Activities of Succinate Dehydrogenase and Lactate Dehydrogenase in Blood Lymphocytes in Yakut Ground Squirrels Spermophilus undulatus During Hibernation and in the Active State.

We studied energy metabolism in blood lymphocytes of Yakut ground squirrels Spermophilus undulatus in active state and during hibernation. Activities of succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH), marker enzymes of mitochondrial respiration and glycolysis, were measured by a cytobiochemical method based on quantitative assessment of a product of NBT reduction to diformazan in blood lymphocytes immobilized on glass. To measure SDH and LDH activities, cytobiochemical staining of immobilized cells was performed with succinate, lactate, and NAD. In the state of hibernation, SDH activity decreased by 3 times and LDH activity decreased by 10 times or more. These results suggest that the decrease in metabolic activity in lymphocytes of ground squirrels during hypothermia is associated with inhibition of glycolysis, rather than with mitochondrial energy supply.

The α2 Na+/K+-ATPase isoform mediates LPS-induced neuroinflammation.

Na+/K+-ATPase is a transmembrane ion pump that is essential for the maintenance of ion gradients and regulation of multiple cellular functions. Na+/K+-ATPase has been associated with nuclear factor kappa B (NFκB) signalling, a signal associated with lipopolysaccharides (LPSs)-induced immune response in connection with activated Toll-like receptor 4 (TLR4) signalling. However, the contribution of Na+/K+-ATPase to regulating inflammatory responses remains elusive. We report that mice haploinsufficient for the astrocyte-enriched α2Na+/K+-ATPase isoform (α2+/G301R mice) have a reduced proinflammatory response to LPS, accompanied by a reduced hypothermic reaction compared to wild type litter mates. Following intraperitoneal injection of LPS, gene expressions of Tnf-α, Il-1ß, and Il-6 was reduced in the hypothalamus and hippocampus from α2+/G301R mice compared to α2+/+ littermates. The α2+/G301R mice experienced increased expression of the gene encoding an antioxidant enzyme, NRF2, in hippocampal astrocytes. Our findings indicate that α2Na+/K+-ATPase haploinsufficiency negatively modulates LPS-induced immune responses, highlighting a rational pharmacological target for reducing LPS-induced inflammation.

Reducing Smad3/ATF4 was essential for Sirt1 inhibiting ER stress-induced apoptosis in mice brown adipose tissue.

Sirtuin 1 (Sirt1) promotes adaptive thermogenesis by controlling the acetylation status of enzymes and transcriptional factors in interscapular brown adipose tissue (iBAT). However, the effects of Sirt1 on endoplasmic reticulum (ER) stress and apoptosis of iBAT remain elusive. In this study, the mRNA levels of Sirt1 and thermogenesis genes were reduced but the genes related with ER stress were elevated in iBAT of high-fat diet (HFD)-induced obese mice. Moreover, ER stress further inhibited mRNA level of Sirt1 and triggered brown adipocyte apoptosis in vitro and in vivo. Further analysis revealed that Sirt1 overexpression alleviated ER stress-induced brown adipocyte apoptosis by inhibiting Smad3 and ATF4. In addition, Smad3 bound to ATF4 promoter region and positively transcriptional regulation of ATF4. Our data also confirmed that Sirt1 reduced early apoptotic cells and blocked the mitochondrial apoptosis pathway by directly interacting with ATF4. Furthermore, Sirt1 attenuated tunicamycin-induced cold intolerance and elevating thermogenesis by inhibiting ER stress and apoptosis in iBAT. In summary, our data collectively revealed Sirt1 reduced ER stress and apoptosis of brown adipocyte in vivo and in vitro by inhibiting Smad3/ATF4 signal. These data reveal a novel mechanism that links Sirt1 to brown adipocyte apoptosis.

Effects of Hypothermia and Self-Warming on Activity of Ca(2+)-Dependent Neutral Proteases in Tissues of Ground Squirrels and Rats.

