Hematology and clinical chemistry parameters in the cat (Felis domesticus).
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Animals↗
Biomedical subjects
M G Murphy
Publications and source records attributed to M G Murphy.
MK-677, an orally active growth hormone secretagogue, reverses diet-induced catabolism.
The reversal of diet-induced negative nitrogen balance by GH suggests a possible therapeutic role for GH treatment in catabolic patients. A double-blind, randomized, placebo-controlled, two-period cross-over study was designed to investigate whether MK-677, an orally active nonpeptide mimic of GH-releasing peptide, can reverse diet-induced protein catabolism. Eight healthy volunteers (ages 24-39 yr) were calorically restricted (18 kcal/kg.day) for two 14-day periods. During the last 7 days of each diet period, subjects received either oral MK-677 25 mg or placebo once daily. There was a 14- to 21-day washout interval between periods. During the first week of caloric restriction (i.e. diet alone), daily nitrogen losses were similar for both treatment groups (mean +/- SE; MK-677 group -2.67 +/- 0.40 g/day vs. placebo group -2.83 +/- 0.26 g/day). During the second week (diet and study drug), mean daily nitrogen balance was 0.31 +/- 0.21 g/day in the MK-677 treatment group compared with -1.48 +/- 0.21 g/day in the placebo group (P < 0.01). MK-677 improved nitrogen balance integrated over the 7 days of treatment; area under the curve day 8-14 nitrogen balance response was +2.69 +/- 5.0 (SE) for MK-677 and -8.97 +/- 5.26 g.day for placebo (P < 0.001). MK-677 produced a peak GH response of 55.9 +/- 31.7 micrograms/L after single dose (day 1 of treatment) and 22.6 +/- 9.3 micrograms/L after a week of dosing compared with placebo treatment peak GH values of approximately 9 (treatment day 1) and approximately 7 micrograms/L (treatment day 7). Following the initial 7-day caloric restriction, insulin-like growth factor-I (IGF-I) declined from 232 +/- 25 to 186 +/- 19 ng/mL in the MK-677 group and from 236 +/- 19 to 174 +/- 23 ng/mL in the placebo group. Mean IGF-I concentration increased significantly during MK-677 to 264 +/- 31 ng/mL (mean for the last 5 days of treatment) compared with 188 +/- 19 ng/mL with placebo (P < 0.01). No significant difference in IGF binding protein-2 was found between the MK-677 and placebo treatments. However, the mean in IGF binding protein-3 for the last 5 days of MK-677 treatment was also significantly increased to 3273 +/- 330 ng/mL (mean +/- SE) compared with placebo 2604 +/- 253 ng/mL (P < 0.01). Neither the serum cortisol nor the PRL response was significantly greater after 7 days of MK-677 dosing compared with 7 days of placebo. MK-677 (25 mg) was generally well tolerated and without clinically significant adverse experiences. In conclusion, MK-677 reverses diet-induced nitrogen wasting, suggesting that if these short-term anabolic effects are maintained in patients who are catabolic because of certain acute or chronic disease states, it may be useful in treating catabolic conditions.
Adult↗
Sequestration of coenzyme A by the industrial surfactant, Toximul MP8. A possible role in the inhibition of fatty-acid beta-oxidation in a surfactant/influenza B virus mouse model for acute hepatic encephalopathy.
