Prostacyclin release and the modulation of some vasoactive hormones

Prostaglandin release into the circulation of the dog was studied by means of the blood-bathed bioassay system of Vane. Bradykinin, angiotensin II and angiotensin I selectively released a prostacyclin-like substance into the calculation, whereas no release was detected with adrenaline, noradrenaline, 5-hydroxytryptamine or acetylcholine. Release induced by bradykinin was mainly of renal origin, whereas that induced by the angiotensins was of more widespread origin, including the lungs and kidneys. No thromboxane A₂ or prostaglandin-like substance could be detected from blood by any of these stimuli. Prostacyclin released by bradykinin contributed to the vascular actions of the kinin as indicated by treatment with cyclo-oxygenase inhibitors. Prostacyclin release also contributed to a reduced pressor effect of angiotensin II. This study indicates that prostacyclin release induced by the vasoactive peptides modulates some of their vascular actions.     

Prostacyclin formation by myometrial & decidual fractions of the pregnant rat uterus

Chopped samples of myometrium, decidua and extrinsic blood vessels from the pregnant rat uterus when incubated at room temperature generated a prostacyclin-like substance. Activity in the incubation mixtures was compared against authentic prostacyclin in two assay systems: relaxation of strips of bovine coronary artery and inhibition of ADP-induced aggregation of rabbit platelet-rich plasma. Results estimated from inhibition of platelet aggregation showed that activity generated by all samples was low on day 12 of pregnancy (less than 0.25 ng/mg). However at the time of delivery (day 22) myometrial synthesis had increased 18.5 fold to over 3 ng/mg of prostacyclin whereas decidual production had only increased 5 times. As there was no increase in synthesis by the extrinsic uterine blood vessels over this period it is proposed that the myometrial muscle cells are the probable source of the prostacyclin-like material.     

Prostacyclin formation by myometrial & decidual fractions of the pregnant rat uterus.

Chopped samples of myometrium, decidua and extrinsic blood vessels from the pregnant rat uterus when incubated at room temperature generated a prostacyclin-like substance. Activity in the incubation mixtures was compared against authentic prostacyclin in two assay systems: relaxation of strips of bovine coronary artery and inhibition of ADP-induced aggregation of rabbit platelet-rich plasma. Results estimated from inhibition of platelet aggregation showed that activity generated by all samples was low on day 12 of pregnancy (less than 0.25 ng/mg). However at the time of delivery (day 22) myometrial synthesis had increased 18.5 fold to over 3 ng/mg of prostacyclin whereas decidual production had only increased 5 times. As there was no increase in synthesis by the extrinsic uterine blood vessels over this period it is proposed that the myometrial muscle cells are the probable source of the prostacyclin-like material.     

Prostacyclin producing activity of human umbilical, placental and uterine vessels

Prostacyclin-like material producing activity of umbilical, placental and uterine vessels was studied.Umbilical arteries and veins were separated at sites 10–15 cm and 1–2 cm from insertion of the umbilical cord to the placenta. Placental arteries and veins were prepared from the first, second and third branches on the chorionic plate. Uterine vessels were obtained at abdominal hysterectomy.After incubation of each specimen in Tris buffer 1 ml (pH8.5, 0.5M) for 30 min at room temperature, the inhibitory effect of the medium on ADP induced platelet aggregation was measured and the prostacyclin-like material was quantified. These procedures were repeated consecutively four times in total for each specimen. Prostacyclin-like material production rate and its total production were calculated.In total prostacyclin-like material production, umbilical arteries and veins were much higher than placental arteries and veins respectively (p<0.001), but there was no significant difference between placental and uterine vessels.These results showed that prostacyclin-like material producing activity of blood vessels declined remarkably at the transitive region from umbilical to placental vessels. It seems that this distribution of vascular prostacyclin-like material producibility in the fetoplacental vascular system correlates with that of vascular reactivity to prostacyclin.     

