Transcriptome Analysis Identifies An ASD-Like Phenotype In Oligodendrocytes And Microglia From C58/J Amygdala That Is Dependent On Sex and Sociability.

Background: Autism Spectrum Disorder (ASD) is a group of neurodevelopmental disorders with higher incidence in males and is characterized by atypical verbal/nonverbal communication, restricted interests that can be accompanied by repetitive behavior, and disturbances in social behavior. This study investigated brain mechanisms that contribute to sociability deficits and sex differences in an ASD animal model. Methods: Sociability was measured in C58/J and C57BL/6J mice using the 3-chamber social choice test. Bulk RNA-Seq and snRNA-Seq identified transcriptional changes in C58/J and C57BL/6J amygdala within which DMRseq was used to measure differentially methylated regions in amygdala. Results: C58/J mice displayed divergent social strata in the 3-chamber test. Transcriptional and pathway signatures revealed immune-related biological processes differ between C58/J and C57BL/6J amygdala. Hypermethylated and hypomethylated genes were identified in C58/J versus C57BL/6J amygdala. snRNA-Seq data in C58/J amygdala identified differential transcriptional signatures within oligodendrocytes and microglia characterized by increased ASD risk gene expression and predicted impaired myelination that was dependent on sex and sociability. RNA velocity, gene regulatory network, and cell communication analysis showed diminished oligodendrocyte/microglia differentiation. Findings were verified using bulk RNA-Seq and demonstrated oxytocin's beneficial effects on myelin gene expression. Limitations: Our findings are significant. However, limitations can be noted. The cellular mechanisms linking reduced oligodendrocyte differentiation and reduced myelination to an ASD phenotype in C58/J mice need further investigation. Additional snRNA-Seq and spatial studies would determine if effects in oligodendrocytes/microglia are unique to amygdala or if this occurs in other brain regions. Oxytocin's effects need further examination to understand its potential as an ASD therapeutic. Conclusions: Our work demonstrates the C58/J mouse model's utility in evaluating the influence of sex and sociability on the transcriptome in concomitant brain regions involved in ASD. Our single-nucleus transcriptome analysis elucidates potential pathological roles of oligodendrocytes and microglia in ASD. This investigation provides details regarding regulatory features disrupted in these cell types, including transcriptional gene dysregulation, aberrant cell differentiation, altered gene regulatory networks, and changes to key pathways that promote microglia/oligodendrocyte differentiation. Our studies provide insight into interactions between genetic risk and epigenetic processes associated with divergent affiliative behavior and lack of positive sociability.

Aging reduces liver resiliency by dysregulating Hedgehog signaling.

Older age is a major risk factor for damage to many tissues, including liver. Aging undermines resiliency and impairs liver regeneration. The mechanisms whereby aging reduces resiliency are poorly understood. Hedgehog is a signaling pathway with critical mitogenic and morphogenic functions during development. Recent studies indicate that Hedgehog regulates metabolic homeostasis in adult liver. The present study evaluates the hypothesis that Hedgehog signaling becomes dysregulated in hepatocytes during aging, resulting in decreased resiliency and therefore, impaired regeneration and enhanced vulnerability to damage. Partial hepatectomy (PH) was performed on young and old wild-type mice and Smoothened (Smo)-floxed mice treated with viral vectors to conditionally delete Smo and disrupt Hedgehog signaling specifically in hepatocytes. Changes in signaling were correlated with changes in regenerative responses and compared among groups. Old livers had fewer hepatocytes proliferating after PH. RNA sequencing identified Hedgehog as a top downregulated pathway in old hepatocytes before and after the regenerative challenge. Deleting Smo in young hepatocytes before PH prevented Hedgehog pathway activation after PH and inhibited regeneration. Gene Ontogeny analysis demonstrated that both old and Smo-deleted young hepatocytes had activation of pathways involved in innate immune responses and suppression of several signaling pathways that control liver growth and metabolism. Hedgehog inhibition promoted telomere shortening and mitochondrial dysfunction in hepatocytes, consequences of aging that promote inflammation and impair tissue growth and metabolic homeostasis. Hedgehog signaling is dysregulated in old hepatocytes. This accelerates aging, resulting in decreased resiliency and therefore, impaired liver regeneration and enhanced vulnerability to damage.

