Buffering characteristics of eel blood at the extreme conditions in the swimbladder

CO₂ binding in whole blood and true plasma of the eel was estimated by measuring CO₂ content and pH in blood aliquots equilibrated with various P_CO2 values over a wide range expected to occur in the swimbladder. Bicarbonate concentration, [HCO₃⁻], was calculated using the CO₂ solubility coefficient, which was measured to average 50 μmol·L⁻¹·Torr⁻¹ (20°C). Buffer lines of non-bicarbonate buffers were obtained in plots of [HCPO₃⁻] against pH, and non-bicarbonate buffer values, β_NB, were obtained by curve. fitting.In the pH range 6.6–8.2, the buffer line for oxygenated whole blood was sigmoid, while that for deoxygenated blood increased its slope monotonously with increasing pH. The β_NB for oxygenated blood displayed a maximum of about 8.6 mmol·L⁻¹pH⁻¹ at pH = 7.4 and dropped down to below 1 mmol·L⁻¹·pH⁻¹ at higher and lower pH. Similar shapes of the buffer lines were obtaned in true plasma; the [HCO₃⁻] levels and β_NB values were, however, somewhat higher than in whole blood.These data are useful fo assess the back-diffusion of CO₂ and HCO₃⁻ in the rete mirabile of the fish swimbladder and to estimate the effects CO₂ back-diffusion exerts on the counter-current enhancement of O₂ in the rete.     

Acidified seawater impacts sea urchin larvae pH regulatory systems relevant for calcification

Calcifying echinoid larvae respond to changes in seawater carbonate chemistry with reduced growth and developmental delay. To date, no information exists on how ocean acidification acts on pH homeostasis in echinoderm larvae. Understanding acid–base regulatory capacities is important because intracellular formation and maintenance of the calcium carbonate skeleton is dependent on pH homeostasis. Using H⁺-selective microelectrodes and the pH-sensitive fluorescent dye BCECF, we conducted in vivo measurements of extracellular and intracellular pH (pH_e and pH_i) in echinoderm larvae. We exposed pluteus larvae to a range of seawater CO₂ conditions and demonstrated that the extracellular compartment surrounding the calcifying primary mesenchyme cells (PMCs) conforms to the surrounding seawater with respect to pH during exposure to elevated seawater pCO₂. Using FITC dextran conjugates, we demonstrate that sea urchin larvae have a leaky integument. PMCs and spicules are therefore directly exposed to strong changes in pH_e whenever seawater pH changes. However, measurements of pH_i demonstrated that PMCs are able to fully compensate an induced intracellular acidosis. This was highly dependent on Na⁺ and HCO₃⁻, suggesting a bicarbonate buffer mechanism involving secondary active Na⁺-dependent membrane transport proteins. We suggest that, under ocean acidification, maintained pH_i enables calcification to proceed despite decreased pH_e. However, this probably causes enhanced costs. Increased costs for calcification or cellular homeostasis can be one of the main factors leading to modifications in energy partitioning, which then impacts growth and, ultimately, results in increased mortality of echinoid larvae during the pelagic life stage.     

The effect of pH on 86Rubidium efflux from pancreatic islet cells

The influence of pH on the K⁺ permeability of pancreatic islet cells was investigated by measuring ⁸⁶Rb⁺ fluxes in isolated rat islets perifused or incubated in the absence of glucose. Acidification of the medium (to pH 6.5), by decreasing the concentration of HCO^?₃ or by increasing pCO₂ at constant HCO^?₃ reversibly reduced the rate of ⁸⁶Rb⁺ efflux from perifused islets. Alkalinization of the medium (to pH 7.8), by decreasing the pCO₂, reversibly increased ⁸⁶Rb⁺ efflux. Similar changes were recorded upon alteration of the pH in a bicarbonate-free, Hepesbuffered medium with or without calcium. Alteration of the CO₂ level at constant external pH — in order to modify internal pH — produced only a small and transient increase in efflux rate when CO₂ was lowered, and a decrease in efflux rate when CO₂ was raised. NH₄Cl reversibly augmented ⁸⁶Rb⁺ efflux in the presence and in the absence of HCO^?₃. At low pH (6.5), ⁸⁶Rb⁺ uptake by islet cells was reduced after 10 min (25%) and 30 min (11%), but not after 60 min, of incubation; it was not significantly affected by high pH (7.8). Calcium uptake and insulin release were reduced at low pH and increased at high pH.These results show that the K⁺ permeability of islet cells is affected by changes in extracellular but not in intracellular pH. They suggest that the endogenous production of protons that accompanies glucose stimulation of islet cells is not the mediator of the decrease in K⁺ permeability induced by the sugar.     

