Rapid molecular characterization of Hb H disease in Chinese by polymerase chain reaction

Summary We have developed a rapid method to molecularly distinguish different types of Hb H disease. The study depended on (a) most of the Hb H disease in Taiwan having an-thalassemia-1 of the Southeast Asia type (-SEA) in one allele and (b) the differences of X box of-globin gene cluster in the other allele. To detect the -SEA allele, we utilized the primers located on either side of the breakpoint to do PCR, then characterized the amplified products. For the other allele, we sequenced part of the X box, and found that bases –2803 to –2461 of the X box of – ^3.7 belonged to the X box of ₂ globin gene. In – ^4.2, the bases belonged to the X box of ₁ globin gene, whereas in ^cs it contained both X boxes of ₁ and ₂ globin genes. There was anMboII site at this region of the X box of ₂ globin gene. We utilized PCR to amplify this region and digested it with restriction enzymeMboII, then combined it with another PCR of different primer pairs to molecularly diagnose different types of Hb H disease. One hundred and one cases of Hb H disease from different families were studied: all of the cases had one allele of -SEA deletion, while the other allele showed that 52/101 were – ^3.7, 41/101 were ^cs , 7/101 were – ^4.2, and 1/101 was – ^G.Taichung. Of 52 cases of Hb H with – ^3.7, 47 were type-I deletion and five were type-II deletion.     

α-Adrenergic responses of isolated canine coronary microvessels

Although -adrenergic activation is known to increase coronary microvascular resistance in vivo, the magnitude of its segmental microvascular consequences is not well understood. Quantification of these effects in vivo is hindered by escape mechanisms that minimize the influences of constrictors, and alterations in flow and pressure, which effect microvascular tone by shear stress-dependent and myogenic mechanisms, respectively. To eliminate these confounding influences, we have studied responses in vitro under conditions with these variables controlled. We evaluated the diameter changes of isolated canine coronary arterioles (110±12 m, n=35) and venules (98±7 m, n=9) in response to -adrenergic activation by norepinephrine (10^–10 to 10^–4 M) in the presence of -adrenergic blockade by alprenolol (10^–6 M). In contrast to the situation in vivo, -adrenergic activation did not constrict isolated coronary arterioles, but constricted isolated coronary venules in a dose-dependent manner over a range of 10^–10 to 10^–4 M (–27 ±3% maximum diameter change). Coronary arteriolar -adrenergic constriction was not promoted by 1) subthreshold or vasoactive doses of the vasoconstrictors KCl, angiotensin II, U46619, endothelin-1, neuropeptide Y or arginine vasopressin, 2) inhibition of the presynaptic uptake of norepinephrine by imipramine (10^–6 M), 3) inhibition of EDRF synthesis by N^g-monomethyl-L-arginine (10^–5 M) or 4) inhibition of prostaglandin synthesis by indomethacin (10^–5 M). Furthermore, -adrenergic activation did not modify microvascular dilatation by adenosine (10^–9 to 10^–4 M) or nitroglycerin (10^–9 to 10^–4 M), suggesting that -adrenergic constriction in vivo is not due to attenuation of cAMP or cGMP-dependent mechanisms of coronary dilatation. In contrast to the lack of constriction in coronary arterioles, canine skeletal muscle arterioles exhibited significant -adrenergic constriction (–80±4%), maximum diameter change). The coronary venular -adrenergic constriction was significantly inhibited by both the ₁-and ₂-adrenergic receptor antagonists, prazosin (10^–8 M) and rauwolscine (10^–7 M), indicating a mixed population of ₁-and ₂-adrenergic receptors. These results suggest that coronary arterioles, but not venules, lose -adrenergic responsiveness during isolation and cannulation, or that the primary coronary microvascular response to -adrenergic activation is venular constriction.     

The Effect of Hydrogen Peroxide in Human Internal Thoracic Arteries: Role of Potassium Channels,Nitric Oxide and Cyclooxygenase Products

