A major role for the EP4 receptor in regulation of renin.

Prostaglandins have been implicated as paracrine regulators of renin secretion, but the specific pathways and receptor(s) carrying out these functions have not been fully elucidated. To examine the contributions of prostanoid synthetic pathways and receptors to regulation of renin in the intact animal, we used a panel of mice with targeted disruption of several key genes: cyclooxygenase-2 (COX-2), microsomal PGE synthases 1 and 2 (mPGES1, mPGES2), EP2 and EP4 receptors for PGE(2), and the IP receptor for PGI(2). To activate the macula densa signal for renin stimulation, mice were treated with furosemide over 5 days and renin mRNA levels were determined by real-time RT-PCR. At baseline, there were no differences in renin mRNA levels between wild-type and the various strains of mutant mice. Furosemide caused marked stimulation of renin mRNA expression across all groups of wild-type control mice. This response was completely abrogated in the absence of COX-2, but was unaffected in mice lacking mPGES1 or mPGES2. The absence of G(s)/cAMP-linked EP2 receptors had no effect on stimulation of renin by furosemide and there was only a modest, insignificant reduction in renin responses in mice lacking the IP receptor. By contrast, renin stimulation in EP4(-/-) mice was significantly reduced by ∼70% compared with wild-type controls. These data suggest that stimulation of renin by the macula densa mechanism is mediated by PGE(2) through a pathway requiring COX-2 and the EP4 receptor, but not EP2 or IP receptors. Surprisingly, mPGES1 or mPGES2 are not required, suggesting other alternative mechanisms for generating PGE(2) in response to macula densa stimulation.

Increased sensitivity to kainic acid in a genetic model of reduced NMDA receptor function.

The pathophysiology of schizophrenia may involve reduced NMDA receptor function and experimental models of NMDA receptor hypofunction have proven useful for characterizing neurobiological abnormalities potentially relevant to schizophrenia. The present study assessed behavioral responses and induction of Fos after administration of kainic acid to wild type mice (NR1(+/+)) and mice with genetically reduced NMDA receptor expression (NR1(neo/neo)). At a dose of 20 mg/kg, kainic acid induced lethal seizures in 100% of the NR1(neo/neo) mice tested but produced no lethal seizures in the wild type mice. The NR1(neo/neo) mice also exhibited enhanced behavioral responses to kainic acid at a dose of 15 mg/kg but no lethal seizures were produced by this dose. A greater induction of Fos was observed in neocortical and limbic cortical regions of the NR1(neo/neo) compared to NR1(+/+) mice after administration of 15 mg/kg kainic acid. In contrast, there were no differences between the genotypes in kainic acid induced Fos in the amygdala, hippocampus, lateral septum, and nucleus accumbens. In order to determine if altered behavioral phenotypes of the NR1(neo/neo) mice could be related to increased sensitivity of kainate receptors to endogenous glutamate, effects of the highly selective kainate antagonist LY382884 were examined. The kainate antagonist reduced the exaggerated acoustic startle responses, deficits in prepulse inhibition of acoustic startle, and motor hyperactivity in the NR1(neo/neo) mice. These findings suggest that selective kainate receptor antagonists could be novel therapeutic candidates for schizophrenia.

Neural activation deficits in a mouse genetic model of NMDA receptor hypofunction in tests of social aggression and swim stress.

Mice with reduced expression of the NR1 subunit of the NMDA receptor (NR1 hypomorphic mice) display altered behavioral phenotypes that may relate to behavioral characteristics of schizophrenia. Altered phenotypes in the NR1 hypomorphs include marked deficits in species-typical behavioral interactions in tests of social aggression and social affiliation. To gain insight into neuroanatomical circuits disrupted by reduced NMDA receptor function, the present work compared regional brain activation in NR1 hypomorphic mice and their wild type controls after a resident-intruder test. Induction of Fos protein was used as an index of neuronal activation. Wild type mice exhibited robust induction of Fos in select brain regions, including specific nuclei of the hypothalamus and amygdala, lateral septum, and widespread regions of the cerebral cortex. Although the behavioral patterns were different for male and female mice, neuroanatomical patterns of Fos induction were remarkably similar for the two sexes. To determine socially specific components of Fos induction by the resident-intruder test, responses were compared for mice assessed in a test of general arousal and stress involving forced swim. Some common brain regions were activated by both tests but regionally specific differences were also found. The NR1 hypomorphic mice tested in the resident-intruder procedure displayed distinctly different behavioral interactions compared to the wild type mice and exhibited a significantly blunted Fos response in almost all brain regions. The mutant mice also exhibited reduced Fos in response to swim stress in specific brain regions. These data suggest that the NR1 hypomorphic mice have functional activation deficits in response to social challenge and swim stress.

Seizure responses and induction of Fos by the NMDA agonist (tetrazol-5-yl)glycine in a genetic model of NMDA receptor hypofunction.

