Effects of enflurane on adrenergic function in canine mesenteric artery and vein.

We examined the pre- and postsynaptic effects of enflurane on the superior mesenteric artery and vein. We measured the release of norepinephrine (NE) from sympathetic nerve terminals caused by electrical stimulation (ES) or potassium and changes in vascular smooth muscle tension. The effect of enflurane was also examined in the presence of alpha 1- and alpha 2-adrenoceptor antagonists. Enflurane (1.4 to 1.6 minimum alveolar anesthetic concentration [MAC] in the dog) did not alter basal tension, but it significantly inhibited tension development caused by 40 mM KCl and ES in the canine mesenteric artery and vein. Enflurane inhibited the KCl- and ES-induced release of NE from sympathetic nerve terminals in the canine mesenteric vein. The alpha 1- and alpha 2-adrenoceptor antagonists, corynanthine or yohimbine, did not modify the effect of enflurane on the ES-induced frequency response curve of the mesenteric artery and vein. These data suggest that enflurane has at least two sites of action in mesenteric vascular beds. One site is presynaptic where it inhibits NE release, and the other is a postsynaptic site distal to the adrenergic receptor where it inhibits the smooth muscle contraction process.

Contractile effects of diaspirin cross-linked hemoglobin (DCLHb) on isolated porcine blood vessels.

The effects of diaspirin cross-linked hemoglobin (DCLHb) on vascular tone were examined in arterial and venous rings isolated from mesenteric, renal, femoral, and pulmonary blood vessels. Rings were placed in organ baths at optimal passive tension in Krebs' solution (37 degrees C) and aerated with a mixture of 95% O2/5% CO2. Vessels were treated with increasing concentrations of DCLHb or an iso-osmotic serum albumin solution. All artery and vein rings contracted in the presence of DCLHb. With the exception of the pulmonary vein, the magnitude of the tension increase with DCLHb was greater in the arteries than in the veins. Removal of the endothelium by scraping or the addition of a nitric oxide synthase inhibitor blocked the DCLHb-induced contraction in the pulmonary artery and vein. DCLHb also significantly reduced basal cyclic guanosine-3-5'-monophosphate in the pulmonary vein. These studies suggest that the constrictor activity of DCLHb is due primarily to inhibition of basal nitric oxide-induced relaxation of the vascular smooth muscle.

Effects of halothane on EDRF/cGMP-mediated vascular smooth muscle relaxations.

BACKGROUND: Halothane has been reported to inhibit endothelium-dependent relaxation in a variety of vessels. These studies were done to determine whether this inhibition is caused by interference with synthesis, release, or action of endothelium-derived relaxing factor (EDRF) on cyclic guanosine monophosphate (cGMP) levels within the vascular smooth muscle. METHODS: Rat aortic rings were suspended in aerated Krebs solution (37 degrees C) and were contracted to a stable plateau with EC60-70 norepinephrine (NE). Relaxations caused by acetylcholine (ACh; 1 x 10(-8)-1 x 10(-6) M), nitric oxide (NO; 5 x 10(-9)-1 x 10(-6) M), or nitroglycerin (NG; 2 x 10(-9)-3 x 10(-7) M) in rings contracted with NE were compared in the presence and absence of halothane. Tissue cGMP contents were measured using a radioimmunoassay method. RESULTS: In the presence of halothane (0.5, 1.0, and 2.0 MAC), the ACh-induced relaxations were significantly attenuated in a concentration-dependent manner, an effect that was reversible. Halothane (2 MAC) significantly attenuated NO-induced relaxations at all concentrations and NG-induced relaxations at low concentrations (5 x 10(-9)-3 x 10(-8) M) but not at higher concentrations (1 x 10(-9)-3 x 10(-7) M) in denuded vessels. Nitric oxide-stimulated (5 x 10(-8)-5 x 10(-6) M) cGMP content was significantly attenuated by halothane (2 MAC) at NO concentrations between 1 x 10(-7) and 5 x 10(-6) M. CONCLUSIONS: Nitric oxide, either endogenous or exogenous, interacts with the enzyme guanylate cyclase to stimulate the production of cGMP. Halothane interfered with the relaxations caused by NO (in rings without endothelium) and decreased the NO-stimulated cGMP content. These results suggest that the site of action of halothane in attenuating endothelium-dependent relaxation in the rat aorta is within the vascular smooth muscle, rather than on the synthesis, release, or transit of the EDRF from the endothelium and that its action may involve an interference with guanylate cyclase activation.

