Intestinal-Type Adenocarcinoma in Head and Neck: Dissecting Oncogenic Gene Alterations Through Whole Transcriptome and Exome Analysis.

Adenocarcinomas of the nasal/paranasal sinuses are uncommon, but intestinal-type adenocarcinomas (ITACs) are important. Due to the rarity of these tumors, their molecular profile is not well known. To further investigate the molecular profile and find potential oncogenic drivers, we compared the whole transcriptome and exome of ITACs at different anatomic locations in the head and neck. Twenty-one head and neck adenocarcinomas were used in this study, divided into 10 sinonasal adenocarcinomas (SNT) and 11 extrasinonasal (T) head and neck adenocarcinomas according to anatomic location and histology. Tumor samples along with normal mucosa were microdissected from formalin-fixed, paraffin-embedded samples, and RNA and DNA were subjected to whole-transcriptome and -exome shotgun sequencing. Analysis of ITACs at sinonasal locations showed 410 subtype-specific differentially expressed (DE) genes and noncoding transcripts compared with the group of other anatomic locations, with 2909 subtype-specific DE genes. The groups shared 872 genes, with 17 highly different or opposing DE genes. Whole-exome mutation analysis revealed the gene MLL3 (KMT2C) to be exhibiting the most frequent loss-of-function mutations in all adenocarcinomas investigated. The results suggest that the head and neck ITACs investigated were mainly caused by loss-of-function mutations in MLL3 that disabled chromatin methylation and remodeling of all MLL3-targeted enhancers in the tumors. This changed the activity of multiple genes/gene clusters, supporting oncogenicity mostly via pathways of signaling, dedifferentiation, proliferation, migration, and immune and inflammatory deregulation, indicating a truly epigenetic event as the root cause for the heterogenous diversity of these enteric types of cancer. The data of this study form the basis for understanding cell fate determination and cellular homeostasis in the normal respiratory mucosa at different anatomic sites and show the contribution of different mucosal components to the etiology/molecular pathology of ITAC.

High-grade sinonasal carcinomas and surveillance of differential expression in immune related transcriptome.

The skull base is the location of a wide variety of malignant tumors. Among them is sinonasal undifferentiated carcinoma (SNUC), a highly aggressive sinonasal neoplasm that was recently reclassified into subgroups of high-grade carcinomas with unique genomic events (e.g., SMARC-deficient carcinoma, nuclear protein in testis NUT carcinoma). Other high-grade carcinomas in this location are neuroendocrine carcinomas, sinonasal adenocarcinomas, and teratocarcinosarcomas. Given the rarity of these tumors, little transcriptomic data is available. The aim of this study was to characterize the immune-oncology gene expression profile in SNUC and other high-grade sinonasal carcinomas. Next-generation sequencing was performed in 30 high-grade sinonasal carcinoma samples using the HTG EdgeSeq Precision Immuno-Oncology Panel. Ingenuity pathway analysis was performed to understand the immunobiology, signaling, and functional perturbations during tumor development. The samples were divided into 3 groups: 21 SNUCs and SMARC-deficient sinonasal carcinomas; 5 high-grade neuroendocrine carcinomas (HGNECs), with small cell and large cell variants; and 4 high-grade sinonasal carcinomas (HGSNCs) of mixed histology (1 NUT carcinoma, 1 teratocarcinosarcoma, and 2 sinonasal adenocarcinomas). PRAME and ASCL1 emerged as upregulated transcripts with strong protein validation for SNUC and HGNEC; other upregulated candidates EZH2 and BRCA1 offer consideration for alternative targeted therapy, and downregulation of major histocompatibility complex molecules and chemokines represent another hurdle in the development of effective immunotherapy. This immune-oncology gene expression analysis of 3 groups of high-grade sinonasal carcinoma with emphasis on SNUC identified a number of differentially expressed transcripts reflecting effects on tumorigenesis. Identification of immune pathways should be further investigated for possible integration of immunotherapy into a multidisciplinary approach to these cancers and personalized treatment.

