Hemodynamic effects of sodium bicarbonate in critically ill neonates.

OBJECTIVE: To analyze the cardiovascular effects of sodium bicarbonate in neonates with metabolic acidosis. DESIGN: Prospective, open, non-randomized, before-after intervention study with hemodynamic measurements performed before and 1, 5, 10, 20, and 30 min after bicarbonate administration. SETTING: Neonatal intensive care unit, tertiary care center. PATIENTS: Sequential sample of 16 paralysed and mechanically ventilated newborn infants with a metabolic acidosis (pH < 7.25 in premature and < 7.30 in term infants, base deficit > -8). INTERVENTION: An 8.4% sodium bicarbonate solution diluted 1:1 with water (final osmolality of 1000 mOsm/l) was administered in two equal portions at a rate of 0.5 mmol/min. The dose in mmol was calculated using the formula "base deficit x body weight (kg) x 1/3 x 1/2". MEASUREMENTS AND RESULTS: Sodium bicarbonate induced a significant but transient rise in pulsed Doppler cardiac output (CO) (+27.7%), aortic blood flow velocity (+15.3%), systolic blood pressure (BP) (+9.3%), (+14.6%), transcutaneous carbon dioxide pressure (PtcCO2) (+11.8%), and transcutaneous oxygen pressure (PtcO2) (+8%). In spite of the PaCO2 elevation, pH significantly improved (from a mean of 7.24 to 7.30), and the base deficit decreased (-39.3%). Calculated systemic vascular resistance (SVR) (-10.7%) and diastolic BP (-11.7%) decreased significantly, while PaO2 and heart rate (HR) did not change. Central venous pressure (CVP) (+6.5%) increased only slightly. By 30 min after bicarbonate administration all hemodynamic parameters, with the exception of the diastolic BP, had returned to baseline. CONCLUSION: Sodium bicarbonate in neonates with metabolic acidosis induces an increase in contractility and a reduction in afterload.

Regulation by aldosterone of Na+,K+-ATPase mRNAs, protein synthesis, and sodium transport in cultured kidney cells.

Transepithelial Na+ reabsorption across tight epithelia is regulated by aldosterone. Mineralocorticoids modulate the expression of a number of proteins. Na+,K+-ATPase has been identified as an aldosterone-induced protein (Geering, K., M. Girardet, C. Bron, J. P. Kraehenbuhl, and B. C. Rossier, 1982, J. Biol. Chem., 257:10338-10343). Using A6 cells (kidney of Xenopus laevis) grown on filters we demonstrated by Northern blot analysis that the induction of Na+,K+-ATPase was mainly mediated by a two- to fourfold accumulation of both alpha- and beta-subunit mRNAs. The specific competitor spironolactone decreased basal Na+ transport, Na+,K+-ATPase mRNA, and the relative rate of protein biosynthesis, and it blocked the response to aldosterone. Cycloheximide inhibited the aldosterone-dependent sodium transport but did not significantly affect the cytoplasmic accumulation of Na+,K+-ATPase mRNA induced by aldosterone.

Proximal sodium reabsorption: An independent determinant of blood pressure response to salt.

The purpose of this study was to evaluate the contribution of renal sodium handling by the proximal tubule as an independent determinant of blood pressure responsiveness to salt in hypertension. We measured blood pressure (BP), renal hemodynamics, and segmental renal sodium handling (with lithium used as a marker of proximal sodium reabsorption) in 38 hypertensive patients and 27 normotensive subjects (15 young and 12 age-matched) on a high and low sodium diet. In control subjects, changing the diet from a low to a high sodium content resulted in no change in BP and increases in glomerular filtration rate (P<0.05), renal plasma flow (P<0.05), and fractional excretion of lithium (FE(Li), P<0.01). In hypertensive patients, comparable variations of sodium intake induced an increase in BP with no change in renal hemodynamics and proximal sodium reabsorption. When analyzed by tertiles of their BP response to salt, salt-insensitive hypertensive patients of the first tertile disclosed a pattern of adaptation of proximal sodium reabsorption comparable to that of control subjects, whereas the most salt-sensitive patients of the third tertile had an inverse pattern with a high FE(Li) on low salt and a lower FE(Li) on high salt, suggesting an inappropriate modulation of proximal sodium reabsorption. The BP response to salt correlated positively with age (r=0.34, P=0.036) and negatively with the changes in FE(Li) (r=-0.37, P=0.029). In a multivariate analysis, the changes in FE(Li) were significantly and independently associated with the salt-induced changes in BP. These results suggest that proximal sodium reabsorption is an independent determinant of the BP response to salt in hypertension.

