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International audience; Background: In the absence of a well-established therapeutic approach, patients with irritable bowel syndrome seek alternative strategies such as probiotics.Aims: The current trial named LAPIBSS aimed to demonstrate the efficacy of a 2-strain mixture of Lactobacillus acidophilus to improve irritable bowel syndrome symptoms.Methods: Eighty patients diagnosed for irritable bowel syndrome were recruited to a multicentre, double-blinded, in parallel groups, placebo-controlled, randomized clinical trial. Patients were provided with a daily dose of two capsules containing either probiotics (5 × 109 cfu/capsule) or placebo for 8 weeks. The primary outcome was abdominal pain score assessed with a 100-mm visual analogue scale. Secondary outcomes included scores of bloating, flatus and rumbling assessed with a 100-mm visual analogue scale, a composite score and bowel habits.Results: Abdominal pain score was significantly improved in both groups at weeks 4 and 8 (P < 0.0001), but no significant differences were found between groups at week 8 (19.0 ± 2.5 vs 25.1 ± 2.6, respectively; LS Means differences = 6.0 ± 3.2; P = 0.06). Significant differences between groups were observed for flatus score at week 4 (P = 0.04) and week 8 (P = 0.03) and composite score (P = 0.04) at week 8.Conclusions: The consumption of the 2-strain mixture of L. acidophilus over 8 weeks is safe and decreases significantly flatus and composite scores.Trial registration number: EudraCT No 2008 A00844-51.

International audience; Since the publication of the Framingham Heart Study, which suggested that uric acid should no longer be associated with coronary heart disease after additional adjustment for cardiovascular disease risk factors, the number of publications challenging this statement has dramatically increased. The aim of this paper was to review and discuss the most recent studies addressing the possible relation between sustained elevated serum uric acid levels and the onset or worsening of cardiovascular and renal diseases. Original studies involving American teenagers clearly showed that serum uric acid levels were directly correlated with systolic and diastolic pressures, which has been confirmed in adult cohorts revealing a 2.21-fold increased risk of hypertension. Several studies involving patients with coronary artery disease support a role for serum uric acid level as a marker and/or predictor for future cardiovascular mortality and long-term adverse events in patients with coronary artery disease. Retrospective analyses have shown an inverse relationship between serum uric acid levels and renal function, and even a mild hyperuricemia has been shown to be associated with chronic kidney disease in patients with type 2 diabetes. Interventional studies, although of small size, showed that uric acid (UA)-lowering therapies induced a reduction of blood pressure in teenagers and a protective effect on renal function. Taken together, these studies support a role for high serum uric acid levels (>6 mg/dL or 60 mg/L) in hypertension-associated morbidities and should bring awareness to physicians with regards to patients with chronic hyperuricemia.

Irritable bowel syndrome is a chronic functional gastrointestinal disorder characterized by abdominal pain/discomfort and altered bowel habits. The use of Lactobacilli as probiotics during irritable bowel syndrome is based on their interesting mechanisms of action and their excellent safety profile but little is known about their clinical efficacy due to the lack of adequately designed clinical trials. The current clinical trial protocol aims to determine the effects of a mixture of Lactobacillus acidophilus NCFM and LAFTI L10 as probiotics to improve irritable bowel syndrome symptoms (LAPIBSS). Eighty patients with a positive diagnosis of irritable bowel syndrome according to Rome III criteria were recruited to a multicentre, double-blinded, in parallel groups, placebo-controlled randomized trial. Patients were provided with a daily dose of two capsules with two strains of Lactobacilli (5x109cfu/capsule) or placebo for 8 weeks on a 1:1 ratio. The primary outcome is to obtain scores of abdominal pain/discomfort assessed with a 100-mm visual analogue scale. The secondary outcome is to obtain scores of bloating, flatus and rumbling tested with a 100-mm visual analogue scale, composite score, stool frequency and stool consistency/appearance assessed with the Bristol Stool Form scale. According to the hypothesis that abdominal pain is mainly the result of a visceral hypersensitivity, the current study protocol aims to provide high quality proof of concept data to elucidate the efficacy of a consumption of a mixture of Lactobacillus acidophilus probiotic strains after 8 weeks, for decreasing abdominal pain. Ethical approval was given by ethics committee French Consultative Committee for the Protection of Individuals in Biomedical Research of the South West (Number CPP08-014a) and ANSM (French National Agency for Medicines and Health Products Safety – Number B80623-40). The findings from LAPBISS will be disseminated through peer-reviewed publications and at scientific conferences. Trial registration: EudraCT N°2008-A00844-51

A few decades ago, the neuromuscular junction (NMJ) concept was reduced to two elements: the nerve ending and the facing muscular zone. This description has since changed substantially based on recent studies conducted on the molecular aspects of neurotransmission. The aim of this paper is to provide a synthetic view of the major morphological, molecular and electrophysiological tools used in the analysis of NMJ architecture and its functional characterization.

STATE OF THE ART In humans, it is currently believed that peripheral nerves remain intact after central nervous system (CNS) injuries. This should lead us to observe a lack of amyotrophy in the peripheral projection areas of CNS damage. Nevertheless, the appearance of amyotrophy, described as underuse amyotrophy, is common in victims of CNS injury. Its pathophysiology remains poorly understood and is currently being debated. Amyotrophy could result directly from the structural deterioration of a nervous fiber in the muscular area corresponding to the CNS injury caused by neuromuscular junction (NMJ) changes. AIMS OF THIS STUDY The aims of this study were to assess the repercussions of a CNS injury on the NMJ and peripheral nerve complex and to evaluate the involvement of peripheral nerves and NMJs in plasticity. METHODOLOGY Peripheral nerve and muscle biopsies were collected from a group of 35 female Wistar rats that had previously undergone a thoracic spinal cord hemisection (15 rats at the T2 level (group 1), 15 rats at the T6 level (group 2), and 5 matched rats used as controls). We studied the localization and expression of the NMJ molecular components in muscle specimens by immunohistochemistry using confocal microscopy. We also searched for signs of nerve and muscle degeneration using light and electron microscopy. RESULTS We observed nonpathologic NMJs coexisting with completely denervated and partially reinnervated NMJs. We also found characteristics of embryonic behavior in rat axons secondary to axonal caliber distortions. Some authors associate this decrease in axonal activity with physiological denervation. CONCLUSION This project was designed to improve the understanding of the mechanisms involved in the interactions between the first and second motoneurons after different types of CNS injuries, with variable functional repercussions. Our results strongly suggest that CNS injuries lead to both morphological and functional repercussions at the NMJ and the peripheral nerve.

From the elaborate information processing that takes place in the brain to the contraction of skeletal muscles, the neurotransmission pathways involve, at least in part, (1) in tissue, Na+, K+-ATPase electrogenesis making action potential (AP) propagation possible and (2) in the cell, the synthesis, maturation, and renewal of an amazing number of molecules concentrated at the neuromuscular junction (NMJ). Our aim is to clarify CNS and peripheral nerve system (PNS) interactions by determining whether the partial motor recovery sometimes observed after a lesion of the first motoneuron is related to (1) changes in active transportation of the ions in peripheral nerve and/or muscle and (2) morphological and/or molecular changes at the NMJ, illustrating a dysfunction. Peripheral nerve surgery is proposed to some spastic patients who have recovered partially after CNS lesions to improve their gait. During these surgical procedures, the nerve and muscle samples that are usually resected can be collected and analyzed. Here, we report on eight patients who showed strictly similar motor recovery 2 years after massive CNS lesions and who underwent a selective tibial neurotomy for a spastic equinus foot. In these eight spastic patients, we performed a pathophysiological, molecular, and metabolic study of their neuromuscular junctions and peripheral nerves to characterize the dysfunction of the neuromuscular transmission after a permanent CNS injury.

