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Manfred Göthert, who had served Naunyn-Schmiedeberg's Arch Pharmacol as Managing Editor from 1998 to 2005, deceased in June 2019. His scientific oeuvre encompasses more than 20 types of presynaptic receptors, mostly on serotoninergic and noradrenergic neurones. He was the first to identify presynaptic receptors for somatostatin and ACTH and described many presynaptic receptors, known from animal preparations, also in human tissue. In particular, he elucidated the pharmacology of presynaptic 5-HT receptors. A second field of interest included ligand-gated and voltage-dependent channels. The negative allosteric effect of anesthetics at peripheral nACh receptors is relevant for the peripheral clinical effects of these drugs and modified the Meyer-Overton hypothesis. The negative allosteric effect of ethanol at NMDA receptors in human brain tissue occurred at concentrations found in the range of clinical ethanol intoxication. Moreover, the inhibitory effect of gabapentinoids on P/Q Ca 2+ channels and the subsequent decrease in AMPA-induced noradrenaline release may contribute to their clinical effect. Another ligand-gated ion channel, the 5-HT 3 receptor, attracted the interest of Manfred Göthert from the whole animal via isolated preparations down to the cellular level. He contributed to that molecular study in which 5-HT 3 receptor subtypes were disclosed. Finally, he found altered pharmacological properties of 5-HT receptor variants like the Arg219Leu 5-HT 1A receptor (which was also shown to be associated with major depression) and the Phe124Cys 5-HT 1B receptor (which may be related to sumatriptan-induced vasospasm). Manfred Göthert was a brilliant scientist and his papers have a major impact on today's pharmacology., (© 2021. The Author(s).)

The serotonin (5-hydroxtryptamine, 5-HT) system plays a role in analgesia and emesis. The aim of this study was to test whether opioids or ketamine inhibit the human 5-HT transporter and whether this increases free plasma 5-HT concentrations. HEK293 cells, stably transfected with the human 5-HT transporter cDNA, were incubated with morphine, hydromorphone, fentanyl, alfentanil, pethidine (meperidine), tramadol, ketamine, and the reference substance citalopram (specific 5-HT transporter inhibitor). The uptake of [(3)H]5-HT was measured by liquid scintillation counting. In a second series of experiments, study drugs were incubated with plasma of ten healthy blood donors and change of 5-HT plasma-concentrations were measured (ELISA). The end point was the inhibition of the 5-HT transporter by different analgesics either in HEK293 cells or in human platelets ex vivo. Tramadol, pethidine, and ketamine suppressed [(3)H]5-HT uptake dose-dependently with an IC50 of 1, 20.9, and 230 μM, respectively. These drugs also prevented 5-HT uptake in platelets with an increase in free plasma 5-HT. Free 5-HT concentrations in human plasma were increased by citalopram 1 μM, tramadol 20 μM, pethidine 30 μM, and ketamine 100 μM to 280 [248/312]%, 269 [188/349]%, and 149 [122/174]%, respectively, compared to controls without any co-incubation (means [95 % CI]; all p < 0.005). No change in both experimental settings was observed for the other opioids. Tramadol and pethidine inhibited the 5-HT transporter in HEK293 cells and platelets. This inhibition may contribute to serotonergic effects when these opioids are given in combination, e.g., with monoamine oxidase inhibitors or selective serotonin reuptake inhibitors.

Besides the three antidepressant-sensitive, Na(+)- and Cl(-)-dependent monoamine transporters, Na(+)-independent organic cation transporters (OCTs) are known to transport monoamines. However, little is known about the interactions of psychotropic drugs with human (h) OCTs. In the present study, a series of diverse antidepressant and antipsychotic drugs were examined for their inhibitory potency at hOCT1, hOCT2 and hOCT3 by measuring inhibition of [(3)H]-MPP(+) uptake into HEK293 cells stably expressing one of the three hOCTs. The inhibitory potencies (IC(50)s) ranged from 1 to 900 μM. Most of the examined drugs showed highest inhibitory potency at hOCT1 which is very sparsely expressed in the brain and mainly involved in renal and hepatic clearance of cationic drugs. At their upper therapeutic plasma concentrations, several drugs are expected to inhibit by more than 20 % hOCT1 and could thus interfere with the pharmacokinetics of hOCT1-transported drugs in the kidney and liver, namely trimipramine, desipramine and fluoxetine (by about 37 %), levomepromazine and nefazodone (by about 32 %), and clozapine and amitriptyline (by about 22 %). At hOCT2 and hOCT3, which are involved in monoamine homeostasis in the brain, IC(50)s of most psychoactive drugs were in the high micromolar range. At their upper plasma concentrations, only three compounds, bupropion, nefazodone and clozapine, showed potential for inhibition, of about 18 % at hOCT2 (bupropion), about 22 % at hOCT3 (nefazodone) and of approximately 10 % at hOCT2 and hOCT3 (clozapine). Thus, under the assumption of a tenfold accumulation in the brain, bupropion, nefazodone and clozapine may notably inhibit the corresponding hOCTs. It remains to be shown whether such a direct inhibition plays a role in the clinical effects of these three drugs.

The muscarinic antagonists oxybutynin and trospium are used as spasmolytic agents for the treatment of overactive urinary bladder disease. Recently, it has been shown that trospium, but not oxybutynin, is a substrate of the multidrug efflux carrier P-glycoprotein, but carrier-mediated drug uptake has not been directly analysed for both drugs. However, trospium has been previously shown to exhibit inhibitory potency for the organic cation transporters (OCTs). The aim of the present study was to examine whether trospium and oxybutynin are substrates, i.e. are transported by the human OCTs (hOCT(1), hOCT(2) and hOCT(3)). Therefore, we measured total and specific (decynium-22-sensitive) uptake, and saturation kinetics of the uptake for [(3)H]oxybutynin and [(3)H]trospium in human embryonic kidney (HEK293) cells transiently transfected with the cDNA of hOCT(1), hOCT(2) or hOCT(3). In addition, we determined IC(50) values for inhibition of hOCT-mediated [(3)H]MPP(+) uptake by unlabelled trospium and oxybutynin. Total uptake of [(3)H]oxybutynin was very high in all transfected HEK293 cells and only a small portion was due to specific, decynium-22-sensitive hOCT-mediated uptake. Oxybutynin inhibited [(3)H]MPP(+) uptake by the three hOCTs with IC(50) values between 20 and 130 μM. Direct determination of transport kinetics was measurable only at hOCT(1) with K (m) of 8 μM and V (max) of 484 pmol/mg protein/min. The rank order of affinity (1/IC(50) or 1/K (m)) of specific oxybutynin uptake was hOCT(1) > hOCT(2) = hOCT(3). The observed high non-specific uptake is obviously a consequence of the high lipophilicity of this uncharged drug. Thus, hOCTs may not play a significant role for the overall pharmacokinetics and tissue distribution of oxybutynin. However, and in contrast to oxybutynin, uptake of [(3)H]trospium, an organic cation, was mainly due to carrier-mediated uptake by the three hOCTs. With IC(50) values of 18, 1.4 and 710 μM (at hOCT(1), hOCT(2) and hOCT(3), respectively) and K (m) values of 17 and 8 μM and about identical V (max) values of about 90 pmol/mg protein/min at hOCT(1) and hOCT(2), respectively; the rank order of affinity (1/IC(50) or 1/K (m)) of specific uptake of trospium was hOCT(2) > hOCT(1) > > hOCT(3). Thus, hOCTs very probably contribute to the active tubular and hepatobiliary secretion of trospium. Furthermore, hOCT(1) and hOCT(3) may be involved in the tissue uptake of this drug in the urinary bladder.

Monoamine neurotransmission is efficiently terminated through synaptic reuptake of released neurotransmitters by high-affinity Na(+)- and Cl(-)-dependent neuronal monoamine transporters of the SLC6A family located in the plasma membrane of presynaptic nerve terminals. Recently, a low-affinity, high-capacity Na(+)- and Cl(-)-independent plasma membrane monoamine transporter (PMAT) belonging to the SLC29 solute carrier family has been cloned. PMAT was shown to transport monoamine neurotransmitters as well as organic cations such as 1-phenyl-4-methyl-pyridinium (MPP(+)). Thus, the PMAT which is highly expressed in the human brain may be involved in the modulation of central monoaminergic neurotransmission and it may be a target for drugs used to treat depression and schizophrenia, i.e., dysregulations of the monoamine homeostasis in the central nervous system (CNS). Therefore, we examined in transfected cells the influence on [(3)H]-MPP(+) transport by the human PMAT (hPMAT) of nine monoamine transport inhibiting antidepressants (ADs) belonging to pharmacologically diverse classes (imipramine, desipramine, amitriptyline, bupropion, fluoxetine, sertraline, paroxetine, reboxetine, and venlafaxine), of the atypical ADs tianeptine and trimipramine and of five antipsychotics (levomepromazine, haloperidol, clozapine, olanzapine, and risperidone). All examined drugs inhibited the hPMAT; however, half-maximum inhibition (IC(50)) was observed at concentrations which were much higher than reported clinical plasma concentrations of these drugs. Thus, inhibition of the hPMAT by these CNS drugs may not (or only marginally) contribute to their therapeutic effects.

