Interaction between glucocorticoids and glucagon in the hormonal modification of calcium retention by isolated rat liver mitochondria.
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ABSTRACT:
SUBMITTER: Hughes BP
PROVIDER: S-EPMC1161052 | biostudies-other | 1979 May
REPOSITORIES: biostudies-other
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Project description:1. Mitochondria isolated from rat liver by centrifugation of the homogenate in buffered iso-osmotic sucrose at between 4000 and 8000g-min, 1h after the administration in vivo of 30mug of glucagon/100g body wt., retain Ca(2+) for over 45min after its addition at 100nmol/mg of mitochondrial protein in the presence of 2mm-P(i). In similar experiments, but after the administration of saline (0.9% NaCl) in place of glucagon, Ca(2+) is retained for 6-8min. The ability of glucagon to enhance Ca(2+) retention is completely prevented by co-administration of 4.2mg of puromycin/100g body wt. 2. The resting rate of respiration after Ca(2+) accumulation by mitochondria from glucagon-treated rats remains low by contrast with that from saline-treated rats. Respiration in the latter mitochondria increased markedly after the Ca(2+) accumulation, reflecting the uncoupling action of the ion. 3. Concomitant with the enhanced retention of Ca(2+) and low rates of resting respiration by mitochondria from glucagon-treated rats was an increased ability to retain endogenous adenine nucleotides. 4. An investigation of properties of mitochondria known to influence Ca(2+) transport revealed a significantly higher concentration of adenine nucleotides but not of P(i) in those from glucagon-treated rats. The membrane potential remained unchanged, but the transmembrane pH gradient increased by approx. 10mV, indicating increased alkalinity of the matrix space. 5. Depletion of endogenous adenine nucleotides by P(i) treatment in mitochondria from both glucagon-treated and saline-treated rats led to a marked diminution in ability to retain Ca(2+). The activity of the adenine nucleotide translocase was unaffected by glucagon treatment of rats in vivo. 6. Although the data are consistent with the argument that the Ca(2+)-translocation cycle in rat liver mitochondria is a target for glucagon action in vivo, they do not permit conclusions to be drawn about the molecular mechanisms involved in the glucagon-induced alteration to this cycle.
| S-EPMC1186292 | biostudies-other
Project description:In response to various pathological stimuli, such as oxidative and energy stress accompanied by high Ca2+, mitochondria undergo permeability transition (PT) leading to the opening of the non-selective PT pores (PTP) in the inner mitochondrial membrane. Opening of the pores at high conductance allows the passage of ions and solutes <1.5 kD across the membrane, that increases colloid osmotic pressure in the matrix leading to excessive mitochondrial swelling. Calcium retention capacity (CRC) reflects maximum Ca2+ overload of mitochondria that occurs just before PTP opening. Quantification of CRC is important for elucidating the effects of different pathological stimuli and the efficacy of pharmacological agents on the mitochondria. Here, we performed a comparative analysis of CRC in mitochondria isolated from H9c2 cardioblasts, and in permeabilized H9c2 cells in situ to highlight the strengths and weaknesses of the CRC technique in isolated cell mitochondria vs. permeabilized cells. The cells were permeabilized by digitonin or saponin, and the Ca2+-sensitive fluorescence probe Calcium Green-5N was used in both preparations. Results demonstrated the interference of dye-associated fluorescence signals with saponin and the adverse effects of digitonin on mitochondria at high concentrations. Analysis of the CRC in permeabilized cells revealed a higher CRC in the saponin-permeabilized cells in comparison with the digitonin-permeabilized cells. In addition, the mitochondrial CRC in saponin-permeabilized cells was higher than in isolated mitochondria. Altogether, these data demonstrate that the quantification of the mitochondrial CRC in cultured cells permeabilized by saponin has more advantages compared to the isolated mitochondria.