Effects of hypothermia and subsequent self-warming on activity of Ca(2+)-dependent neutral proteases were studied in tissues of ground squirrels and rats. Moderate hypothermia did not significantly change activity of Ca(2+)-dependent neutral proteases in the analyzed tissues of ground squirrels, but reduced protease activity in rat heart. Severe hypothermia reduced enzyme activity in the analyzed tissues of rats and ground squirrels. Differences in activity of Ca(2+)-dependent neutral proteases after long-term hypothermia and subsequent self-warming were found only in the heart.

Effects of whole-body heat on male germ cell development and sperm motility in the laboratory mouse.

This study investigated the effects of high temperatures on male germ cell development and epididymal sperm motility of laboratory mice. In Experiment 1, adult males (n=16) were exposed to whole-body heat of 37-38°C for 8h day(-1) for 3 consecutive days, whereas controls (n=4) were left at 23-24°C. In Experiment 2, adult mice (n=6) were exposed to 37-38°C for a single 8-h period with controls (n=6) left at 23-24°C. Experiment 2 was conducted as a continuation of previous study that showed changes in spermatozoa 16h after exposure to heat of 37-38°C for 8h day(-1) for 3 consecutive days. In the present study, in Experiment 1, high temperature reduced testes weights 16h and 14 days after exposure, whereas by Day 21 testes weights were similar to those in the control group (P=0.18). At 16h, 7 and 14 days after exposure, an increase in germ cell apoptosis was noticeable in early and late stages (I-VI and XI-XII) of the cycle of the seminiferous epithelium. However, apoptosis in intermediate stages (VII-X) was evident 16h after heat exposure (P<0.05), without any change at other time periods. By 21 days, there were no significant differences between heat-treated groups and controls. Considerably more caspase-3-positive germ cells occurred in heat-treated mice 16h after heat exposure compared with the control group (P<0.0001), whereas 8h after heat in Experiment 2, sperm motility was reduced with a higher percentage of spermatozoa showing membrane damage. In conclusion, the present study shows that whole-body heat of 37-38°C induces stage-specific germ cell apoptosis and membrane changes in spermatozoa; this may result in reduced fertility at particular times of exposure after heating.

Dopamine treatment attenuates acute kidney injury in a rat model of deep hypothermia and rewarming - The role of renal H2S-producing enzymes.

Hypothermia and rewarming produces organ injury through the production of reactive oxygen species. We previously found that dopamine prevents hypothermia and rewarming-induced apoptosis in cultured cells through increased expression of the H2S-producing enzyme cystathionine ß-Synthase (CBS). Here, we investigate whether dopamine protects the kidney in deep body cooling and explore the role of H2S-producing enzymes in an in vivo rat model of deep hypothermia and rewarming. In anesthetized Wistar rats, body temperature was decreased to 15°C for 3h, followed by rewarming for 1h. Rats (n≥5 per group) were treated throughout the procedure with vehicle or dopamine infusion, and in the presence or absence of a non-specific inhibitor of H2S-producing enzymes, amino-oxyacetic acid (AOAA). Kidney damage and renal expression of three H2S-producing enzymes (CBS, CSE and 3-MST) was quantified and serum H2S level measured. Hypothermia and rewarming induced renal damage, evidenced by increased serum creatinine, renal reactive oxygen species production, KIM-1 expression and influx of immune cells, which was accompanied by substantially lowered renal expression of CBS, CSE, and 3-MST and lowered serum H2S levels. Infusion of dopamine fully attenuated renal damage and maintained expression of H2S-producing enzymes, while normalizing serum H2S. AOAA further decreased the expression of H2S-producing enzymes and serum H2S level, and aggravated renal damage. Hence, dopamine preserves renal integrity during deep hypothermia and rewarming likely by maintaining the expression of renal H2S-producing enzymes and serum H2S.

Endocannabinoid Catabolic Enzymes Play Differential Roles in Thermal Homeostasis in Response to Environmental or Immune Challenge.