The haloalkylamine DSP4 (N[-2-chloroethyl]-N-ethyl-2-bromobenzylamine) is a noradrenergic neurotoxin, which is used for the chemical denervation of noradrenergic neurons, and it has been proposed to be a selective substrate for the neuronal, Na(+)- and Cl(-)-dependent noradrenaline transporter (NAT). In the present study, we investigated whether DSP4 not only interacts with the human NAT (hNAT) but also with other neuronal monoamine transporters such as the transporters for dopamine (hDAT) and serotonin (hSERT) or with nonneuronal (Na(+)-independent) monoamine transporters also known as organic cation transporters (OCTs), such as hOCT(1), hOCT(2), and hOCT(3). Using human embryonic kidney HEK293 cells heterologously expressing the corresponding transporter, we show that DSP4 irreversibly inhibits the hNAT, hDAT, hSERT, and hOCT(3). However, this inhibition includes a reversible component at the hDAT, hSERT, and hOCT(3) but not at the hNAT. The inhibitory potency of DSP4 at the neuronal transporters was highest at the hNAT (IC(50) about 5 microM), and it was about five and 40 times lower at the hSERT and hDAT, respectively. DSP4 inhibited all three hOCTs with high potency (IC(50) about 1 microM) but in a completely reversible manner at hOCT(1) and hOCT(2). Cytotoxicity by 24-h exposure of hNAT- or hOCT-expressing cells to low DSP4 concentrations (<10 microM) could be observed only in hNAT-expressing cells. Thus, DSP4's high-affinity uptake through the NAT together with its completely irreversible mode of interaction with the NAT may contribute to its selectivity as noradrenergic neurotoxin.

Using transfected HEK293 cells that express the human (h) noradrenaline transporter (hNAT), we show differential inhibitory effects of the thiol reagent N-ethylmaleimide (NEM) on [(3)H]NA uptake and [(3)H]nisoxetine binding. Irreversible inhibition of uptake by NEM was complete, faster, and occurred at lower concentrations. Furthermore, hNAT ligands (substrates and inhibitors) prevented NEM-induced inhibition of binding but not that of uptake, indicating different underlying mechanisms of inhibition. NEM-induced uptake inhibition was not primarily due to inhibition of the Na(+)/K(+)-ATPase since ouabain caused only partial inhibition. For the first time, we show that NEM at low concentrations causes a rapid and complete depletion of cellular adenosine triphosphate (ATP) not only in HEK293 cells but also in several other eukaryotic cell lines. Thus, while high NEM concentrations alkylate the NAT protein in a ligand-protectable manner, low concentrations inhibit substrate uptake through a loss of the Na(+) and K(+) gradient as a driving force by depleting cellular ATP.

The cDNA of the murine noradrenaline transporter (mNAT) was cloned from the RNA of the placenta of a C57BL/6 mouse. The cloned mNAT differs from a previously published sequence in two amino acids within the C-terminal region. A cDNA obtained from an inbred mouse strain showed a further amino acid exchange (Ile(505)Val) within the fifth intracellular loop. The pharmacological properties of both, the wild-type mNAT and the variant (mNAT-I(505)V), were studied in human embryonic kidney HEK293 cells transfected with the corresponding cDNA. The kinetic constants for transport (K (m), V (max)) of [(3)H]noradrenaline ([(3)H]-NA) and binding (K (D), B (max)) of the selective NAT inhibitor [(3)H]nisoxetine were not different between the two isoforms; the mean kinetic constants amounted to about 4 microM and 120pmol/mg protein for K (m) and V (max) and 6nM and 18pmol/mg protein for K (D) and B (max), respectively. [(3)H]-NA transport by both isoforms showed the typical properties of an NAT because it was dependent on sodium and chloride and inhibited with almost identical K (i) values by various NAT substrates and inhibitors. The only significant pharmacological difference identified between the two mNAT isoforms was an about threefold higher affinity for cocaine of the very rare mNAT-I(505)V variant.

In view of the high structural and pharmacological similarities between the alpha(2A)-adrenoceptors of humans and other mammalian species, it has been concluded, in particular, from experiments in rabbits that the (2A)-adrenoceptor is the exclusive site of action of central antihypertensive drugs, although the amino acid sequence of the alpha(2A)-adrenoceptor of just this species was unknown. Therefore, the aim of the present investigation was to determine the complete nucleotide sequence of the coding region of the rabbit alpha(2A)-adrenoceptor gene. Degenerate oligonucleotides corresponding to regions of the alpha(2A)-adrenoceptor conserved between rat and man were used in a polymerase chain reaction with genomic DNA prepared from rabbit. A 1,356-base pair product with an open reading frame of 1,353 base pairs was obtained that encodes a protein of 451 amino acids which is similar to the alpha(2A)-adrenoceptors of other mammals (man, pig, rat, mouse, guinea-pig and cattle) but not to their alpha(2B)- and alpha(2C)-adrenoceptor subtypes suggesting its classification as an alpha(2A)-adrenoceptor. However, the degree of amino acid sequence identity is, at best, only 80% and, thus, about 10% less than between the other mammalian species. Compared with the human sequence there are 81 substantial changes of amino acids. In conclusion, rabbit and human alpha(2A)-adrenoceptors substantially differ in their amino acid sequence which may explain the opposite pharmacodynamic properties of the central antihypertensive drug rilmenidine (alpha(2)-adrenoceptor agonism and antagonism, respectively) reported in the literature. Hence, the present study supports the view that experiments with central antihypertensive drugs in rabbits are not reliably predictive for the site of action of such drugs in man.

The pharmacological properties of a specific agmatine uptake mechanism were investigated in the human glioma cell line SK-MG-1 and compared with those of the putrescine transporter expressed by the same cells and with those of several other organic cation transport systems or ion channels reported in the literature. The specific accumulation of [14C]agmatine at 37 degrees C above nonspecific accumulation at 4 degrees C was energy-dependent and saturable with a Vmax of 64.3+/-3.5 nmol/min per mg protein and a Km of 8.6+/-1.4 microM. Specific accumulation was attenuated by replacement of extracellular Na+ by choline by 65%, not affected by lithium and enhanced by replacement by sucrose. Phentolamine, clonidine, 1,3-di(2-tolyl)guanidine, histamine, putrescine, spermine and spermidine were inhibitors of specific [14C]agmatine accumulation. In contrast, corticosterone, desipramine, O-methylisoprenaline, cirazoline, moxonidine, L-arginine, L-lysine, verapamil, nifedipine and CdCl2 at concentrations up to 10 mM failed to inhibit specific [14C]agmatine accumulation, thus excluding that the latter is mediated by amino acid or monoamine carriers, by Ca2+ channels or by the organic cation transporters OCT1, OCT2, OCT3, OCTN1 or OCTN2. The pattern of activity of inhibitory compounds was also different from that determined for specific putrescine accumulation found in the same cells (Km 1.3+/-0.1 microM, Vmax 26.1+/-0.4 nmol/min per mg protein) ruling out an identity of the specific [14C]agmatine and [14C]putrescine accumulation mechanisms. It is concluded that specific accumulation of agmatine in human glioma cells is mediated by a specific transporter whose pharmacological properties are not identical to those of the agmatine transporter previously identified in rat brain synaptosomes and to other so far known carrier mechanisms for organic cations and ion channels. The agmatine uptake system may be important for the regulation of the extracellular concentration of agmatine in man.

Serotonin (5-HT) exerts fast excitatory responses by activation of 5-HT3 receptors, irrespective of whether they are homomerically composed of 5-HT3A subunits or heteromerically assembled of 5-HT3A and 5-HT3B subunits. Here we describe a short, truncated (h5-HT3AT) and a long (h5-HT3AL) splice variant of the human 5-HT3A (hS-HT3A) receptor subunit. The deduced protein of the short isoform consists of 238 amino acids (aa) with a single transmembrane domain (M1). Compared to the known 5-HT3A receptor, the long isoform contains 32 additional aa in the extracellular loop between M2 and M3. Both splice variants are co-expressed together with the 5-HT3A subunit in the amygdala and hippocampus, whereas in the placenta only the short variant is co-expressed. Both splice variants, when expressed in transfected human embryonic kidney (HEK) 293 cells, are not able to form functional homomeric receptors, but modify 5-HT response at heteromeric h5-HT3A receptors. Co-expression of the short variant considerably decelerates the desensitization of the 5-HT3 receptor; thus, heteromeric assemblies of h5-HT3A and the h5-HT3AT subunit exhibit 5-HT-induced cation fluxes which are much larger than those of homomeric hS-HT3A receptors. In contrast, heteromeric complexes containing the h5-HT3AL subunit display reduced cation fluxes. In conclusion, the splice variants increase the functional diversity of 5-HT3 receptors.

