Thyroid disrupting effects of low-dose dibenzothiophene and cadmium in single or concurrent exposure: New evidence from a translational zebrafish model.

Thyroid hormones (THs) are major regulators of biological processes essential for correct development and energy homeostasis. Although thyroid disruptors can deeply affect human health, the impact of exogenous chemicals and in particular mixture of chemicals on different aspects of thyroid development and metabolism is not yet fully understood. In this study we have used the highly versatile zebrafish model to assess the thyroid axis disrupting effects of cadmium (Cd) and dibenzothiophene (DBT), two environmental endocrine disruptors found to be significantly correlated in epidemiological co-exposure studies. Zebrafish embryos (5hpf) were exposed to low concentrations of Cd (from 0.05 to 2 µM) and DBT (from 0.05 to 1 µM) and to mixtures of them. A multilevel assessment of the pollutant effects has been obtained by combining in vivo morphological analyses allowed by the use of transgenic fluorescent lines with liquid chromatography mass spectrometry determination of TH levels and quantification of the expression levels of key genes involved in the Hypothalamic-Pituitary-Thyroid Axis (HPTA) and TH metabolism. Our results underscore for the first time an important synergistic toxic effect of these pollutants on embryonic development and thyroid morphology highlighting differences in the mechanisms through which they can adversely impact on multiple physiological processes of the HPTA and TH disposal influencing also heart geometry and function.

Mitochondrial F(0) F(1) -ATP synthase is a molecular target of 3-iodothyronamine, an endogenous metabolite of thyroid hormone.

BACKGROUND AND PURPOSE: 3-iodothyronamine (T1AM) is a metabolite of thyroid hormone acting as a signalling molecule via non-genomic effectors and can reach intracellular targets. Because of the importance of mitochondrial F(0) F(1) -ATP synthase as a drug target, here we evaluated interactions of T1AM with this enzyme. EXPERIMENTAL APPROACH: Kinetic analyses were performed on F(0) F(1) -ATP synthase in sub-mitochondrial particles and soluble F(1) -ATPase. Activity assays and immunodetection of the inhibitor protein IF(1) were used and combined with molecular docking analyses. Effects of T1AM on H9c2 cardiomyocytes were measured by in situ respirometric analysis. KEY RESULTS: T1AM was a non-competitive inhibitor of F(0) F(1) -ATP synthase whose binding was mutually exclusive with that of the inhibitors IF(1) and aurovertin B. Both kinetic and docking analyses were consistent with two different binding sites for T1AM. At low nanomolar concentrations, T1AM bound to a high-affinity region most likely located within the IF(1) binding site, causing IF(1) release. At higher concentrations, T1AM bound to a low affinity-region probably located within the aurovertin binding cavity and inhibited enzyme activity. Low nanomolar concentrations of T1AM increased ADP-stimulated mitochondrial respiration in cardiomyocytes, indicating activation of F(0) F(1) -ATP synthase consistent with displacement of endogenous IF(1,) , reinforcing the in vitro results. CONCLUSIONS AND IMPLICATIONS: Effects of T1AM on F(0) F(1) -ATP synthase were twofold: IF(1) displacement and enzyme inhibition. By targeting F(0) F(1) -ATP synthase within mitochondria, T1AM might affect cell bioenergetics with a positive effect on mitochondrial energy production at low, endogenous, concentrations. T1AM putative binding locations overlapping with IF(1) and aurovertin binding sites are described.

Parasitoid diversity (Hymenoptera: Braconidae and Figitidae) on frugivorous larvae (Diptera: Tephritidae and Lonchaeidae) at Adolpho Ducke Forest Reserve, Central Amazon Region, Manaus, Brazil / Diversidade de Parasitóides (Hymenoptera: Braconidae e Figitidae) de larvas frugívoras (Diptera: Tephritidae e Lonchaeidae) na Reserva Florestal Adolpho Ducke, Amazônia Ocidental, Manaus, Brasil