Cannabinoid receptor agonists, such as Δ(9)-THC, the primary active constituent of Cannabis sativa, have anti-pyrogenic effects in a variety of assays. Recently, attention has turned to the endogenous cannabinoid system and how endocannabinoids, including 2-arachidonoylglycerol (2-AG) and anandamide, regulate multiple homeostatic processes, including thermoregulation. Inhibiting endocannabinoid catabolic enzymes, monoacylglycerol lipase (MAGL) or fatty acid amide hydrolase (FAAH), elevates levels of 2-AG or anandamide in vivo, respectively. The purpose of this experiment was to test the hypothesis that endocannabinoid catabolic enzymes function to maintain thermal homeostasis in response to hypothermic challenge. In separate experiments, male C57BL/6J mice were administered a MAGL or FAAH inhibitor, and then challenged with the bacterial endotoxin lipopolysaccharide (LPS; 2 mg/kg ip) or a cold (4 °C) ambient environment. Systemic LPS administration caused a significant decrease in core body temperature after 6 h, and this hypothermia persisted for at least 12 h. Similarly, cold environment induced mild hypothermia that resolved within 30 min. JZL184 exacerbated hypothermia induced by either LPS or cold challenge, both of which effects were blocked by rimonabant, but not SR144528, indicating a CB1 cannabinoid receptor mechanism of action. In contrast, the FAAH inhibitor, PF-3845, had no effect on either LPS-induced or cold-induced hypothermia. These data indicate that unlike direct acting cannabinoid receptor agonists, which elicit profound hypothermic responses on their own, neither MAGL nor FAAH inhibitors affect normal body temperature. However, these endocannabinoid catabolic enzymes play distinct roles in thermoregulation following hypothermic challenges.

Cardiac-specific VLCAD deficiency induces dilated cardiomyopathy and cold intolerance.

The very long-chain acyl-CoA dehydrogenase (VLCAD) enzyme catalyzes the first step of mitochondrial ß-oxidation. Patients with VLCAD deficiency present with hypoketotic hypoglycemia and cardiomyopathy, which can be exacerbated by fasting and/or cold stress. Global VLCAD knockout mice recapitulate these phenotypes: mice develop cardiomyopathy, and cold exposure leads to rapid hypothermia and death. However, the contribution of different tissues to development of these phenotypes has not been studied. We generated cardiac-specific VLCAD-deficient (cVLCAD(-/-)) mice by Cre-mediated ablation of the VLCAD in cardiomyocytes. By 6 mo of age, cVLCAD(-/-) mice demonstrated increased end-diastolic and end-systolic left ventricular dimensions and decreased fractional shortening. Surprisingly, selective VLCAD gene ablation in cardiomyocytes was sufficient to evoke severe cold intolerance in mice who rapidly developed severe hypothermia, bradycardia, and markedly depressed cardiac function in response to fasting and cold exposure (+5°C). We conclude that cardiac-specific VLCAD deficiency is sufficient to induce cold intolerance and cardiomyopathy and is associated with reduced ATP production. These results provide strong evidence that fatty acid oxidation in myocardium is essential for maintaining normal cardiac function under these stress conditions.

[Nonspecific effect of Na+/K(+)-ATPase inhibition with strophanthin or under hypothermia in rat heart].

Electron probe microanalysis was applied to study the kinetics of changes in potassium and sodium concentration in muscle cells of isolated heart from Wistar rat during experimental ischemia. Hypoxic perfusion without glucose was shown to evoke the potassium deficiency and sodium accumulation in cardiac myocells. Short-term action (10 min) of strophanthin (0.1 mM/l) recovered Na/K balance in ischemic myocells. Hypothermic perfusion exhibited the opportunity to conserve the cytoplasmic elemental contents in the state corresponding to the beginning of low temperature (4 degrees C) operation.

Deep hypothermia protects against acute hypoxia in vivo in rats: a mechanism related to the attenuation of oxidative stress.

