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Psychostimulant (methamphetamine, cocaine) use disorders have a genetic component that remains mostly unknown. We conducted genome‐wide quantitative trait locus (QTL) analysis of methamphetamine stimulant sensitivity. To facilitate gene identification, we employed a Reduced Complexity Cross between closely related C57BL/6 mouse substrains and examined maximum speed and distance traveled over 30 min following methamphetamine (2 mg/kg, i.p.). For maximum methamphetamine‐induced speed following the second and third administration, we identified a single genome‐wide significant QTL on chromosome 11 that peaked near the Cyfip2 locus (LOD = 3.5, 4.2; peak = 21 cM [36 Mb]). For methamphetamine‐induced distance traveled following the first and second administration, we identified a genome‐wide significant QTL on chromosome 5 that peaked near a functional intronic indel in Gabra2 coding for the alpha‐2 subunit of the GABA‐A receptor (LOD = 3.6–5.2; peak = 34–35 cM [66–67 Mb]). Striatal cis‐expression QTL mapping corroborated Gabra2 as a functional candidate gene underlying methamphetamine‐induced distance traveled. CRISPR/Cas9‐mediated correction of the mutant intronic deletion on the C57BL/6J background to the wild‐type C57BL/6NJ allele was sufficient to reduce methamphetamine‐induced locomotor activity toward the wild‐type C57BL/6NJ‐like level, thus validating the quantitative trait variant (QTV). These studies show the power and efficiency of Reduced Complexity Crosses in identifying causal variants underlying complex traits. Functionally restoring Gabra2 expression decreased methamphetamine stimulant sensitivity and supports preclinical and human genetic studies implicating the GABA‐A receptor in psychostimulant addiction‐relevant traits. Importantly, our findings have major implications for studying psychostimulants in the C57BL/6J strain—the gold standard strain in biomedical research. [ABSTRACT FROM AUTHOR]

Background: Food avoidance secondary to disease or stress can lead to weight loss and rapid deterioration of clinical condition in the common marmoset (Callithrix jacchus). Currently, there are no data supporting the use of any pharmaceuticals as an appetite stimulant in this species; however, benzodiazepines are frequently used for this purpose in other species. Methods: Six marmosets were used in a crossover study design to evaluate the benzodiazepine midazolam as an appetite stimulant and anxiolytic. Total food intake (TFI) and latency to eat (LTE) were measured following administration of oral and injectable midazolam in non‐anxious and anxious states. Results: Injectable midazolam increased TFI and decreased LTE in anxious marmosets, but had no effect in non‐anxious animals. Oral midazolam had no effect on appetite in either state. Conclusions: Injectable midazolam may be an effective treatment for anxiety‐induced inappetence in marmosets. Individual response to both oral and injectable midazolam may vary. [ABSTRACT FROM AUTHOR]

As complexities of addictive behaviors cannot be fully captured in laboratory studies, scientists use simple addiction‐associated phenotypes and measure them in laboratory animals. Locomotor sensitization, characterized by an increased behavioral response to the same dose of the drug, has been extensively used to elucidate the genetic basis and molecular mechanisms of neuronal plasticity. However, to what extent it contributes to the development of addiction is not completely clear. We tested if the development of locomotor sensitization to methamphetamine affects voluntary self‐administration, and vice versa, in order to investigate how two drug‐associated phenotypes influence one another. In our study, we used the genetically tractable model organism, Drosophila melanogaster, and quantified locomotor sensitization and voluntary self‐administration to methamphetamine using behavioral tests that were developed and adapted in our laboratory. We show that flies express robust locomotor sensitization to the second dose of volatilized methamphetamine, which significantly lowers preferential self‐administration of methamphetamine. Naive flies preferentially self‐administer food with methamphetamine over plain food. Exposing flies to volatilized methamphetamine after voluntary self‐administration abolishes locomotor sensitization. We tested period null (per01) mutant flies and showed that they do not develop locomotor sensitization, nor do they show preferential self‐administration of methamphetamine. Our results suggest that there may be partially overlapping neural circuitry that regulates the expression of locomotor sensitization and preferential self‐administration to methamphetamine and that this circuitry requires a functional per gene. [ABSTRACT FROM AUTHOR]