Comparative transcriptome analysis of sinonasal inverted papilloma and associated squamous cell carcinoma: Out-HOXing developmental genes.

BACKGROUND: Sinonasal papilloma has a tendency toward local destruction, recurrence, and malignant transformation. This study aimed to unravel mechanisms in the malignant transformation of sinonasal papillomas using RNA-seq. METHODS: The cohort consisted of 37 consecutive patients; tumor histology included a continuum spectrum (sinonasal papillomas/dysplastic/carcinomas-in-situ/invasive squamous cell carcinomas). These were microdissected and RNA was subjected to whole-transcriptome shotgun sequencing. RESULTS: RNA-seq and pathway analysis showed that the highest expressed genes/potential drivers were development- and differentiation-related genes. The protein expression of six highly upregulated genes (HOXA9, EN1, DUX4, CA9, CD1a, and CK5/6) validated the RNA-seq results. HOXA9 and CA9 were found to be expressed in most of the carcinoma samples but were largely negative in papillomas; all of the CA9-negative carcinomas were recurrent. CONCLUSIONS: We conclude that sinonasal carcinomas arising from papillomas are mainly defined by overexpressed developmental/homeobox genes, which provide the potential for transformation/plasticity, along with differentiation and proliferation behavior of neoplastic cells. Our results support HOXA9 and CA9 as biomarkers for carcinomas, with CA9 emerging as a predictive marker of recurrence.

Magnifying glass on spiradenoma and cylindroma histogenesis and tumorigenesis using systematic transcriptome analysis.

Spiradenoma and cylindroma are related sweat gland tumors. To delineate their histogenesis, gene profiles, and their potential drivers, we performed a whole-transcriptome sequencing analysis of fourteen samples of spiradenoma/cylindroma in comparison to normal samples. A total of 12 spiradenomas, 5 cylindromas, 3 hybrid spiradenomas/cylindromas and 2 adnexal carcinomas were included in this study. 1335 characteristic genes and transcripts expressed over all 14 spiradenoma/cylindroma tumors were identified, and two groups of expression profiles were observed. Highest upregulated top 7 gene signatures characterized benign tumors with developmental and differentiation related genes, and carcinomas with top 7 genes mainly related to signaling, reorganization and metabolism of membranes. Immunohistochemistry of protein expressions validated 4 upregulated genes (ODAM, HOXB13, MYB and SOX10) considered important and as potential biomarkers for spiradenomas and cylindromas. We further compared the transcriptome of eccrine adnexal tumors with the transcriptome of adenoid cystic carcinoma (ACC) to identify the overlapping genes that may indicate histogenesis. There were 36 specific genes overlapping between adnexal carcinomas and the epithelial-dominant subtype of ACC, and 27 specific genes overlapping benign adnexal tumors with the myoepithelial-dominant subtype of ACC, At this point there is no known specific biomarker to aid in the diagnosis of eccrine spiradenoma and cylindroma in small samples or biopsies within the context of morphological overlap with ACC. In conclusion, spiradenomas and cylindromas are characterized by overexpressed developmental genes, where LHX2 and activated WNT signaling possibly drive associated carcinomas.

Detection of a MicroRNA molecular signature of ultraviolet radiation in the superficial regions of melanocytic nevi on sun-exposed skin.

