Mary G. Murphy

Differential effects of docosahexaenoic acid on contractions and L-type Ca2+ current in adult cardiac myocytes

UNLABELLED Beneficial effects of n-3 polyunsaturated fatty acids in Ca2+ overload have been attributed to blockade of L-type Ca2+ current (I(Ca-L)). However, cardiac contractions may be maintained despite block of I(Ca-L). OBJECTIVE This study investigates the cellular basis by which docosahexaenoic acid (DHA), a representative n-3 polyunsaturated fatty acid, inhibits I(Ca-L) while preserving contraction. METHODS Experiments were conducted in adult guinea pig ventricular myocytes with Na+ currents blocked. Contractions initiated by the voltage-sensitive release mechanism (VSRM) and calcium-induced calcium release (CICR) triggered by I(Ca-L), were activated separately with voltage clamp techniques. RESULTS DHA (10 microM) inhibited I(Ca-L) and CICR contractions but not VSRM contractions. CICR contractions exhibited a bell-shaped voltage-dependence. However, in the presence of DHA, only contractions with a sigmoidal voltage-dependence characteristic of the VSRM remained. These contractions exhibited inactivation properties characteristic of the VSRM. DHA abolished I(Ca-L) elicited by test steps from -40 mV. Block was voltage-dependent, as residual I(Ca-L) was elicited by steps from -70 mV. Cd2+ inhibited residual current, but not contractions initiated by the same activation steps. CONCLUSION Preservation of VSRM contractions during block of I(Ca-L), may explain the ability of n-3 polyunsaturated fatty acids to inhibit Ca2+ influx while preserving cardiac contractile function.

Acid:Coenzyme A Ligase in Brain: Fatty Acid Specificity in Cellular and Subcellular Fractions

Abstract: We measured long‐chain fatty acid:coenzyme A (CoA) ligase (EC 6.2.1.3) activity with four fatty acids in brain homogenates, and cellular and subcellular fractions to determine whether there are differences in activity that could be correlated with differences in fatty acid composition and metabolism. In rat brain homogenates, ligase activity varied appreciably with the four acids, with 18:2 > 18:1 > 16:0 > 22:1 (nmol acyl‐CoA formed/min/mg protein; 1.46, 1.20, 0.96, and 0.57, respectively). This order was similar under all incubation conditions tested, including variable pH and fatty acid concentrations. The relative specific activities (RSA, 16:0 = 1.0) with the four substrates were similar in rat brain homogenate, mitochondria, and microsomes, with the highest specific activities in the latter fraction. The RSA were also similar in ox brain homogenates, in rabbit brain microsomes prepared from gray and white matter, in neurons isolated from rat brain, and in cultured neuroblastoma cells. Rat liver homogenates had a significantly different pattern of RSA. These results indicate that the ligase(s) has a preference for certain fatty acids, but suggest that the major control of fatty acid composition and metabolism is a function of subsequent metabolic steps.

The effects of cortical ablation on multiple unit activity in the striatum following dexamphetamine

Bilateral removal of the fronto-parietal cortex of the rat resulted in decreased spontaneous multiple-unit activity recorded in the striatum of freely-moving rats. Cortical ablations changed the neuronal response in the striatum to systemic administration of dexamphetamine (2.5 mg/kg i.p.) from excitation in control animals (88%) to inhibition in ablated animals (61%). Furthermore, catalepsy, induced by haloperidol, but not by morphine, was markedly attenuated after cortical ablation. These changes were accompanied by a 23% decrease in the specific binding of [3H]spiperone in the striatum. The binding of [3H]met-enkephalin was unaffected by the cortical lesions. Levels of glutamate in the striatum decreased from 8.88 +/- 0.5 mumols/g in control animals to 6.93 +/- 0.37 mumols/g after bilateral cortical ablation. On the other hand, cortical ablations did not alter the content of either the gamma-aminobutyric acid or glutamine of the striatum. It is concluded that the excitatory response, observed in striatal neurons in freely-moving animals, is dependent upon an intact cerebral cortex and requires intact cortico-striatal afferents. The results further suggest that neurons in the striatum are under the tonic influence of glutamate, released from cortico-striatal afferents. Lastly, some dopamine D2 binding sites in the striatum are located on cortico-striatal afferent terminals and blockade of these striatal D2 sites may be involved in the induction of catalepsy by neuroleptic drugs.

