ACYL group composition of lipids and the activities of (Na+ + K+)-ATPase, 5'-nucleotidase and gamma-glutamyltranspeptidase in salivary glands and kidneys of rats fed diets containing different dietary fats.

Two nutritional models, an essential fatty acid deficiency model and the feeding of saturated versus unsaturated fats, were used in a feeding study in order to assess the relationship between tissue fatty acid composition and the activities of some membrane-associated enzymes. Purified diets containing 7% hydrogenated coconut oil, 7% corn oil, 10% safflower oil or butter were fed to rats for a total of 49 weeks (1 week of pregnancy, 3 weeks of lactation and 45 weeks post-weaning). Tissue homogenates from submandibular salivary glands and kidneys were analyzed for fatty acid composition of total lipids and phospholipids. Changes in fatty acid patterns typical of essential fatty acid deficiency such as an increase in the levels of 16:1 and 18:1, a decrease in 18:2 and 20:4 and an accumulation of 20:3 omega 9 were observed in salivary glands and kidneys of rats fed the deficient diet. Tissues of rats fed 10% butter also showed fatty acid compositional changes which were somewhat similar to those in essential fatty acid deficiency, but to a lesser degree. The activities of ouabain-sensitive (Na+ + K+)-ATPase were higher in homogenates of salivary glands and kidneys of the deficient rats and those fed butter as compared with their controls. The results suggest a relationship between the double bond index of fatty acids as an indication of membrane lipid fluidity and allosteric modification of (Na+ + K+)-ATPase activity. However, other explanations for the observed changes in (Na+ + K+)-ATPase activity cannot be ruled out. There were no diet-related differences in the activities of gamma-glutamyltranspeptidase or 5'-nucleotidase.

Microsomal fatty acid desaturase activities in vitamin A-deficient rat liver.

The activity of microsomal fatty acid delta 9-desaturase was significantly higher in liver microsomes of vitamin A-deficient rats as compared with their controls. Feeding of vitamin A-supplemented control diet to the deficient rats restored the delta 9-desaturase activity to the control values. The activity of delta 6-desaturase was not affected by vitamin A deficiency.

Activities of fatty acid desaturases and fatty acid composition of liver microsomes in rats fed beta-carotene and 13-cis-retinoic acid.

The fatty acid composition of microsomal lipids and the activities of delta 9- and delta 6-desaturases in liver microsomes of rats fed diets supplemented with beta-carotene and two levels of 13-cis-retinoic acid were studied. Four groups of male, weanling rats were fed semipurified diets containing 0 or 100 mg beta-carotene per kg diet, and 20 or 100 mg 13-cis-retinoic acid per kg diet. After 11 weeks of feeding, the rats were killed, liver microsomes were prepared and assayed for delta 9-desaturase and delta 6-desaturase activities. The activity of delta 9-desaturase was lower in liver microsomes of rats fed beta-carotene-supplemented diet or the diet supplemented with the higher level of 13-cis-retinoic acid. Microsomal delta 6-desaturase activity was, however, higher in liver of rats fed 13-cis retinoic acid; there was no effect of beta-carotene on delta 6-desaturase activity. The fatty acid compositional data on total lipids of liver microsomes were consistent with the diet-induced changes in fatty acid desaturases. Phospholipid composition of liver microsomes was also altered as a result of feeding beta-carotene or 13-cis-retinoic acid-containing diets. The proportions of phosphatidylethanolamine were generally higher, whereas those of phosphatidylcholine were lower in the experimental groups as compared with the control.

Influence of dietary omega-3 fatty acids on transmembrane signalling in rat submandibular salivary gland.

