A twin study of antisocial and neurotic symptoms in childhood

1. 2,2'-Pyridylisatogen tosylate (PIT) has been reported to be an irreversible antagonist of responses to adenosine 5'-triphosphate (ATP) at metabotropic purinoceptors (of the P2Y family) in some smooth muscles. When a recombinant P2Y1 purinoceptor (derived from chick brain) is expressed in Xenopus oocytes, ATP and 2-methylthioATP (2-MeSATP) evoke calcium-activated chloride currents (ICl,Ca) in a concentration-dependent manner. The effects of PIT on these agonist responses were examined at this cloned P2Y purinoceptor. 2. PIT (0.1-100 microM) failed to stimulate P2Y1 purinoceptors directly but, over a narrow concentration range (0.1-3 microM), caused a time-dependent potentiation (2-5 fold) of responses to ATP. The potentiation of ATP-responses by PIT was not caused by inhibition of oocyte ecto-ATPase. At high concentrations (3-100 microM), PIT irreversibly inhibited responses to ATP with a IC50 value of 13 +/- 9 microM (pKB = 4.88 +/- 0.22; n = 3). PIT failed to potentiate inward currents evoked by 2-MeSATP and only inhibited the responses to this agonist in an irreversible manner. 3. Known P2 purinoceptor antagonists were tested for their ability to potentiate ATP-responses at the chick P2Y1 purinoceptor. Suramin (IC50 = 230 +/- 80 nM; n = 5) and Reactive blue-2 (IC50 = 580 +/- 130 nM; n = 6) reversibly inhibited but did not potentiate ATP-responses. Coomassie brilliant blue-G (0.1-3 microM) potentiated ATP-responses in three experiments, while higher concentrations (3-100 microM) irreversibly inhibited ATP-responses. The results indicated that potentiation and receptor antagonism were dissociable and not a feature common to all known P2 purinoceptor antagonists. 4. In radioligand binding assays, PIT showed a low affinity (pKi < 5) for a range of membrane receptors, including: alpha 1, alpha 2-adrenoceptors, 5-HT1A, 5-HT1B, 5-HT2, 5-HT3, D1, D2, muscarinic, central benzodiazepine, H1, mu-opioid, dihydropyridine and batrachotoxin receptors. PIT showed some affinity (pKi = 5.3) for an adenosine (A1) receptor. 5. In guinea-pig isolated taenia caeci, PIT (12.5-50 microM) irreversibly antagonized relaxations to ATP (3-1000 microM); PIT also directly relaxed the smooth muscle and histamine was used to restore tone. Relaxations to nicotine (10-100 microM), evoked by stimulating intrinsic NANC nerves of taenia caeci preparations in the presence of hyoscine (0.3 microM) and guanethidine (17 microM), were not affected by PIT (50 microM, for 25-60 min). 6. These experiments indicate that PIT causes an irreversible antagonism of ATP receptors but, for recombinant chick P2Y1 purinoceptors, this effect is preceded by potentiation of ATP agonism. The initial potentiation by PIT (and by Coomassie brilliant blue-G) of ATP-responses raises the possibility of designing a new class of modulatory drugs to enhance purinergic transmission at metabotropic purinoceptors.     

Differential inhibition by alpha-conotoxin-MII of the nicotinic stimulation of [3H]dopamine release from rat striatal synaptosomes and slices

The presynaptic nicotinic modulation of dopamine release from striatal nerve terminals is well established, but the subtype(s) of neuronal nicotinic acetylcholine receptor (nAChR) underlying this response has not been identified. Recently, alpha-conotoxin-MII has been reported to inhibit potently and selectively the rat alpha3beta2 combination of nAChR subunits. Here we have synthesised the peptide, confirmed its specificity, and examined its effect on the (+/-)-anatoxin-a-evoked release of [3H]dopamine from rat striatal synaptosomes and slices. Alpha-conotoxin-MII (112 nM) completely blocked acetylcholine-evoked currents of alpha3beta2 nAChRs expressed in Xenopus oocytes (IC50 = 8.0 +/- 1.1 nM). Pairwise combinations of other nicotinic subunits were not blocked by 112 nM alpha-conotoxin-MII. On perfused striatal synaptosomes and slices, alpha-conotoxin-MII dose-dependently inhibited [3H]dopamine release evoked by 1 microM (+/-)-anatoxin-a with IC50 values of 24.3 +/- 2.9 and 17.3 +/- 0.1 nM, respectively. The dose-response curve was shifted to the right with increasing agonist concentrations. However, the maximal inhibition of responses achieved by alpha-conotoxin-MII (112 nM) was 44.9 +/- 5.4% for synaptosomes and 25.0 +/- 4.1% for slices, compared with an inhibition by 10 microM mecamylamine of 77.9 +/- 3.7 and 88.0 +/- 2.1%, respectively. These results suggest the presence of presynaptic alpha3beta2-like nAChRs on striatal dopaminergic terminals, but the incomplete block of (+/-)-anatoxin-a-evoked [3H]dopamine release by alpha-conotoxin-MII also supports the participation of nAChRs composed of other subunits. The lower inhibition found in slices is consistent with an additional indirect nicotinic stimulation of dopamine release via an alpha-conotoxin-MII-insensitive nAChR.     

