Stable enhancement of calcium retention in mitochondria isolated from rat liver after the administration of glucagon to the intact animal

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 30μg of glucagon/100g body wt., retain Ca²⁺ 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, Ca²⁺ is retained for 6–8min. The ability of glucagon to enhance Ca²⁺ retention is completely prevented by co-administration of 4.2mg of puromycin/100g body wt. 2. The resting rate of respiration after Ca²⁺ 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 Ca²⁺ accumulation, reflecting the uncoupling action of the ion. 3. Concomitant with the enhanced retention of Ca²⁺ 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 Ca²⁺ 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 Ca²⁺. 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 Ca²⁺-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.     

The effect of glucagon on the kinetics of hepatic mitochondrial calcium uptake

Summary Previous work by this and other laboratories has shown that glucagon administration stimulates calcium uptake by subsequently isolated hepatic mitochondria. This stimulation of hepatic mitochondrial Ca²⁺ uptake byin vivo administration of glucagon was further characterized in the present report. Maximal stimulation of mitochondrial Ca²⁺ accumulation was achieved between 6–10 min after the intravenous injection of glucagon into intact rats. Under control conditions, Ca²⁺ uptake was inhibited by the presence of Mg²⁺ in the incubation medium. Glucagon treatment, however, appeared to obliterate the observed inhibition by Mg²⁺ of mitochondrial Ca²⁺ uptake. Kinetic experiments revealed the usual sigmoidicity associated with initial velocity curves for mitochondrial calcium uptake. Glucagon treatment did not alter this sigmoidal relationship. Glucagon treatment significantly increased the V_max for Ca²⁺ uptake from 292±22 to 377±34 nmoles Ca²⁺ /min per mg protein (n=8) but did not affect the K_0.5, (6.5–8.6 μM). Since the major kinetic change in mitochondrial Ca²⁺ uptake evoked by glucagon is an increase in V_max, the enhancement mechanism is likely to be an increase either in the number of active transport sites available to Ca²⁺ or in the rate of Ca²⁺ carrier movement across the mitochondrial membranes.     

The nature of the changes in liver mitochondrial function induced by glucagon treatment of rats. The effects of intramitochondrial volume,aging and benzyl alcohol

(1) The effects of changes in the intramitochondrial volume, benzyl alcohol treatment and calcium-induced mitochondrial aging on the behaviour of liver mitochondria from control and glucagon-treated rats are reported. (2) The stimulatory effects of glucagon on mitochondrial respiration, pyruvate metabolism and citrulline synthesis could be mimicked by hypo-osmotic treatment of control mitochondria and reversed by calcium-induced aging of mitochondria or by treatment with 20 mM benzyl alcohol. Hypo-osmotic treatment increased the matrix volume whilst aging but not benzyl alcohol decreased this parameter. (3) Liver mitochondria from glucagon and adrenaline-treated rats were shown to be less susceptible to damage by exposure to calcium than control mitochondria and frequently showed slightly (15%) elevated intramitochondrial volumes. (4) Aging, benzyl alcohol and hypo-osmotic media increased the susceptibility of mitochondria to damage caused by exposure to calcium. (5) Glucagon-treated mitochondria were less leaky to adenine nucleotides than control mitochondria. (6) These results suggest that glucagon may exert its action on a wide variety of mitochondrial parameters through a change in the disposition of the inner mitochondrial membrane, possibly by stabilisation against endogenous phospholipase A₂ activity. This effect may be mimicked by an increase in the matrix volume or reversed by calcium-dependent mitochondrial aging.     

