bluuebunny/crossref_metadata_2025 huggingface dataset:
Proton modulation of a Ca(2+)-activated K+ channel from rat skeletal muscle incorporated into planar bilayers.
Authors: Laurido C , Candia S , Wolff D , Latorre R
Published: May 2004

The effect of pH on the activation of a Ca-activated K+ [K(Ca)] channel from rat skeletal muscle incorporated into planar lipid bilayers was studied. Experiments were done at different intracellular Ca2+ and proton concentrations. Changes in pH modified channel kinetics only from the Ca-sensitive face of the channel. At constant Ca2+ concentration, intracellular acidification induced a decrease in the open probability (Po) and a shift of the channel activation curves toward the right along the voltage axis. The displacement was 23.5 mV per pH unit. This displacement was due to a change in the half saturation voltage (Vo) and not to a change in channel voltage dependence. The shifts in Vo induced by protons appeared to be independent of Ca2+ concentration. The slope of the Hill plot of the open-closed equilibrium vs. pH was close to one, suggesting that a minimum of one proton is involved in the proton-driven channel closing reaction. The change in Po with variations in pH was due to both a decrease in the mean open time (To) and an increase in the mean closed time (Tc). At constant voltage, the mean open time of the channel was a linear function of [Ca2+] and the mean closed time was a linear function of 1/[Ca2+]2. Changes in the internal pH modified the slope, but not the intercept of the linear relations To vs. [Ca2+] and Tc vs. 1/[Ca2+]2. On the basis of these results an economical kinetic model of the effect of pH on this channel is proposed. It is concluded that protons do not affect the open-closed reaction, but rather weaken Ca2+ binding to all the conformational states of the channel. Moreover, competitive models in which Ca2+ and H+ cannot bind to the same open or closed state are inconsistent with the data.
A [Na+]o-independent, pHo-dependent mechanism for reduction of intracellular [Ca2+] after influx through Ca2+ channels in mouse pituitary cells.
Authors: Korn S J , Horn R
Published: May 2004

The effect of extracellular pH (pHo) on the duration of calcium-dependent chloride currents (ICl(Ca] was studied in voltage clamped AtT-20 pituitary cells. ICl(Ca) was activated by Ca2+ influx through plasma membrane Ca2+ channels, which were opened by step depolarization to voltages between -20 and +60 mV. Increasing pHo from 7.3 to 8.0 reversibly prolonged ICl(Ca) tail currents in perforated patch recordings from cells bathed in both Na(+)-containing and Na(+)-free solutions. This prolongation was prevented in standard whole cell recordings when the pipette solution contained 0.5 mM EGTA. The effects of raised pHo were not due to alteration of intracellular pH, since tail current prolongation still occurred when intracellular pH was buffered at 7.3 with 80 mM HEPES. The prolongation of ICl(Ca) at pHo 8 could not be accounted for by a direct action on Ca2+ channels, since tail currents were prolonged when pHo was changed rapidly during the tail current, after all Ca2+ channels were closed. The effects of increasing pHo on ICl(Ca) also could not be explained by a direct action on Cl- channels, since changing to pHo 8 did not prolong Cl- tail currents when intracellular Ca2+ concentration [( Ca2+]i) was fixed by EGTA in whole cell recordings. Raising pHo did, however, prolong depolarization-evoked [Ca2+]i transients, measured directly with the Ca2+ indicator dye, fura-2. Taken together, these data demonstrate the presence of a Na(+)-independent, pHo-sensitive mechanism for reduction of [Ca2+]i after influx through Ca2+ channels. This mechanism is associated with the plasma membrane, and is active on a time scale that is relevant to the duration of single action potentials in these cells. We suggest that this mechanism is the plasma membrane Ca2+ ATPase.
Calcium currents in the A7r5 smooth muscle-derived cell line. Increase in current and selective removal of voltage-dependent inactivation by intracellular trypsin.
Authors: Obejero-Paz C A , Jones S W , Scarpa A
Published: May 2004

We studied the effects of trypsin on L-type calcium current in the A7r5 smooth muscle cell line. Intracellular dialysis with trypsin increased the whole-cell current up to fivefold. The effect was concentration dependent, and was prevented by soybean trypsin inhibitor. Ensemble analysis indicated an increase in the number of functional channels, and possibly a smaller increase in the open probability, with no change in the single channel current. The shape of the current-voltage curve was unaffected. Trypsin also nearly eliminated inactivation of currents carried by Ba2+, but had little or no effect on the rapid inactivation process in Ca2+, This indicates that trypsin removes voltage-dependent but not Ca(2+)-dependent inactivation, suggesting the existence of distinct protein domains for these two mechanisms of calcium channel inactivation.
Activation of epithelial Na channels by hormonal and autoregulatory mechanisms of action.
Authors: Els W J , Helman S I , Mencio T
Published: May 2004