Electroresponsive properties and membrane potential trajectories of three types of inspiratory neurons in the newborn mouse brain stem in vitro

1. The electrophysiological properties of inspiratory neurons were studied in a rhythmically active thick-slice preparation of the newborn mouse brain stem maintained in vitro. Whole cell patch recordings were performed from 60 inspiratory neurons within the rostral ventrolateral part of the slice with the aim of extending the classification of inspiratory neurons to include analysis of active membrane properties. 2. The slice generated a regular rhythmic motor output recorded as burst of action potentials on a XII nerve root with a peak to peak time of 11.5 +/- 3.4 s and a duration of 483 +/- 54 ms (means +/- SD, n = 50). Based on the electroresponsive properties and membrane potential trajectories throughout the respiratory cycle, three types of inspiratory neurons could be distinguished. 3. Type-1 neurons were spiking in the interval between the inspiratory potentials (n = 9) or silent with a resting membrane potential of -48.6 +/- 10.1 mV and an input resistance of 306 +/- 130 M omega (n = 15). The spike activity between the inspiratory potentials was burst-like with spikes riding on top of an underlying depolarization (n = 11) or regular with no evidence of bursting (n = 12). Hyperpolarization of the neurons below threshold for spike initiation did not reveal any underlying phasic synaptic activity, that could explain the bursting behavior. 4. Type-1 neurons showed delayed excitation after hyperpolarizing square current pulses or when the neurons were depolarized from a hyperpolarized level. This membrane behavior resembles the response seen in other CNS neurons expressing an IA. The response to 1-s long depolarizing pulses with a large current strength showed signs of activation of an active depolarizing membrane response leading to a transient reduction in the spike amplitude. The relationship between the membrane potential and the amplitude of square current pulses (Vm-I) showed a small upward rectification below -70 mV, and spike adaptation throughout a 1-s pulse had a largely linear time course. 5. Type-1 neurons depolarized and started to fire spikes 398 +/- 102 ms (n = 20) before the upstroke of the integrated XII nerve discharge. The inspiratory potential was followed by fast hyperpolarization, a short fast-repolarizing phase (1,040 +/- 102 ms, n = 5) and a longer slow-repolarizing phase (lasting until the next inspiratory discharge). 6. Type-2 neurons were spiking in the interval between the inspiratory potentials with no evidence of bursting behavior and had an input resistance of 296 +/- 212 M omega (n = 26). The response to hyperpolarizing pulses revealed an initial sag and postinhibitory rebound depolarization. This membrane behavior resembles the response seen in other CNS neurons expressing an Ih. The Vm-I relationship was linear at depolarized potentials and showed a marked upward rectification below -60 mV. Spike trains elicited by 1-s long pulses showed a pronounced early and late adaptation. 7. Type-2 neurons depolarized and started to fire spikes 171 +/- 87 ms (n = 23) before the upstroke of the integrated XII nerve discharge. The inspiratory potential had a variable amplitude from cell to cell and was followed by a short hyperpolarization in the cells displaying a large amplitude inspiratory potential.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of spinal N-methyl-D-aspartate or neurokinin receptors induces long-term potentiation of spinal C-fibre-evoked potentials

The use-dependent increase in synaptic strength between primary afferent C-fibres and second-order neurons in superficial spinal dorsal horn may be an important cellular mechanism underlying central hyperalgesia. This long-term potentiation can be blocked by antagonists of the N-methyl-D-aspartate subtype of glutamate receptor, the neurokinin 1 or the neurokinin 2 receptor. We have tested here whether activation of these receptors by superfusion of the spinal cord with corresponding agonists in the absence of presynaptic activity is sufficient to induce long-term potentiation. In urethane anaesthetized rats C-fibre-evoked field potentials were elicited in superficial laminae of lumbar spinal cord by electrical stimulation of the sciatic nerve. In rats with intact spinal cord, controlled superfusion of the spinal cord at recording segments for 60 min with N-methyl-D-aspartate, substance P or neurokinin A never induced long-term potentiation. Spinal superfusion with a mixture of N-methyl-D-aspartate, substance P and neurokinin A also failed to induce long-term potentiation in four rats tested. In spinalized rats, however, long-term potentiation was induced by either N-methyl-D-aspartate (at 10 microM, to 173 +/- 16% of control) substance P (at 10 microM, to 176 +/- 13% of control) or by neurokinin A (at 1 microM, to 198 +/- .20% of control). The induction of long-term potentiation by N-methyl-D-aspartate, substance P or neurokinin A was blocked by intravenous application of the receptor antagonists dizocilpine maleate (0.5 mg/kg), RP67580 (2 mg/kg) or SR48968 (0.2 mg/kg), respectively. Thus, activation of N-methyl-D-aspartate or neurokinin receptors may induce long-lasting plastic changes in synaptic transmission in afferent C-fibres and this effect may be prevented by tonic descending inhibition.     

