CO2-sensitive neurons in organotypic cultures of the fetal rat medulla

Medullary slices of the fetal rat at gestational day 16 were cultivated (organotypic culture) for up to 20 days and current clamp experiments were performed on outgrowing neurons. CO₂-sensitivity was tested by changing the P_CO2 in the bath solution (equilibrating CO₂ fraction from 0.02 to 0.09). Two groups of CO₂-sensitive neurons were found; one with and the other without intrinsic CO₂-chemosensitivity. Neurons with intrinsic CO₂-sensitivity maintained their spontaneous activity and chemosensitivity after blockade of synaptic transmission. These neurons exhibited action potentials that were preceeded by a spontaneous interspike depolarization and followed by an afterhyperpolarization (beating neurons). Increasing P_CO2 either decreased (inhibited neurons, n=55) or increased the spike frequency of these neurons (stimulated neurons, n=31). The reduced activity of CO₂-inhibited neurons was associated with membrane hyperpolarization and/or decreases in the slope of interspike depolarization. In contrast CO₂-stimulated neurons were depolarized and the slope of their interspike depolarization was augmented during acidosis. In addition, we demonstrated a strong voltage dependence of CO₂-induced effects on membrane potential and spike frequency. Neurons with non-beating activity did not show a spontaneous interspike depolarization and their spike generation and CO₂-sensitivity appeared to be entirely produced through synaptic inputs. The CO₂-mediated changes in electrical properties of these neurons closely resemble those of various CNS neurons, including respiratory neurons, in whole animal or neonatal brainstem-spinal cord preparations.     

Amiloride-sensitive regulation of intracellular pH in B-cells: activation by glucose

Alterations in intracellular pH (pHi) generated by metabolism of glucose has been proposed to be a transduction device for controlling changes in K+ conductance in the plasma membrane of the B-cell leading to depolarization and cyclic variations in the membrane potential associated with spike activity. The influence of permeable weak acids or bases and amiloride inhibition of H+ extrusion by a Na:H exchanger on glucose-induced electrical activity has suggested that the electrical events are pH-sensitive. In order to document that these conditions alter pHi, we determined the influence of glucose, propionic acid, and NH4Cl, in the presence or absence of amiloride on pHi of rat islets using [14C] DMO. Glucose, 2.8 mmol/L decreased pHi by .09 unit compared to the absence of glucose (pHi = 7.08 +/- .01, M +/- SEM) and 16.7 mmol/L glucose reduced pHi by .19 unit. The glucose dose-related decrease in pHi yielded a half-maximal response at 4 mmol/L. The addition of 0.1 mmol/L amiloride had no influence on pHi without glucose and decreased pHi in the presence of 2.8 mmol/L glucose by .14 unit. The addition of 20 mmol/L propionic acid to 2.8 mmol/L glucose reduced pHi to 6.85 +/- .05, whereas 20 mmol/L NH4Cl increased pHi to 7.27 +/- .07. The addition of amiloride did not further lower the reduction in pHi elicited by 20 mmol/L propionic acid or 16.7 mmol/L glucose. These results suggest that the amiloride-sensitive Na:H exchanger plays a major role in regulation of pHi, but another modality for pHi regulation exists to compensate for inhibition of Na:H exchange under conditions of an acid load.     

Intracellular pH regulation of neurons in chemosensitive and nonchemosensitive areas of brain slices.

The role of changes of intracellular pH (pH(i)) as the proximal signal in central chemosensitive neurons has been studied. pH(i) recovery from acidification is mediated by Na(+)/H(+) exchange in all medullary neurons and pH(i) recovery from alkalinization is mediated by Cl(-)/HCO(3)(-) exchange in most medullary neurons. These exchangers are more sensitive to inhibition by changes in extracellular pH (pH(o)) in neurons from chemosensitive regions compared to those from nonchemosensitive regions. Thus, neurons from chemosensitive regions exhibit a maintained intracellular acidification in response to hypercapnic acidosis but they show pH(i) recovery in response to isohydric hypercapnia. A similar pattern of pH(i) response is seen in other CO(2)/H(+)-responsive cells, including glomus cells, sour taste receptor cells, and chemosensitive neurons from snails, suggesting that a maintained fall of pH(i) is a common feature of the proximal signal in all CO(2)/H(+)-sensitive cells. To further evaluate the potential role of pH(i) changes as proximal signals for chemosensitive neurons, studies must be done to: determine why a lack of pH(i) recovery from hypercapnic acidosis is seen in some nonchemosensitive neurons; establish a correlation between hypercapnia-induced changes of pH(i) and membrane potential (V(m)); compare the hypercapnia-induced pH(i) changes seen in neuronal cell bodies with those in dendritic processes; understand why the V(m) response to hypercapnia of many chemosensitive neurons is washed out when using whole cell patch pipettes; and employ knock out mice to investigate the role of certain proteins in the CO(2)/H(+) response of chemosensitive neurons.     

