Comparative Analysis of the Vascular Actions of Diterpenes Isolated from Euphorbia canariensis

We have analysed the effects of 2,3-diepiingol 7,12-diacetate-8-isobutyrate (compound 1 ), ingenol-3-angelate-17-benzoate (compound 2 ), ingenol-3-angelate-17-benzoate-20-acetate (compound 3 ) and 3,5,7,8,9,15-hexahydroxyjatropha-6(17),11-dien-14-one-5,8-bis(2-methylbutyrate)-7-(2-methylpropionate) (compound 4 ), four diterpenes isolated from E. canariensis, on the isometric tension developed by isolated rabbit basilar and carotid arteries. Concentration-response curves to these compounds were obtained cumulatively in both arteries at resting tension and active tone (KCl, 50 mM). At resting tension a concentration-dependent contraction was induced by the four compounds. In the basilar artery the order of potency was 3 = 1 > 2 = 4 , without significant differences between E_max values. In the carotid artery the order of potency was 3 > 2 = 1 = 4 and there were no significant differences between the E_max (maximum effect) values of compounds 1–3 , all of which were higher than that of compound 4 . In pre-contracted basilar artery compounds 1–3 induced concentration-dependent relaxation and compound 4 was almost ineffective; the order of potency was 3 > 2 = 1 without significant differences between E_max values. In the carotid artery with active tone the four compounds tested induced further contractions; the order of potency was 3 > 2 = 4 > 1 without significant differences between E_max values. These results show that the four diterpenes are potent active substances in rabbit basilar and carotid arteries and that there are regional differences between their action. The four compounds tested contract basilar and carotid arteries at resting tension. Compounds 1–3 relax pre-contracted basilar artery but not carotid artery.     

Cycloartane Triterpenes and Ingol Diterpenes Isolated from Euphorbia neriifolia in a Screening Program for Death-Receptor Expression-Enhancing Activity

In our screening program for natural products that increase death-receptor 5 expression, seven new cycloartane triterpenes, euphonerins A-G (1-7), and 3-O-acetyl-8-O-tigloylingol (8), a new ingol diterpene, were isolated from the MeOH extract of Euphorbia neriifolia leaves, together with 3,12-di-O-acetyl-8-O-tigloylingol (9), (24R)-cycloartane-3β,24,25-triol (10), and three known flavonols (11-13). The structures of 1-8 were elucidated by spectroscopic analysis. Among these compounds, 1-11 showed death-receptor 5 expression enhancing activity.     

Release of 3H-5-Hydroxytryptamine from Isolated Rabbit Aorta

Abstract: The main aim of the present investigation was to study systematically the passive and stimulation-evoked release of ³H-5-hydroxytryptamine (³H-5-HT) from rabbit isolated aorta. This was accomplished by preloading rings of aorta with ³H-5-HT (10^–6M) and then monitoring by fractional collection the basal ³H-outflow and stimulation-evoked ³H-overflow. The basal ³H-outflow from aorta preloaded with 10^–6M of either ³H-5-HT or (-)-³H-noradrenaline (³H-NA) leveled off about 100 min. after the onset of wash-out and remained almost constant thereafter (100–240 min.). The basal ³H-outflow from tissue preloaded with ³H-5-HT was almost 3-fold higher (70–240 min.) than that seen after preloading with ³H-NA. Cocaine (3x10^–5M) did not alter the basal ³H-outflow (15–240 min.) from tissue preloaded with ³H-5-HT, while pargyline (5X10^–4M) decreased it by about 66% (100–240 min.). Electrical-field stimulation (S₁S₇, 200 mA, 600 pulses, 0.5 msec, 3 Hz) were applied to the tissue. The initial stimulation-evoked ₃H-overflow from aorta preloaded with ³H-5-HT was higher than the subsequent ones (S₁-S₇: 100, 35, 35, 35, 35, 37, and 40%). Similar results to these were obtained with tissues preloaded with ³H-NA. The stimulation (S₁-S₇; 200 mA; 600 pulses, 0.5 msec, 3 Hz)-evoked ³H-overflow increased in an apparent linear manner with the amount of current used (50–200 mA). This was also the case for number of pulses (100–900) in the stimulus. The stimulation-evoked ³H-overflow depended in part on the stimulation frequency: unchanged at 2–4 Hz; small increase at 8 Hz; and a 15-fold increase at 16 Hz relative to 2 Hz. Tetrodotoxin (10^–6M) decreased the stimulation-evoked ³H-overflow from aorta preloaded with ³H-5-HT (S₂-S₆) by about 60%, while S₁ was not affected. The inhibitory effect of tetrodotoxin was fully reversed by washing the aorta with drug-free salt solution. Omission of Ca²⁺ from the salt solution reduced the stimulation ³H-overflow by 47–57% (S₂-S₆) while S₁ was unaffected. 6-Hydroxydopamine markedly increased the stimulation-evoked ³H-overflow from ³H-5-HT preloaded rings (180–323% of control). Pargyline (5x10^–4M), cocaine (3x10^–5M) and removal of endothelium did not alter the stimulation-evoked ³H-overflow evoked by stimulation (S₁-S₆) of aorta preloaded with ³H-5-HT. It is concluded that ³H-5-HT can be released by electrical-field stimulation as a “false transmitter'’from rabbit isolated aorta. Most of the release is probably of neuronal origin. However, some of the stimulation-evoked ³H-overflow is derived from extraneuronal sites.     

