bluuebunny/crossref_metadata_2025 huggingface dataset:
Noradrenergic Modulation of Methamphetamine‐Induced Striatal Dopamine Depletion
Authors: FORNAI FRANCESCO , ALESSANDRÌ MARIA GRAZIA , TORRAC MARIA TILDE , BASSI LUCIA , SCALORI VERA , CORSINI GIOVANNI UMBERTO
Published: February 2006

ABSTRACT: Noradrenergic (NE) neurons belonging to the locus coeruleus (LC), much more than the A1 and A2 NE areas, are lost in Parkinson's disease (PD). In this study, we reproduced the selective pattern of NE loss involving axons arising from the LC using the selective neurotoxin N‐(‐2‐chloroethyl)‐N‐ethyl‐2‐bromobenzylamine (DSP‐4) (50 mg/kg). In these experimental conditions, we investigated whether NE loss potentiates methamphetamine‐induced striatal dopamine (DA) depletion in mice and in rats. Administration of a moderate dose of methamphetamine to C57B1/6N mice or Sprague‐Dawley rats produced only a partial striatal DA depletion 7 days after drug administration. Pretreatment with DSP‐4, in both animal species, significantly enhanced methamphetamine‐induced striatal DA depletion. Administration of a lower dose of methamphetamine did not decrease striatal DA levels when injected alone, but produced a significant decrease in striatal DA when given to DSP‐4‐pretreated rodents. Moreover, we found that agents reducing the noradrenergic activity (i.e., the alpha‐2 agonist clonidine) enhanced, whereas alpha‐2 antagonists decreased, methamphetamine toxicity. Enhancement of methamphetamine toxicity did not occur if the noradrenergic lesion was produced 12 hr after methamphetamine administration. By contrast, exacerbation of methamphetamine toxicity in NE‐depleted animals was accompanied by increased extracellular DA levels measured with brain dialysis and by a more severe acute DA depletion measured in striatal homogenates.
The Effects of Cocaine on Nonhuman Primate Brain Function Are Age Dependenta
Authors: PAULE MERLE G. , GILLAM MICHAEL P. , MORRIS PAUL
Published: February 2006

ABSTRACT: The effects of acute intravenous (i.v.) cocaine (COC) on several complex brain functions were studied in rhesus monkeys at 1.5, 3 and 10–11 years of age. Subjects performed several operant tasks (for food) that were used to model learning, short‐term memory, color and position discrimination, and motivation, and disruption of performance of these tasks was used to quantitate drug effect. Drug effects were age dependent: The youngest subjects were 3 to 10 times less sensitive than the oldest. Presuming the observed behavioral effects of cocaine were caused primarily via its interaction with dopamine (DA) systems, changes in sensitivity to its effects with age are likely a reflection of the functional status of the DA system. These data, along with preliminary data on levels of DA transporters, suggest that the age‐related differences in sensitivity to cocaine lie in, or ‘downstream’ from, the dopamine receptor.
In Vivo Ibogaine Blockade and In Vitro PKC Action of Cocainea
Authors: ONAIVI EMMANUEL S. , ALI SYED F. , CHAKRABARTI AMITABHA
Published: February 2006

ABSTRACT: Ibogaine may have antiaddiction potential against alcohol, psychostimulant and opiate abuse, but its mechanism of action is unclear. Ibogaine, however, has been demonstrated in numerous studies to have effects in multiple central nervous system (CNS) neurotransmitters systems. We are using in vitro and in vivo systems to study the effects of cocaine and whether these effects can be blocked by ibogaine. For the In Vivo studies, we first determined the acute and subacute effects of ibogaine (1–5.0 mg/kg) in mice using the plus‐maze test. Acutely increasing doses of ibogaine produced a reduced aversion to the open arms. The subacute administration provoked a variable response which was characterized by fluctuations in aversive and antiaversive behavior of the animals to the open arms of the plus‐maze during the 14‐day treatment period. A separate group of mice received 1.0 mg/kg cocaine for 14 days, and upon abrupt cessation from cocaine treatment, ibogaine 2.5 mg/kg was administered to a subgroup of these mice. Ibogaine reversed the withdrawal aversions produced by the abrupt cessation from cocaine administration. For the in vitro studies, the expression and activity of protein kinase C (PKC) isoforms and Ca2+ levels were examined following the incubation of PC 12 cells with cocaine. This is because PKC plays a key role in a number of cellular and neuronal functions. We report that cocaine disrupts signal transduction in PC 12 cells by altering the expression and activity of PKC isoforms and Ca2+ levels. The data obtained suggest (1) that the PC 12 cells may be useful in studying the neurobiology of abused drugs, like cocaine in vitro, (2) that if anxiety is a factor in drug dependency, then the antiaddictive property of ibogaine In Vivo may be associated with modifying the CNS neurotransmission that may be involved in anxiety. It remains to be determined whether the signaling involving PKC is important in the antiaddictive properties of ibogaine.
Effect of Ibogaine on the Various Sites of the NMDA Receptor Complex and Sigma Binding Sites in Rat Braina
Authors: ITZHAK YOSSEF , ALI SYED F.
Published: February 2006