The patch-clamp technique was used on excised (outside-out) patches to characterize h5-HT3A receptors stably transfected in HEK 293 cells and to compare the effects of the barbiturate anaesthetics methohexital and pentobarbital on this ligand-gated cation channel. At negative membrane potentials 5-HT induced inward currents in a concentration-dependent manner (EC50=8.6 microM, Hill coefficient =1.5). The mean peak current induced by 30 microM 5-HT was -110 pA at -100 mV. The 5-HT3A receptor antagonist ondansetron (0.3 nM) reversibly inhibited the 5-HT (30 microM) signal by 70% and at 3 nM it abolished the response. Methohexital and pentobarbital inhibited 5-HT-induced (30 microM) currents in a concentration-dependent manner. The maximal inhibition with a given methohexital or pentobarbital concentration was reached when the respective drug was applied 45 s prior to and during the 2-s 5-HT pulse (IC50 values=95 microM and 127 microM, Hill coefficient = -1.0 and -1.6, respectively). Although the barbiturates were, thus, equipotent, their effects differed substantially with respect to the dependence on the time schedule of application to the patches: the potency of methohexital was virtually maximal when the drug was applied exclusively 45 s before the agonist pulse, but its inhibitory potency decreased considerably when it was exclusively applied during the 2-s 5-HT pulse (IC50=380 microM). Conversely, pentobarbital was almost maximally potent in inhibiting the 5-HT signal when it was exclusively coapplied with this agonist, but its inhibitory potency was considerably lower (IC50 approximately 500 microM) when applied exclusively 45 s before 5-HT. Another difference between both barbiturates involves the rate of inactivation of 5-HT3 receptor-mediated currents: whereas high concentrations of methohexital (> or = 300 microM) were necessary to induce moderate (< or = twofold) acceleration of this parameter, pentobarbital produced such an effect at all concentrations and the extent of acceleration increased with increasing concentration (1.5- to fivefold). In conclusion, two barbiturates, chemically closely related but of different lipophilicity, clearly differ with respect to the kinetics of their effect on 5-HT3 receptor channels; one possible explanation involves drug access to an amphipathic site of action via both an aqueous and a hydrophobic pathway. Pentobarbital, in contrast to methohexital, inhibits hS-HT3A receptor-mediated currents at anaesthetic concentrations (approximately 90 microM).

Nearly 30% of patients treated with metformin experience gastrointestinal side effects. Since release of 5-hydroxytryptamine (5-HT) from the intestine is associated with nausea, vomiting, and diarrhea, we examined whether metformin induces 5-HT release from the intestinal mucosa. In 40% of tissue biopsy specimens of human duodenal mucosa, metformin (1, 10, and 30 microM) caused an increase in 5-HT outflow by 35, 70, and 98%, respectively. Peak increases in 5-HT outflow were observed after 10-15 min exposure to metformin, returning to baseline levels after 25 min. Tetrodotoxin (1 microM) reduced by about 50% the metformin-evoked increase in 5-HT outflow (P<0.05). Metformin-evoked release was not affected by scopolamine + hexamethonium, propranolol, the 5-HT3 receptor antagonist dolasetron, naloxone, or the NK1 receptor antagonist L703606. In the presence of tetrodotoxin (1 microM), somatostatin (1 microM) further reduced metformin-induced 5-HT release by 15-20%. In view of the 5-HT releasing effects of selective 5-HT3 receptor agonists to which metformin (N-N-dimethylbiguanide) is structurally related, we investigated whether metformin directly interacts with 5-HT3 receptors. Receptor binding (inhibition of [3H]-GR65630 binding) and agonist effects (stimulation of [14C]-guanidinium influx) at 5-HT3 receptors were studied in murine neuroblastoma N1E-115 cells, which express functional 5-HT3 receptors. Metformin up to 0.3 mM failed to inhibit [3H]-GR65630 binding and to modify displacement of [3H]-GR65630 binding induced by 5-HT. 5-HT (3 microM) stimulated the influx of [14C]-guanidinium in intact N1E-115 cells. Metformin up to 1 mM failed to modify basal influx, 5-HT-induced influx, and 5-HT+ substance P-induced influx of [14C]-guanidinium. Our results indicate that metformin induces 5-HT3 receptor-independent release of 5-HT from human duodenal mucosa via neuronal and non-neuronal mechanisms. Part of the gastrointestinal side effects observed during treatment with metformin could, thus, be produced by the release of 5-HT and other neurotransmitter substances within the duodenal mucosa.

Effects of some naturally occurring steroids and synthetic analogues on the cation flux through the cation channel of the 5-HT3 receptor and the voltage-gated and tetrodotoxin-sensitive sodium channel were studied in N1E-115 mouse neuroblastoma cells by measuring the 2-min influx of the organic cation [14C]-guanidinium. The cation fluxes in intact cells were either induced by 2 min exposure of the cells to 5-hydroxytryptamine (5-HT, 100microM) or to veratridine (1 mM). Influx of [14C]-guanidinium through both channels was concentration-dependently inhibited by all compounds studied. The rank order of potency for inhibition of the 5-HT3 receptor-induced cation flux was clomiphene approximately/= cyproterone acetate > estradiol > progesterone approximately/= allotetrahydrodeoxycorticosterone > alfaxalone approximately/= testosterone > aldosterone > dexamethasone. With the exception of dexamethasone and testosterone, which were more potent at the voltage-dependent sodium channel, and progesterone and testosterone, which were about nearly equipotent in inhibiting both cation channels, the steroids were twofold (alfaxalone, allotetrahydrodeoxycorticosterone) to 107-fold (cyproterone acetate) more potent at the 5-HT3 receptor channel than at the voltage-gated sodium channel. The potencies of the steroids (and the synthetic analogues) for inhibition of the 5-HT3 receptor-induced [14C]-guanidinium influx were correlated with their lipophilicity (log P values). A similar correlation between log P values and pIC50 values for the steroid-induced inhibition of the veratridine-evoked cation influx through the voltage-gated sodium channel was only found when cyproterone acetate (a compound with extremely low inhibitory potency at this channel) was not included in the regression analysis. The results indicate that both the 5-HT3 receptor channel and the voltage-gated sodium channel are targets for steroids. The relationship between most of the compounds in inhibiting both cation channels and lipophilicity is compatible with a common mechanistic principle in steroid-induced inhibition of the two channels, i.e. a nonspecific hydrophobic interaction with certain membrane lipids in the neighbourhood of the two channels.

Human embryonic kidney (HEK) 293 cells were stably transfected with the cDNA encoding the short splice variant of the mouse 5-HT3 receptor (m5-HT3A(b); isolated by RT-PCR from NG108-15 cells) and its pharmacological properties were compared with those of the native 5-HT3 receptor of the mouse neuroblastoma cell line N1E-115. The m5-HT3A(b) receptor of N1E-115 cells differs from that isolated from NG108-15 cells by one amino acid (Val instead of Ile) at position 52 of the amino acid sequence. Both radioligand binding studies with the selective 5-HT3 receptor antagonist [3H]GR65630 (3-(5-methyl-1H-imidazol-4-yl)-1-(1-methyl-1H-indol-3-yl)-1-propanone) and functional experiments by measurement of [14C]guanidinium influx evoked by 5-HT in the absence and presence of 10 microM substance P were carried out. Binding of [3H]GR65630 to the recombinant receptor in HEK 293 cells and the native receptor in N1E-115 cells was specific and of high affinity (Kd 4.4 and 3.0 nM, respectively) and characterized by Bmax values of 875 and 1414 fmol/mg protein, respectively. At 10 nM [3H]GR65630, specific binding was inhibited by the selective 5-HT3 receptor antagonist ondansetron (Ki 11 and 42 nM, respectively) and by 5-HT (Ki 294 and 563 nM, respectively). In the transfected HEK 293 cells, 5-HT induced an influx of [14C]guanidinium both in the absence (pEC50 5.7) and presence of substance P (pEC50 6.6,) which was counteracted by 0.3 microM ondansetron; in the N1E-115 cells, 5-HT also evoked [14C]guanidinium influx in the absence (pEC50 6.0) and presence of substance P (pEC50 6.0). Both in transfected HEK 293 cells and in N1E-115 cells, the 5-HT receptor ligand RS-056812-198 ((R)-N-(quinuclidin-3-yl)-2-(1-methyl-1 H-indol-3-yl)-2-oxo-acetamide; in the presence of substance P) induced an influx of [14C]guanidinium (pEC50 9.8 and 8.7, respectively) with a maximum of about 70 and 30% of the maximum response to 5-HT, respectively. 5-HT (in the presence of substance P)-induced [14C]guanidinium influx was inhibited by the imidazoline BDF 6143 (4-chloro-2(2-imidazolin-2-ylamino)-isoindoline; pIC50 4.9 and 5.3, respectively) and by the sigma-site ligand (+/-)-ifenprodil (pIC50 5.0 and 5.2, respectively). In conclusion, most of the drugs exhibited practically identical properties at both the recombinant m5-HT3A(b) receptor in HEK 293 cells and the native m5-HT3 receptor of N1E-115 cells. However, the recombinant receptor had a higher affinity for ondansetron, and the potency of 5-HT in inducing cation influx through the recombinant, but not through the native receptor, was increased by substance P. RS-056812-198 was a 10-fold more potent partial agonist at the recombinant than at the native receptor. These differences may be due to cell-specific post-translational modifications of the 5-HT3 receptor protein in the two cell lines, to the expression of other subunits in addition to the m5-HT3A(b) receptor in N1E-115 cells and/or to the difference in the amino acid sequence at position 52 of the short splice variants of the m5-HT3 receptors expressed in the two cell lines.

