Parallel functional assessment of m6A sites in human endodermal differentiation with base editor screens.

N6-methyladenosine (m6A) plays important role in lineage specifications of embryonic stem cells. However, it is still difficult to systematically dissect the specific m6A sites that are essential for early lineage differentiation. Here, we develop an adenine base editor-based strategy to systematically identify functional m6A sites that control lineage decisions of human embryonic stem cells. We design 7999 sgRNAs targeting 6048 m6A sites to screen for m6A sites that act as either boosters or barriers to definitive endoderm specification of human embryonic stem cells. We identify 78 sgRNAs enriched in the non-definitive endoderm cells and 137 sgRNAs enriched in the definitive endoderm cells. We successfully validate two definitive endoderm promoting m6A sites on SOX2 and SDHAF1 as well as a definitive endoderm inhibiting m6A site on ADM. Our study provides a functional screening of m6A sites and paves the way for functional studies of m6A at individual m6A site level.

Rendesivir para tratamento de pacientes com COVID-19 hospitalizados com pneumonia e necessidade de suplementação de oxigênio / Rendesivir for the treatment of patients with COVID-19 hospitalized with pneumonia and need for oxygen supplementation

INTRODUÇÃO: No dia 12 de março de 2021 a ANVISA concedeu o registro para comercialização de rendesivir no Brasil como primeiro e único medicamento com indicação aprovada em bula, para tratamento de pacientes com COVID-19 com pneumonia e necessidade de suplementação de oxigênio (baixo ou alto fluxo e ventilação mecânica não invasiva). A autorização fornecida pela ANVISA para uso do medicamento no Brasil, diverge da recomendação da OMS que desaconselha o uso do rendesivir para tratamento de pacientes hospitalizados por COVID-19, independente de sua gravidade usando como base os resultados interinos do estudo Solidarity (14). Este estudo revelou que o uso do rendesivir, assim como outros antivirais analisados, não foi capaz de reduzir de forma significativa a mortalidade geral ou de nenhum subgrupo estudado, nem retardar o início da ventilação mecânica ou reduzir o tempo de hospitalização. A redução do tempo de internação para a ANVISA foi um critério importante para o contexto brasileiro devido a constante falta de leitos disponíveis para o tratamento dos pacientes acometidos pela COVID-19. TECNOLOGIA: Remdesivir (Veklury®). PERGUNTA: O uso do rendesivir é eficaz e seguro para tratamento de pacientes hospitalizados com COVID-19 e que necessitam de suplementação de oxigênio (de baixo ou alto fluxo e ventilação mecânica não invasiva)? EVIDÊNCIAS CLÍNICAS: As evidências avaliadas baseiam-se em quatro ensaios clínicos randomizados, onde dois compararam rendesivir com placebo e outros dois comparando rendesivir com cuidado padrão. Foram obtidos resultados favoráveis ao rendesivir com maior probabilidade de recuperação no 29º dia (Hazard Ratio [HR] 1,29, 95%IC 1,12-1,49, p<0,001) em um dos estudos. Para o desfecho mortalidade no 29º dia, os resultados do mesmo ensaio foram significativos apenas para o subgrupo de pacientes que necessitavam de suplementação de oxigênio de baixo ou alto fluxo (HR = 0,30, 95%IC 0,14- 0,64). Ao sumarizar os dados dos quatro estudos observou-se que em pacientes hospitalizados com COVID-19, o uso do rendesivir comparado com o grupo controle não resultou em diferenças estatisticamente significativas tanto quanto os desfechos de mortalidade (Risco Relativo [RR]: 0,98, 95%IC 0,84-1,14); necessidade de ventilação mecânica (RR: 0,77, 95%IC 0,48-1,22) e recuperação (RR: 1,09, 95%IC 1,03-1,15), segundo três estudos. O rendesivir comparado com placebo e cuidado padrão pode reduzir em 25% o risco da ocorrência de eventos adversos sérios (RR: 0,75, 95%IC 0,63-0,90). AVALIAÇÃO ECONÔMICA: Os resultados do modelo econômico apresentado pelo demandante têm limitações que tornam incertos os resultados do modelo econômico apresentado, de