Insights into mechanisms underlying the gut and airways modulatory effects of <Emphasis Type="Italic">Swertia chirata</Emphasis>

Swertia chirata is used in folk medicine for the treatment of constipation, colic, diarrhea, and asthma. This study was carried out in order to provide a pharmacological basis for its medicinal use in gastrointestinal and respiratory disorders. Crude extract of Swertia chirata (Sc.Cr) and its fractions were studied using rabbit isolated tissue preparations. In jejunum, Sc.Cr, which tested positive for alkaloids, flavonoids, saponins, tannins, and terpenes, caused stimulation at concentrations of 0.01–1.0 mg/mL, followed by a relaxant effect at higher concentrations. In the presence of atropine, the contractile effect was blocked and only relaxation occurred. Sc.Cr inhibited high K⁺ (80 mM)-induced contractions at 0.01–10 mg/mL and shifted Ca²⁺ concentration–response curves to the right, similar to that caused by verapamil. In trachea, Sc.Cr relaxed the carbachol (1 μM) and high K⁺-induced contractions, in a pattern similar to that of verapamil. Bioassay directed fractionation revealed the separation of spasmogenic and spasmolytic components in aqueous and organic fractions, respectively. The chloroform fraction exhibited a concentration-dependent (0.1–3.0 mg/mL) bronchodilator effect. These results indicate that Swertia chirata exhibits gut excitatory and inhibitory effects, mediated through cholinergic and Ca²⁺ antagonist mechanisms, respectively, as well as bronchodilatation, via Ca²⁺ channel blockade. Thus, this study provides a sound mechanistic background for the therapeutic application of Swertia chirata in gut motility disorders, such as constipation, colic, and diarrhea, and airways hyperactivity disease, such as asthma.     

The inhibition by di(2-ethylhexyl)-phthalate of <Emphasis Type="Italic">erg</Emphasis>-mediated K<Superscript>+</Superscript> current in pituitary tumor (GH<Subscript>3</Subscript>) cells

DEHP (bis(2-ethylhexyl)-phthalate) known to be an endocrine-disrupting chemical is a widely used phthalate. Little information regarding the effects of phthalate esters on ion currents is available. In this study, the effects of DEHP and other phthalate esters (DBEP: di(2-butoxyethyl)-phthalate and DMGP: di(2-methylglycol)-phthalate) on ion currents were investigated in pituitary GH₃ cells. Hyperpolarization-elicited K⁺ currents in GH₃ cells bathed in high-K⁺, Ca²⁺-free solution were examined to evaluate the effects of DEHP, DBEP, and DMGP on the ether-à-go–go-related-gene (erg) K⁺ current (I _K(erg)). Addition of DEHP to GH₃ cells suppressed the amplitude of I _K(erg) in a concentration-dependent manner with an IC₅₀ value of 16.3 μM. With a two-pulse protocol, addition of DEHP shifted the activation curve of I _K(erg) to a depolarized potential by approximately 10 mV with no change in the rate of I _K(erg) deactivation. This compound did not have any effects on delayed rectifier K⁺ current in GH₃ cells, while 4-aminopyridine-3-methanol (100 μM) suppressed this current significantly. DBEP (30 μM) had little or no effect on I _K(erg), while DMGP (30 μM) slightly reduced it. In inside-out configuration, DEHP (30 μM) applied to the bath slightly reduced the activity of large-conductance Ca²⁺-activated K⁺ channels. DEHP (30 μM) increased the frequency of spontaneous action potentials (APs); however, this compound at the same concentration had no effect on AP firing in KCNH2 siRNA-transfected GH₃ cells. The effects described herein can contribute to their actions on functional activity of endocrine or neuroendocrine cells if similar results are found in vivo.     

