Cytotoxicity of caffeine in cultured heart cells

The cytotoxic effect of caffeine on myocardial cells was evaluated in primary cultures of rat heart muscle (M) and endothelioid (E) cells. The cells were exposed to 5, 10, or 20 mM caffeine for 6, 12 or 24 h. As an index of cell injury produced by the treatments, in situ changes in lysosomal membrane permeability were measured by a sensitive cytochemicale technique. The highest concentration of caffeine (20 mM) produced such toxic signs as reduced cell viability, increased vacuolization, and pseudopod formation. At the lower concentrations, caffeine labilized lysosomal membranes of M cells more easily than those of E cells. Pretreatment with hydrocortisone prior to exposure to caffeine protected E and M cell lysosomes from the labilizing effects of caffeine.     

Effects of vasoactive drugs on lysosomal stability in vitro

The effects of methylprednisolone, chlorpromazine, phenoxybenzamine and phentolamine on thermolabilized lysosomes were studied by determining the unsedimentable, fragile lysosomal and total lysosomal activities of β-glucuronidase and acid phosphatase in centrifuged fractions. At concentrations of 10^?5-10^?3 M, methylprednisolone stabilized the lysosomes. Chlorpromazine and phenoxybenzamine at 10^?3 M had a labilizing effect. The changes in the enzyme activities due to phentolamine could not be explained as changes in membrane permeability. The glucocorticoids appear to protect the cell membranes directly and thus counteract the adverse effects of circulatory shock. The same mechanism may explain the salutary effects which chlorpromazine at low concentrations reportedly possesses. The improvement of microcirculation in shock after the administration of phenoxybenzamine or phentolamine is due to their α-adrenergic blocking activity; this in turn leads to vasodilatation and indirectly to a membrane protection.     

Local anaesthetics block hyperpolarization-activated inward current in rat small dorsal root ganglion neurones

(1) Hyperpolarizing voltage steps evoke slowly activating inward currents in a variety of neurones and in cardiac cells. This hyperpolarization-activated inward current (I(h)) is thought to play a significant role in cell excitability, firing frequency, or in setting of the resting membrane potential in these cells. We studied the effects of lidocaine, mepivacaine, QX-314 and bupivacaine as well as its enantiomers on I(h) in the membrane of dorsal root ganglion neurones (DRG). (2) The patch-clamp technique was applied to small dorsal root ganglion neurones identified in 200 micro M thin slices of young rat DRGs. Under voltage-clamp conditions, the whole-cell I(h) current was recorded in the presence of different concentrations of the local anaesthetics. In current-clamp mode the resting membrane potential and the voltage response of DRG neurones to injected current pulses were investigated. (3) I(h) was reversibly blocked by bupivacaine, lidocaine and mepivacaine applied externally in clinically relevant concentrations. Concentration-response curves gave half-maximum inhibiting concentrations of 55, 99 and 190 micro M, respectively. Bupivacaine block of the I(h) current was not stereoselective. No significant effect was observed when QX-314 was applied to the external surface of the membrane. (4) In current-clamp experiments 60 micro M bupivacaine slightly hyperpolarized the membrane. The membrane stimulation by low-amplitude current pulses in the presence of bupivacaine showed an increase of the hyperpolarizing responses. (5) Our findings suggest an important role of the I(h)-block by local anaesthetics in the complex mechanism of drug action during epidural and spinal anaesthesia.     

Studies on gentamicin-induced labilization of rat kidney lysosomes in vitro. Possible protection by selenium

The labilizing effect of gentamicin, an aminoglycoside antibiotic, on isolated rat kidney lysosomes was investigated. The light-scattering behavior of lysosomal suspensions and the release of lysosomal acid hydrolase enzymes (acid phosphatase, beta-glucuronidase and muramidase) from incubated lysosomal suspensions, in the presence of gentamicin, were used as indices of lysosomal membrane labilization. Gentamicin was found to cause a decrease in light absorbance and a release of lysosomal acid hydrolases, which indicate lysosomal membrane swelling. In the presence of selenium, in the form of potassium selenate, the decrease in light absorbance of lysosomal suspensions and the release of lysosomal acid hydrolases from isolated lysosome particles were reduced markedly. This suggests that selenium protects against gentamicin-induced lysosomal membrane labilization. The possible mechanisms of protection by selenium are discussed.     