Introduction We investigated both the effect and the role(s) of potassium channels, nitric oxide (NO) and cyclooxygenase (COX) products in the effect of hydrogen peroxide (H₂O₂) in human internal thoracic artery (ITA) rings. Materials and methods Samples of redundant ITA obtained from patients undergoing a coronary artery bypass graft surgery were cut into 3 mm wide rings and suspended in 20 ml organ baths. Isometric tension was continuously measured with an isometric force transducer connected to a computer-based data acquisition system. Results H₂O₂ (10⁻⁷–10⁻⁴ M) produced concentration-dependent relaxation responses in human ITA precontracted by phenylephrine. The relaxant responses to H₂O₂ did not differ significantly between endothelium-intact and endothelium-denuded preparations. Incubation of human ITA rings with superoxide dismutase (50 U/ml) did not affect the relaxant responses to H₂O₂, while 1,000 U/ml catalase caused a significant decrease. Incubation of endothelium-intact or endothelium-denuded human ITA rings with voltage-dependent potassium channel blocker 4-aminopyridine (5 mM) significantly inhibited the relaxant responses to H₂O₂. COX inhibitor indomethacin (10⁻⁵ M) also caused a significant inhibition. Incubation with ATP-dependent potassium channel blocker glibenclamide (10⁻⁶ M) or Ca²⁺-activated potassium channel blocker iberiotoxin (10⁻⁷ M) or NO synthase (NOS) blocker -nitro-l-arginine methyl ester (10⁻⁴ M) did not alter relaxant responses of ITA rings to H₂O₂. Conclusion The findings of the present study suggested that H₂O₂-induced relaxation responses in human ITA were neither dependant on the endothelium nor blocked by NOS inhibition but they rather seem to depend on the activation of voltage-dependent potassium channels and COX.     

Effects of PAF on cardiac function and eicosanoid release in the isolated perfused rat heart: comparison between normotensive and spontaneously hypertensive rats

The aim of this study was (a) in isolated perfused rat heart to characterize the effects of platelet-activating factor (PAF) on coronary flow, ventricular contractility, and eicosanoid release and (b) to determine whether PAF effects are altered in hearts from spontaneously hypertensive rats (SHR). PAF (10^–10–10^–7 mol) dose-dependently decreased coronary flow and ventricular contractility; concomitantly, coronary effluent concentrations of thromboxane (TX)B₂ and prostaglandin F₂ (PGF₂) were elevated but not those of prostacyclin. The PAF receptor antagonist WEB 2086 (10^–7–10^–5 mol/l) concentration-dependently antagonized these PAF effects. In addition, the cyclo-oxygenase inhibitor indomethacin (5×10^–5 mol/l) prevented PAF (10^–9–10^–7 mol) induced eicosanoid release; in the presence of indomethacin PAF caused coronary constriction and ventricular depression only at the highest dose (10^–7 mol) but had no effect at 10^–9 or 10^–8 mol. Moreover, the TXA₂ antagonist SQ 29,548 (10^–6 mol/l) completely inhibited 10^–8 mol PAF induced ventricular depression but did not effect coronary constriction. In SHR PAF (10^–9–10^–7 mol) evoked decreases in coronary flow and ventricular contractility did not differ from those in normotensive Wistar-Kyoto rats while PAF induced TXA₂ and PGF₂ release was markedly enhanced. In addition, decreases in coronary flow and ventricular contractility induced by the TXA₂ agonist U 46619 (10^–7 mol/l) were markedly depressed in SHR. We conclude that in isolated perfused rat heart PAF causes coronary constriction and depression of ventricular function mainly indirectly through released TXA₂ and/or PGF₂. Moreover, the fact that in SHR the PAF effects on coronary flow and ventricular function are not altered despite markedly enhanced TXA₂ and PGF₂ release supports the view that in the SHR the receptors mediating TXA₂ and/or PGF₂ effects are desensitized.     

Effects of 5-hydroxytryptamine and adenosine on the canine isolated cerebral resistance vessels

We investigated the effects of 5-hydroxytryptamine (5-HT), adenosine, and adenine nucleotides on the canine isolated cerebral resistance arteries ranging from 300 to 500 µm in diameter. Addition of 5-HT and 5-carboxami-dotryptamine (5-CT) produced concentration-dependent contractions in the resistance arteries. Methiothepin (10^–8 M-10^–6 M) caused a parallel shift to the right of the dose-response curves for 5-HT and 5-CT. Neither ketanserin (10^–7 M, 10^–6 M) nor [(1H, 5H)-8-methyl-8 azabicyclo [3,2,1] oct-3-y1]1H-indole-3-carboxylate hydrochloride (ICS 205–930 10^–7M, 10^–6M) caused any significant shift of the dose-response curve for 5-HT. Adenosine, ATP, ADP, and AMP caused concentration-dependent relaxations in the resistance arteries following contraction with PGF2. The adenosine analogs also caused a dose-dependent relaxation of the arterial segments contracted by PGF2. The decreasing order of potency of the agonists was 5-N-ethylcarboxamido adenosine (NECA)>adenosine L-diasteroisomers of phenylisopropyl adenosine (L-PIA). The relaxant response to NECA was competitively antagonized by 8-phenyltheophylline. The pretreatment with 3×10^–5 M N^G-monomethyl-L-arginine (L-NMMA) or 5×10^–5 M aspirin caused no significant effect on the adenosine-induced relaxation in the resistance arteries. These results suggest that 5-HT produces contraction of the canine isolated cerebral resistance arteries, which is mainly mediated via 5-HT₁-like receptors. Adenosine causes an endothelium-independent relaxation of the resistance arteries through the activation of adenosine A₂-receptors.     