Synaptic transmission and hippocampal long-term potentiation in olfactory cyclic nucleotide-gated channel type 1 null mouse

Field potential recording was used to investigate properties of synaptic transmission and long-term potentiation (LTP) at Schaffer collateral-CA1 synapses in both hippocampal slices of mutant mice in which the alpha-subunit of the olfactory cyclic nucleotide-gated channel (alpha3/OCNC)1 was rendered null and also in slices prepared from their wild-type (Wt) littermates. Several measures of basal synaptic transmission were unaltered in the OCNC1 knockout (KO), including maximum field excitatory postsynaptic potential (fEPSP) slope, maximum fEPSP and fiber volley amplitude, and the function relating fiber volley amplitude to fEPSP slope and paired-pulse facilitation. When a high-frequency stimulation protocol was used to induce LTP, similar responses were seen in both groups [KO: 1 min, 299 +/- 50% (mean +/- SE), 60 min, 123 +/- 10%; Wt: 1 min, 287 +/- 63%; 60 min, 132 +/- 19%). However, on theta-burst stimulation, the initial amplitude of LTP was smaller (1 min after induction, 147 +/- 16% of baseline) and the response decayed faster in the OCNC1 KO (60 min, 127 +/- 18%) than in Wt (1 min, 200 +/- 14%; 60 min, 169 +/- 19%). Analysis of waveforms evoked by LTP-inducing tetanic stimuli revealed a similar difference between groups. The development of potentiation throughout the tetanic stimulus was similar in OCNC1 KO and Wt mice when high-frequency stimulation was used, but OCNC1 KO mice showed a significant decrease when compared with Wt mice receiving theta-burst stimulation. These results suggest that activation of cyclic nucleotide-gated channels may contribute to the induction of LTP by weaker, more physiological stimuli, possibly via Ca2+ influx.     

Postnatal development of phase-locked high-fidelity synaptic transmission in the medial nucleus of the trapezoid body of the rat

Synaptic transmission in the medial nucleus of the trapezoid body of rats was analyzed in postnatal days 4-13 (P4-P13) by applying the whole-cell patch-recording technique to brain slices. In P4-P6 animals, evoked EPSCs fluctuated extensively in amplitude and occurred in marked asynchrony, followed by spontaneous EPSCs. With development of animals, the evoked EPSCs increased in amplitude, and the rise time became faster. In addition, the synaptic transmission became phase-locked. The coefficient of variation (CV) of EPSC amplitude decreased with development (0.32 +/- 0.03 for P4-P5 and 0. 05 +/- 0.01 for P9-P11). The amplitude of miniature EPSCs did not change throughout the postnatal days investigated (-30.2 +/- 0.3 pA at -70 mV). The CV was dependent on extracellular Ca2+ concentration ([Ca2+]o) and was reduced with the increase of [Ca2+]o, and this [Ca2+]o dependence was shifted toward lower [Ca2+]o with development. Direct patch recording of the presynaptic terminals demonstrated an increase in Ca2+ currents during these postnatal days. The phase-locked high-fidelity transmission in this synapse is achieved with development likely through the increase of Ca2+ currents and Ca2+ sensitivity of transmitter release mechanisms in the presynaptic terminal.     

Potentiation and inhibition of neuronal nicotinic receptors by atropine: competitive and noncompetitive effects

Atropine, the classic muscarinic receptor antagonist, inhibits ion currents mediated by neuronal nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes. At the holding potential of -80 mV, 1 microM atropine inhibits 1 mM acetylcholine-induced inward currents mediated by rat alpha2beta2, alpha2beta4, alpha3beta2, alpha3beta4, alpha4beta2, alpha4beta4, and alpha7 nicotinic receptors by 12-56%. Inward currents induced with a low agonist concentration are equally inhibited (alpha3beta2, alpha3beta4), less inhibited (alpha2beta4, alpha7), or potentiated (alpha4beta2, alpha4beta4) by 1 microM atropine. Effects on the more sensitive alpha4beta4 nicotinic receptors were investigated in detail by systematic variation of acetylcholine and atropine concentrations and of membrane potential. At high agonist concentration, atropine inhibits alpha4beta4 nicotinic receptor-mediated ion current in a noncompetitive, voltage-dependent way with IC50 values of 655 nM at -80 mV and of 4.5 microM at -40 mV. At low agonist concentration, 1 microM atropine potentiates alpha4beta4 nicotinic receptor-mediated ion current. This potentiating effect is surmounted by high concentrations of acetylcholine, indicating a competitive interaction of atropine with the nicotinic receptor, and potentiation is also reversed at high atropine concentrations. Steady state effects of acetylcholine and atropine are accounted for by a model for combined receptor occupation and channel block, in which atropine acts on two distinct sites. The first site is associated with noncompetitive ion channel block. The second site is associated with competitive potentiation, which appears to occur when the agonist recognition sites of the receptor are occupied by acetylcholine and atropine. The apparent affinity of atropine for the agonist recognition sites of the alpha4beta4 nicotinic acetylcholine receptor is estimated to be 29.9 microM.     