Hepatocyte activity of the cholesterol sensor smoothened regulates cholesterol and bile acid homeostasis in mice.

Cellular cholesterol is regulated by at least two transcriptional mechanisms involving sterol-regulatory-element-binding proteins (SREBPs) and liver X receptors (LXRs). Although SREBP and LXR pathways are the predominant mechanisms that sense cholesterol in the endoplasmic reticulum and nucleus to alter sterol-regulated gene expression, evidence suggests cholesterol in plasma membrane can be sensed by proteins in the Hedgehog (Hh) pathway which regulate organ self-renewal and are a morphogenic driver during embryonic development. Cholesterol interacts with the G-protein-coupled receptor Smoothened (Smo), which impacts downstream Hh signaling. Although evidence suggests cholesterol influences Hh signaling, it is not known whether Smo-dependent sterol sensing impacts cholesterol homeostasis in vivo. We examined dietary-cholesterol-induced reorganization of whole-body sterol and bile acid (BA) homeostasis in adult mice with inducible hepatocyte-specific Smo deletion. These studies demonstrate Smo in hepatocytes plays a regulatory role in sensing and feedback regulation of cholesterol balance driven by excess dietary cholesterol.

CB1 Cannabinoid Receptors Stimulate Gßγ-GRK2-Mediated FAK Phosphorylation at Tyrosine 925 to Regulate ERK Activation Involving Neuronal Focal Adhesions.

CB1 cannabinoid receptors (CB1) are abundantly expressed in the nervous system where they regulate focal adhesion kinase (FAK) and the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase 1 and 2 (ERK1/2). However, the role of CB1-stimulated FAK 925 tyrosine phosphorylation (Tyr-P) in regulating ERK1/2 activation remains undefined. Here, immunoblotting analyses using antibodies against FAK phospho-Tyr 925 and ERK2 phospho-Tyr 204 demonstrated CB1-stimulated FAK 925 Tyr-P and ERK2 204 Tyr-P (0-5 min) which was followed by a decline in Tyr-P (5-20 min). CB1 stimulated FAK-Grb2 association and Ras-mediated ERK2 activation. The FAK inhibitors Y11 and PF 573228 abolished FAK 925 Tyr-P and partially inhibited ERK2 204 Tyr-P. FAK 925 Tyr-P and ERK2 204 Tyr-P were adhesion-dependent, required an intact actin cytoskeleton, and were mediated by integrins, Flk-1 vascular endothelial growth factor receptors, and epidermal growth factor receptors. FAK 925 Tyr-P and ERK2 204 Tyr-P were blocked by the Gßγ inhibitor gallein, a GRK2 inhibitor, and GRK2 siRNA silencing, suggesting Gßγ and GRK2 participate in FAK-mediated ERK2 activation. Together, these studies indicate FAK 925 Tyr-P occurs concurrently with CB1-stimulated ERK2 activation and requires the actin cytoskeleton and Gi/oßγ-GRK2-mediated cross-talk between CB1, integrins, and receptor tyrosine kinases (RTKs).

Hedgehog-YAP Signaling Pathway Regulates Glutaminolysis to Control Activation of Hepatic Stellate Cells.

