Cellular Acidification Research Articles

Transporteurs ioniques dans les pathologies cardiovasculaires : contrôle du pH ou régulation de la concentration calcique intracellulaire

The regulation of the intracellular pH is under tight control by several ion transport systems including the sodium–proton exchanger, the sodium–bicarbonate cotransporter and the chlore–bicarbonate anion exchanger. While the activation of the anion exchange induces a cellular acidification, both the sodium–proton exchanger and the sodium–bicarbonate cotransporter are responsible for a protection against acidosis by extruding protons or importing bicarbonate. These transporters are transmembrane proteins whose activity is regulated by several mechanisms including phosphorylation, calcium binding and which are involved in several pathophysiologic processes such as ischemia, hypertrophy and arrhythmias. Recent studies suggest that the activation of these transporters during various diseases induces an increase in intracellular calcium concentration. Therefore, inhibiting these transporters could represent novel therapeutic strategies for the treatment of cardiovascular diseases.

Cyclic AMP inhibits caspase-8-mediated, pH-dependent, apoptosis by attenuating cellular acidification

Cyclic AMP inhibits caspase-8-mediated, pH-dependent, apoptosis by attenuating cellular acidification

The NHX Family of Na+-H+ Exchangers in Caenorhabditis elegans

Na+-H+ exchangers prevent cellular acidification by catalyzing the electroneutral exchange of extracellular sodium for an intracellular proton. To date, seven Na+-H+ exchangers have been identified in mammals, and although several members of this family have been extensively studied and characterized, it is clear that there are major gaps in our understanding with respect to the remaining family members. To initiate the study of Na+-H+ exchangers in a genomically defined and genetically tractable model system, we have cloned the complete cDNAs and analyzed splice site variation for nine putative homologs from the nematode Caenorhabditis elegans, which we have called NHX-1 through -9. The expression patterns and cellular distributions of the NHX proteins were determined using transcriptional and translational promoter-transgene fusion constructs to green fluorescent protein. Four of the putative exchangers were expressed at the cell surface, whereas five of the exchangers were associated with the membranes of intracellular organelles. Individual isoforms were expressed exclusively in the intestine, seam cells, hypodermal cells of the main body syncytium, and the excretory cell, all of which are polarized epithelial cells, suggesting a role for these proteins in epithelial membrane transport processes in the nematode. Other isoforms were found to express either ubiquitously or in a pan-neural pattern, suggesting a more conserved role in cell pH regulation or neuronal function. Finally, we show that recombinant NHX-4, the ubiquitous nematode Na+-H+ exchanger, mediates Na+-dependent pH recovery after intracellular acidification. NHX-4 has a K(a) for Na+ of approximately 32 mm, is not Cl- -dependent, and is relatively insensitive to the amiloride analog EIPA.

Involvement of Na+-HCO3- cotransporter in mediating cyclic adenosine 3',5'-monophosphate-dependent HCO3- secretion by mouse endometrial epithelium.

The present study investigated the involvement of Na+-HCO3- cotransporter in mediating cAMP-stimulated HCO3- secretion across the cultured mouse endometrial epithelium using the short-circuit current (I(SC)) technique and intracellular pH measurement. Forskolin stimulated a rise in the I(SC), 55.6% and 52.1% of which could be reduced by the removal of extracellular Cl- or by eliminating the contribution of Cl- secretion by bumetanide, an inhibitor of Na+-K+-2Cl- cotransporter, respectively. More than 80% reduction in the forskolin-induced I(SC) was obtained when both Cl- and HCO3- in the bath were removed or in HCO3--free solution with bumetanide, indicating that the I(SC) depended on both Cl- and HCO3-. The presence of the Na+ channel-blocker amiloride in the apical solution did not reduce the forskolin-induced I(SC); however, the I(SC) could be abolished by removing Na+ from the bathing solution, suggesting that the Cl-- and HCO3--dependent I(SC) was also dependent on basolateral Na+. The forskolin-stimulated I(SC) could be reduced 43.6% by removal of HCO3- and 47.9% by a Na+-HCO3--cotransporter inhibitor, dihydrogen-4,4'-didsothiocyanostilbene-2,2'-disulfonic acid (H2DIDS). The inhibitory effect of H2DIDS was observed in Cl--free solution, but not when HCO3- was removed, thus confirming its effect on HCO3--dependent transport. Intracellular pH measurements demonstrated that the recovery from cellular acidification depended on the presence of both basolateral Na+ and HCO3-, further indicating the involvement of Na+-HCO3- cotransporter. Reverse transcription-polymerase chain reaction experiments confirmed the expression of Na+-HCO3- cotransporter in the mouse endometrium. The results suggest that basolaterally located Na+-HCO3- cotransporter is involved in mediating cAMP-stimulated HCO3- secretion across the mouse endometrial epithelium.

