Tracer priming in human protein turnover studies with [15N]glycine

Sixty-three studies in healthy normal volunteers (n = 29), malnourished cancer (n = 8) or non-cancer patients (n = 9), and postoperative radical cystectomy patients (n = 17) were conducted to evaluate the primed constant infusion labeling technique for the estimation of whole-body protein turnover under a variety of dietary conditions. [15N]Glycine was used as the tracer with a prime to infusion ratio of 1300 to 3300 min and a continuous-infusion rate of 0.11 to 0.33 micrograms 15N . kg-1 . min-1 for 24 to 36 hr. The isotopic steady-state enrichment was reached in all subjects both in urinary urea and ammonia between 10 and 26 hr (mean 18 +/- 2). During protein calorie fasting the attainment of isotopic steady state is much quicker (10 to 18 hr) with a primed constant infusion than with a constant infusion alone (approximately 38 hr). A P/I ratio greater or less than 1800 (min) usually resulted in a delay of plateau attainment without affecting the protein turnover values. Reliable estimates of protein kinetics in humans can be made in clinical conditions with a 26-hr infusion of glycine at the rate of 0.28 microgram 15N . kg-1 . min-1 with a P/I ratio of 1800 min, collecting six urine samples every 2 hr from 16 hr and analyzing for both urinary urea and ammonia enrichments.     

Reduced muscle protein breakdown in septic rats following treatment with interleukin-1 receptor antagonist

• 1.1. The role ofinterleukin-1 (IL-1) in sepsis-induced muscle proteolysis was assessed by treating septic rats with recombinant IL-1 receptor antagonist (rIL-Ira).
• 2.2. In initial experiments, we tested the effectiveness of IL-Ira in preventing muscle proteolysis induced by administration of IL-1.
• 3.3. When normal rats were treated with rIL-α (three intraperitoneal doses of 100 μ g/kg body weight each over 16 hr), total and myofibrillar muscle protein breakdown rates, measured as release oftyrosine and 3-methylhistidine, respectively, by incubated extensor digitorum longus muscles, were significantly increased.
• 4.4. This metabolic response to IL-α was completely abolished by rIL-Ira, administered as three intraperitoneal doses of 3 mg/kg body weight each over 16hr.
• 5.5. In subsequent experiments, sepsis was induced in rats by cecal ligation and puncture (CLP); non-septic rats were sham-operated.
• 6.6. Treatment of septic rats over 16hr with a total dose of 25mg/kg body weight of rIL-Ira reduced, but did not normalize, the increased muscle protein breakdown rates seen during sepsis.
• 7.7. When the dose of rIL-Ira was more than doubled and given as a constant infusion at a rate of 4.2 mg/kg body weight/hr for 16 hr, the increased rate of muscle proteolysis in septic rats was normalized.
• 8.8. The present study offers the first direct evidence that IL-1 is involved in the regulation of muscle proteolysis during sepsis.

     

The unfolding and attempted refolding of mitochondrial aspartate aminotransferase from pig heart.

The role of prostaglandins in the regulation of muscle protein breakdown is controversial. We examined the influence of arachidonic acid (5 microM), prostaglandin E2 (PGE2) (2.8 microM) and the prostaglandin-synthesis inhibitor indomethacin (3 microM) on total and myofibrillar protein breakdown in rat extensor digitorum longus and soleus muscles incubated under different conditions in vitro. In other experiments, the effects of indomethacin, administered in vivo to septic rats (3 mg/kg, injected subcutaneously twice after induction of sepsis by caecal ligation and puncture) on plasma levels and muscle release of PGE2 and on total and myofibrillar protein breakdown rates were determined. Total and myofibrillar proteolysis was assessed by measuring production by incubated muscles of tyrosine and 3-methylhistidine respectively. Arachidonic acid or PGE2 added during incubation of muscles from normal rats did not affect total or myofibrillar protein degradation under a variety of different conditions in vitro. Indomethacin inhibited muscle PGE2 production by incubated muscles from septic rats, but did not lower proteolytic rates. Administration in vivo of indomethacin did not affect total or myofibrillar muscle protein breakdown, despite effective plasma levels of indomethacin with decreased plasma PGE2 levels and inhibition of muscle PGE2 release. The present results suggest that protein breakdown in skeletal muscle of normal or septic rats is not regulated by PGE2 or other prostaglandins.     

