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The commercialization of academic research has been promoted by North American policy makers for over 30 years as a means of increasing university financing and to ensure that promising research would eventually find its way to the marketplace. The following issues paper constitutes a reflection on the impact of the Canadian commercialization framework on academic research in the field of genomics. It was written following two workshops and two independent studies organized by academic groups in Quebec (Centre of Genomics and Policy) and Alberta (Health Law Institute). The full sets of recommendations are available upon request to the authors.

Cis-acting variants altering gene expression are a source of phenotypic differences. The cis-acting components of expression variation can be identified through the mapping of differences in allelic expression (AE), which is the measure of relative expression between two allelic transcripts. We generated a map of AE associated SNPs using quantitative measurements of AE on Illumina Human1M BeadChips. In 53 lymphoblastoid cell lines derived from donors of European descent, we identified common cis variants affecting 30% (2935/9751) of the measured RefSeq transcripts at 0.001 permutation significance. The pervasive influence of cis-regulatory variants, which explain 50% of population variation in AE, extend to full-length transcripts and their isoforms as well as to unannotated transcripts. These strong effects facilitate fine mapping of cis-regulatory SNPs, as demonstrated by dissection of heritable control of transcripts in the systemic lupus erythematosus-associated C8orf13-BLK region in chromosome 8. The dense collection of associations will facilitate large-scale isolation of cis-regulatory SNPs.

The mechanism for the association of type 1 diabetes (T1D) with IL2RA remains to be clarified. Neither of the two distinct, transmission-disequilibrium confirmed loci mapping to this gene can be explained by a coding variant. An effect on the levels of the soluble protein product sIL-2RA has been reported but its cause and relationship to disease risk is not clear. To look for an allelic effect on IL2RA transcription in cis, we examined RNA from 48 heterozygous lymphocyte samples for differential allele expression. Of the 48 samples, 32 showed statistically significant allelic imbalance. No known single nucleotide polymorphism (SNP) had perfect correlation with this transcriptional effect but the one that showed the most significant (p = 1.6 × 10−5) linkage disequilibrium with it was the SNP rs3118470. We had previously shown rs3118470 to confer T1D susceptibility in a Canadian dataset, independently of rs41295061 as the major reported locus (p = 5 × 10−3, after accounting for rs41295061 by conditional regression). Lower IL2RA levels consistently originated from the T1D predisposing allele. We conclude that an as yet unidentified variant or haplotype, best marked by rs3118470, is responsible for this independent effect and increases T1D risk through diminished expression of the IL-2R, likely by interfering with the proper development of regulatory T cells.

Common SNPs in the chromosome 17q12-q21 region alter the risk for asthma, type 1 diabetes, primary biliary cirrhosis, and Crohn disease. Previous reports by us and others have linked the disease-associated genetic variants with changes in expression of GSDMB and ORMDL3 transcripts in human lymphoblastoid cell lines (LCLs). The variants also alter regulation of other transcripts, and this domain-wide cis-regulatory effect suggests a mechanism involving long-range chromatin interactions. Here, we further dissect the disease-linked haplotype and identify putative causal DNA variants via a combination of genetic and functional analyses. First, high-throughput resequencing of the region and genotyping of potential candidate variants were performed. Next, additional mapping of allelic expression differences in Yoruba HapMap LCLs allowed us to fine-map the basis of the cis-regulatory differences to a handful of candidate functional variants. Functional assays identified allele-specific differences in nucleosome distribution, an allele-specific association with the insulator protein CTCF, as well as a weak promoter activity for rs12936231. Overall, this study shows a common disease allele linked to changes in CTCF binding and nucleosome occupancy leading to altered domain-wide cis-regulation. Finally, a strong association between asthma and cis-regulatory haplotypes was observed in three independent family-based cohorts (p = 1.78 x 10(-8)). This study demonstrates the requirement of multiple parallel allele-specific tools for the investigation of noncoding disease variants and functional fine-mapping of human disease-associated haplotypes.

High-throughput sequencing is revolutionizing human genetic studies and offers the promise of deciphering the complete sequence of study subjects in the near future (Kuehn 2008; Wheeler et al. 2008) (http://www.1000genomes.org). The massively parallel sequencing technologies are also facilitating large-scale studies targeting genomic loci of biomedical interest. In targeted resequencing of human genomic regions, the goal is to catalog common or rare sequence variation in samples ascertained for a particular phenotype (Altshuler et al. 2008) or a subgroup of samples harboring haplotypes involved in population risk of disease (Lowe et al. 2007; Yeager et al. 2008). Targeted resequencing requires the enrichment of regions of interest by PCR (Brockman et al. 2008) or by capture-based methodologies (Albert et al. 2007; Gnirke et al. 2009). Both can lead to biases in coverage and allele representation. PCR-based enrichment methods often involve pooling of fragments, which are subject to sampling variation. In addition, preferential amplification can occur due to SNPs under primers (Quinlan and Marth 2007). If one allele is amplified preferentially, the ratio of reads derived from each allele will differ from the 1:1 ratio expected. When the preference for one allele is substantial or if there is a weak preference and coverage is low, heterozygous sites on that amplicon are likely to be missed by methods that do not factor in this source of bias. Existing approaches to resequencing data analysis (Quinlan and Marth 2007; Brockman et al. 2008; Wheeler et al. 2008; Yeager et al. 2008; for review, see Stratton 2008) often require the user to set one or more global parameters, yielding results that are either very conservative (high specificity, but missing many heterozygous sites), or very liberal (high sensitivity, but often calling false heterozygous sites). It can be difficult for users to identify appropriate parameter values to achieve a desired tradeoff between high specificity and high sensitivity. Furthermore, a global decision threshold will rarely achieve the same sensitivity level across different regions, since sequence quality, coverage, and amplification bias vary locally. Regions or sites that cannot be accurately called due to low coverage, poor sequence quality, or preferential amplification are typically not distinguished from regions for which there is strong evidence that no polymorphism is present. In this paper, we present an approach for targeted resequencing and analysis of nucleotide substitutions in data generated by massively parallel sequencing. We focus on heterozygous substitutions because they tend to be more difficult to identify than variants in the homozygous state, particularly in regions of low coverage or preferential amplification. We present a probabilistic approach to heterozygous site detection (ProbHD), which provides a heterozygosity probability for each chromosomal position. Providing probabilistic confidence scores allows the user to select an appropriate decision threshold that takes into account the goals of their application and the relative costs of false-positives and false-negatives. ProbHD also allows the identification of preferentially amplified fragments and other poorly sequenced regions. Regions that cannot be called (due to low coverage, preferential amplification, low complexity sequence, or poor alignments) are distinguished from regions that contain no heterozygous sites, thereby allowing the user to identify regions for which additional study is required. The availability of confidence scores also allows for a principled combination of sequencing results from multiple samples. We provide a Bayesian method that combines confidence scores from multiple samples of interest to estimate the probability that a site is heterozygous in all samples. We evaluated ProbHD on genomic regions that we had previously associated with cis-regulatory variation in HapMap CEU or YRI lymphoblastoid cell lines (LCLs). These regions were discovered by allelic expression (AE) mapping as described before (Verlaan et al. 2009). Samples showing an AE phenotype and mapped to a common haplotype must be heterozygous for the variant(s) causing this differential expression. Therefore, in the targeted regions we attempted to identify all heterozygous sites, which represent potential cis-regulatory variants. However, the method may be applied to the study of other phenotypes and also used in general genotype-calling, and therefore the approaches presented below are relevant to a large body of resequencing projects. In our study, we used long-range PCR (LR-PCR) for target preparation and high-throughput picoliter pyrosequencing (454 Life Sciences [Roche] Genome Sequencer FLX [GS-FLX] system platform). The use of HapMap cell lines (Frazer et al. 2007), for which detailed genetic variation information is available, allows for robust estimates of our ability to accurate detect heterozygous sites. Furthermore, we performed additional genotyping assays and comparison to 1000 Genomes Pilot data (http://www.1000genomes.org) to derive accurate performance statistics. The primary performance metrics we use are sensitivity and false call rate (FCR) (the fraction of heterozygous calls that are in error). Finally, we discuss the sources of errors in sample preparation, sequencing, and sequence analysis.