Glucagon Regulation of Oxidative Phosphorylation Requires an Increase in Matrix Adenine Nucleotide Content through Ca2+ Activation of the Mitochondrial ATP-Mg/Pi Carrier SCaMC-3

It has been known for a long time that mitochondria isolated from hepatocytes treated with glucagon or Ca²⁺-mobilizing agents such as phenylephrine show an increase in their adenine nucleotide (AdN) content, respiratory activity, and calcium retention capacity (CRC). Here, we have studied the role of SCaMC-3/slc25a23, the mitochondrial ATP-Mg/P_i carrier present in adult mouse liver, in the control of mitochondrial AdN levels and respiration in response to Ca²⁺ signals as a candidate target of glucagon actions. With the use of SCaMC-3 knock-out (KO) mice, we have found that the carrier is responsible for the accumulation of AdNs in liver mitochondria in a strictly Ca²⁺-dependent way with an S_0.5 for Ca²⁺ activation of 3.3 ± 0.9 μm. Accumulation of matrix AdNs allows a SCaMC-3-dependent increase in CRC. In addition, SCaMC-3-dependent accumulation of AdNs is required to acquire a fully active state 3 respiration in AdN-depleted liver mitochondria, although further accumulation of AdNs is not followed by increases in respiration. Moreover, glucagon addition to isolated hepatocytes increases oligomycin-sensitive oxygen consumption and maximal respiratory rates in cells derived from wild type, but not SCaMC-3-KO mice and glucagon administration in vivo results in an increase in AdN content, state 3 respiration and CRC in liver mitochondria in wild type but not in SCaMC-3-KO mice. These results show that SCaMC-3 is required for the increase in oxidative phosphorylation observed in liver mitochondria in response to glucagon and Ca²⁺-mobilizing agents, possibly by allowing a Ca²⁺-dependent accumulation of mitochondrial AdNs and matrix Ca²⁺, events permissive for other glucagon actions.     

The presence of lysophosphatidylcholine in chromaffin granules

Lysophosphatidylcholine is thought to be a characteristic component of the chromaffin granules in adrenal glands. By the use of a t.l.c. system that resolves minor phospholipids satisfactorily, this subcellular location was confirmed in the present study in bovine glands. However, phospholipid degradation was demonstrated in homogenates of the adrenal medulla and cortex under conditions similar to those of subcellular fractionation (incubation at 4°C for 90min). Phosphatidylethanolamine and cardiolipin were hydrolysed, but the concentration of lysophosphatidylcholine did not change, indicating that the latter was present in the medulla before this treatment. Attempts were made to decrease the time between death of the animal and the extraction of lipids. Lysophosphatidylcholine was easily demonstrable in lipid extracts of the dissected medulla and even in those of the whole bovine gland. For practical reasons it is not possible to decrease further the time lapse before extraction in the case of this animal. Adrenal glands were obtained from anaesthetized and untreated rabbits. These were frozen immediately in liquid N₂ and the lipids were extracted. In a control experiment, the glands from rabbit were dissected and treated in the same manner as with those of ox, and then the lipids were extracted. No lysophosphatidylcholine was detected in the extracts from glands frozen in liquid N₂ but lysophosphatidylcholine was observed in the controls. These results suggest that lysophosphatidylcholine is not a component of chromaffin granules, but is produced if the period between death of the animal and lipid extraction is unduly prolonged. To discover whether lysophosphatidylcholine affected the permeability barrier properties of chromaffin granules, sonicated liposomes of egg phosphatidylcholine alone or with lysophosphatidylcholine (15mol/100mol) were prepared. Both types were shown by electron microscopy to be largely made up of single bilayer vesicles. The exchange diffusion of [¹⁴C]dopamine was measured across their membranes. Both types of liposomes had similar capture volumes (0.5μl/μmol of phospholipid), and the activation energies of the exchange diffusion of dopamine were also similar (31kJ/mol). These results indicate that the presence of this proportion of lysophosphatidylcholine in chromaffin-granule membranes is not likely to influence their barrier properties towards catecholamines.     

