Mechanisms of changes in coronary arterial tone induced by bee venom toxins.

Our study elucidates some mechanisms of contractions or relaxations of isolated porcine left anterior descending coronary artery (LAD) induced by two peptides from the honeybee venom, melittin and apamin. Contractions or relaxations were measured on relaxed or precontracted arteries, respectively. Melittin at lower concentrations (0.1-10 microg/ml) induced transient relaxation, and contraction at higher concentrations (>or=7 microg/ml). The removing of the endothelium diminished the melittin-induced relaxation but did not affect the maximal contraction. The inhibition of prostaglandin and nitric oxide (NO) synthesis (by indomethacin and by N-omega-Nitro-l-arginine, respectively) and the use of K(+) channel inhibitors (apamin and charybdotoxin) showed that melittin evoked relaxation via an endothelium-dependent mechanism (NO production), and by activation of charybdotoxin-sensitive K(+) channels of smooth muscle. Apamin alone did not affect contraction or relaxation, but the inhibition of NO and prostanoid production revealed the involvement of apamin-sensitive K(+) channels of smooth muscle in melittin-induced relaxation. Our data show that melittin and apamin could affect contractility of porcine LAD at concentrations similar to those encountered in multiple honeybee stings in humans. Melittin could directly affect contractility of porcine LAD, whereas apamin acts as a modulator of the relaxant response to melittin.

Direct thrombin inhibitors built on the azaphenylalanine scaffold provoke degranulation of mast cells.

The main structural feature of direct thrombin inhibitor LK-732 responsible for the appropriate interaction at the thrombin active site is a strong basic group. A possibility that a strong basic group of LK-732 might contribute to the mast cell degranulation effect and consequent reduction of tracheal air flow (TAF) and fall of mean arterial blood pressure (MAP) in rats was investigated in the present study. At doses up to 5 mg/kg (i.v.), LK-732 did not cause significant changes of TAF and MAP. At 7 mg/kg (i.v.), a sudden reduction of TAF and a fall of MAP was observed within 5 min after LK-732 administration (75% mortality, p = 0.007). A less basic direct thrombin inhibitor LK-658 (21 mg/kg, i.v.) did not significantly disturb TAF and MAP. A reduction of TAF and a fall of MAP caused by LK-732 (7 mg/kg, i.v.) was almost completely abolished in rats with degranulated mast cells (0% mortality, p = 0.008). LK-732 concentration-dependently degranulated rat peritoneal mast cells in vitro (pEC(50) = 1.92 +/- 0.05 muM). A structure-activity relationship (SAR) study revealed that the terminal basic groups attached to the aromatic ring are responsible for the mast cell degranulation effect. A good correlation was observed between mast cell degranulation and pK(b) of analogues of LK-732 (R(2) = 0.49), but not between mast cell degranulation and thrombin K(i) (R(2) = 0.23). LK-732-induced reduction of TAF, the fall of MAP and high mortality originate from LK-732-induced mast cell degranulation. As judged by the SAR study, this effect could be overcome by reducing the basicity of LK-732.

Long-term effects of elemental mercury on renal function in miners of the Idrija Mercury Mine.

BACKGROUND: The kidneys are one of the main target organs for elemental mercury (Hg(0)). The influence of Hg(0) on kidneys has been extensively studied but the long-term effects on this organ have not yet been determined with certainty. The basic aim of this research was to study the effects of a long-term exposure to Hg(0) vapours on the renal function in miners in the post-exposure period. METHODS: The population studied comprised 53 miners (33 active and 20 retired) from the Idrija Mercury Mine as the exposed miners group and 53 unexposed workers as the control group. On the basis of mine exposure records (air and biological monitoring), the environmental and biological indicators of the past exposure to Hg(0) were calculated for each miner. Kidney function was determined in both groups, i.e. in the exposed miners as well as in the controls. Glomerular kidney function was evaluated by a quantitative analysis of albumin and IgG in urine. Tubular kidney function, however, was determined by a quantitative analysis of alpha(1)-microglobulin in urine and by the enzymatic activity of N-acetyl-beta-d-glucosaminidase (NAG). RESULTS: The mean exposure time in miners was 15 years. The total number of cycles of exposure ranged from 13 to 119. The mean annual time-weighted exposure was 0.29 mg m(-3) and the mean integrated exposure intensity (IEI) was 1413 mg m(-3)-h. Throughout the period of exposure the average urine mercury concentration in miners was 68.24 microg l(-1) and the average sum of peak urine mercury concentrations was 3901 microg l(-1). Albumin, IgG and alpha(1)-microglobulin in urine were significantly elevated in the exposed miners compared with the unexposed controls (t = 2.17, P = 0.03; t = 2.81, P < 0.01; and t = 2.07, P = 0.04). No significant differences were found in the urine NAG activity when the exposed miners and the unexposed workers were compared. Among the indicators of renal function only alpha(1)-microglobulin in the urine correlated significantly with the IEI (r = 0.73; P

Antithrombotic potential of new direct thrombin inhibitors built on the azaphenylalanine scaffold in two rat venous thrombosis models.

