Agonist-induced desensitization of beta-adrenoceptor function in humans. Subtype-selective reduction in beta 1- or beta 2-adrenoceptor-mediated physiological effects by xamoterol or procaterol.

We investigated the effects of beta 2- (procaterol 2 x 50 micrograms/day for 9 days) and beta 1- (xamoterol 2 x 200 mg/day for 14 days) adrenoceptor agonists on lymphocyte beta 2-adrenoceptor density and beta 1- and beta 2-adrenoceptor in vivo function (assessed as isoprenaline-infusion-evoked hemodynamic effects and exercise-induced tachycardia) in healthy volunteers. Procaterol decreased lymphocyte beta 2-adrenoceptor density and all beta 2-adrenoceptor-mediated in vivo effects but did not affect beta 1-adrenoceptor-mediated in vivo effects. In contrast, xamoterol neither affected lymphocyte beta 2-adrenoceptors nor beta 2-adrenoceptor-mediated in vivo effects but decreased beta 1-adrenoceptor-mediated in vivo effects. It is concluded that in humans, generally long-term application of beta 1- or beta 2-adrenoceptor agonists causes desensitization of beta-adrenoceptor function but in a beta-adrenoceptor subtype-selective fashion.

Antiarrhythmic activity of alpha-tocopherol nicotinate and related compounds and their physico-chemical properties.

The effect of alpha-tocopherol nicotinate (Renascin), alpha-tocopherol and dodecanoic acid (lauric acid) on the positive inotropic action of ouabain and digoxin and on cardiac glycoside induced arrhythmias has been tested in isolated guinea-pig left atria and in anaesthetized guinea-pigs. alpha-Tocopherol nicotinate and dodecanoic acid significantly decrease the positive inotropic action of digoxin, but not that of ouabain in isolated guinea-pig atria. Ouabain and digoxin induced arrhythmias are suppressed by the three compounds in isolated guinea-pig atria and in anaesthetized guinea-pigs: alpha-tocopherol nicotinate has the highest antiarrhythmic activity followed by dodecanoic acid and alpha-tocopherol. These results are further evidence for the dissociation between the positive inotropic and arrhythmogenic action of cardiac glycosides. The antiarrhythmic activity of the three compounds and the physico-chemical properties, as measured in liposomes, are compared with that of quinidine and aprindine. As nicotinic acid does not show any effect on cardiac glycoside induced arrhythmias, the ester linkage in alpha-tocopherol nicotinate seems to be of particular importance for its antiarrhythmic activity.

[Bupivacaine and mepivacaine on artificial lipid membranes. A comparison of physicochemical parameters]. / Bupivacain und Mepivacain an künstlichen Lipidmembranen. Ein Vergleich physiko-chemischer Parameter.

Mepivacaine and bupivacaine, frequently used local anesthetics, differ in a variety of clinical effects but show only small differences in chemical structure. In order to learn more about molecular mechanisms of action, we investigated the effects of these two local anesthetics on artificial membranes and liposomes. Methods. Artificial membranes were formed from dipalmitoylphosphatidylcholine - cholesterol or from a phospholipid mixture extracted from rabbit heart to separate into two aqueous phases (100 mM NaCl, 1 mM glycine, pH 7.4). Membrane current and potential were recorded after addition of local anesthetics on one side of the membrane. Electrophoretic mobility of liposomes, formed from phosphatidylcholine and a mixture of phosphatidylcholine with phosphatidylinositol, was measured by laser Doppler techniques. The surface potential (zeta potential) was calculated by Gouy-Chapman theory. Lipid/buffer partition coefficients were determined in phosphatidylcholine liposomes by centrifugation and spectrophotometric measurement of the concentration. Results. Mepivacaine and bupivacaine induce concentration-dependent electrostatic potentials on artificial membranes, resulting from incorporation of the charged drug form. Bupivacaine is more active than mepivacaine. The time of onset is short for mepivacaine and delayed for bupivacaine. The effects are retarded in the presence of glucose and are higher and more rapid for the carbonized form of bupivacaine (CO2 form instead of hydrochloride form). The electrostatic surface potential, measured by electrophoretic liposome mobility, is changed by the local anesthetics.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative studies of cardiodepressant drugs on contraction dynamics and electrophysiological parameters of cardiac tissues at pH 7.4 and 9.

