Bicarbonate-free Medium Research Articles

Effect of High Viscosity on Energy Metabolism and Kinematics of Spermatozoa from Three Mouse Species Incubated under Capacitating Conditions

In order to sustain motility and prepare for fertilization, sperm require energy. The characterization of sperm ATP production and usage in mouse species revealed substantial differences in metabolic pathways that can be differentially affected by capacitation. Moreover, spermatozoa encounter different environments with varying viscoelastic properties in the female reproductive tract. Here, we examine whether viscosity affects sperm ATP levels and kinematics during capacitation in vitro. Sperm from three mouse species (Mus musculus, M. spretus, M. spicilegus) were incubated under capacitating conditions in a modified Tyrode's medium containing bicarbonate, glucose, pyruvate, lactate, and bovine serum albumin (mT-BH) or in a bicarbonate-free medium as a non-capacitating control. Viscosity was increased with the inclusion of polyvinylpyrrolidone. ATP was measured with a bioluminescence kit, and kinematics were examined with a computer-aided sperm analysis system. In M. musculus sperm, ATP declined during capacitation, but no differences were found between non-capacitating and capacitating sperm. In contrast, in M. spretus and M. spicilegus, ATP levels decreased in capacitating sperm. Increasing viscosity in the medium did not modify the timing or proportion of cells undergoing capacitation but did result in additional time- and concentration-dependent decreases in ATP in M. spretus and M. spicilegus under capacitating conditions. Additionally, increased viscosity altered both velocity and trajectory descriptors. The limited impact of capacitation and higher viscosity on M. musculus sperm ATP and kinematics could be related to the low intensity of postcopulatory sexual selection in this species. Responses seen in the other two species could be linked to the ability of their sperm to perform better under enhanced selective pressures.

The CO-releasing molecule CORM-3 protects adult cardiomyocytes against hypoxia-reoxygenation by modulating pH restoration

Several studies have reported that CORM-3, a water-soluble carbon monoxide releasing molecule, elicits cardioprotection against myocardial infarction but the mechanism remains to be investigated. Numerous reports indicate that inhibition of pH regulators, the Na+/H+ exchanger (NHE) and Na+/HCO3− symporter (NBC), protect cardiomyocytes from hypoxia/reoxygenation injury by delaying the intracellular pH (pHi) recovery at reperfusion. Our goal was to explore whether CORM-3-mediated cytoprotection involves the modulation of pH regulation. When added at reoxygenation, CORM-3 (50 μM) reduced the mortality of cardiomyocytes exposed to 3 h of hypoxia and 2 h of reoxygenation in HCO3−-buffered solution. This effect was lost when using inactive iCORM-3, which is depleted of CO and used as control, thus implicating CO as the mediator of this cardioprotection. Interestingly, the cardioprotective effect of CORM-3 was abolished by switching to a bicarbonate-free medium. This effect of CORM-3 was also inhibited by 5-hydroxydecanoate, a mitochondrial ATP-dependent K+ (mKATP) channel inhibitor (500 μM) or PD098059, a MEK1/2 inhibitor (10 μM). In additional experiments and in the absence of hypoxia-reoxygenation, intracellular pH was monitored in cardiomyocytes exposed to cariporide to block NHE activity. CORM-3 inhibited alkalinisation and this effect was blocked by PD098059 and 5-HD. In conclusion, CORM-3 protects the cardiomyocyte against hypoxia-reoxygenation injury by inhibiting a bicarbonate transporter at reoxygenation, probably the Na+/HCO3− symporter. This cardioprotective effect of CORM-3 requires the activation of mKATP channels and the activation of MEK1/2.