forma que a sua utilização para tomada de decisão sobre a tecnologia é duvidosa. ANÁLISE DE IMPACTO ORÇAMENTÁRIO: O impacto orçamentário incremental após a proposta de redução de preço pelo demandante foi estimado em aproximadamente 28 bilhões de reais, como melhor cenário. MONITORAMENTO DO HORIZONTE TECNOLÓGICO: Foram identificadas oito tecnologias potenciais para a indicação clínica. O opaganib consiste no primeiro agente de uma nova classe farmacológica, a dos inibidores de esfingosina-quinases. Outro potencial medicamento para a indicação a ser administrado por via oral e em combinação ao cuidado padrão (não especificado no protocolo do estudo), é o fostamatinib. O reparixin é um análogo do ibuprofeno e sua eficácia para o tratamento de pacientes com pneumonia grave acometidos por COVID-19 está sendo avaliada. O BDB-001 e o ravulizumab têm como alvo farmacológico o fator C5a do sistema complemento. Os anticorpos monoclonais canaquinumab, mavrilimumab e tocilizumabe também estão em desenvolvimento. Além disso, foram identificados três depósitos de patentes relacionados ao rendesivir no Instituto Nacional da Propriedade Intelectual. CONSIDERAÇÕES FINAIS: O balanço entre os aspectos positivos e negativos do rendesivir foi desfavorável ao seu uso no tratamento de pacientes com COVID-19. Apesar do baixo risco de eventos adversos, houve uma baixa confiança na eficácia do medicamento, uma vez que os resultados dos estudos são discrepantes e baseados em uma análise de subgrupo de apenas um estudo. O impacto orçamentário estimado foi considerado elevado. RECOMENDAÇÃO PRELIMINAR DA CONITEC: Diante do exposto, a Conitec, em sua 98ª Reunião Ordinária, realizada no dia 10 de junho de 2021, deliberou que a matéria fosse disponibilizada em consulta pública com recomendação preliminar desfavorável à incorporação no SUS do rendesivir para o tratamento da doença causada pelo coronavírus 2019 (COVID19), em pacientes adultos com pneumonia que requerem suplementação de oxigênio de baixo ou alto fluxo. Foi discutido, emplenária, que a evidência disponívelsobre a tecnologia emavaliação foi baseada emestudos adaptativos heterogêneos, com importantes limitações metodológicas, que podem se traduzir em resultados devidos apenas ao acaso. Além disso, o perfil de segurança do rendesivir, quando comparado aos medicamentos de cuidado padrão, mostrou que o medicamento está associado a um risco aumentado de bradicardia em pacientes diagnosticados com COVID-19. CONSULTA PÚBLICA: Foram recebidas 90 contribuições, sendo 34 contribuições técnico-científicas e 56 de experiência e opinião. Diante das argumentações apresentadas, o plenário da Conitec entendeu que não houve argumentação suficiente para mudança de entendimento acerca de sua recomendação preliminar, com base nas evidências científicas apresentadas, tampouco na redução de preço proposta pelo fabricante. Desse modo, a Comissão, diante das incertezas quanto à eficácia do medicamento, manteve a posição desfavorável à incorporação do rendesivir para o tratamento dos pacientes hospitalizados com pneumonia provocada pelo COVID-19 com necessidade de suplementação de oxigênio. DELIBERAÇÃO FINAL: O Plenário da Conitec, em sua 100ª Reunião Ordinária, no dia 05 de agosto de 2021, deliberou por unanimidade recomendar a não incorporação do rendesivir para o tratamento de pacientes com COVID-19 com necessidade de suplementação de oxigênio (baixo ou alto fluxo, ventilação não invasiva) no âmbito do SUS, com base nas incertezas quanto à eficácia do medicamento. Foi assinado o Registro de Deliberaçã nº 651/2021. DECISÃO: Não incorporar o rendesivir para tratamento de pacientes com Covid-19 hospitalizados com pneumonia e necessidade de suplementação de oxigênio, no âmbito do Sistema Único de Saúde ­ SUS, conforme a Portaria nº 60, publicada no Diário Oficial da União nº 171, seção 1, página 77, em 9 de setembro de 2021.

tiRNAs as a novel biomarker for cell damage assessment in in vitro ischemia-reperfusion model in rat neuronal PC12 cells.

