Tumor Enucleation of Renal Cell Carcinoma in a Solitary Kidney.

We describe the case of a 57 year old man with a solitary kidney after undergoing resection of a Wilm's tumor as a child and a recent left partial colectomy who presents with an incidentally found clinical T1b renal mass. The patient underwent tumor enucleation and had no change in his renal function twelve days after surgery as compared to his preoperative baseline, highlighting the additional nephron-sparing associated with tumor enucleation as compared to partial nephrectomy that includes a gross margin of normal parenchyma.

Research ethics committees in Europe: trials and tribulations.

INTRODUCTION: Ethics committees have been an integral part of clinical research since 1975, when they were introduced through the amendment of the Declaration of Helsinki. Every proposal for clinical research on human subjects has to be submitted to an independent ethics committee for review and approval. The European Clinical Trials Directive 2001/20/EC was implemented in 2004 to harmonise the legislative framework for clinical research in Europe in order to make Europe more competitive in clinical research while at the same time improving the protection of research participants. RESULTS: We have evaluated the situation of ethics committees in Europe five years after the implementation of the new law with special consideration of the number of Ethics Committees per European Member State and the number of members within the specific committees, including the selection of members, also in regard to gender aspects and training requirements, the remuneration or compensation of members in regard of their review obligations, and also issues of conflicts of interest. CONCLUSION: Inadequate remuneration for professional services and gender imbalance are universal concerns across Europe. As the position of ethics committees changes continuously towards greater responsibility, further guidance is needed to uniformly adapt their structures to those needs.

Placebo-controlled trials in major depression are necessary and ethically justifiable: how to improve the communication between researchers and ethical committees.

Despite numerous placebo-controlled clinical trials with antidepressants were conducted in humans and a large amount of data was already published in the last two decades, the members of the 4th European Expert Forum on Ethical Evaluation of Placebo-Controlled Studies in Depression were agreed that placebo-controlled trials with antidepressants also in the future are essential. Placebo-controlled studies measure the effect size in a reliable way and establish sensitivity and internal validity. They are scientifically sound and interpretable in terms of efficacy and are, therefore, clinically more relevant than non-placebo-controlled clinical trials. The "Note of Clarification" of the Declaration of Helsinki opens up where such trials are acceptable. This statement of the members of the 4th European Expert Forum is directed to academia, members of Ethic Committees, regulators, and industry to facilitate their decisions towards clinical studies with antidepressants. "Checklists" for the contents of patients information are given as well as for the investigator.Placebo-controlled clinical trials are scientifically necessary, ethical and feasible. The administration of the placebo is in itself a non-specific treatment and experts agree that there appears to be no increased suicidal risk in the placebo-group of carefully selected and monitored study patients.

Substantial loss of substrate by diffusion during uptake in HEK-293 cells expressing neurotransmitter transporters.

Human embryonic kidney 293 (HEK-293) cells stably transfected with the human serotonin (5-HT) or dopamine transporter (hSERT, hDAT), or the rat GABA transporter GAT-1 were incubated with saturating concentrations of transporter substrates (hSERT: [(3)H]5-HT, [(3)H]N-methyl-phenyl-pyridinium (MPP+); hDAT: [(3)H]dopamine, [(3)H]MPP(+); rGAT: [(3)H]GABA). Uptake velocities decreased significantly over time for [(3)H]5-HT and [(3)H]dopamine (already visible at 1 min), but not for [(3)H]MPP(+) or [(3)H]GABA. In efflux experiments cells were preloaded and substrate diffusion into the medium was studied following the addition of appropriate uptake inhibitors. Fractional effluxes were (% min(-1)) 1.27, 0.72, 0.27 and 0.08 for [(3)H]5-HT, [(3)H]dopamine, [(3)H]MPP(+) and [(3)H]GABA, respectively. The results suggest that in uptake experiments the more lipophilic substrates [(3)H]5-HT and [(3)H]dopamine leave the cells by diffusion already after a short time (1 min) of accumulation.

Quantitative analysis of inward and outward transport rates in cells stably expressing the cloned human serotonin transporter: inconsistencies with the hypothesis of facilitated exchange diffusion.

