Identification and characterization of rostral ventromedial medulla neurons synaptically connected to the urinary bladder afferents in female rats with or without neonatal cystitis.

The neurons in the rostral ventromedial medulla (RVM) play a major role in pain modulation. We have previously shown that early-life noxious bladder stimuli in rats resulted in an overall spinal GABAergic disinhibition and a long-lasting bladder/colon sensitization when tested in adulthood. However, the neuromolecular alterations within RVM neurons in the pathophysiology of early life bladder inflammation have not been elucidated. In this study, we have identified and characterized RVM neurons that are synaptically linked to the bladder and colon and examined the effect of neonatal bladder inflammation on molecular expressions of these neurons. A transient bladder inflammation was induced by intravesicular instillation of protamine sulfate and zymosan during postnatal days 14 through 16 (P14-16) followed by pseudorabies virus PRV-152 and PRV-614 injections into the bladder and colon, respectively, on postnatal day P60. Tissues were examined 96 h postinoculation for serotonergic, GABAergic, and enkephalinergic expressions using in situ hybridization and/or immunohistochemistry techniques. The results revealed that > 50% of RVM neurons that are synaptically connected to the bladder (i.e., PRV-152+) were GABAergic, 40% enkephalinergic, and about 14% expressing serotonergic marker tryptophan hydroxylase 2 (TpH2). Neonatal cystitis resulted in a significant increase in converging neurons in RVM receiving dual synaptic inputs from the bladder and colon. In addition, neonatal cystitis significantly downregulated vesicular GABA transporter (VGAT) with a concomitant increase in TpH2 expression in bladder-linked RVM neurons, suggesting an alteration in supraspinal signaling. These alterations of synaptic connectivity and GABAergic/serotonergic expressions in RVM neurons may contribute to bladder pain modulation and cross-organ visceral sensitivity.

Peripheral antinociceptive effects of a bifunctional µ and δ opioid receptor ligand in rat model of inflammatory bladder pain.

There is a need to develop a novel analgesic for pain associated with interstitial cystitis/painful bladder syndrome (IC/PBS). The use of the conventional µ-opioid receptor agonists to manage IC/PBS pain is controversial due to adverse CNS effects. These effects are attenuated in benzylideneoxymorphone (BOM), a low-efficacy µ-opioid receptor agonist/δ-opioid receptor antagonist that attenuates thermal pain and is devoid of reinforcing effects. We hypothesize that BOM will inhibit bladder pain by attenuating responses of urinary bladder distension (UBD)-sensitive afferent fibers. Therefore, the effect of BOM was tested on responses of UBD-sensitive afferent fibers in L6 dorsal root from inflamed and non-inflamed bladder of rats. Immunohistochemical (IHC) examination reveals that following the induction of inflammation there were significant high expressions of µ, δ, and µ-δ heteromer receptors in DRG. BOM dose-dependently (1-10 mg/kg, i.v) attenuated mechanotransduction properties of these afferent fibers from inflamed but not from non-inflamed rats. In behavioral model of bladder pain, BOM significantly attenuated visceromotor responses (VMRs) to UBD only in inflamed group of rats when injected either systemically (10 mg/kg, i.v.) or locally into the bladder (0.1 ml of 10 mg/ml). Furthermore, oxymorphone (OXM), a high-efficacy µ-opioid receptor agonist, attenuated responses of mechanosensitive bladder afferent fibers and VMRs to UBD. Naloxone (10 mg/kg, i.v.) significantly reversed the inhibitory effects of BOM and OXM on responses of bladder afferent fibers and VMRs suggesting µ-opioid receptor-related analgesic effects of these compounds. The results reveal that a low-efficacy, bifunctional opioid-based compound can produce analgesia by attenuating mechanotransduction functions of afferent fibers innervating the urinary bladder.

Selective Phosphodiesterase 1 Inhibitor BTTQ Reduces Blood Pressure in Spontaneously Hypertensive and Dahl Salt Sensitive Rats: Role of Peripheral Vasodilation.