Vinyl acetate-induced intracellular acidification: implications for risk assessment.

Cancerbioassays have demonstrated the carcinogenic activity of vinyl acetate in rodents. Tumors appear only at the site of contact and mechanistic data suggest that the carcinogenic mechanism involves carboxylesterase-mediated metabolism of vinyl acetate to acetic acid. It has been hypothesized that intracellular formation of acetate causes a reduction of intracellular pH (pH(i)) at noncytotoxic levels, but that prolonged exposure to reduced pH(i) is cytotoxic and/or mitogenic and drives proliferative responses. Coupled with exposure to metabolically formed acetaldehyde at high administered concentrations, nonlinear dose-response curves for epithelial tumors are produced. Freshly isolated rat hepatocytes were used as a model system to test the concept that exposure of cells to vinyl acetate causes a reduction in pH(i). Quantitative fluorescence imaging ratio microscopy showed that exposure of hepatocytes to vinyl acetate concentrations ranging from 10 to 1000 microM caused rapid and sustained reductions of approximately 0.03 to 0.65 pH units. Cellular acidification was rapidly reversed to control pH(i) upon removal of vinyl acetate. There was minimal accumulation of protons during the exposure period, as suggested by minor differences in pH(i) of cells with or without prior exposure to vinyl acetate. The effect of vinyl acetate on pH(i) was attenuated by prior exposure to the carboxylesterase inhibitor bis(p-nitrophenyl)phosphate. These results support the concept that intracellular acidification is a sentinel pharmacodynamic response of cells to vinyl acetate exposure and that pH(i) is an appropriate metric dose for use in quantitative risk assessments of cancer and noncancer human health risk assessment.

Basolateral membrane Cl(-)-, Na(+)-, and K(+)-coupled base transport mechanisms in rat MTALH.

Mechanisms involved in basolateral HCO transport were examined in the in vitro microperfused rat medullary thick ascending limb of Henle (MTALH) by microfluorometric monitoring of cell pH. Removing peritubular Cl(-) induced a cellular alkalinization that was inhibited in the presence of peritubular 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and blunted in the absence of external CO(2)/HCO. The alkalinization elicited by removing peritubular Cl(-) persisted in the bilateral absence of Na(+), together with a voltage clamp. When studied in Cl(-)-free solutions, lowering peritubular pH induced a base efflux that was inhibited by peritubular DIDS or by the absence of external CO(2)/HCO. Removing peritubular Na(+) elicited a cellular acidification that was accounted for by stimulation of a DIDS- and ethylisopropylamiloride (EIPA)-insensitive Na(+)-HCO cotransport and inhibition of a basolateral Na(+)/H(+) exchange. Increasing bath K(+) induced an intracellular alkalinization that was inhibited in the absence of external CO(2)/HCO. At 2 mM, peritubular Ba(2+), which inhibits the K(+)-Cl(-) cotransport, did not induce any change in transepithelial voltage but elicited a cellular alkalinization and inhibited K(+)-induced cellular alkalinization, consistent with the presence of a basolateral, electroneutral Ba(2+)-sensitive K(+)-Cl(-) cotransport that may operate as a K(+)-HCO cotransport. This cotransport was inhibited in the peritubular presence of furosemide, [(dihydroindenyl)oxy]alkanoic acid, 5-nitro-2-(3-phenylpropylamino)benzoate, or DIDS. At least three distinct basolateral HCO transport mechanisms are functional under physiological conditions: electroneutral Cl(-)/HCO exchange, DIDS- and EIPA-insensitive Na(+)-HCO cotransport, and Ba(2+)-sensitive electroneutral K(+)-Cl(-)(HCO) cotransport.

