Neurotensin-induced antinociception and hypothermia in mice: antagonism by TRH and structural analogs of TRH

Intracisternal (IC) administration of neurotensin (NT) in a dose of 10 micrograms produced a significant hypothermia and antinociception in the hot-plate test in mice. Both of these effects of IC NT were completely antagonized by concomitant administration of equimolar doses of thyrotropin-releasing hormone (TRH) and several TRH congeners including 3-methyl-His-TRH (pGlu-3-methyl-His-Pro-NH2), MK-771 (pyro-2-aminoadipyl-histidyl-thiazolidine-4-carboxamide), beta-ala-TRH (pGlu-His-Pro-beta-ala-NH2), and RX-77368 (pGlu-His-dimethyl-Pro-NH2). The antagonism by TRH and TRH analogs on NT-induced hypothermia and antinociception was dose-dependent. Of particular interest was the finding that RX-77368 not only blocked the effects of NT but also produced hyperalgesia. It appears that TRH analogs that are more resistant to biologic degradation are, like TRH, capable of blocking NT-induced behaviors.     

Stimulation of microsomal prostaglandin synthesis by a vasoactive material isolated from blood plasma

Stimulation of prostaglandin synthesis by a material with coronary vasoconstrictor activity extracted from blood plasma was examined. The vasoactive material decreased the Km for arachidonate in the overall synthesis of prostaglandins by rabbit renal microsomal preparations but did not change Vmax. Increases in prostaglandin synthesis caused by the vasoactive material and L-tryptophan or L-epinephrine were additive or synergistic, whereas increases produced by the vasoactive material and hemin or hemoglobin were not. However, hemin and hemoglobin stimulated synthesis of all prostaglandins equally whereas the active material increased the synthesis of prostaglandin F_2α at the expense of other prostaglandins, both in the presence and absence of heme compounds. The increase in prostaglandin F_2α with respect to the other prostaglandins occurred in the presence of reduced glutathione. The vasoactive material attenuated inhibition of prostaglandin synthesis induced by indomethacin or aspirin but not that produced by 5,8,11,14-eicosatetraynoic acid. The interaction of the vasoactive material and indomethacin was competitive whereas hemin attenuated the effects of only low concentrations of indomethacin. Epinephrine enhanced indomethacin inhibition. These data indicate that mode of action of the vasoactive material in prostaglandin synthesis is unlike that of glutathione, aromatic amines, or heme containing compounds.     

Lamb ductus arteriosus: Effect of prostaglandin synthesis inhibitors on the muscle tone and the response to prostaglandin E2

The prostaglandin synthesis inhibitors, indomethacin and eicosa-5,8,11, 14-tetraynoic acid (ETA), have been tested on the isolated lamb ductus arteriosus at low and high PO₂ levels. Both compounds produced a gradual contraction of the hypoxic vessel, and at equal doses the effect of indomethacin was stronger. The maximal tension output of the hypoxic tissue under indomethacin was equal to that of the oxygen-contracted control. ETA- and indomethacin-treated preparations contracted further upon transfer from a low to a high oxygen environment, and the response under indomethacin exceeded significantly control values. Control preparations were relaxed markedly by PGE₂ in low oxygen but showed little or no response in high oxygen. In contrast, preparations pretreated with the inhibitors retained their sensitivity to PGE₂ during exposure to high oxygen. The data are consistent with the idea that E-type prostaglandins play a role in the regulation of the intrinsic tone of the ductus arteriosus during foetal life. It is also suggested that the sensitivity of ductal muscle to E-type prostaglandins is controlled by the rate of endogenous prostaglandin formation.     