Regulation of the peripheral vascular resistance via modulating the vessel diameter has been considered as a main determinant of the arterial blood pressure. Phosphodiesterase enzymes (PDE1-11) hydrolyse cyclic nucleotides, which are key players controlling the vessel diameter and, thus, peripheral resistance. Here, we have tested and reported the effects of a novel selective PDE1 inhibitor (BTTQ) on the cardiovascular system. Normal Sprague Dawley, spontaneously hypertensive (SHR), and Dahl salt-sensitive rats were used to test in vivo the efficacy of the compound. Phosphodiesterase radiometric enzyme assay revealed that BTTQ inhibited all three isoforms of PDE1 in nanomolar concentration, while micromolar concentrations were needed to induce effective inhibition for other PDEs. The myography study conducted on mesenteric arteries revealed a potent vasodilatory effect of the drug, which was confirmed in vivo by an increase in the blood flow in the rat ear arteriols reflected by the rise in the temperature. Furthermore, BTTQ proved a high efficacy in lowering the blood pressure about 9, 36, and 24 mmHg in normal Sprague Dawley, SHR and, Dahl salt-sensitive rats, respectively, compared to the vehicle-treated group. Moreover, additional blood pressure lowering of about 22 mmHg could be achieved when BTTQ was administered on top of ACE inhibitor lisinopril, a current standard of care in the treatment of hypertension. Therefore, PDE1 inhibition induced efficient vasodilation that was accompanied by a significant reduction of blood pressure in different hypertensive rat models. Administration of BTTQ was also associated with increased heart rate in both models of hypertension as well as in the normotensive rats. Thus, PDE1 appears to be an attractive therapeutic target for the treatment of resistant hypertension, while tachycardia needs to be addressed by further compound structural optimization.

Effect of Nearby Construction Activity on Endothelial Function, Sensitivity to Nitric Oxide, and Potassium Channel Activity in the Middle Cerebral Arteries of Rats.

The present study assessed the effect of nearby construction activity on the responses of rat middle cerebral arteries (MCA)to the endothelium-dependent vasodilator acetylcholine and the NO donor sodium nitroprusside (SNP) and the activity of MaxiK potassium channels in MCA smooth muscle cells from male Sprague-Dawley rats. Two monitoring systems were used to assess vibrations in the animal rooms during and immediately after construction activities near the research building where the animal facility is located. One was a commercially available system; the other was a Raspberry-Pi (RPi)-based vibration monitoring system designed in our laboratory that included a small computing unit attached to a rolling sensor (low sensitivity) and a piezoelectric film sensor (high sensitivity). Both systems recorded increased levels of vibration during construction activity outside the building. During the construction period, vasodilator responses to acetylcholine and SNP were abolished, and MaxiK single-channel current opening frequency and open-state probability in cell-attached patches of isolated MCA myocytes were dramatically decreased. Recovery of acetylcholine- and SNP-induced dilation was minimal in MCA from rats studied after completion of construction but housed in the animal facility during construction, whereas responses to acetylcholine and SNP were intact in rats purchased, housed, and studied after construction. Baseline levels of vibration returned after the completion of construction, concomitant with the recovery of normal endothelium-dependent vasodilation to acetylcholine and of NO sensitivity assessed by using SNP in MCA from animals obtained after construction. The results of this study indicate that the vibration associated with nearby construction can have highly disruptive effects on crucial physiologic phenotypes.

Evaluation of Cerebral Blood Flow Autoregulation in the Rat Using Laser Doppler Flowmetry.

When investigating the body's mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained using laser Doppler flowmetry (LDF). This paper demonstrates a closed skull preparation that allows cerebral blood flow to be assessed without penetrating the skull or installing a chamber or cerebral window. To evaluate autoregulatory mechanisms, a model of controlled blood pressure reduction via graded hemorrhage can be utilized while simultaneously employing LDF. This enables the real time tracking of the relative changes in the blood flow in response to reductions in arterial blood pressure produced by the withdrawal of circulating blood volume. This paradigm is a valuable approach to study cerebral blood flow autoregulation during reductions in arterial blood pressure and, with minor modifications in the protocol, is also valuable as an experimental model of hemorrhagic shock. In addition to evaluating autoregulatory responses, LDF can be used to monitor the cortical blood flow when investigating metabolic, myogenic, endothelial, humoral, or neural mechanisms that regulate cerebral blood flow and the impact of various experimental interventions and pathological conditions on cerebral blood flow.

Fatty Acid-Treated Induced Pluripotent Stem Cell-Derived Human Cardiomyocytes Exhibit Adult Cardiomyocyte-Like Energy Metabolism Phenotypes.

Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) (iPSC-CMs) are a promising cell source for myocardial regeneration, disease modeling and drug assessment. However, iPSC-CMs exhibit immature fetal CM-like characteristics that are different from adult CMs in several aspects, including cellular structure and metabolism. As an example, glycolysis is a major energy source for immature CMs. As CMs mature, the mitochondrial oxidative capacity increases, with fatty acid ß-oxidation becoming a key energy source to meet the heart's high energy demand. The immaturity of iPSC-CMs thereby limits their applications. The aim of this study was to investigate whether the energy substrate fatty acid-treated iPSC-CMs exhibit adult CM-like metabolic properties. After 20 days of differentiation from human iPSCs, iPSC-CMs were sequentially cultured with CM purification medium (lactate+/glucose-) for 7 days and maturation medium (fatty acids+/glucose-) for 3-7 days by mimicking the adult CM's preference of utilizing fatty acids as a major metabolic substrate. The purity and maturity of iPSC-CMs were characterized via the analysis of: (1) Expression of CM-specific markers (e.g., troponin T, and sodium and potassium channels) using RT-qPCR, Western blot or immunofluorescence staining and electron microscopy imaging; and (2) cell energy metabolic profiles using the XF96 Extracellular Flux Analyzer. iPSCs-CMs (98% purity) cultured in maturation medium exhibited enhanced elongation, increased mitochondrial numbers with more aligned Z-lines, and increased expression of matured CM-related genes, suggesting that fatty acid-contained medium promotes iPSC-CMs to undergo maturation. In addition, the oxygen consumption rate (OCR) linked to basal respiration, ATP production, and maximal respiration and spare respiratory capacity (representing mitochondrial function) was increased in matured iPSC-CMs. Mature iPSC-CMs also displayed a larger change in basal and maximum respirations due to the utilization of exogenous fatty acids (palmitate) compared with non-matured control iPSC-CMs. Etomoxir (a carnitine palmitoyltransferase 1 inhibitor) but not 2-deoxyglucose (an inhibitor of glycolysis) abolished the palmitate pretreatment-mediated OCR increases in mature iPSC-CMs. Collectively, our data demonstrate for the first time that fatty acid treatment promotes metabolic maturation of iPSC-CMs (as evidenced by enhanced mitochondrial oxidative function and strong capacity of utilizing fatty acids as energy source). These matured iPSC-CMs might be a promising human CM source for broad biomedical application.

Stem Cell Therapies in Cardiovascular Disease.

Despite considerable advances in medicine, cardiovascular disease is still rising, with ischemic heart disease being the leading cause of death and disability worldwide. Thus extensive efforts are continuing to establish effective therapeutic modalities that would improve both quality of life and survival in this patient population. Novel therapies are being investigated not only to protect the myocardium against ischemia-reperfusion injury but also to regenerate the heart. Stem cell therapy, such as potential use of human mesenchymal stem cells and induced pluripotent stem cells and their exosomes, will make it possible not only to address molecular mechanisms of cardiac conditioning, but also to develop new therapies for ischemic heart disease. Despite the studies and progress made over the last 15 years on the use of stem cell therapy for cardiovascular disease, the efforts are still in their infancy. Even though the expectations have been high, the findings indicate that most of the clinical trials generally have been small and the results inconclusive. Because of many negative findings, there is certain pessimism that cardiac cell therapy is likely to yield any meaningful results over the next decade or so. Similar to other new technologies, early failures are not unusual and they may be followed by impressive success. Nevertheless, there has been considerable attention to safety by the clinical investigators because the adverse events of stem cell therapy have been impressively rare. In summary, although regenerative biology might not help the cardiovascular patient in the near term, it is destined to do so over the next several decades.

Hyperoxia Causes Mitochondrial Fragmentation in Pulmonary Endothelial Cells by Increasing Expression of Pro-Fission Proteins.