How melanocytes transform into melanoma cells remains largely unknown. However, prolonged ultraviolet radiation exposure is linked with melanoma, and the DNA of melanomas arising in chronically sun-exposed skin is characterized by an elevated number of pyrimidine transitions, mainly C>T (predominantly caused by ultraviolet B), and transversions of GC>TA or AT>CG (caused by ultraviolet A over indirect mechanisms). Since ultraviolet penetrates mostly only the superficial dermis, we sought to determine the extent to which superficial and deep melanocytes of nevi in sun-exposed skin differ in their miRNA expression and consider the changes as likely secondary to ultraviolet radiation-induced damage. The differentially expressed miRNAs were analyzed for known potential oncomiRs or linked to potential oncogenes or tumor suppressors. Superficial and deep melanocytes were microdissected from the nevi of 14 patients. The suspensions were processed for hybridization to a ribonucleotide protection system with 2280 total probes, including 2256 miRNA probes targeting 2083 human miRNAs. A comprehensive analysis of all human miRNAs registered in miRBase 11.0 was performed using the HTG Molecular Diagnostic database. Statistical analysis of these data yielded for 14 samples a statistically relevant profile of 39 miRNA species at FDR<0.1 that were differentially expressed between superficial and deep melanocytes. Ingenuity Pathway Analysis based on the expression data of these 39 miRNAs suggested the gene transcripts AR, MDM2, SMAD2/3, and YBX1 as the most probable miRNA targets, which were validated at the protein level. Our findings suggest that superficial ultraviolet radiation-damaged melanocytes can be differentiated from deep melanocytes on the basis of the expression of 39 miRNAs, the most probable gene transcript and protein targets of which are AR, MDM2, SMAD2/3, and YBX1, with YBX1 expression validating the best the molecular signature in immunohistochemical analysis.

Transcriptome comparison identifies potential biomarkers of spine and skull base chordomas.

Chordomas are rare, slowly growing, locally aggressive bone neoplasms that arise from embryonic remnants of the notochord, showing dual epithelial-mesenchymal differentiation. The high plasticity probably is the main reason for the high variety in phenotypes of chordoma, from its high heterogeneity on a cellular level to its subtype variations depending on tissue location, with its potential to develop from an inactive quiescent form to an aggressive cancer with extreme adaptability and resistance to drugs and other treatments. Gene expression profiles of formalin-fixed, paraffin-embedded skull chordoma, spine chordoma, and normal tissue specimens were generated and compared. Using strict criteria, we identified 222 differentially expressed transcripts unique to skull base chordoma, 261 unique to spine chordoma, and 192 common to both chordoma subtypes. Further analysis of these three groups of transcripts allowed the selection of three subsets of highly differentially expressed genes as potential biomarkers, disease drivers, and therapeutic targets in both chordoma subtypes. Immunohistochemistry revealed LMX1A to be dominant in skull base chordoma, SALL3 to be unique to spine chordoma, and T to be common to both chordoma subtypes. In both chordoma subtypes, the genes with the highest expression were predominantly development-related genes, mostly transcription factors. Our findings indicate that these developmental genes play important oncogenic roles in chordoma, mainly causing high plasticity and resistance to therapy in both these cancer subtypes but also determining their differentiation status and proliferation activity, pointing to features expected of heterogeneous stem cell-like tissues with similarities to their notochord origins.

Whole-transcriptome analysis of chordoma of the skull base.

Fourteen skull base chordoma specimens and three normal specimens were microdissected from paraffin-embedded tissue. Pools of RNA from highly enriched preparations of these cell types were subjected to expression profiling using whole-transcriptome shotgun sequencing. Using strict criteria, 294 differentially expressed transcripts were found, with 28 % upregulated and 72 % downregulated. The transcripts were annotated using NCBI Entrez Gene and computationally analyzed with the Ingenuity Pathway Analysis program. From these significantly changed expressions, the analysis identified 222 cancer-related transcripts. These 294 differentially expressed genes and non-coding RNA transcripts provide here a set to specifically define skull base chordomas and to identify novel and potentially important targets for diagnosis, prognosis, and therapy of this cancer. Significance Genomic profiling to subtype skull base chordoma reveals potential candidates for specific biomarkers, with validation by IHC for selected candidates. The highly expressed developmental genes T, LMX1A, ZIC4, LHX4, and HOXA1 may be potential drivers of this disease.

In-depth characterization of the salivary adenoid cystic carcinoma transcriptome with emphasis on dominant cell type.