Na/K-ATPase electrogenic activity and its indispensable role in maintaining gradients suggest that the modifications in isoform distribution and the functioning of the sodium pump have a major influence on both the neuronal functions, including excitability, and motor efficiency. This article proposes to clarify the involvement of Na/K-ATPase in the transmission of nerve influx within the peripheral nerve and then in the genesis, the maintenance, and the physiology of muscle contraction by comparing the data found in the literature with our work on neuron and muscle characterization of Na/K-ATPase activity and isoforms.

Neuromuscular transmission results from a double signal transduction from electric impulses to chemical messengers, taking place at a highly differentiated region, the neuromuscular junction (NMJ). A nerve cell responds to a specific stimulus by modifications of its plasmic membrane properties, generating an action potential (AP). This electric signal is transmitted along the axon to the NMJ, where it induces the voltage-gated calcium channels to open. Intracellular calcium entry leads to acetylcholine release in the synaptic space at the active zones but all scientists do not consider it the major release factor. Acetylcholine binding with its receptor at the muscle membrane generates an endplate potential when the induced depolarization is greater than the sodium voltage channel opening threshold. Muscle AP causes a muscle contraction involving the three phases. This paper will successively review the electrophysiological and molecular mechanisms involved at the pre-, inter- and postsynaptic levels. The last part of the article will discuss electromechanical considerations directly affecting the mechanical properties of the muscle fiber, most particularly the factors influencing the predetermined involvement of motor units, motor neuron electrical properties determining responses to synaptic inputs and finally the impact of recruited motor neurons and their electrical impulse rates on muscle contraction strength and velocity.

It has been shown that the onset of a central nervous system lesion in the rat results in morphological modifications of the peripheral nerves and the underlying neuromuscular junctions, without suggesting a functional correlation between recuperation of motor functions and sublesional metabolic activity. Using double lesion localization (T2 and T6) in a spinal rat model has nevertheless pointed out the functional importance of the T2-T6 metameric interval in the reinnervation phenomena observed, raising the problem of spinal generation in locomotor movements. Motivated by electrophysiological data that have given support to the concept of an anatomic substrate for these intramedullary rhythm generators, we attempted to establish a relation between the functional recuperation possible after a central nervous system lesion and modifications within the metabolism of the underlying neuromuscular system. We notably focused on Na/K-ATPase, whose crucial role in neuromuscular transmission has been evidenced. This paper proposes to demonstrate the involvement in the mechanisms of metabolic regulation after trans-synaptic denervation, i.e., a central nervous system lesion. Our study includes the Na/K-ATPase activity analysis on the sublesional peripheral nerve and the combined analysis of the expression of different RNA messengers within the corresponding muscle groups. We have also investigated the spatiotemporal organization of the compensating processes of the nerves underlying the lesion using magnetic resonance spectroscopy.

Acute renal failure (ARF) induced by Ischemia-Reperfusion (I-R) is associated with a significant impairment of tubular sodium reabsorption. Na⁺,K⁺-ATPase has a crucial role in tubular sodium reabsorption and maintenance of water and ion homeostasis. In this study, we examined whether renal I-R affects Na⁺,K⁺-ATPase activity in a renal auto graft model (Large White Pig) with controlateral nephrectomy. Kidney samples were obtained from medulla (n = 4) and cortex (n = 3) after one hour of warm ischemia in autotransplanted models. These experimental groups were compared with a controlateral nephrectomy group (Control). Na⁺,K⁺-ATPase activity in purified membrane fractions was measured as ouabain-sensitive K⁺ -stimulated paranitrophenyl phosphatase (pNPPase) activity. The Na⁺,K⁺-ATPase activities of control, ischemia and ischemia -reperfusion were significantly higher in the medulla than in the cortex, irrespective of the experimental group (P0.05). In cortices, Na⁺,K⁺-ATPase activity increased significantly (P0.05) by 35% after ischemia compared to control group. In medullae, there was only a trend toward an activation of Na⁺,K⁺-ATPase activity after Ischemia (increased by 25%) and after Ischemia-Reperfusion (increased by 30%) compared to control group, and these differences did not reach statistical significance. These data suggest that renal ischemia induces a significant alteration of Na⁺,K⁺-ATPase by increasing its ouabain-sensitive K⁺-stimulated paranitrophenyl phosphatase (pNPPase) activity in isolated membrane from cortical renal tissues after one hour of ischemia. This change of activity seems to be a new regulation mechanism of an important membrane ion transporter during renal I-R in the large pig model of renal transplantation.

Spinal cord injuries (SCI) induce a loss of skeletal muscle mass and functional capacity. The muscle excitability and contractility depend on the plasma membrane potential, regulated by transmembrane ion gradients, and thus necessarily on the Na⁺,K⁺-ATPase activity. The aim of this work was to evaluate the consequences of a spinal cord transection (SCT) on the skeletal muscle Na⁺,K⁺-ATPase and the impact of collateral GlyceroPhosphoLipids enriched in DocosaHexaenoic Acid (GPL-DHA) administration. The Na⁺,K⁺-ATPase activity and membrane expression of Na⁺,K⁺-ATPase α1, α2 and β1 isoforms were assessed by K⁺-stimulated paranitrophenyl phosphatase (pNPPase) measurements and Western Blotting, respectively. The results show that spinal cord transection increased significantly (p0.05) Na⁺,K⁺-ATPase activity in muscle by 25% and decreased the amounts of α1 isoform and α2 isoform expressions by 50% (p0.05) respectively compared to controls. The results also show that early injection of GPL-DHA after SCT decreases in membrane skeletal muscle the α1 and α2 isoforms expression but increases the membrane Na⁺,K⁺-ATPase activity. This treament partially restores the membrane expression of the β1 subunit of the Na⁺,K⁺-ATPase. These data suggest that the increase of β1 subunit expression is probably the main trigger to the membrane Na⁺,K⁺-ATPase activation following a trans-synaptic denervation.

In dogs, it has been reported that acute ischemia or severe and terminal heart failure results in a selective reduction of myocardial alpha 3 isoform of Na, K-ATPase activity. The aim of this study was to evaluate if a similar change in the two canine digitalis receptor isoforms occurs following 4 weeks of rapid ventricular pacing-induced heart failure without profound necrosis. Heart failure was induced in dogs by rapid ventricular pacing (240 beats x min-1). Digitalis receptors were quantitated by [3H]-ouabain binding with isolated microsomal membranes from sham-operated (n = 3) and heart failure dogs (n = 4) and by Western blot analysis using specific alpha 1 and alpha 3 polyclonal antibodies. In kinetic studies, similar dissociation rates of 19 to 22 x 10(-4) s-1 and 1.3 to 2.4 x 10(-4) s-1 corresponding to high and low affinity sites respectively, were found in sham-operated and CHF dogs. Immunoblotting showed similar abundance of alpha 1 isoform in the two groups; however, levels of alpha 3 were increased by at least 50% in pacing-induced heart failure animals. In conclusion, heart failure selectively modulates the expression of cardiac alpha 3 isoform in dogs.