OBJECTIVE: We explored mechanisms that alter mitochondrial structure and function in pulmonary endothelial cells (PEC) function after hyperoxia. APPROACH AND RESULTS: Mitochondrial structures of PECs exposed to hyperoxia or normoxia were visualized and mitochondrial fragmentation quantified. Expression of pro-fission or fusion proteins or autophagy-related proteins were assessed by Western blot. Mitochondrial oxidative state was determined using mito-roGFP. Tetramethylrhodamine methyl ester estimated mitochondrial polarization in treatment groups. The role of mitochondrially derived reactive oxygen species in mt-fragmentation was investigated with mito-TEMPOL and mitochondrial DNA (mtDNA) damage studied by using ENDO III (mt-tat-endonuclease III), a protein that repairs mDNA damage. Drp-1 (dynamin-related protein 1) was overexpressed or silenced to test the role of this protein in cell survival or transwell resistance. Hyperoxia increased fragmentation of PEC mitochondria in a time-dependent manner through 48 hours of exposure. Hyperoxic PECs exhibited increased phosphorylation of Drp-1 (serine 616), decreases in Mfn1 (mitofusion protein 1), but increases in OPA-1 (optic atrophy 1). Pro-autophagy proteins p62 (LC3 adapter-binding protein SQSTM1/p62), PINK-1 (PTEN-induced putative kinase 1), and LC3B (microtubule-associated protein 1A/1B-light chain 3) were increased. Returning cells to normoxia for 24 hours reversed the increased mt-fragmentation and changes in expression of pro-fission proteins. Hyperoxia-induced changes in mitochondrial structure or cell survival were mitigated by antioxidants mito-TEMPOL, Drp-1 silencing, or inhibition or protection by the mitochondrial endonuclease ENDO III. Hyperoxia induced oxidation and mitochondrial depolarization and impaired transwell resistance. Decrease in resistance was mitigated by mito-TEMPOL or ENDO III and reproduced by overexpression of Drp-1. CONCLUSIONS: Because hyperoxia evoked mt-fragmentation, cell survival and transwell resistance are prevented by ENDO III and mito-TEMPOL and Drp-1 silencing, and these data link hyperoxia-induced mt-DNA damage, Drp-1 expression, mt-fragmentation, and PEC dysfunction.

Epoxyeicosatrienoic acids pretreatment improves amyloid ß-induced mitochondrial dysfunction in cultured rat hippocampal astrocytes.

Amyloid-ß (Aß) has long been implicated as a causative protein in Alzheimer's disease. Cellular Aß accumulation is toxic and causes mitochondrial dysfunction, which precedes clinical symptoms of Alzheimer's disease pathology. In the present study, we explored the possible use of epoxyeicosatrienoic acids (EETs), epoxide metabolites of arachidonic acid, as therapeutic target against Aß-induced mitochondrial impairment using cultured neonatal hippocampal astrocytes. Inhibition of endogenous EET production by a selective epoxygenase inhibitor, MS-PPOH, caused a greater reduction in mitochondrial membrane potential in the presence of Aß (1, 10 µM) exposure versus absence of Aß. MS-PPOH preincubation also aggravated Aß-induced mitochondrial fragmentation. Preincubation of the cells with either 14,15- or 11,12-EET prevented this mitochondrial depolarization and fragmentation. EET pretreatment also further improved the reduction observed in mitochondrial oxygen consumption in the presence of Aß. Preincubation of the cells with EETs significantly improved cellular respiration under basal condition and in the presence of the protonophore, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP). The uncoupling of ATP synthase from the electron transfer chain that occurred in Aß-treated cells was also prevented by preincubation with EETs. Lastly, cellular reactive oxygen species production, a hallmark of Aß toxicity, also showed significant reduction in the presence of EETs. We have previously shown that Aß reduces EET synthesis in rat brain homogenates and cultured hippocampal astrocytes and neurons (Sarkar P, Narayanan J, Harder DR. Differential effect of amyloid beta on the cytochrome P450 epoxygenase activity in rat brain. Neuroscience 194: 241-249, 2011). We conclude that reduction of endogenous EETs may be one of the mechanisms through which Aß inflicts toxicity and thus supplementing the cells with exogenous EETs improves mitochondrial dynamics and prevents metabolic impairment.

Redox signaling via oxidative inactivation of PTEN modulates pressure-dependent myogenic tone in rat middle cerebral arteries.