BACKGROUND & AIMS: Cirrhosis results from accumulation of myofibroblasts derived from quiescent hepatic stellate cells (Q-HSCs); it regresses when myofibroblastic HSCs are depleted. Hedgehog signaling promotes transdifferentiation of HSCs by activating Yes-associated protein 1 (YAP1 or YAP) and inducing aerobic glycolysis. However, increased aerobic glycolysis alone cannot meet the high metabolic demands of myofibroblastic HSCs. Determining the metabolic processes of these cells could lead to strategies to prevent progressive liver fibrosis, so we investigated whether glutaminolysis (conversion of glutamine to alpha-ketoglutarate) sustains energy metabolism and permits anabolism when Q-HSCs become myofibroblastic, and whether this is controlled by hedgehog signaling to YAP. METHODS: Primary HSCs were isolated from C57BL/6 or Smoflox/flox mice; we also performed studies with rat and human myofibroblastic HSCs. We measured changes of glutaminolytic genes during culture-induced primary HSC transdifferentiation. Glutaminolysis was disrupted in cells by glutamine deprivation or pathway inhibitors (bis-2-[5-phenylacetamido-1,2,4-thiadiazol-2-yl] ethyl sulfide, CB-839, epigallocatechin gallate, and aminooxyacetic acid), and effects on mitochondrial respiration, cell growth and migration, and fibrogenesis were measured. Hedgehog signaling to YAP was disrupted in cells by adenovirus expression of Cre-recombinase or by small hairpin RNA knockdown of YAP. Hedgehog and YAP activity were inhibited by incubation of cells with cyclopamine or verteporfin, and effects on glutaminolysis were measured. Acute and chronic liver fibrosis were induced in mice by intraperitoneal injection of CCl4 or methionine choline-deficient diet. Some mice were then given injections of bis-2-[5-phenylacetamido-1,2,4-thiadiazol-2-yl] ethyl sulfide to inhibit glutaminolysis, and myofibroblast accumulation was measured. We also performed messenger RNA and immunohistochemical analyses of percutaneous liver biopsies from healthy human and 4 patients with no fibrosis, 6 patients with mild fibrosis, and 3 patients with severe fibrosis. RESULTS: Expression of genes that regulate glutaminolysis increased during transdifferentiation of primary Q-HSCs into myofibroblastic HSCs, and inhibition of glutaminolysis disrupted transdifferentiation. Blocking glutaminolysis in myofibroblastic HSCs suppressed mitochondrial respiration, cell growth and migration, and fibrogenesis; replenishing glutaminolysis metabolites to these cells restored these activities. Knockout of the hedgehog signaling intermediate smoothened or knockdown of YAP inhibited expression of glutaminase, the rate-limiting enzyme in glutaminolysis. Hedgehog and YAP inhibitors blocked glutaminolysis and suppressed myofibroblastic activities in HSCs. In livers of patients and of mice with acute or chronic fibrosis, glutaminolysis was induced in myofibroblastic HSCs. In mice with liver fibrosis, inhibition of glutaminase blocked accumulation of myofibroblasts and fibrosis progression. CONCLUSIONS: Glutaminolysis controls accumulation of myofibroblast HSCs in mice and might be a therapeutic target for cirrhosis.

New Insights into the Mechanism of Action of Soluble Klotho.

The klotho gene encodes a type I single-pass transmembrane protein that contains a large extracellular domain, a membrane spanning segment, and a short intracellular domain. Klotho protein exists in several forms including the full-length membrane form (mKl) and a soluble circulating form [soluble klotho (sKl)]. mKl complexes with fibroblast growth factor receptors to form coreceptors for FGF23, which allows it to participate in FGF23-mediated signal transduction and regulation of phosphate and calcium homeostasis. sKl is present in the blood, urine, and cerebrospinal fluid where it performs a multitude of functions including regulation of ion channels/transporters and growth factor signaling. How sKl exerts these pleiotropic functions is poorly understood. One hurdle in understanding sKl's mechanism of action as a "hormone" has been the inability to identify a receptor that mediates its effects. In the body, the kidneys are a major source of sKl and sKl levels decline during renal disease. sKl deficiency in chronic kidney disease makes the heart susceptible to stress-induced injury. Here, we summarize the current knowledge of mKl's mechanism of action, the mechanistic basis of sKl's protective, FGF23-independent effects on the heart, and provide new insights into the mechanism of action of sKl focusing on recent findings that sKl binds sialogangliosides in membrane lipid rafts to regulate growth factor signaling.

CB1 cannabinoid receptors promote maximal FAK catalytic activity by stimulating cooperative signaling between receptor tyrosine kinases and integrins in neuronal cells.

