A mixed-methods study on evaluating an updated, francophone version of ETAT+ training in Madagascar.

Background: Madagascar needs major efforts to achieve the UN Sustainable Development Goals, despite the considerable reduction of child mortality during past years. In this context, implementation of emergency triage assessment and treatment (ETAT) plays an important role. In recent years, ETAT training activities rarely took place in Madagascar. To strengthen ETAT in Madagascar, a pilot training course was conducted in December 2019 at the University Hospital Mahajanga. Objectives: This study aims to evaluate if the ETAT+ pilot training content matches clinical needs in Madagascar and whether participants achieved their learning objectives. Methods: In this cross-sectional mixed-methods study, a 41-item questionnaire was used at the end of the ETAT+ training to evaluate their learning experience from the 12 participants (paediatricians, physicians, nurses and midwives). Six weeks after the training, guided interviews were conducted among five participants to describe how training content could be transferred into clinical practice in five health facilities. Results: Results suggest that this pilot project designed to contribute to the re-establishment of ETAT in Madagascar meets participants' needs and is adapted to clinical realities in terms of transmitted knowledge, skills and competencies. However, results also show that considerable multi-disciplinary efforts are needed to advance ETAT+ implementation in Madagascar. Conclusion: Implementation processes of ETAT training programmes need re-evaluation to assure their validity to contribute to quality of care improvements efficiently. Further operational research is required to evaluate sustainable, innovative implementation strategies adapted to contexts in Madagascar. Contributions of the study: This study aims to evaluate an updated Malagasy version of the Emergency Triage Assessment and Treatment Plus (ETAT+). The training met the participants' needs and was adapted to the clinical realities in Madagascar relating to transmitted knowledge, skills and competencies.

Intensive-care management of snakebite victims in rural sub-Saharan Africa: An experience from Uganda.

Background: Antivenom is rarely available for the management of snakebites in rural sub-Saharan Africa(sSA). Objectives: To report clinical management and outcomes of 174 snakebite victims treated with basic intensive-care interventions in a rural sSA hospital. Methods: This cohort study was designed as a retrospective analysis of a database of patients admitted to the intensive care unit (ICU) of St. Mary's Hospital Lacor in Gulu, Uganda (January 2006 - November 2017). No exclusion criteria were applied. Results: Of the 174 patients admitted to the ICU for snakebite envenomation, 60 (36.5%) developed respiratory failure requiring mechanical ventilation (16.7% mortality). Results suggest that neurotoxic envenomation was likely the most common cause of respiratory failure among patients requiring mechanical ventilation. Antivenom (at probably inadequate doses) was administered to 22 of the 174 patients (12.6%). The median (and associated interquartile range) length of ICU stay was 3 (2 - 5) days, with an overall mortality rate of 8%. Of the total number of patients, 67 (38.5%) were younger than 18 years. Conclusion: Results suggest that basic intensive care, including mechanical ventilation, is a feasible management option for snakebite victims presenting with respiratory failure in a rural sSA hospital, resulting in a low mortality rate, even without adequate antivenom being available. International strategies which include preventive measures as well as the strengthening of context-adapted treatment of critically ill patients at different levels of referral pathways, in order to reduce deaths and disability associated with snakebites in sSA are needed. Provision of efficient antivenoms should be integrated in clinical care of snakebite victims in peripheral healthcare facilities. Snakebite management protocols and preventive measures need to consider specific requirements of children. Contributions of the study: It is estimated that up to 138 000 people die each year following snakebites. Currently, reliable provision of efficient snake-bite antivenom is challenging in many rural health facilities in sub- Saharan Africa (sSA). Our results suggest that basic intensive-care interventions, including mechanical ventilation, is a feasible management option for critically ill snakebite victims in a rural sSA hospital, resulting in a low mortality rate, even without adequate antivenom doses being available.

Non-invasive ventilation with bubble CPAP is feasible and improves respiratory physiology in hospitalised Malawian children with acute respiratory failure.