Bicarbonate is required for proliferation of pulmonary endothelial cells

RationaleThe second messenger cAMP is widely known to regulate proliferation in numerous cell types. Many of these studies implicate cAMP generated by membrane‐bound cyclases (AC1‐9). While these membrane‐bound ACs generate cAMP in the near membrane compartment, cAMP can also be generated in unique subcellular compartments by the soluble adenylyl cyclase (sAC or AC10). Soluble AC localizes to the cytosol as well as organelles, such as the mitochondria, and is uniquely stimulated by bicarbonate to generate cAMP. Recently, AC10 was observed to be overexpressed in prostate carcinoma, suggesting a role for AC10 in cellular proliferation; however, the independent role of bicarbonate in proliferation has not been investigated. Thus, we tested the hypothesis that bicarbonate is required for proliferation of pulmonary endothelial cells.MethodsPulmonary microvascular endothelial cells (PMVECs) and pulmonary artery endothelial cells (PAECs) were seeded at 100, 000 cells per well in media with and without bicarbonate. The cells were counted for seven consecutive days to generate growth curves and blood gas analysis performed to determine pH, pO2, pCO2, and HCO3− concentrations of the media. Additionally, a separate set of PMVECs were seeded at 100, 000 cells per well in bicarbonate free media and bicarbonate added back to the media after 24, 48, 72, 96, 120, 144, and 168 hours and counted for five days following bicarbonate add back.ResultsGrowth curves revealed that PMVECs and PAECs seeded in the bicarbonate‐containing media entered into log phase growth followed by a plateau phase at cell confluence; however, PMVECs and PAECs seeded in the bicarbonate free media did not enter log phase growth nor reach confluence. When bicarbonate was added back to PMVECs initially grown in a bicarbonate free environment, the cells proliferated to numbers comparable to PMVECs seeded in bicarbonate containing media throughout the growth curve; however, if PMVECs were incubated in bicarbonate free media for more than 96 hours, these cells did not enter into log phase growth and did not form a confluent monolayer.ConclusionThese data reveal a requirement for bicarbonate for proliferation of pulmonary endothelial cells, since neither PAECs nor PMVECs formed confluent monolayers in the absence of bicarbonate.Support or Funding InformationNIH 121513This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cystic Fibrosis Transmembrane Conductance Regulator Regulates Pannexin 1 Channel Opening for Acidosis‐induced ATP Release from Cardiomyocytes

ATP is released in the heart under a variety of conditions, including hypoxia, ischaemia and catecholamine stimulation. Under physiological conditions, extracellular ATP, and its breakdown product, adenosine, contribute to local vasodilation and regulation of coronary blood flow. We investigated the mechanism of ATP release in cardiomyocytes isolated from adult rat heart.Treatment with lactic acid produced pH depression and enhanced ATP release from cardiomyocytes. On the other hand, simulated ischaemia (metabolic inhibition plus anoxia) lowered ATP release. The acidosis‐induced ATP release was abolished by the specific CFTR inhibitor, CFTRinh‐172, CFTR pore blocker, GlyH‐101, or CFTR siRNA, suggesting that CFTR was involved. Forskolin and IBMX, agents that activate CFTR by elevating the intracellular cAMP, also increased ATP release, confirming the role of CFTR. However, blockade of Pannexin1 channels by Fast Green FCF, Brilliant Blue FCF, or pannexin1 siRNA, attenuated the ATP release during acidosis or forskolin treatment, and further lowered the ATP release during simulated ischaemia, suggesting that Pannexin1 functions as the ATP release channel. Immunofluorescence imaging suggested that CFTR and Pannexin1 were not co‐localized. Inhibition of either the Na+/H+ exchanger (NHE) with amiloride or the Na+/Ca2+ exchanger (NCX) with SN6 or KB‐R7943 abolished acidosis‐induced ATP release. FAK inhibitor 14, FAK autophosphorylation inhibitor, can attenuate the forskolin or lactic‐acid‐stimulated ATP release. Furthermore, forskolin or lactic‐acid‐stimulated ATP release was abolished by bicarbonate free medium or caspase inhibitor. Thus, we have demonstrated that the acidosis‐induced ATP release was involved in calcium induced CFTR open. The cyclic AMP, which activates mitochondrial PKA, is generated within mitochondria by the bicarbonate‐regulated soluble adenylyl cyclase (sAC). Hence, we propose that the CFTR Cl channel opens during acidosis, allowing the movement of bicarbonate into the cell, which activate the HCO3‐sAC‐cAMP‐PKA (mito‐sAC) signaling cascade and indirectly modulates Pannexin1 gating by activation of caspase.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evaluation of intracellular pH regulation and alkalosis defense mechanisms in preimplantation embryos