BACKGROUND: Adenoid cystic carcinoma (ACC), 1 of the most common salivary gland malignancies, arises from the intercalated ducts, which are composed of inner ductal epithelial cells and outer myoepithelial cells. The objective of this study was to determine the genomic subtypes of ACC with emphasis on dominant cell type to identify potential specific biomarkers for each subtype and to improve the understanding of this disease. METHODS: A whole-genome expression study was performed based on 42 primary salivary ACCs and 5 normal salivary glands. RNA from these specimens was subjected to expression profiling with RNA sequencing, and results were analyzed to identify transcripts in epithelial-dominant ACC (E-ACC), myoepithelial-dominant ACC (M-ACC), and all ACC that were expressed differentially compared with the transcripts in normal salivary tissue. RESULTS: In total, the authors identified 430 differentially expressed transcripts that were unique to E-ACC, 392 that were unique to M-ACC, and 424 that were common to both M-ACC and E-ACC. The sets of E-ACC-specific and M-ACC-specific transcripts were sufficiently large to define and differentiate E-ACC from M-ACC. Ingenuity pathway analysis identified known cancer-related genes for 60% of the E-ACC transcripts, 69% of the M-ACC transcripts, and 68% of the transcripts that were common in both E-ACC and M-ACC. Three sets of highly expressed candidate genes-distal-less homeobox 6 (DLX6) for E-ACC; protein keratin 16 (KRT16), SRY box 11 (SOX11), and v-myb avian myeloblastosis viral oncogene homolog (MYB) for M-ACC; and engrailed 1 (EN1) and statherin (STATH), which are common to both E-ACC and M-ACC)-were further validated at the protein level. CONCLUSIONS: The current results enabled the authors to identify novel potential therapeutic targets and biomarkers in E-ACC and M-ACC individually, with the implication that EN1, DLX6, and OTX1 (orthodenticle homeobox 1) are potential drivers of these cancers. Cancer 2016;122:1513-22. © 2016 American Cancer Society.

Global transcriptome and sequenome analysis of formalin-fixed salivary epithelial-myoepithelial carcinoma specimens.

Diverse microarray and sequencing technologies have been widely used to characterize molecular changes in malignant epithelial cells in salivary neoplasms. Such gene expression studies to identify markers and targets in tumor cells are, however, compromised by the cellular heterogeneity of these tumors and by the difficulties to accrue matching controls representing normal salivary glands. Seventeen samples of primary salivary epithelial-myoepithelial carcinoma along with tissue from six normal major salivary glands were microdissected from paraffin-embedded tissue. Pools of RNA from highly enriched preparations of these cell types were subjected to expression profiling using a whole-transcriptome shotgun sequencing experiment. In parallel, extracted genomic DNA was used for the 50 gene hotspot panel sequenome. KRAS mutations in three patients (18%), NRAS mutations in one patient (6%), but no HRAS, MET, PIK3CA, or BRAF mutations. Using strict and conservative criteria, 220 differentially expressed transcripts were found, with 36% up- and 64% downregulated. The transcripts were annotated using NCBI Entrez Gene, and computationally analyzed with the Ingenuity Pathway Analysis program. From these significantly changed expressions, the analysis identified 26 cancer-related transcripts and 16 transcripts related to mitochondrial dysfunction overlapping with three cancer-related genes. These 220 differentially expressed genes including microRNAs provide here a sufficiently large set to specifically define epithelial-myoepithelial carcinoma and to identify novel and potentially important targets for diagnosis, prognosis, and therapy of this cancer.

