Ernst A. Singer

Carrier-mediated release, transport rates, and charge transfer induced by amphetamine, tyramine, and dopamine in mammalian cells transfected with the human dopamine transporter.

Abstract: Amphetamine and related substances induce dopamine release. According to a traditional explanation, this dopamine release occurs in exchange for amphetamine by means of the dopamine transporter (DAT). We tested this hypothesis in human embryonic kidney 293 cells stably transfected with the human DAT by measuring the uptake of dopamine, tyramine, and d‐ and l‐amphetamine as well as substrate‐induced release of preloaded N‐methyl‐4‐[3H]phenylpyridinium ([3H]MPP+). The uptake of substrates was sodium‐dependent and was inhibited by ouabain and cocaine, which also prevented substrate‐induced release of MPP+. Patch‐clamp recordings revealed that all four substrates elicited voltage‐dependent inward currents (on top of constitutive leak currents) that were prevented by cocaine. Whereas individual substrates had similar affinities in release, uptake, and patch‐clamp experiments, maximal effects displayed remarkable differences. Hence, maximal effects in release and current induction were ∼25% higher for d‐amphetamine as compared with the other substrates. By contrast, dopamine was the most efficacious substrate in uptake experiments, with its maximal initial uptake rate exceeding those of amphetamine and tyramine by factors of 20 and 4, respectively. Our experiments indicate a poor correlation between substrate‐induced release and the transport of substrates, whereas the ability of substrates to induce currents correlates well with their releasing action.

Serotonin-transporter mediated efflux: A pharmacological analysis of amphetamines and non-amphetamines

The physiological function of neurotransmitter transporter proteins like the serotonin transporter (SERT) is reuptake of neurotransmitter that terminates synaptic serotoninergic transmission. SERT can operate in reverse direction and be induced by SERT substrates including 5-HT, tyramine and the positively charged methyl-phenylpyridinium (MPP(+)), as well as the amphetamine derivatives para-chloroamphetamine (pCA) and methylene-dioxy-methamphetamine (MDMA). These substrates also induce inwardly directed sodium currents that are predominantly carried by sodium ions. Efflux via SERT depends on this sodium flux that is believed to be a prerequisite for outward transport. However, in recent studies, it has been suggested that substrates may be distinct in their properties to induce efflux. Therefore, the aim of the present study was a pharmacological characterization of different SERT substrates in uptake experiments, their abilities to induce transporter-mediated efflux and currents. In conclusion, the rank order of affinities in uptake and electrophysiological experiments correlate well, while the potencies of the amphetamine derivatives for the induction of efflux are clearly higher than those of the other substrates. These discrepancies can be only explained by mechanisms that can be induced by amphetamines. Therefore, based on our pharmacological observations, we conclude that amphetamines distinctly differ from non-amphetamine SERT substrates.

Inhibition of vasopressin-stimulated flank marking behavior by V1-receptor antagonists

Flank marking, a form of olfactory communication displayed by hamsters, is dependent upon vasopressin-sensitive neurons in the anterior hypothalamus. In the present study two vasopressin type-1 (V1) receptor antagonists, d(CH2)5Tyr(Me)AVP and dPTyr(Me)AVP were tested for their ability to block flank marking stimulated by the microinjection of arginine vasopressin (AVP) into the anterior hypothalamus. Dose-response curves were established for AVP and flank marking in the presence or absence of different concentrations of each antagonist. DPTyr(Me)AVP was microinjected into the anterior hypothalamus 1 h before the microinjection of AVP while d(CH2)5Tyr(Me)AVP and AVP were prepared together and delivered as a single microinjection. This procedure was necessary because dPTyr(Me)AVP, but not d(CH2)5Tyr(Me)AVP, had agonist activity when initially injected into the anterior hypothalamus in concentrations ranging from 0.90-900 microM. The ED50 values (microM) for dPTyr(Me)AVP and AVP were 17.9 and 0.90, respectively. The initial agonist activity of dPTyr(Me)AVP was always followed by blocker activity. Both V1-receptor antagonists caused a dose-dependent decrease in AVP-stimulated flank marking. Maximal inhibition of AVP-stimulated flank marking was produced with approximately 1.0 mM of either antagonist. Both antagonists blocked AVP-stimulated flank marking behavior for over 12 h following their microinjection.

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.