We have previously shown the incorporation of dietary omega-3 and omega-6 fatty acids from menhaden oil and corn oil, respectively, into membrane phospholipids of submandibular salivary gland (SMSG) of rat [Alam S. Q. and Alam B. S. (1988) Arch. Oral Biol. 33, 295-299]. We now demonstrate the influence of such incorporation on the regulation of G proteins and adenylate cyclase activity. Cholera toxin ribosylated three protein peptides (Mr 42,000, 44,000 and 46,000) to different extents in the two groups. We found 4.9- and 2.6-fold higher and 0.4-fold lower ribosylation of Mr 42,000, 44,000 and 46,000 peptides, respectively, in SMSG membranes of rats fed a diet containing 10% menhaden oil (group II) compared to those fed 10% corn oil (group I). Functional distinctions between different forms of these peptides are not known. Cholera toxin also exhibited radiolabelling of three peptides in the SMSG membranes from normal or fasting rats. In these membranes inhibitory G proteins were not detected by pertussis toxin dependent ADP ribosylation or by a low concentration of guanylyl 5-imidodiphosphate (10(-8) M), which selectively activates inhibitory G proteins which inhibit forskolin stimulated activity of adenylate cyclase. In group II membranes both basal and fluoride stimulated activities of adenylate cyclase were found to be significantly higher than the corresponding values in group I (P less than 0.02). In cholera toxin dependent ribosylated membranes of group I, basal and fluoride stimulated activities of adenylate cyclase were significantly higher than those obtained in the absence of cholera toxin (P less than 0.02). Surprisingly, corresponding values were found to be lower in ribosylated membranes of group II. This could be due either to conformational changes in heavily ribosylated G proteins, which alters coupling with the catalytic subunit of adenylate cyclase, or due to dissociation of excessive inhibitory beta gamma complex from alpha beta gamma complex upon the activation of G proteins.

Effect of essential fatty acid deficiency on G-proteins, cAMP-dependent protein kinase activity and mucin secretion in the rat submandibular salivary glands.

Studies were conducted to determine whether beta-adrenergic cell signalling is altered in submandibular salivary glands (SMSG) is essential fatty acid (EFA) deficiency. Three groups of rats were fed diets which were deficient in EFA (EFAD), marginally deficient in EFA (MEFAD) or contained sufficient amount of EFA (Control). Rats were killed after 20 wk on diets, SMSG were dissected out and cyclic AMP-dependent protein kinase (PKA) activity was measured. The specific enzyme activities were higher in the homogenates and supernatant fractions of the gland from EFAD and MEFAD rats compared with the controls. The relative levels of guanine nucleotide-binding regulatory proteins (Gs and Gi) were also measured in the SMSG membranes of rats fed the 3 diets. The levels of Gs were significantly higher in the EFAD and MEFAD groups than in the controls. No significant differences were observed in the secretion of trichloroacetic acid-phosphotungstic acid (TCA-PTA) precipitable glycoproteins from the SMSG slices among the 3 dietary groups.

Prostaglandin E2 production in submandibular salivary glands of rats in essential fatty acid deficiency.

Three groups of rats were fed an essential fatty acid (EFA)-deficient diet (EFAD), marginally EFA-deficient diet (MEFAD) or a control diet. Arachidonic acid levels in total phospholipids and the ex-vivo production of prostaglandin E2 (PGE2) in the presence of calcium ionophore were measured at 5 and 9 weeks in the submandibular salivary glands (SMSG). The arachidonic acid levels were significantly different among the 3 groups of rats fed the respective diets for 9 weeks. Ex-vivo PGE2 production was significantly decreased in the EFAD group but not in the MEFAD group as compared to the control group. The changes in fatty acid composition, arachidonic acid levels and ex-vivo production of PGE2 were reversed after 5 weeks of feeding the control diet to the EFAD or the MEFAD rats. Since arachidonic acid and PGE2 are involved in signal transduction pathways in the SMSG, an EFA deficiency is likely to modify these pathways.

The concentrations of free fatty acids, triglycerides and phospholipids in submandibular salivary glands of rats in essential fatty acid deficiency.

EFA deficiency was created in young weanling rats by feeding for 16 weeks purified diets containing 0% fat or 7% hydrogenated coconut oil (HCO). Control rats were fed similar diets which contained 7% corn oil or 5% HCO + 2% corn oil. Submandibular salivary gland (SMSG) lipids were extracted and analyzed for the concentration of the free fatty acids (FFA), triglycerides (TG), total lipid P and the proportions of various phospholipids. In EFA-deficient SMSG, the concentrations of FFA and TG fractions were lower compared to those of the controls. Total lipid P was either unaffected or slightly increased; the proportion of phospholipids was not changed. Possible reasons for these changes in SMSG lipids are discussed.

Effect of isoprenaline on salivary gland lipids of rats fed a choline-deficient diet.