Salicylic acid blocks indomethacin-induced cyclooxygenase inhibition and lesion formation in rat gastric mucosa

Salicylic acid has been shown to decrease gastric mucosal lesions induced by indomethacin in the rat. In vitro, it has also been shown to counteract the inhibitory effect of indomethacin and aspirin on the cyclooxygenase enzyme system in seminal vesicle microsomes and in platelets and vascular tissue. The hypothesis that the mechanism of salicylic acid "protection" against indomethacin-induced gastric lesions involves interference with indomethacin-induced mucosal cyclooxygenase inhibition was tested. Male, fasted rats were treated with intragastric salicylic acid in doses of 50, 100, 200, 300, or 400 mg/kg concomitantly with a sc injection of 20 mg/kg of indomethacin. Gastric mucosal lesions and mucosal cyclooxygenase activity (as measured by ex vivo prostaglandin F2 alpha synthesis) were examined 3 hr later. Intragastric salicylic acid, 200-400 mg/kg, significantly reduced indomethacin-induced lesion formation, while counteracting significantly indomethacin inhibition of prostaglandin synthesis. Salicylic acid alone did not significantly change cyclooxygenase activity. It is concluded that topical salicylic acid can decrease indomethacin-induced gastric mucosal lesion in the rat, in part, by counteracting the inhibitory effect of indomethacin at the cyclooxygenase level.     

Fever is not responsible for the elevated glucose kinetics in sepsis

Previous studies have suggested that alterations in the classical neuroendocrine system may not be responsible for the increased glucose metabolism observed during hypermetabolic sepsis. The purpose of the present study was to determine whether inhibition of the cyclooxygenase pathway with indomethacin, which prevents the production of arachidonic acid metabolites by this pathway and the sepsis-induced increase in body temperature, would abolish the increases in glucose appearance (Ra), recycling, and hyperlactacidemia. Sepsis was induced in chronically catheterized conscious rats by multiple injections of live Escherichia coli via a subcutaneous catheter. Septic animals received iv injections of indomethacin (5 mg/kg) every 6-8 hr to block the cyclooxygenase pathway. Glucose kinetics were assessed in 24-hr fasted rats using a constant iv infusion of [6-3H]- and [U-14C]glucose. Treatment with indomethacin prevented the 1-2 degrees C increase in body temperature observed in septic animals. Septic rats exhibited an elevated plasma lactate concentration and increased rates of glucose appearance and recycling. The sepsis-induced alterations in these variables were not attenuated by indomethacin. These results suggest that neither elevated body temperature nor the generation of arachidonic acid metabolites of the cyclooxygenase pathway is responsible for increasing glucose production in hypermetabolic septic rats.     

Aspirin inhibits rat megakaryocyte thromboxane synthesis

This study examines the question of whether the aspirin-induced delay in the recovery of platelet cyclooxygenase pathway activity, as measured by RIA of thromboxane B₂, results from a direct effect on megakaryocyte cyclooxygenase. From our measurement of recovery of TXB₂ and information on megakaryocyte transit time in rats, we propose that thromboxane synthesis may represent a relatively late step in the differentiation of megakaryocytes. Megakaryocyte thromboxane production was depressed by 70% and that of platelets by 85% at two hr after 20 mg/kg oral aspirin dissolved in DMSO. Full megakaryocyte thromboxane recovery occurred by 72 hr and preceded complete platelet thromboxane recovery by 24 hr. Whereas megakaryocyte thromboxane synthesis showed substantial recovery by 36 hr after aspirin, platelet recovery did not begin for 24 hr and achieved a maximal recovery rate over the following 12 hr. This finding is consistent with predictions based upon human data for both megakaryocyte labeling studies and post-aspirin platelet recovery. We conclude from our data and from estimates of megakaryocyte maturation times in marrow, that thromboxane synthesis develops in rat megakaryocytes after approximately 48 hr of cytoplasmic differentiation toward platelet shedding. This metabolic capacity therefore serves as a marker of megakaryocyte differentiation.     