Neurovascular effects of reactive oxygen intermediates produced by photoradiation.

These experiments examined the effect of reactive oxygen intermediates, produced by laser illumination of the photosensitizer hematoporphyrin derivative, on the accumulation and release of norepinephrine from sympathetic nerve terminals. Using an isolated, spirally cut, superfused caudal artery of the rat, basal overflow of norepinephrine (NE) was significantly increased both during and after generation of reactive oxygen intermediates. Generation of reactive oxygen intermediates increased overflow of NE in vascular preparations in which release of NE had previously been elevated by the continuous superfusion of Krebs' solution, containing high concentrations of potassium (40 mM). Calcium free solutions did not block the overflow of norepinephrine augmented by reactive oxygen intermediates. This increase in overflow was due both to an increase in release of NE and an inhibition of accumulation of NE.

Effects of bufalin on norepinephrine turnover in canine saphenous vein.

Abundant experimental data suggest that an endogenous digitalislike factor is responsible for some essential hypertension. Some forms of hypertension have also been associated with increased levels of catecholamines. We therefore designed experiments to investigate the role of digitalislike factors in the regulation of norepinephrine turnover in the neurovascular junction. We chose bufalin, an amphibian-derived compound that shares many of the physiological properties postulated as characteristic of digitalislike compounds, as a model of the mammalian compound. In vitro experiments in canine saphenous veins showed that, in addition to inhibiting norepinephrine uptake, bufalin increased norepinephrine overflow by an amount larger than could be explained solely by uptake inhibition. The effect of bufalin on norepinephrine overflow is inhibited by tetrodotoxin, which suggests a dependence of this response on Na+ influx through the neuronal membranes. We propose that Na+,K(+)-ATPase inhibition resulting in neuronal depolarization is responsible for the augmented norepinephrine turnover caused by bufalin and that these indirect effects of norepinephrine on the cardiovascular system may play a role in the etiology of hypertension.

Vascular interactions of calcium and reactive oxygen intermediates produced following photoradiation.

This study was designed to examine vascular smooth muscle contractile properties following enhanced production of reactive oxygen intermediates (ROIs), which were produced by pretreating rat caudal arteries and aortas with a photosensitizer, hematoporphyrin derivative, 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 bathing solution; however, verapamil (5.5 microM) and nifedipine (10 microM) 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 ROI-induced 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 pathological 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.

Contractile properties of isolated vascular smooth muscle after photoradiation.

The purpose of this study was to characterize the responses of various types of vascular smooth muscle to conditions that would be encountered during photodynamic therapy, namely laser illumination of photosensitizer-pretreated tissue. Vascular smooth muscle obtained from representative canine, rodent, and rabbit vascular beds was cut into rings and placed in organ baths (37 degrees C, aerated with 95% O2-5% CO2). These vessels were pretreated for 30 min with the photosensitizer hematoporphyrin derivative (HpD, 3-30 micrograms/ml) washed, and then exposed to red laser light (633 nm, 1-3.5 mW) for up to 20 min. Under basal tension conditions laser illumination of HpD-pretreated vessels resulted in an increase in tension, whereas laser illumination of vessels not exposed to HpD did not contract. This sustained contraction was not reversed by washing the tissue with fresh Krebs-Ringer solution. Responses to norepinephrine, transmural electrical stimulation, and elevated concentrations of KCl were reduced in blood vessels tested after HpD laser illumination. Laser-induced contractions of canine carotid arteries did not require the presence of an intact vascular endothelium. Vascular effect of these photosensitizers appears to involve the formation of oxygen-derived radicals. This preparation could provide a good model for examining the effects of free radicals on vascular physiology.