Polyunsaturated fatty acids (PUFA) supplementations modify cell lipid composition leading to a change in cell function. However, the effect of PUFA supplementations in renal model cell on the kidney epithelial cells membrane fatty acid profile is not known. Therefore, the purpose of this study was to determine the effects of PUFAs with different ω6/ω3 ratios supplementations in the kidney epithelial cells and the type of supplementation that can be used as cellular protection during kidney transplantation. For that, we used as model the LLCPK1 cell and determined their membrane fatty acid (FA) composition after supplementation with three different commercial food supplements. These supplements consist of S1: docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) with ω6/ω3 ratio = 0.1, S2: DHA, EPA, linoleic acid (LA) and γ-linoleic acid (GLA) with ω6/ω3 ratio = 2.5, or S3: α-linolenic acid (ALA) and LA with ω6/ω3 ratio near 1. Cells were incubated for 24 hr with 30 μM of ω3 fatty acids from each supplement. Fatty acid composition of control and experimental groups was analysed by gas chromatography after extraction of lipids and fatty acids methylation. The efficiency of cell PUFA supplementation was achieved by showing 2 to 4 fold increases in cell PUFA incorporation. Whatever the supplementation used, the cell saturated fatty acids (SFA) were decreased by 50% following the three supplementations used (p0.001) as compared to control group. These decreases in SFA were compensated in part by increasing monounsaturated fatty acid levels. All these changes were observed with constant of cell ω6/ω3 ratio whatever the supplementations used. These data suggest that the supplements, with long chain polyunsaturated fatty acids or their precursors, lead to important regulation in the lipidome (desaturases and elongases) associated to preserved ω6/ω3 ratios. The fatty acids remodeling may represent an interesting new mechanism by which renal FA homoestasis could occurred.

The first definition of food supplements in France was established by decree 96-307 of April 10th 1996. In 2002, the European Community adopted a regulation for food supplements (European Directive 2002/46/CE June 10th). This was an important event in the regulation of food supplements. The European regulation was adopted in France, with some modifications, by decree 2006-352 of March 20th 2006. The European Regulation on food supplements is more defined than those for any other food types and is exemplary. The Regulation on addition of vitamins and minerals to food differs from the regulation on the addition of other substances such as amino acids, essential fatty acids, fibers, carbohydrates, various plant, and herbal extracts. While the Regulation includes vitamins and minerals to the positive list of supplements, other substances are included in the negative list of supplements. According to the Regulation, substances added to food supplements must have a nutritional or physiological effect. The increased use of food supplements led to the creation of a department specialized in the safety of food supplement. The safety of food supplements is a permanent concern for sanitary authorities. These authorities have recently combined scientific methodological approaches and a collective expertise to implement and monitor simple and useful rules that insure consumer's safety. Safety laws aim to protect the consumers of food supplements.

In skeletal muscle the relationship between Na+,K+-ATPase activity and isoform content remains controversial (9,6). It could be due to the fiber-type content, membrane isolation and analytical methods. We investigated the distribution of subunit α1 and α2 Na+,K+-ATPase catalytic isoforms and the Na+,K+-ATPase activity in isolated membranes from white ( type I and glycolitic fibers) and red (type II and oxidative fibers) skeletal muscles. Red Gastrocnemius and White Gastrocnemius muscles were sampled from 8 week-old female Wistar rats and crude membranes were performed. The Na+,K+-ATPase activity and membrane distribution of Na+,K+-ATPase α1 and α2 isoforms were assessed by ouabain sensitive K-phosphatase (Kpase) measurements and Western Blot respectively. The Na+,K+-ATPase activity was 6 fold lower in White Gastrocnemius membranes than in Red Gastrocnemius membranes. The α1 and α2-isoform levels are higher in RG than in White Gastrocnemius. The α1 and α2-subunit Red Gastrocnemius content was significantly higher than in WG. The correlation between crude membrane Kpase activity and both catalytic α-subunit of the Na+,K+-ATPase exist.These data suggest that the Na+,K+-ATPase phosphatase activity correlates with the α1 and α2 isoforms levels in Red Gastrocnemius and White Gastrocnemius and confirms the fiber-specific Na+,K+-ATPase catalytic α-subunits and α2-isoform as the major catalytic isoform in rat skeletal muscle.

As cholesterol, oxysterols, can insert the cell membrane and thereby modify the functions of membrane-bound proteins. The Na,K-ATPase is very sensitive to its lipid environment, seems to be involved in important endothelial functions as the regulation of nitric oxide (NO) release. The effects of 7-ketocholesterol , an oxysterol present in oxidized LDL, was investigated on Na,K-ATPase in isolated human endothelial cells. Cells were incubated 24h with lecithin-, cholesterol- or 7-ketocholesterol liposomes (6 μg/ml). K+-stimulated paranitrophenyl phosphatase activity, reflecting Na,K-ATPase activity, was evaluated as well as cell viability and lipoperoxidation. The expression of Na,K-ATPase subunits mRNAs and membrane fluidity were also investigated. As Na,K-ATPase and nitric oxide seem to be related, we determined the production of NO and the expression of endothelial NO synthase mRNAs. Na,K-ATPase activity was strongly decreased by 7-ketocholesterol. This decrease, not related to lipoperoxidation, was correlated with a decreased expression of the Na,K-ATPase α1-subunit messengers and with rigidity of plasma membranes. Cholesterol induced similar effects but was less potent than 7-ketocholesterol. Basal NO production and expression of endothelial NO synthase mRNAs were not modified by 7-ketocholesterol. Our new findings demonstrate that 7-ketocholesterol, used at non toxic doses, was very potent to disrupt the transport of ions by Na,K-ATPase and perturb membrane structure. These data demonstrate that 7-ketocholesterol induces endothelial dysfunction without cell death that may contribute to early events in atherosclerosis.

Recently, Na,K-ATPase isoforms with differential affinities for digitalis have been identified that may contribute to different toxicity profiles. Our objectives were to localize them and to define tissue receptor patterns by examining the effect of different glycosides on the Na,K-ATPase activity. The digitalis derivatives used exhibit variation in lipophilicity and rate of enzyme inhibition. Membrane fractions enriched in Na,K-ATPase were prepared from canine heart, brain, aorta and peripheral nerves. The inhibition of enzyme activities indicates a pattern of differential sensitivities with IC50 values starting from 3 nM in heart and 30 nM in brain. Therefore, high and low affinity active forms of the Na,K-ATPase enzyme coexist in these tissues. The data also suggest the existence of two Na,K-ATPase isoforms in aorta and peripheral nerves as identified by the action of digitoxigenin and LND 796 where the predominant expression is that of a high affinity form. The comparison of the patterns of digitalis sensitivities in these different tissues, suggests a more complex molecular interaction than that which can be explained by the presence of only two forms.