Differential sensitivity to neurotensin-induced hypothermia, but not analgesia, in Fischer and Lewis rats

Neurotensin (NT) produces behavioral and physiological effects, including analgesia and hypotheria, when administered into the CNS. Fischer and Lewis rats exhibit differential behavioral responses to central NT receptor activation. To further characterize these differences, we assessed central NT-induced analgesia and hypothermia in independent groups of rats from each strain. Fischer and Lewis rats showed a similar dose-orderly analgesic response in a hot-plate test. Such an isosensitivity was not observed for NT-induced hypothermia. Although NT produced a dose-orderly decrease in mean rectal temperature in both strains, the magnitude of the hypothermic response was significantly smaller in Fischer than in Lewis rats. These findings provide further evidence of genetic differences in central neurotensinergeric neurotransmission in these two strains.     

Alpha adrenergic stimulation modified prostaglandins release in vas deferens

Methoxamine enhanced contractions of mouse vasa deferentia through alpha adrenoceptor stimulation. Additionally, the generation of PGF-like material by the tissue was increased by methoxamine, whereas that of PGE-like material was decreased. The effects of methoxamine on prostaglandin output (stimulation and depression) were antagonized by yohimbine and phenoxybenzamine. Moreover, the stimulating influence of methoxamine on the contractile activity of the preparations was attenuated by the prostaglandin synthesis inhibitors indomethacin and acetylsalicylic acid. The results suggest that methoxamine is able to modify prostaglandin synthesis and output in mouse vasa deferentia, probably through an alpha adrenergic mediated mechanism coupled to the prostaglandin synthesizing system. The fact that indomethacin and acetylsalicylic acid blocked alpha stimulatory responses to methoxamine, suggest a modulatory role for endogenous prostaglandins. Since methoxamine increased PGF levels, this prostaglandin might potentiate the contractile influence of methoxamine.     

Block of oxytocin-induced parturition and oviposition by prostaglandin inhibitors

The possibility that oxytocin-induced labor is mediated, at least in part, by endogenous release of prostaglandins was investigated in two animal models using inhibitors of prostaglandin synthesis. Term pregnant rabbits failed to deliver after an i.v. oxytocin challenge (100 mU) when they were pretreated with an oral dose of indomethacin (10–25 mg) 45–60 minutes prior to oxytocin, while control rabbits began to deliver within a few minutes. Similarly, oxytocin- but not prostaglandin E₁-induced oviposition in the coturnix quail was inhibited by indomethacin. 5,8,11,14-Eicosatetraynoic acid, another inhibitor of prostaglandin synthesis, was also effective in blocking oxytocin-promoted oviposition. Based on these observations on two quite diverse species, it is suggested that prostaglandins may play a universal role in the expulsion of the uterine content.     

Biosynthesis of prostaglandin E by rat superior cervical ganglia.

Abstract— —The biosynthesis of immunoreactive prostaglandin E (iPGE) was examined in homogenates of rat superior cervical ganglia and in isolated intact ganglia incubated in vitro. Ganglia homogenates produced iPGE from exogenous arachidonic acid. Prostaglandin synthesis by the homogenates was inhibited by the prostaglandin synthetase inhibitors, eicosatetraynoic acid, indomethacin and sodium meclofenamate and was stimulated by norepinephrine and dopamine. Whole ganglia incubated in Krebs-bicarbonate solution also synthesized iPGE which was released into the incubation bath in a time-dependent manner. As observed in the homogenates, norepinephrine and dopamine enhanced iPGE formation by the intact tissue. Phospholipase A also stimulated iPGE synthesis by the whole ganglia. The effect of phospholipase A was antagonized by dibutyryl cyclic AMP but not by dibutyryl cyclic GMP. The results suggest that neuronally synthesized prostaglandins may be available for modulating adrenergic neuron function and that endogenous neuronal constituents such as catecholamines and cyclic AMP may influence the activity of the prostaglandin synthetase system.     