Active control of intracellular pH.

When the interior of a squid giant axon is loaded with acid, the cell responds by extruding that acid in an energy-requiring process. Our results indicate that this acid extrusion is accomplished by exchanging external HCO3- for internal Cl- at the expense of ATP.     

HCO3(-)-dependent intracellular pH regulation in the premature myocardium.

This study investigated developmental changes in Na(+)-H+ exchange and HCO3(-)-Cl- exchange activities in newborn and adult rabbit hearts. pHi was measured using the fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in isolated myocytes. Myocardial mechanical function was measured in the isolated ventricular preparation. Intracellular acidosis with normal pHo was induced by an NH4Cl (10 mM) prepulse technique. Upon removal of NH4Cl, pHi fell transiently and then recovered toward the control level. In the HCO3-/CO2-buffered solution, the rate of recovery of pHi in the newborn was greater than in the adult. In the HCO3-/CO2-buffered solution, 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of Na(+)-H+ exchange, inhibited the recovery of pHi completely in the adult. In the newborn, however, significant recovery of pHi was observed in the presence of EIPA. In the presence of both EIPA and 4-acetamido-4'-isothiocyanatostilbene-2',2'-disulfonic acid (SITS), an inhibitor of HCO3(-)-Cl- exchange, the recovery of pHi was not observed in the two age groups. In the HEPES-buffered solution that did not contain HCO3-/CO2, the rate of recovery of pHi after NH4Cl removal was similar in the two age groups. In the HEPES-buffered solution, the recovery of pHi was completely inhibited by EIPA in the two age groups. In the presence of EIPA in the HCO3-/CO2-buffered solution, contractile function decreased during acidosis after NH4Cl removal and did not recover in the adult. In the newborn, significant recovery of contractile function was observed after NH4Cl removal in the presence of EIPA. The recovery of mechanical function observed in the presence of EIPA in the newborn was inhibited by SITS. These data suggest that, although there is no developmental change in the Na(+)-H+ exchange activity, HCO3(-)-Cl- exchange is more active in the premature myocardium. The presence of the HCO3(-)-Cl- exchanger is important in maintaining myocardial contractile function during acidosis, especially when Na(+)-H+ exchange is inhibited and may partly explain the greater resistance of the premature myocardium to acidosis.     

Expression and regulation of intercellular adhesion molecule-1 (ICAM-1) in organotypic cultures of rat liver tissue

Background/Aims: The objective of the present study was to analyze the expression and regulation of intercellular adhesion molecule-1 (ICAM-1) in organotypic cultures of rat liver slices, which preserve the normal microenvironment of liver cells.Methods: Rat liver slices were maintained in culture for 15 min to 24 h and examnied for ICAM-1 expression by immunohistochemistry and Western blotting in basal conditions and after stimulation with 1000 IU/ml interferon-γ (IFNγ), 1000 IU/ml tumor necrosis factor-α (TNFα) and 50 μg/ml endotoxin. Immunohistochemical results were evaluated using a semiquantitative scoring system.Results: In uncultured slices, ICAM-1 was not detected on hepatocytes. In unstimulated liver slices maintained in organotypic culture, ICAM-1 was induced at the surface of scattered hepatocytes (score at 15 min, 0.33±0.47 and at 24 h, 1.17±0.69). After 4 h of stimulation, a significant increase in ICAM-1 expression by hepatocytes and adjacent sinusoidal cells, but not by intra-hepatic biliary epithelial cells, was observed for IFNγ (score: 2.35±0.47) and endotoxin (score: 2.67±0.47), but not with TNFα (score: 0.66±0.47). After 24 h of stimulation, a further increase in the extent of ICAM-1 expression by hepatocytes was observed for IFNγ (score: 3.67±0.47) and endotoxin (score: 4.0±0.0), and a significant overexpression of ICAM-1 by hepatocytes was detectable after treatement with TNFα (score: 3.67±0.47).Conclusions: In rat liver organotypic cultures, TNFα, IFNγ and endotoxin induce the expression of ICAM-1 in hepatocytes and adjacent sinusoidal endothelial cells, but not in portal tracts.     