μ-Calpain Activation and Calpain-Mediated Cytoskeletal Proteolysis Following Traumatic Brain Injury

Abstract: Increasing evidence suggests that excessive activation of the calcium-activated neutral protease μ-calpain could play a major role in calcium-mediated neuronal degeneration after acute brain injuries. To further investigate the changes of the in vivo activity of μ-calpain after unilateral cortical impact injury in vivo, the ratio of the 76-kDa activated isoform of μ-calpain to its 80-kDa precursor was measured by western blotting. This μ-calpain activation ratio increased to threefold in the pellet of cortical samples ipsilateral to the injury site at 15 min, 1 h, 3 h, and 6 h after injury and returned to control levels at 24–48 h after injury. We also investigated the effect of μ-calpain activation on proteolysis of the neuronal cytoskeletal protein α-spectrin. Immunoreactivity for α-spectrin breakdown products was detectable within 15 min after injury in cortical samples ipsilateral to the injury site. The levels of α-spectrin breakdown products increased in a biphasic manner, with a large increase between 15 min and 6 h after injury, followed by a smaller increase between 6 and 24 h after the insult. No further accumulation of α-spectrin breakdown products was observed between 24 and 48 h after injury. Histopathological examinations using hematoxylin and eosin staining demonstrated dark, shrunken neurons within 15 min after traumatic brain injury. No evidence of μ-calpain autolysis, calpain-mediated α-spectrin degradation, or hematoxylin and eosin neuronal pathology was detected in the contralateral cortex. Although μ-calpain autolysis and cytoskeletal proteolysis occurred concurrently with early morphological alterations, evidence of calpain-mediated proteolysis preceded the full expression of evolutionary histopathological changes. Our results indicate that rapid and persistent μ-calpain activation plays an important role in cortical neuronal degeneration after traumatic brain injury. Our data also suggest that specific inhibitors of calpain could be potential therapeutic agents for the treatment of traumatic brain injury in vivo.     

Evidence that lysosomes are not involved in the degradation of myofibrillar proteins in rat skeletal muscle.

To examine the role of lysosomes in the degradation of skeletal-muscle myofibrillar proteins, we measured the release of N tau-methylhistidine from perfused muscle of starved and fed rats in the presence or absence of agents that inhibit lysosomal proteinase activity. After 1 day of starvation, the release of N tau-methylhistidine by perfused muscle of 4-, 8- and 24-week-old rats increased by 322, 159 and 134% respectively. On the other hand, total protein breakdown, assessed by tyrosine release, increased by 62, 20 and 20% respectively. Inhibitors of lysosomal proteinases as well as high concentrations of insulin or amino acids failed to diminish the release of N tau-methylhistidine by perfused muscle of starved and fed rats, despite a 25-35% inhibition of total protein breakdown. The data strongly suggest that the complete breakdown of myofibrillar proteins occurs via a non-lysosomal pathway. They also suggest that total proteolysis, which primarily reflects non-myofibrillar protein breakdown, occurs at least in part within lysosomes.     