Luminal dopamine modulates canine ileal water and electrolyte transport

Previous studies have suggested that dopamine stimulates active ileal ion absorption via ₂-adrenergic or dopaminergic receptor activation. Identification of a dopamine 1a receptor on rat enterocytes located in intestinal crypts prompted this investigation of the effect of luminally administered dopamine on water and ion transport in the canine ileum. Absorption studies (n=27) were performed in dogs with 25-cm ileal Thiry-Vella fistulas. Perfusion with [¹⁴C PEG was used to calculate absorption of water and electrolytes from the Thiry-Vella fistula. Experiments consisted of three 1-hr periods: basal, luminal drug infusion at 10^–4 M, and recovery. Agonists used included dopamine (DOP: -adrenergic, D₁ and D₂ receptor) and SKF 38393 (D₁ receptor). Antagonists used included terazosin (TZ: ₁) and yohimbine (YOH: ₂). DOP caused significant increases in water and electrolyte absorption. TZ and YOH prevented the dopamine-induced proabsorptive response. Luminal DOP may serve as a proabsorptive modulator of ileal transport, acting via ₁, ₂, and dopaminergic receptors. The development of more potent proabsorptive dopamine analogs, which maintain the ability to broadly activate mucosal receptors, may be useful in such clinical situations as diabetic diarrhea, short gut syndrome, or following small bowel transplantation.Presented in part as a poster presentation at the Annual Meeting of the American Gastroenterological Association, New Orleans, May 14–18, 1994, and published in abstract form inGastroenterology 106:A432, 1994.Supported in part by National Institutes of Health grant R29-DK 41178 (C.J.Y.).     

Bronchodilator action of an agonist for histamine H3-receptors in guinea pig perfused bronchioles and lung parenchymal strips

Isolated guinea pig perfused bronchioles and lung parenchymal strips were examined as an in vitro model for assessment of the direct effect of pharmacologic agents on the airway smooth muscle. The experiments were performed with a perfusion technique in bronchioles, the input pressure being measured as an index of the state of dilation, while changes in tension of the lung parenchymal strips were measured with an isometric force transducer. In both preparations, histamine and acetylcholine elicited dose-related contractile responses whereas fenoterol induced a concentration-dependent relaxation. After the 3 agonists' activities were compared in these 2 preparations, we tested the intrinsic effects of a specific H₃ agonist, (R)-methylhistamine ([R]-MeHA). Statistical analysis was by Student's t test on the E_max (expressed as a percentage of 10^–4 M papaverine relaxation), EC₅₀, and slopes of regression lines calculated from the concentration-response curves plotted for (R)-MeHA alone or in presence of antagonists. Our results showed that (R)-MeHA induced a concentration-dependent relaxation of perfused bronchioles and lung parenchymal strips competitively inhibited by 10^–7 M thioperamide (H₃-antagonist), whereas 10^–5 M cimetidine (H₂-antagonist) failed to prevent this effect. These results suggest the presence of H₃-histaminergic dilatory receptors in the guinea pig airway. Offprint requests to: J.-L. Burgaud     

Vascular relaxing mechanism of denopamine in isolated canine coronary,femoral, mesenteric,and renal arteries

Summary We investigated the mechanism of vascular relaxation produced by denopamine (deno), an oral positive inotropic agent that has selective ₁-adrenergic action. Deno concentration-dependently (0.1 µM–30 µM) relaxed ring segments of canine femoral, mesenteric, and renal arteries which were partially precontracted with 1 µm phenylephrine or norepinephrine, but did not relax those precontracted with 5 µM prostaglandin F₂ or 40 mM K⁺. The relaxation was not significantly inhibited by pretreatment with 10 µM propranolol or metoprolol. Deno produced a parallel rightward shift in concentration-response curves to phenylephrine in femoral and renal arteries. The Schild plot yielded linear regressions of slopes of 1.301 ± 0.106 and 0.823 ± 0.122, respectively, which were not significantly different from unity. The pA₂ values of Deno against phenylephrine in femoral and renal arteries were 5.41 ± 0.03 and 5.76 ± 0.06, respectively.On the other hand, Deno concentration-dependently (10 nM–10 µM) relaxed ring segments of canine coronary arteries which were partially precontracted with 5 µM prostaglandin F₂. The relaxation was significantly inhibited by pretreatment with 10 µM metoprolol.In conclusion, vascular smooth muscle relaxation by Deno was mediated through ₁-adrenergic action in canine coronary arteries and through the blocking effect of -adrenoceptors in canine femoral, mesenteric, and renal arteries.     

