[Involvement of the Piezo1 and TRPV4 stretch-activated channels in pulmonary hypertension]. / Implication des canaux mécanosensibles Piezo1 et TRPV4 dans l'hypertension pulmonaire.

Pulmonary hypertension is a pulmonary circulation pathology characterized by remodelling and hyperreactivity of the pulmonary arteries. Vasodilatation/vasoconstriction balance is modified in favour of constriction via, among other things, the proliferation of smooth muscle cells and the development of endothelial dysfunction. In addition, the pulmonary arteries undergo modification of mechanical forces, inducing modified activation of stretch-activated channels (SAC) such as Piezo1 and TRPV4. These ionic channels are sensitive to stretch and their activation can induce various cellular physiological responses, which strongly contribute to development and continuation of the pathology.

Stress and recovery in sports: Effects on heart rate variability, cortisol, and subjective experience.

This study aimed to investigate (1) the time-variations and (2) the repeated measures relationship between training load (TL) and psychological and physiological parameters and performance. Data were collected around 12-weeks of training in fifteen national swimmers. Psychological states were assessed using the RESTQ-36-R-Sport Questionnaire and the Sport Emotion Questionnaire. Subjects collected four saliva samples throughout the day at 1) 7 a.m. immediately after waking, 2) 30 min after waking, 3) 60 min after waking, and 4) 8 p.m., allowing us to calculate the area under the curve with respect to ground (AUCg) and the sAA over cortisol ratio (AOC). Finally, heart rate variability was computed using a submaximal 5'-5' running test. Time variations were analyzed throughout repeated measures ANOVA and repeated measures correlations were run using the "rmcorr" R package. Recovery-stress states and emotional markers showed quadratic curves, while parasympathetic markers showed linear trajectories over time. Significant associations over time were found between TL and recovery, stress, emotional states, lnRMSSD and the AOC. Taken together, these results provided evidence that psychological and physiological states do not follow the same dynamics (i.e., linear vs. quadratic vs. no variation) in the functional training periodization condition. Our study also provided evidence that recovery-stress states, emotional states, lnRMSSD, and the AOC were of interest due to their intra-individual associations around the time with TL.

[Interest of transcranial stimulation in pelvic and perineal disorders]. / Intérêt de la stimulation transcrânienne dans les troubles pelvi-périnéaux.

OBJECTIVE: The aim of this article was to describe the diagnostic and therapeutic value of transcranial stimulation in pelvic and perineal disorders. METHODS: A literature review (Medline database and Google scholar) with no time limit was performed using keywords: "transcranial direct stimulation", "transcranial magnetic stimulation", "neurogenic bladder", "urinary incontinence", "Parkinson disease", "multiple sclerosis", "stroke", "muscle spasticity", "pelvic pain", "visceral pain". RESULTS: Twelve articles have been selected. Transcranial magnetic or electrical stimulation is a noninvasive neuromodulation technique widely used to establish brain maps to highlight causal relationships between brain and function. Regarding pelvic-perineal disorders, repeated transcranial stimulation has shown significant effects for the treatment of overactive bladder in Parkinson's disease (P<0.05) and multiple sclerosis, but also for the treatment of refractory chronic pelvic pain (P=0.026). Finally, therapeutic effects have also been demonstrated in irritable bowel syndrome. No evidence of efficacy was found on genito-sexual disorders. CONCLUSION: Data from the literature suggest that transcranial stimulation is a noninvasive treatment that may have a role in the management of pelvic and perineal disorders. Its promising field of action would require prospective and randomized studies on a larger scale.

Elite swimmers' internal markers trajectories in ecological training conditions.

This study examined the trajectories of elite swimmers' recovery-stress states and cardiac vagal-related markers during a 3-month training period preceding the national championship and their within-person relationships with perceived control. A Multilevel Growth Curve Analysis (MGCA) approach was used with 21 male elite swimmers. Four waves of assessments of psychological (stress, recovery, perceived control) and physiological (heart rate recovery, heart rate variability) markers were completed during a 3month training preparation leading to a major competition. Results of MGCA revealed (a) a significant positive linear effect of time (ie, linear increase over time) and a significant negative quadratic effect of time (ie, inverted U shape over time) on perceived stress whereas the opposite pattern of results was observed for perceived recovery; and (b) a significant positive linear effect of time for nHRR60. Both at level 1 (within-person level of analysis) and 2 (between-person level of analysis), perceived control was (a) positively associated with athletes' perceived recovery and parasympathetic markers (ie, MeanRR; pNN50); and (b) negatively related to swimmers' perceived stress. Results also indicated that within-person interactions of perceived control with time reached significance for general recovery and HRV. Finally, within-person interaction of perceived control with squared time reached significance for subjective sport-specific and total stress. Overall, this study provided insights into the key role played by perceived control on both psychological and physiological markers related to recovery-stress states' levels during the 3-month training period preceding the national championship.