Effect of wortmannin and phorbol ester on Paramecium fluid-phase uptake in the presence of transferrin

The kinetics of the uptake of the fluid phase marker Lucifer Yellow (LY), and its alteration by wortmannin, an inhibitor of phosphatidylinositol-3 kinase (PI-3K), and the PKC modulators: GF 109203 X, an inhibitor, and phorbol ester, an activator was studied in eukaryotic model Paramecium aurelia. Spectrophotometric quantification of LY accumulation was performed in the presence or absence of transferrin, a marker of receptor-mediated endocytosis. Internalization of LY showed a curvilinear kinetics: the high initial rate of LY uptake (575 ng LY/mg protein/hr) decreased almost 5-fold within 15 min, reaching plateau at 126 ng/mg protein/hr. Transferrin induced a small increase (7.5%) in the fluid phase uptake rate (after 5 min) followed by a small decrease at longer incubation times. Lucifer Yellow and transferrin (visualized by streptavidin-FITC) were localized in Paramecium by 3-D reconstruction by confocal microscopy. LY showed a scattered, diffuse fluorescence typical of fluid phase uptake whereas transferrin accumulated in membrane-surrounded endosomes. Wortmannin did not affect LY accumulation but decreased it when transferrin was present in the incubation medium. This suggests an effect on the transferrin uptake pathway, presumably on the stage of internalization in "mixing" endosomes to which transferrin and LY were targeted. Phorbol ester diminished LY accumulation by 22% and this effect persisted up to 25 min of incubation. PKC inhibitor did not affect LY uptake. However, in the presence of transferrin, the LY uptake increased within the first 15 minutes followed by a rapid 20% decrease in comparison to the control. Such an effect of PKC modulators suggests that PMA action on fluid phase uptake is not directly mediated by PKC.     

Relative role of the glutaminase, glutamate dehydrogenase, and AMP-deaminase pathways in hepatic ureagenesis: studies with 15N.

We have studied the relative roles of the glutaminase versus glutamate dehydrogenase (GLDH) and purine nucleotide cycle (PNC) pathways in furnishing ammonia for urea synthesis. Isolated rat hepatocytes were incubated at pH 7.4 and 37 degrees C in Krebs buffer supplemented with 0.1 mM L-ornithine and 1 mM [2-15N]glutamine, [5-15N]glutamine, [15N]aspartate, or [15N]glutamate as the sole labeled nitrogen source in the presence and absence of 1 mM amino-oxyacetate (AOA). A separate series of incubations was carried out in a medium containing either 15N-labeled precursor together with an additional 19 unlabeled amino acids at concentrations similar to those of rat plasma. GC-MS was utilized to determine the precursor product relationship and the flux of 15N-labeled substrate toward 15NH3, the 6-amino group of adenine nucleotides ([6-15NH2]adenine), 15N-amino acids, and [15N]urea. Following 40 min incubation with [15N]aspartate the isotopic enrichment of singly and doubly labeled urea was 70 and 20 atom % excess, respectively; with [15N]glutamate these values were approximately 65 and approximately 30 atom % excess for singly and doubly labeled urea, respectively. In experiments with [15N]aspartate as a sole substrate 15NH3 enrichment exceeded that in [6-NH2]adenine, indicating that [6-15NH2]adenine could not be a major precursor to 15NH3. Addition of AOA inhibited the formation of [15N]glutamate, 15NH3 and doubly labeled urea from [15N]aspartate. However, AOA had little effect on [6-15NH2]adenine production. In experiments with [15N]glutamate, AOA inhibited the formation of [15N]aspartate and doubly labeled urea, whereas 15NH3 formation was increased. In the presence of a physiologic amino acid mixture, [15N]glutamate contributed less than 5% to urea-N. In contrast, the amide and the amino nitrogen of glutamine contributed approximately 65% of total urea-N regardless of the incubation medium. The current data indicate that when glutamate is a sole substrate the flux through GLDH is more prominent in furnishing NH3 for urea synthesis than the flux through the PNC. However, in experiments with medium containing a mixture of amino acids utilized by the rat liver in vivo, the fraction of NH3 derived via GLDH or PNC was negligible compared with the amount of ammonia derived via the glutaminase pathway. Therefore, the current data suggest that ammonia derived from 5-N of glutamine via glutaminase is the major source of nitrogen for hepatic urea-genesis.     