The effects of propranolol, amethocaine (tetracaine), lignocaine (lidocaine), procaine and benzocaine on force of contraction and action potential parameters of guinea-pig left atria and papillary muscles have been investigated at pH 7.4 and 9. All the drugs decreased the force of contraction of both preparations. Their biological activities correlated with their lipid/buffer partition coefficients and with their ability to change the surface potential of liposomes. The sequence of activities is as follows: propranolol greater than amethocaine greater than lignocaine greater than benzocaine greater than procaine. Uncharged benzocaine had no effect on the liposome surface charge. Biological and physicochemical effects obtained at pH 9 were higher than those observed at pH 7.4. Action potential amplitude and Vmax were decreased by all the drugs except benzocaine. At pH 9, the action potential duration was generally shortened, except by amethocaine and procaine; both caused a prolongation of the action potential at 90% repolarization.

Effect of praziquantel on the cardiotonic action of cardiac glycosides.

The effect of praziquantel on isolated left guinea-pig atria has been studied. Praziquantel caused a concentration-dependent increase in the force of contraction of the isolated left atria, an effect which may be due to an increase in Ca++ fluxes. Praziquantel antagonized the cardiotonic effect of cardiac glycosides on guinea-pig atria. This antagonism is neither due to an alteration of Ca++ concentration in the cell, nor to a direct action on Na+, K+-ATPase, nor to inhibition of the binding of cardiac glycosides to heart tissue. This interaction may involve an enhancement of K+ fluxes in cardiac cells under the influence of praziquantel.

Pharmacodynamic specification and membrane stabilising action of a homologous series of silasympathomimetics.

The pharmacodynamic effect of a homologous series of aminomethyldimethyl-(methoxyphenyl)-silane derivatives have been tested on isolated guinea-pig left atria. All molecules increase the force of contraction to different degrees by a partly direct and partly indirect mechanism (measured on atria isolated from reserpine treated guinea-pigs). The positive inotropic effect is reduced by bupranolol. Furthermore the compounds show an antagonistic effect to orciprenaline. The membrane stabilising action is lowest for those compounds showing the highest inotropic effects and vice versa.

Comparative studies of local anesthetic effects on artificial membranes and isolated cardiac tissues.

Drugs with local anesthetic properties like propranolol, tetracaine, lidocaine, procaine, induce concentration dependent potentials in artificial membranes. The size of the drug induced potential correlates with cardiodepressant activity, measured as decrease in force of contraction of guinea-pig atria and papillary muscles. Drugs inducing high potentials like propranolol and tetracaine, show low EC50 values in cardiac tissues. Propranolol in comparison to lidocaine diffuses more rapidly across the lipid membrane as derived from the time dependent decline in membrane potential and from radioactive tracer measurements. After application of a single drug concentration on cardiac tissues a longer time is required for propranolol to reach steady state than for example for lidocaine. Effects are higher at pH 9 than at pH 7 and 7.4, respectively. The results suggest that the site of action of propranolol primarily are cytoplasmic cell structures whereas that of lidocaine and procaine is the outer membrane surface.

The effect of different surface chemical groups on drug binding to liposomes.

