UCL logo

UCL Discovery

UCL home » Library Services » Electronic resources » UCL Discovery

RELATIVE MITOCHONDRIAL-MEMBRANE POTENTIAL AND [CA2+]I IN TYPE-I CELLS ISOLATED FROM THE RABBIT CAROTID-BODY

DUCHEN, MR; BISCOE, TJ; (1992) RELATIVE MITOCHONDRIAL-MEMBRANE POTENTIAL AND [CA2+]I IN TYPE-I CELLS ISOLATED FROM THE RABBIT CAROTID-BODY. J PHYSIOL-LONDON , 450 33 - 61.

Full text not available from this repository.

Abstract

1. In the accompanying paper (Duchen & Biscoe, 1992) we have described graded changes in autofluorescence derived from mitochondrial NAD(P)H in type I cells of the carotid body in response to changes of P(O2) over a physiologically significant range. These observations suggest that mitochondrial function in these cells is unusually sensitive to oxygen and could play a role in oxygen sensing. We have now explored further the relationships between hypoxia, mitchondrial membrane potential (DELTA-psi(m)) and [Ca2+]i.2. The fluorescence of Rhodamine 123 (Rh 123) accumulated within mitochondria is quenched by DELTA-psi(m). Mitochondrial depolarization thus increases the fluorescence signal. Blockade of electron transport (CN-, anoxia, rotenone) and uncoupling agents (e.g. carbonyl cyanide p-trifluoromethoxy-phenylhydrazone; FCCP) increased fluorescence by up to 80-120%, while fluorescence was reduced by blockade of the F0 proton channel of the mitochondrial ATP synthase complex (oligomycin).3. DELTA-psi(m) depolarized rapidly with anoxia, and was usually completely dissipated within 1-2 min. The depolarization of DELTA-psi(m) with anoxia (or CN-) and repolarization on reoxygenation both followed a time course well characterized as the sum of two exponential processes. Oligomycin (0.2-2-mu-g/ml) hyperpolarized DELTA-psi(m) and abolished the slower components of both the depolarization with anoxia and of the subsequent repolarization. These data (i) illustrate the role of the F1-F0 ATP synthetase in slowing the rate of dissipation of DELTA-psi(m) on cessation of electron transport, (ii) confirm blockade of the ATP synthetase by oligomycin at these concentrations, and (iii) indicate significant accumulation of intramitochondrial ADP during 1-2 min of anoxia.4. Depolarization of DELTA-psi(m) was graded with graded changes in P(O2) below about 60 mmHg. The stimulus-response curves thus constructed strongly resemble those for [Ca2+]i and NAD(P)H with P(O2). The change is DELTA-psi(m) closely followed changes in P(O2) with time.5. The rate of rise of [Ca2+]i in response to anoxia is strongly temperature sensitive. The rate of depolarization of DELTA-psi(m) with anoxia similarly increased at least two- to fivefold on warming from 22 to 36-degrees-C. The change with FCCP was not significantly altered by temperature.6. These data show that the mitochondrial membrane potential changes over a physiological range of P(o2) values in type I cells. This contrasts with the behaviour in dissociated chromaffin cells and sensory neurons, in which no change was measurable until the P(O2) fell close to zero.7. Intracellular [Ca2+]i shows a graded increase with hypoxia over a similar range of P(O2) values to the changes in DELTA-psi(m), and is released, at least in part, from an intracellular store. The source of that calcium remains uncertain. We have previously suggested it is derived from mitochondria, and have now examined this proposal further.8. Rotenone, which blocks mitochondrial electron transport at complex I, depolarized DELTA-psi(m) and raised [Ca2+]i. Any further change in [Ca2+]i in response to anoxia was occluded. Tetramethyl-p-phenylene diamine (TMPD) bypasses the rotenone-induced block, directly reducing cytochrome c. TMPD reversed both the depolarization of DELTA-psi(m) and the rise in [Ca2+]i CN- depolarized DELTA-psi(m) and raised [Ca2+]i despite the presence of TMPD, confirming this mechanism.9. The changes in mitochondrial function with hypoxia are not secondary to the changes in [Ca2+]i. K+-induced depolarization of the cells raises [Ca2+]i more than does anoxia, but produced only small changes in DELTA-psi(m) which were abolished by removal of extracellular Ca2+ while the response to hypoxia was unchanged.10. The simplest mechanisms that link changes in [Ca2+]i with DELTA-psi(m) are (i) direct release of mitochondrial Ca2+ and (ii) release from a pool sensitive to the ATP/ADP. P(i) ratio. Oligomycin, which blocks the F1-F0 ATP synthetase while hyperpolarizing DELTA-psi(m), initiates a rapid and sustained rise of [Ca2+]i to levels similar to those seen with anoxia, FCCP or CN-. Subsequent responses to these stimuli were much reduced in amplitude. In the absence of external Ca2+, responses to oligomycin were present but were transient rather than sustained.11. These results together suggest that [Ca2+]i is entrained to DELTA-psi(m) during hypoxia in these cells and that depolarization of DELTA-psi(m), graded with P(O2), therefore appears central to transduction in the carotid body. The effect of oligomycin suggests that alterations in ATP/ADP.P(i) ratio could provide a link between changes in DELTA-psi(m) and [Ca2+]i.12. The increase in [Ca2+]i is thus probably derived from at least two intracellular sources, mitochondrial and non-mitochondrial.

Type: Article
Title: RELATIVE MITOCHONDRIAL-MEMBRANE POTENTIAL AND [CA2+]I IN TYPE-I CELLS ISOLATED FROM THE RABBIT CAROTID-BODY
Keywords: RAT-LIVER MITOCHONDRIA, LIVING CELLS, OXIDATIVE-PHOSPHORYLATION, ENERGY-METABOLISM, OXYGEN DEPENDENCE, RHODAMINE-123, CHEMORECEPTION, FLUORESCENCE, RESPONSES, CYANIDE
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Cell and Developmental Biology
URI: http://discovery.ucl.ac.uk/id/eprint/121775
Downloads since deposit
0Downloads
Download activity - last month
Download activity - last 12 months
Downloads by country - last 12 months

Archive Staff Only

View Item View Item