The Uptake of Bupivacaine in an in situ Isolated Perfused Rabbit Lung Preparation

Abstract: A double indicator technique has been used in an in situ isolated perfused rabbit lung model to examine the first pass effect of the lung on systemic bupivacaine concentrations. Bupivacaine (0.5 mg/kg) was given in two consecutive boluses to six in situ isolated perfused New Zealand White rabbit lung preparations. The mean recovery (first bolus) of bupivacaine was 62.6% ± 6.3 (S.E.M.), and 63.7% ± 10.2 (second bolus), suggesting bupivacaine accumulation in the lung. The average mean transit time for bupivacaine was 280.5% ± 24.1 and 264.8% ± 36.7 longer than ICG (Indocyanine Green) following the first and second boluses respectively (P < 0.01). There were no differences in the first pass effect of the lung between the first and second boluses of bupivacaine. The profiles of the bupivacaine concentrations suggest that uptake is followed by accumulation and later back diffusion. This has implications for conditions that decrease the uptake and therefore increase the risk of systemic toxicity.     

Biochemical and Morphological Characterization of Subcellular Fractions Isolated from Rabbit Colon Muscle

Abstract From a homogenate of rabbit colon muscle subcellular fractions were isolated by differential centrifugation. The crude microsomal fraction could be separated into subfractions, a fraction of vesicular microsomes at 35% sucrose, a fraction containing sarcolemma, mitochondrial fragments and microsomal vesicles at 35–45% sucrose and a small protein fraction at 45–55% sucrose. Their biochemical properties and their morphological characterization were investigated. The cholesterol and the phospholipid content was equally distributed between the microsomal fractions 35% and 35–45% while the RNA was localized to the mitochondria and the microsomal fraction 35%. The enzyme cytochrome c oxidase was found to be concentrated in the mitochondria while a high contamination was found in the microsomal fractions 35–45%. The NADH-oxidase activity was highest in the 35% fraction and the 5′-nucleotidase activity in the 40,000 × g supernatant. The microsomal subfractions contained the enzymes ATPase, adenylate cyclase and phosphodiesterase. In the 35% fraction Ca stimulated the hydrolysis of ATP. The binding of [³H]-ouabain and the incorporation of [³H]-leucine was most pronounced in the 35% fraction. In a K⁺ -free Krebs Ringer medium the binding of the glucoside was stimulated in all the fractions. From these results we concluded that the fraction 35% sucrose may be mainly derived from the endoplasmic reticulum and the plasma membrane while the 35–45% originates from the plasma membrane, mitochondria and to a lesser extent the endoplasmic reticulum.     

Aloe-Emodin Acetate, an Anthraquinone Derivative from Leaves of Kniphofia foliosa

The leaves of K. FOLIOSA yielded aloe-emodin acetate in addition to chrysophanol and knipholone. Aloe-emodin acetate is reported here in Liliaceae family for the first time.     

Vasodilator Action of the Nitroxylated Cyanoamidine Derivative,KRN2391, in Rabbit Basilar Artery