Early-life stress (ELS) is associated with negative consequences, including maladaptive long-lasting brain effects. These alterations seem to increase the likelihood of developing substance use disorders. However, the molecular consequences of ELS are poorly understood. In the present study, we tested the impact of ELS induced by maternal separation with early weaning (MSEW) in CD1 male mice at different phases of cocaine self-administration (SA). We also investigated the subsequent alterations on GluR2, GluR1, cAMP response element-binding (CREB), and CREB-phosphorylation (pCREB) in ventral tegmental area (VTA) and nucleus accumbens (NAc) induced by both MSEW and cocaine SA. Our results show that MSEW animals expressed a higher cocaine intake, an increased vulnerability to the acquisition of cocaine SA, and incapacity to extinguish cocaine SA behaviour. MSEW mice showed decreased GluR2 and increased GluR1 and pCREB in NAc. Also, results displayed reduction of basal levels of GluR1 and CREB and an elevation of GluR1/GluR2 ratio in the VTA. Such results hint at an enhanced glutamatergic function in NAc and increased excitability of VTA DA neurons in maternally separated mice. Altogether, our results suggest that MSEW induces molecular alterations in the brain areas related to reward processing, increasing the vulnerability to depression and cocaine-seeking behaviour. [ABSTRACT FROM AUTHOR]

Recurrent relapse is a major problem in treating opiate addiction. Pavlovian conditioning plays a role in recurrent relapse whereby exposure to cues learned during drug intake can precipitate relapse to drug taking. α7 nicotinic acetylcholine receptors (nAChRs) have been implicated in attentional aspects of cognition and mechanisms of learning and memory. In this study we have investigated the role of α7 nAChRs in morphine-conditioned place preference (morphine-CPP). CPP provides a model of associative learning that is pertinent to associative aspects of drug dependence. The α7 nAChR antagonist methyllycaconitine (MLA; 4 mg/kg s.c.) had no effect on the acquisition, maintenance, reconsolidation or extinction of morphine-CPP but selectively attenuated morphine-primed reinstatement of CPP, in both mice and rats. Reinstatement of morphine-CPP in mice was accompanied by a selective increase in [3 H]-AMPA binding (but not in [3 H]-MK801 binding) in the ventral hippocampus that was prevented by prior treatment with MLA. Administration of MLA (6.7 μg) directly into the ventral hippocampus of rats prior to a systemic priming dose of morphine abolished reinstatement of morphine-CPP, whereas MLA delivered into the dorsal hippocampus or prefrontal cortex was without effect. These results suggest that α7 nAChRs in the ventral hippocampus play a specific role in the retrieval of associative drug memories following a period of extinction, making them potential targets for the prevention of relapse. [ABSTRACT FROM AUTHOR]

Tobacco use leads to 6 million deaths every year due to severe long-lasting diseases. The main component of tobacco, nicotine, is recognized as one of the most addictive drugs, making smoking cessation difficult, even when 70 percent of smokers wish to do so. Clinical and preclinical studies have demonstrated consistently that nicotine seeking is a complex behavior involving various psychopharmacological mechanisms. Evidence supports that the population of smokers is heterogeneous, particularly as regards the breadth of motives that determine the urge to smoke. Here, we review converging psychological, genetic and neurobiological data from clinical and preclinical studies supporting that the mechanisms controlling nicotine seeking may vary from individual to individual. It appears timely that basic neuroscience integrates this heterogeneity to refine our understanding of the neurobiology of nicotine seeking, as tremendous progress has been made in modeling the various psychopharmacological mechanisms driving nicotine seeking in rodents. For a better understanding of the mechanisms that drive nicotine seeking, we emphasize the need for individual-based research strategies in which nicotine seeking, and eventually treatment efficacy, are determined while taking into account individual variations in the mechanisms of nicotine seeking. [ABSTRACT FROM AUTHOR]