The common genetic variants associated with complex traits typically lie in noncoding DNA and may alter gene regulation in a cell type-specific manner. Consequently, the choice of tissue or cell model in the dissection of disease associations is important. We carried out an expression quantitative trait loci (eQTL) study of primary human osteoblasts (HOb) derived from 95 unrelated donors of Swedish origin, each represented by two independently derived primary lines to provide biological replication. We combined our data with publicly available information from a genome-wide association study (GWAS) of bone mineral density (BMD). The top 2000 BMD-associated SNPs (P < ∼10−3) were tested for cis-association of gene expression in HObs and in lymphoblastoid cell lines (LCLs) using publicly available data and showed that HObs have a significantly greater enrichment (threefold) of converging cis-eQTLs as compared to LCLs. The top 10 BMD loci with SNPs showing strong cis-effects on gene expression in HObs (P = 6 × 10−10 − 7 × 10−16) were selected for further validation using a staged design in two cohorts of Caucasian male subjects. All 10 variants were tested in the Swedish MrOS Cohort (n = 3014), providing evidence for two novel BMD loci (SRR and MSH3). These variants were then tested in the Rotterdam Study (n = 2090), yielding converging evidence for BMD association at the 17p13.3 SRR locus (Pcombined = 5.6 × 10−5). The cis-regulatory effect was further fine-mapped to the proximal promoter of the SRR gene (rs3744270, r2 = 0.5, P = 2.6 × 10−15). Our results suggest that primary cells relevant to disease phenotypes complement traditional approaches for prioritization and validation of GWAS hits for follow-up studies.

Regulatory cis-acting variants account for a large proportion of gene expression variability in populations. Cis-acting differences can be specifically measured by comparing relative levels of allelic transcripts within a sample. Allelic expression (AE) mapping for cis-regulatory variant discovery has been hindered by the requirements of having informative or heterozygous single nucleotide polymorphisms (SNPs) within genes in order to assign the allelic origin of each transcript. In this study we have developed an approach to systematically screen for heritable cis-variants in common human haplotypes across >1000 genes. In order to achieve the highest level of information per haplotype studied, we carried out allelic expression measurements by using both intronic and exonic SNPs in primary transcripts. We used a novel RNA pooling strategy in immortalized lymphoblastoid cell lines (LCLs) and primary human osteoblast cell lines (HObs) to allow for high-throughput AE. Screening hits from RNA pools were further validated by performing allelic expression mapping in individual samples. Our results indicate that >10% of expressed genes in human LCLs show genotype-linked AE. In addition, we have validated cis-acting variants in over 20 genes linked with common disease susceptibility in recent genome-wide studies. More generally, our results indicate that RNA pooling coupled with AE read-out by second generation sequencing or by other methods provides a high-throughput tool for cataloging the impact of common noncoding variants in the human genome.

We developed a HPLC method on a novel continuous bed matrix (UNO Q, Bio-Rad) for the direct quantification of adenoviral type 5 (Ad5) particles produced in 293S Human Embryonic Kidney cells and compared this with an existing HPLC method on a conventional ion-exchange resin (Resource Q, Pharmacia). The 293S cell extract contained large amounts of DNA. This contaminated the viral peak on the Resource Q column and only after Benzonase treatment was it possible to quantify the viral particles in the cell extract. In contrast, the virus peak on the UNO Q column was resolved from the DNA which eliminates the need for pretreatment of the sample with Benzonase. Cross-analysis of the Ad5 fraction from the UNO Q column using a size-exclusion HPLC column revealed no additional contaminating peaks. We conclude that the purity of the Ad5 virus peak on the continuous bed matrix UNO Q column was superior to the purity of the virus on the conventional Resource Q column, which is essential for reliable quantification.

Although the impact of environmental contaminants on human health has been widely studied, few reports in the Canadian literature have focussed on the specific vulnerability of children. Because of their rapid growth, physiologic and metabolic immaturity, the fetus and child are often at increased risk from toxic substances in their environments. Furthermore, greater air, food and fluid intakes relative to body weight compared with the adult, increase the child–s potential for excessive exposures. The crawling stage of infancy, the play patterns and short stature of toddlers also serve to increase their exposure to dust and heavy and volatile substances which accumulate near the floor. This article provides an overview of some of the developmental physiologic, anatomic and behavioural features of the fetus, infant and child which increase their vulnerability to environmental contaminants in comparison with adults. Specific examples are given.

High dietary Na+ intake enhances pressor and sympathoexcitatory responses in spontaneously hypertensive rats (SHR) but not Wistar-Kyoto (WKY) rats. To evaluate the possible contribution of central ouabain-like activity (OLA), brain and peripheral OLA was assessed in SHR vs. WKY rats at 4 wk of age and after 2 and 4 wk of high vs. control Na+ intake started at 4 wk of age. In SHR, hypertension developed with maturation and was exacerbated by high Na+ intake. With control Na+ intake, SHR showed higher OLA at 4, 6, and 8 wk of age in the pituitary and hypothalamus and also by 8 wk in the adrenals and left ventricle but not in plasma. High Na+ intake increased OLA in all tissues examined in both WKY rats and SHR. After 2 wk on high Na+, only OLA in hypothalamus and pituitary was higher in SHR vs. WKY rats; after 4 wk on high Na+, peripheral (i.e., adrenals, left ventricle, and plasma) OLA was also higher. These results indicate that in SHR the development of hypertension is associated early on with increases in central OLA and in a later phase with increases in peripheral OLA as well. High Na+ intake increases OLA in both SHR and WKY rats, but the higher OLA may affect sympathetic activity and blood pressure only in SHR.

