Biomedical subjects

S Huck

Publications and source records attributed to S Huck.

At least 37 records · Page 2Linked to original sources

PubMed1996

The human T-cell receptor gamma variable pseudogene V10 is a distinctive marker of human speciation.

The V10 variable gene of the human T-cell receptor gamma locus (TCRG-V10), the only member of the subgroup III, has a structural defect which inhibits the splicing of the leader intron. We show that there is a single point mutation in the V10 leader donor splice site responsible for this situation and that this mutation is found in the different populations tested, indicating that V10 corresponds to a pseudogene in humans. We restored the splice site by mutagenesis and obtained correct splicing in vitro. Analysis of the V10 germline gene in different primates reveals functional splice sites in the closest human apes, the chimpanzee and the gorilla. The splice competence of TCRG-V10 in higher primates was addressed in peripheral blood lymphocytes from chimpanzee by specific cDNA amplification, and correct splicing of the TCRG-V10 leader intron was found as well as a majority of in frame rearrangements involving only the TCRG-J1 or J2 segments. These results suggest that V10(+)gamma /delta T cells may represent an important subset in the non-human higher primates, contrary to the situation observed in the human.

Amino Acid Sequence↗

PubMed1996

A somatostatin receptor inhibits noradrenaline release from chick sympathetic neurons through pertussis toxin-sensitive mechanisms: comparison with the action of alpha 2-adrenoceptors.

The effects of somatostatin and analogues were investigated in cultures of chick sympathetic neurons. Electrically evoked tritium overflow from cultures labelled with [3H]noradrenaline was reduced by somatostatin-14 in a concentration-dependent manner, with half maximal effects at 0.3 nM and a maximum of 45% inhibition. Somatostatin-28 was equipotent to somatostatin-14 (half maximal concentration at 0.5 nM), and seglitide was less potent, the effects being half maximal at 4.2 nM. The inhibitory action of somatostatin-14 on stimulation-evoked overflow desensitized within minutes at 100 nM, but not at 10 nM, and was abolished by a pretreatment of neurons with pertussis toxin. All somatostatin analogues reduced voltage-activated Ca2+ currents recorded in the whole-cell configuration of the patch-clamp technique, with somatostatin-14 being equipotent to somatostatin-28, but more potent than seglitide. However, the inhibition of Ca2+ currents occurred at concentrations more than ten-fold higher than those required for the reduction of stimulation evoked 3H overflow. The action of somatostatin upon Ca2+ currents was also abolished by pertussis toxin and desensitized within minutes. In preceding experiments, alpha 2-adrenoceptor activation had been found to reduce transmitter release and Ca2+ currents of chick sympathetic neurons through a pertussis toxin-sensitive mechanism. In the present study, the alpha 2-adrenergic agonist UK 14,304 completely occluded the inhibition of Ca2+ currents and of electrically evoked overflow by somatostatin-14. Neither UK 14,304 nor somatostatin affected the resting membrane potential or voltage-dependent K+ currents. These results demonstrate that chick sympathetic neurons possess SRIF1 type somatostatin receptors which control transmitter release. This effect is mediated by pertussis toxin-sensitive GTP binding proteins and apparently involves an inhibition of voltage-activated Ca2+ channels, but not a modulation of K+ channels. Since alpha 2-adrenergic agonists share all of these actions and occlude the effects of somatostatin, alpha 2-adrenoceptors and SRIF1 receptors seem to regulate sympathetic transmitter release via common signalling mechanisms.

Adrenergic alpha-Agonists↗

PubMed1996

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

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

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1996

Inhibition of N-type calcium channels: the only mechanism by which presynaptic alpha 2-autoreceptors control sympathetic transmitter release.

Alpha 2-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 alpha 2-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 10 microM nicotine, or by adding Ca2+ to otherwise Ca(2+)-free medium when cells had been made permeable by the calcium ionophore A23187 or by alpha-latrotoxin. Pretreatment of neurons with the N-type Ca2+ channel blocker omega-conotoxin GVIA and application of the alpha 2-adrenergic agonist UK 14304 reduced the overflow elicited by electrical pulses, K+ or nicotine, but not the overflow caused by Ca2+ after permeabilization with alpha-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 alpha-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 omega-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 omega-conotoxin GVIA, which reduced mean amplitude from 0.95 to 0.23 nA, entirely prevented alpha 2-adrenergic effects. Our results indicate that the alpha 2-autoreceptor-mediated modulation of noradrenaline release from chick sympathetic neurons relies exclusively on the inhibition of omega-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.

