Adel B. Elmoselhi

Status of myocardial antioxidants in ischemia-reperfusion injury.

BACKGROUND Myocardial ischemia-reperfusion represents a clinically relevant problem associated with thrombolysis, angioplasty and coronary bypass surgery. Injury of myocardium due to ischemia-reperfusion includes cardiac contractile dysfunction, arrhythmias as well as irreversible myocyte damage. These changes are considered to be the consequence of imbalance between the formation of oxidants and the availability of endogenous antioxidants in the heart. OBSERVATIONS An increase in the formation of reactive oxygen species during ischemia-reperfusion and the adverse effects of oxyradicals on myocardium have now been well established by both direct and indirect measurements. Although several experimental studies as well as clinical trials have demonstrated the cardioprotective effects of antioxidants, some studies have failed to substantiate the results. Nonetheless, it is becoming evident that some of the endogenous antioxidants such as glutathione peroxidase, superoxide dismutase, and catalase act as a primary defense mechanism whereas the others including vitamin E may play a secondary role for attenuating the ischemia-reperfusion injury. The importance of various endogenous antioxidants in suppressing oxidative stress is evident from the depression in their activities and the inhibition of cardiac alterations which they produce during ischemia-reperfusion injury. The effects of an antioxidant thiol containing compound, N-acetylcysteine, and ischemic preconditioning were shown to be similar in preventing changes in the ischemic-reperfused hearts. CONCLUSIONS The available evidence support the role of oxidative stress in ischemia-reperfusion injury and emphasize the importance of antioxidant mechanisms in cardioprotection.

Helicobacter pylori VacA Disrupts Apical Membrane-Cytoskeletal Interactions in Gastric Parietal Cells

Helicobacter pylori persistently colonize the human stomach and have been linked to atrophic gastritis and gastric carcinoma. Although it is well known that H. pylori infection can result in hypochlorhydria, the molecular mechanisms underlying this phenomenon remain poorly understood. Here we show that VacA permeabilizes the apical membrane of gastric parietal cells and induces hypochlorhydria. The functional consequences of VacA infection on parietal cell physiology were studied using freshly isolated rabbit gastric glands and cultured parietal cells. Secretory activity of parietal cells was judged by an aminopyrine uptake assay and confocal microscopic examination. VacA permeabilization induces an influx of extracellular calcium, followed by activation of calpain and subsequent proteolysis of ezrin at Met469-Thr470, which results in the liberation of ezrin from the apical membrane of the parietal cells. VacA treatment inhibits acid secretion by preventing the recruitment of H,K-ATPase-containing tubulovesicles to the apical membrane of gastric parietal cells. Electron microscopic examination revealed that VacA treatment disrupts the radial arrangement of actin filaments in apical microvilli due to the loss of ezrin integrity in parietal cells. Significantly, expression of calpain-resistant ezrin restored the functional activity of parietal cells in the presence of VacA. Proteolysis of ezrin in VacA-infected parietal cells is a novel mechanism underlying H. pylori-induced inhibition of acid secretion. Our results indicate that VacA disrupts the apical membrane-cytoskeletal interactions in gastric parietal cells and thereby causes hypochlorhydria.

Ischemia–reperfusion alters gene expression of Na+–K+ ATPase isoforms in rat heart

The present study investigated whether oxidative stress plays a role in ischemia-reperfusion-induced changes in cardiac gene expression of Na(+)-K(+) ATPase isoforms. The levels of mRNA for Na(+)-K(+) ATPase isoforms were assessed in the isolated rat heart subjected to global ischemia (30 min) followed by reperfusion (60 min) in the presence or absence of superoxide dismutase (5 x 10(4)U/L) plus catalase (7.5 x 10(4)U/L), an antioxidant mixture. The levels of mRNA for the alpha(2), alpha(3), and beta(1) isoforms of Na(+)-K(+) ATPase were significantly reduced in the ischemia-reperfusion hearts, unlike the alpha(1) isoform. Pretreatment with superoxide dismutase+catalase preserved the ischemia-reperfusion-induced changes in alpha(2), alpha(3), and beta(1) isoform mRNA levels of the Na(+)-K(+) ATPase, whereas the alpha(1) mRNA levels were unaffected. In order to test if oxidative stress produced effects similar to those seen with ischemia-reperfusion, hearts were perfused with an oxidant, H(2)O(2) (300 microM), or a free radical generator, xanthine (2mM) plus xanthine oxidase (0.03 U/ml) for 20 min. Perfusion of hearts with H(2)O(2) or xanthine/xanthine oxidase depressed the alpha(2), alpha(3), and beta(1) isoform mRNA levels of the Na(+)-K(+) ATPase, but had lesser effects on alpha(1) mRNA levels. These results indicate that Na(+)-K(+) ATPase isoform gene expression is altered differentially in the ischemia-reperfusion hearts and that antioxidant treatment appears to attenuate these changes. It is suggested that alterations in Na(+)-K(+) ATPase isoform gene expression by ischemia-reperfusion may be mediated by oxidative stress.