Recent advances in genome-wide genotyping together with new technologies provide unprecedented opportunities for multiplex screening. These advances provide insight into diseases, hold promises to improve clinical practices to address lifestyle changes, inform reproductive decisions, identify newborns at risk as well as possibly move genetic screening out of the realm of the clinics into direct-to-consumer market forces. Advances in multiplex genetic screening raise ethical issues with regard to consent. Some of the concerns that may arise include how to: manage the incidental and excess information; integrate information about susceptibility testing into the clinic, given the complexity of the information; address the psychosocial impact and educate health professionals about the meaning of the results. If multiplex screening is used in genomic research and made available in the clinic, each ethical issue deserves consideration in the consent process and should be discussed. Key concepts: Become aware of new technologies that are being used to expand screening. Understand the distinction between testing and screening. Describe a spectrum of social, ethical issues that are involved in multiplex testing and screening. Know why multiplex screening/testing requires informed consent. Familiarize the reader with the concept of broad consent. Identify the emerging issues relevant to multiplex screening/testing. Consider whether multiplex screening/testing will shift the clinical approach towards a ‘direct-to-consumer’ model. Keywords: multiplex screening; genome-wide association; consent; broad consent; healthcare; genetic

The energetic role of inosine (INO) remains controversial. The aim of the present study was first to test whether endogenous INO consumption/production correlates with regional myocardial contractile performance and second to test whether locally increased levels of INO influence contractility and blood flow in severely ischemic myocardium. Fentanyl-anesthetized dogs with implanted sonomicrometry crystals and independently perfused left anterior descending coronary arteries were studied. Two relatively load-independent indexes of regional myocardial contractility derived from left ventricular pressure-segment length loops were used: the regional stroke work-end-diastolic segment length relationship (W r /L ed ) and the end-systolic pressure-segment length relationship (P lv /L es ). Very good correlations between myocardial contractile performance (as measured by the slope of the regional W r /L ed relationship) and endogenous INO consumption/production under both nonischemic and ischemic conditions were found. Ischemia severely depressed contractility, significantly shifting rightward the W r /L ed and P lv /L es relationships. INO infused into the left anterior descending bypass, in a concentration of 600 to 800 μmol/L, partially restored contractile performance as evidenced by a significant leftward displacement of both relationships. W r , measured at a common maximum L ed , increased significantly by 61 ± 5%. Border-zone collateral flow (microspheres) increased by 35 ± 7% within the endocardial segments and by 34 ± 9% in the epicardial segments, but no increase in flow in the ischemic region was measureable. With the current emphasis on recanalization with thrombolytic therapy and considering the apparent safety of INO, this naturally occurring nucleoside might prove to be a useful adjunctive agent in the treatment of acute myocardial ischemia.

In humans, high sodium intake not only increases the blood pressure, and thus can cause left ventricular hypertrophy (LVH), but also appears to increase LVH independent of this increase in blood pressure. In both normo- and hyper-tensive rats the hypertrophic effect of increased dietary sodium intake on the heart has been clearly established. In normotensive rats, this effect is strain and age dependent, and seems independent of hemodynamic effects of high sodium intake. In both rats and humans, dietary sodium appears to increase wall thickness, resembling pressure overload rather than an increased left ventricular diameter as expected of volume overload. The mechanisms through which high dietary sodium induces hypertrophy are still unknown. It is possible that dietary sodium increases either adrenergic stimulation and (or) enhances sensitivity for adrenergic stimulation and that this hypertrophic response mainly acts via stimulation of α1-adrenergic receptors. Stimulation of the α1-adrenergic receptors will increase the inositol phosphate – diacyl glycerol pathway and enhance the Na+/H+ exchange. The activity of this exchanger might play an important role in the development of dietary sodium induced cardiac hypertrophy.Key words: heart, dietary sodium, left ventricular hypertrophy, hemodynamics, sympathetic activity.

In hypertension, increased systolic blood pressure (BP) and the resulting increase in systolic wall stress are major determinants of the degree of left ventricular hypertrophy (LVH). Antihypertensive drugs all decrease BP, but different classes of these drugs may activate other trophic mechanisms and therefore may have different effects on LVH. Angiotensin-converting enzyme (ACE) inhibitors decrease the major cardiac growth-promoting factors such as systolic wall stress, diastolic wall stress, and cardiac sympathetic and renin activity, and consistently cause regression of LVH. On the other end of the drug spectrum, arterial vasodilators may decrease systolic wall stress, but increase diastolic wall stress and cardiac sympathetic and renin activity, resulting in either the absence of regression or even progression of LVH. Other classes of antihypertensive drugs nonuniformly change neural, humoral, or mechanical stimuli, so that the net effect ranges from full regression, partial regression, to none. Age and reactivity of sympathetic and/or renin activity may play a major role in determining the response of cardiac mass to BP lowering.

Osteoblasts are key players in bone remodeling. The accessibility of human primary osteoblast-like cells (HObs) from bone explants makes them a lucrative model for studying molecular physiology of bone turnover, for discovering novel anabolic therapeutics, and for mesenchymal cell biology in general. Relatively little is known about resting and dynamic expression profiles of HObs, and to date no studies have been conducted to systematically assess the osteoblast transcriptome. The aim of this study was to characterize HObs and investigate signaling cascades and gene networks with genomewide expression profiling in resting and bone morphogenic protein (BMP)-2- and dexamethasone-induced cells. In addition, we compared HOb gene expression with publicly available samples from the Gene Expression Omnibus. Our data show a vast number of genes and networks expressed predominantly in HObs compared with closely related cells such as fibroblasts or chondrocytes. For instance, genes in the insulin-like growth factor (IGF) signaling pathway were enriched in HObs ( P = 0.003) and included the binding proteins (IGFBP-1, -2, -5) and IGF-II and its receptor. Another HOb-specific expression pattern included leptin and its receptor ( P < 10−8). Furthermore, after stimulation of HObs with BMP-2 or dexamethasone, the expression of several interesting genes and pathways was observed. For instance, our data support the role of peripheral leptin signaling in bone cell function. In conclusion, we provide the landscape of tissue-specific and dynamic gene expression in HObs. This resource will allow utilization of osteoblasts as a model to study specific gene networks and gene families related to human bone physiology and diseases.

