Effect of beta adrenergic receptor agonists and blockaders on the action of bradykinin

A study was made of the effect of beta-adrenomimetics (isoprenaline, orciprenaline, inoline) and beta-adrenoblockers (propranolol, pindolol, oxprenolol, atenolol and practolol) on changes in the tone of smooth muscles of an isolate ileum of guinea-pigs, increase in microvascular permeability and depressor reaction in rats induced by bradykinin. beta-Adrenomimetics decreased spasmogenic and microcirculatory effects of bradykinin. Depending on the selectivity and presence of partial agonistic activity, beta-adrenoblockers exerted different influence on changes in the tone of extravasal muscles and permeability induced by bradykinin. In doses of 0.1, 0.5 and 1 mg/kg (intravenously) beta-adrenoblockers potentiated and prolonged the depressor effect of bradykinin.     

Two different tetrodotoxin-separable inward sodium currents in the membrane of isolated cardiomyocytes

Isolated ventricular myocytes of 3 to 5 weeks old rats were studied under conditions of intracellular perfusion and voltage clamp. The existence of two inward sodium currents with different TTX-sensitivities and different properties was shown. The fast sodium current was more sensitive to TTX (Kd about 8 X 10(-8) mol/l). The block of the slow sodium current by TTX was less specific (Kd about 7 X 10(-6) mol/l). There was an about four fold difference in the inactivation time constants between these currents. The maximum on the I-V curve of the slow sodium current was shifted along the voltage axis by about 15 mV in the positive direction as compared with that of the fast sodium current. A slow current carried by calcium ions was observed in sodium-free solution. The kinetics and TTX-sensitivity of this current were similar to those of the slow sodium current. The amplitude of this current was 15 to 20 times lower as compared with the slow sodium current observed in Na-containing solution with 10(-6) mol/l TTX (a concentration which completely blocked the fast sodium current). It is suggested that the slow voltage-gated TTX-sensitive channels described are not highly selective and pass both sodium and calcium ions.     

Effect of forskolin on action potential, slow inward current and tension of frog atrial fibers

The new nonhormonal activator of adenylate cyclase forskolin was studied on frog atrial trabeculae by current clamp and voltage clamp methods using a double sucrose gap technique. Forskolin (5 X 10(-6) M to 2 X 10(-5) M) dose-dependently increased action potential duration, the height of the plateau and twitch tension. The time constant for inactivation of the slow inward current and the steady state kinetic variables of calcium channels d infinity and f infinity remained uneffected. Forskolin increased the amplitude of slow inward calcium current isi and of the phasic tension related to it. The maximal conductance gsi increased. These effects were indistinguishable from those obtained earlier on cardiac fibers with hormonal and nonhormonal activators of cyclic AMP-dependent phosphorylation. The beta-adrenoreceptor antagonist propranolol 10(-6)M did not decrease the effect of forskolin. Forskolin had no effect when slow inward current was previously increased by saturating concentrations of the beta-adrenergic agonist isoproterenol (10(-4)M). Our results are in favour of the hypothesis that cyclic AMP-dependent phosphorylation of membrane proteins modulates the Ca-entry in the heart cells through the membrane slow calcium channels.     

Effect of beta-adrenoblockaders on the smooth muscles of the trachea and bronchi

It has been shown in experiments on anesthetized guinea-pigs that (+/-) and (+) propranolol produced a dose-dependent increase in the resistance of the air ways. Meanwhile oxprenolol, trimepranol and atenolol had a poor bronchoconstrictor effect, and labetalol evoked no changes in the tone of the bronchi. On an isolated trachea of the guinea-pig both forms of propranolol, as well as trimepranol and oxprenolol produced contractions, atenolol did not cause any changes in the tone of the smooth muscles, while labetalol made the smooth muscles relax. It has been also demonstrated on an isolated trachea that pretreatment with atropine, diphenhydramine, and diethylamide of lysergic acid did not lead to any noticeable changes in the bronchoconstrictive action of propranolol. At the same time verapamil and Ca2+-free Krebs solution reversed the propranolol effect.     

Heterogeneity among beta-adrenoreceptor blockers in the modulation of energy-dependent uptake of the organic cation amantadine by rat renal tubules.