Dietary antioxidants are frequently proposed as protective agents for the vascular endothelium during the onset of atherosclerosis. This protection may occur at two distinct levels. First, they prevent oxidative modification of atherogenic lipoproteins (LDL). Second, they can provide a cellular protection against oxidized LDL-mediated endothelium dysfunction, although this mechanism remains poorly considered in many instances. To gain insight into the mechanism underlying such cellular protection against oxidized LDL, we examined the impact of a popular traditional medicine, an extract from Ginkgo biloba with well-known antioxidant properties, on two endothelial cells properties: cell adhesion and ionic homeostasis. Cellular lipoperoxides levels were also measured as a marker of cellular oxidative stress. Human umbilical-vein endothelial cells were exposed to native (nat-) or oxidized (ox-) LDL, the latter prepared to be compatible with clinically observed levels of oxidation. Although nat-LDL had little effect, ox-LDL increased endothelial adhesive properties (35%, p0.01) and lipoperoxidation (45%, p0.01). Na,K-ATPase activity, a key regulator of ionic homeostasis, was significantly decreased after exposure to nat-LDL (30%, p0.01) and dramatically depressed after exposure to ox-LDL (65%, p0.001). The standardized preparation of Ginkgo biloba EGb-761 totally protected adhesive properties and endothelial lipoperoxide levels. Moreover, it limited the decrease in Na,K-ATPase activity induced by ox-LDL to levels similar to nat-LDL. This suggests that EGb-761 protects endothelial adhesive properties and helps prevent the disruption of ionic homeostasis. The EGb-761-mediated inhibition of ox-LDL-induced lipoperoxide levels in endothelial cells appears to be an important mechanism by which Ginkgo biloba extract protects endothelial properties.

Na/K-ATPase electrogenic activity and its indispensable role in maintaining gradients suggest that the modifications in isoform distribution and the functioning of the sodium pump have a major influence on both the neuronal functions, including excitability, and motor efficiency. This article proposes to clarify the involvement of Na/K-ATPase in the transmission of nerve influx within the peripheral nerve and then in the genesis, the maintenance, and the physiology of muscle contraction by comparing the data found in the literature with our work on neuron and muscle characterization of Na/K-ATPase activity and isoforms.

It has been shown that the onset of a central nervous system lesion in the rat results in morphological modifications of the peripheral nerves and the underlying neuromuscular junctions, without suggesting a functional correlation between recuperation of motor functions and sublesional metabolic activity. Using double lesion localization (T2 and T6) in a spinal rat model has nevertheless pointed out the functional importance of the T2-T6 metameric interval in the reinnervation phenomena observed, raising the problem of spinal generation in locomotor movements. Motivated by electrophysiological data that have given support to the concept of an anatomic substrate for these intramedullary rhythm generators, we attempted to establish a relation between the functional recuperation possible after a central nervous system lesion and modifications within the metabolism of the underlying neuromuscular system. We notably focused on Na/K-ATPase, whose crucial role in neuromuscular transmission has been evidenced. This paper proposes to demonstrate the involvement in the mechanisms of metabolic regulation after trans-synaptic denervation, i.e., a central nervous system lesion. Our study includes the Na/K-ATPase activity analysis on the sublesional peripheral nerve and the combined analysis of the expression of different RNA messengers within the corresponding muscle groups. We have also investigated the spatiotemporal organization of the compensating processes of the nerves underlying the lesion using magnetic resonance spectroscopy.

In humans, it is currently believed that peripheral nerves remain intact after central nervous system (CNS) injuries. This should lead us to observe a lack of amyotrophy in the peripheral projection areas of CNS damage. Nevertheless, the appearance of amyotrophy, described as underuse amyotrophy, is common in victims of CNS injury. Its pathophysiology remains poorly understood and is currently being debated. Amyotrophy could result directly from the structural deterioration of a nervous fiber in the muscular area corresponding to the CNS injury caused by neuromuscular junction (NMJ) changes.The aims of this study were to assess the repercussions of a CNS injury on the NMJ and peripheral nerve complex and to evaluate the involvement of peripheral nerves and NMJs in plasticity.Peripheral nerve and muscle biopsies were collected from a group of 35 female Wistar rats that had previously undergone a thoracic spinal cord hemisection (15 rats at the T2 level (group 1), 15 rats at the T6 level (group 2), and 5 matched rats used as controls). We studied the localization and expression of the NMJ molecular components in muscle specimens by immunohistochemistry using confocal microscopy. We also searched for signs of nerve and muscle degeneration using light and electron microscopy.We observed nonpathologic NMJs coexisting with completely denervated and partially reinnervated NMJs. We also found characteristics of embryonic behavior in rat axons secondary to axonal caliber distortions. Some authors associate this decrease in axonal activity with physiological denervation.This project was designed to improve the understanding of the mechanisms involved in the interactions between the first and second motoneurons after different types of CNS injuries, with variable functional repercussions. Our results strongly suggest that CNS injuries lead to both morphological and functional repercussions at the NMJ and the peripheral nerve.

From the elaborate information processing that takes place in the brain to the contraction of skeletal muscles, the neurotransmission pathways involve, at least in part, (1) in tissue, Na+, K+-ATPase electrogenesis making action potential (AP) propagation possible and (2) in the cell, the synthesis, maturation, and renewal of an amazing number of molecules concentrated at the neuromuscular junction (NMJ). Our aim is to clarify CNS and peripheral nerve system (PNS) interactions by determining whether the partial motor recovery sometimes observed after a lesion of the first motoneuron is related to (1) changes in active transportation of the ions in peripheral nerve and/or muscle and (2) morphological and/or molecular changes at the NMJ, illustrating a dysfunction. Peripheral nerve surgery is proposed to some spastic patients who have recovered partially after CNS lesions to improve their gait. During these surgical procedures, the nerve and muscle samples that are usually resected can be collected and analyzed. Here, we report on eight patients who showed strictly similar motor recovery 2 years after massive CNS lesions and who underwent a selective tibial neurotomy for a spastic equinus foot. In these eight spastic patients, we performed a pathophysiological, molecular, and metabolic study of their neuromuscular junctions and peripheral nerves to characterize the dysfunction of the neuromuscular transmission after a permanent CNS injury.

Neuromuscular transmission results from a double signal transduction from electric impulses to chemical messengers, taking place at a highly differentiated region, the neuromuscular junction (NMJ). A nerve cell responds to a specific stimulus by modifications of its plasmic membrane properties, generating an action potential (AP). This electric signal is transmitted along the axon to the NMJ, where it induces the voltage-gated calcium channels to open. Intracellular calcium entry leads to acetylcholine release in the synaptic space at the active zones but all scientists do not consider it the major release factor. Acetylcholine binding with its receptor at the muscle membrane generates an endplate potential when the induced depolarization is greater than the sodium voltage channel opening threshold. Muscle AP causes a muscle contraction involving the three phases. This paper will successively review the electrophysiological and molecular mechanisms involved at the pre-, inter- and postsynaptic levels. The last part of the article will discuss electromechanical considerations directly affecting the mechanical properties of the muscle fiber, most particularly the factors influencing the predetermined involvement of motor units, motor neuron electrical properties determining responses to synaptic inputs and finally the impact of recruited motor neurons and their electrical impulse rates on muscle contraction strength and velocity.

A few decades ago, the neuromuscular junction (NMJ) concept was reduced to two elements: the nerve ending and the facing muscular zone. This description has since changed substantially based on recent studies conducted on the molecular aspects of neurotransmission. The aim of this paper is to provide a synthetic view of the major morphological, molecular and electrophysiological tools used in the analysis of NMJ architecture and its functional characterization.

