Biomedical subjects

A Teschemacher

Publications and source records attributed to A Teschemacher.

7 recordsLinked to original sources

PubMed1999

Kinetic analysis of drug-receptor interactions of long-acting beta2 sympathomimetics in isolated receptor membranes: evidence against prolonged effects of salmeterol and formoterol on receptor-coupled adenylyl cyclase.

The long-acting beta2 sympathomimetics salmeterol and formoterol have been presumed to exert their prolonged action either by binding to an accessory binding site ("exo-site") near the beta2 adrenoceptor or by their high affinity for beta2 adrenoceptors and correspondingly slow dissociation. Whereas most studies with salmeterol had been done in intact tissues, which have slow diffusion and compartmentation of drugs in lipophilic phases, that restrict drug access to the receptor biophase, we used purified receptor membranes from rat lung and disaggregated calf tracheal myocytes as model systems. Binding experiments were designed to measure the slow dissociation of agonists by means of delayed association of (-)-[125I]iodopindolol. Rat lung membranes were pretreated with high concentrations of agonists (salmeterol, formoterol, isoprenaline) before dissociation was induced by 50-fold dilution. Half-times of association of (-)-[125I]iodopindolol remained unchanged compared with untreated controls, indicating that dissociation of agonists occurred in less than 2 min. Adenylyl cyclase experiments were designed to determine the on and off kinetics of agonists to beta2 adrenoceptors by measuring the rate of receptor-induced cyclic AMP (cAMP) formation. Experiments were performed in tracheal membranes characterized by high Vmax values of cAMP formation. Adenylyl cyclase activation occurred simultaneously with the addition of the agonist, continued linearly with time for 60 min, and ceased immediately after the antagonist was added. Similarly, when receptor membranes were preincubated in a small volume with high salmeterol concentrations, there was a linear increase in cAMP formation, which was immediately interrupted by a 100-fold dilution of the reaction mixture. This militates against the exo-site hypothesis. On the other hand, dissociation by dilution was much less when membranes were preincubated with a large volume of salmeterol at the same concentration, indicating that physicochemical effects, and not exo-site binding, underlie its prolonged mode of action.

Adenylyl Cyclases↗

PubMed1998

Do parasympatholytic effects of long-acting beta 2-sympathomimetics contribute to their relaxant effects in airway smooth muscle cells?

To address the hypothesis of whether functional antagonistic effects of long-acting beta 2-sympathomimetics (formoterol and salmeterol) might be supported by direct antagonistic effects on muscarinic acetylcholine receptors (mAChR), we performed radioligand binding experiments with 3H-quinuclidinyl-benzilate in membranes of airway smooth muscle cells (calf tracheal myocytes). beta 2-Sympathomimetics (short- and long-acting) were compared to catechol-ethanolamines and catechol-ethylamines. Tracheal myocytes were characterized by a high density of mAChR 1017 +/- 17 fmol/mg, which exceeds that of beta 2-adrenoceptors 20-fold. The affinities of drugs were determined by competition binding. Dissociation constants ¿pKD-values) of formoterol (5.04 +/- 0.05) and salmeterol (5.24 +/- 0.04) matched that of ACh (5.37 +/- 0.03) and were significantly higher than that of the mAChR-agonist carbachol (4.65 +/- 0.03). pKD-values of mAChR-agonists were strictly dependent on GTP-concentration (> 50-fold difference between high- and low-affinity states), in contrast to those of formoterol, thereby characterizing formoterol as an mAChR-antagonist. A 10-fold lower affinity of the related compound fenoterol (3.94 +/- 0.02) hinted at the formyl-amino moiety of formoterol as a structural determinant of high-affinity whereas a 100-fold lower affinity of salbutamol as compared to salmeterol suggested the aliphatic side chain was a structural determinant of high affinity. The high affinity of dobutamine (4.96 +/- 0.02) and dopexamine (6.20 +/- 0.02) provided evidence that high affinity can be found not only for catechol-ethanolamines with long side chains but also for catechol-ethylamines. The question of whether high local concentrations after inhalation of long-acting beta 2-sympathomimetics could contribute to their therapeutical effects by antagonism with ACh on mAChR remains to be answered.