Adrenergic nerve function and contractile activity of the caudal artery of the streptozotocin diabetic rat.

The adrenergic nerve function and contractile responses of the densely innervated caudal artery of the 8-week streptozotocin (SZ) (65 mg/kg i.p.) diabetic rat were investigated. Segments of this artery were removed from diabetic and control rats, placed in Krebs-filled tissue baths (37 degrees C) and isometric tension recorded. Contractile responses to sympathetic nerve activation by electrical stimulation and to cumulative concentrations of norepinephrine (NE) and tyramine were recorded. In order to determine NE content, the NE was extracted from the caudal artery, isolated by adsorption chromatography, and quantified by HPLC with electrochemical detection. NE accumulation and release were also studied by quantifying the amount of tritiated NE [( 3H]NE) and its metabolites in extracts of the tissue or incubation medium. The responses of the caudal artery of SZ diabetic rats to electrical stimulation (4-16 Hz) and to tyramine (1 X 10(-5)-1 X 10(-4) M) were significantly less than those of arteries from control rats and the NE content reduced by 41%, while sensitivity to NE was unchanged. Diabetic arteries also accumulated and released more [3H]NE than did arteries from control rats. These results establish that neurovascular function of the isolated caudal artery of the 8-week SZ diabetic rat is abnormal and suggest that problems in the ability of adrenergic nerves to store and release NE may contribute to this dysfunction. Such changes may play a role in the cardiovascular disturbances associated with diabetic autonomic neuropathy.

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 degrees 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.

Further isolation of endogenous factors in canine and human plasma that inhibit [(3)H]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.

Plasma histamine and catecholamine levels during hypotension induced by morphine and compound 48/80.

Histamine receptors are present in adrenergic terminals, and histamine is reported to inhibit release of the neurotransmitter norepinephrine (NE) at certain neuroeffector junctions. However, a physiological role for histamine in modifying adrenergic neurotransmission has not been established. To examine the interaction of elevated plasma histamine and catecholamine release, two compounds that release histamine, morphine (3 mg/kg), and compound 48/80 (0.5 mg/kg), were administered intravenously (i.v.). Plasma norepinephrine (NE) levels were used to monitor sympathetic nervous system activity, and plasma epinephrine (Epi) levels were used to monitor adrenal activity. Both morphine and compound 48/80 caused an immediate and marked increase in plasma histamine. Simultaneous with this increase, a marked decrease in mean arterial pressure occurred. Plasma NE levels increased in animals administered compound 48/80, but in morphine-treated animals, plasma NE levels did not change from pretreatment values. Plasma Epi levels increased in both groups, but the magnitude and duration of the responses differed. The results indicate that elevated plasma catecholamines can increase in response to histamine-induced hypotension but this effect can be suppressed by the central actions of morphine.

Neurovascular function in the rat during pregnancy.

Activity of the vascular neuroeffector junction was examined in pregnant (PG) and nonpregnant (NPG) rats to determine whether changes could account for the reported alterations in sympathetic control of the maternal circulation. Caudal and mesenteric arteries were removed from NPG and 19-21 day PG rats and prepared for isometric tension recording. Frequency-response measurements were obtained, followed by norepinephrine (NE) and tyramine concentration-response measurements. The caudal artery developed more tension in response to NE, tyramine, and electrical stimulation than did the mesenteric artery; however, there were no differences between vessels from NPG and PG rats. NE content, [3H]NE accumulation, and effects of plasma on [3H]NE accumulation of NPG and PG caudal arteries were also compared and found to be similar. Therefore, vascular neuroeffector functions of NE release, receptor sensitivity, and NE accumulation are not modified in the rat during pregnancy. Changes in sympathetic control of the maternal circulation are likely to be dependent on alterations at sites other than the neuroeffector junction.

