Destabilization of light NREM sleep by thalamic PLCß4 deletion impairs sleep-dependent memory consolidation.

Sleep abnormality often accompanies the impairment of cognitive function. Both rapid eye movement (REM) and non-REM (NREM) sleep have associated with improved memory performance. However, the role of composition in NREM sleep, consisting of light and deep NREM, for memory formation is not fully understood. We investigated how the dynamics of NREM sleep states influence memory consolidation. Thalamocortical (TC) neuron-specific phospholipase C ß4 (PLCß4) knockout (KO) increased the total duration of NREM sleep, consisting of destabilized light NREM and stabilized deep NREM. Surprisingly, the longer NREM sleep did not improve memory consolidation but rather impaired it in TC-specific PLCß4 KO mice. Memory function was positively correlated with the stability of light NREM and spindle activity occurring in maintained light NREM period. Our study suggests that a single molecule, PLCß4, in TC neurons is critical for tuning the NREM sleep states and thus affects sleep-dependent memory formation.

Phospholipase C-ß1 potentiates glucose-stimulated insulin secretion.

PLC-ß exerts biologic influences through GPCR. GPCRs are involved in regulating glucose-stimulated insulin secretion (GSIS). Previous studies have suggested that PLC-ßs might play an important role in pancreatic ß cells. However, because of a lack of the specific inhibitors of PLC-ß isozymes and appropriate genetic models, the in vivo function of specific PLC-ß isozymes in pancreatic ß cells and their physiologic relevance in the regulation of insulin secretion have not been studied so far. The present study showed that PLC-ß1 was crucial for ß-cell function by generation of each PLC-ß conditional knockout mouse. Mice lacking PLC-ß1 in ß cells exhibited a marked defect in GSIS, leading to glucose intolerance. In ex vivo studies, the secreted insulin level and Ca2+ response in Plcb1f/f; pancreas/duodenum homeobox protein 1 (Pdx1)-Cre recombinase-estrogen receptor T2 (CreERt2) islets was lower than those in the Plcb1f/f islets under the high-glucose condition. PLC-ß1 led to potentiate insulin secretion via stimulation of particular Gq-protein-coupled receptors. Plcb1f/f; Pdx1-CreERt2 mice fed a high-fat diet developed more severe glucose intolerance because of a defect in insulin secretion. The present study identified PLC-ß1 as an important molecule that regulates ß cell insulin secretion and can be considered a candidate for therapeutic intervention in diabetes mellitus.-Hwang, H.-J., Yang, Y. R., Kim, H. Y., Choi, Y., Park, K.-S., Lee, H., Ma, J. S., Yamamoto, M., Kim, J., Chae, Y. C., Choi, J. H., Cocco, L., Berggren, P.-O., Jang, H.-J., Suh, P.-G. Phospholipase Cß1 potentiates glucose-stimulated insulin secretion.

High-Frequency Neuronal Oscillatory Abnormalities in the Phospholipase C-ß1 Knockout Mouse Model of Schizophrenia.

BACKGROUND: Schizophrenia is a complex neuropsychiatric disorder characterized by psychoses, socioaffective disturbances, and cognitive deficits. The phosphodiesterase enzyme phospholipase C-ß1 has been reported to be reduced in postmortem tissue of schizophrenia patients. Dysregulation of neuronal oscillations, particularly those in the higher frequency range such as beta (12-30 Hz) and gamma (30-80 Hz), are also associated with this disorder. We investigated the influence of phospholipase C-ß1 gene deletion on cortical oscillatory activity and sensorimotor gating behavior. METHODS: Adult phospholipase C-ß1 knockout and wild-type C57Bl/6J control mice (total n = 26) underwent surgical implantation of extradural electrodes to allow electrocorticography recordings. Electrocorticography was recorded during prepulse inhibition behavior sessions to measure ongoing and auditory-evoked electrophysiological responses. Mice were also pretreated with antipsychotic drugs haloperidol (0.25 mg/kg), clozapine (2.5 mg/kg), and olanzapine (5 mg/kg). RESULTS: Phospholipase C-ß1 knockout mice exhibited reduced prepulse inhibition and diminished power and phase synchrony of beta and gamma oscillatory responses to auditory stimuli as well as elevated ongoing beta oscillations. Reductions in prepulse inhibition were highly correlated with the power and phase synchrony of evoked oscillations. Clozapine and olanzapine ameliorated the prepulse inhibition deficit in phospholipase C-ß1 knockout mice, but not the electrophysiology abnormalities. CONCLUSIONS: Phospholipase C-ß1 reduction leads to disturbances to beta and gamma oscillatory dynamics and prepulse inhibition behavior. The strong relationships between these measures demonstrate the importance of event-related oscillatory activity to sensorimotor gating behavior. However, dissociation of these measures observed in the drug studies suggests that abnormalities in neuronal networks may not necessarily need to be corrected for behavioral improvement.

