Analysis of the adverse effects of drugs at the cellular and subcellular levels]

A release of histamine after the lipophilic betablockers exaprolol and propranolol correlates with their capability of displacing the bound membrane Ca2+ and increasing the disorder of phospholipidic membranes of the isolated mast cells. Electron microscopy confirmed intracellular displacement of histamine from granules of mast cells after exaprolol without marked structural changes on the plasmatic membrane. Hydrophilic and selective atenolol, which does not possess a histamine-liberating effect, decreases spontaneous transfer of the intracellular calcium, decreases the disorder of the mast-cell membranes, and together with exaprolol and propranolol inhibits, in dose-dependence way, the gain of extracellular histamine in cells. The inhibitory effect of EDTA, tetrodotoxine and suramine on histamine release after exaprolol explains the non-receptor mechanism of exaprolol effect, which confirms a possibility of induction of adverse effects of blockers of the beta-adrenergic receptor in the development of a bronchospasm.     

Effect of chloroquine on isolated mast cells.

Chloroquine liberated a relatively low amount of histamine from isolated rat mast cells. In a dose-dependent way, this drug inhibited histamine liberation from mast cells stimulated with compound 48/80, A23187, concanavalin A plus phosphatidylserine (Con A + PS) and abolished histamine liberation induced by exaprolol. The degranulation was decreased in cells stimulated with 48/80, Con A + PS and exaprolol. Chloroquine significantly inhibited the formation of thromboxane B2 in mast cells stimulated with 48/80, Con A + PS and A23187. We assume that chloroquine interferes with mast cells at a plasmic membrane site as well as intracellularly.     

Evidence for intracellular histamine liberation in isolated rat mast cells

The beta-adrenoceptor blocking drug exaprolol liberated histamine from isolated rat mast cells in a dose- and time-dependent way. Histamine was liberated within seconds and was not followed by a parallel granule liberation. The inhibition of histamine liberation was induced with low temperature, low pH, high concentration of Ca2+, TTD, suramin and EDTA. Subcellular distribution of 3H-exaprolol demonstrated a quantitative relationship between histamine depletion against exaprolol uptake in isolated rat mast cell granules. A nonspecific mechanism of action in the effect of exaprolol on mast cells is discussed. It is proposed that the drug acts on mast cells due to the direct and indirect ion exchange mechanism resulted in disproportion between histamine and granule liberation.     

Atenolol, exaprolol and mast cell membranes.

Lipophilic exaprolol and hydrophilic atenolol differ in their interaction with mast cell membranes. Exaprolol, as compared with atenolol, significantly decreased 32P incorporation into, but increased arachidonic acid liberation from, membrane phospholipids. Moreover, exaprolol significantly decreased phosphate incorporation in compound 48/80 and ConA-PS treated cells and decreased thromboxane formation in stimulated cells. On the other hand, atenolol decreased significantly only arachidonate liberation from stimulated mast cells. These results corroborate to some extent the effect of exaprolol and atenolol on histamine liberation which correlates with their membrane perturbing properties.     

Membrane perturbing activity of beta-adrenoceptor blocking drugs in isolated rat mast cells

Beta-adrenoceptor blocking (BAB) drugs perturb the membranes of isolated rat mast cells. Membrane fluidisation was temperature dependent and was determined by the liposolubility of the BAB drugs. The secretory index, evaluated as the ratio between histamine liberation and degranulation, correlated with the membrane order parameter of the mast cell membranes. The rank order of potency for mast cell activation and membrane fluidisation was: exaprolol greater than propranolol greater than metipranolol greater than atenolol.     

The effect of beta-adrenoceptor blocking drugs on histamine liberation, prostaglandin synthesis and phospholipid turnover in isolated mast cells stimulated with concanavalin A.

Exaprolol, metipranolol and propranolol decreased significantly histamine liberation, degranulation, 45Ca uptake and thromboxane B2 formation in isolated rat mast cells stimulated with concanavalin A and phosphatidylserine. Moreover, exaprolol and metipranolol decreased 32P incorporation into membrane phospholipids and metipranolol and propranolol reduced the liberation of arachidonic acid from membrane phospholipids of stimulated mast cells. Exaprolol significantly increased the arachidonic acid liberation from these cells. Possible mechanisms of interaction of beta-adrenoceptor blocking drugs with isolated mast cells are discussed.     

