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H Acker

Publications and source records attributed to H Acker.

At least 55 records · Page 3Linked to original sources
PubMed1994

Evidence for redistribution-associated intracellular pK shifts of the pH-sensitive fluoroprobe carboxy-SNARF-1.

Properties and peculiarities of the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1 (carboxy-SNARF-1), in view of pHi measurements in single cells, were evaluated using confocal laser scanning microscopy. It was found that in human malignant glioma cells (U 118 MG) grown in multicellular spheroid culture, intracellular calibration curves (nigericin method) varied from one cell to another despite emission ratioing of the fluorescence signals. In addition, considerable deviations between indicator calibration in cell-free solution and intracellular calibration were observed. Microspectrofluorometric measurements revealed that these deviations are attributable to intracellular pK shifts of the indicator rather than to spectral changes of the fluorescence emission. The observed pK shifts are probably due to intracellular redistribution of the indicator between cytosol and lipophilic cell compartemants, e.g. plasma membrane, since the indicator can even be loaded efficiently into the cells via its active acid form (instead of the acetoxymethyl ester form). An approximate theoretical derivation of a cellular calibration curve confirms that a reversible, pH-dependent intracellular redistribution of the protonated indicator component results in an apparent pK shift of delta pK = log(1 + epsilon.P), with P the partition coefficient and epsilon a factor that depends on the different mean layer thicknesses of the cytosol and plasma membrane. Since the apparent pK shift amounts to about 1 pH unit in tumour cells of spheroids, the intracellular pH measuring range of carboxy-SNARF-1 is almost restricted to alkaline pH values. Further consequences of the redistribution phenomenon are discussed with special respect to intracellular ion imaging.

Acid-Base Equilibrium↗
PubMed1994

Mechanisms and meaning of cellular oxygen sensing in the organism.

Oxygen sensors in the body induce various cell activities to avoid any mismatch between oxygen demand and oxygen supply and to maintain an optimal level of oxygen partial pressure (PO2) in various organs. Oxygen sensing seems to be a well conserved process among procaryontic and eucaryontic cells. The molecular mechanism of oxygen sensing is unknown, but it has been suggested that a hemeprotein is involved that does not participate in the mitochondrial energy production. As examplified on the carotid body and on erythropoietin producing HepG2 cells, a cytochrome b was described for the NAD(P)H oxidase of neutrophiles might be an attractive candidate for this hemeprotein. It is hypothesised that hydrogen peroxide (H2O2) produced by this cytochrome b in direct correlation with cellular PO2, serves as a second messenger to regulate potassium channels or gene expression. One might forsee, that this new concept of oxygen sensing could have an impact on all processes in physiology and pathophysiology which are dealing with reactive oxygen intermediates.

Animals↗
PubMed1993

Photometric characteristics of haem proteins in erythropoietin-producing hepatoma cells (HepG2).

Erythropoietin (Epo)-producing hepatoma cells (HepG2) reveal, in addition to the cytochromes of the respiratory chain, a photometrically measurable haem signal with absorbance maxima at 559 nm and 427 nm, suggesting the presence of a b-type cytochrome. This activity exhibited a low midpoint potential, CO-binding spectra and reduction which was insensitive to both cyanide and antimycin. This haem possessed a 22 kDa subunit and might be part of an electron transfer chain similar to the NADPH oxidase, since the NADPH oxidase cytosolic activating factor (p47) could be identified by Western blot analysis. H2O2, which was detected inside the cells by confocal microscopy, might therefore be produced by the suggested electron transfer chain. This cyanide- and antimycin-insensitive but hypoxia-sensitive cytochrome b would be an attractive candidate for controlled Epo production in response to pO2.

Blotting, Western↗
PubMed1993

Depth profiles of pH and PO2 in the isolated brain stem-spinal cord of the neonatal rat.

