Beauty is in the soul of the beholder: psychological implications of beauty and African American women

An in vitro receptor binding and in vivo microdialysis study was performed to further investigate the modulation of dopamine (DA) D2 receptors by neurotensin (NT) peptides. Saturation experiments with the D2 agonist [3H]NPA (N-propylnorapomorphine) showed that 10 nM of NT, 10 nM of neuromedin N (NN) and 1 nM of the C-terminal NT-(8-13) fragment significantly increased the KD values by 125%, 181%, and 194%, respectively without significantly affecting the Bmax value of the [3H]NPA binding sites in coronal sections of rat ventral forebrain mainly containing the nucleus accumbens (Acb) and the olfactory tubercle. In line with the previous findings that NT can increase GABA release in the Acb and that NT receptors are not found on DA terminals in this brain region, the present in vivo microdialysis study demonstrated that local perfusion of NT (1 nM) counteracted the D2 agonist pergolide (2 mu M) induced inhibition of GABA, but not of DA release in the rat Acb. This result indicates that NT counteracts the D2 agonist induced inhibition of GABA release in the rat Acb, via an antagonistic postsynaptic NT/D2 receptor interaction as also suggested by the inhibitory regulation of D2 receptor affinity in the Acb by the NT peptides demonstrated in the present receptor binding experiments. Thus, the neuroleptic and potential antipsychotic profile of the NT peptides may involve an antagonistic NT/D2 receptor regulation in the ventral striatum.     

Relation of exocytotic release of gamma-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the gamma-aminobutyric acid (GABA) carrier, NNC-711, (1-[(2-diphenylmethylene)amino]oxyethyl)- 1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride, blocks the Ca(2+)-independent release of [3H]GABA from rat brain synaptosomes induced by 50 mM K+ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca(2+)-dependent release of [3H]GABA in the total absence of carrier-mediated release. Reversal of the Na+/Ca2+ exchanger was used to increase the intracellular free Ca2+ concentration ([Ca2+]i) to test whether an increase in [Ca2+]i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca2+]i may rise to values above 400 nM, as a result of Na+/Ca2+ exchange, without inducing release of [3H]GABA, but subsequent K+ depolarization immediately induced [3H]GABA release. Thus, a rise of only a few nanomolar Ca2+ in the cytoplasm induced by 50 mM K+ depolarization, after loading the synaptosomes with Ca2+ by Na+/Ca2+ exchange, induced exocytotic [3H]GABA release, whereas the rise in cytoplasmic [Ca2+] caused by reversal of the Na+/Ca2+ exchanger was insufficient to induce exocytosis, although the value for [Ca2+]i attained was higher than that required for exocytosis induced by K+ depolarization. The voltage-dependent Ca2+ entry due to K+ depolarization, after maximal Ca2+ loading of the synaptosomes by Na+/Ca2+ exchange, and the consequent [3H]GABA release could be blocked by 50 microM verapamil. Although preloading the synaptosomes with Ca2+ by Na+/Ca2+ exchange did not cause [3H]GABA release under any conditions studied, the rise in cytoplasmic [Ca2+] due to Na+/Ca2+ exchange increased the sensitivity to external Ca2+ of the exocytotic release of [3H]GABA induced by subsequent K+ depolarization. Thus, our results show that the vesicular release of [3H]GABA is rather insensitive to bulk cytoplasmic [Ca2+] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca2+ entry through Ca2+ channels near the active zones.     

Mechanism of endothelium-dependent relaxation induced by thrombin in the pig coronary artery

The present study describes the characterization of the binding properties and autoradiographic distribution of a new nonpeptide antagonist of neurotensin receptors, [3H]SR 142948A (2-[[5-(2,6-dimethoxyphenyl)-1-(4-(N-(3-dimethylaminopropyl)-N-methyl carbamoyl)-2-isopropylphenyl)-1H-pyrazole-3-carbonyl]-amino]-ad amantane-2-carboxylic acid, hydrochloride), in the rat brain. The binding of [3H]SR 142948A in brain membrane homogenates was specific, time-dependent, reversible and saturable. [3H]SR 142948A bound to an apparently homogeneous population of sites, with a Kd of 3.5 nM and a Bmax value of 508 fmol/mg of protein, which was 80% higher than that observed in saturation experiments with [3H]neurotensin. [3H]SR 142948A binding was inhibited by SR 142948A, the related nonpeptide receptor antagonist, SR 48692 (2-[[1-(7-chloroquinolin-4-yl)-5-(2,6-dimethoxyphenyl)-1H-pyrazole -3-carbonyl]amino]-adamantane-2-carboxylic acid) and neurotensin. Saturation and competition studies in the presence or absence of the histamine H1 receptor antagonist, levocabastine, revealed that [3H]SR 142948A bound with similar affinities to both the levocabastine-insensitive neurotensin NT1 receptors (20% of the total binding population) and the recently cloned levocabastine-sensitive neurotensin NT2 receptors (80% of the receptors) (Kd = 6.8 and 4.8 nM, respectively). The regional distribution of [3H]SR 142948A binding in the rat brain closely matched the distribution of [125I]neurotensin binding. In conclusion, these findings indicate that [3H]SR 142948A is a new potent antagonist radioligand which recognizes with high affinity both neurotensin NT1 and NT2 receptors and represents thus an excellent tool to study neurotensin receptors in the rat brain.     