Effects of the intravenous anaesthetic ketamine on the desipramine-sensitive noradrenaline transporter (NAT) were examined in cultured bovine adrenal medullary cells and in transfected Xenopus laevis oocytes expressing the bovine NAT (bNAT). Incubation (1-3 h) of adrenal medullary cells with ketamine (10-300 microM) caused an increase in appearance of catecholamines in culture medium. Ketamine (10-1000 microM) inhibited desipramine-sensitive uptake of [3H]noradrenaline (NA) (IC50=97 microM). Saturation analysis showed that ketamine reduced Vmax of [3H]NA uptake without changing Km, indicating a non-competitive inhibition. Other inhibitors of NAT, namely cocaine and desipramine, showed a competitive inhibition of [3H]NA uptake while a derivative of ketamine, phencyclidine, showed a mixed type of inhibition. Ketamine (10-1000 microM) also inhibited the specific binding of [3H]desipramine to plasma membranes isolated from bovine adrenal medulla. Scatchard analysis of [3H]desipramine binding revealed that ketamine increased Kd without altering Bmax, indicating a competitive inhibition. In transfected Xenopus oocytes expressing the bNAT, ketamine attenuated [3H]NA uptake with a kinetic characteristic similar to that of cultured adrenal medullary cells. These findings are compatible with the idea that ketamine non-competitively inhibits the transport of NA by interacting with a site which partly overlaps the desipramine binding site on the NAT.

The patch-clamp technique was applied in out-side-out patches of N1E-115 mouse neuroblastoma cells to investigate the effects of ifenprodil [(+/-) erythreo-ifenprodil tartratel, a drug with neuroprotective properties in cerebral ischemia, on the inward currents through 5-HT3 receptor channels. A high time resolution was achieved by using a rapid solution exchange system (exchange rate <1 ms). Ifenprodil inhibited the peak currents evoked by 30 microM 5-HT in a concentration-dependent but voltage-independent manner. The effect was most potent when ifenprodil was continuously applied to the patches 45 s before and during the 2-s administration of 5-HT (IC50=16 microM) and it was only slightly less potent when it was applied during the 45 s prior to 5-HT only (IC50=29 microM). When applied in this manner, ifenprodil also produced a concentration-dependent increase of the onset time constant (tauON) of the 5-HT (30 microM)-induced currents. When the drug was exclusively co-applied with 5-HT, ifenprodil was least potent in inhibiting the peak currents (IC50=98 microM), and it had no effect on the current onset kinetics. All protocols of ifenprodil application accelerated current inactivation as reflected by a decrease of the current inactivation time constant (tauOFF). All effects of ifenprodil were reversible after washout periods of 2-5 min. In conclusion, the potency of ifenprodil in inhibiting the inward current through 5-HT3 receptor channels is strongly dependent on the application protocol: presence of the drug before the agonist-induced activation of the 5-HT3 receptor channels is necessary for a relatively potent inhibition of the 5-HT-induced peak current and is a prerequisite for the prolongation of tauON; in addition, a weak but fast inhibitory effect on the current amplitude and decay constant of the 5-HT-induced current was revealed by the experiments in which ifenprodil was exclusively present during exposure to 5-HT. Three alternatives compatible with the components of the ifenprodil effect have been discussed: (1) different effects of the two enantiomers, (2) action via two different mechanisms, and (3) operation via a single mechanism only.

Transporters for the monoamine neurotransmitters, including noradrenaline, 5-hydroxytryptamine [5-HT] and dopamine, have twelve transmembrane spanning regions and cotransport Na+ and Cl- ions. Another family of Na(+)-dependent transporters is that containing the Na+/glucose and Na+/proline cotransporters that are found in the epithelial cells of renal and intestinal brush border membranes. It has been shown that various trivalent lanthanides can substitute for Na+ for transport of glucose and proline. The aim of this study was to determine the effects of lanthanides on the activities of the human noradrenaline, 5-HT and dopamine transporters. Cultured cells were incubated for 2 min with 10 nM 3H-noradrenaline (SK-N-SH-SY5Y human neuroblastoma cells), 3H-5-HT (JAR human placental choriocarcinoma cells) or 3H-dopamine (COS-7 cells transfected with the cDNA of the human dopamine transporter). Specific amine uptake was determined as the difference between accumulation of the amine in the cells in the absence and presence of a corresponding uptake inhibitor. Under both isotonic (150 mM NaCl or LiCl or 90 mM lanthanide salt) and hypertonic (150 mM NaCl +100 mM LiCl, 250 mM LiCl or 150 mM lanthanide salt) conditions, replacement of Na+ by Li+, La3+, Eu3+ or Sm3+ abolished the specific uptake of noradrenaline in SK-N-SH-SY5Y cells and replacement of Na+ by Li+ or Eu3+ decreased the specific uptake of 5-HT in JAR cells by 94-100% and that of dopamine in transfected COS-7 cells by 95-99%. The direct effects of Eu3+ (with Na+ present) on the human noradrenaline transporter in SK-N-SH-SY5Y cells were also examined. Eu3+ inhibited noradrenaline uptake into the cells (IC50 2.6 mM) and nisoxetine binding to crude membranes of SK-N-SH-SY5Y cells (IC50 4.7 mM) with similar potencies. Further experiments showed that 4.5 mM EuCl3 in the presence of 150 mM Na+ caused a 3.5-fold increase in the K(m) of noradrenaline and no change in the maximal rate of noradrenaline uptake. EuCl3 (4.5 mM) also caused a pronounced inhibition of the Na(+)-dependent stimulation of noradrenaline uptake by SK-N-SH-SY5Y cells. It can be concluded from these data that, in contrast with the Na+/glucose and Na+/proline cotransporters, the lanthanides cannot substitute for Na+ in the transport of substrates by the monoamine neurotransmitter transporters and that the lanthanides inhibit the latter transporters by interacting with sites of the transporters involved in amine and Na+ binding.

The influence of several imidazolines and sigma-site ligands on cation influx through the 5-HT3 receptor channel in N1E-115 mouse neuroblastoma cells was studied by measuring the 2-min influx of the organic cation [14C] guanidinium induced by 1 microM 5-HT (in the presence of 10 microM substance P in all experiments). In addition, we determined specific binding of [3H]DTG (1,3-di(2-tolyl)-guanidine), a selective sigma-site radioligand, and [3H] GR65630 (3-(5-methyl-1H-imidazol-4-yl)-1-(1-methyl-1H-indol-3-yl)-1- propanone), a selective 5-HT3 receptor antagonist, to membranes prepared from N1E-115 cells. The 5-HT-induced [14C]guanidinium influx was inhibited by the imidazolines, ondansetron, antazoline, idazoxan, BDF 6143 (4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline), cirazoline, naphazoline, clonidine and by the guanidine agmatine, but not by the catecholamine adrenaline. The inhibitory effect of the imidazolines on cation influx through the 5-HT3 receptor channel was mimicked by the sigma-site ligands, (+/-)-ifenprodil, (+)-3-PPP ((R)-3-(3-hydroxyphenyl)-N-propylpiperidine), DTG (1,3-di-tolyl-guanidine). haloperidol, dizocilpine, and ketamine as well as by the polyamines, arcaine and spermidine.-Ondansetron inhibited [3H]GR65630 binding with high affinity, whereas inhibition of binding of this radioligand to the 5-HT3 receptor by antazoline, BDF 6143, idazoxan, cirazoline, (+/-)-ifenprodil, (+)-3-PPP, DTG and haloperidol occurred in the high micromolar range. In the competition experiments with [3H]DTG, (+/-)-ifenprodil, haloperidol, unlabelled DTG, BDF 6143 and (+)-3-PPP inhibited binding of the radioligand at moderate affinity (Ki values in the range of 1 microM or lower), whereas ondansetron, antazoline, idazoxan, cirazoline, naphazoline, clonidine, tolazoline, efaroxan, RX821002 (2-[2-(2-methoxy-1,4-benzodioxanyl)]imidazoline), ketamine and spermidine exhibited affinity, in the high micromolar or millimolar range only. Comparison of the potencies of the ligands (pIC50% values) in inhibiting 5-HT-induced [14C]guanidinium influx with their affinities (pKi values) at the 5-HT recognition sites of the 5-HT3 receptor and at the sigma 2-sites of the N1E-115 cells by means of multiple regression analysis revealed a significant correlation with the affinities at both sites. In conclusion, our data suggest that imidazolines and sigma-ligands, which as a rule possess low affinity for the 5-HT recognition site of the 5-HT3 receptor, may be assumed to exert their inhibitory effect on cation influx through the 5-HT3 receptor channels, at least in part, by interacting with sigma 2-binding sites.