Effects of the direct NMDA agonist (tetrazol-5-yl)glycine (TZG) were examined in a genetic mouse model of reduced NMDA receptor function. In this model, expression of the NR1 subunit is reduced but not eliminated and the mice are therefore designated as NR1 hypomorphic. Previous work suggested that the reduced NR1 subunit expression produced a functional subsensitivity as judged by a blunted Fos induction response to a sub-seizure dose of TZG. In the present study seizure threshold doses of TZG were tested in the wild type and mutant mice. Surprisingly, there was no difference in the seizure sensitivity between the wild type mice and mice presumed to express very low levels of the NR1 subunit. An extensive neuroanatomical analysis of Fos induction was conducted after the threshold seizure doses of TZG. The results demonstrate that some brain regions of the NR1 -/- mice exhibit much lower Fos induction in comparison to the NR1 +/+ mice. These regions include hippocampus, amygdala, and cerebral cortical regions. However, in other regions, similar induction of Fos was observed in both genotypes in response to the NMDA agonist. Regions showing similar Fos induction in the NR1 +/+ and NR1 -/- mice include the lateral septum, nucleus of the solitary tract, and medial hypothalamic regions. The results suggest that the NMDA receptor hypofunction in the NR1 -/- mice is not global but regionally specific and that subcortical structures are responsible for the seizure-inducing effects of TZG.

Expression and function of NPSR1/GPRA in the lung before and after induction of asthma-like disease.

A genetic contribution to asthma susceptibility is well recognized, and linkage studies have identified a large number of genes associated with asthma pathogenesis. Recently, a locus encoding a seven-transmembrane protein was shown to be associated with asthma in founder populations. The expression of the protein GPRA (G protein-coupled receptor for asthma susceptibility) in human airway epithelia and smooth muscle, and its increased expression in a mouse model of asthma, suggested that a gain-of-function mutation in this gene increased the disease risk. However, we report here that the development of allergic lung disease in GPRA-deficient mice is unaltered. A possible explanation for this finding became apparent upon reexamination of the expression of this gene. In contrast to initial studies, our analyses failed to detect expression of GPRA in human lung tissue or in mice with allergic lung disease. We identify a single parameter that distinguishes GPRA-deficient and wild-type mice. Whereas the change in airway resistance in response to methacholine was identical in control and GPRA-deficient mice, the mutant animals showed an attenuated response to thromboxane, a cholinergic receptor-dependent bronchoconstricting agent. Together, our studies fail to support a direct contribution of GPRA to asthma pathogenesis. However, our data suggest that GPRA may contribute to the asthmatic phenotype by altering the activity of other pathways, such as neurally mediated mechanisms, that contribute to disease. This interpretation is supported by high levels of GPRA expression in the brain and its recent identification as the neuropeptide S receptor.

Effects of haloperidol, clozapine, and quetiapine on sensorimotor gating in a genetic model of reduced NMDA receptor function.

RATIONALE: Reduced N-methyl D-aspartate (NMDA) receptor function is hypothesized to contribute to the pathophysiology of schizophrenia. In order to model chronic and developmental NMDA receptor hypofunction, a mouse line was developed that expresses low levels of the NMDA R1 (NR1) subunit of the NMDA receptor. These mice show increased acoustic startle reactivity and deficits in prepulse inhibition (PPI) of acoustic startle. OBJECTIVES: The present study tested the hypothesis that these altered acoustic startle responses in the NR1 hypomorphic (NR1-/-) mice would be affected by antipsychotic drug treatment. METHODS: Mice were injected with drugs 30 min before assessment of acoustic startle responses with and without prepulse stimuli. RESULTS: Haloperidol (0.5 or 1.0 mg/kg) did not reduce the increased startle reactivity in the NR1-/- mice, but did increase PPI in both the mutant and wild type mice. Clozapine (3 mg/kg) and quetiapine (20 mg/kg) reduced startle magnitude and increased PPI in both the wild type and mutant mice. The antidepressant drug imipramine (10 and 20 mg/kg) had minimal effects on startle amplitude in NR1-/- or wild type mice. However, for the 20-mg/kg dose of imipramine, a significant increase in PPI was observed in the wild type animals, but not in the mutant mice. CONCLUSIONS: The results demonstrate that PPI can be increased in a mouse model of chronic NMDA receptor hypofunction by typical and atypical antipsychotic drugs. The similar effects of typical and atypical antipsychotic drugs to increase PPI in the wild type and mutant mice indicates that the assessment of behavior of the NR1 hypomorphic mice in the PPI paradigm offers no advantage over the wild type controls for identifying new clozapine-like drugs.

Proinflammatory actions of thromboxane receptors to enhance cellular immune responses.

Metabolism of arachidonic acid by the cyclo-oxygenase (COX) pathway generates a family of prostanoid mediators. Nonsteroidal anti-inflammatory drugs (NSAIDs) act by inhibiting COX, thereby reducing prostanoid synthesis. The efficacy of these agents in reducing inflammation suggests a dominant proinflammatory role for the COX pathway. However, the actions of COX metabolites are complex, and certain prostanoids, such as PGE(2), in some circumstances actually inhibit immune and inflammatory responses. In these studies, we examine the hypothesis that anti-inflammatory actions of NSAIDs may be due, in part, to inhibition of thromboxane A(2) synthesis. To study the immunoregulatory actions of thromboxane A(2), we used mice with a targeted disruption of the gene encoding the thromboxane-prostanoid (TP) receptor. Both mitogen-induced responses and cellular responses to alloantigen were substantially reduced in TP(-/-) spleen cells. Similar attenuation was observed with pharmacological inhibition of TP signaling in wild-type splenocytes, suggesting that reduced responsiveness was not due to subtle developmental abnormalities in the TP-deficient mice. The absence of TP receptors reduced immune-mediated tissue injury following cardiac transplant rejection, an in vivo model of intense inflammation. Taken together, these findings show that thromboxane augments cellular immune responses and inflammatory tissue injury. Specific inhibition of the TP receptor may provide a more precise approach to limit inflammation without some of the untoward effects associated with NSAIDs.