Effects of dietary saturated, N-6 and N-3 polyunsaturated fats on blood pressure and prostaglandins outflow from perfused mesenteric vascular bed in rats

Effects of the dietary administration of saturated fat and of n-6 and n-3 polyunsaturates on blood pressure, prostaglandin metabolism in small vessels, tissue fatty acid distribution and urinary PGE2 excretion were compared. Rats were divided into three groups. Diets contained 10% hydrogenated coconut oil (HCO), 10% safflower oil (SFO) or 10% cod liver oil (CLO) added to a basic fat free diet for 10 weeks. Systolic blood pressure was increased in the CLO group animals. Urinary PGE2 excretion was decreased in the HCO and CLO groups as compared to that in the SFO group animals. PGE2, 6-keto-PGF1 alpha and thromboxane (Tx) B2 outflow from isolated perfused mesenteric arterial beds were extremely decreased in the CLO group animals, and to a lesser extent in the HCO group as compared to the SFO animals. In the tissue phospholipid, 20:3n-9/20:4n-6 ratios were increased in the HCO group indicating essential fatty acid deficiency, and n-6 and n-3 polyunsaturates were elevated in the SFO and the CLO group animals respectively. Arachidonic acid concentration was highest in the SFO group, while there was no significant differences between the HCO and the CLO group. These results suggest that dietary fatty acid manipulation affects urinary PGE2 excretion and PGI2, PGE2 and TxA2 synthesis in mesenteric arterial beds and also changes the tissue fatty acid distribution. Furthermore, n-3 polyunsaturates caused an extreme reduction of 2-series PGs synthesis in small resistance vessels.     

Effects of dietary polyunsaturated fats and vitamin E on aging and peroxidative damage to DNA

Peroxidative damage to DNA was studied in rats fed either a diet with 10% tocopherol-stripped corn oil and 30 IU DL-alpha-tocopherol acetate/kg (group A), the same diet without vitamin E (group B), a diet with 24% corn oil without vitamin E (group C), or the diet of group A for 10 months and then the diet of group C for 4 months (group D). After 3, 6, 9, and 14 months of feeding the diets, body weights, motoric activities, testicular weights, and lipid-soluble fluorophores in testes were measured. Groups A and B had higher hepatic DNA template activities at 9 and 14 months than group C, and group A had higher testicular DNA template activities than groups B and C at 6, 9, and 14 months. Hepatic DNA template activity of group C decreased from 6 to 9 and from 9 to 14 months. Group C hepatic DNA transcribed less long RNA than that of groups B and D, and more short RNA than groups B and D. Group A testicular DNA transcribed more medium-length RNA than that of groups B and D, and less short RNA than that of groups B, C, and D. DNA-bound tryptophan and DNA crosslinking were inversely related to DNA template activities. DNA damage correlated with other biochemical and physiological changes that are characteristic of cellular impairment in aging and disease.     

Influence of dietary selenium on the mutagenic activity of perfusate and bile from rat liver, perfused with 1,1-dimethylhydrazine

The mutagenic effect of 1,1-dimethylhydrazine (UDMH) was studied in the liver perfusion/cell culture system. Male Wistar rats, fed a selenium-deficient diet with or without selenium supplementation in the drinking water, were used as liver donors. UDMH caused an increased mutation frequency in Chinese hamster V79 cells exposed in the perfusate. The effect was statistically significant with both selenium-deficient and selenium-supplemented livers. With selenium-deficient livers, a significant mutagenic effect was also obtained when V79 cells were treated with bile collected after the administration of UDMH. Bile flow and bile acid excretion were not affected by UDMH treatment of selenium-deficient or selenium-supplemented livers. There was a tendency towards reduced C-oxygenation of N,N-dimethylaniline in microsomes from selenium-deficient livers perfused with UDMH. The lactate/pyruvate ratio in the perfusate was increased by UDMH, the effect being more pronounced with selenium-deficient than selenium-supplemented livers.     

Effects of prostaglandins and catecholamines on rat spleen adenylate cyclase in vitro

The results reported here show some characteristics of adenylate cyclase (EC 4.6.1.1) derived from homogenates of rat spleen, and describe the in vitro stimulation of this enzyme by prostaglandins, nucleotides, and F⁻ under conditions where cyclic nucleotide degradative pathways are effectively inhibited.Particulate fractions from rat spleen homogenates contain high adenylate cyclase activities, and the highest specific activity is recovered in a particulate fraction prepared by low speed (1200 × g) centrifugation. Activity found in all particulate fractions is stimulated by fluoride, prostaglandins E₁ and E₂, catecholamines, and purine nucleotides. No stimulation is caused by prostaglandins F_1α and F_2α. Stimulation by prostaglandin E₁ or E₂ is augmented by GTP and other purine nucleotides, and stimulation by the combination of GTP and prostaglandin E₁ is equal to that caused by optimal fluoride concentrations. Stimulation c caused by L-isoproterenol is additive to that caused by GTP but is not increased by GTP.     