ABSTRACT: Although the alkaloid ibogaine is a potent hallucinogenic agent some indications suggest that it may be useful for the treatment of opioid and cocaine addiction. The neurochemical mechanism(s) underlying ibogaine effects remain unclear. In the present study we investigated the interaction of ibogaine with the phencyclidine (PCP) site located in the ionophore of the N‐methyl‐d‐aspartate (NMDA) receptor complex, with the NMDA receptor binding site, and with sigma binding sites. In well‐washed membrane preparations of rat cortex and cerebellum, the PCP sites were labeled with [3H]MK‐801 or [3H]1‐[1(2‐theinyl)‐cyclohexyl]‐piperidine ([3H]TCP), and the NMDA receptor with [3H]‐CGP 39653. The sigma‐1 and sigma‐2 binding site in rat cortex and cerebellum were labeled with [3H]pentazocine and [3H]1,3‐di‐o‐tolyl‐guanidine ([3H]DTG), respectively. Results indicated that ibogaine interacts with high‐ and low‐affinity PCP binding sites in the cortex: Ki(H) = 0.01–0.05 μM; Ki(L) = 2–4μM, and only with low‐affinity sites in the cerebellum: Ki = 2–4, μM. In contrast, ibogaine (>100 μM) had no affinity for [3H]‐CGP 39653 binding sites (cortex and cerebellum). The affinity of ibogaine for sigma‐1 and ‐2 binding sites in cortex and cerebellum ranged from 1.5–3μM. Since NMDA receptor antagonists (e.g., MK‐801) are thought to attenuate opioid withdrawal symptoms and cocaine sensitization, it is possible that binding of ibogaine to the PCP sites contributes to its potential ‘endabuse’ properties. In turn, ibogaine interaction with sigma binding sites may be associated with its adverse effects.
Neurochemical and Neuroendocrine Effects of Ibogaine in Rats: Comparison to MK‐801
Authors: BAUMANN MICHAEL H. , ROTHMAN RICHARD B. , ALI SYED F.
Published: February 2006

ABSTRACT: Ibogaine (IBO) is a naturally‐occurring indole compound that is being evaluated as a potential medication for substance use disorders. Although the precise mechanism of IBO action is unclear, recent in vitro data show this drug displays properties similar to the noncompetitive N‐methyl‐d‐aspartate (NMDA) antagonist MK‐801. The purpose of the present work was to compare In Vivo neurobiological effects of IBO and MK‐801 in rats. Groups of male rats (n = 6–8/group) were decapitated 30 and 60 min after receiving intraperitoneal (i.p.) IBO (10 & 100 mg/kg), MK‐801 (0.1 & 1.0 mg/kg) or vehicle. Trunk blood was collected for the analysis of plasma prolactin and corticosterone; brains were harvested and dissected for determination of dopamine (DA), serotonin (5‐HT) and their metabolites. Both IBO and MK‐801 increased corticosterone secretion, but only IBO elevated plasma prolactin. IBO produced dramatic reductions in tissue DA levels with concurrent increases in the metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). This profile of IBO‐induced changes in DA transmission was observed in the striatum, olfactory tubercle, and hypothalamus. The effects of MK‐801 on DA metabolism did not mimic IBO, as MK‐801 tended to increase DA and its metabolites. Neither drug appreciably affected 5‐HT systems. Our results suggest that the effects of IBO on neuroendocrine function and DA transmission are not due to MK‐801‐like properties of IBO. Thus, the In Vivo mechanism of IBO action cannot be explained simply on the basis of antagonism at NMDA receptors.
Alteration in Electroencephalogram and Monoamine Concentrations in Rat Brain following Ibogaine Treatment
Authors: BINIENDA ZBIGNIEW , BEAUDOIN MICHAEL A. , THORN BRETT T. , PRAPURNA D. REBECCA , JOHNSON JOHN R. , FOGLE C. MATTHEW , SLIKKER WILLIAM , ALI SYED F.
Published: February 2006

ABSTRACT: Ibogaine (IBO) is a psychoactive indole alkaloid that has antiaddictive properties. However, treatment with IBO may lead to neurotoxicity, since IBO and its metabolites interact persistently with many neurotransmitter systems. Here, we recorded cortical electroencephalogram (EEG) signals from rats anesthetized with isoflurane. The heart rate (HR) was monitored via electrocardiogram (EKG) electrodes. After the baseline EEG was recorded, rats received one intraperitoneal (i.p.) dose of 50 mg/kg IBO. EEG signals were recorded for 2 hr. Rats were then sacrificed and brains dissected into frontal cortex (FC), caudate nucleus (CN), hippocampus (HIP), and brain stem (BS). The level of dopamine (DA), serotonin (5‐HT), and their metabolites were determined by high‐performance liquid chromatography with electrochemical detection (HPLC‐ECD). Compared with baseline, a decrease in HR immediately after IBO injection and a decrease in δ, θ, α, and β power spectra frequency bands (1–4, 4–8, 8–13, 13–32Hz) during the first 30 min after IBO administration was observed. EEG recovered within the next 15 min. In CN, the level of DA decreased and DA turnover rate increased significantly. The levels of 5‐HT increased in FC. The pattern of EKG and EEG response to IBO may be due to multiple receptor interactions of IBO.
Repeated Neonatal Phencyclidine Treatment Impairs Performance of a Spatial Task in Juvenile Ratsa
Authors: SIRCAR RATNA , RUDY JERRY W.
Published: February 2006

ABSTRACT: The present study was designed to determine whether repeated postnatal blockade of N‐methyl‐d‐aspartate (NMDA) channel produces cognitive deficit in juvenile rats. Rats received phencyclidine (PCP) intraperitoneally (i.p.) from postnatal Day 5 and continued daily till Day 15. On Day 28, saline‐ and PCP‐treated rats were trained in the Morris water maze task. PCP‐treated rats performed as well as the saline‐treated rats on the first day of testing, but on the second day of testing they did significantly poorly compared to saline‐treated controls. These data suggest that chronic postnatal NMDA channel blockade by PCP impairs processes that enable rats to retain spatial information.
Uterine Position Determines the Extent of Dopamine Reduction after Chronic Prenatal Cocaine Exposure
Authors: LIPTON JACK W. , ROBIE HEATHER S. , LING ZAODUNG , WEESE‐MAYER DEBRA E. , CARVEY PAUL M.
Published: February 2006