Possible role of prostaglandins as negative regulators in growth stimulation by tumor necrosis factor and epidermal growth factor in human fibroblasts

Recombinant tumor necrosis factor (TNF), epidermal growth factor (EGF), and transforming growth factor beta (TGF-beta) stimulated growth of confluent human diploid fibroblasts (FS-4 cells) in the presence of fetal calf serum. TGF-beta synergistically enhanced both the TNF- and EGF-stimulated cell growth, whereas synergism between the mitogenic action of EGF and that of TNF was not observed. When indomethacin or acetylsalicylic acid, an inhibitor of prostaglandin production, was added to FS-4 cells, cell growth stimulated by EGF or TNF was increased, suggesting that prostaglandins induced by these mitogens antagonize their growth stimulatory actions. In contrast, neither indomethacin nor acetylsalicylic acid had a significant effect on the TGF-beta-induced growth of FS-4 cells. Mitogenic responses of indomethacin-treated cells to EGF, TNF, and TGF-beta were similarly suppressed by the addition of exogenous prostaglandin D2 (PGD2). Other prostaglandins such as PGE2 and PGF2 produced less inhibition of the cell growth.     

Regulation of B cell tolerance by macrophage-derived mediators: antagonistic effects of prostaglandin E2 and interleukin 1

A role for prostaglandins in the mechanism of B cell tolerance induction in normal adult mouse spleen cells was examined. Two inhibitors of the cyclooxygenase pathway of arachidonic acid metabolism, indomethacin and acetylsalicylic acid, abrogated hapten-specific B cell tolerance induction by trinitrophenyl-human gamma-globulin. Tolerance was fully restored by the addition of prostaglandin E2 (PGE2) at a concentration of greater than or equal to 6 nM. T cell-depleted spleen cells produced comparable amounts of PGE2 in culture, indicating that the tolerance promoting activity of PGE2 occurred with physiologically relevant concentrations. Depletion and reconstitution experiments indicated that macrophages in the spleen cell preparations completely accounted for both PGE2 production and the effects of indomethacin and acetylsalicylic acid on B cell tolerance induction. The macrophage product interleukin 1 (IL 1) was also found to alter B cell susceptibility to tolerance induction. Thus, human IL 1 containing monocyte supernatants and purified IL 1 were found to interfere with B cell tolerance induction when added to macrophage- and T cell-depleted splenic B cells. Tolerance was restored in such cultures by the addition of 10 nM PGE2. These experiments demonstrate that within mixed lymphoid populations macrophages through the release of mediators modulate B cell susceptibility to tolerance induction.     

Enhancement of natural and antibody-dependent lymphocyte cytotoxicity by drugs which inhibit prostaglandin production by tumor target cells

Our previous observations suggested that the production of prostaglandins by tumor cells exposed to lymphocytes might constitute a mechanism by which the tumor cells Could subvert the effects of a cellular immune response directed against them. The present experiments tested this hypothesis by determining whether inhibition of prostaglandin production permitted enhanced expression of natural and antibody-dependent lymphocyte cytotoxicity against the target cells. Cell lines T₂₄ and HCV₂₉ were labelled with ⁵¹Chromium and incubated with purified lymphocytes obtained from venous blood of normal donors. Antiserum to T₂₄ and varying concentrations of inhibitors of prostaglandin synthetase (indomethacin, fenclozic acid, acetylsalicylic acid, and 2,6-xylenol) were added at the onset of incubation and assay tubes were incubated for varying times at 37 °C. In some experiments, lymphocytes or labeled target cells were preincubated with inhibitors and then washed prior to their addition to the assay tubes. Cytotoxicity was determined by measuring ⁵¹Chromium release and assessing any differences that might reflect the presence of the various drugs. Each prostaglandin synthetase inhibitor significantly enhanced both natural and antibody-dependent lymphocyte cytotoxicity. Enhancement appeared to reflect an effect on the target cells, presumeably by an inhibition of prostaglandin production. No increase in spontaneous ⁵¹Chromium release was apparent. The inhibitors did not appear to activate lymphocytes. This evidence supports the suggestion of a mechanism in which tumor cells may prevent the effect of a cellular immune response by producing inhibitory levels of prostaglandins. These results also suggest that manipulation of this mechanism can enhance the effectiveness of the lymphocyte response and may be a consideration in assessing lymphocyte/tumor cell interaction in vitro and in vivo.     