Animals↗

PubMed1995

alpha 2-Adrenoreceptor-mediated inhibition of acetylcholine-induced noradrenaline release from rat sympathetic neurons: an action at voltage-gated Ca2+ channels.

[3H]Noradrenaline release was studied in cultured sympathetic neurons derived from superior cervical ganglia of neonatal rats. Acetylcholine elicited a concentration- and time-dependent increase in 3H outflow which was half-maximal at about 300 microM and within 5 s. The overflow induced by 10 s exposure to 300 micro A acetylcholine was reduced by the nicotinic antagonist hexamethonium, but increased by the muscarinic antagonist atropine. Cd2+ (300 microM) prevented the overflow evoked by electrical field stimulation, but reduced acetylcholine-induced overflow by less than 50%. Removal of extracellular Ca2+ abolished stimulation-evoked tritium overflow irrespective of the stimulus. The selective alpha2-adrenoceptor agonist UK 14,304 inhibited acetylcholine-evoked overflow to a significantly smaller extent (approximately 25% maximal inhibition) than electrically induced overflow ( > or = 45% maximal inhibition). These inhibitory effects were antagonized by the alpha2-adrenoceptor antagonist yohimbine. Noradrenaline (0.1 microM) reduced acetylcholine-evoked overflow to the same extent as did UK 14,304 (0.1 microM). UK 14,304 had no effect when 3H overflow was evoked by acetylcholine in the presence of 300 microM Cd2+. Currents through nicotinic acetylcholine receptors and voltage-activated Ca2+ currents were studied with the whole-cell variant of teh patch-clamp technique. UK 14,304 reduced nicotinic acetylcholine receptor currents and voltage-activated Ca2+ currents with similar potency and efficacy. Yohimbine, however, antagonized only the inhibition of voltage-activated Ca2+ currents, but not the effects of UK 14,304 on nicotinic receptor currents. Furthermore, yohimbine per se reduced currents through nicotinic receptors. Noradrenaline (10 microM) inhibited voltage-dependent Ca2+ currents just as did UK 14,304 (10 microM), but failed to reduce currents through nicotinic acetylcholine receptor channels. Cd2+ (300 microM) abolished voltage-activated Ca2+ currents and reduced nicotinic acetylcholine receptor currents by 65%. These results indicate that acetylcholine evokes noradrenaline release from rat sympathetic neurons by activation of nicotinic receptors and restricts this release via muscarinic receptors. The acetylcholine-induced transmitter release is based on two mechanisms, one involving and the other one bypassing voltage-dependent Ca2+ channels. alpha2-Adrenoceptor activation reduces voltage-activated Ca2+ currents and effects exclusively the component of acetylcholine-induced release which involves voltage-dependent Ca2+ channels. These results support the hypothesis that voltage-activated Ca2+ channels are the sole site of autoinhibitory alpha2-adrenergic effects on transmitter release from rat sympathetic neurons. The inhibitory effects of alpha2-adrenoceptor agonists and antagonists on currents through nicotinic acetylcholine receptors are not mediated by an alpha2-adrenoceptor.

Acetylcholine↗

PubMed1995

Rapid, agonist-induced desensitization of alpha 2-autoreceptors modulating transmitter release.