The antithrombotic potential of new direct thrombin inhibitors built on the azaphenylalanine scaffold (LK-732, LK-639 and LK-731) and their amidoxime prodrugs (LK-658, LK-633 and LK-730) was studied in comparison to argatroban and nadroparin in two rat models of venous thrombosis, induced either by complete stasis combined with hypercoagulability (model 1) or by partial stasis combined with vessel injury (model 2). In initial experiments LK-732 was established as the most promising antithrombotic of the LK inhibitors and as such was further tested. In model 1, intravenous bolus administration of LK-732 produced a dose-dependent inhibition of thrombus formation with an ID50 value of 1.3 mg/kg. This ID50 value was approximately four times higher than the ID50 value of argatroban (0.3 mg/kg; p=0.011). However, in model 2, LK-732 and argatroban decreased thrombus weight by 50% at similar ID50 values (3.8 mg/kg vs 3.0 mg/kg, respectively; p=0.726). The ex vivo anticoagulant effect of LK-732 was substantially weaker compared to argatroban at doses that produced comparable antithrombotic effects. After subcutaneous administration, in vivo thrombus weight reduction of LK inhibitors (10 mg/kg) ranged between 22 to 48%. However, their oral antithrombotic effect at a dose of 30 mg/kg was rather low. LK amidoxime prodrugs failed to produce a substantial antithrombotic effect after subcutaneous (10 mg/kg) as well as after oral administration (30 mg/kg). In conclusion, thrombin inhibitors built on the azaphenylalanine scaffold represent a new group of intravenously effective antithrombotics. However, optimisation of the oral antithrombotic effect of amidoxime prodrug LK-658 of the lead inhibitor LK-732 is required for justifying further development of these inhibitors.

Mibefradil is more effective than verapamil for restoring post-ischemic function of isolated hearts of guinea pigs with acute renal failure.

The deleterious intracellular Ca(2+) overload in the ischemic-reperfusion injury of the heart can be even more expressed in subjects with acute renal failure in whom maintenance of intracellular Ca(2+) has already been disturbed in normoxia. To study the influence of acute renal failure in ischemic-reperfusion injury on the heart, we used isolated Langendorff's hearts of guinea pigs with gentamicin-induced acute renal failure. We examined arrhythmias, heart contractility and myocardial cell damage during reperfusion. Two specific Ca(2+) channel antagonists, mibefradil (0.1 and 1 microM) and verapamil (0.1 microM), were used to test the possible involvement of T-type and L-type Ca(2+) channels in these processes. We exposed hearts to 50 min of zero-flow global ischemia and 60 min of reperfusion. During reperfusion, unrecoverable ventricular fibrillation appeared more often in hearts of animals with acute renal failure than in control hearts (80% vs. 0%, respectively). Mibefradil, but not verapamil, applied either pre- or post-ischemically, terminated ventricular fibrillation in all hearts of animals with acute renal failure. Mibefradil (0.1 microM only) improved contractility in hearts of animals with acute renal failure during reperfusion by 30%. During reperfusion, lactate dehydrogenase (LDH) release rate increased less in hearts of guinea pigs with acute renal failure than in control hearts and only verapamil decreased it additionally. Thus, our results suggest a more important role of T- than of L-type Ca(2+) channels in ischemic-reperfusion injury in isolated guinea pig hearts with acute renal failure.

Tezosentan inhibits both equinatoxin II and endotelin-1 induced contractions of isolated porcine coronary artery in a similar way.

In the present study we examined the endothelium-dependent mechanism in the constriction of the isolated porcine coronary artery induced by Equinatoxin II (EqT II). EqT II is a polypeptide isolated from the sea anemone (Actinia equina, L.). Contractions induced by endothelin-1 (ET-1) were compared with the contractions induced by EqT II. The force of contraction induced by 100 nM EqT II reached only 30% of the force of contraction induced by 100 nM ET-1. EC50 for ET-1 was 5.14 nM, and for EqT II 101.1 nM. The effects of tezosentan, an endothelin ETA/B receptor antagonist, on contractions induced by either ET-1 or EqT II were compared. Tezosentan inhibited both ET-1 and, to a lesser extent, EqT II-induced contractions of isolated porcine coronary artery. Our present results confirm the involvement of endothelium in the EqT II-induced contractions of coronary arteries. The mode of action of tezosentan upon EqT II-induced contractions indicate that besides its pore-forming effect in the membranes, endothelium, and specifically endothelin-dependent mechanisms, are very important components of the toxin constrictory effects.