Eight representative beta-adrenoreceptor blocking drugs, acebutolol, atenolol, labetalol, metoprolol, nadolol, pindolol, propranolol, and timolol, were studied in vitro with respect to their potential to block energy-dependent uptake of [3H]amantadine into proximal and distal rat renal tubule fragments in the presence and absence of bicarbonate. Five of the eight beta-adrenoreceptor blockers showed a dose-dependent inhibition of renal tubule accumulation of amantadine: labetalol, metoprolol, pindolol, propranolol, and timolol. Labetalol was the only beta-adrenoreceptor blocker with greater inhibitory potency in phosphate-based buffer than in bicarbonate-based buffer. Propranolol was the most potent inhibitor of renal tubule amantadine accumulation with IC50 values of 15 +/- 10 and 31 +/- 11 microM for proximal and distal tubule fragments, respectively, in a bicarbonate-based buffer environment. Inhibition in a phosphate-based buffer was less potent only in proximal tubules, with an IC50 of 76 +/- 30 microM. Kinetic studies of propranolol inhibition of amantadine uptake were consistent with both uncompetitive and competitive inhibition mechanisms in bicarbonate-based buffer in both proximal and distal renal tubule segments. There was no chiral preference between the R and S forms of propranolol for the inhibitory effects observed. These data suggest that there is potential for selection among the beta-adrenoreceptor blocking drugs to minimize or restrict the inhibition of amantadine energy-dependent uptake at the organic cation ion uptake sites characterized by amantadine in the presence and absence of bicarbonate.     

A new mechanism of action of vasopressin on the neuronal membrane]

It is shown in this work that vasopressin at the concentrations of 1 x 10(-16) to 1 x 10(-6) mol/l decreased statistically significant the amplitude of the electrosensitive sodium and calcium ionic currents of the mollusc's Lymnaea stagnalis neuronal membrane. This peptide increased the amplitude of the fast potassium current at the concentration of 1 x 10(-16) and 1 x 10(-15) mol/l. It decreased the fast potassium current and did not change the slow potassium current at the concentrations more than 1 x 10(-9) mol/l.     

Calcium modulation of inositol 1,4,5-trisphosphate-induced calcium release from neuroblastoma x glioma hybrid (NG108-15) microsomes

Subcellular fractions of neuroblastoma x glioma (NG108-15) hybrid cells were used to study the mechanism of inositol 1,4,5-trisphosphate-induced calcium release. A microsomal fraction, enriched in endoplasmic reticulum and plasma membranes and almost devoid of mitochondria, was the most active in inositol trisphosphate- or GTP-dependent release of calcium. Neither GTP nor inositol 1,4,5-trisphosphate affected the calcium efflux mediated by the other reagent, suggesting that inositol trisphosphate and GTP act on different calcium-sequestrating vesicles. The stimulation of calcium release by GTP was relatively slow (t1/2 = 90 s), dependent on polyethyleneglycol, and greater at 2 X 10(-5) M calcium (5 nmol X min-1 X mg-1) than at 10(-6) M calcium (0.8 nmol X min-1 X mg-1). The inositol trisphosphate-induced calcium efflux was not mimicked by inositol monophosphate; it was fast (t1/2 less than 10 s) and unaffected by 3% polyethyleneglycol. The amount of calcium released by inositol trisphosphate was greatest at 10(-6) M external calcium (1 nmol X min-1 X mg-1) and it was undetectable at 2 X 10(-5) M calcium. A feedback inhibition of the inositol trisphosphate-induced calcium release by cytoplasmic calcium provides a safety mechanism preventing deleterious effects of abnormally high calcium levels.     

Effect of nonachlazine on transmembrane ionic currents in the frog atrial trabeculae

The effect of the antianginal drug nonachlazine displaying antiarrhythmic properties on transmembrane ionic currents in the frog atrial fibers was studied in experiments on isolated trabeculae of the frog atria. The transmembrane ionic currents were measured by a voltage clamp technique based on a double sucrose gap arrangement. Nonachlazine (1.03 X 10(-5) mol/l) decreased the amplitude of the fast inward current whatever the magnitude of membrane potential. The drug inhibited the slow inward current and prevented the adrenaline-increased permeability of the slow sodium-calcium channel if external sodium ions were replaced by choline chloride. Nonachlazine (1.03 X 10(-5) mol/l) diminished the amplitude of the inward ionic current in a calcium-free medium as well. The stimulatory effect of prostacycline (2 X 10(-7) mol/l) on the fast inward ionic current was inhibited by nonachlazine. The data obtained suggest that the antiarrhythmic effect of nonachlazine might be linked with the inhibition of the fast sodium inward current and the slow calcium inward current.     

An insulin-stimulated cation channel in skeletal muscle. Inhibition by calcium causes oscillation.