Effect of hydroalcoholic extract of <Emphasis Type="Italic">Acorus calamus</Emphasis> on tibial and sural nerve transection-induced painful neuropathy in rats

This study was designed to investigate the therapeutic potential of hydroalcoholic extracts of Acorus calamus (AC) in tibial and sural nerve transection (TST)-induced neuropathic pain in rats. The hot plate, paw heat allodynia, acetone drop, and pinprick tests were performed to assess the degree of heat hyperalgesia, heat and cold allodynia, and mechanical hyperalgesia, respectively, at different time intervals, i.e., day 0, 1, 3, 6, 9, 12, 15, 18, and 21. The tissue superoxide anion and total calcium were measured as markers of oxidative stress. Tissue myeloperoxidase activity was measured as a specific marker of inflammation. Histopathological evaluation was also performed in the nervous tissue to assess the axonal degeneration. Pregabalin served as positive control in this study. TST in rats significantly induced thermal hyperalgesia and allodynia, mechanical hyperalgesia, and increased the levels of superoxide anion, total calcium, and myeloperoxidase (MPO) activity. Moreover significant histological changes were also observed. Oral administration of AC hydroalcoholic extract (100 and 200 mg/kg for 14 days) attenuated TST-induced behavioral, biochemical, and histological changes. Acorus calamus has ameliorative potential in TST-induced painful neuropathy, and this effect may be attributed to its multiple actions including anti-inflammatory, antioxidant, and neuroprotective actions.     

Suitability of <Emphasis Type="Italic">Daphnia similis</Emphasis> as an alternative organism in ecotoxicological tests: implications for metal toxicity

The acute toxicity of metals to Daphnia similis was determined and compared to other daphnid species to evaluate the suitability of this organism in ecotoxicology bioassays. To verify the performance D. similis in toxicity tests, we also investigated the effect of Pseudokirchneriella subcapitata at 1 × 10⁵ and 1 × 10⁶ cells ml⁻¹ on Cd and Cr acute toxicity to the cladoceran. Daphnid neonates were exposed to a range of chromium and cadmium concentrations in the absence and presence of the algal cells. Metal speciation calculations using MINEQL⁺ showed that total dissolved metal concentrations in zooplankton culture corresponded to 96.2% free Cd and 100% free Cr concentrations. Initial total dissolved metal concentrations were used for 48 h-LC₅₀ determination. LC₅₀ for D. similis was 5.15 × 10⁻⁷ mol l⁻¹ dissolved Cd without algal cells, whereas with 1 × 10⁵ cells ml⁻¹, it was significantly higher (7.15 × 10⁻⁷ mol l⁻¹ dissolved Cd). For Cr, the 48 h-LC₅₀ value of 9.17 × 10⁻⁷ mol l⁻¹ obtained for the cladoceran in tests with 1 × 10⁶ cells ml⁻¹ of P. subcapitata was also significantly higher than that obtained in tests without algal cells (5.28 × 10⁻⁷ mol l⁻¹ dissolved Cr). The presence of algal cells reduced the toxicity of metals to D. similis, as observed in other studies that investigated the effects of food on metal toxicity to standard cladocerans. Comparing our results to those of literature, we observed that D. similis is as sensitive to metals as other standardized Daphnia species and may serve as a potential test species in ecotoxicological evaluations.     

Effect of nitroxyalkyl derivatives of fullerenylproline on the activity of CA<Superscript>3+</Superscript>-ATPase of sarcoplasmic reticulum

The effect of nitroxyalkyl derivatives of fullerenylproline methyl ester on the enzymatic activity of Ca²⁺-ATPase of sarcoplasmic reticulum (SR) has been studied. It is shown that hybrid derivatives of C₆₀ fullerene are capable of inhibiting the activity of Ca²⁺-ATPase of SR. The mononitrate inhibits the hydrolytic activity of the enzyme with K _i = 1.92 × 10⁻⁶ M; active Ca²⁺ transport, with K _i = 3.79 × 10⁻⁶ M. The dinitrate inhibits ATP hydrolysis with K _i = 2.38 × 10⁻⁸ M; Ca²⁺ transport, with K _i  = 3.08 × 10⁻⁸ M. Fullerenylproline methyl ester does not affect the enzymatic activity of Ca²⁺-ATPase. Based on these data it is possible to predict the possible fields of application for hybrid fullerene derivatives as potential drugs.     