Abstract: KRN2391 is a cyanoamidine derivative with a pyridine ring and a nitroxyl group. This gives the molecule a dual pharmacological action as both an ATP-sensitive K channel (K_ATP) opener and an organic nitrate. In cerebrovascular disease with endothelial dysfunction, such a compound could be advantageous to prevent the negative consequences of a reduced synthesis of endogenous nitric oxide and endothelium-derived hyperpolarizing factor. The objective of this study was to characterise the vasodilator action of KRN2391 in a cerebral artery. As shown in the rabbit basilar artery contracted by endothelin-1 KRN2391 elicited a concentration-dependent relaxation. KRN2391 was unable to relax arteries contracted by a 60 mM K solution. The KRN2391-induced relaxation of endothelin-1-contracted arteries was unaffected by N^G-nitro-L-arginine (0.1 mM), indomethacin (10 μM) or removal of the endothelium. The guanylate cyclase inhibitors ODQ (10 μM) and LY53583 (10 μM), and the cGMP phosphodiesterase inhibitor zaprinast (10 μM) each had no effect on the KRN2391-induced relaxation. Glibenclamide (1 μM), a blocker of KATP, caused a rightward shift of the concentration-response curve for KRN2391. The relaxation induced by the prototype K_ATP opener levcromakalim was inhibited to a similar extent by glibenclamide. Addition of ODQ or LY53583, or the calcium-sensitive K channel blockers apamin (0.1 μM) and charybdotoxin (0.1 μM) in the presence of glibenclamide did not produce a significant further inhibition of the KRN-induced relaxation. KRN2391 (10 μM) did not influence the content of cGMP in the basilar artery, whereas the nitric oxide donor 3-morpholino-sydnonimine (0.1 mM) increased the cGMP level three-fold. Thus, KRN2391 is an effective vasodilator of the rabbit basilar artery, acting mainly through opening of K_ATP. The nitro-moiety of the molecule does not seem to contribute to the relaxant effect in this artery.     

Effects of Amitriptyline and Clomipramine in the Isolated,Perfused Rabbit Heart

Abstract The cardiac effects of supratherapeutic concentrations of two tricyclic antidepressants were studied in isolated rabbit hearts, which were perfused with a modified Krebs-Henseleit solution containing 0.25 or 0.50 μg ml^-1 of amitriptyline or 0.28μg ml^-1 of clomipramine. The followingparameters were continuously recorded: heart rate, amplitude and rate of contraction, coronary flow rate, myocardial oxygen consumption and ECG. The lowest concentration of amitriptyline caused a time correlated decrease (20%) in the frequency of spontaneous beating and a pronounced decrease in the amplitude (62%) and rate of cardiac contraction (58%). Maximum increases of the PQ-interval of about 46% and of the QRS-complex of about 100% were observed. At the higher amitriptyline concentration these effects further increased. Clomipramine 0.28 μg ml^-1 also had a very pronounced and time correlated negative inotropic effect, but the effects upon the conduction velocities were substantially lesser than those produced by the equimolar concentration of amitriptyline. The compounds caused only insignificant changes in coronary flow. The oxygen consumption did not decrease in proportion to the decrease in contractility, as an expression of decreased myocardial efficiency. The effects of the drugs are discussed in relation to their myocardial accumulation pharmacokinetics and influence upon the membraneous sodium and calcium flux and intracellular metabolism.     

Effects of Terbutaline on α-Adrenergic Responses and Ca2+ Influx in Isolated Rabbit Aorta

Effects of terbutaline applied in vivo or in vitro on α-adrenergic receptors in the rabbit aorta in normal and Ca²⁺–free solution, and on basal, high potassium-, and phenylephrine-stimulated Ca²⁺ uptake into aorta were investigated. Three day terbutaline administration (25 mg/kg, subcutaneously three times daily) to rabbits increased the pK_B for phentolamine in aorta rings (control 7.3 + 0.2, n = 9; terbutaline 7.8 +0.2, n = 15). It also depressed phenylephrine-stimulated contractions of aorta rings in Ca²⁺–free but not those in normal Krebs solution. It did not significantly depress the basal, or phenylephrine-evoked Ca²⁺ influx into aorta rings, but decreased high potassium-induced Ca²⁺–influx (control 0.58 + 0.05 umoles/g aorta; n = 3, terbutaline 0.41 +0.06 umoles/g aorta, n = 3). In vitro application of 50 μM terbutaline did not significantly alter phenylephrine-stimulated contractions of aorta rings in Ca²⁺–free Krebs solution or significantly depress basal or phenylephrine-induced Ca²⁺ influx into aortas, but did decrease high potassium-stimulated Ca²⁺–influx. Thus, 3-day terbutaline administration increased the affinity of α-adrenergic receptors for phentolamine and had a tendency to increase contractions of aorta rings to phenylephrine. It also decreased high potassium-stimulated Ca²⁺ influx, and depressed phenylephrine-induced contractions in Ca²⁺–free Krebs solution, while in vitro terbutaline application also decreased potassium-induced Ca²⁺ influx.     