BACKGROUND: In low-income countries and those with a high prevalence of HIV, respiratory failure is a common cause of death in children. However, the role of non-invasive ventilation with bubble continuous positive airway pressure (bCPAP) in these patients is not well established. METHODS: A prospective observational study of bCPAP was undertaken between July and September 2012 in 77 Malawian children aged 1 week to 14 years with progressive acute respiratory failure despite oxygen and antimicrobial therapy. RESULTS: Forty-one (53%) patients survived following bCPAP treatment, and an HIV-uninfected single-organ disease subgroup demonstrated bCPAP success in 14 of 17 (82%). Compared with children aged ≧60 months, infants of 0-2 months had a 93% lower odds of bCPAP failure (odds ratio 0·07, 95% confidence interval 0·004-1·02, P  =  0·05). Following commencement of bCPAP, respiratory physiology improved, the average respiratory rate decreased from 61 to 49 breaths/minute (P  =  0·0006), and mean oxygen saturation increased from 92·1% to 96·1% (P  =  0·02). CONCLUSIONS: bCPAP was well accepted by caregivers and patients and can be feasibly implemented into a tertiary African hospital with high-risk patients and limited resources.

Analysis of the anorectic efficacy of HMR1426 in rodents and its effects on gastric emptying in rats.

OBJECTIVES: Pharmacodynamics of HMR1426 in rodents. SUBJECTS: Male and female rats and male mice. MEASUREMENTS: 24 h feed consumption was measured. From the time curves IC(50) values of HMR1426 were calculated. Microanalysis of feeding behavior was determined. Macronutrient preference was measured, by offering rats three different diets. Gastric emptying was measured after liquid gastric loads or solid meals. In rats with gastric cannulas, milk consumption was measured with closed or open cannulas. Diabetes-related parameters and thyroid hormones were measured. RESULTS: HMR1426 inhibited feed consumption dose-dependently in rodents. Microstructural analysis of feeding after HMR1426 differed from central acting anorectics. HMR1426 inhibited consumption of fat- and carbohydrate-enriched diets. Gastric emptying was dose- and time-dependently delayed. Gastric emptying correlated with the time course of the anorectic effect. In sham-fed rats, HMR1426 had no anorectic effect with open cannulas. Anorectic effect occurred with closed cannulas. We proved that HMR1426 is not a CCK(A) agonist. CONCLUSION: The correlation between anorectic properties of HMR1426 and gastric emptying suggests that gastric emptying may cause the anorectic properties of HMR1426. The differences in microstructural feeding behavior between HMR1426 and centrally active anorectics makes it unlikely that HMR1426 acts via the CNS. Evidence for a peripheral mode of action is derived from sham-fed rats with open gastric fistula. When the milk fed was drained, HMR1426 was ineffective. HMR1426 is not a CCK(A) agonist. The molecular action of HMR1426 causing gastric emptying and its anorectic properties are under investigation.

Cretaceous (Late Albian) coniferales of Alexander Island, Antarctica. 2. Leaves, reproductive structures and roots.

Coniferous foliage from the Albian of Alexander Island, Antarctica, is assigned to the Araucariaceae, Podocarpaceae, and Taxodiaceae based on attached or associated fertile remains. Araucarian foliage represented by Araucaria alexandrensis sp. nov. and A. chambersii sp. nov. is associated with ovulate cone scales described as Araucarites wollemiaformis sp. nov. and A. citadelbastionensis sp. nov., respectively. The Podocarpaceae is represented by Bellingshausium willeyii sp. nov. and the Taxodiaceae by Athrotaxites ungeri, both with attached cones. Sterile foliage is widespread belonging to the form genera Podozamites, Elatocladus, Brachyphyllum and Pagiophyllum. The conifers in this Albian southern high-latitude flora make up ca. 15% of the species diversity. Evidence from leaf litter distribution on palaeosols and leaf morphology suggest that the majority of conifers were large canopy-forming trees, although a few were probably small understorey shrubs.

Properties and function of KCNQ1 K+ channels isolated from the rectal gland of Squalus acanthias.