ABSTRACT: Most studying the consequences of prenatal cocaine (COC) exposure employ rodents or other multiparous organisms in their models. We have previously shown that when pregnant Sprague‐Dawley albino rats are administered a 30 mg/kg subcutaneous (s.c.) injection on embryonic day 15 (E15), fetal brain COC levels show a proximal‐to‐distal (in relation to the cervix) gradient that can vary by as much as 350%. The present study sought to determine whether this gradient translated into a similar gradient in brain dopamine (DA) levels. Pregnant rats were administered COC or saline (SAL) (30 mg/kg COC or 1 ml/kg SAL, b.i.d., E7‐E19). On E20, dams were anesthetized with halothane, the fetuses immediately removed, their brains excised, frozen and subsequently processed for DA, dihydroxyphenylacetic acid (DOPAC) or homovanillic acid (HVA). High‐performance liquid chromatography (HPLC) analysis revealed a proximal‐to‐distal gradient for DA in both COC‐ and SAL‐exposed fetuses. Average fetal DA levels per litter were significantly lower in COC‐exposed litters (57.39 ± 3.67 ng/hemibrain SAL; 48.29 ± 3.87 ng/hemibrain COC F7,1 = 11.66, p <0.05). The gradients for DA were in opposite directions such that COC litters showed the lowest levels of DA in the most distal uterine positions, whereas SAL‐exposed litters showed the highest DA levels in the same location. These data suggest that a gradient in brain dopamine normally exists for fetuses based upon uterine position, and that cocaine can have selectively greater effects on this level as a function of fetal location.
The Effects of Gestational Cocaine Exposure on Pregnancy Outcome, Postnatal Development, Cognition and Locomotion in Ratsa
Authors: CHOI SUN‐JO , MAZZIO ELIZABETH , SOLIMAN KARAM F.A.
Published: February 2006

ABSTRACT: Pregnant female Sprague‐Dawley rats were injected with either 40 mg/kg (−)cocaine hydrochloride or an equivalent volume of 0.9% saline, subcutaneously (s.c.), once daily from gestational day 12 (GD 12) to GD 21. Gestational cocaine exposure had no effect on maternal weight gain, length of gestation, birthweight, fetal mortality, postnatal weight gain or locomotive activity in offspring. There was an unusual reduction in the male/female offspring ratio in the treated group (1:0.65) verses controls (1:1.04) (p <0.01). Male offspring scored significantly lower on memory and learning tasks on postnatal day 30 (PND30) as determined by the water maze test (p <0.01). Exposure to cocaine in utero had no effect on postnatal female sexual maturation or cognitive function. The present study indicates that gestational cocaine exposure can lead to cognitive impairment selectively in male offspring, without any apparent postnatal physical abnormalities or adverse effects on maternal health status.
Gestational Cocaine Exposure Alters Postnatal Pituitary‐Adrenal Axis Activity and Stress Endurance in Ratsa
Authors: CHOI SUN‐JO , MAZZIO ELIZABETH A. , REAMS RENEE R. , SOLIMAN KARAM F.A.
Published: February 2006

ABSTRACT: Female pregnant Sprague‐Dawley rats were injected once daily with 40 mg/kg cocaine hydrochloride or 0.9% saline from gestational day 12 (GD 12) to GD 21. From postnatal day 21 (PND 21) to PND 60, both male and female offspring were examined for stress response. Treated male and female offspring demonstrated a diminished tolerance to stress as determined by a cold water stress test performed at PND 21, 30 and 40. Base hormonal levels of adrenocorticotropin hormone (ACTH) and corticosterone were not affected by prenatal cocaine exposure in male offspring at PND 30. However, immobilization for 1 hr caused a significant sustained elevation of corticosterone levels at both PND 30 and PND 60 in male treated offspring as compared to the control group. Plasma ACTH levels were also significantly sustained after 1 hr of immobilization at PND 60 for the cocaine‐treated male offspring. These results indicate both a diminished capacity to respond to stress and an abnormal heightened reactivity of the pituitary‐adrenal axis in offspring exposed to cocaine in utero.
Repeated Administration of Cocaine Alters Dopamine Uptake and Release in the Striatum Nucleus Accumbensa
Authors: CLAY LYNNE H. , MAZZIO ELIZABETH A. , KOLTA MALAK G. , SOLIMAN KARAM F.A.
Published: February 2006

ABSTRACT: Male Sprague‐Dawley rats were administered 25 mg/kg, intraperitoneally (i.p.) cocaine‐HCl twice daily for 14 consecutive days (total of 50 mg/kg), while control animals received an equivalent volume of 0.9% saline. After three days of withdrawal, the animals were sacrificed for dissection of striatal (STR) and nucleus accumbens (NA) brain regions. The treated group demonstrated a dose‐dependent reduction for in vitro cocaine inhibition of [3H]dopamine (DA) uptake in the NA tissue verses controls. There were no significant differences amongst the treated and control groups for in vitro cocaine inhibition of [3H]DA in the STR. In vitro d‐amphetamine (1, 5 and 10 μM)‐stimulated DA release from STR tissue was not significantly different between the treated and the control groups. However, there was a significant decline in basal STR DA release and a significant enhancement of d‐amphetamine (1 and 5 μM)‐stimulated DA release in the NA for the treatment group versus controls. The results from the present study indicates sensitization to cocaine is primarily related to DA uptake and release in the NA.
Discovery of Phospholamban: A Personal History
Authors: KATZ ARNOLD M.
Published: February 2006

ABSTRACT: Early efforts to identify mechanisms by which sympathetic stimulation increases myocardial contractility led to studies of effects of β‐adrenergic agonists and cyclic AMP on the cardiac contractile proteins and sarcoplasmic reticulum (SR); initial positive reports, however, could not be confirmed. The discovery that cyclic AMP‐dependent protein kinases (PK‐A) mediated intracellular actions of cyclic AMP led at least four groups to test the hypothesis that phosphorylation of the cardiac SR played a role in the actions of β‐adrenergic agonists. Three of them (Wollenberger, Wray et al., and LaRaia & Morkin) demonstrated that cardiac SR was a substrate for PK‐A phosphorylation; however, the lability of the Ca2+ pump in these membranes made it difficult to demonstrate a functional significance of this finding. Our group, which had extensive experience in measuring SR Ca2+ transport, began by examining the ability of PK‐A to activate the SR Ca2+ pump “poised” at half‐saturating Ca2+ concentrations. Our initial positive result led to the discovery that a 22,000‐dalton protein, named phospholamban by Phyllis B. Katz, mediated effects on Ca2+ transport by the SR that could explain both the inotropic and lusitropic effects of sympathetic stimulation.
Comparative Ultrastructure of Ca2+ Release Units in Skeletal and Cardiac Muscle
Authors: FRANZINI‐ARMSTRONG CLARA , PROTASI FELICIANO , RAMESH VENKAT
Published: February 2006