The apoptotic members CD95, BclxL, and Bcl-2 cooperate to promote cell migration by inducing Ca(2+) flux from the endoplasmic reticulum to mitochondria.

Metalloprotease-processed CD95L (cl-CD95L) is a soluble cytokine that implements a PI3K/Ca(2+) signaling pathway in triple-negative breast cancer (TNBC) cells. Accordingly, high levels of cl-CD95L in TNBC women correlate with poor prognosis, and administration of this ligand in an orthotopic xenograft mouse model accelerates the metastatic dissemination of TNBC cells. The molecular mechanism underlying CD95-mediated cell migration remains unknown. Here, we present genetic and pharmacologic evidence that the anti-apoptotic molecules BclxL and Bcl-2 and the pro-apoptotic factors BAD and BID cooperate to promote migration of TNBC cells stimulated with cl-CD95L. BclxL was distributed in both endoplasmic reticulum (ER) and mitochondrion membranes. The mitochondrion-localized isoform promoted cell migration by interacting with voltage-dependent anion channel 1 to orchestrate Ca(2+) transfer from the ER to mitochondria in a BH3-dependent manner. Mitochondrial Ca(2+) uniporter contributed to this flux, which favored ATP production and cell migration. In conclusion, this study reveals a novel molecular mechanism controlled by BclxL to promote cancer cell migration and supports the use of BH3 mimetics as therapeutic options not only to kill tumor cells but also to prevent metastatic dissemination in TNBCs.

Adenylyl cyclase 8 is central to glucagon-like peptide 1 signalling and effects of chronically elevated glucose in rat and human pancreatic beta cells.

AIMS/HYPOTHESIS: Glucose and incretins regulate beta cell function, gene expression and insulin exocytosis via calcium and cAMP. Prolonged exposure to elevated glucose (also termed glucotoxicity) disturbs calcium homeostasis, but little is known about cAMP signalling. We therefore investigated long-term effects of glucose on this pathway with special regard to the incretin glucagon-like peptide 1 (GLP-1). METHODS: We exposed INS-1E cells and rat or human islets to different levels of glucose for 3 days and determined functional responses in terms of second messengers (cAMP, Ca(2+)), transcription profiles, activation of cAMP-responsive element (CRE) and secretion by measuring membrane capacitance. Moreover, we modulated directly the abundance of a calcium-sensitive adenylyl cyclase (ADCY8) and GLP-1 receptor (GLP1R). RESULTS: GLP-1- or forskolin-mediated increases in cytosolic calcium, cAMP-levels or insulin secretion were largely reduced in INS-1E cells cultured at elevated glucose (>5.5 mmol/l). Statistical analysis of transcription profiles identified cAMP pathways as major targets regulated by glucose. Quantitative PCR confirmed these findings and unravelled marked downregulation of the calcium-sensitive adenylyl cyclase ADCY8 also in rat and in human islets. Re-expression of ADCY8, but not of the GLP1R, recovered GLP-1 signalling in glucotoxicity in INS-1E cells and in rat islets. Moreover, knockdown of this adenylyl cyclase showed that GLP-1-induced cAMP generation, calcium signalling, activation of the downstream target CRE and direct amplification of exocytosis by cAMP-raising agents (evaluated by capacitance measurement) proceeds via ADCY8. CONCLUSIONS/INTERPRETATION: cAMP-mediated pathways are modelled by glucose, and downregulation of the calcium-sensitive ADCY8 plays a central role herein, including signalling via the GLP1R.

Centropontine myelinolysis after correction of hyponatremia: role of associated hypokalemia.