Adrenergic beta-Agonists↗

PubMed1997

Presynaptic action of the neurosteroid pregnenolone sulfate on inhibitory transmitter release in cultured hippocampal neurons.

The effects of the neurosteroid pregnenolone sulfate (PS) were studied in 3- to 9-week-old hippocampal cultures from neonatal rats. Cells were voltage clamped using CsCl filled electrodes, while action potentials and excitatory glutamatergic currents were abolished by superfusing with a combination of tetrodotoxin, 6-cyano-7-nitroquinoxaline (CNQX) and 2-amino-5-phosphonopentanoic acid (AP-5). Under these conditions spontaneous GABAergic inhibitory postsynaptic currents (sIPSCs) were seen as inward currents at a holding potential of -70 mV. Their amplitude distributions were skewed without clearly detectable peaks. PS at 1-50 microM concentrations decreased the frequency of sIPSCs, with 1 microM being the most effective concentration. The effect appeared after 10-15 min of steroid application and the magnitude of the reduction increased during the early wash period. No recovery of sIPSC frequency was found after 30 min of washing with steroid-free medium. sIPSC amplitudes were not significantly changed at the time the effect of PS on sIPSC frequency was observed. The slow onset of this effect and its duration suggest a novel presynaptic action of the neurosteroid PS on GABAergic inhibition in the mammalian brain.

Animals↗

PubMed1996

Corticosterone-induced decrease of inhibitory postsynaptic potentials in rat hippocampal pyramidal neurons in vitro depends on cytosolic factors.

Previous studies using high-resistance sharp electrodes demonstrated that corticosterone (CORT) reduced GABAergic synaptic inhibition in CA1 neurons of the rat hippocampus in vitro. In the present study we used whole-cell gigaseal recordings to investigate the possible role of cytosolic factors in the transduction mechanism underlying this action. The perturbation of the intracellular milieu that occurs under these recording conditions abolished the CORT-induced decrease in inhibitory postsynaptic conductance. CORT actually increased GABAA receptor-mediated conductances in about 50% of the neurons tested when the whole-cell recording mode was employed. As in the high-resistance microelectrode studies, CORT did not change the resting membrane potential, action potential amplitude or input resistance. The results suggest that the reduction of GABAergic synaptic inhibition in hippocampal CA1 pyramidal neurons induced by CORT critically depends on the presence of cytosolic factors, which wash out during whole-cell gigaseal recordings.

Animals↗

PubMed1996

Is inhibition of oxygen radical production of neutrophils by sympathomimetics mediated via beta-2 adrenoceptors?

Despite their beneficial effects on cardiovascular derangements in patients with severe sepsis, high doses of sympathomimetics might contribute to an impaired neutrophil function. This study was conducted to examine whether various sympathomimetics [(-)-epinephrine (EPI), dopamine (DA) and dobutamine (DOB)] differ in their potency to suppress the formation of oxygen radicals by neutrophils and whether this potency correlates with their affinity to or intrinsic activity for beta-2 adrenoceptors (beta-2 AR). Oxygen radical production of human neutrophils was induced by N-formyl-methionyl-leucyl-phenyl-alanine and detected by chemiluminescence measurements. Dose-response curves for the inhibition of chemiluminescence by sympathomimetics were measured in the absence and presence of 0.1 microM CGP 20,712 A (1-[2(3-carbamoyl-4-hydroxy phenoxy)-ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl) phenoxy]-2-propanol methanesulfonate) and 0.1 microM ICI 118,551 (erythro-(+/-)-1-(7-methylindan-4-yloxy)-3 isopropylaminobutan-2-ol hydrochloride) to selectively antagonize beta-1 AR and beta-2 AR, respectively. Inhibition of chemiluminescence of neutrophils by EPI was approximately 100-fold more potent than that by DA and DOB. Only the inhibition curve by EPI exhibited two components, one at nanomolar and one at micromolar concentrations. The nanomolar component was sensitive against beta-2 AR blockade, whereas the micromolar one was insensitive against both beta AR antagonists. Dose-response curves for DA and DOB exhibited a simple hyperbolic shape at micromolar concentrations and were insensitive against both beta AR antagonists. Maximum inhibition by DA and DOB was equipotent to that by EPI. However, the EC50 for DA was much lower than its dissociation constants, KD, assayed in membrane preparations by radioligand binding, whereas the EC50 of DOB matched KD. This difference could not be explained by a different efficiency of signal transduction, which was determined in receptor-coupled adenylate cyclase activity and which only showed a slightly higher efficiency of DA (51%) than of DOB (34%). Therefore, sympathomimetics were also investigated in a cell-free system, in which chemiluminescence was generated by horseradish peroxidase with hydrogen peroxide as substrate. Surprisingly, all of the sympathomimetics suppressed chemiluminescence with micromolar concentrations. We conclude that sympathomimetics with high affinity and high intrinsic activity (EPI) inhibit neutrophil function via occupation of beta-2 AR, whereas sympathomimetics with low affinity (DA) or low intrinsic activity (DOB) may act by direct scavenging of oxygen radicals.