Modulation of nitrate reductase activity in cucumber ( Cucumis sativus) roots

Nitrate reductase (NR) (EC 1.6.6.1) activity and NR activation state, i.e. activity in the presence of Mg 2+ relative to activity in the absence of Mg 2+, in cucumber ( Cucumis sativus) leaves increased in the light and decreased in the dark. In contrast to leaves, NR activation state in the roots did not show light/dark-dependent changes. Root NR was activated by anoxia or by addition of uncoupler (CCCP) or mannose. These treatments decreased ATP levels in root tissue. On the contrary, high oxygen supply promoted some NR inactivation. When an extract from anoxic roots was preincubated with ATP, NR was gradually inactivated. Subsequent addition of 5′-AMP resulted in a remarkable reactivation of the enzyme. NR extracted from hyperoxygenated roots was activated by preincubation with 5′-AMP, and the process was reversed by ATP. These results suggest the participation of adenine nucleotides on the in vivo modulation of NR activity in cucumber roots. NR was activated in vivo by cellular acidification and inactivated by alkalinisation. The acid-induced activation of NR was greatly prevented by okadaic acid, a protein phosphatase inhibitor. Our data indicate that, as in barley roots, anoxia, uncouplers, and mannose feeding activate cucumber root NR, at least partly, by enhancing NR dephosphorylation via a decrease in the internal level of ATP and a concomitant cellular acidification.

Simultaneous measurement of cellular respiration and acidification with a single CMOS ISFET

In vivo, the pH value and oxygen partial pressure are the most important physico–chemical parameters in the microenvironment of human tissues. In vitro, the extracellular acidification rate of cell cultures is an indicator of global cellular metabolism, while the rate of oxygen consumption is a measure of mitochondrial activity. Earlier approaches had the disadvantage that these two values had to be measured with two separate sensors at different loci within the tissue or cell culture. Furthermore, conventional Clark-type oxygen sensors are not very compatible for miniaturisation, making it impossible to measure at small cell volumes or even at the single cell level. We have, therefore, developed an ISFET based sensor structure which is able to measure both pH and oxygen partial pressure. This sensor structure was tested in vitro for simultaneous records of cellular acidification and respiration rates at the same site within the cell culture. This sensor is manufactured by a CMOS-process.

Ortho-substituted PCB95 alters intracellular calcium signaling and causes cellular acidification in PC12 cells by an immunophilin-dependent mechanism.

ortho-Substituted PCBs mobilize Ca2+ from isolated brain microsomes by interaction with FKBP12/RyR complexes. Investigation into the cellular importance of this mechanism was undertaken using PC12 cells by fluoroimaging the actions of specific PCB congeners on [Ca2+]i and pH. RyR and IP3R share a common intracellular Ca2+ store in PC12 cells. Perfusion of nM to low microM PCB95 caused a transient rise of [Ca2+]i that was not completely dependent on extracellular Ca2+. Pre-incubation of the cells with ryanodine or FK506 completely eliminated PCB95 responses, suggesting a primary action on the FKPP12/RyR-sensitive store. PCB95, but not PCB126, induced a gradual decrease in cytosolic pH that could be completely eliminated by FK506 pre-incubation of the cells. Direct respiration measurement using isolated brain mitochondria demonstrated that neither of the PCBs directly altered any stage of mitochondrial respiration. These results revealed that PCB95 disrupts intracellular Ca2+ signaling in PC12 cells by interaction with the FKBP12/RyR complex that in turn accelerated cellular metabolism, possibly affecting signaling between ER and mitochondria. Since ortho-substituted PCBs have been shown to be neurotoxic and may affect neurodevelopment, studies on the molecular mechanism by which they alter cellular signaling may provide valuable information on the physiological roles of FKPB12 and RyR on neuronal functions.

Internalization of adenovirus by alveolar macrophages initiates early proinflammatory signaling during acute respiratory tract infection.