Exercise training,indomethacin, and isoproterenol-induced myocardial necrosis in the rat

Summary It was hypothesized that endurance exercise training would attenuate isoproterenol-induced myocardial necrosis in the rat by increasing the concentration of prostacyclin in the myocardial vasculature. Rats were randomly assigned to exercise and control groups. Exercisers ran on a motorized treadmill 1 h · d^–1, 5 d · week^–1 for 14 weeks. Immediately following the training program subgroups of rats were injected with 4 mg · kg^–1 indomethacin or saline. One day later, all rats were given a subcutaneous injection of isoproterenol (20 mg · kg^–1); after another 24h they were sacrificed. A decrease of myocardial creatine kinase (CK) activity was used as a marker for myocardial necrosis. Endurance exercise training attenuated the isoproterenol-induced decrease in myocardial CK relative to control by approximately 37% (exercise: 16.4 ± 0.6 U · mg^–1 protein; control: 10.5 ± 0.6 U · mg^–1 protein; p < 0.05). Pretreatment with indomethacin decreased myocardial CK in the exercise-trained rats (indomethacin: 15.4 ± 0.8 U · mg^–1 protein; saline: 17.7 ± 0.7 U · mg^–1 protein; p < 0.05), but not in the controls (indomethacin: 10.3 ± 1.0 U · mg^–1 protein; saline: 10.8 ± 0.6 U · mg^–1 protein; p > 0.05). The concentration of myocardial 6-keto-PGF₁ a marker for prostacyclin, was not altered by exercise but, as expected, was reduced by indomethacin pretreatment (p < 0.05). Thus, exercise training reduces myocardial damage caused by isoproterenol, but the evidence does not support the hypothesis that prostacyclin mediated this effect of training. Further research is needed to determine the extent to which exercise training-induced alterations in sensitivity to PGI₂ or TXA₂ affect myocardial damage from isoproterenol. The effect of any exercise-induced alteration in cardiac -adrenoceptor number or binding characteristics which result in myocardial resistance to isoproterenol also cannot be overlooked.     

Cardiovascular effects of Ekebergia capensis Sparrm (Meliaceae) ethanolic leaf extract in experimental animal paradigms

Summary

The purpose of this study was to examine the in vivo effects of Ekebergia capensis leaf ethanolic extract (EKE) on the blood pressure of anaesthetised normotensive male Wistar rats and conscious weanling Dahl salt-sensitive (DSS) rats, which develop hypertension as they age. To investigate possible mechanism(s) of the extract’s hypotensive effects, the contractile or relaxant responses to EKE in the absence or presence of reference drugs were evaluated in Wistar rat isolated aortic rings precontracted with methoxamine hydrochloride (ME, 10 μM).Acute intravenous administration of EKE elicited hypotensive responses in anaesthetised animals, while sub-chronic treatment with the extract averted the development of high blood pressure in weanling DSS rats. Isometric recordings of methoxamine hydrochloride (ME) pre-contracted, isolated, endothelium-intact and -denuded aortic rings revealed concentration-dependent relaxation responses to EKE (1–160 mg/ml). The potency was significantly less in the endothelium-denuded rings. Inhibitors of endothelium-derived relaxing factor (EDRF), L-NAME, methylene blue and indomethacin significantly reduced EKE-evoked vasorelaxations in endothelium-intact aortic rings.These results indicate that the vasorelaxant effect of EKE was in part mediated via EDRF-dependent or -independent pathways. These observations suggest that the hypotensive effect of EKE was in part mediated via modulation of total peripheral resistance of the vascular smooth muscles.     

Alterations of GTP-binding Proteins (Gsα and Gq/11α) in Gastric Smooth Muscle Cells from Streptozotocin-Induced and WBN/Kob Diabetic Rats

We investigated possible impairment of the signal transduction system in gastric myocytes of streptozotocin-induced diabetic (STZ) and spontaneous diabetic WBN/Kob (WBN/Kob) rats. Gastric motility 10 weeks after the onset of diabetes mellitus was significantly reduced in both diabetic rats compared with control, and the decreased motility was not recovered by the administration of insulin to maintain normal blood glucose levels. There was no significant difference between both types of diabetic rats and control rats in total number of [³H]quinuclidinyl benzilate ([³H]QNB) binding sites (B _max: 545–587 fmol/mg protein) on gastric smooth muscle cell membranes or in the affinity of [³H]QNB for the binding sites(K _d : 0.06–0.07 nM). Immunoblot analysis using polyclonal anti-G-protein antibodies indicated increased expression of G_s in gastric smooth muscle cell membranes, but no significant change in G_i or G_q/₁₁ expression in STZ rats, and decreased expression of G_q/₁₁ with no significant change in G_s and G_i in WBN/Kob rats. The cAMP production in gastric smooth muscle cell membranes was augmented in the absence and presence of 100 M isoproterenol, and 100 M forskolin in STZ rats, whereas no significant change of cAMP production was observed in WBN/Kob rats irrespective of the presence of the stimulants. These findings suggest that long-standing diabetes may induce alterations in signal transduction at downstream receptors in gastric myocytes, resulting in the impairment of gastric motility, although the mechanism of reduced contractile activity may differ between STZ and WBN/Kob rats.     