Nicardipine diminished equinatoxin II-induced decrease of coronary flow in isolated rat and pig hearts.

Equinatoxin II (EqT II) is a basic, cardiotoxic polypeptide. The vasoconstrictory effect of the toxin on isolated porcine coronary arteries was diminished by nicardipine, an L-type calcium channel antagonist. A comparison was made of the effects of EqT II alone and EqT II in the presence of nicardipine on the coronary flow in porcine and rat hearts isolated according to Langendorff's method. In both models EqT II decreased coronary flow in a dose-dependent manner and there were no statistically significant differences between the two models (p>0.05). However, 1 M nicardipine diminished the effects of EqT II on coronary flow in isolated porcine hearts more than in isolated rat hearts (p<0.05). The results suggest that the activation of L-type calcium channels is one of the mechanisms involved in the lowering of coronary flow induced by EqT II.

Effects of mibefradil and verapamil on ischemic-reperfusion in the hearts of guinea pigs with acute renal failure.

Two parameters indicating the ischemic-reperfusion myocardial injury, coronary flow and lactate dehydrogenase (LDH) release rate, were evaluated in guinea pigs with gentamicine-induced acute renal failure (ARF) and compared with those of healthy animals. Isolated Langendorff's hearts were exposed to 50 min of zero-flow global ischemia and 60 min of reperfusion. The influences of calcium channel antagonists (of T- and L-type antagonist mibefradil and of L-type antagonist verapamil) in reperfusion solution were evaluated. Our results showed coronary dilatation and higher LDH release rate in ARF than in control hearts before ischemia. Recovery of coronary flow during reperfusion was better and LDH release rate lower in ARF vs. control hearts. Perfusion with mibefradil and verapamil did not additionally increase coronary flow, however 0.1 µM mibefradil and verapamil decreased LDH release rate during reperfusion in ARF hearts in comparison to control hearts. Our results showed some protective effects of 0.1 µM mibefradil and verapamil on LDH release rate during reperfusion, but not on coronary flow in guinea pigs hearts with ARF.

Lacidipine decreases the honeybee venom-induced vasoconstriction of the isolated porcine coronary artery.

The venom of the honeybee Apis mellifera induces cardiovascular dysfunction. As its effects on coronary arteries have not yet been described, we studied the effects of the whole honeybee venom (non-volatile part) in the isolated porcine left anterior descending coronary artery (LAD) and the influence of L-type Ca2+ channel blocker, lacidipine, upon the venom effects in LAD. The venom produced concentration dependent contractions (7 - 70 µg/ml) of the porcine LAD; maximal effect of the venom was approximately the same as the effect of 30 mM KCl. Lacidipine concentration dependently (0.1-10 µM) and significantly (P ≤ 0.05) decreased the venom-induced vasoconstriction. The results indicate the involvement of L-type Ca2+ channels in coronary contraction, induced by bee venom.

Nicardipine dose-dependently reduces the effect of equinatoxin II on coronary flow in isolated porcine heart.

Death after i.v. administration of equinatoxin II (EqT II) has been attributed to the circulatory failure resulting from cardiotoxic effects. The mechanism of action is unknown. The aim of the present work was to study the effects of the toxin on vascular tone in the isolated porcine coronary artery and on coronary flow in the isolated pig heart. EqT II caused concentration-dependent contractions of rings of the isolated epicardial porcine coronary artery with an EC50 value of 89±5 nM (n=5-6) and maximal effect of about 140% of the contraction induced by 20 nM KCl. On Langendorff's porcine heart preparation EqT II caused a dose-dependent decrease of coronary flow. At EqT II doses lower than 0.05 µmol/100 g of heart weight there were no measurable effects of the toxin. At dose 0.5 µmol/100 g the toxin decreased coronary flow to less than 9.8±2.5 % of the control value. The constrictory effect of the toxin on isolated porcine coronary arteries was diminished by the L-type calcium channel antagonist nicardipine (NC). NC in 1 µM concentration almost completely abolished the effect of EqT II on coronary flow. Our results confirmed involvement of L-type calcium channels in the vasoconstrictory effects of EqT II on epicardial coronary arteries.

Action of mibefradil and lacidipine on the isolated human anterior tibial artery.