Micromolar changes in lysophosphatidylcholine concentration cause minor effects on mitochondrial permeability but major alterations in function

Mice deficient in group 1b phospholipase A₂ have decreased plasma lysophosphatidylcholine and increased hepatic oxidation that is inhibited by intraperitoneal lysophosphatidylcholine injection. This study sought to identify a mechanism for lysophosphatidylcholine-mediated inhibition of hepatic oxidative function. Results showed that in vitro incubation of isolated mitochondria with 40–200 μM lysophosphatidylcholine caused cyclosporine A-resistant swelling in a concentration-dependent manner. However, when mitochondria were challenged with 220 μM CaCl₂, cyclosporine A protected against permeability transition induced by 40 μM, but not 80 μM lysophosphatidylcholine. Incubation with 40–120 μM lysophosphatidylcholine also increased mitochondrial permeability to 75 μM CaCl₂ in a concentration-dependent manner. Interestingly, despite incubation with 80 μM lysophosphatidylcholine, the mitochondrial membrane potential was steady in the presence of succinate, and oxidation rates and respiratory control indices were similar to controls in the presence of succinate, glutamate/malate, and palmitoyl-carnitine. However, mitochondrial oxidation rates were inhibited by 30–50% at 100 μM lysophosphatidylcholine. Finally, while 40 μM lysophosphatidylcholine has no effect on fatty acid oxidation and mitochondria remained impermeable in intact hepatocytes, 100 μM lysophosphatidylcholine inhibited fatty acid stimulated oxidation and caused intracellular mitochondrial permeability. Taken together, these present data demonstrated that LPC concentration dependently modulates mitochondrial microenvironment, with low micromolar concentrations of lysophosphatidylcholine sufficient to change hepatic oxidation rate whereas higher concentrations are required to disrupt mitochondrial integrity.     

Starvation reduces pyruvate dehydrogenase phosphate phosphatase activity in rat kidney

Pyruvate dehydrogenase complex (PDC) from rat kidney or pig heart previously inactivated by phosphorylation (PDHP) was activatedin vitro by PDHP phosphatase from kidneys of starved or fed rats. Starvation for 48 h of the rats from which the PDC was prepared led to a decrease in the rate of activation of PDC at early time periods (<2 min), particularly at submaximal concentrations of Mg²⁺. Using intact permeable kidney mitochondria incubated for 15 sec, it was found that starvation of rats more than doubled the Mg²⁺ concentration at which the half maximal increment of PDC activity (PDC_a) was observed. Reduction of PDHP phosphatase activity due to starvation was also apparent when phosphatase was separated from PDC and recombined with PDC from the same or different animals.

Intraperitoneal injection of insulin and glucose 1 h before sacrifice of starved rats prevented the reduction of PDHP phosphatase activity whether or not protein synthesis was inhibited. The effect of insulin in restoration of PDHP phosphatase activity of starved rats was not mimicked by 5-methylpyrazole 3-carboxylic acid, an inhibitor of lipolysis.

When renal PDHP phosphatase was incubated with pig heart PDC in the presence of 10 mM Mg²⁺ and 0.1 mM Ca²⁺ the increment in PDC_a, in 1 min was 30% of fully activated PDC activity (PDC₁) observed after 15 min. Removal of divalent cations did not affect the increment in 1 min but prevented further increments. Conversely okadaic acid diminished 1 min increment but did not disturb PDC_t. It is suggested that the different behaviour of renal PDC from fed and starved animals may partly be due to different divalent cation independent PDHP phosphatase activity.

     

Effects of short-term ozone exposure on lung mitochondrial oxidative and energy metabolism

Ozone effects on lung mitochondrial oxidative metabolism were examined after short-term exposure of rats and monkeys to O₃. Exposure of animals to 2 ppm O₃ for 8 hr or to 4 ppm O₃ for 4 hr caused a 15–27% (P < 0.05) depression of lung mitochondrial O₂ consumption, using 2-oxoglutarate, succinate, and glycerol-1-phosphate. but not ascorbate plus Wurster's blue as substrates. Under these exposure conditions (4 ppm 4 hr) the ADP:O ratios dropped 25–36% (P < 0.05) and the respiratory control indices decreased 27–33% (P < 0.02) for oxidation of all substrates examined. Lung mitochondria from control animals were relatively impermeable to added NADH, but those from O₃-exposed animals showed an increased permeability as judged from NADH oxidation at a rate 3-fold higher than the control. Likewise, added cytochrome c caused a 22% (P < 0.01) stimulation of succinate oxidation in exposed lung mitochondria as against 5% (nonsignificant) in controls. Ozone exposure also caused a 20% (P < 0.01) oxidation of thiol groups in lung mitochondria, but no lipid peroxidation products were detectable in O₃-exposed lung tissue. The depression of substrate utilization, coupled phosphorylation and respiratory control observed in lung mitochondria of O₃-exposed animals might be related to alteration of membrane permeability, and inhibition of respiratory enzymes (dehydrogenases) due to oxidation of functional thiol groups.     