CORONARY ENDOTHELIAL FUNCTION IN OPEN HEART SURGERY

1. During open heart surgery, the heart is arrested and protected by hyperkalaemic cardioplegia. The coronary endothelium may be damaged by ischaemia-reperfusion and cardioplegia. Subsequently, this may affect cardiac function immediately after cardiac surgery and cause mortality or morbidity. 2. Our studies have investigated coronary endothelial function after exposure to hyperkalemi. (K⁺ 20 or 50 mmol/L). Endothelium-dependent relaxation and hyperpolarization of the coronary smooth muscle and intracellular free calcium concentration in the endothelial cell were measured with regard to K⁺ exposure. 3. Endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation to A23187, bradykinin, and substance P in the presence of either U46619 (10 nmol/L)- o. K⁺ (25 mmol/L)-induced contraction was reduced after exposure to either 20 or 50mmol/LK.⁺ 4. The hyperpolarization of the membrane potential in response to the endothelium-derived relaxing factor (EDRF) stimuli was also reduced by exposure t. K⁺. 5. The intracellular free calcium concentration remained unchanged after exposure t. hyperkalemia. 6. We conclude that the EDHF-mediated coronary endothelial function is impaired after exposure to hyperkalaemic cardioplegia. The impairment of this function is due to the changed effect of EDHF on the smooth muscle cell, probably through partially depolarizing the membrane and affecting K⁺ channels rather than alteration of its biosynthesis/release in the endothelial cell. It may be of use to search for a new cardioplegia that preserves this endothelial function during open heart surgery.     

Pharmacological explanation for the medicinal use of <Emphasis Type="Italic">Juniperus excelsa</Emphasis> in hyperactive gastrointestinal and respiratory disorders

Crude extract of Juniperus excelsa (JeExt), which tested positive for the presence of anthraquinone, flavonoids, saponins, sterols, terpenes and tannin, exhibited a protective effect against castor oil-induced diarrhoea in mice at 100–1000 mg/kg. In rabbit jejunum preparations, JeExt (0.01–1.0 mg/mL) caused relaxation of spontaneous and K⁺ (80 mM)-induced contractions at similar concentrations to papaverine, whereas verapamil was relatively more potent against K⁺. JeExt (0.03–0.3 mg/mL) shifted Ca²⁺ concentration–response curves to the right, like papaverine or verapamil. JeExt (0.003–0.01 mg/mL) caused a leftward shift of isoprenaline-induced inhibitory concentration–response curves, similar to papaverine. JeExt (1.0–30 mg/kg) caused suppression of carbachol (CCh, 100 μg/kg)-induced increase in inspiratory pressure of anaesthetized rats. In guinea-pig trachea, JeExt (0.001–3.0 mg/mL) relaxed CCh (1 μM)- and high K⁺-induced contractions and shifted isoprenaline-induced inhibitory curves to the left. This study suggests that Juniperus excelsa possibly exhibits a combination of Ca²⁺ antagonist and phosphodiesterase inhibitory effects, which provides a pharmacological basis for its traditional use in disorders of gut and airways hyperactivity, such as diarrhoea, colic and asthma.     