Tritiated methylphenylpyridinium ([3H]MPP+), a substrate of the neuronal and extraneuronal noradrenaline transporter (uptake1 and uptake2, respectively) and of the organic cation transporter (OCT1), was used to characterize the amine transport system of the established human glioma cell line SK-MG-1. Uptake of [3H]MPP+ (25 nM) into SK-MG-1 cells increased linearly with time for up to 15 min. Selective uptake1 inhibitors (e.g. (+)oxaprotiline) or omission of Na+ or Cl-ions did not affect [3H]MPP+ uptake, whereas uptake2 inhibitors such as O-methyl-isoprenaline (OMI) or corticosterone as well as depolarizing concentrations of K+ or Ba2+ strongly reduced [3H]MPP+ uptake. Initial rates of OMI(100 microM)-sensitive [3H]MPP+ uptake were saturable, with a K(m) of about 17 microM and a maximal rate of about 50 pmol/(min x mg protein). IC50 (or Ki) values for inhibition of [3H]MPP+ uptake by substrates and inhibitors of uptake2 or OCT1 were highly significantly correlated with published IC50 values for inhibition of uptake2 but not with corresponding values for inhibition of OCT1. The results presented here clearly demonstrate that human glioma cells express an uptake2 transporter. Thus, glial cells in the human central nervous system endowed with this transporter are likely to contribute to the inactivation of neuronally released noradrenaline.

The cDNA from a schizophrenic patient heterozygous for a mutation of the 5-HT1A receptor gene was used to clone the variant and wild-type DNA into a eukaryotic expression vector. The mutation was characterized by a base pair substitution (A --> G) at the first position of codon 28, leading to an Ile --> Val amino acid exchange. COS-7 cells were transfected with the cDNA of either the wild type or the variant 5-HT1A receptor. The potencies of the 5-HT1A receptor agonists 8-hydroxy-2-(di-n-propylamino) tetraline (8-OH-DPAT), 5-HT and roxindole, and of the antagonists methiothepin and spiperone in inhibiting specific binding of [3H]8-OH-DPAT of the mutant and wild-type 5-HT1A receptor ligands concentration-dependently inhibited specific [3H]8-OH-DPAT binding to both the wild-type and the variant 5-HT1A receptor. The rank order of potency of the ligands in inhibiting [3H]8-OH-DPAT binding was identical at both receptors and was roxindole > 8-OH-DPAT > 5-HT> methiothepin > spiperone. This rank order is characteristic for 5-HT1A receptors. The negative logarithms of the concentrations required for 50% inhibition (pIC50 values) of the ligands at the mutant 5-HT receptor correlated highly significantly with those at the wildtype receptor (r = 0.995). It is concluded that the pharmacological profile of the mutant 5-HT1A receptor does not differ from that of the wild-type 5-HT1A receptor.

N1E-115 mouse neuroblastoma cells were used to study the influence of ethanol on the 5-HT- and veratridine-induced influx of 14C-guanidinium via the 5-HT3 receptor channel and the fast sodium channel, respectively. Ethanol (10-100 mM) concentration-dependently increased the 5-HT-induced 14C-guanidinium influx, leaving the basal and veratridine (100 microM)-induced influx unaffected. The increasing effect of ethanol (100 mM) was observed at all 5-HT concentrations investigated; accordingly, ethanol increased the maximum response to 5-HT. Whereas in the absence of ethanol the concentration-response curve for 5-HT was bell-shaped, this was no longer the case when ethanol (100 mM) was present in the incubation buffer; the descending branch of the concentration-response curve for 5-HT at concentrations above 300 microM was virtually no longer observed. When, in the presence of substance P (10 microM) the 5-HT-induced 14C-guanidinium influx was already enhanced, the ability of ethanol (100 mM) to increase the 5-HT-induced influx was considerably diminished (by 72%). Preincubation of N1E-115 cells with 5-HT caused a decay of the subsequent 5-HT response ("desensitization") which was dependent on the duration of preincubation; ethanol (100 mM) did not affect the rate of this decay of the 5-HT response. The 5-HT (30 microM)-induced 14C-guanidinium influx was also increased by methanol (100 mM) and n-propanol (100 mM). The rank order of the increasing effect of the n-alkanols (at 100 mM) was: methanol < ethanol < n-propanol; i.e. the degree of enhancement increased with the lipophilicity of the alcohols.(ABSTRACT TRUNCATED AT 250 WORDS)

Imidazolines bind with high affinity not only to alpha-adrenoceptors but also to specific imidazoline binding sites (IBS) labelled by either [3H]clonidine or [3H]idazoxan and termed I1- and I2-IBS, respectively. Since bovine adrenal chromaffin cells lack alpha 2-adrenoceptors, we investigated the pharmacological characteristics of [3H]clonidine binding sites in the bovine adrenal medulla. The binding of [3H]clonidine was rapid, reversible, partly specific (as defined by naphazoline 0.1 mmol/l; 55% specific binding at [3H]clonidine 10 nmol/l), saturable and of high affinity. The specific binding of [3H]clonidine to bovine adrenal medullary membranes was concentration-dependently inhibited by various imidazolines, guanidines and an oxazoline derivative but not, or with negligible affinity, by rauwolscine and (-)-adrenaline. In most cases, the competition curves were best fitted to a two-site model. The rank order of affinity for the high affinity site (in a few cases the single detectable site) was as follows: naphazoline > or = BDF 7579 (4-chloro-2-isoindolinyl guanidine) > or = clonidine > or = cirazoline > or = BDF 6143 (4-chloro-2-(2-imidazoline-2-ylamino)-isoindoline hydrochloride) > BDF 7572 (4,7-chloro-2-(2-imidazolin-2-ylamino)-isoindoline) > moxonidine = rilmenidine > BDF 6100 (2-(2-imidazoline-2-ylamino)-isoindoline) = idazoxan > phentolamine > aganodine = guanabenz > amiloride > histamine. This rank order is compatible with the pharmacological properties of the I1-IBS. The non-hydrolysable GTP-analogue Gpp(NH)p (5'guanylylimidodiphosphate; 100 mumol/l) inhibited specific [3H]clonidine binding by about 50%. Equilibrium [3H]clonidine binding was also significantly reduced by K+ and Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)

The neuronal Na(+)- and Cl(-)-dependent transporter for the neurotransmitter noradrenaline (NA) is the primary target for the tricyclic antidepressant desipramine. The NA-transporter belongs to a new gene family of structurally related Na(+)- and Cl(-)-dependent neurotransmitter transporters. In this study, the chromosomal localization for the gene encoding the human NA-transporter (h-NAT) was determined. By hybridization of a panel of somatic cell hybrids and by fluorescent in situ hybridization to metaphase chromosomes, the hNAT gene was localized on chromosome 16q12.2. In addition, evidence is presented for an intron-exon structure of the gene.

The influence of local and general anaesthetics on cation influx through the fast, voltage-dependent sodium channel and the 5-HT3 receptor cation channel was studied in N1E-115 mouse neuroblastoma cells by measuring 2-min influx of the organic cation 14C-guanidinium induced by either veratridine (1 mmol/l) or 5-HT (100 mumol/l). The veratridine-induced influx of 14C-guanidinium was potentiated by scorpion toxin and inhibited by tetrodotoxin. The 5-HT-induced 14C-guanidinium influx was not affected by tetrodotoxin but it was inhibited by nanomolar concentrations of the selective 5-HT3 receptor antagonists ondansetron and ICS 205-930; at high micromolar concentrations these compounds also inhibited the veratridine-induced influx of 14C-guanidinium. The 14C-guanidinium influx through both channels was inhibited by local and general anaesthetics. The rank order of potency for inhibition of veratridine-induced influx by local anaesthetics was tetracaine > bupivacaine > cocaine > lidocaine > procaine and that for inhibition of the 5-HT3 receptor channel was tetracaine > bupivacaine > cocaine > procaine > lidocaine. With the exception of procaine and cocaine, which were equipotent at both channels, the local anaesthetics were 4.4-fold (lidocaine) to 25-fold (tetracaine) more potent at the fast sodium channel than at the 5-HT3 receptor channel. The rank order of potency for general anaesthetics was propofol > etomidate = alfaxalone = ketamine > thiopental = methohexital at the fast sodium channel, and propofol > or = etomidate > alfaxalone = methohexital > thiopental > ketamine at the 5-HT3 receptor channel.(ABSTRACT TRUNCATED AT 250 WORDS)

The specific (i.e. nisoxetine-sensitive) binding of [3H]desipramine was studied in membranes prepared from bovine adrenal medullae. (1) [3H]desipramine bound reversibly and with high affinity (KD = 2.8 nmol/l) to a single class of non-interacting binding sites (Hill coefficient = 0.96); the maximal number of binding sites (Bmax) was 2.1 pmol/mg protein. (2) Binding of [3H]desipramine was dependent on [Na+] and [Cl-]. Increasing the concentrations of these ions increased binding. (3) Substrates and inhibitors of the neuronal noradrenaline transport system (uptake1) inhibited binding of [3H]desipramine with a rank order of potency typical for an interaction with the uptake1 carrier. The characteristics of [3H]desipramine binding remained essentially unchanged after solubilization of adrenomedullary membranes with the non-ionic detergent digitonin. The results indicate that the plasma membrane of bovine adreno-medullary cells is endowed with the neuronal uptake1 transporter.