BACKGROUND: Patients with severe hyponatremia have a high risk for centropontine myelinolysis (CPM) during treatment, but the incidence rate and risk factors have not been well-assessed. METHODS: This study was conducted in a medical intensive care unit (ICU) of a university teaching hospital. All patients with a serum sodium concentration < 120 mmol/l and a serum osmolality level < 250 mosmol/kg upon ICU admission were enrolled in this prospective study and were included if they underwent a baseline brain computerized tomography scan (CT scan) and a follow-up brain magnetic resonance imaging 1 month after admission. The diagnosis of CPM was based on cerebral magnetic resonance imaging findings, i.e. T1-weighted images with T2-weighted images showing hyperintense signal in the corresponding areas which were not apparent on the initial cerebral CT scan. RESULTS. Of the 22 patients included, 12 were considered as having acute hyponatremia and 8 were chronic alcoholics. In 12 patients, the increase in serum sodium level was < 12 mmol/I in any 24-hour period. CPM was diagnosed in 7/22 patients (31.8%) and was asymptomatic in 4 of them. CPM was present in 4 patients with acute hyponatremia and in 4 chronic alcoholics. It was associated with a lower baseline potassium level (p = 0.05) and NaCl administration during the first 24 hours (p = 0.005). However, non-acute hyponatremia, chronic alcoholism and rapid correction of serum sodium did not appear as risk factors. CONCLUSION: The incidence rate of CPM following severe hyponatremia is high and can develop even when there is a slow correction of serum sodium level. Hypokalemia is a predisposing factor.

[Prospective assessment of information programme for blood transfusion in critical care patients]. / Evaluation prospective d'une procédure d'information des patients transfusés en réanimation.

OBJECTIVES: Assessment of oral and written information programme for blood transfusion in critical care patients and study of factors associated with the biological follow-up. STUDY DESIGN: Prospective study in one intensive care unit. PATIENTS AND METHODS: All blood recipients in 2000 were orally informed of transfusion process. In regard to the French haemovigilance regulation, written paper was given to the patient or passed on to the next hospital physician. Four months later, a questionnaire was proposed to the patients. RESULTS: In 2000, blood transfusion was performed for 102 patients and data were obtained for 59 patients (34 number of survivor patients, follow-up was impossible for eight and one refused the questionnaire). Quality assurance programme permit a global orally and written information of critical care patients. However, thirty-eight patients (64%) remembered oral information. Patients were younger (55 +/- 22 vs 68 +/- 13, OR = 1.04; p = 0.03). Biological follow-up was obtained for 22 patients (37%), particularly when written information was directly given to the patient (22/24 vs 2/35; OR = 335; p < 0.0001). CONCLUSION: Biological follow-up was obtained for only one third of blood recipients, because, in first, only 64% of critical care patients remembered oral information, particularly the younger patients. Perhaps, written information systematically given to the patient could permit a high biological follow-up. This study confirmed the lack of sensitivity of all hospital physician for haemovigilance.

Optical analysis of displacement and strain fields on human skin.

BACKGROUND/AIMS: The aim of this article is to propose a new way to measure the mechanical behaviour of skin by using optical analysis software. Some examples on scars and stretch marks show the validity and the prospects of such a method. METHODS: Software is used to compare two states of deformation. The user takes two photos of the skin surface he wants to study, before and after the deformation is applied. The software is used to process the data. RESULTS: The software gives a mapping of the displacement, and strain fields of the area studied. Investigations can be realised in vitro or in vivo, and it is possible to study, for example, cutaneous lesions like scars and stretch marks. CONCLUSIONS: The method presented gives convincing results, which open many prospects in dermatology, surgery and cosmetology.

Intrathecal baclofen after traumatic brain injury: early treatment using a new technique to prevent spasticity.