Methods of blocker-induced noise analysis were used to investigate the way in which forskolin and vasopressin stimulate Na transport at apical membranes of short-circuited frog skin transporting Na at spontaneous rates of transport. Experiments were done under conditions where the apical Ringer solution contained either 100 mM Na or a reduced Na concentration of 5 or 10 mM Na and buffered with either HCO3 or HEPES. Reduction of apical solution Na concentration caused a large autoregulatory increase of Na channel density (NT) similar in magnitude to that observed previously in response to blocker (amiloride) inhibition of apical membrane Na entry. Forskolin at 2.5 microM caused maximal and reversible large increases of NT, which were larger than could be elicited by 30 mU/ml vasopressin. In both the absence and presence of the autoregulatory increase of NT (caused by reduction of apical Na concentration), forskolin caused large increases of NT. Although the fractional increases of NT in response to forskolin were roughly similar, the absolute increases of NT were considerably larger in those tissues studied at reduced Na concentration and where baseline values of NT were markedly elevated by reduction of apical Na concentration. Because the effects on NT were additive, it is likely that the cAMP-dependent and autoregulatory mechanism that lead to changes of NT are distinct. We speculate that autoregulation of NT may involve change of the size of a cytosolic pool of Na-containing vesicles that are in dynamic balance with the apical membranes. cAMP-dependent regulation of NT may involve change of the dynamic balance between vesicles and the apical membranes of these epithelial cells. Alternative hypotheses cannot at present be ruled out, but will require incorporation of the idea that regulation of NT can occur both by hormonal and nonhormonal (autoregulatory) mechanisms of action.
Calmodulin activation of the Ca2+ pump revealed by fluorescent chelator dyes in human red blood cell ghosts.
Authors: James-Kracke M R
Published: May 2004

Ca2+ transport in red blood cell ghosts was monitored with fura2 or quin2 incorporated as the free acid during resealing. This is the first report of active transport monitored by the fluorescent intensity of the chelator dyes fura2 (5-50 microM) or quin2 (250 microM) in hemoglobin-depleted ghosts. Since there are no intracellular compartments in ghosts and the intracellular concentrations of all assay chelator substances including calmodulin (CaM), the dyes, and ATP could be set, the intracellular concentrations of free and total Ca [( Cafree]i and [Catotal]i) could be calculated during the transport. Ghosts prepared with or without CaM rapidly extruded Ca2+ to a steady-state concentration of 60-100 nM. A 10(4)-fold gradient for Ca2+ was routinely produced in medium containing 1 mM Ca2+. During active Ca2+ extrusion, d[Cafree]i/dt was a second order function of [Cafree]i and was independent of the dye concentration, whereas d[Catotal]i/dt increased as a first order function of both the [Cafree]i and the concentration of the Ca:dye complex. CaM (5 microM) increased d[Catotal]i/dt by 400% at 1 microM [Cafree]i, while d[Cafree]i/dt increased by only 25%. From a series of experiments we conclude that chelated forms of Ca2+ serve as substrates for the pump under permissive control of the [Cafree]i, and this dual effect may explain cooperativity. Free Ca2+ is extruded, and probably also Ca2+ bound to CaM or other chelators, while CaM and the chelators are retained in the cell.
Role of GTP-binding proteins in the regulation of mammalian cardiac chloride conductance.
Authors: Hwang T C , Horie M , Nairn A C , Gadsby D C
Published: May 2004

Beta-Adrenoceptor agonists activate a time- and voltage-independent Cl- conductance in mammalian cardiac myocytes. To characterize the cellular signaling pathways underlying its regulation, wide-tipped pipettes fitted with a pipette perfusion device were used to record whole-cell current and to introduce nucleotides to the interior of guinea pig ventricular myocytes. Replacement of pipette GTP with GDP beta S prevented activation of the Cl- conductance by Iso, suggesting a requirement for G protein turnover. With GTP in the pipette, the effect of Iso could be abolished by the beta-adrenoceptor antagonist propranolol, and mimicked by histamine or forskolin. These actions of Iso and forskolin are mediated exclusively via cAMP-dependent protein kinase (PKA), because (a) maximal activation of the Cl- conductance by forskolin or pipette cAMP occluded the effect of Iso, and (b) switching to pipette solution containing a synthetic peptide inhibitor (PKI) of PKA completely abolished the Cl- conductance activated by Iso and prevented the action of forskolin, but had no further effect. These results argue against basal activation of the Cl- conductance, and make it extremely unlikely that the stimulatory G protein, Gs, has any direct, phosphorylation-independent influence. The muscarinic receptor agonists acetylcholine (ACh) and carbachol diminished, in a reversible manner, Cl- conductance activated by Iso or forskolin, but not that elicited by cAMP. The muscarinic inhibition was abolished by replacing pipette GTP with GDP beta S, or by preincubating cells with pertussis toxin (PTX), and was therefore mediated by an inhibitory G protein, presumably Gi, influencing adenylyl cyclase activity. Nonhydrolyzable GTP analogues (GTP gamma S or GppNHp) applied via the pipette did not themselves activate Cl- conductance, but rendered Cl- current activation by brief exposures to Iso or histamine, but not to forskolin, irreversible. The Cl- conductance persistently activated by Iso was insensitive to propranolol or ACh, but could still be abolished by pipette application of PKI. The data indicate that stimulation of beta-adrenergic or histaminergic receptors in the presence of nonhydrolyzable GTP analogues causes persistent activation of Gs and uncouples it from the receptors. We conclude that autonomic regulation of cardiac Cl- conductance reflects accurately the underlying modulation of adenylyl cyclase activity and, hence, that this system is a suitable mammalian model for in situ studies of the interactions between adenylyl cyclase, Gs, Gi, and forskolin.
Light-activated ion channels in solitary photoreceptors of the scallop Pecten irradians.
Authors: Nasi E , Gomez M P
Published: May 2004