PLCB3 Loss of Function Reduces Pseudomonas aeruginosa-Dependent IL-8 Release in Cystic Fibrosis.

The lungs of patients with cystic fibrosis (CF) are characterized by an exaggerated inflammation driven by secretion of IL-8 from bronchial epithelial cells and worsened by Pseudomonas aeruginosa infection. To identify novel antiinflammatory molecular targets, we previously performed a genetic study of 135 genes of the immune response, which identified the c.2534C>T (p.S845L) variant of phospholipase C-ß3 (PLCB3) as being significantly associated with mild progression of pulmonary disease. Silencing PLCB3 revealed that it potentiates the Toll-like receptor's inflammatory signaling cascade originating from CF bronchial epithelial cells. In the present study, we investigated the role of the PLCB3-S845L variant together with two synthetic mutants paradigmatic of impaired catalytic activity or lacking functional activation in CF bronchial epithelial cells. In experiments in which cells were exposed to P. aeruginosa, the supernatant of mucopurulent material from the airways of patients with CF or different agonists revealed that PLCB3-S845L has defects of 1) agonist-induced Ca2+ release from endoplasmic reticulum and rise of Ca2+ concentration, 2) activation of conventional protein kinase C isoform ß, and 3) induction of IL-8 release. These results, besides identifying S845L as a loss-of-function variant, strengthen the importance of targeting PLCB3 to mitigate the CF inflammatory response in bronchial epithelial cells without blunting the immune response.

The thalamic mGluR1-PLCß4 pathway is critical in sleep architecture.

The transition from wakefulness to a nonrapid eye movement (NREM) sleep state at the onset of sleep involves a transition from low-voltage, high-frequency irregular electroencephalography (EEG) waveforms to large-amplitude, low-frequency EEG waveforms accompanying synchronized oscillatory activity in the thalamocortical circuit. The thalamocortical circuit consists of reciprocal connections between the thalamus and cortex. The cortex sends strong excitatory feedback to the thalamus, however the function of which is unclear. In this study, we investigated the role of the thalamic metabotropic glutamate receptor 1 (mGluR1)-phospholipase C ß4 (PLCß4) pathway in sleep control in PLCß4-deficient (PLCß4-/-) mice. The thalamic mGluR1-PLCß4 pathway contains synapses that receive corticothalamic inputs. In PLCß4-/- mice, the transition from wakefulness to the NREM sleep state was stimulated, and the NREM sleep state was stabilized, which resulted in increased NREM sleep. The power density of delta (δ) waves increased in parallel with the increased NREM sleep. These sleep phenotypes in PLCß4-/- mice were consistent in TC-restricted PLCß4 knockdown mice. Moreover, in vitro intrathalamic oscillations were greatly enhanced in the PLCß4-/- slices. The results of our study showed that thalamic mGluR1-PLCß4 pathway was critical in controlling sleep architecture.

Knockdown of phospholipase C-ß1 in the medial prefrontal cortex of male mice impairs working memory among multiple schizophrenia endophenotypes.

BACKGROUND: Decreased expression of phospholipase C-ß1 (PLC-ß1) has been observed in the brains of patients with schizophrenia, but, to our knowledge, no studies have shown a possible association between this altered PLC-ß1 expression and the pathogenesis of schizophrenia. Although PLC-ß1-null (PLC-ß1(-/-)) mice exhibit multiple endophenotypes of schizophrenia, it remains unclear how regional decreases in PLC-ß1 expression in the brain contribute to specific behavioural defects. METHODS: We selectively knocked down PLC-ß1 in the medial prefrontal cortex (mPFC) using a small hairpin RNA strategy in mice. RESULTS: Silencing PLC-ß1 in the mPFC resulted in working memory deficits, as assayed using the delayed non-match-to-sample T-maze task. Notably, however, other schizophrenia-related behaviours observed in PLC-ß1-/- mice, including phenotypes related to locomotor activity, sociability and sensorimotor gating, were normal in PLC-ß1 knockdown mice. LIMITATIONS: Phenotypes of PLC-ß1 knockdown mice, such as locomotion, anxiety and sensorimotor gating, have already been published in our previous studies. Further, the neural mechanisms underlying the working memory deficit in mice may be different from those in human schizophrenia. CONCLUSION: These results indicate that PLC-ß1 signalling in the mPFC is required for working memory. Importantly, these results support the notion that the decrease in PLC-ß1 expression in the brains of patients with schizophrenia is a pathogenically relevant molecular marker of the disorder.