Slow intravenous administration of low dose aspirin inhibits both vascular and platelet cyclooxygenase activity: an experimental study in the rat

Aspirin irreversibly inhibits cyclooxygenase, thus preventing thromboxane (Tx)A2 production in platelets and prostacyclin in vascular cells. While it is generally accepted that the inhibitory effect of low dose aspirin is cumulative on platelet cyclooxygenase, it is still a matter of debate whether a similar phenomenon also occurs on vascular cyclooxygenase. We have measured in anesthetized rats the inhibitory effect of two doses of aspirin (2.5 and 5.0 mg/kg), given intravenously either as a bolus or as a continuous infusion (for 30 min), on platelet TxB2 and 6-ketoprostaglandin F1 alpha generation by different vascular segments. Aspirin significantly inhibited both platelet and vascular cyclooxygenase independently of the rate of drug administration. The aspirin peak plasma levels at the end of bolus injection was about 170 times higher than the average level measured during the slow infusion (1.21 +/- 0.15 micrograms/ml). At this concentration aspirin did not affect in vitro either platelet or vascular cyclooxygenase activity. Thus the inhibitory effect of aspirin on both platelet and vascular cyclooxygenase seems to be related to total exposure of the enzyme to the drug rather than to the maximal drug concentration attainable in the systemic circulation. These findings may be relevant to the current debate on optimal conditions for the biochemical selectivity of aspirin as an antithrombotic drug.     

Effects of β-hydroxy-β-methylbutyrate treatment in different types of skeletal muscle of intact and septic rats

β-Hydroxy-β-methylbutyrate (HMB) is a leucine metabolite that may have a positive effect in protein catabolic conditions. Therefore, we hypothesized that HMB treatment could attenuate the sepsis-induced protein catabolic state. The aims of our study were to elucidate the effect of HMB in healthy and septic animals and to evaluate the differences in the action of HMB in different muscle types. Intact and septic (5 mg endotoxin/kg i.p.) rats were administered with HMB (0.5 g/kg/day) or saline. After 24 h, extensor digitorum longus (EDL) and soleus (SOL) muscles were isolated and used for determination of total and myofibrillar proteolysis, protein synthesis, leucine oxidation, activity of cathepsins B and L, chymotrypsin-like activity, and expression of α-subunits of proteasome. Our results indicate that the catabolic state induced by the endotoxin treatment was caused both by increase in protein breakdown (due to activation of proteasome system) and by attenuation of protein synthesis. The EDL (muscle composed of white, fast-twitch fibers) was more susceptible to these changes than the SOL (muscle composed of red, slow-twitch fibers). The HMB treatment had no effect in healthy animals but counteracted the changes in septic animals. The action of HMB was mediated by attenuation of proteasome activity and protein breakdown, not by stimulation of protein synthesis. More pronounced effect of the HMB treatment on myofibrillar proteolysis was observed in the SOL.     

Kinetics and mechanisms of recombinant human interleukin 1 and tumor necrosis factor-alpha-induced changes in circulating numbers of neutrophils and lymphocytes