The effect of etidocaine on spontaneous and evoked release of norepinephrine from rat brain synaptosomes.

Local anesthetics have been shown to have an effect on neurotransmission. In this study we examined the effect of a local anesthetic, etidocaine, on the uptake, efflux, and release of norepinephrine (NE) from central nerve terminals. The studies were performed on synaptosomes and vesicles prepared from rat brains. Etidocaine 10(-4) M inhibited synaptosomal accumulation of [3H]NE and did not significantly effect vesicular accumulation of this neurotransmitter. This concentration of etidocaine also augmented efflux of norepinephrine from synaptosomal preparations. This augmented efflux was primarily due to an increase in the deaminated metabolite 3,4-dihydroxyphenylglycol (DOPEG). The presence of etidocaine did not significantly alter the release of NE from synaptosomes superfused with high potassium (40 mM), a calcium-dependent exocytotic release process. These results indicate that in the central nervous system, as previously demonstrated in the peripheral nervous system, high concentrations of etidocaine alter vesicular storage of NE, resulting in more NE leaking into the cytoplasm where it is metabolized to an inactive metabolite.

Plasma histamine and hemodynamic responses following administration of nalbuphine and morphine.

A comparative study of plasma histamine levels following administration or morphine and nalbuphine in pentobarbital anesthetized dogs was performed. Two concentrations, 3 mg/kg and 0.3 mg/kg of these drugs were investigated. High dose morphine caused an immediate marked increase in plasma histamine from 5.0 +/- 0.4 to 340 +/- 72 ng/ml. Simultaneous with this increase in plasma histamine was a marked decrease in mean arterial blood pressure within the first minute. In contrast significant alterations in plasma histamine levels were not observed with high or low doses of nalbuphine. A low dose of morphine (0.3 mg/kg) did not increase plasma histamine levels. Heart rate was not changed by any drug treatment. The use of compound 48/80 a specific mast cell degranulating agent allowed for the identification of a specific pool of mast cells capable of responding to morphine. In vitro exposure of purified dog leukocytes to high doses of morphine did not result in histamine release. These results indicate that nalbuphine does not increase plasma histamine, while morphine does, and that the source of the increase in plasma histamine is from tissue mast cells.

Partial isolation of an endogenous norepinephrine uptake inhibitor in canine plasma.

We have previously reported that homologous plasma inhibits the accumulation of [3H]norepinephrine ( [3H]NE) by the canine saphenous vein. The purpose of this study was to extract the inhibitory substance(s) from plasma and to examine some of its properties. The net accumulation of tritium by saphenous vein strips following incubations in [3H]NE was inhibited 34.4 +/- 6.1% by a 60% plasma in Krebs solution. An acetone extract of this plasma reconstituted to 83% of its original unextracted volume also significantly inhibited net tritium accumulation by 24.5 +/- 4.7%. After lyophilization of this extract, the inhibitor was not readily solubilized in dilute acetic acid but was soluble in dilute sodium bicarbonate solutions. This extract solution, reconstituted to 66% of the original plasma volume, significantly inhibited the accumulation of [3H]NE by 35.0 +/- 9.8%. Kinetic analysis using this preparation suggests that the inhibition may be competitive in nature. The activity was not sensitive to heat but was abolished by the neuronal uptake blocker cocaine. Further purification with gel chromatography produced a small molecular weight fraction that inhibited tritium accumulation. When this sample was reconstituted in a volume equivalent to that of the unextracted plasma, the contractile response of the isolated canine saphenous vein to electrical stimulation was significantly enhanced. Thus the inhibitory activity of plasma on the uptake of [3H]NE by the canine saphenous vein is due in part to a heat-stable small molecule that inhibits the initial rate of [3H]NE uptake.

The effects of morphine, nalbuphine, and butorphanol on adrenergic function in canine saphenous veins.

