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 oleate,palmitate, and octanoate on gluconeogenesis in isolated rabbit liver cells

The effect of oleate, palmitate, and octanoate on glucose formation was studied with lactate or pyruvate as substrate. Octanoate was much more quickly oxidized and utilized for ketone body production than were oleate and palmitate. Among fatty acids studied, only octanoate resulted in a marked increase of the 3-hydroxybutyrate/acetoacetate (3-$\text{OHB}\text{AcAc}$) ratio. Each of the fatty acids studied stimulated glucose synthesis from pyruvate. The enhancement of gluconeogenesis by long-chain fatty acids was abolished after the addition of ammonia. As concluded from the “crossover” plot, the stimulatory effect of fatty acids was due to: (i) a stimulation of pyruvate carboxylation, (ii) a provision of reducing equivalents for glyceraldehyde phosphate dehydrogenase, and (iii) an acceleration of flux through hexose diphosphatase. Moreover, palmitate and oleate resulted in an increased generation of mitochondrial phosphpenolpyruvate, while in the presence of octanoate, the activity of mitochondrial phosphoenolpyruvate carboxykinase was diminished. When lactate was used as the glucose precursor, palmitate and oleate increased glucose production by about 50% but did not affect the contribution of mitochondrial phosphoenolpyruvate carboxykinase to gluconeogenesis. In contrast, in spite of the stimulation of both pyruvate carboxylase and hexose diphosphatase, as judged from the crossover plot, the addition of octanoate resulted in a marked inhibition of both glucose formation and mitochondrial generation of phosphoenolpyruvate. The inhibitory effect of octanoate was reversed by ammonia. Results indicate that fatty acids and ammonia are potent regulatory factors of both the rate of glucose formation and the contribution of mitochondrial phosphoenolpyruvate carboxykinase to gluconeogenesis in hepatocytes of the fasted rabbit.     

Effects of sulfhydryl reagents on phagocytosis and exocytosis in rabbit polymorphonuclear leukocytes

The effect of sulfhydryl reagents on phagocytosis and concomitant enzyme release and on ionophore A 23187 + Ca2+-induced exocytosis in rabbit polymorphonuclear leukocytes (PMN's) was studied. Membrane-penetrating sulfhydryl reagents such as cytochalasin A and N-naphthylmaleimide in micromolar concentrations inhibit both phagocytosis and exocytosis. Poorly penetrating reagents such as p-chloromercuribenzene sulfonate (pCMBS) and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), inhibit only in high concentrations (pCMBS), or they are ineffective as inhibitors (DTNB). Inhibition by pCMBS is not reversed by glutathione or dithiothreitol; this suggests that some pCMBS probably enters the cell. Specific intracellular sulfhydryl compounds appear to be essential in the cellular apparatus involved in phagocytosis and exocytosis; various possibilities are considered. A concentration of N-naphthylmaleimide which completely inhibits phagocytosis and exocytosis leaves cellular ATPase activity intact.     

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 cocunut 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 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 effects 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.     

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.     

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.     

Effects of tension and transmural stimulation on prostaglandin production by estrous rabbit oviductal isthmus

The effect of longitudinal and circular stretch on the amounts of Prostaglandin F (PGF) and Prostaglandin E (PGE) found in the fluid bathing rabbit oviductal isthmus has been investigated. It was found that the amounts of PGE nad PGF measured in the bathing fluid of longitudinally or circularly stretched tissues were negatively correlated to the maturity of the animal. Prostaglandin E increased with time in the tissues under longitudinal and circular tension. Prostaglandin F also increased with time under longitudinal tension but remained fairly constant under circular tension. Increasing the load from 0.5 to 2.0 g had no significant effect on PGE found under longitudinal or circular tension or on PGF found under longitudinal tension. Under circular tension, PGF found increased. Transmural stimulation at 20 Hz increased PGE 8-fold over control values while PGF increased only 1 to 3-fold. It is suggested that distension of the rabbit oviductal isthmus results in increased PGF production, which could be important in ovum transport.     