KCNQ1 (KVLQT1) K+ channels play an important role during electrolyte secretion in airways and colon. KCNQ1 was cloned recently from NaCl-secreting shark rectal glands. Here we study the properties and regulation of the cloned sKVLQT1 expressed in Xenopus oocytes and Chinese hamster ovary (CHO) cells and compare the results with those obtained from in vitro perfused rectal gland tubules (RGT). The expression of sKCNQ1 induced voltage-dependent, delayed activated K+ currents, which were augmented by an increase in intracellular cAMP and Ca2+. The chromanol derivatives 293B and 526B potently inhibited sKCNQ1 expressed in oocytes and CHO cells, but had little effect on RGT electrolyte transport. Short-circuit currents in RGT were activated by alkalinization and were decreased by acidification. In CHO cells an alkaline pH activated and an acidic pH inhibited 293B-sensitive KCNQ1 currents. Noise analysis of the cell-attached basolateral membrane of RGT indicated the presence of low-conductance (<3 pS) K+ channels, in parallel with other K+ channels. sKCNQ1 generated similar small-conductance K+ channels upon expression in CHO cells and Xenopus oocytes. The results suggest the presence of low-conductance KCNQ1 K+ channels in RGT, which are probably regulated by changes in intracellular cAMP, Ca2+ and pH.

Synthesis and activity of novel and selective I(Ks)-channel blockers.

Since the discovery of the I(Ks)-potassium channel as the slowly activating component of the delayed rectifier current (I(k)) in cardiac tissue, the search for blockers of this current has been intense. During the screening of K(ATP)-channel openers of the chromanol type we found that chromanol 293B was able to block I(Ks). Chromanol 293B is a sulfonamide analogue of the K(ATP)-channel openers but had no activity on this target. Experiments were initiated to improve the activity and properties based on this lead compound. As a screening model we used Xenopus oocytes injected with human minK (KCNE1). Variations of the aromatic substituent and the sulfonamide group were prepared, and their activity was evaluated. We found that the greatest influence on activity was found in the aromatic substituents. The most active compounds were alkoxy substituted. We chose HMR1556 ((3R, 4S)-(+)-N-[-3-hydroxy-2,2-dimethyl-6-(4,4,4-trifluorobutoxy)chroman-4-yl]-N-methyl-ethanesulfonamide) 10a for development as an antiarrhythmic drug. The absolute configuration, resulting from an X-ray single-crystal structure analysis, was determined.

DCPIB is a novel selective blocker of I(Cl,swell) and prevents swelling-induced shortening of guinea-pig atrial action potential duration.

1. We identified the ethacrynic-acid derivative DCPIB as a potent inhibitor of I(Cl,swell), which blocks native I(Cl,swell) of calf bovine pulmonary artery endothelial (CPAE) cells with an IC(50) of 4.1 microM. Similarly, 10 microM DCPIB almost completely inhibited the swelling-induced chloride conductance in Xenopus oocytes and in guinea-pig atrial cardiomyocytes. Block of I(Cl,swell) by DCPIB was fully reversible and voltage independent. 2. DCPIB (10 microM) showed selectivity for I(Cl,swell) and had no significant inhibitory effects on I(Cl,Ca) in CPAE cells, on chloride currents elicited by several members of the CLC-chloride channel family or on the human cystic fibrosis transmembrane conductance regulator (hCFTR) after heterologous expression in Xenopus oocytes. DCPIB (10 microM) also showed no significant inhibition of several native anion and cation currents of guinea pig heart like I(Cl,PKA), I(Kr), I(Ks), I(K1), I(Na) and I(Ca). 3. In all atrial cardiomyocytes (n=7), osmotic swelling produced an increase in chloride current and a strong shortening of the action potential duration (APD). Both swelling-induced chloride conductance and AP shortening were inhibited by treatment of swollen cells with DCPIB (10 microM). In agreement with the selectivity for I(Cl,swell), DCPIB did not affect atrial APD under isoosmotic conditions. 4. Preincubation of atrial cardiomyocytes with DCPIB (10 microM) completely prevented both the swelling-induced chloride currents and the AP shortening but not the hypotonic cell swelling. 5. We conclude that swelling-induced AP shortening in isolated atrial cells is mainly caused by activation of I(Cl,swell). DCPIB therefore is a valuable pharmacological tool to study the role of I(Cl,swell) in cardiac excitability under pathophysiological conditions leading to cell swelling.