Nuclear magnetic resonance (NMR) spectroscopy was described in 1946 (1,2), initially as a method that had appeal only for nuclear physicists who used it to accurately determine nuclear magnetic moments. Thissituation changed rapidly, however, when it was demonstrated that the NMR frequency for the same nucleus in different chemical compounds was different (3). For example, two separate signals are observed in a 14N NMR spectrum of a solution of NH,NO,, representing the NH: and NO; ions, respectively (4). Since individual atoms within one molecule also give rise to resolved signals (5) it became clear that the NMR technique held great analytical potential, in particular since the spectra can be recorded in such a way that the area under a signal is directly proportional to its concentration. Such phenomena and various theoretical aspects of NMR are currently quite well understood (6,7). Because of these features NMR has become the foremost spectroscopic method for the analysis of all sorts of chemical compounds.

Inter-individual variation in gene expression has proven to be in part governed by genetic determinants, which may be trans- or cis-acting. The underlying cause of cis-acting regulatory variation has been identified in only a handful of the hundreds of genes shown to display differential allelic expression. In this report, we describe a systematic effort to map common cis-acting variants in 64 genes, using association methods in HapMap samples. We identified 16 loci (25%), each of which harbors common haplotypes that affect total expression of a gene, and a further 17 loci (27%) with evidence of haplotypes affecting relative allelic expression in heterozygote samples. Our survey suggests that detailed mapping of allele-specific in vivo expression will provide a rich source of regulatory SNPs or haplotypes that should be given high priority in association studies of human phenotypes.

Cis-acting allelic variation in gene regulation is a source of phenotypic variation. Consequently, recent studies have experimentally screened human genes in an attempt to initiate a catalog of genes possessing cis-acting variants. In this study, we use human EST data in dbEST as the source of allelic expression data, and the HapMap database to provide expected allele frequencies in human populations. We demonstrate a greater concordance of allele frequencies estimated from human ESTs in dbEST with those derived from the CEPH HapMap sample representing Caucasians from northern and western Europe, than population samples obtained in Asia and Africa. Deviations between allele frequencies observed in EST databases and the ones obtained from the CEPH HapMap samples may result from common heritable cis-acting variants altering the relative allele distribution in RNA. We provide in silico as well as experimental evidence that this strategy does allow significant enrichment of genes harboring common heritable cis-acting polymorphisms in linkage disequilibrium with expressed alleles.

The identification of human sequence polymorphisms that regulate gene expression is key to understanding human genetic diseases. We report a survey of human genes that demonstrate allelic differences in gene expression, reflecting the presence of putative allele-specific cis-acting factors of either genetic or epigenetic nature. The expression of allelic transcripts in heterozygous samples is assessed directly by relative quantitation of intragenic marker alleles in messenger or heteronuclear RNA derived from cells or tissues. This survey used 193 single-nucleotide polymorphisms (SNPs) from 129 genes expressed in lymphoblastoid cell lines, to identify 23 genes (18%) with common allele-specific transcripts whose expression deviated from the expected equimolar ratio. A subset of these deviations, or “allelic imbalances,” can be observed in multiple samples derived from reference CEPH (“Centre d’Etude du Polymorphisme Humain”) pedigrees and demonstrate a spectrum of patterns of transmission, including cosegregation of allelic skewing across generations compatible with Mendelian inheritance as well as random monoallelic expression for three genes ( IL1A, HTR2A, and FGB). Additional studies for BTN3A2 provide evidence of SNPs and haplotypes in complete linkage disequilibrium with high- and low-expressing transcripts. The pipeline described herein offers tools for efficient identification and characterization of allelic expression allowing identification of regulatory sequence variants as well as epigenetic variation affecting human gene expression.

High sodium diet (HS, 8% NaCl) induces left ventricular hypertrophy (LVH) in normotensive rats without an increase in pressure or volume load or in resting cardiac sympathetic activity. HS may affect LV adrenoceptors density or affinity or their postreceptor pathways, thereby causing LVH. We therefore assessed the effects of HS with and without blockade of alpha1- or beta-adrenoceptors by terazosin or nadolol, alone or in combination, on resting hemodynamics, LV and right ventricular (RV) weights, and LV dimensions of male WKY rats. HS increased LV weight by 14% to 17%, and the ratio of LV wall thickness to radius by 18% to 23%. Singly or in combination, the adrenoceptor antagonists did not prevent HS-induced LVH, but instead aggravated it. The increased ratio of LV wall thickness to radius and of LV to RV were attenuated by terazosin or nadolol alone. Neither the resting LV peak-systolic or end-diastolic pressures nor the right atrial pressure was changed by HS, either alone or in combination with the blockers. The failure of chronic alpha1- or beta-blockade to prevent HS-induced LVH suggests that adrenoceptor activation is not important in evoking the LVH. However, the blockers shifted the LVH from a concentric to an eccentric form, suggesting an involvement of additional trophic factors during adrenoceptor blockade.

The search for expression quantitative trait loci has traditionally centred entirely on the process of transcription, whereas variants with effects on messenger RNA translation have not been systematically studied. Here we present a high-throughput approach for measuring translational cis-regulation in the human genome. Using ribosomal association as proxy for translational efficiency of polymorphic messenger RNAs, we test the ratio of polysomal/non-polysomal messenger RNA level as a quantitative trait for association with single nucleotide polymorphisms on the same messenger RNA transcript. We identify one important ribosomal distribution effect, from rs1131017 in the 5'-untranslated region of RPS26, that is in high linkage disequilibrium with the 12q13 locus for susceptibility to type 1 diabetes. The effect on translation is confirmed at the protein level by quantitative western blots, both ex vivo and after in vitro translation. Our results are a proof-of-principle that allelic effects on translation can be detected at a transcriptome-wide scale.

Objectives.This study evaluated technetium-99m (Tc-99m) teboroxime kinetics in postischemic and partially necrotic myocardium with complete and low flow reperfusion using an isolated perfused rat heart model.Background.Technetium-99m teboroxime has been proposed for use in the early diagnosis of reperfusion after thrombolysis on the basis of models of myocardial necrosis with complete reperfusion. Clinically, however, reperfusion is frequently incomplete, resulting in a mixture of necrotic, ischemic and postischemic tissue.Methods.Hearts were classified into five groups: group 1 (n = 8, control); group 2 (n = 7, 30 min of no flow with complete reperfusion); group 3 (n = 12, 60 min of no flow to induce partial necrosis, followed by complete reperfusion); group 4 (n = 8, continuous low flow without flow interruption); and group 5 (n = 9, 60 min of no flow with low flow reperfusion). Buffer containing Tc-99m teboroxime was perfused for 15 min, followed by tracerfree buffer for 35 min, to evaluate uptake and clearance, respectively.Results.Uptake slopes for groups 1 to 5 were (mean ± SD) 3.0 ± 0.7, 2.6 ± 0.8, 2.1 ± 0.5, 0.8 ± 0.2 and 0.8 ± 0.3, respectively (p ≤ 0.0005 for groups 1, 2 and 3 vs. groups 4 and 5, and p = 0.003 for group 3 vs. groups 1 and 2). Clearance curves from groups 1 to 3 were best fit by a biexponential function (p < 0.001); those from groups 4 and 5 were monoexponential. In groups 1, 2 and 3, the initial clearance rate constants (k1) (0.9 ± 0.5 × 10−3; 1.0 ± 0.2 × 10−3; 1.1 ± 0.5 × 10−3s−1, respectively) and the monoexponential rate constants (kmono) (2.0 ± 0.3 × 10−4; 2.2 ± 0.4 × 10−4; 2.1 ± 0.2 × 10−4s−1, respectively) were significantly greater than those in groups 4 and 5 (0.9 ± 0.5 × 10−4; 1.2 ± 0.3 × 10−4s−1, respectively, p ≤ 0.005).Conclusions.The uptake and initial clearance kinetics of Tc-99m teboroxime depend mainly on myocardial flow in this model. The presence of partial necrosis and postischemic injury has little effect on the initial clearance but leads to some reduction in uptake at normal flow rates. Evaluation of Tc-99m teboroxime kinetics may permit early noninvasive detection of inadequate reperfusion in acute myocardial infarction.