Long-chain fatty acid:CoA ligase in rat brain in vitro: a comparison of activities with oleic and cis-vaccenic acids.

Long‐chain fatty acid:CoA ligase (EC 6.2.1.3) has been examined in vitro with brain preparations from 12‐ to 16‐day‐old rats using oleic (18: 1[n‐9]) and cis‐vaccenic (18: 1[n‐7]) acids. A novel assay system, which permits measurement of product as [14C]fatty acyl‐[3H]CoA, [14C]fatty acyl‐CoA, or fatty acyl‐[3H]CoA, was used. With the double‐label assay, reaction conditions were established that gave a molar ratio of [3H]CoA to [14C]fatty acid in the product of 1.06 ± 0.12 (mean ±s.d., n= 14). This indicates that the method is specific for the fatty acid added to the incubation mixture. The assay is sensitive to the formation of approximately 0.02 nmol fatty acyl‐CoA. With oleic acid, specific activity of the ligase was highest in a microsomal fraction (3.91 ± 0.55 nmol oleoyl‐CoA formed/min/mg protein; n= 16). Optimal activity was obtained at concentrations of fatty acid, CoA, ATP and Mg2+ of 50 μM, 50 μM, 10 mm, and 8 mm, respectively. The apparent Kms (approximate) for oleic acid and coenzyme A were 20 and 6 μM, respectively. With cis‐vaccenic acid (18:1[n‐7]), a naturally occurring isomer of oleate, the maximum reaction rate was higher than that with oleate, as were the apparent Kms for both fatty acid (105 μM) and coenzyme A (23 μM).

Dietary menhaden, seal, and corn oils differentially affect lipid and ex vivo eicosanoid and thiobarbituric acid-reactive substances generation in the guinea pig.

This investigation was carried out to characterize the effects of specific dietary marine oils on tissue and plasma fatty acids and their capacity to generate metabolites (prostanoids, lipid peroxides). Young male guinea pigs were fed nonpurified diet (NP), or NP supplemented (10%, w/w) with menhaden fish oil (MO), harp seal oil (SLO), or corn oil (CO, control diet) for 23 to 28 d. Only the plasma showed significant n−3 polyunsaturated fatty acid (PUFA)-induced reductions in triacylglycerol (TAG) or total cholesterol concentration. Proportions of total n−3 PUFA in organs and plasma were elevated significantly in both MO and SLO dietary groups (relative to CO), and in all TAG fractions levels were significantly higher in MO-than SLO-fed animals. The two marine oil groups differed in their patterns of incorporation of eicosapentaenoic acid (EPA). In guinea pigs fed MO, the highest levels of EPA were in the plasma TAG, whereas in SLO-fed animals, maximal incorporation of EPA was in the heart polar lipids (PL). In both marine oil groups, the greatest increases in both docosahexaenoic acid (22∶6n−3, DHA) and docosapentaenoic acid (22∶5n−3, DPA) relative to the CO group, were in plasma TAG, although the highest proportions of DHA and DPA were in liver PL and heart TAG, respectively. In comparing the MO and SLO groups, the greatest difference in levels of DHA was in heart TAG (MO>SLO, P<0.005), and in levels of DPA was in heart PL (SLO>MO, P<0.0001). The only significant reduction in proportions of the major n−6 PUFA, arachidonic acid (AA), was in the heart PL of the SLO group (SLO>MO=CO, P<0.005). Marine oil feeding altered ex vivo generation of several prostanoid metabolites of AA, significantly decreasing thromboxane A2 synthesis in homogenates of hearts and livers of guinea pigs fed MO and SLO, respectively (P<0.04 for both, relative to CO). Lipid peroxides were elevated to similar levels in MO- and SLO-fed animals in plasma, liver, and adipose tissue, but not in heart preparations. This study has shown that guinea pigs respond to dietary marine oils with increased organ and plasma n−3 PUFA, and changes in potential synthesis of metabolites. They also appear to respond to n−3 PUFA-enriched diets in a manner that is different from that of rats.