The disruption of appropriate cellular stress responses is implicated in the pathogenesis of different neurological disorders including ischemic injury. Early diagnosis and treatment are often associated with better prognosis in ischemic stroke patients. Thus, there is an urgent need to improve the speed and accuracy of stroke diagnosis by developing highly sensitive stroke biomarkers. We recently reported that transfer RNA (tRNA) was involved in cell stress response pathways. Under cell stress conditions, mature tRNA is cleaved by a specific ribonuclease, angiogenin, generating tRNA-derived stress-induced RNA (tiRNA). To study tiRNA generation in an in vitro model of ischemic-reperfusion injury, we used the rat neuronal cell line, PC12, in combination with analysis of SYBR staining and immuno-northern blotting using anti-1-methyladenosine antibody, which detects 1-methyladenosine (m1A) modification of tRNA. We demonstrated that oxygen-glucose deprivation induced tRNA cleavage and tiRNA generation. Time course analysis showed a dramatic up-regulation of tiRNA generation by oxygen-glucose deprivation (OGD) which started a few minutes after reperfusion. Minocycline, a neuroprotective antibiotic, treatment protected PC12 cells against OGD-reperfusion cell damage resulting in a marked down-regulation of the generated tiRNA. Our findings show that cleavage of tRNA and tiRNA generation in rat neuronal PC12 cells occurs with reperfusion injury and the detection of tiRNA could be used as a potential cell damage marker and treatment effect indicator for this type of injury.

Methodical Challenges and a Possible Resolution in the Assessment of Receptor Reserve for Adenosine, an Agonist with Short Half-Life.

The term receptor reserve, first introduced and used in the traditional receptor theory, is an integrative measure of response-inducing ability of the interaction between an agonist and a receptor system (consisting of a receptor and its downstream signaling). The underlying phenomenon, i.e., stimulation of a submaximal fraction of receptors can apparently elicit the maximal effect (in certain cases), provides an opportunity to assess the receptor reserve. However, determining receptor reserve is challenging for agonists with short half-lives, such as adenosine. Although adenosine metabolism can be inhibited several ways (in order to prevent the rapid elimination of adenosine administered to construct concentration-effect (E/c) curves for the determination), the consequent accumulation of endogenous adenosine biases the results. To address this problem, we previously proposed a method, by means of which this bias can be mathematically corrected (utilizing a traditional receptor theory-independent approach). In the present investigation, we have offered in silico validation of this method by simulating E/c curves with the use of the operational model of agonism and then by evaluating them using our method. We have found that our method is suitable to reliably assess the receptor reserve for adenosine in our recently published experimental setting, suggesting that it may be capable for a qualitative determination of receptor reserve for rapidly eliminating agonists in general. In addition, we have disclosed a possible interference between FSCPX (8-cyclopentyl-N³-[3-(4-(fluorosulfonyl)benzoyloxy)propyl]-N¹-propylxanthine), an irreversible A1 adenosine receptor antagonist, and NBTI (S-(2-hydroxy-5-nitrobenzyl)-6-thioinosine), a nucleoside transport inhibitor, i.e., FSCPX may blunt the effect of NBTI.

Cancer Immunotherapy: Whence and Whither.