The effect of DSP4-induced destruction of noradrenergic neurones on 3H-3-(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid (3H-CPP) binding to N-methyl-D-aspartate (NMDA) receptors and on 3H-desipramine (3H-DMI) binding to the neuronal noradrenaline carrier was investigated in rat brain cortex buffy coat membranes. 3H-DMI bound with high affinity to a single site at the neuronal noradrenaline carrier (KD = 5.26 +/- 1.67 nmol/l) whereas the binding of 3H-CPP to the NMDA receptor was of intermediate affinity (KD = 274 +/- 45 nmol/l). Fourteen days after a single-dose treatment with DSP4 (1) the Bmax value for 3H-DMI binding was reduced by 74%, (2) the Bmax value for 3H-CPP binding only tended to be decreased (by 24%; not statistically significant), (3) the endogenous noradrenaline content was reduced by 70% compared to untreated controls and, (4) the absolute amount of the NMDA-evoked 3H-noradrenaline overflow but not the fractional release was reduced by 55%. It is concluded that in the rat cerebral cortex presynaptic NMDA-receptors on noradrenergic nerve endings, which have previously been detected in release experiments with NMDA on cortical synaptosomes preincubated with 3H-noradrenaline, cannot be identified in radioligand binding experiments. Obviously, the cerebral cortical NMDA receptors are predominantly located on postsynaptic neuronal membranes and potentially on non-noradrenergic nerve terminals as well.

RNA isolated from both rat phaeochromocytoma (PC12) cells and bovine adrenal medulla was size-selected and injected into oocytes of Xenopus laevis. Expression of the neuronal noradrenaline transporter was assayed 3 days after injection by measuring the nisoxetine-sensitive noradrenaline uptake. A 2 kilobase fraction of either total RNA derived from both tissues or of polyadenylated mRNA obtained from bovine adrenal medulla caused Na(+)-dependent transport of 3H-noradrenaline in the injected oocytes.

The muscarine receptors of PC12 (rat phaeochromocytoma) cells were studied in functional and binding experiments. The catecholamine stores of PC12 cells were labelled by incubation of the cells with tritiated noradrenaline. Muscarinic agonists elicited concentration-dependent release of tritium which consisted overwhelmingly of unchanged 3H-noradrenaline. The rank order of potency was: oxotremorine greater than acetylcholine greater than muscarine = methacholine greater than carbachol greater than bethanechol. The release evoked by carbachol (0.1 mmol/l) was inhibited with high potency by the M1-selective antagonist telenzepine (pKi = 8.82), with intermediate potency by pirenzepine (pKi = 7.00) and with low potency by the M2-selective antagonist AF-DX 116 (pKi = 5.74). The binding of 3H-N-methylscopolamine to PC12 membranes was inhibited by various non-selective and subtype-selective muscarinic antagonists with the following rank order of potency: telenzepine = atropine greater than 4-DAMP greater than dicyclomine greater than pirenzepine greater than HHSiD much greater than AF-DX 116. A similar rank order was obtained for the inhibition by these compounds of 3H-telenzepine binding to M1-receptors in membranes of the cerebral cortex of the guinea pig. The Hill coefficients for inhibition of 3H-N-methylscopolamine binding (to PC12 membranes) by pirenzepine, telenzepine and AF-DX 116 were below unity. Specific binding of both 3H-telenzepine and 3H-N-methylscopolamine to muscarine receptors of PC12 membranes was saturable and of high affinity; the maximal number of binding sites was higher for 3H-N-methylscopolamine than for 3H-telenzepine (calculated for the active (+)enantiomer). PC12 cells are presumably endowed with more than one subtype of muscarine receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

The distribution of 3H-isoprenaline in the perfused rat heart was re-examined. After initial loading with 3H-isoprenaline hearts were washed out with amine-free solution; the efflux curves were subjected to the peeling technique, and half times for efflux and compartment sizes were determined. In contrast to earlier reports from his department (Bönisch et al. 1974;l Bönisch 1978), 3H-isoprenaline was found to distribute mainly into one extra-neuronal compartment, irrespective of whether COMT was intact or inhibited (by the presence of U-0521). It was also not influenced by pretreatment of the animals with reserpine. This type of distribution was influenced neither by the concentration of isoprenaline nor by the duration of the loading of the tissue with the amine. The one major extra-neuronal distribution compartment of 3H-isoprenaline has the characteristics of the "old" compartment III: it has a relatively short half time for the efflux of 3H-isoprenaline and it has a high activity of COMT. Moreover, corticosterone inhibits the inward and outward flux of 3H-isoprenaline into and from compartment III. The Ki for the inhibition by corticosterone of the efflux of 3H-isoprenaline (2 mumol/l) is very similar to the Ki for impairment of uptake2 (determined by Bönisch 1978). Apart from the major distribution compartment III, two minor distribution compartments were detected: On the one hand, experiments with hearts which had an intact COMT revealed that a minor distribution compartment IV (characterized by a long half time for efflux and by an absence of COMT activity) may exist, although its magnitude does not exceed one tenth of the former compartment IV. In addition, part of the quickly equilibrating (and rather small) compartment II was corticosterone-sensitive. When the results of Azevedo et al. (1983 are considered together with the present results, compartment III appears to represent the uptake of 3H-isoprenaline into myocardial cells, while it is likely that radioactivity accumulated in the smooth muscle of blood vessels may constitute the corticosterone-sensitive part of compartment II.

1. Isolated superior cervical ganglia of the rat were incubated for 2--30 min (37 degrees C) in Krebs' solution or tissue culture medium (BGJb) containing 22Na and then washed for 30 min in ice-cold 22Na-free Krebs' solution (to clear extracellular space). The radioactivity remaining in the ganglia was taken as a measure of 22Na influx into the intracellular compartment of the ganglion. 2. Addition of cholinomimetics (100 microM nicotine or 100 microM carbachol) to the incubation led to an increase in 22Na influx. This increase reached maximal values after 10 min of incubation; it was more pronounced after incubation in Krebs' solution than in BGJb medium. 3. While chlorisondamine (3 microM) or dopamine (100 microM) greatly reduced the carbachol-induced 22Na influx, tetrodotoxin (2 microM) did not have any effect. 4. In ganglia obtained from animals treated with 6-hydroxydopamine in the early postnatal phase (resulting in an extensive destruction of peripheral sympathetic neurons) neither carbachol (100 microM) nor nicotine (100 microM) produced an increase in 22Na influx demonstrating that the intraneuronal compartment is responsible for this enhanced influx. 5. The effects of dopamine, chlorisondamine and tetrodotoxin on the carbachol-induced 22Na uptake into superior cervical ganglia are similar to their effects on carbachol-mediated induction of tyrosine hydroxylase in superior cervical ganglia kept in tissue culture (Thoenen and Otten 1977b). It is concluded that the induction of tyrosine hydroxylase via nicotinic receptors is closely linked to the enhanced sodium influx into the adrenergic neurons mediated by the same receptors.

The inhibition of N-ethylmaleimide (NEM) of uptake1 and desipramine binding was studied on clonal rat phaeochromocytoma cells (PC12 cells) in different experimental settings: (1) 3H-noradrenaline uptake into intact PC12 cells; (2) 3H-noradrenaline uptake into isolated PC12 plasma membrane vesicles; (3) 3H-desipramine binding to isolated PC12 plasma membrane vesicles. In plasma membrane vesicles, NEM inhibited 3H-desipramine binding and 3H-noradrenaline uptake with similar potency (the IC50's were 1.36 mmol/l and 1.04 mmol/l, respectively). However, in intact cells, NEM was about 75 times more potent in inhibiting 3H-noradrenaline uptake (IC50 = 0.014 mmol/l). The increased potency of NEM in intact cells is probably due to an inhibition of the Na+/K+-ATPase and not to a direct interaction with the noradrenaline carrier. The inactivation by NEM of 3H-desipramine binding to PC12 plasma membrane vesicles was irreversible. Both an inhibitor (cocaine, 1 mmol/l) and a substrate of uptake1 (amezinium, 1 mmol/l) protected desipramine binding from inactivation. These results are compatible with the hypothesis of a common binding site for substrates and inhibitors of the neuronal noradrenaline carrier.