Effects of systemic and intrafollicular injections of LH,prostaglandins, and indomethacin on the luteinization of rabbit Graafian follicles

The possibility that initiation of luteinization in ovarian follicles by luteinizing hormone (LH) is mediated by prostaglandins (PG's) was investigated in rabbits. Estrous rabbits, given an ovulatory dose of LH (50 μg) intravenously, were administered indomethacin (IM), an inhibitor of PG biosynthesis, by various routes. Progesterone levels in the serum and in the induced corpora lutea (CL) were subsequently measured by radioimmunoassay. Continued daily subcutaneous injections of IM from 2 days before through 2 days after LH treatment reduced the corpus luteal level, measured at 72 hours post-LH, of PGF from 208 ± 43 to 98 ± 20 pg/CL (P < 0.025) and that of PGE from 272 ± 31 to 115 ± 9 pg/CL (P < 0.005). At the same time, progesterone levels were 72 ± 12 and 93 ± 10 ng/CL (P > 0.05) in the oil-treated and IM-treated rabbits, respectively. Serum progesterone continued to rise in a linear fashion during the period from 24 to 72 hours following LH treatment, whether IM was injected or not. Intrafollicular treatment with LH (100 ng/follicle) raised the progesterone content in the treated follicles 72 hours later from 1.1 ± 0.5 to 50.1 ± 13.5 ng. (P < 0.01). This progesterone content reached 21.5 ± 15.8 ng (P < 0.05) in follicles similarly treated with PGE₂ (5 μg/follicle), but remained meagre at lower doses of PGE₂ (100 ng/follicle and 2 ng/follicle). Serum progesterone increased from 0.5 ± 0.1 to 1.2 ± 0.1 ng/ml (P < 0.005) within 72 hours in rabbits treated intrafollicularly with LH, but remained unaltered in those similarly treated with PGE₂ (P > 0.1). Intrafollicular injections with PGF_2α failed to induce changes in either level of progesterone. It is concluded that prostaglandins probably do not mediate the luteinizing action of LH in rabbit Graafian follicles, although some degree of luteinization can be induced by high levels of exogenous PGE₂.     

Differential effects of prazosin and rauwolsin on the release of prostaglandins I2 and E2 from the perfused mesenteric arterial bed of the rabbit following nerve stimulation

Sympathetic nerve stimulation of the perfused mesenteric arterial bed of the rabbit, , increase the secretion of prostaglandin (PG)I₂ and PGE₂. Prazosin (4.8 × 10⁻⁶), and α₁ adrenergic receptor antagonist, inhibited this inrease in release of PGI₂ but not of PGE₂ whereas rauwolsin (10⁻⁷ M), an α₂ adrenergic receptor antagonist, inhibited the increase in release of PGE₂ but not of PGI₂. Prazosin (10⁻⁶ M) completely blocked the vasoconstrictor response to nerve stimulation, and to norepinephrine and phenylephrine administration, suggesting there to be little of an α₂ adrenergic receptor component in this response. It is concluded that the increase in PGI₂ release follows the activation of α₁ adrenergic receptors and is therefore post-junctional in origin, whereas the increase in PGE₂ release follows the activation of α₂ adrenergic receptors and may be pre- and/or post-junctional in origin.Indomethacin (2.8 × 10⁻⁷, 5.6 × 10⁻⁷ and 1.12 × 10^{−6 M} did not affect the vasoconstrictor responses to nerve stimulation at 10 Hz, whereas rauwolsin (10⁻⁷ M) in the presence of indomethacin substantially increased them. These results indicate that PGE₂ does not regulate norepinephrine release following nerve stimulation at 10 Hz to rabbit mesenteric arteries, and that the inhibition of norepinephrine release following stimulation of α₂ pre-junctional receptors is independent of PG involvement.     