We have measured depth profiles of extracellular pH (pHECR) and PO2 (PtO2) as well as the kinetics of changes of pHECR in the isolated brain stem-spinal cord preparation of the neonatal rat using pH and PO2 microelectrodes that entered from the ventral surface. When the preparation was superfused with control mock cerebrospinal fluid (Control mock CSF; pH = 7.5, PO2 = 630 Torr, PCO2 = 28 Torr, at 27 degrees C), the pH in the medulla diminished with a nearly constant gradient from the surface to a depth of about 1000 microns, the slope being about 0.1 pH unit per 100 microns. A similar gradient in the 200 to 300 microns of the CSF above the surface suggested existence of unstirred layers despite continuously flowing superfusate. The pH gradient in the spinal cord was somewhat smaller than that in the medulla. The PO2 gradients in both medulla and spinal cord were about 100 Torr per 100 microns from 200 microns above to 100 to 200 microns below the surface; PO2 reached zero at about 450 (medulla) to 600 microns (spinal cord). Although the preparation was anoxic and acidic except for a small layer below the surface, respiratory activity was recorded for several hours in C4 phrenic roots. The kinetics of changes in pHECF were recorded at 100 and 200 microns depth while rapidly replacing the control mock CSF by more acidic CSF, either with increased PCO2 ("Respiratory acidosis") or by adding fixed acid ("Metabolic acidosis"). The changes in pHECF were smaller than those in pHCSF, particularly during respiratory acidosis, as a result of the buffering of the brain tissue. Our results show the importance of superficial layers of the ventral medulla in producing respiratory rhythmicity; they further suggest that somewhat alkaline CSF (pH about 7.8) should be used in this preparation to ensure physiologic surface pH values despite unstirred surface layers.

Animals↗
PubMed1992

Influence of glucose on metabolism and growth of rat glioma cells (C6) in multicellular spheroid culture.

The metabolism and growth of rat glioma C6 cells in multicellular spheroid culture depended strongly on the glucose supply. A low glucose level (0.1 g/l) in the culture medium reduced lactate production, increased oxygen consumption and diminished hydrogen ion production under normoxia as well as hypoxia. A high glucose level (10 g/l glucose) increased lactate production, had no significant influence on oxygen consumption and increased the hydrogen ion production under hypoxia. Hydrogen ion release from cells under normoxic and hypoxic conditions could be significantly diminished by amiloride (l mM), indicating the involvement of the Na+/H+ exchanger. The growth of the C6 spheroids was enhanced under low glucose conditions, possibly due to the more physiological extracellular pH in the deeper regions of the spheroids. The growth was inhibited under high glucose conditions, which seemed to be toxic due to a massive hydrogen production giving acidosis. The glucose supply strongly influenced the local hydrogen ion production inside the C6 spheroids and this might in turn lead to changes in the response to different therapeutic modalities.

Animals↗
PubMed1992

The meaning of H2O2 generation in carotid body cells for PO2 chemoreception.

The rat carotid body is able to generate H2O2 in type-I cells with the aid of an electron-transferring chain with cytochrome b as the major component as it can be detected by spectrophotometry as well as confocal laser-microscopy. This cytochrome b is reducible by hypoxia, but not by cyanide, indicating that it does not participate in the energy production by the respiratory chain. The carotid body possesses a glutathione peroxidase (GPO) which scavenges H2O2 and other organic hydroperoxides. The nervous chemoreceptor discharge can be inhibited by external application of hydroperoxides with a similar half maximal value (60-80 microM) as used to stimulate GPO. A hypothetical signal chain is described which suggests the involvement of cytochrome b as an O2 sensor in PO2 chemoreception of the carotid body and the degradation of H2O2 by glutathione to control the K(+)-conductivity of carotid body cells.

Animals↗
PubMed1991

Effects of amiloride treatment on U-118 MG and U-251 MG human glioma and HT-29 human colon carcinoma cells.

Human glioma (U-118 MG, U-251 MG) and human colon carcinoma (HT-29) spheroids and monolayers were continuously exposed to amiloride under physiological Na+ and HCO3- conditions. Amiloride in concentrations of 0.1-0.2 mM inhibited growth, while 0.5 mM or higher induced disintegration of the glioma spheroids within 4-6 days. Growth retardation of the HT-29 spheroids was achieved at concentrations of 0.4-0.5 mM and total growth inhibition and disintegration were achieved at 1.0 mM. Monolayer cultures of glioma cells were also more sensitive to amiloride than those of colon carcinoma cells. The higher amiloride concentrations induced pyknotic nuclei mainly in the central areas of the spheroids where the extracellular pH (pHe) was low. The amiloride-sensitive glioma spheroids had lower pHe than the colon carcinoma spheroids. The intracellular pH (pHi), measured in monolayers, was higher (7.11-7.18) in glioma cells than in colon carcinoma cells (6.94). High concentrations of amiloride, 1.0 mM for 1 h in combination with low Na+ concentrations, caused a strong pHi decrease in glioma cells but only a slight decrease in the colon carcinoma cells. The pHi measurements in glioma monolayers were carried out after 2-6 days of continuous exposure to 0.1 mM amiloride at physiological levels of Na+ and HCO3- to simulate the conditions during growth inhibition. After several days this caused, when growth already was inhibited, an acidification of pHi. Parallel measurements with X-ray microanalysis showed an increase of intracellular sodium and a decrease of intracellular potassium in the gliomas, while no such changes were seen in the colon carcinoma cells under identical conditions. It is concluded that the two glioma cell lines were more sensitive to amiloride, both as monolayers and spheroids, than the corresponding cultures of the colon carcinoma cell line. The inhibition of proliferation by amiloride seemed not to have a clear connection to pHi regulation.