Intracellular pH (pHi) regulation is an important homeostatic function of cells. There are three major pHi-regulatory mechanisms: HCO3−/Cl− exchanger (anion exchanger [AE]), which alleviates alkalosis, and the Na+/H+ and Na+,HCO3−/Cl− exchangers, both of which alleviate acidosis. We hypothesized that there would be developmental changes in pHi-regulatory activity in preimplantation embryos as conditions in the oviduct are alkaline but acidic in the uterus. This study focused on the AE mechanism in pronuclear (PN) zygotes, two-cell (2-c), four-cell (4-c), morula, and blastocyst stage embryos from Balb/c mice. Microspectrofluorometry was used to monitor changes in pHi in embryos subjected to Cl−–free media in presence or absence of an AE inhibitor, DIDS, and in embryos recovering from NH4Cl-induced alkalosis. Real-time polymerase chain reaction was used to identify AE isoforms. The pHi changes were greatest in PN zygotes (0.086 ± 007 pHU/min) but fell as embryos developed to the 2-c, 4-c, morula, and blastocyst stages (0.063 ± 006; 0.035 ± 007; 0.024 ± 004, and 0.014 ± 004 pHU/min, respectively). DIDS significantly reduced the rise in pHi caused by Cl− removal in all embryos; the finding pointed out that this pHi changes are due to AE activity. But DIDS only inhibited the recovery responses of PN zygote, 2-c and 4-c embryos but not morula or blastocyst stage embryos. In bicarbonate-containing medium, all embryos recovered from induced alkalosis but only the morula and blastocyst stages could fully compensate from ammonium induced–alkalosis in bicarbonate-free medium. The finding showed that commonly used ammonium pulse method to investigate AE function against alkalosis is not suitable for morula and blastocyst embryonic stages. All embryos expressed SLC4A2 and SLC4A4 coding for AE-2 and AE-4, but none expressed either AE-1 or AE-3. The gradual change in the response to alkalosis in preimplantation embryos may be adaptations to their normal in vivo environment, where the early embryos are located in the alkaline oviduct, whereas the morula and blastocyst move into the acidic uterus.

HCO3--Dependent Impact of Na+,K+,2Cl- Cotransport in Vascular Smooth Muscle Excitation-Contraction Coupling

In smooth muscles, inhibition of Na⁺,K⁺,2Cl^- cotransport (NKCC) by bumetanide decreased intracellular Cl^- content ([Cl^-]_i) and suppressed the contractions triggered by diverse stimuli. This study examines whether or not bicarbonate, a regulator of several Cl^- transporters, affects the impact of NKCC in excitation-contraction coupling. Addition of 25 mM NaHCO₃ attenuated the inhibitory action of bumetanide on mesenteric artery contractions evoked by 30 mM KCl and phenylephrine (PE) by 5 and 3-fold, respectively. In cultured vascular smooth muscle cells, NaHCO₃ almost completely abolished inhibitory actions of bumetanide on transient depolarization and [Ca²⁺]_i elevation triggered by PE. In bicarbonate-free medium, bumetanide decreased [Cl^-]_i by ∼15%; this effect was almost totally abrogated by NaHCO₃. The addition of NaHCO₃ resulted in 2-fold inhibition of NKCC activity and 3-fold attenuation of [Cl^-]_i. These data strongly suggest that extracellular HCO₃^- diminishes the NKCC-sensitive component of excitation-contraction coupling via suppression of this carrier.

Evaluation of the functionality of cooled stored sperm with three commercial extenders

s / Theriogenology 70 (2008) 1384–1404 1393 and parameters of sperm motility. Furthermore, analysis of data according to the total sperm number per dose revealed a reduction of sperm motility with the decreasing number of spermatozoa. Doses with 1.1 10 cells had a lower percentage of motile spermatozoa (SpermVision) after 72 h of storage than doses with 2.2 10 cells. In doses of 0.6 10 cells, a significant drop was already detectable after 24 h. A thermoresistance test (incubation for 300 min at 38 8C) with 72-h old semen samples containing 0.6 10 cells revealed a lower motility (P 70% motility and normal acrosome at 2 d preservation of liquid semen at 4 8C were defined as cold shock resistant (Boar A), whereas sperm with 75%, and rate of affected acrosomes was <20%. Over all storage time points, sperm had decreasing absolute values of motility parameters in bicarbonate-free medium. Nevertheless, sperm motility still could be stimulated by bicarbonate in terms of specific kinematic parameters (e.g. path velocities).

