Sigismund Huck

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

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

Are GABAA Receptors Containing α5 Subunits Contributing to the Sedative Properties of Benzodiazepine Site Agonists

Classical benzodiazepines (BZs) exert anxiolytic, sedative, hypnotic, muscle relaxant, anticonvulsive, and amnesic effects through potentiation of neurotransmission at GABAA receptors containing α1, α2, α3 or α5 subunits. Genetic studies suggest that modulation at the α1 subunit contributes to much of the adverse effects of BZs, most notably sedation, ataxia, and amnesia. Hence, BZ site ligands functionally inactive at GABAA receptors containing the α1 subunit are considered to be promising leads for novel, anxioselective anxiolytics devoid of sedative properties. In pursuing this approach, we used two-electrode voltage clamp experiments in Xenopus oocytes expressing recombinant GABAA receptor subtypes to investigate functional selectivity of three newly synthesized BZ site ligands and also compared their in vivo behavioral profiles. The compounds were functionally selective for α2-, α3-, and α5-containing subtypes of GABAA receptors (SH-053-S-CH3 and SH-053-S-CH3-2′F) or essentially selective for α5 subtypes (SH-053-R-CH3). Possible influences on behavioral measures were tested in the elevated plus maze, spontaneous locomotor activity, and rotarod test, which are considered primarily predictive of the anxiolytic, sedative, and ataxic influence of BZs, respectively. The results confirmed the substantially diminished ataxic potential of BZ site agonists devoid of α1 subunit-mediated effects, with preserved anti-anxiety effects at 30 mg/kg of SH-053-S-CH3 and SH-053-S-CH3-2′F. However, all three ligands, dosed at 30 mg/kg, decreased spontaneous locomotor activity, suggesting that sedation may be partly dependent on activity mediated by α5-containing GABAA receptors. Hence, it could be of importance to avoid substantial agonist activity at α5 receptors by candidate anxioselective anxiolytics, if clinical sedation is to be avoided.

UTP‐ and ATP‐triggered transmitter release from rat sympathetic neurones via separate receptors

In rat cultured sympathetic neurones, UDP, UTP and ATP at micromolar concentrations triggered Ca2+‐dependent and tetrodotoxin‐sensitive [3H]‐noradrenaline release. The overflow evoked by UTP or ATP was similar at 100 μmol 1−1, the concentration used in all subsequent experiments. Pre‐exposure of the neurones to 100 μmol 1−1 UTP significantly reduced ensuing secretory effects of UTP but not of ATP. Conversely, pre‐exposure to ATP diminished the overflow due to ATP but not that due to UTP. In the presence of 10 μmol 1−1 pyridoxal‐5″‐phosphate or 30 μmol 1−1 suramin, the secretory response to ATP was reduced, but the effect of UTP was unaltered. Zn2+ (10 μmol 1−1) reduced the overflow triggered by UTP, but increased the overflow due to ATP. These results indicate the presence of separate receptors for pyrimidine nucleotides and for purine nucleotides which both trigger transmitter release.