Amiloride↗
PubMed1991

Local blood flow velocities in the carotid body of fetal sheep and newborn lambs.

Elastically-suspended microelectrodes were used in the vascularly isolated blood-perfused carotid body of fetal and newborn lambs as well as of 6-7-day-old lambs to measure local blood flow velocities by means of hydrogen clearance. Fetal sheep (n = 9) carotid bodies elicited mean local blood velocity values between 0.008 and 0.11 cm.s-1, whereas newborn lamb carotid bodies (n = 7) showed values between 0.008 and 0.067 cm.s-1 at a perfusion pressure range between 30 and 150 mmHg. The 6-7-day-old lamb carotid bodies (n = 5) were characterized by values of 0.003 and 0.049 cm.s-1 over the same perfusion pressure range. Fetal carotid body values were statistically significantly higher than the values of the 6-7-day-old lamb carotid bodies, whereas the newborn carotid body values showed no significant difference to both other groups. The flow velocity/perfusion pressure relationship peaked at perfusion pressure values between 100 and 150 mmHg in all groups with a reduced steepness in the lamb carotid body. It is concluded that local blood flow velocities in the carotid body are similar to that in other organs, and that after birth local blood flow velocities in the carotid body decrease during the first week of life, probably induced by vasoconstriction, changed blood gas values, and/or increasing shunt flow.

Animals↗
PubMed1991

Hypoxia increases the cyclic AMP content of the cat carotid body in vitro.

The cyclic AMP content of cat carotid bodies in vitro measured with a radioimmunoassay under control conditions (PO2: 230 torr) was 0.79 +/- 0.10 pmol/carotid body (n = 10). Lowering medium PO2 to 20 torr for 2 min significantly increased cyclic AMP content to 1.13 +/- 0.14 pmol/carotid body (n = 10). This increase was inhibited neither by propranolol (34 microM) nor by propranolol plus haloperidol (27 microM). Inhibition of the cyclic nucleotide phosphodiesterase with 1-methyl-3-isobutylxanthine (0.8 mM) provoked a fast and large increase in cyclic AMP during both control and hypoxic conditions. The cyclic AMP increase induced by hypoxia was still observed when extracellular Ca2+ was absent. Inhibition of the adenylate cyclase by N-(cis-2-phenylcyclopentyl)azacyclotridecan-2-imine hydrochloride (MDL 12330A; 20-1,000 microM) under zero-Ca2+ conditions irreversibly inhibited the cyclic AMP increase produced by hypoxia. Similarly, inhibition of the Ca2(+)-calmodulin complex by trifluoperazine (0.2 mM) or calmidazolium (R 24571; 50-200 microM) prevented the cyclic AMP response. These results suggest that cyclic AMP may be involved in the PO2-sensing mechanism of the carotid body. Hypoxia appears to activate adenylate cyclase directly and independent of any hormone-receptor interactions.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Involvement of an NAD(P)H oxidase as a pO2 sensor protein in the rat carotid body.

The rat carotid body tissue reveals a photometrically measurable haem signal with absorbance maxima at 560 nm, 518 nm and 425 nm, suggesting the presence of a b-type cytochrome; this was confirmed by pyridine haemochrome and CO spectra. The quantity of cytochrome b was estimated to be 310 pmol.mg of protein-1. This haem is capable of H2O2 formation, which can be inhibited by 10 microM-diphenyliodonium (DPI). The hypoxia-induced increase in nervous chemoreceptor discharge and the reduction of FAD and NAD(P)+ were also inhibited by DPI (10 microM). These results suggest that an oxidase such as the NAD(P)H oxidase of neutrophils may act as a pO2 sensor protein in the rat carotid body, probably inducing the pO2 chemoreceptor process by H2O2 formation.

Animals↗
PubMed1990

Aerobic glycolysis in the retina of the crab Ocypode ryderi.

