Acute adaptations of the coronary circulation to exercise

Coronary blood flow is tightly coupled to myocardial oxygen consumption to maintain a consistently high level of myocardial oxygen extraction. This tight coupling has been proposed to depend on periarteriolar, oxygen tension, signals released from cardiomyocytes (adenosine acting on K _ATP ⁺ channels), and/or the endothelium (prostanoids, nitric oxide, endothelin [ET]) and autonomic influences (catecholamines), but the contribution of each of these regulatory pathways and their interactions are still incompletely understood. Until recently, experimental studies into the regulation of coronary blood flow during exercise were principally performed in the dog. We have performed several studies on the regulation of vasomotor tone in coronary resistance vessels in chronically instrumented exercising swine. These studies have shown that the coronary resistance vessels in swine lack significant α-adrenergic control, but that these vessels are subject to β-adrenergic feed-forward control during exercise, which is aided by a parasympathetic withdrawal. In addition, withdrawal of an ET-mediated vasoconstrictor influence also contributes to exercise-induced coronary vasodilation. Coronary blood flow regulation by endothelial and metabolic vasodilator pathways contributes to resting vasomotor tone regulation but does not appear to contribute to the exercise-induced coronary vasodilation. Furthermore, blockade of one vasodilator pathway is not compensated by an increased contribution of the other vasodilator mechanisms, suggesting that porcine coronary vasomotor control by endothelial and metabolic factors occurs in a linear additive rather than a nonlinear synergistic fashion.     

The role of the 132-160 region in prion protein conformational transitions

The native conformation of host-encoded cellular prion protein (PrP(C)) is metastable. As a result of a post-translational event, PrP(C) can convert to the scrapie form (PrP(Sc)), which emerges as the essential constituent of infectious prions. Despite thorough research, the mechanism underlying this conformational transition remains unknown. However, several studies have highlighted the importance of the N-terminal region spanning residues 90-154 in PrP folding. In order to understand why PrP folds into two different conformational states exhibiting distinct secondary and tertiary structure, and to gain insight into the involvement of this particular region in PrP transconformation, we studied the pressure-induced unfolding/ refolding of recombinant Syrian hamster PrP expanding from residues 90-231, and compared it with heat unfolding. By using two intrinsic fluorescent variants of this protein (Y150W and F141W), conformational changes confined to the 132-160 segment were monitored. Multiple conformational states of the Trp variants, characterized by their spectroscopic properties (fluorescence and UV absorbance in the fourth derivative mode), were achieved by tuning the experimental conditions of pressure and temperature. Further insight into unexplored conformational states of the prion protein, likely to mimic the in vivo structural change, was obtained from pressure-assisted cold unfolding. Furthermore, salt-induced conformational changes suggested a structural stabilizing role of Tyr150 and Phe141 residues, slowing down the conversion to a beta-sheet form.     

O-raffinose crosslinked hemoglobin lacks site-specific chemistry in the central cavity: structural and functional consequences of beta93Cys modification

Reacting human deoxyHbA0 with oxidized raffinose (O-raffinose), a trisaccharide, results in a low oxygen affinity "blood substitute," stabilized in a noncooperative T-conformation and possesses readily oxidizable rhombic heme. In this study, we fractionated the O-raffinose-modified HbA0 heterogeneous polymer (O-R-PolyHbA0) into six distinct fractions with a molecular weight distribution ranging from 64 to approximately 600 kDa using size-exclusion chromatography (SEC). Oxygen equilibrium and kinetics binding parameters of all fractions were nearly identical, reflecting a lack of heterogeneity in ligand binding properties among O-R-PolyHbA0 species (Hill coefficient n equal to 1.0). Several mass spectrometry techniques were used to evaluate undigested and digested HbA0, O-R-PolyHbA0, and O-R-PolyHbA0 fractions. Proposed sites of intramolecular crosslinking (i.e., beta1Lys82, beta2Lys82, and beta1Val1) were not found to be the predominant site of crosslinking within the central cavity. Intermolecular crosslinking with O-raffinose results in no discernible site of amino acids modifications with the exception of beta93Cys and alpha104Cys. Based on accessible surface area (ASA) calculations in intact deoxyHbA0, slight conformational changes are required to allow for the S on alpha104Cys to be modified during the reaction with O-raffinose or its partially oxidized product(s). The stabilization of HbA0 in the T-conformation may not be a direct correlate of O-raffinose induced changes, but an indirect consequence of changing hydration in the water-filled central cavity and/or the distal heme pocket leading in the latter case to accelerated iron oxidation. Structural data presented here when taken together with the oxidative instability of O-R-PolyHbA0 may provide some basis for the reported toxicity of this oxygen carrier.     