The effect of prostaglandins E1 and E2 on the human erythrocyte as monitored by spin labels

The effects of the prostaglandins PGE₁ and PGE₂ on the deformability of the human erythrocyte were studied using spin-labeled erythrocytes. Two magnetic resonance parameters were measured: (1) The orientation relaxation time, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, for the erythrocyte, and (2) the order parameter, S, for a fatty acid spin label bound to the membrane. Prostaglandins PGE₁ and PGE₂ exhibited opposite effects on both $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ and S. PGE₂ made the cell less deformable (increases of $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ and S) and PGE₁ made the erythrocyte more deformable (decrease of $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ and S).     

Effects of steroidal hormones on sexual, attack, and search behavior in the isolated male chick

Immature male chickens were treated with testosterone (1 mg/day), Δ₄-androstenedione (1 mg/day), 5α-dihydrotestosterone (5α-DHT; 1 mg/day), 5α-androstanedion (1 mg/day), or estradiol (100 μg/day) in order to assess the effects of these steroids on copulatory behavior, agonistic behavior, and attentional processes. Testosterone, estradiol, and 5α-DHT were most effective in stimulating male copulatory behavior above that of oil-treated controls; whereas Δ₄-androstenedione and 5α-androstanedione had less, but nevertheless significant, effects on this behavior. Testosterone and 5α-DHT facilitated agonistic behavior; however, estradiol, 5α-androstanedione, and Δ₄-androstenedione were ineffective in this capacity. The persistence of response to a given stimulus type was increased by testosterone and decreased by 5α-DHT: 5α-Androstanedione had no discernible effect on this behavior. These findings suggest that in the male chicken the neural structures regulating male copulatory and aggressive behavior as well as attentional processes are differentially sensitive to sex steroids. The effects of all these steroids on somatic structures were assessed.     

Isolation and characterization of the rabbit prt gene product

The product of the rabbit prt gene (PRT), a gene linked to the immunoglobulin κ-light chain gene ab, was purified from rabbit serum by precipitation with ammonium sulfate and by chromotography on DEAE-Sephadex and Sephacryl S300. Analysis of PRT indicated that it was associated rabbit hemopexin; the molecular weight of PRT (i.e., 68,000), as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was similar to the reported molecular weight of rabbit hemopexin; the PRT phenotypes correlated with the phenotypes of a hematin binding protein; PRT itself bound hematin; and the amino acid composition of PRT was similar to the amino acid composition of rabbit hemopexin. The prt gene, however, need not be the structural gene for hemopexin; it may encode a glycosyl transferase responsible in part for the carbohydrate associated with the protein.     

Effect of prostaglandins on ornithine decarboxylase activity in the testis of immature rat

Intratesticular injection of prostaglandin E₂(PGE₂) and F_2α (PGF_2α) caused stimulation of ornithine decarboxylase (ODC) activity in the testis of immature rats. PGE₂ at a dose of 10 μg per testis was maximally effective 2 hours after the injection. Dibutyryl cyclic AMP (cAMP) and 1 methyl, 3-isobutyl xanthine (MIX), a phosphodiesterase inhibitor, also stimulated ODC activity. Simultaneous injection of PGE₂ and FSH or LH caused additional stimulation of ODC activity. Similarly injection of PGE₂ in addition to cAMP or MIX also caused increased stimulation of ODC. Indomethacin (IM, 60 μg/testis) inhibited LH, FSH or cAMP induced ODC activity. However, IM at the same dose inhibited the synthesis of total proteins. These results suggest that PGE₂ and PGF_2α stimulate the activity of ODC. The action of prostaglandins may be independent of the action of gonadotropic hormones. cAMP appears to mediate the action of prostaglandins in the testis of rat.     

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.     