EFFECT AND MECHANISM OF CARDIOPLEGIC ARREST ON THE CORONARY ENDOTHELIUM-SMOOTH MUSCLE INTERACTION

1. During cardiac surgery, the heart is arrested and protected by hyperkalaemic cardioplegia. The coronary endothelium may be damaged by ischaemia-reperfusion and cardioplegia. Subsequently, this may affect cardiac function immediately after cardiac surgery and cause mortality and morbidity. 2. We investigated coronary endothelium-smooth muscle interaction after exposure to depolarizing (hyperkalaemic; K⁺ 20 or 50 mmol/L) and hyperpolarizing (the K⁺ channel opener aprikalim) cardioplegia and organ preservation solution (University of Wisconsin (UW) solution). Endothelium-dependent relaxation and hyperpolarization of the coronary smooth muscle were studied in the porcine and human large conductance and micro-coronary arteries. Intracellular free calcium concentration in endothelial cells was also measured. 3. The endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation to A23187, bradykinin, and substance Pin arteries contracted by either U46619 (10nmol/L) or K⁺ (25 mmol/L) was reduced after exposure to either high K⁺ or UW solution, but was maximally preserved after exposure to aprikalim. The hyperpolarization of the membrane potential in response to the above endothelium-derived relaxing factor stimuli was also reduced by exposure to depolarizing cardioplegia. Studies in microcoronary arteries are in accordance with findings in large arteries. The intracellular free calcium concentration remained unchanged after exposure to hyperkalaemia. 4. We concluded that: (i) during cardiac surgery, the function of coronary circulation may be changed due to exposure to depolarizing cardioplegia or preservation solutions; (ii) the functional change in the coronary circulation is related to the altered interaction between the endothelium and smooth muscle; (iii) depolarizing (hyperkalaemia) cardioplegia or hyperkalaemic organ preservation solutions affect endothelium-smooth muscle interaction through the EDHF pathway; (iv) EDHF relaxes the porcine large and microcoronary arteries through multiple K⁺ channels; and (v) that hyperpolarizing vasodilators (K⁺ channel openers) may protect EDHF-mediated endothelial function when used as cardioplegia.     

Reduced nitric oxide formation causes coronary vasoconstriction and impaired dilator responses to endogenous agonists and hypoxia in dogs

We investigated the relative contribution of basal and agonist stimulated EDRF/NO release to the adjustment of coronary tone and myocardial perfusion in conscious dogs by inhibiting coronary endothelial NO formation with N^G-nitro-l-arginine methyl ester (l-NAME). Chronically instrumented conscious dogs (n = 9) were prepared for measurement of mean arterial blood pressure (MAP), heart rate (HR), coronary blood flow (CF) and diameter of the left circumflex (CD_LC) and left anterior descending (CD_LAD) coronary artery, respectively. Intracoronary infusions of l-NAME (30.3 mM; 0.25 ml × min^–1) caused significant increases in MAP and decreases in HR. CD_LC decreased by 3.8% from 3.01 ± 0.04 to 2.90±0.04 mm and CF decreases by 30% from 12.9 ± 0.2 to 9.1 ± 0.2 (aU). Peak reactive hyperemia (CF_max) evoked by 20-s-lasting occlusions of the left circumflex coronary artery decreased from 29.9 ± 0.8 to 25.8 ± 1.0 aU and maximal flow-dependent coronary dilation were reduced from 2.04 ± 0.08 to 0.91±0.12% after inhibition of NO-synthesis. Intracoronary infusions of acetylcholine (ACh), Adenosinc (Ado), bradykinin (Bk), and papaverine (Pap) caused dose-dependent increases in CD_LC and CE Infusion of l-NAME nearly abolished the dilator effect of Ado on CD_LC and reduced those to ACh, Bk and Pap. Increases in CF to ACh, Ado and Bk but not to Pap were reduced by l-NAME. Subsequent intracoronary infusions of l-arginine (303 mM; 0.25 ml × min^–1) reduced l-NAME-induced CF-changes partly, but did not reverse coronary constriction. These results suggest that inhibition of the continuous release of nitric oxide markedly reduces myocardial perfusion in vivo. Endogenous dilator mechanisms are likewise impaired. Thus, in the heart, nitric oxide deficiency probably cannot be fully compensated for by counter-regulating mechanisms. Correspondence to: E. Bassenge at the above address     

Acute and chronic effects of sodium and potassium on the tropical freshwater cladoceran <Emphasis Type="Italic">Pseudosida ramosa</Emphasis>