BACKGROUND: In the early course of severe head trauma, the clinical value of intrathecal administration of baclofen to reduce autonomic disorders and spasticity has not been established. METHODS: We studied four patients (Glasgow Coma Scale score 3 or 4) with autonomic disorders and spasticity who failed to respond to conventional treatment during the early course of head injury. Baclofen (25 microg/mL) was infused continuously through an intrathecal catheter inserted at patient bedside and subcutaneously tunneled. When this treatment was successful, the spinal catheter was removed and surgically replaced by another catheter connected to a subcutaneous pump. Clinical follow-up was obtained at 6 months after the head injury. RESULTS: Mean delay for the initiation of intrathecal baclofen was 25 days (range, 21 to 31 days), and optimal dose was 385 +/- 185 microg/day. In all patients, the Ashworth score was consistently reduced (3.5 +/- 0.5 vs. 4.5 +/- 0.5 for upper limbs and 2 +/- 0.5 vs. 4.5 +/- 0.5 for lower limbs), as were both the frequency and intensity of autonomic disorders. The spinal catheters were used during a mean period of 9.5 +/- 1.7 days without complications. All three survivors were equipped with a programmable pump and had a lower Ashworth score at 6 months. Autonomic disorders had disappeared in two patients and remained modest in the remaining patient. CONCLUSION: Continuous administration of baclofen via the intrathecal route using this new technique seems to reduce autonomic disorders and spasticity during the early course of severe traumatic head injury.

Imaging of Ca(2)+ intracellular dynamics with a third-harmonic generation microscope.

We describe the promising development of third-harmonic generation (THG) in laser scanning microscopy for study of the functional imaging of live biological cells. The dynamics of Ca(2+) in biological cells is shown. The Ca(2+) signal consists of a transient increase in the intracellular concentration. THG microscopy allows one to temporally visualize the release of Ca(2+) from internal stores and (or) calcium influx.

Role of protein kinases in the prolactin-induced intracellular calcium rise in Chinese hamster ovary cells expressing the prolactin receptor.

There is still only limited understanding of the early steps of prolactin signal transduction in target cells. It has been shown that prolactin actions are associated with cell protein phosphorylation, Ca2+ increases, and so on. However, the link between the activation of kinases and calcium influx or intracellular Ca2+ mobilization has not yet been clearly established. Chinese hamster ovary (CHO) cells, stably transfected with the long form of rabbit mammary gland prolactin receptor (PRL-R) cDNA were used for PRL-R signal transduction studies. Spectrofluorimetric techniques were used to measure intracellular calcium ([Ca2+]i) in cell populations with Indo1 as a calcium fluorescent probe. We demonstrate that, although protein kinase C activation (PMA or DiC8) caused a calcium influx in CHO cells, prolactin-induced PKC activation was not responsible for the early effect of prolactin on [Ca2+]i. Activation of protein kinase A (PKA) or protein kinase G did not modify [Ca2+]i and inhibition of PKA pathway did not affect the prolactin response. In the same way, phosphatidylinositol-3 kinaseinhibition had no effect on the prolactin-induced Ca2+ increase. On the other hand, tyrosine kinase inhibitors (herbimycin A, lavendustin A, and genistein) completely blocked the effect of prolactin on [Ca2+]i (influx and release). W7, a calmodulin-antagonist, and a specific inhibitor of calmodulin kinases (KN-62), only blocked prolactin-induced Ca2+ influx but had no significant effect on Ca2+ release. Using pharmacological agents, we present new data concerning the involvement of protein phosphorylations in the early effects of prolactin on ionic channels in CHO cells expressing the long form of PRL-R. Our results suggest that, at least in the very early steps of prolactin signal transduction, serine-threonine phosphorylation does not participate in the prolactin-induced calcium increase. On the other hand, tyrosine phosphorylation is a crucial, very early step, since it controls K+ channel activation, calcium influx, and intracellular calcium mobilization. Calmodulin acts later, since its inhibition only blocks the prolactin-induced Ca2+ influx.

Twenty-four-hour patterns of hormones and metabolites in week 9 and 19 of lactation in high-yielding dairy cows fed triglycerides and free fatty acids.

Effects of feeding crystalline triglycerides (TGA), free fatty acids (FFA), and a starch-rich ration (STA) on metabolite and hormone concentrations in blood plasma were studied in high-yielding dairy cows over a 24-h period in week 9 and 19 of lactating. Energy-corrected milk production in the three groups was similar, but was lower in week 19 than in week 9. Energy and protein intakes were greater in week 9 than in week 19, but energy and protein balances in the three groups and in weeks 9 and 19 were similar. Plasma glucose and triglyceride concentrations were lower in week 9 than in week 19. In cows fed FFA, glucose concentrations were highest in week 9. Plasma triglyceride, phospholipid and cholesterol concentrations were highest, whereas beta-hydroxybutyrate concentrations were lowest in FFA-fed cows in weeks 9 and 19. Concentrations of insulin-like growth factor-I in week 19 were lower in cows fed TGA and FFA than in those fed the starch-rich ration. Post-prandial responses were usually greater following morning than afternoon meals. Fructosamine, albumin, urea, growth hormone, thyroxine, and 3,5,3'-triiodothyronine concentrations were similar in weeks 9 and 19 and were not influenced by dietary treatment or feeding times. In conclusion, there were distinct metabolic and endocrine effects of feeding TGA and FFA compared with STA and the concentrations as well as the 24-h changes of various metabolic and endocrine traits in weeks 9 and 19 of lactation were also different.