1. The release of previously incorporated [3H]-noradrenaline was investigated in cultures of dissociated chick or rat sympathetic neurones and in cerebrocortical slices from neonatal or adult rats. Noradrenaline, in the presence of 10 mumol l-1 of the uptake inhibitor, cocaine, or the selective alpha 2-adrenoceptor agonist, 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK 4,304), was applied for different periods of time in order to detect a possible time-dependence of the alpha 2-adrenoceptor-mediated inhibition of electrically evoked tritium outflow. 2. In chick sympathetic neurones, stimulation-evoked overflow was reduced to 30%, 42%, or 56% of control when noradrenaline (1 mumol l-1) was present for 2, 8, or 16 min, respectively. Likewise, UK 14,304 (1 mumol l-1) present for these periods of time reduced 3H overflow to 35%, 51%, and 53% of control, respectively. Addition of 1 nmol l-1 to 10 mumol l-1 UK 14,304 for either 2 or 16 min did not produce significantly different IC50 values, but the inhibitory effects were smaller with 16 min as compared to 2 min exposure at concentrations > or = 10 nmol l-1. 3. In rat sympathetic neurones, noradrenaline (100 nmol l-1) reduced stimulation-evoked overflow to 33%, 56%, or 57% of control, when present for 2, 8, or 16 min, respectively. Addition of UK 14,304 (1 mumol l-1) for these periods of time caused inhibition to 11%, 41%, and 46% of control. Applying UK14,304 for either 2 or 16 min did not result in significantly different IC5o values, but the inhibition induced by 16 min as compared to 2 min exposure was smaller at concentrations > 10 nmol 1-1.4. In cerebrocortical slices from either neonatal or adult rats, exposure to 0.1 to 1.0 micromol 1-1 UK14,304 for 16 min never caused a smaller inhibition than a corresponding 3 min exposure, although various experimental conditions were investigated.5 The results demonstrate that alpha 2-adrenoceptors which regulate noradrenaline release from sympathetic neurones undergo agonist-induced desensitization within minutes. Such rapid desensitization of alpha 2-autoreceptors was not detected in brain slice preparations.

Adrenergic alpha-Agonists↗

PubMed1995

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 Ca(2+)-dependent and tetrodotoxin-sensitive [3H]-noradrenaline release. The overflow evoked by UTP or ATP was similar at 100 mumol l-1, the concentration used in all subsequent experiments. Pre-exposure of the neurones to 100 mumol l-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 mumol l-1 pyridoxal-5'-phosphate or 30 mumol l-1 suramin, the secretory response to ATP was reduced, but the effect of UTP was unaltered. Zn2+ (10 mumol l-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.

Adenosine Triphosphate↗

PubMed1995

Fibrin sealant matrix supports outgrowth of peripheral sensory axons.

It has been suggested that fibrin-based matrix has an important role during the early stage of nerve regeneration. A fibrin sealant matrix, which was made by combining diluted human fibrinogen and thrombin, was used as a substrate for in vitro elongation of neurites and in vivo regeneration of axons. In the in vitro experiment, dissociated embryonic chick sensory neurons were cultured on dishes coated with fibrin sealant matrix and compared with the solution of thrombin/calcium chloride, or with poly-D-lysine (PDLctr). After 16 hours, cultures were stained immunohistochemically with a monoclonal antineurofilament antibody. The neurons survived well, and an abundant network of neurites, qualitatively similar to that on PDLctr, developed on the fibrin sealant matrix. The percentage of neurons that had outsprout at 16 hours was the same both in the fibrin sealant matrix and PDLctr groups. By contrast, all the neurons plated on the dishes treated with the solution of thrombin/calcium chloride were dead after 16 hours. Immunohistochemical staining of fibrinogen also showed an even distribution of fibrin matrix over the culture dishes. For the in vivo experiments, 48 rat sciatic nerves were cut and reconnected with two epineurial stitches. Fibrin sealant matrix or phosphate buffer solution was applied to the transsected and repaired region. Pinch reflex test showed that the regeneration of the leading sensory fibre was significantly faster in the fibrin sealant matrix group than in the control group at 3 and 4 days. These results indicate that fibrin sealant matrix accelerates regeneration of axons in vivo during the early phase, and also supports elongation of neurites in vitro.

Animals↗

PubMed1995

Isolation of T cell receptor gamma/delta chain constant genes from minke whale.

The polymerase chain reaction using oligonucleotides based on consensus sequences from other species allowed us to amplify minke whale (Balaenoptera acutorostrata) T cell receptor (TcR) gamma and delta constant genes. Two types of Cgamma clones were obtained which only differ by one mismatch. These minke whale Cgamma clones showed 83% nucleotide and 70% amino acid sequence similarity to the corresponding region of the human TRGC genes. The minke whale Cdelta sequences were all identical, and showed 86% nucleotide and 77.8% amino acid sequence similarity to the human TRDC gene. The whale Cgamma and Cdelta clones were used as probes for Southern blot analysis. The data confirmed that there is a unique Cdelta gene in minke whale. Two different Cgamma genes were detected, one of them also cross-hybridizing with a human Cgamma probe, suggesting that the Cgamma locus in minke whale consists of at least two different constant genes.