Necrosis of the tongue and unilateral blindness in temporal arteritis.

While blindness is one of the typical clinical presentations of temporal arteritis, tongue necrosis, on the other hand, is an unusual complication of the disease. An 80 year old male patient presenting a sudden massive swelling of the tongue was admitted to the Hospital of Yverdon. The swelling rapidly progressed to a complete necrosis of the tongue within a few days. The clinical presentation, the dramatic evolution of the necrosis, and sudden unilateral blindness despite prompt treatment confirmed our diagnosis of temporal arteritis. However, all the examinations, including biopsy of the right temporal artery, remained non-specific for the disease. Our diagnosis was based on the unusual clinical presentation of the disease.

A novel inhibitor of the Na+/H+ exchanger type 3 activates the central respiratory CO2 response and lowers the apneic threshold.

Cultured CO2-sensitive neurons from the ventrolateral medulla of newborn rats enhanced their bioelectric activity upon intracellular acidification induced by inhibition of the Na+/H+ exchanger type 3 (NHE3). Now we detected NHE3 also in the medulla oblongata of adult rabbits. Therefore, this animal model was employed to determine whether NHE3 inhibition also affects central respiratory chemosensitivity in vivo. Seven anesthetized (pentobarbital), vagotomized, paralyzed rabbits were artificially ventilated with O2-enriched air. From the phrenic nerve compound discharge, integrated burst amplitude (IPNA), respiratory rate (fR), and phrenic minute activity (IPNA. fR) were taken as measures of central respiratory rhythm and drive. Effects of potent NHE3 inhibition with the novel brain permeant substance S8218 were studied by comparing respiratory characteristics before and after up to 9.2 +/- 1.1 mg/kg cumulative drug application, yielding average plasma concentrations of 0.9 +/- 0.2 microg/ml. In response to S8218, the baseline level of IPNA. fR was significantly enhanced by an average of 51.0 +/- 6.4% (n = 27, p < 0.0001). The influence of NHE3 inhibition on the respiratory CO2 response was studied at plasma concentrations of S8218 maintained in the range of 0.3 microg/ml (10(-6) M). Although the metabolic acid-base status thereby remained widely unchanged, the group mean apneic threshold PaCO2 was significantly lowered by 0.45 +/- 0.11 kPa (n = 7, p < 0.01), whereby in four of seven animals even strong hyperventilation failed to suppress phrenic nerve rhythmicity completely. Likewise, S8218 significantly augmented IPNA. fR, in the range of PaCO2 between 1 and 6 kPa above threshold, by an average of 38.0 +/- 8.5% (n = 35, p < 0.0001). These in vivo results are compatible with the effects of NHE3 inhibition on chemosensitive brainstem neurons in vitro. Moreover, rhythmogenesis is supported through NHE3 inhibition by lowering the threshold PCO2 for central apnea.

The cardiac K+ channel KCNQ1 is essential for gastric acid secretion.

BACKGROUND & AIMS: Gastric H+ secretion via the H+/K+-adenosine triphosphatase is coupled to the uptake of K+. However, the molecular identity of luminal K+ channels enabling K+ recycling in parietal cells is unknown. This study was aimed to investigate these luminal K+ channels. METHODS: Acid secretion was measured in vivo and in vitro; KCNQ1 protein localization was assessed by immunofluorescence, and acid-sensitivity of KCNQ1 by patch-clamp. RESULTS: We identified KCNQ1, which is mutated in cardiac long QT syndrome, as a K+ channel located in tubulovesicles and apical membrane of parietal cells, where it colocalized with H+/K+-adenosine triphosphatase. Blockade of KCNQ1 current by 293B led to complete inhibition of acid secretion. The putative KCNQ1 subunits, KCNE2 and KCNE3, were abundant in human stomach; KCNE1, however, was absent. Coexpression of KCNE3/KCNQ1 in COS cells led to an acid-insensitive current; KCNE2/KCNQ1 was activated by low extracellular pH. CONCLUSIONS: We identified KCNQ1 as the missing luminal K+ channel in parietal cells and characterized its crucial role in acid secretion. Because KCNE3 and KCNE2 are expressed in human stomach, one or both are candidates to coassemble with KCNQ1 in parietal cells. Thus, stomach- and subunit-specific inhibitors of KCNQ1 might offer new therapeutical perspectives for peptic ulcer disease.

