Selective Inhibition of 2-Oxoglutarate and 2-Oxoadipate Dehydrogenases by the Phosphonate Analogs of Their 2-Oxo Acid Substrates

Phosphonate analogs of pyruvate and 2-oxoglutarate are established specific inhibitors of cognate 2-oxo acid dehydrogenases. The present work develops application of this class of compounds to specific in vivo inhibition of 2-oxoglutarate dehydrogenase (OGDH) and its isoenzyme, 2-oxoadipate dehydrogenase (OADH). The isoenzymes-enriched preparations from the rat tissues with different expression of OADH and OGDH are used to characterize their interaction with 2-oxoglutarate (OG), 2-oxoadipate (OA) and the phosphonate analogs. Despite a 100-fold difference in the isoenzymes ratio in the heart and liver, similar Michaelis saturations by OG are inherent in the enzyme preparations from these tissues (KmOG = 0.45 ± 0.06 and 0.27 ± 0.026 mM, respectively), indicating no significant contribution of OADH to the OGDH reaction, or similar affinities of the isoenzymes to OG. However, the preparations differ in the catalysis of OADH reaction. The heart preparation, where OADH/OGDH ratio is ≈ 0.01, possesses low-affinity sites to OA (KmOA = 0.55 ± 0.07 mM). The liver preparation, where OADH/OGDH ratio is ≈ 1.6, demonstrates a biphasic saturation with OA: the low-affinity sites (Km,2OA = 0.45 ± 0.12 mM) are similar to those of the heart preparation; the high-affinity sites (Km,1OA = 0.008 ± 0.001 mM), revealed in the liver preparation only, are attributed to OADH. Phosphonate analogs of C5-C7 dicarboxylic 2-oxo acids inhibit OGDH and OADH competitively to 2-oxo substrates in all sites. The high-affinity sites for OA are affected the least by the C5 analog (succinyl phosphonate) and the most by the C7 one (adipoyl phosphonate). The opposite reactivity is inherent in both the low-affinity OA-binding sites and OG-binding sites. The C6 analog (glutaryl phosphonate) does not exhibit a significant preference to either OADH or OGDH. Structural analysis of the phosphonates binding to OADH and OGDH reveals the substitution of a tyrosine residue in OGDH for a serine residue in OADH among structural determinants of the preferential binding of the bulkier ligands to OADH. The consistent kinetic and structural results expose adipoyl phosphonate as a valuable pharmacological tool for specific in vivo inhibition of the DHTKD1-encoded OADH, a new member of mammalian family of 2-oxo acid dehydrogenases, up-regulated in some cancers and associated with diabetes and obesity.

New Data on Cylindrospermopsin Toxicity

Cylindrospermopsin (CYN) is a widely spread cyanotoxin that can occur in fresh water and food. This research aims to investigate CYN toxicity by studying the effects of drinking 0.25 nM of CYN-contaminated water from a natural source, and of the direct application of moderate concentrations of CYN on different animal targets. The chosen structures and activities are rat mitochondria inner membrane permeability, mitochondrial ATP synthase (ATPase) and rat liver diamine oxidase (DAO) activities (EC 1.4.3.22.), the force of the contraction of an excised frog heart preparation with functional innervation, and the viability of a human intestinal epithelial cell line (HIEC-6). The oral exposure to CYN decreased the reverse (hydrolase) activity of rat liver ATPase whereas its short-term, in vitro application was without significant effect on this organelle, DAO activity, heart contractions, and their neuronal regulation. The application of CYN reduced HIEC-6 cells’ viability dose dependently. It was concluded that CYN is moderately toxic for the human intestinal epithelial cells, where the regeneration of the epithelial layer can be suppressed by CYN. This result suggests that CYN may provoke pathological changes in the human gastrointestinal tract.