To assess the possible contribution of brain ouabain-like activity (OLA) to the pressor effects of high-sodium intake in Dahl salt-sensitive (Dahl S) rats, we assessed the effects of high (8%) on blood pressure (BP) and peripheral and brain OLA in Dahl on blood pressure (BP) and peripheral and brain OLA in Dahl S and Dahl salt-resistant (Dahl R) rats. On regular sodium intake, Dahl S and R had similar BP; however, by 7 wk of age adrenal and plasma OLA were 15–30% higher in Dahl S vs. R, whereas central OLA remained similar. On high-sodium intake, in Dahl S both peripheral and central OLA increased within 1 wk with additional increases after 3 wk. These increases preceded the rise in BP. In Dahl R rats, high sodium did not increase BP. However, 3 wk of high sodium did increase peripheral as well as central OLA, the latter to a lesser extent compared with Dahl S and not in the hypothalamus. These results are consistent with the concept that central OLA may be involved in the pressor responses to high sodium in Dahl S. Circulating OLA may play a role in the regulation of renal function to excrete excess sodium in both strains.

We evaluated, firstly, the sensitivity to cardiac ischemic ATP breakdown during the development of hypertension and cardiac hypertrophy in Spontaneously Hypertensive Rats (SHR) v Wistar Kyoto (WKY) controls, and secondly, the effects of short-term (8 days) and prolonged (3 months) antihypertensive treatment with the angiotensin converting enzyme inhibitor enalapril on hypertrophy and sensitivity to global ischemia. In isolated perfused hearts, ischemia was induced by a stepwise lowering of the perfusion pressure and the appearance of the ATP breakdown products (purines) in the coronary effluent was assessed as a measure of ischemia. Hearts from 2.5- and 4-month-old SHR started to release purines at a higher perfusion pressure than hearts of WKY, associated with a higher maximum concentration in the coronary effluent. This increased ischemic ATP breakdown in 2.5- and 4-month-old SHR could be attributed to a decreased flow at a given perfusion pressure, because of a two-fold increase in coronary vascular resistance (CVR). In contrast, the maximal purine concentration in the coronary effluent in hearts of 7-month-old SHR was reduced compared to the younger SHR and only slightly higher than 7-month-old WKY, despite a persistent increase in CVR. Enalapril normalized the blood pressure, but only prolonged treatment, significantly prevented and regressed cardiac hypertrophy, and reduced CVR. Whereas enalapril did not influence ATP breakdown in WKY, in SHR both short- and long-term treatment normalized it to the pattern observed in WKY. We conclude that during the early phase of cardiac hypertrophy the hearts of SHR become more sensitive to ischemic ATP breakdown solely because of an increase in CVR, whereas during the established hypertrophic phase, the hearts appear to adapt metabolically, resulting in normalized purine release. Enalapril normalized the transient increase in sensitivity to ischemic ATP breakdown during the development of hypertension in SHR, independent of effects on cardiac hypertrophy, apparently by improving coronary flow at low perfusion pressures.

Both the adrenal glands and the hypothalamus have been proposed to produce compound(s) with ouabainlike activity (OLA). To evaluate the contribution of the adrenal glands, 4-wk-old spontaneously hypertensive rats (SHR) were sham operated or adrenalectomized. The adrenalectomized SHR were given daily injections of corticosterone and aldosterone. Subsequently, rats were randomized to control or high (8%) dietary Na+, and after 2.5 wk, blood pressure and OLA in plasma, hypothalamus, and pituitary were evaluated. Hypertension developed somewhat less in adrenalectomized vs. sham-operated SHR. On control Na+ intake, adrenalectomy caused only minor decreases in circulating and central OLA. Adrenalectomy did not prevent the 50-90% increases in plasma, hypothalamus, and pituitary OLA caused by high Na+ intake for 2.5 wk. These findings are consistent with the concept that, at least in SHR, the central nervous system may represent the major source of both central and peripheral OLA.

To evaluate whether changes in central ouabain-like activity (OLA) play a role in the effects of dietary sodium on sympathetic outflow and blood pressure (BP), the effects of high vs. control sodium intake on BP, brain OLA, and responsiveness to exogenous ouabain were studied in young spontaneously hypertensive rats (SHRs) vs. Wistar-Kyoto (WKY) rats. At 4 weeks of age, the BP of SHRs was already significantly increased compared with that of WKY rats, and hypertension developed further with maturation. High sodium intake exacerbated the hypertension. Compared with WKY rats, in SHRs OLA in the hypothalamus was already increased at 4 weeks of age. High sodium intake increased brain OLA in both SHRs and WKY rats. However, high dietary sodium decreased pressor and sympathoexcitatory responses to exogenous ouabain only in SHRs. These results may indicate that in WKY rats on control sodium, endogenous OLA already exerts its maximal central effects and a further increase in OLA by high sodium intake does not cause hemodynamic changes, whereas in young SHRs the increase in brain OLA by high sodium intake may play a functional role in the pressor and sympathoexcitatory responses to high sodium.

The cardiotoxic effect of cyclosporine in the inbred rat was investigated in the isolated perfused heart model. Diastolic and systolic function was studied in 5 groups of (Lewis x Brown Norway) F1 rats treated with different doses of cyclosporine for different time intervals and in a group which received the vehiculum cremophor. Of the cyclosporine-treated rats, one group was studied four weeks after drug discontinuation to assess reversibility of the effect. To control for the nephrotoxic side effects of cyclosporine, a group of animals that had undergone 1.5-1.75 renal ablation was included. Left ventricular pressures were measured with a balloon catheter; stepwise increases in preload were obtained by small increments in the balloon volume. Over the range of left ventricular volumes studied, no differences were found in diastolic pressure between the groups. Peak systolic pressures were significantly lower in hearts obtained from cyclosporine treated rats which had received 15 or 7.5 mg/kg/24 h subcutaneously for three weeks (P less than 0.05). Our results indicate that cyclosporine treatment resulted in a reversible decrease in myocardial contractile force apparently unrelated to decreases in renal function or to changes in mean arterial pressure.