Saphenous vein rings mounted in organ chambers containing Krebs-Ringer solution were used to determine if the venodilator effects of morphine, nalbuphine, and butorphanol are the result of interference with adrenergic neurotransmission or are caused by direct depressant actions on venous smooth muscle cells. Morphine (5 X 10(-5) M and 2 X 10(-4) M) caused a dose-dependent depression of the contractile response to transmural electrical stimulation. H1- and H2- histamine antagonists did not attenuate the inhibitory effect of morphine. Concentrations of morphine and nalbuphine lower than 5 X 10(-5) M had no effect, whereas 5 X 10(-6) M butorphanol significantly depressed the evoked tension response to electrical stimulation. The contractile responses of the veins to exogenous norepinephrine (NE) were not altered by morphine, indicating a presynaptic site of action rather than a direct action on the venous smooth muscle. Transmural electrical stimulation was used to evoke release of endogenous NE. Morphine (5 X 10(-5) M and 2 X 10(-4) M), nalbuphine (2 X 10(-4) M), and butorphanol (4 X 10(-6) M) significantly decreased release of NE. Naloxone did not alter NE release and did not attenuate the inhibition of NE release observed with the opiates, indicating that the effect of morphine on this neuroeffector junction is not mediated by a naloxone-sensitive opiate receptor. Blockade of presynaptic alpha receptors by phenoxybenzamine or phentolamine augments NE release caused by transmural electrical stimulation; morphine inhibited this augmentation. The results of these experiments indicate that high concentrations of morphine may decrease NE release, an effect that may contribute to the venodilation and hypotension observed following administration of high doses of morphine in humans. In the usual analgesic doses, the venodilatory effects of morphine cannot be explained by local action on either NE release or venous smooth muscle contractility.

Accumulation of [3H]norepinephrine in canine saphenous vein: influence of plasma.

This study was designed to systematically characterize the neuronal and extraneuronal uptake systems for norepinephrine (NE) in isolated nerve-rich vascular tissue and then to explore the possibility that circulating factors may alter these uptake systems. Strips from canine saphenous vein were incubated in Krebs-Ringer solution containing L-[3H]NE, and both tissue and medium were analyzed for total tritium, [3H]NE, and [3H]metabolite content. Under these conditions neuronal uptake (uptake1) played the dominant role in NE disposition. The addition of cocaine, ouabain, or potassium or the reduction of sodium decreased the activity of uptake1, and extraneuronal uptake (uptake 2) then played a major role in NE metabolism. The addition of 30, 60, and 90% plasma reduced NE accumulation and 3,4-dihydroxyphenylglycol (DOPEG) formation to 71 and 62% of control values, respectively; the formation of normetanephrine (NMN) and O-methylated deaminated compounds (OMDA) was not significantly different. In the presence of cocaine and uptake 1 inhibitor, 30% plasma did not cause a further significant decrease in neuronal accumulation of NE. The inhibitory activity of plasma on NE accumulation was not significantly affected by boiling or standing at room temperature for 45 min. These studies show that in the isolated canine saphenous vein the addition of plasma, like low sodium, cocaine, or ouabain, decreases neuronal uptake. The identity of the component in plasma responsible for inhibition of NE uptake appears to be a nonprotein, heat-stable molecule.

Ionic and osmotic influence on prostaglandin release from the gill tissue of a marine bivalve, Modiolus demissus.

1. Prostaglandin E2 (PGE2) was identified in Modiolus demissus gill tissue on the basis of solvent extraction, thin layer and column chromatography, bioassay, and radioimmunoassay. The presence of PGE2 was detected in both tissue and sea water incubate surrounding the tissue. 2. Both hyposmotic stress and magnesium-free sea water significantly increased release of prostaglandins into sea water. Hyposmotic stress also significantly increased prostaglandin synthesis. 3. Examination of tissues revealed that homogenates of the mantle and lower visceral mass contained significantly fewer nanograms immunoreactive prostaglandins per gram wet weight than homogenates of the gill, posterior adductor muscle, upper visceral mass, or siphon tissue. 4. Prostaglandin release could be increased by addition of arachidonic acid, and inhibited by addition of acetylsalicylic acid or indomethacin.