Electrophysiological effects mediated by the stimulation of cardiac β2-adrenoceptors with tulobuterol

Summary We studied the electrophysiological effects of the specific ₂-agonist tulobuterol in the guinea-pig sinus node and in sheep cardiac Purkinje fibers. Stimulation of ₂-adrenoceptors by tulobuterol resulted in a slight increase in the rate of firing of the sinus node. In Purkinje fibers, however, automaticity was not affected up to concentrations of 10^–6 M. Consistently, tulobuterol (10^–8–10^–6 M) did not affect the pacemaker current studied under voltage-clamp conditions. In the same range of concentrations (10^–8–10^–6 M) tulobuterol dose-dependently increased the contractile force of driven Purkinje fibers. Tulobuterol, at a very high concentration (10^–5 M), had membrane depressant effects as demonstrated by the block of automaticity induced in the spontaneously beating Purkinje fibers and by the reduction of the maximum rate of depolarization in driven preparations.Our results suggest that stimulation of ₂-adrenoceptors with tulobuterol in sheep Purkinje fibers is associated with an inotropic rather than a chronotropic effect. On the whole, the data confirm the lack of cardiac side effects of tulobuterol.     

Relationship of gastric mucosal damage induced in pigs by antiinflammatory drugs to their effects on prostaglandin production

Experiments were performed in pigs to examine the relationship between the effects of various nonsteroid antiinflammatory drugs on gastric (fundic) mucosal content of prostaglandin (PG) E₂ and 6-keto-PGF₁, and the development of damage to the fundic mucosa under acute and chronic dosage conditions. Oral administration of a single dose of indomethacin (5 mg/kg) caused an almost immediate reduction in mucosal potential difference, followed at 5–15 min by ultrastructurally observed damage to mucosal capillaries, mucous, and parietal cells; efflux of Na⁺, K⁺, and Cl^– ions into the gastric lumen with an apparent loss of luminal H⁺ ions; and a statistically significant reduction (from 10–60 min) in fundic prostaglandin content. Thus, under acute dosage conditions, development of mucosal damage by indomethacin was paralleled by the reduction in prostaglandin production. Repeated oral dosage of aspirin, indomethacin, sulindac, or diclofenac to pigs for 10 days significantly reduced gastric mucosal as well as plasma prostaglandin levels, coincident with the development of severe gastric mucosal damage. The relatively less irritant drugs, flufenamic acid, azapropazone and fenclofenac, failed to significantly decrease gastric mucosal content of prostaglandins, although these drugs have been reported to inhibit prostaglandin synthesisin vitro and also were found to reduce the prostaglandin plasma levels in animals receiving these drugs. Another drug with low irritancy, meseclazone, markedly decreased both mucosal and plasma levels of prostaglandins. The results show that while ulcerogenic drugs reduce the mucosal and plasma prostaglandins levels in parallel with their ulcerogenicity, this relationship does not always hold for drugs with low ulcerogenic activity.     

Effects of hemorrhagic shock on alkaline secretion and mucosal tolerance to acid in rat duodenum

The effects of hemorrhagic shock (HE) on duodenal HCO₃ ^– secretion and mucosal tolerance to acid were investigated in anesthetized rats and compared with those of indomethacin. HE was performed by bleeding from the carotid artery to reduce arterial blood pressure to about 50 mm Hg (3 ml bleeding per 200 g of body weight) with a significant decrease in arterial pH and [HCO₃ ^–], and indomethacin was given subcutaneously in a dose of 5 mg/kg. The proximal duodenum (1.7 cm) secreted HCO₃ ^– at the rate of 1.5–1.8 eq/15 min (3.5–4.2 eq/cm/hr), and responded to luminal acid (10 mM HCl for 10 min) by a significant rise in HCO₃ ^– output. Indomethacin had no effect on basal HCO₃ ^– output but significantly inhibited the acid-induced HCO₃ ^– secretion, while under HE conditions duodenal HCO₃ ^– output significantly declined and failed to increase in response to luminal acidification. Subcutaneously administered 16, 16-dmPGE₂ (30 g/kg) significantly increased HCO₃ ^– secretion in the presence of indomethacin but had less effect on the impaired HCO₃ ^– output caused by HE. In contrast, intravenous infusion of NaHCO₃ (3 mmol/kg/hr) ameliorated the acid-base imbalance caused by HE, and significantly restored the impaired HCO₃ ^– responses induced by HE but not by indomethacin. Both HE and indomethacin induced extensive damage in the mucosa when the duodenal loop was perfused with 50 mM HCl for 1.5 hr, and these lesions were significantly reduced by NaHCO₃ infusion and 16, 16-dmPGE₂, respectively. These results suggest that HE impaired duodenal HCO₃ ^– secretion and reduced the tolerance of the mucosa to acid. This effect may be mainly a result of a decrease of HCO₃ ^– availability, but it is not accounted for by a deficiency of endogenous prostaglandins.     