1. The carrier-mediated uptake of labelled 5-hydroxytryptamine (3H-5-HT) in rabbit platelets (defined as the difference between uptake observed in the absence and presence of 10 mumol l-1 imipramine) was studied after inhibition of monoamine oxidase and after a 1:13 dilution of the platelet-rich plasma (PRP) with Tris-containing buffer. 2. Irrespective of whether the rabbits were pretreated with reserpine or not, initial rates of 3H-5-HT uptake were maintained for at least 15 s. 3. Analysis of the saturation kinetics of 3H-5-HT uptake using Hill's equation yielded Km, Vmax and nH values of 130 nmol l-1, 116 pmol 10(8) platelets-1 min-1 and 1.40, respectively. Pretreatment of the animals with reserpine did not affect any of these kinetic parameters, but depleted more than 99% of the platelets' 5-HT stores. 4. The nH value remained greater than unity when the duration of incubation with 3H-5-HT was extended from 15 to 30 s and when the uptake of 3H-5-HT was inhibited by the presence of imipramine (10-40 nmol l-1). However, it was reduced to unity (with a consequential increase in Km) when 300 nmol l-1 ketanserin was present. This concentration of ketanserin did not affect 3H-5-HT uptake at substrate concentrations far below Km. 5. Imipramine inhibited 3H-5-HT uptake by increasing the Km for 3H-5-HT without changing Vmax. The Ki for this interaction was 18 nmol l-1.(ABSTRACT TRUNCATED AT 250 WORDS)

The specific (i.e., nisoxetine-sensitive) binding of 3H-desipramine was studied in purified plasma membranes of PC-12 cells (rat phaeochromocytoma cells). 3H-desipramine bound reversibly and with high affinity (KD = 4.5 nmol/l) to a single, non-interacting site (Hill coefficient = 1.04); the maximal number of binding sites (Bmax) was 19.6 pmol/mg protein. Like the uptake of noradrenaline (by uptake1), the binding of 3H-desipramine was dependent on both sodium and chloride. The stimulation of binding by chloride and sodium was characterized by a Hill coefficient of about 1 and 2, respectively. Both, chloride and sodium, slowed the rate of dissociation of bound 3H-desipramine. Increasing concentrations of sodium decreased the KD of 3H-desipramine binding without altering the Bmax. The binding of 3H-desipramine was inhibited by tricyclic antidepressants and other noradrenaline uptake blockers. There was a highly significant correlation between the potencies of a series of drugs for the inhibition of 3H-desipramine binding and for the inhibition of 3H-noradrenaline uptake into intact PC-12 cells. Both, binding of 3H-desipramine and uptake of 3H-noradrenaline, were stereoselectively inhibited by the enantiomers of cocaine and oxaprotiline. However, for most of the substrates of uptake1 the IC50 for inhibition of 3H-desipramine binding was much higher than that for inhibition of 3H-noradrenaline uptake. Nevertheless, noradrenaline competitively inhibited 3H-desipramine binding and unmasked dissociation of bound 3H-desipramine. Thus, 3H-desipramine probably binds to the substrate recognition site.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasa deferentia of either untreated or reserpine (R) and/or pargyline (P) pretreated rats were incubated with 3H-noradrenaline and then washed with amine- and Ca2+-free solution until (after 100 min) the efflux of radioactivity largely originated from adrenergic nerve endings; COMT was inhibited by U-0521 (U). After 110 min of wash out, the sodium chloride in the wash-out solution was replaced by an equimolar concentration of either Tris-HCl or LiCl. This caused a desipramine-sensitive (i.e., carrier-mediated) efflux of tritiated noradrenaline. The initial increase of the "low Na+"-induced efflux dependent on the experimental conditions: it was most pronounced when the axoplasmic concentration of noradrenaline was high (RPU) and relatively small when MAO and vesicular storage were intact (U). The effects of Li+ and Tris+ differed with regard to the time course of the efflux of tritium: under all three experimental conditions (RPU, PU, U), Tris+ caused the rate of efflux of tritium to increase gradually within the 30 min period of observation, while Li+ either had a "peak-effect" (RPU, PU) or a "plateau-effect" (U). Under "U-conditions" Tris+ caused a slowly increasing, pronounced increase with time of the efflux of both, 3H-noradrenaline and 3H-DOPEG; whereas Li+ caused only a small and sustained increase of the efflux of 3H-noradrenaline and a decrease in the efflux of 3H-DOPEG.(ABSTRACT TRUNCATED AT 250 WORDS)

The veratridine-induced release of 3H-noradrenaline from noradrenergic neurones was examined in the isolated vas deferens of either untreated or reserpine plus pargyline-pretreated rats. The rat vas deferens, whose catechol O-methyltransferase was inhibited, was first incubated with 0.4 mumol/l 3H-(-)noradrenaline (30 min) and then washed repeatedly with amine-free solution. After 120 min (i.e., well after the efflux of tritium from the tissue had reached a steady level and was predominantly of neuronal origin), washout was continued in the presence of veratridine for further 10-15 min. In vasa deferentia of untreated rats, veratridine (1-100 mumol/l) caused a concentration-dependent increase in the efflux of tritium. At high concentrations of the drug (30 or 100 mumol/l), this increase in efflux was peak-like during the first 3 min ("peak response") and then fell to a plateau ("plateau response"). In the presence of veratridine, unchanged 3H-noradrenaline accounted for about 75% of the tritium efflux (the rest being represented by deaminated 3H-catechol metabolites). The "peak response" to veratridine (100 mumol/l) was abolished by tetrodotoxin (TTX; 1 mumol/l) or the absence of external Ca2+. Cocaine (10 mumol/l) affected neither the "peak response" as such nor the contribution by 3H-noradrenaline to the efflux of tritium during that response. Hence, the "peak response" was due to exocytotic release of 3H-noradrenaline from the neurone. The "plateau response" to veratridine (100 mumol/l) was unaffected by the absence of external Ca2+, largely resistant to TTX (1 mumol/l) and moderately reduced by cocaine.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of a decrease of the K+ gradient on the extraneuronal inward transport and outward movement of catecholamines were studied in rat heart, rabbit aortic rings and guinea-pig trachealis smooth muscle. Elevation of the extracellular K+ concentration caused a) inhibition of the corticosteroid-sensitive extraneuronal uptake (uptake2) of 3H-isoprenaline in rat heart and of 3H-noradrenaline in rabbit aorta, and b) acceleration of efflux of 3H-isoprenaline from rat heart, 3H-noradrenaline from rabbit aorta and adrenaline (measured by microphotometry) from guinea-pig trachealis muscle. In rat heart and rabbit aorta, the acute omission of one or the other of the ions Na+, Cl-, K+ or Ca2+ from the perfusion of incubation medium had no effect on initial rates of uptake2 of catecholamines, except that the absence of K+ had a small inhibitory effect in the rat heart. The prolonged absence of Na+, Ca2+ or K+ from the perfusion or incubation medium caused a marked inhibition of uptake2 of catecholamines. These inhibitory effects developed more quickly in rat heart than in rabbit aorta. These results are compatible with the possibility that either the K+ gradient across the cell membrane or the resting membrane potential is the force driving uptake2.

The adrenergic nerve endings of vasa deferentia of either untreated or reserpine (R) and/or pargyline (P) pretreated rats were loaded with 3H-noradrenaline; COMT was inhibited by U-0521 (U). After 100 min of wash-out with Ca2+-free solution, the efflux of tritium (and of 3H-noradrenaline) from the tissue was largely of neuronal origin and remained constant with time (when expressed as fractional rate of loss; FRL). After 110 min of wash-out the effect of inhibition of the Na+,K+-ATPase (by low K+ or ouabain) on basal and on sympathomimetic amine-induced efflux of tritium (or 3H-noradrenaline, under the condition U) was studied in paired experiments. Inhibition of the Na+,K+-ATPase caused a time-dependent increase in the efflux of tritium (or 3H-noradrenaline) which was inhibited by desipramine. Inhibition of the Na+,K+-ATPase also caused a time-dependent reduction of the initial rate of neuronal uptake of 3H-noradrenaline. The effectiveness of the sympathomimetic amines tyramine and amphetamine in inducing "release" (i.e., outward-transport) of noradrenaline depended on the experimental condition: it was most pronounced under the condition RPU, followed by the condition PU and lowest under the condition U (i.e., in tissue of untreated rats). Inhibition of the Na+,K+-ATPase caused an early and transient enhancement of the "release" of noradrenaline induced by tyramine or amphetamine. This enhancement was seen already within the first min after inhibition of the ATPase, i.e., before a pronounced inhibition of uptake (of noradrenaline) and before a pronounced increase of the basal efflux was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

The uptake of 3H-noradrenaline into reserpine-pretreated PC-12 cells (a clonal cell line which possesses "uptake1") was abolished when at high extracellular Cl- all the extracellular Na+ was replaced by Tris+ and when at high extracellular Na+ all the extracellular Cl- was replaced by isethionate. Increases in the external Cl- concentration (at a fixed high Na+ concentration) progressively increased the uptake of 3H-noradrenaline. The same was found with increase in the external Na+ concentration (at a fixed high Cl- concentration). From the anions tested only Br- and SCN- were able to partially mimic the transport-stimulating effect of Cl- (with about 40% and 20% effectiveness, respectively). When chloride was replaced by nitrate or larger anions such as sulphate, methylsulphate or isethionate, virtually no transport of 3H-noradrenaline was observed. The initial rate of uptake of 3H-noradrenaline showed saturation with increasing concentrations of noradrenaline when determined at several fixed concentrations of either Na+ or Cl-. The apparent Km for noradrenaline transport (KmNA) progressively decreased and the VmaxNA increased with increases in the concentration of Na+ (at a high concentration of Cl-) or Cl- (at a high concentration of Na+). The stimulation of the initial rate of uptake of 3H-noradrenaline by increasing concentrations of either Na+ or Cl- obeyed saturation kinetics when determined at several concentrations of noradrenaline. The concentration of Na+ (or Cl-) which caused half-maximal stimulation of uptake (i.e., the apparent KmNa+ and the apparent KmCl-) decreased with increases in the concentration of noradrenaline. These results strongly suggest that Na+ and Cl- are co-transported with noradrenaline.