This study aimed to identify parasitoid species of frugivorous larvae and to describe the tritrophic interactions involving wild fruits, frugivorous insects and their natural enemies at Adolpho Ducke Forest Reserve (RFAD) (Manaus, AM, Brazil). Collections were performed in four 1 km² quadrants in the corners of the RFAD. The wild fruits were collected inside the forest in access trails leading to each collection area and in trails that surrounded the quadrants, up to five metres from the trail on each side. The fruits were placed in plastic containers covered with thin fabric, with a vermiculite layer on the base to allow the emergence of flies or parasitoids. Seven Braconidae species were collected, distributed among Opiinae: Doryctobracon areolatus (Szépligeti, 1911), Utetes anastrephae (Viereck, 1913), and Opius sp., and Alysiinae: Asobara anastrephae (Muesebeck, 1958), Phaenocarpa pericarpa Wharton and Carrejo, 1999, Idiasta delicata Papp, 1969, and Asobara sp. Parasitism rates by braconids and figitids are presented. Doryctobracon areolatus was the most frequent, parasitizing the highest number of fly species, and showing the highest parasitism percentage in larvae feeding on Micropholis williamii fruits. The collected figitids belong to Aganaspis nordlanderi Wharton, 1998 and A. pelleranoi (Brethes, 1924). All 15 tritrophic associations are new records for the Brazilian Amazon region. The RFAD is an important natural reservoir of frugivorous larvae parasitoids.

Este trabalho teve por objetivo identificar as espécies de parasitóides de larvas frugívoras e descrever as interações tritróficas desses inimigos naturais com seus insetos hospedeiros e frutos silvestres da Reserva Florestal Adolpho Ducke (RFAD). As coletas foram realizadas em quatro quadrantes de 1 km², ocupando áreas próximas aos cantos da RFAD. Os frutos silvestres foram coletados no interior da floresta nas trilhas de acesso a cada área de coleta e nas trilhas que delimitavam os quadrantes, até cinco metros em cada lado. Os frutos foram acondicionados em recipientes plásticos, cobertos com tecido fino, contendo camada de vermiculita, para obtenção das moscas ou dos parasitóides, que eram individualizados, contados, sexados e fixados em álcool 70 por cento. As taxas de parasitismo por braconídeos e figitídeos são apresentadas. Foram coletadas sete espécies de Braconidae, distribuídas em Opiinae: Doryctobracon areolatus (Szépligeti, 1911), Utetes anastrephae (Viereck, 1913), Opius sp. e Alysiinae: Asobara anastrephae (Muesebeck, 1958), Phaenocarpa pericarpa Wharton e Carrejo, 1999, Idiasta delicata Papp, 1969 e Asobara sp. Doryctobracon areolatus foi a espécie mais frequente, parasitando o maior número de espécies de moscas e apresentando a maior percentagem de parasitismo em larvas em frutos de Micropholis williamii. OS figitídeos pertenciam a Aganaspis nordlanderi Wharton, 1998 e A. pelleranoi (Brethes, 1924). Todas as 15 associações tritróficas obtidas representam novos registros para a região Amazônica brasileira.

Diversity and seasonality of fruit flies (Diptera: Tephritidae and Lonchaeidae) and their parasitoids (Hymenoptera: Braconidae and Figitidae) in orchards of guava, loquat and peach / Diversidade e variação sazonal de moscas-das-frutas (Diptera: Tephritidae, Lonchaeidae) e seus parasitóides (Hymenoptera: Braconidae, Figitidae) em pomares de goiaba, nêspera e pêssego

This work was carried out in orchards of guava progenies, and loquat and peach cultivars, in Monte Alegre do Sul, SP, Brazil, in 2002 and 2003. Guavas and loquats were bagged and unbagged bi-weekly and weekly, respectively, for assessment of the infestation period. Peach was only bagged weekly. The assays started when the fruits were at the beginning of development, but still green. Ripe fruits were taken to the laboratory and placed individually into plastic cups. McPhail plastic traps containing torula yeast were hung from January 2002 to January 2004 to assess the fruit fly population in each orchard, but only the Ceratitis capitata population is here discussed. Five tephritid species were reared from the fruits: Anastrepha bistrigata Bezzi, A. fraterculus (Wiedemann), A. obliqua (Macquart), A. sororcula Zucchi, and C. capitata, in addition to six lonchaeid species: Neosilba certa (Walker), N. glaberrima (Wiedemann), N. pendula (Bezzi), N. zadolicha McAlpine and Steyskal, Neosilba sp. 4, and Neosilba sp. 10 (both species are in the process of being described by P. C. Strikis), as well as some unidentified Neosilba species. Ten parasitoid species were obtained from fruit fly puparia, of which five were braconids: Asobara anastrephae (Muesebeck), Doryctobracon areolatus (Szépligeti), D. brasiliensis (Szépligeti), Opius bellus Gahan, and Utetes anastrephae (Viereck), and five figitids: Aganaspis pelleranoi (Brèthes), Dicerataspis grenadensis Ashmead, Lopheucoila anastrephae (Rhower), Leptopilina boulardi (Barbotin, Carlton and Kelner-Pillaut), and Trybliographa infuscata Diaz, Gallardo and Uchôa. Ceratitis capitata showed a seasonal behavior with population density peaking at the second semester of each year. Anastrepha and Neosilba species remained in the orchards throughout both years.