Intracellular pH regulation in heart

Intracellular pH (pH_i) is an important modulator of cardiac excitation and contraction, and a potent trigger of electrical arrhythmia. This review outlines the intracellular and membrane mechanisms that control pH_i in the cardiac myocyte. We consider the kinetic regulation of sarcolemmal H⁺, OH⁻ and HCO₃⁻ transporters by pH, and by receptor-coupled intracellular signalling systems. We also consider how activity of these pH_i effector proteins is coordinated spatially in the myocardium by intracellular mobile buffer shuttles, gap junctional channels and carbonic anhydrase enzymes. Finally, we review the impact of pH_i regulatory proteins on intracellular Ca²⁺ signalling, and their participation in clinical disorders such as myocardial ischaemia, maladaptive hypertrophy and heart failure. Such multiple effects emphasise the fundamental role that pH_i regulation plays in the heart.     

Duodenal acidity “sensing” but not epithelial HCO3? supply is critically dependent on carbonic anhydrase II expression

Carbonic anhydrase (CA) is strongly expressed in the duodenum and has been implicated in a variety of physiological functions including enterocyte HCO₃⁻ supply for secretion and the “sensing” of luminal acid and CO₂. Here, we report the physiological role of the intracellular CAII isoform involvement in acid-, PGE_2, and forskolin-induced murine duodenal bicarbonate secretion (DBS) in vivo. CAII-deficient and WT littermates were studied in vivo during isoflurane anesthesia. An approximate 10-mm segment of the proximal duodenum with intact blood supply was perfused under different experimental conditions and DBS was titrated by pH immediately. Two-photon confocal microscopy using the pH-sensitive dye SNARF-1F was used to assess duodenocyte pH_i in vivo. After correction of systemic acidosis by infusion of isotonic Na₂CO₃, basal DBS was not significantly different in CAII-deficient mice and WT littermates. The duodenal bicarbonate secretory response to acid was almost abolished in CAII-deficient mice, but normal to forskolin- or 16,16-dimethyl PGE₂ stimulation. The complete inhibition of tissue CAs by luminal methazolamide and i.v. acetazolamide completely blocked the response to acid, but did not significantly alter the response to forskolin. While duodenocytes acidified upon luminal perfusion with acid, no significant pH_i change occurred in CAII-deficient duodenum in vivo. The results suggest that CA II is important for duodenocyte acidification by low luminal pH and for eliciting the acid-mediated HCO₃⁻ secretory response, but is not important in the generation of the secreted HCO₃⁻ ions.     

The effect of 4,4′-diisothiocyanato-stilbene-2,2′-disulfonate on CO2 permeability of the red blood cell membrane

It has long been assumed that the red cell membrane is highly permeable to gases because the molecules of gases are small, uncharged, and soluble in lipids, such as those of a bilayer. The disappearance of ¹²C¹⁸O¹⁶O from a red cell suspension as the ¹⁸O exchanges between labeled CO₂ + HCO₃⁻ and unlabeled HOH provides a measure of the carbonic anhydrase (CA) activity (acceleration, or A) inside the cell and of the membrane self-exchange permeability to HCO₃⁻ (P_m,HCO⁻3). To test this technique, we added sufficient 4,4′-diisothiocyanato-stilbene-2,2′-disulfonate (DIDS) to inhibit all the HCO₃⁻/Cl⁻ transport protein (Band III or capnophorin) in a red cell suspension. We found that DIDS reduced P_m,HCO⁻3 as expected, but also appeared to reduce intracellular A, although separate experiments showed it has no effect on CA activity in homogenous solution. A decrease in P_m,CO2 would explain this finding. With a more advanced computational model, which solves for CA activity and membrane permeabilities to both CO₂ and HCO₃⁻, we found that DIDS inhibited both P_m,HCO⁻3 and P_m,CO2, whereas intracellular CA activity remained unchanged. The mechanism by which DIDS reduces CO₂ permeability may not be through an action on the lipid bilayer itself, but rather on a membrane transport protein, implying that this is a normal route for at least part of red cell CO₂ exchange.     