Mazindol, nomifensine and desmethylimipramine inhibit potassium-induced release of dopamine: effect of stimulus strength

Catecholamine uptake blockers exhibit a second action: They suppress the potassium (K+)-stimulated release of [3H]-dopamine (DA) from rat striatal tissue in vitro. In the present study, a K+ dose-response curve was obtained for the release of both [3H]-DA and endogenous DA from striatal tissue, in the absence and presence of catecholamine uptake blockers. Three drugs were used, namely, mazindol, nomifensine, and desmethylimipramine. The data showed that diminished release in the presence of mazindol or nomifensine was dependent upon the concentration of K+. Marked inhibition of stimulus-induced release of either 3H-DA or endogenous DA was observed at low concentrations of K+ (15 and 20 mM), but not at higher concentrations of K+ (40 and 60 mM). In contrast, the diminished release of DA in the presence of desmethylimipramine persisted over the K+ range of 20-60 mM. These data show that the drug action observed previously was not restricted to a specific neuronal pool of DA that was labelled with [3H]-DA. Further, the data emphasize the importance of the concentration of the depolarizing stimulus when evaluating the effects of drugs on release of catecholamines.     

Molecular cloning, functional expression, and mutagenesis of cDNA encoding a cysteine proteinase inhibitor from sunflower seeds

1. Ouabain, an inhibitor of Na+/K+ ATPase induces the release of acetylcholine from central and myenteric cholinergic neurones principally due to partial depolarization of the cell membrane. The effect of ouabain has been examined on neurogenic contractions in the guinea-pig ileum arising from either electrical field stimulation or from naloxone in morphine-exposed preparations. 2. Guinea-pig isolated ileum preparations were stimulated transmurally (0.1 Hz, 0.3 ms, 200 mA) to elicit contractions of the myenteric plexus-longitudinal smooth muscle. 3. Incubation with morphine (0.3 microM, 60 min) was followed by naloxone (1 microM) which produced withdrawal contractions in 16/26 preparations (median of 10.7 [2.2-40.0]% of a maximal contracture to KCl (60 mM)). 4. In parallel experiments, ouabain (1 microM) was added to the tissue before exposure to morphine (0.3 microM, 60 min). Naloxone (1 microM) subsequently displayed a withdrawal contraction in all 26/26 tissues (57.9 [30.5-151.7]% of a maximal contracture to KCl (60 mM). 5. Ouabain neither affected the concentration-dependent contractions of guinea-pig ileum produced by carbachol nor the inhibition of electrically-evoked contraction produced by morphine (0.3 microM). 6. The muscarinic antagonist atropine (0.1 microM) antagonized control naloxone withdrawal responses. The atropine resistant component, evident in ouabain-treated tissues, was blocked by SR140333((S)1-[2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenyla cetyl)piperidin-3-yl]ethyl]-4-phenyl-1-azoniabicyclo[2.2. 2]-octane, chloride), a substance P antagonist. 7. Clonidine (alpha2-adrenoceptor agonist) inhibited electrically-evoked contractions. Exposure to the alpha2-adrenoceptor antagonist RX811059 (2-(2-ethoxy-1,4-benzodioxan-2-yl)-2-imidazoline), resulted in a contracture which was not significantly enhanced by ouabain (1 microM). 8. Ouabain selectively potentiates the naloxone-induced withdrawal contraction following acute exposure to morphine the major components of which are mediated by both acetylcholine and substance P.     