ABSTRACT: The sarcoplasmic reticulum (SR) of striated muscle fibers interacts with exterior membranes (surface membrane and transverse tubules) to form junctions that are involved in the internal release of calcium during excitation‐contraction coupling. Release of calcium through the ryanodine receptors (RyRs) or calcium release channels of the SR is under the control of the L type calcium channels or dihydropyridine receptors (DHPRs) of exterior membranes. Interacting clusters of the two proteins constitute calcium release units. The cytoplasmic domains of RyRs are visible as large electron‐dense structures (the feet) with four identical subunits in the junctional gap separating SR from exterior membranes. In freeze‐fracture replicas of skeletal muscle, large intramembrane particles are grouped into clusters of tetrads in the exterior membranes, and the tetrads are located in correspondence of the four subunits of the feet. Lack of tetrads in dysgenic muscle fibers with a null mutation for DHPRs and appearance of the tetrads after transfection with cDNA for DHPR indicate identity of tetrads with four DHPRs. In cardiac muscle, DHPRs are located at the sites of SR‐surface junctions, but they are not grouped into tetrads. This is consistent with a possible direct DHPR‐RyR interaction in skeletal but not in cardiac muscle. The size and distribution of SR‐surface junctions in skeletal and cardiac muscles provide further clues to their function.
Structural Studies on Phospholamban and Implications for Regulation of the Ca2+‐ATPase
Authors: MORTISHIRE‐SMITH RUSSELL J. , BROUGHTON HOWARD , GARSKY VICTOR M. , MAYER ERNEST J. , JOHNSON ROBERT G.
Published: February 2006

ABSTRACT: The cardiac sarcoplasmic reticulum (SR) protein phospholamban (PLB) is an endogenous inhibitor of the SR Ca2+‐ATPase. Phosphorylation of PLB relieves this inhibition and up‐regulates calcium transport. PLB has proved remarkably difficult to study by conventional solution‐state nuclear magnetic resonance (NMR) methods, due primarily to the extreme hydrophobic nature of the protein and its propensity to form pentamers. That the C‐terminal domain of PLB is helical and membrane spanning is now well established; the structure of the cytoplasmic domain is relatively ill defined. In order to discern the effect of phosphorylation on the structure of the cytoplasmic domain, we have characterized a variety of model peptides in several structure‐inducing and/or lipid‐mimicking environments using circular dichroism and solution‐state NMR. The resolution of peptide structures obtained in aqueous trifluoroethanol was markedly improved by the incorporation of 15 N labels into the peptide backbone, allowing a variety of isotope edited, filtered, and resolved techniques to be applied. Molecular dynamics simulations on the full‐length protein were combined with an analysis of published data to suggest a revised model for the structure of PLB.
Phosphorylation States of Phospholambana
Authors: COLYER JOHN
Published: February 2006

ABSTRACT: Phospholamban is a small integral membrane protein of cardiac, smooth, and slow‐twitch skeletal muscle sarcoplasmic reticulum that interacts with the Ca2+ pump of these organelles and inhibits Ca2+‐pump activity while in the dephosphorylated form. Three sites of Ser/Thr phosphorylation have been identified in the primary sequence of phospholamban, at Ser‐10, Ser‐16, and Thr‐17. In vitro studies indicate that these residues are phosphorylated by PKC (Ser‐10), PKA, PKG or PKC (Ser‐16), and CaM kinase II (Thr‐17). Phosphorylation of Ser‐16 (or Thr‐17) is accompanied by an increase in Ca2+ pump activity in direct proportion to the stoichiometry of phosphorylation. Dual phosphorylation of both Ser‐16 and Thr‐17 does not cause any further stimulation of pump function over that achieved by stoichiometric phosphorylation of a single site. Examination of the pattern of phosphorylation in vivo has been aided by the generation of polyclonal antibodies specific for the phosphorylated forms of phospholamban. β‐Adrenergic stimulation of cardiac muscle results in phosphorylation of both Ser‐16 and Thr‐17. The time course of Ser‐16 phosphorylation precedes Thr‐17. The spatial distribution of Ser‐16 and Thr‐17 phosphorylated forms of phospholamban is not identical; phospholamban located in the nuclear membrane of a cardiac myocyte is phosphorylated exclusively on Ser‐16, whereas phospholamban molecules in the SR membrane of the same cell are phosphorylated on Ser‐16 and/or Thr‐17. Finally, we have identified a novel stimulus for the phosphorylation of phospholamban. Ca2+ store depletion, achieved by exposure of myocytes to SERCA inhibitors, prompts the phosphorylation of phospholamban on Ser‐16. This would be expected to increase Ca2+ uptake by the SR in an attempt to achieve the refilling of the SR.
High‐Level Coexpression of the Canine Cardiac Calcium Pump and Phospholamban in Sf21 Insect Cells
Authors: AUTRY JOSEPH M. , JONES LARRY R.
Published: February 2006