In this study, the toxicities of sodium and potassium to the tropical freshwater cladoceran Pseudosida ramosa were assessed. Acute toxicity tests on this species showed that the 48-h LC₅₀ of Na⁺ was 556 mg l⁻¹, while that of K⁺ was 17.7 mg l⁻¹. Long-term exposure of female P. ramosa to sodium reduced the total number of survivors from 10 to 6 at a concentration of 249 mg l⁻¹, 21-day fecundity from 20.4 to 14.3 eggs female⁻¹ at concentrations ranging from 72 to 249 mg l⁻¹, 21-day fertility from 20.1 to 6.5 neonates female⁻¹ at concentrations ranging from 25 to 249 mg l⁻¹. Furthermore, fecundity of each brood from the second to the fifth was significantly lower at 249 mg l⁻¹ and fertility of each brood from the first to the fifth at concentrations ranging from 25 to 249 mg l⁻¹. A significant decrease in fertility was associated with an increase in the number of aborted eggs. Long-term exposure to potassium decreased the 21-day fecundity of P. ramosa from 14.2 to 10.8 eggs female⁻¹ at a concentration of 11 mg l⁻¹ and fertility (fourth brood only) at 6.2 and 11 mg l⁻¹. Tropical reservoirs located near areas where the soil is overloaded with fertilizers and ferti-irrigation with vinasse already show concentrations of Na⁺ and K⁺ very close to those producing sub-lethal long-term effects on P. ramosa. A possible consequence is that organisms of the aquatic biota cannot adapt and freshwater taxa may become locally extinct, transferring dominance to salt-tolerant taxa.     

Effects of an antiarrhythmic drug A-2545 on canine ventricular arrhythmia models; comparison with mexiletine and flecainide

We investigated effects of a new Na⁺ channel blocking antiarrhythmic drug, A-2545, N-3 (2,2,5,5-tetramethyl-3-pyrroline-3-carboxamido)-propyl-phthalimide-hydrochloride, on various canine ventricular automaticity arrhythmias induced by two-stage coronary ligation, digitalis and adrenaline, and compared them with those of mexiletine. A-2545 showed antiarrhythmic effects, significantly decreasing the arrhythmic ratio of 24-h and 48-h coronary ligation-, digitalis- and adrenaline-induced automaticity arrhythmias. The antiarrhythmic plasma concentrations (IC₅₀) of A-2545, 2 mg kg^–1 10 min^–1, i.v., for 24-h and 48-h coronary ligation-, digitalis- and adrenaline-induced arrhythmias were 1.8, 1.3, 5.8 and 3.7 μg ml^–1, respectively, and that calculated for oral A-2545 (25 mg kg^–1) in 24-h coronary ligation-induced arrhythmia was 1.8 μg ml^–1. A-2545 is specifically potent in suppressing coronary ligation-induced arrhythmias, i.e., decreasing the arrhythmic ratio nearly to zero by oral administration, and among the intravenously given experiments A-2545 was effective at lower concentrations than other arrhythmia models; A-2545, 2 mg kg^–1 10 min^–1, was equipotent to 5 mg kg^–1 10 min^–1 mexiletine in suppressing 24-h coronary ligation-induced arrhythmia, indicating that A-2545 is more potent than mexiletine. In order to determine whether A-2545 has arrhythmogenic effects, we used programmed electrical stimulation (PES)-induced reentry arrhythmias in dogs with old myocardial infarction and compared effects of A-2545 and flecainide. A-2545, 2 and 5 mg kg^–1 10 min^–1, significantly suppressed the PES-induced arrhythmias in all six dogs without aggravating them. These arrhythmias were not markedly suppressed by flecainide either with 1 or 3 mg kg^–1 10 min^–1; moreover even in one out of six dogs aggravation of arrhythmia was noted after 1 mg kg^–1 10 min^–1. In conclusion, A-2545 suppressed various canine ventricular arrhythmias, and the antiarrhythmic effect of A-2545 was more potent than that of mexiletine, and A-2545 did not show arrhythmogenic effects compared to flecainide. Received: 16 February 1998 / Accepted: 7 October 1998     

