Ca/calmodulin-dependent protein kinase II contributes to intracellular pH recovery from acidosis via Na/H exchanger activation

The Na⁺/H⁺ exchanger (NHE-1) plays a key role in pH_i recovery from acidosis and is regulated by pH_i and the ERK1/2-dependent phosphorylation pathway. Since acidosis increases the activity of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) in cardiac muscle, we examined whether CaMKII activates the exchanger by using pharmacological tools and highly specific genetic approaches. Adult rat cardiomyocytes, loaded with the pH_i indicator SNARF-1/AM were subjected to different protocols of intracellular acidosis. The rate of pH_i recovery from the acid load (dpH_i/dt)—an index of NHE-1 activity in HEPES buffer or in NaHCO₃ buffer in the presence of inhibition of anion transporters—was significantly decreased by the CaMKII inhibitors KN-93 or AIP. pH_i recovery from acidosis was faster in CaMKII-overexpressing myocytes than in overexpressing β-galactosidase myocytes (dpH_i/dt: 0.195 ± 0.04 vs. 0.045 ± 0.010 min^− 1, respectively, n = 8) and slower in myocytes from transgenic mice with chronic cardiac CaMKII inhibition (AC3-I) than in controls (AC3-C). Inhibition of CaMKII and/or ERK1/2 indicated that stimulation of NHE-1 by CaMKII was independent of and additive to the ERK1/2 cascade. In vitro studies with fusion proteins containing wild-type or mutated (Ser/Ala) versions of the C-terminal domain of NHE-1 indicate that CaMKII phosphorylates NHE-1 at residues other than the canonical phosphorylation sites for the kinase (Ser648, Ser703, and Ser796). These results provide new mechanistic insights and unequivocally demonstrate a role of the already multifunctional CaMKII on the regulation of the NHE-1 activity. They also prove clinically important in multiple disorders which, like ischemia/reperfusion injury or hypertrophy, are associated with increased NHE-1 and CaMKII.     

PRL and GH synthesis and release from the sea bream (Sparus auratus L.) pituitary gland in vitro in response to osmotic challenge

The endocrine factors prolactin (PRL) and growth hormone (GH) are believed to have counteracting effects in the adaption of fish to changes in environmental salinity. In order to further investigate this interaction sea bream were challenged with full seawater (SW) or freshwater (FW) for 7 days and the response of pituitary glands cultured in vitro to an osmotic challenge (230, 275 and 320 mOsm/kg) was assessed. In vitro PRL secretion from pituitaries of SW-adapted fish was unaltered in response to an osmotic challenge, while GH secretion increased in the lowest osmolality (230 mOsm/kg). In contrast, both GH and PRL secretion by pituitaries from FW challenged fish was significantly increased (p < 0.01) over that of pituitaries from SW fish at the highest osmolality (320 mOsm/kg). After FW challenge pituitary PRL content and de novo synthesised and released PRL were significantly increased (p < 0.01), while total PRL secretion was not different from SW animals. GH pituitary content decreased in FW animals while total secretion and secretion of de novo synthesised protein were significantly increased (p < 0.01). In addition, after transfer of fish to FW expression of PRL and GH increased 3- and 2-fold, respectively. Despite the increase in PRL expression, no increase in total PRL secretion occurred and although in gills a 2-fold increase in the osmoregulatory marker, Na⁺/K⁺-ATPase activity was detected, profound haemodilution and a cumulative mortality of 40% occurred in sea bream placed in FW. Taken together the results suggest that the sea bream pituitary gland fails to respond appropriately to the osmotic challenge caused by low salinity and the physiological response evoked in vivo is not enough to allow this species to withstand and adapt to FW.     

Extensive heterozygosity in flanking microsatellites of Plasmodium falciparum Na/H exchanger (pfnhe-1) gene among Indian isolates

Plasmodium falciparum Na⁺/H⁺ exchanger-1 (pfnhe-1) gene has been proposed to be a possible marker for quinine resistance. Here, we describe the sequence analysis of the flanking microsatellites of the pfnhe-1 gene among 108 Indian P.falciparum isolates. Among the parasite population, a high degree of polymorphism was observed at all the 10 microsatellite loci within ±40 kb region of the pfnhe-1 gene where the number of alleles varied from 2 to 16 with a high expected heterozygosity ranging from 0.43 to 0.91 at these loci. Also, higher levels of heterozygosity have been observed in P.falciparum isolates collected from both low and high transmission and drug resistant areas. Furthermore, there was no association between QN resistance associated DNNND repeats in PFNHE-1 and the flanking microsatellite haplotypes. In conclusion, the observed high level of microsatellite polymorphism and absence of selective sweep in the flanking ±40 kb region of the pfnhe-1 gene could be an indication that there is no strong selection pressure on this target gene.     