Adenovirus is a common respiratory pathogen which causes a broad range of distinct clinical syndromes and has recently received attention for its potential for in vivo gene delivery. Although adenovirus respiratory tract infection (ARTI) results in dose-dependent, local inflammation, the pathogenesis of this remains unclear. We hypothesized that alveolar macrophages (AMphi) rapidly internalize adenovirus following in vivo pulmonary administration and then initiate inflammatory signaling within the lung. To evaluate the role of AMphi in the induction of lung inflammation during ARTI in vivo, we directly assessed adenovirus uptake by murine AMphi and correlated uptake with the initiation of proinflammatory gene expression. Stimulation of cytokine (tumor necrosis factor alpha [TNF-alpha], interleukin-6 [IL-6], macrophage inflammatory protein-2 [MIP-2], and MIP-1alpha) expression in the lung was evaluated at the level of mRNA (by reverse transcription-PCR [RT-PCR]) and protein (by enzyme-linked immunosorbent assay) and by identification of cells expressing TNF-alpha and IL-6 mRNA in lung tissues (by in situ hybridization) and isolated lung lavage cells (by RT-PCR). Adenovirus, labeled with the fluorescent dye (Cy3), was rapidly and widely distributed on epithelial surfaces of airways and alveoli and was very rapidly ( approximately 1 min) localized within AMphi. At 30 min after infection AMphi but not airway epithelial or vascular endothelial cells expressed mRNA for TNF-alpha and IL-6, thus identifying AMphi as the cell source of initial cytokine signaling. IL-6, TNF-alpha, MIP-2, and MIP-1alpha levels progressively increased in bronchoalveolar lavage fluid after pulmonary adenovirus infection, and all were significantly elevated at 6 h (P < 0.05). To begin to define the molecular mechanism(s) by which adenovirus initiates the inflammatory signaling in macrophages, TNF-alpha expression from adenovirus-infected RAW264.7 macrophages was evaluated in vitro. TNF-alpha expression was readily detected in adenovirus-infected RAW cell supernatant with kinetics similar to AMphi during in vivo infection. Blockage of virus uptake at specific cellular sites, including internalization (by wortmannin), endosome acidification and/or lysis (by chloroquine) or by Ca(2+) chelation (by BAPTA) completely blocked TNF-alpha expression. In conclusion, results showed that during ARTI, (i) AMphi rapidly internalized adenovirus, (ii) expression of inflammatory mediators was initiated within AMphi and not airway epithelial or other cells, and (iii) the initiation of inflammatory signaling was linked to virion uptake by macrophages occurring at a point after vesicle acidification. These results have implications for our understanding of the role of the AMphi in the initiation of inflammation following adenovirus infection and adenovirus-mediated gene transfer to the lung.

Sensitivity of Mechanical and Metabolic Functions to Changes in Coronary Perfusion: A Metabolic Basis of Perfusion–Contraction Coupling

Experimental evidence indicates a metabolic basis of contraction–perfusion coupling during an increase in cardiac work load. This study aims to characterize adjustment of myocardial energy metabolism in response to acute low flow ischemia (LFI), and to determine its involvement in perfusion–contraction coupling. Intracellular parameters were measured in isolated rat hearts by NMR spectroscopy and biochemical methods during 30 min of graded LFI and reperfusion as compared to continuous perfusion (control). Oxygen pressure was set to reach maximal oxygen extraction at 70% coronary flow rate (CFR), therefore oxygen limitation was proportional to coronary underperfusion. At 69, 38 and 10% CFR left ventricular pressures decreased to 71, 43 and 25% of pre-ischemic values respectively (P<0.005 v 97% in control) without an increase in diastolic tone, and recovered to 92±3% after 30 min of reperfusion. Despite hydrolysis of high energy phosphates and cellular acidification, ADP concentrations were stable in underperfused hearts. At 69, 38 and 10% CFR, cytosolic phosphorylation potentials (PP) decreased from 74±10 m M−1during pre-ischemia to 40±6, 25±4 and 14±4 m M−1respectively (P<0.05 v 63±9 m M−1in control), and lactate efflux increased to 256±18, 386±22 and 490±43 μ mol/gdw respectively (P<0.005 v 186±22 μ mol/gdw in control). Glycogen contents decreased (P<0.005 v control) and accounted for 27–30% of lactate efflux. These results indicate: (a) proportionate depression of contraction force and glycogen contents, and increased glucose uptake and anaerobic energy production in the underperfused myocardium. Coordinated modulation of these parameters attributes cytosolic PP a regulatory function; (b) resetting of cytosolic PP to lower levels mediates perfusion–contraction coupling during graded LFI. The data are consistent with the concept that glycolytic energy production improves myocardial tolerance to ischemia.