Amino Acid Sequence↗

PubMed1994

T cell receptor gamma cDNA in human fetal liver and thymus: variable regions of gamma chains are restricted to V gamma I or V9, due to the absence of splicing of the V10 and V11 leader intron.

Although complete in-frame transcripts of the human T cell receptor gamma V10 and V11 genes have been described, the corresponding gamma chains have never been found in gamma delta T cell receptors. In this study, we show that the leader intron of all V10 and V11 cDNA isolated from fetal thymus, fetal liver and adult peripheral blood lymphocytes are unspliced. We demonstrate that, due to the absence of splicing, V10 and V11 are pseudogenes and cannot be expressed in gamma chains. They are the first pseudogenes of this type described in a rearranging T cell receptor/immunoglobulin locus. Therefore the gamma repertoire at the protein level is limited to subgroup V gamma I and to V9. By analysis of the gamma polymerase chain reaction products from total cDNA, we find that the gamma locus is active in early ontogeny (8 weeks), as shown by the presence of rearranged V9 and V10 gene transcripts in the liver. At 13 weeks, the V gamma I genes as well as V9 and V10 have undergone productive rearrangements in the liver, and in the thymus. Most rearrangements, if not all, involve the T cell receptor gamma C1 region (JP1, JP, J1 segments) in both tissues, confirming the accessibility of the C1 region in early stages of development.

Amino Acid Sequence↗

PubMed1994

Developmental regulation of multiple nicotinic AChR channel subtypes in embryonic chick habenula neurons: contributions of both the alpha 2 and alpha 4 subunit genes.

Habenula neurons from both early and late stage embryonic chickens express multiple subtypes of nicotinic acetylcholine receptor channels (nAChRs). The channel subtypes expressed by habenula neurons are similar in functional properties, but apparently distinct in subunit composition, from their peripheral counterparts in autonomic ganglia. Early in development, nicotine activates four classes of neuronal bungarotoxin (nBGT)-sensitive channels (approx. conductance = 15, 30, 50, 60pS) that are intermingled on the surface of habenula neuronal somata. In neurons removed from older animals, nAChR channel activity has increased 4- to 40-fold and channel subtypes have become spatially segregated from one another. Analysis of the profile of nAChR subunit gene expression by polymerase chain reaction indicates that several of the alpha-type subunit genes, including alpha 2,3,4,5,7, and alpha 8, as well as both beta 2 and beta 4, are expressed. Treatment of the neurons with subunit specific antisense oligonucleotides reveals that the alpha 2 and alpha 4 (but not alpha 3) subunits contribute to the functional profile of native nAChRs expressed by habenula neurons. Consideration of the functional properties and apparent subunit composition of autonomic ganglion nAChRs in the chick suggests that habenula neurons may utilize a very distinct set of subunit combinations to produce an array of nAChR channel subtypes similar in both conductance and pharmacological profile to those expressed by sympathetic neurons.

Acetylcholine↗

PubMed1994

Alpha 2-adrenoceptor-mediated inhibition of electrically evoked [3H]noradrenaline release from chick sympathetic neurons: role of cyclic AMP.