Este trabalho foi realizado em três pomares em Monte Alegre do Sul, SP, em 2002 e 2003, representados por coleção de progênies de goiabeiras, de cultivares de nespereiras e de cultivares de pessegueiros. O período de infestação foi determinado por meio de ensacamento e desensacamento quinzenal e semanal de goiabas e nêsperas, respectivamente, e pelo ensacamento semanal de pêssegos. Os ensaios iniciaram-se com os frutos verdes (princípio de desenvolvimento). Os frutos maduros foram levados ao laboratório e acondicionados individualmente em copos plásticos. A flutuação populacional de Ceratitis capitata (Wiedemann) foi avaliada por meio de armadilhas plásticas modelo McPhail com torula em cada pomar, de janeiro/2002 a janeiro/2004. Dos frutos foram obtidas cinco espécies de tefritídeos: Anastrepha bistrigata Bezzi, A. fraterculus (Wiedemann), A. obliqua (Macquart), A. sororcula Zucchi e C. capitata e seis de lonqueídeos: Neosilba certa (Walker), N. glaberrima (Wiedemann), N. pendula (Bezzi), N. zadolicha McAlpine and Steyskal, Neosilba sp. 4 e Neosilba sp. 10, além de algumas espécies não-identificadas. Foram obtidas 10 espécies de parasitóides, cinco da família Braconidae - Asobara anastrephae (Muesebeck), Doryctobracon areolatus (Szépligeti), D. brasiliensis (Szépligeti), Opius bellus Gahan e Utetes anastrephae (Viereck) - e cinco da família Figitidae - Aganaspis pelleranoi (Brèthes), Dicerataspis grenadensis Ashmead, Lopheucoila anastrephae (Rhower), Leptopilina boulardi (Barbotin, Carlton and Kelner-Pillaut) e Trybliographa infuscata Diaz, Gallardo and Uchôa. Ceratitis capitata apresentou comportamento sazonal com picos populacionais durante o segundo semestre dos dois anos. As espécies de Anastrepha e de Neosilba permaneceram nos pomares durante os dois anos.

Colony membership is reflected by variations in cuticular hydrocarbon profile in a Neotropical paper wasp, Polistes satan (Hymenoptera, Vespidae)

Nestmate recognition is one the most important features in social insect colonies. Although epicuticular lipids or cuticular hydrocarbons have both structural and defensive functions in insects, they also seem to be involved in several aspects of communication in wasps, bees and ants. We analyzed and described for the first time the cuticular hydrocarbons of a Neotropical paper wasp, Polistes satan, and found that variation in hydrocarbon profile was sufficiently strong to discriminate individuals according to their colony membership. Therefore, it seems that small differences in the proportion of these compounds can be detected and used as a chemical-based cue by nestmates to detect invaders and avoid usurpation.

Ghrelin tissue distribution: comparison between gene and protein expression.

Ghrelin, the natural ligand of the GH secretagogue (GHS) receptor, was originally isolated from the stomach and detected in several tissues, but a systematic study of its tissue distribution has not been performed. In the present investigation, we evaluated ghrelin gene expression (by RT-PCR technique) and ghrelin protein concentration (by enzyme immunoassay technique) in tissues obtained from control rats as well as in rats subjected to 48-h fasting. The ghrelin gene was expressed in stomach, small intestine, brain, cerebellum, pituitary, heart, pancreas, salivary gland, adrenal, ovary and testis, with maximum expression occurring in the stomach, while no significant expression was detected by standard RT-PCR in liver, lung, kidney and skeletal muscle. Ghrelin protein was detected in stomach, small intestine, brain, cerebellum, pituitary, lung, skeletal muscle pancreas, salivary gland, adrenal, ovary and testis, at concentrations ranging from 0.05 to 1.43 ng/mg of homogenate protein (the highest concentration occurred in the lung, followed by the brain). Ghrelin was not detectable in the heart, liver and kidney. Therefore, gene and protein expression were dissociated. Fasting did not produce significant changes in ghrelin gene expression, while the distribution of ghrelin between different tissues was significantly modified: protein concentration increased in the brain, cerebellum, lung and salivary gland, while it decreased in the stomach.