Diabetes-induced changes in retinal NAD-redox status: pharmacological modulation and implications for pathogenesis of diabetic retinopathy.

Diabetes-induced changes in retinal metabolism and function have been linked to increased aldose reductase activity, hypoxia or 'pseudohypoxia' (increase in NADH/NAD+ attributed to increased sorbitol dehydrogenase activity). To address this controversy, we evaluated the effects of two vasoactive compounds, alpha(1)-adrenoceptor antagonist prazosin and antioxidant DL-alpha-lipoic acid, as well as sorbitol dehydrogenase inhibitor (SDI-157) and aldose reductase inhibitor (sorbinil) on retinal free mitochondrial and cytosolic NAD+/NADH ratios in streptozotocin-diabetic rats. Diabetes-induced decrease in mitochondrial and cytosolic NAD+/NADH ratios was completely or partially corrected by prazosin and DL-alpha-lipoic acid (despite the fact that prazosin did not affect and DL-alpha-lipoic acid even further increased sorbitol pathway activity) as well as by sorbinil, whereas SDI-157 was totally ineffective. Hypoxia-like metabolic changes in the diabetic retina originate from aldose reductase, but not sorbitol dehydrogenase activity.

Inhibition of sorbitol dehydrogenase exacerbates autonomic neuropathy in rats with streptozotocin-induced diabetes.

We have developed an animal model of diabetic autonomic neuropathy that is characterized by neuroaxonal dystrophy (NAD) involving ileal mesenteric nerves and prevertebral sympathetic superior mesenteric ganglia (SMG) in chronic streptozotocin (STZ)-diabetic rats. Studies with the sorbitol dehydrogenase inhibitor SDI-158, which interrupts the conversion of sorbitol to fructose (and reactions dependent on the second step of the sorbitol pathway), have shown a dramatically increased frequency of NAD in ileal mesenteric nerves and SMG of SDI-treated versus untreated diabetics. Although lesions developed prematurely and in greater numbers in SDI-treated diabetics, their distinctive ultrastructural appearance was identical to that previously reported in long-term untreated diabetics. An SDI effect was first demonstrated in the SMG of rats that were diabetic for as little as 5 wk and was maintained for at least 7.5 months. As in untreated diabetic rats, rats treated with SDI i) showed involvement of lengthy ileal, but not shorter, jejunal mesenteric nerves; ii) demonstrated NAD in paravascular mesenteric nerves distributed to myenteric ganglia while sparing adjacent perivascular axons ramifying within the vascular adventitia; and, iii) failed to develop NAD in the superior cervical ganglia (SCG). After only 2 months of SDI-treatment, tyrosine hydroxylase immunolocalization demonstrated marked dilatation of postganglionic noradrenergic axons in paravascular ileal mesenteric nerves and within the gut wall versus those innervating extramural mesenteric vasculature. The effect of SDI on diabetic NAD in SMG was completely prevented by concomitant administration of the aldose reductase inhibitor Sorbinil. Treatment of diabetic rats with Sorbinil also prevented NAD in diabetic rats not treated with SDI. These findings indicate that sorbitol pathway-linked metabolic imbalances play a critical role in the development of NAD in this model of diabetic sympathetic autonomic neuropathy.

Inhibition of Jurkat-T-lymphocyte Na+/H+-exchanger by CD95(Fas/Apo-1)-receptor stimulation.