Male adult rats were fed choline deficient and a choline-supplemented diet for 3 weeks. Half the rats from each group were injected with isoprenaline (16 mg/kg body weight) and the remainder with saline. After 2 and 8 hours, rats were sacrificed, their submandibular salivary glands were disected out, and extracted for lipids. The fatty acid composition of total lipids and some neutral and phospholipid fractions were determined. Isoprenaline administration resulted in a slightly higher level of oleic acid in SMSG of rats fed choline-deficient but not the control diet. There was an increase in the free fatty acid (FFA) concentrations of the gland after 2 hours of isoprenaline treatment in rats fed the control diet; no such increase was observed in the choline-deficient group. The fatty acid composition of FFA fraction was also slightly changed as a result of isoprenaline treatment, but only in the choline-deficient group. The fatty acid composition of triglyceride (TG), phosphatidyl choline (PC), and phosphatidyl ethanolamine (PE) fractions was not changed.

In-vivo incorporation of omega 3 fatty acids into membrane lipids of rat salivary glands and changes in adenylate-cyclase activity.

Dietary omega 3 fatty acids from menhaden oil were incorporated into membrane phospholipids of submandibular salivary glands (SMSG). Eicosapentaenoic (20:5), docosapentaenoic (22:5) and docosahexaenoic (22:6) acids constituted about 20 per cent of the total fatty acids in phospholipids, phosphatidylcholine and phosphatidylethanolamine fractions of the SMSG plasma membranes of rats fed for 6 weeks a diet containing 10 per cent menhaden oil. The changes in fatty-acid composition of the membrane phospholipids were accompanied by higher adenylate-cyclase activity in the SMSG membranes of rats fed 10 per cent menhaden oil than in rats fed 10 per cent corn oil or 8 per cent coconut oil + 2 per cent corn oil. However, there were no diet-related differences in the fold-stimulation of adenylate-cyclase activity.

Effect of 13-cis retinoic acid on fatty acid composition and membrane-associated enzymes in rat submandibular salivary glands.

Weanling rats were fed for 4 weeks semipurified diets containing 0, 100 or 300 mg 13-cis retinoic acid per kg diet. The activities of (Na+ + K+)-ATPase, Mg2+-ATPase and gamma-glutamyltranspeptide were measured in submandibular salivary gland (SMSG) homogenates. The activity of (Na+ + K+)-ATPase in the SMSG was reduced in rats fed 13-cis retinoic acid. There was no effect on the activities of the other two enzymes. Fatty acid composition of total lipids in SMSG suggest that decrease in (Na+ + K+)-ATPase activity may be associated with changes in fatty acid composition of total lipids.

Fatty acid incorporation into membranes of dispersed rat submandibular salivary gland cells and their effect on adenylate cyclase activity.

Dispersed cells from rat submandibular salivary glands were incubated in medium supplemented with different fatty acids, such as stearic (18:0), oleic (cis 18:1 omega 9) and elaidic acid (trans 18:1 omega 9). The exogenous fatty acids were incorporated into the membrane lipids. A comparison of the adenylate cyclase activity in membranes of cells incubated with different fatty acids revealed the following order (from highest to lowest specific activity): stearic, elaidic, oleic. The findings suggest that changes in adenylate cyclase activity in these dispersed membranes enriched with different fatty acids may be related to the possible changes in membrane fluidity. The synthesis of glycoproteins was studied by measuring the incorporation of [1-14C]-glucosamine into TCA-PTA-precipitable material in the presence or absence of fatty acids. No effect of the exogenous fatty acids on glycoprotein synthesis was observed.

Reversal of diet-induced changes in adenylate cyclase activity and fatty acid composition of rat submandibular salivary gland lipids.

This study sought to determine if the diet-induced changes in submandibular salivary glands can be reversed. Two groups of male weanling Sprague-Dawley rats were fed purified diets containing 9% butter + 1% corn oil (group I, control) or 9% ethyl ester concentrate of n-3 fatty acids + 1% corn oil (group II, experimental). After 5 weeks of feeding the respective diets, rats in group I were divided into two subgroups: Ia, which was maintained on the control diet, and Ib, which was shifted to the experimental diet for the reversal study. The rats in the experimental group were kept on their original diet. After five further weeks of feeding, the rats were killed, and membranes from submandibular glands were prepared and assayed for adenylate cyclase activity and for the fatty acid composition of total phospholipids. Changes characteristic of feeding n-3 fatty acids, including a significant increase in membrane fluidity as measured by the degree of unsaturation of fatty acids, were observed in the total phospholipids of membranes from the experimental group. The adenylate cyclase activity was two- to three-fold higher in membranes of rats fed the experimental diet (group II) than the control diet (group Ia). Whereas the diet-induced changes in fatty acid composition and membrane fluidity were largely reversed (group Ib, reversal study), changes in adenylate cyclase activity were only partially reversed. The results suggest that, in addition to the fatty acid composition and membrane fluidity, other factors may also be important in modifying adenylate cyclase activity.