PROPERTIES OF THE SARCOPLASMIC RETICULUM CA2+-PUMP IN CORONARY ARTERY SKINNED SMOOTH MUSCLE

Pig coronary artery cultured smooth muscle cells were skinned using saponin. In the presence of an ATP-regenerating system and oxalate, the skinned cells showed an ATP-dependent azide insensitive Ca2+-uptake which increased linearly with time for >1 h. The Ca2+-uptake occurred with Km values of 0.20±0.03 μM for Ca2+ and 400±34 μM for MgATP2−. Thapsigargin and cyclopiazonic acid inhibited this uptake with IC50 values of 0.13±0.02 and 0.56±0.04 μM, respectively. These properties of SR Ca2+-pump are similar to those reported for membrane fractions isolated from fresh smooth muscle of coronary artery and other arteries. However, optimum pH of the uptake in the skinned cells (6.2) was lower than that reported previously using isolated membranes (6.4–6.8).

Effects of peroxide on contractility of coronary artery rings of different sizes

Reactive oxygen species (ROS, free radicals) produced during cardiac ischemia and reperfusion can damage the contractile functions of arteries. The sarcoplasmic reticulum (SR) Ca2+ pump in coronary artery smooth muscle is very sensitive to ROS. Here we show that contractions of de-endothelialized rings from porcine left coronary artery produced by the hormone Angiotensin II and by the SR Ca2+ pump inhibitors cyclopiazonic acid and thapsigargin correlate negatively with the tissue weight. In contrast, the contractions due to membrane depolarization by high KCl correlate positively. Peroxide also produces a small contraction which correlates negatively with the tissue weight. When artery rings are treated with peroxide and washed, their ability to contract with Angiotensin II, cyclopiazonic acid and thapsigargin decreases. Thus, the SR Ca2+ pump may play a more important role in the contractility of the smaller segments of the coronary artery than in the larger segments. These results are consistent with the hypothesis that ROS which damage the SR Ca2+ pump affect the contractile function of the distal segments more adversely than of the proximal segments.

Endothelin contraction in pig coronary artery: Receptor types and Ca2+-mobilization

Endothelin is one of the most potent vasoconstrictors known. It plays an important role in the regulation of vascular tone and in the development of many cardiovascular diseases. This study focuses on the receptor types and the Ca2+ mobilization responsible for endothelin-1 (ET-1) contraction in de-endothelialized pig coronary artery rings. ET-1 contracted the artery rings with an EC50 = 6.5 +/- 1 nM and a maximum contraction which was 98.6 +/- 9% of the contraction produced by 60 mM KCl. BQ123 (5 microM), an ETA antagonist, reversed 78 +/- 3% of the ET-1 contraction (50 nM). IRL1620, a selective ETB agonist, produced 23 +/- 3% of the total ET-1 contraction with an EC50 = 12.7 +/- 2 nM. More than 85% of the contraction due to 100 nM IRL 1620 was inhibited by 200 nMBQ788, an ETB antagonist. Therefore, approximately 80% of the ET-1 contraction in this artery occurred via ETA receptors, and the other 20% was mediated by ETB receptors. To assess the Ca2+ pools utilized during the ET-1 response, ET-1 contraction was also examined in medium containing an L-type Ca2+ channel blocker nitrendipine, and in Ca2+ free medium containing 0.2 mM EGTA. In Ca2+ containing medium the contraction elicited by ET-1 was 98.6 +/- 9% of the KCl contraction, however, in the presence 10 microM nitrendipine the ET-1 induced contraction was 54 +/- 7% of the KCl contraction, and in Ca(2+)-free medium it was 13 +/- 2%. Similarly, the IRL 1620 contractions in Ca2+ containing medium, in the presence of nitrendipine and in Ca(2+)-free medium were 22.4 +/- 3%, 12 +/- 3% and 11 +/- 2% of the KCl response respectively. Thus, both ETA and ETB contractions utilize extracellular Ca2+ pools via L-type Ca2+ channels and other undefined route(s), as well as intracellular Ca2+ pools. In the pig coronary artery smooth muscle, ET-1 contractions occur predominantly via ETA receptors, with ETB receptors using similar Ca2+ mobilization pathways, but the ETB receptors appear to use the intracellular Ca2+ stores to a greater extent.