The recent development of whole genome association studies has lead to the robust identification of several loci involved in different common human diseases. Interestingly, some of the strongest signals of association observed in these studies arise from non-coding regions located in very large introns or far away from any annotated genes, raising the possibility that these regions are involved in the etiology of the disease through some unidentified regulatory mechanisms. These findings highlight the importance of better understanding the mechanisms leading to inter-individual differences in gene expression in humans. Most of the existing approaches developed to identify common regulatory polymorphisms are based on linkage/association mapping of gene expression to genotypes. However, these methods have some limitations, notably their cost and the requirement of extensive genotyping information from all the individuals studied which limits their applications to a specific cohort or tissue. Here we describe a robust and high-throughput method to directly measure differences in allelic expression for a large number of genes using the Illumina Allele-Specific Expression BeadArray platform and quantitative sequencing of RT-PCR products. We show that this approach allows reliable identification of differences in the relative expression of the two alleles larger than 1.5-fold (i.e., deviations of the allelic ratio larger than 60∶40) and offers several advantages over the mapping of total gene expression, particularly for studying humans or outbred populations. Our analysis of more than 80 individuals for 2,968 SNPs located in 1,380 genes confirms that differential allelic expression is a widespread phenomenon affecting the expression of 20% of human genes and shows that our method successfully captures expression differences resulting from both genetic and epigenetic cis-acting mechanisms., Author Summary We describe a new methodology to identify individual differences in the expression of the two copies of one gene. This is achieved by comparing the mRNA level of the two alleles using a heterozygous polymorphism in the transcript as marker. We show that this approach allows an exhaustive survey of cis-acting regulation in the genome; we can identify allelic expression differences due to epigenetic mechanisms of gene regulation (e.g. imprinting or X-inactivation) as well as differences due to the presence of polymorphisms in regulatory elements. The direct comparison of the expression of both alleles nullifies possible trans-acting regulatory effects (that influence equally both alleles) and thus complements the findings from gene expression association studies. Our approach can be easily applied to any cohort of interest for a wide range of studies. It notably allows following up association signals and testing whether a gene sitting on a particular haplotype is over- or under-expressed, or can be used for screening cancer tissues for aberrant gene expression due to newly arisen mutations or alteration of the methylation patterns.

OBJECTIVE: To evaluate the sensitivity to ischaemia of rat hearts made hypertrophic by pressure overload [two-kidney, one clip (2-K,1C) rats], volume overload (aortocaval arteriovenous shunt), minoxidil or isoproterenol. METHODS: Ischaemia was induced in the isolated perfused hearts by a stepwise lowering of the perfusion pressure; at each step the coronary effluent was assessed for the products of ATP breakdown. RESULTS: Hypertension increased cardiac weight by 35 and 56% after 2.5 and 12 weeks, respectively. Volume overload increased heart weight by 25 and 55% after 1 and 12 weeks, respectively. Minoxidil (for 5 weeks) or isoproterenol (for 1 week) increased cardiac weight by 22 and 16%, respectively. The hearts from 2-K,1C rats started to release ATP catabolites in the coronary effluent at a substantially higher perfusion pressure, and with significantly higher maximal levels, than the control hearts. In contrast, in volume overload cardiac ATP breakdown was similar to that in the controls, and isoproterenol administration caused significantly lower levels of ATP breakdown. At identical flow rates, normalized per gram dry tissue, the purine concentration in the coronary effluent was similar in all of the models of cardiac hypertrophy studied and in the respective controls, and was even lower in the long-term volume-overloaded and isoproterenol-induced hypertrophic hearts. CONCLUSIONS: We conclude that hearts from hypertensive rats are more sensitive to ischaemic ATP breakdown during lowering of perfusion pressure than hearts from volume-overloaded or control rats. This is independent of the duration of the hypertrophic process, and can be explained by a lower coronary flow per gram heart tissue at a given perfusion pressure. This conclusion is strengthened by the observation that hypertrophic hearts from volume-overloaded rats had similar amounts of cardiac hypertrophy to the hearts from the hypertensive rats, without a change in flow, coronary vascular resistance or ischaemic sensitivity, whereas the hearts from isoproterenol-treated rats had lower ischaemic sensitivity and coronary vascular resistance.

High-energy phosphates in heart muscle deprived of oxygen are rapidly broken down to purine nucleosides and oxypurines. We studied the role of xanthine oxidase/dehydrogenase (EC 1.2.3.2/EC 1.2.1.37) in this process with novel high-pressure liquid chromatographic techniques. Under various conditions, including ischemia and anoxia, the isolated perfused rat heart released adenosine, inosine and hypoxanthine, and also substantial amounts of xanthine and urate. Allopurinol, an inhibitor of xanthine oxidase, greatly enhanced the release of hypoxanthine. From the purine release we calculated that the rat heart contained about 18 mU xanthine oxidase per g wet weight. Subsequently, we measured a xanthine oxidase activity of 9 mU/g wet wt. in rat-heart homogenate. When endogenous low molecular weight inhibitors were removed by gel-filtration, the activity increased to 31 mU/g wet wt. Rat myocardial xanthine oxidase seems to be present mainly in the dehydrogenase form, which upon storage at -20 degrees C is converted to the oxidase form.

The coronary vasodilator adenosine can be formed in the heart by breakdown of AMP or S-adenosylhomocysteine (SAdoHcy). The purpose of this study was to get insight into the relative importance of these routes of adenosine formation in both the normoxic and the ischemic heart. A novel HPLC method was used to determine myocardial adenosine and SAdoHcy. Accumulation of SAdoHcy was induced in isolated rat hearts by perfusion with L-homocysteine thiolactone or L-homocysteine. The release of adenosine, inosine, hypoxanthine, xanthine and uric acid was determined. Additional in vitro experiments were performed to determine the kinetic parameters of S-adenosylhomocysteine hydrolase. During normoxia the thiolactone caused a concentration-dependent increase in SAdoHcy. At 2000 microM of the thiolactone an SAdoHcy accumulation of 0.49 nmol/min per g wet weight was found during normoxia. L-Homocysteine (200 microM) caused an increase of 0.37 and 4.17 nmol SAdoHcy/min per g wet weight during normoxia and ischemia, respectively. The adenosine concentration in ischemic hearts was significantly lower when homocysteine was infused (6.2 vs. 11.5 nmol/g; P less than 0.05). Purine release was increased 4-fold during ischemia. The Km for hydrolysis of SAdoHcy was about 12 microM. At in vitro conditions favoring near-maximal SAdoHcy synthesis (72 microM adenosine, 1.8 mM homocysteine), the synthesis rate in homogenates was 10 nmol/min per g wet weight. From the combined in vitro and perfusion studies, we conclude that S-adenosylhomocysteine hydrolase can contribute significantly to adenosine production in normoxic rat heart, but not during ischemia.