Retinas from the scallop Pecten irradians were enzymatically dispersed, yielding a large number of isolated photoreceptors suitable for tight-seal recording. Whole-cell voltage clamp measurements demonstrated that the phototransducing machinery remained intact: quantum bumps could be elicited by dim illumination, while brighter flashes produced larger, smooth photocurrents. Single-channel currents specifically activated by light were recorded in cell-attached patches, and were almost exclusively confined to the rhabdomeric region. Their density is sufficiently high to account for the macroscopic photoresponse. Channel activation is graded with stimulus intensity in a range comparable to that of the whole-cell response, and can be recorded with illumination sufficiently dim to evoke only quantum bumps. Light-dependent channel openings are very brief, on average 1 ms or less at 20-22 degrees C, apparently not because of blockage by extracellular divalent cations. The mean open time does not change substantially with stimulus intensity. In particular, since dwell times are in the millisecond range even with the dimmest lights, the channel closing rate does not appear to be the rate-limiting step for the decay kinetics of discrete waves. The latency of the first opening after light onset is inversely related to light intensity, and the envelope of channel activity resembles the time course of the whole-cell photocurrent. Unitary currents are inward at resting potential, and have a reversal voltage similar to that of the macroscopic light response. Voltage modulates the activity of light-sensitive channels by increasing the opening rate and also by lengthening the mean open times as the patch is depolarized. The unitary conductance of the predominant class of events is approximately 48 pS, but at least one additional category of smaller-amplitude openings was observed. The relative incidence of large and small events does not appear to be related in a simple way to the state of adaptation of the cell.
Contribution of sarcolemmal sodium-calcium exchange and intracellular calcium release to force development in isolated canine ventricular muscle.
Authors: Bouchard R A , Bose D
Published: May 2004

The aim of this work was to determine the relationship between peak twitch amplitude and sarcoplasmic reticulum (SR) Ca2+ content during changes of stimulation frequency in isolated canine ventricle, and to estimate the extent to which these changes were dependent upon sarcolemmal Na(+)-Ca2+ exchange. In physiological [Na+]o, increased stimulation frequency in the 0.2-2-Hz range resulted in a positive inotropic effect characterized by an increase in peak twitch amplitude and a decrease in the duration of contraction, measured as changes in isometric force development or unloaded cell shortening in intact muscle and isolated single cells, respectively. Action potentials recorded from single cells indicated that the inotropic effect was associated with a progressive decrease of action potential duration and a marked reduction in average time spent by the cell near the resting potential during the stimulus train. The frequency-dependent increase of peak twitch force was correlated with an increase of Ca2+ uptake into and release from the SR. This was estimated indirectly using the phasic contractile response to rapid (less than 1 s) lowering of perfusate temperature from 37 degrees C to 0-2 degrees C and changes of twitch amplitude resulting from perturbations in the pattern of electrical stimulation. Lowering [Na+]o from 140 to 70 mM resulted in an increase of contractile strength, which was accompanied by a similar increase of apparent SR Ca2+ content, both of which could be abolished by exposure to ryanodine (1 x 10(-8) M), caffeine (3 x 10(-3) M), or nifedipine (2 x 10(-6) M). Increased stimulation frequency in 70 mM [Na+]o resulted in a negative contractile staircase, characterized by a graded decrease of peak isometric force development or unloaded cell shortening. SR Ca2+ content estimated under identical conditions remained unaltered. Rate constants derived from mechanical restitution studies implied that the depressant effect of increased stimulation frequency in 70 mM [Na+]o was not a consequence of a decreased rate of refilling of a releasable pool of Ca2+ within the cell. These results demonstrate that frequency-dependent changes of contractile strength and intracellular Ca2+ loading in 140 mM [Na+]o require the presence of a functional sarcolemmal Na(+)-Ca2+ exchange process. The possibility that the negative staircase in 70 mM [Na+]o is related to inhibition of Ca(2+)-induced release of Ca2+ from the SR by various cellular mechanisms is discussed.
On the mechanism of G protein beta gamma subunit activation of the muscarinic K+ channel in guinea pig atrial cell membrane. Comparison with the ATP-sensitive K+ channel.
Authors: Ito H , Tung R T , Sugimoto T , Kobayashi I , Takahashi K , Katada T , Ui M , Kurachi Y
Published: May 2004