The binding of four secondary and tertiary amine drugs with local anesthetic activity (propranolol, tetracaine, lidocaine, procaine) to liposomes containing charged surface groups of different chemical composition has been investigated. Binding is determined by measurement of partition coefficients and of drug induced zeta potential changes of the liposomes. For propranolol 30% of the total amount of drug dissolved in phosphatidylcholine is located as protonated form in the liposome surface. Fifty percent of tetracaine and 13% of procaine contribute to the surface charges. Negative surface charges (phosphatidylserine) facilitate drug binding and drug protonization in the liposome surface. Positive surface charges (hexadecyltrimethylammonium) prevent the protonization of the drugs. Different chemical groups of single negatively charged phospholipids or of electrostatically neutral lipids have no significant effect on drug binding which proves that binding is not influenced by steric and bulky head group configurations. The drugs interact hydrophobically with the lipid phase in such a way that the drug amine protonizes in the presence of the negatively charged phosphate oxygen of the phospholipid. Hexadecanoic acid is located deeper within the liposome surface than other negatively charged phospholipids. Correspondingly the drug action is weaker and drug protonization is prevented.

Drug-induced surface potential changes of lipid vesicles and the role of calcium.

The effects of four different drugs with local anesthetic properties were investigated on the surface potential of phospholipid vesicles composed of electrostatically neutral lipids (phosphatidylcholine), negatively charged lipids (phosphatidylserine) and a mixture of acidic and neutral lipids (soy bean lipids). Propranolol, tetracaine, lidocaine and procaine decrease the negative surface potential of phosphatidylserine and soy bean liposomes and increase that of phosphatidylcholine liposomes. The drugs interact with the liposomes in such a way that the protonated amine groups point towards the polar head groups of the phospholipids and the rest of the molecule is probably incorporated into the hydrophobic core of the lipid bilayer. The same sequence in drug activity normally measured in biological tissues (propranolol greater than tetracaine greater than lidocaine greater than procaine) is found for the surface potential change of the phospholipids. Calcium prevents the binding of the drugs to phosphatidylserine, especially the binding of lidocaine and procaine. Because of its high affinity for negative surface charges, Ca2+ chelates with phosphatidylserine and blocks the incorporation of the drug molecule. Vice versa, when incorporated into the liposomal bilayer, the drug blocks the interaction of calcium. These antagonistic effects are only observed in liposomes made from acidic phospholipids and not in those made from pure electrostatically neutral lipids like phosphatidylcholine.

Effect of pH and different substrates on the electrokinetic properties of (Na+, K+)-ATPase vesicles.

Some biophysical properties of a (Na+, K+)-ATPase preparation from guinea-pig kidney have been analysed. The recently developed technique of laser Doppler spectroscopy was applied to measure particle mobility under electrophoretic conditions. The following results were obtained: 1. magnesium ions at pH 7.3 decrease the mobility of the ATPase containing vesicles by binding to negatively charged surface groups. At pH 3.3 the competitive binding of protons causes a shift of the mobility vs. [Mg2+] curve to higher values of [Mg2+], 2. binding of ATP at pH 7.3 (Kd = 0.9 X 10(-4) M for (mM 1 NaCl, 0.2 KCl, 0.1 MgCl2, 0.1 Tris) was measured as an increase in particle mobility depending also on [Mg2+]. At pH 3.3 also unspecific ATP-binding occurred, 3. ITP and GTP had the same Kd value as ATP; ADP a slightly lower one (Kd = 1.2 X 10(-4) M). Tris-H3PO4 (Kd = 2.6 X 10(-4) M) was also able to increase particle mobility, but only at higher concentrations and not to the same extent as ATP; AMP induced only very small changes, 4. from the mobility-pH curve an isoelectric point of 4.1 is derived (buffer: 1 mM NaCl, 0.2 mM KCl, 0.1 mM MgCl2, 0.1 mM Tris). In the presence of 0.9 mM ATP the isoelectric point is shifted to 3.2. As the electrophoretic mobility is directly proportional to the net charge of the vesicles, the results may be interpreted as changes in surface charge density, originating from both a conformational change of the ATPase polypeptide and a decrease in vesicle size.

Drug-induced zeta potential changes in liposomes studied by laser Doppler spectroscopy.