| S-EPMC8688924 | biostudies-literature
Project description:1. The administration of glucagon to fed rats by intraperitoneal injection, or the perfusion of livers from fed rats with glucagon by the method of Mortimore [Mortimore (1963) Am.J. Physiol. 204, 699--704] was associated with increases of 15- and 5-fold respectively, in the time for which a given load of exogenous Ca2+ is retained by mitochondria subsequently isolated from the liver. This effect of glucagon was (a) also induced by N6O2'-dibutyryl cyclic AMP, (b) completely blocked by cycloheximide, (c) relatively slow in onset (15--60 min) and (d) associated with a stimulation of about 20% in the rates of ADP-stimulated oxygen utilization and Ca2+ transport measured in the presence of succinate. 2. Perfusion of livers with glucagon resulted in the isolation of mitochandria which showed a 50% increase, no significant change and a 40% increase in the concentrations of endogenous Ca, Mg and Pi respectively, when compared with mitochondria isolated from control perfused livers. 3. The administration of insulin or adrenaline to fed rats induced increases of 10- and 8-fold respectively, in the time for which Ca2+ is retained by isolated liver mitochondria. Perfusion of livers with insulin had no effect on mitochondrial Ca2+ retention time. 4. The perfusion of livers from starved rats with glucagon, or the administration of either glucagon or insulin to starved rats, increased by about 2.5- and 15-fold respectively, the time for which isolated mitochondria retain Ca2+. 5. Mechanisms which may be responsible for the observed alterations in Ca2+-retention time are discussed.
| S-EPMC1186228 | biostudies-other
Project description:1. Added Ca(2+) stimulates the translocation of ATP by isolated rat liver mitochondria. 2. The apparent K(m) for added Ca(2+) in stimulating the translocation of 200mum-ATP is approx. 160mum (75mum ;free' Ca(2+)). 3. The greatest stimulation of ATP translocation by Ca(2+) occurs at the lower concentrations of ATP. 4. Sr(2+) (and to a lesser extent Ba(2+)) can replace Ca(2+) whereas Mg(2+) and Mn(2+) have only little ability to stimulate ATP translocation. 5. Translocation of dATP is also stimulated by Ca(2+) whereas that of ADP is stimulated to only a relatively small degree. 6. Studies with metabolic inhibitors and uncouplers provide evidence that stimulation by Ca(2+) and by uncouplers is additive and that the mechanism of Ca(2+) stimulation does not seem to involve the high-energy intermediate of oxidative phosphorylation. 7. In the presence of Ca(2+), ATP is able to effectively compete with ADP for translocation. 8. Added K(+) further enhances the ability of Ca(2+) to stimulate ATP translocation. 9. These findings are discussed in relation to the potential involvement of Ca(2+) in modifying enzymic reactions involved in the regulation of cell metabolism.
| S-EPMC1174085 | biostudies-other
Project description:1. Liver mitochondria suspended in an iso-osmotic buffered potassium chloride medium containing an oxidizable substrate and phosphate accumulated added Ca(2+). During this process H(+) appeared in the medium and the mitochondrial suspension showed increased light-scattering. Respiration was markedly stimulated. 2. The addition of excess of Ca(2+), respiratory inhibitors or uncoupling agents caused extensive mitochondrial swelling associated with release of Ca(2+) into the suspending medium. When the suspension became anaerobic extensive swelling also occurred. Only under conditions when the addition of uncoupling agents would have produced high rates of electron transport, e.g. in the presence of succinate, was the structural integrity of the mitochondrion maintained after Ca(2+) accumulation. 3. Conditions that prevented respiration-dependent Ca(2+) accumulation also prevented Ca(2+)-induced swelling. Bovine plasma albumin was without effect, indicating that U-factor was not involved. Oligomycin together with ADP or ATP partially stabilized the mitochondria against Ca(2+)-induced swelling. 4. It is suggested that a ;high-energy' intermediate generated by coupled electron transport is required to prevent the mitochondrial swelling that results as a consequence of Ca(2+) accumulation.