Comparison between a linear ion trap and a triple quadruple MS in the sensitive detection of large peptides at femtomole amounts on column.

In addition to the importance of sample preparation and extract separation, MS detection is a key factor in the sensitive quantification of large undigested peptides. In this article, a linear ion trap MS (LIT-MS) and a triple quadrupole MS (TQ-MS) have been compared in the detection of large peptides at subnanomolar concentrations. Natural brain natriuretic peptide, C-peptide, substance P and D-Junk-inhibitor peptide, a full D-amino acid therapeutic peptide, were chosen. They were detected by ESI and simultaneous MS(1) and MS(2) acquisitions. With direct peptide infusion, MS(2) spectra revealed that fragmentation was peptide dependent, milder on the LIT-MS and required high collision energies on the TQ-MS to obtain high-intensity product ions. Peptide adsorption on surfaces was overcome and peptide dilutions ranging from 0.1 to 25 nM were injected onto an ultra high-pressure LC system with a 1 mm id analytical column and coupled with the MS instruments. No difference was observed between the two instruments when recording in LC-MS(1) acquisitions. However, in LC-MS(2) acquisitions, a better sensitivity in the detection of large peptides was observed with the LIT-MS. Indeed, with the three longer peptides, the typical fragmentation in the TQ-MS resulted in a dramatic loss of sensitivity (> or = 10x).

The contact region between three domains of the extracellular loop of ASIC1a is critical for channel function.

Acid-sensing ion channels are members of the epithelial Na(+) channel/degenerin family. They are neuronal nonvoltage-gated Na(+) channels that are activated by extracellular acidification. In this study, we investigated the role of a highly conserved region of the extracellular part of ASIC1a that forms the contact between the finger domain, the adjacent beta-ball, and the upper palm domain in ASIC1a. The finger domain contributes to the pH-dependent gating and is linked via this contact zone to the rest of the protein. We found that mutation to Cys of residues in this region led to decreased channel expression and current amplitudes. Exposure of the engineered Cys residues to Cd(2+) or to charged methane thiosulfonate sulfhydryl reagents further reduced current amplitudes. This current inhibition was not due to changes in acid-sensing ion channel pH dependence or unitary conductance and was likely due to a decrease of the probability of channel opening. For some mutants, the effect of sulfhydryl reagents depended on the pH of exposure in the range 7.4 to 6.8, suggesting that this zone undergoes conformational changes during inactivation. Our study identifies a region in ASIC1a whose integrity is required for normal channel function.

Role of the epithelial sodium channel (ENaC) and its regulator CAP1/Prss8 in colon