Adenylyl Cyclases↗

PubMed1995

The neuroactive steroid 5 alpha-tetrahydrodeoxycorticosterone increases GABAergic postsynaptic inhibition in rat neocortical neurons in vitro.

The neuroactive steroid 5 alpha-pregnane-3 alpha, 21-diol-20-one (5 alpha-tetrahydrodeoxycorticosterone; 5 alpha-THDOC) has been shown to potentiate GABA-induced chloride currents in cell cultures and subcellular preparations. In this study, we recorded from pyramidal neurons in an in vitro slice preparation of the adult rat frontal neocortex using intracellular microelectrodes. 5 alpha-THDOC (10 microM) increased and prolonged the inhibitory postsynaptic potential (IPSP). The mean maximal synaptic conductance of the early, GABAA receptor-mediated, IPSP was enhanced to more than 700%, the one at the maximum of the late, partially GABAB receptor-mediated, IPSP to approximately 400%. The progesterone/glucocorticoid receptor antagonist RU 38486 did not prevent the IPSP increase. At a concentration of 1 microM 5 alpha-THDOC increased only the early IPSP to about 125%. Responses to the iontophoretically applied specific GABAA receptor agonist muscimol but not to the specific GABAB receptor agonist L-baclofen were enhanced by 5 alpha-THDOC (10 microM). In the giga-seal whole-cell configuration when the GABAB receptor-mediated IPSP component was absent due to intracellular perfusion, 5 alpha-THDOC (10 microM) increased IPSPs to a similar extent as in the conventional microelectrode recordings. Excitatory postsynaptic potentials, resting membrane potential, input resistance and action potential amplitude were not affected by 5 alpha-THDOC (10 microM). These data demonstrate that in neocortical tissue of the rat 5 alpha-THDOC enhances GABAergic inhibition by interacting with postsynaptic GABAA receptors while synaptic excitation and parameters of electric excitability remain unchanged.

Animals↗

PubMedDate not supplied

[Calcium-independent inhibition of the spontaneous release of GABA by baclofen in the rat hippocampus].

The mechanism of calcium-independent spontaneous GABA release in a long-living (3-10 weeks) rat hippocampal neuron culture was studied. It was found that Cd2+ (100 microM), a Ca2+ channel blocker, reversibly decreased the frequency and amplitude of GABAergic spontaneous miniature inhibitory postsynaptic currents (smIPSCs). Besides, Cd2+ decreased the currents evoked by muscimol application onto the neurons, which is evidence of an additional postsynaptic effect. The GABAB receptor agonist baclofen (0.1-50 microns) produced a concentration-dependent decrease in the smIPSC frequency, while not affecting the current amplitude. The baclofen effect was blocked by the pertussis toxin. The baclofen efficacy both in the presence of Cd2+ (presumably compete blocking of Ca2+ channels) and in the absence of this agent were similar and could be completely accounted for by the loss of an equivalent smIPSC fraction. Thus, the presynaptic baclofen-induced inhibition of the spontaneous GABA release can be entirely independent of the calcium channel modulation (the latter playing a decisive role in a mediator release induced by the action potential). Therefore, the smIPSC measurements (widely used in the past decade for the study of presynaptic drug activity) may inadequately reflect the drug effect in the intact brain.

Animals↗