We have investigated the mechanistic basis of our recent observation that exposing young mice to an industrial surfactant potentiates the inhibition of fatty-acid beta-oxidation that occurs with subsequent virus infection (Murphy et al., Biochim. Biophys. Acta 1315, 208-216, 1996). In our mouse model for acute hepatic encephalopathy (AHE), neonatal mice were painted on their abdomens from birth to postnatal day 12 with nontoxic amounts of the industrial surfactant, Toximul MP8 (Tox), and then infected with a sublethal dose (LD30) of mouse-adapted human Influenza B (Lee) virus (FluB). Mortality in mice treated with Tox + FluB was significantly higher than that in mice treated with FluB alone. In vitro assays of hepatic beta-oxidation of [1-(14)C]palmitic and [1-(14)C]octanoic acids in the presence or absence of exogenous coenzyme A (CoA) indicated that Tox-mediated inhibition of oxidation was masked when CoA was added to the assays. FluB also inhibited beta-oxidation by 20-30%, however this effect was independent of exogenous CoA which suggested that it involved a different mechanism. Tox-mediated potentiation of the inhibitory effect was most obvious (> 80% inhibition) when assays were done without added CoA. Analysis of hepatic CoA and its esters indicated that levels of both free CoA and acetyl-CoA were significantly lower in mice that were painted with Tox for 12 days. Tox-dependent reductions of acetyl-CoA were transient and returned to normal values after cessation of painting, whereas those of CoA persisted. FluB infection alone significantly reduced hepatic acetyl-CoA and the magnitude of this reduction (> 30%) was not affected by pre-exposing the mice to Tox. Relative to control mice, levels of acid insoluble acyl-CoA esters were elevated significantly in FluB and Tox + FluB treated mice. Activation of both [1-(14)C]palmitic and [1-(14)C]octanoic acids was reduced in Tox-exposed mice at experimental day 12, but only when exogenous CoA was not included in the assay media; this effect appeared to persist after cessation of painting. Collectively, these data support the concept that Tox and FluB have independent effects on hepatic CoA metabolism that are associated with abnormalities in fatty-acid beta-oxidation. However, these do not fully explain the synergistic effect of the virus and chemical on beta-oxidation inhibition, which is a candidate co-mechanism for potentiation of mortality in this mouse model of AHE.
3-Hydroxyacyl CoA Dehydrogenases↗
Diets enriched in menhaden fish oil, seal oil, or shark liver oil have distinct effects on the lipid and fatty-acid composition of guinea pig heart.
The purpose of this investigation was to determine whether diets supplemented with oils from three different marine sources, all of which contain high proportions of long-chain n-3 polyunsaturated fatty acids (PUFA), result in qualitatively distinct lipid and fatty acid profiles in guinea pig heart. Albino guinea pigs (14 days old) were fed standard, nonpurified guinea pig diets (NP) or NP supplemented with menhaden fish oil (MO), harp seal oil (SLO) or porbeagle shark liver oil (PLO) (10%, w/w) for 4-5 weeks. An n-6 PUFA control group was fed NP supplemented with corn oil (CO). All animals appeared healthy, with weight gains marginally lower in animals fed the marine oils. Comparison of relative organ weights indicated that only the livers responded to the diets, and that they were heavier only in the marine-oil fed guinea pigs. Heart total cholesterol levels were unaffected by supplementing NP with any of the oils, whereas all increased the triacylglycerol (TAG) content. The fatty-acid profiles of total phospholipid (TPL), TAG and free fatty acid (FFA) fractions of heart lipids showed that feeding n-3 PUFA significantly altered the proportions of specific fatty-acid classes. For example, all marine-oil-rich diets were associated with increases in total monounsaturated fatty acids in TPL (p < 0.05), and with decreases in total saturates in TAG (p < 0.05). Predictably, the n-3 PUFA enriched regimens significantly increased the cardiac content of n-3 PUFA and decreased that of n-6 PUFA, although the extent varied among the diets. As a result, n-6/n-3 ratios were significantly lower in all myocardial lipid classes of marine-oil-fed guinea