Background: Previous studies have investigated α1GABAA and α5GABAA receptor mechanisms in the behavioral effects of ethanol (EtOH) in monkeys. However, genetic studies in humans and preclinical studies with mutant mice suggest a role for α2GABAA and/or α3GABAA receptors in the effects of EtOH. The development of novel positive allosteric modulators (PAMs) with functional selectivity (i.e., selective efficacy) at α2GABAA and α3GABAA receptors allows for probing of these subtypes in preclinical models of the discriminative stimulus and reinforcing effects of EtOH in rhesus macaques. Methods: In discrimination studies, subjects were trained to discriminate EtOH (2 g/kg, intragastrically) from water under a fixed‐ratio (FR) schedule of food delivery. In oral self‐administration studies, subjects were trained to self‐administer EtOH (2% w/v) or sucrose (0.3 to 1% w/v) under an FR schedule of solution availability. Results: In discrimination studies, functionally selective PAMs at α2GABAA and α3GABAA (HZ‐166) or α3GABAA (YT‐III‐31) receptors substituted fully (maximum percentage of EtOH‐lever responding ≥80%) for the discriminative stimulus effects of EtOH without altering response rates. Full substitution for EtOH also was engendered by a nonselective PAM (triazolam), an α5GABAA‐preferring PAM (QH‐ii‐066) and a PAM at α2GABAA, α3GABAA, and α5GABAA receptors (L‐838417). A partial (MRK‐696) or an α1GABAA‐preferring (zolpidem) PAM only engendered partial substitution (i.e., ~50 to 60% EtOH‐lever responding). In self‐administration studies, pretreatments with the functionally selective PAMs at α2GABAA and α3GABAA (XHe‐II‐053 and HZ‐166) or α3GABAA (YT‐III‐31 and YT‐III‐271) receptors increased EtOH, but not sucrose, drinking at doses that had few, or no, observable sedative‐motor effects. Conclusions: Our results confirm prior findings regarding the respective roles of α1GABAA and α5GABAA receptors in the discriminative stimulus effects of EtOH and, further, suggest a key facilitatory role for α3GABAA and potentially α2GABAA receptors in several abuse‐related effects of EtOH in monkeys. Moreover, they reveal a potential role for these latter subtypes in EtOH's sedative effects. [ABSTRACT FROM AUTHOR]

Extrasynaptic δ subunit‐containing γ‐aminobutyric acid type A receptors (δ‐GABAARs) are emerging as targets for a number of neuropsychopharmacological drugs, including the direct GABA site agonist gaboxadol and neuroactive steroids. Among other regions, these δ‐GABAARs are functionally expressed in the ventral tegmental area (VTA), the cell body region of mesocorticolimbic dopamine (DA) system important for motivated behaviours, and in the target region, the nucleus accumbens. Gaboxadol and neurosteroids induce VTA DA neuron plasticity ex vivo, by inhibiting the VTA GABA neurons, and aversive place conditioning, which are absent in the δ‐GABAAR knockout mice (δ‐KO). It is not known whether δ‐GABAARs are important for the effects of other drugs, such as opioids (that also inhibit GABA neurons) and stimulants (that primarily elevate monoamine levels). Here, we used δ‐KO mice and conditioned place preference (CPP) test to study the rewarding effects of morphine (20 mg/kg), methamphetamine (1 mg/kg) and mephedrone (5 mg/kg). Morphine‐induced nociception was also assessed using tail‐flick and hot‐plate tests. We found that the δ‐KO mice failed to express morphine‐induced CPP, but that they were more sensitive to morphine‐induced analgesia in the tail‐flick test. In contrast, stimulant‐induced CPP in the δ‐KO mice was similar to that in the wild‐type controls. Thus, the conditioned rewarding effect by opioids, but not that of stimulants, was impaired in the absence of δ‐GABAARs. Further studies are warranted to assess the potential of δ‐GABAAR antagonists as possible targets for reducing morphine reward and potentiating morphine analgesia. [ABSTRACT FROM AUTHOR]

Withdrawal from extended-access cocaine self-administration leads to progressive intensification ('incubation') of cocaine craving. After prolonged withdrawal (1-2 months), when craving is high, expression of incubation depends on strengthening of excitatory inputs to medium spiny neurons (MSN) of the nucleus accumbens (NAc). These excitatory inputs interact with the intra-NAc GABAergic 'microcircuit', composed of MSN axon collaterals and GABAergic interneurons. Here, we investigated whether the increased glutamatergic neurotransmission observed after prolonged withdrawal is accompanied by altered GABAergic neurotransmission, focusing on NAc core. Rats self-administered cocaine or saline (6 hours/day) and then underwent >40 days of withdrawal. First, we investigated parvalbumin positive (PV+) interneurons, GABAergic fast-spiking interneurons that regulate MSN activity. Immunohistochemical studies revealed no significant change in PV signal intensity or the number of PV+ cells in cocaine rats versus saline controls. We then screened PV and other interneuron markers using immunoblotting. We detected no changes in levels of PV, calretinin, calbindin or neuronal nitric oxide synthase. Because expression of these markers is activity dependent, our results suggest no marked changes in interneuron activity. Finally, we utilized local field potential recording, which can detect GABA-mediated alterations at the circuit level, to investigate potential changes in two circuits implicated in cocaine craving: prelimbic prefrontal cortex to NAc core and basolateral amygdala to NAc core. We detected differential adaptations in these circuits, some of which may involve GABA. Overall, our results suggest that alterations in GABA transmission may accompany incubation of cocaine craving, but they are circuit specific and less pronounced than alterations in glutamate transmission. [ABSTRACT FROM AUTHOR]