Identification of exchange mechanisms for the regulation of intracellular pH in rat thyroid FRTL-5 cells.

pH is maintained in cells by plasma membrane exchange mechanisms. In the absence of HCO3- ions, FRTL-5 cells regulate intracellular pH (pHi) by an Na+/H+ antiport but HCO3(-)-dependent exchangers cannot operate. We have investigated pHi regulation (by microfluorimetry and the pH sensitive dye 2',7'-bis(2-carboxyethyl)-5(6')-carboxyfluorescein) in small groups (five to six cells) of FRTL-5 thyroid cell monolayers held in Krebs-Ringer buffer (pH 7.4) with or without HCO3- ions. The exchangers were investigated with inhibitors (amiloride or its derivative dimethylamiloride for the Na+/H+ antiporter and the stilbene derivative disodium 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) for HCO3(-)-dependent mechanisms), ionic substitution and by NH4+/NH3 (10 mM) acid loading. Basal pHi was lower in the presence (7.3 +/- 0.058, mean +/- S.D., n = 14) than in the absence (7.59 +/- 0.078, n = 10) of HCO3- ions. In HCO3(-)-free media, cells recovered from acid load by 0.34 +/- 0.04 pH units in the first 2 min and finally reached a pHi of 7.35 +/- 0.06. This recovery was Na(+)-dependent and blocked by dimethylamiloride during the 15 min following intracellular acidification. In HCO3(-)-containing media, cells recovered from an acid load at a similar rate, but reached 99 +/- 10% (n = 9) of the baseline pH; this recovery was also dependent on Na+ ions. Moreover, although dimethylamiloride and DIDS reduced the rate of recovery to 0.06 +/- 0.02 and 0.18 +/- 0.04 pH units respectively during the 2-min period, the cells returned to the basal pHi within 15 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neutrophil intracellular pH and Na+/H+ exchanger activity in pre-eclampsia

Elevated Na(+)/H(+) exchanger activity and intracellular acidosis have previously been demonstrated in white blood cells isolated from women who have suffered from a pre-eclamptic pregnancy. The mechanisms underlying this abnormality and the implications in pre-eclamptic pregnancies are, at present, unclear. In this study, we used neutrophils from third trimester pre-eclamptic patients and third trimester normotensive pregnant controls to determine Na(+)/H(+) exchanger isoform-1 (NHE-1) activity and intracellular pH. This was performed using a well-validated technique involving flurometry and a pH sensitive dye, 2,7'Bis-(carboxyethyl) 5.6 carboxyfluorescein acetomethyl ester (BCECF-AM). Time course experiments were performed to assess the contribution of plasma factors to intracellular pH measurements. Plasma digoxin-like factor (DLF) was assessed in both patients and normotensive controls. Neutrophil intracellular pH was significantly lower in the pre-eclamptic patients (7.15 +/- 0.050) compared with the normotensive pregnant controls (7.36 +/- 0.027; P<.001). NHE-1 activity (in mmol/L/min) was significantly higher in the pre-eclamptics (32.4 +/- 1.9) compared with the normotensive neutrophils (27.1 +/- 1.6; P =.038). Times course experiments showed that mean pre-eclamptic intracellular pH increased from 7.11 +/- 0.049 to 7.25 +/- 0.043 after 2 hours of incubation. DLF, measured as amount of inorganic phosphate liberated from adenosine triphosphate (ATP), was significantly lower when plasma from the pre-eclamptic patients was incubated with the enzyme compared with plasma from the normotensive pregnant women (54.9 +/- 2.6 nmol/mL plasma v 63.91 +/- 1.7 nmol/mL plasma, n = 6, P =.018 unpaired Student's t test). The results suggest that elevated NHE-1 activity and intracellular acidosis are intermediate phenotypes in women who have pre-eclampsia. Intracellular pH may have been affected by plasma as shown in the time course experiments. DLF, an inhibitor of Na(+)/K(+)ATPase, may contribute to this intracellular acidosis in pre-eclamptic neutrophils.     

Weak acids, weak bases and intracellular pH.

Against the background of classical observations made 50 years ago, a brief review is offered of some of the work performed in the author's laboratory on the behavior of weak acids and bases towards living animal cells. The significance of membrane permeability of the charged partner of these electrolytes is pointed out, and the existence of an active process of H+ extrusion (or its equivalent) in response to acid loading is demonstrated. The effect of intracellular inhomogeneity on weak acid and base transmembrane distribution is examined. The significance of these variables for weak acid- or base-derived intracellular pH is discussed.     