This study explores the role of cyclic AMP in electrically evoked [3H]noradrenaline release and in the alpha 2-adrenergic modulation of this release in chick sympathetic neurons. Along with an increase in stimulation-evoked tritium overflow, applications of forskolin enhanced the formation of intracellular cyclic AMP. Both effects of forskolin were potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. The forskolin-induced increase in overflow was abolished by the Rp-diastereomer of cyclic AMP-thioate, an antagonist at cyclic AMP-dependent protein kinases, and 1,9-dideoxy-forskolin, an inactive analogue at adenylyl cyclase, had no effect on the evoked overflow. A 24-h pretreatment with either cholera toxin or forskolin reduced the subsequent forskolin-induced accumulation of cyclic AMP and inhibited the stimulation-evoked release. Basal cyclic AMP production, however, remained unaltered after forskolin treatment and was enhanced after 24 h of cholera toxin exposure. The alpha 2-adrenergic agonist bromoxidine did not affect the formation of cyclic AMP stimulated by forskolin but reduced electrically evoked release. However, effects of bromoxidine on 3H overflow were attenuated by forskolin as well as by 8-bromo-cyclic AMP. Effects of bromoxidine on [3H]noradrenaline release were paralleled by an inhibition of voltage-activated Ca2+ currents, primarily through a delayed time course of current activation. This effect was abolished when either forskolin or 8-bromo-cyclic AMP was included in the pipette solution. Both substances, however, failed to affect Ca2+ currents in the absence of bromoxidine. These results suggest that the signaling cascade of the alpha 2-adrenergic inhibition of noradrenaline release involves voltage-activated Ca2+ channels but not cyclic AMP.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗

PubMed1993

Methoxyverapamil reduction of nicotine-induced catecholamine release involves inhibition of nicotinic acetylcholine receptor currents.

The mechanism by which the putative Ca2+ channel blocker methoxyverapamil (D600) inhibits nicotine-induced catecholamine release was investigated in bovine adrenal chromaffin cells and in neurons from paravertebral sympathetic ganglia of chick embryos. We found D600 to prevent catecholamine release evoked by 30 s applications of nicotine with a significantly higher potency than the release induced either by 30 s K+ depolarizations or by electrical field stimulation of sympathetic neurons. Like the use-dependent action of D600 upon Ca2+ channels, the magnitude of inhibition of the K(+)-evoked secretion depended on the duration of stimulation (10 s to 5 min). Data on catecholamine release were supplemented by patch-clamp recordings. We found whole-cell currents in chromaffin cells evoked by (extrapolated) 0.5 s applications of nicotine to be significantly more sensitive to D600 than Ca2+ currents induced by a 0.5 s depolarization from -80 to 0 mV. In both instances, the potency of D600 depended on the duration of the (nicotinic and depolarizing) stimuli. Our data suggest that D600 inhibits nicotine-induced catecholamine release by reducing nicotinic acetylcholine receptor currents rather than voltage-gated Ca2+ currents. Hence, in chromaffin cells as well as in sympathetic neuronal preparations, D600 does not appear to be a suitable tool to investigate the part voltage-activated Ca2+ currents play in cellular events induced by nicotine.

Adrenal Medulla↗

PubMed1992

Pertussis toxin abolishes the inhibition of Ca2+ currents and of noradrenaline release via alpha 2-adrenoceptors in chick sympathetic neurons.

Effects of alpha 2-adrenoceptor agonists on whole-cell Ca2+ currents and 3H-noradrenaline release were investigated by applying the patch-clamp technique and electrical field stimulation to cultured embryonic chick sympathetic neurons. A 24-h exposure of the sympathetic neurons to pertussis toxin (100 ng/ml) abolished both the alpha 2-adrenoceptor-mediated inhibition of Ca2+ currents and the modulation of noradrenaline release caused by noradrenaline (1 mumol/l; in the presence of 10 mumol/l cocaine) or the alpha 2-adrenoceptor agonists 5-bromo-6-(2-imidazolin-2- ylamino)quinoxaline (UK 14,304, 10 mumol/l) and clonidine (10 mumol/l). These results suggest that the alpha 2-autoreceptor-mediated inhibition of noradrenaline release from chick sympathetic neurons operates through the modulation of Ca2+ channels via pertussis-toxin-sensitive GTP-binding-proteins.

Adrenergic alpha-Agonists↗

PubMed1992

Intergenerational continuity of parental rejection and depressed affect.

Using survey and observational data from a sample of 451 intact families, this study used structural equation modeling to examine the intergenerational continuity of depressed mood and rejecting parenting. The model partially replicated the intergenerational model of Elder, Caspi, and Downey (1986) that indicates a cyclical transmission process by which parents' personality traits affect parent-child interaction, which, in turn, increases the propensity for developmental problems among offspring. The results indicated a pattern of intergenerational transmission of depressed mood through parental rejection of offspring. Multiple reporters and multiple indicators strengthen previous intergenerational findings by reducing some of the method variance biases that have been problematic in prior studies.