Oxygen and Light Accelerate Recovery from SO 2-induced Inhibition of Leaf Photosynthesis and from Cytoplasmic Acidification

Leaves of several plant species were illuminated either in air or in a gas atmosphere with reduced oxygen content, and photosynthesis and transpiration were recorded after brief exposure to high concentrations of SO2. Inhibition of photosynthesis by SO2 was similar in air and at reduced oxygen concentrations or, in some experiments, even larger under low oxygen. Recovery from inhibition was always faster in high than in low oxygen. In leaves of Pelargonium zonale, stomata remained open or closed only slowly after fumigation, whereas in spinach and potato leaves stomata closed rapidly under the influence of high SO2. After cessation of fumigation, stomata always reopened. pH-indicating fluorescent dyes were employed to monitor SO2-dependent pH changes in the apoplast and the cytosol of leaf tissue. In the cytosol, acidification was weaker in the dark than in the light. In the light, it was stronger in 21 % oxygen than in 1% oxygen. Cytosolic acidification could not fully be attributed to the hydration and oxidation of SO2 but was also caused by a transient breakdown of transmembrane proton gradients that resulted in the influx of acid from acidic leaf compartments into the cytosol. This was concluded from opposite pH changes in the cytosol and the apoplast under the influence of SO2. Even chloroplasts were transiently acidified by SO2 as shown by the kinetics of 505 nm absorption, which reflect the pH-sensitive interconversion of violaxanthin to zeaxanthin. Differences in the kinetics of SO2-dependent inhibition of photosynthesis and of cytosolic acidification in high and low oxygen suggest combined nucleophilic attack of sulfite, and of radicals generated in the light, on sensitive cellular constituents. However, the reversibility of photosynthesis inhibition and of acidification, particularly in the presence of air levels of oxygen, demonstrates fast repair. The observations suggest that detrimental effects on plants caused by long-term exposure to ambient concentrations of SO2 which are several hundredfold lower than used in the present investigation, can neither be explained by radical damage nor by immediate effects of cellular acidification. Both radical detoxification and pH regulation are highly effective in healthy plants.

Relation between intracellular acidification and camptothecin-induced apoptosis in leukemia cells

Leukemia cells (HL-60 and P388) treated with the topoisomerase I inhibitor camptothecin (CPT) undergo rapid apoptosis as judged from internucleosomal degradation of genomic DNA, morphological changes and flow cytometry analysis. The intracellular free calcium concentration is not affected by the treatment with a high dose of CPT. In contrast, fluorescence measurements of cells loaded with the pH indicator BCECF-AM indicate that the intracellular pH decreases significantly. Incubation of the leukemia cells with a high drug concentration for 5 h or with lower drug concentrations for 15 h results in a pronounced intracellular acidification. Measurements with the whole cell population show a decrease of 0.3–0.4 pH units. The extent of the acidic shift is proportional to the drug concentration and the period of incubation. No such effects were observed with P388CPT5 cells resistant to CPT. The results support the hypothesis that apoptosis induced in leukemia cells by CPT is associated with decreased intracellular pH. Modification of intracellular pH by topoisomerase inhibitors is viewed as an essential event responsible for the induction and/or propagation of apoptosis. The role of CPT-induced cellular acidification in the mechanism of action of the drug is discussed.

