Correlation between the myotropic activity of leukotriene A4 on guinea-pig lung, trachea and ileum and its biotransformation in situ

Leukotrienes A4 and D4 displayed equivalent myotropic activity on guinea pig lung parenchyma strips. However, on the trachea, the activity of LTD4 was much higher than that of LTA4. The potencies of these two leukotrienes were also different on strips of longitudinal muscles of the ileum where LTD4 was very active whereas LTA4 was inactive. Since the activities of both leukotrienes were blocked by FPL-55712, our results suggested that the transformation of LTA4 by the smooth muscle preparations was a prerequisite to its biological activity. LTA4 was then incubated for 10 min with homogenates of guinea pig lung parenchyma, trachea and longitudinal muscles of ileum, and the metabolites were analysed by bioassay using strips of guinea pig ileum and lung parenchyma in a cascade superfusion system and also by reversed phase high performance liquid chromatography (RP-HPLC). Homogenates of lung parenchyma rapidly transformed LTA4 to LTB4, LTC4, LTD4 and LTE4. Incubation of LTA4 with homogenates of trachea or of the longitudinal muscles of ileum showed the formation of LTB4 and its isomers but no significant amount of peptido-leukotrienes were detected. These findings reveal that LTA4 undergoes distinctly different metabolic transformations in these tissues which correspond to the biological activities of the products recovered. These results strongly suggest that the myotropic activity and potency of LTA4 is related to the tissue levels of enzymes which catalyse its biotransformation.     

Evidence for negative feedback control of cholesterogenesis from mevalonate in liver: Absence in the intestine of guinea pigs fed A 0,5 % cholesterol diet

The in vitro rate of incorporation of [2-¹⁴C]-acetate and [2-¹⁴C]-mevalonate into cholesterol of liver, ileum and caecum was determined in guinea pigs. In control animals, contrary to the situation observed when acetate was used as precursor, the rate of conversion of mevalonate to cholesterol was higher in liver than in intestine. In this latter tissue, the cholesterogenesis varied depending on the portion tested. The distribution of radiolabel derived from mevalonate between esterified and unesterified cholesterol differed among the various tissues. In cholesterol-fed guinea pigs, the plasma, liver, intestine and aorta cholesterol contents increased significantly. In addition, a negative feedback control existed for hepatic cholesterol synthesis for mevalonate and acetate. This control was absent in intestinal tissues.     

Tonic activity of submucosal neurons influences basal ion transport

The influence of tonically active submucosal neurons on basal ion transport was studied using sheets of guinea pig ileum set up in flux chambers. Tetrodotoxin evoked an immediate and sustained decrease in short-circuit current that was sustained for 60 minutes compared with control tissues in which basal currents gradually decreased over time. Time-dependent changes in basal short-circuit currents in tissues treated with atropine were not significantly different from control tissues. The decrease in short-circuit current after tetrodotoxin resulted from a greater increase in net chloride absorption than sodium absorption. Changes in net sodium and chloride transport were due to an increase in the mucosal-to-serosal fluxes of these ions. The results suggest that tonic activity of submucosal neurons limits the absorptive capacity of the guinea pig ileum.     

Correlation between the myotropic activity of leukotriene A4 on guinea-pig lung, trachea and ileum and its biotransformation in situ