To our knowledge, there is little reference in literature with regards to alpha3-isoform of Na+,K+-ATPase in human peripheral nerves. The aim of this study was to determine the expression of the neuronal alpha3-isoform of Na+,K+-ATPase in human sural nerves from patients with a permanent medullary central nervous system injury.We studied the immunolocalization of alpha3-isoform of Na+,K+-ATPase using a polyclonal antibody against the amino sequence near the phosphorylation site of the alpha3-isoforms of Na+,K+-ATPase using immunohistochemistry and confocal laser scanning microscopy. An antibody specific for alpha2-isoform of Na+,K+-ATPase was used to label the Schwann cells.Morphometric analysis of longitudinal section of human sural nerves showed that the alpha3-isoform of Na+,K+-ATPase was distributed along the length of axolemma. The myelin sheath of the Schwann cells showed clearly a distribution of alpha3- but not alpha2-isoforms of Na+,K+-ATPase at the level of Schmidt-Lanterman incisures.The human sural nerve shows a specific localization of the Na+,K+-ATPase alpha3-isoform in the Schmidt-Lanterman incisures of Schwann cells in addition to its localization in axonal membranes.

Diabetes mellitus type 2 is a major factor for cardiovascular diseases. The toxic effects of chronic hyperglycemia involve many alterations in the vascular tissue, including atherosclerosis. The pathogenesis of atherosclerosis in the diabetic syndrome (DS-II) has not been fully elucidated. A better understanding of the progress of DS-II at the level of the aorta could help us to identify inhibitors of atherosclerosis. Studies have shown that obesity and high calorie diet (HCD) are associated with the development of DS-II, however the lack of naturally occurring experimental models of DS-II have impaired to directly address these issues. We hypothesize that a HCD induces DS-II. This study (15 months duration) is designed to determine if HCD induces DS-II in the desert sand rat (Psammomys obesus; P. obesus). We also evaluated the histopathology of the aorta in animals fed with a laboratory chow pellets ad libitum (hypercaloric) and in control (animals fed with the naturally occurring hypocaloric diet; halophile plants). The weight and blood chemistry (glucose, lipids, and insulin levels) were evaluated periodically (once per week), and the histology of the aortas of these animals were assessed every 3 months for up to 12 months, during the development of DS-II. This study demonstrates that 40% of the animals in HCD develop DS-II at 3 months. Histological characterization demonstrates the typical alterations observed in atherosclerosis, i.e., alteration of the elastic fibers of the media layer and enrichment in collagen and glucosaminoglycans. This study demonstrates that P. obesus is an excellent animal model to study the progression of DS-II and the development of atherosclerosis.

Hypertension and diabetes are commonly associated and strongly predispose to renal injuries. In general, antihypertensive therapies protect from these damages, but the effect of cicletanine, a new type of antihypertensive drug, is unknown. This study examines the effects of cicletanine on renal failure in spontaneously hypertensive rats with diabetes (SHRD). Diabetes mellitus was induced with streptozotocin in uninephrectomized SHR. Rats received the vehicle, 10 mg or 50 mg/kg per day of cicletanine for 6 weeks. Age-matched untreated Wistar-Kyoto rats were used as controls. Systolic blood pressure (SBP), microalbuminuria and proteinuria were assessed throughout the treatment. At the end of the study, creatinine clearance measurements and histological analysis of kidneys were performed. Cicletanine did not affect SBP but decreased the elevated albuminuria of diabetic SHR in a dose-dependent manner. Similar results were obtained for proteinuria. Treatment with the high dose of cicletanine also normalized the altered creatinine clearance of diabetic SHR. These results indicate that cicletanine has a renal-protective action, probably blood pressure-independent, in a model combining hypertension and diabetes. The mechanism of renal-protection of cicletanine is not clearly established but may be due to the stimulation of arterial prostacyclin synthesis and/or to the reduction of intraglomerular capillary pressure.

Long duration ischemia in hypothermic conditions followed by reperfusion alters membrane transport function and in particular Na,K-ATPase. We compared the protective effect of two well-described cardioplegic solutions on cardiac Na,K-ATPase activity during reperfusion after hypothermic ischemia. Isolated perfused rat hearts (n = 10) were arrested with CRMBM or UW cardioplegic solutions and submitted to 12 hr of ischemia at 4 degrees C in the same solution followed by 60 min of reperfusion. Functional recovery and Na,K-ATPase activity were measured at the end of reperfusion and compared with control hearts and hearts submitted to severe ischemia (30 min at 37 degrees C) followed by reflow. Na,K-ATPase activity was not altered after 12 hr of ischemia and 1 hr reflow when the CRMBM solution was used for preservation (55 +/- 2 micromolPi/mg prot/hr) compared to control (53 +/- 2 micromol Pi/mg prot/hr) while it was significantly altered with UW solution (44 +/- 2 micromol Pi/mg prot/hr, p0.05 vs control and CRMBM). Better preservation of Na,K-ATPase activity with the CRMBM solution was associated with higher functional recovery compared to UW as represented by the recovery of RPP, 52 +/- 12% vs 8 +/- 5%, p0.05 and coronary flow (70 +/- 2% vs 50 +/- 8%, p0.05). The enhanced protection provided by CRMBM compared to UW may be related to its lower K+ content.

The current treatment for heart failure, i.e., carvedilol administration, has brought about a significant reduction in cardiovascular morbidity and mortality. The European Cardiology Association task-force has recently recalled that the treatment for heart failure should be considered differently according to the regions within Europe. However, most studies have involved North American or Australasian subjects. The results of these studies have shown that carvedilol improves the symptomatic status of patients with mild to moderate chronic heart failure, but their extrapolation to the situation in Europe is questionable. In the present study, the authors have reported on the use of this beta-blocker over a 6-month period in 48 European subjects (an older patient population, and more frequently given digitalis treatment) with mild to moderate stage II and III chronic heart failure (mean NYHA functional stage = 2.75 +/- 0.2; mean left ventricular ejection fraction = 32.4 +/- 3.4). Six patients (12.5%) were obliged to withdraw from the study due to adverse reactions to carvedilol, i.e., 2 subjects during the test-dose (4.2%) and 4 others who were subsequently unable to withstand the progressive stepping up of drug dosage (8.3%). In the remaining 42 patients, the mean NYHA scores dropped significantly from 2.75 +/- 0.2 to 1.8 +/- 0.1 (p0.05) at the end of the study period, indicating an increase in symptomatic improvement with a favorable hemodynamic tolerance profile, even in cases of combined therapy with digitalis. This study confirms the positive action of carvedilol on functional symptomatology in southern European patients presenting with mild to moderate chronic heart failure. However, it should be emphasized that this beta-blocker should only be administered under strict and specialized medical surveillance as its effect of significantly slowing down the heart rate could also result in cardiac insufficiency.

