Search

Carotid body physiology meets cytochrome c oxidase crystallography Commentary to Ortega-Sáenz P, López-Barneo J. Physiology of the Carotid Body: From Molecules to Disease. Changing gas composition of the super-fusion medium from 20% O SB 2 sb , 3% CO SB 2 sb , 77% N SB 2 sb (normoxia) to 97% N SB 2 sb , 3% CO SB 2 sb (full hypoxia) is followed by a characteristic hypoxia-induced increased chemoreceptor sinus nerve activity (registration in black) serving as control. 1 CcO crystallographic changes of about 3 Ångström are displayed by CB chemoreceptor discharge. a Chemoreceptor discharge under normoxic and hypoxic CO application (red registration line) The effect of CO is shown as a percentage of peak chemoreceptor activity induced by 4-min hypoxia. [Extracted from the article]

Cellular retention and processing of a radiolabelled monoclonal anti-prostate antibody were evaluated after binding to prostatic adenocarcinoma DU 145 cells. An endocytosis assay revealed that the rate of release of radioactivity from the cells had an initially rapid phase within the first hours after antibody incubation, which was presumably due to release of monovalently bound antibodies. This was followed by a slower phase, with the possible release of intact bivalently bound antibodies and excretion of degraded internalized antibodies. The relative amount of released radioactivity of high molecular mass was high, indicating that the major part of the antibodies were released without being internalized and degraded. However, when only the radiolabelled antibody that remained cell-associated after 2 h and longer was considered, a substantial part was found to be internalized and radioactive degradation products were excreted. About 30% of the initially cell-associated radioactivity still remained associated with the cells after 48 h, indicating a rather slow antibody processing, which is favourable if the antibody is to be used for targeted radiotherapy. The retention of cell-associated radioiodine was very similar irrespective of whether the antibodies were radiolabelled with the direct chloramine-T method or the indirect ATE (succinimidyl based reagent) method. Since the ATE method can be used to form stable antibody constructs with the therapeutically relevant alpha-emitting radionuclide, astatine-211, this was an interesting finding that will be further evaluated in the future. [ABSTRACT FROM AUTHOR]

A new method was tested for studies of penetration of substances into tumorlike tissue. The penetration of the ions K, Cl, and Ca through several layers of tumor cells was demonstrated by using double barrelled, ion sensitive microelectrodes with extra thin tip diameters. Spheroids consisting of human glioma, U-118 MG, and human thyroid cancer, HTh-7, cells were used as models of tumor tissue. A microelectrode was inserted into the center of a spheroid. Thereafter, the concentration of the test substance was increased in the surrounding medium. The change in concentration inside the spheroid was recorded and the penetration pattern evaluated. All three types of tested ions penetrated easily through the spheroids. The K ions penetrated most efficiently, and the Ca ions showed the slowest penetration. The Ca ions penetrated somewhat more slowly in the U-118 MG spheroids (which had rather small extracellular spaces) than in the HTh-7 spheroids (which had larger extracellular spaces). Ion sensitive electrodes, which are easily available, were used in this study only to demonstrate the principle. We hope that the method described can be used for penetration studies of various substances. For example, all substances that can be detected by enzyme microelectrodes could be studied. The main advantage of the method is that the complete penetration pattern can be studied as a function of time in individual spheroids. Previously described methods require histological procedures for each analyzed penetration time. [ABSTRACT FROM AUTHOR]

Small intensely fluorescent (SIF) cells are paraganglionic cells derived from sympathicoblasts which may serve as interneurons, endo-/paracrine cells or arterial chemoreceptors within sympathetic ganglia. Like paraganglionic cells of other locations, e.g., carotid body glomus cells, they are responsive to hypoxia. Recent studies on glomus cells and other hypoxia-sensing cells suggested the involvement of a b 558-type cytochrome and intracellular generation of H2O2 in the process of oxygen sensing. In the present study, we demonstrate the occurrence of the small subunit of cytochrome b 558, p22phox, in SIF cells of guinea-pig sympathetic ganglia by immunohistochemistry using two different antisera. H2O2 production was monitored in explanted intact superior cervical ganglia of 2-day-old rats by confocal laser scanning analysis of rhodamine 123 fluorescence generated due to oxidation of dihydrorhodamine 123 by H2O2. Using this technique, SIF cell clusters appeared as sites of highest H2O2 production within the ganglia. Thus, SIF cells exhibit two key features of an oxidase system generating reactive oxygen species. This may be involved in the proposed chain of events in oxygen sensing, but alternative cellular functions of this system have also to be considered. [ABSTRACT FROM AUTHOR]