A cation channel has been identified in the plasma membrane of skeletal muscle that oscillates open and closed in a regular manner. In an experimental system of patch-clamped reconstituted plasma membrane in phospholipid bilayers, the oscillations are calcium-dependent and constitute regular closing events due to inhibition of the channel by calcium with a Ki of 2.2 +/- 1 x 10(-6) M, followed by reopening. There are 3.7 +/- 1 calcium binding sites/channel. With sodium as the current vehicle, conductance is increased by voltage, insulin (Km = 5 +/- 0.6 x 10(-9) M), and hydrolyzable guanine nucleotides. Cyclic GMP alone with increase the conductance with a Km of 3.7 +/- 0.6 x 10(-7) M. In the absence of calcium, the unitary conductance with insulin + GTP or cGMP at 150 mM NaCl is 153 picosiemens. Sodium current is insensitive to 10(-5) M tetrodotoxin but inhibited by mu-conotoxin (Ki = 5 x 10(-8) M). These findings in the reconstituted system were verified in patch-clamped whole muscle cells where an insulin and cGMP-dependent sodium current inhibited by mu-conotoxin could be demonstrated. In the whole cell experiments, slow calcium-dependent oscillations of the sodium current were also detected.     

Ca2+-antagonistic properties of phospholipase A2 inhibitors, mepacrine and chloroquine

The effects of putative phospholipase A2 inhibitors mepacrine and chloroquine on membrane ionic currents were studied in intact frog atrial trabeculae. Both agents decreased slow calcium channel current Isi and fast sodium channel current If. Isi was affected twice at least in comparison to If. Half-block of Isi was observed at approximately 10(-6) mol/l mepacrine and at approximately 10(-5) mol/l chloroquine. These effects on transmembrane ionic transport should be considered when using the above agents as phospholipase inhibitors or antiarrhythmic drugs.     

Experimental study of the interaction of nonsteroidal anti-inflammatory agents--voltaren and acetylsalicylic acid--with beta-adrenoblockers

It has been demonstrated in rat experiments that the beta-adrenoblockers propranolol and pindolol differ in the influence on the therapeutic and toxic effects of voltaren and acetylsalicylic acid. Propranolol has an analgetic action of its own, reducing the analgetic and antiinflammatory effects of voltaren and acetylsalicylic acid. It potentiates the antipyretic effect of voltaren and ulcerogenic action of both nonsteroidal antiinflammatory drugs. Pindolol exerts both analgetic and antiinflammatory action and does not affect the antipyretic effect of voltaren and ulcerogenic action of nonsteroidal antiinflammatory drugs. The difference in the action of the beta-adrenoblockers under study is likely to be linked with the characteristics of their pharmacological action spectrum.     

Effect of beta-adrenergic antagonists on bioluminescence control in three species of brittlestars (Echinodermata: Ophiuroidea)

The role of adrenaline in the nervous control of bioluminescence in three brittlestar species, Amphiura filiformis, Amphipholis squamata, and Ophiopsila aranea, was assessed by testing two different beta-adrenergic antagonists (propranolol and labetalol) over a wide concentration range (10(-10)-10(-3)M). We compared the effects of analogues (active vs. inactive) of the same substance (L- and D-enantiomers of propranolol). Propranolol presented both specific and nonspecific effects: (i) nonspecific effects were observed at the higher concentrations tested (10(-4) and 10(-3)M) in all three species; (ii) specific effects were detected only at the lower concentrations tested (10(-6)-10(-5)M). In A. squamata, the involvement of adrenaline in the nervous control of luminescence is supported by propranolol and labetolol specific inhibition. The neuropharmacological implications of nonspecific effects, the involvement of adrenaline and the interspecific differences in the brittlestar nervous control of bioluminescence are discussed.     

Effect of beta-blockers on sodium and potassium content of human erythrocytes.

Recently, hyperpotassemia was reported in patients treated with propranolol, but the mechanism has not yet been delineated. We have investigated the effects of various beta-adrenergic blockers and of D-propranolol on the Na+ and K+ distribution intra- and extracellularly in human erythrocytes. K+ loss and Na+ gain by cells was demonstrated at drug concentrations of 10(-4) M or greater. D-Propranolol was more effective than L-propranolol, whereas pindolol was ineffective. Practolol increased Na+ content but did not influence K+. The results suggest that electrolyte redistribution across cell membranes is not a likely explanation for hyperpotassemia in patients treated with propranolol, or for the local anaesthetic effect of this drug.     