Kinetic analysis of75selenium uptake by mitochondria of germinating vigna radiata of different selenium status

Earlier studies in our laboratory demonstrated the beneficial role of Se inVigna radiata, a Se-deficient legume, during germination, as reflected in growth-related parameters and specific uptake of⁷⁵Se. Uptake of Na₂ ⁷⁵SeO₃, added in vitro by mitochondria isolated from seedlings germinated in control (without Se), and Se-supplemented groups (0.5, 1.0, and 2.0 ppm Se) indicated a proportional increase in the uptake with added Na₂ ⁷⁵SeO₃, in concentrations up to 25 γM. The uptake of⁷⁵Se, increased linearly with time up to 15 min and a definite efflux followed at 30 min. The results were indicative of cooperative effects during Se transport. Kinetic analyses of the uptake of⁷⁵Se during time intervals of 15 and 30 min were carried out both in the whole mitochondria and the mitochondrial protein fractions. Graphical analyses using Lineweaver-Burk plot, Hill plot, log [v] vs log [A] and Scatchard plot confirmed the existence of negative cooperativity during⁷⁵Se uptake. Hill coefficient (nH) values were estimated to be around 0.7–0.8. Scatchard plots for⁷⁵Se uptake were biphasic, suggesting the probable presence of two classes of binding sites. The number of high and low affinity binding sites were estimated to be around 4–7 and 26–30 nmol/mg protein, respectively. Studies with mitochondrial respiratory inhibitors indicated about 10–20% of the total⁷⁵Se uptake to be energy dependent. Inhibition of⁷⁵Se uptake by about 60–70% by sulfate and sulfite (5–25 γM) implies the involvement of dicarboxylate port in Se transport. A decrease in the uptake of⁷⁵Se by 40–60% effected by CdCl₂, HgCl₂, mersalyl, and NEM confirmed the interaction of thiols in the process. Evidence for the regulatory nature of⁷⁵Se uptake by mitochondria ofV. radiata emerges from the present study.     

Intestinal lipid alterations occur prior to antibody-induced prostaglandin E2 production in a mouse model of ischemia/reperfusion

Ischemia/reperfusion (IR) induced injury results in significant tissue damage in wild-type, but not antibody-deficient, Rag-1^−/− mice. However, Rag-1^−/− mice sustain intestinal damage after administration of wild-type antibodies or naturally occurring, specific anti-phospholipid related monoclonal antibodies, suggesting involvement of a lipid antigen. We hypothesized that IR initiates metabolism of cellular lipids, resulting in production of an antigen recognized by anti-phospholipid antibodies. At multiple time points after Sham or IR treatment, lipids extracted from mouse jejunal sections were analyzed by electrospray ionization triple quadrupole mass spectrometry. Within 15 min of reperfusion, IR induced significantly more lysophosphatidylcholine (lysoPC), lysophosphatidylglycerol (lysoPG) and free arachidonic acid (AA) production than Sham treatment. While lysoPC, lysoPG, and free AA levels were similar in C57Bl/6 (wild-type) and Rag-1^−/− mice, IR led to Cox-2 activation and prostaglandin E₂ (PGE₂) production in wild-type, but not in the antibody-deficient, Rag-1^−/− mice. Administration of wild-type antibodies to Rag-1^−/− mice restored PGE₂ production and intestinal damage. These data indicate that IR-induced intestinal damage requires antibodies for Cox-2 stimulated PGE₂ production but not for production of lysoPC and free AA.     