ET(B)-mediated contraction differs between left descending coronary artery and its next branch.

Pig left descending coronary artery (main artery) and its next branch (branch arteries) differ in many properties. Here we report on the receptor types and the Ca2+ pools utilized for endothelin (ET) contraction in 3 mm long de-endothelialized rings of the main (weight 7.38 ± 0.38 mg) and the branch (1.07 ± 0.03 mg) arteries. KCl (60 mM) contracted the main and the branch arteries with force of 41.8 ± 3.1 and 16.9 ± 1.0 mN (millinewton), respectively. Force of contraction for all the other agents was normalized taking the KCl value as 100%. We determined the total ET-induced responses using ET-1 and those mediated by ETB using IRL1620. In Ca2+-containing solutions, ET-1 contracted the main arteries with pECB = 8.2 ± 0.1 and a maximum force of 98 ± 5%. The branch arteries also gave similar values of pEC50 (8.4 ± 0.1) and maximum force (99 ± 14%). IRL1620 contracted the main and the branch arteries with pEC50 = 7.9 ± 0.1 but the maximum force was significantly higher in the branch arteries (44 ± 3%) than in the main (15 ± 2%). In Ca2+-free solutions, the pEC50 values for ET-1 or IRL-1620 did not change but the maximum force of contraction was diminished considerably in both main and branch arteries. Thus, the left coronary artery and its next branch differ in that the role of ETB receptors is greater in the latter.

Clinically oriented laboratory illuminates challenging respiratory concepts to the pre-clinical medical students.

As the pre-clinical curriculum in medical schools evolves toward active learning and clinical applications, traditional physiology laboratories have been trying to find their niche. Since most medical students perceive the mastery of respiratory physiological concepts as formidable, we constructed a hands-on spirometer laboratory to equip our first-year medical students to proficiently comprehend and apply some of the basic concepts in respiration. In this lab, students record and interpret Forced Vital Capacity (FVC) test in real time. In the last 4 years the laboratory was conducted in clinical examination rooms using IQmark digital spirometer machines (Midmark Diagnostics Group, Versailles, Ohio). In each year, the class of approximately 54 students was divided into six groups, each with its own machine. By performing the test on peers, students collected the following parameters: FVC, FEV1 (Forced Expiratory Volume at 1 second), the FEV1/FVC ratio and the subject’s height. Then each student drew a figure and briefly discussed the relationship between height and these parameters. Furthermore, to simulate restrictive lung diseases, a bandage was tightly wrapped around a volunteer student’s chest and an FVC test was performed. Finally, the students completed a survey to assess the value of the lab in their comprehension of theoretical concepts learned in the classroom and the relevance to their medical education. On an average, 88% of the students rated the lab as extremely or very useful in helping them understand physiological concepts of the pulmonary function test in normal and pathological conditions. Although these evaluations are more subjective based on the students’ perception, the responses were consistent in the last four consecutive years. The majority of the class (485%) correctly showed the expected linear correlation between the height and the FVC and FEV1 while the FEV1/FVC ratio did not change, as well as the simulation of restrictive lung diseases on these parameters. The clinically oriented setting of the lab energized the students and enhanced their efforts to link the challenging abstractions of the classroom to practical applications. As previous reports indicate (Euliano 2000), this method appears to improve students’ proficiency in challenging concepts and needs to be implemented more frequently in the pre-clinical curriculum.