The response of myocardial lactate and hypoxanthine metabolism during percutaneous transluminal coronary angioplasty was studied in a series of 15 patients undergoing this procedure. A minimum of 4 balloon inflations was performed per patient with an average duration per occlusion of 49 +/- 11 seconds (mean +/- standard deviation) for a total occlusion time of 192 +/- 40 seconds. Thermodilution coronary venous blood flow measured in the great cardiac vein decreased from control values of 72 +/- 4 ml/min (mean +/- standard error of the mean) to 47 +/- 10 ml/min with the fourth coronary occlusion (p less than 0.005). Arteriovenous lactate and hypoxanthine showed peak differences during the reactive hyperemia after the first 2 occlusions which did not increase after subsequent occlusions. Within minutes after the procedure, lactate and hypoxanthine efflux was no longer seen, demonstrating the reversibility of the metabolic disturbances after repeated ischemia. The results of this study indicate that there is no permanent alteration in lactate or hypoxanthine metabolism after percutaneous transluminal coronary angioplasty with 4 coronary occlusions of 40 to 60 seconds' duration, with a total occlusion time of 192 +/- 40 seconds.

Adenosine kinase (EC 2.7.1.20) in a cytoplasmic fraction of rat heart was subjected to 5′-AMP-Sepharose 4B chromatography. The enzyme showed affinity for the column in contrast to adenosine deaminase, and was eluted with adenosine plus MgATP. Fractions containing adenosine kinase were put on a column of DEAE-Sephacel and eluted with a gradient. The enzyme was purified up to 3000-fold (yield 10%). The specific activity exceeded 8000 units per gram of protein and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed only one band. We conclude that the method presented is a simple, quick, and elegant way of purifying myocardial adenosine kinase to virtual homogeneity.

Quantitative determination of myocardial adenosine formation and breakdown is necessary to gain insight into the mechanism and regulation of its physiological actions. Deamination of adenosine was studied in isolated perfused rat hearts by infusion of adenosine (1 to 20 mumol X litre-1). All catabolites in the perfusates (inosine, hypoxanthine, xanthine and uric acid) were measured, as well as unchanged adenosine. Apparent uptake of adenosine was determined; it increased linearly with the concentration of adenosine infused. Adenosine was predominantly deaminated, even at low (1 mumol X litre-1) concentration. The inhibitory capacity of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) was determined, while 5 mumol X litre-1 adenosine was infused. EHNA inhibited the apparent adenosine deaminase activity for 62 and 92% at 5 and 50 mumol X litre-1, respectively. When 50 mumol X litre-1 EHNA was infused into normoxic hearts, release of adenosine was significantly elevated, as was coronary flow. Induction of ischaemia increased total purine release four-to fivefold. Infusion of EHNA into ischaemic hearts did not alter total purine release, but adenosine release increased from 15 to 60% of total purines. However, when EHNA was present, a large part of total purine release still existed of inosine, hypoxanthine, xanthiner and uric acid. This was 83% during normoxia and 40% during ischaemia. These results suggest significant contribution of IMP and GMP breakdown to purine release from isolated perfused rat hearts.

1. 1. Cytosol from pig skeletal muscle, but not heart, contains an inhibitor of AMP-deaminase (AMP-D, EC 3.5.4.6) which reduces AMP-D activity 8-fold. 2. 2. Heart and skeletal muscle AMP-D have been purified to apparent homogeneity by cellulose phosphate and DEAE-Sephacel chromatography. 3. 3. AMP-D from skeletal muscle is inhibited more severely than the heart enzyme by an increase in adenylate energy charge to levels exceeding 0.4. Nevertheless both enzymes seem to be regulated by the energy charge, which contrasts with reports for rabbit heart AMP-D.

Until recently the assessment of alteration in myocardial metabolism in man early after an abrupt occlusion of a major coronary artery has not been feasible. Percutaneous transluminal coronary angioplasty (PTCA), however, now provides a unique opportunity to study the time course of these metabolic changes during the transient interruption of coronary flow by the balloon occlusion sequence in patients with single-vessel disease and without angiographically demonstrable collateral circulation [1, 2]. The need to detect any persisting metabolic or mechanical dysfunction becomes of even greater concern as the number of dilated vessels and the duration of balloon inflation tend to increase, thereby enhancing both the extent and the severity of ischemia. The risk exists that the damage induced by the intervention may exceed its benefit.

An isocratic high pressure liquid chromatographic system was developed for the estimation of purine nucleosides and oxypurines in blood. Use was made of a reversed-phase column. Nucleotides derived from erythrocytes affected the separation; these compounds were removed with A12O3. The recovery of the whole clean-up procedure exceeded 75%, and the lower detection limit of the assay for blood metabolites was 0.1 mumol/l. In 6 healthy volunteers, non-resting, the following blood concentrations (mean values +/- S.D. in mumol/l) were observed: adenosine (less than 0.1), inosine (0.2 +/- 0.1), hypoxanthine (2.2 +/- 1.3) and xanthine (0.2 +/- 0.1). In plasma and serum the total amount of these compounds was 1.9 and 5.4 times higher, respectively, presumably due to nucleotide breakdown during blood processing. The myocardial arterial-venous differences of blood purine nucleosides, oxypurines and lactate were subsequently measured in blood samples from 13 patients with angiographically documented ischemic heart disease, undergoing an atrial pacing stress test. No significant release of adenosine, inosine and xanthine by the heart was detectable in this study. The myocardial arterial-venous difference of lactate changed from 0.01 +/- 0.03 mmol/l (mean +/- SEM) at rest, to -0.10 +/- 0.04 mmol/l during pacing (p less than 0.002). Relatively larger changes were observed for hypoxanthine: pacing increased the arterial-venous difference from -0.01 +/- 0.05 to -0.51 +/- 0.17 mumol/l (p less than 0.02). We conclude that the high pressure liquid chromatographic assay of blood hypoxanthine is a useful tool in the diagnosis of ischemic heart disease.

Myocardial contraction depends fully on purine nucleotides (ATP) and creatine phosphate (CrP). The oxygen dependent formation and the breakdown of ATP are in a delicately regulated balance. During ischemia oxygen supply is lowered, ATP-levels fall and the heart releases catabolites of purine nucleotides, probably to maintain an adequately high energy charge. Released are uric acid (UA), xanthine (X), hypoxanthine (Hx), inosine (Ino) and adenosine (Ado). Amongst these, adenosine is very important because of its coronary vasodilating properties (1). Maintenance of adequate levels of purine nucleotides occurs not only by (slow) de novo synthesis, but also by phosphorylation of Ado and salvage of Hx.