Insulin-dependent whole-body glucose utilization and insulin-responses to glucose in week 9 and week 19 of lactation in dairy cows fed rumen-protected crystalline fat or free fatty acids.

Whole-body insulin-dependent glucose utilization and insulin responses to glucose in euglycemic-hyperinsulinemic clamps (EHGC) and in hyperglycemic clamps (HGC) were evaluated in high yielding dairy cows fed rumen-protected fat (triglycerides), free fatty acids, or a starch-rich ration (n = 5 per group) in Week 9 and Week 19 of lactation. The experiment was performed under conditions of nonsignificant differences in energy and protein balances in Week 9 and Week 19 and in the three groups. Basal (pre-infusion) concentrations of glucose were lower (P < 0.05) in Week 9 than in Week 19 of lactation and were higher (P < 0.05) in Week 9 in cows fed free fatty acids than in cows fed the starch-rich ration. In EHGC, glucose infusion rates were similar in Week 9 and Week 19 and in the different groups, indicating similar insulin-dependent glucose utilization. Furthermore, because insulin concentrations in EHGC in Week 9 and Week 19 and in the three groups were very similar, metabolic clearance rates of insulin were not affected by stage of lactation and feeding. In addition, insulin responses to the same glucose increments in HGC were not different in Week 9 and Week 19 and in the three groups, indicating that insulin secretion was not affected by stage of lactation and feeding. In conclusion, insulin secretion, insulin metabolic clearance rate, and insulin-dependent glucose utilization between Week 9 and Week 19 of lactation were stable. Furthermore, feeding rumen-protected triglycerides or free fatty acids did not significantly modify insulin secretion, insulin metabolic clearance rate and glucose-dependent glucose utilization compared with starch-rich feeding.

Distinct cytoplasmic regions of the prolactin receptor are required for prolactin-induced calcium entry.

Two cytoplasmic regions of the prolactin (PRL) receptor are well documented for their participation in PRL signal transduction, the membrane proximal box 1 and the COOH-terminal region. In order to study the role of these regions in PRL-induced Ca2+ increase, we use Chinese hamster ovary (CHO) cells stably transfected with mutated PRL receptor cDNA. These cells express the long form of PRL receptor deleted from box 1 (CHO Delta1 cells) or the 141 amino acids of the COOH-terminal region (CHO H3 cells). The patch-clamp technique in "whole-cell" configuration and microfluorimetric techniques were used singly or in combination. Data obtained for these cells were compared with those we have recently published using CHO cells expressing the wild-type long form of the PRL receptor (CHO TSE32). In contrast to CHO TSE32 cells, exposure of CHO Delta1 or H3 cells to PRL (0.05-50 nM) did not modify [Ca2+]i. We have previously shown that the PRL-induced calcium influx via voltage-insensitive, Ca2+ channels was due to the activation of tyrosine kinase-dependent K+ channels that hyperpolarize the CHO TSE32 cell membrane (hyperpolarization-driven Ca2+ influx). Therefore, two events are involved in PRL-induced Ca2+ changes (i) JAK2-activation of K+ channels and (ii) intracellular messenger-opening of Ca2+ channels. In CHO Delta1 cells, PRL (0.05-50 nM) neither hyperpolarized the membrane potential nor stimulated the JAK2-dependent K+ current, confirming the pivotal role played by box 1/JAK2 in the PRL-induced activation of K+ channels. However, when these cells were voltage-clamped below the resting membrane potential, application of 5 nM PRL resulted in an increase in Ca2+ influx. Therefore, box 1/JAK2 was not involved in the opening of these Ca2+ channels. In CHO H3 cells, 5 nM PRL activated the K+ current and hyperpolarized the membrane potential without any effect on [Ca2+]i. Moreover, PRL was also ineffective on CHO H3 cells voltage-clamped below the resting membrane potential. Therefore, the COOH-terminal region is involved in the production of the intracellular messenger that opens voltage-independent Ca2+ channels. We conclude from these findings that box 1 and COOH-terminal regions are both needed for PRL-induced Ca2+ changes.