The mechanism of G protein beta gamma subunit (G beta gamma)-induced activation of the muscarinic K+ channel (KACh) in the guinea pig atrial cell membrane was examined using the inside-out patch clamp technique. G beta gamma and GTP-gamma S-bound alpha subunits (G alpha *'s) of pertussis toxin (PT)-sensitive G proteins were purified from bovine brain. Either in the presence or absence of Mg2+, G beta gamma activated the KACh channel in a concentration-dependent fashion. 10 nM G beta gamma almost fully activated the channel in 132 of 134 patches (98.5%). The G beta gamma-induced maximal channel activity was equivalent to or sometimes larger than the GTP-gamma S-induced one. Half-maximal activation occurred at approximately 6 nM G beta gamma. Detergent (CHAPS) and boiled G beta gamma preparation could not activate the KACh channel. G beta gamma suspended by Lubrol PX instead of CHAPS also activated the channel. Even when G beta gamma was pretreated in Mg(2+)-free EDTA internal solution containing GDP analogues (24-48 h) to inactivate possibly contaminating G i alpha *'s, the G beta gamma activated the channel. Furthermore, G beta gamma preincubated with excessive GDP-bound G o alpha did not activate the channel. These results indicate that G beta gamma itself, but neither the detergent CHAPS nor contaminating G i alpha *, activates the KACh channel. Three different kinds of G i alpha * at 10 pM-10 nM could weakly activate the KACh channel. However, they were effective only in 40 of 124 patches (32.2%) and their maximal channel activation was approximately 20% of that induced by GTP-gamma S or G beta gamma. Thus, G i alpha * activation of the KACh channel may not be significant. On the other hand, G i alpha *'s effectively activated the ATP-sensitive K+ channel (KATP) in the ventricular cell membrane when the KATP channel was maintained phosphorylated by the internal solution containing 100 microM Mg.ATP. G beta gamma inhibited adenosine or mACh receptor-mediated, intracellular GTP-induced activation of the KATP channel. G i alpha *'s also activated the phosphorylated KATP channel in the atrial cell membrane, but did not affect the background KACh channel. G beta gamma subsequently applied to the same patch caused prominent KACh channel activation. The above results may indicate two distinct regulatory systems of cardiac K+ channels by PT-sensitive G proteins: G i alpha activation of the KATP channel and G beta gamma activation of the KACh channel.
DRD2 Gene Transfer Into the Nucleus Accumbens Core of the Alcohol Preferring and Nonpreferring Rats Attenuates Alcohol Drinking
Authors: Thanos Panayotis K. , Taintor Nicholas B. , Rivera Seth N. , Umegaki Hiroyuki , Ikari Hiroyuki , Roth George , Ingram Donald K. , Hitzemann Robert , Fowler Joanna S. , Gatley S. John , Wang Gene‐Jack , Volkow Nora D.
Published: May 2004

Abstract: Background: Transient overexpression of the dopamine D2 receptor (DRD2) gene in the nucleus accumbens (NAc) using an adenoviral vector has been associated with a significant decrease in alcohol intake in Sprague Dawley rats. This overexpression of DRD2 reduced alcohol consumption in a two‐bottle‐choice paradigm and supported the view that high levels of DRD2 may be protective against alcohol abuse.

Methods: Using a limited access (1 hr) two‐bottle‐choice (water versus 10% ethanol) drinking paradigm, we examined the effects of the DRD2 vector in alcohol intake in the genetically inbred alcohol‐preferring (P) and ‐nonpreferring (NP) rats. In addition, micro–positron emission tomography imaging was used at the completion of the study to assess in vivo the chronic (7 weeks) effects of ethanol exposure on DRD2 levels between the two groups.

Results: P rats that were treated with the DRD2 vector (in the NAc) significantly attenuated their alcohol preference (37% decrease) and intake (48% decrease), and these measures returned to pretreatment levels by day 20. A similar pattern of behavior (attenuation of ethanol drinking) was observed in NP rats. Analysis of the [11C]raclopride micro–positron emission tomography data after chronic (7 weeks) exposure to ethanol revealed clear DRD2 binding differences between the P and NP rats. P rats showed 16% lower [11C]raclopride specific binding in striatum than the NP rats.

Conclusions: These findings further support our hypothesis that high levels of DRD2 are causally associated with a reduction in alcohol consumption and may serve as a protective factor against alcoholism. That this effect was seen in P rats, which are predisposed to alcohol intake, suggests that they are protective even in those who are genetically predisposed to high alcohol intake. It is noteworthy that increasing DRD2 significantly decreased alcohol intake but did not abolish it, suggesting that high DRD2 levels may specifically interfere with the administration of large quantities of alcohol. The significantly higher DRD2 concentration in NP than P rats after 7 weeks of ethanol therefore could account for low alcohol intake.
Gender Differences in the Relationships Between Multiple Measures of Alcohol Consumption and Physical and Mental Health
Authors: Green Carla A. , Perrin Nancy A. , Polen Michael R.
Published: May 2004

Abstract: Background: Epidemiological research examining health consequences of alcohol consumption generally relies on average volume consumed, yet examinations of drinking patterns show different dimensions of use associated with different health outcomes. Gender differences in metabolism and body composition may lead to gender‐specific consequences of drinking frequency, quantity consumed per occasion, average amount consumed, and drinking pattern. Inconsistent results suggest gender differences are not well understood.

Methods: Participants were 3069 male and 2600 female health maintenance organization survey respondents. Gender differences in relationships between alcohol consumption and health were examined using analyses of covariance adjusting for age alone and for age, ethnicity, marital status, body water index, and smoking. Past‐year alcohol consumption (frequency, quantity per occasion, average drinks per month, and drinking pattern) and health measures (Short Form‐36 general health, physical functioning, mental health subscales) were examined.

Results: Gender × drinking frequency and drinking quantity interactions were significant in age‐adjusted and fully adjusted models of general health and physical functioning. Gender interactions for drinking pattern were significant in the age‐adjusted model and marginally significant in the fully adjusted model. No gender × drinking measure interactions were found for mental health. Fully adjusted models attenuated but did not eliminate gender differences for health and magnified relationships for functioning, the latter after adjusting for body water. For both genders, light to moderate consumption and more frequent drinking were associated with better health and functioning; relationships were stronger among women than men.