Intraspecific variation in Trichogramma bruni Nagaraja, 1983 (Hymanoptera: Trichogrammatidae) associated with different hosts

Trichogramma bruni is an insufficienty studied South American species whose limits are still not well defined. Thus, the objective of the present study was to characterize T. bruni taxonomically and to determine the association between morphological variations as well as host and habitat, based on morphological and biological studies. Specimens from the Escola Superior de Agricultura "Luiz de Queiroz" (ESALQ) collection, and from the University of California Riverside (UCR) and specimens collected from the vegetation of forest parks with native areas planted with eucalyptus in Piracicaba and Itatinga, State of Säo Paulo, were also analyzed. The holotype deposited at Univeridade Federal de Minas Gerais (UFMG) collection was also examined. The variability in the genital capsule of T. bruni observed both among individuals of the same progeny and among specimens from different hosts is remarkable and is mainly related to the dorsal lamina. Therefore, an association of diagnostic characters rather than the dorsal lamina alone should be used for the identification of T. bruni and intraspecific variations should be considered. The intraspecific variation observed for T. bruni is a factor that should be considered for its identification, since the influence of the environment (habitat + host) and the variation among individuals itself is responsible for the plasticity observed in the genital capsule. Heliconius erato phyllis, Hamadryas feronia, Erosina hyberniata and Mechanitis lysiminia are new hosts of T. bruni

Intraspecific variation in Trichogramma bruni Nagaraja, 1983 (Hymanoptera: Trichogrammatidae) associated with different hosts

Trichogramma bruni is an insufficienty studied South American species whose limits are still not well defined. Thus, the objective of the present study was to characterize T. bruni taxonomically and to determine the association between morphological variations as well as host and habitat, based on morphological and biological studies. Specimens from the Escola Superior de Agricultura "Luiz de Queiroz" (ESALQ) collection, and from the University of California Riverside (UCR) and specimens collected from the vegetation of forest parks with native areas planted with eucalyptus in Piracicaba and Itatinga, State of São Paulo, were also analyzed. The holotype deposited at Univeridade Federal de Minas Gerais (UFMG) collection was also examined. The variability in the genital capsule of T. bruni observed both among individuals of the same progeny and among specimens from different hosts is remarkable and is mainly related to the dorsal lamina. Therefore, an association of diagnostic characters rather than the dorsal lamina alone should be used for the identification of T. bruni and intraspecific variations should be considered. The intraspecific variation observed for T. bruni is a factor that should be considered for its identification, since the influence of the environment (habitat + host) and the variation among individuals itself is responsible for the plasticity observed in the genital capsule. Heliconius erato phyllis, Hamadryas feronia, Erosina hyberniata and Mechanitis lysiminia are new hosts of T. bruni.

Trichogramma bruni é uma espécie sul-americana pouco estudada e que ainda não tem seus limites bem definidos. Assim, o objetivo deste trabalho foi realizar uma caracterização taxonômica de T. bruni e analisar a associação das variações morfológicas com os hospedeiros e habitats, com base em estudos morfológicos e biológicos. Foram examinados espécimes da coleção da Escola Superior de Agricultura "Luiz de Queiroz" (ESALQ) e da coleção da University of California Riverside (UCR) e exemplares coletados na vegetação de hortos florestais com áreas nativas e plantadas com eucalipto em Piracicaba e Itatinga, Estado de São Paulo. O holótipo (Universidade Federal de Minas Gerais) foi examinado. A variabilidade na cápsula genital de T. bruni, tanto entre os indivíduos de uma mesma progênie como nos espécimes provenientes dos diferentes hospedeiros, é notória e relacionada principalmente à lâmina dorsal. Desta forma, para a identificação de T. bruni, uma associação de caracteres diagnósticos deve ser utilizada e as variações intra-específicas devem ser consideradas. Avariação intra-específica apresentada por T. bruni deve ser considerada em seu reconhecimento, pois a influência do ambiente (habitat + hospedeiro) e a própria variação entre indivíduos são responsáveis pela plasticidade observada na cápsula genital. Heliconius erato phyllis, Hamadryas feronia, Erosina hyberniata e Mechanitis lysiminia são novos registros de hospedeiros.