The current concepts and practice of cancer immunotherapy evolved from classical experiments that distinguished "self" from "non-self" and the finding that humoral immunity is complemented by cellular immunity. Elucidation of the biology underlying immune checkpoints and interactions between ligands and ligand receptors that govern the immune system's ability to recognize tumor cells as foreign has led to the emergence of new strategies that mobilize the immune system to reverse this apparent tolerance. Some of these approaches have led to new therapies such as the use of mAbs to interfere with the immune checkpoint. Others have exploited molecular technologies to reengineer a subset of T cells to directly engage and kill tumor cells, particularly those of B-cell malignancies. However, before immunotherapy can become a more effective method of cancer care, there are many challenges that remain to be addressed and hurdles to overcome. Included are manipulation of tumor microenvironment (TME) to enhance T effector cell infiltration and access to the tumor, augmentation of tumor MHC expression for adequate presentation of tumor associated antigens, regulation of cytokines and their potential adverse effects, and reduced risk of secondary malignancies as a consequence of mutations generated by the various forms of genetic engineering of immune cells. Despite these challenges, the future of immunotherapy as a standard anticancer therapy is encouraging. Mol Cancer Res; 15(6); 635-50. ©2017 AACR.

Safety and efficacy of intracoronary nicorandil as hyperaemic agent for invasive physiological assessment: a patient-level pooled analysis.

AIMS: Our aim was to evaluate the safety and efficacy of intracoronary (IC) nicorandil as an alternative choice of hyperaemic agent for invasive physiologic studies. METHODS AND RESULTS: A total of 480 intermediate coronary lesions from 429 patients enrolled from six Japanese and Korean centres were analysed. IC nicorandil showed earlier achievement of hyperaemia (time to the lowest FFR: 18.0 s [1st and 3rd quartile value 15.6-21.5] vs. 44.0 s [36.0-60.0], p<0.001) with similar hyperaemic efficacy, compared with intravenous (IV) adenosine/ATP (FFR 0.82 [0.75-0.87] vs. 0.82 [0.74-0.88], p=0.207). FFR measurements with both agents showed excellent correlation and classification agreement (CA) for FFR ≤0.80 (r=0.941, ICC 0.980, CA 90.8%, kappa=0.814, AUC of nicorandil 0.980, all p<0.001). Only three patients (0.7%) showed changes in classification across the grey zone (0.75-0.80). IC nicorandil produced fewer changes in blood pressure (BP) and heart rate (HR) and showed less chest pain than IV adenosine/ATP (all p<0.001). When comparing ΔFFR according to ΔBP or ΔHR between IV adenosine/ATP and IC nicorandil, there were no correlations, either between ΔFFR and ΔBP (r=-0.114, p=0.091), or between ΔFFR and ΔHR (r=1.000, p=0.151). CONCLUSIONS: Nicorandil IC bolus injection is a simple, safe and effective hyperaemic method for FFR measurement and can be used as a substitute for adenosine.

Studies on the adenosine deaminase-catalyzed conversion of adenosine and nucleoside prodrugs by different capillary electrophoresis modes.

Four kinds of fast and efficient capillary electrophoresis modes, i.e., immobilized enzymatic reactor (IER), electrophoretically mediated microanalysis (EMMA), capillary zone electrophoresis (CZE), and micellar electrokinetic chromatography (MEKC), were first developed to study the adenosine deaminase (ADA)-catalyzed conversion of adenosine and nucleoside prodrugs, which is critical for releasing prodrugs into the intracellular compartment for phosphorylation. The enzyme-activated prodrug approach is a strategy that has been successfully employed to improve physicochemical and pharmacokinetic properties of potential therapeutic agents, especially in the search for antiviral nucleoside analogues. Adenosine, amino-ddG, and amino-D4G could be converted by ADA to different extents under our experimental conditions. Steady-state parameters K(m), V(max), and k(cat) were also determined. The substrate efficiencies (k(cat)/K(m)) of adenosine, amino-ddG, and amino-D4G were 0.19±0.01, 0.047±0.005, and 0.017±0.010 µM(-1) s(-1), respectively. The enzymatic reaction could be performed at a nanoliter scale and all manipulation steps were combined into a fully automated assay in on-line modes, which opened the possibilities of high-throughput screening of large libraries of synthetic nucleoside analogues for biological activity and a relative mechanism study of nucleoside and its analogues.