The Insecticidal Activity of Rhinella schneideri (Werner, 1894) Paratoid Secretion in Nauphoeta cinerea Cocroaches

Rhinella schneideri is a common toad found in South America, whose paratoid toxic secretion has never been explored as an insecticide. In order to evaluate its insecticidal potential, Nauphoeta cinerea cockroaches were used as an experimental model in biochemical, physiological and behavioral procedures. Lethality assays with Rhinella schneideri paratoid secretion (RSPS) determined the LD50 value after 24 h (58.07µg/g) and 48 h exposure (44.07 µg/g) (R2 = 0.882 and 0.954, respectively). Acetylcholinesterase activity (AChE) after RSPS at its highest dose promoted an enzyme inhibition of 40%, a similar effect observed with neostigmine administration (p < 0.001, n= 5). Insect locomotion recordings revealed that RSPS decreased the distance traveled by up to 37% with a concomitant 85% increase in immobile episodes (p < 0.001, n = 36). RSPS added to in vivo cockroach semi-isolated heart preparation promoted an irreversible and dose dependent decrease in heart rate, showing a complete failure after 30 min recording (p < 0.001, n ≥ 6). In addition, RSPS into nerve-muscle preparations induced a dose-dependent neuromuscular blockade, reaching a total blockage at 70 min at the highest dose applied (p < 0.001, n ≥ 6). The effect of RSPS on spontaneous sensorial action potentials was characterized by an increase in the number of spikes 61% (p < 0.01). Meanwhile, there was 42% decrease in the mean area of those potentials (p < 0.05, n ≥ 6). The results obtained here highlight the potential insecticidal relevance of RSPS and its potential biotechnological application.

Dexmedetomidine enhances tolerance to bupivacaine cardiotoxicity in the isolated rat hearts: alpha 2 adrenoceptors were not involved

Background Dexmedetomidine was proved to mitigate bupivacaine-induced cardiotoxicity but mechanism of this ability is still unclear. This study was designed to investigate the direct effects of dexmedetomidine on cardiotoxicity induced by bupivacaine on Langendorff rat heart preparation and the role of alpha 2 adrenoceptors in this process was explored. Methods Hearts of rat were isolated, mounted on a Langendorff system. Five experimental groups were assessed after 10 min Krebs-Henseleit buffer (KHB) infusions as follow: (1) Group Con, only KHB was perfused; (2) Group Dex, KHB was perfused for 5 min, then dexmedetomidine (10 nmol/L) was added; (3) Group Bupi, KHB was perfused for 25 min, then bupivacaine (50 μmol/L) was added; (4) Group Bupi + Dex, KHB was perfused for 5 min, then the dexmedetomidine (10 nmol/L) was added for 20 min, at last a mixture of KHB + dexmedetomidine + bupivacaine were perfused; (5) Group Bupi + Dex + Yoh, a combination of KHB + yohimbine (alpha 2 adrenoceptor antagonists, 1 μmol/L) was perfusion for 5 min, then dexmedetomidine (10 nmol/L) was added for 20 min, at last a mixture of KHB + yohimbine + dexmedetomidine + bupivacaine was perfused. The experimental perfusion was maintained for 35 min in group Con and group Dex, and the experimental perfusion was sustained until asystole in the other three groups. Results Compared with group Bupi, dexmedetomidine significantly increased the time to first arrhythmia (P < 0.001) and time to asystole (P < 0.001) in group Bupi + Dex. In addition, dexmedetomidine also significantly increased the time to 25, 50 and 75% reductions in heart rate (P < 0.001) and the time to 25, 50 and 75% reductions in rate-pressure product (P < 0.001) in group Bupi + Dex. Dexmedetomidine increased the cardiac tissue bupivacaine content when asystole (Bupi + Dex vs. Bupi, 58.5 ± 6.3 vs. 46.8 ± 5.6 nmol/g, P = 0.003). The benefit of dexmedetomidine on bupivacaine-induced cardiotoxicity were not eliminated by yohimbine. Conclusions Dexmedetomidine could delay the occurrence of bupivacaine-induced arrhythmia and asystole in the isolated rat hearts, but the alpha 2 adrenoceptors were not involved in this process.