3H-Desipramine binding sites of the plasma membranes of rat phaeochromocytoma cells (PC12-cells) were solubilized with the nonionic detergent digitonin (0.5%). With the method described here, the binding characteristics of the desipramine binding site were essentially unaltered by solubilization. Binding of 3H-desipramine to the solubilized binding site showed the following characteristics: (1) 3H-desipramine bound with high affinity (KD = 16.6 nmol/l) to a single class of noninteracting (Hill-coefficient = 1.01) binding sites; (2) binding was reversible; (3) binding of unlabelled desipramine had the same dissociation constant as had 3H-desipramine; (4) increasing concentrations of sodium- and chloride-ions stimulated the binding of 3H-desipramine; (5) binding was inhibited by various inhibitors and substrates of neuronal uptake of noradrenaline; and (6) inhibition of binding by the optical isomers of cocaine, oxaprotiline, and amphetamine showed marked stereoselectivity (with preference for (-)cocaine, (+)oxaprotiline, and (+)amphetamine). The finding that the binding of 3H-desipramine to the solubilized binding site was dependent on sodium and chloride, as the neuronal uptake of noradrenaline is, and the finding that all substrates of uptake inhibited the binding of 3H-desipramine, is consistant with the view that desipramine binds to the substrate recognition site of the neuronal carrier for noradrenaline.

PC-12 cells (a clonal line of rat phaeochromocytoma cells) take up noradrenaline by a transport system which is identical with the neuronal amine transport system ("uptake1"). The uptake of 3H-noradrenaline into reserpine-pretreated PC-12 cells (monoamine oxidase inhibited) was saturable (Km = 0.6 +/- 0.1 mumol/l), dependent on sodium and chloride, and competitively inhibited by (+)-amphetamine (Ki = 0.18 +/- 0.04 mumol/l), cocaine (Ki = 0.55 +/- 0.15 mumol/l) and desipramine (Ki = 4.3 +/- 0.6 nmol/l). The uptake and accumulation of 3H (+)-amphetamine showed characteristics comparable to those of 3H-noradrenaline, since the uptake of 3H (+)-amphetamine (0.1 mumol/l) was reduced by omission of sodium or chloride from the incubation medium. The sodium-sensitive component of uptake and accumulation of 3H (+)-amphetamine was fully inhibited by cocaine and desipramine. The IC50 of desipramine for inhibition of the sodium-sensitive component of the 1-min uptake of 3H (+)-amphetamine (20 nmol/l) was about 2 nmol/l, i.e., identical with the Ki for inhibition of uptake of 3H-noradrenaline. At concentrations above 1 mumol/l, desipramine additionally caused an inhibition of the sodium-independent permeation of 3H (+)-amphetamine into PC-12 cells. Hence, by using a homogeneous population of cells endowed with "uptake1", it is possible to demonstrate - besides a pronounced lipophilic entry - a carrier-mediated uptake of 3H (+)-amphetamine.

1. The neuronal release by 100 mumol/l veratridine of preloaded 3H-noradrenaline was studied in the rat vas deferens, the MAO, COMT and vesicular uptake of which were inhibited. To prevent any exocytotic release of the 3H-amine, all solutions were calcium-free. Veratridine induced an early and a late peak of tritium efflux. The early peak was abolished by the presence of 1 mumol/l desipramine, the late peak was abolished by 1 mumol/l tetrodotoxin (administered subsequently to the first peak). The administration of veratridine plus 1 mmol/l ouabain resulted in only the early peak of efflux. 2. The peak response to veratridine plus ouabain was increased by a very early administration of veratridine plus ouabain (after 40 min of wash-out instead of the usual 130 min) (i.e., when the relative size of the axoplasmic distribution compartment was increased). However, very high axoplasmic 3H-noradrenaline levels (after loading with 37 instead of the usual 0.2 mumol/l) reduced the height of the peak (when expressed as a FRL). 3. Substantially similar responses to veratridine plus ouabain were obtained after loading with 3H-noradrenaline, 3H-adrenaline or 3H-dopamine. 4. As the second peak of veratridine-induced release is ouabain-sensitive, it appears to be caused by exhaustion of neuronal ATP stores; this, in turn, raises the intravesicular pH and induces efflux of 3H-noradrenaline from the vesicles into the axoplasm. The first peak, on the other hand, represents outward transport of 3H-noradrenaline from the axoplasmic compartment. Evidently, a pronounced vesicular distribution of 3H-noradrenaline takes place even after inhibition by reserpine of the vesicular uptake. 5. In preparations with intact vesicular uptake (MAO and COMT inhibited) a plateau-response was obtained; in the presence of 10 mumol/l Ro 4-2184 (a reserpine-like compound) a peak response was restored after loading with 0.2 mumol/l 3H-noradrenaline, less so after loading with 37 mumol/l. 6. It is confirmed that veratridine (plus ouabain) exerts a reserpine-like effect when applied to tissues with intact vesicular uptake and intact MAO.

Bönisch et al. (1974) identified kinetically two extraneuronal compartments into which 3H-isoprenaline distributes in the perfused rat heart: compartment III (characterized by a half time for the efflux of 3H-isoprenaline of about 10 min) had about the same size as compartment IV (half time for efflux: 23 min). These authors suggested that compartment III might be located in the vascular smooth muscle, while compartment IV might be located in myocardial cells. The present study was carried out to validate or refute this suggestion. Rat hearts were perfused for 5, 20 or 60 min with 1 mumol/l 3H-isoprenaline; additional hearts were perfused with 1 mumol/l 3H-isoprenaline for 30 min in the presence of either 20 mumol/l corticosterone or 20 mumol/l corticosterone plus 30 mumol/l cocaine. COMT was inhibited in all experiments (by the presence of 100 mumol/l U-0521). Quantitative autoradiography revealed in all groups that the silver grain density (grains/mm2) was greater over small blood vessels (arterioles and venules) than over myocardial cells. However, total silver grains over myocardial cells greatly exceeded those over small blood vessels (by a factor of 6 to 9). Thus, the suggestion of Bönisch et al. (1974) is untenable. Autoradiographic results obtained with small specimens of ventricular muscle are representative of the whole heart, since "silver grains over total tissue" (per mm2) were highly significantly correlated with the 3H-isoprenaline content of the homogenized hearts (in pmol/g). While corticosterone reduced the accumulation of 3H-isoprenaline in myocardial cells, it failed to affect the appearance of silver grains over Purkinje cells. However, cocaine prevented this type of accumulation. Thus, uptake in Purkinje cells appears to resemble neuronal rather than extraneuronal uptake.

The neuronal noradrenaline uptake mechanism (uptake1) has been further characterized. For a number of substrates of uptake1 the half-saturating concentration (Km) and the maximal initial transport rate (Vmax) were determined. Furthermore, the dissociation constants (KD) for binding of these substrates to the desipramine binding site of the neuronal noradrenaline carrier were measured. The uptake experiments were done on rat phaeochromocytoma cells (PC12 cells), the binding experiments on purified plasma membranes of PC12 cells. The substrates differed markedly in respect of Vmax, Km, and KD. Neither Km and Vmax nor KD and Vmax were found to be correlated. However, the discrepancy between Km and KD expressed as the ratio, Km/KD, was negatively correlated with Vmax (r = -0.9315, n = 7, p less than 0.01). For the interpretation of these results a model on the basis of the steady-state assumption has been proposed for uptake1. From the mathematics of that model the following conclusions can be drawn. (1) The half-saturating substrate concentration (Km) is not identical with the dissociation constant for the binding of a substrate to the substrate recognition site (KD). (2) The discrepancy between Km and KD is expected to be negatively correlated with the maximal initial transport rate of the substrate (Vmax). The experimental results are in good agreement with the proposed model for uptake1. Especially the negative correlation between Km/KD and Vmax supports the hypothesis that desipramine inhibits uptake1 via binding to the substrate recognition site of the neuronal noradrenaline carrier.

The effects of choline+ (10-40 mmol/l) on 3H-noradrenaline uptake by, and 3H-noradrenaline efflux from, noradrenergic neurones were studied in vasa deferentia of reserpine-pretreated rats at an external Na+ concentration of 100 mmol/l. Monoamine oxidase and catechol-O-methyltransferase were inhibited. Choline+ (20 and 40 mmol/l) competitively inhibited the neuronal uptake of 3H-noradrenaline. From the choline+-induced changes in the apparent Km for 3H-noradrenaline transport, a Ki of 35 mmol/l was obtained. Choline+ (10, 20 and 40 mmol/l) accelerated the neuronal efflux of 3H-noradrenaline in a concentration-dependent manner. This acceleration of efflux was greatly reduced in the presence of 1 mumol/l desipramine, indicating that choline+ is capable of eliciting "accelerative exchange diffusion". Choline+ (40 mmol/l) and (-)noradrenaline (4.5 mumol/l) (i.e., concentrations about equivalent to the Ki and Km for choline+ and (-)noradrenaline, respectively) produced virtually identical increases in the neuronal efflux of 3H-noradrenaline. Choline+ (3-300 mmol/l) inhibited the specific binding of 3H-desipramine to plasma membranes derived from cultured rat phaeochromocytoma (PC-12) cells. The Ki for this interaction was 48 mmol/l. This results suggest that choline+ acts as alternative substrate of the neuronal noradrenaline transport system and should, therefore, not be used in transport studies with noradrenaline as substitute for Na+ in Na+-deficient media.