Human recombinant interleukins 1 alpha and 1 beta (rIL-1 alpha and -1 beta) both induced monophasic peripheral neutrophilia and lymphopenia in Lewis rats 1.5 hr after i.v. injection. The kinetics of rIL-1 alpha- and -1 beta-induced neutrophilia were similar to those induced by human monocyte-derived IL-1, IL-1 alpha, and IL-1 beta, and the peripheral neutrophilia was accompanied by a marked decrease in marrow neutrophils. Arachidonic acid metabolites are implicated as biochemical intermediates in the production of the neutrophilia but not lymphopenia, since indomethacin and dexamethasone both completely abrogated IL-1-induced neutrophilia but did not affect the IL-1-induced lymphopenia. Acetylsalicylic acid, a cyclooxygenase inhibitor, did not inhibit IL-1-induced neutrophilia, suggesting that products of the lipoxygenase rather than the cyclooxygenase pathway of arachidonate metabolism may contribute to the neutrophilia. Human recombinant tumor necrosis factor-alpha (rTNF) administered i.v. to Lewis rats induced peripheral neutropenia, two peaks of neutrophilia, and lymphopenia. A wide range of doses of rTNF resulted in an initial neutropenia at 0.5 hr after injection followed by a first peak of neutrophilia at 1.5 hr and a second peak of neutrophilia at 6 hr. The initial neutropenia and the first peak of neutrophilia were not inhibited by pretreatment of rats with dexamethasone, indomethacin, or aspirin. The second peak of neutrophilia was inhibited by both dexamethasone and indomethacin, but was not at all inhibited by aspirin, suggesting that the second peak of neutrophilia is mediated by the release of endogenous cytokines, especially by IL-1, since exogenous IL-1-induced neutrophilia is also completely inhibited by dexamethasone and indomethacin but not by aspirin. The TNF-induced peripheral neutrophilia is also accompanied by a significant depletion of bone marrow neutrophils, indicating that the source of increased circulating neutrophils is, at least in part, via recruitment of marrow neutrophils. Systemic blood pressure was not affected by IL-1 or rTNF at the dosages employed, showing that the changes in circulating leukocyte subsets were not attributable to hemodynamic changes nor to the hemodynamic change-related release of adrenal hormones. Adrenalectomy did not alter the IL-1- or rTNF-induced neutrophilia or lymphopenia, also demonstrating that neither monokine mediates its hematologic effects on peripheral blood leukocytes via the release of adrenal hormones.(ABSTRACT TRUNCATED AT 400 WORDS)     

Age-related growth of the spleen in normal and glucocorticoid treated rats

1. Age-related changes in the growth, nucleic acid content and protein turnover of the spleen have been studied in normal male rats. 2. A rapid and marked atrophy of the spleen, was found 24 hr after exposure to cortisone or dexamethasone; increased rates of protein breakdown being primarily responsible. 3. Nonetheless, the total amount of protein synthesized in the spleen (measured in vivo) was significantly decreased (30-50%) 24 hr after exposure to these steroids. 4. This compared with only a 15% decrease in whole body protein synthesis, indicating a more pronounced hormonal effect on the spleen than on most other body tissues.     

Lipopolysaccharides decrease angiotensin converting enzyme activity expressed by cultured human endothelial cells.

Angiotensin converting enzyme (ACE) is present on endothelial cells and plays a role in regulating blood pressure in vivo by converting angiotensin I to angiotensin II and metabolizing bradykinin. Since ACE activity is decreased in vivo in sepsis, the ability of lipopolysaccharide (LPS) to suppress endothelial cell ACE activity was tested by culturing human umbilical vein endothelial cells (HUVEC) for 0-72 hr with or without LPS and then measuring ACE activity. ACE activity in intact HUVEC monolayers incubated with LPS (10 micrograms/ml) decreased markedly with time and was inhibited by 33%, 71%, and 76% after 24 hr, 48 hr, and 72 hr, respectively, when compared with control, untreated cells. The inhibitory effect of LPS was partially reversible upon removal of the LPS and further incubation in the absence of LPS. The LPS-induced decrease in ACE activity was dependent on the concentrations of LPS (IC50 = 15 ng/ml at 24 hr) and was detectable at LPS concentrations as low as 1 ng/ml. That LPS decreased the Vmax of ACE in the absence of cytotoxicity and without a change in Km suggests that LPS decreased the amount of ACE present on the HUVEC cell membrane. While some LPS serotypes (Escherichia coli 0111:B4 and 055:B5, S. minnesota) were more potent inhibitors of ACE activity than others (E. coli 026:B6 and S. marcescens), all LPS serotypes tested were inhibitory. These finding suggest that LPS decreases endothelial ACE activity in septic patients; in turn, this decrease in ACE activity may decrease angiotensin II production and bradykinin catabolism and thus play a role in the pathogenesis of septic shock.     

