Ernst Agneter

Effects of antiglaucoma drugs on ocular hemodynamics in healthy volunteers

There is evidence that ocular blood flow plays a critical role in the clinical course of glaucoma. Hence a reduction in ocular blood flow due to topical antiglaucoma treatment should be avoided. The purpose of this study was to characterize the effect of antiglaucoma drugs on ocular hemodynamics.

Sustained dopamine release induced by secretoneurin in the striatum of the rat : a microdialysis study

Abstract: Secretoneurin (SN) is a neuropeptide derived from secretogranin II that is found in brain and endocrine tissues. The aim of the present study was to determine the influence of this novel peptide on dopamine (DA) release from rat striatum using the microdialysis technique. Rat SN (1–30 µmol/L added to the dialysis buffer) enhanced DA outflow of awake rats in a concentration‐dependent way without marked effects on the outflow of 3,4‐dihydroxyphenylacetic acid or homovanillic acid. The increase in extracellular DA content caused by the peptide was observed throughout the entire period of administration (up to 4 h). Human SN and its 15‐amino‐acid C‐terminal sequence also increased DA outflow, but the effects were smaller than those of rat SN. Two other peptides derived from secretogranin II were without effect on DA efflux. These results establish that SN has a pronounced effect on DA release under in vivo conditions.

Induction by low Na+ or Cl− of cocaine sensitive carrier‐mediated efflux of amines from cells transfected with the cloned human catecholamine transporters†

COS‐7 cells transfected with the cDNA of the human dopamine transporter (DAT cells) or the human noradrenaline transporter (NAT cells) were loaded with [3H]‐dopamine or [3H]‐noradrenaline and superfused with buffers of different ionic composition. In DAT cells lowering the Na+ concentration to 0, 5 or 10 mM caused an increase in 3H‐efflux. Cocaine (10 μM) or mazindol (0.3 μM) blocked the efflux at low Na+, but not at 0 Na+. Lowering the Cl− concentration to 0, 5 or 10 mM resulted in an increased efflux, which was blocked by cocaine or mazindol. Desipramine (0.1 μM) was without effect in all the conditions tested. In NAT cells, lowering the Na+ concentration to 0, 5 or 10 mM caused an increase in 3H‐efflux, which was blocked by cocaine or mazindol. Desipramine produced a partial block, its action being stronger at 5 or 10 mM Na+ than at 0 mM Na+. Efflux induced by 0, 5 or 10 mM Cl− was completely blocked by all three uptake inhibitors. In cross‐loading experiments, 5 mM Na+‐ or 0 Cl−‐induced efflux was much lower from [3H]‐noradrenaline‐loaded DAT, than NAT cells and was sensitive to mazindol, but not to desipramine. Efflux from [3H]‐dopamine‐loaded NAT cells elicited by 5 mM Na+ or 0 Cl− was blocked by mazindol, as well as by desipramine. Thus, cloned catecholamine transporters display carrier‐mediated efflux of amines if challenged by lowering the extracellular Na+ or Cl−, whilst retaining their pharmacological profile. The transporters differ with regard to the ion dependence of the blockade of reverse transport by uptake inhibitors.

α2‐Adrenoceptor‐Mediated Inhibition of Electrically Evoked [3H]Noradrenaline Release from Chick Sympathetic Neurons: Role of Cyclic AMP