There is growing interest in using hypothermia to prevent hypoxic damage in clinical and experimental models, although the mechanisms regulated by hypothermia are still unclear. As reactive oxygen and nitrogen species are the main factors causing cellular damage, our objective was to study the scope of hypothermia in preventing hypoxia-induced oxidative damage. We analysed systemic and hepatic indicators of oxidative stress after an acute hypoxic insult (10% oxygen in breathing air) in normothermic (37°C body temperature) and hypothermic conditions (22°C) in rats. Exposure to hypoxia resulted in tissue damage (aspartate aminotransferase increased from 54.6 ± 6.9 U l(-1) in control animals to 116 ± 1.9 U l(-1) in hypoxia, and alanine aminotransferase increased from 19 ± 0.8 to 34 ± 2.9 U l(-1)), oxidative stress (nitric oxide metabolites increased from 10.8 ± 0.4 µM in control rats to 23 ± 2.7 µM in hypoxia, and thiobarbituric reactive substances increased from 3.3 ± 0.2 to 5.9 ± 0.4 nm) and antioxidant consumption (reduced/oxidized glutathione ratio changed from 9.8 ± 0.3 to 6.8 ± 0.3). In contrast, when hypothermia was applied prior to hypoxia, the situation was reversed, with a reduction in aspartate aminotransferase (from 116 ± 1.9 in hypoxic animals to 63 ± 7.8 U l(-1) in animals exposed to hypothermia followed by hypoxia), alanine aminotransferase (from 34 ± 2.9 to 19 ± 0.9 U l(-1)), oxidative stress (nitric oxide metabolites decreased from 23 ± 2.7 to 17.8 ± 1.9 µM and thiobarbituric acid-reactive substances decreased from 5.9 ± 0.4 to 4.3 ± 0.2 nm) and antioxidant preservation (reduced/oxidized glutathione ratio changed from 6.8 ± 0.3 to 11.1 ± 0.1). Hypoxia induced a decrease in liver enzymatic antioxidant activities even during hypothermia. Both treatments, hypoxia and hypothermia, produced a similar increase in hepatic caspase-3 activity. In conclusion, hypothermia prevented the tissue damage and oxidative stress elicited by hypoxia. Our results provide new evidence concerning the protective mechanism of hypothermia in vivo.

Hypothermia-induced hyperphosphorylation: a new model to study tau kinase inhibitors.

Tau hyperphosphorylation is one hallmark of Alzheimer's disease (AD) pathology. Pharmaceutical companies have thus developed kinase inhibitors aiming to reduce tau hyperphosphorylation. One obstacle in screening for tau kinase inhibitors is the low phosphorylation levels of AD-related phospho-epitopes in normal adult mice and cultured cells. We have shown that hypothermia induces tau hyperphosphorylation in vitro and in vivo. Here, we hypothesized that hypothermia could be used to assess tau kinase inhibitors efficacy. Hypothermia applied to models of biological gradual complexity such as neuronal-like cells, ex vivo brain slices and adult non-transgenic mice leads to tau hyperphosphorylation at multiple AD-related phospho-epitopes. We show that Glycogen Synthase Kinase-3 inhibitors LiCl and AR-A014418, as well as roscovitine, a cyclin-dependent kinase 5 inhibitor, decrease hypothermia-induced tau hyperphosphorylation, leading to different tau phosphorylation profiles. Therefore, we propose hypothermia-induced hyperphosphorylation as a reliable, fast, convenient and inexpensive tool to screen for tau kinase inhibitors.

[Ishemic-reperfusion syndrome prophylaxis in general hypothermia in experiment].

The influence of general supercooling on rats were studied. The action of corvitin and pentoxiphillin on the prooxidant-antioxidant homeostasis for conditions of general supercooling were analysed. On the based of investigations results analysis of lipids peroxide oxidation indexes and antioxidant protection system in animals it were established, that corvitin in difference of pentoxiphillin give more significant protectory effect in conditions of ishemic-reperfusion syndrome.