Ouabain-induced stimulation of sodium-hydrogen exchange in rat optic nerve astrocytes

Sodium-dependent transporters are inhibited indirectly by the Na-K-ATPase inhibitor ouabain. Here we report stimulation of sodium-hydrogen exchange (NHE) in ouabain-treated cells. BCECF was used to measure cytoplasmic pH in cultured rat optic nerve astrocytes. Ammonium chloride was applied to acid load the cells. On removal of ammonium chloride, cytoplasmic pH fell abruptly, then gradually recovered toward baseline. Ouabain (1 microM) did not change cell sodium content, but the rate of pH recovery increased by 68%. Ouabain speeded pH recovery both in the presence and absence of bicarbonate. In bicarbonate-free medium, dimethylamiloride, an NHE inhibitor, eliminated the effect of 1 microM ouabain on pH recovery. Western blot analysis showed an NHE1 immunoreactive band but not NHE2, NHE3, or NHE4. Immunoprecipitation studies showed phosphorylation of NHE1 in cells treated with 1 microM ouabain. Ouabain evoked an increase of cAMP, and the effect of 1 microM ouabain on pH recovery was abolished by H-89, a protein kinase A inhibitor. 8-Bromoadenosine-cAMP increased the pH recovery rate, and this recovery was not further increased by ouabain. Although 1 microM ouabain did not alter cytoplasmic calcium concentration, it stimulated calcium entry after store depletion, a response abolished by 2-APB. Ouabain-induced stimulation of pH recovery was suppressed by inhibitors of capacitative calcium entry, SKF-96365, and 2-APB, as well as the cytoplasmic calcium chelator BAPTA. The cAMP increase in ouabain-treated cells was abolished by BAPTA and 2-APB. Taken together, the results are consistent with increased capacitative calcium entry and subsequent cAMP-PKA-dependent stimulation of NHE1 in ouabain-treated cells.

Effect of heparin on in vitro capacitation of boar sperm

Chlortetracycline (CTC) fluorescent pattern, the ability to undergo acrosome reaction (AR) upon exposure to 10 microM calcium ionophore A23187 and vitality estimation were used to investigate the effect of the sulfated glycosaminoglycan heparin on the in vitro capacitation of porcine spermatozoa. Sperm incubation in capacitating medium (CM) supplemented with 10 mM heparin for up to 120 min, showed an increase in the number of capacitated sperm (B pattern) and acrosome reacted sperm (AR pattern), without affecting their viability. In this condition, spermatozoa were incubated in CM depleted of albumin, calcium, bicarbonate or combinations, in the presence of heparin. In either calcium or bicarbonate-free media, capacitation was only basal and did not show variations in the presence of heparin. In absence of albumin the presence of calcium and bicarbonate stimulated capacitation, which was further increased by the addition of heparin. These results suggest that heparin enhances in vitro capacitation of porcine sperm only under capacitating conditions. Additionally, when sperm were incubated with 100 microg/ml biotinylated heparin in the presence or absence of unlabeled heparin, we observed that heparin binding sites were located mostly on the acrosomal region of boar sperm in an specific and saturable manner. The in vitro effect of heparin described in this work indicates that sulfated glycosaminoglycans, which are normally present in the female reproductive tract, might play an important role in the fertilization process in porcines.

Exposure of actin on the surface of the human sperm head during in vitro culture relates to sperm morphology, capacitation and zona binding

The aim of this study was to determine the relationship between the proportion of motile sperm with actin exposed on the surface of the head and sperm function. Semen samples were obtained from normozoospermic men and sperm function tests were performed. Motile sperm selected by swim-up were incubated with actin monoclonal antibody (A-mAb, 1:100) for 2 h, then anti-mouse IgG Dynabeads were used to detect sperm-bound A-mAb. Sperm capacitation was increased by phorbol myristate acetate (PMA) and decreased by bicarbonate-free medium. The proportion of sperm with exposed actin increased with time for up to 2 h incubation. Bicarbonate-free medium significantly decreased the proportion of sperm with exposed actin. PMA significantly enhanced this phenomenon. Sperm bound to zona pellucida (ZP) had a significantly higher proportion with exposed actin than did sperm remaining in medium. Of the 79 samples studied, an average of 9.4% (range 1-27%) of motile sperm had exposed actin after 2 h incubation and this was significantly correlated with sperm normal morphology and ZP binding. Exposure of actin on the surface of the sperm head during in vitro culture may be related to membrane modification during sperm capacitation and hence may be a useful marker for this subpopulation of sperm.