Prostacyclin producing activity of human umbilical, placental and uterine vessels

Prostacyclin-like material producing activity of umbilical, placental and uterine vessels was studied. Umbilical arteries and veins were separated at sites 10-15cm and 1-2cm from insertion of the umbilical cord to the placenta. Placental arteries and veins were prepared from the first, second and third branches on the chorionic plate. Uterine vessels were obtained at abdominal hysterectomy. After incubation of each specimen in Tris buffer 1 ml (pH8.5, 0.5M) for 30 min at room temperature, the inhibitory effect of the medium on ADP induced platelet aggregation was measured and the prostacyclin-like material was quantified. These procedures were repeated consecutively four times in total for each specimen. Prostacyclin-like material production rate and its total production were calculated. In total prostacyclin-like material production, umbilical arteries and veins were much higher than placental arteries and veins respectively (p less than 0.001), but there was no significant difference between placental and uterine vessels. These results showed that prostacyclin-like material producing activity of blood vessels declined remarkably at the transitive region from umbilical to placental vessels. It seems that this distribution of vascular prostacyclin-like material producibility in the fetoplacental vascular system correlates with that of vascular reactivity to prostacyclin.     

Ovarian hormones inhibit the release of prostacyclin-like material from isolated rat uterus

The influence of sex steroids on the production of prostacyclin (PGI₂) like material by the isolated rat uterus incubated in a buffer medium was explored by monitoring its ability to inhibit ADP-induced platelet aggregation. Chopped uterine strips from rats in natural estrus can generate an unstable substance that inhibits platelet aggregation and suggest to be prostacyclin. This capacity was significantly enhanced in preparations from spayed animals. The injection of 17-beta estradiol; progesterone or both diminished the production of the prostacyclin-like material by the uterus from ovariectomized rats. The already existing notion that ovarian steroids are able to regulate the synthesis of stable prostaglandins is discussed together with the present results suggesting in addition a depressive effect of sex hormones on the uterine PGI₂ synthetase system.     

Generation of a leukotriene-like substance from porcine vascular and other tissues

When chopped porcine vascular tissue was incubated with A23187 in the presence of a cyclo-oxygenase inhibitor, a substance with the biological properties of a leukotriene was released into the supernatant. The highest concentrations of leukotriene-like material were released by coronary and pulmonary arteries and the adventitia removed from these vessels, the greatest amount being released by the adventitia. Smaller quantities of leukotriene-like substance were released from other blood vessels but not from aorta, atrial or ventricular smooth muscle. Release of the leukotriene-like material was time-dependent. The activity of the leukotriene-like substance was antagonised by FPL 55712 and its release was inhibited by BW 755c. Leukotriene-like material was also generated from guinea-pig, rat, rabbit and dog blood vessels incubated with A23187 and from guinea-pig vessels during antigen challenge.     

Generation of a leukotriene-like substance from porcine vascular and other tissues

When chopped porcine vascular tissue was incubated with A23187 in the presence of a cyclo-oxygenase inhibitor, a substance with the biological properties of a leukotriene was released into the supernatant. The highest concentrations of leukotriene-like material were released by coronary and pulmonary arteries and the adventitia removed from these vessels, the greatest amount being released by the adventitia. Smaller quantities of leukotriene-like substance were released from other blood vessels but not from aorta, atrial or venticular smooth muscle. Release of the leukotriene-like material was time-dependent. The activity of the leukotriene-like substance was antagonised by FPL 55712 and its release was inhibited by BW 755c. Leukotriene-like material was also generated from guinea-pig, rat, rabbit and dog blood vessels incubated with A23187 and from guinea-pig vessels during antigen challenge.     

Enzymatically activated microencapsulated liposomes can provide pulsatile drug release

A system for the delayed or pulsed release of biologically active substances was achieved by encapsulating liposomes containing the substance of interest inside microcapsules. The microcapsules retain the liposomes but allow controlled diffusion of the active substance when it is released from the liposomes. Furthermore, by coating the liposomes with phospholipase A2 (an enzyme that removes an acyl group from the 2 position of phospholipids) before placing them within the microcapsule, a pulsatile release pattern was achieved both in vitro and in vivo. The time of onset of the pulse as well as the release rate can be controlled by the amount of phospholipase A2, the molecular weight of the poly(L-lysine) that is used to coat the microencapsulated liposomes, and the composition of the phospholipid bilayer membrane. Even at 37 degrees C the system would protect a model enzyme (horseradish peroxidase). When not placed inside the microencapsulated liposomes, the enzyme lost its activity in solution at 37 degrees C in a few days, whereas it retained 40% of the initial activity after 30 days of incubation at 37 degrees C inside the microencapsulated liposomes.     