Na+/H+ exchange in isolated hamster enterocytes. Its major role in intracellular pH regulation

The intracellular pH (pHi) of isolated hamster intestinal cells was determined with three techniques: the null-point method with digitonin, the distribution of the weak acid 5,5-dimethyloxazolidine-2,4-dione, and the trapped fluorescent indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. In physiologic saline, the pHi determined with the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein was 7.00 +/- 0.03 (n = 20) but was 6.68 +/- 0.04 (n = 25) when there was no Na+ in the medium, indicating an important role of external Na+ in maintaining pHi at neutral level. When Na+ was added to an incubation medium lacking the ion, the pHi increased. The time-course of this alkalinization depended on the Na+ concentration, whereas K+ had only a slight effect. Amiloride (1 mM) completely inhibited the Na+ effect, thereby showing the basic role of the Na+/H+ antiport in the regulation of pHi. The effect of 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and furosemide showed that there also exists an anionic component in the process, probably of the Cl-/OH- (HCO3-) exchange type. These results show that the amiloride-sensitive Na+/H+ antiport plays an important but not exclusive role in maintaining the pHi neutral or slightly alkaline in isolated intestinal cells from the hamster.     

Leucocyte intracellular pH and Na+/H+ antiport activity in human hypertension

Hypertension is associated with thickening of the wall of resistance vessels, but the cellular or genetic basis of this is unclear. Cell proliferation and intracellular alkalinization via increased Na+/H+ exchange are linked in the response of tissues to growth factors. To define a possible cellular basis for vascular medial thickening in hypertension, we studied leucocyte intracellular pH, buffering power and Na+/H+ antiport activity in 17 hypertensive and 17 age-, sex- and weight-matched normotensive subjects. The cells from hypertensive subjects were significantly more alkaline [median (range): 7.49 (7.26-7.95) versus 7.39 (7.25-7.53); P less than 0.01], and had a lower buffering power [8.95 (3.05-17.98) versus 12.57 (7.44-19.95) mmol/l per pH unit; P less than 0.02] than those from normotensive subjects. Moreover, the activity of the Na+/H+ antiport was higher when cells were acid-loaded to an intracellular pH of 6.7. The presence of a similar increased activity in vascular smooth muscle cells may be associated with increased cellular proliferation resulting in a thickened media or increased vascular smooth muscle contractility.     

Method for preparing cultures of central neurons: cytochemical and immunochemical studies.

We report a simplified method for culturing fetal central nervous system cells predominantly inducing neurons that grow, differentiate, and live in vitro for as long as 10 weeks. These central nervous system cells form a confluent cell culture in which about 80% of the cells are fully differentiated neurons producing interconnecting axons and dendrite processes and live upon a sparse underlying population of fibrillary and protoplasmic astrocytes, oligodendrocytes, and fibroblasts. Morphological and cytochemical characteristics of these cell types, based on immunofluorescent cell specific markers and silver staining of neurons, are presented.     

H+ ion activation and inactivation of the ventricular gap junction: a basis for spatial regulation of intracellular pH

H(+) ions are powerful modulators of cardiac function, liberated during metabolic activity. Among their physiological effects is a chemical gating of cell-to-cell communication, caused by H(+)-mediated closure of connexin (Cx) channels at gap junctions. This protects surrounding tissue from the damaging effects of local intracellular acidosis. Cx proteins (largely Cx-43 in ventricle) form multimeric pores between cells, permitting translocation of ions and other solutes up to approximately 1 kDa. The channels are essential for electrical and metabolic coordination of a tissue. Here we demonstrate that, contrary to expectation, H(+) ions can induce an increase of gap-junctional permeability. This occurs during modest intracellular acid loads in myocyte pairs isolated from mammalian ventricle. We show that the increase in permeability allows a local rise of [H(+)](i) to dissipate into neighboring myocytes, thereby providing a mechanism for spatially regulating intracellular pH (pH(i)). During larger acid loads, the increased permeability is overridden by a more familiar H(+)-dependent inhibition (H(+) inactivation). This restricts cell-to-cell H(+) movement, while allowing sarcolemmal H(+) transporters such as Na(+)/H(+) exchange, to extrude the acid from the cell. The H(+) sensitivity of Cx channels therefore defines whether junctional or sarcolemmal mechanisms are selected locally for the removal of an acid load. The bell-shaped pH dependence of permeability suggests that, in addition to H(+) inactivation, an H(+) activation process regulates the ensemble of Cx channels open at the junction. As well as promoting spatial pH(i) regulation, H(+) activation of junctional permeability may link increased metabolic activity to improved myocardial coupling, the better to meet mechanical demand.