| S-EPMC1214334 | biostudies-other
Project description:Ageing is associated with an increase in the incidence of heart failure, even if the existence of a real age-related cardiomyopathy remains controversial. Effective contraction and relaxation of cardiomyocytes depend on efficient production of ATP (handled by mitochondria) and on proper Ca2+ supply to myofibrils during excitation-contraction (EC) coupling (handled by Ca2+ release units, CRUs). Here, we analyzed mitochondria and CRUs in hearts of adult (4 months old) and aged (≥24 months old) mice. Analysis by confocal and electron microscopy (CM and EM, respectively) revealed an age-related loss of proper organization and disposition of both mitochondria and EC coupling units: (a) mitochondria are improperly disposed and often damaged (percentage of severely damaged mitochondria: adults 3.5 ± 1.1%; aged 16.5 ± 3.5%); (b) CRUs that are often misoriented (longitudinal) and/or misplaced from the correct position at the Z line. Immunolabeling with antibodies that mark either the SR or T-tubules indicates that in aged cardiomyocytes the sarcotubular system displays an extensive disarray. This disarray could be in part caused by the decreased expression of Cav-3 and JP-2 detected by western blot (WB), two proteins involved in formation of T-tubules and in docking SR to T-tubules in dyads. By WB analysis, we also detected increased levels of 3-NT in whole hearts homogenates of aged mice, a product of nitration of protein tyrosine residues, recognized as marker of oxidative stress. Finally, a detailed EM analysis of CRUs (formed by association of SR with T-tubules) points to ultrastructural modifications, i.e., a decrease in their frequency (adult: 5.1 ± 0.5; aged: 3.9 ± 0.4 n./50 μm2) and size (adult: 362 ± 40 nm; aged: 254 ± 60 nm). The changes in morphology and disposition of mitochondria and CRUs highlighted by our results may underlie an inefficient supply of Ca2+ ions and ATP to the contractile elements, and possibly contribute to cardiac dysfunction in ageing.
| S-EPMC8395047 | biostudies-literature
Project description:1. The excessive accumulation of Ca(2+) by mitochondria suspended in an iso-osmotic buffered potassium chloride medium containing oxidizable substrate and phosphate led to extensive swelling and release of accumulated Ca(2+) from the mitochondria. When the Ca(2+) was removed from the medium by chelation with ethylene glycol bis(aminoethyl)tetra-acetate, the swelling was reversed in a respiration-dependent contraction. The contracted mitochondria were shown to have regained some degree of respiratory control. 2. The respiration-dependent contraction could be supported by electron transport through a restricted portion of the respiratory chain, and by substrates donating electrons at different levels in the respiratory chain. 3. Respiratory inhibitors appropriate to the substrate present completely inhibited the contraction. Uncoupling agents, and the inhibitors oligomycin and atractyloside, were without effect. 4. When the reversal of swelling had been prevented by respiratory inhibitors, the addition of ATP induced a contraction of the mitochondria. In the absence of added chelating agent the contraction was very slow. The ATP-induced contraction was completely inhibited by oligomycin and atractyloside, was incomplete in the presence of uncoupling agents and was unaffected by respiratory inhibitors. 5. The relationship between the energy requirements of respiration-dependent contraction and the requirements of ion transport and other contractile systems are discussed.
| S-EPMC1214335 | biostudies-other
Project description:The data presented in this article are related to the research article entitled "Rotenone decreases ischemia-induced injury by inhibiting mitochondrial permeability transition in mature brains" (Rekuviene et al., 2017) [1]. Data in this article present the direct effects of rotenone on calcium retention capacity (CRC) in isolated normal cortex and cerebellum mitochondria, effects of rotenone intravenous infusion on leak and phosphorylating respiration rates of isolated cortex and cerebellum mitochondria, on activities of respiratory chain complexes I and II in freezed-thawed/sonicated cortex and cerebellum mitochondria after brain ischemia. In addition, detailed experimental procedures of isolation of brain mitochondria, measurements of CRC, respiration, activities of respiratory chain complexes and H2O2 generation in cortex and cerebellum mitochondria are described.