The effect of glucagon treatment and starvation of virgin and lactating rats on the rates of oxidation of octanoyl-l-carnitine and octanoate by isolated liver mitochondria

1. Oxygen-consumption rates owing to oxidation of octanoate or octanoylcarnitine by isolated mitochondria from livers of fed, starved and glucagon-treated virgin or 12-day-lactating animals were measured under State-3 and State-4 conditions, in the presence or absence of l-malate and inhibitors of tricarboxylic acid-cycle activity (malonate and fluorocitrate). 2. Mitochondria from fed lactating animals had a slightly lower rate of octanoylcarnitine oxidation than did those of fed virgin animals, whereas the rates of octanoate oxidation were unaffected. 3. Starvation of virgin animals for 24h or 48h resulted in a large (70–100%) increase in mitochondrial octanoylcarnitine oxidation; rates of octanoate oxidation were either unaffected (24 and 48h starvation in the absence of malonate and fluorocitrate) or diminished by 30% (48h starvation in the presence of inhibitors). In lactating animals, 24h starvation resulted in a smaller increase in the rate of octanoylcarnitine oxidation than that obtained for mitochondria from virgin rats. 4. Glucagon treatment (by intra-abdominal injection) of fed virgin and lactating rats increased the rate of mitochondrial oxidation of both octanoylcarnitine and octanoate. Injection of glucagon into 48h-starved virgin rats did not increase further the already elevated rate of octanoylcarnitine oxidation, but reversed the inhibition of octanoate β-oxidation observed for these mitochondria in the presence of malonate and fluorocitrate. 5. It is suggested that glucagon activates octanoylcarnitine oxidation by increasing the activity of the carnitine/acylcarnitine transport system [Parvin & Pande (1979) J. Biol. Chem. 254, 5423–5429] and that the increase in octanoate oxidation by mitochondria from glucagon-treated animals is caused by the increased rate of ATP synthesis in these mitochondria. 6. The results are discussed in relation to the increased capacity of the liver to oxidize long-chain fatty acids and carnitine esters of medium-chain fatty acids under conditions characterized by increased ketogenesis.     

Lack of acute zinc effects in glucose metabolism in healthy and insulin-dependent diabetes mellitus patients

Acute or chronic zinc administration may cause hyperglycemia in experimental animals. These findings are attributed to permissive actions of glucocorticoids and glucagon upon hepatic gluconeogenesis and glycogenolysis. The effect of Zn⁺⁺ on plasma glucose, C-peptide, glucagon, and cortisol was investigated in healthy and insulin-dependent diabetes mellitus (IDDM) patients. Ten normal individuals (5 of each sex, aged 24.10 ± 1.96) and 10 IDDM (5 of each sex, aged 25.20 ± 8.10) were tested at 7:00 AM after 12-h fast. Twenty-five mg of Zn⁺⁺ were administered intravenously during 1 min, and blood samples were collected from the contralateral arm at 0, 3, 30, 60, 90 and 120 min after Zn⁺⁺ injection. The plasma levels of glucose, C-peptide, and glucagon remained constant throughout the experimental period in both groups studied. Plasma cortisol levels decreased significantly, which is consistent with our previous findings. These results suggest that, in contrast to experimental animals, acute Zn⁺⁺ administration, despite decreasing cortisol levels, does not change carbohydrate metabolism in human beings.     

Vitamin A inhibits lipoperoxidation ascorbate-Fe++ dependent of rat kidney microsomes and mitochondria

In the present study it was investigated if Vitamin A supplementation could protect rat kidney microsomes and mitochondria from in vitro lipoperoxidation. After incubation of rat kidney microsomes and mitochondria in an ascorbate-Fe⁺⁺ system, at 37°C during 60 min, it was observed that the total cpm/mg protein originated from light emission (chemiluminescence) was lower in those organelles obtained from the control group when compared with the vitamin A supplemented group. The fatty acid composition of microsomes and mitochondria from control group was profoundly modified when subjected to nonenzymatic lipoperoxidation with a considerable decrease of arachidonic acid, C20:4 (n-6) and docosapentaenoic acid, C22:5 (n–3) in mitochondria and docosahexaenoic acid C22:6 (n-3) in microsomes.As a consequence the peroxidizability index, a parameter based on the maximal rate of oxidation of specific fatty acids was higher in the supplemented animals than in those used as control. These results indicate that Vitamin A may act as antioxidant protecting rat kidney microsomes and mitochondria from deleterious effect.     