Regulation of prostaglandin synthesis during differentiation of cultured mouse myeloid leukemia cells

Mouse myeloid leukemia cells (Ml) were induced to differentiate into mature macrophages and granulocytes by various inducers. The differentiated Ml cells synthesized and released prostaglandins, whereas untreated Ml cells did not. When the cells were prelabelled with [¹⁴C]arachidonate, the major prostaglandins released into the culture media were found to be prostaglandin E₂, D₂, and F_2α in an early stage of differentiation, but the mature cells produced predominantly prostaglandin E₂. The synthesis and release of prostaglandins were completely inhibited by indomethacin. Dexamethasone, a potent inducer of differentiation of Ml cells, did not induce production of prostaglandins in resistant Ml cells that could not differentiate even with a high concentration of dexamethasone. These results suggest that production of prostaglandins in Ml cells is closely associated with differentiation of the cells. Homogenates of dexamethasone-treated Ml cells converted arachidonate to prostaglandins, but this conversion was scarcely observed with homogenates of untreated Ml cells. Dexamethasone and the other inducers stimulated the release of arachidonate from phospholipids. Therefore, induction of prostaglandin synthesis during differentiation of Ml cells may result from induction of prostaglandin synthesis activity and stimulation of the release of arachidonate from cellular lipids. Lysozyme activity, which is a typical biochemical marker of macrophages, was induced in Ml cells by prostaglandin E₂ or D₂ alone, as well as by inducers of differentiation of the cells, but it was not induced by arachidonate or prostaglandin F_2α. These results suggest that prostaglandin synthesis is important in differentiation of myeloid leukemia cells.     

Synergistic inhibitory effect of ascorbic acid and acetylsalicylic acid on prostaglandin E2 release in primary rat microglia

Ascorbic acid (vitamin C) has been suggested to protect cerebral tissue in a variety of pathophysiological situations such as head trauma, ischemia or Alzheimer's disease. Most of these protective actions have been attributed to the antioxidative capacity of ascorbic acid. Besides the presence of elevated levels of oxygen radicals, prostaglandins produced by neurones and microglial cells seem to play an important role in prolonged tissue damage. We investigated whether ascorbic acid alone inhibits prostaglandin E2 (PGE2) synthesis and may augment the inhibitory effect of acetylsalicylic acid on prostaglandin synthesis. Ascorbic acid dose-dependently inhibited PGE2 synthesis in lipopolysaccharide-treated primary rat microglial cells (IC50 = 3.70 micro m). In combination with acetylsalicylic acid (IC50 = 1.85 micro m), ascorbic acid augmented the inhibitory effect of acetylsalicylic acid on PGE2 synthesis (IC50 = 0.25 micro m in combination with 100 micro m ascorbic acid). Ascorbic acid alone or in combination with acetylsalicylic acid did not inhibit cyclooxygenase-2 (COX-2) protein synthesis but inhibited COX-2 enzyme activity. Our results show that ascorbic acid and acetylsalicylic acid act synergistically in inhibiting PGE2 synthesis, which may help to explain a possible protective effect of ascorbic acid in various brain diseases.     

Prostaglandins and skin tumor promotion: inhibition of tumor promoter-induced ornithine decarboxylase activity in epidermis by inhibitors of prostaglandin synthesis.