Atrial fibrillation (AF) is accompanied by various changes in ion channels that cause atrial electrophysiological remodeling. The enzyme Na,K-ATPase is also a major cellular mechanism for the regulation of ion homeostasis. During AF, Na,K-ATPase may be regulated by synthesis of its alpha- and beta-subunits as well as changes in membrane fluidity. To test this hypothesis, we studied the effect of pacing-induced AF in sheep on atrial Na,K-ATPase alpha- and beta-subunits and on membrane fluidity as well.A group of six sheep (AF group) was subjected to overdrive electrical stimulation of the right atrium in order to induce AF. A group of six sham operated sheep served as control. All paced sheep developed multiple episodes of sustained AF with a mean total duration of 110 min over a 2-hours period. Protein expression of Na,K-ATPase alpha- and beta-subunits in atrial microsomal membranes was assayed by Western blotting analysis. When significant changes in membrane expression were observed, transcriptional regulation was analysed by Northern blotting. Membrane fluidity was assessed on atrial microsomal fractions by anisotropy measurements using the fluorescent probe diphenylhexatriene.Atrial fibrillation enhanced the expression of the Na,K-ATPase beta1-subunit at both membrane and mRNA levels. Anisotropy values were higher in AF group than in control group, indicating a decreased fluidity of the membranes isolated from paced sheep atria.These data are the first evidence for an enhanced Na,K-ATPase beta1-subunit expression in membrane during AF. Membrane rigification represents a new factor of tachycardia-induced atrial remodeling.

The endothelial Na,K-ATPase is an active component in maintaining a variety of normal vascular functions. The enzyme is characterized by a complex molecular heterogeneity that results from differential expression and association of multiple isoforms of both its alpha- and beta-subunits. The aim of the present study was to determine which isoforms of the Na,K-ATPase are expressed in human endothelial cells. HUVEC (human umbilical vein endothelial cells) were used as a model of well known human endothelial cells. The high sensitive method RT-PCR was used with primers specific for the various isoforms of the alpha- and beta-subunits of the Na,K-ATPase. The results show that HUVEC express alpha1-, but not alpha2-, alpha3- or alpha4-isoforms of the catalytic subunit and that beta3- but not beta2- or beta1-isoforms is present in these cells. These findings are in contradiction with our previous detection of Na,K-ATPase isoforms in HUVEC using antibodies (14). Such results raise the technical problem of the specificity of the available antibodies directed against the different isoforms as well as the question of the physiological relevance of the diversity of the Na,K-ATPase isoforms.

The Na,K-ATPase is a heterodimer composed of an alpha-catalytic and a beta-glycoprotein subunit. At present, three different alpha-polypeptides (alpha1, alpha2, alpha3) and two distinct beta-isoforms (beta1 and beta2) have been detected in human heart. The aim of the present study was to determine whether or not the beta3-isoform of the Na,K-ATPase can be detected in human heart. Using the highly sensitive method of RT-PCR, we here show that human heart expresses the beta3-isoform of the Na,K-ATPase. Given the differences in pharmacological properties of the nine different Na,K-ATPase isoenzymes (containing all combinations of the subunit isoforms), the study of beta3-isoform regulation in human heart may be of interest in understanding the altered response of human myocardium to digitalis therapy during heart failure.

The effects of digitalis on the baroreflexes in human chronic heart failure have been well studied. Similarly, since it has been recently shown that chemoregulation remains generally effective during cardiac failure, the goal of this study was to evaluate the effects of a chronic administration of digoxin on the chemoreflexes. Hemodynamic and blood gas parameters were assessed in 7 patients with chronic congestive heart failure before and after chronic administration for 10 days of digoxin therapy (0.25 mg daily). In both situations measurements were performed 1/ in baseline conditions at room air and, 2/ after inhalation of pure O2 for 30 min, in order to inhibit the activation of the chemoreflexes. At room air, acute O2 inhalation resulted in a significant decrease in heart rate and cardiac output. After digoxin therapy, comparatively to pre-treatment values, cardiac output, stroke volume and PaO2 were significantly higher while heart rate, systemic resistance and pulmonary wedge pressure were lower. Furthermore, acute O2 inhalation did not modify heart rate or any hemodynamic variables. These results suggest that after digoxin therapy chemoreflex was no more activated in these patients. This effect may be related to the sympatho-inhibitory and to the positive inotropic effects of digoxin: improving hemodynamic and blood gas parameters may result in the inactivation of the reflex.

Na+/K+-ATPase during diabetes may be regulated by synthesis of its alpha and beta subunits and by changes in membrane fluidity and lipid composition. As these mechanisms were unknown in liver, we studied in rats the effect of streptozotocin-induced diabetes on liver Na+/K+-ATPase. We then evaluated whether fish oil treatment prevented the diabetes-induced changes. Diabetes mellitus induced an increased Na+/K+-ATPase activity and an enhanced expression of the beta1 subunit; there was no change in the amount of the alpha1 and beta3 isoenzymes. Biphasic ouabain inhibition curves were obtained for diabetic groups indicating the presence of low and high affinity sites. No alpha2 and alpha3 isoenzymes could be detected. Diabetes mellitus led to a decrease in membrane fluidity and a change in membrane lipid composition. The diabetes-induced changes are not prevented by fish oil treatment. The results suggest that the increase of Na+/K+-ATPase activity can be associated with the enhanced expression of the beta1 subunit in the diabetic state, but cannot be attributed to changes in membrane fluidity as typically this enzyme will increase in response to an enhancement of membrane fluidity. The presence of a high-affinity site for ouabain (IC50 = 10-7 M) could be explained by the presence of (alphabeta)2 diprotomeric structure of Na+/K+-ATPase or an as yet unknown alpha subunit isoform that may exist in diabetes mellitus. These stimulations might be related, in part, to the modification of fatty acid content during diabetes.

Diabetic neuropathy is a degenerative complication of diabetes accompanied by an alteration of nerve conduction velocity and Na,K-ATPase activity. This decrease in Na,KATPase activity has been implicated in the pathogenesis of electrophysiological abnormalities characterizing neuropathic complication in different animal models and in humans (1). Na,K-ATPase is a membrane-bound enzyme consisting of three catalytic isoenzymes (~1, ct2, ~3) and a large lipid core (2). It plays a fundamental rote in Na + and K + transport by maintaining the ionic gradients necessary for nervous excitability. A decrease in this process leads to an increase in the intracellular concentration of sodium and a corresponding decrease in membrane sodium potential. The mechanism responsible for impairment of Na,K-ATPase during hyperglycemia is still uncleal, but alterations of the kinetic parameters of the enzyme and/or of subunit expression are likely (3,4). In recent years there has been great interest in the effect of n-3 essential fatty acids, found particularly in fish oil, on the prevention of atherosclerosis and hypertension in animal models and patients with vascular disease (5). Fish oil treatment as MaxEPA was recently reported to improve vascular function in diabetic patients (6), and prevent c~2 isoenzyme activity of Na, K-ATPase in diabetic cardiomyopathy (7). Aim o f the study. The present study in rats was designed, first, to measure diabetes-induced abnormalities in Na,KATPase acyivity, isoenzyme expression, fatty acid content in sciatic membranes, and nerve conduction velocity and second, to assess the preventive ability of a fish-oil rich diet (rich in n-3 fatty acids) on these abnormalities. Methods. Diabetes was induced by intravenous streptozotocin injection. Diabetic animals (D) and nondiabetic control animals (C) were fed the standard rat chow either without supplementation or supplemented with either fish oil (DM, CM) or olive oil (DO, CO) at a daily dose of 0.5 g/kg by gavage during 8 wk.