RECEPTORS CONTROLLING TRANSMITTER RELEASE FROM SYMPATHETIC NEURONS IN VITRO

Primary cultures of postganglionic sympathetic neurons were established more than 30 years ago. More recently, these cultures have been used to characterize various neurotransmitter receptors that govern sympathetic transmitter release. These receptors may be categorized into at least three groups: (1) receptors which evoke transmitter release: (2) receptors which facilitate; (3) receptors which inhibit, depolarization-evoked release. Group (1) comprises nicotinic and muscarinic acetylcholine receptors, P2X purinoceptors and pyrimidinoceptors. Group (2) currently harbours beta-adrenoceptors, P2 purinoceptors, receptors for PACAP and VIP, as well as prostanoid EP1 receptors. In group (3), muscarinic cholinoceptors, alpha 2- and beta-adrenoceptors, P2 purinoceptors, and receptors for the neuropeptides NPY, somatostatin (SRIF1) and LHRH, as well as opioid (delta and kappa) receptors can be found. Receptors which regulate transmitter release from neurons in cell culture may be located either at the somatodendritic region or at the sites of exocytosis, i.e. the presynaptic specializations of axons. Most of the receptors that evoke release are located at the soma. There ionotropic receptors cause depolarizations to generate action potentials which then trigger Ca(2+)-dependent exocytosis at axon terminals. The signalling mechanisms of metabotropic receptors which evoke release still remain to be identified. Receptors which facilitate depolarization-evoked release appear to be located preferentially at presynaptic sites and presumably act via an increase in cyclic AMP. Receptors which inhibit stimulation evoked release are also presynaptic origin and most commonly rely on a G protein-mediated blockade of voltage-gated Ca2+ channels. Results obtained with primary cell cultures of postganglionic sympathetic neurons have now supplemented previous data about neurotransmitter receptors involved in the regulation of ganglionic as well as sympatho-effector transmission. In the future, this technique may prove useful to identify yet unrecognized receptors which control the output of the sympathetic nervous system and to elucidate underlying signalling mechanisms.

Inhibition of N‐type Calcium Channels: The Only Mechanism by which Presynaptic α2‐Autoreceptors Control Sympathetic Transmitter Release

α2‐Adrenoceptors are known to inhibit voltage‐dependent Ca2+ channels located at neuronal cell bodies; the present study investigated whether this or alternative mechanisms, possibly downstream of Ca2+ entry, underlie the presynaptic α2‐adrenergic modulation of transmitter release from chick sympathetic neurons. Using chick sympathetic neurons, overflow of previously incorporated [3H]noradrenaline was elicited in the presence of extracellular Ca2+ by electrical pulses, 25 mM K+ or 10μM nicotine, or by adding Ca2+ to otherwise Ca2+‐free medium when cells had been made permeable by the calcium ionophore A23187 or by α‐latrotoxin. Pretreatment of neurons with the N‐type Ca2+ channel blocker ω‐conotoxin GVIA and application of the α2‐adrenergic agonist UK 14304 reduced the overflow elicited by electrical pulses, K+ or nicotine, but not the overflow caused by Ca2+ after permeabilization with α‐latrotoxin or A23187. In contrast, the L‐type Ca2+ channel blocker nitrendipine reduced the overflow due to K+ and nicotine, but not the overflow following electrical stimulation or α‐latrotoxin‐ and A23187‐permeabilization. The inhibition of electrically evoked overflow by UK 14304 persisted in the presence of nitrendipine and the L‐type Ca2+ channel agonist BayK 8644, which per se enhanced overflow. In ω‐conotoxin GVIA‐treated cultures, electrically evoked overflow was also enhanced by BayK 8644 and almost reached the value obtained in untreated neurons. However, UK 14304 lost its effect under these conditions. Whole‐cell recordings of voltage‐activated Ca2+ currents corroborated these results: UK 14304 inhibited Ca2+ currents by 33%, nitrendipine caused a 7% reduction, and BayK 8644 increased the currents by 30%. Moreover, the dihydropyridines failed to abolish the inhibition by UK 14304, but pretreatment with ω‐conotoxin GVIA, which reduced mean amplitude from 0.95 to 0.23 nA, entirely prevented α2‐adrenergic effects. Our results indicate that the α2‐autoreceptor‐mediated modulation of noradrenaline release from chick sympathetic neurons relies exclusively on the inhibition of ω‐conotoxin GVIA‐sensitive N‐type Ca2+ channels. Mechanisms downstream of these channels and voltage‐sensitive Ca2+ channels other than N‐type appear not to be important.