Constitutive Protease-activated Receptor-2-mediated Migration of MDA MB-231 Breast Cancer Cells Requires Both β-Arrestin-1 and -2

Protease-activated receptor-2 (PAR-2) is activated by trypsin-like serine proteases and can promote cell migration through an ERK1/2-dependent pathway, involving formation of a scaffolding complex at the leading edge of the cell. Previous studies also showed that expression of a dominant negative fragment of beta-arrestin-1 reduces PAR-2-stimulated internalization, ERK1/2 activation, and cell migration; however, this reagent may block association of many proteins, including beta-arrestin-2 with clathrin-coated pits. Here we investigate the role of PAR-2 in the constitutive migration of a metastatic breast cancer cell line, MDA MB-231, and use small interfering RNA to determine the contribution of each beta-arrestin to this process. We demonstrate that a trypsin-like protease secreted from MDA MB-231 cells can promote cell migration through autocrine activation of PAR-2 and this correlates with constitutive localization of PAR-2, beta-arrestin-2, and activated ERK1/2 to pseudopodia. Addition of MEK-1 inhibitors, trypsin inhibitors, a scrambled PAR-2 peptide, and silencing of beta-arrestins with small interfering RNA also reduce base-line migration of MDA MB-231 cells. In contrast, a less metastatic PAR-2 expressing breast cancer cell line does not exhibit constitutive migration, pseudopodia formation, or trypsin secretion; in these cells PAR-2 is more uniformly distributed around the cell periphery. These data demonstrate a requirement for both beta-arrestins in PAR-2-mediated motility and suggest that autocrine activation of PAR-2 by secreted proteases may contribute to the migration of metastatic tumor cells through beta-arrestin-dependent ERK1/2 activation.

Regulation of human airway ciliary beat frequency by intracellular pH.

pHi affects a number of cellular functions, but the influence of pHi on mammalian ciliary beat frequency (CBF) is not known. CBF and pHi of single human tracheobronchial epithelial cells in submerged culture were measured simultaneously using video microscopy (for CBF) and epifluorescence microscopy with the pH-sensitive dye BCECF. Baseline CBF and pHi values in bicarbonate-free medium were 7.2 +/- 0.2 Hz and 7.49 +/- 0.02, respectively (n = 63). Alkalization by ammonium pre-pulse to pHi 7.78 +/- 0.02 resulted in a 2.2 +/- 0.1 Hz CBF increase (P < 0.05). Following removal of NH4Cl, pHi decreased to 7.24 +/- 0.02 and CBF to 5.8 +/- 0.1 Hz (P < 0.05). Removal of extracellular CO2 to change pHi resulted in similar CBF changes. Pre-activation of cAMP-dependent protein kinase (10 microM forskolin), broad inhibition of protein kinases (100 microM H-7), inhibition of PKA (10 microM H-89), nor inhibition of phosphatases (10 microM cyclosporin + 1.5 microM okadaic acid) changed pHi-mediated changes in CBF, nor were they due to [Ca2+]i changes. CBF of basolaterally permeabilized human tracheobronchial cells, re-differentiated at the air-liquid interface, was 3.9 +/- 0.3, 5.7 +/- 0.4, 7.0 +/- 0.3 and 7.3 +/- 0.3 Hz at basolateral i.e., intracellular pH of 6.8, 7.2, 7.6 and 8.0, respectively (n = 18). Thus, intracellular alkalization stimulates, while intracellular acidification attenuates human airway CBF. Since phosphorylation and [Ca2+]i changes did not seem to mediate pHi-induced CBF changes, pHi may directly act on the ciliary motile machinery.

Determination of carbon dioxide production rates for mammalian cells in 24-well plates.