The group I mGlu receptor agonist DHPG induces a novel form of LTD in the CA1 region of the hippocampus

The group I specific metabotropic glutamate (mGlu) receptor agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) (100 microM, 10 min) induced long-term depression (LTD) of synaptic transmission in the CA1 region of adult rat hippocampal slices, measured using a grease-gap recording technique. In "normal" (1 mM Mg2+-containing) medium, LTD (measured 30 min after washout of DHPG) was small (13+/-3%), but LTD was enhanced if DHPG was applied when the tissue was made hyperexcitable, either by omitting Mg2+ from the perfusate (35+/-3%) or by adding the GABA(A) receptor antagonist picrotoxin (29+/-2%). The N-methyl-D-aspartate (NMDA) receptor antagonist AP5 (100 microM) substantially reduced the generation of DHPG-induced LTD in Mg2+-free medium, but had little effect on LTD induced in the presence of picrotoxin. In Mg2+-free medium, the threshold concentration of DHPG required to induce LTD was between 1 and 3 microM. Neither agonists specific for group II (100 nM DCG-IV or 1 microM LY354740) or group III (10 microM L-AP4) mGlu receptors or a combined group I and II agonist (30-100 microM (1S,3R)-ACPD) induced LTD. However, an agonist (1 mM CHPG) which activates mGlu5 but not mGlu1 receptors did induce LTD. Surprisingly, DHPG-induced LTD was reversed by mGlu receptor antagonists, applied hours after washout of DHPG. DHPG-induced LTD did not occlude with LTD induced by synaptic activation (1200 stimuli delivered at 2 Hz), in Mg2+-free medium. These data show that activation of group I mGlu receptors (probably mGlu5) can induce LTD and that this mGlu receptor-mediated LTD may, or may not, require activation of NMDA receptors, depending on the experimental conditions.     

Role of GABAB receptors in the control of hypoglossal motoneurons in vivo

There is little information on GABAB receptor-mediated effects on orofacial motoneurons. We recorded the inspiratory activity from both hypoglossal (XII) nerves in urethane-anesthetized, paralyzed, vagotomized and artificially ventilated rats. A GABAB receptor agonist, baclofen, or antagonist, CGP-35348, was microinjected into one XII nucleus. Baclofen rapidly reduced the XII nerve activity in a dose-dependent manner by over 50%. The antagonist caused a delayed suppression of activity by 40%. We conclude that: (1) GABAB receptors within the XII nucleus may suppress the activity of inspiratory XII motoneurons, but they are not tonically active under the conditions of our experiment; (2) there is a net endogenous excitatory effect in XII motoneurons that is mediated by GABAB receptors located in the reticular formation surrounding the XII nucleus.     

Requirement of protein synthesis for group I mGluR-mediated induction of epileptiform discharges

Picrotoxin (50 microM) elicited rhythmic synchronized bursting in CA3 pyramidal cells in guinea pig hippocampal slices. Addition of the selective group I metabotropic glutamate receptor (mGluR) agonist (S)-3,5-dihydroxyphenylglycine (25 microM) elicited an increase in burst frequency. This was soon followed by a slowly progressive increase in burst duration (BD), converting the brief 250-520 ms picrotoxin-induced synchronized bursts into prolonged discharges of 1-5 s in duration. BD was significantly increased within 60 min and reached a maximum after 2-2.5 h of agonist exposure. The protein synthesis inhibitors anisomycin (15 microM) or cycloheximide (25 microM) significantly impeded the mGluR-mediated development of the prolonged bursts; 90-120 min of agonist application failed to elicit the expected burst prolongation. By contrast, the mGluR-mediated enhancement of burst frequency progressed unimpeded. Furthermore, protein synthesis inhibitors had no significant effect on the frequency or duration of fully developed mGluR-induced prolonged discharges. These results suggest that the group I mGluR-mediated prolongation of synchronized bursts has a protein synthesis-dependent mechanism.     