Effects of Vanadate on Responses of Guinea-pig Isolated Trachea to Spasmogens

Abstract— The effects of vanadate on the contractility of the guinea-pig isolated trachea was examined. Vanadate (0·1 Mm ) produced a sustained contraction that was abolished in Ca²⁺-free EGTA (0·1 Mm )-containing physiological salt solution but was resistant to verapamil (1 μm ). Vanadate (0·1 Mm ) depressed tracheal responses to CaCl₂ (in Ca²⁺-free depolarizing solution), KCl, acetylcholine, histamine and 5-hydroxytryptamine. For vanadate (10 μm ), the inhibition of spasmogenic responses only reached statistical significance for histamine and 5-hydroxytryptamine. Caffeine (1 Mm )-induced spasm (trachea at 20°C in the presence of indomethacin (2·8 μm )) was not affected by vanadate (10 μm -0·1 Mm ). Vanadate (0·1 Mm ) slightly depressed the responses to KCl (50 Mm ), acetylcholine (1 Mm ), histamine (1 Mm ) or 5-hydroxytryptamine (0·1 Mm ) observed in Ca²⁺-free EGTA (0·1 Mm )-containing physiological salt solution. Vanadate (0·5 Mm ) depressed Ca²⁺ (20 μm )-induced contraction of trachea which had been chemically skinned of its plasmalemmal membranes. The mechanism of the inhibitory effect of vanadate on tracheal responses to a variety of spasmogens remains obscure, but, under in-vitro conditions, vanadate clearly does not induce hyper-reactivity of airway smooth muscle to spasmogens.     

Contractile Responses and Structure of Small Bronchi Isolated from Rats after 20 Months' Exposure to Ozone

Short-term exposure to high concentrations of ozone has beenshown to increase airway responsiveness in normal humans andin all laboratory animal species studied to date. While ourknowledge concerning the pulmonary effects of single exposuresto ozone has increased rapidly over recent years, the effectsof repeated exposures are less understood. The goal of the presentstudy was to determine whether airway responsiveness is increasedafter near-lifetime exposure to ozone. Airway segments representingapproximately eighth generation airways were isolated from Fischer344 rats of both genders that had been exposed for 6 hr perday, 5 days per week for 20 months to 0, 0.12, 0.5, or 1.0 partsper million (ppm) ozone. Circtimferential tension developmentwas measured in isolated airways in response to bethanechol,acetylcholine, and electrical field stimulation. Responsivenessof the airways to the contractile stimuli was described by theeffective dose or frequency that elicited half-maximum contraction(ED50) and the maximum response. Since ozone exposure is associatedwith remodeling of peripheral airways, smooth muscle area wasdetermined and tension responses were normalized to the areameasurements. Before normalization of tension data to smoothmuscle area, neither the ED50 nor maximum response of smallbronchi to the contractile stimuli was altered after chronicozone exposure. Smooth muscle area was greater in airways isolatedfrom animals that had been exposed to 0.5 ppm ozone. After accountingfor smooth muscle area, maximum responses of the small bronchiisolated from male rats were significantly reduced after 0.12and 0.5 ppm ozone. Although not significant statistically, asimilar trend was observed in airways isolated from female rats.These results suggest that the increase in airway responsivenessassociated with acute ozone exposure does not persist duringnear-lifetime exposure. Although the mechanism responsible forthe adaptation to the effects of 03 on airway responsivenessis unknown, the results indicate that smooth muscle cell functionwas compromised by the chronic exposure. The mechanism(s) responsiblefor mediating this effect and the relevance of these resultsto humans remains to be determined.     

Myocardial infrastructure in the Isolated Rabbit Heart Exposed to Dopamine,Dobutamine, Isoprenaline,G-Strofanthin,Xamoterol and Hypoxia

Abstract: The isolated spontaneously beating heart was used for comparing the effects of hypoxia and positive inotropic drugs on myocardial ultrastructure. Hypoxia gives a significant decrease in the volume fractions of mitochondrial cristae relative to the total mitochondrial volume (V_vmcristae) and a significant increase in the volume fraction of mitochondrial matrix relative to the total mitochondrial volume (V_vmmatrix), but changes in volume fractions of mitochondria (V_vmitochondria) and myofibrils (V_vmyofibrils) were absent. Significant changes in ultrastructure in hearts treated with dopamine (0.6 μM), dobutamine (90 nM), G-strofanthin (0.25 μM), xamoterol (32 nM) and isoprenaline (0.15 μM or 15 nM) were absent. Furthermore, myocardial effects in the isolated rabbit heart without exposure to any treatment showed a significantly decrease in oxygen consumption after 90 min. and a significant decrease in frequency of contractions after 120 min. perfusion time, but no change in contractility was seen. We conclude that this experimental model is useful in studies of positive inotropic drugs.     