Essential metal (Cu,Zn) exposures alter the activity of ATPases in gill,kidney and muscle of tilapia <Emphasis Type="Italic">Oreochromis niloticus</Emphasis>

An acute (96 h—0.1, 0.5, 1.0, 1.5 μg/ml) and chronic (up to 30 days—0.05 μg/ml) protocols of Cu and Zn were applied to freshwater fish Oreochromis niloticus to investigate these essential metal effects on the activities of gill, kidney and muscle Na⁺/K⁺-ATPase, Mg²⁺-ATPase and Ca²⁺-ATPase. In vitro effects of both metals (20 min—0.1, 0.5, 1.0, 1.5 μg/ml) were also measured to be able to compare both exposure routes. Data showed that ATPase activities, in general, decreased following all the exposure conditions, though there were some increases especially in Mg²⁺-ATPase activity. Among the enzymes, Na⁺/K⁺-ATPase and Ca²⁺-ATPase appeared to be more sensitive than Mg²⁺-ATPase to the metals. The data also indicated that effects of Cu on ATPase activity in the tissues of O. niloticus were stronger than the effects of Zn, possibly due to higher toxic effects of Cu. In vivo and in vitro exposures of metals showed similar trends with a few exceptions, especially in the gill. Variability of ATPase activity is determined by tissue type, metal species, concentration and duration. This work showed that even essential metals can alter significantly activities of ATPases in fish and thus suggests using them as a sensitive biomarker in metal contaminated waters.     

The catalase activity of Nα‐acetyl‐microperoxidase‐8

Abstract: N ^α ‐Acetylated microperoxidase‐8 (Ac‐MP‐8) is a water soluble, ferric heme model for peroxidases. We report here that Ac‐MP‐8 catalyzes catalase‐type reaction in addition to peroxidase‐type and cytochrome P450‐type reactions. The catalase activity of Ac‐MP‐8 was determined by the Clark oxygen electrode, which measures the production of oxygen in solution. The K_m and k_cat of the decomposition of hydrogen peroxide (H₂O₂) catalyzed by Ac‐MP‐8 are 40.9 mm and 4.1 per s, respectively. The specificity constant (k_cat/K_m) of Ac‐MP‐8 in catalase‐type reaction of H₂O₂ is 100.2,/m /s, which is 5‐ to 12‐ and 50‐ to 100‐fold less than those of MPs in cytochrome P450‐type reaction of aniline/H₂O₂ and peroxidase‐type reaction of o‐methoxyphenol/H₂O₂, respectively. These results indicate that Ac‐MP‐8 can catalyze three different types of reactions, and the relative catalytic specificities of Ac‐MP‐8 with a histidyl ligand exhibit the following orders: peroxidase‐type > cytochrome P450‐type > catalase‐type reactions. Comparisons of the enzyme activities of Ac‐MP‐8 suggest that the fifth ligands of hemoproteins influence the ratio of the three types of reactions.     

Metal sensitivity of the embryonic development of the ramshorn snail <Emphasis Type="Italic">Marisa cornuarietis</Emphasis> (Prosobranchia)