Physostigmine-induced hypothermic response in rats and its relationship with endogenous arginine vasopressin.

AIMS: It is well known that physostigmine (PHY) and other anticholinesterase (anti-ChE) agents induce hypothermia in rodents but little is known about the mechanism of action. Because arginine vasopressin (AVP) has been found to be an endogenous antipyretic molecule in the CNS, we determined if PHY-induced hypothermia is linked to the endogenous release of AVP. MAIN METHODS: Core temperature and motor activity were monitored by telemetry in rats maintained at an ambient temperature of 25 degrees C. Tail skin temperature was also measured at 30min intervals to estimate nonevaporative heat loss. The central cholinergic antagonist, scopolamine (1mg/kg; ip) and an AVP V(1) receptor antagonist (30microg/kg; ip) were administered during the period of PHY (200microg/kg; sc) induced hypothermia at 10am. Plasma AVP concentration and plasma cholinesterase (ChE) activity were measured at 50min after administration of PHY or scopolamine, respectively. KEY FINDINGS: PHY led to a rapid reduction in core temperature concomitant with a marked increase in heat loss from the tail. The hypothermic response of PHY was blocked by the AVP V(1) receptor antagonist. Administration of scopolamine also reversed the hypothermic responses and led to marked elevations in motor activity. Plasma AVP levels increased markedly at 50min after PHY and plasma ChE activity was significantly reduced by PHY. SIGNIFICANCE: The results clearly demonstrate that PHY-induced hypothermia was blocked by the AVP V(1) antagonist and associated with elevations in plasma AVP, suggesting a novel role for AVP in the mechanism of action of anti-ChE agents.

Ketorolac alters blood flow during normothermia but not during hyperthermia in middle-aged human skin.

In young healthy humans full expression of reflex cutaneous vasodilation is dependent on cyclooxygenase (COX)- and nitric oxide synthase (NOS)-dependent mechanisms. Chronic low-dose aspirin therapy attenuates reflex cutaneous vasodilation potentially through both platelet and vascular COX-dependent mechanisms. We hypothesized the contribution of COX-dependent vasodilators to reflex cutaneous vasodilation during localized acute COX inhibition would be attenuated in healthy middle-aged humans due to a shift toward COX-dependent vasoconstrictors. Four microdialysis fibers were placed in forearm skin of 13 middle-aged (53 +/- 2 yr) normotensive healthy humans, serving as control (Ringer), COX-inhibited (10 mM ketorolac), NOS-inhibited (10 mM N(G)-nitro-l-arginine methyl ester), and combined NOS- and COX-inhibited sites. Red blood cell flux was measured over each site by laser-Doppler flowmetry as reflex vasodilation was induced by increasing oral temperature (T(or)) 1.0 degrees C using a water-perfused suit. Cutaneous vascular conductance was calculated (CVC = flux/mean arterial pressure) and normalized to maximal CVC (CVC(max); 28 mM sodium nitroprusside). CVC(max) was not affected by localized microdialysis drug treatment (P > 0.05). Localized COX inhibition increased baseline (18 +/- 3%CVC(max); P < 0.001) compared with control (9 +/- 1%CVC(max)), NOS-inhibited (7 +/- 1%CVC(max)), and combined sites (10 +/- 1%CVC(max)). %CVC(max) in the COX-inhibited site remained greater than the control site with DeltaT(or) < or = 0.3 degrees C; however, there was no difference between these sites with DeltaT(or) > or = 0.4 degrees C. NOS inhibition and combined COX and NOS inhibition attenuated reflex vasodilation compared with control (P < 0.001), but there was no difference between these sites. Localized COX inhibition with ketorolac significantly augments baseline CVC but does not alter the subsequent skin blood flow response to hyperthermia, suggesting a limited role for COX-derived vasodilator prostanoids in reflex cutaneous vasodilation and a shift toward COX-derived vasoconstrictors in middle-aged human skin.