Mitogenic factors are known to stimulate the Na+/H+-exchanger (NHE), leading to cytosolic alkalinization and/or cell swelling. Conversely, a hallmark of apoptosis is cell shrinkage and CD95-induced apoptosis has been reported to be paralleled by cytosolic acidification. To assess whether the CD95-receptor regulates NHE activity in Jurkat T-lymphocytes, we performed conventional BCECF fluorescence measurements and SNARF flow cytometric analysis (FACS). The recoveries from acidifications following application of butyrate or a NH3 pulse were both abolished by a specific NHE-inhibitor, HOE694, indicating that they fully depend on NHE activity. Thus they were taken as a measure of NHE activity. CD95-receptor stimulation caused a cytosolic acidification and blunted the recovery from acidification following application of butyrate or a NH3 pulse. Moreover, the NHE-dependent alkalinization following osmotic cell shrinkage was almost abolished by CD95-receptor stimulation. As apparent from the effect of osmotic cell shrinkage, inhibition of the NHE by CD95-receptor stimulation was absent in Lck56-deficient J-CaM1.6 cells and restored by retransfection of J-CaM1.6-cells with Lck56. CD95-receptor stimulation led within 4 h to a decrease of cellular ATP which could contribute to NHE inhibition. Treatment of Jurkat cells with the NHE inhibitor HOE694 accelerated CD95-induced DNA fragmentation. In conclusion, CD95-receptor stimulation inhibits NHE activity through a mechanism that depends directly or indirectly on the activation of the Src-like kinase Lck56. This effect contributes to CD95-induced cytosolic acidification, DNA fragmentation and cell shrinkage.

Interaction between osmotic and oxidative stress in diabetic precataractous lens: studies with a sorbitol dehydrogenase inhibitor.

Both sorbitol accumulation-linked osmotic stress and "pseudohypoxia" [increase in NADH/NAD+, similar to that in hypoxic tissues, and attributed to increased sorbitol dehydrogenase (1-iditol:NAD+ 5-oxidoreductase; EC 1.1.1.14; SDH) activity] have been invoked among the mechanisms underlying oxidative injury in target tissues for diabetic complications. We used the specific SDH inhibitor SDI-157 [2-methyl-4(4-N,N-dimethylaminosulfonyl-1-piperazino)pyrimid ine] to evaluate the role of osmotic stress versus "pseudohypoxia" in oxidative stress occurring in diabetic precataractous lens. Control and diabetic rats were treated with or without SDI-157 (100 mg/kg/day for 3 weeks). Lens malondialdehyde (MDA) plus 4-hydroxyalkenals (4-HA), MDA, GSH, and ascorbate levels, as well as the GSSG/GSH ratios, were similar in SDI-treated and untreated control rats, thus indicating that SDI-157 was not a prooxidant. Intralenticular osmotic stress, manifested by sorbitol levels, was more severe in SDI-treated diabetic rats (38.2+/-6.8 vs 21.2+/-3.5 micromol/g in untreated diabetic and 0.758+/-0.222 micromol/g in control rats, P<0.01 for both), while the decrease in the free cytosolic NAD+/NADH ratio was partially prevented (120+/-16 vs 88+/-11 in untreated diabetic rats and 143+/-13 in controls, P<0.01 for both). GSH and ascorbate levels were decreased, while MDA plus 4-HA and MDA levels were increased in diabetic rats versus controls; both antioxidant depletion and lipid aldehyde accumulation were exacerbated by SDI treatment. Superoxide dismutase (superoxide:superoxide oxidoreductase; EC 1.15.1.1), GSSG reductase (NAD[P]H:oxidized-glutathione oxidoreductase; EC 1.6.4.2), GSH transferase (glutathione S-transferase; EC 2.5.1.18), GSH peroxidase (glutathione:hydrogen-peroxide oxidoreductase; EC 1.11.1.9), and cytoplasmic NADH oxidase activities were increased in diabetic rats versus controls, and all the enzymes but GSH peroxidase were up-regulated further by SDI. In conclusion, sorbitol accumulation and osmotic stress generated oxidative stress in diabetic lens, whereas the contribution of "pseudohypoxia" was minor. SDIs provide a valuable tool for exploring mechanisms of oxidative injury in sites of diabetic complications.

Rate- and site-dependent effects of propafenone, dofetilide, and the new I(Ks)-blocking agent chromanol 293b on individual muscle layers of the intact canine heart.