Central chemoreceptor stimulus in the terrestrial,pulmonate snail,Helix aspersa

We studied the effect of hypercapnic and fixed acid central chemoreceptor stimulation on the pneumostone in the pulmonate snail, Helix aspersa. We found that focal stimulation of the central chemoreceptor area of the pulmonate snail brain with hypercapnic solutions more effectively increased the pneumostonal area than did fixed acid stimulation at the same extracellular pH. Disrupting intracellular pH regulation by inhibiting Cl⁻ transport, either pharmacologically (DIDS) or by ion substitution (Cl⁻-free perfusate), enhanced pneumostomal responses to CO₂. While maintaining a constant perfusate pH, addition of NH₄Cl to the perfusate resulted in pneumostomal closure; whereas removal of NH₄Cl from the bath resulted in pneumostomal opening. In conclusion, the ventilatory response to CO₂ in H. aspersa does not require Cl⁻ transport or conductance. Furthermore, changing pH_i alone is an adequate stimulus for the central chemoreceptors in the snail.     

Effect of acidosis on contraction, intracellular pH, and calcium in the newborn and adult rabbit aorta

Summary  This study investigated the effect of acidosis on intracellular pH (pH_i), intracellular calcium concentration ([Ca]_i), and vascular contraction in the aorta of the newborn and adult rabbit. Isometric tension, pH_i, and [Ca]_i were measured in an isolated ring preparation. After the vascular contraction was induced with 50 mM KCl, the effect of respiratory acidosis produced by elevation of PCO₂ was studied. Respiratory acidosis caused a transient depression followed by a recovery of contractile tension. The decrease in developed tension was greater in the newborn than in the adult. The decrease in pH_i during acidosis was similar in the two age groups. [Ca]_i increased during acidosis and the increase was greater in the newborn than in the adult. These data show that the vasorelaxant effect of acidosis in the newborn aorta is greater than that in the adult aorta. The greater vasodilation in the newborn cannot be explained by the difference in pH_i or [Ca]_i.     

Venous blood gas and metabolite response to low-intensity muscle contractions with external limb compression

The effect of low-intensity resistance exercise with external limb compression (100 [EC100] and 160 [EC160] mm Hg) on limb blood flow and venous blood gas-metabolite response was investigated and compared with that of high-intensity resistance exercise (no external compression). Unilateral elbow flexion muscle contractions were performed at 20% (75 repetitions, 4 sets, 30-second rest intervals) and 70% of 1-repetition maximum (1-RM; 3 sets, each set was until failure, 3-minute rest intervals). Precontraction brachial arterial blood flow (Doppler ultrasound) was reduced with EC100 or EC160 (56% and 39% of baseline value, respectively) compared with no external compression (control). At 20% 1-RM, brachial arterial blood flow increased after contractions performed with EC160 (190%), but not with the others. Decreases in venous oxygen partial pressure (P_vO₂) and venous oxygen saturation (S_vO₂) were greater during EC100 and EC160 than control (mean [SE]: P_vO₂, 28 [3] vs 26 [2] vs 33 [2] mm Hg; S_vO₂, 41% [5%] vs 34% [4%] vs 52% [5%], respectively). Changes in venous pH (pH_v), venous carbon dioxide partial pressure (P_vCO₂), and venous lactate concentration ([L⁻]_v) were greater with EC160 than EC100 and/or control (pH_v, 7.19 [0.01] vs 7.25 [0.01] vs 7.27 [0.02]; P_vCO₂, 72 [3] vs 64 [2] vs 60 [3] mm Hg; [L⁻]_v, 5.4 [0.6] vs 3.7 [0.4] vs 3.0 [0.4] mmol/L, respectively). Seventy percent 1-RM contractions resulted in greater changes in pH_v (7.14 [0.02]), P_vCO₂ (91 [5] mm Hg), and [L⁻]_v (7.0 [0.5] mmol/L) than EC100 and EC160, but P_vO₂ (30 [4] mm Hg) and S_vO₂ (40% [3%]) were similar. In conclusion, changes in pH_v, P_vCO₂, and [L⁻]_v, but not in P_vO₂ and S_vO₂, are sensitive to changes in relative, “internal” intensity of low-intensity muscle contractions caused by reduced blood flow (EC160) or high-intensity muscle contractions. Given the magnitude of the changes in pH_v, P_vCO₂, and [L⁻]_v, it appears plausible that they may be involved in stimulating the observed increase in muscle activation via group III and IV afferents.     