In isolated human anterior tibial artery the effects of two different types of calcium channel antagonists, mibefradil (a selective T-type Ca2+ channel antagonist) and lacidipine (a 1,4 dihydropyridine Ca2+ channel antagonist, acting at L- and T-type, but binds preferentially at L-type Ca2+ channels) were compared. Both drugs reduced the contractions of isolated arteries induced by 60 mM KCl. The potency (IC50) of mibefradil was 6.5 µM and of lacidipine 82.4 nM. The potencies of both Ca2+ channel antagonists differed significantly (p<0.001 at 0.1 and 1 µM; p<0.01 at 10 µM). Lacidipine was 79-times more effective than mibefradil in reducing the vasoconstriction in isolated human anterior tibial artery. One of the reasons for higher potency of lacidipine could be a higher density of L-type than of T-type Ca2+ channels in tissue of the human anterior tibial artery.

Thermal conductivity of the porcine heart tissue.

The porcine heart was used as a model for studying the thermal changes in myocardium at cooling and re-warming during open heart surgery. A section of the heart septum was excised and tissue was cut into two similar square slices. The same shape of the tissue, cut from the surface from the upper lateral wall of the left ventricle, covered with epicardium and fat, was taken for another measurement. A thin (<0.5 mm) square thermal source of the same length of the side as the tissue samples was put between the two slices of tissue. This set was placed in the middle of two identical copper cylinders (2r=50 mm, height=55 mm) used to keep the outer side of the specimen at controlled room temperature. Thermal conductivity of the heart tissue was determined at controlled thermal power, and known difference of the temperature at the edge of the tissue and at the middle of the heater, when steady state was reached. Thermal conductivity calculated from the temperature difference and the geometry of heater and samples was 0.75 W/m.K for septal heart tissue, and 0.60 W/m.K for the lateral wall ventricle tissue with epicardium and fat.

Comparison of effects of nitrendipine, lacidipine and mibefradil on postischaemic myocardial damage in isolated rat hearts.

During ischaemia and reperfusion increased cytosolic Ca2+ is one of the important causes for ischaemic-reperfusion myocardial injury. In the present study we compared effects of preferentially L-type Ca2+ antagonists nitrendipine (NT) and lacidipine (LP), and of mibefradil (MB) a Ca2+ antagonist with higher affinity to T- than to L-type channels on myocardial function during reperfusion. Coronary flow (CF), heart rate (HR), left ventricular pressure (LVP), lactate dehydrogenase (LDH) release rate and ECG were registered during 40 min of reperfusion following 30 min of global zero flow ischaemia in Langendorff's isolated rat hearts. Either NT (100 nmol/L) or LP (10 nmol/L) or MB (100 nmol/L) was added to Krebs-Henseleit solution 10 min before ischaemia till the end of experiments. All three drugs influenced CF, HR and LVP. All of them decreased LDH release rate (P < 0.05, in µkat/g.min) when compared with control hearts (53.2 ± 5.1): MB (19.4 ± 4.3) > LP (30.7 ± 6.6) > NT (43.3 ± 2.8). NT reduced the duration of continuous arrhythmias at the beginning of reperfusion (to 59.1 ± 6.1 % of ischaemic controls) as well as the number of single arrhythmic events arising during the whole period of reperfusion (to 26.1 ± 6.0 % of ischaemic controls). MB diminished only single arrhythmic events during reperfusion to 39.1 ± 17.3 % of ischaemic controls. LP did not affect the onset of arrhythmias. Results of our experiments indicate a relatively greater importance of T-type than of L-type Ca2+ channels in the arising of postischaemic myocardial damage.

Lowering of the coronary flow in isolated rat heart by equinatoxin II depends upon extracellular Ca2+ concentration.

Equinatoxin II (EqT II) is a basic polypeptide toxin from the sea anemone Actinia equina (L.). Its LD50 in mice is 33 g/kg. The cause of death after intravenous application has been attributed to the circulatory failure resulting in the cardiotoxic effects. In Langendorff's rat and guinea-pig heart preparations EqT II caused dose dependent decrease in the coronary flow (CF). Morphologic changes of different cell cultures incubated with EqT II are the result of Ca2+ entry through the newly formed discrete pores. Pores in the cell membranes are composed of the toxin oligomeres. In the present study we tried to evaluate the dependence of vasoconstrictor effects of EqT II on isolated rat hearts upon the Ca2+ concentration in the perfusion solution. EqT II did not affect the CF in the group without Ca2+. The strongest effect was observed in the group with 1.5 mM Ca2+ where the CF decreased to 7.7±7 %. The results of our experiments indicate that the effects of EqT II on CF depend on Ca2+ concentration in the extracellular solution.