Somatostatin stimulates prostaglandin production by rat gastric epithelial cellsin vitro,but is not cytoprotective

Whether somatostatin stimulates prostaglandin synthesis by gastric cells is controversial. Also, it is unknown whether somatostatin protects gastric cells against exogenous injury in conditions independent of systemic factors and of inhibition of gastric acid secretion. The present study was undertaken (1) to evaluate the effect of somatostatin on prostaglandin production by rat gastric epithelial cells in monolayer culture and (2) to assess whether somatostatin protects gastric cells against taurocholate- and indomethacin-induced damage in vitro. Somatostatin at concentrations of 10^–5 M and 10^–4 M significantly stimulated PGE₂ and 6-keto-PGF¹ production by rat gastric epithelial cells but was not able to prevent damage induced by sodium taurocholate and indomethacin to rat gastric cells in monolayer culture. These results suggest that: (1) somatostatin stimulates prostaglandin synthesis by cultured rat gastric epithelial cells, but (2) is not directly protective to rat gastric epithelial cell monolayers against drug-induced damage in vitro.This work was supported by the Medical Research Service of the Veterans Administration.     

Contractile effect of Sclerocarya birrea (A Rich) Hochst (Anacardiaceae) (Marula) leaf aqueous extract on rat and rabbit isolated vascular smooth muscles

Backround

Sclerocarya birrea (Anacardiaceae) is traditionally used for treating hypertension. The pharmacological effects of S birrea leaf aqueous extract (SBE) on rabbit and rat vascular smooth muscles were investigated in this study.

Methods

Fresh S birrea leaves (1 kg) were air dried at 26 ± 1°C, milled, macerated in 2.5 l of distilled water for 48 hours, filtered, and the filtrate was concentrated in a rotary evaporator. Rat isolated portal vein preparations, as well as rabbit isolated endothelium-denuded and endothelium-intact descending thoracic aortic ring preparations were mounted in 30-ml Ugo Basile organ baths under physiological conditions, and challenged with SBE (50–400 mg/ml). The contractile effects of SBE and/or other reference drugs on the isolated vascular smooth muscle preparations were recorded by means of Ugo Basile’s force–displacement transducers and Gemini recorders.

Results

SBE (50–400 mg/ml) caused a significant, concentration-dependent upward shift in baseline tone in the aortic ring preparations (p < 0.01–0.001). Indomethacin (20 µM) markedly attenuated the contractile effects of SBE in both the endothelium-intact and -denuded aortic rings, while N^G-nitro-_L-arginine methyl ester (L-NAME, 100 µM) significantly (p < 0.05) increased the contractile tension of the endothelium-intact aortic rings. Verapamil (1–3 µg/ml) partially inhibited the contractile effects of SBE. SBE also elicited significant (p < 0.05–0.01) increases in the amplitude of the myogenic contractions of the portal veins. These contractions were abolished by verapamil (1–3 µg/ml) in a concentration-dependent manner, while prazosin (1–3 µg/ml) did not affect the SBE-induced contractions.

Conclusion

SBE possessed spasmogenic effects on vascular smooth muscle preparations in vitro. It may induce and/or exacerbate hypertension, contrary to the folkloric, ethnomedical use of S birrea.     

Interferonγ and tumour necrosis factorα induce a synergistic antiproliferative response in human Ewing's sarcoma cells in vitro

Three human cell lines derived from Ewing's sarcoma (RM-82, VH-64, and WE-68) were investigated to establish the influence of recombinant human interferon (rhIFN) and tumour necrosis factor (rhTNF) on cell proliferation and survival and to characterize IFN and TNF receptor expression. Incorporation of [³H]thymidine into cells was inhibited by rhIFN after 24 h of incubation. Half-maximal inhibition was observed with 10–80 U/ml rhIFN. A maximal effect (50%–70% inhibition of cell proliferation) was achieved by treatment of cells with 250 U/ml rhIFN. The influence of rhTNF on proliferation was found to differ among cell lines and varied with the concentration and the duration of exposure of cells to this cytokine. In WE-68 and VH-64 cells [³H]thymidine incorporation was not affected by rhTNF up to 2000 U/ml after 96 h of incubation, where-as in RM-82 cells the incorporation was inhibited by 35% after 48 h of incubation with 100 U/ml rhTNF. However, all cell lines showed a synergistic antiproliferative response to the combination of rhIFN and rhTNF after 24 h of incubation. The human recombinant cytokines interleukin(IL)-1, IL-1, IL-2, IL-3, IL-4, IL-6 and granulocyte/macrophagecolony-stimulating factor, tested alone and in combination with rhIFN and rhTNF, had no influence on cell proliferation. Binding studies in the cell lines with¹²⁵I-rhIFN revealed a dissociation constant (K _d ) of 160–306 pM and approximately 8000–13500 receptors/cell. Binding experiments with¹²⁵I-rhTNF indicated 430–1250 receptors/cell withK _d ranging from 13 pM to 162 pM. These data indicate that, among various cytokines, only IFN and TNF are capable of potently reducing Ewing's sarcoma cell growth in vitro. Our data suggest that IFN alone or in combination with TNF may be useful in the design of novel strategies in Ewing's sarcoma therapy.     