Conclusions: Gender × drinking measure interactions in health outcomes suggest analyses should include such interactions except, possibly, for mental health. Adjusting for potential confounders can attenuate (general health) or magnify (physical functioning) gender differences. Functional status appears a sensitive measure for evaluating gender differences in alcohol's health effects, adjusting for body water. Women's health may benefit proportionally more from moderate drinking than men's.
Alcohol‐Induced Lipid and Morphological Changes in Chick Retinal Development
Authors: Aguilera Yolanda , Ruiz‐Gutiérrez Valentina , Prada Francisco A. , Martínez Juan J. , Quesada Adela , Dorado Manuel E.
Published: May 2004

Abstract: Background: Alcohol exposure causes alterations in the lipid content of different organs and a reduction of long‐chain fatty acids. During embryo development, the central nervous system is extremely vulnerable to the teratogenic effects of alcohol, and the visual system is particularly sensitive.

Methods: White Leghorn chick embryos were injected with 10‐ and 20‐μl alcohol doses into the yolk sac at day 6 of incubation. The lipid composition of the retina was analyzed in embryos at day 7 of incubation (E7), E11, E15, and E18. The percentages of phospholipids, free cholesterol, esterified cholesterol, diacylglycerides, and free fatty acids were estimated by using an Iatroscan thin layer chromatography flame ionization detector. Gas chromatography and mass spectrometry were used to determine fatty acid composition. The morphological study was performed at E7, E11, and E19 by means of semithin and immunohistochemical techniques.

Results: In the retina, alcohol causes the total lipid content to change, with a remarkable increase in free cholesterol and a dramatic decrease in esterified cholesterol. Diacylglycerides and free fatty acids tend to increase. Phosphatidylcholine and phosphatidylethanolamine decrease, whereas phosphatidylserine, sphingomyelin, and phosphatidylinositol increase. The main fatty acids of the retina also undergo changes. At E7, myriotic acid increases, and oleic acid and polyunsaturated fatty acids such as arachidonic acid and docosahexaenoic acid decrease. From E18 onward, there is some recovery, except for fatty acids, which recover earlier. From a morphological point of view, alcohol effects on retinal development are various: increase of intercellular spaces in all cell layers, pyknosis with loss of cellularity in the inner nuclear cell layer and ganglion cell layer, retarded or disorderly cell migration, early cell differentiation, and loss of immunoreactivity for myelin oligodendrocyte–specific protein.

Conclusions: Acute alcohol exposure during embryo development causes the lipid composition of the retina to change, with a trend to recovery in the last stages. These alterations are in line with the changes observed at a morphological level.
Long‐Lasting Alterations of the Mesolimbic Dopamine System After Periadolescent Ethanol Drinking by Alcohol‐Preferring Rats
Authors: Sahr A. E. , Thielen R. J. , Lumeng L. , Li T.‐K. , McBride W. J.
Published: May 2004

Abstract: Background: This study tested the hypothesis that ethanol consumption by alcohol‐preferring (P) rats during the periadolescent period causes persistent alterations in the mesolimbic dopamine (DA) system. After ethanol drinking during periadolescence, P rats were examined for alterations in basal locomotor activity, changes in extracellular DA levels and extraction fraction in the nucleus accumbens (NAc) by using no‐net‐flux (NNF) microdialysis, and changes in the response of the mesolimbic DA system to ethanol.

Methods: Male P rat pups were given 24‐hr free‐choice access to 15% (v/v) ethanol from postnatal day (PD) 30 through PD 60. On PD 70, rats were assessed for locomotor activity. On PD 70 to 80, rats were implanted with bilateral guide cannulas aimed above the NAc. After at least 5 days, microdialysis probes were inserted bilaterally; on the following day, NNF microdialysis experiments were conducted. On the day after the NNF experiment, conventional microdialysis experiments were conducted to measure extracellular levels of DA in response to intraperitoneal injection of saline or ethanol 2.5 g/kg.

Results: Compared with the ethanol‐naive group, ethanol drinking by P rats during periadolescence did not alter basal locomotor activity, nor did it alter the basal extracellular concentration of DA. There was, however, a significant increase in the extraction fraction of DA of ethanol‐drinking animals relative to the controls (57.4 ± 2.7% and 45.8 ± 2.3%, respectively). Additionally, compared with controls, P rats with exposure to ethanol during the periadolescent period showed a prolonged increase in the extracellular levels of DA after a challenge dose of ethanol.

Conclusions: The results of the microdialysis experiments suggest that periadolescent ethanol drinking by P rats increases basal DA neurotransmission (as indicated by higher DA clearance while maintaining the same extracellular DA concentrations) and prolongs the response of DA neurotransmission to ethanol.
Acoustic Startle Reactivity During Acute Alcohol Withdrawal in Rats That Differ in Genetic Predisposition Toward Alcohol Drinking: Effect of Stimulus Characteristics
Authors: Chester Julia A. , Blose Annette M. , Froehlich Janice C.
Published: May 2004

Abstract: Background: We have previously reported an association between greater alcohol withdrawal magnitude after a single alcohol exposure and a genetic predisposition toward low alcohol drinking in rats selectively bred for differences in alcohol intake when acoustic startle reactivity to a tone stimulus was used to index acute alcohol withdrawal. The purpose of this study was to examine whether the quality of the acoustic startle stimulus (noise versus tone) is important for detecting a genetic relationship between alcohol withdrawal magnitude and alcohol drinking behavior.