Ambient salinity modifies the action of triiodothyronine in the air-breathing fish Anabas testudineus Bloch: Effects on mitochondria-rich cell distribution, osmotic and metabolic regulations

The hydromineral and metabolic actions of thyroid hormone on osmotic acclimation in fish is less understood. We, therefore, studied the short-term action of triiodothyronine (T₃), the potent thyroid hormone, on the distribution and the function of gill mitochondria-rich (MR) cells and on the whole body hydromineral and metabolic regulations of air-breathing fish (Anabas testudineus) adapted to either freshwater (FW) or acclimated to seawater (SA; 30 g L⁻¹). As expected, 24 h T₃ injection (100 ng g⁻¹) elevated (P < 0.05) plasma T₃ but classically reduced (P < 0.05) plasma T₄. The higher Na⁺, K⁺-ATPase immunoreactivity and the varied distribution pattern of MR cells in the gills of T₃-treated FW and SA fish, suggest an action of T₃ on gill MR cell migration, though the density of these cells remained unchanged after T₃ treatment. The ouabain-sensitive Na⁺, K⁺-ATPase activity, a measure of hydromineral competence, showed increases (P < 0.05) in the gills of both FW and SA fish after T₃ administration, but inhibited (P < 0.05) in the kidney of the FW fish and not in the SA fish. Exogenous T₃ reduced glucose (P < 0.05) and urea (P < 0.05) in the plasma of FW fish, whereas these metabolites were elevated (P < 0.05) in the SA fish, suggesting a modulatory effect of ambient salinity on the T₃-driven metabolic actions. Our data identify gill MR cell as a target for T₃ action as it promotes the spatial distribution and the osmotic function of these cells in both fresh water and in seawater. The results besides confirming the metabolic and osmotic actions of T₃ in fish support the hypothesis that the differential actions of T₃ may be due to the direct influence of ambient salinity, a major environmental determinant that alters the osmotic and metabolic strategies of fish.     

Angiotensin II inhibits the electrogenic Na/HCO3 cotransport of cat cardiac myocytes

The Na⁺/HCO₃⁻ cotransporter (NBC) plays an important role in intracellular pH (pH_i) regulation in the heart. In the myocardium co-exist the electrogenic (eNBC) and electroneutral (nNBC) isoforms of NBC. We have recently reported that angiotensin II (Ang II) stimulated total NBC activity during the recovery from intracellular acidosis through a reactive oxygen species (ROS) and ERK-dependent pathway. In the present work we focus our attention on eNBC. In order to study the activity of the eNBC in isolation, we induced a membrane potential depolarization by increasing extracellular K⁺ [K⁺]_o from 4.5 to 45 mM (K⁺ pulse). This experimental protocol enhanced eNBC driving force leading to intracellular alkalization (0.19 ± 0.008, n = 6; data expressed as an increase of pH_i units after 14 min of applying the K⁺ pulse). This alkalization was completely abrogated by the NBC blocker S0859 (− 0.004 ± 0.016*, n = 5; * indicates p < 0.05 vs control) but not by the Na⁺/H⁺ exchanger blocker HOE642 (0.185 ± 0.04, n = 4), indicating that we are exclusively measuring eNBC. The K⁺ pulse induced alkalization was canceled by 100 nM Ang II (− 0.008 ± 0.018*; n = 5). This inhibitory effect was prevented when the myocytes were incubated with losartan (AT₁ receptor blocker, 0.18 ± 0.02; n = 4) or SB202190 (p38 MAP kinase inhibitor, 0.25 ± 0.06; n = 5). Neither chelerythrine (PKC inhibitor, − 0.06 ± 0.04*; n = 4), nor U0126 (ERK inhibitor, − 0.07 ± 0.04*; n = 4) nor MPG (ROS scavenger, − 0.02 ± 0.05*; n = 8) affected the Ang II-induced inhibition of eNBC. The inhibitory action of Ang II on eNBC was corroborated with perforated patch-clamp experiments, since no impact of the current produced by eNBC on action potential repolarization was observed in the presence of Ang II. In conclusion, we propose that Ang II, binding to AT₁ receptors, exerts an inhibitory effect on eNBC activity in a p38 kinase-dependent manner.     