Application of 12-O-tetradecanoylphorbol-13-acetate to mouse skin led to a dramatic induction of epidermal ornithine decarboxylase (EC 4.1.1.17; L-ornithine carboxy-lyase) activity. The degree of induction was remarkably depressed by prior treatment of skin with indomethacin, acetylsalicylic acid or flufenamic acid, inhibitors of prostaglandin synthesis. In contrast, dexamethasone, a steroidal anti-inflammatory drug, was ineffective. The inhibition of tumor promoter-induced ornithine decarboxylase activity by the non-steroidal anti-inflammatory drugs was completely counteracted by treatment with prostaglandin E₁ and E₂ but not with prostaglandin F_1α or F_2α.     

Effects of prostaglandin antagonist SC-19220 on body temperature and on hyperthermic responses to prostaglandin E1 and leukocytic pyrogen in the cat

Intravenous injection of SC-19220 (3–9 mg/kg) caused dose-related hypothermic responses in cats. Repeated administration of SC-19220 resulted in tolerance to its hypothermic action. During SC-19220-induced hypothermia, the hyperthermic activity of both prostaglandin E₁ and leukocytic pyrogen was reduced or abolished. Neither prostaglandin E₁ nor leukocytic pyrogen was antagonized when given shortly after recovery from SC-19220-induced hypothermia or by doses of SC-19220 which did not cause hypothermia. Although these results may indicate a role of prostaglandins in normal physiological thermoregulation, it is also possible that production of hypothermia by SC-19220 is unrelated to prostaglandin antagonism.     

Effects of prostaglandin antagonist SC-19220 on body temperature and on hyperthermic responses to prostaglandin E-1 and leukocytic pyrogen in the cat.

Intravenous injection of SC-19220 (3-9 mg/kg) caused dose-related hypothermic responses in cats. Repeated administration of SC-19220 resulted in tolerance to its hypothermic action. During SC-19220-induced hypothermia, the hyperthermic activity of both prostaglandin E-1 and leukocytic pyrogen was reduced or abolished. Neither prostaglandin E-1 nor leukocytic pyrogen was antagonized when given shortly after recovery from SC-19220-induced hypothermia or by doses of SC-19220 which did not cause hypothermia. Although these results may indicate a role of prostaglandins in normal physiological thermoregulation, it is also possible that production of hypothermia by SC-19220 is unrelated to prostaglandin antagonism.     

A synthetic analog of prostaglandin E2 is able to induce in vivo theta antigen on spleen cells of adult thymectomized mice

The effect of prostaglandins on the in vivo induction of theta antigen in splenic spontaneous rosette-forming cells derived from adult thymectomized mice was studied. A long-acting synthetic analog of prostaglandin E₂, di-M-PGE₂, mimicked the effects of thymic hormone and was active when mice were treated with as little as 0.1 μg ip. In addition, indomethacin, a potent inhibitor of prostaglandin biosynthesis, was able to reverse the inductive effects of exogenous thymic hormone and inhibit the expression of theta antigen in normal mice, presumably by interfering with the effect of endogenous thymic factors. Finally, indomethacin also partially suppressed the stimulatory effects of exogenously administered di-M-PGE₂, suggesting that this agent is effective, at least in part, because it stimulates endogenous prostaglandin biosynthesis. Possible mechanisms of action for the effects of prostaglandins are presented.     

Effect of prostaglandin synthesis inhibitors on neurotensin and sodium salicylate-induced gastric cytoprotection in rats

Sodium salicylate (SA) has been reported to inhibit the formation of gastric ulcerations induced by aspirin, indomethacin, and absolute ethanol. In this study, SA dose-dependently inhibited gastric ulcers induced by three hours of cold-restraint stress (CRS); SA-induced cytoprotection was prevented by both acetylsalicylic acid (aspirin) and indomethacin pretreatment. Neurotensin (NT), which has previously been demonstrated to prevent the development of CRS-induced gastric ulcerations after intracisternal administration, was found to be ineffective in animals pre-treated with aspirin, and with indomethacin, as previously described. These data suggest that in the CRS model both NT- and SA-induced gastric cytoprotection require a functionally intact gastrointestinal prostaglandin synthetic pathway.     