High salt recruits aversive taste pathways.

In the tongue, distinct classes of taste receptor cells detect the five basic tastes; sweet, sour, bitter, sodium salt and umami. Among these qualities, bitter and sour stimuli are innately aversive, whereas sweet and umami are appetitive and generally attractive to animals. By contrast, salty taste is unique in that increasing salt concentration fundamentally transforms an innately appetitive stimulus into a powerfully aversive one. This appetitive-aversive balance helps to maintain appropriate salt consumption, and represents an important part of fluid and electrolyte homeostasis. We have shown previously that the appetitive responses to NaCl are mediated by taste receptor cells expressing the epithelial sodium channel, ENaC, but the cellular substrate for salt aversion was unknown. Here we examine the cellular and molecular basis for the rejection of high concentrations of salts. We show that high salt recruits the two primary aversive taste pathways by activating the sour- and bitter-taste-sensing cells. We also demonstrate that genetic silencing of these pathways abolishes behavioural aversion to concentrated salt, without impairing salt attraction. Notably, mice devoid of salt-aversion pathways show unimpeded, continuous attraction even to very high concentrations of NaCl. We propose that the 'co-opting' of sour and bitter neural pathways evolved as a means to ensure that high levels of salt reliably trigger robust behavioural rejection, thus preventing its potentially detrimental effects on health.

Optogenetic stimulation of escape behavior in Drosophila melanogaster.

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO(2) avoidance, proboscis extension and giant-fiber mediated startle response. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-ß, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Implantation failure in mice with a disruption in Phospholipase C beta 1 gene: lack of embryonic attachment, aberrant steroid hormone signalling and defective endocannabinoid metabolism.

Phospholipase C beta 1 (PLCß1) is a downstream effector of G-protein-coupled receptor signalling and holds central roles in reproductive physiology. Mice with a disruption in the Plcß1 gene are infertile with pleiotropic reproductive defects, the major reproductive block in females being implantation failure. Here, PLCß1 was demonstrated at the luminal and glandular epithelia throughout the pre- and peri-implantation period, with transient stromal expression during 0.5-1.5 days post coitum (dpc). Examination of implantation sites at 4.5 dpc showed that in females lacking functional PLCß1 (knock-out (KO) females), embryos failed to establish proper contact with the uterine epithelium. Proliferating luminal epithelial cells were evident in KO implantation sites, indicating failure to establish a receptive uterus. Real-time PCR demonstrated that KO implantation sites had aberrant ovarian steroid signalling, with high levels of estrogen receptor α, lactoferrin and amphiregulin mRNA, while immunohistochemistry revealed very low levels of estrogen receptor α protein, possibly due to rapid receptor turnover. KO implantation sites expressed markedly less fatty acid amide hydrolase and monoacylglycerol lipase, indicating that endocannabinoid metabolism was also affected. Collectively, our results show that PLCß1 is essential for uterine preparation for implantation, and that defective PLCß1-mediated signalling during implantation is associated with aberrant ovarian steroid signalling and endocannabinoid metabolism.

Increased adult hippocampal neurogenesis and abnormal migration of adult-born granule neurons is associated with hippocampal-specific cognitive deficits in phospholipase C-ß1 knockout mice.