On the interaction of beta-adrenoceptor-blocking drugs with isolated mast cells

The interaction of beta-adrenoceptor blocking drugs (BAB drugs) with isolated mast cells resulted, according to the compound, in either a liberation of biogenic amines or an inhibition of stimulated amine release. The liberatory drugs exaprolol and K? 1124 decreased the level of cAMP, stimulated the activity of cyclic nucleotide-phosphodiesterase, decreased the incorporation of orthophosphate into membrane phospholipids and rapidly displaced calcium from binding sites in mast cells. The inhibitory drugs alprenolol, metipranolol, oxprenolol, practolol and propranolol, possessing lower liposolubility, produced opposite effects. Drugs from both groups displaced histamine from binding sites in isolated mast cell granules. The interaction of BAB drugs with mast cells is a result of non-specific rather than specific receptor interactions. Inhibitory drugs interfere with mast cells at membrane sites while liberatory drugs penetrate the membrane, thus acting both at the level of membrane and intracellularly.     

Histamine liberation and membrane fluidisation of mast cells exposed to the beta-adrenoceptor blocking drug propranolol

Propranolol liberates histamine from isolated mast cells and decreases the uptake of extracellular histamine in a dose-dependent way. Histamine liberation due to propranolol is accompanied by calcium displacement from intracellular storage sites. The significant increase in membrane fluidity due to propranolol is temperature dependent. The perturbation of membranes is most probably the explanation of propranolol's interaction with isolated rat mast cells which results in altered histamine transportation.     

Histamine H1 receptors in C6 glial cells are coupled to calcium-dependent potassium channels via release of calcium from internal stores

We investigated the action of histamine on C6-astroglioma cells using patch clamp recording and intracellular calcium measurement. Application of 100 μM histamine hyperpolarized the resting membrane potential and increased free intracellular calcium. Membrane hyperpolarization was accompanied by a decrease in input resistance. The effect of histamine was reversible and responses persisted following repeated applications. In voltage clamp experiments histamine elicited an outward current associated with a conductance increase and a reversal potential near the Nernst potential for potassium. The action of histamine was blocked by mepyramine but not by cimetidine or thioperamide suggesting that a H₁ receptor mediated the response. Quinidine and charybdotoxin, but not apamin, blocked the hyperpolarization. Buffering internal calcium with BAPTA diminished the activation of the potassium channel, suggesting a calcium-dependent K⁺-channel, which was also found to be regulated by protein kinase C and phosphatases. The increase in intracellular calcium was not dependent on external calcium or sensitive to pertussis toxin, cholera toxin, forskolin or 8-bromo-cAMP. Both the hyperpolarization and the increase in intracellular calcium were blocked by thapsigargin or the phospholipase C inhibitor U73122. These results indicate that histamine liberates calcium from internal stores by activation of phospholipase C which in turn leads to an increase of intracellular Ca²⁺ and thereby to the activation of a calcium-dependent potassium channel in C6 glial cells. Received: 8 August 1996 / Accepted: 22 January 1997     

Comparison of the effect of eleven beta-adrenoceptor blocking drugs in perturbing lipid membrane: an ESR spectroscopy study

The perturbation effect of the beta-adrenoceptor blocking drugs atenolol, propranolol, practolol, oxprenolol, doberol, pronethanol, metipranolol, alprenolol, K?-1124, pindolol, and exaprolol on rat brain lipid membrane was investigated by ESR spectroscopy using the spin probe method. Using stearic acids spin labeled at the 5th, 12th, and 16th positions, it was found that lipophilic drugs disorder the membrane and their effect is about 5-10 times higher at the 16th carbon membrane depth than at the 5th depth. Exaprolol induced nonlamellar phases in the bovine brain lipid membrane as detected by 31P NMR spectroscopy. The relative potencies of the drugs at 10 mmol/liter concentration to disorder the lipid membrane at the 16th carbon depth were in the order: exaprolol greater than alprenolol approximately equal to propranolol greater than metipranolol approximately equal to doberol greater than control sample greater than pindolol approximately equal to practolol approximately equal to atenolol. This order qualitatively corresponds with some of their nonspecific biological membrane activities but is not related to their beta-adrenoceptor blocking potencies. The inequality of the membrane perturbation propensities of the drugs indicates that they perturb the lipid membrane in a structure-dependent manner, i.e., that each induces a specific rather than a nonspecific membrane perturbation.     

Histamine-induced ion secretion across rat distal colon: involvement of histamine H1 and H2 receptors