Myocardial Pharmacokinetics of Amitriptyline and Clomipramine in the Isolated,Perfused Rabbit Heart

Abstract The myocardial pharmacokinetics of amitriptyline and clomipramine were investigated in isolated rabbit hearts, which were perfused with a modified Krebs-Henseleit solution containing the equimolar concentrations 0.25 or 0.28 μg ml^-1 of the compounds, respectively. The rate of myocardial uptake of the drugs as a function of time was indirectly followed by determinations of the concentrations of the compounds in fractional samples of the coronary output of perfusate. The time course of disposition of amitriptyline from the myocardium was similarly followed after changing from amitriptyline perfusion to perfusion with drug-free liquid. The amitriptyline accumulation and disposition processes were found to fit bi-exponential functions indicating myocardial two-compartment characteristics of the compound. Clomipramine did only exhibit one-compartment myocardial characteristics. The biological half-life of amitriptyline in the myocardium was about 37.7 min. and a pronounced cardiac accumulation of about 340 μg of the compound at steady state was evidenced. The myocardial half-life of clomipramine was about 106 min. and the accumulated amount at steady state was calculated to be 1055 μg. After amitriptyline perfusion an increase in the pharmacokinetic rate constants k₁₀ and k₁₂ and a decrease in the apparent central volume of distribution was observed.     

Role of Cyclic AMP in Stimulation-Evoked Release of 3H-Noradrenaline from Rabbit Isolated Ear Artery

Abstract: The possible involvement of cyclic AMP in the stimulation-evoked ³H-overflow from rabbit isolated ear artery preloaded with ³H-noradrenaline was studied. Cyclic AMP (10^–5–3x10^–4M), 8–bromo-cyclic AMP (3x10^–4M) and adenosine (10^–5–3X10^–4M) enhanced stimulation-evoked ³H-overflow. Dibutyryl-cyclic AMP (10^–5–3x10^–4M) had no effect. Theophylline (3X10^–5M) and the phosphodiesterase inhibitor AH 21–132 (3X10^–5M) did not alter the enhancement of ³H-overflow caused by either cyclic AMP or adenosine. Forskolin (3X10^–6M) and the phosphodiesterase inhibitors ICI 63 197 (10^–4M) and AH 21–132 (3x10^–6–3x10^–5M) increased stimulation-evoked ³H-overflow. Forskolin (10^–6M) enhanced the effect of ICI 63 197 (3x10^–5M) but it did not alter the effect of AH 21–132. It is concluded that cyclic AMP is involved in the stimulation-evoked release of noradrenaline from postganglionic sympathetic nerves in the rabbit ear artery.     

Cigarette Smoke Inhibits Contractile Responses of Rat Isolated Colon to Angiotensin II

Abstract: The contractile response of isolated superfused rat colons to angiotensin II was inhibited almost totally when cigarette smoke was in contact with the serosal surface of the colon. Filtration of visible particles from the smoke diminished but did not abolish this inhibitory action. An inhibitory factor was also retained by the filter papers. One inhibitor, which was dissolved in Krebs buffer, behaved like nicotine. Carbon monoxide and dioxide were effective inhibitors but only at higher concentrations than they are present in cigarette smoke. Removal of carbon dioxide from cigarette smoke did not abolish the inhibitory action. Neither of the gases nor nicotine alone could fully mimic the action of cigarette smoke. It is possible that the inhibitory action of cigarette smoke was due to combined action of these compounds. It is, however, also possible, that cigarette smoke contains still other inhibitory compounds. Cigarette smoke probably had its effect directly on the smooth muscle cells, but activation of inhibitory nerves could not be excluded.     

Biological Responses of Isolated Macrophages to Cobalt Metal and Tungsten Carbide-Cobalt Powders

A previous study from this laboratory, using morphological and biochemical (LDH release) parameters, has shown that tungsten carbide-cobalt dust exhibits a greater cytotoxicity toward isolated macrophages than cobalt metal powder alone. The present study extends this comparison by examining additional biological parameters. Glucose uptake and superoxide anion production by isolated macrophages were significantly more depressed by the tungsten carbidecobalt mixture (WC—Co) than by cobalt alone (Co) while pure tungsten carbide (WC) had no effect or even stimulated the cells. For glucose-6-phosphate dehydrogenase and cell-associated plasminogen activator (PA) activities, no difference between Co and WC—Co dusts was observed. These observations add further evidence to our previous findings regarding the different biological reactivity of cobalt metal alone or mixed with tungsten carbide.