Methods: Alcohol‐naive male rats selectively bred for high alcohol intake [alcohol‐preferring (P), high‐alcohol‐drinking (HAD)1, and HAD2] or low alcohol intake [alcohol‐nonpreferring (NP), low‐alcohol‐drinking (LAD)1, and LAD2] received a single intragastric infusion of water or alcohol (4.0 g/20.3 ml/kg; 25% v/v), and acoustic startle test sessions were given at 14, 16, 18, 20, and 24 hr after infusion. Each test session consisted of a 5‐min acclimation period followed by random presentation of various white noise stimuli (90, 100, 110, and 120 dB.)

Results: Line differences in acoustic startle magnitude under control conditions were present in all three pairs of selectively bred lines; P rats showed a greater startle magnitude relative to NP rats, whereas both LAD lines showed a greater startle magnitude relative to both HAD lines. During alcohol withdrawal, the P, HAD1, and HAD2 lines showed enhanced startle magnitude compared with their water‐treated controls. No change in startle magnitude during alcohol withdrawal was found in the NP, LAD1, or LAD2 lines.

Conclusions: In contrast to our prior findings, these results showed a genetic association between high alcohol drinking and a greater startle response magnitude to a noise stimulus during alcohol withdrawal. It seems that the genetic association between alcohol drinking and alcohol withdrawal, as assessed by the acoustic startle response, depends on the quality of the acoustic startle stimulus.
Alcohol Use Disorders Among Emergency Department–Treated Older Adolescents: A New Brief Screen (RUFT‐Cut) Using the AUDIT, CAGE, CRAFFT, and RAPS‐QF
Authors: Kelly Thomas M. , Donovan John E. , Chung Tammy , Cook Robert L. , Delbridge Theodore R.
Published: May 2004

Abstract: Background: Early identification of alcohol use disorders (AUD) among emergency department (ED)‐treated patients is important for facilitating intervention and further evaluation outside EDs. A number of brief screening instruments have been developed for identifying patients with AUD, but it is not clear whether they are practical and perform well with older adolescents in an ED setting. This study contrasted four brief screening instruments for detecting DSM‐IV–defined AUD and tested a newly developed brief screen for use among ED‐treated older adolescents.

Methods: The Alcohol Use Disorders Identification Test (AUDIT), the CAGE, the CRAFFT, and a modified RAPS‐QF were given to 93 alcohol‐using older adolescents (55% men; aged 18–20 years) in an ED. Receiver operator characteristic analyses were used to evaluate the performance of brief screens against the criterion of a lifetime DSM‐IV alcohol abuse or dependence diagnosis.

Results: Of existing instruments, the AUDIT had the best overall performance in identifying AUD (sensitivity, 82%; specificity, 78%). A new, shorter screening instrument composed of two AUDIT items, two CRAFFT items, and one CAGE item (RUFT‐Cut) performed as well as the AUDIT (sensitivity, 82%; specificity, 78%).

Conclusions: Among existing alcohol screening instruments, the AUDIT performed best for identifying ED‐treated older adolescents with alcohol use disorders. The RUFT‐Cut is a brief screening instrument for AUD that shows promise for identifying ED‐treated older adolescents who are in need of intervention or further evaluation. Future research should focus on use of the RUFT‐Cut in other settings with larger, more diverse samples of adolescents.
Alanine Aminopeptidase Activity in Urine: A New Marker of Chronic Alcohol Abuse?
Authors: Taracha Ewa , Habrat Boguslaw , Lehner Malgorzata , Wislowska Aleksandr , Woronowicz Bohdan T. , Bogulas Marta , Charewicz Jolanta , Markuszewski Cezary , Plaźnik Adam
Published: May 2004

Abstract: Background: The purpose of this study was to evaluate the activity of urinary alanine aminopeptidase (AAP), the enzyme released from the brush border membranes of renal proximal tubules, as a new biological marker of chronic alcohol abuse.

Methods: The AAP activity was assayed and compared between a group of 76 alcoholics undergoing detoxification and a group of 37 alcoholics abstaining from alcohol for at least 6 weeks. In all patients, the enzyme activity was measured both in untreated urine (uAAP) and after removal of endogenous AAP inhibitors by molecular filtration on Sephadex (eAAP).

Results: There was a correlation between the uAAP and eAAP activities in both groups of patients (r= 0.61 and r= 0.81 in abstinent alcoholics and in alcoholics undergoing detoxification, respectively), and both the uAAP activity and the eAAP activity were significantly and markedly higher in alcoholics being detoxified than in their abstinent counterparts. As revealed by receiver operating characteristic analysis, the discriminative power of the eAAP activity assay was higher than that of the uAAP activity assay. The area under the corresponding receiver operating characteristic curves was 0.84 ± 0.04 and 0.78 ± 0.05 (mean ± SE), respectively.

Conclusions: The results of this study demonstrate that the assays of urinary AAP activity, which relate to the nephrotoxic effects of alcohol abuse, could be a valuable complement to the other presently used markers of chronic alcohol abuse that are generally based on ethanol hepatotoxicity. Compared with the uAAP activity test, the eAAP activity test is of clear diagnostic advantage.
Single‐Channel Analyses of Ethanol Modulation of Neuronal Nicotinic Acetylcholine Receptors
Authors: Zuo Yi , Nagata Keiichi , Yeh Jay Z. , Narahashi Toshio
Published: May 2004

Abstract: Background: We have previously reported that ethanol potentiates the acetylcholine‐induced currents of the α4β2 neuronal nicotinic acetylcholine receptors in rat cortical neurons and of those that are stably expressed in human embryonic kidney cells. The potentiation of the maximal currents evoked by high concentrations of acetylcholine suggests that ethanol affects the channel gating.