Key points The functional importance of residues in loop G of the GABAA receptor has not been investigated. D43 and T47 in the α1 subunit are of particular significance as their structural modification inhibits activation by GABA., While the T47C substitution had no significant effect, non-conservative substitution of either residue (D43C or T47R) reduced the apparent potency of GABA., Propofol potentiated maximal GABA-evoked currents mediated by α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors. Non-stationary variance analysis revealed a reduction in maximal GABA-evoked Popen, suggesting impaired agonist efficacy., Further analysis of α1(T47R)β2γ2 receptors revealed that the efficacy of the partial agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol) relative to GABA was impaired., GABA-, THIP- and propofol-evoked currents mediated by α1(T47R)β2γ2 receptors deactivated faster than those mediated by α1β2γ2 receptors, indicating that the mutation impairs agonist-evoked gating., Spontaneous gating caused by the β2(L285R) mutation was also reduced in α1(T47R)β2(L285R)γ2 compared to α1β2(L285R)γ2 receptors, confirming that α1(T47R) impairs gating independently of agonist activation., Abstract The modification of cysteine residues (substituted for D43 and T47) by 2-aminoethyl methanethiosulfonate in the GABAA α1 subunit loop G is known to impair activation of α1β2γ2 receptors by GABA and propofol. While the T47C substitution had no significant effect, non-conservative substitution of either residue (D43C or T47R) reduced the apparent potency of GABA. Propofol (1 μ m), which potentiates sub-maximal but not maximal GABA-evoked currents mediated by α1β2γ2 receptors, also potentiated maximal currents mediated by α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors. Furthermore, the peak open probabilities of α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors were reduced. The kinetics of macroscopic currents mediated by α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors were characterised by slower desensitisation and faster deactivation. Similar changes in macroscopic current kinetics, together with a slower activation rate, were observed with the loop D α1(F64C) substitution, known to impair both efficacy and agonist binding, and when the partial agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol) was used to activate WT or α1(T47R)β2γ2 receptors. Propofol-evoked currents mediated by α1(T47R)β2γ2 and α1(F64C)β2γ2 receptors also exhibited faster deactivation than their WT counterparts, revealing that these substitutions impair gating through a mechanism independent of orthosteric binding. Spontaneous gating caused by the introduction of the β2(L285R) mutation was also reduced in α1(T47R)β2(L285R)γ2 compared to α1β2(L285R)γ2 receptors, confirming that α1(T47R) impairs gating independently of activation by any agonist. These findings implicate movement of the GABAA receptor α1 subunit's β1 strand during agonist-dependent and spontaneous gating. Immobilisation of the β1 strand may provide a mechanism for the inhibition of gating by inverse agonists such as bicuculline. [ABSTRACT FROM AUTHOR]

Background Alcohol use disorders are associated with single-nucleotide polymorphisms in GABRA2, the gene encoding the GABAA receptor α2-subunit in humans. Deficient GABAergic functioning is linked to impulse control disorders, intermittent explosive disorder, and to drug abuse and dependence, yet it remains unclear whether α2-containing GABAA receptor sensitivity to endogenous ligands is involved in excessive alcohol drinking. Methods Male wild-type (Wt) C57 BL/6J and point-mutated mice rendered insensitive to GABAergic modulation by benzodiazepines (BZD; H101R), allopregnanolone (ALLO) or tetrahydrodeoxycorticosterone (THDOC; Q241M), or high concentrations of ethanol (EtOH) (S270H/L277A) at α2-containing GABAA receptors were assessed for their binge-like, moderate, or escalated chronic drinking using drinking in the dark, continuous access (CA) and intermittent access (IA) to alcohol protocols, respectively. Social approach by mutant and Wt mice in forced alcohol abstinence was compared to approach by Et OH-naïve controls. Social deficits in forced abstinence were treated with allopregnanolone (0, 3.0, 10.0 mg/kg, intraperitoneal [i.p.]) or midazolam (0, 0.56, 1.0 mg/kg, i.p.). Results Mice with BZD-insensitive α2-containing GABAA receptors (H101R) escalated their binge-like drinking. Mutants harboring the Q241M point substitution in Gabra2 showed blunted chronic intake in the CA and IA protocols. S270H/L277A mutants consumed excessive amounts of alcohol but, unlike wild-types, they did not show forced abstinence-induced social deficits. Conclusions These findings suggest a role for: (i) H101 in species-typical binge-like drinking, (ii) Q241 in escalated chronic drinking, and (iii) S270 and/or L277 in the development of forced abstinence-associated social deficits. Clinical findings report reduced BZD-binding sites in the cortex of dependent patients; the present findings suggest a specific role for BZD-sensitive α2-containing receptors. In addition, amino acid residue 241 in Gabra2 is necessary for positive modulation and activation of GABAA receptors by ALLO and THDOC; we postulate that neurosteroid action on α2-containing receptor may be necessary for escalated chronic EtOH intake. [ABSTRACT FROM AUTHOR]