Voltage-activated calcium channels involved in veratridine-evoked [3H]dopamine release in rat striatal slices

The present study explored the role of different sub-types of voltage-activated Ca2+ channels (VACCs) in mediating veratridine-evoked [3H]dopamine (DA) release from rat striatal slices. The release of [3H]DA evoked by veratridine (25 microM) decreased by 50.6+/-2.9% (n=8) in the absence of calcium and was completely abolished by 1 microM tetrodotoxin. The L-type Ca2+ channel blockers nifedipine (10 microM), nitrendipine (10 microM), diltiazem (10 microM) and verapamil (10 microM) did not modulate this release. Similarly, [3H]DA release was affected neither by the N-type VACC blocker omega-conotoxin-GVIA (1 microM) nor by the selective P-type channel blockers omega-agatoxin-IVA and omega-agatoxin-TK at low nM concentrations (30 nM), indicating no involvement of N- and P-type Ca2+ channels. In contrast, higher concentrations of omega-agatoxin-IVA that would also inhibit Q-type VACCs, blocked the release of [3H]DA by 27.9+/-8.1% (n=5) and 37.5+/-13.6% (n=3) at 0.3 and 1 microM, respectively. In addition, application of the Q-type Ca2+ channel blocker omega-conotoxin-MVIIC (0.01-3 degrees M) reduced [3H]DA release in a concentration-dependent manner, with maximum inhibition of 35.3+/-4.1% at 3 microM (n=5). On the basis of these results, it is concluded that the Ca2+ channels that participate in veratridine-evoked [3H]DA release are Q-type Ca2+ channels.     

Margatoxin increases dopamine release in rat striatum via voltage-gated K+ channels

The distribution of iodinated margatoxin ([125I]margatoxin) binding sites in rat was investigated by autoradiography. Rat striatum expresses a high density of margatoxin binding sites and, therefore, the effects of margatoxin, charybdotoxin and iberiotoxin have been studied on [3H]dopamine release from rat striatal slices in vitro. Margatoxin (0.1-100 nM) and charybdotoxin (10-1000 nM), but not iberiotoxin increased the spontaneous and the electrically evoked [3H]dopamine release. [3H]dopamine release by margatoxin was inhibited by tetrodotoxin and omega-conotoxin GVIA, but not by atropine, naloxone, N(omega)-nitro-L-arginine and neurokinin or neurotensin receptor antagonists. In the buffer solution used for release experiments, [125I]margatoxin labels a maximum of 0.12 pmol of sites/mg protein in rat striatal membranes with a Kd of 5 pM. [125I]margatoxin binding was inhibited by margatoxin (Ki of 4 pM), charybdotoxin (Ki of 162 pM) but not by iberiotoxin. We conclude that inhibition of margatoxin-sensitive voltage-gated K+ channels increases [3H]dopamine release demonstrating their role in repolarization of nigrostriatal projections. In contrast, iberiotoxin-sensitive, high-conductance Ca2+-activated K+ channels are not involved in release of [3H]dopamine.     

SK&F 96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenylethyl]-1H- imidazole hydrochloride) stimulates phosphoinositide hydrolysis in human U373 MG astrocytoma cells

SK&F 96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenylethyl]-1H-imi dazole hydrochloride) stimulated the accumulation of [3H]inositol monophosphates ([3H]IP1) in human U373 MG astrocytoma cells prelabelled with [3H]inositol (EC50 15 +/- 1 microM, Hill coefficient 3.8 +/- 0.4). SK&F 96365-induced accumulation of [3H]IP1 increased linearly with time, but there was no initial rapid formation of [3H]IP3. SK&F 96365 also stimulated [3H]IP1 accumulation in human HeLa cells, but only to a small extent in slices of rat cerebral cortex and guinea-pig cerebellum. SK&F 96365-induced accumulation of [3H]IP1 in U373 MG cells increased as extracellular Ca2+ was increased from nominally zero to 4 mM, but there was no evidence that SK&F 96365 induced any marked entry of Ca2+ into cells; only an inhibition of store-refilling-induced Ca2+ entry was apparent. Further, the response to SK&F 96365 was additive with that to the Ca2+ ionophore ionomycin. Depolarization of the cells with raised K+ produced only a small stimulation of phosphoinositide hydrolysis. SK&F 96365 caused the release of Ca2+ from intracellular stores in U373 MG cells (EC50 26 +/- 14 microM), but thapsigargin induced only a small accumulation of [3H]IP1. Miconazole, another N-substituted imidazole, also stimulated [3H]IP1 accumulation in U373 cells.     

Assessment of the role of TRH in the release of [3H]-dopamine from rat nucleus accumbens-lateral septum slices

We have studied [3H]-dopamine ([3H]-DA) release from rat nucleus accumbens lateral septum slices in response to various paradigms aimed at increasing endogenous or exogenous thyrotropin releasing hormone (TRH) concentrations in the extracellular space. High KCl concentrations significantly enhanced [3H]-DA release by fourfold. TRH (10(-4) or 5 x 10(-4) M) did not affect [3H]-DA release. The release of [3H]-DA was not stimulated by TRH either in the presence of N-1-carboxy-2-phenylethyl (N(im)benzyl)-histidyl-beta naphthylamide, a specific pyroglutamyl peptidase II inhibitor, or that of specific inhibitors of prolyl endopeptidase and pyroglutamyl peptidase I. None of the peptidase inhibitors modified the [3H]-DA release by themselves. These results suggest that the TRH stimulation of [3H]-DA release in vitro observed in previous studies is not due to peptide inactivation but may be due to a nonspecific effect. TRH enhancement of DA release in nucleus accumbens in vivo may not be the result of a direct effect of TRH on DA terminals.     