ABSTRACT: Phospholamban is a pentameric transmembrane phosphoprotein that regulates the activity of the Ca2+‐transporting ATPase (SERCA2a) in cardiac sarcoplasmic reticulum. To better understand the structure and function of phospholamban and its mode of regulation of the ATPase, phospholamban and SERCA2a were coexpressed at high levels in Sf21 insect cells using the baculovirus expression system. SERCA2a was expressed as a functionally active Ca2+ pump, accounting for ≥ 20% of the total protein in Sf21 cell microsomes. Wild‐type phospholamban, as well as phospholamban with different point mutations in the transmembrane region, inhibited both Ca2+ transport and ATP hydrolysis by the recombinant Ca2+ pump. The inhibition of SERCA2a activity was reversed by an anti‐phospholamban monoclonal antibody. The phospholamban molecules studied decreased the apparent Ca2+ affinity of the Ca2+ pump, but had no effect on enzyme velocity measured at saturating Ca2+ concentration. Monomeric phospholamban produced by mutations in the leucine/isoleucine zipper domain decreased the apparent Ca2+ affinity the most, giving stronger inhibition of the Ca2+ pump than even wild‐type phospholamban. Thus, the baculovirus cell expression system is ideally suited for examining functional interactions between phospholamban and SERCA2a. The results obtained suggest that the phospholamban monomer may be the active species inhibiting the Ca2+ pump in the cardiac sarcoplasmic reticulum membrane.
Co‐reconstitution and Co‐crystallization of Phospholamban and Ca2+‐ATPase a
Authors: YOUNG HOWARD S. , REDDY LAXMA G. , JONES LARRY R. , STOKES DAVID L.
Published: February 2006

Significant advances have recently been made in understanding the regulation of Ca2+‐ATPase by phospholamban and in modeling their structures. However, these insights would be furthered by determining the 3‐D structure of both proteins within the membrane, thus revealing the structural basis for their interaction. To this end, we have developed methods for reconstituting purified Ca2+‐ATPase with recombinant phospholamban. After reconstitution at high lipid‐to‐protein ratios, we have verified their functional association by measuring calcium transport and ATPase activity. Furthermore, we have grown co‐crystals after reconstitution at low lipid‐to‐protein ratios. The structure of Ca2+‐ATPase has recently been solved by cryoelectron microscopy at 8‐Å resolution, thus revealing transmembrane α‐helices. Using a variety of constraints, we have associated these helices with the predicted transmembrane sequences to produce a detailed model for the packing of transmembrane helices. Structure determination of the co‐crystals is currently underway, which we hope will eventually reveal the interaction of phospholamban with Ca2+‐ATPase at a similar level of detail.
Potential for Pharmacology of Ryanadine Receptor/Calcium Release Channelsa
Authors: TRIPATHY LE XU ASHUTOSH , PASEK DANIEL A. , MEISSNER GERHARD
Published: February 2006

ABSTRACT:Calcium release channels, known also as ryanodine receptors (RyRs), play an important role in Ca2+signaling in muscle and nonmuscle cells by releasing Ca2+from intracellular stores. Mammalian tissues express three different RyR isoforms comprising four 560‐kDa (RyR polypeptide) and four 12‐kDa (FK506 binding protein) subunits. The large protein complexes conduct monovalent and divalent cations and are capable of multiple interactions with other molecules. The latter include small diffusible endogenous effector molecules including Ca2+, Mg2+, adenine nucleotides, sufhydryl modifying reagents (glutathione, NO, and NO adducts) and lipid intermediates, and proteins such as protein kinases and phosphatases, calmodulin, immunophilins (FK506 binding proteins), and in skeletal muscle the dihydropyridine receptor. Because of their role in regulating intracellular Ca2+levels and their multiple ligand interactions, RyRs constitute an important, potentially rich pharmacological target for controlling cellular functions. Exogenous effectors found to affect RyR function include ryanoids, toxins, xanthines, anthraquinones, phenol derivatives, adenosine and purinergic agonists and antagonists, NO donors, oxidizing reagents, dantrolene, local anesthetics, and polycationic reagents.
Factors That Control Sarcoplasmic Reticulum Calcium Release in Intact Ventricular Myocytesa
Authors: BERS DONALD M. , LI LI , SATOH HIROSHI , McCALL EILEEN
Published: February 2006

ABSTRACT: Much has been discovered studying sarcoplasmic reticulum (SR) Ca release channels in SR vesicles and lipid bilayers. We have focused on how SR Ca release is regulated in intact mammalian ventricular myocytes, using fluorescent Ca indicators, voltage clamp, and confocal microscopy. Three major factors appear to contribute to the probability of spontaneous localized SR Ca release events (or Ca “sparks”) in resting myocytes: (1) cytosolic [Ca], (2) SR Ca content, and (3) time after previous activity (i.e., recovery from adapted or inactivated state). These same three factors function during excitation‐contraction (E‐C) coupling and can explain rest potentiation of twitches, increased fractional SR Ca release at higher SR Ca loads, and Ca overload. Since SR Ca release is sensitive to both ICa and SR Ca load, we have controlled (and measured) these parameters. At constant SR Ca load and ICA in intact cells we have found that SR Ca release is increased by Ca‐calmodulin‐dependent protein kinase (CaMKII) and FK506 (which may interfere with the interaction between the Ca release channel and the FK binding protein) and is reduced by the Ca channel agonist Bay K 8644, CaMKII inhibitors, and during ventricular hypertrophy. Thus the regulation of the SR Ca release channel in the intact cell is an important factor in cellular cardiac function.
Alterations in Heart Failure of Cyclic Amp‐Dependent Inotropic and Lusitropic Properties of Cardiac and Skeletal Muscle
Authors: BISHOP AMY , TRAVERS KERRY E. , GROSSMAN JESSICA , JOHNSON HEATHER , PERREAULT CYNTHIA , WOOLF JOHN H. , CITTADINI ANTONIO , GONZALES‐SERRATOS HUGO , MORGAN JAMES P
Published: February 2006

ABSTRACT: A central working hypothesis in our laboratory is that deficient cellular cyclic AMP concentrations may be responsible, at least in part, for striated muscle dysfunction, both cardiac and skeletal, in heart failure. These results suggest that therapy aimed at restoring cyclic AMP to normal levels may be effective with regard to improving systolic and diastolic function in the heart and may decrease the development of fatigue in skeletal muscle of patients with failure. The use of cyclic AMP‐dependent drugs in clinical practice has been limited by side effects associated with raising total cellular content of this cyclic nucleotide. However, evidence suggesting that separate pools of cyclic AMP may exist within the cell raises the possibility that those pools associated with excitation/contraction coupling could serve as more specific therapeutic targets.
cAMP‐Mediated Signal Transduction and Sarcoplasmic Reticulum Function in Heart Failure
Authors: MOVSESIAN MATTHEW A.
Published: February 2006