Modulation of sarcoplasmic reticulum function: a new strategy in cardioprotection?

This article reviews the experimental evidence suggesting that cytosolic Ca(2+) overload plays a major role in the development of myocardial injury during ischemia-reperfusion and that Ca(2+) release from the sarcoplasmic reticulum (SR) is of crucial importance in the early phase of ischemia. It is suggested that interventions able to deplete the SR Ca(2+) pool and/or to reduce the rate of SR Ca(2+) release should be cardioprotective. This thesis is supported by the review of experimental studies in which modulators of the SR Ca(2+)-ATPase or SR Ca(2+) release channel (ryanodine receptor) have been used. In addition, the role of the SR in ischemic preconditioning and in some instances of toxic myocardial injury (particularly, anthraquinone-induced injury) is discussed.

A3 adenosine receptor stimulation modulates sarcoplasmic reticulum Ca(2+) release in rat heart.

OBJECTIVE: Stimulation of A3 adenosine receptors has been shown to protect cardiac myocytes from ischemic injury, but the mechanism of this action is unknown. We evaluated the effect of adenosine agonists and antagonists on the sarcoplasmic reticulum (SR) Ca(2+) channels. METHODS: Isolated rat hearts were perfused with control buffer or different adenosine agonists and antagonists. Hearts were then homogenized and used to determine SR Ca(2+)-induced Ca(2+) release, assayed by quick filtration technique after loading with 45Ca(2+), and the binding of [3H]ryanodine, a specific ligand of the SR Ca(2+) release channel. In parallel experiments, hearts were challenged with 30 min of global ischemia and 120 min of reperfusion, and the extent of tissue necrosis was evaluated by triphenyltetrazolium chloride staining. RESULTS: Perfusion with the A1>A3 agonist R-PIA and the A3>A1 agonist IB-MECA was associated with reduced [3H]ryanodine binding, due to reduced B(max) (by about 20%), whereas K(d) and Ca(2+)-dependence of the binding reaction were unaffected. These actions were abolished by the A3 antagonist MRS 1191, while they were not affected by A1 and A2 antagonists. The rate constant of SR Ca(2+) release decreased by 25-30% in hearts perfused with R-PIA or IB-MECA. Tissue necrosis was significantly reduced in the presence of R-PIA or IB-MECA. Protection was removed by MRS 1191, and it was not affected by A1 and A2 antagonists. Hearts were also protected by administration of dantrolene, a ryanodine receptor antagonist. In the presence of dantrolene, no further protection was provided by IB-MECA. CONCLUSION: A3 adenosine receptor stimulation modulates the SR Ca(2+) channel. This action might account for the protective effect of adenosine.

Effect of MEN 10755, a new disaccharide analogue of doxorubicin, on sarcoplasmic reticulum Ca(2+) handling and contractile function in rat heart.

1. The use of anthraquinone antineoplastic agents is limited by their cardiac toxicity, which is largely due to activation of the sarcoplasmic reticulum (SR) Ca(2+) release channel (ryanodine receptor). MEN 10755 is a new disaccharide analogue of doxorubicin. We have evaluated its effects on SR function and its toxicity in isolated working rat hearts. 2. In rat SR vesicles, doxorubicin stimulated [(3)H]-ryanodine binding by increasing its Ca(2+)-sensitivity. At 1 microM Ca(2+), ryanodine binding increased by 15.3+/-2.5 fold, with EC(50)=20.6 microM. Epirubicin produced a similar effect, i.e. 9.7+/-0.6 fold stimulation with EC(50)=11.1 microM. MEN 10755 increased ryanodine binding by 1.9+/-0.3 fold (P:<0.01 vs doxorubicin and epirubicin), with EC(50)=38.9 microM. 3. Ca(2+)-induced Ca(2+) release experiments were performed by quick filtration technique, after SR loading with (45)Ca(2+). At 2 microM Ca(2+), doxorubicin (50 microM) increased the rate constant of Ca(2+) release to 82+/-5 s(-1) vs a control value of 22+/-2 s(-1) (P:<0.01), whereas 50 microM MEN 10755 did not produce any significant effect (24+/-3 s(-1)). 4. Ca(2+)-ATPase activity and (45)Ca(2+)-uptake were not significantly affected by doxorubicin, its 13-dihydro-derivative, epirubicin, MEN 10755 and the 13-dihydro-derivative of MEN 10755, at concentrations < or =100 microM. 5. In isolated heart experiments, administration of 30 microM doxorubicin or epirubicin caused serious contractile impairment, whereas 30 microM MEN 10755 produced only minor effects. 6. In conclusion, in acute experiments MEN 10755 was much less cardiotoxic than equimolar doxorubicin or epirubicin. This result might be accounted for by reduced activation of SR Ca(2+) release.