Sodium overload through voltage-dependent Na + channels induces necrosis and apoptosis of rat superior cervical ganglion cells in vitro

Using the failure to exclude trypan blue as a criterion for cell death, we found that veratridine, the voltage-dependent Na + channel activator, exerted its toxicity to cultured sympathetic neurons in a dose-dependent manner (half-maximal toxicity occurred at 2 μM). The co-presence of tetrodotoxin completely reversed the toxicity only at concentrations of veratridine <20μM. Veratridine neurotoxicity was due to the influx of Na +; a medium low in Na + (36 mM) completely abolished its neurotoxicity, whereas a Ca 2+-free medium did not attenuate its neurotoxicity. Furthermore, the buffering action of 1,2-Bis-(2-aminophenoxy)ethane-N,N,N′,N′,-tetraacetate (BAPTA) on veratridine-induced increase in intracellular Ca 2+ levels neither blocked veratridine neurotoxicity in normal medium, nor attenuated the low Na + effect. Elevated K + effectively blocked veratridine neurotoxicity in a Ca 2+-dependent manner. Cytoplasmic pH measurements using a fluorescent pH indicator demonstrated that cellular acidification (from pH 7.0 to pH 6.5) occurred upon treatment with veratridine. Both veratridine-induced acidification and cell death were ameliorated by 5-(N-ethyl-N-isopropyl)amiloride, the specific inhibitor of the Na +/H + exchanger (IC 50 = 0.5 μM). Finally, necrosis occurred predominantly in veratridine neurotoxicity, but both staining with bis-benzimide and TUNEL analysis showed nuclear features of apoptosis in sympathetic neurons undergoing cell death.

Efficient Limitation of Intracellular Edema and Sodium Accumulation by Cardioplegia is Dissociated from Recovery of Rat Hearts from Cold Ischemic Storage

Energy deficiency and disturbances of sodium and water homeostasis are considered as mechanisms of injury during hypothermic preservation of cardiac muscle. The present study attempts to characterize the effect of potassium (K+) and magnesium (Mg2+) cardioplegia on these mechanisms. Cellular parameters were measured by multinuclear NMR spectroscopy in isolated rat hearts during 12 h of ischemia at 4°C and 2 h of normothermic reperfusion with an isoosmotic Krebs-Henseleit (KH) solution. Potassium and magnesium cardioplegia (a) reduced the rate of ATP hydrolysis and cellular acidification during early stages of ischemia; (b) caused an early cessation of the phase of fast sodium influx after 40 min (P<0.001 vs 120 min with KH); (c) reduced intracellular sodium accumulation to 148–165μmol/gdw after 12 h (P<0.01 vs 268±15μmol/gdw with KH); (d) decreased ischemic volumes to 2.7±0.1 and 2.8±0.1 ml/gdw after 8 and 12 h of storage, respectively (P<0.005 v 3.0 and 3.3 ml/gdw with KH). Quantitative analysis of these parameters showed that both hypothermia and cardioplegia increased the relative contribution of sodium to intracellular water accumulation by a factor of 2–2.5. In view of the marked reduction in absolute sodium and water contents, the data indicate that cold cardioplegia limits the increase in intracellular osmolarity. Myocardial mechanical and metabolic recoveries, and cellular viability deteriorated during prolongation of the ischemic period from 8 to 12 h in all experimental groups (P<0.005). Reperfusion was efficient in reversing intracellular sodium and water accumulation in hearts stored with cardioplegia, in contrast to hearts stored in KH. Magnesium, but not potassium cardioplegia, lowered interstitial water contents (P<0.01 v KH), increased intracellular magnesium concentrations (P<0.001), improved mechanical and metabolic recoveries (P<0.01) and cellular viability (P<0.001). These results indicate (a) cardioplegia reduces intracellular sodium (by 46%) and water accumulation (by 66%) during cold ischemia; (b) both hypothermia and cardioplegia limit the rise in intracellular osmolarity and increase the contribution of sodium to cellular swelling; (c) intracellular sodium and water contents were dissociated from myocardial viability and recovery from cold ischemia in potassium and magnesium cardioplegic solutions. It is concluded that intracellular sodium and water accumulation are not dominant factors in determination of cardiac outcome from ischemia.