The present study examined the level of generation of reactive oxygen species (ROS) and roles of inactivation of the phosphatase PTEN and the PI3K/Akt signaling pathway in response to an increase in intramural pressure-induced myogenic cerebral arterial constriction. Step increases in intraluminal pressure of cannulated cerebral arteries induced myogenic constriction and concomitant formation of superoxide (O2 (.-)) and its dismutation product hydrogen peroxide (H2O2) as determined by fluorescent HPLC analysis, microscopic analysis of intensity of dihydroethidium fluorescence and attenuation of pressure-induced myogenic constriction by pretreatment with the ROS scavenger 4,hydroxyl-2,2,6,6-tetramethylpiperidine1-oxyl (tempol) or Mito-tempol or MitoQ in the presence or absence of PEG-catalase. An increase in intraluminal pressure induced oxidation of PTEN and activation of Akt. Pharmacological inhibition of endogenous PTEN activity potentiated pressure-dependent myogenic constriction and caused a reduction in NPo of a 238 pS arterial KCa channel current and an increase in [Ca(2+)]i level in freshly isolated cerebral arterial muscle cells (CAMCs), responses that were attenuated by Inhibition of the PI3K/Akt pathway. These findings demonstrate an increase in intraluminal pressure induced increase in ROS production triggered redox-sensitive signaling mechanism emanating from the cross-talk between oxidative inactivation of PTEN and activation of the PI3K/Akt signaling pathway that involves in the regulation of pressure-dependent myogenic cerebral arterial constriction.

Antinociception produced by 14,15-epoxyeicosatrienoic acid is mediated by the activation of beta-endorphin and met-enkephalin in the rat ventrolateral periaqueductal gray.

Cytochrome P450 genes catalyze formation of epoxyeicosatrienoic acids (EETs) from arachidonic acid. The effects of 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET microinjected into the ventrolateral periaqueductal gray (vlPAG) on the thermally produced tail-flick response were studied in male Sprague-Dawley rats. 14,15-EET microinjected into vlPAG (3-156 pmol) dose-dependently inhibited the tail-flick response (ED50 = 32.5 pmol). In contrast, 5,6-EET, 8,9-EET, and 11,12-EET at a dose of 156 pmol were not active when injected into the vlPAG. 14,15-EET failed to displace the radiobinding of [3H][D-Ala2,NHPe4, Gly-ol5]enkephalin (mu-opioid receptor ligand) or [3H]naltrindole (delta-opioid receptor ligand) in crude membrane fractions of rat brain. Tail-flick inhibition produced by 14,15-EET from vlPAG was blocked by intra-vlPAG pretreatment with antiserum against beta-endorphin or Met-enkephalin or the mu-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) or the delta-opioid receptor antagonist naltrindole but not with dynorphin A[1-17] antiserum or the kappa-opioid receptor antagonist nor-binaltorphimine. In addition, tail-flick inhibition produced by 14,15-EET treatment was blocked by intrathecal pretreatment with Met-enkephalin antiserum, naltrindole, or CTOP but not with beta-endorphin antiserum. It is concluded that 1) 14,15-EET itself does not have any affinity for mu- or delta-opioid receptors and 2) 14,15-EET activates beta-endorphin and Met-enkephalin, which subsequently act on mu- and delta-opioid receptors to produce antinociception.

(+)-Morphine attenuates the (-)-morphine-produced conditioned place preference and the mu-opioid receptor-mediated dopamine increase in the posterior nucleus accumbens of the rat.

An unbiased conditioned place preference paradigm and the microdialysis technique was used to evaluate the effect of (+)-morphine pretreatment on the conditioned place preference produced by (-)-morphine and the increased release of the dopamine produced by mu-opioid ligand endomorphin-1, respectively, in the posterior nucleus accumbens shell of the male CD rat. (-)-Morphine (2.5-10 microg) microinjected into the posterior nucleus accumbens shell dose-dependently produced the conditioned place preference. Pretreatment with (+)-morphine (0.1-10 pg) given into the posterior accumbens shell for 45 min dose-dependently attenuated the conditioned place preference produced by (-)-morphine (5 microg) given into the same posterior accumbens shell. However, higher doses of (+)-morphine (0.1 and 1 ng) were less effective in attenuating the (-)-morphine-produced conditioned place preference. Thus, like given systemically, (+)-morphine given into the posterior nucleus accumbens shell also induces a U-shaped dose-response curve for attenuating the (-)-morphine-produced conditioned place preference. Microinjection of mu-opioid agonist endomorphin-1 (1-10 microg) given into the ventral tegmental area dose-dependently increased the release of the extracellular dopamine in the posterior nucleus accumbens shell in the urethane-anesthetized rats. The increased dopamine caused by endomorphin-1 (10 microg) was completed blocked by the (+)-morphine (10 pg) pretreatment given into ventral tegmental area. It is concluded that (+)-morphine attenuates the (-)-morphine-produced conditioned place preference and the mu-opioid receptor-mediated increase of extracellular dopamine in the posterior nucleus accumbens shell of the rat.