Specific p38 inhibition in stimulated endothelial cells: a possible new anti-inflammatory strategy after hypothermia and rewarming.

To protect immature organ systems during corrective cardiac surgery, patients are cooled to a minimal temperature of 17 degrees C during cardiopulmonary bypass (CPB). However hypothermic CPB triggers the whole body inflammatory response and results in unwanted prolonged inflammation. The present study was designed to clarify the hypothermia and rewarming induced mechanisms and examine interventional pharmacological strategies that could prevent prolonged inflammation. Stimulated primary human umbilical vein endothelial cells (HUVECs) were exposed to a dynamic temperature protocol analogous to clinical settings. Furthermore endothelial cells were pretreated with methylprednisolone and/or tacrolimus as well as with MAPK inhibitors (SB203580, U0126 and SP600125). Cell viability, expression of IL-6 and ERK 1/2, p38 and SAPK/JNK were investigated. Stimulated endothelial cells secreted significantly higher IL-6 protein 2h after rewarming in comparison to normothermic control cells. Moreover, dynamic temperature changes lead to increased MAPK phosphorylation. Only the combined pre-treatment with MP and TAC served to inhibit the IL-6 secretion. As intracellular signalling pathway we could demonstrate that SB203580 as specific p38 inhibitor most effectively down regulated the unwanted IL-6 release after cooling and rewarming. Therefore inhibition of p38 or components of the p38 pathway could be a promising and selective antiinflammatory therapeutic target after hypothermic CPB.

Traumatic injury activates MAP kinases in astrocytes: mechanisms of hypothermia and hyperthermia.

Hyperthermia is common following traumatic brain injury (TBI) and has been associated with poor neurologic outcome, and hypothermia has emerged as a potentially effective therapy for TBI, although its mechanism is still unclear. In this study we investigated the effects of temperature modulations on astrocyte survival following traumatic injury and the involved MAPK pathways. Trauma was produced by scratch injury of a monolayer of confluent astrocytes in culture, followed by incubation at hypothermia (308 degree C), normothermia (378 degree C), or hyperthermia (398 degree C). The activation of MAPK pathways including extracellular signal-regulated protein kinase (ERK), c-Jun NH(2)-terminal kinase ( JNK), and p38 MAPK were measured at 0, 15, 30, 60, and 120 min after traumatic injury followed by temperature modulation. Apoptosis of astrocytes was assessed by quantitation of cleaved caspase-3 expression 24 h after injury. Our findings showed that only JNK activation at 15 min after trauma was reduced by hypothermia, and this was associated with a marked reduction in apoptosis. Hyperthermia activated both ERK and JNK and increased apoptosis. The specific JNK inhibitor, SP60025, markedly reduced JNK-induced apoptosis at normothermia and hyperthermia, and showed a dose-dependent effect. In conclusion, the JNK pathway appears to mediate traumatic injury-induced apoptosis in astrocytes. Prolonged hyperthermia as a secondary insult worsens apoptosis by increasing JNK activation. Hypothermia protects against traumatic injury via early suppression on JNK activation and subsequent prevention of apoptosis. Manipulation of the JNK pathway in astrocytes may represent a therapeutic target for ameliorating the devastating progression of tissue injury and cell death after TBI.

[Functioning of tyrosine protein kinases and phosphatases in gastric mucosa cells under conditions of oxidative and nitrosative stress in gastric lesions].