pigs. Analyses of the profiles of individual PUFA indicated that quantitatively, the fatty acids of the three marine oils were metabolized and/or incorporated into TPL, TAG and FFA in a diet-specific manner. In animals fed MO-enriched diets in which eicosapentaenoic acid (EPA) > docosahexacnoic acid (DHA), ratios of DHA/EPA in the hearts were 1.2, 2.2 and 1.5 in TPL, TAG and FFA, respectively. In SLO-fed guinea pigs in which dietary EPA approximately DHA, ratios of DHA/EPA were 0.9, 3.4 and 2.1 in TPL, TAG and FFA, respectively. Feeding NP + PLO (DHA/EPA = 4.8), resulted in values for DHA/EPA in cardiac tissue of 2.1, 10.6 and 2.9 in TPL, TAG and FFA, respectively. In the TAG and FFA, proportions of n-3 docosapentaenoic acid (n-3 DPA) were equal to or higher than EPA in the SLO- and PLO-fed animals. The latter group exhibited the greatest difference between the DHA/n-3 DPA ratio in the diet and in cardiac TAG and FFA fractions (7, 3.4 and 3.1, respectively). Quantitative analysis indicated that > or = 85% of the n-3 PUFA were in TPL, 7-11% were in TAG, and 2-6% were FFA. Specific patterns of distribution of EPA, DPA and DHA depended on the dietary oil. Both the qualitative and quantitative results of this study demonstrated that in guinea pigs, n-3 PUFA in different marine oils are metabolized and/or incorporated into cardiac lipids in distinct manners. In support of the concept that the diet-induced alterations reflect changes specifically in cardiomyocytes, we observed that direct supplementation of cultured guinea pig myocytes for 2-3 weeks with EPA or DHA produced changes in the PUFA profiles of their TPL that were qualitatively similar to those observed in tissue from the dietary study. The factors that regulate specific deposition of n-3 PUFA from either dietary oils or individual PUFA are not yet known, however the differences that we observed could in some manner be related to cardiac function and thus their relative potentials as health-promoting dietary fats.
Animals↗
Abnormalities in hepatic fatty-acid metabolism in a surfactant/influenza B virus mouse model for acute encephalopathy.
Abnormalities in fatty-acid metabolism are believed to play a role in nonspecific acute encephalopathy (AE) with hepatomegaly, although the specific nature of these abnormalities and their temporal relationship to the pathology are not well defined. We have examined hepatic fatty-acid beta-oxidation and metabolism in a mouse model for AE in which neonatal mice were exposed dermally to nontoxic doses of the industrial surfactant, Toximul MP8 (Tox), daily from days 1 to 12 after birth, and then infected with a sublethal dose (LD30) of mouse-adapted human influenza B (Lee) virus (FluB). The number of deaths in the group treated with Tox + FluB were significantly higher than those in the group infected with virus alone. Under optimal in vitro assay conditions, beta-oxidation of [1-14C]palmitic acid was approximately 15% higher in liver homogenates from mice painted with Tox for 12 days (P < 0.02); catabolism of [1-14C]octanoic acid to 14C-labelled water-soluble products (14C-WSP) and 14CO2 was unaltered by Tox. Infecting Tox-free mice with FluB inhibited beta-oxidation of both [1-14C]palmitate and [1-14C]octanoate by 20-30% (P < 0.001). On days 18-19, when most Tox + FluB-dependent deaths occurred, the inhibition of oxidation was increased to approximately 50% in mice given the combined treatment. Treatment of the mice with Tox/FluB also altered the pattern of incorporation of fatty acids into complex lipids. Hepatic levels of thiobarbituric acid reactive substance (TBARS), a marker for lipid peroxides, were approximately 15% higher in Tox-painted than in control mice (P < 0.01); FluB alone had no effect. In Tox + FluB-treated animals, TBARS levels were > 2-fold higher than in any other experimental group (P < 0.001). These studies demonstrated that nasally-administered FluB has profound effects on hepatic fatty-acid metabolism, particularly beta-oxidation. Exacerbation of this and related effects by exposing young animals to xenobiotic surfactants could be the basis of surfactant-mediated potentiation of virus-induced mortality.
Adenosine Triphosphate↗
Human pharmacokinetics and tolerability of L-365,260, a novel cholecystokinin-B antagonist.