Developmental transcription factor EN1--a novel biomarker in human salivary gland adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC), a rare and progressive salivary malignancy, is characterized by histogenetic, morphologic, and clinical heterogeneity. Extensive efforts to characterize the molecular events associated with these tumors have included the identification of biomarkers for prognostication and post-therapy assessment. In a previous study of genome-wide methylation screening, the authors of the current report identified a limited number of differentially methylated gene regions in ACC, and significant hypermethylation was observed at the transcriptional start sites of genes that encode for the transcription factor engrailed homeobox 1 (EN1). Clinicopathologic correlation analyses indicated that EN1 methylation status is correlated with histologic tumor grade, tumor location, and final patient outcome. To ascertain definitively whether aberrant EN1 expression accompanies human salivary ACC, the authors used an immunohistochemical technique to directly evaluate EN1 protein expression in ACC of the salivary gland. The data revealed increased EN1 protein expression in solid type ACC, which was correlated with a significantly lower survival rate. The current results validated EN1 as a potential biomarker in a large cohort of patients with salivary ACC. Immunohistochemical analysis of EN1 in biopsy specimens obtained for diagnostic purposes and/or surgically resected material may reveal that EN1 is a biologic predictor of poor prognosis in patients with salivary ACC.

Nuclear plakoglobin is essential for differentiation of cardiac progenitor cells to adipocytes in arrhythmogenic right ventricular cardiomyopathy.

RATIONALE: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a disease of desmosome proteins characterized by fibroadipogenesis in the myocardium. We have implicated signaling properties of junction protein plakoglobin (PG) in the pathogenesis of ARVC. OBJECTIVE: To delineate the pathogenic role of PG in adipogenesis in ARVC. METHODS AND RESULTS: We generated mice overexpressing PG, either a wildtype (PG(WT)) or a truncated (PG(TR)), known to cause ARVC, in the heart; and PG null (PG⁻/⁻) embryos. PG(WT) and PG(TR) mice exhibited fibro-adiposis, cardiac dysfunction, and premature death. Subcellular protein fractionation and immunofluorescence showed nuclear localization of PG(WT) and PG(TR) and reduced membrane localization of PG(TR). Coimmunoprecipitation showed reduced binding of PG(TR) but not PG(WT) to desmosome proteins DSP and DSG2. Transgene PG(WT) and PG(TR) were expressed in c-Kit+:Sca1+ cardiac progenitor cells (CPCs) isolated from the hearts of PG(WT) and PG(TR) by fluorescence activated cell sorting. CPCs isolated from the transgenic hearts showed enhanced adipogenesis, increased levels of adipogenic factors KLF15, C/EBP-α and noncanonical Wnt5b, and reduced level of CTGF, an inhibitor of adipogenesis. Treatment with BIO activated the canonical Wnt signaling, reversed the proadipogenic transcriptional switch and prevented adipogenesis in a dose-dependent manner. Moreover, c-Kit+ CPCs, isolated from PG⁻/⁻ embryos, were resistant to adipogenesis, expressed high mRNA levels of CTGF and other canonical Wnt signaling targets. CONCLUSIONS: Nuclear PG provokes adipogenesis in c-Kit+ CPCs by repressing the canonical Wnt signaling and inducing a proadipogenic gene expression. The findings suggest that adipocytes in ARVC, at least in part, originate from c-Kit+ CPCs.

CpG island methylation profiling in human salivary gland adenoid cystic carcinoma.