Abstract: This study explores the role of cyclic AMP in electrically evoked [3H]noradrenaline release and in the α2‐adrenergic modulation of this release in chick sympathetic neurons. Along with an increase in stimulation‐evoked tritium overflow, applications of forskolin enhanced the formation of intracellular cyclic AMP. Both effects of forskolin were potentiated by the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine. The forskolin‐induced increase in overflow was abolished by the Rp‐diastereomer of cyclic AMP‐thioate, an antagonist at cyclic AMP‐dependent protein kinases, and 1,9‐dideoxy‐forskolin, an inactive analogue at adenylyl cyclase, had no effect on the evoked overflow. A 24‐h pretreatment with either cholera toxin or forskolin reduced the subsequent forskolin‐induced accumulation of cyclic AMP and inhibited the stimulation‐evoked release. Basal cyclic AMP production, however, remained unaltered after forskolin treatment and was enhanced after 24 h of cholera toxin exposure. The α2‐adrenergic agonist bromoxidine did not affect the formation of cyclic AMP stimulated by forskolin but reduced electrically evoked release. However, effects of bromoxidine on 3H overflow were attenuated by forskolin as well as by 8‐bromo‐cyclic AMP. Effects of bromoxidine on [3H]noradrenaline release were paralleled by an inhibition of voltage‐activated Ca2+ currents, primarily through a delayed time course of current activation. This effect was abolished when either forskolin or 8‐bromo‐cyclic AMP was included in the pipette solution. Both substances, however, failed to affect Ca2+ currents in the absence of bromoxidine. These results suggest that the signaling cascade of the α2‐adrenergic inhibition of noradrenaline release involves voltage‐activated Ca2+ channels but not cyclic AMP. Elevated levels of cyclic AMP, however, antagonize this α2‐adrenergic reduction, apparently through a disinhibition of Ca2+ channels.

Comparison of the Effects of Ketamine and Memantine on Prolactin and Cortisol Release in Men: A Randomized, Double-blind, Placebo-controlled Trial

N-methyl D-aspartate (NMDA)-antagonists decrease neurotoxicity by inhibiting Ca2+ influx which is of interest for the treatment of acute cerebrovascular insults and chronic neurodegenerative disorders. Currently, there is no surrogate marker for quantification of NMDA-receptor–mediated drug effects, which hampers dose-finding clinical studies. As prolactin and cortisol liberation is in part influenced through NMDA-receptors we investigated whether the elevation of prolactin or cortisol plasma levels is a class effect of NMDA-antagonists and might be an appropriate marker for studying NMDA-antagonistic potency. Fifteen healthy male volunteers participated in this placebo-controlled, randomized, three-way crossover trial. Ketamine (0.5mg/kg), memantine (0.16 mg/kg; i.e., a well tollerated standard dose) or placebo were infused over 60 min. Ketamine increased serum prolactin and cortisol levels (p < 0.001), whereas memantine and placebo did not affect hormone levels. Further studies are needed to define whether higher doses of memantine or other NMDA antagonists can induce hormone release.

Amphetamine reverses or blocks the operation of the human noradrenaline transporter depending on its concentration: superfusion studies on transfected cells.

Whether amphetamine enhances noradrenergic activity by uptake blockade or a releasing action is still a matter of debate. In order to gain insight into the interaction of amphetamine with the noradrenaline transporter its cDNA was transfected into COS-7 cells (NAT-cells) or cotransfected with the cDNA of the vesicular monoamine transporter (NAT/VMAT-cells); cells were loaded with [3H]noradrenaline, superfused and the efflux analysed for total tritium and [3H]noradrenaline. In NAT-cells amphetamine stimulated [3H]noradrenaline efflux concentration-dependently when added to the superfusion buffer at 0.01, 0.1 and 1 microM. By contrast, 10 or 100 microM amphetamine stimulated efflux to a smaller extent or not at all; however, on switching back to amphetamine-free buffer a prompt increase of efflux was observed. Cocaine did not increase efflux per se and blocked the amphetamine-induced efflux. In NAT/VMAT-cells amphetamine stimulated efflux in a concentration-dependent manner. The effect showed saturation at 1 microM and was not suppressed at higher concentrations. Cocaine also elicited efflux from NAT/VMAT-cells concentration-dependently; the maximum was reached at approximately 1 microM and amounted to only about half of the amphetamine-induced efflux. It is concluded that amphetamine can induce noradrenaline transporter mediated release only at high nanomolar to low micromolar concentrations. At higher concentrations it blocks the noradrenaline transporter; in this case, the releasing action of amphetamine, like that of cocaine, is dependent on a vesicular pool of noradrenaline.