Cortisol promotes and integrates the osmotic competence of the organs in North African catfish (Clarias gariepinus Burchell): Evidence from in vivo and in situ approaches

The short-term in situ and long-term in vivo effects of cortisol were examined in North African catfish (Clarias gariepinus) to identify how this major corticosteroid integrates the osmotic competence of fish organs. In the in situ approach, the hydromineral effects of cortisol perfusion (75-300 ng ml⁻¹) for 20 min were tested and the indices of hydromineral and metabolic regulations were measured in our in vivo experimental fish after three alternate intraperitoneal cortisol injections (40 and 200 ng g⁻¹ body mass) for 5 days. Na⁺, K⁺-ATPase activity, a measure of cellular osmotic competence, responded to in situ and in vivo cortisol treatments. In situ cortisol delivery increased the Na⁺, K⁺-ATPase activity in the gill (P < 0.001) and kidney (P < 0.001) but decreased (P < 0.01) in the liver and showed no effect on intestine. In vivo cortisol treatment, on the contrary, increased Na⁺, K⁺-ATPase activity in the gills (P < 0.01), intestine (P < 0.05) and liver (P < 0.01) but decreased (P < 0.05) in the kidney. As expected, plasma cortisol increased (P < 0.001) with increasing doses of cortisol injections which produced direct effects on the metabolites and the mineral contents including the elevations of glucose (P < 0.05), lactate (P < 0.05) and Mg²⁺ (P < 0.05) and reductions of urea (P < 0.05), Na⁺ (P < 0.05) and K⁺ (P < 0.05) in the plasma. A decline of triiodothyronine (P < 0.01) occurred in the catfish after in vivo cortisol treatment and that implies a direct cortisol action on the homeostatic integration in this fish. Evidence is thus presented that in catfish cortisol regulates the whole body hydromineral and metabolite homeostasis by promoting and integrating the osmotic and metabolic functions of the multiple organ systems including liver.     

cGMP/PKG pathway mediates myocardial postconditioning protection in rat hearts by delaying normalization of intracellular acidosis during reperfusion

Ischemic postconditioning has been demonstrated to limit infarct size in patients, but its molecular mechanisms remain incompletely understood. Low intracellular pH (pHi) inhibits mitochondrial permeability transition, calpain activation and hypercontracture. Recently, delayed normalization of pHi during reperfusion has been shown to play an important role in postconditioning protection, but its relation with intracellular protective signaling cascades is unknown. The present study investigates the relation between the rate of pHi normalization and the cGMP/PKG pathway in postconditioned myocardium. In isolated Sprague-Dawley rat hearts submitted to transient ischemia both, postconditioning and acidic reperfusion protocols resulted in a similar delay in pHi recovery measured by ³¹P-NMR spectroscopy (3.6 ± 0.2 min and 3.5 ± 0.2 min respectively vs. 1.4 ± 0.2 min in control group, P < 0.01) and caused equivalent cardioprotection (48% and 41% of infarct reduction respectively, P < 0.01), but only postconditioning increased myocardial cGMP levels (P = 0.02) and activated PKG. Blockade of cGMP/PKG pathway by the addition of the guanylyl cyclase inhibitor ODQ or the PKG inhibitor KT5823 during reperfusion accelerated pHi recovery and abolished cardioprotection in postconditioned hearts, but had no effect in hearts subjected to acidic reperfusion suggesting that PKG signaling was upstream of delayed pHi normalization in postconditioned hearts. In isolated cardiomyocytes the cGMP analog 8-pCPT-cGMP delayed Na⁺/H⁺-exchange mediated pHi normalization after acidification induced by a NH₄Cl pulse. These results demonstrate that the cGMP/PKG pathway contributes to postconditioning protection at least in part by delaying normalization of pHi during reperfusion, probably via PKG-dependent inhibition of Na⁺/H⁺-exchanger.     

Activation of the cardiac Na-Ca exchanger by sorcin via the interaction of the respective Ca-binding domains

Sorcin is a penta-EF-hand protein that interacts with intracellular target proteins after Ca²⁺ binding. The sarcolemmal Na⁺/Ca²⁺ exchanger (NCX1) may be an important sorcin target in cardiac muscle. In this study, RNAi knockdown of sorcin, purified sorcin or sorcin variants was employed in parallel measurements of: (i) NCX activity in isolated rabbit cardiomyocytes using electrophysiological techniques and (ii) sorcin binding to the NCX1 calcium binding domains (CBD1 and (iii) using surface plasmon resonance and gel overlay techniques. Sorcin is activated by Ca²⁺ binding to the EF3 and EF2 regions, which are connected by the D helix. To investigate the importance of this region in the interaction with NCX1, three variants were examined: W105G and W99G, mutated respectively near EF3 and EF2, and E124A that does not bind Ca²⁺ due to a mutation at EF3. Downregulation of sorcin decreased and supplementation with wt sorcin (3 μM) increased NCX activity in isolated cardiomyocytes. The relative stimulatory effects of the sorcin variants were: W105G > wt sorcin > Sorcin Calcium Binding Domain (SCBD) > W99G > E124A. Sorcin binding to both CBD1 and 2 was observed. In the presence of 50 µM Ca²⁺, the interaction with CBD1 followed the order W105G > SCBD > wt sorcin > W99G > E124A. In sorcin, the interacting surface can be mapped on the C-terminal Ca²⁺-binding domain in the D helix region comprising W99. The fast association/dissociation rates that characterize the interaction of sorcin with CBD1 and 2 may permit complex formation/dissociation during an excitation/contraction cycle.     