A study was conducted to examine the tolerability and pharmacokinetics of single and multiple oral doses of L-365,260, a novel antagonist for type B cholecystokinin (CCK) receptors and to quantify effects of selective blockade of type B CCK receptors through treatment with L-365,260 on measures of anxiety, hunger, and cognitive performance. Healthy volunteers were given single oral doses of up to 50 mg of L-365,260 and multiple oral doses of up to 25 mg every 6 hours for 10 days. Plasma concentrations of L-365,260 were quantified by means of high-performance liquid chromatography. Anxiety and hunger were assessed by visual analog scale and the Spielberger State Anxiety Index. Cognitive testing was used to evaluate attention level and short-term memory. L-365,260 was rapidly absorbed and a biphasic pattern of elimination was demonstrated with a terminal half-life (t1/2) of 8 to 12 hours. The mean (n = 6) values for peak plasma concentration (C(max)) and time to peak concentration (t(max)) of L-365,260 were 503 ng/mL and 1.25 hours, respectively, after a single 50-mg oral dose. Accumulation of L-365,260 plasma concentrations was seen after the prescribed multiple-dose regimens. Steady state was achieved after 3 days of oral administration. L-365,260 had an acceptable tolerability profile after oral administration. No changes in measures of anxiety, hunger, or short-term memory were observed at doses of L-365,260 shown to have antagonist activity at the CCK-B receptor.
Administration, Oral↗
Selective blockade of cholecystokinin type B receptors with L-365,260 does not impair gallbladder contraction in normal humans.
OBJECTIVES: To evaluate the effect of selective blockade of type B cholecystokinin receptors on gall bladder contraction in normal humans and to compare methods for quantitative analysis of gall bladder contraction. METHODS: L-365,260, a novel, nonpeptide cholecystokinin antagonist shown to be selective for type B cholecystokinin receptors, was administered every 6 h over a 5-7 day period. Plasma levels of L-365,260 were determined by high pressure liquid chromatography. Gallbladder contraction after a standardized fatty meal was measured by ultrasonography, and results were calculated by ellipsoid or sum of cylinders methods. RESULTS: L-365,260 levels were comparable to levels in earlier studies demonstrating inhibition of pentagastrin-stimulated acid secretion in normal subjects and blockade of anxiogenic effects of cholecystokinin injections in patients with panic disorder. Regardless of the method used for estimating gallbladder size, none of the L-365,260 doses studied inhibited gallbladder contraction. Gallbladder size was most consistently estimated by the ellipsoid method using measurements normalized to individual values for minimum and maximum gallbladder dimensions. CONCLUSIONS: Multiple oral doses of L-365,260 do not alter ultrasonographically assessed gallbladder contraction at doses shown to be clinically effective in earlier studies. Despite being more difficult to implement, the sum of cylinders method for estimating gall bladder size offers no advantage over the ellipsoid method.
Adult↗
Effects of exogenous linoleic acid on fatty acid composition, receptor-mediated cAMP formation, and transport functions in rat astrocytes in primary culture.
We have examined the effects of culturing neonatal rat-brain astrocytes in medium containing delipidated serum, with or without added linoleic acid (LA, 18:2 omega 6), on membrane fatty-acid composition and functions. After 18-21 days in culture, polyunsaturated fatty acids (PUFA) constituted approximately equal to 24 mol% of the total fatty acids in the astrocytes grown in delipidated media ("controls'); these proportions were increased by 35-40% to approximately equal to 33 mol% when the cells were supplemented with 35 microM LA. Notable differences in the PUFA profiles of the cells cultured with or without added LA included: (a) higher proportions of omega 6 PUFA in the LA-supplemented astrocytes (approximately equal to 25%, relative to approximately equal to 10% in controls) that were accompanied by an increase in the ratio of omega 6/omega 3 PUFA (from < 2 in controls to approximately equal to 5), and (b) higher proportions of 20:3 omega 9 and 22:3 omega 9 in the control astrocytes (> 5%) relative to the LA-supplemented cells (approximately equal to 1%). The major metabolites in the omega 6 PUFA-enriched cells were arachidonic (20:4 omega 6), adrenic (22:4 omega 6) and docosapentaenoic (22:5 omega 6) acids (15, 5 & 3 mol%, respectively). Enrichment of the astrocytes in omega 6 PUFA did not alter basal levels of cAMP, nor did it affect the amounts of cAMP formed in response to forskolin, isoproterenol, adenosine or histamine. However, dopamine-dependent increases in cAMP formation in the presence of the phosphodiesterase inhibitor, Ro 20-1724, were reduced by approximately equal to 25% relative to those in controls. LA supplementation modified uptake of [3H]adenosine into the astrocytes; values for Kt for a high affinity transport were increased relative to controls, and maximum capacity of a lower affinity process was reduced. Uptake of [3H]glutamate was not altered in the omega 6 PUFA-enriched astrocytes. This study demonstrated that cultured astrocytes take up exogenous linoleic acid and incorporate its metabolites into phospholipid, and that the resulting changes in membrane PUFA composition modify only specific cell functional properties.