THE SLOPE PARAMETER OF CONCENTRATION-RESPONSE CURVES USED AS A TOUCHSTONE FOR THE EXISTENCE OF SPARE RECEPTORS

Abstract The present work was stimulated by findings of a large reserve of presynaptic α2-autoreceptors in rat neocortex by different investigators and our own group, using classical models of receptor agonism. The mathematical background of these classical models seems erroneous since the asymmetry that spare receptors introduce into concentration-response curves is not considered appropriately. This asymmetry leads to a steepening of curve fits based on the logistic function. Therefore, the slope parameter c of a logistically fitted concentration-response curve can be used as a touchstone for the existence of spare receptors. Spare receptors induce a c > 1. Concentration-response data of the α2-autoreceptor-mediated inhibition of evoked [3H]-noradrenaline release in rat neocortex slices were re-analysed. The estimates of the slope parameter c of logistically fitted concentration-response curves obtained after treatment of rats with either vehicle or N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) to achieve an irreversible inactivation of α2-autoreceptors, were not compatible with the existence of a large receptor reserve. A model for nonlinear regression analysis developed under the a priori assumption of spare receptors confirmed the absence of spare receptors.

Estimation of the biophase concentration of noradrenaline at presynaptic α2-adrenoceptors in brain slices

SummaryThe aim of the present study was to determine the local concentrations of noradrenaline existing at presynaptic α2-adrenoceptors during electrical pulse train stimulation of brain slices at different frequencies. The experiments are based on the assumption that the concentration of released noradrenaline at the α2-adrenoceptors exerting a certain autoinhibition should be equal to the concentration of exogenous noradrenaline causing the same inhibition under conditions in which any influence of the released transmitter is excluded. In order to avoid autoinhibition, hippocampus and cortex slices of the rabbit and the rat, prelabelled with [3H]noradrenaline and superfused in presence of an uptake inhibitor, were electrically stimulated using 4 pulses delivered at 100 Hz (POP stimulation). Exogenous noradrenaline diminished the overflow of tritium elicited by POP stimulation in a concentration-dependent manner. In rabbit brain tissues the EC50 value and maximum inhibition of noradrenaline release were found to be approximately 6 nmol/l and more than 95%, respectively, whereas in rat tissues the corresponding values were between 20 and 30 nmol/l and approximately 90%. When electrical stimulation was performed with trains of 36 pulses delivered at 0.1, 0.3 or 3 Hz in absence or presence of an uptake inhibitor, the α2-adrenoceptor antagonist yohimbine (1 or 10 μmol/l) enhanced the evoked tritium overflow in a manner which was dependent on the frequency of stimulation and on blockade of the re-uptake mechanism. The facilitatory effects of yohimbine reflected an extent of autoinhibition which was between 53% (36 pulses/0.1 Hz, no uptake inhibitor) and 85% (36 pulses/3 Hz, uptake inhibitor present) in rabbit and between 16% (36 pulses/0.3 Hz, no uptake inhibitor) and 71% (36 pulses/3 Hz, uptake inhibitor present) in rat brain slices. Accordingly, the corresponding estimated biophase concentrations of noradrenaline were generally higher in rat than in rabbit tissues (they were between 32.5 and 74.5 or 5.1 and 51.6 nmol/l in the presence or absence of an uptake inhibitor, respectively, in the rat, and between 15 and 23.1 or 6.1 and 18.6 nmol/l in the rabbit). The observed frequency dependence of the effect of re-uptake blockade on the calculated biophase concentrations of noradrenaline would be compatible with the idea of a dependence of the effectiveness of the re-uptake mechanism on the firing rate of the neurone in being more effective at lower frequencies. Moreover, the stikingly low biophase concentrations of noradrenaline suggest that also in brain tissue noradrenaline causes lateral inhibition of release as has recently been shown for guinea-pig vas deferens.