Effect of ageing on adenylate cyclase activity and G-proteins in rat submandibular salivary glands.

Membranes were prepared from the submandibular salivary glands of male young (3-months-old) and aged (24-months-old), Fisher 344 rats, and assayed for adenylate cyclase activity and for the ribosylation of G-proteins in the presence of [32P]-NAD+ and cholera toxin. Adenylate cyclase activity (basal, fluoride-, forskolin-, and isoproterenol-stimulated) was significantly lower (p less than 0.01) in the glands of aged rats. The pattern of ribosylation was different between the 2 groups. Two cholera toxin-specific bands (Mr 42,000 and 44,000) were ribosylated in the glands of young rats whereas in aged rats, only an Mr 42,000 band was clearly observed. The incorporation of 32P from NAD+ into the Mr 42,000 and 44,000 bands increased with the time of ribosylation (7.5-90 min). In aged rats, however, the incorporation of 32P into the Mr 42,000 band was much slower and did not increase much after 60 min of ribosylation. At each time point, the extent of ribosylation was lower in the membranes from the aged rats. These findings suggest that the decrease in adenylate cyclase activity in the submandibular salivary glands of aged rats may be due partly to the changes in their levels of Gs as a result of ageing.

[3H]-forskolin binding sites in rat submandibular salivary glands.

Specific [3H]-forskolin binding was linear as a function of the membrane protein concentration and reached steady-state by 30-40 min of incubation at 37 degrees C. Such binding was saturable and the bound radioligand could be readily dissociated by an excess of unlabelled forskolin. There was only one type of binding site, with a dissociation constant (Kd) of 0.16 microM. The concentration of the binding sites (Bmax) was 0.73 pmol/mg protein. There was no significant difference in the [3H]-forskolin binding characteristics in membranes of rats fed for 6 weeks on diets containing 10% corn oil, 8% coconut oil + 2% corn oil, or 10% menhaden fish oil.

Membrane fluidity and adenylate cyclase activity in the lacrimal glands of rats fed diets containing trans fatty acids.

The effects of feeding a diet containing trans fatty acids on the fatty acid composition of plasma membrane phospholipids, fluorescence polarization of diphenylhexatriene (DPH) and adenylate cyclase activity in the exorbital lacrimal glands of rats were studied. Three groups of male, weanling rats were fed semipurified diets containing 20% corn oil (CO), 20% partially hydrogenated soybean oil (PHSBO) (a source of trans fatty acids) and 19% PHSBO + 1% CO. Plasma membranes of the lacrimal glands from rats fed 20% PHSBO showed higher adenylate cyclase activity and lower fluidity as shown by a lower double bond index of the fatty acids of their phospholipids and higher fluorescence polarization of DPH. When 1% CO was included with the diet containing PHSBO, the adenylate cyclase activity and membrane fluidity tended to be normal. The results suggest that feeding of a diet containing trans fatty acids in the absence of sufficient linoleic acid (18:2) can result in a decrease in membrane fluidity and an increase in adenylate cyclase activity in the lacrimal glands.

Lung surfactant and fatty acid composition of lung tissue and lavage of rats fed diets containing different lipids.

Two nutritional models, essential fatty acid (EFA) deficiency and the feeding of saturated vs unsaturated fats, were used to determine the effects of dietary lipids on the fatty acid composition of rat lung and lavage. Semipurified diets containing 7% corn oil, 7% hydrogenated coconut oil (EFA-deficient), 10% butter or 10% safflower oil were fed to dams during lactation and thereafter to their offspring for a total of 24 weeks. Lipids were extracted from the lung lavage and lung tissue and their fatty acid composition was determined. The content of dipalmitoylphosphatidylcholine (DPPC), the main surfactant in the lungs, was also determined. The results show that the levels of DPPC in the lungs of rats fed 10% butter decreased although the decrease in the EFA-deficient rats was greater. Comparing rats fed butter with those fed corn oil, there were also modifications in the fatty acid composition of the total lipids and phospholipids of lung tissue and lavage as well as in phosphatidyl-choline, phosphatidylethanolamine and phosphatidylinositol + phosphatidylserine fractions isolated from the lung tissue. The changes in fatty acid composition were somewhat fewer in rats fed butter then in those fed an EFA-deficient diet. The results suggest that a marginal EFA deficiency produced in rats by long-term feeding of 10% butter may account for the reduction in DPPC levels and in the changes in fatty acid composition in the lung tissue and lavage.