We investigated the effects of metal ions on the embryonic development of the ramshorn snail, Marisa cornuarietis, by exposing embryos to varying concentrations of copper (0, 50, 100, and 250 μg Cu²⁺/L), lead (0, 5, 10, and 15 mg Pb²⁺/L), lithium (0, 1, 2.5, and 3 mg Li⁺/L), or palladium (0, 50, 100, and 500 μg Pd²⁺/L). Effects of these metals were examined by recording mortality, the rate of tentacles and eyes formation, heart rate, hatching success, and weight after hatching. Compared to the control, we found a significant delay in the formation of tentacles and eyes after treatment with 100 μg Cu²⁺/L, 15 mg Pb²⁺/L, 2.5 mg Li⁺/L or 500 μg Pd²⁺/L. The heart rate decreased significantly at 500 μg Pd²⁺/L. At 10 mg Pb²⁺/L, 2.5 mg Li⁺/L, or 500 μg Pd²⁺/L, hatching was delayed significantly; 50 μg Cu²⁺/L induced a significantly earlier hatching, and reduced body weight. The LC₅₀ values were calculated to be about 50 μg Cu²⁺/L, 500 μg Pd²⁺/L, 2500 μg Li⁺/L, and 10000 μg Pb²⁺/L. These results show that the embryonic development of M. cornuarietis is about as sensitive to copper and lithium, compared to the most sensitive fishes used in embryo toxicity testing. Even though the MariETT is a laboratory-based assay focusing on toxicological endpoints of a selected model species, future application is envisaged to include testing of “natural” samples such as stream water or sediment interstitial water.     

Increased inward rectifier K+ current of coronary artery smooth muscle cells in spontaneously hypertensive rats; partial compensation of the attenuated endothelium-dependent relaxation via Ca2+-activated K+ channels

Endothelium-dependent vasorelaxation is partly mediated by small-conductance (SK3) and intermediate-conductance Ca²⁺-activated K⁺ channels (SK4) in the endothelium that results in endothelium-dependent hyperpolarization (EDH). Apart from the electrical propagation through myoendothelial gap junctions, the K⁺ released from the endothelium facilitates EDH by increasing inward rectifier K⁺ channel (Kir) conductance in smooth muscle cells. The EDH-dependent relaxation of coronary artery (CA) and Kir current in smooth muscle cells (CASMCs) of hypertensive animals are poorly understood despite the critical role of coronary flow in the hypertrophic heart. In spontaneously hypertensive (SHR) and control (WKY) rats, we found attenuation of the CA relaxation by activators of SK3 and SK4 (NS309 and 1-EBIO) in SHR. In isolated CASMCs, whole-cell patch-clamp study revealed larger I_Kir in SHR than WKY, whereas the myocytes of skeletal and cerebral arteries showed smaller I_Kir in SHR than WKY. While the treatment with I_Kir inhibitor (0.1 mmol/L Ba²⁺) alone did not affect the WKY-CA, the SHR-CA showed significant contractile response, suggesting relaxing influence of the higher I_Kir in the CASMCs of SHR. Furthermore, the attenuation of NS309-induced relaxation of CA by the combined treatment with 0.1 mmol/L Ba²⁺ was more prominent in SHR than WKY. Our study firstly shows a distinct increase of I_Kir in the CASMCs of SHR, which could partly compensate for the attenuated relaxation via endothelial SK3 and SK4.     

Cardiotoxicity of adrenochrome in isolated rabbit hearts assessed by epicardial NADH fluorescence

Noradrenaline in a micromolar concentration has recently been shown to contribute to ischemic tissue injury by direct cardiotoxic effects independent of functional alterations. Oxygen free radicals, generated during the auto-oxidation of catecholamines, are important mediators of catecholamine cardiotoxicity. However, the role of the oxidative products (aminochromes) is still unclear. We examined the effects of adrenochrome on functional parameters and on regional myocardial ischemia (MI) in isolated electrically-driven rabbit hearts with depleted catecholamine stores (reserpine 7.0 mg/kg i.p. 16 – 24 h before preparation, Langendorff, constant pressure: 70 cm H₂O, Tyrode solution, Ca⁺⁺ 1.8 mmol/l, 37°C). Repetitive MI, separated by a reperfusion period of 50 min, was induced by coronary artery branch ligature, and MI was quantitated from epicardial NADH fluorescence photography. Adrenochrome-treatment (10^– 6 M or 10^– 4 M) was started after a reperfusion period of 20 min. The left ventricular pressure (LVP) was significantly enhanced by adrenochrome (p <0.05), but it fell thereafter to below its initial value in hearts treated with adrenochrome 10^– 4 M. The global coronary flow (CF) was not affected by adrenochrome 10^– 6 M (P >0.05), but it was significantly decreased by adrenochrome 10^– 4 M (P <0.05). The relative CF (= CF/LVP × heart-rate) was numerically decreased by adrenochrome 10^– 6 M (p >0.05) and more markedly by adrenochrome 10^– 4 M (p <0.05). Whereas epicardial NADH fluorescence was similar after repetitive coronary artery occlusions in controls and in hearts treated with adrenochrome 10^– 6 M (p >0.05), it was significantly enhanced by adrenochrome 10^– 4 M (p <0.05). In isolated rabbit hearts, adrenochrome possesses deleterious effects on MI only at a very high concentration but not in a micromolar concentration. Therefore, it seems that aminochromes may be less cardiotoxic than catecholamines. Received: 28 February 1994 / Accepted: 2 May 1994     