Leukotrienes A₄ and D₄ displayed equivalent myotropic activity on guinea pig lung parenchyma strips. However, on the trachea, the activity of LTD₄ was much higher than that of LTA₄. The potencies of these two leukotrienes were also different on strips of longitudinal muscles of the ileum where LTD₄ was very active whereas LTA₄ was inactive. Since the activities of both leukotrienes were blocked by FPL-55712, our results suggested that the transformation of LTA₄ by the smooth muscle preparations was a prerequisite to its biological activity. LTA₄ was then incubated for 10 min with homogenates of guinea pig lung parenchyma, trachea and longitudinal muscles of ileum, and the metabolites were analysed by bioassay using strips of guinea pig ileum and lung parenchyma in a cascade superfusion system and also by reversed phase high performance liquid chromatography (RP-HPLC). Homogenates of lung parenchyma rapidly transformed LTA₄ to LTB₄, LTC₄, LTD₄ and LTE₄. Incubation of LTA₄ with homogenates of trachea or of the longitudinal muscles of ileum showed the formation of LTB₄ and its isomers but no significant amount of peptido-leukotrienes were detected. These findings reveal that LTA₄ undergoes distinctly different metabolic transformations in these tissues which correspond to the biological activites of the products recovered. These results strongly suggest that the myotropic activity and potency of LTA₄ is related to the tissue levels of enzymes which catalyse its biotransformation.     

Release of leukotrienes from guinea pig lung stimulated by C5ades Arg anaphylatoxin

The complement anaphylatoxins C5a and C5Ades Arg contract guinea pig peripheral airway preparations and trachea by a mechanism largely independent of histamine release. In trachea the contractions are inhibited by FPL 55712, a relatively specific inhibitor of slow-reacting substance of anaphylaxis (SRS-A). SRS-A is now known to be a mixture of leukotrienes C4, D4, and E4 (LTC4, LTD4, LTE4). These data suggest that C5-derived anaphylatoxins stimulate production and release of leukotrienes in pulmonary tissues. To define these observations more precisely, fragments of guinea pig lung were incubated with porcine C5ades Arg, and the supernatant fluids were analyzed for leukotrienes by using both pharmacologic and chemical methods. In addition to histamine, a smooth muscle contracting activity characteristic of SRS-A was released from C5a-treated lung preparations. The contractile substance was identified as a leukotriene based on: 1) the characteristic contraction of guinea pig ileum, 2) inhibition of the contractile activity by FPL 55712, 3) enhanced release of activity in the presence of indomethacin or L-cysteine, 4) chromatographic behavior of ethanol-extracted active material on Amberlite XAD-7 resin, and 5) cochromatography of the active material on reverse-phase, high performance liquid chromatography with standard LTD4. We therefore concluded the humoral factor C5ades Arg induces a leukotriene release reaction in guinea pig lung tissue. This particular response of pulmonary tissue to anaphylatoxin has not been appreciated previously as an immediate effect of complement activation on the pathophysiology of the lung.     

Extrahepatic synthesis of apolipoprotein E

Apolipoprotein E (apoE) synthesis has been examined in rat and guinea pig tissues using in vitro translation and [35S]methionine labeling of tissue slices. A number of tissues not involved in lipoprotein synthesis synthesize a protein very similar to apoE, including the spleen, adrenal, kidney, testis, ovary, heart, and lung. Although the intestine is involved in lipoprotein synthesis, apoE synthesis could not be detected in intestinal mucosa. The protein synthesized by the extrahepatic tissues was identified as apoE by its electrophoretic mobility, its immunologic reactivity with a monospecific antibody and by limited proteolysis mapping with Staphylococcus aureus V8 protease. ApoE represented between 0.02 and 0.7% of the total protein synthesized in the extrahepatic tissues, indicating that apoE mRNA is a fairly abundant mRNA in these tissues. ApoE mRNA was also detected by hybridization with a rat apoE cDNA clone, which hybridized to a single mRNA 1250 nucleotides in length in rat liver and in extrahepatic tissues. Hybridization of the apoE clone to rat genomic DNA demonstrated that the apoE gene was more heavily methylated in intestinal mucosa, which did not synthesize apoE, than in liver, testis, or kidney. 35S labeling of peritoneal macrophages revealed that both rat and guinea pig macrophages synthesized and secreted apoE in vitro. Rhesus aortic smooth muscle cells also synthesized and secreted apoE. The possible functions of apoE synthesized in the peripheral tissues are considered.     