U73122 and U73343 inhibit receptor-mediated phospholipase D activation downstream of phospholipase C in CHO cells

We investigated the effects of nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine and sodium nitroprusside on basal and K+-evoked release of [3H]noradrenaline from superfused synaptosomes from the rat cerebral cortex. Both substances produced concentration-dependent increases in the release of the labeled transmitter under basal and depolarized conditions. The effects of the donors on basal release were Ca2+-independent but were not inhibited by the carrier-uptake blocker, desipramine; the effects were abolished by hemoglobin (an NO scavenger). Thirty-five minutes after stimulation with sodium nitroprusside, the synaptosomes were still responsive to KCl stimulation, indicating that the donor's effects were not caused by damage to the synaptosome membrane. The cGMP analogue, 8-bromo-cGMP, had no effect on basal release, and the enhanced release produced by sodium nitroprusside was not inhibited by the specific inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one, indicating that NO's effects on basal release of the neurotransmitter are guanylate cyclase-independent. Both of the NO donors had more marked effects on release of [3H]noradrenaline during K+-stimulated depolarization. The NO-mediated increase in this case was partially antagonized by 10 microM LH-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one, and 8-Br-cGMP was also capable of producing concentration-dependent increases in the K+-stimulated release of the transmitter. These findings indicate that the effects of the NO donors on [3H]noradrenaline release during depolarization are partially mediated by the activation of guanylate cyclase.     

Posttraumatic stress and maladjustment among adult burn survivors 1 to 2 years postburn. Part II: the interview data

Using native plasma membrane vesicle suspensions from the rat cerebral cortex under conditions designed to alter intravesicular [Ca2+], we found that Ca2+ induced 47 +/- 5% more influx of [3H]GABA, [3H]D-aspartate and [3H]glycine at 37 degrees C with half-times 1.7 +/- 0.5, 1.3 +/- 0.4 and 1.3 +/- 0.4 min, respectively. We labelled GABA transporter sites with the uptake inhibitor, [3H]-(R,S)-N-[4,4-bis(3-methyl-2-thienyl)but-3-en-1-yl]nipecotic acid and found that Ca2+ induced a partial dissociation of the bound inhibitor from GABA transporter sites with a similar half-time. By means of rapid kinetic techniques applied to native plasma membrane vesicle suspensions, containing synaptic vesicles stained with the amphipathic fluorescent styryl membrane probe N-(3-triethylammoniumpropyl)-4-[4-(dibutylamino)styryl]pyrid inium dibromide, we have measured the progress of the release and reuptake of synaptic vesicles in response to Ca2+ and high-[K+] depolarization in the 0.0004-100 s range of time. Synaptic vesicle exocytosis, strongly influenced by external [Ca2+], appeared with the kinetics accelerated by depolarization. These results are consistent with the potential involvement of Ca2+ in taking low-affinity transporters to the plasma membrane surface via exocytosis.     

Nitric oxide involvement in regulating the dopamine transport in the striatal region of rat brain

Spontaneous [3H]dopamine ([3H]DA) overflow was measured from striatal slices in the presence of different glutamate (Glu) receptor agonists such as N-methyl-D-aspartate (NMDA), kainate (KA) and quisqualate (QA) and their corresponding antagonists, Dizocilpine maleate (MK-801), D-gamma-glutamyl-aminomethanesulfonic acid (GAMS) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively. [3H]DA uptake and release in the presence of L-Arginine (L-Arg) and NG-nitro-arginine (L-N-Arg), an inhibitor of nitric oxide (NO) synthesis were also evaluated. L-N-Arg alone or combined with L-Arg significantly reduced [3H]DA uptake at 10 and 100 microM from 33% to 44% from striatal slices. Whereas, in brain synaptosomal fractions L-Arg induced a biphasic effect on that [3H]DA uptake in a dose dependent manner, and L-N-Arg showed an absolute inhibition in 80-90% of this [3H]DA uptake at 1-500 microM. The amino acids, lysine, valine and histidine (100 microM) had a little effect inhibitory on [3H]DA uptake from synaptosomal fractions. Glu agonists, NMDA (10 microM) and KA (10 microM) importantly increased the spontaneous [3H]DA overflow, which was blocked by MK-801 (10 microM) and GAMS (10 microM), respectively. QA had no effect on [3H]DA release. L-Arg (10-200 microM) potentiated the spontaneous [3H]DA overflow in a dose dependent fashion from striatal slices, being reverted by 10 microM L-N-Arg alone or in combination with all other compounds; whereas, lysine, histidine and valine did not modify that spontaneous [3H]DA overflow. Results support the hypothesis related to the participation of NO on DA transport possibly synthesized at the dopaminergic (DAergic) terminals in the striatum; also that L-Arg concentration may determine alternative mechanisms to regulate the DAergic activity at the striatum.     