Benzodiazepine and barbiturate ligands modulate responses of cultured hippocampal neurones to the GABAA receptor partial agonist, 4-PIOL

We have previously characterized 5-(4-piperidyl)isoxazol-3-ol (4-PIOL) as a non-desensitizing partial agonist at GABAA receptors and shown that the responses are mediated by short-duration channel openings consonant with single-ligand gated openings of the Cl- channels. We presently investigate whether responses of cultured rat hippocampal neurones to 4-PIOL are modulated by benzodiazepine (BDZ) and barbiturate receptor ligands. Whole-cell patch-clamp recordings of maximal responses to 1 mM 4-PIOL were comparable in size to responses evoked by 10 microM of the full GABAA agonist, isoguvacine. The BDZ receptor inverse agonist, DMCM (1 microM) reduced responses to isoguvacine (to 65.7 +/- 11.0%) and 4-PIOL (to 69.3 +/- 3.5%) to a similar extent. The BDZ agonist, midazolam (0.1 microM) potentiated responses to both agonists, and resulted in responses with an early peak with later fading. Potentiation of the peak response to 4-PIOL (to 163 +/- 14%) was significantly less than for isoguvacine (215 +/- 11%). Pentobarbital (50 microM) caused a very marked, but variable, potentiation of the peak response to 4-PIOL (to 484 +/- 93%), which was significantly greater than the potentiation of the peak response to isoguvacine (to 304 +/- 46%), and induced fading. This suggests that a relatively larger number of the 4-PIOL-induced channel openings can be transformed to longer duration openings by pentobarbital. In conclusion, responses to 4-PIOL and isoguvacine are modulated by BDZ and barbiturate ligands in a qualitatively similar manner, but with a number of quantitative differences which cannot be readily explained by the kinetic model of Macdonald and Twyman (1992). Investigation of these responses at the single-channel level could provide further insight into the operation of the GABAA receptor-ionophore complex.     

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.     

5-HT1A receptor-mediated inhibition of long-term potentiation in rat visual cortex

We investigated the effect of 8-hydroxy-2-(N,N-dipropylamino)tetralin (8-OH-DPAT), a 5-HT1A receptor agonist, on the induction of long-term potentiation in rat visual cortex slices. Perfusion of 8-OH-DPAT (0.1-10 microM) did not affect layer II/III field potentials evoked by test stimulation of layer IV, but significantly reduced long-term potentiation induced by tetanic stimulation. The inhibitory effect of 8-OH-DPAT was blocked by the 5-HT1A receptor antagonist, pindolol (10 microM), but not by the 5-HT2,7 receptor antagonist, ritanserin (100 microM), nor by the 5-HT3,4 receptor antagonist, MDL72222 (100 microM). These results suggest that the rat visual cortex long-term potentiation is inhibited by 5-HT1A receptor stimulation.     

Neuroprotection with Bcl-2(20-34) peptide against trauma

We tested the neuroprotective potential of the Bcl-2(20-34) peptide sequence in hippocampal slices. Treatment with Bcl-2 after fluid percussion trauma significantly improved recovery of CA1 antidromic PS to a mean of 92%+/-1 of initial amplitude, compared with only 16%+/-2 in unmedicated slices. The EC50 for trauma protection was 84 microM Bcl-2(20-34). Protection with Bcl-2(20-34) also extended to long-term potentiation. No protection was seen with the reverse sequence of Bcl-2(20-34). Treatment with Bcl-2(20-34) also protected against hypoxic damage, with treated slices recovering to 98%+/-2, while unmedicated slices recovered to 14%+/-2. Similar protection was seen against AMPA, NMDA and nitric oxide. These findings indicate that Bcl-2(20-34) provides specific neuroprotection against acute CA1 neuronal injury.     

Role of PKC in the effects of alpha1-adrenergic stimulation on Ca2+ transients, contraction and Ca2+ current in guinea-pig ventricular myocytes

The effects of alpha1-adrenoceptor stimulation on intracellular Ca2+ transients, contractility and L-type Ca2+ current (ICa,L) were studied in single cells isolated from ventricles of guinea-pig hearts. The aim of our study was to elucidate the mechanisms of the positive inotropic effect of alpha1-adrenergic stimulation by focussing on the role of protein kinase C (PKC). Phenylephrine, an alpha1-adrenergic agonist, at concentrations of 50-100 microM elicited a biphasic inotropic response: a transient negative inotropic response (22.9+/-6.0% of control) followed by a sustained positive inotropic response (61.0+/-8.4%, mean+/-SE, n=12). The Ca2+ transient decreased by 10.2+/-3.9% during the negative inotropic phase, while it increased by 67.7+/-10% (n=12) during the positive inotropic phase. These effects were inhibited by prazosin (1 microM), a alpha1-adrenergic antagonist. Phenylephrine increased the ICa,L by 60.8+/-21% (n=5) during the positive inotropic phase. To determine whether activation of PKC is responsible for the increases in Ca2+ transients, contractile amplitude and ICa,L during alpha1-adrenoceptor stimulation, we tested the effects of 4beta-phorbol 12-myristate 13-acetate (PMA), a PKC activator, and of bisindolylmaleimide I (GF109203X) and staurosporine, both of which are PKC inhibitors. PMA mimicked phenylephrine's effects on Ca2+ transients, contractile amplitude and ICa,L. PMA (100 nM) increased the Ca2+ transient, contractile amplitude and ICa,L by 131+/-17%, 137+/-25% (n=8), and 81.1+/-26% (n=5), respectively. Prior exposure to GF109203X (1 microM) or staurosporine (10 nM) prevented the phenylephrine-induced increases in Ca2+ transients, contractile amplitude and ICa,L. Our study suggests that during alpha1-adrenoceptor stimulation increase in ICa,L via PKC causes an increase in Ca2+ transients and thereby in the contractile force of the ventricular myocytes.     