Effects of Monatepil, a Novel Calcium Antagonist with α1-Adrenergic Blocking Activity, on the Low-Density Lipoprotein Receptor in Human Skin Fibroblasts

To investigate the mechanisms of the hypolipidemic effect ofmonatepil, a new class of calcium antagonists with₁-adrenergic blocking activity, we examined theeffects of the drug on low-density lipoprotein (LDL) receptor activity andthe level of LDL receptor mRNA present in cultured human skin fibroblasts.At concentrations of 2 × 10^–5M, monatepilincreased the binding (248 ± 43% mean ± SD),internalization (374 ± 18%), and degradation (145 ±2%) of ¹²⁵I-LDL in human skin fibroblasts (n =3, p<0.05). Treatment of human skin fibroblasts with 2 × 10^–5 M of monatepil for 6 hours resulted in an increasein LDL receptor mRNA to 163% of the control level (n = 2), asshown by Northern blot analysis. Our results suggest that the hypolipidemicclinical effects of monatepil may be due to increased LDL receptoractivity.     

Increases in plasma lysosomal enzymes in Type 1 (insulin-dependent) diabetes mellitus: relationship to diabetic complications and glycaemic control

Summary Lysosomal enzymes degrade membrane glycoconjugates, and increased circulating enzyme activity may be an important mechanism in the pathogenesis of diabetic microangiopathy. We have assayed a profile of seven lysosomal enzyme activities (nmol·h^–1·ml^–1) in platelet-free plasma from 54 Type 1 (insulin-dependent) diabetic subjects (median age 31 years) and 42 matched normal control subjects. A significant increase in median (interquartile range) enzyme activity was measured in diabetic compared to control subjects for -d-glucuronidase, 121 (97.7–171) vs 88.8 (62.8–113), p <0.001; -d-Nacetylglucosaminidase, 693 (568–799) vs 568 (462–686), p <0.001; -d-mannosidase, 23.8 (16.7–28.9) vs 14.5 (10.1–20.0), p <0.001; and -d-galactosidase, 6.94(6.11–9.99) vs 6.66(4.78–8.33), p <0.04. In contrast, -l-fucosidase, -d-galactosidase and -d-mannosidase activities were similar in diabetic and control subjects. None of the enzyme activities differed significantly (p <0.05) between 24 diabetic patients with clinical complications and 30 complication-free diabetic patients with similar glycaemic control which does not support the hypothesis that enzyme increases in diabetes arise simply by leakage from damaged tissues. In the diabetic subjects HbA₁, median (interquartile range) 9.10 (7.40–10.60), was significantly related to -d-glucuronidase (r _s = 0.56, p <0.001) and -d-Nacetylglucosaminidase (r _s = 0.55, p <0.001). We have therefore demonstrated in diabetic subjects an increase in certain lysosomal glycosidases, that correlates with glycaemic control. Such increased enzyme activities acting on endothelial and basement membrane substrates may reduce glycoconjugate content, modify charge density, and thereby influence membrane permeability, a hallmark of microangiopathy.     

Die Wirkung von N1,n-Butylbiguanid auf den Stoffwechsel der isolierten perfundierten Leber normaler und alloxandiabetischer ketotischer Ratten