ABSTRACT: There is evidence that the effects of β‐adrenergic receptor agonists on myocardial contractility result principally from the phosphorylation of phospholamban by cAMP‐dependent protein kinase and the consequent deinhibition of SERCA2 activity and stimulation of sarcoplasmic reticulum Ca2+ transport. An impairment in β‐adrenergic receptor‐stimulated cAMP generation, attributable to down‐regulation of β1‐adrenergic receptors and increased activity of Gαi and G protein‐coupled receptor kinase, has long been recognized in failing human myocardium. This impairment is associated with a compartment‐specific decrease in sarcoplasmic reticulum cAMP content that may selectively reduce phospholamban phosphorylation. Published and preliminary results indicate that two plausible explanations for this compartment‐specific decrease‐a reduction in sarcoplasmic reticulum‐associated cAMP‐dependent protein kinase or an increase in sarcoplasmic reticulum‐associated cAMP phosphodiesterase‐are unlikely. Instead, there is reason to believe that the selective reduction in β1‐adrenergic receptor density in failing myocardium is causally related to this compartment‐specific decrease in cAMP content through an as‐yet‐undetermined mechanism. The fact that the modulation of SERCA2 activity by phospholamban is preserved in failing human myocardium offers an opportunity for improvement in the therapy of heart failure.
Pharmacology of the Cardiac Sarcoplasmic Reticulum Calcium ATPase‐Phospholamban Interaction
Authors: JOHNSON ROBERT G.
Published: February 2006

ABSTRACT: Accumulating evidence points to the critical role of phospholamban (PLB) regulation of the cardiac sarcoplasmic reticulum (SR) calcium ATPase in influencing the kinetics of calcium handling within the cardiac myocyte under normal and pathological conditions. Based on the data, it has been hypothesized that PLB inhibitors (e.g., calcium ATPase stimulators) would be of potential importance as positive lusitropes and inotropes in the treatment of heart failure. Experiments measuring tension transients in saponin‐permeabilized cardiac muscles from genetically engineered mice under a variety of SR calcium loading conditions provide evidence of the functional alterations that can be achieved by manipulation of the degree of PLB inhibition of the calcium pump. Testing of the above hypothesis will ultimately require a selective, high‐affinity, membrane‐permeable small molecule stimulator of the cardiac calcium pump. Screening for cardiac calcium pump activators has produced a series of agents exerting apparently different mechanisms of action; some may be tools to help to elucidate the nature of the PLB‐calcium ATPase interaction(s). The rationale for PLB as a drug target, the optimal profile of a PLB inhibitor, and the properties of several low‐molecular‐weight compounds will be explored.
Interleukin‐1β, Interleukin‐18, and the Interleukin‐1β Converting Enzymea
Authors: DINARELLO CHARLES A.
Published: February 2006

ABSTRACT: When injected intravenously into humans and animals, interleukin‐1β (IL‐1β) is perhaps the most potent of the endogenous pyrogens. However, IL‐1β is initially synthesized as a relatively inactive precursor molecule (proIL‐1β) which lacks a signal peptide and hence remains inside the cell. To be active as a fever‐producing molecule, proIL‐1β must first be processed to an active mature molecule and secreted. Although several enzymes associated with inflammatory tissues are capable of processing proIL‐1β into an active molecule in the extracellular compartment, the IL‐1β converting enzyme (ICE, also called caspase‐1) cuts intracellular proIL‐1β after the aspartic acid residue in position 116, resulting in a highly active mature IL‐1β that is secreted into the extracellular space. IL‐18 is also initially synthesized as an inactive precursor molecule (proIL‐18) lacking a signal peptide. IL‐18 is a member of the IL‐1 family, and like IL‐1β, proIL‐18 is cleaved by ICE to yield an active molecule. However, unlike IL‐1β, IL‐18 is not an endogenous pyrogen following intraperitoneal injection into mice. Nevertheless, IL‐18 may contribute to inflammation and fever because IL‐18 is a potent inducer of tumor necrosis factor, chemokines, and interferon‐γ production.
Interleukin‐11: A gp130 Cytokine
Authors: TREPICCHIO WILLIAM L. , DORNER ANDREW J.
Published: February 2006

ABSTRACT: Interleukin (IL)‐11 is a member of the gp130 family of cytokines. Comparison of IL‐11 with another gp130 family member, IL‐6, indicates that these two cytokines share many overlapping signal transduction mechanisms. However, unlike IL‐6, treatment of patients with a recombinant human form of IL‐11 (rhIL‐11) does not increase body temperature, suggesting significant differences in the in vivo function of these two molecules. Recent studies demonstrate that IL‐11 has potent anti‐inflammatory activity in a variety of preclinical animal models of disease. rhIL‐11 reduces production of proinflammatory mediators such as tumor necrosis factor‐α and IL‐12 from activated macrophages. This effect on proinflammatory cytokine production is mediated at the transcriptional level by inhibition of the transcription factor, NF‐ΚB. To further understand the anti‐inflammatory mechanisms of action of rhIL‐11 and to elucidate differences between IL‐11 and IL‐6 signal transduction pathways, the effects of these two cytokines on in vitro macrophage function were compared.
Immunomodulatory Properties of Interferon‐γ: An Updatea
Authors: BILLIAU ALFONS , HEREMANS HUBERTINE , VERMEIRE KURT , MATTHYS PATRICK
Published: February 2006

ABSTRACT:During the early aspecific phase of host defense, production of interferon (IFN)‐γ by natural killer cells plays an important role in bringing about acute inflammation, mainly because of the activating effects of IFN‐γ on adhesive properties of endothelial cells and on mediator production by mononuclear phagocytes (MPCs). In the subsequent antigen‐specific phase of the immune response, IFN‐γ acts as a regulator of antigen presentation and of proliferation and differentiation of lymphocyte populations. Immunosuppressive as well as immunostimulatory effects may result from these actions.