Effect of ezetimibe-simvastatine over endothelial dysfunction in dyslipidemic patients: assessment by 13N-ammonia positron emission tomography.

BACKGROUND: Dyslipidemias constitute an independent risk factor for the development of atherogenesis and they also predispose to the development of endothelial dysfunction (ED). Using PET with (13)N-ammonia, it is possible to quantify myocardial blood flow (MBF) in mL/min/g and to quantitatively evaluate ED. With the use of lipid lowering therapy it is possible to reduce ED and increase the MBF and the endothelial-dependent vasodilation index (ENDEVI). In this study, we aimed to evaluate with (13)N-ammonia PET the benefic effects of the combined treatment ezetimibe/simvastatine on the endothelial function of dyslipidemic patients after 8 weeks of treatment. MATERIAL AND METHODS: Fourteen consecutive patients with dyslipidemia diagnosis and 17 healthy volunteers were studied with a three phase [rest, Cold Pressor Test (CPT), and adenosine-induced hyperemia] (13)N-ammonia PET for MBF quantification assessment. A second PET study was performed in the dyslipidemic group after 8 weeks of treatment with ezetimibe/simvastatine (10/40 mg). Myocardial flow reserve (MFR), ENDEVI, and %ΔMBF were calculated. RESULTS: Total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides concentrations were markedly altered in the dyslipidemic group and after 8 weeks of treatment these values improved. Dyslipidemic patients showed endothelial dysfunction when compared with the control group, (MFR 2.79 ± 0.94 vs 3.15 ± 0.48, P < 0.05 ; ENDEVI 1.28 ± 0.25 vs 1.53 ± 0.24, P < 0.05; and %ΔMBF 29.08 ± 24.62 vs 53 ± 24.60%, P < 0.05, respectively). After 8 weeks of treatment, we found a significant increase in all the endothelial function markers (MFR: 3.14 ± 0.86, P < 0.05, ENDEVI 1.65 ± 0.23, P < 0.05; %ΔMBF: 65.21 ± 23.43, P < 0.05). CONCLUSIONS: Dyslipidemic patients show endothelial dysfunction measured with (13)N-ammonia PET. Treatment with ezetimibe/simvastatine was effective improving the lipid profile as well as the endothelial function of these patients. PET may be a useful tool to monitor vascular reactivity and regression/progression of coronary atherosclerosis after pharmacologic interventions.

ADAR1 is involved in the development of microvascular lung injury.

Deamination of adenosine on pre-mRNA to inosine is a recently discovered process of posttranscription modification of pre-mRNA, termed A-to-I RNA editing, which results in the production of proteins not inherent in the genome. The present study aimed to identify a role for A-to-I RNA editing in the development of microvascular lung injury. To that end, the pulmonary expression and activity of the RNA editase ADAR1 were evaluated in a mouse model of endotoxin (15 mg/kg IP)-induced microvascular lung injury (n=5) as well as in cultured alveolar macrophages stimulated with endotoxin, live bacteria, or interferon. ADAR1 expression and activity were identified in sham lungs that were upregulated in lungs from endotoxin-treated mice (at 2 hours). Expression was localized to polymorphonuclear and monocytic cells. These events preceded the development of pulmonary edema and leukocyte accumulation in lung tissue and followed the local production of interferon-gamma, a known inducer of ADAR1 in other cell systems. ADAR1 was found to be upregulated in alveolar macrophages (MH-S cells) stimulated with endotoxin (1 to 100 microg/mL), live Escherichia coli (5x10(7) colony-forming units), or interferon-gamma (1000 U/mL). Taken together, these data suggest that ADAR1 may play a role in the pathogenesis of microvascular lung injury possibly through induction by interferon.

Simultaneous assessment of ecto- and cytosolic-5'-nucleotidase activities in brain micropunches.