Spin label study of the perturbation effect of the local anaesthetics tetracaine and dibucaine on synaptosomes at pharmacological concentrations

The method of electron spin resonance spectroscopy of spin probes was used to determine the lowest concentrations of the local anaesthetics dibucaine and tetracaine exerting perturbations on synaptosome membranes. The perturbation depends on the temperature and the membrane depth, as well as on the concentration and the structure of the anaesthetics. Using spin labelled stearic acid at the 5th carbon position a negligible effect of the anaesthetics on the order parameter was found in the membrane both at 1 degree and 22 degrees, within the buffer concentration 0.01-10 mmol/l, but at 37 degrees and concentrations higher than 0.1 mmol/l the disordering effect was significant and of comparable efficiency for dibucaine and tetracaine. Employing stearic acid labelled at the 16th carbon position, disordering of the hydrocarbon core of the membrane caused by tetracaine or dibucaine was detected at 1 degree and 22 degrees, as well as at 37 degrees. The disordering effect occurred at buffer concentrations higher than 0.01 mmol/l for dibucaine, and higher than 0.1 mmol/l for tetracaine. At equal anaesthetic membrane concentrations and at the 16th carbon membrane depth, dibucaine was approximately twice as effective as tetracaine in perturbing synaptosomes. Tetracaine induced nonlamellar phases in the rat brain lipid membrane as detected by 31P NMR spectroscopy. The dynamic and structural perturbation effects of the local anaesthetics was found in that concentration range at which the anaesthetics influence various activities of biological membranes.     

The effect of D-penicillamine on the release of acid hydrolases from rat liver lysosomes induced by gamma-hexachlorcyclohexan.

d-Penicillamine administered p.o. at a dose of 800 mg/kg, exerts in vivo a stabilizing effect on lysosomal membrane in rat liver. This was demonstrated in normal rats and in the animals whose lysosomes had been rendered fragile by an intraperitoneal injection of 40 mg/kg gamma-HCH. Under these experimental conditions, D-penicillamine protected rat liver against cell necrosis induced by gamma-HCH and prevented, therefore, the efflux β-glycerophosophatase from the organ to the blood plasma. D-Penicillamine also significantly relieved the in vitro labilizing effect of gamma-HCH upon lysosomal membrane in the lysosome-enriched liver suspension. On the other hand. D-penicillamine did not significantly affect the activities of the three acid hydrolases, β-glycerophosphatase β-glucosidase and cathepsin D, chosen as lysosomal marker enzymes, nor their total activities in rat liver. The possible mechanisms by which D-penicillamine exerts protective action on gamma-HCH-induced alterations of rat liver lysosomes are discussed.     

Local anaesthetics inhibit influx of calcium, sodium and potassium into rat ileum, diaphragm and human isolated saphenous vein

The effects of 5 different local anaesthetics, lignocaine, prilocaine, etidocaine, mepivacaine, bupivacaine, on uptake of Ca2+, Na+ and K+ were studied, using a Kone Microlyte Analyzer, into 3 different types of muscle fibres, the rat ileum, rat diaphragm and human isolated saphenous vein. The aim of the present experiments was to see if local anaesthetics had a calcium antagonistic activity, like that produced by calcium antagonists, e.g. verapamil. The results showed that local anaesthetics in moderate and high concentrations (more than 100 microM) depressed uptake of Ca2+, as well as the Na+ and K+, although the depression of the uptake of Ca2+ was 7 and 2 times less than those of Na+ and K+. In low concentrations (1-10 microM), the local anaesthetics had no or little effect on Ca2+ uptake, although they still significantly reduced Na+ and K+ uptake. It was concluded that the effect of local anaesthetics on uptake of Ca2+, Na+ and K+ into rat ileum, diaphragm and human saphenous vein, is concentration-dependent, and that only at high concentrations do they have a calcium antagonistic activity in muscle. Furthermore, there was no clear evidence that the effect of local anaesthetics was species-dependent, since the uptake of Ca2+ was depressed by only about 17% more in smooth muscle than in skeletal muscle.     