Animals↗
Wear of the polyethylene of Harris-Galante acetabular components inserted without cement.
The amount and rate of linear wear of the polyethylene of eighty Harris-Galante acetabular components, implanted without cement during a primary or revision total hip replacement in seventy-two patients, were determined by a comparison of the thicknesses of the cup as measured on the initial and most recent follow-up radiographs. The measurements were corrected for magnification error by direct measurement of the diameter of the femoral head on each radiograph. The mean duration of follow-up was sixty-eight months (range, fifty-six to ninety-six months). Linear wear averaged 0.77 +/- 0.51 millimeter (range, zero to 1.83 millimeters) over-all, with a mean rate of 0.14 +/- 0.09 millimeter (range, zero to 0.35 millimeter) per year. An increased rate of wear per year correlated with a decreased age of the patient and with regular exercise, but there was no relationship with the weight or sex of the patient, the initial thickness of the polyethylene, or the angle of abduction of the cup.
Acetabulum↗
Short-term exposure of intact ventricular myocytes to isoproterenol results in desensitization of adenylate cyclase.
We have examined the effects of short-term exposure to low (50 nM) levels of the beta-adrenoceptor agonist isoproterenol on the ability of intact rat ventricular myocytes to increase their intracellular levels of cyclic AMP (cAMP) in response to subsequent isoproterenol exposure. Exposing freshly isolated, nonstimulated myocytes (which contained 3.5 +/- 0.3 pmol cAMP/mg protein) to isoproterenol resulted in rapid, dose-dependent increases in cAMP formation; 5-min exposure raised intracellular cAMP content to 6.5 +/- 0.7 pmol/mg protein and 54.6 +/- 0.9 pmol/mg protein, respectively, in the absence and presence of the phosphodiesterase inhibitor 4-(3-butoxy-4-methoxybenzyl)imiazolidin-2-one (RO 20-1724). In myocytes incubated for 5-20 min with isoproterenol, washed twice to remove the agonist, and then rechallenged for 20 min with the agonist in the presence of Ro 20-1724, a 10-min preexposure to the agonist resulted in a significant reduction (approximately 20%; P < 0.05) in the receptors' responsiveness to the rechallenge, and increasing the preexposure time to 20 min resulted in the maximum attenuation in responsiveness (approximately 30%; P < 0.001). Myocytes preincubated with forskolin (1 microM), which directly activates adenylate cyclase contained 9.0 +/- 0.7 pmol cAMP/mg protein after 5 min exposure, and up to 20 min preexposure to forskolin did not alter the cells' responsiveness to subsequent challenge with isoproterenol. The results of this study indicate that exposing freshly isolated rat ventricular myocytes to nanomolar levels of isoproterenol for as little as 10 min desensitizes them to subsequent challenge with agonist.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenylyl Cyclases↗
Interaction between dietary intake and ovariectomy on concentrations of insulin-like growth factor-I, GH and LH in plasma of heifers.