Schizophrenia is a devastating psychiatric illness with a complex pathophysiology. We have recently documented schizophrenia-like endophenotypes in phospholipase C-ß1 knockout (PLC-ß1(-/-)) mice, including deficits in prepulse inhibition, hyperlocomotion, and cognitive impairments. PLC-ß1 signals via multiple G-protein coupled receptor pathways implicated in neural cellular plasticity; however, adult neurogenesis has yet to be explored in this knockout model. In this study, we employed PLC-ß1(-/-) mice to elucidate possible correlates between aberrant adult hippocampal neurogenesis (AHN) and schizophrenia-like behaviors. Using stereology and bromodeoxyuridine (BrdU) immunohistochemistry we demonstrated a significant increase in the density of adult-generated cells in the granule cell layer (GCL) of adult PLC-ß1(-/-) mice compared with wild-type littermates. Cellular phenotype analysis using confocal microscopy revealed these cells to be mature granule neurons expressing NeuN and calbindin. Increased neuronal survival occurred concomitant with reduced caspase-3(+) cells in the GCL of PLC-ß1(-/-) mice. Stereological analysis of Ki67(+) cells in the subgranular zone suggested that neural precursor proliferation is unchanged in PLC-ß1(-/-) mice. We further showed aberrant migration of mature granule neurons within the GCL of adult PLC-ß1(-/-) mice with excessive adult-generated mature neurons residing in the middle and outer GCL. PLC-ß1(-/-) mice exhibited specific behavioral deficits in location recognition, a measure of hippocampal-dependent memory, but not novel object recognition. Overall, we have shown that PLC-ß1(-/-) mice have a threefold increase in net AHN, and have provided further evidence to suggest a specific deficit in hippocampal-dependent cognition. We propose that abnormal cellular plasticity in these mice may contribute to their schizophrenia-like behavioral endophenotypes.

A PLCß/PI3Kγ-GSK3 signaling pathway regulates cofilin phosphatase slingshot2 and neutrophil polarization and chemotaxis.

Neutrophils, in response to a chemoattractant gradient, undergo dynamic F-actin remodeling, a process important for their directional migration or chemotaxis. However, signaling mechanisms for chemoattractants to regulate the process are incompletely understood. Here, we characterized chemoattractant-activated signaling mechanisms that regulate cofilin dephosphorylation and actin cytoskeleton reorganization and are critical for neutrophil polarization and chemotaxis. In neutrophils, chemoattractants induced phosphorylation and inhibition of GSK3 via both PLCß-PKC and PI3Kγ-AKT pathways, leading to the attenuation of GSK3-mediated phosphorylation and inhibition of the cofilin phosphatase slingshot2 and an increase in dephosphorylated, active cofilin. The relative contribution of this GSK3-mediated pathway to neutrophil chemotaxis regulation depended on neutrophil polarity preset by integrin-induced polarization of PIP5K1C. Therefore, our study characterizes a signaling mechanism for chemoattractant-induced actin cytoskeleton remodeling and elucidates its context-dependent role in regulating neutrophil polarization and chemotaxis.

Bidirectional modulation of fear extinction by mediodorsal thalamic firing in mice.

The mediodorsal thalamic nucleus has been implicated in the control of memory processes. However, the underlying neural mechanism remains unclear. Here we provide evidence for bidirectional modulation of fear extinction by the mediodorsal thalamic nucleus. Mice with a knockout or mediodorsal thalamic nucleus-specific knockdown of phospholipase C ß4 exhibited impaired fear extinction. Mutant mediodorsal thalamic nucleus neurons in slices showed enhanced burst firing accompanied by increased T-type Ca(2+) currents; blocking of T channels in vivo rescued the fear extinction. Tetrode recordings in freely moving mice revealed that, during extinction, the single-spike (tonic) frequency of mediodorsal thalamic nucleus neurons increased in wild-type mice, but was static in mutant mice. Furthermore, tonic-evoking microstimulations of the mediodorsal thalamic nucleus, contemporaneous with the extinction tones, rescued fear extinction in mutant mice and facilitated it in wild-type mice. In contrast, burst-evoking microstimulation suppressed extinction in wild-type mice, mimicking the mutation. These results suggest that the firing mode of the mediodorsal thalamic nucleus is critical for the modulation of fear extinction.

Phospholipase C-ß3 regulates FcɛRI-mediated mast cell activation by recruiting the protein phosphatase SHP-1.