Experimental study on prediction of drug toxicity. Drug-induced hemolysis and lysosomes lysis in vitro]

The effect of drugs on isolated rat liver lysosomes, erythrocytes and some erythrocyte enzymes was studied in vitro. Many tranquilizers, antihistaminics and antidepressants, which are known to have the greatest number of adverse reactions in clinical use, had a lytic effect on the particle membranes at concentrations above 2 X 10(-4) M. The present study revealed a good correlation between the p.o. LD50 (rat and mouse) and H30 (molar concentration of drug causing 30% hemolysis). It is suggested, therefore, that the labilizing effect of drugs on rat erythrocytes is useful for testing the lytic action of drugs in vivo.     

The effects of opioids, local anesthetics and adjuvants on isolated pregnant rat uterine muscles

Local anaesthetics, opioids and adjuvants are often used for managing labor pain. Some others of these agents are reported to cause alterations on uterine contractility during labor. However, there are controversies and the effects of some others are unknown. In the present study, we aimed to elucidate the effects of opioids such as alfentanyl, meperidine, remifentanyl; local anesthetics such as mepivacaine, ropivacaine, bupivacaine; and adjuvants such as clonidine and midazolam on isolated pregnant rat uterine muscle. Strips of longitudinal uterine smooth muscle obtained from rats pregnant for 18-21 days were suspended in 20 ml organ baths. Isometric tension was continuously measured with an isometric force transducer connected to a computer-based data acquisition system. The effects of cumulative concentrations of alfentanyl, meperidine, remifentanyl, mepivacaine, ropivacaine, bupivacaine, clonidine and midazolam (10(-8) - 10(-4) M, for all) on contractions induced by oxytocin (1 mU/ml) were studied. Alfentanyl (10(-5) M), meperidine (10(-5) M), remifentanyl (10(-4) M), bupivacaine (10(-4) M), ropivacaine (10(-4) M) and midazolam (3 x 10(-5) M) caused significant decreases in contractile responses of uterine strips to oxytocin. Contrastingly, mepivacaine increased (33.1% +/- 7.2%) oxytocin-induced contractions of uterine strips while clonidine exerted no significant effect. The sensitivity of myometrial preparations to tested local anesthetics or opioids did not differ significantly. The findings of the present study demonstrated that some local anesthetics, opioids and adjuvants caused significant and agent-specific alterations on contractility of the pregnant rat myometrium. Therefore, they seemed to have a potential to influence uterine contractility during clinical management of pain during labor. However, further research is needed to extrapolate these finding to clinical practice.     

Increasing Membrane Interactions of Local Anaesthetics as Hypothetic Mechanism for Their Cardiotoxicity Enhanced by Myocardial Ischaemia

While myocardial ischaemia enhances the cardiotoxicity of local anaesthetics, the pharmacological background remains unclear. Cardiolipin (CL) localized in mitochondrial membranes is possibly the site of cardiotoxic action of local anaesthetics and peroxynitrite is produced by cardiac ischaemia and reperfusion. We verified the hypothetic mechanism that local anaesthetics may interact with CL‐containing biomembranes to change the membrane biophysical property and their membrane interactions may be increased by peroxynitrite. Biomimetic membranes were prepared with different phospholipids and cholesterol of varying compositions. The membrane preparations were reacted with peroxynitrite of pathologically relevant concentrations and local anaesthetics (bupivacaine and lidocaine) of a cardiotoxic concentration separately or in combination. Changes in membrane fluidity were determined by measuring fluorescence polarization. Peroxynitrite decreased the fluidity of biomimetic membranes at 0.1–10 μM with the relative potency being CL>1‐stearoyl‐2‐arachidonoylphosphatidylcholine>1,2‐dipalmitoylphosphatidylcholine‐constituting membranes, indicating the lipid peroxidation‐induced membrane rigidification determined by the unsaturation degree of membrane lipids. When treated with 0.1–10 μM peroxynitrite, biomimetic membranes were more rigid with elevating the CL content from 0% to 30 mol%, suggesting that CL is a primary target of peroxynitrite. Bupivacaine and lidocaine fluidized at 200 μM biomimetic membranes containing 10 mol% CL and their effects were increased by pre‐treating the membranes with 0.1 and 1 μM peroxynitrite. Cardiotoxic bupivacaine and lidocaine increasingly interact with CL‐containing mitochondria model membranes which are relatively rigidified by peroxynitrite. Such an increasing membrane interaction may be, at least in part, responsible for the local anaesthetic cardiotoxicity enhanced by myocardial ischaemia.     