Tyrosine phosphorylation (Tyr-P) of focal adhesion kinase (FAK) regulates FAK activation. Phosphorylated FAK Tyr 397 binds Src family kinases (Src), which in turn directly phosphorylate FAK Tyr 576/577 to produce maximal FAK enzymatic activity. CB1 cannabinoid receptors (CB1) are abundantly expressed in the nervous system and influence FAK activation by presently unknown mechanisms. The current investigation determined that CB1-stimulated maximal FAK catalytic activity is mediated by Gi/o proteins in N18TG2 neuronal cells, and that G12/13 regulation of Rac1 and RhoA occurs concomitantly. Immunoblotting analyses using antibodies against FAK phospho-Tyr 397 and phospho-Tyr 576/577 demonstrated that the time-course of CB1-stimulated FAK 576/577 Tyr-P occurred in three phases: Phase I (0-2 min) maximal Tyr-P, Phase II (5-20 min) rapid decline in Tyr-P, and Phase III (>20 min) plateau in Tyr-P at submaximal levels. In contrast, FAK 397 Tyr-P was monophasic and significantly lower in magnitude. FAK 397 Tyr-P and Phase I FAK 576/577 Tyr-P involved protein tyrosine phosphatase (PTP1B and Shp1/Shp2)-mediated Src activation, Protein Kinase A (PKA) inhibition, and integrin activation. Phase I maximal FAK 576/577 Tyr-P also required cooperative signaling between receptor tyrosine kinases (RTKs) and integrins. The integrin antagonist RGDS peptide, Flk-1 vascular endothelial growth factor receptor (VEGFR) antagonist SU5416, and epidermal growth factor receptor (EGFR) antagonist AG 1478 blocked Phase I FAK 576/577 Tyr-P. CB1 agonists failed to stimulate FAK Tyr-P in the absence of integrin activation upon suspension in serum-free culture media. In contrast, cells grown on the integrin ligands fibronectin and laminin displayed increased FAK 576/577 Tyr-P that was augmented by CB1 agonists and blocked by the Src inhibitor PP2 and Flk-1 VEGFR antagonist SU5416. Taken together, these studies have identified a complex integrative pathway utilized by CB1 to stimulate maximal FAK 576/577 Tyr-P in neuronal cells.

Striatal CB1 and D2 receptors regulate expression of each other, CRIP1A and δ opioid systems.

Although biochemical and physiological evidence suggests a strong interaction between striatal CB1 cannabinoid (CB1 R) and D2 dopamine (D2 R) receptors, the mechanisms are poorly understood. We targeted medium spiny neurons of the indirect pathway using shRNA to knockdown either CB1 R or D2 R. Chronic reduction in either receptor resulted in deficits in gene and protein expression for the alternative receptor and concomitantly increased expression of the cannabinoid receptor interacting protein 1a (CRIP1a), suggesting a novel role for CRIP1a in dopaminergic systems. Both CB1 R and D2 R knockdown reduced striatal dopaminergic-stimulated [(35) S]GTPγS binding, and D2 R knockdown reduced pallidal WIN55212-2-stimulated [(35) S]GTPγS binding. Decreased D2 R and CB1 R activity was associated with decreased striatal phosphoERK. A decrease in mRNA for opioid peptide precursors pDYN and pENK accompanied knockdown of CB1 Rs or D2 Rs, and over-expression of CRIP1a. Down-regulation in opioid peptide mRNAs was followed in time by increased DOR1 but not MOR1 expression, leading to increased [D-Pen2, D-Pen5]-enkephalin-stimulated [(35) S]GTPγS binding in the striatum. We conclude that mechanisms intrinsic to striatal medium spiny neurons or extrinsic via the indirect pathway adjust for changes in CB1 R or D2 R levels by modifying the expression and signaling capabilities of the alternative receptor as well as CRIP1a and the DELTA opioid system.

Cannabinoid CB1 receptors transactivate multiple receptor tyrosine kinases and regulate serine/threonine kinases to activate ERK in neuronal cells.

