pulmonary circulation.

Interactions of Volatile Anesthetics and Reactive Oxygen Intermediates on Vascular Smooth Muscle

Reactive oxygen intermediates (ROIs, also referred to as oxygen-derived free radicals or reactive oxygen metabolites) have been implicated in several physiological and pathological processes.1–3 In particular, they have been reported to be associated with several vascular abnormalities, including those that occur during hypertension,4 reperfusion injury,5 transplant rejection,3 inflammation, premature aging,6 radiation injury, diabetes7 and endotoxic shock.8 An increase in PaO2, especially after hypoxia, is a major stimulus for production of ROIs.9

Further isolation of endogenous factors in canine and human plasma that inhibit [3H]norepinephrine accumulation

We have previously shown that both homologous canine plasma and a crude extract of this plasma contain substances that inhibit accumulation of [(3)H]norepinephrine ([(3)H]NE) by the canine saphenous vein. The purpose of this study was to further purify these substances and to determine if similar factors were present in human plasma. Crude extracts of plasma were purified with a Folch extraction in which most of the biological activity was recovered in the bottom or organic phase. This phase significantly inhibited [(3)H]NE uptake by the canine saphenous vein (23.5 +/- 7.6% by concentrate from 9.1 ml of original plasma/ml incubate solution) and increased development of tension following transmural electrical stimulation by 91.5 +/- 23.3% (extract from 1 ml of plasma/ml bath solution). The Folch extracts obtained from 100ml of plasma were purified by column and thin layer (TLC) chromatography. Samples were applied to a silicic acid column and eluted with chloroform, acetone, and methanol. The [(3)H]NE uptake inhibitory activity was primarily recovered in the methanol fraction. TLC of the methanol fraction of canine plasma on silica gel G plates (with pre-absorbent) resulted in five zones which were then assayed for their ability to inhibit [(3)H]NE accumulation by the saphenous vein. In the first zone (concentrate from 27.5 ml plasma/ml bath solution) there was significantly greater inhibitory activity (55.4 +/- 8.3%), than in the corresponding zone obtained from solvent blanks (20.7 +/- 4.1%). These results indicate that there is a factor or possibly factors in canine and human plasma that have thin layer chromatographic properties of a polar lipid, which inhibit [(3)H]NE accumulation and enhance the contractile response of vascular smooth muscle to transmural electrical stimulation.