SOMATIC AND PREJUNCTIONAL NICOTINIC RECEPTORS IN CULTURED RAT SYMPATHETIC NEURONES SHOW DIFFERENT AGONIST PROFILES

1 The release of [3H]‐noradrenaline ([3H]‐NA) in response to nicotinic acetylcholine receptor (nAChR) agonists was compared with agonist‐induced currents in cultured rat superior cervical ganglion (SCG) neurones. 2 [3H]‐NA release in response to high concentrations of nicotinic agonists was reduced, but not fully inhibited, by the presence of either tetrodotoxin (TTX) or Cd2+ to block voltage‐gated Na+ or Ca2+ channels, respectively. We used the component of transmitter release that remained in the presence of these substances (named TTX‐ or Cd2+‐insensitive release) to pharmacologically characterize nAChRs in proximity to the sites of vesicular exocytosis (prejunctional receptors). Prejunctional nAChRs were activated by nicotinic agonists with a rank order of potency of dimethylphenylpiperazinium iodide (DMPP) > nicotine > cytisine > ACh, and with EC50 values ranging from 22 μM (DMPP) to 110 μM (ACh). 3 [3H]‐NA release in response to low concentrations of nAChR agonists was fully inhibited by the presence of either TTX or Cd2+ (named TTX‐ or Cd2+‐sensitive release). TTX‐sensitive release was triggered by nicotinic agonists with a rank order of potency of DMPP > cytisine ≈ nicotine ≈ ACh, which due to its similarity to TTX‐insensitive release indicates that it might also be triggered by prejunctional‐type nAChRs. The EC50 values for TTX (Cd2+)‐sensitive release were less than 10 μM for all four agonists. 4 By contrast to transmitter release, somatic nAChRs as seen by patch clamp recordings were most potently activated by cytisine, with a rank order of potency of cytisine > nicotine ≈ DMPP > ACh. EC50 values for the induction of currents exceeded 20 μM for all four agonists. 5 The nicotinic antagonist mecamylamine potently inhibited all transmitter release in response to nicotine. α‐Bungarotoxin (α‐BuTX) was, on the other hand, without significant effect on nicotine‐induced TTX‐insensitive release. The competitive antagonist dihydro‐β‐erythroidine (DHβE) caused rightward shifts of the dose‐response curves for both TTX‐sensitive and TTX‐insensitive transmitter release as well as for currents in response to nicotine, with pA2 values ranging from 4.03 to 4.58. 6 Due to clear differences in the pharmacology of agonists we propose that nAChRs of distinct subunit composition are differentially targeted to somatic or axonal domains.

Organic cation transporter mRNA and function in the rat superior cervical ganglion

Reuptake of extracellular noradrenaline (NA) into superior cervical ganglion (SCG) neurones is mediated by means of the noradrenaline transporter (NAT, uptake 1). We now demonstrate by single‐cell RT‐PCR that mRNA of the organic cation transporter 3 (OCT3, uptake 2) occurs in rat SCG neurones as well. Furthermore, our RT‐PCR analyses reveal the presence of mRNA for novel organic cation transporters 1 and 2 (OCTN1 and OCTN2), but not for OCT1 or OCT2 in the ganglion. Making use of the NAT as a powerful, neurone‐specific transporter system, we loaded[3H]‐N‐methyl‐4‐phenylpyridinium ([3H]‐MPP+) into cultured rat SCG neurones. The ensuing radioactive outflow from these cultures was enhanced by desipramine and reserpine, but reduced (in the presence of desipramine) by the OCT3 inhibitors cyanine 863, oestradiol and corticosterone. In contrast, cyanine 863 enhanced the radioactive outflow from cultures preloaded with [3H]‐NA. Two observations suggest that a depletion of storage vesicles by cyanine 863 accounts for the latter phenomenon: first, the primary radioactive product isolated from supernatants of cultures loaded with [3H]‐NA was the metabolite [3H]‐DHPG; and second, inhibition of MAO significantly reduced the radioactive outflow in response to cyanine 863. The outflow of [3H]‐MPP+ was significantly enhanced by MPP+, guanidine, choline and amantadine as potential substrates for OCT‐related transmembrane transporters. However, desipramine at a low concentration essentially blocked the radioactive outflow induced by all of these substances with the exception of MPP+, indicating the NAT and not an OCT as their primary site of action. The MPP+‐induced release of [3H]‐MPP+ was fully prevented by a combined application of desipramine and cyanine 863. No trans‐stimulation of [3H]‐MPP+ outflow was observed by the OCTN1 and OCTN2 substrate carnitine at 100 μM. Our observations indicate an OCT‐mediated transmembrane transport of [3H]‐MPP+. Amongst the three OCTs expressed in the SCG, OCT3 best fits the profile of substrates and antagonists that cause trans‐stimulation and trans‐inhibition, respectively, of [3H]‐MPP+ release.