Quantitative aspects of inward and outward transport of substrates by the human plasmalemmal serotonin transporter (hSERT) were investigated. Uptake and superfusion experiments were performed on human embryonic kidney 293 cells permanently expressing the hSERT using [(3)H]serotonin (5-HT) and [(3)H]1-methyl-4-phenylpyridinium (MPP(+)) as substrates. Saturation analyses rendered K(m) values of 0.60 and 17.0 microM for the uptake of [(3)H]5-HT and [(3)H]MPP(+), respectively. Kinetic analysis of outward transport was performed by prelabeling the cells with increasing concentrations of the two substrates and exposing them to a saturating concentration of p-chloroamphetamine (PCA; 10 microM). Apparent K(m) values for PCA induced transport were 564 microM and about 7 mM intracellular [(3)H]5-HT and [(3)H]MPP(+), respectively. Lowering the extracellular Na(+) concentrations in uptake and superfusion experiments revealed differential effects on substrate transport: at 10 mM Na(+) the K(m) value for [(3)H]5-HT uptake increased approximately 5-fold and the V(max) value remained unchanged. The K(m) value for [(3)H]MPP(+) uptake also increased, but the V(max) value was reduced by 50%. When efflux was studied at saturating prelabeling conditions of both substrates, PCA as well as unlabeled 5-HT and MPP(+) (all substances at saturating concentrations) induced the same efflux at 10 mM and 120 mM Na(+). Thus, notwithstanding a 50% reduction in the V(max) value of transport into the cell, MPP(+) was still able to induce maximal outward transport of either substrate. Thus, hSERT-mediated inward and outward transport seems to be independently modulated and may indicate inconsistencies with the classical model of facilitated exchange diffusion.

Oligomerization of the human serotonin transporter and of the rat GABA transporter 1 visualized by fluorescence resonance energy transfer microscopy in living cells.

Recent biochemical studies indicate that the serotonin transporter can form oligomers. We investigated whether the human serotonin transporter (hSERT) can be visualized as an oligomer in the plasma membrane of intact cells. For this purpose, we generated fusion proteins of hSERT and spectral variants of the green fluorescent protein (cyan and yellow fluorescent proteins, CFP and YFP, respectively). When expressed in human embryonic kidney 293 cells, the resulting fusion proteins (CFP-hSERT and YFP-hSERT) were efficiently inserted into the plasma membrane and were functionally indistinguishable from wild-type hSERT. Oligomers were visualized by fluorescence resonance energy transfer microscopy in living cells using two complementary methods, i.e. ratio imaging and donor photobleaching. Interestingly, oligomerization was not confined to hSERT; fluorescence resonance energy transfer was also observed between CFP- and YFP-labeled rat gamma-aminobutyric acid transporter. The bulk of serotonin transporters was recovered as high molecular weight complexes upon gel filtration in detergent solution. In contrast, the monomers of CFP-hSERT and YFP-hSERT were essentially undetectable. This indicates that the homo-oligomeric form is the favored state of hSERT in living cells, which is not significantly affected by coincubation with transporter substrates or blockers. Based on our observations, we conclude that constitutive oligomer formation might be a general property of Na(+)/Cl(-)-dependent neurotransmitter transporters.

Transporter-mediated release: a superfusion study on human embryonic kidney cells stably expressing the human serotonin transporter.

HEK 293 cells stably expressing the human serotonin transporter (hSERT) were grown on coverslips, preincubated with [(3)H]5-hydroxytryptamine (5-HT), and superfused. Substrates of the hSERT [e.g., p-chloroamphetamine (PCA)], increased the basal efflux of [(3)H]5-HT in a concentration-dependent manner. 5-HT reuptake blockers (e.g., imipramine, paroxetine) also raised [(3)H]5-HT efflux, reaching approximately one-third of the maximal effect of the hSERT substrates. In uptake experiments, both groups of substances inhibited [(3)H]5-HT uptake. Using the low-affinity substrate [(3)H]N-methyl-4-phenylpyridinium (MPP(+)) to label the cells in superfusion experiments, reuptake inhibitors failed to enhance efflux. Similar results were obtained using human placental choriocarcinoma (JAR) cells that constitutively express the hSERT at a low level. By contrast, PCA raised [(3)H]MPP(+) efflux in both types of cells, and its effect was inhibited by paroxetine. The addition of the Na(+),K(+)-ATPase inhibitor ouabain (100 microM) to the superfusion buffer enhanced basal efflux of [(3)H]5-HT-loaded hSERT cells by approximately 2-fold; the effect of PCA (10 microM) was strongly augmented by ouabain, whereas the effect of imipramine was not. The Na(+)/H(+) ionophore monensin (10 microM) also augmented the effect of PCA on efflux of [(3)H]5-HT as well as on efflux of [(3)H]MPP(+). In [(3)H]5-HT-labeled cells, the combination of imipramine and monensin raised [(3)H]5-HT efflux to a greater extent than either of the two substances alone. In [(3)H]MPP(+)-labeled cells, imipramine had no effect on its own and fully reversed the effect of monensin. The results suggest that the [(3)H]5-HT efflux caused by uptake inhibitors is entirely due to interrupted high-affinity reuptake, which is ongoing even under superfusion conditions.