BACKGROUND: Recent in vitro studies have demonstrated regional differences in electrophysiological properties of individual left ventricular muscle layers. Controversy exists on the relevance of these findings for the situation in vivo. Thus, this study was designed to determine whether the in vivo canine heart exhibits regional differences in left ventricular refractoriness and in the susceptibility to sodium and potassium channel blockers. METHODS AND RESULTS: In 16 dogs, 36 needle electrodes (12 mm long, 4 bipolar electrodes, interelectrode distance 2.5 mm) were inserted into the left ventricular wall. By use of a computerized multiplexer-mapping system, the spread of activation in epicardial, endocardial, and midmyocardial muscle was reconstructed during ventricular pacing at 300- and 850-ms basic cycle length (BCL). Effective refractory periods (ERPs) were determined at baseline and after application of propafenone (2 mg/kg), dofetilide (30 microg/kg), or chromanol 293b (10 mg/kg) by the extrastimulus technique (BCL 300 and 850 ms). At baseline, activation patterns and ERPs were uniform in all muscle layers. Propafenone homogeneously decreased conduction velocity and moderately prolonged ERPs without any regional differences. Dofetilide and chromanol 293b did not affect the spread of activation. Dofetilide exhibited reverse use-dependent effects on ERP, still preserving transmural homogeneity of refractoriness. Chromanol 293b led to a regionally uniform but more pronounced increase in local ERPs at faster than at slower pacing rates. CONCLUSIONS: At the heart rates applied, the in vivo canine heart does not exhibit regional differences in electrophysiological properties. Given the homogeneity of antiarrhythmic drug effects, induction of local gradients of refractoriness is obviously not a common mechanism of proarrhythmia in normal hearts.

Evaluation of a sorbitol dehydrogenase inhibitor on diabetic peripheral nerve metabolism: a prevention study.

AIMS/HYPOTHESIS: Studies of the role of sorbitol dehydrogenase in nerve functional deficits induced by diabetes reported contradictory results. We evaluated whether sorbitol dehydrogenase inhibition reduces metabolic abnormalities and enhances oxidative stress characteristic of experimental diabetic neuropathy. METHODS: Control and streptozotocin-diabetic rats were treated with or without sorbitol dehydrogenase inhibitor (SDI)-157 (100 mg x kg(-1) x day(-1), in the drinking water, for 3 weeks). Sciatic nerve free mitochondrial (cristae and matrix) and cytosolic NAD(+): NADH ratios were calculated from the beta-hydroxybutyrate, glutamate and lactate dehydrogenase systems. Concentrations of metabolites, e. g. sorbitol pathway intermediates and variables of energy state were measured in individual nerves spectrofluorometrically by enzymatic procedures. RESULTS: The flux through sorbitol dehydrogenase (manifested by nerve fructose concentrations) was inhibited by 53 % and 74 % in control and diabetic rats treated with SDI compared with untreated control and diabetic groups. Free NAD(+):NADH ratios in mitochondrial cristae, matrix and cytosol were decreased in diabetic rats compared with controls and reduction in either of the three variables was not prevented by sorbitol dehydrogenase inhibitor. Phosphocreatine concentrations and phosphocreatine:creatine ratios were decreased in diabetic rats compared with controls and were further reduced by the inhibitor. Malondialdehyde plus 4-hydroxyalkenals concentration was increased and reduced gluthathione concentration was reduced in diabetic rats compared with the control group, and changes in both variables were further exacerbated by sorbitol dehydrogenase inhibitor. Neither NAD-redox and energy states nor lipid aldehyde and reduced gluthathione concentrations were affected by treatment with the inhibitor in control rats. CONCLUSION/INTERPRETATION: Inhibition of sorbitol dehydrogenase does not offer an effective approach for prevention of oxidation and metabolic imbalances in the peripheral nerve that is induced by diabetes and is adverse rather than beneficial. [Diabetologia (1999) 42: 1187-1194]

Selective inhibition of the Na+/H+ exchanger type 3 activates CO2/H+-sensitive medullary neurones.