Mast cells are major effectors in high-affinity IgE receptor (FcɛRI)-dependent allergic reactions. Here we show that phospholipase C (PLC)-ß3 is crucial for FcɛRI-mediated mast cell activation. Plcb3(-/-) mice showed blunted FcɛRI-dependent late-phase, but not acute, anaphylactic responses and airway inflammation. Accordingly, FcɛRI stimulation of Plcb3(-/-) mast cells exhibited reduced cytokine production but normal degranulation. Reduced cytokine production in Plcb3(-/-) cells could be accounted for by increased activity of the negative regulatory Src family kinase Lyn and reduced activities of the positive regulatory protein kinases MAPKs. Mechanistically, PLC-ß3 constitutively interacts with FcɛRI, Lyn, and SHP-1 (protein phosphatase). SHP-1 probably recognizes its substrates Lyn and MAPKs via the recently described kinase tyrosine-based inhibitory motif, KTIM. Consistent with PLC-ß3- and SHP-1-mediated repression of Lyn activity by dephosphorylation at Tyr396, FcɛRI-mediated phenotypes were similar in Plcb3(-/-) and SHP-1 mutant mast cells. Thus, we have defined a PLC-ß3- and SHP-1-mediated signaling pathway for FcɛRI-mediated cytokine production.

TRPV1-expressing primary afferents generate behavioral responses to pruritogens via multiple mechanisms.

The mechanisms that generate itch are poorly understood at both the molecular and cellular levels despite its clinical importance. To explore the peripheral neuronal mechanisms underlying itch, we assessed the behavioral responses (scratching) produced by s.c. injection of various pruritogens in PLCbeta3- or TRPV1-deficient mice. We provide evidence that at least 3 different molecular pathways contribute to the transduction of itch responses to different pruritogens: 1) histamine requires the function of both PLCbeta3 and the TRPV1 channel; 2) serotonin, or a selective agonist, alpha-methyl-serotonin (alpha-Me-5-HT), requires the presence of PLCbeta3 but not TRPV1, and 3) endothelin-1 (ET-1) does not require either PLCbeta3 or TRPV1. To determine whether the activity of these molecules is represented in a particular subpopulation of sensory neurons, we examined the behavioral consequences of selectively eliminating 2 nonoverlapping subsets of nociceptors. The genetic ablation of MrgprD(+) neurons that represent approximately 90% of cutaneous nonpeptidergic neurons did not affect the scratching responses to a number of pruritogens. In contrast, chemical ablation of the central branch of TRPV1(+) nociceptors led to a significant behavioral deficit for pruritogens, including alpha-Me-5-HT and ET-1, that is, the TRPV1-expressing nociceptor was required, whether or not TRPV1 itself was essential. Thus, TRPV1 neurons are equipped with multiple signaling mechanisms that respond to different pruritogens. Some of these require TRPV1 function; others use alternate signal transduction pathways.

Sensitivity to MK-801 in phospholipase C-ß1 knockout mice reveals a specific NMDA receptor deficit.

Phospholipase C-ß1 (PLC-ß1) is a critical component of multiple signalling pathways downstream of neurotransmitter receptors. Mice lacking this enzyme display a striking behavioural phenotype with relevance to human psychiatric disease. Glutamatergic dysfunction is strongly associated with several abnormal behavioural states and may underlie part of the phenotype of the phospholipase C-ß1 knockout (KO) mouse. A heightened response to glutamatergic psychotomimetic drugs is a critical psychosis-related endophenotype, and in this study it was employed as a correlate of glutamatergic dysfunction. Control (n=8) and PLC-ß1 KO mice (n=6) were treated with MK-801, a NMDA receptor (NMDAR) antagonist, following either standard housing or environmental enrichment, and the motor function and locomotor activity thus evoked was assessed. In addition, MK-801 binding to the NMDAR was evaluated through radioligand autoradiography in post-mortem tissue (on a drug-naive cohort). We have demonstrated a significantly increased sensitivity to the effects of the NMDA antagonist MK-801 in the PLC-ß1 KO mouse. In addition, we found that this mouse line displays reduced hippocampal NMDAR expression, as measured by radioligand binding. We previously documented a reversal of specific phenotypes in this mouse line following housing in an enriched environment. Enrichment did not alter this heightened MK-801 response, nor NMDAR expression, indicating that this therapeutic intervention works on specific pathways only. These findings demonstrate the critical role of the glutamatergic system in the phenotype of the PLC-ß1 KO mouse and highlight the role of these interconnected signalling pathways in schizophrenia-like behavioural disruption. These results also shed further light on the capacity of environmental factors to modulate subsets of these phenotypes.

PLC-beta1 knockout mice as a model of disrupted cortical development and plasticity: behavioral endophenotypes and dysregulation of RGS4 gene expression.