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 microM) to or the omission of K+ from the superfusion buffer accelerated basal efflux. The effect of PCA (10 microM) 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.

Ion dependence of carrier-mediated release in dopamine or norepinephrine transporter-transfected cells questions the hypothesis of facilitated exchange diffusion.

The mechanism of release mediated by the human dopamine and norepinephrine transporter (DAT and NET, respectively) was studied by a superfusion technique in human embryonic kidney 293 cells stably transfected with the respective transporter cDNA and loaded with the metabolically inert substrate [(3)H]1-methyl-4-phenylpyridinium. Release was induced by amphetamine, dopamine, and norepinephrine or by lowering the sodium or chloride concentration in the superfusion buffer (iso-osmotic replacement by lithium and isethionate, respectively). Efflux of [(3)H]1-methyl-4-phenylpyridinium was analyzed at 30-s time resolution. In both transporters, release induced by the substrates amphetamine, dopamine, and norepinephrine followed the same time course as release induced by the removal of chloride and was faster than that caused by the removal of sodium. In the presence of low sodium (DAT: 10 mM; NET: 5 mM) none of the substrates was able to induce release from either type of cell, but adding back sodium to control conditions promptly restored the releasing action. In the presence of low chloride (DAT: 3 mM; NET: 2 mM), however, amphetamine as well as the catecholamines stimulated release from both types of cell. In contrast with the ion dependence of release observed in superfusion experiments, uptake initial rates of substrates at concentrations used in release experiments were the same or even higher at low sodium than at low chloride. The results indicate a decisive role of extracellular sodium for carrier-mediated release unrelated to the sodium-dependent uptake of the releasing substrate, and suggest a release mechanism different from simple exchange diffusion considering only the amines as substrates.

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.

Twenty years of the ethics committee at the medical faculty of the University of Vienna. An interim report.

The organisation and assessment procedures of the ethics committee of the University of Vienna medical faculty are described. Data concerning the work of the committee from 1993 through to 1997 are given in detail. Finally, the results of a survey among physicians on the acceptance of the committee's work are presented. In conclusion, the workload of an ethics committee in a large medical faculty can only be handled by efficient management of assessment procedures. However, it is difficult to achieve sufficient acceptance in a field governed by various interests.

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

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 approximately 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.

Effects of histamine and nitric oxide synthase inhibition on plasma levels of von Willebrand factor antigen.

Endothelial cells release von Willebrand factor (vWf) either constitutively or by a regulated pathway. Based on various studies in vitro, we hypothesized that the stimulatory action of histamine on vWf release could also be induced in vivo and that it may be inhibited by endogenous production of nitric oxide (NO). Nine healthy subjects received placebo or one of two dosages of a primed constant infusion of the NO-synthase inhibitor N-monomethyl-L-arginine (L-NMMA) in a randomized, double-blind, three-way crossover trial. Histamine was coinfused for 15 minutes at 0.16 microg/kg/min after 30 minutes of pretreatment with either placebo or L-NMMA. Thirty minutes after either the low or the high L-NMMA dose was started, which caused, respectively, a 40% decrease and a 60% decrease in exhaled end expiratory NO level (p = 0.008), there was no increase in von Willebrand factor antigen (vWf-Ag) level (p > 0.05). Histamine caused an 11% (95% confidence interval (CI): 0.4% to 22%; p = 0.011) increase in vWf-Ag level at 125 minutes. After pretreatment with the low and the high L-NMMA doses, vWf-Ag level increased by 18% (Cl: 5% to 31%; p = 0.011) and by 29% (CI: 15% to 42%; p = 0.008), respectively. At 125 minutes, vWf-Ag level was significantly higher after either L-NMMA pretreatment when compared with the results after histamine alone (p < 0.05). In conclusion, the infusion of histamine increased vWf-Ag level, and the inhibition of NO-synthase enhanced this effect, whereas it did not by itself elevate vWf-Ag level. Thus endogenously produced NO may dampen the regulated pathway of vWf secretion; it will be interesting to investigate whether endogenous NO production also inhibits vWf release caused by other stimulators.

The slope parameter of concentration-response curves used as a touchstone for the existence of spare receptors.

The present work was stimulated by findings of a large reserve of presynaptic alpha2-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 alpha2-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 alpha2-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. Evaluation methods which neglect the alteration of the geometrical form of concentration-response curves due to non-proportionality between receptor occupation and relative response do not seem appropriate to quantify spare receptors. These methods may detect spare receptors where they do not exist.

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.

Nitric oxide synthase inhibition in the histamine headache model.