NhaA antiporter functions using 10 helices,and an additional 2 contribute to assembly/stability

The Escherichia coli Na⁺/H⁺ antiporter (Ec-NhaA) is the best-characterized of all pH-regulated Na⁺/H⁺ exchangers that control cellular Na⁺ and H⁺ homeostasis. Ec-NhaA has 12 helices, 2 of which (VI and VII) are absent from other antiporters that share the Ec-NhaA structural fold. This α-hairpin is located in the dimer interface of the Ec-NhaA homodimer together with a β-sheet. Here we examine computationally and experimentally the role of the α-hairpin in the stability, dimerization, transport, and pH regulation of Ec-NhaA. Evolutionary analysis (ConSurf) indicates that the VI–VII helical hairpin is much less conserved than the remaining transmembrane region. Moreover, normal mode analysis also shows that intact NhaA and a variant, deleted of the α-hairpin, share similar dynamics, suggesting that the structure may be dispensable. Thus, two truncated Ec-NhaA mutants were constructed, one deleted of the α-hairpin and another also lacking the β-sheet. The mutants were studied at physiological pH in the membrane and in detergent micelles. The findings demonstrate that the truncated mutants retain significant activity and regulatory properties but are defective in the assembly/stability of the Ec-NhaA dimer.Living cells are critically dependent on processes that regulate intracellular pH, Na⁺, and volume (1), and Na⁺/H⁺ antiporters play a primary role in these homeostatic mechanisms (reviewed in ref. 2). These antiporters are found in the cytoplasmic and intracellular membranes of most organisms (reviewed in refs. 3–6), and they have long been human drug targets (7).The principal Na⁺/H⁺ antiporter in Escherichia coli, Ec-NhaA, is responsible for intracellular Na⁺ and H⁺ homeostasis (8), and homologs have recently been implicated in the virulence of pathogenic bacteria (9). In humans, orthologs have been suggested to be involved in essential hypertension (10), as well as diabetes (11).Ec-NhaA is characterized by exceptionally high transport activity (12), a stoichiometry of 2H⁺/Na⁺ (13), and strong pH dependence (12), a property shared with other prokaryotic (8) and eukaryotic Na⁺/H⁺ antiporters (reviewed in refs. 3–6). Crystal structures of down-regulated Ec-NhaA at acidic pH (14) (Fig. 1) reveal a unique structural fold shared by a growing number of secondary transporters (15, 16, 17).Open in a separate windowFig. 1.(A) Evolutionary conservation analysis of Ec-NhaA. The crystal structure of Ec-NhaA, in ribbon representation, as seen in the plane of the membrane, represented with gray outlines, with the intracellular side facing up. ConSurf analysis (consurf.tau.ac.il) was carried out using PDB ID code 1ZCD (14). The amino acids are colored by their conservation grades using the color-coding bar, with cyan through maroon indicating variable through conserved. Overall, helices VI and VII, denoted with roman numerals, are of the least conserved TM helices with average conservation scores of 1 and 5, respectively, according to the ConSurf scoring method. The sites of the three single amino acid mutations Q47C, S246C, and V254C and the two sites in which Ec-NhaA was truncated (A182 on and A218 on) are shown as spheres (B) Ec-NhaA dimer. The crystal structure of the Ec-NhaA dimer (27), PDB ID code 4AU5, in ribbon representation as seen from the cytoplasm, with TM helices numbered as in A. The first monomer is colored light blue and the second colored wheat. The α-hairpin (composed of helices VI and VII) and β-sheet of one monomer are highlighted in marine and in the other monomer in orange. The red dashed line marks the boundary between the two functional domains of the transporter, the core domain to the left and the panel/dimer interface to the right. As can be seen the α- and β-hairpins constitute the dimer interface. (C) Close-up view on the interaction between the monomers. The Ec-NhaA dimer interface is shown as ribbon representation, with monomers colored as in B. Although the β-hairpin contributes the majority of interactions between subunits from the extracellular side, there is also a small interface between the cytoplasmic ends of helix VII of one monomer and helix IX of the other. This interaction probably involves hydrogen bond and hydrophobic contacts and forms a zipper-like structure consisting of R204 and L210 of one monomer and V254 and W258 of the other (27). The figures were generated using PyMol (PyMOL Molecular Graphics System, version 1.7.4; Schrödinger, LLC) (47, 48).Omitting transmembrane segments (TMs) VI and VII, the remaining 10 TMs of monomeric Ec-NhaA are organized into a highly conserved, densely packed core domain composed of two structurally related helix bundles (TMs III, IV, and V and TMs X, XI, and XII) that are topologically inverted with respect to each other (Fig. 1B) (14). TMs IV and XI are each interrupted by an unwound chain that crosses the other chain in the middle of the membrane, leaving two short helices oriented toward the cytoplasm (c) or the periplasm (p) (IVc, IVp and XIc, XIp, respectively; Fig. 1) (14). This noncanonical TM assembly—the NhaA fold—creates a delicately balanced electrostatic environment in the middle of the membrane at the ion binding site(s), which likely plays a critical role in cation exchange activity. The other TMs—the dimer interface domain—comprise a bundle along the dimer interface and also contain inverted-topology repeats (TMs I, II and VIII, IX; Fig. 1B) (18).Interestingly, the Na⁺/H⁺ antiporter structures that share the NhaA fold are characterized by different numbers of TMs from 12 to 13 (16, 17, 19) and are dimeric like Ec-NhaA. However, two prokaryotic ASBT (apical sodium-dependent bile acid transporter, also known as SLC10A2) symporters share the NhaA fold but have only 10 TMs; they lack the Ec-NhaA equivalents of helices VI and VII (20, 21). The ASBTs also differ from Ec-NhaA in that they are Na⁺/bile acid symporters. In addition, the ASBTs are monomeric. Taken together, the data raise questions regarding the role of TMs VI and VII in Ec-NhaA.In Ec-NhaA, helices VI and VII, which form an α-hairpin, are located in the dimer interface (Fig. 1 B and C). Therefore, these substructures may contribute to NhaA dimerization and/or stability; however, data in this regard are not available. In the current study, we constructed an NhaA mutant deleted of TMs VI and VII and studied its properties at physiological pH. The mutant devoid of TMs VI and VII is defective with respect to dimerization and/or stability but exhibits decreased but significant transport activity, as well as pH regulation.     