Effects of local anaesthetics on responses of human saphenous vein and bovine coronary artery to neurotransmitters, acetylcholine, noradrenaline and 5-hydroxytryptamine

The effects of 5 different local anaesthetics, lignocaine, etidocaine, prilocaine, mepivacaine, bupivacaine, and 3 different neurotransmitters, acetylcholine (ACh), noradrenaline (NA), 5-hydroxytryptamine (5-HT) on tone and contractility of human saphenous vein and bovine coronary artery were studied and compared in vitro. The experiments were carried out to see if there were species or tissue differences in the action of local anaesthetics and neurotransmitters at vascular smooth muscle, i.e. saphenous vein and coronary artery. Another important aspect was to see if local anaesthetics modified cholinergic (ACh), adrenergic (NA) and serotoninergic (5-HT) responses in vascular smooth muscle. Among the local anaesthetics studied, lignocaine and etidocaine produced prolonged relaxations, whereas prilocaine, mepivacaine, bupivacaine produced contractions in the saphenous vein and coronary artery. High concentrations of the latter drugs produced relaxations in the blood vessels. Among the local anaesthetics, lignocaine produced differential effects on saphenous vein and coronary artery, its effect on the latter was 3 times larger than on the saphenous vein. ACh, NA and 5-HT contracted the saphenous vein. In the coronary artery, ACh and 5-HT contracted whereas NA relaxed the vessel. On a molar basis, ACh was less effective than NA in contracting the saphenous vein. 5-HT was equipotent on both the saphenous vein and coronary artery. Lignocaine reduced ACh, NA and 5-HT-induced contractions in the saphenous vein and those of ACh and 5-HT in the coronary artery, and shifted the control curves, non-competitively, to the right. The relaxation produced by NA in the coronary artery was enhanced by lignocaine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions Between Salicylic Acid and Pyridy;-3-methanol: Anti-inflammatory and Teratogenic Effects

Anti-inflammatory and teratogenic effects of nicotinic acid, pyridyl-3-methanol and salicylic acid were studied. Tests were also performed on mixtures of salicylic acid and pyridyl-3-methanol and on an ester of pyridyl-3-methanol and salicylic acid (S-2063). Anti-inflammatory effects were studied on carrageenin-in-duced rat paw oedema and stabilizing effects were studied on rat liver lysosomes. Teratological studies were carried out on NMRI mice. Nicotinic acid was found to be very potent in the anti-inflammatory test and in stabilizing rat liver lysosomes. Pyridyl-3-methanol had less marked anti-inflammatory effects and had no significant stabilizing effect on the lysosomes. No foetal damage was caused. Salicylic acid had anti-inflammatory effects that were even less marked than those of pyridyl-3-metha-nol, but had slight stabilizing effects on the lysosomal membranes. Salicylate-induced skeletal malformations were obtained after treatment in early organogenesis. Given during late pregnancy a high incidence of foetal death and premature birth was observed. S-2063 had anti-inflammatory effects that were approximately the same as that of an equimolar mixture of salicylic acid and pyridyl-3-methanol. S-2063 caused a decreased membrane permeability of the lysosomal membranes for β-glu-curonidase but not for acid phosphatases. The teratogenic effect was not much increased as could be expected from the pronounced anti-inflammatory effect of S-2063.     

Effects of local anaesthetics on short-term desensitization of guinea-pig taenia caecum to histamine.

1 Short-term desensitization to histamine was induced by incubating guinea-pig taenia caecum with 10(-4)M histamine for 30 min (desensitizing incubation) in normal Locke-Ringer solution or Ca-free Locke-Ringer solution containing 0.2 mM EGTA. This desensitization was measured as a reduction of the maximal contractile response. 2 The effects of the presence of local anaesthetics during the desensitizing incubation were examined. Results showed that tetracaine, procaine, procainamide, oxybuprocaine and lignocaine inhibited the desensitization, whereas dibucaine, benzocaine and mepivacaine did not. 3 The inhibitory effects of these drugs on the desensitization were not correlated with their lipid solubility nor with the potency of their known effects, such as membrane stabilization, Ca channel blockade, calmodulin antagonism, or inhibition of C-kinase. 4 It is concluded that the inhibitory effects of local anaesthetics on the desensitization are not due to their non-specific membrane-stabilizing effects per se, but to some other action.     