Myocardial ischemic preconditioning and mitochondrial F1F0-ATPase activity.

A short period of ischemia followed by reperfusion (ischemic preconditioning) is known to trigger mechanisms that contribute to the prevention of ATP depletion. In ischemic conditions, most of the ATP hydrolysis can be attributed to mitochondrial F1F0-ATPase (ATP synthase). The purpose of the present study was to examine the effect of myocardial ischemic preconditioning on the kinetics of ATP hydrolysis by F1F0-ATPase. Preconditioning was accomplished by three 3-min periods of global ischemia separated by 3 min of reperfusion. Steady state ATP hydrolysis rates in both control and preconditioned mitochondria were not significantly different. This suggests that a large influence of the enzyme on the preconditioning mechanism may be excluded. However, the time required by the reaction to reach the steady state rate was increased in the preconditioned group before sustained ischemia, and it was even more enhanced in the first 5 min of reperfusion (101 +/- 3.0 sec in preconditioned vs. 83.4 +/- 4.4 sec in controls, p < 0.05). These results suggest that this transient increase in activation time may contribute to the cardioprotection by slowing the ATP depletion in the very critical early phase of post-ischemic reperfusion.

Sarcoplasmic reticulum calcium uptake in human myocardium subjected to ischemia and reperfusion during cardiac surgery.

We evaluated the effect of ischemia and reperfusion on sarcoplasmic reticulum Ca uptake in patients subjected to cardiac surgery. Our series included 16 patients (seven female, nine male, age 63 +/- 2 years): five were subjected to aortic valve replacement, five to aortic and mitral valve replacement, six to coronary artery bypass graft. In each case no clinical, electrocardiographic or echocardiographic evidence of perioperative infarction was observed. Biopsies were obtained from the right atrium of each patient before starting extracorporeal circulation, and after the recovery of spontaneous contractile activity, i.e. after cardioplegia-ischemia-reperfusion. The tissue was homogenized, and oxalate-supported Ca uptake, which represents sarcoplasmic reticulum Ca uptake, was measured in the unfractionated homogenate. The assay was performed under basal conditions and in the presence of 900 microM ryanodine, in order to block sarcoplasmic reticulum Ca release channels. Under basal conditions at pCa = 5.85 the rate of sarcoplasmic reticulum Ca uptake averaged 4.76 +/- 0.37 nmol/min per mg of protein in the pre-ischemic samples, and decreased significantly in the post-ischemic samples (3.09 +/- 0.29 nmol/min per mg, P < 0.01). A significant decrease of Ca uptake after ischemia and reperfusion was observed also in the presence of ryanodine (3.53 +/- 0.48 nmol/min per mg) compared to pre-ischemic values (5.98 +/- 0.56 nmol/min per mg, P < 0.01). Additional experiments showed no change in the Ca sensitivity of Ca uptake in the postischemic samples (Kca = 0.48 +/- 0.02 microM, no significant difference after ischemia and reperfusion). In conclusion, active sarcoplasmic reticulum Ca transport was impaired in human atrial myocardium after reversible ischemia and reperfusion.

[Protection of the ischemic myocardium]. / La protezione del miocardio ischemico.