(+)-Morphine and (-)-morphine stereoselectively attenuate the (-)-morphine-produced tail-flick inhibition via the naloxone-sensitive sigma receptor in the ventral periaqueductal gray of the rat.

We have previously demonstrated that (+)-morphine and (-)-morphine pretreated spinally for 45 min stereoselectively attenuates the tail-flick inhibition produced by (-)-morphine given spinally in the mouse. The present study is then undertaken to determine if the same phenomenon observed in the mouse spinal cord can also take place in the ventral periaqueductal gray of the rat. Pretreatment with (+)-morphine for 45 min at 0.3 to 3.3 fmol dose-dependently attenuated the tail-flick inhibition produced by (-)-morphine (9 nmol) given into the ventral periaqueductal gray. Likewise, pretreatment with (-)-morphine for 45 min at a higher dose (3-900 pmol), which given alone did not affect the baseline tail-flick latency, also dose-dependently attenuated the tail-flick inhibition produced by (-)-morphine. Thus, (+)-morphine is approximately 270,000-fold more potent than (-)-morphine in attenuating the (-)-morphine-produced tail-flick inhibition. The attenuation of the (-)-morphine-produced tail-flick inhibition induced by (+)-morphine or (-)-morphine was dose-dependently reversed by (+)-naloxone (27.5 to 110 pmol) pretreatment for 50 min given into the ventral periaqueductal gray. Pretreatment with the sigma receptor antagonist BD1047 (N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine dihydrobromide) (11-45 nmol) for 45 min given into the ventral periaqueductal gray also reversed dose-dependently the attenuation of the (-)-morphine-produced tail-flick inhibition induced by (+)-morphine or (-)-morphine, indicating that the effects are mediated by the activation of the sigma receptors. Since (+)-morphine, (-)-morphine and (+)-naloxone do not have any affinity for the naloxone-inaccessible sigma receptors, we therefore propose that (+)-morphine and (-)-morphine attenuate the (-)-morphine-produced tail-flick inhibition via the activation of the naloxone-sensitive sigma receptor originally proposed by Tsao and Su [Tsao, L.T., Su, T.P., 1997. Naloxone-sensitive, haloperidol-sensitive, [(3)H](+)-SKF-1047-binding protein partially purified from rat liver and rat brain membranes: an opioid/sigma receptor. Synapse 25, 117-124].

dextro-Morphine attenuates the morphine-produced conditioned place preference via the sigma(1) receptor activation in the rat.

An unbiased conditioned place preference paradigm was used to evaluate the effect of dextro-morphine on the morphine-produced reward in male CD rats. Morphine sulfate (1-10 mg/kg) given intraperitoneally dose-dependently produced the conditioned place preference. Pretreatment with dextro-morphine at a dose from 0.1 to 3 microg/kg given subcutaneously dose-dependently attenuated the morphine-produced conditioned place preference. However, dextro-morphine at a higher dose 100 microg/kg did not affect the morphine-produced conditioned place preference. Thus, dextro-morphine pretreatment induces a U-shaped dose-response curve for attenuating the morphine-produced conditioned place preference. The attenuation of the morphine-produced conditioned place preference was reversed by the pretreatment with the sigma(1) receptor antagonist BD1047 (N-[2-(3,4-Dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine dihydrobromide. dextro-Morphine or BD1047 given alone did not affect the baseline place conditioning. It is concluded that dextro-morphine attenuated the morphine-produced conditioned place preference via the sigma(1) receptor activation.

Paradoxical hyperalgesia induced by mu-opioid receptor agonist endomorphin-2, but not endomorphin-1, microinjected into the centromedial amygdala of the rat.