Structure-conformation-activity studies of glucagon and semi-synthetic glucagon analogs

Summary Examination of glucagon structure-activity relationships and their use for the development of glucagon antagonists (inhibitors) have been hampered until recently by the lack of high purity of semisynthetic glucagon analogs and inadequate study of full dose-response curves for these analogs in sensitive bioassay systems. Recently a number of highly purified glucagon fragments and semi-synthetic analogs have been prepared and their full dose-response activities examined over a wide concentration range using the hepatic membrane adenylate cyclase assay, the hepatic membrane receptor binding assay, and glycogenolytic activity in isolated rat hepatocytes. The results of these studies have enabled us to identify and dissociate the structural (and in some cases conformational) features of glucagon important for binding from those most responsible for biological activity (transduction). Key findings in these studies were the observation that: (1) the C-terminal region of glucagon is primarily of importance for hormone binding to receptors; (2) glucagon_1–21 and glucagon_1–6 have low potency, but are essentially fully active glucagon derivatives; and (3) highly purified glucagon_2–29 ([1-des-histidine]-glucagon), [1-N-carbamoylhistidine]-glucagon and [1-N-carbamoylhistidine, 12-N-carbamoyllysine]-glucagon are all partial agonists.These and other findings led us to synthesize several semisynthetic analogs of glucagon which were found to possess no intrinsic biological activity in the hepatic adenylate cyclase assay system, but which could block the effect of glucagon (competitive inhibitors) in activating adenylate cyclase in this system. Two of these highly purified analogs [1-des-histidine] [2-N-trinitrophenylserine, 12-homoarginine]-glucagon and [1-N-trinitrophenylhistidine, 12-homoarginine]-glucagon were quite potent glucagon antagonists (inhibitors) with pA₂ values of 7.41 and 8.16 respectively. The latter compound has also been demonstrated to decrease dramatically blood glucose levels of diabetic animals in vivo. These results demonstrate that glucagon is a major contributor to the hyperglycemia of diabetic animals.Examination of the known and calculated conformational properties of glucagon provide insight into the structural and conformational properties of glucagon and its analogs most responsible for its biological activity. Consideration of these features and the mechanism of glucagon action at the membrane receptor level provide a framework for further developing glucagon analogs for theoretical and therapeutic applications.     

Role of catalase in myocardial protection against ischemia in heat shocked rats

It was recently reported that in rats exposure to heat shock leads to appearance of a myocardial heat shock protein (HSP 70) and to an increase in myocardial catalase activity. This correlated with an improvement in post-ischemic function either in Langendorff-perfused hearts after low-flow ischemia or in working hearts after short-term, no-flow ischemia. We investigated the effect of the same hyperthermic treatment on functional recovery from no-flow ischemia of various durations in isolated working rat hearts performing at high or low external workloads. Rats were heated to core temperature of 42° C for 15 min. No significant protein oxidation (% oxidized methionine) was observed 2.5 hr after treatment. A protein with migration characteristics similar to HSP 70 was observed in hearts of heat shocked rats 24 hr after this treatment while their myocardial catalase activity was not increased. Hearts of similarly treated rats were excised 24 hr after hyperthermia and perfused in a working mode with Krebs-Henseleit buffer (1.25 mM Ca²⁺, 11 mM glucose). At 15 cm H₂O preload and 100 cm H₂O afterload after 30 min no-flow ischemia, control hearts recovered to 36.9%, 2%, 47.6%, and 21.5% of the preischemic values of heart rate-peak systolic pressure product (RPP), aortic output, coronary flow, and cardiac output, respectively. After only 25 min of ischemia the respective recovered values were 61.6%, 11.5%, 58.7%, and 33.5%. Throughout the recovery period these hemodynamic values were consistently higher in hearts of heat shocked animals than in those of control hearts but the differences were not statistically significant. After 25 min ischemia only 2 out of 7 control hearts recovered some aortic output, whereas in the heat shocked animals all 5 hearts recovered. After only 20 min of no-flow ischemia and at a lower workload (12.5 cm H₂O preload and 75 cm H₂O afterload), control hearts recovered to 85.1% of RPP, 54.1% of aortic output, and 68.3% of cardiac output. None of these variables was significantly improved by heat shock pretreatment. In summary, we were unable to demonstrate a similar degree of protective effect of heat shock pretreatment as compared to other reports where both HSP 70 and increased catalase activity were present. The reason(s) could be related to lack of induction of myocardial catalase activity in our study.     