Direct Effects of Dopamine on Colonic Mucosal pH: Implications for Tonometry

Tonometric measurements of colonic and gastric mucosa pH are used as indirect determinants of splanchnic perfusion in shocked patients or those undergoing aortic cross-clamp. Mucosal acidification in response to splanchnic vasodilators such as dopamine has been assumed to signify ischemia. However, cellular acidification may occur independent of oxygenation and the direct effects of dopamine on mucosal acid-base are unknown.We examined the effects of dopamine on cellular pH (independent of oxygenation) of intestinal mucosain vitro.Crypts isolated from the distal colon of Sprague-Dawley rats were loaded with a pH-sensitive fluorescent probe, perfused with a Hepes-buffered Ringers solution, and imaged with confocal laser scanning microscopy. In separate experiments, crypts were loaded with a calcium-sensitive probe (Fura-2) and concentrations of free cytosolic calcium were measured with fluorescence imaging.Dopamine perfusion produced a reversible cytosolic acidification of crypts which was not significantly affected by (i) the nominal absence of bicarbonate, (ii) α- and β-adrenergic receptor blockade, or (iii) protein kinase C inhibition. Dopamine did not significantly affect intracellular calcium concentrations. However, dopamine-induced acidification was inhibited by (a) blocking sodium-hydrogen exchange with amiloride, (b) prior exposure to adenosine 3′, 5′-cyclic monophosphate (cAMP), or (c) protein kinase A blockade (allP< 0.01).Dopamine directly acidifies mucosal crypt cells in a mechanism that involves a cAMP-mediated inhibition of sodium-hydrogen exchange. This finding accounts for the acidification of intestinal mucosa during low-dose dopamine infusion despite a demonstrable improvement in splanchnic perfusion. Direct mucosal effects of pharmacological agents must be considered in the evaluation of perfusion parameters based on tonometric data.

Effect of acidification on the location of H+-ATPase in cultured inner medullary collecting duct cells.

In previous studies, our laboratory has utilized a cell line derived from the rat inner medullary collecting duct (IMCD) as a model system for mammalian renal epithelial cell acid secretion. We have provided evidence, from a physiological perspective, that acute cellular acidification stimulates apical exocytosis and elicits a rapid increase in proton secretion that is mediated by an H+-ATPase. The purpose of these experiments was to examine the effect of acute cellular acidification on the distribution of the vacuolar H+-ATPase in IMCD cells in vitro. We utilized the 31-kDa subunit of the H+-ATPase as a marker of the complete enzyme. The distribution of this subunit of the H+-ATPase was evaluated by immunohistochemical techniques (confocal and electron microscopy), and we found that there is a redistribution of these pumps from vesicles to the apical membrane. Immunoblot evaluation of isolated apical membrane revealed a 237 +/- 34% (P < 0.05, n = 9) increase in the 31-kDa subunit present in the membrane fraction 20 min after the induction of cellular acidification. Thus our results demonstrate the presence of this pump subunit in the IMCD cell line in vitro and that cell acidification regulates the shuttling of cytosolic vesicles containing the 31-kDa subunit into the apical membrane.

Nitrate reductase in higher plants: A case study for transduction of environmental stimuli into control of catalytic activity

In higher plants, cytosolic NAD(P)H‐nitrate reductase (NR) is rapidly modulated by environmental conditions such as light, CO2, or oxygen availability. In leaves, NR is activated by photosynthesis, reaching an activation state of 60–80%. In the dark, or after stomatal closure, leaf NR is inactivated down to 20 or 40% of its maximum activity. In roots, hypoxia or anoxia activate NR, whereas high oxygen supply inactivates NR. Spinach leaf NR is inactivated by phosphorylation of serine 543 and subsequent Mg2+‐dependent binding of 14‐3‐3 proteins at, or close to, this phosphorylation site. At least three different protein kinases (NR‐PK) have been identified in spinach leaves that are able to phosphorylate NR on serine 543. Two of them show up as calmodulin‐like domain protein kinases (CDPKs), and one as a SNF1‐like protein kinase. Dephosphorylation of serine 543 is catalyzed by a Mg2+‐dependent protein phosphatase and by a type 2A protein phosphatase (NR‐PP), which is regulated by a trimer/dimer interconversion. The NR‐PKs, NR‐PPs, and 14‐3‐3s are present even in NR‐depleted plant tissues. Artificial activation of NR in vivo is achieved by cellular acidification, by respiratory inhibitors, or by mannose feeding. As for anoxia, these treatments seem to act, at least in part, via cytosolic acidification, mediated by low cytosolic ATP levels. Activation is also achieved by ionophore‐induced release of divalent cations from the cytosol. In addition, cytosolic AMP and phosphate esters seem to regulate NR‐PK and NR‐PP activities, thereby adapting NR activity within minutes to the changing environment.