The effects of endomorphin-2 or endomorphin-1 microinjected into the centromedial amygdala on the thermally-induced tail-flick response were studied in male CD rats. Microinjection of endomorphin-2 (8.7-35.0 nmol) given into the centromedial amygdala time- and dose-dependently decreased the tail-flick latencies. On the other hand, endomorphin-1 (8-32.6 nmol) given into the same site did not cause any change of the tail-flick latency. However, endomorphin-1 (32.6 nmol) or endomorphin-2 (35.0 nmol) given into the basolateral site of amygdala did not affect the tail-flick latency. Pretreatment with the antiserum against dynorphin A(1-17) (200 microg) significantly reversed the decrease of the tail-flick latency induced by endomorphin-2. The decrease of the tail-flick latency induced by endomorphin-2 was also blocked by the endomorphin-2 selective micro-opioid receptor antagonist 3-methoxynaltrexone (6.4 pmol) and by the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (30 nmol), but not by the kappa-opioid receptor antagonist nor-binaltorphimine (6.6 nmol). It is concluded that endomorphin-2, but not endomorphin-1, given into the centromedial amygdala stimulates a 3-methoxynaltrexone-sensitive mu-opioid receptor subtype to induce the release of dynorphin A(1-17), which then acts on the NMDA receptor, but not kappa-opioid receptor for producing hyperalgesia. This conclusion is further supported by the additional findings that dynorphin A(1-17) (2.3 nmol) given into the centromedial amygdala also caused the decrease of the tail-flick latency, which was similarly blocked by the NMDA receptor antagonist MK-801 (30 nmol), but not kappa-opioid receptor antagonist nor-binaltorphimine (6.6 nmol).

dextro-Naloxone or levo-naloxone reverses the attenuation of morphine antinociception induced by lipopolysaccharide in the mouse spinal cord via a non-opioid mechanism.

Glial stimulation by intrathecal injection of lipopolysaccharide (LPS) attenuated the tail-flick inhibition produced by morphine given intrathecally in the spinal cord of the male CD-1 mice. The phenomenon has been defined as antianalgesia. The effects of dextro-naloxone or levo-naloxone on the attenuation of morphine-produced tail-flick inhibition induced by LPS were then studied. Pretreatment with dextro-naloxone or levo-naloxone reversed the attenuation of the morphine-produced tail-flick inhibition induced by LPS. Pretreatment with dextro-naloxone or levo-naloxone alone did not affect the morphine-produced tail-flick inhibition. It is concluded that dextro-naloxone and levo-naloxone block the LPS-induced antianalgesia against morphine antinociception via a non-opioid mechanism.

Reactive oxygen species cerebral autoregulation in health and disease.

Reactive oxygen species (ROS) are a family of oxygen-derived free radicals that are produced in mammalian cells under normal and pathologic conditions. Many ROS, such as the superoxide anion (O2-) and hydrogen peroxide (H2O2), act as cellular signaling molecules within blood vessels, altering mechanisms mediating mechanical signal transduction and autoregulation of cerebral blood flow. This article focuses on the actions of ROS, such as O2.- and H2O2, and how they influence mechanisms responsible for the modulation of pressure-induced myogenic tone in the cerebral circulation and blood flow autoregulation in response to elevated arterial pressure. ROS may be a key target for therapeutic interventions in pediatric patients who have hypoxic injury or altered cerebral metabolism induced by trauma or infection.

Antianalgesia: stereoselective action of dextro-morphine over levo-morphine on glia in the mouse spinal cord.