LND-623 is a new aminosteroid analog of ouabain, with a greater separation between efficacy and toxicity than ouabain. To determine its mechanism of action, we studied its biochemical and physiological effects on human red blood cell sodium transports on different cellular structures regarded as sites of contractile control, and we compared its relative efficacy to ouabain in rat heart preparations and membrane-bound Na, K-ATPase isoenzymes. The response to ouabain was evaluated in Langendorff-perfused hearts and on purified membrane-bound Na, K-ATPase. LND-623 is 6.8-fold more efficient than ouabain in inhibiting the human Na+ pump (IC50 = 0.098 +/- 0.001 microM vs. 0.67 +/- 0.02 microM); (P0.0001). LND-623 had no effect on the following cellular functions: Na-Ca exchange, Na-K cotransport, Ca-ATPase, slow calcium channels, adenylate cyclase system, phosphodiesterase, and calcium sensitivity of the contractile protein system. The dose-response curve for the positive inotropic and inhibitory effects on rat cardiac isoenzymes produced by LND-623 were clearly biphasic. The amplitude of the maximum inotropic effect, without any toxic effect, was up to three-fold higher with LND-623 than with the same maximum dose of ouabain used. The strong positive inotropic effect of LND-623 in rats could be related to a specific inhibition of the two rat cardiac isoforms of the Na, K-ATPase.

Three isoenzymes of digitalis receptors (alpha 1, alpha 2, alpha 3) in the brain and only one in the kidney (alpha 1) can be distinguished by their ouabain affinities and their responsiveness to sodium. Since we have reported modulations for these digitalis receptors by their fatty acid membrane environment, anaesthesics could bind on and modulate either directly these receptors or indirectly by disturbing membrane lipids. The aim of this study was to evaluate this anaesthetic action on apparent ouabain affinities and sodium dependence of cerebral and renal Na+, K(+)-ATPase isoenzymes activities. Rat brain and kidney membrane fractions with pentobarbital-induced anaesthetized state were compared to an unanaesthetized state for their (1) fatty acid composition of total membrane phospholipids, (2) responsiveness to ouabain and (3) Na+ dependence of digitalis receptors. An anaesthesia period of 10 minutes induced (1) a fatty acid modification of brain membranes and (2) a significant sensibilization to ouabain for the alpha 2 and alpha 3 isoforms of digitalis receptors (alpha 2, IC50; 8.2 +/- 0.5 x 10(-7) mol/l vs 4.5 +/- 0.2 x 10(-7) mol/l; alpha 3, IC50; 6.0 +/- 0.3 x 10(-8) mol/l vs 2.5 +/- 0.1 x 10(-8). In contrast, the ouabain affinity of the alpha 1 subunit expressed in kidney and brain membranes was unaltered. No anaesthetic effect was observed on the Na+ dependence of the alpha 1 isoenzyme in the brain (4 mmol/l) and the kidney (8 mmol/l). Pentobarbital induced a desensibilization for alpha 2-receptors (8.3 +/- 0.5 vs 16.0 +/- 1.4 mmol/l Na+) and a sensibilization for alpha 3-receptors (14.4 +/- 0.8 vs 10 +/- 1.3 mmol/l Na+). These altered properties could be related to a selective modification of the fatty acid composition and/or to the presence of a specific binding site for pentobarbital on these two neuronal digitalis receptors.

Na+/K+-ATPase consists of an active complex of two noncovalently linked subunits (a catalytic α and a glycosilated β subunit) surrounded by a lipid bulk of 50% of the mass molecular (5). In vertebrate (rat, human, chicken), it has been clearly shown that they are three independent genes for the α subunit, known as α1, α2 and α3. If their best characterized difference, ouabain affinity, is not informative of their physiological relevance, their first real physiological significance is their functional difference in response to Na+ (4, 7, 11–13). These specific characteristics can be related to their divergence in aminoacids. However, the specificity of the surrounding environment and its functional implication is not yet understood. This article summarize ouabain and Na+ sensitivities of the brain and kidney isoenzymes under different experimental conditions modulating their membrane environment (anaesthesia and fatty acid diets).

Three alpha (α1, α2, α3) and three beta (s1, s2, s3) subunits of Na+/K+-ATPase have been identified from cDNA cloning (9). The expression of only α1 isoform has been shown in the kidney in all the mammalian species considered. In the brain, two other isoforms (α2 and α3) are concomitantly expressed (as reviewed in 15). However, each of them can be expressed separately in other tissues such as: α2 in the cardiac and skeletal muscles and α3 in the pineal gland (14), and macrophages (16). In dog heart, we have previously reported the existence of α1 and α+ isoforms. Since it was immunologically demonstrated that the α+ form could contain a mixture of α2 and; α3 isoforms (15), it was our objective to further investigate in dog heart and brain the nature and function of these two isoforms. We have used the panel of antibodies specific for the three rat α subunit isoforms (13) in Western blot analysis of canine microsomal fractions from kidney, brain and heart. In fliese 3 tissues, functional differences were investigated in terms of sensitivity to digitoxigenin (DGN) and Na+since differences in Na+ affinity have also been associated with isoform properties (4–7, 14).

The oligomeric structure of Na+/K+-ATPase assumed two polypetides: α catalytic (ouabain receptor) and s glycoprotein subunits. Each of these subunits are encoded from a multigene family relevant of three α (α1, α2, α3) and two β (β1, β2) subunit isoforms (7). In adult rat brain, these three different α entities described as express by Northern and Western analysis (7) were considered functional since three inhibitory processes were decomposed from ouabain dose-response curves (3). In the heart, until recently, two classes of binding and inhibitory sites have been demonstrated in human (5), dog (8,9) and guinea pig (4) and well correlated with two α entities (4, 9) except for human. Indeed, the PCR analysis of gene transcription and Northern blot analysis demonstrated that adult human ventricles express mRNA for all three α1, α2 and α3 isoenzymes (1,2,11,12). However these informations need to be correlated to protein properties like Western blot analysis and functional detections. Thus, we have investigated the expression of Na+,K+-ATPase α2, α3 and β2 subunits by immunobloting as well as the degree of heterogeneity of Na+,K+-ATPase activity in term of ouabain sensitivity. These characteristics were compared to those we previously described in the dog and guinea pig. Moreover, the understanding of digitalis drugs-induced inotropic action in heart failure requires the study of properties of human heart Na+/K+-ATPase from different states of congestive heart failure (CHF). Thus, displacement of [3H] ouabain (13) has been used to compare the density and affinity for ouabain affinity binding sites in membrane preparations from different pathophysiological states of CHF.

Cordil-LND796 is a new cardiotonic glycoside under development. In rat brain microsomes where three isoforms of the Na,K-ATPase with differential affinities for cardiac glycosides have been identified, Cordil had higher affinity for the alpha 3 (IC50 = 0.02 microM) than for the alpha 2 (IC50 = 0.6 microM) and the alpha 1 (IC50 = 30 microM) isozymes. Cordil is potentially a selective inhibitor for both alpha 2 and alpha 3 Na,K-ATPase isoforms. Using inside out vesicles we have shown that Cordil binds to and inhibits Na,K-ATPase at an extracellular site. The dissociation kinetic rates (k-1) from the ATPase and the phosphatase activity (K-dependent dephosphorylation) of the Na,K-ATPase were similar for Cordil. Despite these similarities to ouabain comparison of the kinetics of the Na,K-ATPase inhibition by ouabain and Cordil revealed marked differences in their association rates (k+1 = 0.7 l mol-1 min-1 and k+1 = 6 x 10(-3) l mol-1 min-1 respectively) and their dissociation rates (k-1 = 1.3 +/- 0.2 x 10(-4) s-1 and k-1 = 69 +/- 7 x 10(-4) s-1 respectively). Both binding association and dissociation rates were enhanced for Cordil. These data are compatible with a stabilizing effect of Cordil on the E2P conformational state of Na,K-ATPase.