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.     

Regulation of Intracellular pH in Periportal and Perivenular Hepatocytes Isolated from Ethanol-Treated Rats

The aim of this study was to gain information on intracellular pH (pH_i) regulation in periportal (PP) and perivenular (PV) hepatocytes isolated from rats pair-fed liquid diets with either ethanol (T rats) or isocaloric carbohydrates (C rats). pH_i was analyzed by the pH-sensitive dye BCECF in perfused sub-confluent hepatocyte monolayers. Cells were acid-loaded by pulse exposure to NH₄Cl and were alkali-loaded by suddenly reducing external C0₂ and HC0₃^-(from 10% and 50 mm , respectively, to 5% and 25 mm ) at constant pH_out. In cells from C rats: (a) steady-state pH_i was higher in PP than in PV hepatocytes in the presence, but not in the absence, of bicarbonate; (b) pH_i recovery from an acid load was 35% higher in PP than in PV cells in the presence of HC0₃^-, whereas it was similar in HC0₃^--free experiments; and, on the contrary, (c) pH_i recovery from an alkaline load was 30% higher in PV than in PP cells. In cells from T rats: (a) steady-state pH_i was always lower than in cells isolated from pair-fed animals; (b) steady-state pH_i was similar in PP and PV hepatocytes either in the presence or absence of bicarbonate in the perfusate; (c) pH_i recovery from an acid load was not significantly different in PP and PV cells either in the presence of HC0₃^- or in HC0₃^--free experiments; and (d) pH_i recovery from an alkaline load was similar in PP and PV cells. Our data suggest that chronic ethanol treatment selectively modifies pH, by affecting the activity of ion transport mechanisms regulating pH_i in PP and PV hepatocytes isolated from rat liver.     

Intracellular Levels of Na+ and TTX-sensitive Na+ Channel Current in Diabetic Rat Ventricular Cardiomyocytes

Intracellular Na⁺ ([Na⁺] _i ) is an important modulator of excitation–contraction coupling via regulating Ca²⁺ efflux/influx, and no investigation has been so far performed in diabetic rat heart. Here, we examined whether any change of [Na⁺] _i in paced cardiomyocytes could contribute to functional alterations during diabetes. Slowing down in depolarization phase of the action potential, small but significant decrease in its amplitude with a slight depolarized resting membrane potential was traced in live cardiomyocytes from diabetic rat, being parallel with a decreased TTX-sensitive Na⁺ channel current (I _Na) density. We recorded either [Na⁺] _i or [Ca²⁺] _i by using a fluorescent Na⁺ indicator (SBFI-AM or Na-Green) or a Ca²⁺ indicator (Fura 2-AM) in freshly isolated cardiomyocytes. We examined both [Na⁺] _i and [Ca²⁺] _i at rest, and also [Na⁺] _i during pacing with electrical field stimulation in a range of 0.2–2.0 Hz stimulation frequency. In order to test the possible contribution of Na⁺/H⁺ exchanger (NHE) to [Na⁺] _i , we examined the free cytoplasmic [H⁺] _i changes from time course of [H⁺] _i recovery in cardiomyocytes loaded with SNARF1-AM by using ammonium prepulse method. Our data showed that [Na⁺] _i in resting cells from either diabetic or control group was not significantly different, whereas the increase in [Na⁺] _i was significantly smaller in paced diabetic cardiomyocytes compared to that of the controls. However, resting [Ca²⁺] _i in diabetic cardiomyocytes was significantly higher than that of the controls. Here, a lower basal pH _i in diabetics compared with the controls correlates also with a slightly higher but not significantly different NHE activity and consequently a similar Na⁺ loading rate at resting state with a leftward shift in pH sensitivity of NHE-dependent H⁺-flux. NHE protein level remained unchanged, while protein levels of Na⁺/K⁺ ATPase and Na⁺/Ca²⁺ exchanger were decreased in the diabetic cardiomyocytes. Taken together, the present data indicate that depressed I _Na plays an important role in altered electrical activity with less Na⁺ influx during contraction, and an increased [Ca²⁺] _i load in these cells seems to be independent of [Na⁺] _i . The data with insulin treatment suggest further a recent balance between Na⁺ influx and efflux proteins associated with the [Na⁺] _i , particularly during diabetes.     