Histamine has been widely used experimentally to induce headache in healthy subjects and migraine in migraineurs. There is evidence that the vascular effects of histamine are at least partially mediated by nitric oxide (NO). Hence we hypothesized that subjective symptoms and hemodynamic effects of histamine could be reduced by systemic NO-synthase inhibition. We therefore studied the effect of pretreatment with N-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of NO-synthase, or placebo on headache, flush and discomfort scores during histamine infusion. Additionally, blood flow velocities in the middle cerebral and the ophthalmic artery and ocular fundus pulsations were measured. Whereas L-NMMA blunted the effect of histamine in the ophthalmic artery and the ocular circulation, NO-synthase inhibition did not mitigate subjective symptoms. Histamine did not affect mean blood flow velocities in the middle cerebral artery. Hence, we conclude that NO-synthase inhibition reduces the histamine-induced vascular effects in the ocular circulation, but is not sufficient to attenuate or abort the subjective symptoms provoked by histamine infusion.

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

1. 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. 2. In DAT cells lowering the Na+ concentration to 0, 5 or 10 mM caused an increase in 3H-efflux. Cocaine (10 microM) or mazindol (0.3 microM) 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 microM) was without effect in all the conditions tested. 3. 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. 4. 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. 5. 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.

Cerebral and ocular hemodynamic effects of sumatriptan in the nitroglycerin headache model.

BACKGROUND AND PURPOSE: Sumatriptan is a selective 5-hydroxytryptamine1d (5-HT1d)-receptor agonist, highly effective in the short-term treatment of migraine headaches. However, the mechanism underlying the action of sumatriptan is not yet completely understood. To further characterize the vascular effects of sumatriptan, we studied the effects on cerebral and ocular circulation in the well-established nitroglycerin headache model. METHODS: In a double-blind, placebo-controlled, randomized, two-way crossover study in 10 healthy male subjects, we administered either placebo plus nitroglycerin or sumatriptan plus nitroglycerin. Blood flow velocity in the middle cerebral artery and the ophthalmic artery, as well as ocular fundus pulsations and systemic hemodynamic parameters, were measured after sumatriptan and placebo and during the following infusion of nitroglycerin. RESULTS: After infusion of nitroglycerin, blood flow velocity in the middle cerebral decreased by -13.3% versus baseline after placebo pretreatment, but by only -2.2% after sumatriptan (treatment effect, +10.8%; p < 0.05). In contrast, sumatriptan had no effect in the ophthalmic artery. Ocular fundus pulsations, which estimate local pulsatile ocular blood flow, were slightly reduced after sumatriptan. Moreover, sumatriptan partially prevented the increase in fundus pulsations during nitroglycerin infusion (treatment effect, -5.4%; p < 0.05). CONCLUSIONS: Sumatriptan prevents the effect of nitroglycerin-induced vasodilation in the middle cerebral artery but not in the ophthalmic artery, which strongly supports the concept that sumatriptan directly vasoconstricts distended basal cerebral arteries. Measurement of ocular fundus pulsations indicates that sumatriptan also has a small vasoconstrictor action on resistance vessels.

Cholera toxin induces cyclic AMP-independent down-regulation of Gs alpha and sensitization of alpha 2-autoreceptors in chick sympathetic neurons.

The role of the stimulatory GTP-binding protein (Gs) in the alpha 2-autoinhibitory modulation of noradrenaline release was investigated in cultured chick sympathetic neurons. The alpha 2-adrenoceptor agonist UK 14,304 caused a concentration-dependent reduction of electrically evoked [3H] noradrenaline release with half-maximal effects at 14.0 +/- 5.5 nM. In neurons treated with 100 ng/ml cholera toxin for 24 h, the half-maximal concentration was lowered to 3.2 +/- 1.4 nM without changes in the maximal effect of UK 14,304. The pretreatment with cholera toxin also increased the inhibitory action of 10 nM UK 14,304 when compared with the inhibition of noradrenaline release in untreated cultures derived from the same cell population. In cultures treated with either 10 microM forskolin or 100 microM 8-bromo-cyclic AMP, neither the half-maximal concentration nor the maximal effect of UK 14,304 was altered. Cholera toxin, forskolin, and 8-bromo-cyclic AMP all induced an increase in spontaneous outflow and a reduction in electrically evoked overflow, effects not observed after a pretreatment with dideoxyforskolin. Exposure of neurons to cholera toxin, but not to forskolin or 8-bromo-cyclic AMP, induced a translocation of alpha-subunits of Gs (Gs alpha) from particulate to soluble fractions and led ultimately to a complete loss of Gs alpha from the neurons. In contrast, no effect was seen on the distribution of either alpha-subunits of Gi- or Ga-type G proteins or of beta-subunits. These results indicate that cholera toxin causes a selective, cyclic AMP-independent down-regulation of Gs alpha. This down-regulation of Gs alpha is associated with the sensitization of alpha 2-autoreceptors.