Enhanced Serotonin Transporter Function during Depression in Seasonal Affective Disorder

Decreased synaptic serotonin during depressive episodes is a central element of the monoamine hypothesis of depression. The serotonin transporter (5-HTT, SERT) is a key molecule for the control of synaptic serotonin levels. Here we aimed to detect state-related alterations in the efficiency of 5-HTT-mediated inward and outward transport in platelets of drug-free depressed patients suffering from seasonal affective disorder (SAD). 5-HTT turnover rate, a measure for the number of inward transport events per minute, and tyramine-induced, 5-HTT-mediated outward transport were assessed at baseline, after 4 weeks of bright light therapy, and in summer using a case–control design in a consecutive sample of 73 drug-free depressed patients with SAD and 70 nonseasonal healthy controls. Patients were drug-naive or medication-free for at least 6 months prior to study inclusion, females patients were studied in the follicular phase of the menstrual cycle. All participants were genotyped for a 5-HTT-promoter polymorphism (5-HTTLPR) to assess the influence of this polymorphism on 5-HTT parameters. Efficiency of 5-HTT-mediated inward (p=0.014) and outward (p=0.003) transport was enhanced in depressed patients. Both measures normalized toward control levels after therapy and in natural summer remission. Changes in outward transport showed a clear correlation with treatment response (ρ=0.421, p=0.001). Changes in inward transport were mediated by changes in 5-HTT transport efficiency rather than affinity or density. 5-HTTLPR was not associated with any of the 5-HTT parameters. In sum, we conclude that the 5-HTT is in a hyperfunctional state during depression in SAD and normalizes after light therapy and in natural summer remission.

Reduction of [3H]muscimol binding sites in rat dorsal spinal cord after neonatal capsaicin treatment

Two-day-old rats were pretreated with 50 mg/kg of capsaicin. After 3--4 months, specific binding of [3H]muscimol and [3H]strychnine was measured in membrane preparations from dorsal spinal cord. A 20-30% decrease of the number of [3H]muscimol binding sites was observed after capsaicin treatment. In contrast, [3H]strychnine binding was unchanged. The results provide indirect evidence for a presynaptic location of GABA receptors on capsaicin-sensitive primary afferent neurons.

Stimulation of luteinizing hormone release after stereotaxic microinjection of neurotensin into the medial preoptic area of rats.

Neurons immunoreactive to luteinizing hormone-releasing hormone (LHRH) are localized to the medial preoptico-septal region of the rat hypothalamus, an area known to be essential for the regulation of the surge of luteinizing hormone (LH) that precedes ovulation. The effect of neurotensin (NT) on circulating LH levels following its discrete microinjection into this region was evaluated. Within 30 min after the stereotaxic microinjection of 40 ng of NT in 50 nl into the medial preoptic area (MPOA) just rostral to the organum vasculosum of the lamina terminalis in ovariectomized (OVX), anesthetized rats, plasma LH levels rose from 417 +/- 36 to 923 +/- 113 ng/ml (p less than 0.001). There was also a significant increase (p less than 0.001) in plasma LH levels following the microinjection of 50 ng of norepinephrine in 50 nl in this same site. Similarly, levels of LH increased from 48 +/- 7 to 110 +/- 19 ng/ml (p less than 0.004) following the microinjection of NT into the rostral MPOA on the morning of proestrus in anesthetized rats. In contrast, saline, substance P, leu-enkephalin and LHRH microinjected in the rostral MPOA of OVX rats had no significant effect on circulating levels of LH. The microinjection of NT 0.4 mm rostral or caudal to this area was also without effect. The presence of two binding sites with KdS of 4.2 and 29 nM and site concentrations of 0.11 and 0.68 pmol/mg in tissue homogenates of the rostral MPOA were also established. These data suggest a role for NT in the central regulation of LH release.