Both nifedipine a calcium antagonist, and propranolol a beta-adrenergic blocker, are used as protective agents of the ischemic myocardium. In the clinical setting, the combination of the two drugs is used successfully although several case reports indicate potential dangers of the combination. For this reason we decided to study the combined effect of nifedipine and DL-propranolol in the isolated rat heart made ischemic for a short period of time. Apex displacement was taken as a measure of contractility. Release of the AMP catabolites adenosine, inosine, (hypo)xanthine and uric acid was used as a marker of ATP breakdown. Contractility during ischemia was not affected by the drugs. DL-Propranolol (30 or 150 micrograms/l) had no effect on ischemic myocardial purine release, while nifedipine (15 micrograms/l) reduced purine release during ischemia by 33% (P less than 0.02). The combination of 15 micrograms/l nifedipine and 150 micrograms/l DL-propranolol decreased purine release by 53% (P less than 0.005 vs. nifedipine). We conclude from these results that propranolol has a synergistic effect, adding to the beneficial action of nifedipine on ischemic myocardium.

An ATP-sparing effect has been demonstrated for a number of calcium antagonists. Nifedipine probably has a similar action, but data supporting this view are limited. Therefore we decided to study the effect of nifedipine on high-energy phosphate (and carbohydrate) metabolism in the ischemic rat heart. Langendorff preparations were made ischemic for less than 15 min. The reduction in coronary flow was 60 or 70%. Apex displacement during ischemia, a measure of contractility, was comparable for nifedipine-treated and untreated hearts. Ischemia caused a considerable release of the AMP catabolites adenosine, inosine and (hypo)xanthine, and of lactate. Nifedipine (10-100 micrograms/l) prevented this in a dose-dependent way. The highest dose reduced the release of purines and lactate by 90% (P less than 0.01) and 60% (P less than 0.001), respectively. The drug acted in a similar way during reperfusion. Due to ischemia, the adenylate energy charge (ATP + 0.5 ADP)/(ATP + ADP + AMP), decreased 15% (P less than 0.001); nifedipine at a concentration of 100 micrograms/l prevented this decrease (P less than 0.05). We conclude that nifedipine exerts a beneficial effect on myocardial adenine nucleotide metabolism during ischemia and reperfusion.

The effects of ischemia on contraction and relaxation of cardiac muscle function are still largely an enigma, understandably so because of the complexity of the effects of interruption of the flow through the coronary bed. The effects of stasis of coronary flow are evidently both to block the supply of nutrients and oxygen to the myocardium and to prevent washout of metabolites. Hence the ischemic insult is at least composed of the effects of impeded regulation of the extracellular milieu and of the derailment of the intracellular milieu as a consequence of the blockade of oxidative phosphorylation. Anoxia, which is often used as a model of ischemia, differs from ischemia itself because membrane-permeable metabolites are washed out of the extracellular space and hence also accumulate less inside the cell.

During myocardial ischemia, ATP is broken down to the AMP-cata-bolites adenosine, inosine, (hypo)xanthine and uric acid. These apolar compounds can easily pass the cell membrane and enter the blood stream1. After reperfusion, energy charge and CrP are restored, but ATP levels remain low, because of purine loss2. To restore ATP levels, 3 main pathways exist: 1. adenosine phosphorylation, 2. inosine/hypoxanthine salvage, 3. de novo synthesis.

Both nifedipine the calcium channel blocker and propranolol the β-blocker have been proposed for the treatment of ischemic heart disease and hypertension. The combination of nifedipine and propranolol has recently been tested in patients, in general with favourable results. In a few case reports, however, potential dangers with this therapy have been described. Few studies have been conducted to test the effect of nifedipine on high-energy phosphate metabolism in the ischemic heart, although it is generally assumed that the drug prevents breakdown of adenosine 5′-triphosphate (ATP). In addition, it is surprising that the action of nifedipine plus propranolol on ischemic heart has not been studied in animals. We induced ischemia in isolated perfused rat hearts (60 or 75% reduction in coronary flow in the absence of drugs). With a novel high performance liquid chromatographic assay we were able to measure the effect of nifedipine and propranolol on purine release, a sensitive index for myocardial ATP breakdown. The calcium antagonist prevented, in a dose dependent manner, the release of purine nucleosides and oxypurines from the ischemic heart, indicating decreased ATP breakdown. Nifedipine (100 μg/l) reduced, for instance, adenosine release by 93% ( P

To determine the importance of intracellular reactive oxygen metabolites in ischaemia and reperfusion, rat hearts were depleted of glutathione, a major intracellular antioxidant, prior to total global or low flow ischaemia. Chronic pretreatment of rats with up to 7 mmol.kg-1 L-buthionine-S,R-sulphoximine over a period of 96 hours resulted in 85% depletion of total myocardial glutathione. Subsequent recovery of glutathione after treatment with the sulphoximine was slow, reaching only 54% of control levels, 96 hours after the final dose. Depletion of myocardial glutathione by 70% did not increase the metabolic consequences of either total global or low flow ischaemia in the isolated perfused heart, as determined by 31P NMR spectroscopy and high performance liquid chromatographic analysis of purine release. During low flow ischaemia glutathione depletion caused a significant reduction in purine release. On reperfusion, functional recovery was depressed compared to controls. Hearts depleted of glutathione also showed a decrease in developed tension during normoxic perfusion prior to low flow ischaemia. There was no difference between the two groups before or after total global ischaemia. These results do not support the hypothesis of free radical mediated reperfusion injury; however, the method described here for the depletion of glutathione should prove a useful tool for further investigation into the role of glutathione in cardiac metabolism and function.

Nifedipine* is a blocker of slow Ca-channels1,2, which is presently used for the treatment of angina pectoris and hypertension. Nifedipine -like other Ca-antagonists- is thought to have an ATP-sparing effect, but the data available are conflicting3–5. We studied the effect of nifedipine on nucleotide metabolism in the isolated (ischemic) rat heart. We used release of adenosine (catabolites) as a sensitive indicator for adenine nucleotide breakdown6. Nifedipine decreased the release of adenosine, inosine and (hypo)-xanthine during ischemia and reperfusion.

AMP-deaminase (AMP-D) (E.C. 3.5.4.6) catalyzes the hydrolytic deamination of AMP to IMP and NH3. AMP-D plays an important role in skeletal muscle and liver (1,2). We purified AMP-D by adsorption chromatography on cellulose phosphate and ion-exchange chromatography on DEAE-Sephacel. We examined the kinetic constants and the influence of changing energy charges on this enzyme and investigated the difference with the enzyme purified from pig skeletal muscle.