The Authors review several pharmacological interventions aimed at protecting the ischemic myocardium. Drugs which have been widely used in the treatment of ischemic heart diseases, such as beta-blockers, nitrates and calcium-antagonists, are able to delay the development of ischemic injury if administered before the beginning of ischemia, but their clinical effectiveness is limited. The new drugs which are presently investigated are designed to counteract the molecular mechanisms which mediate irreversible tissue injury, namely cytosolic calcium overload, cellular hyperosmolarity, and free radical production. In particular, interventions able to interfere with the release of calcium from its intracellular stores would be of major importance. In this regards, it is interesting to point out that derivatives of phenylalkylamine calcium-antagonists have been reported to modulate the opening probability of sarcoplasmic reticulum calcium channels.

Energy metabolism in myocardial stunning.

We investigated the effect of reversible ischemia, leading to persistent contractile dysfunction (stunning), on myocardial energy metabolism. The balance of energy metabolism is expressed by the phosphorylation state of cytosolic nucleotides. This variable cannot be measured directly because of nucleotide compartmentation, but in the isolated heart it can be estimated by the release of purine catabolites. We have previously shown that increased energy consumption or impaired energy production cause purine release to increase, while primary reduction in energy consumption has the opposite effect. Isolated working rat hearts were reperfused after 10 min of global ischemia, measuring hemodynamic variables, tissue high energy phosphate compounds and purine release. In post-ischemic recovery, aortic flow and minute work decreased to 82 +/- 3% and 77 +/- 4% of control, adenine nucleotide pool was reduced by 4.6 mumol/g dry wt, phosphocreatine to creatine ratio increased significantly and purine release decreased to 42 +/- 6% (P < 0.01). The rate of purine salvage, as evaluated by the incorporation of exogenous 3H-adenosine and 14C-hypoxanthine into tissue nucleotides, was much lower than net purine release, and was unchanged after ischemia and reperfusion. The adenine nucleotide pool could be depleted to the same extent as in the stunned myocardium by prolonged (60 min) aerobic perfusion. In this group the hemodynamic variables were unchanged and purine release averaged 87 +/- 9% of control (P = NS). In other experiments prolonged perfusion was combined with preload reduction in order to decrease energy demand. This protocol reproduced the effects of ischemia-reperfusion: aortic flow and minute work averaged 79 +/- 4% and 73 +/- 9% of control, adenine nucleotide depletion was 4.4 mumol/g dry wt and purine release decreased to 38 +/- 5% (P < 0.01). Our findings support the view that stunning is not due to adenine nucleotide depletion or to impairment in energy production, which would cause purine release to increase, but rather to primary reduction in energy utilization.

[Variations in the purine metabolism of the reperfused heart]. / Variazioni del metabolismo purinico nel cuore riperfuso.

Isolated hearts were subjected to 30 min of aerobic perfusion followed by 10 min of global normothermic ischemia and 40 min of reperfusion. We determined the release of purine catabolites (adenosine, inosine, hypoxanthine, xanthine, uric acid) and the incorporation of exogenous 3H-adenosine and 14C-hypoxanthine into cellular nucleotides. Ischemia-reperfusion produced remarkable reduction in the release of purine catabolites, with no significant variation in the incorporation of adenosine and hypoxanthine.

Effects of verapamil, gallopamil, diltiazem and nifedipine on sarcoplasmic reticulum function in rat heart.

We investigated the effect of the calcium antagonists verapamil, gallopamil, diltiazem and nifedipine on cardiac sarcoplasmic reticulum function. In a cell-free homogenate from rat hearts, oxalate-supported Ca uptake was stimulated by verapamil, gallopamil and diltiazem at concentrations in the order of 10 nM to 100 nM, while higher concentrations were ineffective. Nifedipine was also ineffective. Peak stimulation of Ca uptake averaged 15-20% of control. Ca uptake is the difference between active Ca transport by Ca-ATPase and passive efflux through sarcoplasmic reticulum channels. In the presence of 300 microM ryanodine, which blocks sarcoplasmic reticulum channels, Ca uptake increased by 50%, but no further stimulation was produced by the addition of any calcium antagonist, at concentrations ranging from 1 nM to 100 microM. In a fraction enriched in sarcoplasmic reticulum, no drug affected the activity of Ca-ATPase at concentrations able to stimulate Ca uptake. We conclude that low concentrations of verapamil, gallopamil and diltiazem reduce Ca efflux through the Ca channels of the sarcoplasmic reticulum. Such an action might contribute to the clinical effect of these drugs.