Estimation ofpA2 values at presynaptic α2-autoreceptors in rabbit and rat brain cortex in the absence of autoinhibition

SummaryAn attempt was made to determinepA2 values of antagonists at the presynaptic, release-inhibiting α2-autoreceptorsof rabbit and rat brain cortex under conditions when there was very little released noradrenaline in the autoreceptor biophase and, hence,pA2 values were not distorted by endogenous autoinhibition. Cortex slices were preincubated with3H-noradrenaline and then superfused and stimulated by trains of 4 pulses delivered at 100 Hz or, in a few cases, by trains of 36 pulses at 3 Hz. The α-adrenoceptor agonists clonidine, noradrenaline, and α-methylnoradren-aline concentration-dependently decreased the stimulation-evoked overflow of tritium. The a-adrenoceptor antagonists yohimbine, rauwolscine and idazoxan did not increase the overflow of tritium elicited by 4 pulses/100 Hz in rabbit brain slices and increased it only slightly in rat brain slices. In contrast, the antagonists increased markedly the overflow at 36 pulses/3 Hz. All antagonists caused parallel shifts to the right of the concentration-response curves of clonidine, noradrenaline, and α-methylnoradrenaline.pA2 values were calculated either from linear regression of log [agonist concentration ratio − 1] on log [antagonist concentration] or from sigmoid curve fitting. The slopes of the linear regression lines were close to unity, and thepA2 values calculated by the two methods agreed well. There was no consistent preferential antagonism of any antagonist to any agonist.pA2 values determined with stimulation by 4 pulses/100 Hz were by 0.53–0.80 log units higher than those determined with stimulation by 36 pulses/3 Hz. ThepA2 values (4 pulses/100 Hz) of yohimbine and rauwolscine in rabbit brain slices (approximately 7.9 and 8.2, respectively), were slightly higher than in rat brain slices (approximately 7.6 and 7.7, respectively), whereas thepA2 value of idazoxan in the rabbit. (about 7.1) was lower than itspA2 value in the rat (about 8.0). The experiments confirm thatpA2 values determined under conditions of autoinhibition are too low. Stimulation with short (30 ms) bursts of pulses permits the estimation ofpA2 values at presynaptic a2-autoreceptors without (rabbit) or almost without (rat) the complication of autoinhibition. The values suggest that α2-adrenoceptors in rabbit brain cortex differ slightly from those in rat brain cortex.

Characterization of Carrier‐Mediated Efflux in Human Embryonic Kidney 293 Cells Stably Expressing the Rat Serotonin Transporter: A Superfusion Study

Human embryonic kidney 293 cells stably transfected with the rat plasmalemmal serotonin transporter (rSERT) were incubated with 5‐[3H]hydroxytryptamine ([3H]5‐HT) and superfused. Substrates of the rSERT, such as p‐chloroamphetamine (PCA) or methylenedioxymethamphetamine, concentration‐dependently increased basal efflux of [3H]5‐HT. 5‐HT reuptake blockers (e.g., imipramine, citalopram) also caused an enhancement of [3H]5‐HT efflux, reaching about half the maximal effect of the rSERT substrates. In uptake experiments, both groups of substances concentration‐dependently inhibited 5‐HT uptake. EC50 values obtained in superfusion experiments significantly correlated with IC50 values from uptake studies (r2 = 0.92). Addition of the Na+,K+‐ATPase inhibitor ouabain (100 μM) to or the omission of K+ from the superfusion buffer accelerated basal efflux. The effect of PCA (10 μM) was markedly enhanced by both measures, whereas the effect of uptake inhibitors remained unchanged. When [3H]MPP+, a substrate with low affinity for the rSERT, was used instead of [3H]5‐HT for labeling the cells, uptake inhibitors failed to augment efflux. By contrast, PCA accelerated [3H]MPP+ efflux, and its effect was strongly enhanced in the presence of ouabain. The results suggest that the [3H]5‐HT efflux caused by substrates of rSERT is carrier‐mediated, whereas efflux induced by uptake inhibitors is a consequence of interrupted high‐affinity reuptake that is ongoing even under superfusion conditions.

Implications of the EU directive on clinical trials for emergency medicine.

A laudable attempt by the European Union to implement good clinical practice in the conduct of clinical trials on drugs for human use will, unless amended, make impossible a range of potentially life saving studies after May 2004. Directive 2001/20/EC, adopted in April last year, is an important and comprehensive document.1 It is a cornerstone of a Europe-wide harmonisation of the provisions governing clinical trials and can be expected to foster and facilitate multinational clinical research. It will be adopted by member states before 1 May 2003, and its provisions will be applied from 1 May 2004 at the latest. Several articles in the directive deal with the protection of clinical trial subjects. Article 5 outlines the conditions for research in incapacitated patients unable to give informed consent. The article, however, is framed to address the needs of individuals who are incapacitated for long periods, many even permanently. A clinical trial can only be done if “informed consent of the legal representative has been obtained.” …