Effects of ionophore A23187 on calcium fluxes from cultured adrenal cells

The effect of the calcium ionophore A23128 on calcium fluxes from Y-1 adrenal cortical cells was investigated. Conditions were chosen which are known to result in an inhibition of steroidogenesis (6 . 10(-6) M ionophore and 3 . 10(-4) M extracellular calcium). Calcium efflux from Y-1 cells exhibited two distinct phases. A fast phase which was insensitive to the mitochondrial poison sodium azide and a slow, azide-sensitive phase. The ionophore brought about a rapid increase in the rate of calcium efflux and an 84% reduction in the size of the calcium pool which was associated with the slow efflux phase as well as a reduction in its rate constant. A decrease in the size of the rapidly exchanging calcium pool was also detected. Ethanol, the solvent which was used for the ionophore, slightly increased the rate constant of the rapidly exchanging pool. Conditions which resulted in diminished steroidogenic capacity also brought about a reduction in the size of an energy dependent, intracellular pool. The data is interpreted as being consistent with a hypothesis that the ionophore-induced inhibition of steroidogenesis may be causatively related to the loss of intracellular calcium or to the mechanism which brings about the loss.     

Lipophilic beta-blockers inhibit monocyte and endothelial cell-mediated modification of low density lipoproteins.

The effects of propranolol, pindolol and metoprolol on the modification of low density lipoprotein (LDL) by U937 monocyte-like cells, endothelial cells and copper ions were studied by determination of the lipid peroxidation product content and measurement of the relative electrophoretic mobility of the particle. Propranolol and pindolol inhibited LDL oxidation by U937 cells in a dose-dependent manner from 10 to 100 microM, whereas metoprolol had no effect. In the case of LDL modification by endothelial cells, all the three beta-blockers were efficient within the same range of concentrations, and the order of potency was propranolol greater than pindolol greater than metoprolol. In vitro oxidation of LDL in the presence of copper ions was also inhibited by propranolol; pindolol and metoprolol had no significant protective effect in this system. These results concerning the inhibitory action of beta-blockers were confirmed by testing the degradation of modified LDL by J774 macrophages. Although the concentrations of the drugs utilized in this study are relatively high, in long-term treatment beta-blockers might accumulate in target tissues, and the protective effect of propranolol against LDL oxidation might be involved in its inhibitory action on atherosclerosis previously reported in animal models.     

Direct characterization of beta-adrenoceptors in membranes of immature red blood cells from rats.

By means of the radioactive antagonist ligand (3H)(-) dihydroalprenolol (DHAP) specific binding sites were identified in membrane preparations from red blood cells from rats. These specific sites were characterized as beta-adrenoceptors because of the following reasons: Specific binding of DHAP (in contrast to unspecific binding) was dependent on temperature and time of incubation. Furthermore, specific binding of DHAP showed saturability, temperature-dependent reversibility and high affinity (KD-value of DHAP = 6.51 nM). Specific binding of DHAP was competitively inhibited by beta-adrenergic antagonists (pindolol greater than alprenolol greater than or equal to propranolol greater than practolol) and agonists (isoprenaline greater than adrenaline). The (-) enantiomers of pindolol and isoprenaline showed pronounced higher affinities for the receptor sites than the respective (+) enantiomers. The receptor density in the membrane preparations (pmoles/mg protein) was strongly dependent on the degree of reticulocytosis: The Bmax-values increased more than 4 to 5 fold without alteration of the respective KD-values when reticulocyte counts were enhanced from 3 to 80% treatment of the animals with increasing doses of acetyl phenylhydrazine.     

Ionic currents in dispersed chemoreceptor cells of the mammalian carotid body

Ionic currents of enzymatically dispersed type I and type II cells of the carotid body have been studied using the whole cell variant of the patch-clamp technique. Type II cells only have a tiny, slowly activating outward potassium current. By contrast, in every type I chemoreceptor cell studied we found (a) sodium, (b) calcium, and (c) potassium currents. (a) The sodium current has a fast activation time course and an activation threshold at approximately -40 mV. At all voltages inactivation follows a single exponential time course. The time constant of inactivation is 0.67 ms at 0 mV. Half steady state inactivation occurs at a membrane potential of approximately -50 mV. (b) The calcium current is almost totally abolished when most of the external calcium is replaced by magnesium. The activation threshold of this current is at approximately -40 mV and at 0 mV it reaches a peak amplitude in 6-8 ms. The calcium current inactivates very slowly and only decreases to 27% of the maximal value at the end of 300-ms pulses to 40 mV. The calcium current was about two times larger when barium ions were used as charge carriers instead of calcium ions. Barium ions also shifted 15-20 mV toward negative voltages the conductance vs. voltage curve. Deactivation kinetics of the calcium current follows a biphasic time course well fitted by the sum of two exponentials. At -80 mV the slow component has a time constant of 1.3 +/- 0.4 ms whereas the fast component, with an amplitude about 20 times larger than the slow component, has a time constant of 0.16 +/- 0.03 ms. These results suggest that type I cells have predominantly fast deactivating calcium channels. The slow component of the tails may represent the activity of a small population of slowly deactivating calcium channels, although other possibilities are considered. (c) Potassium current seems to be mainly due to the activity of voltage-dependent potassium channels, but a small percentage of calcium-activated channels may also exist. This current activates slowly, reaches a peak amplitude in 5-10 ms, and thereafter slowly inactivates. Inactivation is almost complete in 250-300 ms. The potassium current is reversibly blocked by tetraethylammonium. Under current-clamp conditions type I cells can spontaneously fire large action potentials. These results indicate that type I cells are excitable and have a variety of ionic conductances. We suggest a possible participation of these conductances in chemoreception.     