Dans les cellules épithéliales sensibles à l'aldostérone, le canal sodique épithélial (ENaC) joue un rôle critique dans le contrôle de l'équilibre sodique, le volume sanguin, et la pression sanguine. Le rôle d'ENaC est bien caractérisé dans le rein et les poumons, cependant le rôle d'ENaC et son régulateur positif la protéase activatrice de canal 1 (CAP1 /Prss8) sur le transport sodique dans le côlon reste en grande partie inconnu. Nous avons étudié l'importance d'ENaC et de CAPMPrss8 dans le côlon. Les souris déficientes pour la sous- unité aENaC (souris ScnnlaKO) dans les cellules superficielles intestinales étaient viables et ne montraient pas de létalité embryonnaire ou postnatale. Sous diète normale (RS) ou pauvre en sodium (LS), la différence de potentiel rectale sensible à l'amiloride (APDamii) était drastiquement diminuée et son rythme circadien atténué. Sous diète normale (RS) ou diète riche en sodium (HS) ou fort chargement de potassium, le sodium et le potassium plasmatique et urinaire n'étaient pas significativement changé. Cependant, sous LS, les souris Senni aK0 perdaient des quantités significativement augmentées de sodium dans leurs fèces, accompagnées par de très hauts taux d'aldostérone plasmatique et une rétention urinaire en sodium augmentée. Les souris déficientes en CAPl/PmS (Prss8K0) dans les cellules superficielles intestinales étaient viables et ne montraient pas de létalité embryonnaire ou postnatale. Sous diètes RS et HS cependant, les souris Prss8KO montraient une diminution significative du APDamil dans l'après-midi, mais le rythme circadien était maintenu. Sous diète LS, la perte de sodium par les fèces était accompagnée par des niveaux d'aldostérone plasmatiques plus élevés. Par conséquent, nous avons identifié la protéase activatrice de canal CAP 1 IPrss8 comme un régulateur important d'ENaC dans le côlon in vivo. De plus, nous étudions l'importance d'ENaC et de CAPIIPrss8 dans les conditions pathologiques comme les maladies inflammatoires chroniques de l'intestin (MICI). Le résultat préliminaire out montre qu'une déficience d'Prss8 mènait à la détérioration de la colite induite par le DSS comparé aux modèles contrôles respectifs. En résumé, l'étude a montré que sous restriction de sel, l'absence d'ENaC dans Pépithélium de surface du côlon était compensée par 1'activation du système rénine-angiotensine- aldostérone (RAAS) dans le rein. Ceci a mené à un pseudohypoaldostéronisme de type I spécifique au côlon avec résistance aux minéralocorticoïdes sans signe d'altération de rétention de potassium. - In aldosterone-responsive epithelial cells of kidney and colon, the epithelial sodium channel (ENaC) plays a critical role in the control of sodium balance, blood volume, and blood pressure. The role of ENaC is well characterized in kidney and lung, whereas role of ENaC and its positive regulator channel-activating protease 1 (CAPl/PrasS) on sodium transport in colon is largely unknown. We have investigated the importance of ENaC and CAPI/Prss8 in colon for sodium and potassium balance. Mice lacking the aENaC subunit (Scnnla mice) in intestinal superficial cells were viable and did not show any fetal or perinatal lethality. Under regular (RS) or low salt (LS) diet, the amiloride sensitive rectal potential difference (APDamii) was drastically decreased and its circadian rhythm blunted. Under regular salt (RS) or high salt (HS) diets or under potassium loading, plasma and urinary sodium and potassium were not significantly changed. However, upon LS, the ScnnlaK0 mice lost significant amounts of sodium in their feces, accompanied by very high plasma aldosterone and increased urinary sodium retention. Mice lacking the CAPl/PrasS (Prss8K0) in intestinal superficial cells were viable and did not show any fetal or perinatal lethality. Upon RS and HS diets, however, Prss8K0 exhibited a significantly reduced APDamii in the afternoon, but its circadian rhythm was maintained. Upon LS diet, sodium loss through feces was accompanied by higher plasma aldosterone levels. Thus, we have identified the channel-activating protease CAPl/Prss8 as an important in vivo regulator of ENaC in colon. Furthermore, we are investigating the importance of ENaC and CAPI/Prss8 in pathological conditions like inflammatory bowel disease (IBD). Preliminary data showed that PmS-deficiency led to worsening of DSS-induced colitis as compared to their respective controls. Overall, the present study has shown that under salt restriction, the absence of ENaC in colonic surface epithelium was compensated by the activation of renin-angiotensin- aldosterone (RAAS) system in the kidney. This led to a colon specific pseudohypoaldosteroni sm type 1 with mineralocorticoid resistance without evidence of impaired potassium retention.