The objective of this study was to determine if alterations in dietary intake and(or) ovariectomy influence plasma concentrations of IGF-I, GH and LH in heifers. Cyclic heifers (n = 23) were individually fed for 10 wk either 1) 1.8% of body weight in dry matter per day (GAIN; n = 7) 2) 1.1% of body weight in dry matter per day (MAINT; n = 8); or 3) 0.7% of body weight in dry matter per day (LOSE; n = 8). After 10 wk of dietary treatment, heifers were ovariectomized 36 to 40 h following the second injection of prostaglandin F2alpha analog (2 injections 11 d apart). Heifers weighed 444 +/- 13, 387 +/- 8, and 349 +/- 9 kg in the GAIN, MAINT and LOSE groups, respectively, at the time of ovariectomy; the average daily weight gains during the 10-wk period were 0.96, 0.17 and -0.31 kg, respectively (P < 0.001), for the 3 groups. Blood plasma was collected for 6 h at 15-min intervals 1 d before and 2 wk after ovariectomy. The MAINT group of heifers had greater IGF-I concentrations than either the LOSE or GAIN groups; IGF-I decreased (P < 0.05) by 23 and 35% after ovariectomy in the MAINT and GAIN groups, respectively, but did not change (P > 0.10) in the LOSE groups. Dietary restriction tended to increase (P < 0.10) GH pulse frequency and mean GH. Ovariectomy had no effect (P > 0.10) on mean GH or GH pulse frequency but increased (P < 0.05) GH pulse amplitude in the GAIN groups. Dietary treatment had no effect (P > 0.10) on mean LH, or LH pulse amplitude and frequency. However, across dietary treatments, ovariectomy increased mean LH and LH pulse frequency but did not affect (P > 0.10) LH pulse amplitude. In summary, dietary restriction increased GH secretion while ovariectomy increased LH secretion. There appears to be a dichotomy of response between GH and IGF-I in the way heifers respond to dietary treatment and(or) ovariectomy.
Journal Article↗
The gastrin-receptor antagonist L-365,260 inhibits stimulated acid secretion in humans.
We investigated the effect of a novel gastrin-cholecystokinin-B receptor antagonist, L-365,260 [(3R)-3(N'-3-methylphenyl)ureido)-1,3-dihydro-5-phenyl- 2H-1,4-benzodiazepin-2-one], on gastric acid secretion in humans. In a double-blind, four-period crossover study, eight subjects received single oral doses of placebo or of 2.5, 10, or 50 mg L-365,260, followed by an intravenous infusion of pentagastrin at doses of 0.05, 0.4, and 2 micrograms/kg/hr for successive 30-minute periods. L-365,260 caused a dose-dependent inhibition of pentagastrin-stimulated gastric acid secretion. A single oral dose of 50 mg L-365,260 produced 50% inhibition of the gastric acid output response to pentagastrin (0.4 micrograms/kg/hr) when the mean (+/- SD) plasma L-365,260 concentration was 502 +/- 108 ng/ml. Plasma L-365,260 concentrations (all doses combined) and the inhibition of gastric acid output were correlated with a correlation coefficient of r = 0.45 (p < 0.05). Single oral doses of L-365,260 up to 50 mg did not inhibit basal gastric acid output or alter plasma gastrin concentrations. L-365,260 was well tolerated at oral doses up to 50 mg. These findings show that L-365,260 is an orally active antagonist at gastrin-cholecystokinin-B receptors in humans.
Adolescent↗
Effect of inhibitors of eicosanoid metabolism on release of [3H]noradrenaline from the human neuroblastoma, SH-SY5Y.
Nordihydroguaiaretic acid (NDGA; a lipoxygenase inhibitor), LY-270766 (an inhibitor of 5-lipoxygenase), and the diacylglycerol lipase inhibitor RG 80267 completely eliminated potassium-evoked release of [3H]-noradrenaline ([3H]NA) from the human neuroblastoma clone SH-SY5Y with IC50 values of 10, 15, and 30 microM, respectively. In contrast, these inhibitors only partially inhibited carbachol-evoked release and had little effect on the calcium ionophore A23187-evoked release of NA in this cell line. Arachidonic acid partially inhibited potassium- and A23187-evoked release but did not reverse the inhibition of potassium-evoked release observed in the presence of RG 80267. These studies suggest that arachidonic acid (or its lipoxygenase products) are not important intermediates in the regulation of exocytosis in SH-SY5Y. This conclusion is strengthened by our studies in which SH-SY5Y cells were grown in medium supplemented with bovine serum albumin-linoleic acid (50 microM). Under these conditions there was a selective increase in content of membrane polyunsaturated fatty acids of the omega 6 series, including arachidonic acid; however, these changes did not effect potassium-, veratridine-, carbachol-, or calcium ionophore-evoked release of [3H]NA.