We investigated the changes in dopamine ( DA), glutamate and γ-aminobutyric acid ( GABA) during cocaine self-administration in rats implanted with guide cannulae into the nucleus accumbens and ventral pallidum. After stabilized cocaine self-administration, separate groups of rats underwent extinction (10 days) procedure in which cocaine infusion was replaced by saline injections. With using a 'yoked' procedure, the effects of cocaine or its withdrawal on the level of neurotransmitters were evaluated by dual-probe microdialysis. Repeated cocaine administration reduced basal glutamate levels in the nucleus accumbens and ventral pallidum, whereas it did not affect basal accumbal DA levels. Only rats that self-administered cocaine had increased basal GABA overflow in both examined brain structures. Active or passive cocaine administration elevated extracellular accumbal DA, however, the extent of cocaine-evoked DA level was significantly higher in rats that self-administered cocaine while both groups of animals showed also an attenuation of GABA level in the nucleus accumbens. On day 10 of extinction training, rats previously given cocaine revealed decreases in the basal accumbal concentration of glutamate while the basal GABA levels were significantly enhanced as compared with baseline of saline-yoked controls. Potassium depolarization delayed the reduction of the accumbal and pallidal extracellular glutamate levels in the active and passive cocaine groups. The present data indicate that changes in DA and GABA neurotransmission during maintenance phase mirror the motivational aspects of cocaine intake. Depending on acute (24 hours) or late (10 days) cocaine withdrawal, different neurotransmitter systems (i.e. glutamate or GABA) seem to be involved. [ABSTRACT FROM AUTHOR]

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • The microdose administration of novel drug candidates to humans in early development is currently undergoing evaluation as a cost-efficient approach to the early assessment of pharmacokinetics (PK) before the commitment of resources required to support formal phase 1 studies. • The microdose approach assumes that PK can be extrapolated linearly over the full range of exposures achieved with sub-pharmacological doses up to the therapeutic dose range. • Few microdose studies have been undertaken in the context of pharmaceutical drug development and their precise role in the selection of candidates for further clinical evaluation at therapeutic doses has yet to be fully substantiated. WHAT THIS STUDY ADDS • The present study describes the elective application of a human microdose study with a novel EP1 receptor antagonist, GSK269984A, to address a critical development liability posed by uncertainty with respect to the predicted human PK profile. • Microdose data revealed a favourable PK profile, consistent with a clinically acceptable dosing regimen. These data support the value of undertaking a microdose study early in the drug discovery process to facilitate risk evaluation and to enable decision-making. AIM The primary objective was to evaluate the pharmacokinetics (PK) of the novel EP1 antagonist GSK269984A in human volunteers after a single oral and intravenous (i.v.) microdose (100 µg). METHOD GSK269984A was administered to two groups of healthy human volunteers as a single oral ( n= 5) or i.v. ( n= 5) microdose (100 µg). Blood samples were collected for up to 24 h and the parent drug concentrations were measured in separated plasma using a validated high pressure liquid chromatography-tandem mass spectrometry method following solid phase extraction. RESULTS Following the i.v. microdose, the geometric mean values for clearance (CL), steady-state volume of distribution ( Vss) and terminal elimination half-life ( t1/2) of GSK269984A were 9.8 l h−1, 62.8 l and 8.2 h. Cmax and AUC(0,∞) were 3.2 ng ml−1 and 10.2 ng ml−1 h, respectively; the corresponding oral parameters were 1.8 ng ml−1 and 9.8 ng ml−1 h, respectively. Absolute oral bioavailability was estimated to be 95%. These data were inconsistent with predictions of human PK based on allometric scaling of in vivo PK data from three pre-clinical species (rat, dog and monkey). CONCLUSION For drug development programmes characterized by inconsistencies between pre-clinical in vitro metabolic and in vivo PK data, and where uncertainty exists with respect to allometric predictions of the human PK profile, these data support the early application of a human microdose study to facilitate the selection of compounds for further clinical development. [ABSTRACT FROM AUTHOR]