Tubocurarine blocks a calcium-dependent potassium current in rat tumoral pituitary cells.

We investigated the effects of potassium channel inhibitors on electrical activity, membrane ionic currents, intracellular calcium concentration ([Ca2+]i) and hormone release in GH3/B6 cells (a line of pituitary origin). Patch-clamp recordings show a two-component after hyperpolarization (AHP) following each action potential (current clamp) or a two-component tail current (voltage-clamp). Both components can be blocked by inhibiting Ca2+ influx. Application of D-tubocurarine (dTc) (20-500 microM) reversibly suppressed the slowly decaying Ca2+-activated K+ tail current (I AHPs) in a concentration-dependent manner. On the other hand, low doses of tetraethylammonium ions (TEA+) only blocked the rapidly decaying voltage- and Ca2+-activated K+ tail current (I AHPf). Therefore, GH3/B6 cells exhibit at least two quite distinct Ca2+-dependent K+ currents, which differ in size, voltage- and Ca2+-sensitivity, kinetics and pharmacology. These two currents also play quite separate roles in shaping the action potential. d-tubocurarine increased spontaneous Ca2+ action potential firing, whereas TEA increased action potential duration. Thus, both agents stimulated Ca2+ entry. I AHPs is activated by a transient increase in [Ca2+]i such as a thyrotrophin releasing hormone-induced Ca2+ mobilization. All the K+ channel inhibitors we tested: TEA, apamin, dTC and charybdotoxin, stimulated prolactin and growth hormone release in GH3/B6 cells. Our results show that I AHPs is a good sensor for subplasmalemmal Ca2+ and that dTc is a good pharmacological tool for studying this current.

17 beta-estradiol stimulates a rapid Ca2+ influx in LNCaP human prostate cancer cells.

Prostate growth is known to be controlled by steroids such as androgens and estradiol. For this reason steroids (estradiol, adrenal androgens) or steroid inhibitors are commonly used as palliative treatments for prostate carcinoma. In view of the pivotal role played by Ca2+ ions in cell proliferation, we decided to investigate the effects of 17 beta-estradiol (E2) on intracellular calcium concentration ([Ca2+]i) in a human prostate tumor cell line. LNCaP. In this study, we show that E2 induced a dose-dependent (0.1-100 nmol/l) influx of Ca2+ in these cells. These effects occurred rap dly after the beginning of the ejection and were maintained in the presence of the hormone (plateau phase). Estradiol-induced Ca2+ influx was unaffected by the saturation of the androgen receptor with pure antiandrogen flutamide. The use of tamoxifen, an antiestrogen binding to nuclear receptors, or E2 covalently linked to bovine serum albumin that cannot penetrate the cell membrane, did not block the ([Ca2+]i) response. Our results suggest the existence of E2 binding sites at the plasma membrane surface of LNCaP cells, linked to calcium signalling and, more specifically, Ca2+ channels.

Dual Effect of Prolactin on Protein Kinase C Activity in CHO Cells Expressing Functional Prolactin Receptors.

We investigated the effects of prolactin (PRL) on the protein kinase C (PKC) activity in Chinese hamster ovary (CHO-E32) cells stably transfected with rabbit mammary gland PRL receptor cDNA. These cells express a functional long form of PRL-R. A 10-min to 2-hour treatment with 5 nM PRL resulted in the translocation of PKC activity from the cytosol to the membrane. Longer treatment (10-24 h) with the same concentration of PRL decreased the PKC activity in both particulate and cytoplasmic fractions. The PRL effect was dose dependent: maximal action was obtained with 1-10 nM. The PRL-induced activation of PKC was blocked by 20 nM staurosporine, a PKC inhibitor. Two inhibitors of tyrosine kinase, herbimycin A (1.75 &mgr;M) and genistein (100 &mgr;M), had no effect on PRL-induced activation of PKC. Copyright 1996 S. Karger AG, Basel