Effects of Plasmalemmal V-ATPase Activity on Plasma Membrane Potential of Resident Alveolar Macrophages

The acid-base status and functional responses of alveolar macrophages (m) are influenced by the activity of plasmalemmal V-type H⁺-pump (V-ATPase), an electrogenic H⁺ extruder that provides a possible link between intracellular pH (pH_i) and plasma membrane potential (E_m). This study examined the relationships among E_m, pH_i, and plasmalemmal V-ATPase activity in resident alveolar m from rabbits. E_m and pH_i were measured using fluorescent probes. E_m was –46 mV and pH_i was 7.14 at an extracellular pH (pH_o) of 7.4. The pH_i declined progressively at lower pH_o values. Decrements in pH_o also caused depolarization of the plasma membrane, independent of V-ATPase activity. The pH effects on E_m were sensitive to external K⁺, and hence, probably involved pH-sensitive K⁺ conductance. H⁺ were not distributed at equilibrium across the plasma membrane. V-ATPase activity was a major determinant of the transmembrane H⁺ disequilibrium. Pump inhibition with bafilomycin A₁ caused cytosolic acidification, due most likely to the retention of metabolically generated H⁺. V-ATPase inhibition also caused depolarization of the plasma membrane, but the effects were mediated indirectly via the accompanying pH_i changes. V-ATPase activity was sensitive to E_m. E_m hyperpolarization (valinomycin-clamp) reduced V-ATPase activity, causing an acidic shift in baseline pH_i under steady-state conditions and slowing pH_i recovery from NH₄Cl prepulse acid-loads. The findings indicate that a complex relationship exists among E_m, pH_i, and pH_o that was partially mediated by plasmalemmal V-ATPase activity. This relationship could have important consequences for the expression of pH- and/or voltage-sensitive functions in alveolar m.     

Effect of the diphosphonate ethane-I-hydroxy-I, I-diphosphonate (EHDP) on hemoglobin oxygen affinity of diabetic and healthy subjects.

In order to study the effect of prolonged elevation of plasma inorganic phosphate (P₁) on red cell metabolism and function, oxyhemolobin dissociation curves (ODC) from zero to full saturation were performed on whole blood from 14 insulintreated, nonacidotic diabetics and 5 healthy volunteers following oral administration of disodium ethane-l-hydroxy-l, l-diphosphonate (EHDP; 20 mg kg^?1 day^?1 or placebo for 28 days. EHDP significantly increased mean P_i (diabetics, 1.18 to 1.67 mmol/liter; P < 0.001; controls 1.14 to 1.69 mmol/liter; P < 0.005 and P₅₀ at in vivo pH of the ODC (diabetics, 25.6 to 26.7 mm Hg, P < 0.025; controls, 27.6 to 29.5 mm Hg, P < 0.02). P_i and P₅₀ were correlated in both diabetics (r = 0.58, P < 0.01) and controls (r = 0.69, P < 0.05). Mean P₅₀ in diabetics was significantly lower than normal in spite of normal red cell 2,3-diphosphoglycerate (2,3-DPG) (diabetics, 15.2μmol/g of Hb; controls, 14.3 μmol/g of Hb). 2,3-DPG increased when the diabetics were on EHDP (15.2 to 16.3 μmol/g of Hb, P < 0.005), while no significant changes occurred in hemoglobin concentration, oxygen saturation, or blood pH. The study emphasizes the importance of P_i on red cell function and indicates that an elevation of P_i tends to counteract the defect in oxygen-release capacity of the red cells in diabetic subjects.     