Methods: We performed single‐channel patch‐clamp experiments to elucidate the detailed mechanism of ethanol modulation of the α4β2 receptor that is stably expressed in human embryonic kidney cells.

Results: At least two conductance states, 40.5 pS and 21.9 pS, were activated by acetylcholine. Acetylcholine at 30 nM predominantly induced the high conductance state currents (85% of total). Ethanol did not affect the single‐channel conductance but selectively modulated the high‐conductance state currents. The high‐conductance state currents exhibited two open time constants. Both time constants were increased by 100 mM ethanol, from 1.9 msec to 2.8 msec and from 9.0 msec to 15.5 msec, respectively. Ethanol also prolonged the burst duration and the open time within burst and increased the probability of channel opening.

Conclusions: These changes in single‐channel parameters indicate that ethanol stabilizes the α4β2 receptor‐channel in the opening state, explaining how the maximum acetylcholine‐induced whole‐cell currents are further potentiated by ethanol.
Illegal Alcohol Sales to Obviously Intoxicated Patrons at Licensed Establishments
Authors: Toomey Traci L. , Wagenaar Alexander C. , Erickson Darin J. , Fletcher Linda A. , Patrek William , Lenk Kathleen M.
Published: May 2004

Abstract: Background: Early studies assessing propensity of alcohol sales to underage youth found that, before intervention, likelihood of licensed alcohol establishments selling alcohol to underage youth was 50% or higher across many communities. Community‐wide interventions successfully lowered underage alcohol sales rates in several communities. Across studies assessing propensity for alcohol sales to obviously intoxicated patrons, sales rate estimates ranged from 58% to 85% for on‐premise establishments (e.g., bars). No previous studies have assessed likelihood of alcohol sales to obviously intoxicated patrons in off‐premise establishments (e.g., liquor stores). One goal of this study was to assess propensity for illegal alcohol sales to obviously intoxicated patrons at on‐ and off‐premise establishments. Another goal was to assess whether server and/or establishment characteristics were related to the likelihood of illegal sales. Results may inform future interventions to reduce illegal alcohol sales at licensed alcohol establishments.

Methods: Trained actors attempted to purchase alcohol while acting out signs of obvious intoxication at a census of on‐ and off‐premise alcohol establishments (n= 372) in 11 communities. The outcome variable was whether an establishment sold alcohol to a buyer. Independent variables included age and gender of server/clerk, type of establishment, area, exterior maintenance, type of license, and time of purchase attempt. Bivariate and multivariate analyses were conducted.

Results: Seventy‐nine percent of the establishments sold alcohol to a buyer (83% and 76% at off‐ and on‐premise establishments, respectively). Servers/clerks who appeared younger than age 31 and off‐premise establishments were significantly more likely than older appearing servers and on‐premise establishments to sell alcohol to buyers.

Conclusions: The likelihood of alcohol sales to obviously intoxicated patrons in licensed alcohol establishments is very high. Interventions to reduce illegal alcohol sales to intoxicated patrons are needed.
Differential Neurosensitivity to the Discriminative Stimulus Properties of Ethanol in C57BL/6J and C3H/He Mice
Authors: Becker Howard C. , Crissman Alicia M. , Studders Shannon , Kelley Brian M. , Middaugh Lawrence D.
Published: May 2004

Abstract: Background: A large body of evidence suggests that the interoceptive cue associated with ethanol intoxication is complex and dependent on a number of environmental and biological factors. Despite the fact that mice have been widely used to study genetic influences on sensitivity to various actions of ethanol, few studies have used mice to examine sensitivity to the discriminative stimulus effects of ethanol. The purpose of this study was to compare sensitivity to the discriminative stimulus effects of ethanol in two inbred mouse strains, namely C57BL/6J and C3H/He mice.

Methods: Adult male C57BL/6J and C3H/He mice were trained to discriminate between ethanol and saline using a two‐lever food reinforcement operant procedure. Once criterion discrimination performance was achieved, dose‐response functions were determined from generalization tests. Additional experiments were conducted to determine whether differences in discrimination performance were related to differential blood/brain ethanol levels in the two mouse strains.

Results: A greater proportion of C57BL/6J mice acquired the discrimination and required fewer trials to achieve criterion performance compared with C3H/He mice with a 1.0 g/kg ethanol training dose. This deficit in acquisition was overcome when the training dose was increased to 2.0 g/kg for C3H/He mice. In a second experiment, a 1.5 g/kg training dose of ethanol was used for both strains. Again, a greater proportion of C57BL/6J mice acquired the discrimination and required fewer training trials to achieve criterion performance compared with C3H/He mice. Blood ethanol levels did not differ between the strains after administration of the 1.5 g/kg training dose. However, blood and brain ethanol levels did differ between the strains after doses of ethanol were administered that produced equivalent discrimination performance.