Na/K-ATPase inhibition by ouabain induces CaMKII-dependent apoptosis in adult rat cardiac myocytes

The positive inotropic effect produced by Na⁺/K⁺-ATPase inhibition has been used for the treatment of heart failure for over 200 years. Recently, administration of toxic doses of ouabain has been shown to induce cardiac myocyte apoptosis. However, whether prolonged administration of non-toxic doses of ouabain can also promote cardiac myocyte cell death has never been explored. The aim of this study was to assess whether non-toxic doses of ouabain can induce myocyte apoptosis and if so, to examine the underlying mechanisms. For this purpose, cardiac myocytes from rat and cat, two species with different sensitivity to digitalis, were cultured for 24 h in the presence or absence of 2 µM (rat) and 25 nm-2 µM ouabain (cat). Cell viability and apoptosis assays showed that ouabain produced, in the rat, a 43 ± 5% decrease in cell viability due to apoptosis (enhanced caspase-3 activity, increased Bax/Bcl-2 and TUNEL-positive nuclei) and necrosis (LDH release and trypan blue staining). Similar results were obtained with 25 nM ouabain in the cat. Ouabain-induced reduction in cell viability was prevented by the NCX inhibitor KB-R7943 and by the CaMKII inhibitors, KN93 and AIP. Furthermore, CaMKII overexpression exacerbated ouabain-induced cell mortality which in contrast was reduced in transgenic mice with chronic CaMKII inhibition. However, KN93 failed to affect ouabain-induced inotropy. In addition, whereas ERK½ inhibition with PD-98059 had no effect on cell mortality, PI3K inhibition with wortmannin, exacerbated myocyte death. We conclude that ouabain triggers an apoptotic cascade that involves NCX and CaMKII as a downstream effector. Ouabain simultaneously activates an antiapoptotic cascade involving PI3K/AKT which is however, insufficient to completely repress apoptosis. The finding that KN93 prevents ouabain-induced apoptosis without affecting inotropy suggests the potential use of CaMKII inhibitors as an adjunct to digitalis treatment for cardiovascular disease.     