Effects of the antiparkinsonian drug budipine on neurotransmitter release in central nervous system tissue in vitro

The effects of the antiparkinsonian drugs budipine and biperiden on spontaneous and electrically evoked release of dopamine (DA), acetylcholine (ACh), GABA or noradrenaline (NA) were studied in caudate nucleus or cortex slices, respectively, of the rabbit brain. Whereas both drugs (1-10 microM) strongly increased spontaneous [3H]outflow in caudate nucleus slices preincubated with [3H]DA, budipine inhibited but biperiden facilitated the evoked DA release. In the presence of the DA-reuptake inhibitor nomifensine, a significant part of the budipine-induced basal [3H] outflow consisted of unmetabolized DA. Synaptosomal high-affinity uptake of [3H]DA was only weakly affected by budipine and biperiden (IC50 values, 11 and 9 microM, respectively). Budipine enhanced also basal [3H]outflow from cortex slices prelabeled with [3H]NA, however this outflow consisted mainly of NA metabolites even in the presence of cocaine. The evoked release of [3H]ACh in rabbit caudate nucleus slices preincubated with [3H] choline was almost unaffected by budipine but enhanced by biperiden in the absence of further drugs. In the presence of nomifensine, however, budipine inhibited, but biperiden still enhanced, the evoked ACh release. Moreover, both drugs showed antimuscarinic properties in the presence of the ACh esterase inhibitor physostigmine, i.e., they facilitated the evoked ACh release, exhibiting pA2 values of about 6.9 (budipine) and 8.3 (biperiden). Addition of the D2 receptor antagonist domperidone diminished all inhibitory effects of budipine on the evoked ACh release. The evoked overflow of [3H] in caudate nucleus slices preincubated with [3H]GABA was reduced by both budipine and biperiden. It is concluded that both anticholinergic and indirect dopaminomimetic properties contribute to the antiparkinsonian effects of budipine, whereas biperiden exhibits mainly anticholinergic effects. Moreover, both drugs might disinhibit GABA controlled neurons in the central nervous system.     

Survey of antibodies against various infectious disease agents in racing camels in Abu Dhabi, United Arab Emirates

1. The roles of both Ca2+ and adenosine 3':5'-cyclic monophosphate (cyclic AMP) in carbachol and K(+)-stimulated [3H]-noradrenaline release from SH-SY5Y human neuroblastoma cells were examined. 2. Both carbachol and K+ caused a time- and dose-related stimulation of [3H]-noradrenaline release. The release event in perfused cells was monophasic. Half-maximum stimulation measured in statically incubated (3 min) cells was 38 +/- 4 microM and 63 +/- 4 mM respectively. K+ (100 mM, added)-evoked release was greater than that produced by carbachol (1 mM). 3. Both carbachol and K+ caused a time- and dose (measured at 3 min)-related stimulation of cyclic AMP formation with half-maximum stimulation occurring at 5 +/- 1 microM and 49 +/- 2 mM respectively. In contrast to its effects on release, carbachol produced a greater stimulation of cyclic AMP formation than K+. 4. K(+)-stimulated [3H]-noradrenaline release was entirely dependent on Ca2+ entry as 2.5 mM Ni2+ abolished release. However, carbachol-evoked (1 mM) release appeared to be unaffected by Ni2+ pretreatment. 5. These data suggest that in SH-SY5Y cells, elevated cyclic AMP levels are not directly involved in [3H]-noradrenaline release. In addition, carbachol-stimulated release is largely independent of extracellular Ca2+ possibly implying a role for intracellular stored Ca2+ in the release process.     

Electrical activity of the proventriculus of the polychaete worm Syllis spongiphila