The method of heterotopic rat heart transplantation for investigation the cardioprotective efficacy of cardioplegic solutions

This article describes in detail the modified method of heterotopic heart transplantation in rats. The methods of animal anesthesia, surgical features of the removal of a donor heart and its implantation to a recipient rat are described in detail. The advantages and disadvantages of donor heart preservation methods are described, as well as methods for evaluating a donor heart preparation, the quality of preservation and the effectiveness of cardioprotection. Experimental approaches to the study of the effectiveness of cardioplegic and preservative solutions using this technique are proposed. The data of our own pilot study on a comparative analysis of the effectiveness of cardioprotection using preservative solutions using this experimental model are presented.

Nerol Attenuates Ouabain-Induced Arrhythmias

Nerol (C10H18O) is a monoterpene found in many essential oils, such as lemon balm and hop. In this study, we explored the contractile and electrophysiological properties of nerol and demonstrated its antiarrhythmic effects in guinea pig heart preparation. Nerol effects were evaluated on atrial and ventricular tissue contractility, electrocardiogram (ECG), voltage-dependent L-type Ca2+current (ICa,L), and ouabain-triggered arrhythmias. Overall our results revealed that by increasing concentrations of nerol (from 0.001 to 30 mM) there was a significant decrease in left atrium contractile force. This effect was completely and rapidly reversible after washing out (~ 2 min). Nerol (at 3 mM concentration) decreased the left atrium positive inotropic response evoked by adding up CaCl2in the extracellular medium. Interestingly, when using a lower concentration of nerol (30μM), it was not possible to clearly observe any significant ECG signal alterations but a small reduction of ventricular contractility was observed. In addition, 300μM nerol promoted a significant decrease on the cardiac rate and contractility. Important to note is the fact that in isolated cardiomyocytes, peak ICa,Lwas reduced by 58.9 ± 6.31% after perfusing 300μM nerol (n=7, p<0.05). Nerol, at 30 and 300μM, delayed the time of onset of ouabain-triggered arrhythmias and provoked a decrease in the diastolic tension induced by the presence of ouabain (50μM). Furthermore, nerol preincubation significantly attenuated arrhythmia severity index without changes in the positive inotropism elicited by ouabain exposure. Taken all together, we may be able to conclude that nerol primarily by reducing Ca2+influx through L-type Ca2+channel blockade lessened the severity of ouabain-triggered arrhythmias in mammalian heart.

Maximum cardiac performance of Antarctic fishes that lack haemoglobin and myoglobin: exploring the effect of warming on nature’s natural knockouts

Antarctic notothenioids, some of which lack myoglobin (Mb) and/or haemoglobin (Hb), are considered extremely stenothermal, which raises conservation concerns since Polar regions are warming at unprecedented rates. Without reliable estimates of maximum cardiac output ($\dot{Q}$), it is impossible to assess their physiological scope in response to warming seas. Therefore, we compared cardiac performance of two icefish species, Chionodraco rastrospinosus (Hb−Mb+) and Chaenocephalus aceratus (Hb−Mb−), with a related notothenioid, Notothenia coriiceps (Hb+Mb+) using an in situ perfused heart preparation. The maximum $\dot{Q}$, heart rate (fH), maximum cardiac work (WC) and relative ventricular mass of N. coriiceps at 1°C were comparable to temperate-water teleosts, and acute warming to 4°C increased fH and WC, as expected. In contrast, icefish hearts accommodated a higher maximum stroke volume (VS) and maximum $\dot{Q}$ at 1°C, but their unusually large hearts had a lower fH and maximum afterload tolerance than N. coriiceps at 1°C. Furthermore, maximum VS, maximum $\dot{Q}$ and fH were all significantly higher for the Hb−Mb+ condition compared with the Hb−Mb− condition, a potential selective advantage when coping with environmental warming. Like N. coriiceps, both icefish species increased fH at 4°C. Acutely warming C. aceratus increased maximum $\dot{Q}$, while C. rastrospinosus (like N. coriiceps) held at 4°C for 1 week maintained maximum $\dot{Q}$ when tested at 4°C. These experiments involving short-term warming should be followed up with long-term acclimation studies, since the maximum cardiac performance of these three Antarctic species studied seem to be tolerant of temperatures in excess of predictions associated with global warming.