Phospholipid composition of liver in rats fed high levels of 13-cis retinoic acid.

The composition of liver phospholipids was studied in rats fed for 4 weeks diets containing 0, 100 or 300 mg 13-cis retinoic acid per kg diet. There was a significant decrease in phosphatidylcholine content, whereas the levels of phosphatidylethanolamine were slightly increased in liver phospholipids of rats fed 13-cis retinoic acid. The fatty acid composition of total phospholipids, PC, PE, and P1 + PS fractions revealed a general increase in the levels of 18:2 and 20:3 omega 6, whereas the levels of 20:4 omega 6 and C22 fatty acids were reduced in most of the hepatic phospholipids isolated from rats fed 13-cis retinoic acid containing diets. A decrease in the double-bond index of fatty acids was also observed in phospholipids of rats fed 13-cis retinoic acid. The data suggest that high levels of 13-cis retinoic acid may possibly be influencing the activities of microsomal desaturating and chain-elongating enzymes in the liver.

Pulmonary lipid peroxides and fatty acids of rats fed different lipids and exposed to oxygen at hyperbaric pressure.

Semipurified diets containing different lipids were fed to rat dams during lactation and subsequently to their pups for 33 weeks post-weaning. Some rats within each group were exposed to oxygen at hyperbaric pressure (OHP). Lipid peroxide levels were lower in lungs of rats fed 7% hydrogenated coconut oil or 10% butter as compared with their controls, fed 7% corn oil or 10% safflower oil, respectively. Exposure to OHP increased lung peroxide levels. This increase varied with the type of fat in the diet. Studies of the fatty acid composition indicate that lipid peroxide levels generally increased with an increase in the levels of 18:2 in lung total lipids. The results suggest that the type of dietary lipid may alter the susceptibility of the animal to pulmonary oxygen toxicity.

Incorporation of trans fatty acids into submandibular salivary gland lipids.

Three groups of rats were fed diets containing 20% corn oil, 20% margarine stock (MS) or 19% MS + 1% corn oil. Diets were fed for 12 weeks, 1 week of pregnancy, 3 weeks of lactation and 8 weeks post-weaning. The incorporation of trans-octadecenoate into various lipids of the submandibular salivary gland (SMSG) homogenates and plasma membranes was studied. Trans octadecenoate was incorporated into all the lipid fractions studied. Its levels were the highest in phosphatidylethanolamine. The double bond index of phospholipid fatty acids in the plasma membranes of the SMSG was substantially lower in the group fed 20% MS. The fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) was generally higher in the membranes of SMSG from rats fed MS than that of the other two groups, thus indicating lower fluidity. Also, the breakpoints in fluorescence polarization were at a higher temperature in the membranes from rats fed MS as compared with those fed corn oil. Lower fluidity of plasma membranes of SMSG observed in rats fed 20% MS may result in modification of the activities of membrane-bound enzymes.

[3H]forskolin- and [3H]dihydroalprenolol-binding sites and adenylate cyclase activity in heart of rats fed diets containing different oils.

The characteristics of the cardiac adenylate cyclase system were studied in rats fed diets containing fish oil (menhaden oil) and other oils. Adenylate cyclase activity generally was higher in cardiac homogenates and membranes of rats fed diet containing 10% menhaden oil than in the other oils. The increase in enzyme activity, especially in forskolin-stimulated activity, was associated with an increase in the concentration of the [3H] forskolin-binding sites in cardiac membranes of rats fed menhaden oil. The beta-adrenergic receptor concentration was not significantly altered although the affinity for [3H]dihydroalprenolol-binding was lower in membranes of rats fed menhaden oil than those fed the other oils. omega-3 fatty acids from menhaden oil were incorporated into the cardiac membrane phospholipids. The results suggest that the observed increase in myocardial adenylate cyclase activity of rats fed menhaden oil may be due to an increase in the number of the catalytic subunits of the enzyme or due to a greater availability of the forskolin-binding sites.