Further studies on the in vivo effect of cephaloridine on the stability of rat kidney lysosomes

The stabilizing effect of cephaloridine, an antibiotic, on rat kidney lysosomal membranes was tested by a single subcutaneous injection. The release of two lyososomal enzymes, acid phosphatase and muramidase, was used as an index of lysosome membrane integrity. The levels of these enzymes in the kidney extracts as well as in the isolated kidney lysosome fractions were found to be raised considerably, compared to the controls. In rats treated with cephaloridine, the supernatant fraction obtained from the kidney homogenates, after centrifugation at 15,000 g_av, contained lower enzyme activities than were found in the control animals. It is suggested that cephaloridine may inhibit the release of acid phosphatase and muramidase from rat kidney lysosomes and, therefore, may exert a stabilizing effect on the lysosomal membrane system. The possible mechanism of interaction of this antibiotic with rat kidney lysosomal membranes is proposed.     

Interaction of local anaesthetics with lipid membranes under inflammatory acidic conditions

The clinical fact that local anaesthetics do not successfully work in the patients with inflammation has been generally interpreted on the basis of inflamed tissue acidification. In order to verify this hypothesis, the interaction of local anaesthetics with lipid membranes was studied by determining the drug-induced changes of membrane physicochemical property (membrane fluidity) at different pH covering inflammatory acidic conditions. At clinically relevant concentrations, lidocaine, procaine, prilocaine and bupivacaine fluidized 1,2-dipalmitoylphosphatidylcholine membranes with the potency decreased with lowering the pH from 7.9 to 5.9. When treated as the aqueous acidic solution (pH 4.0) similar to marketed injection solutions, lidocaine showed more pronounced pH dependence, so the reduction of its membrane-fluidizing effects at acidic pH theoretically correlated to that of its non-ionized membrane-interactive concentrations. Unlike phosphatidylcholine membranes, however, nerve cell model membranes consisting of different phospholipids and cholesterol were fluidized by lidocaine at pH 6.4–6.9 corresponding to the acidity of inflamed tissues. Cationic lidocaine was effective in fluidizing anionic phosphatidylserine and cardiolipin membranes at pH 6.4, but not zwitterionic phospholipid membranes, whereas it was ineffective on any membranes at pH 2.0 where membrane acidic phospholipids were not ionized. Local anaesthetics are considered to form the ion-pairs specifically with counter-ionic phospholipids and act on the membranes of nerve cells even under inflammatory acidic conditions. The drug and membrane interaction causable in inflamed tissue acidification does not support the conventional theory on the local anaesthetic failure associated with inflammation. Received 3 November 2006; revised version accepted 31 January 2007     

Enhancement of delayed-rectifier potassium conductance by low concentrations of local anaesthetics in spinal sensory neurones

Combining the patch-clamp recordings in slice preparation with the 'entire soma isolation' method we studied action of several local anaesthetics on delayed-rectifier K(+) currents in spinal dorsal horn neurones. Bupivacaine, lidocaine and mepivacaine at low concentrations (1 - 100 microM) enhanced delayed-rectifier K(+) current in intact neurones within the spinal cord slice, while exhibiting a partial blocking effect at higher concentrations (>100 microM). In isolated somata 0.1 - 10 microM bupivacaine enhanced delayed-rectifier K(+) current by shifting its steady-state activation characteristic and the voltage-dependence of the activation time constant to more negative potentials by 10 - 20 mV. Detailed analysis has revealed that bupivacaine also increased the maximum delayed-rectifier K(+) conductance by changing the open probability, rather than the unitary conductance, of the channel. It is concluded that local anaesthetics show a dual effect on delayed-rectifier K(+) currents by potentiating them at low concentrations and partially suppressing at high concentrations. The phenomenon observed demonstrated the complex action of local anaesthetics during spinal and epidural anaesthesia, which is not restricted to a suppression of Na(+) conductance only.     

Stabilization of rat liver lysosomes by (+)-cyanidanol-3 in vivo.