In this report, we describe a method for the quantitative determination of carbon dioxide production rates of mammalian cells. Custom-made, reusable, optically clear plugs are used to seal the wells of a 24-well plate. These plugs prevent the loss of CO2 produced by the mammalian cells cultured in bicarbonate-free medium. Measurements of pH, total liquid phase CO2, and viable cell density are used to estimate the average CO2 production rate during a 6-h incubation period. Using this method, four chemicals well-characterized in regards to toxicity, 2,4-dinitrophenol, antimycin A, rotenone, and cyanide, were found to elicit significant changes in CO2 production for given concentrations within 6 h, without inducing a decline in culture viability. Over longer exposure times, similar concentrations caused growth inhibition but not cell death. An assay based on metabolic change corresponding to growth inhibition that is more sensitive than traditional measures of cell death is a feasible complement to existing methods in drug discovery and toxicity testing.

Reduction of intracellular pH inhibits constitutive expression of cyclooxygenase-2 in human colon cancer cells.

Cyclooxygenase-2 (COX-2) over-expression is critically involved in tumor formation. Intracellular pH (pHi) has been shown to be alkaline in cancer cells, and to be an important trigger for cell proliferation. This study therefore analyzed the relationship between pHi and COX-2 expression. HRT-18 and Caco-2 cells cultured in medium with bicarbonate maintained a pHi of approximately 7.6, which is higher than that of non-neoplastic cells. Cells grown in bicarbonate-free medium with a pH at 6.8 showed a reduction in pHi to approximately 7.0. Importantly, reduction of pHi resulted in a complete inhibition of COX-2 mRNA and protein expression. When cells were grown in bicarbonate-supplemented medium at pH 6.8, pHi maintained at approximately 7.6 and COX-2 expression was not inhibited. Additionally, analysis utilizing protein synthesis inhibitor cycloheximide demonstrated that pHi mediated inhibition of COX-2 mRNA expression requires de novo protein synthesis of regulatory protein(s). These data strongly suggest that an alkaline pHi is an important trigger for constitutive COX-2 expression. Defining pHi-mediated mechanisms that govern the constitutive COX-2 expression may help in developing new strategies to block COX-2 over-expression in cancer cells.

Hypertrophic responsiveness of cardiomyocytes to alpha- or beta-adrenoceptor stimulation requires sodium-proton-exchanger-1 (NHE-1) activation but not cellular alkalization.

The influence of the sodium-proton-exchanger-1 (NHE-1) inhibitor HOE694 on alpha- or beta-adrenoceptor mediated stimulation of protein synthesis was investigated in cultured ventricular cardiomyocytes from adult rat pre-treated with fetal calf serum to induce hypertrophic responsiveness to beta-adrenoceptor stimulation. Stimulation of alpha-adrenoceptors with phenylephrine (10 microM) in bicarbonate-free medium caused cellular alkalization (Delta pH(i): + 0.17 +/- 0.02, n = 5, P < 0.05). HOE694, an NHE-1 inhibitor, completely abolished this effect. [14C]phenylalanine incorporation into cellular protein mass increased in the presence of phenylephrine by 23+/-8%, and this effect was also abolished in the presence of HOE694. HOE694 (1 mciroM) neither influenced basal protein synthesis nor interfered with alpha-adrenoceptor mediated activation of ERK2. Phorbol myristate acetate, a direct stimulator of protein kinase C, mimicked the effect of alpha-adrenoceptor stimulation in regard to protein synthesis, but did not lead to cellular alkalization. Protein synthesis increased in the presence of isoprenaline, a beta-adrenoceptor agonist also. Again, HOE694 attenuated the stimulation of protein synthesis although isoprenaline did not cause cellular alkalization. In conclusion, the growth response to different hypertrophic stimuli, namely alpha- or beta-adrenoceptor stimulation, is attenuated in the presence of the NHE-1 inhibitor HOE694 and this inhibition is independent from cellular alkalization.