Acute alterations in sodium flux in vitro lead to decreased myofibrillar protein breakdown in rat skeletal muscle.

Myofibrillar protein breakdown was evaluated by measuring the release of N tau-methylhistidine by isolated rat skeletal muscles or perfused rat muscles in the presence of a variety of agents known to affect Na+ flux. Total cell proteolysis was evaluated simultaneously by measuring tyrosine release by muscles after the inhibition of protein synthesis with cycloheximide. Treatment of muscles with the Na+ ionophore monensin or inhibitors of Na+-K+ ATPase (ouabain, digoxin or vanadate) decreased N tau-methylhistidine release by muscles by 21-35%. A phorbol ester (phorbol 12-myristate 13-acetate) as well as a synthetic diacylglycerol known to activate protein kinase C and a Na+/H+ antiport also decreased N tau-methylhistidine release by muscles. Removal of extracellular Na+ blocked the ability of these agents to attenuate N tau-methylhistidine release by muscles, suggesting that their effectiveness required a change in Na+ flux. In contrast with N tau-methylhistidine release by muscles, these agents, except for monensin, did not effect the release of tyrosine, suggesting that they attenuate specifically the breakdown of myofibrillar proteins. Overall these results indicate a link between Na+ and the regulation of protein breakdown in rat skeletal muscle, whereby an influx of Na+ can result in a decrease in myofibrillar proteolysis. Left unresolved is whether phospholipid hydrolysis is involved in this scheme.     

Effect of propionate on the utilization of nitrogen from 15NH4Cl for urea synthesis in hepatocytes isolated from sheep liver

1. The effect of ornithine (2.0 mM) and propionate (5.0 mM) on the utilization of N from 15NH4Cl (5.0 mM) for urea synthesis in hepatocytes isolated from sheep liver was investigated. 2. The capacity of sheep hepatocytes to utilize [15N]ammonia in the absence of the other exogenous substrates was very low and amounted 132 +/- 37.3 mumol/hr per 1 g dry wt. 3. Ornithine failed to affect the total [15N]ammonia uptake and total urea synthesis, but at the same time it markedly increased the utilization of [15N]ammonia for ureagenesis and diminished the rate of urea synthesis from endogenous sources. 4. Propionate markedly increased total [15N]ammonia utilization and total urea formation; this increase resulted from the rise of ammonia utilization for urea synthesis and it was similar in the presence or absence of ornithine. 5. The capacity of sheep liver cells to utilize ammonia in the presence of propionate (in the presence or absence of ornithine) amounted to 256 mumol/hr per 1 g dry wt, thus being similar to the values in vivo. 6. It is concluded that in sheep hepatocytes both ornithine and propionate stimulate the utilization of ammonia for urea synthesis and these effects take place independently and occur by different mechanisms.     

Effects of aspirin and indomethacin on cerebral circulation in the conscious rat: Evidence for a physiological role of endogenous prostaglandins

The purpose of this work was to evaluate the effects of equipotent doses of two different inhibitors of cyclo-oxygenase, indomethacin and aspirin, on cerebral blood flow and cerebral vascular resistances in the conscious undisturbed rat, using the reference sample radioactive microsphere method. We found that both, aspirin (50 mg/kg) and indomethacin (5 mg/kg) at 3, 15 and 60 min after their intravenous administration, increased cerebral vascular resistances and decreased cerebral blood flow to a similar extent. Both drugs completely abolished the hypotensive effect of 5 mg/kg i.v. arachidonic acid and they did not change arterial PO₂, PCO₂ or pH values. We conclude that the pharmacological inhibition of cyclooxygenase in the conscious undisturbed rat leads to a cerebral vasoconstriction and consequently to a decrease in cerebral blood flow. Our results evidence that prostaglandins are a physiological factor that actively contributes to the maintenance of cerebral circulation.