Background: This pilot feasibility study examined the role of genetics in laboratory-induced cocaine craving. Methods: Thirty-four African American, cocaine-depend- ent male subjects underwent a baseline assessment, cue-exposure session, and genetic analysis. Subjects were classified as either cue-reactive or nonreactive. Results: Among single nucleotide polymorphism markers in 13 candidate genes examined for association with cocaine cue-reactivity, two were statistically significant: GABRA2 (coding for GABA-A receptor alpha-2 subunit; rs11503014, nominal p= .001) and OPRM1 (coding for mu opioid receptor; rs2236256, nominal p= .03). Conclusions: These pilot results suggest that cocaine craving shows variability among cocaine-dependent subjects, and that GABRA2 and OPRM1 polymorphisms have differential influences on cocaine cue-reactivity, warranting studies in future research. (Am J Addict 2012;21:411-415) [ABSTRACT FROM AUTHOR]

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N-methyl-D-aspartate receptors (NMDARs) are ion channels gated by glutamate, the major excitatory neurotransmitter in the mammalian central nervous system (CNS). They are widespread in the CNS and are involved in numerous physiological and pathological processes including synaptic plasticity, chronic pain and psychosis. Aberrant NMDAR activity also plays an important role in the neuronal loss associated with ischaemic insults and major degenerative disorders including Parkinson's and Alzheimer's disease. Agents that target and alter NMDAR function may, thus, have therapeutic benefit. Interestingly, NMDARs are endowed with multiple extracellular regulatory sites that recognize ions or small molecule ligands, some of which are likely to regulate receptor function in vivo. These allosteric sites, which differ from agonist-binding and channel-permeation sites, provide means to modulate, either positively or negatively, NMDAR activity. The present review focuses on allosteric modulation of NMDARs containing the NR2B subunit. Indeed, the NR2B subunit confers a particularly rich pharmacology with distinct recognition sites for exogenous and endogenous allosteric ligands. Moreover, NR2B-containing receptors, compared with other NMDAR subtypes, appear to contribute preferentially to pathological processes linked to overexcitation of glutamatergic pathways. The actions of extracellular H+, Mg2+, Zn2+, of polyamines and neurosteroids, and of the synthetic compounds ifenprodil and derivatives ('prodils') are presented. Particular emphasis is put upon the structural determinants and molecular mechanisms that underlie the effects exerted by these agents. A better understanding of how NR2B-containing NMDARs (and NMDARs in general) operate and how they can be modulated should help define new strategies to counteract the deleterious effects of dysregulated NMDAR activity. [ABSTRACT FROM AUTHOR]

1 ATP receptors present on rat alveolar macrophages (NR8383 cells) were identified by recordings of membrane current, measurements of intracellular calcium. RT PCR and inmunocytochemislry. 2 In whole-cell recordings with a sodium-based internal solution, ATP evoked an inward current at —60 mV. This reversed at 0 mV. The EC[sub50] for ATP was 18 μM in normal external solution (calcium 2 mM. magnesium 1 mM). The currents evoked by 2′,3-O-(4-benzoyl]henzoyl-ATP were about five-fold smaller than those observed with ATP. ADP. UTP and αβ-methylene-ATP (αβmeATP) (up to 100 μM) had no effect. ATP-evoked currents were potentiated up to ten-fold by ivermectin and were unaffected by suramin (30-100 μM), pyridoxal-phosphate-6-azophenyl-(2.4-sulphonic acid) (30-100 &muM). and brilliant blue G (1 μM). 3 In whole-cell recordings with 11 potassium-based internal solution and low ECTA (0.01 mM), ATP evoked an inward current at —60mV that was followed by larger outward current. ADP and UTP (1— 100 μM) evoked only outward currents: these reversed polarity at the potassium equilibrium potential and were blocked by apamin (10nM). Outward currents were also blocked by the phospholipase C inhibitor U73I22 (l μM). and they were not seen with higher intracellular EGTA (l0mM). Suramin (30 μM) blocked the outward currents evoked by ATP and UTP, but not that evoked by ADP. PPADS (10 μM) blocked the ADP-evoked outward current without altering the ATP or UTP currents. 4 RT—PCR showed transcripts for P2X subunits 1,4 and 7 (not 2, 3, 5, 6) and P2Y receptors 1, 2, 4 and 12 (not 6). Immunocytochemistry showed strong P2X[sub4] receptor expression partly associated with the membrane, weak P2X[sub7], staining that was not associated with the cell membrane, and no P2X[sub1] receptor immunoreactivity. 5 We conclude that rat alveolar macrophages express (probably hornomeric) P2X[sub4] receptors, but find no evidence for other functional P2X subtypes. The P2Y receptors are most likely P2Y[sub1] and P2Y[sub2] and these couple through phospholipase C to an increase in intracellular calcium and the opening of SK. type potassium channels. [ABSTRACT FROM AUTHOR]