BACKGROUND: DNA methylation is a fundamental epigenetic event associated with physiologic and pathologic conditions, including cancer. Hypermethylation of CpG islands at active gene promoters leads to transcriptional repression, whereas hypomethylation is associated with gene overexpression. The aim of this study was to identify genes in adenoid cystic carcinoma (ACC) of salivary gland strongly deregulated by epigenetic CpG island methylation, to validate selected genes by conventional techniques, and to correlate the findings with clinicopathologic factors. METHODS: The authors analyzed 16 matched normal and tumor tissues for aberrant DNA methylation using the methylated CpG island amplification and microarray method and the pyrosequencing technique. RESULTS: Microarray analysis showed hypomethylation in 7 and hypermethylation in 32 CpG islands. Hypomethylation was identified in CpG islands near FBXO17, PHKG1, LOXL1, DOCK1, and PARVG. Hypermethylation was identified near genes encoding predominantly transcription factors (EN1, FOXE1, GBX2, FOXL1, TBX4, MEIS1, LBX2, NR2F2, POU3F3, IRX3, TFAP2C, NKX2-4, PITX1, NKX2-5), and 13 genes with different functions (MT1H, EPHX3, AQPEP, BCL2L11, SLC35D3, S1PR5, PNLIPRP1, CLIC6, RASAL, XRN2, GSTM5, FNDC1, INSRR). Four CpG islands by EN1, FOXE1, TBX4, and PITX1 were validated by pyrosequencing. CONCLUSIONS: The highly methylated genes in tumor versus normal tissue are linked to developmental, apoptotic, and other fundamental cellular pathways, suggesting that down-regulation of these genes is associated with ACC development and progression. With EN1 hypermethylation showing potential as a possible biomarker for ACC in salivary gland, the biological and therapeutic implications of these findings require further preclinical investigations.

Genetic fate mapping identifies second heart field progenitor cells as a source of adipocytes in arrhythmogenic right ventricular cardiomyopathy.

The phenotypic hallmark of arrhythmogenic right ventricular cardiomyopathy, a genetic disease of desmosomal proteins, is fibroadipocytic replacement of the right ventricle. Cellular origin of excess adipocytes, the responsible mechanism(s) and the basis for predominant involvement of the right ventricle are unknown. We generated 3 sets of lineage tracer mice regulated by cardiac lineage promoters alpha-myosin heavy chain (alphaMyHC), Nkx2.5, or Mef2C. We conditionally expressed the reporter enhanced yellow fluorescent protein while concomitantly deleting the desmosomal protein desmoplakin in cardiac myocyte lineages using the Cre-LoxP technique. Lineage tracer mice showed excess fibroadiposis and increased numbers of adipocytes in the hearts. Few adipocytes in the hearts of alphaMyHC-regulated lineage tracer mice, but the majority of adipocytes in the hearts of Nkx2.5- and Mef2C-regulated lineage tracer mice, expressed enhanced yellow fluorescent protein. In addition, rare cells coexpressed adipogenic transcription factors and the second heart field markers Isl1 and Mef2C in the lineage tracer mouse hearts and in human myocardium from patients with arrhythmogenic right ventricular cardiomyopathy. To delineate the responsible mechanism, we generated transgenic mice expressing desmosomal protein plakoglobin in myocyte lineages. Transgene plakoglobin translocated to nucleus, detected by immunoblotting and immunofluorescence staining and coimmunoprecipitated with Tcf7l2, a canonical Wnt signaling transcription factor. Expression levels of canonical Wnt/Tcf7l2 targets bone morphogenetic protein 7 and Wnt5b, which promote adipogenesis, were increased and expression level of connective tissue growth factor, an inhibitor of adipogenesis, was decreased. We conclude adipocytes in arrhythmogenic right ventricular cardiomyopathy originate from the second heart field cardiac progenitors, which switch to an adipogenic fate because of suppressed canonical Wnt signaling by nuclear plakoglobin.

Differential interactions of thin filament proteins in two cardiac troponin T mouse models of hypertrophic and dilated cardiomyopathies.