New insights into receptor theory, as provided by an artificial partial agonist made-to-measure

In the present study a mixture of a full agonist (noradrenaline) and a full antagonist (yohimbine) was used to mimic the effects of a partial agonist (clonidine) on α2-autoreceptor-mediated regulation of noradrenaline release in order to learn more about the shape of concentration-response curves in the absence and presence of spare receptors. The sigmoidal shape of the cloud of single experimental data points may be reflected by different curve fits based on either descriptive or mechanistic mathematical models. Only mechanistic models allow the interpretation of the relationship between occupancy of receptors and induced response. The experiments were performed in rat neocortex and in rabbit hippocampus tissue where electrical field stimulation with 4 pulses/100 Hz of slices prelabelled with [3H]noradrenaline elicited the release of noradrenaline.A receptor reserve was found in the rabbit hippocampus and quantified from the concentration-response curve of noradrenaline in this tissue using a mechanistic general response function, developed to reflect the condition of spare receptors. The mixture of noradrenaline and yohimbine, NAYoh, (three parts to one part), corrected by the different affinities to the α2-autoreceptors, was designed to mirror the quantified proportion of 75% non-spare and 25% spare α2-autoreceptors. In the spare receptor-free rat cortex NAYoh acted like a typical partial agonist, as clonidine, with nearly the same EC50 (= Kd in this case) as the full agonist noradrenaline, but with a maximum effect significantly lower than that of noradrenaline. In the rabbit hippocampus, however, the same maximum effect was obtained with NAYoh, noradrenaline and clonidine. In this tissue the EC50 of the logistic fit of the concentration-response relationship of noradrenaline was lower than that of NAYoh and the corresponding slope parameter of the noradrenaline curve was significantly higher than unity, indicating the descriptive character of this curve fit which does not allow an interpretation of the link between occupation and response. NA-Yoh and clonidine, however, yielded slope parameters of around unity. Therefore, the logistic EC50s of NA-Yoh and of clonidine may be interpreted mechanistically as Kds which was confirmed by the estimation of their Kds using the general response function. In contrast, the general response function yielded a Kd of noradrenaline significantly higher than its logistic EC50. The observation that the mixture of a pure agonist and a pure antagonist shows the properties of a partial agonist, suggests that the efficacy of the agonist-antagonist continuum is the conditional probability p (0≤p≤ 1) to activate a receptor given it occupies this receptor, where p is unity for a full agonist and zero for a full antagonist. Activation of a receptor, however, leads to a response only when this receptor is a non-spare receptor.In a system with a possible receptor reserve the use of both an (artificial) partial agonist and a full agonist, which induce different slope parameters in the logistic fits of their concentration-response curves, allows us to quantify the receptor reserve of the full agonist by nonlinear regression analysis of the concentration-response curve of the full agonist with the general response function.

In vivo drug-response measurements in target tissues by microdialysis.

To study the suitability of the microdialysis technique for the measurement of target tissue pharmacodynamics in humans, the model compounds theophylline, milrinone, and compound 48/80 were administered locally by means of reversed microdialysis to the interstitial space of skeletal muscle or skin in 24 healthy volunteers. Simultaneously, interstitial concentrations of cyclic adenosine monophosphate (cAMP; as an indicator of phosphodiesterase activity) were measured in skeletal muscle, and interstitial concentrations of histamine (as an indicator of mast cell release) were measured in skin. In muscle, reversed microdialysis with milrinone led to a dose‐dependent increase in interstitial cAMP concentrations (n = 8), whereas no significant effect on cAMP was observed for theophylline versus placebo (1.63 ± 0.53 nmol/L; n = 6), even at local concentrations exceeding those attained after therapeutic doses. In skin, reversed microdialysis with compound 48/80 increased interstitial histamine concentration dose dependently versus placebo (5.99 ± 2.74 nmol/L; n = 10). From our experiments in human skeletal muscle and skin, we concluded that microdialysis was a suitable technique for the characterization of in vivo drug response at the relevant target site. Extension of these measurements to several other human tissues is readily feasible.