The technique of laser Doppler spectroscopy is used to measure the electrophoretic mobility of liposomes under the influence of one beta-blocking agent and three local anesthetics. All four drugs decrease the mobility (i.e., the zeta potential) of negatively charged phospholipids (soybean lipids, phosphatidylserine and cardiolipin). The mobility of electrostatically neutral pure phosphatidylcholine (zero mobility under control conditions at pH 7 and 4) is increased linearly with the logarithm of drug concentration, indicating binding and incorporation of positively charged drug molecules. The sequence of strength of activity, measured by zeta-potential changes, corresponds to that found in biological tissues: propranolol greater than tetracaine greater than lidocaine greater than procaine. For purely negatively charged lipids (phosphatidylserine, cardiolipin) the activity of the drug is higher at acidic pH, (pH 4), while for electrostatically neutral (phosphatidylcholine) or partly neutral (soybean) lipid liposomes drug activity is about the same at pH 9, 7 and 4. A Hill plot of the data reveals noncooperative drug binding. From the line width of the scattering power spectrum the mean particle radius and the average interparticle distance in the samples are determined.

Electrokinetic properties of (Na+, K+)-ATPase vesicles as studied by laser Doppler spectroscopy.

The technique of laser Doppler electrophoresis was applied for the study of the surface charge properties of (NA+, K+)-ATPase containing microsomal vesicles derived from guinea-pig kidney. The influence of pH, the screening and binding of uni- and divalent cations and the binding of ATP show: (1) one net negative charge per protein unit with a pK = 3.9; (2) deviation from the Debye relation between surface potential and ionic strength for univalent cations, with no difference in the effect of Na+ and K+; (3) Mg2+ binds with an association constant of Ka = 1.1. 10(2) M-1 while ATP binds with an apparent Ka = 1.1.10(4) M-1 for 1 mM NaCl, 0.2 mM KCI, 0.1 mM MgCl2, 0.1 mM Tris-HCl2, 0.1 mM Tris-HCl (pH 7.3). The binding is weaker at higher Mg2+ concentrations. There is no ATP binding in the absence of Mg2+. In addition, the average vesicle size derived from the linewidth of the quasielastic light scattering spectrum is 203.7 +/- 15.2 nm. In the presence of ATP a reduction in size is observed.

Influence of vanadate on electrophysiological and contractile parameters of atrial myocardium.

Whereas vanadate increases isometric force of contraction in stimulated rat left atria (EC50: 36.6 mumol/1), it decreases force of contraction in guinea-pigs (EC50: 4.7 mumol/1). Action potential duration at 90 and 30% repolarisation is decreased in guinea-pig atria by 70 and 60%, but in rat atria by only 40 and 7% respectively. Whereas the time course of the negative inotropic action is rapid and correlates with that of the shortening of the action potential, there is no relationship between action potential changes and the slowly developing positive intropic effect. Negative and positive inotropic actions seem to be the result of two superimposed processes, whose kinetics suggest an extra- and intracellular site of action. Na+, K+-ATPase prepared from rat and guinea-pig atria and ventricle is inhibited by vanadate to a similar extent: IC50 (mumol/1) rat: atria 1.05, ventricle 0.49; guinea-pig: atria 0.75, ventricle 0.62. This indicates that the discrepancy in inotropy cannot be explained by the different sensitivity of the Na+, K+-ATPase to vanadate.

Effect of angiotensin II on artificial lipid membranes.

(5-Isoleucine)-angiotensin II applied to black lipid membranes produced current fluctuations varying between deltaG=5-10(-11) omega-1 and 3.5-10(-10) omega(-1). These fluctuations depend on the voltage and the hydrostatic pressure. The membrane resistance is lowered by deltaR=6.1-10(7) omega-cm2. With (5-isoleucine, 8-leucine)-angiotensin II the jumps are of a single amplitude (deltaG=2-10(-10) omega(-1)). In both cases water and ions are transported across the membrane.