After perfusion stop a $$P_{{\text{O}}_{\text{2}} } $$ decrease has been measured in the carotid body tissue by means of elastically suspended $$P_{{\text{O}}_{\text{2}} } $$ needle electrodes. With the formula $${\text{U}}_{{\text{O}}_{\text{2}} } = \frac{\alpha }{{760}} \cdot \frac{{\Delta P_{{\text{O}}_{\text{2}} } }}{{\Delta t}} \cdot 10^5 $$ it is possible to calculate the oxygen consumption from this $$P_{{\text{O}}_{\text{2}} } $$ decrease. Our experiments have shown that the oxygen consumption of the non-perfused carotid body of the cat depends linearly on local $$P_{{\text{O}}_{\text{2}} } $$ . The $$\frac{{\Delta P_{{\text{O}}_{\text{2}} } }}{{\Delta t}}$$ measured in situ (Krebs-Henseleit perfusion) is with 6 Torr/s at a tissue $$P_{{\text{O}}_{\text{2}} } $$ of 100 Torr (37° C) two times higher than the values measured in vitro (Krebs-Henseleit-perfusion). The $$\frac{{\Delta P_{{\text{O}}_{\text{2}} } }}{{\Delta t}}$$ in vivo corresponds to an oxygen consumption of 1.2 ml O/100 g min at a $$P_{{\text{O}}_{\text{2}} } $$ of 100 Torr at 37° C. The $$P_{{\text{O}}_{\text{2}} } $$ dependence of the oxygen consumption of the carotid body might be caused by a special oxidase which may be involved in the chemoreceptive process. [ABSTRACT FROM AUTHOR]

Local $$\frac{{\Delta P_{O_2 } }}{{\Delta t}}$$ in the carotid body after perfusion stop depends on the oxygen consumption of the tissue and the apparent O solubility coefficient α of the blood. Oxygen consumption of the carotid body tissue can be determined by measuring the local $$\frac{{\Delta P_{O_2 } }}{{\Delta t}}$$ with $$P_{O_2 } $$ needle electrodes after Krebs-Henseleit perfusion. Assuming the same $$\frac{{\Delta P_{O_2 } }}{{\Delta t}}$$ for the blood-perfused carotid body the actual $$P_{O_2 } $$ decrease can be used to estimate the hemoglobin content of the tissue. The influence of hemoglobin is described by the factor α/α. Control values measured in the Krebs-Henseleit-perfused carotid body yielded values of α/α = 0.8 −3. In a series of 11 blood-perfused carotid body preparations with 105 perfusion stops the quotient α/α changed with arterial $$P_{O_2 } $$ values higher than 100 Torr α/α was between 0.8 and 2. This value thus was in the same range as the control values. Below 100 Torr higher values up to 11 were observed. Since a blood-perfused tissue (15 g % Hb) would have an α/α of about 128, we conclude that the carotid body is mainly perfused by plasma, and that with decreasing arterial $$P_{O_2 } $$ more red cells flowed through the carotid body. [ABSTRACT FROM AUTHOR]

Isolated rat hepatocytes maintained in primary culture on gas permeable membrane for 20 h form monolayers and establish at their cell borders a network of canaliculi (approximate diameter 3.5 μm). In the presence of the known choleretic bile acid dehydrocholate, dilation of canaliculi occurs. When nonfluorescent carboxyfluorescein diacetate ester is added to the culture medium, fluorescent carboxyfluorescein appears in the intracanalicular space. In the dilated state, fluid containing the fluorescent compound could be collected from the canaliculi by puncture with a micropipette. The intracanalicular space shows a negative electrical potential difference of 31 mV in reference to the bath solution and is 13.5 mV more positive with reference to recordings from the cytosol of cultured rat hepatocytes. Cultured rat hepatocytes grown on gas permeable membrane are energetically stable over 3 d. On Day 4, ATP levels increase markedly, whereas Na−K-ATPase activity declines. Ionic composition of hepatocytes, as measured by electronprobe element analysis on cryosection samples, does not change markedly during monolayer formation. With formation of bile canaliculi, the activity of alkaline phosphatase rapidly increases within 24 h and is stable for the next 3 d. Within that time the activity of γ-glutamyltranspeptidase, however, increases steadily, reaching a 1.6-fold higher activity than freshly isolated hepatocytes. Bile acids appear in the culture supernatant after 1 d. When unconjugated [C]cholic acid is added to the cultures the supernatant contains also [C]tauro- and [C]glycocholic acid, indicating the preservation of conjugation capacity in these cultures. Total bile acid concentrations in the supernatant increase from 5 to 26 μ M on Day 4. The cultures do not secrete α-fetoprotein. Monolayer cultures of hepatocytes in the presence of choleretic bile acids seem to be a suitable model system to collect and to analyze the composition of primary bile. In conjunction with the electrical parameters, it is possible to describe directly properties of bile secretion at the canalicular pole of the intact hepatocyte. [ABSTRACT FROM AUTHOR]

A PCO electrode working on the principle of electrical conductivity is described. The calibration curve can be linearized according to the formula $$G = G_0 + b\sqrt {P{\text{CO}}_2 } $$ . This linearity has been tested in the PCO range of 0.93-9.33 kPa (7-70 Torr). For the experiments electrodes are used which have conductivity values of about 50 nS and drifts of maximally 5%/h at a PCO of 5.33 kPa (40 Torr). The response time ( T) is about 20 s. The temperature sensitivity is 2.4 nS/1 K between 298K-310K. The standard error of the measurements is σ=0.33 nS. With these electrodes tissue PCO can be measured on the surface of various organs. [ABSTRACT FROM AUTHOR]