Metabolic products and pathways of fluorotelomer alcohols in isolated rat hepatocytes

Fluorotelomer alcohols (FTOHs; CF(3)(CF(2))(x)C(2)H(4)OH; where x=3, 5, 7, 9) are a novel class of polyfluorinated contaminants, recently detected in the North American atmosphere, that are possible precursors to the series of perfluoroalkyl carboxylates (PFCAs) in human blood. An in vivo rat study validated earlier independent work that poly- and per-fluoroalkyl carboxylates were metabolites of FTOHs, but our detection of several novel metabolites prompted us to examine their pathways in greater detail using isolated rat hepatocytes. Using 8:2 FTOH (i.e. where x=7) as a model compound, the metabolic products formed by isolated rat hepatocytes were identified, and three synthesized intermediates were incubated separately to elucidate the metabolic pathways. For 8:2 FTOH, a major fate was direct conjugation to form the O-glucuronide and O-sulfate. Using 2,4-dinitrophenylhydrazine (DNPH) trapping, the immediate oxidation product of 8:2 FTOH was identified as 8:2 fluorotelomer aldehyde (8:2 FTAL; CF(3)(CF(2))(7)CH(2)C(H)O). 8:2 FTAL was transient and eliminated HF non-enzymatically to yield 8:2 fluorotelomer alpha,beta-unsaturated aldehyde (8:2 FTUAL; CF(3)(CF(2))(6)CFCHC(H)O) which was also short-lived and reacted GSH and perhaps other endogenous nucleophiles. Four polyfluorinated acid intermediates were also detected, including 8:2 fluorotelomer carboxylate (8:2 FTCA; CF(3)(CF(2))(7)CH(2)C(O)O(-)), 8:2 fluorotelomer alpha,beta-unsaturated carboxylate (8:2 FTUCA; CF(3)(CF(2))(6)CFCHC(O)O(-)), tetrahydroperfluorodecanoate (CF(3)(CF(2))(6)(CH(2))(2)CO(2)(-)), and dihydroperfluorodecenoate (CF(3)(CF(2))(6)CHCHCO(2)(-)). The pathways leading to 8:2 FTCA and FTUCA involve oxidation of 8:2 FTAL, however, the pathways leading to the latter two polyfluorinated acids remain inconclusive. The fate of the unsaturated metabolites, 8:2 FTUAL and FTUCA, included conjugation with GSH and dehydrofluorination to yield alpha,beta-unsaturated GSH conjugates, and GS-8:2 FTUAL which was subsequently reduced to the corresponding alcohol. Perfluorooctanoate (PFOA) and minor amounts of perfluorononanoate (PFNA) were confirmed as metabolites of 8:2 FTOH, and the respective roles of beta- and alpha-oxidation mechanisms are discussed. The analogous acids, aldehydes, and conjugated metabolites of 4:2, 6:2, and 10:2 FTOH (i.e. where x=3, 5, and 9, respectively) were also detected, and metabolite profiles among FTOHs generally differed only in the length of their perfluoroalkyl chains. Preincubation with aminobenzotriazole, but not pyrazole, inhibited the formation of metabolites from all FTOHs, suggesting that their oxidation was catalyzed by P450, not alcohol dehydrogenase.     

Sonic hedgehog is required for cardiac outflow tract and neural crest cell development

The Hedgehog signaling pathway is critical for a significant number of developmental patterning events. In this study, we focus on the defects in pharyngeal arch and cardiovascular patterning present in Sonic hedgehog (Shh) null mouse embryos. Our data indicate that, in the absence of Shh, there is general failure of the pharyngeal arch development leading to cardiac and craniofacial defects. The cardiac phenotype results from arch artery and outflow tract patterning defects, as well as abnormal development of migratory neural crest cells (NCCs). The constellation of cardiovascular defects resembles a severe form of the human birth defect syndrome tetralogy of Fallot with complete pulmonary artery atresia. Previous studies have demonstrated a role for Shh in NCC survival and proliferation at later stages of development. Our data suggest that SHH signaling does not act directly on NCCs as a survival factor, but rather acts to restrict the domains that NCCs can populate during early stages (e8.5-10.5) of cardiovascular and craniofacial development.     