Existence of Three Subtypes of Bradykinin B2 Receptors in Guinea Pig

We describe the binding of [3H]bradykinin to homogenates of guinea pig brain, lung, and ileum. Analysis of [3H]bradykinin binding kinetics in guinea pig brain, lung, and ileum suggests the existence of two binding sites in each tissue. The finding of two binding sites for [3H]bradykinin in ileum, lung, and brain was further supported by Scatchard analysis of equilibrium binding in each tissue. [3H]Bradykinin binds to a high-affinity site in brain, lung, and ileum (KD = 70-200 pM), which constitutes approximately 20% of the bradykinin binding, and to a second, lower-affinity site (0.63-0.95 nM), which constitutes the remaining 80% of binding. Displacement studies with various bradykinin analogues led us to subdivide the high- and lower-affinity sites in each tissue and to suggest the existence of three subtypes of B2 receptors in the guinea pig, which we classify as B2a, B2b, and B2c. Binding of [3H]bradykinin is largely to a B2b receptor subtype, which constitutes the majority of binding in brain, lung, and ileum and represents the lower-affinity site in our binding studies. Receptor subtype B2c constitutes approximately 20% of binding sites in the brain and lung and is equivalent to the high-affinity site in brain and lung. We suggest that a third subtype of B2 receptor (high-affinity site in ileum), B2a, is found only in the ileum. All three subtypes of B2 receptors display a high affinity for bradykinin, whereas they show different affinities for various bradykinin analogues displaying agonist or antagonist activities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic nucleotide levels during carbachol-induced smooth muscle contractions.

Cyclic nucleotide levels and tension were measured at various times during carbachol-induced smooth muscle contractions. Cyclic GMP levels were markedly increased during contractions of rat vas deferens, guinea pig myometrium and guinea pig taenia coli, but were unchanged during contractions of rat uterus or guinea pig ileum. No significant changes in cyclic GMP levels could be detected in estrogen-primed rat uteri at any of the times or drug concentrations studied. Even in tissues in which large increases in cyclic GMP levels could be detected during carbachol-induced contractions (i.e. guinea pig myometrium and taenia coli) the contractions appeared to precede the cyclic GMP increases by several seconds. No significant changes in cyclic AMP levels were observed during carbachol-induced contractions in any of the smooth muscles studied. Thus, changes in tissue levels of the cyclic nucleotides do not appear to be responsible for the initiation of carbachol-induced smooth muscle contractions.     

Distribution in tissues and effects by acute hypoxia on angiotensin I converting enzyme activity in guinea pig and chicken

Angiotensin I converting enzyme activity was measured in homogenates of guinea pig and chicken organs (lung, kidney, heart, ileum, diaphragm and liver), using a spectrophotometric assay for hydrolysis of hippuryl-L-histidyl-L-leucine. High specific activities were found in lung, kidney and diaphragm, but the highest corresponded to guinea pig lung and chicken kidney. Acute hypoxia decreased angiotensin I converting enzyme activity in guinea pig lung and chicken diaphragm, but the changes in kidney were considered non-significant in both the guinea pig and chicken.     

Dietary regulation of maternal and fetal cholesterol metabolism in the guinea pig

Studies to determine the effects of pre-natal interventions on maternal and fetal cholesterol homeostasis were carried out in the guinea pig. Guinea pig dams were fed either non-purified guinea pig diet or diet supplemented with either 1.1% of the bile acid binding resin cholestyramine or 0.25% cholesterol. Whole body rates of endogenous cholesterol synthesis were determined by quantitation of [3H]water incorporation into digitonin precipitable sterols in non-pregnant animals and at 40 and 60 days of gestation in the dam and fetus. Maternal hepatic cholesterol synthesis was reduced 87% by dietary cholesterol and was increased 3.5-fold with cholestyramine feeding. Fetal hepatic and peripheral tissue cholesterol synthesis rates peaked at 40 days gestation when peripheral tissue cholesterol synthesis was 5.7-fold higher and hepatic synthesis 6.2-fold greater than the near adult levels observed at 60 days. Cholesterol synthesis in the fetus was relatively insensitive to dietary manipulations; however, maternal cholestyramine treatment did result in a 1.4-fold increase in fetal carcass cholesterol synthesis at 60 days gestation. These data demonstrate that maternal cholesterogenic systems maintain responsiveness to dietary regulation during pregnancy; whereas fetal cholesterol homeostasis is relatively insensitive to dietary cholesterol throughout gestation yet may respond to induction by maternal cholestyramine treatment during the late gestation period.     