Mitochondrial dysfunction as a mediator of hippocampal apoptosis in a model of hepatic encephalopathy

In this study, we describe the presence of apoptosis, associated with a mitochondrial dysfunction in the hippocampus of animals in an experimental model defined as minimal hepatic encephalopathy (MHE). This experimental model was studied after 10 days of induced portal vein calibrated stricture, leading to portal hypertension and to a moderate hyperammonemia, without the presence of other evident central nervous system changes. The molecular mechanisms here proposed indicate the presence of apoptotic intrinsic pathways that point to hippocampal mitochondria as an important mediator of apoptosis in this experimental model. In this model of MHE, the presence of DNA fragmentation is documented by 2.3-times increased number of TUNEL-positive cells. These findings together with a higher ratio of the Bcl-2 family members Bax/Bcl-xL in the outer mitochondrial membrane of the MHE animals together with 11% of cytochrome c release indicate the presence of apoptosis in this experimental model. A detailed analysis of the hippocampal mitochondrial physiology was performed after mitochondrial isolation. The determination of the respiratory rate in the presence of malate plus glutamate and ADP showed a 45% decrease in respiratory control in MHE animals as compared with the sham group. A marked decrease of cytochrome oxidase (complex IV of the electron transport chain) was also observed, showing 46% less activity in hippocampal mitochondria from MHE animals. In addition, mitochondria from these animals showed less ability to maintain membrane potential (ΔΨ _m) which was 13% lower than the sham group. Light scattering experiments showed that mitochondria from MHE animals were more sensitive to swell in the presence of increased calcium concentrations as compared with the sham group. In addition, in vitro studies performed in mitochondria from sham animals showed that mitochondrial permeability transition (MPT) could be a mitochondrial mediator of the apoptotic signaling in the presence of NH₄ ⁺ and calcium.     

Vasopressin and angiotensin II stimulate ureogenesis through increased mitochondrial citrulline production.

Vasopressin, angiotensin II, glucagon and epinephrine (through a cAMP-independent, alpha₁adrenergic mechanism), stimulate ureogenesis in isolated rat hepatocytes. Mitochondria, isolated from hepatocytes which were previously treated with these hormones, displayed an enhanced rate of citrulline synthesis in the presence of NH₄Cl as the nitrogen source. When mitochondria were incubated with glutamine as the nitrogen source, only those mitochondria isolated from hepatocytes previously treated with epinephrine or glucagon displayed an enhanced capacity to synthesize citrulline.When cells were incubated in the absence of extracellular calcium, the effects of vasopressin and angiotensin II on urea synthesis were abolished, whereas those of epinephrine and glucagon were only diminished. Mitochondria isolated from cells incubated under these conditions, showed that the effect of all these hormones on citrulline synthesis could still be observed. However, the effects of glucagon and epinephrine plus propranolol were larger than those of angiotensin II or vasopressin.Phosphatidylinositol labeling was significantly increased by epinephrine, vasopressin and angiotensin II both in the absence or presence of calcium. Cyclic AMP levels were significantly increased by glucagon or epinephrine but not by vasopressin or angiotensin II. The effect of epinephrine on cyclic AMP levels was blocked by propranolol both in the absence or presence of calcium.     