Hypercapnia as well as lowered intracellular pH (pHi) increase the bioelectric activity of CO2/H+-sensitive neurones (VLNcs) of the ventrolateral medulla oblongata. Here we describe that immunoreactive Na+/H+ exchanger (NHE3) is present in ventrolateral neurones from medullary organotypic cultures (obex level). To test whether VLNcs can be acidified and thereby activated by inhibition of NHE3, we used the novel high-affinity NHE3-inhibitors S1611 and S3226. Both drugs raised the firing rates of VLNcs to at least 150% of the control values, and depolarized membrane potential by up to 15 mV at concentrations (0.5-1 micromol/l) suitable for selective inhibition of NHE3. The changes in bioelectric activity strongly resembled the responses to hypercapnia (PCO2: 60-100 mmHg). In BCECF-AM-loaded cultures a subfraction of ventrolateral VLNcs was found to be intracellularly acidified by 0.05-0.1 pH units following treatment with S1611; the time course of this acidification was similar to that evoked by hypercapnia. All drug effects were sustained and readily reversible upon washing. Non-CO2/H+-responsive medullary neurones as well as hippocampal CA3 neurones were unaffected by up to 20 micromol/l S1611. It is concluded that the selective inhibition of NHE3 acidifies and activates CO2/H+-sensitive neurones within the ventrolateral medulla oblongata.

Cl--dependent and Cl--independent Na+/ HCO3- acid extrusion in cultured rat cerebellar astrocytes.

It is still uncertain whether the Na+-dependent Cl--HCO3- exchanger (NCBE) is expressed in mammalian astrocytes. Using fluorescent indicators to monitor the intracellular pH (pHi) and intracellular Na+ or Cl- levels, the NCBE in cultured rat cerebellar astrocytes was examined in detail. In nominally bicarbonate-free (Hepes-buffered) medium, a marked pHi recovery from internal acid load was seen which could be blocked completely by 30 microM HOE 694, a specific Na+-H+ exchanger isoform 1(NHE-1) inhibitor, at a pHi above 6.9. These conditions were therefore used to block NHE activity in CO2/HCO3-buffered media when the NCBE was being studied at pHi above 6.9. After internal acid loading in completely Cl--free bicarbonate-buffered medium (containing HOE 694), the rates of pHi recovery and transient Na+ influx were considerably slowed, and the Cl--dependent acid extrusion was both Na+- and 4,4-diisothiocyano-stilbene-disulphonic acid (DIDS)-sensitive. Moreover, a HCO3-dependent Cl- efflux during internal acid injection was seen. These results suggest that the NCBE is present in astrocytes. Following repetitive internal acid loading by addition of 5% CO2 to internal Cl- depleted cells, a similar rate of pHi recovery was consistently seen, suggesting Cl--independent pHi regulation also occurred in astrocytes. Moreover, this pHi recovery was completely blocked in the absence of sodium or on addition of DIDS, confirming that the Na+-HCO3 cotransporter (NBC) is present. Thus, the present study provides evidence that both the NCBE and NBC play important roles in acid extrusion in cultured mammalian astrocytes.

Expression, functional characterization and tissue distribution of a Na+/H+ exchanger cloned from Xenopus laevis oocytes (XL-NHE).

We examined the functional properties of a Na+/H+ exchanger cloned from Xenopus laevis oocytes (XL-NHE) upon stable transfection into PS120 fibroblasts which lack endogenous Na+/H+ exchange. In contrast to untransfected cells, XL-NHE-transfected cells displayed Na+-dependent alkalinization upon acidification with nigericin. XL-NHE activity was inhibited by amiloride, ethylisopropylamiloride, HOE694 [(3-methylsulphonyl-4-piperidinobenzoyl)-guanidine methanesulphonate] and HOE642 [4-isopropyl-3-methylsulphonylbenzoyl)-guanidine methanesulphonate], Ki values being calculated at 5 micromol/l, 25 nmol/l, 300 nmol/l and 180 nmol/l, respectively. The Na+ dependence of pHi recovery was compatible with simple Michaelis-Menten kinetics, the Km for Na+ being 22.0+/-3.2 mmol/l and the Hill coefficient for Na+ being approximately 1. XL-NHE was activated by phorbol ester, whereas forskolin exerted no effect, suggesting the involvement of phospholipase C/protein kinase C signalling pathways rather than protein kinase A signalling pathways in XL-NHE stimulation. Using reverse transcription polymerase chain reaction, XL-NHE message could be detected in various Xenopus tissues including heart, brain, skeletal muscle, reticulocytes, A6-kidney cells and oocytes.