Arachidonic Acid↗
Beta-oxidation of [1-14C]palmitic acid by mouse astrocytes in primary culture: effects of agents implicated in the encephalopathy of Reye's syndrome.
beta-Oxidation of [1-14C]palmitic acid was examined in homogenates of astrocytes cultured from neonatal mouse brain. Under optimal reaction conditions (< or = 50 micrograms protein, 10 min at 37 degrees C), oxidation increased as a function of palmitate concentration (15 microM to 2 mM) and reached a maximum rate of 1.98 +/- 0.29 nmol/min/mg protein (mean +/- SEM) at 0.2 mM substrate. Eadie-Hofstee analysis of data from four experiments yielded apparent values for Vmax of 1.87 nmol/min/mg protein, and for Km, 35-40 microM. There were no dramatic changes in the oxidation rate in cells between 10 and 36 days in culture. During the 10-min assays, less than 0.05% of the radioactivity was converted to 14CO2 by the astrocytes; water-soluble products accounted for 1-2% of the total substrate added. Studies with KCN indicated that 60-70% of the total activity occurred in the mitochondria. We have been studying the structural and functional changes associated with the cerebral encephalopathy of Reye's syndrome (RS). Three-week-old astrocytes exposed to serum from RS children for the final 7 days of culture exhibited minor mitochondrial pleomorphism and had increased numbers of other intracellular organelles. Examination of the effects of agents implicated in RS indicated that oxidation of [1-14C]palmitate was not altered by Na+ salicylate (1-3 mM), but was inhibited by the industrial surfactant, Toximul MP-8 (> or = 10 micrograms/ml), 4-pentenoic acid (> or = 0.1 microM), or with 4 days' exposure to ammonia (10 nM). The latter treatment also resulted in an increase in protein synthesis, cell volume, and malondialdehyde formation. These results suggest that some of the "toxins" implicated in RS inhibit fatty-acid oxidation in the astrocytes and produce other lipid-related abnormalities that could be related to encephalopathy.
Ammonia↗
Non-eicosanoid functions of essential fatty acids: regulation of adenosine-related functions in cultured neuroblastoma cells.
Studies have demonstrated that augmenting the omega 6 polyunsaturated-fatty-acid (PUFA) content of N1E-115 neuroblastoma cells by media supplementation with linoleic acid results in greater than or equal to 2-fold increases in basal levels of intracellular cyclic AMP (cAMP). Data suggested some involvement of increased production of adenosine from endogenous metabolites; however, increases in adenosine were not related to increased activity of 5'-nucleotidase or decreased uptake of extracellular adenosine. PUFA-dependent elevations in basal cAMP were evident within 1 min of exposure to a phosphodiesterase inhibitor; this phenomenon did not appear to be due to PUFA-dependent changes in Ca2+ uptake or to increases in sensitivity of adenylate cyclase to Ca2+. Forskolin-stimulated cAMP formation was 3-fold higher in PUFA-enriched cells than in control cells, which suggested a direct effect on the functioning of the catalytic unit. Linoleic acid supplementation resulted in a 2-fold increase in the maximum amounts of cAMP produced in response to the stable adenosine analogue, 5'-N'ethylcarboxy-amidoadenosine (NECA). The altered stimulatory response did not involve eicosanoid formation, but may have been related to an increase in the number of stimulatory adenosine receptors, as judged by binding of [3H]NECA. These studies indicate that membrane PUFA modulate adenosine-related functions in neuroblastoma cells, and suggest that a complex series of mechanisms is involved in this regulation.