Simultaneous extraction and assay of cyclic nucleotides, prostaglandins, and DNA from cat alveolar bone

A method for the simultaneous extraction of cAMP, cGMP, PGE₂, PGF_2α, and DNA from a small sample of mineralized bone and the subsequent assay of these substances is described. Various solvents were tested for efficiency of extraction for the fatty acids, and water or 40% ethanol was found to extract more than 90% of labeled prostaglandin. In order to avoid enzymatic degradation, the substances were extracted at ?5°C requiring a solvent which would not freeze during extraction. Frozen alveolar cat bone samples were homogenized in 40% ethanol in the presence of 5 mm EDTA to inhibit phosphodiesterase. Small aliquots of the homogenate were withdrawn for the spectrofluorophotometric assay of DNA. After centrifugation, the supernatant was extracted first with petroleum ether, in order to take out neutral lipids, followed by ethyl acetate partition. The ethyl acetate layer was dired with N₂ gas, reconstituted with assay buffer, and assayed for PGE₂ and PGF_2α. A portion of the aqueous fraction was used for cAMP binding assay, while the rest was column chromatographed to elute the cGMP for radioassay. On the basis of per microgram of DNA, values for each of the following in cat alveolar bone were: 0.346 ± 0.049 pmol for cAMP, 0.026 ± 0.001 pmol for cGMP, 5.52 ± 1.46 pg for PGE₂, and 1.00 ± 0.29 pg for PGF_2α. Values calculated after the dilution of the sample aliquots or addition of standards to cAMP, cGMP, or PGE₂ showed no significant difference (P < 0.05) to their respective values. Within the limits of the sensitivity for each of the assay systems, it is feasible to measure cAMP, cGMP, PGE₂, and PGF_2α in alveolar bone from the same sample.     

Platelet activating factor intravenous infusion in rats stimulates vascular lysosomal hydrolase secretion independent of blood neutrophils

Intravenous infusion of platelet activating factor into rats at doses of 5-20 nmoles/kg results in a rapid and dose-dependent increase in the plasma level of several lysosomal hydrolases, notably glucosaminidase. This hydrolase secretion occurs concomitantly with the increased vascular permeability but subsequent to the neutropenia and hypotensive responses to platelet activating factor. The glucosaminidase release in vivo does not exhibit any lasting desensitization, does not require cytochalasin B, and is quantitatively the same in rats made neutropenic with anti-neutrophil serum, and thus is different from the published in vitro degranulating effect of this lipid with neutrophils. Therefore, lysosomal hydrolase secretion may be an important pathophysiologic response to very low blood levels of platelet activating factor.     

Rate sensitivity of blood pressure to hypoxia

A biochemical kinetic model is used to describe changes in mean arterial blood pressure in dogs to three different rates of fall of arterial partial pressure of oxygen. The model is a linear loop with one variable rate coefficient (parametric control) which has been previously shown to characterize the rate sensitivity to presented stimuli. A three component model was identified under a least squares criterion and it showed that a unique (stimulation independent) representation can be obtained which can serve as a conceptual framework for the study of this phenomenon.     

Rapid preparation of a relatively stable, partially purified lipoprotein lipase from perfused rat heart

Rat hearts, extensively washed with cold 0.15 M NaCl solution, were perfused with 5 ml of 0.15 M NaCl containing 16 U of heparin and 10% glycerol to release endothelium-bound lipoprotein lipase. Approximately 100 mU of enzyme activity could be released from each heart (weighing about 1.7 g). Several hearts could be sequentially perfused with the same heparin solution to enrich it in lipase activity. When compared with other equally rapid and frequently used sources of rat lipoprotein lipase (such as heart acetone powder or postheparin plasma), our enzyme preparation had a much higher specific activity suggesting that a greater purification level had been already achieved in a single step. In addition, this lipoprotein lipase preparation contained only trace amounts of lipids, was stable for an hour at 37 degrees C and retained 75% of its activity after 10 days at 4 degrees C. The described procedure is a quick way to prepare a soluble, partially purified and relatively stable lipoprotein lipase that may be useful especially for the in vitro preparation of triacylglycerol-rich lipoprotein remnants.     

Phospholipase A2 modulation by calmodulin, prostaglandins and cyclic nucleotides

Phospholipase A2 in the presence of Ca2+ was stimulated by calmodulin and by prostaglandin F2 alpha. Prostaglandin E2, cyclic-AMP and cyclic-GMP inhibited phospholipase A2 in the presence or absence of calmodulin. Dimethylsuberimidate cross-linking of phospholipase A2 with calmodulin was found to be Ca2+ dependent. These results indicate that phospholipase A2 is directly regulated by a host of key intracellular regulators and is one of the calmodulin-regulated enzymes.     