Two cytosolic,Ca2+-dependent,neutral proteinases from rabbit liver: Purification and properties of the proenzymes

Two Ca²⁺-requiring proteinases have been purified from rabbit liver cytosol and shown to be present in isolated hepatocytes. They differ in relative molecular mass, with the major and minor forms, M_r = 150,000 and M_r = 200, 000, accounting for 75 and 18% of the total cytosolic neutral proteinase activity, respectively. Both are recovered as inactive proenzymes that can be converted to the active, low-Ca²⁺-requiring proteinases by incubation with Ca²⁺ and substrate [S. Pontremoli, E. Melloni, F. Salamino, B. Sparatore, M. Michetti, and B. L. Horecker (1984) Proc. Natl. Acad. Sci. USA81, 53–56. Each proenzyme is composed of two subunits, with molecular masses of 80 and 100 kDa, respectively. Activation of the proenzymes was found to correlate with their dissociation into subunits. The optimum pH for conversion of the proenzymes to the active proteinases in the presence of 5 mm Ca²⁺ and 2 mg/ml of denatured globin was approximately 7.5, and the same pH optimum was observed for the digestion of denatured globin by the activated proteinases. Following activation, each proteinase was observed to undergo autolytic inactivation at rates that were dependent on the concentration of both Ca²⁺ and the digestible substrate. A model is proposed for the activation of the proenzymes and the subsequent inactivation of the active proteinases.     

LH biosynthesis and secretion in rat anterior pituitary cell cultures: stimulation of LH glycosylation and secretion by GNRH and an agonistic analogue and blockade by an antagonistic analogue.

In rat anterior pituitary cell cultures GnRH (1nM) stimulated a progressive increase in LH release into the medium from 1 to 8 h of incubation, while cellular LH showed a corresponding decrease. GnRH (1nM) neither modified the uptake nor the incorporation of [³H]-glucosamine and [³H]-proline into total protein. The incorporation of [³H]-proline into cellular LH also was unaffected by GnRH. In contrast, GnRH stimulated a 3 to 4-fold increase in [³H]-glucosamine incorporation into cellular LH. The agonistic analogue, [des GlyNH₂¹⁰]-LHRH ethylamide, mimicked the GnRH effects and was 5 to 6 times more potent than GnRH. The antagonistic analogue, [D-Phe², D-Phe⁶]-LHRH blocked the GnRH-stimulated effects. These results suggest that GnRH and agonistic analogues may preferentially regulate turnover or synthesis of the carbohydrate moiety of LH.     

Synthesis of prostaglandins by the ductus arteriosus of the bovine fetus

Previous studies demonstrated that prostaglandins are local or tissue hormones which can be released from blood vessel walls. In the present study, we investigated the capacity of bovine ductus arteriosus to synthetize prostaglandins . After incubation of slices of ductus arteriousus in Krebs' solution with (1-¹⁴C) arachidonic acid for 3 hours, more than 40% of the radiolabeled material recovered from the incubating medium were metabolites of arachidonic acid. The major product was indistinguisable from 6 keto-PGF_1α as determined by its chromatographic mobility and resistance to alkaline conversion to PGB.The PGI₂ synthetic capacity of the ductus arteriosus, as revealed by the predominance of its major metabolite 6 keto-PGF_1α, suggests that this metabolic pathway of arachidonic acid may contribute to the hemodynamic changes occurring during fetal life and at birth.     

Binding of monoclonal antibody to cathepsin M located on the external surface of rabbit lysosomes

A monoclonal antibody raised against rabbit liver cathepsin M binds to intact rabbit liver lysosomes. The binding is specific and is abolished by treating the lysosomes with trypsin, which has previously been shown to digest the membrane-bound cathepsin M [S. Pontremoli, E. Melloni, M. Michetti, F. Salamino, B. Sparatore, and B. L. Horecker (1982) Biochem, Biophys. Res. Commun. 106, 903-909]. Rabbit liver lysosomes are adsorbed onto Sepharose 4B coupled to anti-cathepsin M, but not to Sepharose 4B itself or to Sepharose coupled to a nonspecific antibody. The results confirm the location of membrane-bound cathepsin M on the outer surface of the lysosomal membrane.     