We propose a new methodology for simultaneous assessment of ecto- and cytosolic-5'-nucleotidase that can be utilized in brain to measure the activity of these enzymes in micropunches of tissues. It is based on the differential sensitivity of both enzymes to alpha,beta-methyleneadenosine 5'-diphosphate (AMP-CP) and the requirements for magnesium as a co-factor. The design of assay protocol contains an internal validation by allowing comparisons between total level of 5'-nucleotidase activity with that calculated from the sum of individual activities of the ecto- and cytosolic-5'-nucleotidases. We have applied this new approach to assess the activity of ecto- and cytosolic-5'-nucleotidase in the brain regions relevant to sleep regulation. The level of both enzymes was significantly lower in the cerebral cortex than other brain regions tested.

Determinants of the S-adenosylhomocysteine (SAH) technique for the local assessment of cardiac free cytosolic adenosine.

The S-adenosylhomocysteine (SAH) technique allows the estimation of the free cytosolic adenosine concentration using the kinetic properties of the enzyme SAH-hydrolase (adenosine+homocysteine reversible SAH+H2O). Besides the cytosolic adenosine concentration, the local SAH signal may also depend on the local homocysteine availability, the continuous production of SAH from S-adenosylmethionine (SAM-->SAH+CH3) and the activity of the enzyme SAH-hydrolase. These variables were studied with high spatial resolution (sample dry mass 25 mg) in left ventricular myocardium from 26 anesthetized open-chest dogs in which heart rate averaged 86 +/- 14 beats/min and mean aortic pressure 96 +/- 17 mmHg. Homocysteine infusion (48 mg/kg i.v.) increased the normal plasma homocysteine concentration from 5.0 +/- 0.8 to 586 +/- 40 microM after 30 min when the average tissue concentration was 94% of the plasma concentration and similar in low and high flow areas (flow range 0.04 to 1.91 ml/min/g). Local SAH content was 1.18 +/- 0.48 nmol/g under control conditions and increased to 4.33 +/- 0.59 nmol/g within 60 min following competitive blockage of the SAH-hydrolase by adenosine dialdehyde (10 mumol/kg i.v.). This increase of the SAH content was slightly more in high than in low-flow areas (P < 0.01). Regional SAH-hydrolase activity (9.0 +/- 0.5 nmol/min/g) was comparable in high and low flow areas. All three variables exhibited an observed variability which was larger than the methodical variability suggesting significant spatial heterogeneity in the myocardium. A regrouping analysis indicated that between four and five samples taken from distant sites should be averaged to obtain a robust estimate of the above metabolic parameters. Reconciling the measurements with a mathematical model of cardiac adenosine metabolism and fitting of the measured SAH tissue levels gave an estimate of 72 pmol/min/g for the mean transmethylation rate. Estimates of the cytosolic adenosine concentration of cardiomyocytes and endothelial cells under control physiological conditions were 24 and 7 microM, respectively. Thus, the present measurements provide a basis for the quantitative assessment of the local cytosolic adenosine concentration in relation to blood flow.

In vitro assessment of susceptibility of Acanthamoeba polyphaga to drugs using combined methods of dye-binding assay and uptake of radiolabelled adenosine.

A technique based on binding of eosin dye to cell was applied to quantitate Acanthamoeba trophozoites in culture. Using this technique in combination with the uptake of radiolabelled adenosine, we assessed the activities of triazole (saperconazole), imidazole (ketoconazole, miconazole) and diamidine (berenil, pentamidine, dibromopropamidine) compounds against A. polyphaga trophozoites. The quantity of dye bound to the trophozoites correlated (r = 0.99) with the number of organisms per well. When inhibition of the growth of Acanthamoeba was the only parameter used to measure the effectiveness of these drugs, saperconazole was found to be most active with IC50 (concentration of drug that inhibited by 50%, the growth of A. polyphaga trophozoites incubated at 28 degrees C for 48 h) of 0.95 microM. The IC50s of the other drugs ranged from > or = 1500 microM for micronazole, the least active, to 3.0 microM for berenil. However, when efficacy was assessed by combining inhibition of growth with the uptake of [14C-8]adenosine by the drug treated organism, the diamidines, particularly berenil and pentamidine were considered most potent.