The aim of the present study was to investigate the effect of histamine, a product of e.g. mast cells, on short-circuit current (I(sc)) across rat distal colon. Histamine concentration-dependently stimulated an increase in I(sc), which often was preceded by a transient negative current. Neither a release of neurotransmitters nor a release of prostaglandins contributed to the histamine response. The histamine-induced increase in I(sc) was blocked by the histamine H(1) antagonist, pyrilamine, but was resistant against the histamine H(2) antagonist, cimetidine. Conversely, the histamine H(1) agonist, TMPH (2-(3-trifluoromethylphenyl)histamine), exclusively evoked an increase in I(sc), whereas the histamine H(2) agonist, amthamine, evoked only a decrease in I(sc) suggesting that stimulation of different types of histamine receptors is responsible for the two phases of the response evoked by native histamine. Histamine induces the opening of glibenclamide-sensitive Cl(-) channels and of charybdotoxin-sensitive K(+) channels in the apical membrane as demonstrated by experiments at basolaterally depolarized epithelia. A further action site is the basolateral membrane, because histamine stimulates a charybdotoxin- and tetrapentylammonium-sensitive K(+) conductance in this membrane as observed in tissues, in which the apical membrane was permeabilized with an ionophore, nystatin. The increase in I(sc) evoked by histamine was blocked after depletion of intracellular Ca(2+) stores with cyclopiazonic acid and after blockade of inositol 1,4,5-trisphosphate (IP(3)) receptors, suggesting a release of stored Ca(2+). This was confirmed by the observation that the histamine H(1) agonist TMPH induced an increase in the fura-2 ratio signal of epithelial cells within isolated colonic crypts. Consequently, the mediator histamine seems to stimulate both histamine H(1) and H(2) receptors, from which the former seems to be prominently involved in the induction of epithelial chloride secretion.     

Beta-adrenoceptor blocking drugs and calcium transport in isolated rat mast cells

The beta-adrenoceptor blocking (BAB) drugs exaprolol (EXA), metipranolol (MET) and propranolol (PRO) inhibited histamine liberation and degranulation from isolated rat mast cells stimulated with the calcium ionophore A23187. MET was the most and EXA the least active. Atenolol (ATE) had no effect. Inhibition by BAB drugs of secretion induced with A23187 was not accompanied by any change in 45Ca uptake. On the other hand, EXA, MET and PRO significantly decreased 45Ca uptake by mast cells stimulated with 48/80. The effect of BAB drugs on inhibition of A23187-induced secretion from isolated mast cells was dependent on the lipid solubility of the studied drugs.     

Analysis of histamine actions by intra- and extracellular recording in hippocampal slices of the rat

Extra- and intracellular recordings from CA1 pyramidal and dentate granule cells in hippocampal slices of the rat revealed a depressant effect of histamine which was usually accompanied by a hyperpolarization but no changes in conductance of the cell membrane. Synaptic potentials were unaffected. It is concluded that histamine has a post-synaptic action on ionic conductance in the dendrites, that allows a modulation of quick information transfer but no rapid synaptic transmission.     

Regulatory role of calcium on histamine secretion

Calcium seems to have two opposing effects on histamine secretion from mast cells. A rise in the cytosol calcium concentration initiates the chain of reactions leading to histamine secretion. On the other hand, calcium appears to have a regulatory role, limiting the secretion. Removal of cell surface calcium enhances histamine secretion. The present work demonstrates an inhibitory effect of calcium in the medium, using low concentrations of compound 48/80 as the secretagogue. Histamine secretion in response to compound 48/80 primarily utilizes intracellular calcium. When low concentrations of compound 48/80 were used (usually 20-50 ng/ml), calcium (1 mM) inhibited the secretion, the inhibition being more pronounced as the pH was increased from 6.5 to 8.5. The higher pH conceivably promotes the binding of calcium to the phospholipids in the cell membrane. Calcium at this site seems to depress the efflux of calcium from the intracellular stores to the cytosol. The possibility that the removal of calcium from the cell surface causes increased sodium permeability was considered. However, the sodium channel blocker tetrodotoxin (10(-5) M) was equally ineffective in influencing histamine release in the presence and absence of calcium, indicating that a change of sodium permeability was not involved. Antigen-induced (anaphylactic) histamine secretion depends mainly on extracellular calcium, although some secretion occurs in a calcium-free medium. Addition of calcium alone to the medium caused only slight increase in the secretion, but when both phosphatidylserine and calcium were added histamine secretion was remarkably stimulated, apparently through the effect of phosphatidylserine on calcium transport across the plasma membrane.     

Disposition of exaprolol, a new beta-blocker, in rats

Pharmacokinetics of tritium-labeled exaprolol, 1-(2-cyclohexyl[2,4-3H]-3-isopropylamino-2-propanol, a potent beta-adrenergic blocker, was studied in rats. The assay of the parent drug in plasma and excreta was based on extraction into toluene and TLC. In rats elimination half-lives of exaprolol after iv and oral administration of 26.8 and 40.1 hr, respectively, were found; the bioavailability was 26.7%. After oral administration to rats, the drug was rapidly and completely absorbed. The long retention of exaprolol in tissues of iv injected rats was reflected by the steady-state distribution volume of 59.38 liters/kg and the clearance rate of 1.44 liters/hr/kg. The total 3H excreted in urine within 96 hr after iv or oral administration to rats amounted to 47.9 and 47.1% of dose, respectively, and in feces to 34.2 and 29.8%, respectively. The ratio of 3H-exaprolol to total 3H concentration in plasma, urine, and feces of rats indicated a moderately extensive metabolism of the parent drug. The substance was about 50% bound to human serum albumin. The rat erythrocyte/plasma partition coefficient rose with exaprolol concentration.     