Crosstalk between cytosolic pH and intracellular calcium in human lymphocytes:: Effect of 4-aminopyridin, ammoniun chloride and ionomycin

Stimulation of lymphocytes by specific antigens is followed by the activation of different signal transduction mechanisms, such as alterations in the cytoplasmic levels of Ca 2+, H + and variations in membrane potential. To study interrelationships among these parameters, changes in pHi and Ca 2+ were measured with the fluorescent probes BCECF and Fura-2 in freshly isolated blood human lymphocytes. Moreover, membrane potential qualitative alterations were recorded with the fluorescent dye bis-oxonol. In a bicarbonate-free medium, cell alkalinization with NH 4Cl slightly decreased intracellular Ca 2+ concentration ([Ca 2+] i) due to efflux of Ca 2+ from the cell. In contrast, an elevation of pHi induced with 4-AP increased [Ca 2+] i, either in the presence or absence of external Ca 2+. The increase in Ca 2+-free medium is likely to be due to Ca 2+ release from thapsigargin and caffeine-independent intracellular stores. Both 4-AP or NH 4Cl induced a plasma membrane depolarisation, although with different kinetics. The ionosphere ionomycin increased pHi, Ca 2+ levels and also induced membrane depolarisation. Together, these observations demonstrate a lack of correlation between the magnitude of changes in pHi and Ca 2+.

Sarcolemmal Na+/H+ exchanger activity and expression in human ventricular myocardium

OBJECTIVESTo determine sarcolemmal Na+/H+ exchanger (NHE) activity and expression in human ventricular myocardium.BACKGROUNDAlthough the sarcolemmal NHE has been implicated in various physiological and pathophysiological phenomena in animal studies, its activity and expression in human myocardium have not been studied.METHODSVentricular myocardium was obtained from unused donor hearts with acute myocardial dysfunction (n = 5) and recipient hearts with chronic end stage heart failure (n = 11) through a transplantation program. Intracellular pH (pHi) was monitored in enzymatically isolated single ventricular myocytes by microepifluorescence. As the index of sarcolemmal NHE activity, the rate of H+ efflux at a pHi of 6.90 (JH6.9) was determined after the induction of intracellular acidosis in bicarbonate-free medium. Na+/H+ exchanger isoform 1 (NHE1) expression in ventricular myocardium was determined by immunoblot analysis.RESULTSHuman ventricular myocytes exhibited readily detectable sarcolemmal NHE activity after the induction of intracellular acidosis, and this activity was suppressed by the NHE1-selective inhibitor HOE-642 (cariporide) at 1 μmol/L. Sarcolemmal NHE activity of myocytes was significantly greater in recipient hearts (JH6.9 = 1.95 ± 0.18 mmol/L/min) than it was in unused donor hearts (JH6.9 = 1.06 ± 0.15 mmol/L/min). In contrast, NHE1 protein was expressed in similar abundance in ventricular myocardium from both recipient and unused donor hearts.CONCLUSIONSSarcolemmal NHE activity of human ventricular myocytes arises from the NHE1 isoform and is inhibited by HOE-642. Sarcolemmal NHE activity is significantly greater in recipient hearts with chronic end-stage heart failure than it is in unused donor hearts, and this difference is likely to arise from altered posttranslational regulation.

Importance of bicarbonate transport for protection of cardiomyocytes against reoxygenation injury.

Isolated cardiomyocytes from adult rats were incubated in anoxic bicarbonate-buffered media at extracellular pH (pH(o)) 6.4 until a cytosolic Ca(2+) overload and intracellular pH (pH(i)) of 6.4 were reached. On reoxygenation, the pH of the medium was changed to 7.4 to activate the Na(+)/H(+)exchanger (NHE) and the Na(+)-HCO(-)(3) symporter (NBS). The reoxygenation was performed in the absence or presence of the NHE inhibitor HOE-642 (3 micromol/l) and/or the NBS inhibitor DIDS (0.5 mmol/l), as in bicarbonate-free media. In reoxygenated control cells pH(i) rapidly recovered to the preanoxic level, and a burst of spontaneous oscillations of cytosolic Ca(2+) occurred, accompanied by the development of hypercontracture. When NBS and NHE were simultaneously inhibited during reoxygenation, pH(i) recovery was prevented, Ca(2+) oscillations were attenuated, and hypercontracture was abolished. Sole inhibition of NBS or NHE showed no protection against hypercontracture. In the absence of cytosolic acidosis, HOE-642 or DIDS did not prevent hypercontracture induced by Ca(2+) overload. The results demonstrate that simultaneous inhibition of NHE and NBS is needed to protect myocardial cells against reoxygenation-induced hypercontracture.