Studies of the Regulation of Basal Adenylate Cyclase Activity by Membrane Polyunsaturated Fatty Acids in Cultured Neuroblastoma

The role of membrane polyunsaturated fatty acids (PUFAs) in the regulation of basal adenylate cyclase activity was examined in intact N1E‐115 neuroblastoma cells. Addition of linoleic acid (50 μM) to the culture medium for 48 h resulted in a significant increase in phospholipid PUFA content and in a two‐ to fivefold increase in basal accumulation of cyclic AMP (cAMP). Both phenomena were reversed on removal of linoleate from the medium. PUFA enrichment stimulated cell proliferation by —20% without altering the relative proportion of cellular protein. The supplemented cells synthesized significantly larger amounts of prostaglandin (PG) E and D than did the controls; however, blockade of PG synthesis by indomethacin or ibuprofen did not alter cAMP formation. Supplemented cells contained higher levels of malondialdehyde (MDA) than did controls, and MDA formation was reduced by coculture with α‐tocopherol; however, its inclusion in the medium did not affect cAMP accumulation. Linoleate‐supplemented cells responded to cyclase‐activating agonists to the same extent as did control cells. Responses to inhibitory agonists (e.g., isoproterenol and carbamylcholine) were altered, but not to a sufficient extent to account for the PUFA‐dependent increases in basal adenylate cyclase activity.

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 totalphospholipid (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 ≈ 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 ≥ 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.

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.

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, 18:2Ω6), on membrane fatty-acid composition and functions. After 18–21 days in culture, polyunsaturated fatty acids (PUFA) constituted≈24 mol% of the total fatty acids in the astrocytes grown in delipidated media (‘controls’); these proportions were increased by 35–40% to≈33 mol% when the cells were supplemented with 35μM LA. Notable differences in the PUFA profiles of the cells cultured with or without added LA included: (a) higher proportions of Ω6 PUFA in the LA-supplemented astrocytes (≈25%, relative to≈10% in controls) that were accompanied by an increase in the ratio of Ω6/Ω3 PUFA (from <2 in controls to ≈5), and (b) higher proportions of 20:3Ω9 and 22:3Ω9 in the control astrocytes (>5%) relative to the LA-supplemented cells (≈1%). The major metabolites in the Ω6 PUFA-enriched cells were arachidonic (20:4Ω6), adrenic (22:4Ω6) and docosapentaenoic (22:5Ω6) acids (15, 5 & 3 mol%, respectively). Enrichment of the astrocytes in Ω6 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 ≈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 Ω6 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 membrans PUFA composition modify only specific cell functional properties.

Serum lipid abnormalities in a chemical/viral mouse model for Reye's syndrome

Neonatal mice given nontoxic dermal applications of an industrial surfactant, Toximul MP8 (Tox), and subsequently infected with sublethal doses of mouse-adapted human Influenza B (Lee) virus (FluB) develop many of the biochemical features of Reyes Syndrome (RS). To determine whether these also include abnormal circulating lipid, we examined serum lipid profiles in the mouse model throughout the treatment course using Iatroscan-TH10. Following 10 days of exposure to surfactant, serum phospholipid and cholesterol levels were significantly reduced relative to control animals. These reductions were transient; however, four days following virus administration, significant differences in serum lipid were again evident. These abnormalities coincided and correlated with increased animal mortality. Animals that received combined Tox + virus treatment had significant decreases in serum total lipids relative to control animals, a reflection of a reduction in all lipid classes, including phospholipid, cholesterol, neutral glycerides (triglycerides plus diglycerides) and free fatty acids. Phospholipid (specifically phosphatidylcholine and lysophosphatidylcholine) and free fatty acid levels in the Tox + virus group were also significantly lower than those in animals that received virus alone. This study has demonstrated that suckling mice given chemical/viral treatment have the serum hypopanlipidemia but not the freefattyacidemia that are characteristic of RS.