BACKGROUND AND PURPOSE: Signalling networks that regulate the progression of cannabinoid CB(1) receptor-mediated extracellular signal-regulated kinase (ERK) activation in neurons are poorly understood. We investigated the cellular mechanisms involved in CB(1) receptor-stimulated ERK phosphorylation in a neuronal cell model. EXPERIMENTAL APPROACH: Murine N18TG2 neuronal cells were used to analyse the effect of specific protein kinase and phosphatase inhibitors on CB(1) receptor-stimulated ERK phosphorylation. The LI-COR In Cell Western assay and immunoblotting were used to measure ERK phosphorylation. KEY RESULTS: The time-course of CB(1) receptor-stimulated ERK activation occurs in three phases that are regulated by distinct cellular mechanisms in N18TG2 cells. Phase I (0-5 min) maximal ERK phosphorylation is mediated by CB(1) receptor-stimulated ligand-independent transactivation of multiple receptor tyrosine kinases (RTKs). Phase I requires G(i/o) ßγ subunit-stimulated phosphatidylinositol 3-kinase activation and Src kinase activation and is modulated by inhibition of cAMP-activated protein kinase A (PKA) levels. Src kinase activation is regulated by the protein tyrosine phosphatases 1B and Shp1. The Phase II (5-10 min) rapid decline in ERK phosphorylation involves PKA inhibition and serine/threonine phosphatase PP1/PP2A activation. The Phase III (>10 min) plateau in ERK phosphorylation is mediated by CB(1) receptor-stimulated, ligand-independent, transactivation of multiple RTKs. CONCLUSIONS AND IMPLICATIONS: The complex expression of CB(1) receptor-stimulated ERK activation provides cellular selectivity, modulation of sensitivity to agonists, and coincidence detection with RTK signalling. RTK and PKA pathways may provide routes to novel CB(1) -based therapeutic interventions in the treatment of addictive disorders or neurodegenerative diseases. LINKED ARTICLES This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

CB(1) cannabinoid receptors and their associated proteins.

CB1 receptors are G-protein coupled receptors (GPCRs) abundant in neurons, in which they modulate neurotransmission. The CB(1) receptor influence on memory and learning is well recognized, and disease states associated with CB(1) receptors are observed in addiction disorders, motor dysfunction, schizophrenia, and in bipolar, depression, and anxiety disorders. Beyond the brain, CB(1) receptors also function in liver and adipose tissues, vascular as well as cardiac tissue, reproductive tissues and bone. Signal transduction by CB(1) receptors occurs through interaction with Gi/o proteins to inhibit adenylyl cyclase, activate mitogen-activated protein kinases (MAPK), inhibit voltage-gated Ca(2+) channels, activate K(+) currents (K(ir)), and influence Nitric Oxide (NO) signaling. CB(1) receptors are observed in internal organelles as well as plasma membrane. beta-Arrestins, adaptor protein AP-3, and G-protein receptor-associated sorting protein 1 (GASP1) modulate cellular trafficking. Cannabinoid Receptor Interacting Protein1a (CRIP1a) is an accessory protein whose function has not been delineated. Factor Associated with Neutral sphingomyelinase (FAN) regulates ceramide signaling. Such diversity in cellular signaling and modulation by interacting proteins suggests that agonists and allosteric modulators could be developed to specifically regulate unique, cell type-specific responses.

Signal transduction via cannabinoid receptors.

The endocannabinoids anandamide and 2-arachidonoylglycerol are lipid mediators that signal via CB(1) and CB(2) cannabinoid receptors and Gi/o-proteins to inhibit adenylyl cyclase and stimulate mitogen-activated protein kinase. In the brain, CB(1) receptors interact with opioid receptors in close proximity, and these receptors may share G-proteins and effector systems. In the striatum, CB(1) receptors function in coordination with D(1) and D(2) dopamine receptors, and combined stimulation of CB(1)-D(2) receptor heteromeric complexes promotes a unique interaction to stimulate cAMP production. CB(1) receptors also trigger growth factor receptor signaling cascades in cells by engaging in cross-talk or interreceptor signal transmission with the receptor tyrosine kinase (RTK) family. Mechanisms for CB(1) receptor-RTK transactivation can include stimulation of signal transduction pathways regulated by second messengers such as phospholipase C, metalloprotease cleavage of membrane-bound precursor proteins such as epidermal growth factor which activate RTKs, RTK autophosphorylation, and recruitment of non-receptor tyrosine kinases. CB(1) and CB(2) receptors are expressed in peripheral tissues including liver and adipose tissue, and are induced in pathological conditions. Novel signal transduction resulting from endocannabinoid regulation of AMP-regulated kinase and peroxisome proliferator-activated receptors have been discovered from studies of hepatocytes and adipocytes. It can be predicted that drug discovery of the future will be based upon these novel signal transduction mechanisms for endocannabinoid mediators.

Improvements in the safety of patient care can help end the medical malpractice crisis in the United States.