A comparative study between crosslinked sodium hyaluronate (healaflow glaucoma implant) and high molecular weight sodium hyaluronate (healon 5) as subconjunctival spacers injected into failed filtration blebs during needling with MMC

Purpose: To compare the efficacy and safety of a slowly resorbable glaucoma implant, Healaflow (HF) and Healon 5 (H5) as spacers injected into failing filtration blebs at the time of bleb needlings, augmented with MMC Methods: Eighty-four glaucoma patients with filtration bleb failure following glaucoma surgery (deep sclerectomy/ trabeculectomy) underwent bleb needling using either HF (n=44) or H5 (n=40) as an intrableb viscoelastic spacer. All needlings were augmented by MMC and performed in the operating room, by a single surgeon. The choice between HF and H5 was randomised. Postoperative data for IOP, number of glaucoma medications (GMs), antimetabolite injections (AMIs), bleb morphology and all complications were recorded at day 1 (D1), weeks 1 (W1), 4 (W4), month 6 (M6) and last visit. Success was defined as IOP≤21mmHg and 20% reduction in IOP from baseline Results: Age was comparable between groups (HF vs H5; 66 vs 72 years; p=0.13*). There were no differences in mean postoperative IOP or GMs at any time point between groups (mean IOP = HF vs H5; prior to needling=19.8mmHg vs 18.7mmHg, p=0.48*; D1=10.4mmHg vs 10.2mmHg, p=0.85*; W1=12.3mmHg vs 13.8mmHg, p=0.33*; W4=15.7mmHg vs 15.1mmHg, p=0.69*; M6=14.3mmHg vs 13.8mmHg, p=0.69*. Mean GMs= HF vs H5; prior to needling=1.7 vs 1.7, p=0.96*; M6=0.9 vs 1.1, p=0.66*). Success rates were comparable between HF and H5 groups (74% vs 71%). Requirements for AMIs were similar between groups; post filtration surgery and prior to needling (mean AMIs 1.54 vs 1.65 p=0.82*) and within the first six months following needling ( 0.77 vs 0.65 p=0.70*). Complication rates were infrequent in both groups (9%, HF; 3%, H5) * two-sample t-test Conclusions: The early to mid-term success rates of needlings using intrableb spacers were high. However there were no differences observed between the use of crosslinked and high molecular weight sodium hyaluronate

Scnn1 sodium channel gene family in genetically engineered mice.

The amiloride-sensitive epithelial sodium channel is the limiting step in salt absorption. In mice, this channel is composed of three subunits (alpha, beta, and gamma), which are encoded by different genes (Scnn1a, Scnn1b, and Scnn1c, respectively). The functions of these genes were recently investigated in transgenic (knockout) experiments, and the absence of any subunit led to perinatal lethality. More defined phenotypes have been obtained by introducing specific mutations or using transgenic rescue experiments. In this report, these approaches are summarized and a current gene-targeting strategy to obtain conditional inactivation of the channel is illustrated. This latter approach will be indispensable for the investigation of channel function in a wide variety of organ systems.

Selective ion changes during spontaneous mitochondrial transients in intact astrocytes.

The bioenergetic status of cells is tightly regulated by the activity of cytosolic enzymes and mitochondrial ATP production. To adapt their metabolism to cellular energy needs, mitochondria have been shown to exhibit changes in their ionic composition as the result of changes in cytosolic ion concentrations. Individual mitochondria also exhibit spontaneous changes in their electrical potential without altering those of neighboring mitochondria. We recently reported that individual mitochondria of intact astrocytes exhibit spontaneous transient increases in their Na(+) concentration. Here, we investigated whether the concentration of other ionic species were involved during mitochondrial transients. By combining fluorescence imaging methods, we performed a multiparameter study of spontaneous mitochondrial transients in intact resting astrocytes. We show that mitochondria exhibit coincident changes in their Na(+) concentration, electrical potential, matrix pH and mitochondrial reactive oxygen species production during a mitochondrial transient without involving detectable changes in their Ca(2+) concentration. Using widefield and total internal reflection fluorescence imaging, we found evidence for localized transient decreases in the free Mg(2+) concentration accompanying mitochondrial Na(+) spikes that could indicate an associated local and transient enrichment in the ATP concentration. Therefore, we propose a sequential model for mitochondrial transients involving a localized ATP microdomain that triggers a Na(+)-mediated mitochondrial depolarization, transiently enhancing the activity of the mitochondrial respiratory chain. Our work provides a model describing ionic changes that could support a bidirectional cytosol-to-mitochondria ionic communication.