Comparison of Glycerol, Other Polyols, Trehalose, and Raffinose to Provide a Defined Cryoprotectant Medium for Mouse Sperm Cryopreservation

Most procedures for mouse sperm cryopreservation have utilized raffinose to provide hypertonicity for cell desiccation prior to freezing and glycerol to block intracellular ice formation. Trehalose has been shown in other cell systems to provide positive protection to the plasma membrane and so was examined as a replacement for raffinose. Comparison of 3 and 6% glycerol and 7.5 and 20% sugar showed that 6% glycerol and 7.5% sugar gave maximal protection consistently and so were adopted as standard. Comparison of raffinose and trehalose at this concentration showed trehalose to give significantly better recovery of intact cells: 48 ± 6% for trehalose, 36 ± 9% for raffinose (± SE,n= 5; arc sine transformed data;P< 0.01). Less hydrophilic polyols should prove more permeant to the membrane than glycerol, enter the cell rapidly, and so possibly inhibit lethal intracellular ice formation effectively. We hypothesized that one of these polyols plus glycerol would be a more effective cryoprotectant than glycerol alone. The polyols tested as supplements to 6% glycerol were propane-1,2-diol, propane-1,3-diol, 1,1,1-tris-(hydroxymethyl)ethane (THME), and 2-ethyl-2-(hydroxymethyl)-propane-1,3-diol (EHMP). With 6% glycerol and 7.5% raffinose or trehalose, the two diols and THME gave less cryoprotection than with glycerol alone, and EHMP reduced postthaw membrane integrity to nil, thus invalidating the hypothesis. Comparison of bicarbonate-containing medium MJB to bicarbonate-free medium NTP, both with 6% glycerol/7.5% trehalose, showed no difference in recovery of membrane-intact cells. For ease of pH maintenance, NTP was chosen for studies of addition prefreeze and removal postthaw of 6% glycerol/7.5% trehalose cryoprotectant within vitrofertilization as endpoint. Three protocols for cryoprotectant handling were tested: serial addition/dilution; dialysis addition and removal; and dialysis addition and direct insemination without cryoprotectant removal. The last proved significantly superior (P< 0.01), giving 62% fertilized eggs, normalized to controls, compared to 21% for dialysis addition and removal and 32% for serial addition and dilution. The glycerol/trehalose combination thus provides a defined cryoprotectant which, when used with addition by dialysis prefreeze and direct insemination postthaw, yields a satisfactory yield of fertilized eggs in anin vitrofertilization system.

Alpha1-adrenergic stimulation of sarcolemmal Na+-H+ exchanger activity in rat ventricular myocytes: evidence for selective mediation by the alpha1A-adrenoceptor subtype.

Alpha1-adrenoceptor (alpha1-AR) stimulation increases sarcolemmal Na+-H+ exchanger (NHE) activity. The present study was designed to determine the role(s) of alpha1-AR subtype(s) in mediating this response. As an index of NHE activity, acid efflux rates (JHs) were determined in single rat ventricular myocytes loaded with the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1 after 2 consecutive intracellular acid pulses in bicarbonate-free medium. JH at pHi 6.90 did not change significantly during the second pulse relative to the first in control cells but increased in a dose-dependent manner when the second pulse occurred in the presence of phenylephrine (nonselective alpha1-AR agonist) or A61603 (alpha1A-AR-selective agonist), with EC50 values of 1.24 micromol/L and 3.6 nmol/L, respectively (both agonists given together with 1 micromol/L atenolol). Stimulation of NHE activity by 10 micromol/L phenylephrine was inhibited in a dose-dependent manner by the competitive antagonists prazosin, WB4101, and 5-methylurapidil, with IC50 values of 12, 32, and 149 nmol/L, respectively. Analyses of the relative EC50 and IC50 values obtained (and Ki values estimated from the antagonist IC50s) in relation to the relative potencies of these agents at native rat alpha1-AR subtypes and their relative affinities for recombinant rat alpha1-ARs suggest that alpha1-adrenergic stimulation of sarcolemmal NHE activity is likely to be mediated selectively by the alpha1A-AR.