The publication of To Err is Human: Building a Safer Health System by the Institute of Medicine (IOM) in 1999 made the general public aware of the large number of patients that suffer preventable medical injuries in hospitals throughout the United States. Improvements in patient safety are needed to reduce this high incidence of medical error and must include the establishment of a culture of safety in every healthcare facility. A culture of safety is characterized by honesty, transparent error communication, and a systems analysis approach to medical error prevention. This type of medical culture can serve as the foundation for sustained improvements in patient safety and will help provide permanent relief from the medical malpractice crisis. Health policymakers should create policies that encourage hospital executives to establish and maintain cultures of safety in their institutions.

Prolonged recovery rate of CB1 receptor adaptation after cessation of long-term cannabinoid administration.

Long-term cannabinoid administration produces region-dependent CB1 receptor desensitization and down-regulation. This study examined the time course for normalization of CB1 receptors and G-protein activation using 3H-labeled N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR141716A) and guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS binding), respectively, in hippocampus and striatum/globus pallidus (GP). Mice were treated with escalating doses of Delta9-tetrahydrocannabinol (Delta9-THC) or R+-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate (WIN55,212-2) for 15 days, and tissue was collected 1, 3, 7, or 14 days after final injection. [3H]SR141716A and WIN55,212-2-stimulated [35S]GTPgammaS binding were decreased in both regions 1 day after treatment. WIN55,212-2-stimulated G-protein activation in striatum/GP returned to control level at 3 days after cessation of treatment with either drug but did not return to control level in hippocampus until 14 days. CB1 receptor binding did not recover to control levels until day 7 or 14 after treatment in striatum/GP and hippocampus, respectively. The mechanism of CB1 binding site down-regulation was investigated after long-term Delta9-THC treatment. Analysis of CB1 receptor mRNA in hippocampus and striatum/GP showed that transcriptional regulation could not explain prolonged recovery rates from CB1 receptor down-regulation. In contrast, CB1 receptor protein, as determined by immunoblot analysis, matched the down-regulation and recovery rates of CB1 receptor binding sites relatively closely. These data demonstrate that cannabinoid-induced decreases in CB1 receptor function persist for relatively long time periods after cessation of long-term drug treatment and that CB1 receptor signaling recovers more quickly in striatum/GP than hippocampus. Moreover, down-regulation of CB1 receptor binding sites does not seem to result mainly from transcriptional regulation, suggesting that adaptive regulation of CB1 receptors in brain primarily occurs at the protein level.

Protein kinase inhibitor peptide (PKI): a family of endogenous neuropeptides that modulate neuronal cAMP-dependent protein kinase function.

Signal transduction cascades involving cAMP-dependent protein kinase are highly conserved among a wide variety of organisms. Given the universal nature of this enzyme it is not surprising that cAMP-dependent protein kinase plays a critical role in numerous cellular processes. This is particularly evident in the nervous system where cAMP-dependent protein kinase is involved in neurotransmitter release, gene transcription, and synaptic plasticity. Protein kinase inhibitor peptide (PKI) is an endogenous thermostable peptide that modulates cAMP-dependent protein kinase function. PKI contains two distinct functional domains within its amino acid sequence that allow it to: (1) potently and specifically inhibit the activity of the free catalytic subunit of cAMP-dependent protein kinase and (2) export the free catalytic subunit of cAMP-dependent protein kinase from the nucleus. Three distinct PKI isoforms (PKIalpha, PKIbeta, PKIgamma) have been identified and each isoform is expressed in the brain. PKI modulates neuronal synaptic activity, while PKI also is involved in morphogenesis and symmetrical left-right axis formation. In addition, PKI also plays a role in regulating gene expression induced by cAMP-dependent protein kinase. Future studies should identify novel physiological functions for endogenous PKI both in the nervous system and throughout the body. Most interesting will be the determination whether functional differences exist between individual PKI isoforms which is an intriguing possibility since these isoforms exhibit: (1) cell-type specific tissue expression patterns, (2) different potencies for the inhibition of cAMP-dependent protein kinase activity, and (3) expression patterns that are hormonally, developmentally and cell-cycle regulated. Finally, synthetic peptide analogs of endogenous PKI will continue to be invaluable tools that are used to elucidate the role of cAMP-dependent protein kinase in a variety of cellular processes throughout the nervous system and the rest of the body.