Presents a list of topics on the effect of various chemicals on general physiology of human beings. Effect of acute and chronic nicotine on lipogenesis in mouse adipose tissues; Contribution of plasma extravasation in a model of murine joint inflammation; Influence of endogenously produced reactive oxygen species on the inotropic and chronotropic effects of adrenoreceptors.

Studies the consequences of reduced nitric-oxide production on the vascular reactivity of porcine coronary arteries after angioplasty. Key issues of interest; Analysis of pertinent topics and relevant issues; Implications on pharmacology.

1 Previous studies have reported discrepancies in the potencies of A[sub1] adenosine receptor agonists at mouse motor nerve terminals. In addition, conflicting results on the role of protein kinase A (PKA) IB mediating the inhibitory effects of A[sub1] receptor agonists have been published. We thus decided to investigate the possibility of endogenous control of adenosine receptor sensitivity by protein kinases. using a variety of protein kinase inhibitors in conjunction with the adenosine receptor agonist 2-chloroadenosine (CADO). 2 CADO, at the concentration employed previously to study spontaneous ACh release in the mouse (1 μM), did not inhibit spontaneous ACh release in our experiments. However, a higher concentration of CADO (10 μM) produced highly statistically-significant reductions in spontaneous ACh release. 3 In the presence of the non-selective protein kinase inhibitor, H7 (50 μM), the potency of CADO was increased such that 1 μM CADO now reduced spontaneous quantal ACh release to approximately 63% of control. 4 Both H7, and the selective PKA inhibitor, KT5720 (500 nM) prevented increases in ACh release produced by CPT cyclic AMP (250 μM), suggesting these kinase inhibitors were blocking PKA. In contrast to H7, however, KT5720. did not reveal an inhibitory effect of 1 μM CADO. A number of other non-selective PKA inhibitors also failed to increase the potency of CADO. 5 The results suggest that an endogenous H7-sensitive process modulates the sensitivity of the mouse A[sub1] adenosine receptor and that the inhibitory effects of CADO are independent of cyclic AMP accumulation or PKA inhibition. [ABSTRACT FROM AUTHOR]

The effects of ambient and elevated ozone levels on growth and photosynthesis of beech (Fagus sylvatica) were studied by exposing seedlings in open-top chambers for one growing season to three treatments: charocoal-filtered (CF), non-filtered (NF) and non-filtered air with addition of ozone (30 ppb ozone) on clear days for 8-10 h d-1(NF+). Ambient levels were relatively low and accumulated to an AOT40 (accumulated exposure over a threshold of 40 ppb) of 4055 ppb h (for the period 23 Apr-30 Sept). The NF+ chambers received an AOT40 of 8880 ppb h. Throughout the growing season we measured growth and photsynthetic properties. The treatments did not cause strong effects: measurements of gas exchange (light-saturated assimilation rate, CO2 and light-response curves) and chlorophyll fluorescence showed slight and mostly non-significant reductions of several parameters. No significant differences were found for growth, through in the NF+ treatment (AOT40 8880 ppb h) the relative growth rate for diameter increment was times reduced by 12% compared with the control treatment. [ABSTRACT FROM AUTHOR]