Isolation and characterization of minor glycosaminoglycans in the rabbit vitreous body

Protein synthesis in the microvascular system of the rabbit brain was inhibited following the elevation of body temperature by 2?2.5°C. $\text{In}$,$\text{vivo}$ labeling studies indicated that hyperthermia also induced the synthesis of a 74K protein in cerebral microvessels which is similar in molecular weight to one of the major heat shock proteins previously reported in tissue culture systems following elevation of ambient temperature. The present results suggest that a physiologically relevant increase in body temperature, similar to that attained during fever reactions, can induce the synthesis of a heat shock protein in the cerebral microvascular system of the intact mammal.     

Phorbol esters inhibit LH stimulated steroidogenesis by mouse Leydig cells in vitro

The effect of phorbol esters on the stimulation of testosterone production in response to LH was studied in mouse Leydig cells incubated in vitro. The tumor promoting phorbol esters, Phorbol-12-myristate-13-acetate and Phorbol-12-13-didecanoate at nanomolar concentrations effectively inhibited testosterone production by Leydig cells in response to stimulation by LH, whereas non-tumor promoting phorbol esters were ineffective. When the cells were stimulated by 8Br-cAMP, instead of LH, the testosterone production was stimulated similarly as in the presence of LH, but phorbol esters were without any effect. This suggests that the tumor promoting phorbol esters may act in the Leydig cells by suppressing the stimulation of cAMP production in response to hormonal activation and/or by interfering with the hormone-receptor interaction.     

Thymosin beta arg10, a major variant of thymosin beta 10 in rabbit tissues

Two homologous peptides, designated thymosin beta 4 and thymosin beta 10, respectively, have been shown to be widely distributed in mammalian cells and tissues (S. Erickson-Viitanen, S. Ruggieri, P. Natalini, and B.L. Horecker (1983) Arch. Biochem. Biophys. 221, 570-576; S. Erickson-Viitanen, S. Ruggieri, P. Natalini, and B.L. Horecker, (1983) Arch. Biochem. Biophys. 225, 407-413). In the rabbit, thymosin beta 4 is replaced by a variant, thymosin beta ala4, that contains alanine in place of serine at the blocked NH2-terminus. It is reported that in rabbit tissues thymosin beta 10 is also replaced by a variant, designated thymosin beta arg10, that contains an additional amino acid, arginine, inserted following lysine-38. The rabbit tissues analyzed also differ from those of other mammals in the relative quantities of thymosin beta ala4 and beta arg10, which are nearly equal, compared to tissues from other mammals where the quantities of thymosin beta 10 are only one-third to one-tenth those of thymosin beta 4.     

Metabolic behavior of acetyl glyceryl ether phosphorylcholine on interaction with rabbit platelets

The metabolic fate of 1-O-[³H]alkyl-2-acetyl-sn-glycero-3-phosphorylcholine ([³H]-AGEPC) upon interaction with rabbit platelets was investigated. [³H]AGEPC was converted to a product identified as the long-chain fatty acyl analog. The reaction was unaffected by extracellular calcium. After a lag time of 30 to 60 s the kinetics of the conversion was linear. The rate of the reaction was found to be a function of platelet and AGEPC concentrations. Of the [³H]AGEPC (10^?9m) 85 ± 5% was processed into the-long chain fatty acyl analog within 1 h when incubated at 37 2C with a 1.25 × 10⁹ platelets per milliliter suspension. A maximal number of 1200 to 3600 [³H]AGEPC molecules were converted to the long-chain fatty acyl derivative per minute per platelet in the presence of 2 mm EDTA. Under similar conditions the 1-O-[³H]alkyl-2-(lyso)-sn-glycero-3-phosphorylcholine ([³H]lysoGEPC) also was transformed to a comparable long-chain fatty acyl derivative at a much slower rate and to a lower extent. No significant increase in lysoGEPC was noted in incubation mixtures containing [³H]AGEPC. The possible direct transacylation of AGEPC upon interaction with platelets is discussed as well as the possible involvement of this reaction in directly triggering the platelet response to AGEPC stimuli.