AIM: Mutations in a sarcomeric protein can cause hypertrophic cardiomyopathy (HCM) or dilated cardiomyopathy (DCM), the opposite ends of a spectrum of phenotypic responses of the heart to mutations. We posit the contracting phenotypes could result from differential effects of the mutant proteins on interactions among the sarcomeric proteins. To test the hypothesis, we generated transgenic mice expressing either cardiac troponin T (cTnT)-Q92 or cTnT-W141, known to cause HCM and DCM, respectively, in the heart. METHODS AND RESULTS: We phenotyped the mice by echocardiography, histology and immunoblotting, and real-time polymerase chain reaction. We detected interactions between the sarcomeric proteins by co-immunoprecipitation and determined Ca2+ sensitivity of myofibrillar protein ATPase activity by Carter assay. The cTnT-W141 mice exhibited dilated hearts and decreased systolic function. In contrast, the cTnT-Q92 mice showed smaller ventricles and enhanced systolic function. Levels of cardiac troponin I, cardiac alpha-actin, alpha-tropomyosin, and cardiac troponin C co-immunoprecipitated with anti-cTnT antibodies were higher in the cTnT-W141 than in the cTnT-Q92 mice, as were levels of alpha-tropomyosin co-immunoprecipitated with an anti-cardiac alpha-actin antibody. In contrast, levels of cardiac troponin I co-immunoprecipitated with an anti-cardiac alpha-actin antibody were higher in the cTnT-Q92 mice. Ca2+ sensitivity of myofibrillar ATPase activity was increased in HCM but decreased in DCM mice compared with non-transgenic mice. CONCLUSION: Differential interactions among the sarcomeric proteins containing cTnT-Q92 or cTnT-W141 are responsible for the contrasting phenotypes of HCM or DCM, respectively.

Isoflurane modulates genomic expression in rat amygdala.

General anesthesia, at a minimum, provides amnesia and unresponsiveness. Although anesthetics have many modulatory effects on neuronal ionophore protein complexes, it is not clear that the resulting electrophysiologic changes are the sole mechanisms of clinical anesthetic action. Cells respond to environmental changes in several ways, including alterations in DNA transcription leading to changes in the cell's proteins. We sought to expose the changes in global genomic expression, seeking potential targets involved in the processes of anesthetic-induced amnesia, and persistent long-term side effects of general anesthesia, including nausea and postoperative cognitive decline. Using Affymetrix GeneChips, we surveyed changes in expression across the entire expressed genome of Sprague-Dawley rat (n = 10 baseline, n = 6 isoflurane) basolateral amygdala 6 h after exposure to 15 min of 2% (1.4 MAC) isoflurane. Isoflurane administration was associated with altered expression in 269 unique genes possessing functional annotation. Affected genes were related to DNA transcription, protein synthesis, metabolism, signaling cascades, cytoskeletal structural proteins, and neural-specific proteins, among others. Even brief exposure to isoflurane leads to widespread changes in the genetic control in the amygdala 6 h after exposure. Gene expression is a dynamic process that may explain some long-term effects of anesthesia and that has the potential to modulate some of those effects using specific molecular therapeutics.

Presence of morphine in rat amygdala: evidence for the mu3 opiate receptor subtype via nitric oxide release in limbic structures.

BACKGROUND: We have identified a novel mu opiate receptor, p3, which is expressed in several human tissues, is selective for opiate alkaloids, insensitive to opioid peptides, and also is coupled to constitutive nitric oxide release. We, and others, have also demonstrated the presence of opiate alkaloids as endogenous substances in various nerve tissues taken from mammals, man and invertebrates. MATERIAL/METHODS: Morphine isolation and identification was achieved by high pressure liquid chromatography coupled to electrochemical detection. This material was finally identified by nano-electrospray ionization quadruple time-of-flight tandem mass spectrometry (Q-TOF MS/MS). Morphine's ability to release nitric oxide from limbic tissues was determined in real-time via an amperometric probe. RESULTS: We demonstrate the presence of morphine in rat brain amygdala at 12.7 +/- 5.4 ng/g wet tissue. Morphine was able to stimulate the release of nitric oxide from hippocampus and amygdalar tissues in a naloxone and L-NAME sensitive manner. Furthermore, rat chow, incubation medium etc, did not contain morphine, eliminating the possibility of contamination. CONCLUSION: This finding provides evidence that morphine biosynthesis occurs in mammalian neural tissues. It also demonstrates that morphine releases nitric oxide in limbic tissues. Given the limbic system involvement in modulating emotion, including experiences related to pain perception, it appears that morphine is involved with this activity.