The straited myoepithelial cells of the proventriculus of Syllis spongiphila are composed of only one or two sarcomeres that may reach 40 mum in length. Experiments were performed to study some of their electrophysiological properties and their synaptic control. The mean resting potentials recorded in two different bathing media were 59-1 +/- 5-5 mV (S.D., n=91) and 62-5 +/- 6-3 mV (S.D., n=98). At rest the membrane potential is determined largely by permeability of the membrane to K+ ions, but the membrane is also permeable to other ions. On a semilogarithmic plot of membrane potential v. [K]o the mean slope of the data points from 9 to 90 mM-[K]o was 48 +/- 3 mV for a 10-fold change in [K]o. The anterior end of the animal was stimulated with a suction electrode to elicit activity of nerve fibres that innervate the proventriculus. Single indirect stimuli usually evoked hyperpolarizing or biphasic responses, and occasionally depolarizing responses, from the myoepithelial cells. The depolarizing synaptic potentials exhibited a faster time course than the hyperpolarizing ones. The rise time to peak ranged from 20 to 35 ms for simple depolarizations (n=32) and 25-75 ms for simple hyperpolarizations (n=103). Time to decay to half amplitude ranged from 20 to 55 ms for depolarizations (n=29) and 62-135 ms for hyperpolarizations (n=87). Low frequency (is less than or equal to 4 Hz) trains of indirectly applied stimuli elicited mainly hyperpolarizing responses; higher frequency (5-40 Hz) trains elicited complex responses composed of hyperpolarizations and depolarizations. Hyperpolarizations were selectively and reversibly abolished in chloride-free solutions. The reversal potential of the hyperpolarizing synaptic potential was -104 +/- 3 mV (S.D., n=8, 2 preparations). In calcium-free solution both hyperpolarizations and depolarizations were almost completely abolished. 4 mM-Mn2+ added to the bath almost completely abolished the depolarization but not the hyperpolarization. It was not clear whether Mn2+ acted at the presynaptic membrane, the postsynaptic membrane or both. The myoepithelial cells are electrically coupled. The mean space constant of five preparations was 0-52 mm (range 0-40-0-66 mm).     

Characteristics of electrically evoked somatodendritic dopamine release in substantia nigra and ventral tegmental area in vitro

Somatodendritic dopamine (DA) release from neurons of the midbrain represents a nonclassical form of neuronal signaling. We assessed characteristics of DA release during electrical stimulation of the substantia nigra pars compacta (SNc) in guinea pig midbrain slices. With the use of parameters optimized for this region, we compared stimulus-induced increases in extracellular DA concentration ([DA]o) in medial and lateral SNc, ventral tegmental area (VTA), and dorsal striatum in vitro. DA release was monitored directly with carbon-fiber microelectrodes and fast-scan cyclic voltammetry. Detection of DA in SNc was confirmed by electrochemical, pharmacological, and anatomic criteria. Voltammograms of the released substance had the same peak potentials as those of DA obtained during in vitro calibration, but different from those of the indoleamine 5-hydroxytryptamine. Similar voltammograms were also obtained in the DA-rich striatum during local electrical stimulation. Contribution from the DA metabolite 3,4-dihydroxyphenylacetic acid to somatodendritic release was negligible, as indicated by the lack of effect of the monoamine oxidase inhibitor pargyline (20 microM) on the signal. Lastly, DA voltammograms could only be elicited in regions that were subsequently determined to be positive for tyrosine hydroxylase immunoreactivity (TH-ir). The frequency dependence of stimulated DA release in SNc was determined over a range of 1-50 Hz, with a constant duration of 10 s. Release was frequency dependent up to 10 Hz, with no further increase at higher frequencies. Stimulation at 10 Hz was used in all subsequent experiments. With this paradigm, DA release in SNc was tetrodotoxin insensitive, but strongly Ca2+ dependent. Stimulated [DA]o in the midbrain was also site specific. At the midcaudal level examined, DA efflux was significantly greater in VTA (1.04 +/- 0.05 microM, mean +/- SE) than in medial SNc (0.52 +/- 0.05 microM), which in turn was higher than in lateral SNc (0.35 +/- 0.03 microM). This pattern followed the apparent density of TH-ir, which was also VTA > medial SNc > lateral SNc. This report has introduced a new paradigm for the study of somatodendritic DA release. Voltammetric recording with electrodes of 2-4 microns tip diameter permitted highly localized, direct detection of endogenous DA. The Ca2+ dependence of stimulated release indicated that the process was physiologically relevant. Moreover, the findings that somatodendritic release was frequency dependent across a range characteristic of DA cell firing rates and that stimulated [DA]o varied markedly among DA cell body regions have important implications for how dendritically released DA may function in the physiology and pathophysiology of substantia nigra and VTA.     

Effects of the dopamine D3 antagonist PD 58491 and its interaction with the dopamine D3 agonist PD 128907 on brain dopamine synthesis in rat