Adolescent↗

PubMed1992

Uptake mechanism of technetium-99m-d, 1-HMPAO in cell cultures of the dissociated postnatal rat cerebellum.

The accumulation and retention mechanisms of 99mTc-d, 1-hexamethylpropyleneamine oxime (99mTc-d, 1-HMPAO) were investigated in cultures of the dissociated rat cerebellum. Our experiments indicate a linear dependency of the uptake on incubation time and on the concentration of the radioligand. Upon chloroform extraction and distribution between the lipophilic and the hydrophilic phases, we located 69.1% of the retained radioactivity in the hydrophilic phase, 24.1% in a bound state and 6.8% in the lipophilic phase. The water-soluble, unbound radioactive contents of the cultures were identified as 99mTcO4- by HPLC analysis. Treatment of cultures with diethyl maleate (DEM) inhibited the accumulation of radioactivity along with a reduction of the GSH contents of the cultures. However, even in the absence of GSH, significant amounts of radioactivity were accumulated. DEM reduced the radioactive contents of cultures predominantly by diminishing the aqueous phase of the chloroform-extracted material. By contrast, the metabolic state, manipulated by treating the cultures with oligomycin B or 2,4-dinitrophenol, had no significant effect on the accumulation of radioactivity. Our experiments suggest two major mechanisms for the retention of radioactivity following the exposure of neuronal tissue to 99mTc-d, 1-HMPAO: Conversion of the lipophilic complex to the hydrophilic product, 99mTcO4-, and binding to non-diffusible cell components.

2,4-Dinitrophenol↗

PubMed1991

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

Sympathetic 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 mumol/l). The alpha 2-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline (UK-14,304) caused a concentration-dependent decrease in overflow, whereas the alpha 1-adrenoceptor agonist methoxamine was ineffective at up to 1 mumol/l. The concentration-response curve of UK-14,304 was shifted to the right by the alpha 2-adrenoceptor antagonist yohimbine (0.03 mumol/l). Yohimbine on its own caused no significant change. The noradrenaline reuptake inhibitor cocaine (10 mumol/l) caused a small (20%) increase in evoked overflow. The results indicate that cultured chick sympathetic neurons possess release-modulating alpha 2-adrenoceptors and that the electrically induced overflow of transmitter occurs under conditions virtually free of autoinhibition.

Adrenergic alpha-Agonists↗

PubMed1991

Modulation of calcium currents via alpha 2-adrenoceptors in embryonic chick sympathetic neurons.

In order to gain insight into the mechanism of the autoinhibition of noradrenaline release, the present study explores the effects of substances acting at various adrenoceptor-subtypes on voltage-activated Ca2+ currents. Experiments were carried out on cultured embryonic chick sympathetic neurons using the patch clamp technique. Ca2+ currents associated with a (fully activating) depolarizing 150 ms voltage step to 0 mV were reduced by noradrenaline and the two alpha 2-adrenoceptor agonists UK 14,304 and clonidine, predominantly during the early phase of activation. We quantified these effects by measuring Ca2+ current amplitudes in the absence and presence of substances 10 ms after the beginning of the depolarization. Noradrenaline effects were maximal at 5 mumol/l, causing a 28% depression of the current. Half-maximal effects (IC50) were apparent at 0.7 mumol/l. UK 14,304 was equipotent to noradrenaline (IC50: 0.5 mumol/l; maximal effect: 26% depression). Clonidine, while active in the same range of concentration (IC50: 0.6 mumol/l), had a smaller maximal effect (20% depression). Methoxamine and isoprenaline, on the other hand, did not significantly reduce the Ca2+ current at 10 mumol/l. The noradrenaline-induced inhibition was attenuated by yohimbine (1 mumol/l). Neither prazosin (1 mumol/l) nor propranolol (1 mumol/l) interfered with the effect of noradrenaline. These results indicate a reduction of Ca2+ influx via alpha 2-adrenoceptors and suggest that the autoreceptor-mediated inhibition of transmitter release in embryonic chick sympathetic neurons operates through the modulation of Ca2+ channels.

Adrenergic alpha-Agonists↗