We have previously shown that the naturally occurring levo-morphine at a subanalgesic picomolar dose pretreated i.t. induces antianalgesia against levo-morphine-produced antinociception. We now report that the synthetic stereo-enantiomer dextro-morphine, even at an extremely low femtomolar dose, induces antianalgesia against levo-morphine-produced antinociception using the tail-flick (TF) test in male CD-1 mice. Intrathecal pretreatment with dextro-morphine (33 fmol) time-dependently attenuated the i.t. levo-morphine-produced TF inhibition for 4 h and returned to the preinjection control level at 24 h. Intrathecal pretreatment with dextro-morphine (0.3-33 fmol), which injected alone did not affect the baseline TF latency, dose-dependently attenuated the TF inhibition produced by i.t.-administered levo-morphine (3.0 nmol). The ED(50) value for dextro-morphine to induce antianalgesia was estimated to be 1.07 fmol, which is 71,000-fold more potent than the ED(50) value of levo-morphine, indicating the high stereoselective action of dextro-morphine over levo-morphine for the induction of antianalgesia. Like levo-morphine, the dextro-morphine-induced antianalgesia against levo-morphine-produced TF inhibition was dose-dependently blocked by the nonopioid dextro-naloxone and its stereo-enantiomer levo-naloxone, a nonselective mu-opioid receptor antagonist. The antianalgesia induced by levo-morphine and dextro-morphine is reversed by the pretreatment with the glial inhibitor propentofylline (3.3-65 nmol), indicating that the antianalgesia is mediated by glial stimulation. The findings strongly indicate that the antianalgesia induced by levo-morphine and dextro-morphine is mediated by the stimulation of a novel nonopioid receptor on glial cells.

Differential mechanisms of antianalgesia induced by endomorphin-1 and endomorphin-2 in the ventral periaqueductal gray of the rat.

The effects of pretreatment with endomorphin-1 (EM-1) and endomorphin-2 (EM-2) given into the ventral periaqueductal gray (vPAG) to induce antianalgesia against the tail-flick (TF) inhibition produced by morphine given into the vPAG were studied in rats. Pretreatment with EM-1 (3.5-28 nmol) given into vPAG for 45 min dose-dependently attenuated the TF inhibition produced by morphine (9 nmol) given into vPAG. Similarly, pretreatment with EM-2 (1.7-7.0 nmol) for 45 min also attenuated the TF inhibition induced by morphine; however, a high dose of EM-2 (14 nmol) did not attenuate the morphine-produced TF inhibition. The attenuation of morphine-produced TF inhibition induced by EM-2 or EM-1 pretreatment was blocked by pretreatment with mu-opioid antagonist (-)-naloxone (55 pmol) but not nonopioid (+)-naloxone (55 pmol). However, pretreatment with a morphine-6beta-glucuronide-sensitive mu-opioid receptor antagonist 3-methoxynaltrexone (6.4 pmol) selectively blocked EM-2- but not EM-1-induced antianalgesia. Pretreatment with dynorphin A(1-17) antiserum reversed only EM-2- but not EM-1-induced antianalgesia. Pretreatment with antiserum against beta-endorphin, [Met(5)]enkephalin, [Leu(5)]enkephalin, substance P or cholecystokinin, or with delta-opioid receptor antagonist naltrindole (2.2 nmol) or kappa-opioid receptor antagonist norbinaltorphimine (6.6 nmol) did not affect EM-2-induced antianalgesia. It is concluded that EM-2 selectively releases dynorphin A(1-17) by stimulation of a novel subtype of mu-opioid receptor, tentatively designated as mu(3) in the vPAG to induce antianalgesia, whereas the antianalgesia induced by EM-1 is mediated by the stimulation of another subtype of mu(1)- or mu(2)-opioid receptor.

Increased release of immunoreactive dynorphin A1-17 from the spinal cord after intrathecal treatment with endomorphin-2 in anesthetized rats.

We previously demonstrated pretreatment with antiserum against dynorphin A1-17 attenuates endomorphin-2-induced analgesia and antianalgesia, suggesting that these endomorphin-2 effects are mediated by the release of dynorphin A1-17. Lumbar-cisternal spinal perfusion was used to measure the release of immunoreactive dynorphin A1-17 into spinal perfusates from urethane-anesthetized rats following endomorphin-2 or endomorphin-1 treatment within the perfusion solution. Treatment with endomorphin-2 (5-50 nmol) for 3 min caused a dose-dependent increase of immunoreactive dynorphin A1-17 in spinal perfusates, with a maximal increase detected between 24 and 48 min after endomorphin-2 treatment, while levels returned to baseline within 60 min. Endomorphin-2-induced release of immunoreactive dynorphin A1-17 was attenuated by pretreatment with mu-opioid receptor antagonist naloxone or 3-methoxynaltrexone. Endomorphin-1 induced a slight increase in immunoreactive dynorphin1-17 as well, but only at the highest dose used (50 nmol). Our results suggest that endomorphin-2 stimulated a specific subtype of mu-opioid receptor to induce the release of immunoreactive dynorphin A1-17 in spinal cords of rats.