Studies on the lack of glucagon response to clycogenolysis and gluconeogensis and decrease in protein synthesis in isolated hepatocytes from hypophysectomized animals.

Effect of various concentrations of glucagon on gluconeogenesis and glycogenolysis was studied in isolated hepatocytes obtained from normal and hypophysectomized animals. Addition of glucagon (10^?10 to 10^?6M) stimulated glycogenolysis and gluconeogenesis by 2–3 fold in normal hepatocytes. However, this concentration of glucagon had only a slight effect in isolated hepatocytes obtained from hypophysectomized animals. This lack of glucagon response was not due to reduction in glycogen levels in isolated hepatocytes obtained from hypophysectomized animals. Studies on the incorporation of¹⁴C-alanine,¹⁴C-leucine and¹⁴C-valine showed a 3–5 fold decrease in the incorporation of these amino acids into protein in hypophysectomized animals compared to normal controls.     

On the mechanism of the glucagon-induced inhibition of hepatic protein synthesis.

The intraperitoneal administration of glucagon (200 μg) to rats produced a transient increase of the hepatic polypeptide chain completion time, the increase being maximum at 5 min returning to control values at 20 min. This inhibitory effect was sustained when glucagon was constantly supplied by continuous infusion. Postmitochondrial supernatants from livers of the control group or rats treated with glucagon for 5 min showed no difference in their protein synthetic activity. After 20 min of intraperitoneal administration of the hormone, that is, when the effect on protein synthesis had vanished, the levels of cAMP were still 40% above those of the control group, and the ribosomal proteins were 110% more phosphorylated. These results suggest that the observed effect of glucagon is not due to its direct action on the protein synthesis machinery. On the other hand, the variations in the hepatic amino acid content brought about by glucagon do not appear to be quantitatively significant to account for the observed inhibition of protein synthesis. The effect of glucagon was always paralleled by a decrease in the $\text{[ATP]}\text{[ADP]}$ ratio which may be responsible for the observed decrease in the rates of elongation and/or termination steps of protein synthesis. Glucagon also produced a rise in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ ratio in both cellular compartments, cytosol and mitochondria, as reflected by the rise in the lactate to pyruvate and the β-hydroxybutyrate to acetoacetate ratios. This shift of the NAD⁺ couple to a more reduced state seems to be the result of an increased mobilization and oxidation of fatty acids brought about by the hormone. It is postulated then that the primary effect of glucagon leading to a decrease in protein synthesis is probably to increase the state of reduction of the hepatic nicotinamide nucleotide system. This point of view is supported by the fact that the nicotinamide and adenine nucleotide systems in rat liver are in equilibrium through cytosolic equilibrium reactions, so that a decrease in the $\text{[ATP]}\text{[ADP]}$ ratio brought about by glucagon may be secondary to the increase in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ ratio. This hypothesis is supported by the fact that glucagon was not effective in inhibiting hepatic protein synthesis in rats pretreated with a drug, 2-benzene-sulfonamido-5-(β-methoxy-ethoxy)pyrimidine, that prevents fatty acid mobilization and the subsequent changes in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ and $\text{[ATP]}\text{[ADP]}$ ratios. Furthermore, the administration of exogenous fatty acid brings about an inhibition of the rate of hepatic protein synthesis accompanied by a decrease in the ATP levels and an increase in the state of reduction of the NAD⁺ system.     

Enzymatic properties of mitochondria isolated from normal and vitamin B12-deficient rats.

The mitochondria from livers of normal and vitamin B₁₂-deprived animals were compared. The rate of oxygen uptake (states 3 and 4) of mitochondria from vitamin B₁₂-deprived livers was higher than that of normal mitochondria while the respiratory control index of B₁₂-deprived mitochondria was lower than normal. Most Krebs cycle enzyme activities were increased in the hepatic mitochondria from B₁₂-deprived animals. Two-dimensional gel electrophoresis of mitochondrial matrix proteins also indicated that an increase in Krebs cycle enzyme quantities occurred but the increase was not general for all mitochondrial proteins.