The dopamine (DA) D3 receptor antagonist PD 58491 [3-[4-[1-[4-[2-[4-(3-diethylaminopropoxy)phenyl]benzoimidazol++ +-1-yl-butyl]-1H-benzoimidazol-2-yl]phenoxy]propyl]diethylamine] bound with high affinity and selectivity to recombinant human DA D3 versus D2L and D4.2 receptors transfected into Chinese hamster ovary cells: Ki values of 19.5 nM versus 2,362 and >3,000 nM, respectively. In contrast, the putative DA D3 receptor antagonist (+)-AJ76 displayed low affinity and selectivity for D3 versus D2L and D4.2 receptors (91 nM vs. 253 and 193 nM, respectively). In vitro, PD 58491 (1 nM-1 microM) exhibited D3 receptor antagonist activity, reversing the quinpirole (10 nM)-induced stimulation of [3H]thymidine uptake in D3 CHOpro-5 cells, but did not have any significant intrinsic activity by itself in this assay. PD 58491 did not decrease the gamma-butyrolactone-induced increase in DA synthesis (L-3,4-dihydroxyphenylalanine accumulation) in rat striatum, indicating that the compound possessed no in vivo DA D2/D3 receptor agonist action at DA autoreceptors. PD 58491 (3-30 mg/kg, i.p.) generally did not alter DA or serotonin synthesis in either the striatum or mesolimbic region of rat brain. The D3-preferring agonist PD 128907 decreased DA synthesis in striatum and mesolimbic regions, and this effect was attenuated by pretreatment with PD 58491. These findings support the hypothesis that DA D3 autoreceptors may in part modulate the synthesis and release of DA in striatum and mesolimbic regions.     

Dopamine inhibition: enhancement of GABA activity and potassium channel activation in hypothalamic and arcuate nucleus neurons

Dopamine (DA) decreases activity in many hypothalamic neurons. To determine the mechanisms of DA's inhibitory effect, whole cell voltage- and current-clamp recordings were made from primary cultures of rat hypothalamic and arcuate nucleus neurons (n = 186; 15-39 days in vitro). In normal buffer, DA (usually 10 microM; n = 23) decreased activity in 56% of current-clamped cells and enhanced activity in 22% of the neurons. In neurons tested in the presence of glutamate receptor antagonists D,L-2-amino-5-phosphonovalerate (AP5; 100 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM), DA application (10 microM) revealed heterogeneous effects on electrical activity of cells, either hyperpolarization and decrease in activity (53% of 125) or depolarization and increase in spontaneous activity (22% of 125). The DA-mediated hyperpolarization of membrane potential was associated with a decrease in the input resistance. The reversal potential for the DA-mediated hyperpolarization was -97 mV, and it shifted in a positive direction when the concentration of K+ in the incubating medium was increased, suggesting DA activation of K+ channels. Because DA did not have a significant effect on the amplitude of voltage-dependent K+ currents, activation of voltage-independent K+ currents may account for most of the hyperpolarizing actions of DA. DA-mediated hyperpolarization and depolarization of neurons were found during application of the Na+ channel blocker tetrodotoxin (1 microM). The hyperpolarization was blocked by the application of DA D2 receptor antagonist eticlopride (1-20 microM; n = 7). In the presence of AP5 and CNQX, DA (10 microM) increased (by 250%) the frequency of spontaneous inhibitory postsynaptic currents (IPSCs) in 11 of 19 neurons and evoked IPSCs in 7 of 9 cells that had not previously shown any IPSCs. DA also increased the regularity and the amplitude (by 240%) of spontaneous IPSCs in 9 and 4 of 19 cells, respectively. Spontaneous and DA-evoked IPSCs and inhibitory postsynaptic potentials were blocked by the gamma-aminobutyrate A (GABA(A)) antagonist bicuculline (50 microM), verifying their GABAergic origin. Pertussis toxin pretreatment (200 ng/ml; n = 15) blocked the DA-mediated hyperpolarizations, but did not prevent depolarizations (n = 3 of 15) or increases in IPSCs (n = 6 of 10) elicited by DA. Intracellular neurobiotin injections (n = 21) revealed no morphological differences between cells that showed depolarizing or hyperpolarizing responses to DA. Immunolabeling neurobiotin-filled neurons that responded to DA (n = 13) showed that GABA immunoreactive neurons (n = 4) showed depolarizing responses to DA, whereas nonimmunoreactive neurons (n = 9) showed both hyperpolarizing (n = 6) and depolarizing (n = 3) responses. DA-mediated hyperpolarization, depolarization, and increases in frequency of postsynaptic activity could be detected in embryonic hypothalamic or arcuate nucleus neurons after only 5 days in vitro, suggesting that DA could play a modulatory role in early development. These findings suggest that DA inhibition in hypothalamic and arcuate nucleus neurons is achieved in part through the direct inhibition of excitatory neurons, probably via DA D2 receptors acting through a Gi/Go protein on K+ channels, and in part through the enhancement of GABAergic neurotransmission.     