Interaction of positive allosteric modulators with human and Drosophila recombinant GABA receptors expressed in Xenopus laevis oocytes

1. A comparative study of the actions of structurally diverse allosteric modulators on mammalian (human alpha 3 beta 2 gamma 2L) or invertebrate (Drosophila melanogaster Rdl or a splice variant of Rdl) recombinant GABA receptors has been made using the Xenopus laevis oocyte expression system and the two electrode voltage-clamp technique. 2. Oocytes preinjected with the appropriate cRNAs responded to bath applied GABA with a concentration-dependent inward current. EC50 values of 102 +/- 18 microM; 152 +/- 10 microM and 9.8 +/- 1.7 microM were determined for human alpha 3, beta 1 gamma 2L, Rdl splice variant and the Rdl receptors respectively. 3. Pentobarbitone enhanced GABA-evoked currents mediated by either the mammalian or invertebrate receptors. Utilizing the appropriate GABA EC10, the EC50 for potentiation was estimated to be 45 +/- 1 microM, 312 +/- 8 microM and 837 +/- 25 microM for human alpha 3, beta 1 gamma 2L, Rdl splice variant and Rdl receptors respectively. Maximal enhancement (expressed relative to the current induced by the EC10 concentration of GABA where this latter response = 1) at the mammalian receptor (10.2 +/- 1 fold) was greater that at either the Rdl splice variant (5.5 +/- 1.3 fold) or Rdl (7.9 +/- 0.8 fold) receptors. 4. Pentobarbitone directly activated the human alpha 3 beta 1 gamma 2L receptor with an EC50 of 1.2 +/- 0.03 mM and had a maximal effect amounting to 3.3 +/- 0.4 fold of the response evoked by the EC10 concentration of GABA. Currents evoked by pentobarbitone were blocked by 10-30 microM picrotoxin and potentiated by 0.3 microM flunitrazepam. Pentobarbitone did not directly activate the invertebrate GABA receptors. 5. 5 alpha-Pregnan-3 alpha-ol-20-one potentiated GABA-evoked currents mediated by the human alpha 3 beta 1 gamma 2L receptor with an EC50 of 87 +/- 3 nM and a maximal enhancement of 6.7 +/- 0.8 fold of that produced by the GABA EC10 concentration. By contrast, relatively high concentrations (3-10 microM) of this steroid had only a modest effect on the Rdl receptor and its splice variant. 6. A small direct effect of 5 alpha-pregnan-3 alpha-ol-20-one (0.3-10 microM) was detected for the human alpha 3 beta 1 gamma 2L receptor (maximal effect only 0.08 +/- 0.01 times that of the GABA EC10). This response was antagonized by 30 microM picrotoxin and enhanced by flunitrazepam (0.3 microM). 5 alpha-Pregnan-3 alpha-ol-20-one did not directly activate the invertebrate GABA receptors. 7. Propofol enhanced GABA-evoked currents mediated by human alpha 3 beta 1 gamma 2L and Rdl splice variant receptors with EC50 values of 3.5 +/- 0.1 microM and 8 +/- 0.3 microM respectively. The maximal enhancement was similar at the two receptor types (human 11 +/- 1.8 fold; invertebrate 8.8 +/- 1.4 fold that of the GABA EC10). 8. Propofol directly activated the human alpha 3 beta 1 gamma 2L receptor with an EC50 of 129 +/- 10 microM, and at a maximally effective concentration, evoked a current amounting to 3.5 +/- 0.5 times that elicited by a concentration of GABA producing 10% of the maximal response. The response to propofol was blocked by 10-30 microM picrotoxin and enhanced by flunitrazepam (0.3 microM). Propofol did not directly activate the invertebrate Rdl splice variant receptor. 9. GABA-evoked currents mediated by the human alpha 3 beta 1 gamma 2L receptor were potentiated by etomidate (EC50 = 7.7 +/- 0.2 microM) and maximally enhanced to 8 +/- 0.8 fold of the response to an EC10 concentration of GABA. By contrast, the Rdl, or Rdl splice variant forms of the invertebrate GABA receptor were insensitive to the positive allosteric modulating actions of etomidate. Neither the mammalian nor the invertebrate receptors, were directly activated by etomidate. 10. delta-Hexachlorocyclohexane enhanced GABA-evoked currents with EC50 values of 3.4 +/- 0.1 microM and 3.0 +/- 0.1 microM for the human alpha 3 beta 1 gamma 2L receptor and the Rdl splice variant receptor respectively. The maximal enhancement was 4.5     