Purification and Characterisation of a Newly Isolated Stable Long-Life Tannase produced by F. subglutinans (Wollenweber and Reinking) Nelson et al.

A stable long-life tannase was synthesised by Fusarium subglutinans and the fermentation processing parameters were optimised. Maximum enzyme production (9.38 U/ml) was recorded after 96 h of incubation at 35°C, initial pH 5, in submerged culture (200 rpm) utilising 2% (w/v) tannic acid as a sole carbon source. The tannase produced was purified to electrophoretic homogeneity through two-step column chromatography and the purified form remained stable in a pH range of 6–8. Its midpoint of thermal inactivation (T _m) was recorded at 70°C after 60 min of exposure. Maximum tannase activity was enabled at pH 6 and 40°C. Ca²⁺, K⁺, Mg²⁺ and Mn⁺ showed a stimulatory effect while Ba²⁺, Co²⁺, Cu²⁺, Fe³⁺ and Zn⁺ showed a competitive inhibitory effect on enzyme activity. Values of K _m, V _max, K _cat and the molecular mass of the purified enzyme were 0.116 μM ml⁻¹ min⁻¹, 3.57 mM, 1.16 μM ml⁻¹ min⁻¹ and 150 kDa, respectively. The participation of the SH group and carbohydrates in the enzyme structure was also suggested by the results. The stability of the purified and partially purified enzyme at −15°C extended to 13 months.     

Effect of temperature on phytoextraction of hexavalent and trivalent chromium by hybrid willows

The removal of hexavalent and trivalent chromium from hydroponic solution by plants to changes in temperature was investigated. Pre-rooted hybrid willows (Salix matsudana Koidz × alba L.) were exposed to a nutrient solution spiked with potassium chromate (K₂CrO₄) or chromium chloride (CrCl₃) for 4 days. Ten different temperatures were tested ranging from 11 to 32°C. Total Cr in solutions and in plant materials were all analyzed quantitatively. The results revealed that large amounts of the applied Cr were removed from the hydroponic solution in the presence of the plants. Significantly faster removal of Cr(III) than Cr(VI) was achieved by hybrid willows from the hydroponic solutions at all temperatures (P < 0.01). The removal rates of both chemical forms of Cr by plants increased linearly with the increase of temperatures. The highest removal rate of Cr(VI) was found at 32°C with a value of 1.99 μg Cr/g day, whereas the highest value of Cr(III) was 3.55 μg Cr/g day at the same temperature. Roots were the main sink for Cr accumulation in plants at all temperatures. Translocation of both chemical forms of Cr from roots to lower stems was only found at temperatures ≥24°C. The temperature coefficient values (Q ₁₀) were 2.41 and 1.42 for Cr(VI) and Cr(III), respectively, indicating that the removal of Cr(VI) by hybrid willows was much more susceptible to changes in temperature than that of Cr(III). This information suggests that changes in temperature have a substantial influence on the uptake and accumulation of both chemical forms of Cr by plants.