Effect of dilute solutions of biologically active substances on cell membranes

The article presents data on changes in physicochemical properties of different biological membranes (plasmatic, microsomal, synaptosomes) under the action of biologically active substances, which are different in their chemical structure and the mechanism of action (natural and synthetic antioxidants, thyrotropin - releasing hormone, phorbol esters), in the wide range of concentrations (10^?22?10^?3 M). Dose dependences of the effect of biologically active substances on the activity of membrane-bound enzymes, lipid peroxidation, the structural state of the various regions of the lipid bilayer of membranes have been obtained and analyzed in terms of their formal generality of polymodality, number and position of the maxima, a sign change of the effect. An attempt to explain the mechanism of each of the observed peaks in these curves has been made. The maximum in the range of relatively high “physiological” concentrations (10^?3–10^?7 M) is associated with introduction of biologically active substances into biomembranes. In this study maxima in the range of ultra-low doses (10^?11–10^?16 M) and “apparent” concentrations (10^?18 M), where the presence of biologically active substance molecule in a reaction volume is probabilistic in nature, are explained by physicochemical properties of diluted biologically active substances solutions. This conclusion is based on our data on the changes in IR spectra of aqueous solutions of biologically active substances and the results obtained by academician A.I. Konovalov et al. concerning the physicochemical properties of dilute solutions of biologically active substances (conductivity, surface tension, charge), due to the formation of so-called “nanoassociates” from biologically active substance molecule and numerous number of water molecules. The nanoassociates formation and biological effect disappear if the low concentration solutions are kept in a special shielded permalloy container protecting its contents from external electromagnetic field. Thus, nanoassociates are the material carriers of the unique ability of the ultra-low doses of biologically active substances to exhibit biological effects.     

Specific binding of an immunoreactive and biologically active 125I-labeled N(1)acylated substance P derivative to parotid cells

A biologically active ¹²⁵I-substance P derivative (I¹²⁵-BH-substance P), prepared by conjugation of substance P with [^125I]Bolton-Hunter reagent, binds specifically to isolated rat parotid cells. The Kd is 4 nM for I-BH-substance P, 5 nM for substance P, 0.18 μM for substance P octa(4–11)peptide, and 1.6 μM for substance P [pyroglutamyl⁶]hexa(6–11)peptide. Substance P free acid and substance P penta(7–11)peptide are much weaker competitors and the C-terminal tri(9–11)peptide has no effect at 30 μM. The binding is also inhibited by 1 μM physalaemin, eledoisin and substance P methyl ester, but not by unrelated peptides. The selective inhibition of the binding by the biologically active analogs and fragments of substance P indicates that the ¹²⁵I-labeled N(1)acylated substance P derivative may interact with a substance P receptor on parotid cells.     

Arachidonic acid strongly stimulates prostaglandin I3 (PGI3) production from eicosapentaenoic acid in human endothelial cells

Eicosapentaenoic acid (EPA) is a prominent polyunsaturated fatty acid in fish oil which inhibits blood platelet aggregation and thromboxane A2 formation but not prostacyclin-like material generation from vascular endothelium. In this study we investigated interaction between EPA and arachidonic acid (AA) during their oxygenation by cultured endothelial cells. As measured by gas chromatography-mass spectrometry (GC-MS), AA increased markedly prostaglandin I3 (PGI3) production from EPA while that of PGI2 from AA was decreased by EPA. However, increasing the ratio AA/EPA over one almost suppressed the inhibition of PGI2 formation by EPA, and the stimulation of PGI3 production by AA was even higher. The effect of AA on EPA conversion to minor prostaglandins like PGE3 and PGF3 alpha was similar then confirming the stimulating effect and suggesting it is occurring at the cyclooxygenase instead of the prostacyclin synthase level. Altogether these data indicate that, in certain nutritional states where the liberation of EPA from endothelial cells will be accompanied with that of endogenous AA, substantial amounts of PGI3 could contribute to the prostacyclin-like activity of the vessel wall in addition to PGI2.     

Chemical mediator of insulin action stimulates lipid synthesis and down regulates the insulin receptors in primary cultures of rat hepatocytes

Incubation of rat liver particulate fraction with insulin causes the release of a substance that stimulates lipid synthesis and down regulates the insulin receptor in primary cultures of isolated rat hepatocytes. This substance may be similar to putative mediator(s) of insulin action which has been shown to modulate the activity of key enzymes of lipid and carbohydrate metabolism in various cell free systems. Our data demonstrate that the mediator of insulin is also biologically active in an intact cell system. Down regulation of the insulin receptor by the mediator supports the concept that this phenomenon is a post binding event of insulin action.     