Zusammenfassung An isolierten perfundierten Lebern von normalen und von alloxandiabetischen ketotischen Ratten wurde der Effekt von N₁,n-Butylbiguanid auf den Leberstoffwechsel untersucht. — N₁,n-Butylbiguanid in einer Konzentration von 2.55 · 10^–5 M im Perfusionsmedium hatte folgende Effekte. — 1. Die Aminosäurebilanz normaler und diabetischer Lebern wurde in positiver Richtung verändert. Die in Kontrollversuchen feststellbare kontinuierliche Nettoabgabe von -Aminosäuren nahm unter Butylbiguanid signifikant ab oder es kam sogar zu einer Nettoaufnahme von -Aminosäuren. — 2. Die Nettoabgabe von anorganischem Phosphat durch normale und diabetische Lebern wurde durch Butylbiguanid signifikant vermindert. — 3. Die erhöhte Nettoharnstoffabgabe diabetischer Lebern wurde durch Butylbiguanid signifikant vermindert. Die Harnstoffbilanz normaler Lebern blieb unbeeinflußt. — 4. Die Nettoabgabe von Kalium durch diabetische Lebern wurde durch Butylbiguanid signifikant vermindert. — 5. Der in Experimenten mit diabetischen Lebern erhöhte Lactat/Pyruvat-Quotient im Perfusionsmedium normalisierte sich bei Butylbiguanidzusatz rascher. — 6. Die Gallebildung normaler und diabetischer Lebern ist unter Butylbiguanid gesteigert. Die Steigerung ist in den Experimenten mit diabetischen Lebern signifikant. — N₁,n-Butylbiguanid in einer Konzentration von 2.55 · 10^–5 M hatte keinen signifikanten Effekt auf die Nettoglucosebilanz, die Aufnahme unveresterter Fettsäuren, die Ketonkörperbildung, die Leberdurchblutung und die Änderungen des Leberglykogengehaltes in Experimenten mit normalen und diabetischen Lebern. — N₁,n-Butylbiguanid in einer 4-fach höheren Konzentration (1.02 · 10^–4 M) hatte in Experimenten mit normalen Lebern folgende Wirkung: 1. Der positive Effekt der kleineren Biguanidkonzentration (2.55 · 10^–5 M) auf die Aminosäurebilanz war nicht mehr nachweisbar. — 2. Die Lactatkonzentrationen im Medium waren deutlich erhöht. — 3. Der Lactat/Pyruvat-Quotient im Medium lag deutlich höher als in den Kontrollexperimenten. — Bei einer Butylbiguanidkonzentration von 2.55 · 10^–5 M befand sich das zugegebene Biguanid ausschließlich im Plasmaanteil des Perfusionsmediums. Bei einer Butylbiguanidkonzentration von 1.02 · 10^–4 M war der Verteilungsraum des Butylbiguanids größer, als dem Plasmavolumen entsprach. Weder bei Verwendung der niedrigen, noch bei Verwendung der hohen Biguanidkonzentration trat ein meßbarer Konzentrationsabfall im Medium während der dreistündigen Perfusion ein. Die Biguanidausscheidung mit der Galle war minimal.

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The effect of N₁,n-butylbiguanide on the metabolism of isolated perfused livers of normal and alloxan-diabetic ketotic rats

Summary The effect of N₁, n-butylbiguanide on the metabolism of isolated perfused livers of normal and alloxan-diabetic ketotic rats was studied. — N₁, n-butylbiguanide in a concentration of 2.55 · 10^–5 M in the perfusion medium had the following effects: 1. The amino acid balance of normal and diabetic livers was altered in a positive direction. The continuous net output of -amino acids seen in control experiments decreased significantly under butylbiguanide and a net uptake of -amino acids took place. 2. The net output of inorganic phosphate by normal and diabetic livers was significantly diminished by butylbiguanide. 3. The elevated net output of urea by diabetic livers decreased significantly under butylbiguanide. The urea balance of normal livers remained unchanged. 4. The net output of potassium by diabetic livers was significantly diminished by butylbiguanide. 5. The lactate/ pyruvate-ratio, which was elevated in experiments with diabetic livers became normalized more rapidly in presence of butylbiguanide. 6. Bile production of normal and diabetic livers was stimulated by butylbiguanide. This stimulation was significant in experiments with diabetic livers. — In experiments with normal and diabetic livers N₁,n-butylbiguanide in a concentration of 2.55 · 10^–5 M did not have any significant effect on the glucose balance, the uptake of non-esterified fatty acids, the production of ketone bodies, the liver blood flow, or liver glycogen content. — N₁,n-butylbiguanide at a concentration four times greater (1.02 · 10^–4 M) had the following effects in experiments with normal livers: 1. The positive effect of the smaller biguanide concentration (2.55 · 10^–5 M) on the amino acid balance could not be demonstrated with the higher concentration. 2. The concentration of lactate in the medium showed a significant increase. 3. The lactate/pyruvate-ratio was significantly higher than in control experiments. — At a concentration of 1.02 · 10^–4 M the equilibration volume for butylbiguanide was greater than the plasma volume. A measurable decrease in the medium concentration of butylbiguanide did not occur during the 3 hours of perfusion with either a low or a high concentration. The biguanide excretion with bile was minimal.

Teilergebnisse wurden auf dem II. Internat. Symposion über Diabetesfragen (1963) vorgetragen (s. Mohnike, G., ed., II. Internat. Sympos. über Diabetesfragen, S. 221ff., Karlsburg b. Greifswald).