4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone↗
Further studies of the mechanism(s) of polyunsaturated-fatty-acid-mediated increases in intracellular cAMP formation in N1E-115 neuroblastoma cells.
Following earlier observations that increasing the polyunsaturated fatty-acid (PUFA) content of N1E-115 neuroblastoma cells elevated basal and adenosine (Ado)-stimulated intracellular cyclic AMP (cAMP) formation, we carried out studies to determine the mechanism(s) by which PUFA exerted their modulatory effects. Basal increases in cAMP in the PUFA-enriched (PUFA+) cells were evident with short (60 sec) exposure to a phosphodiesterase inhibitor (Ro 20-1724), and increased to a maximum at 20 min; they were not observed in the absence of Ro 20-1724. Forskolin-stimulated cAMP formation in the presence of the Ro compound was 2- to 3-fold higher in the PUFA+ cells. Basal elevations in cAMP were reduced by approximately 70% by exposing the PUFA+ cells to Ado deaminase (ADA) or to an Ado antagonist, and were further increased by inhibiting ADA, which suggested that they could be producing endogenous Ado that activated stimulatory Ado receptors. However, this did not appear to involve PUFA-mediated stimulation of 5'-nucleotidase activity or inhibition of [3H]Ado uptake. Overall, the results of this study indicated that multiple mechanisms are involved in PUFA modulation of cAMP formation.
4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone↗
Adenosine-dependent regulation of cyclic AMP accumulation in primary cultures of rat astrocytes and neurons.
The regulation of intracellular cyclic AMP (cAMP) formation by adenosine (Ado) and its analogues has been examined in primary cultures of rat-brain astrocytes and neurons. In the presence of the phosphodiesterase inhibitor, Ro 20-1724, basal levels of cAMP ranged from 40-120 pmol/mg protein in both cell types. Levels were not altered by treating the cells with Ado deaminase, which suggested that they did not produce appreciable amounts of endogenous Ado under standard culture conditions. In the astrocytes, microM quantities of agonists increased cAMP up to 30-fold higher than basal values; the relative potencies were typical of an A2 Ado receptor (NECA greater than Ado greater than R-PIA). Neuron-enriched cultures exhibited a maximum fourfold increase in cAMP in response to NECA; this was decreased a further eightfold when the cultures had prolonged exposure to the antimitotic agent, c-Ara, to eliminate greater than 98% of the nonneuronal cells. Low (nM) amounts of the Ado agonists inhibited cAMP formation in both cell types. In the astrocytes, the order of potency of inhibition of isoproterenol-stimulated cAMP formation was typical of an A1 receptor (R-PIA greater than Ado greater than NECA); maximum inhibition was 55-65%. Isoproterenol did not increase cAMP in the neuronal cultures. However, forskolin-stimulated formation was effectively (approximately 50%) inhibited by A1 Ado agonists; inhibition was not affected by prolonged treatment with c-Ara. From this study we tentatively concluded that rat astrocytes and neurons both contain inhibitory A1 Ado receptors, but that the stimulatory "A2" subtype is localized mainly on astrocytes.
Adenosine↗
Surfactant-potentiated increases in intracranial pressure in a mouse model of Reye's syndrome.
Severe encephalopathy, the usual cause of death in Reye's syndrome (RS), is characterized by cerebral edema with associated increases in intracranial pressure (ICP). In previous studies, we have shown that exposure of neonatal mice to nontoxic doses of an industrial surfactant and subsequent infection with mouse-adapted influenza B (Lee) virus result in a significant increase in mortality rate and that this is associated with several of the characteristic features of human RS. In the present study we have measured ICP in the young mice undergoing their version of the disease, and we now report that the animals treated with surfactant plus virus experience increases in intracranial pressure that are significantly in excess of those in any of the three control groups. These findings support our hypothesis that this and the other abnormal biochemical and morphological responses in RS are related in some manner to a chemically compromised host.
Animals↗