Three types of slow inward currents as distinguished by melittin in 3-day-old embryonic heart

Membrane slow inward currents of 3-day-old embryonic chick single heart cells were investigated using the whole-cell patch clamp technique. In a solution containing only Na+ ions and in the presence of tetrodotoxin and Mn2+, the inward current-voltage relationship presented two maxima, confirming the existence of two different voltage-dependent slow inward currents. The first type, a fast transient slow inward current (Isi (ft], was activated from a holding potential of -80 mV and showed fast activation and inactivation. This current was highly sensitive to melittin (10(-8) M) and insensitive to low concentrations of desmethoxyverapamil [-)D888, 10(-9)-10(-6) M). Depolarizing voltage steps from a holding a potential of -50 mV activated two components of the slow inward current, i.e., a slow and a sustained current (Isi(sts] that showed a slow inactivation followed by a slow inactivation and a sustained component. Melittin at a high concentration (10(-4)M) completely blocked the slow transient component (Isi(st] and left unblocked the sustained component (Isi(s]. Both components (Isi(st) and Isi(s] were blocked by verapamil (10(-5)M) and low concentrations of (-)D888 (10(-8)-10(-6)M).     

Role of chemical structure in stereoselective recognition of beta-blockers by cyclodextrins in capillary zone electrophoresis

Most of the β-blocking drugs for treating diseases of the cardiovascular system are chiral aryloxy–propanolamine derivatives. Tipically, the S(−) enantiomers are more active than the R(+) enantiomers. Only some of them (for example timolol) are used as single enantiomers, the others are employed as racemates. For the determination of the enantiomeric purity of timolol European Pharmacopoeia prescribes an HPLC method using chiral stationary phase. However, the use of chiral capillary zone electrophoresis for the determination of the enantiomeric purity is of pharmaceutical interest. This study describes the application of various cyclodextrin derivatives, hydroxypropyl-β-cyclodextrin, randomly methylated β-cyclodextrin, sulphated β-cyclodextrin and sulphated -cyclodextrin for the stereoselective analyses of β-blockers. Baseline separation was obtained for bopindolol, carvedilol, mepindolol, pindolol and alprenolol, while only partial separation was observed for sotalol, propranolol, oxprenolol, atenolol, bisoprolol, bupranolol, and metoprolol. The uneven molecular recognition of the enantiomers of the β-blockers, especially of the optical isomers of labetalol and nadolol, showed the importance of the chemical nature of the separators and the analytes.     

Isolated rat cardiac myocytes as an experimental model to study calcium overload: the effect of calcium-entry blockers

Calcium overload and the effect of a series of calcium-entry blockers were studied in isolated adult cardiac myocytes from the rat challenged with veratrine. The isolation procedure resulted in a high yield of individual rod shaped, calcium tolerant myocytes. After incubation with veratrine, an alkaloid which induces both sodium and calcium influx, 93% of the myocytes became calcium intolerant: the quiescent rod shaped cells vigorously contracted after 30 sec of contact with veratrine and contracture (round cells) ensued within 1 min. Exposure for 30 min to various doses of calcium-entry blockers prior to veratrine addition resulted in the prevention of contracture, the degree of protection depending on the type and the concentration of calcium-entry blocker. Among the different calcium-entry blockers tested, the diarylalkylpiperazines lidoflazine, cinnarizine and flunarizine were protective from the 10(-7) M concentration onwards. Nicardipine was protective at the 10(-6) M and 10(-5) M concentrations, verapamil at 10(-5)M only while other blockers of the "slow channel" type (diltiazem and nifedipine) were not protective in the concentration range tested. This study shows that isolated myocytes represent a valid model for pharmacological investigations. The results with the calcium-entry blockers stress the heterogeneity of the different series of calcium-entry blockers.