Effect of zolpidem on miniature IPSCs and occupancy of postsynaptic GABAA receptors in central synapses

GABAA-mediated miniature IPSCs (mIPSCs) were recorded from layer V pyramidal neurons of the visual cortex using whole-cell patch-clamp recording in rat brain slices. At room temperature, the benzodiazepine site agonist zolpidem enhanced both the amplitude (to 138 +/- 26% of control value at 10 microM) and the duration (163 +/- 14%) of mIPSCs. The enhancement of mIPSC amplitude was not caused by an increase of the single-channel conductance of the postsynaptic receptors, as determined by peak-scaled non-stationary fluctuation analysis of mIPSCs. The effect of zolpidem on fast, synaptic-like (1 msec duration) applications of GABA to outside-out patches was also investigated. The EC50 for fast GABA applications was 310 microM. In patches, zolpidem enhanced the amplitude of currents elicited by subsaturating GABA applications (100-300 microM) but not by saturating applications (10 mM). The increase of mIPSC amplitude by zolpidem provides evidence that the GABAA receptors are not saturated during miniature synaptic transmission in the recorded cells. By comparing the facilitation induced by 1 microM zolpidem on outside-out patches and mIPSCs, we estimated the concentration of GABA seen by the postsynaptic GABAA receptors to be approximately 300 microM after single vesicle release. We have estimated a similar degree of receptor occupancy at room and physiological temperature. However, at 35 degreesC, zolpidem did not enhance the amplitude of mIPSCs or of subsaturating GABA applications on patches, implying that, in these neurons, zolpidem cannot be used to probe the degree of receptor occupancy at physiological temperature.     

A1 adenosine receptor modulation of electrically-evoked contractions in the bisected vas deferens and cauda epididymis of the guinea-pig

1. The effects of adenosine receptor agonists upon both electrically-evoked and phenylephrine-induced contractile responses were investigated in the bisected vas deferens and the cauda epididymis of the guinea-pig. Electrical field-stimulation (10 s trains of pulses at 9 Hz, 0.1 ms duration, supramaximal voltage) elicited biphasic and monophasic contractile responses from preparations of bisected vas deferens and cauda epididymis, respectively; these responses were abolished by tetrodotoxin (300 nM). 2. In the prostatic half of the vas deferens the A1 selective adenosine receptor agonists, N6-cyclopentyladenosine (CPA) and (2S)-N6-[2-endo-norbornyl]adenosine ((S)-ENBA) and the non-selective A1/A2 adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA) inhibited electrically-evoked contractions (pIC50+/-s.e.mean values 6.15+/-0.24, 5.99+/-0.26 and 5.51+/-0.24, respectively). The responses to CPA were blocked by the A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine, DPCPX (100 nM). 3. In the epididymal half of the vas deferens NECA potentiated (at < or = 100 nM) and inhibited (at > or = 1 microM) electrically-evoked contractions. In the presence of the non-selective alpha-adrenoceptor antagonist phentolamine (3 microM), the alpha1-adrenoceptor antagonist, prazosin (100 nM), or at a reduced train length (3 s) NECA inhibited electrically-evoked contractions (pIC50 values 6.05+/-0.25, 5.97+/-0.29 and 5.71 +/-0.27, respectively). CPA (at 10 microM) also inhibited electrically-evoked contractions in this half of the vas deferens. In the presence of prazosin (100 nM), CPA also inhibited electrically-evoked contractions (pIC50 6.14+/-0.67); this effect was antagonized by DPCPX (30 nM, apparent pK(B) 8.26+/-0.88). In the presence of the P2 purinoceptor antagonist, suramin (300 microM), CPA (up to 1 microM) potentiated electrically-evoked contractions. 4. NECA, CPA and APNEA potentiated electrically-evoked contractions in preparations of cauda epididymis (pEC50 values 7.49+/-0.62, 7.65+/-0.74 and 5.84+/-0.86, respectively), the response to CPA was competitively antagonized by DPCPX (100 nM) with an apparent pK(B) value of 7.64+/-0.64. 5. The alpha1-adrenoceptor agonist phenylephrine elicited concentration-dependent contractile responses from preparations of bisected vas deferens and cauda epididymis. NECA (1 microM) potentiated responses to phenylephrine (< or = 1 microM) in the epididymal, but not in the prostatic half of the vas deferens. In preparations of epididymis NECA (1 microM) shifted phenylephrine concentration response curves to the left (4.6 fold). In the presence of a fixed concentration of phenylephrine (1 microM), NECA elicited concentration-dependent contractions of preparations of the epididymal half of the vas deferens and of the epididymis (pEC50 values 7.57+/-0.54 and 8.08+/-0.18, respectively). NECA did not potentiate responses to ATP in either the epididymal half of the vas deferens or the epididymis. 6. These studies are consistent with the action of stable adenosine analogues at prejunctional A1 and postjunctional A1-like adenosine receptors. The prejunctional A1 adenosine receptors only inhibit the electrically-evoked contractions of purinergic origin (an effect predominant in the prostatic half of the vas deferens). At the epididymis, where electrically-evoked contractions are entirely adrenergic, the predominant adenosine receptor agonist effect is a potentiation of alpha1-adrenoceptor-, but not of ATP-induced contractility.     