Trypanosoma cruzi: distinct patterns of infection in the sibling caviomorph rodent species Thrichomys apereoides laurentius and Thrichomys pachyurus (Rodentia, Echimyidae)

Thrichomys apereoides, a caviomorph rodent species common in a highly endemic area for Chagas disease in Brazil, may act as reservoir of the parasite. However, no information is available concerning its sibling species Thrichomys pachyurus, found in the Pantanal region, where Trypanosoma cruzi is found only in the enzootic cycle. We followed up the cross infection of these cryptic species with two isolates derived from naturally infected T. pachyurus and Thrichomys apereoides laurentius. No regional co-adaptation between Thrichomys species and the regional isolates were noticed. However, significant differences in the outcome of the infection were observed. T. a. laurentius was more resistant than T. pachyurus, as expressed by lower parasitemia and less histopathological damage. The routine biochemical markers used for laboratory rodents were unsuitable for follow up of infection in Thrichomys spp, since they did not correlate with the histopathological findings or allowed the kinetic follow-up of tissue colonization by the parasite.     

Effects of voltage-gated Na+ channel toxins from Tityus serrulatus venom on rat arterial blood pressure and plasma catecholamines

Scorpion toxins interact with ionic channels of excitable cells, leading to a massive release of neurotransmitters. Voltage-gated Na+ channel toxins are mainly responsible for the toxic effects of scorpion envenoming and can be classified into two classes: alpha- and beta-neurotoxins. TsTX-V and TsTX-I from Tityus serrulatus venom (TsV) are, respectively, examples of these toxins. In this work, we compared the effects of these toxins on mean arterial pressure (MAP) and catecholamines release in rats. Toxins were isolated by ion exchange chromatography (TsTX-I) followed by RP-HPLC (TsTX-V). All experiments were performed on conscious unrestrained rats previously catheterised. The toxins (15 and 30 microg/kg) and TsV (50 and 100 microg/kg) were injected intravenously. MAP was continuously monitored through femoral catheter. Epinephrine (E) and norepinephrine (NE) levels were determined by RP-HPLC with electrochemical detection, at 10 min before and 2.5, 30 and 90 min after treatments. Maximal pressor effects were observed at 2.5-3.5 min. TsV induced intense long lasting increase in MAP, as did TsTX-I. TsTX-V showed the lowest pressor effects. TsV showed the highest effects on catecholamines release, followed by TsTX-I and TsTX-V with maximal effect at 2.5 min, followed by a gradual reduction, however remaining higher than controls. Although both toxins act on Na+ channels, TsTX-I displayed significant and more intense effects on catecholamines release and blood pressure than TsTX-V. It seems that the toxicity of TsTX-V is not related only with its ability to release catecholamines, indicating that other neurotransmitters, may be involved in its toxicity.     

The proteome and secretome of human arterial smooth muscle cells

Smooth muscle cells (SMCs) play a crucial role in cardiovascular disorders. A differential proteomic approach should help to elucidate SMC dysfunctions involved in these diseases. With this goal in mind, we plotted the first 2-dimensional (2-D) maps of the proteome and secretome of human arterial smooth muscle cell (ASMC). Intracellular and secreted proteins were extracted from a primary culture of SMCs obtained from patients undergoing coronary artery bypass surgery (n = 11) and separated by 2-dimensional gel electrophoresis. Silver-stained gels were analyzed using Progenesis software. A high level of between-gel reproducibility was obtained, allowing us to generate two protein patterns specific to the ASMC proteome and secretome, respectively. A total of 121 and 40 distinct intracellular and secreted polypeptide spots, corresponding to 83 and 18 different proteins, respectively, were identified by matrix-assisted laser desorption/ionization mass spectrometry. The 2-D reference maps and database resulting from this study confirm that SMCs are involved in a wide range of biological functions. They could constitute a useful tool for a wide range of investigators involved in vascular biology, allowing them to investigate SMC protein changes associated with cardiovascular disorders or environmental stimuli.