Comparison of neurotensin levels, receptors and actions in LS/Ibg and SS/Ibg mice

Neurotensin (NT), injected centrally, markedly enhances sensitivity to ethanol-induced anesthesia in SS but not in LS mice (4). Since LS and SS mice were bred selectively for differential sensitivity to ethanol, these findings suggest that neurotensinergic neuronal processes mediate some of ethanol's actions and that LS and SS mice might differ genetically in neurotensinergic systems. Indeed, in biochemical studies it was shown that LS and SS mice differ in NT-like immunoreactivity in specific brain regions, i.e., hypothalamus, and in NT receptor densities (Bmax) in frontal cortex and striatum. In other experiments LS and SS mice differed in behavioral responses to centrally administered NT. Intracerebroventricular (ICV) administration of NT produced dose-dependent changes in motor activity, hypothermia, and analgesia in both LS and SS mice. SS mice appeared to be more sensitive than LS to NT-induced analgesia but not hypothermia. Neurotensin increased or decreased locomotor activity in both SS and LS mice following intraventral tegmental area or ICV administration, respectively. The results indicate that LS and SS mice, which were selectively bred for differences in ethanol sensitivity, differ genetically in NT concentrations, receptor densities in specific brain regions, and in some receptor-mediated behavioral responses to NT.     

Evidence of a cyclooxygenase-related prostaglandin synthesis in coral. The allene oxide pathway is not involved in prostaglandin biosynthesis

Certain corals are rich natural sources of prostaglandins, the metabolic origin of which has remained undefined. By analogy with the lipoxygenase/allene oxide synthase pathway to jasmonic acid in plants, the presence of (8R)-lipoxygenase and allene oxide synthase in the coral Plexaura homomalla suggested a potential metabolic route to prostaglandins (Brash, A. R., Baertshi, S. W., Ingram, C.D., and Harris, T. M. (1987) J. Biol. Chem. 262, 15829-15839). Other evidence, from the Arctic coral Gersemia fruticosa, has indicated a cyclooxygenase intermediate in the biosynthesis (Varvas, K., Koljak, R., J?rving, I., Pehk, T., and Samel, N. (1994) Tetrahedron Lett. 35, 8267-8270). In the present study, active preparations of G. fruticosa have been used to identify both types of arachidonic acid metabolism and specific inhibitors were used to establish the enzyme type involved in the prostaglandin biosynthesis. The synthesis of prostaglandins and (11R)-hydroxyeicosatetraenoic acid was inhibited by mammalian cyclooxygenase inhibitors (indomethacin, aspirin, and tolfenamic acid), while the formation of the products of the 8-lipoxygenase/allene oxide pathway was not affected or was increased. The specific cyclooxygenase-2 inhibitor, nimesulide, did not inhibit the synthesis of prostaglandins in coral. We conclude that coral uses two parallel routes for the initial oxidation of polyenoic acids: the cyclooxygenase route, which leads to optically active prostaglandins, and the lipoxygenase/allene oxide synthase metabolism, the role of which remains to be established. An enzyme related to mammalian cyclooxygenases is the key to prostaglandin synthesis in coral. Based on our inhibitor data, the catalytic site of this evolutionary early cyclooxygenase appears to differ significantly from both known mammalian cyclooxygenases.     

Ethacrynic acid induced release of prostaglandin E to increase renal blood flow

Ethacrynic acid administered to anesthetized dogs was found to increase the level of prostaglandin E as determined by radioimmunoassay in renal venous blood at the time when renal blood flow was increased by this agent. No change was found in the renal venous level of prostaglandin F. When ethacrynic acid was administered after treatment with indomethacin, which blocks the increase in renal blood flow induced by the natriuretic agent, no increase in the renal venous level of prostaglandin E was seen. Thus, the dilation of the renal vasculature would appear to be caused by a stimulation of synthesis and release of prostaglandin E by ethacrynic acid.