Activation of the cloned human NK3 receptor in Chinese Hamster Ovary cells characterized by the cellular acidification response using the Cytosensor microphysiometer.

1. The aim of the present study was to validate the Cytosensor microphysiometer, a novel system that measures the extracellular acidification rate as a reliable index of the integrated functional response to receptor activation, as a method for studying NK3 receptor pharmacology, and then to use this system to assess the functional activity of novel compounds at this receptor. 2. The selective NK3 agonist senktide caused reproducible, concentration-related increases in acidification ratein CHO-NK3 cells, with a pEC50 value of 8.72+/-0.11 (n=15). [Beta-Ala8]NKA(4-10), the selective NK2 agonist, elicited a much weaker response (pEC50=6.68+/-0.08, n=4), while the NK1-selective agonist substance P methylester only caused a very weak response at concentrations > or =3 microM (n=2). The rank order of potency for the endogenous tachykinins NKB>NKA>substance P (n=3) confirmed the response was mediated by the NK3 receptor. Moreover, the actual potencies obtained were consistent with affinities measured in radioligand binding studies. 3. The novel compounds PD156319-121 (0.3-1 microM), PD161182 (10-300 nM), PD168001 (10-100 nM) and PD168073 (10-100 nM) all acted as surmountable antagonists of the senktide-induced acidification response, with pA2 values of 7.49, 8.67, 9.17 and 9.25 respectively (n=3-5). In comparison the known NK3 antagonist SR142801 (10-100 nM) had a pA2 value of 8.83 (n=8) for the interaction with senktide. Again, these values are consistent with the radioligand binding data. 4. Amiloride (1 mM) inhibited the senktide-induced acidification response by 68.3+/-3.3 (n=4), indicating that the Na+/H+ antiporter plays an important role in this response, and this is consistent with the importance of this antiporter in other acidification responses. 5. Inhibition of protein kinase C with staurosporine (0.1 microM), or depletion of the intracellular Ca2+ stores with thapsigargin (1 microM), both resulted in a reduction in the maximum response to senktide (63.3+/-1.7 and 68.9+/-3.2% respectively, n=3-5), and co-application of these inhibitors abolished the response (n=3). This strongly suggested that the NK3 receptor was coupling via phospholipase C (PLC), as would be expected, although this could not be confirmed by the use of the putative PLC/PLA2 inhibitor U73122. 6. In conclusion, we have demonstrated the utility of the Cytosensor in the characterization of functional responses to agonists, and assessment of the affinities of antagonists in CHO cells expressing the human NK3, and have shown that our series of novel compounds are non-peptide NK3 antagonists of high affinity, as exemplified by PD168073.

Effect of ambient SO2 levels on S fractions in Pinus sylvestris foliage growing in the subarctic

The total S and SO4‐S concentrations of Scots pine (Pinus sylvestris L.) needles growing at a distance of 10–80 km from the metallurgical complex in Monchegorsk in subarctic Russia were measured, and organic S was calculated as the difference between them, in order to study the impact of SO2 (3–28 μg m−3 a−1) on foliar S fractions. Total S concentrations of currentand previous‐year needles collected in April 1991 and July 1992 amounted to 658–2548 ppm, SO4‐S concentrations to 99–1297 ppm and organic S concentrations to 447–1599 ppm. The SO4‐S concentrations and the S/N ratios (0.032–0.113 on a gram atom basis) show that S in excess of the growth requirements of trees was entering the needles at all the five sites studied. Foliar K and Ca, but not Mg, increased with increasing SQ4‐S. It is hypothesized that exposure to SO2 concentrations of ≥2 μg m−3 as a growing season mean together with pollution episodes of ∼ 100 μg m−3 h−1 result in an oxidative stress high enough to damage Scots pine needles, which derive SO2 effectively from the atmosphere, and that cellular acidification is a secondary effect.