1. The impact of a selective fungal pathogen, Colletotrichum coccodes, on above- ground biomass and development of height hierarchies in monoculture populations of Abutilon theophrasti (velvetleaf) was examined over an 11-week period, at each of three densities (125, 250 and 375 plants m-2). over three field seasons. 2. Fungal infection caused significant reductions (30-44%) in unit area above-ground biomass within 5 weeks of inoculation in 2 of the 3 years. However, 8 weeks following C. coccodes inoculation, A. theophrasti biomass within inoculated plots did not differ significantly from biomass within uninoculated control plots in any of the three years. As expected. mean biomass per plant generally declined with increasing monoculture density regardless of inoculation treatment and harvest date In all three years. biomass per unit area increased significantly with planting density 2 weeks after inoculation (WAI), In subsequent harvests (5 and 8 WAI). however, A. theophrasti unit area biomass was generally little affected by increasing planting density regardless of inoculation treatment. 3. Height frequency distributions of control and inoculated A. theophrasti populations within each of the three panting densities were generally positively skewed (L-shaped) by the first harvest (2 WAI) and negatively skewed (J-shaped) 3 weeks later. A positive skewness indicates the presence of a large number of short A. theophrasti individuals and a relatively few, taller plants within a population. The opposite trend is observed for a negatively skewed population structure. By the third harvest (8 WAI), however, height frequency distributions at the two lowest densities remained negatively skewed regardless of inoculation treatment. while at the highest density, frequency distributions typically reverted to being positively skewed. [ABSTRACT FROM AUTHOR]

Studies the effects of increased nutrient and organic loading on a community dominated by submersed macrophytes and gastropods in a drainage channel in the Lewes Brooks. Increased organic loading resulting in significant increases in conductivity, decreases in oxygen, pH and chlorophyll-a values in the water column and increases and decreases in the percentage cover of Potamogeton crispus and Groeblandia densa.

Studies the fruiting phenology and seed dispersal in a non-native succulent, Carpobrotus edulis which is invading many coastal plant assemblages in California. Description of the fruit produced by the plant; Animals that feed on the fruits; Frequency of seeds in the soil beneath adult plants than away from them.

Lipid peroxidation was induced in rat liver membrane fractions in vitro upon NADPH-dependent metabolic activation of the anesthetic agent halothane at low O2 concentrations. Halothane-induced lipid peroxidation was dependent on time, concentration of halothane, and the calculated O2 concentrations present in the system. Lipid peroxidation was inducible at increasing O2 concentrations up to 12 μM, decreased at higher O2 concentrations up to 48 μM, and was not detectable at normoxic conditions. Leukotriene B4 (LTB4) was identified as a product arising upon lipid peroxidation by reverse-phase high-pressure liquid chromatography combined with a radioimmunoassay. LTB4 formation was maximal under conditions of maximal lipid peroxidation at a calculated O2 concentration of 12 μM. Even at high concentration, the 5-lipoxygenase inhibitors MK886 (10 μM), ZD2138 (20 μM), and ZM230487 (20 μM) were not inhibitory in halothane-induced lipid peroxidation nor in the associated formation of LTB4. Synthetic LTB4 was transformed into its 20-hydorxy derivative by co-oxidation in an O2-concentration-dependent manner, being considerably reduced at the low O2 concentrations that maximally promoted lipid peroxidation. The collective evidence of these data raises the possibility that exposure to halothane might lead to peroxidation-associated net synthesis of LTB4 through 5-lipoxygenase-independent escape routes in liver tissue under physiologically or pathophysiologically low O2 concentrations. [ABSTRACT FROM AUTHOR]

Histone acetylation has previously been correlated with both chromosome replication and transcription. We present evidence that (a) confirms both correlations in the true slime mold, Physarum polycephalum and (b) shows that quite a different pattern of acetate turnover is associated with replication compared with transcription. The pattern associated with replication involves turnover of acetate on all four core histones on species containing one or two acetates per molecule. This pattern was resolved from the transcription-associated pattern by three different procedures: (a) detailed analysis of gels of histones pulse-labelled with acetate; (b) the pattern of acetylation of histones pulse-labelled with [³H]lysine; and (c) the pattern of acetylation of soluble histones. The pattern associated with transcription is restricted to histones H3 and H4 and occurs mostly on highly acetylated species. This pattern was resolved by (a) analysis of gels of histones pulse-labelled with acetate; (b) the pattern of histone acetylation in G2 phase of the ceil cycle; and (c) the pattern of histone acetylation in the presence of cycloheximide. [ABSTRACT FROM AUTHOR]