Activities of enzymes that metabolize platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in neutrophils and eosinophils from humans and the effect of a calcium ionophore

Enzymatic systems in human blood cells are described for the activation and inactivation of a biologically active phospholipid (1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine) with hypotensive, platelet-aggregating, and inflammatory properties. The results document the presence of alkyldihydroxyacetone-phosphate synthase (forms the $\text{O}$-alkyl linkage in lipids), 1-alkyl-2-lyso-$\text{sn}$-glycero-3-phosphocholine:acetyl-CoA acetyltransferase (produces the biologically active molecule), and 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine: acetylhydrolase (destroys the biological activity) in human neutrophils and eosinophils. Both the acetyltransferase and acetylhydrolase activities are increased severalfold after treatment of normal neutrophils with ionophore A23187; however, alkyldihydroxyacetone-phosphate synthase activity is not influenced by the ionophore. Eosinophils isolated from patients with eosinophilia have significantly greater activities of all the enzymes studied than the eosinophils isolated from normal individuals. Our results indicate the acetyltransferase responsible for 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine synthesis may serve an important role in human blood cells that release this biologically active phospholipid. Moreover, the acetyltransferase activity was found to be dramatically influenced by calcium flux.     

Modulation of lipopolysaccharide-induced production of tumor necrosis factor, interleukin 1, and interleukin 6 by synthetic precursor Ia of lipid A

Abstract Endotoxin (lipopolysaccharide, LPS) induces the production of mediators of inflammation, which exerts pathophysiological effects such as fever or shock in mammals. In the present study we have investigated the modulation of LPS by the synthetic non-active tetraacylated precursor Ia of lipid A (compound 406) in the induction of tumor necrosis factor (TNF), interleukin 1 (IL-1) and interleukin 6 (IL-6) in human peripheral blood mononuclear cells (PBMC) and in human peripheral blood monocytes (PBMo). PBMC stimulated with LPS released TNF in a concentration dependent manner. Release of biologically active TNF, IL-1 and IL-6 was first detectable 4 h after LPS stimulation. Compound 406 alone in all concentrations tested did not induce TNF, IL-1 or IL-6 release, intracellular TNF or IL-1β, or mRNA for TNF or IL-1. Added to PBMC 1 h before LPS compound 406 enhanced or suppressed TNF release and suppressed IL-1 and IL-6 release depending on the ratio of concentrations between stimulator (LPS) and modulator (compound 406). In contrast to LPS stimulation alone TNF, IL-1 and IL-6 release in presence of compound 406 was delayed and first detectable after 6 to 8 h. Compound 406 was able to suppress LPS-induced intracellular TNF and IL-1β in PBMC. Added to PBMo 1 h before LPS it totally inhibited the production of mRNA for TNF and IL-1. When added to PBMC 1 h after LPS, TNF release was suppressed in a concentration-dependent way and release of biologically active TNF, IL-1 and IL-6 could again be detected for the first time after 4 h. Compound 406 was not able to inhibit phorbol 12-myristate 13-acetate (PMA)-induced TNF and IL-1 release in PBMo which suggests that its modulating effect is LPS-specific. This study provides evidence that the modulating effect of compound 406 on the LPS induction of TNF, IL-, 1 and IL-6 could be due to competitive binding.     

Total synthesis of hypothalamic "somatostatin"

The synthesis of the tetradecapeptide somatostatin (somatotropin release inhibiting factor, SRIF) by conventional methods of peptide synthesis is described. The synthetic material was found to be biologically active at the pico-gram/ml dose level.     

Neurotensin elevates hematocrit and plasma levels of the leukotrienes, LTB4, LTC4, LTD4 and LTE4, in anesthetized rats.