High‐level production of IFN‐γ during this phase of host defense is now classically seen as a hallmark of a T‐helper 1 (TH1)‐type reaction, characterized by activation of antimicrobial activity of macrophages and by inflammatory reactions with a DTH character. Development of TH1‐type lymphocyte populations producing IFN‐γ is regulated by other cytokines including interleukin (IL)‐12. In many systems IL‐12 and IFN‐γ act in a similar fashion, and a current subject of debate is the question of whether all activities of IL‐12 are mediated by IFN‐γ. Another question is whether IFN‐γ, by its ability to potentiate MPCs' ability to produce IL‐12, plays a role in bringing about or stabilizing TH1 type responses. In two model systems of autoimmune disease, experimental autoimmune encephalomyelitis and collagen‐induced arthritis, IL‐12 and IFN‐γ were found to act independently.
Delineation of the Proinflammatory Cytokine Cascade in Fever Inductiona
Authors: ZETTERSTRÖM MARIA , SUNDGREN‐ANDERSSON ANNA K. , ÖSTLUND PERNILLA , BARTFAI TAMAS
Published: February 2006

ABSTRACT: Bacterial lipopolypolysaccharide (LPS)‐induced fever involves induction of the proinflammatory cytokines interleukin (IL)‐1α, IL‐1β, tumor necrosis factor‐α (TNF‐α), and IL‐6, both in the periphery and in the brain. These molecules can induce expression of each other and also regulate expression of their own receptors in a complex manner. The functional hierarchy of these highly inducible proteins is therefore difficult to determine. Using mice strains carrying the null mutations of IL‐1β, IL‐1RI, IL‐1RAcP, or IL‐6, respectively, we show that LPS‐induced fever involves IL‐1β, which acts at a complex consisting of the type I IL‐1 receptor and the IL‐1RAcP. This action occurs prior to central IL‐6 release, which has been shown to be a necessary component of fever responses induced by LPS, IL‐1β, and also TNF‐α. In the absence of IL‐1β, as in IL‐1β‐deficient mice, LPS, IL‐1α, and IL‐1β cause hyperresponsive fevers when exogenously applied. Murine TNF‐α is a poor pyrogen in mice even when mice are kept at thermoneutral temperature (30°C). TNF‐α‐mediated fever depends on central IL‐6 expression.
Prostaglandin E2 in the Pathogenesis of Fever: An Updatea
Authors: COCEANI FLAVIO , AKARSU EYUP S.
Published: February 2006

ABSTRACT: Prostaglandin E2 (PGE2) is recognized as a key intermediate in the sequence of events leading to fever. Normally undetectable or barely detectable in brain, it rises selectively on exposure to an infectious noxa and the attendant generation of pyrogenic cytokines outside and, in the case of interleukin (IL)‐6, inside the brain. The mechanism by which pyrogens in the circulation promote the appearance of PGE2 within the confines of brain is not clear, and it is not known how PGE2 activation is selective with IL‐6 being induced in brain. We have found that the cerebral microvasculature is not suitable as a source of PGE2 in response to blood‐borne pyrogens. In addition, we show that IL‐6 differs from other pyrogens in being able to stimulate specifically PGE2 synthesis. Nevertheless, brain‐derived IL‐6 does not appear to be necessary for PGE2 activation and the attendant fever. We conclude that signal‐transducing mechanisms operating across the blood‐brain barrier are most critical for the development of the febrile reponse to a systemic noxa.
Cytokines and Fever: Mechanisms and Sites of Action
Authors: LUHESHI GIAMAL N.
Published: February 2006

ABSTRACT: The cytokines interleukin (IL)‐1, IL‐6, and tumor necrosis factor (TNF)‐α induce increases in body temperature via direct and indirect actions on the brain and are believed to act as endogenous pyrogens. We studied the mechanisms of action of these cytokines on fever in rats. Local administration of lipopolysaccharide (LPS) into a subcutaneous air pouch elicits marked fever, accompanied by increases in the levels of TNF‐α, IL‐1, and IL‐6 in the pouch, but only IL‐6 in the plasma. Thus, TNF‐α and IL‐1 probably act locally to stimulate the release of a secondary circulating mediator(s) (e.g., IL‐6) that can interact with the brain. Neural afferents have also been implicated in relaying messages to the brain. Pyrogenic responses are reportedly attenuated by subdiaphragmatic vagotomy; however, we failed to observe inhibition of fever in vagotomized rats injected with either LPS or a pyrogenic dose of IL‐1, although behavioral responses are abolished.
Afferent Pathways of Pyrogen Signalinga
Authors: BLATTEIS CLARK M. , SEHIC ELMIR , LI SHUXIN
Published: February 2006

ABSTRACT: We and others recently showed that fever induced by intravenously or intraperitoneally injected lipopolysaccharide (LPS) may involve brain signaling via hepatic vagal afferents. This suggests that LPS fever may be initiated by mediators released mainly by cells in the liver, presumably macrophages (Kupffer cells, Kc). To verify this possibility, we disabled the Kc of conscious guinea pigs with gadolinium chloride and monitored their core temperature and associated preoptic prostaglandin E2 (PGE2) responses to iv LPS. Gadolinium chloride pretreatment significantly attenuated both the febrile and PGE2 rises, thus supporting the hypothesis. Additionally, fluorescein‐labeled LPS was detected in Kc 15 minutes after its iv administration. Paradoxically, however, the label was also present in gadolinium chloride‐pretreated guinea pigs. Thus, either Kc are not the primary source of pyrogenic mediators or LPS does not provide the stimulus for their production. Because the iv injection of LPS elicits virtually immediately the production of complement fragments, and Kc express their receptors and produce various mediators on their activation, we hypocomplemented guinea pigs with cobra venom factor. The core temperature rises produced by iv LPS were reduced by complement depletions >60%. LPS iv per se decreased complement, that is, complement was consumed by 12% within 10 minutes. Thus, the onset of LPS fever may involve complement system and Kc activation, but their precise roles await clarification.
Hypothalamic Neurons: Mechanisms of Sensitivity to Temperature a
Authors: BOULANT J. A.
Published: February 2006