( + )-Cyanidanol-3. [(+)-catechin], administered by subcutaneous injection at a dose of 100 to 200 mg/kg/day for 6–20 days, exerts in vivo a stabilizing effect on lysosomal membranes in rat liver. This was demonstrated in normally fed rats and in animals whose lysosomes had been rendered fragile by intoxication with galactosamine and ethanol. Under these circumstances, the discharge of acid phosphatase, β-glucuronidase and β-N-acetylglucosaminidase by the lysosomes was significantly reduced, by 15–20 percent, compared to the controls. Moreover, in vitro, products of the oxidation of (+)-cyanidanol-3 exert a strong stabilizing effect on the lysosomal membrane. Such stabilization of the lysosomes may have a beneficial effect in various hepatic disorders involving abnormal fragility of the lysosomes.     

Studies on the nephrotoxicity of aminoglycoside antibiotics and protection from these effects (4). Effects of tobramycin alone and in combination with latamoxef on the stability of rat kidney lysosomal membranes

Effects of tobramycin (TOB) alone and in combination with latamoxef (LMOX) on the stability of rat kidney lysosomal membranes were investigated. Rats were injected with doses of TOB (90 mg/kg/day, s.c.) alone. LMOX (2,000 mg/kg/day, s.c.) alone or TOB (90 mg/kg/day, s.c.) and LMOX (2,000 mg/kg/day, s.c.) for 5 consecutive days. The rat kidney lysosomes were isolated on the 1st, 3rd and 5th days and incubated in a 0.25 M sucrose solution containing 1 mM EDTA (pH 7.0) at 37 degrees C for 20 min. After incubation, the activity of N-acetyl-beta-D-glucosaminidase (NAG) released from lysosomes was measured, and the percent NAG release was calculated as an index of the stability of lysosomal membranes. The percent releases of NAG from lysosomes of TOB alone-treated rats were 40 and 50% greater than those of normal rats on the 1st and 3rd days, respectively. On the other hand, treatment with TOB and LMOX suppressed the NAG release from lysosomes with TOB alone by about 80 to 100%. There were insignificant slight increases in the percent NAG release in LMOX alone-treated rats on the 3rd and 5th days. In addition, the in vitro study indicated that incubation of the lysosomal fraction from kidneys of normal rats with TOB (30 micrograms/ml) significantly increased the NAG release, compared with that of the non-treated lysosomal fraction. However, the preincubated mixture of TOB (30 micrograms/ml) and LMOX (50 micrograms/ml) in vitro significantly suppressed the release of NAG from lysosomes by 85%. These results suggest that the suppression of the releases of NAG from lysosomes by the combination of TOB with LMOX may contribute to the protective effect of LMOX against TOB nephrotoxicity.     

Effects of mefloquine on the release of marker enzymes from the rat liver crude lysosomal fraction

The effects of the antimalarial agent mefloquine on the release of marker enzymes, acid phosphatase and beta glucuronidase, from the rat liver lysosomes in a crude lysosomal preparation were investigated and compared with that of chloroquine whose membrane effects have been well-documented in the literature. At 10 microM, mefloquine decreased significantly the release of marker enzymes when compared to the control, but at the higher concentrations of 100 microM and 500 microM, it markedly accelerated the release of enzymes. This suggested that mefloquine exerted a concentration-dependent biphasic effect of membrane stabilization and labilization. In comparison, chloroquine diminished the release of enzymes over the concentration range of 10-500 microM, that is showing a membrane stabilizing effect similar to other reports. Since serum levels of 1.6-3.2 microM are reported after a weekly dose of mefloquine, the present results suggest that a predominant stabilization effect should prevail under these conditions. However, higher drug concentrations may result in labilization with undesirable consequences.     

Effect of aflatoxin on rat liver lysosomes

The effect of aflatoxin on the activities of 5 rat liver lysosomal enzymes (acid deoxyribonuclease, arylsulfatases A and B, β-glucuronidase, β-glucosidase and β-galactosidase) has been investigated in vivo and in vitro. Three hours after the administration of aflatoxin, activities of most of the enzymes increased significantly reaching maximal levels at 48 hr. The activity of acid DNase increased the most (276 per cent of the control level). At the same period (48 hr) the activity of the soluble lysosomal enzymes greatly increased. Aflatoxin had a labilizing effect on lysosomal membranes, causing the release of lysosomal enzymes into the supernatant. The data are discussed in connection with the biochemical mechanism of aflatoxin intoxication.