Mechanism of hCG-induced spermiation in the toad Rhinella arenarum (Amphibia, Anura)

In Rhinella arenarum spermiation occurs as a consequence of LH/FSH increase during the amplexus or by a single dose of hCG, among other gonadotropins. The present study employs an in vitro system to study the mechanism of action of hCG in the spermiation of R. arenarum. Testicular fragments were incubated for 2 h at 28 °C in the presence or absence of 20 IU hCG with or without different PKA/PKC inhibitors and activators as well as ouabain and amiloride as Na⁺/K⁺ ATPase and transcellular Na⁺ transport inhibitors, respectively. Ouabain did not induce spermiation in absence of hCG and inhibited hCG-induced spermiation in a dose-dependent manner, reaching 90% inhibition with the higher concentration. In contrast, amiloride neither affected spermiation nor steroidogenesis. Activation of PKA with 8Br-cAMP induced spermiation in the absence of hCG while its inhibition with H89 blocked hCG action. On the other hand, PKC inhibition with Bi or STP did not affect hCG-induced spermiation although PKC activation significantly decreased hCG-dependent sperm release. These results suggest that PKC inhibits spermiation but also that the inhibition exerted by the kinase could be blocked by hCG. Taken together, these observations could indicate that PKA is involved in the mechanism of the gonadotropin action, mechanism also requiring the activation of a non-pumping Na⁺/K⁺ ATPase pathway.     

Cytosolic pH regulation in density-defined subpopulations of bronchoalveolar macrophages

Bronchoalveolar macrophages (m) represent a heterogeneous population of morphologically and functionally distinct cells. In mixed populations of bronchoalveolar m, cytosolic pH (pH _i ) regulation has been shown to involve both Na⁺-dependent and -independent mechanisms for H⁺ extrusion, i.e., passive H⁺ extrusion in exchange for extracellular Na⁺ (Na⁺-H⁺ exchange or NHE) and active H⁺ extrusion by plasmalemmal vacuolar-type H⁺-ATPase (V-ATPase), respectively. The present studies explored the possibility that individual subpopulations of bronchoalveolar m possess distinct ensembles of H⁺ extrusion mechanisms. Rabbit bronchoalveolar m were separated into five density-defined subpopulations using a discontinuous density gradient. Scanning and transmission electron microscopy revealed morphological differences between the subpopulations. The number of plasmalemmal projections and electron-dense inclusions increased with increments in cell density. The subpopulations were also functionally distinct. Fe receptor-mediated phagocytosis increased in the increasing density subpopulations. Despite these differences, all subpopulations displayed Na⁺-dependent and -independent mechanisms for pH _i recovery from intracellular acid loads (ammonia prepulse technique). We conclude that NHE and V-ATPase activities were present in each subpopulation. These findings support the use of mixed populations to study pH _i homeostasis in bronchoalveolar m. Offprint requests to: Akhil Bidani     

The sting of salt on an old,but open,wound--is Na(+) the cause of mitochondrial and myocardial injury during ischemia/reperfusion?

D. B. Sawyer, T. M. Suter and C. S. Apstein. The Sting of Salt on an Old, but Open, Wound—is Na⁺ the Cause of Mitochondrial and Myocardial Injury During Ischemia/Reperfusion? Journal of Molecular and Cellular Cardiology (2002) 34, 699-702.     

Estrogen exerts concentration-dependent pro-and anti-hypertrophic effects on adult cultured ventricular myocytes. Role of NHE-1 in estrogen-induced hypertrophy

Estrogen has been shown to protect the heart and attenuate myocardial hypertrophy and left ventricular remodelling through as yet to be defined mechanisms. In the present study we examined concentration-dependent effects of estrogen on hypertrophy of adult rat cardiomyocytes, potential underlying mechanisms related to intracellular pH (pH_i) and possible sex-dependent responses. Cardiomyocytes were isolated from adult male and female Sprague-Dawley rats and used immediately for pH_i determinations or cultured and subsequently treated for 24 h with 17β-estradiol to assess hypertrophic responses. Fluorometric measurements with the pH_i-sensitive dye BCECF demonstrated that at 1 pM 17β-estradiol increased pH_i (+ 0.05 pH units in females and + 0.12 pH units in males, P < 0.05) by a rapid non-genomic mechanism that was blocked by the sodium-hydrogen exchange isoform 1 (NHE-1) specific inhibitor AVE-4890 (AVE, 5 μM). Treatment with 1 pM 17β-estradiol for 24 h increased cell size (females: 20%, P < 0.05; males: 29%, P < 0.05) and ANP expression (females: 414%, P < 0.05; males: 497%, P < 0.05) in a NHE-1-, and ERK1/2 MAPK-dependent manner. At 1 nM, 17β-estradiol decreased pH_i (females: − 0.24 pH units, P < 0.05; males: − 0.07 pH units, P < 0.05) which was also prevented by AVE, although at this concentration the hormone had no direct hypertrophic effect but instead prevented hypertrophy induced by phenylephrine. Our results show that low levels of estrogen produce cardiomyocyte hypertrophy through ERK/NHE-1 activation and intracellular alkalinization whereas an antihypertrophic effect is seen at high concentrations. These effects may further our understanding of the role of estrogen in heart disease particularly associated with hypertrophy.     