Electrically evoked acetylcholine release from hippocampal slices is inhibited by the cannabinoid receptor agonist, WIN 55212-2, and is potentiated by the cannabinoid antagonist, SR 141716A

This study examined the effect of the cannabinoid receptor agonist, WIN 55212-2, on the electrically evoked release of [14C]acetylcholine (ACh) from superfused brain slices from the hippocampus, a region with a high density of cannabinoid receptors. A comparison was also made with [14C]ACh release from the nucleus accumbens, which has relatively fewer cannabinoid receptors. In the hippocampal slices, WIN 55212-2 produced a dose-dependent inhibition of [14C]ACh release, with an EC50 of 0.03 microM and a maximal inhibition of 81% at 1 microM. In the nucleus accumbens slices, WIN 55212-2 produced a weak inhibition of [14C]ACh release, which did not quite reach statistical significance. The inhibition of electrically evoked hippocampal [14C]ACh release by WIN 55212-2 could be prevented by the cannabinoid receptor antagonist, SR 141716A (EC50, 0.3-1.0 microM). In addition to antagonizing the effects of WIN 55212-2, SR 141716A alone produced a 2-fold potentiation of the electrically stimulated [14C]ACh release in this region (EC50, 0.1-0.3 microM). By contrast, in nucleus accumbens slices, no potentiation of the stimulated release of [14C]ACh release by SR 141716A was observed. Basal [14C]ACh release was unaffected by WIN 55212-2 or SR 141716A in either area. These results suggest that cannabinoid receptor activation can produce a strong inhibition of ACh release in the hippocampus. Furthermore, the potentiation of ACh release in the hippocampus by SR 141716A alone suggests either that this compound is an inverse agonist at cannabinoid receptors or it is antagonizing the actions of an endogenous ligand acting on these receptors.     

Effects of exogenous phytohormones on plastid tRNA modifications in ragi coleoptiles

The influence of adenosine and selective A1 and A2 agonists and antagonists was investigated on the cholinergic and the excitatory non-cholinergic (e-NC) contractions induced by electrical field stimulation in the guinea-pig bronchi. Adenosine (10 nM-1 mM) induced a concentration-dependent inhibition of the e-NC contraction (EC50 = 90 +/- 14 microM), whereas the cholinergic peak was only slightly affected. Preincubation of the tissue with the adenosine uptake blocker dipyridamole (10 microM) significantly shifted the concentration-inhibition curve to adenosine to the left (EC50 = 10 +/- 1 microM), suggesting an interaction with extracellular adenosine receptors of A1 and/or A2 subtype. To characterize the receptor type involved in this effect, selective adenosine derivatives were studied. The agonist to both A1 and A2 adenosine receptors, 5'-N-ethylcarboxamidoadenosine (NECA) was more potent than the selective A1 agonist, (-)-R-6-phenylisopropyladenosine (R-PIA), in inhibiting the e-NC contraction (EC50 = 0.10 +/- 0.04 and 0.60 +/- 0.12 microM, respectively, with a maximal inhibition of 70 and 45%, respectively). The concentration-response curve to NECA was shifted to the right by the A2 receptor selective antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) (10 microM) (EC50 = 1.4 +/- 0.5 microM) as well as by the specific A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (10 microM) (EC50 = 0.7 +/- 0.3 microM). The inhibitory effect induced by the association of both antagonists, DPCPX and DMPX, was considerably potentiated (EC50 > 22 +/- 2.5 microM). The effect of R-PIA was also shifted to the right by DPCPX (EC50 = 8.2 +/- 1.6 microM) but was not modified by DMPX. The contractile response to exogenous substance P was unaffected by NECA pretreatment (0.3 microM). Altogether, these results suggest that adenosine-induced inhibition of e-NC contraction of guinea-pig bronchi is mediated through activation of both A1 and A2 adenosine receptors linked to inhibition of the release of neuropeptides from C-fibre nerve endings.