Acute cold stress caused lesions of gastric mucosa as a result of its attack by active oxygen and nitrogen compounds. The tissue regeneration is regulated by a cascade of tyrosine protein kinases. Gastric ulceration leads to a decrease in activity of tyrosine protein kinases and phosphatases, following by fall in phosphotyrosine content in proteins of plasma membranes of gastric mucosa cells. No changes in superoxide dismutase activity, slight increase in catalase activity, inhibition of glutathione peroxydase, significant increase in OH* content and decrease in zinc level were observed in the gastric mucosa cells of stressed rats. That increased oxidative damage can lead to inactivation of protein tyrosine phosphatases. Nitric oxide synthase activity was three times higher in gastric mucosa cells after the cold stress. That can promote nitrosylation of tyrosine residues. During following days nitric oxide synthase activity remains high. Superoxide dismutase is activated on the 4 and 5th day after the stress. Catalase activity normalizes after second day. Tyrosine protein kinase activity increases in membranes with maximum on the 4th day, and remains inhibited in cytosole. Tyrosine protein phosphatases keep inhibited as well. Gluthatione peroxydase activity and zinc level decreased on the 5th day. Obtained results can be the evidence of violations in signal transduction through protein tyrosine kinase cascades, due to the reduction in tyrosine phosphorylation, as a result of increase in the content of active oxygen and nitrogen species.

Increased neuronal injury in transgenic mice with neuronal overexpression of human cyclooxygenase-2 is reversed by hypothermia and rofecoxib treatment.

Cyclooxygenase-2 (COX-2) is up-regulated during ischemia. However, the role of COX-2 in neuronal injury is still unclear. In this study we tested whether neuronal overexpression of human COX-2 in a transgenic mouse model potentiates neuronal injury after global ischemic insult. Further, we tested whether the neuronal injury could be ameliorated by intra-ischemic mild hypothermia (33-34 degrees C) alone or in combination with diet treatment of rofecoxib, a COX-2 specific inhibitor. Global ischemia with intra-ischemic normothermia (36-37 degrees C) resulted in significantly higher neuronal damage in the CA1 region of hippocampus of transgenic mice than in wild type controls, confirming a deleterious role of COX-2 in ischemic neuronal damage. Hypothermia significantly reduced neuronal damage in both transgenic mice and wild type controls to the same extent, suggesting that the aggravating effect of COX-2 could be largely eliminated by hypothermia. When hypothermia was combined with rofecoxib treatment, neuronal damage was further reduced in response to global ischemia. The results suggest that COX-2 inhibition by prophylactic treatment with rofecoxib coupled with hypothermia at the time of acute stroke insult could be an effective therapeutic approach in early stages of stroke treatment in high risk patients.

The role of protein kinase A in acute ethanol-induced neurobehavioral actions in rats.

BACKGROUND: cAMP-dependent protein kinase (PKA) signaling pathways are involved in the regulation of ethanol-induced sedative effects in knockout mouse models. In the present study, we examined the role of PKA on the behavioral action caused by ethanol in Sprague Dawley rats. METHODS: A loss of righting reflex (LORR) test was used to study the acute sedative effects of intraperitoneally injected ethanol. Rotarod performance was used to study the motor impairment caused by ethanol. Convulsions induced by intracerebroventricular (ICV) N-methyl-d-aspartate (NMDA) were used to evaluate ethanol's effect on NMDA receptors. Western blot analysis was used to assay protein levels for NR1 and phosphoserine 897 on NR1 subnuits. RESULTS: ICV pretreatment with H-9 (a nonspecific PK inhibitor) or KT 5720 (a specific PKA inhibitor) dose-dependently attenuated ethanol-induced sleeping time as assessed by LORR. ICV KT 5720 did not reduce ketamine or pentobarbital-induced sleeping time. Pretreatment with forskolin (an activator of adenylyl cyclase) or chelerythrine (a selective PKC inhibitor) had no effect on ethanol-induced LORR. Ethanol-induced motor impairment was also attenuated after pretreatment with KT 5720. Ethanol significantly inhibited NMDA-induced convulsions; the inhibitory effects of ethanol were reduced by prior ICV KT 5720, which had no significant effects on the levels of phosphoserine 897 on NMDA NR1 subunits in the several brain areas we examined. CONCLUSIONS: Our results suggest that the PKA pathway may participate in ethanol-induced neurobehavioral changes and that NMDA receptors may be involved in the PKA regulation of ethanol's actions.