Assessment of the enantiomeric purity of R- and S-N6-phenylisopropyladenosine (PIA): implications for adenosine receptor subclassification.

A chiral column high-performance liquid chromatographic method was developed for the assessment of the enantiomeric purity of the stereoisomers of N6-phenylisopropyladenosine (PIA). The observed chiral purity of R-PIA was greater than 99.9%, whereas S-PIA was found to contain 4.4% of the R-enantiomer. In radioligand binding studies, the observed affinity of S-PIA for the adenosine A1 receptor (IC50 240 nM) could entirely be attributed to its content of R-PIA (IC50 7.8 nM). Calculation of a theoretical IC50 of pure S-PIA for the A2 receptor yielded a value of 6700 nM, which was 35-fold higher than for R-PIA (190 nM). Concludingly, the utilization of enantiomeric impure S-PIA in the definition of adenosine receptor subclasses is questionable.

A low-priced computer-assisted densitometer and analysis software for quantitative autoradiography.

A computer-assisted densitometer which consists of a darkroom enlarger, a black-and-white exposure meter, an amplifier, an analog-to-digital converter, and a microcomputer with a monitor and a graphic printer can be utilized for the quantitative analysis of autoradiograms. QUANTAR, a computer program written in BASIC, records the density measurements and stores the data in a file that can be easily retrieved by commercially available spreadsheet software. A spreadsheet template was designed to convert the digital readings into concentration of ligand in tissue. A variety of spreadsheet templates can be created to analyze data for a standard curve, a Scatchard plot, or a binding competition curve. This system, which is easy to assemble, may be useful to the frugal investigator with a modest equipment budget.

In vivo induced malignant hyperthermia in pigs. I. Physiological and biochemical changes and the influence of dantrolene sodium.

The effects of an induced malignant hyperthermia (MH) crisis have been studied in the intact pig. Both physiological and biochemical changes in skeletal muscle were studied. MH was induced with 3% halothane plus a bolus injection of succinylcholine. In the prechallenge period a significant difference was observed in the concentration of certain muscle metabolites, comparing the MH-susceptible (MH+) with the non-susceptible (MH-) pigs. A lower level was measured for phosphocreatine (PCr), inosine monophosphate (IMP) and an increased level of lactate and creatine (Cr) in the susceptible pigs (MH+). The challenge caused a significant reduction of the level of PCr and adenosine in MH+ pigs, compared to the prechallenge period. After administration of dantrolene sodium, a significant decrease was measured in the level of lactate, compared to the prechallenge period as well as during the challenge. In contrast, in the control pigs no significant changes were observed in muscle metabolites, either after induction of MH or after the administration of dantrolene sodium. Enzyme activity determinations of muscle adenylate kinase and adenosine monophosphate (AMP)-deaminase did not show any difference in activity either before or during the MH crisis or after treatment with dantrolene sodium. The earliest physiological change during an induced MH crisis in our study was the rapid increase of the end-tidal CO2. Within 5 min after MH induction, end-tidal CO2 was doubled. It is concluded that the monitoring of the end-tidal CO2 is essential to diagnose MH at a very early stage.

Assessment of vasoactive metabolites released from the isolated guinea pig during heart hypoxia and beta-adrenergic stimulation.

Two isolated guinea pig hearts (H1, H2) were perfused in series in order to bioassay in the recipient heart (H2) the release of vasoactive metabolites. Due to an effective reoxygenation system transit time between H1 and H2 as only 20 sec. Hypoxic perfusion (30% O2) of H1 caused relaxation of the coronary vessels of H2, and this effect could be completely abolished by adenosine deaminase. Similar results were obtained when H1 was stimulated by isoproterenol while H2 was protected by propranolol. From our findings it is concluded that adenosine is the primary vasodilating substance released by the heart into the coronary circulation.