Effects of glucagon and N6O2′-dibutyryladenosine 3′:5′-cyclic monophosphate on calcium transport in isolated rat liver mitochondria

Abstract

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.

A kinetic analysis of the effects of adrenaline on calcium distribution in isolated rat liver parenchymal cells

“…The cells were then suspended in 2 ml sucrose-HEPES-EGTA medium and homogenized for 60 s at setting 2-5 using an Ultra-Turrax TP ION homogenizer. Control experiments, similar to those described by Hughes & Barritt (1978), showed that the inclusion of 0-5 mM-EGTA in the isolation medium prevents the uptake of endogenous 45Ca by mitochondria. Subcellular fractions were isolated by centrifugation of: the homogenate at 200 g for 5 min (the precipitate was discarded), the supernatant from the previous step at 4600 g for 5 min (the precipitate = the mitochondrial fraction), the supernatant from the mitochondrial precipitate at 15,600 g for 10 min (the precipitate, an intermediate fraction, was discarded), and the supernatant from the previous step at 70,000 g for 90 min (the precipitate = the microsomal fraction).…”

“…The respiratory control ratio (the ratio of the ADP-stimulated to ADP-depleted rates of oxygen utilization) of mitochondria prepared from isolated hepatocytes was found to be 3'0 + 0 5 (n = 19) when measured in the presence of 12-5 mM-succinate as described by Hughes & Barritt (1978). 4"Ca exchange by isolated mitochondria.…”

“…4"Ca exchange by isolated mitochondria. Mitochondria which sedimented between 3000 and 22500 g min were isolated from the livers of fed rats, and their integrity and protein content measured, as described by Hughes & Barritt (1978). Mitochondria (1-5 mg protein/ml) were incubated at 37 Cin a medim which contained 150 mM-KCl, 10 mM-HEPES-KOH (pH 7 4), 2 mM-K phosphate, 2 mM-MgCl2, 10 mM-nitrilotriacetic acid, 2 mM-succinate, 3 mm-ATP and 25 or 10/SMtotal Ca.…”

“…Cells were separated from the incubation medium and extracted with 0 3 M-HClO4 containing 1 mM-LaCl3 as described by Blackmore et al (1978). The amount of Ca present was determined by atomic absorption spectroscopy as described previously (Hughes & Barritt, 1978).…”

α‐adrenergic mobilization of hepatic mitochondrial calcium

“…Another factor was the further reduction of calcium due to the inclusion of EGTA in the homogenization medium to prevent calcium re-uptake during mitochondrial isolation. Treatment of liver mitochondria with EGTA or EDTA has been shown to markedly decrease the calcium content [ 14,151.…”