International union of basic and clinical pharmacology. XCI. structure, function, and pharmacology of acid-sensing ion channels and the epithelial Na+ channel.

The epithelial Na(+) channel (ENaC) and the acid-sensing ion channels (ASICs) form subfamilies within the ENaC/degenerin family of Na(+) channels. ENaC mediates transepithelial Na(+) transport, thereby contributing to Na(+) homeostasis and the maintenance of blood pressure and the airway surface liquid level. ASICs are H(+)-activated channels found in central and peripheral neurons, where their activation induces neuronal depolarization. ASICs are involved in pain sensation, the expression of fear, and neurodegeneration after ischemia, making them potentially interesting drug targets. This review summarizes the biophysical properties, cellular functions, and physiologic and pathologic roles of the ASIC and ENaC subfamilies. The analysis of the homologies between ENaC and ASICs and the relation between functional and structural information shows many parallels between these channels, suggesting that some mechanisms that control channel activity are shared between ASICs and ENaC. The available crystal structures and the discovery of animal toxins acting on ASICs provide a unique opportunity to address the molecular mechanisms of ENaC and ASIC function to identify novel strategies for the modulation of these channels by pharmacologic ligands.

Role of reactive hyperreninemia in blood pressure changes induced by sodium depletion in patients with refractory hypertension

Sixteen patients with refractory hypertension were submitted to vigorous sodium depletion while cardiovascular homeostasis was monitored with measurements of hormonal and hemodynamic parameters and repeat saralasin tests. This regimen resulted in a negative sodium balance by an average of 300 mEq. The loss of sodium closely correlated to the decrease of body weight (r = 0.70, p less than 0.005). Blood pressure (BP) decreased from 176/166 +/- 8/3 to 155/109 +/-6/3 mm Hg. There was a significant correlation between percent increments in plasma renin activity (PRA) and the rise in plasma norepinephrine (r = 0.68, p less than 0.05) and a close negative correlation between percent increase in PRA and the ratio of fall in mean blood pressure (MAP) per unit of weight loss (r = -0.73, p less than 0.005). Thus, patients with the least percent increase in PRA demonstrated the greatest fall in BP per unit of weight loss, indicating that relative rather than absolute elevation of renin may be the factor limiting antihypertensive efficacy of sodium depletion. Sodium depletion induced increase in peripheral resistance and decrease in cardiac output, both mostly attributable to relative hyperreninemia. Indeed, the adverse hemodynamic changes were reversed by angiotensin inhibition, during which BP normalized. It is concluded that vigorous sodium depletion complemented by angiotensin blockade or suppression with sympatholytic agents improves management of otherwise refractory hypertension.

A combined computational and functional approach identifies new residues involved in pH-dependent gating of ASIC1a.

Acid-sensing ion channels (ASICs) are key receptors for extracellular protons. These neuronal nonvoltage-gated Na(+) channels are involved in learning, the expression of fear, neurodegeneration after ischemia, and pain sensation. We have applied a systematic approach to identify potential pH sensors in ASIC1a and to elucidate the mechanisms by which pH variations govern ASIC gating. We first calculated the pK(a) value of all extracellular His, Glu, and Asp residues using a Poisson-Boltzmann continuum approach, based on the ASIC three-dimensional structure, to identify candidate pH-sensing residues. The role of these residues was then assessed by site-directed mutagenesis and chemical modification, combined with functional analysis. The localization of putative pH-sensing residues suggests that pH changes control ASIC gating by protonation/deprotonation of many residues per subunit in different channel domains. Analysis of the function of residues in the palm domain close to the central vertical axis of the channel allowed for prediction of conformational changes of this region during gating. Our study provides a basis for the intrinsic ASIC pH dependence and describes an approach that can also be applied to the investigation of the mechanisms of the pH dependence of other proteins.