Possible involvement of cyclooxygenase products in the actions of platelet-activating factor and of lipoxygenase products in the vascular effects of epinephrine in perfused rat liver

Platelet-activating factor (PAF) stimulates glycogenolysis and induces vasoconstriction in perfused rat liver. The effect of PAF was rapid but transient and it was blocked by indomethacin and bromophenacyl bromide which suggests a role of cyclooxygenase metabolites in its action. The homologous desensitization of glycogenolysis produced by PAF and the sensitivity of its actions to inhibitors of cyclooxygenase and phospholipase A2 markedly differentiate the mechanism of action of this agent with that of alpha 1-adrenergic agents, vasopressin or angiotensin II. No effect of PAF in isolated hepatocytes was observed which suggest that cells other than hepatocytes could be involved in its action in perfused liver. In addition nordihydroguaiaretic acid and bromophenacyl bromide abolished the vascular effect (but not the glycogenolysis) produced by epinephrine which suggest a role for lipoxygenase products in this effect.     

Effects of anions on ADP-stimulated aggregation of bovine blood platelets

ADP-stimulated aggregation of bovine blood platelets was observed in media containing isotonic potassium salts of various monovalent anions. The aggregation depended on the anion in the medium, the order of aggregation being Cl^?, Br^?>I^?>SCN^?, $\text{ClO}{}_4{}^{\mathrm{?}}$. After 30-min incubation, the extent of aggregation of platelets in Cl^? or Br^? medium was little changed, whereas, that in SCN^? or $\text{ClO}{}_4{}^{\mathrm{?}}$ medium was remarkably decreased. This anion dependency of aggregation may be due to change in the membrane potential.     

Kinetic measurements of the release of prostaglandins from perfused rat lungs

Prostaglandins released from isolated, ventilated and perfused rat lungs were measured by a simple modification of the Vane technique using the rat stomach fundus as a continuous bioassay tissue. Exogenously supplied arachidonic acid was converted mainly to PGF₂ which was determined by bioassay. A novel method for mixing a stream of inhibitors with the perfusate was used to determine PGF₂ in the presence of substrate amounts of arachidonic acid. Using this system the apparent K_m for PGF₂ production with arachidonic acid as the substrate was found to be 1.90 × 10⁻⁴M, while the K_i for aspirin was found to be 2.47 × 10⁻⁴M. These kinetic parameters are close to those reported for cell free systems and subcellular fractions suggesting that both substrate and inhibitor have ready access to the site of prostaglandin synthesis. The method appears to be generally useful to determine the effect of drugs and environmental factors on the release of prostaglandins by the lung.     

Effects of several calcium antagonists and dipyridamole in the isolated perfused guinea pig heart

Three calcium (Ca) antagonists and dipyridamole were examined in the isolated perfused guinea pig heart at perfusate Ca concentrations of 1.25 and 3.75 mM. The Ca antagonists: FR 7534, nifedipine and D600 produced similar dose-dependent decreases in left ventricular dp/dt and myocardial oxygen consumption (MV?O₂) at both Ca concentrations. However, dose response curves were shifted significantly to the right by increased perfusate Ca requiring six to ten times more Ca antagonist to produce equivalent effects. Dipyridamole produced only slight negative inotropic effects which appeared to be less dependent on external Ca concentration. All four agents significantly increased coronary blood flow at 1.25 mM Ca but not at 3.75 mM Ca. The Ca antagonists decreased heart rate at 3.75 mM Ca whereas dipyridamole had strong negative chronotropic effects at both perfusate Ca concentrations. These experiments provide evidence that FR 7534 acts as a Ca antagonist. In addition, Ca antagonists of different structure had similar effects on the isolated heart distinct from those of dipyridamole.     