Biochemical and functional properties of distinct nicotinic acetylcholine receptors in the superior cervical ganglion of mice with targeted deletions of nAChR subunit genes

Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission in ganglia of the autonomic nervous system. Here, we determined the subunit composition of hetero‐pentameric nAChRs in the mouse superior cervical ganglion (SCG), the function of distinct receptors (obtained by deletions of nAChR subunit genes) and mechanisms at the level of nAChRs that might compensate for the loss of subunits. As shown by immunoprecipitation and Western blots, wild‐type (WT) mice expressed: α3β4 (55%), α3β4α5 (24%) and α3β4β2 (21%) nAChRs. nAChRs in β4 knockout (KO) mice were reduced to < 15% of controls and no longer contained the α5 subunit. Compound action potentials, recorded from the postganglionic (internal carotid) nerve and induced by preganglionic nerve stimulation, did not differ between α5β4 KO and WT mice, suggesting that the reduced number of receptors in the KO mice did not impair transganglionic transmission. Deletions of α5 or β2 did not affect the overall number of receptors and we found no evidence that the two subunits substitute for each other. In addition, dual KOs allowed us to study the functional properties of distinct α3β4 and α3β2 receptors that have previously only been investigated in heterologous expression systems. The two receptors strikingly differed in the decay of macroscopic currents, the efficacy of cytisine, and their responses to the α‐conotoxins AuIB and MII. Our data, based on biochemical and functional experiments and several mouse KO models, clarify and significantly extend previous observations on the function of nAChRs in heterologous systems and the SCG.

Electrically evoked noradrenaline release from cultured chick sympathetic neurons: modulation via presynaptic α2-adrenoceptors and lack of autoinhibition

SummarySympathetic neurons from twelve day old chick embryos were plated on polystyrol discs and kept in culture for five days. After incubation with 3H-noradrenaline the discs were transferred to small chambers and superfused. Electrical field stimulation (36 pulses at 3 Hz) increased the outflow of tritium. The evoked overflow of tritium was abolished in the absence of extracellular calcium and was diminished by about 90% in the presence of tetrodotoxin (1 μmol/l). The α2-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline (UK-14,304) caused a concentration-dependent decrease in overflow, whereas the α1-adrenoceptor agonist methoxamine was ineffective at up to 1 μmol/l. The concentration-response curve of UK-14,304 was shifted to the right by the α2-adrenoceptor antagonist yohimbine (0.03 μmol/l). Yohimbine on its own caused no significant change. The noradrenaline reuptake inhibitor cocaine (10 μmol/l) caused a small (20%) increase in evoked overflow. The results indicate that cultured chick sympathetic neurons possess release-modulating α2-adrenoceptors and that the electrically induced overflow of transmitter occurs under conditions virtually free of autoinhibition.

Serum-free medium for cultures of the postnatal mouse cerebellum: only insulin is essential.

Serum-free culture conditions for dissociated postnatal mouse cerebellar cells were investigated. This study demonstrates that among various supposed growth-promoting factors only insulin is required as an additive to the basic medium. If viewed by phase contrast microscopy, cultures kept in insulin-supplemented basic medium looked identical to those maintained in the presence of a mixture of growth-promoting factors (insulin, putrescine, transferrin, progesterone, triiodothyronine, selenium). In addition, quantitative evidence is provided indicating that cellular survival is supported to the same extent by insulin as by this admixture. Insulin concentrations required ranged between 0.3-20 micrograms/ml. By contrast to serum-supplemented culture conditions, no significant proliferation of non-neuronal cells was observed in serum-free culture media. It is expected that by these findings attention will be focused again to the exact role insulin plays in the central nervous system.