Rostral hypothalamic neurons are influenced by endogenous factors that affect thermoregulation and fever. Intracellular recordings reveal the synaptic and intrinsic mechanisms responsible for neuronal thermosensitivity. Many temperature‐sensitive and temperature‐insensitive neurons display a depolarizing prepotential that precedes action potentials. Temperature has little effect on the prepotential of insensitive neurons; however, in warm‐sensitive neurons, the prepotential's depolarization is elevated by warming, and this increases the firing rate. Intracellular cAMP can increase neuronal thermosensitivity by enhancing the thermal response of the prepotential, most likely by thermosensitive ionic conductances. Warm‐sensitive neurons also receive inhibitory synaptic input (IPSPs) from temperature‐insensitive neurons, enhancing the thermosensitivity of some neurons, because cooling increases IPSP amplitude and duration. Therefore, even though IPSP frequencies do not change, cooling can decrease firing rates by increasing IPSP amplitudes. Because endogenous factors change neuronal firing rate and thermosensitivity, these changes likely occur both post‐ and presynaptically as well as by ionic conductances that determine the time interval between action potentials.
Tolerance to Pyrogens
Authors: ZEISBERGER EUGEN , ROTH JOACHIM
Published: February 2006

ABSTRACT: In humans or experimental animals, the repeated confrontation with lipopolysaccarides (LPS) from gram‐negative bacteria, but not with muramyl dipeptide (MDP) from gram‐positive bacteria, leads to attenuation of almost all pathophysiologic effects mediated by proinflammatory cytokines. Our experiments in guinea pigs and rats demonstrate that attenuation of the febrile response during the development of LPS tolerance is associated with a reduced production of cytokines rather than a decrease in responsiveness to cytokines. Cross‐tolerance experiments demonstrate that different stimuli influencing LPS‐induced tumor necrosis factor (TNF) release and nitric oxide (NO) synthesis can modify the development of tolerance. On the other hand, the lack of cross‐tolerance between LPS and MDP indicates that MDP can activate the cytokine cascade and induce the febrile response in animals tolerant to LPS. This may indicate distinct receptors and signal pathways for LPS and MDP, leading to activation of the cytokine cascade. LPS tolerance has also been demonstrated in ex vivo and in vitro studies. In cultures of monocytes, diminished synthesis of TNF and NO reported after LPS restimulation could be prevented and reversed by interferon and granulocyte‐macrophage colony‐stimulating factor. These findings add an additional hypothesis in tolerance development.
Sleep: A Physiologic Role for IL‐1β and TNF‐αa
Authors: KRUEGER JAMES M. , FANG JIDONG , TAISHI PING , CHEN ZUTANG , KUSHIKATA TETSUYA , GARDI JANOS
Published: February 2006

ABSTRACT: Interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) are involved in physiologic sleep regulation. Administration of exogenous IL‐1β or TNF‐α induces increased non‐rapid eye movement sleep (NREMS). Inhibition of IL‐1 or TNF reduces spontaneous sleep. There is a diurnal rhythm of TNF‐α mRNA and IL‐1β mRNA in brain with highest levels occurring during peak sleep periods. Mice lacking either the TNF 55‐kD receptor or the IL‐1 type I receptor sleep less than do strain controls. IL‐1β and TNF‐α are part of a larger biochemical cascade involved in sleep regulation; other somnogenic substances in this cascade include growth hormone‐releasing hormone and nitric oxide. Several additional substances are involved in inhibitory feedback mechanisms, some of which inhibit IL‐1 and TNF. A major challenge to sleep research is to define how and where these molecular steps produce sleep.
Heat Shock Proteins and the Inflammatory Response
Authors: MOSELEY POPE L.
Published: February 2006

ABSTRACT: The heat shock proteins (HSPs) are important in the cellular response to stress and in cellular homeostatic functions such as protein synthesis and protein transport across membranes. An emerging body of data supports a role for HSPs in the inflammatory response, suggesting that HSPs participate in cytokine signal transduction and in the control of cytokine gene expression, that HSPs enhance antigen presentation to T lymphocytes, and that HSPs displayed on the surface of cells are important in targeting cytotoxic cells. Because changes in HSP accumulation occur in the whole organism under physiologic conditions, understanding the role of HSPs in the immune/inflammatory response may aid in understanding the organism's response to danger.
Benefits and Risks of Antipyretic Therapya
Authors: MACKOWIAK PHILIP A. , PLAISACNE KAREN I.
Published: February 2006

ABSTRACT: Physicians have used various forms of antipyretic therapy since antiquity to lower the temperature of febrile patients. Nevertheless, it has yet to be determined whether the benefits of antipyretic therapy outweigh its risks. It is not known, for example, if core temperatures encountered during the febrile state ever reach levels that are intrinsically noxious (and therefore merit antipyretic intervention) or when, if ever, fever's metabolic costs exceed its physiologic benefits, or if the benefits of symptomatic relief afforded by antipyretic drugs consistently exceed their toxicologic cost. Whereas preliminary experimental and clinical observations suggest that antipyretic therapy has the potential to increase the duration and/or severity of certain infections, such data are as yet too fragmentary to draw firm conclusions regarding their validity. Finally, although clinicians have long suspected that bacteremia and other severe infections might induce fevers that are less responsive to antipyretic therapy than are those associated with self‐limited infections, this concept has not held up under scientific scrutiny. Thus, despite over 2.5 millennia of clinical experience, important questions regarding the risks and benefits of antipyretic therapy remain to be answered.