Our experiments on the isolated and superfused crab retina reveal that pronounced gradients for PO2 and pH exist in this tissue. The PgO2 profiles and a delayed recovery of the PgO2 after hypoxia seem to be a consequence of the oxygen consumption inside the tissue, as much as both characteristics can be abolished by impairment of electron transport in the respiratory chain after application of antimycin A. The pH profile is obviously created by a production and steady release of lactate, which could be measured in the superfusate. As this lactate release is occurring inspite of a sufficient oxygen supply and consumption, it can be concluded that this tissue performs aerobic glycolysis.

Aerobiosis↗
PubMed1990

Hypoxia and ion activities within the brain stem of newborn rabbits.

Eleven rabbits between the 1st and 28th days of life were anesthetized (ketamine 40 mg/kg and acepromazine 3 mg/kg im) thoracotomized, paralyzed, and artificially ventilated with 50% O2 and 10% O2 in N2 or 100% N2. Three-barreled ion-sensitive microelectrodes were used to measure direct-current potentials, potassium (aK+o) and calcium (aCa2+o) activities, and tissue PO2. During control, mean levels of aK+o and aCa2+o were 4.4 +/- 1.1 and 1.3 +/- 0.3 mM, respectively. During hypoxia, changes in aCa2+o were inconsistent, and aK+o revealed three phases: slow (phase I) and fast (phase II) rate of rise and a saturation level (phase III) at the group mean of 6.8 +/- 2.3 mM. Durations of phases I and II decreased, and their slopes increased with maturation. Hypoxia-related excitation of phrenic nerve activity (PHR) occurred during phase I, and gasplike PHR and/or apnea occurred during phases II and III. During recovery after hypoxia, PHR was independent of aK+o levels. Vagal nerve stimulation caused a rapid increase in aK+o followed by a continuous decay even though stimulation continued. Hypoxia had no significant effect on maximal aK+o increase. We concluded that ion homeostasis is less sensitive to the reduced availability of O2 shortly after birth than it is later in life. This age dependence may have an important role in the high resistance to lack of O2 during the early postnatal period in mammals.

Animals↗
PubMed1989

Indications to an NADPH oxidase as a possible pO2 sensor in the rat carotid body.

The rat carotid body superfused with low pO2 exhibited an optical absorbance spectrum which resembles the reduced spectrum of the NADPH oxidase in neutrophils. Diphenylene iodonium (DPI) as a specific inhibitor of the oxidase attenuated the reduced absorbance spectrum in the carotid body. Also absorbance bleaching by low doses of cyanide (50 and 100 microM) was inhibited by DPI, whereas higher doses of cyanide (300 microM) caused an absorbance spectrum typical for reduced cytochromes. It is concluded that an NADPH oxidase acts as a pO2 sensor in the carotid body with low affinity for oxygen and high affinity for cyanide.

Animals↗
PubMed1989

Ionic currents on type-I cells of the rabbit carotid body measured by voltage-clamp experiments and the effect of hypoxia.

Type-I cells (from rabbit embryos) in primary culture were studied in voltage-clamp experiments using the whole cell arrangement of the patch-clamp technique. With a pipette solution containing 130 mM K+ and 3 mM Mg-ATP, large outward currents were obtained positive to a threshold of about -30 mV by clamping cells from -50 mV to different test pulses (-80 to 50 mV). Negative to -30 mV, the slope conductance was low (outward rectification). The outward currents were blocked by external Cs+ (5 mM) and partially blocked by TEA (5 mM) and Co2+ (1 mM). The initial part of the outward currents during depolarizing voltage pulses exhibited a transient Ca2+ inward component partially superimposed to a Ca2+-dependent outward current. Inward currents were further characterized by replacing K+ with Cs+ in the intra- and extracellular solution in order to minimize the outward component and by using 1.8 mM Ca2+, 10.8 mM Ca2+ or 10.8 mM Ba2+ as charge carrier. Slow-inactivating inward currents were recorded at test potentials ranging from -50 to 40 mV (holding potential -80 mV). The maximal amplitude, measured at 10 mV in the U-shaped I-V curve, amounted to 247 +/- 103 pA (n = 3). This inward current was insensitive to 3 microM TTX, but blocked by 1 mM Co2+ and partially reduced by 10 microM D600 and 3 microM PN 200-100. In contrast to outward currents, the inward currents exhibited a 'run-down' within about 10 min. Lowering the pO2 from the control of 150 Torr (air-gassed medium) to 28 Torr had no apparent effect on inward currents, but depressed reversibly outward currents by 28%. In conclusion, it is suggested that type-I cells possess voltage-activated K+ and Ca2+ channels which might be essential for chemoreception in the carotid body.

Adenosine Triphosphate↗