20R 14 beta-amino 3 beta-rhamnosyl 5 beta-pregnan 20 beta-ol (LND 623, CAS 90520-42-6) was investigated and compared to digoxin in anesthetized dogs. The hemodynamic profiles showed: a) a pure positive inotropic action of LND-623; b) its potency was four-fold higher than that of digoxin and more marked in heart failure; c) its duration of action was maintained for at least 6 h. The onset and reversal of the inotropic effects of a single dose (3.3 nmol.kg-1.min-1) were faster with LND-623 than those of digoxin. This reversal is consistent with the faster dissociation profile observed at the level of the high affinity cardiac Na+,K(+)-ATPase receptor form. The advantage of LND-623 over digoxin resides in its larger therapeutic index (ratio of arrhythmogenic to inotropic responses) in anesthetized dogs with propranolol-induced heart failure. This index was 6 for LND-623 and 2 for digoxin.

Digitalis is clinically effective in heart failure, but it has an extremely narrow therapeutic index. Inclusion of fish oil (FO) in the diet may have cardioprotective effects by different mechanisms (1,2): decreased heart rate and occurrence of arrhythmias, increased mechanical activity, and ejection fraction. The aim of this study was to investigate if dietary FO with incorporation of n-3 polyunsaturated fatty acids in membranes could promote positive inotropy of ouabain. Methods. Twenty-five male Wistar rats were fed for 60 d: (i) supplemented with FO concentrate [180 mg eicosapentaenoic acid (EPA) + 120 mg docosahexaenoic acid (DHA)/g oil] at a dose of 0.5 g/kg/d (group FO), (ii) without supplementation (group C). Inotropic and toxic responses to cumulative doses of ouabain (from 100 nM to 0.3 raM) were evaluated in the isolated perfused rat heart and the relative variations in cardiac energetic metabolism, and pH were obtained using 3tp magnetic resonance spectroscopy. Activity, contribution, affinity, and expression of Na,K-ATPase isoforms were determined on purified membrane-bound Na,K-ATPase. Fatty acid composition of cardiac membrane fractions was analyzed by gas chromatography. Results. The rat is the only mammalian species with a well-characterized biphasic inotropic response to ouabain which is coupled to consecutive inhibition of the cx2- and o~[isoforms of high and low affinity for ouabain, respectively. Results show that (i) there is a twofold increase in the lowaffinity response to ouabain (0.1 mM) (% inotropy = 40 _+ 3 in group C vs. 79 -+ 7 in group FO, P < 0.001), (ii) there is a lower inhibition of ctl Na,K-ATPase activity (ICs0 = 7.2 _+ 2.0 × 10 -5 M in group C and 3.4 _+ 0.8 × 10 -4 M in group FO, P < 0.05), (iii) the higher inotropic response to ouabain is not associated with significant changes in energetics, (iv) alteration of function and energetics in response to the toxic dose of 0.3 mM is delayed in the FO group, (v) there are no differences in overall Na,K-ATPase activity or in the proportion of its isoforms, and (vi) fatty acid composition of rat hearts submitted to FO diet shows that the proportion of EPA and DHA had significantly risen. In conclusion, improving ouabain efficacy may represent an additional mechanism whereby FO exert their cardioprotective action in rats and possibly in humans. Acknowledgment. This work was supported in part by grants of the French Ministry of Health (PHRC, UF t638 and UF 1673), by MRT (grant 650994), CNRS (UMR 6612 and URA 1829), and the Assistance Publique-H6pitaux de Marseille.

It is generally accepted that the primary target of the cardiac glycosides is the sarcolemma-bound Na/K-ATPase activity. The inhibition of this function by ouabain occurs in the same range of concentrations as pharmacologically active doses in various species including rat. In this species, it has been shown that the sensitivity of Na/K-ATPase to ouabain was heterogeneous with high and low sensitivity forms (KD 10−7, 10−8 M and 10−6, 10−5 M, respectively). According to experimental and clinical reviews the cardiac glycosides were inotropic in both normal and failing hearts. However, none of these studies were comparative.

Canine cardiac myocytes contain two distinct molecular forms of the Na,K-ATPase catalytic subunit. They are resolved by gel electrophoresis and identified using immunological techniques. The apparent molecular weights of the catalytic subunits are 95,000 (alpha) and 98,000 (alpha +). As judged by [3H]ouabain-binding measurements and Na,K-ATPase assays, the two forms are active and differ by a factor of 150 in their respective affinity for digitalis (ouabain and digitoxigenin). The dissociation constant of the high affinity form (alpha +) is KD, 2 nM, and that of the low affinity molecular form (alpha) is KD, 300 nM. According to both enzymatic and binding assays, up to 70% of maximum inhibition is caused by occupation of the high affinity sites (alpha +). Inasmuch as the pharmacological and toxic concentrations of digitalis in dog are 1 and 200 nM, respectively, and as maximum inhibition of Na+ pump in vivo should not exceed 80% to avoid toxicity (Akera, T. and Brody, T. (1982) Annu. Rev. Physiol. 44, 375-388), it appears that the high affinity molecular form (alpha +) is the pharmacological receptor exclusively related to positive inotropy, whereas the low affinity form (alpha) is mainly associated with toxicity.

Recently, it has been demonstrated that more than one receptor type would be responsible for the isotropic effect of ouabain. This effect is linked to an increased availability of Cain. In rat heart two classes of ouabain binding sites have been described: a high affinity (H. A.) class with a low capacitiy, and a low affinity (L. A.) class with a high capacity. Although both sites are involved in the inotropic response, these H. A. sites may not be related to inhibition of Na/K-ATPase activity. However, we have reported that a Ca-free perfusion of rat heart revealed a Na/K-ATPase activity highly sensitive to ouabain by modifying the sarcolemmal calcium pool.

The adaptation of the myocardium to mechanical overload which results in cardiac hypertrophy involves several membrane functions. The digitalis receptor in sarcolemma vesicles from hypertrophied rat hearts is characterized by binding of [3H]ouabain and ouabain-induced inhibition of (Na+,K+)-ATPase. The results show the existence of two families of ouabain binding sites with apparent dissociation constants (Kd) of 1.8-3.2 X 10(-8) M and 1-8 X 10(-6) M, respectively, which are similar to those found in normal hearts. The presence of the high affinity receptor in hypertrophied rat heart is correlated to a detectable inhibition of the (Na+,K+)-ATPase (IC50 = 1-3 X 10(-8) M). However, the high and low affinity sites in hypertrophied hearts bind and release ouabain at 4-5-fold slower rates than the corresponding sites in normal hearts. These properties are similar to that we observed in newborn rat cardiac preparations. Taken together with the expression of myosin isoforms (Schwartz, K., Lompre, A.M., Bouveret, P., Wisnewsky, C., and Whalen, R.G. (1982) J. Biol. Chem. 23, 14412-14418), our data show that the physiological adaptation of the heart also involves the resurgence of the neonatal forms of the digitalis receptor.