The complexity of the genetics underlying schizophrenia is highlighted by the multitude of molecular pathways that have been reported to be disrupted in the disorder including muscarinic, serotonergic, and glutamatergic signaling systems. It is of interest, therefore, that phospholipase C-beta1 (PLC-beta1) acts as a point of convergence for these pathways during cortical development and plasticity. These signaling pathways, furthermore, are susceptible to modulation by RGS4, one of the more promising candidate genes for schizophrenia. PLC-beta1 knockout mice were behaviorally assessed on tests including fear conditioning, elevated plus maze, and the Y maze. In situ hybridization was used to assess RGS4 expression. We found that PLC-beta1 knockout mice display abnormal anxiety profiles on some, but not all measures assessed, including decreased anxiety on the elevated plus maze. We also show memory impairment and a complete absence of acquisition of hippocampal-dependent fear conditioning. Furthermore, at a molecular level, we demonstrate dramatic changes in expression of RGS4 mRNA in selective regions of the PLC-beta1 knockout mouse brain, particularly the CA1 region of the hippocampus. These results validate the utility of the PLC-beta1 knockout mouse as a model of schizophrenia, including molecular and cellular evidence for disrupted cortical maturation and associated behavioral endophenotypes.

Phospholipase C-beta1 knockout mice exhibit endophenotypes modeling schizophrenia which are rescued by environmental enrichment and clozapine administration.

Phospholipase C-beta1 (PLC-beta1) is a rate-limiting enzyme implicated in postnatal-cortical development and neuronal plasticity. PLC-beta1 transduces intracellular signals from specific muscarinic, glutamate and serotonin receptors, all of which have been implicated in the pathogenesis of schizophrenia. Here, we present data to show that PLC-beta1 knockout mice display locomotor hyperactivity, sensorimotor gating deficits as well as cognitive impairment. These changes in behavior are regarded as endophenotypes homologous to schizophrenia-like symptoms in rodents. Importantly, the locomotor hyperactivity and sensorimotor gating deficits in PLC-beta1 knockout mice are subject to beneficial modulation by environmental enrichment. Furthermore, clozapine but not haloperidol (atypical and typical antipsychotics, respectively) rescues the sensorimotor gating deficit in these animals, suggesting selective predictive validity. We also demonstrate a relationship between the beneficial effects of environmental enrichment and levels of M1/M4 muscarinic acetylcholine receptor binding in the neocortex and hippocampus. Thus we have demonstrated a novel mouse model, displaying disruption of multiple postsynaptic signals implicated in the pathogenesis of schizophrenia, a relevant behavioral phenotype and associated gene-environment interactions.

Protective roles of adenosine A1, A2A, and A3 receptors in skeletal muscle ischemia and reperfusion injury.

Although adenosine exerts cardio-and vasculoprotective effects, the roles and signaling mechanisms of different adenosine receptors in mediating skeletal muscle protection are not well understood. We used a mouse hindlimb ischemia-reperfusion model to delineate the function of three adenosine receptor subtypes. Adenosine A(3) receptor-selective agonist 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (Cl-IBMECA; 0.07 mg/kg ip) reduced skeletal muscle injury with a significant decrease in both Evans blue dye staining (5.4 +/- 2.6%, n = 8 mice vs. vehicle-treated 28 +/- 6%, n = 7 mice, P < 0.05) and serum creatine kinase level (1,840 +/- 910 U/l, n = 13 vs. vehicle-treated 12,600 +/- 3,300 U/l, n = 14, P < 0.05), an effect that was selectively blocked by an A(3) receptor antagonist 3-ethyl-5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS-1191; 0.05 mg/kg). The adenosine A(1) receptor agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA; 0.05 mg/kg) also exerted a cytoprotective effect, which was selectively blocked by the A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 0.2 mg/kg). The adenosine A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680; 0.07 mg/kg)-induced decrease in skeletal muscle injury was selectively blocked by the A(2A) antagonist 2-(2-furanyl)-7-[3-(4-methoxyphenyl)propyl]-7H-pyrazolo[4,3-e] [1,2,4]triazolo[1,5-C]pyrimidin-5-amine (SCH-442416; 0.017 mg/kg). The protection induced by the A(3) receptor was abrogated in phospholipase C-beta2/beta3 null mice, but the protection mediated by the A(1) or A(2A) receptor remained unaffected in these animals. The adenosine A(3) receptor is a novel cytoprotective receptor that signals selectively via phospholipase C-beta and represents a new target for ameliorating skeletal muscle injury.