The action of local anaesthetics on histamine release.

The effect of the local anaesthetics lidocaine, procaine and tetracaine on compound 48/80-induced histamine release from isolated rat mast cells has been investigated. They inhibited histamine release in a dose-dependent manner; at a concentration of 20 mM there was almost total inhibition of histamine release by lidocaine and about 75% inhibition by procaine. Tetracaine exerted a biphasic effect: at concentrations below 1 mM it inhibited, but at concentrations above 1 mM it potentiated histamine release. The inhibitory effect of lidocaine on compound 48/80-evoked histamine release was dependent upon the time of preincubation of mast cells with this anaesthetic and it persisted after washing the cells and resuspension in a lidocaine-free medium. An increase of calcium ions antagonized the inhibitory action of lidocaine. These results can be explained by (1) blockade of membrane receptors for calcium binding which leads to a decrease in intracellular calcium concentration and (2) increase of cellular cyclic AMP content which subsequently inhibits the releasing process.     

Effect of beta-adrenoceptor blocking drugs on 32P incorporation into and arachidonic acid liberation from phospholipids in stimulated rat mast cells

The lipophilic beta-adrenoceptor blocking (BAB) drugs metipranolol, propranolol and exaprolol significantly decreased 48/80- and A23187-induced 32P incorporation into rat mast cell phospholipids. Exaprolol was the most active, followed by propranolol and metipranolol. Atenolol and metipranolol significantly decreased the 48/80-stimulated, and metipranolol and exaprolol the A23187-stimulated 3H-arachidonic acid liberation from isolated mast cells.     

Some effects of histamine in the depolarized rat uterus

1. The relaxant effect of histamine in the isolated rat uterus remained after the preparation was depolarized in a potassium Ringer.2. The effect was abolished by the calcium-chelating agent, ethyleneglycol bis-aminoethyl ether-tetraacetic acid (EGTA).3. Histamine caused relaxation during calcium-induced contractures in a depolarized uterus treated with EGTA, but did not produce relaxation during barium-induced contractures.4. While responses of a normally polarized rat uterus to acetylcholine were inhibited by histamine, those of a depolarized uterus were enhanced by histamine. The inhibitory effects of isoprenaline and papaverine on acetylcholine responses were maintained in depolarized preparations.5. Both the enhancing effect of histamine on responses to acetylcholine and the relaxation of calcium-induced contractures were abolished by burimamide, indicating that H(2)-receptors mediate the effects of histamine in the depolarized tissue.6. A reduction in the rate of exchange of calcium across the depolarized cell membrane was demonstrated with high concentrations of histamine.7. The results are consistent with the hypothesis that in the rat uterus, the stimulation of H(2)-receptors by histamine is accompanied by a reduction in calcium exchange across the membrane which may result in a decrease in the concentration of free intracellular calcium available to stimulate contraction of the myofilaments. Histamine may act by increasing the binding of calcium within the cell.     

EFFECTS OF VARIOUS DRUGS ON THE OSMOTIC LYSIS OF RAT MAST CELLS

1. Rat mast cells were exposed to low osmotic pressures to produce a ‘nonspecific’ disruption of the mast cell, with release of histamine along with other intracellular contents. 2. The effect of non-steroidal anti-inflammatory drugs and various other drugs upon osmotically induced histamine release was examined. 3. Representative non-steroidal anti-inflammatory drugs indomethacin, phenylbutazone and flufenamic acid, the acidic compounds ethacrynic acid, iopanoic acid and probenecid, and the local anaesthetic lignocaine, all caused a dose-dependent facilitation of osmotically induced histamine release compared to controls. All drugs were active at 0.1 mmol/1. 4. The previously observed inhibition of compound 48/80 and the antigen-induced histamine release from rat mast cells by similar concentrations of the drugs used in the present study are unlikely to be due to mast cell plasma membrane stabilization.     

Substance P-induced histamine release from rat peritoneal mast cells and its inhibition by antiallergic agents and calmodulin inhibitors.

Substance P-induced histamine release and Ca2+ release from the intracellular Ca store of rat peritoneal mast cells were inhibited by both antiallergic drugs and microtubule inhibiting agents. It was found that in the case of antiallergic compounds, histamine release inhibition may be intimately related to the inhibition of Ca2+ release from the intracellular store in which the microtubules play an important role. When mast cells were pretreated with either theophylline or dibutyryl cAMP, the inhibition of histamine release was closely related to the inhibition of Ca2+ release from the intracellular Ca store. Calmodulin inhibitors were also effective in inhibiting histamine release from mast cells induced by substance P. The inhibitory potencies of calmodulin inhibitors on histamine release from mast cells were closely correlated with those exerted on calmodulin activity.