Inhibition of Na+/H+ exchange preserves viability,restores mechanical function,and prevents the pH paradox in reperfusion injury to rat neonatal myocytes

Summary Rat neonatal myocytes exposed to 2.5 mM CaCN and 20 mM 2-deoxyglucose at pH 6.2 (chemical hypoxia) quickly lose viability when pH is increased to 7.4, with or without washout of inhibitors — a pH paradox. In this study, we evaluated the effect of two Na⁺/H⁺ exchange inhibitors (dimethylamiloride and HOE694) and a Na⁺/Ca²⁺ exchange inhibitor (dichlorobenzamil) on pH-dependent reperfusion injury. Intracellular free Ca²⁺ and electrical potential were monitored by laser scanning confocal microscopy of rat neonatal cardiac myocytes grown on coverslips and co-loaded with Fluo-3 and tetramethylrhodamine methylester. After 30–60 min of chemical hypoxia at pH 6.2, mitochondria depolarized and Ca²⁺ began to increase uniformly throughout the cell. Free Ca²⁺ reached levels estimated to exceed 2 M by 4h. Washout of inhibitors at pH 7.4 (reperfusion), with or without dichlorobenzamil, killed most cells within 60 min, despite a marked reduction of Ca²⁺ in dichloroben zamil-treated cells. Reperfusion at pH 7.4 in the presence of 75 M dimethylamiloride or 20 M HOE694, or at pH 6.2, prevented cell death. HOE694-treated cells placed into culture medium recovered mitochondrial membrane potential. In most cells, this occurred before normal Ca²⁺ was restored. Contracted myocytes re-extended over a 24-h-period. By 48 hours, most cells contracted spontaneously and showed normal Ca²⁺ transients. Our results indicate that Na⁺/H⁺ exchange inhibition protects against pH-dependent reperfusion injury and facilitates full recovery of cell function.Abbreviations DCB dichlorobenzamil - DMA dimethylamiloride - 2-DOG 2-deoxy-D-glucose - HOE694 3-methylsulfonyl-4-piperidinobenzoyl guanidine hydrochloride - KRH Krebs-Ringer-HEPES buffer containing 115 mM NaCl, 5 mM KCl, 1 mM KH₂PO₄, 1,2 mM MgSO₄, 2 mM CaCl₂, and 25 mM Na-HEPES buffer - PI propidium iodide - TMRM tetramethylrhodamine methylester - electrical potential difference - electric potential This work was supported, in part, by Grant N00014-89-J-1433 from the Office of Naval Research and Grants AG07218 an DK37034 from the National Institutes of Health. I.S.H. was the recipient of a Post-Doctoral Scholarship from the Medical Research Council of South Africa. A preliminary report of portions of this work was presented at Experimental Biology '93, New Orleans, March 28–April 1, 1993 (15)     

Evidence for apical Na+/H+ exchanger in bovine main pancreatic duct

The finding of a high Pco ₂ in basally secreted pancreatic juice of man and dog raises the hypothesis of proton secretion from ductal epithelial cells presumably through a Na⁺/H⁺ exchanger. To test this possibility, H⁺ luminal secretion and Na⁺ movements were measuredin vitro on samples of bovine pancreatic ducts mounted in Ussing-type chambers. The rate of luminal acidification measured by the pH stat method, using bicarbonate-free media gassed with 100% O₂, reached 2.75 Eq/cm²/hr. Proton secretion was blocked in the presence of 1 mM amiloride or in the absence of Na⁺ (replaced by choline) in the mucosal solution. Study of transepithelial²²Na fluxes in short-circuited tissue, bathed on both sides by control Ringer solution, gassed by 95% O₂-5% CO₂ demonstrated a net sodium transport from the mucosal to the interstitial side of the duct (net²²Na flux=3.23±0.8 Eq/cm²/hr). This net sodium transport was electroneutral and blocked by mucosal amiloride (0.5–1 mM/liter) or by interstitial ouabain (1 mM/liter). These results are consistent with the existence of a Na⁺/H⁺ exchanger on the luminal side of the bovine main pancreatic duct.     

Cardiovascular risk in obesity: Different activation of inflammation and immune system between obese and morbidly obese subjects

Background

Both inflammation and immunity are involved in the development and progression of atherosclerosis. Obesity is considered a major modifiable cardiovascular risk factor, however, the correlation between increasing degrees of obesity and cardiovascular risk is not clear yet. Aim of our study was to investigate how different degrees of obesity are associated with inflammation and immune system responses.