Characterization of a novel cell damage model induced by acid and pepsin using rat gastric epithelial cells: Protective effect of sucralfate

We have established a new model for rat gastric epithelial cell (RGM1) damage caused by both acid and pepsin. Exposure of RGM1 to an acidified medium (pH 3.5–5.0) for 10-50 min decreased cell viability in a time- and pH-dependent manner. Pepsin (0.5–1.0 mg/mL) at pH 4.5 potentiated cell damage in a concentration-dependent manner. Based on these results, two types of cell damage models caused by incubation of cells at pH 4.0 and with pepsin (0.75 mg/mL) at pH 4.5 for 30 min, respectively, were established. The intracellular pH (pH) gradually decreased with a decrease in medium pH and an increase in exposure time. At pH ≤ 4.0, pH_i reached approximately pH 6.3. Pepsin at pH 4.5 caused a further reduction in pH_i compared with the acidified medium alone. Pepsin pre-incubated with pepstatin did not induce any cell damage. Pretreatment with sucralfate (0.1 -3 mg/mL) for 2 h significantly prevented cell damage caused at both pH 4.0 and with pepsin at pH 4.5 in a concentration-dependent manner. Sucralfate (3 mg/mL) significantly prevented the reduction in pH_i at pH 4.0 or with pepsin at pH 4.5. 16,16-Dimethyl prostaglandin E₂ (30 μg/mL) had no effect on either cell damage or pH_i. These cell damage models involving RGM1 are useful for studying the mechanism underlying cell damage and for the screening of cytoprotective drugs.     

Effects of Concanavalin A on Na+-Dependent and Na+-Independent Mechanisms for H+ Extrusion in Alveolar Macrophages

Alveolar macrophages (mφ) possess two parallel mechanisms for plasmalemmal H⁺ extrusion: a V-type H⁺ pump (V-ATPase) and a Na⁺/H⁺ exchanger (NHE). To investigate the coordinated functioning of these H⁺ extruders for mφ intracellular pH (pH_i) regulation, we investigated the effects of the plant lectin concanavalin A (ConA) on resident alveolar mφ from rabbits. ConA (1 μM, 30-min pretreatment) activated the mφ for phagocytosis of opsonized Escherichia coli. ConA activation did not affect the baseline pH_i of mφ or the initial rate of pH_i recovery (dpH_i/dt) from an intracellular acid load (acid-loaded pH_i nadir ≈ 6.9). However, the contributions of Na⁺-independent H⁺ transport (i.e. V-ATPase activity) and Na⁺-dependent H⁺ transport (i.e. NHE activity) to dpH_i/dt were altered significantly. The lectin stimulated Na⁺/H⁺ exchange and inhibited V-ATPase activity. In control mφ, V-ATPase-mediated H⁺ extrusion was responsible for >80% of dpH_i/dt. Conversely, in ConA-treated mφ, Na⁺/H⁺ exchange was responsible for ∼65% of dpH_i/dt, and V-ATPase activity was responsible for only 35% of dpH_i/dt. These results underscore the complex mechanisms and signaling pathways that coordinate the activities of cellular acid-base transporters in mφ pH_i regulation. Accepted for publication: 18 March 1997     

Effects of Lidocaine on Cytosolic pH Regulation and Stimulus-Induced Effector Functions in Alveolar Macrophages