| S-EPMC5512184 | biostudies-literature
Project description:Under high Ca(2+) load conditions, Ca(2+) concentrations in the extra-mitochondrial and mitochondrial compartments do not display reciprocal dynamics. This is due to a paradoxical increase in the mitochondrial Ca(2+) buffering power as the Ca(2+) load increases. Here we develop and characterize a mechanism of the mitochondrial Ca(2+) sequestration system using an experimental data set from isolated guinea pig cardiac mitochondria. The proposed mechanism elucidates this phenomenon and others in a mathematical framework and is integrated into a previously corroborated model of oxidative phosphorylation including the Na(+)/Ca(2+) cycle. The integrated model reproduces the Ca(2+) dynamics observed in both compartments of the isolated mitochondria respiring on pyruvate after a bolus of CaCl2 followed by ruthenium red and a bolus of NaCl. The model reveals why changes in mitochondrial Ca(2+) concentration of Ca(2+) loaded mitochondria appear significantly mitigated relative to the corresponding extra-mitochondrial Ca(2+) concentration changes after Ca(2+) efflux is initiated. The integrated model was corroborated by simulating the set-point phenomenon. The computational results support the conclusion that the Ca(2+) sequestration system is composed of at least two classes of Ca(2+) buffers. The first class represents prototypical Ca(2+) buffering, and the second class encompasses the complex binding events associated with the formation of amorphous calcium phosphate. With the Ca(2+) sequestration system in mitochondria more precisely defined, computer simulations can aid in the development of innovative therapeutics aimed at addressing the myriad of complications that arise due to mitochondrial Ca(2+) overload.
| S-EPMC3615037 | biostudies-literature
Project description:Key pointsSympathetic outflow and circulating glucogenic hormones both regulate liver function by increasing cytosolic calcium, although how these calcium signals are integrated at the tissue level is currently unknown. We show that stimulation of hepatic nerve fibres or perfusing the liver with physiological concentrations of vasopressin only will evoke localized cytosolic calcium oscillations and modest increases in hepatic glucose production. The combination of these stimuli acted synergistically to convert localized and asynchronous calcium responses into co-ordinated intercellular calcium waves that spread throughout the liver lobule and elicited a synergistic increase in hepatic glucose production. The results obtained in the present study demonstrate that subthreshold levels of one hormone can create an excitable medium across the liver lobule, which allows global propagation of calcium signals in response to local sympathetic innervation and integration of metabolic regulation by multiple hormones. This enables the liver lobules to respond as functional units to produce full-strength metabolic output at physiological levels of hormone.AbstractGlucogenic hormones, including catecholamines and vasopressin, induce frequency-modulated cytosolic Ca2+ oscillations in hepatocytes, and these propagate as intercellular Ca2+ waves via gap junctions in the intact liver. We investigated the role of co-ordinated Ca2+ waves as a mechanism for integrating multiple endocrine and neuroendocrine inputs to control hepatic glucose production in perfused rat liver. Sympathetic nerve stimulation elicited localized Ca2+ increases that were restricted to hepatocytes in the periportal zone. During perfusion with subthreshold vasopressin, sympathetic stimulation converted asynchronous Ca2+ signals in a limited number of hepatocytes into co-ordinated intercellular Ca2+ waves that propagated across entire lobules. A similar synergism was observed between physiological concentrations of glucagon and vasopressin, where glucagon also facilitated the recruitment of hepatocytes into a Ca2+ wave. Hepatic glucose production was significantly higher with intralobular Ca2+ waves. We propose that inositol 1,4,5-trisphosphate (IP3 )-dependent Ca2+ signalling gives rise to an excitable medium across the functional syncytium of the hepatic lobule, co-ordinating and amplifying the metabolic responses to multiple hormonal inputs.
| S-EPMC6647271 | biostudies-literature