Methods

One-hundred healthy individuals were divided into 3 groups according to body mass index (BMI): 22 overweight (OW), 26 obese (O) and 52 morbidly obese (MO). High-sensitivity C-Reactive Protein (hs-CRP, immunonephelometry), leptin (radio-immunoassay) and CD4⁺CD28^nullT-lymphocytes (flow-cytometry), a particular subset of T-lymphocytes with pro-atherogenic and plaque-destabilizing properties, were assessed.

Results

hs-CRP levels were significantly higher in O vs OW (p = 0.036), in MO vs OW (p < 0.001) and in MO vs O (p = 0.012). Similarly, leptin levels were higher in O vs OW (p = 0.02), in MO vs OW (p < 0.001) and in MO vs O (p < 0.001). CD4⁺CD28^nullT-lymphocytes were higher in O vs OW (p < 0.001), in O vs MO (p = 0.03) and in MO vs OW (p = 0.01). hs-CRP and leptin levels significantly correlated each other (r = 0.39; p < 0.001) and with waist circumference (r = 0.52; p < 0.001; r = 0.64; p < 0.001) and BMI (r = 0.60; p < 0.001; r = 0.74; p < 0.001).

Conclusions

Our study demonstrates that, notwithstanding higher levels of inflammation, MO are characterized by less detrimental immune activation, as shown by the reduced CD4⁺CD28^nullT-cells expansion as compared to OW and O, which might translate in less immune vascular injury. These findings suggest that MO might represent a particular population, in which different pathophysiological mechanisms take part if compared with “classic” obesity.     

Impact of constitutional polymorphisms in VCAM1 and CD44 on CD34+ cell collection yield after administration of granulocyte colony-stimulating factor to healthy donors

Background

The number of CD34⁺ cells mobilized from bone marrow to peripheral blood after administration of granulocyte colony-stimulating factor varies greatly among healthy donors. This fact might be explained, at least in part, by constitutional differences in genes involved in the interactions tethering CD34⁺ cells to the bone marrow.

Design and Methods

We analyzed genetic characteristics associated with CD34⁺ cell mobilization in 112 healthy individuals receiving granulocyte colony-stimulating factor (filgrastim; 10 μg/kg; 5 days).

Results

Genetic variants in VCAM1 and in CD44 were associated with the number of CD34⁺ cells in peripheral blood after granulocyte colony-stimulating factor administration (P=0.02 and P=0.04, respectively), with the quantity of CD34⁺ cells ×10⁶/kg of donor (4.6 versus 6.3; P<0.001 and 7 versus 5.6; P=0.025, respectively), and with total CD34⁺ cells ×10⁶ (355 versus 495; P=0.002 and 522 versus 422; P=0.012, respectively) in the first apheresis. Of note, granulocyte colony-stimulating factor administration was associated with complete disappearance of VCAM1 mRNA expression in peripheral blood. Moreover, genetic variants in granulocyte colony-stimulating factor receptor (CSF3R) and in CXCL12 were associated with a lower and higher number of granulocyte colony-stimulating factor-mobilized CD34⁺ cells/μL in peripheral blood (81 versus 106; P=0.002 and 165 versus 98; P=0.02, respectively) and a genetic variant in CXCR4 was associated with a lower quantity of CD34⁺ cells ×10⁶/kg of donor and total CD34⁺ cells ×10⁶ (5.3 versus 6.7; P=0.02 and 399 versus 533; P=0.01, respectively).

Conclusions

In conclusion, genetic variability in molecules involved in migration and homing of CD34⁺ cells influences the degree of mobilization of these cells.     

Functional and Cellular Regulation of the Myocardial Na+/H+ Exchanger

The Na⁺/H⁺ exchanger is a pH-regulatory protein present in the plasma membrane of cardiomyocytes and other cell types. In response to intracellular acidosis, the protein removes one intracellular proton in exchange for an extracellular sodium. The protein consists of a membrane transporting domain and a regulatory cytosolic domain. The regulatory cytosolic domain mediates the stimulation of the membrane domain. Hormonal stimulation of myocardial cells results in activation of the antiporter, possibly through protein kinases and other regulatory proteins. Several hormones and growth factors have been shown to stimulate the antiporter in the myocardium, including endothelin, thrombin, angiotensin II, and ₁-adrenergic stimulation. The exact mechanisms involved in this stimulation are as yet unclear, and may be important in regulation of the Na⁺/H⁺ exchanger during ischemia and reperfusion.