Protein Kinase A activity is increased in mouse lumbar spinal cord but not brain following morphine antinociceptive tolerance for 15 days.

The present study investigated the effect of morphine antinociceptive tolerance on Protein Kinase A (PKA) activity in mouse brain (periaqueductal gray (PAG), thalamus, medulla) and lumbar spinal cord (LSC). A model was developed in which mice expressed a 21-fold level of morphine antinociceptive tolerance following implantation of a 75-mg morphine pellet for 15 days. Cytosolic and particulate PKA activity was measured directly in homogenates from the PAG, thalamus, medulla and LSC which studies have shown play a role in morphine-induced analgesia. In addition, a kinetic analysis of cytosolic and particulate PKA activity in homogenates from these regions was conducted and PKA V(max) and K(m) values were determined. Results demonstrated that chronic morphine treatment did not alter PKA activity or PKA kinetics in mouse brain. Moreover, particulate PKA activity/kinetics were not altered in LSC. However, cytosolic PKA activity was significantly increased in LSC following morphine treatment for 15 days. Furthermore, an increase in cytosolic PKA V(max) was observed in LSC. These results suggest that spinal and supraspinal PKA activity are differentially altered during morphine tolerance in mice. Thus, neurons in mouse brain and LSC that comprise the pain pathway descending from the brainstem and ending in the spinal cord respond differently to chronic morphine treatment.

Chronic Delta9-tetrahydrocannabinol treatment produces antinociceptive tolerance in mice without altering protein kinase A activity in mouse brain and spinal cord.

The present study investigated the effect of different levels of Delta-9-tetrahydrocannabinol (Delta(9)-THC) antinociceptive tolerance on Protein Kinase A (PKA) activity in mouse brain and spinal cord. To strengthen this investigation, a positive control was developed to demonstrate the assay utilized in this study was sensitive enough to detect an increase in PKA activity in the anatomical regions utilized in this study. The membrane-permeant and phosphodiesterase-resistant cAMP analog 8-Bromoadenosine-3',5'-cyclic monophosphorothioate, Sp-isomer (Sp-8-Br-cAMPS) was utilized for the development of this positive control and this compound produced an increase in PKA activity in several mouse brain regions (i.c.v.) and lumbar spinal cord (i.t.) following its administration. Models were then developed in which mice expressed either a 13-fold or 49-fold level of Delta(9)-THC antinociceptive tolerance following chronic treatment with 10mg/kg Delta(9)-THC or 80mg/kg Delta(9)-THC for 6.5 days. Basal and total cytosolic and particulate PKA activities were measured directly in homogenates from the striatum, hippocampus, cerebellum, cortex and lumbar spinal cord. Results from this study indicate that chronic exposure to Delta(9)-THC does not produce an increase in PKA activity in these mouse brain regions or spinal cord. Future work is needed to determine the role of PKA in cannabinoid tolerance in mice.

Alterations in brain Protein Kinase A activity and reversal of morphine tolerance by two fragments of native Protein Kinase A inhibitor peptide (PKI).

Two peptide fragments of native Protein Kinase A inhibitor (PKI), PKI-(6-22)-amide and PKI-(Myr-14-22)-amide, significantly reversed low-level morphine antinociceptive tolerance in mice. The inhibition of Protein Kinase A (PKA) activity by both peptide fragments was then measured in specific brain regions (thalamus, periaqueductal gray (PAG), and medulla) and in lumbar spinal cord (LSC), which in previous studies have been shown to play a role in morphine-induced analgesia. In drug naive animals, cytosolic PKA activity was greater than particulate PKA activity in each region, while cytosolic and particulate PKA activities were greater in thalamus and PAG compared to medulla and LSC. The addition of both peptides to homogenates from each region completely abolished cytosolic and particulate PKA activities in vitro. Following injection into the lateral ventricle of the brain of drug naive mice and morphine-tolerant mice, both peptides inhibited PKA activity in the cytosolic, but not the particulate fraction of LSC. In addition, cytosolic and particulate PKA activities were inhibited by both peptides in thalamus. These results demonstrate that the inhibition of PKA reverses morphine tolerance. Moreover, the inhibition of PKA activity in specific brain regions and LSC from morphine-tolerant mice by PKI analogs administered i.c.v. is evidence that PKA plays a role in morphine tolerance.