Conclusions: Results indicate that ethanol discrimination was more readily acquired and maintained in C57BL/6J mice than C3H/He mice. Ethanol dose‐response functions generated from generalization tests also clearly demonstrated greater sensitivity to the discriminative stimulus properties of ethanol in C57BL/6J mice compared with the C3H/He strain. This differential sensitivity to the interoceptive cue produced by ethanol does not seem to be related to learning or pharmacokinetic differences between the two inbred strains.
A Double‐Blind, Placebo‐Controlled Study of Olanzapine in the Treatment of Alcohol‐Dependence Disorder
Authors: Guardia José , Segura Lidia , Gonzalvo Begoña , Iglesias Lisbeth , Roncero Carlos , Cardús María , Casas Miguel
Published: May 2004

Abstract: Background: A 12‐week, double‐blind, randomized, parallel‐group clinical trial, comparing olanzapine and placebo treatment together with cognitive‐behavioral psychotherapy, was carried out to determine the efficacy, safety, and tolerability of olanzapine in the treatment of alcoholism.

Methods: A total of 60 alcohol‐dependent patients were assigned to 12 weeks’ treatment with either olanzapine or placebo. The primary variable relapse to heavy drinking rate was evaluated by means of intention‐to‐treat analyses. Alcohol consumption, craving, adverse events, and changes in the biochemical markers of heavy drinking and possible toxicity were also evaluated.

Results: We did not find significant differences in the survival analysis between placebo and olanzapine‐treated patients (Kaplan‐Meier log rank = 0.46, df= 1, p= 0.50). Eleven (37.9%) patients treated with olanzapine relapsed compared with 9 (29%) of those receiving placebo (χ2= 0.53, df= 1, p= 0.5). Although some adverse events (weight gain, increased appetite, drowsiness, constipation, and dry mouth) were found more frequently in the olanzapine group, differences did not reach statistical significance in comparison with the placebo group.

Conclusions: Olanzapine was well tolerated, as the rate of adverse events was low, and it was safe, because it did not interfere with the normalization of biochemical markers of heavy drinking or alter liver function markers. Alcohol‐dependent patients showed good adherence and compliance with the treatment protocol, but we found no differences in relapse rate or other drinking variables when comparing olanzapine with placebo‐treated patients.
Fetal Learning With Ethanol: Correlations Between Maternal Hypothermia During Pregnancy and Neonatal Responsiveness to Chemosensory Cues of the Drug
Authors: Abate Paula , Pepino M. Yanina , Spear Norman E. , Molina Juan C.
Published: May 2004

Abstract: Background: Fetuses learn about ethanol odor when the drug is present in the amniotic fluid. Prenatal learning comprising ethanol's chemosensory cues also suggests an acquired association between ethanol's chemosensory and postabsorptive properties. Ethanol‐related thermal disruptions have been implicated as a significant component of the drug's unconditioned properties. In the present study, ethanol‐induced thermal changes were analyzed in pregnant rats subjected to a moderate ethanol dose. This thermal response was later tested for its correlation with the responsiveness of the progeny to ethanol and nonethanol chemosensory stimuli.

Methods: During gestational day (GD) 14, pregnant rats were subjected to a minor surgical procedure to place a subcutaneous telemetric thermal sensor in the nape of the neck. During GDs 17 to 20, females received a daily intragastric administration of ethanol (2 g/kg) or water, using solutions kept at room temperature. Maternal body temperatures were recorded before and after (4 consecutive hours) the administration of water or ethanol. Newborns representative of both prenatal treatments were tested in terms of behavioral activity elicited by the smell of ethanol or of a novel odorant (cineole). A third group of pups were tested in response to unscented air stimulation.

Results: Ethanol administration during late gestation induced reliable maternal hypothermia, a thermal disruption greater than that observed in water‐treated females. It was systematically observed that maternal ethanol‐induced hypothermia negatively correlated with neonatal motor reactivity elicited by ethanol olfactory stimulation. No other significant correlations were observed in terms of responsiveness to cineole or to unscented air in animals prenatally exposed to ethanol or water.

Conclusions: In conjunction with prior research, the present results indicate that fetal ethanol exposure may yield learning of an association between ethanol's sensory and unconditioned properties. Ethanol‐induced hypothermia during late gestation seems to represent a significant component of ethanol's unconditioned consequences. Specifically, ethanol‐related thermal disruptions in the womb are highly predictive of neonatal responsiveness to ethanol's chemosensory cues that are known to be processed by the near‐term fetus.
Influence of Ethanol Dependence and Methionine Enkephalin Antisense on Serum Endomorphin‐1 and Methionine Enkephalin Levels
Authors: Banks William A. , Kumar Vijaya B. , Morley John E.
Published: May 2004

Abstract: Background: Opiate peptides are involved in the physical dependence on ethanol. Levels of methionine enkephalin (MEnk), for example, are affected by ethanol. No study on the effect of ethanol on endomorphin, the endogenous ligand for the μ‐opiate receptor, has yet been conducted.

Methods: We examined the effect of ethanol ingestion on serum endomorphin (EM)‐1 and MEnk levels. We also determined the effect of antisense directed at MEnk on serum levels of EM‐1 and MEnk.

Results: Serum EM‐1 levels steadily decreased about 20% during 56 days of ethanol ingestion in liquid feed, whereas a similar decrease in serum MEnk levels was not statistically significant. Serum MEnk levels decreased about 20% by 48 hr after antisense injection and then returned to baseline, whereas serum EM‐1 levels increased by about 80% and remained elevated for about 2 weeks. In mice not treated with antisense or alcohol, there was no correlation between the serum levels of EM‐1 and MEnk.

Conclusions: These results show that serum levels of EM‐1 are decreased by physical dependence on ethanol and that this effect is not directly mediated through MEnk.