Repression of Drosophila photoreceptor cell fate through cooperative action of two transcriptional repressors Yan and Tramtrack

Using the endogenous cannabinoid receptor agonist anandamide, the synthetic agonist CP 55940 [[1alpha,2beta(R)5alpha]-(-)-5-(1,1-dimethylheptyl+ ++)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]phenol], and the specific antagonist SR 141716 [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide hydrochloride], second messenger activation of the central cannabinoid receptor (CB1) was examined in rat striatal and cortical slices. The effects of these cannabinoid ligands on electrically evoked dopamine (DA) release from [3H] dopamine-prelabelled striatal slices were also investigated. CP 55940 (1 microM) and anandamide (10 microM) caused significant reductions in forskolin-stimulated cyclic AMP accumulation in rat striatal slices, which were reversed in the presence of SR 141716 (1 microM). CP 55940 (1 microM) had no effect on either KCl- or neurotransmitter-stimulated 3H-inositol phosphate accumulation in rat cortical slices. CP 55940 and anandamide caused significant reductions in the release of dopamine after electrical stimulation of [3H]dopamine-prelabelied striatal slices, which were antagonised by SR 141716. SR 141716 alone had no effect on electrically evoked dopamine release from rat striatal slices. These data indicate that the CB1 receptors in rat striatum are negatively linked to adenylyl cyclase and dopamine release. That the CB1 receptor may influence dopamine release in the striatum suggests that cannabinoids play a modulatory role in dopaminergic neuronal pathways.     

Noradrenaline release from rat sympathetic neurones triggered by activation of B2 bradykinin receptors

1. The role of bradykinin receptors in the regulation of sympathetic transmitter release was investigated in primary cultures of neurones dissociated from superior cervical ganglia of neonatal rats. These cultures were loaded with [3H]-noradrenaline and the outflow of radioactivity was determined under continuous superfusion. 2. Bradykinin (100 nmol l[-1] applied for 10 min) caused a transient increase in tritium outflow that reached a peak within four minutes after the beginning of the application and then declined towards the baseline, despite the continuing presence of the peptide. ATP (100 micromol l[-1]) and nicotine (10 micromol l[-1]) caused elevations in 3H outflow with similar kinetics, whereas outflow remained elevated during a 10 min period of electrical field stimulation (0.5 ms, 50 mA, 50 V cm[-1], 1.0 Hz). 3. When bradykinin was applied for periods of 2 min, the evoked 3H overflow was half-maximal at 12 nmol l(-1) and reached a maximum of 2.3% of cellular radioactivity. The preferential B1 receptor agonist des-Arg9-bradykinin failed to alter 3H outflow. The B2 receptor antagonists, [D-Phe7]-bradykinin (1 micromol l[-1]) and Hoe 140 (10 nmol l[-1]), per se did not alter 3H outflow, but shifted the concentration-response curve for bradykinin-evoked 3H overflow to the right by a factor of 7.9 and 4.3, respectively. 4. Bradykinin-induced overflow was abolished in the absence of extracellular Ca2+ and in the presence of either 1 micromol l(-1) tetrodotoxin or 300 micromol l(-1) Cd2+, as was electrically-induced overflow. Activation of alpha2-adrenoceptors by 1 micromol l(-1) UK 14,304 reduced both bradykinin- and electrically-triggered overflow. The Ca2+-ATPase inhibitor thapsigargin (0.3 micromol l[-1]) failed to alter either type of stimulated overflow. Caffeine (10 mmol l[-1]) enhanced bradykinin-induced overflow, but reduced overflow triggered by electrical field stimulation. 5. Inclusion of Ba2+ (0.1 to 1 mmol l[-1]) in the superfusion medium enhanced electrically induced overflow by approximately 100% and potentiated bradykinin-triggered overflow by almost 400%. Application of 1 mmol l(-1) Ba2+ for periods of 2 min triggered 3H overflow, and this overflow was abolished by 1 micromol l(-1) tetrodotoxin and enhanced by 10 mmol l(-1) caffeine. In contrast, inclusion of tetraethylammonium (0.1 to 1 mmol l[-1]) in the superfusion buffer caused similar increases of bradykinin- and electrically evoked 3H overflow (by about 100%), and tetraethylammonium, when applied for 2 min, failed to alter 3H outflow. 6. Treatment of cultures with 100 ng ml(-1) pertussis toxin caused a significant increase in bradykinin-, but not in electrically-, evoked tritium overflow. Treatment with 100 ng ml(-1) cholera toxin reduced both types of stimulated 3H overflow. 7. These data reveal bradykinin as a potent stimulant of action potential-mediated and Ca2+-dependent transmitter release from rat sympathetic neurones in primary cell culture. This neurosecretory effect of bradykinin involves activation of B2-receptors, presumably linked to pertussis- and cholera toxin-insensitive G proteins, most likely members of the Gq family. Results obtained with inhibitors of muscarinic K+ (KM) channels, like caffeine and Ba2+, indicate that the secretagogue action of bradykinin probably involves inhibition of these K+ channels.