Observations on prostaglandins in normal and leukemic human lymphocytes

Prostaglandins E (PGE) and F₂ (PGF₂) were measured in lymphocytes of normal subjects, children with acute lymphocytic leukemia (ALL), and adults with chronic lymphocytic leukemia (CLL). In ALL lymphocytes PGE increased from a normal value of 25 pgrams to 270 pgrams/10⁶ cells, and PGF₂ increased from a normal value of 31 pgrams to 482 pgrams/10⁶ cells. In CLL lymphocytes, levels of PGE and PGF₂ were normal or low. When normal lymphocytes were stimulated with phytohemagglutinin (PHA), the level of PGE and PGF₂ fluctuated, followed by corresponding changes in the level of cyclic nucleotides. In cultured ALL lymphocytes, the level of PGE remained high, while cyclic 3′:5′-adenosine monophosphate (c-AMP) level was constantly low, and the initial high level of PGF₂ fluctuated in relation to similar oscillations of cyclic 3′:5′-guanosine monophosphate (c-GMP). These values were lower, although not significantly, when ALL lymphocytes were stimulated with PHA. When CLL lymphocytes were stimulated with PHA, the level of PGE remained low (20 pgrams), as did that of c-AMP. The level of PGF₂, after a brief initial increase (130 pgrams), returned to and remained at a lower level (60 pgrams) while the level of c-GMP was persistently high. These results suggest: (1) prostaglandins may indirectly influence the cell cycle, possibly through modulation of cyclase activity and levels of cyclic nucleotides; and (2) some derangement of this regulatory mechanism may be present in leukemic lymphocytes.     

Dipeptide metabolism in the isolated perfused rat kidney

Renal handling of glycyl-proline was studied in the isolated perfused rat kidney. Glycyl-proline disappeared from the perfusate as a function of time. The dipeptide was freely filtered at the glomerulus but only 6% of the filtered load was excreted in the urine as the intact peptide. More than 90% of the filtered dipeptide was reabsorbed as the intact peptide and/or its hydrolytic products. Non-filtration mechanisms were also involved to a significant extent in the clearance of the peptide. Hydrolysis at intratubular, intracellular and peritubular sites all contribute to the disappearance of the dipeptide from the perfusate, though the relative contributions of each mechanism are not known. Significant metabolic conversions, especially the conversion of glycine to serine, were also observed during perfusion.     

Effect of parathyroid hormone on Na+-dependent phosphate transport and cAMP-dependent 32P phosphorylation in brush border vesicles from isolated perfused canine kidneys

Concentrative uptake of 32Pi induced by the dissipation of a Na+ gradient (overshoot) was demonstrated in brush border membrane vesicles obtained from isolated perfused canine kidneys. Na+-dependent 32Pi transport was decreased in brush border vesicles from isolated kidneys perfused with parathyroid hormone (PTH) for 2 h compared to uptake measured in vesicles from kidneys perfused without PTH. Cyclic AMP-dependent 32P phosphorylation of a 62,000 Mr protein band was demonstrable on autoradiograms of sodium dodecyl sulfate-polyacrylamide gels of membrane suspensions from kidneys perfused +/- PTH. Evidence that perfusion with PTH resulted in cAMP-dependent phosphorylation in isolated kidneys from parathyroidectomized dogs (decreased cAMP-dependent 32P phosphorylation of the 62,000-Mr band in brush border vesicles) was obtained after 2-h perfusion with PTH. Decreased 32P phosphorylation was not observed if membranes were allowed to dephosphorylate prior to 32P phosphorylation in vitro. We conclude that brush border vesicles from isolated perfused canine kidneys can be used to study the action of PTH on Na+-Pi cotransport in brush border membranes and on cAMP-dependent phosphorylation of the membrane. It is strongly suggested that PTH effects changes in Na+-dependent 32Pi transport in isolated brush border vesicles and changes in 32P phosphorylation of vesicles via a direct action on the renal cortical cell rather than as a consequence of extrarenal actions of the hormone.     

Effects of hormones on the activity of glucose-6-phosphatase in primary cultures of rat hepatocytes

Although the activity of glucose-6-phosphatase in rat liver is altered markedly following the administration of a variety of hormones in vivo, it is not certain whether the hormones act directly on the hepatocyte. To study this problem hepatocytes were isolated by a collagenase-perfusion technique and cultured on collagen gel/nylon mesh membranes. The activity of glucose 6-phosphatase in cells cultured with fetal calf serum and with Dulbecco's modified Eagle's medium or Leibovitz L-15 medium decreased to less than 10-30% of the activity in freshly isolated cells by 96 h. However, when L-15 plus newborn or fetal calf serum was supplemented with glucagon (10(-6)M), epinephrine (10(-6)M), triiodothyronine (10(-6)M), and dexamethasone (10(-5)M) (L-15-GETD), the activity of glucose-6-phosphatase was maintained so that, after 144 h, the activity was at least 80% of that detected in freshly isolated cells. In cells cultured in L-15 plus serum for 72 or 96 h and then in L-15-GETD, glucose-6-phosphatase increased 30-50% over that in control cultures after 24 h. Insulin, which decreases glucose-6-phosphatase activity when administered to intact animals, also decreased the glucose-6-phosphatase activity in cultured hepatocytes to 20-50% of that in controls.