Local anesthetics influence a variety of stimulus-induced effector functions in leukocytes. The present study determined the effects of lidocaine on intracellular pH (pH_i) regulation, superoxide production, and tumor necrosis factor-α (TNF-α) release in alveolar macrophages (mφ). Resident mφ were obtained by bronchoalveolar lavage of rabbits. The cells were subjected to an intracellular acid load, and subsequent pH_i recovery was followed in the presence or absence of bafilomycin A₁, a specific inhibitor of V-type H⁺-translocating ATPase (V-ATPase) or amiloride, an inhibitor of Na⁺/H⁺ exchange. Lidocaine slowed pH_i recovery in a dose-dependent manner. Pretreatment (1 h) with 2.5 mM lidocaine abolished Na⁺/H⁺ exchange and reduced the V-ATPase-mediated component of pH_i recovery. Lidocaine also significantly depressed the superoxide production induced by phorbol ester. TNF-α release induced by endotoxin was not affected significantly by the local anesthetic. Macrophage viability (trypan blue exclusion) and cellular ATP concentration were unaffected. These results indicate that lidocaine inhibits pH_i regulatory mechanisms in alveolar mφ. This disruption of pH_i regulation could contribute to inhibitory actions of lidocaine on mφ effector functions. Accepted for publication: 28 January 1997     

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.     

A link between polymorphonuclear leukocyte intracellular calcium,plasma insulin,and essential hypertension

BackgroundIntracellular ionized calcium ([Ca²⁺]_i) is a key mediator in the activation and oxidant production by peripheral polymorphonuclear leukocytes (PMN). Primed PMN contribute to oxidative stress (OS) and inflammation in essential hypertension (EH). Elevated [Ca²⁺]_i has been described in insulin-resistant states and in various cell types in EH but not in EH PMN. The aim of this study was to evaluate the levels of [Ca²⁺]_i in peripheral EH PMN in relation to plasma insulin levels and blood pressure (BP).MethodsThe PMN were separated from blood of 20 nonsmoking, nonobese untreated EH patients, age range 20 to 60 years and from 20 age- and gender-matched healthy individuals (NC). Plasma glucose and insulin levels 2 h after a 75-g oral glucose load, reflected insulin resistance. PMN [Ca²⁺]_i was measured by flow cytometry in isolated cells stained with Fluo-3.ResultsThe EH PMNs showed significantly increased [Ca²⁺]_i compared to NC PMN. Eighty percent of EH patients showed significantly higher plasma insulin levels after glucose load. Linear regression analysis showed significant correlation between 1) PMN [Ca²⁺]_i and mean arterial pressure (MAP) (r = 0.5, P < .006); 2) PMN [Ca²⁺]_i and fasting plasma insulin (r = 0.7, P < .005); and 3) fasting plasma insulin and MAP (r = 0.4, P < .04).ConclusionsThis study adds PMN to previously described cells exhibiting elevated [Ca²⁺]_i, contributing to OS and inflammation. The correlation of individual BP with both PMN [Ca²⁺]_i and plasma insulin levels, together with the fact that elevated [Ca²⁺]_i mediates PMN priming, suggest that elevated [Ca²⁺]_i and insulin are involved in the pathogenesis of hypertension-induced vascular injury in EH.     

Low Density Lipoprotein Receptors Mediate Intracellular Calcium Signals in Microalbuminuric Hypertensives

Hyperlipidemia is an established risk factor for progressive renal damage and proteinuria. Platelets and vascular smooth muscle cells (VSMC) are known to possess low density lipoprotein (LDL) cholesterol receptors. We used platelet LDL receptors to investigate the hypothesis that elevated lipids could activate intracellular calcium [Ca²⁺]_i signals, leading to altered vascular tone and permeability. We divided essential hypertensives into microalbuminuric positive (MA+) and negative (MA−) groups and measured baseline and LDL stimulated levels of [Ca²⁺]_i. The MA+ group demonstrated a significantly higher mean baseline [Ca²⁺]_i level (119.0 ± 24.5 v 86.2 ± 25.4 μmol/mL, P = .001). The MA+ group also displayed greater elevations in [Ca²⁺]_i levels after stimulation with LDL in concentrations of 10 and 25 μg/mL (100.9 ± 54.8 v 40.9 ± 20.2, P = .04 and 111.6 ± 51.0 v 52.9 ± 39.9 μmol/mL, P = .03, respectively). Our data indicate that hypertensive patients with early glomerular capillary injury display exaggerated influx of [Ca²⁺]_i after LDL receptor stimulation. Heightened LDL receptor sensitivity may facilitate LDL mediated [Ca²⁺]_i signals, leading to increased VSMC tone and proliferation and progressive renal disease.