The IV injection of neurotensin (NT) into anesthetized rats produced a marked increase in hematocrit, labored breathing and peripheral blood stasis with cyanosis. This effect could also be produced by the NT-related peptides, neuromedin-N and xenopsin; however, it was not observed when nine other biologically active peptides, including bradykinin and substance P, were tested. Associated with these responses were increases in the plasma levels of histamine (measured radioenzymatically) and the leukotrienes, LTB4, LTC4, LTD4, and LTE4 (measured by RIA and HPLC). The increment in hematocrit after varying doses of NT correlated to the increase in plasma levels of LTC4. Histamine and LTC4 were both capable of elevating hematocrit when given IV; however, LTC4 was approximately 1000 times more potent than histamine and active doses of histamine elevated LTC4 levels. Furthermore, the effects of NT on plasma LTC4 and hematocrit were reduced by pretreating animals with antagonists to histamine and serotonin. Pretreatment with the specific mast cell degranulating agent, compound 48/80, also blocked NT's ability to elevate plasma levels of histamine, LTB4 and LTC4 and prevented the increased hematocrit and cyanosis. These results indicate that NT-related peptides are very potent and specific stimulators of leukotriene release and that this action is mediated by mast cells and associated with loss of plasma volume and blood stasis. A working hypothesis is that histamine, released from mast cells in response to NT, stimulates LTC4 production by other cells.     

Purification and properties of blood group A-active glycoprotein from oyster viscera

A blood group A active substance was isolated from an acetone-dried powder of oyster viscera by extraction with 0.1 M NaCl after heating a homogenate with extraction medium, in boiling water. After the removal of the acidic fraction with cetylpyridinium chloride, the separated neutral fraction was digested successively with α-amylase and amyloglucosidase to remove glycogen. The blood group A-active portion was eluted from a Sepharosé 4B column and purified by DEAE-Sephadex column chromatography. The purified active substance was homogeneous by polyacrylamide gel electrophoresis, and its molecular weight was estimated as 100 000 by sedimentation equilibrium.The sugar content of the purified active substance, expressed in percentage of dry weight, was galactosamine, 16.6; galactose, 12.8; fucose, 9.9; glucosamine, 4.6; and glucose, 3.3. Sialic acid was not detected. Total amino acid content was 23.0% and the main constituents were threonine, proline and serine. The ORD spectrum indicated that the hexosamines were N-acetylated. Absence of glycolipid was confirmed by the analysis of fatty acid and sphingosine base.This active substance had a strong blood group A activity (0.04 μg/ml) but neither B nor H activity; it interacted with lima bean lectin but not with concanavalian. A.     

Cloning and sequence analysis of zooA,a Streptococcus zooepidemicus gene encoding a bacteriocin-like inhibitory substance having a domain structure similar to that of lysostaphin

The nucleotide sequence has been determined for zooA, a gene encoding the bacteriocin-like inhibitory substance zoocin A in Streptococcus zooepidemicus strain 4881. The zooA gene product corresponds to the 285-amino acid (aa) zoocin A pre-peptide from which a leader sequence is cleaved to form the 262-aa biologically active molecule of estimated molecular mass 27 877 Da. Expression of zooA in a Gram-negative host was shown by the extracellular release from Escherichia coli, containing cloned zooA, of a biologically active peptide having an identical range of anti-bacterial activity to that of zoocin A, purified from S. zooepidemicus strain 4881. Data base searches revealed sequences having homologies with known muralytic proteins produced by both Gram-positive and Gram-negative bacteria and indicate a `mix and match' blending of domain-type structures, the C-terminal putative receptor-recognition region of the molecule being joined by a threonine-proline-rich linker to an N-terminal putative catalytic region having homology with several known endopeptidases, including lysostaphin.     

Effects of prostacyclin analogs on cyclic adenosine monophosphate and superoxide formation in human polymorphonuclear leukocytes stimulated by formyl-methionyl-leucyl-phenylalanine

The modulation of cyclic AMP levels and superoxide release in isolated FMLP-stimulated human PMN by two oxacyclic analogs and one carbacyclic analog of PGI2 and by PGE1 is investigated over a wide range of concentrations of the test compounds. The prostacyclin analogs only marginally increase the cyclic AMP levels in unstimulated PMN but like PGE1 potentiate the FMLP-induced rise in cyclic AMP. The concentration dependency is bell-shaped with a maximum effect at about 10 microM of the prostanoids. In contrast, all prostanoids dose-dependently inhibit FMLP-induced superoxide release almost to completion. The relative inhibitory potency of the prostacyclin analogs corresponds to their prostacyclin-like action in other systems. It is therefore suggested that PMN contain prostacyclin receptors, which, however, have weaker affinities than those in platelets. The lack of correlation between inhibition of superoxide formation and modulation of the cyclic AMP system rules out the possibility that cyclic AMP can simply be considered the second messenger of prostacyclins in PMN. The potential biological relevance of the effects of prostacyclin-like compounds on PMN functions is discussed.