Specificity of Ethylcholine Mustard Aziridinium as an Irreversible Inhibitor of Choline Transport in Cholinergic and Noncholinergic Tissue

The sensitivity of choline transport to inhibition by ethylcholine mustard aziridinium (ECMA) was studied in several tissues. Choline transport was found to be inhibited irreversibly by ECMA in guinea pig and rat synaptosomes but not inhibited in erythrocytes or kidney slices. If this finding can be extended to other tissues ECMA sensitivity may provide a simple criterion for identifying the choline carrier associated with cholinergic tissue.     

Hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesterol 7 alpha-hydroxylase activity in neonatal guinea pig.

Optimal assay conditions for hepatic HMG-CoA reducatase activity and cholesterol 7 alpha-hydroxylase activity in the guinea pig were determined. These two enzyme activities were studied in the liver of newborn guinea pigs during the first three postnatal weeks. Hepatic HMG-CoA reductase activity of neonatal guinea pigs was similar to that of adult animals. However, cholesterol 7 alpha-hydroxylase activity of newborns was about one-third of that in adult guinea pigs. This finding suggests that the system for bile acid synthesis in the neonatal guinea pigs is underdeveloped.     

Autoradiographic localization of binding sites for atrial natriuretic factor

The distribution of atrial natriuretic factor (ANF) binding sites in Wistar rat tissues, as well as tissues from other species was studied. Using autoradiography of slide mounted tissue sections incubated with 125I-labelled ANF, high densities of binding sites were found in the renal glomeruli and papilla, aortic smooth muscle, iliac vein, choroid plexus, anterior pituitary, lung, and adrenal zona glomerulosa. Results from renal, aortic, adrenal, and lung tissues from spontaneously hypertensive rats did not differ from those of Wistar rats. Binding sites were also observed in guinea pig glomeruli, renal medulla, and aorta as well as in the rabbit aorta.     

Selective inhibition of cholesterol synthesis in liver versus extrahepatic tissues by HMG-CoA reductase inhibitors

Hepatic specificity of inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase may be achieved by efficient first-pass liver extraction resulting in low circulating drug levels, as with lovastatin, or by lower cellular uptake in peripheral tissues, seen with pravastatin. BMY-21950 and its lactone form BMY-22089, new synthetic inhibitors of HMG-CoA reductase, were compared with the major reference agent lovastatin and with the synthetic inhibitor fluindostatin in several in vitro and in vivo models of potency and tissue selectivity. The kinetic mechanism and the potency of BMY-21950 as a competitive inhibitor of isolated HMG-CoA reductase were comparable to the reference agents. The inhibitory potency (cholesterol synthesis assayed by 3H2O or [14C]acetate incorporation) of BMY-21950 in rat hepatocytes (IC50 = 21 nM) and dog liver slices (IC50 = 23 nM) equalled or exceeded the potencies of the reference agents. Hepatic cholesterol synthesis in vivo in rats was effectively inhibited by BMY-21950 and its lactone form BMY-22089 (ED50 = 0.1 mg/kg p.o.), but oral doses (20 mg/kg) that suppressed liver synthesis by 83-95% inhibited sterol synthesis by only 17-24% in the ileum. In contrast, equivalent doses of lovastatin markedly inhibited cholesterol synthesis in both organs. In tissue slices from rat ileum, cell dispersions from testes, adrenal, and spleen, and in bovine ocular lens epithelial cells, BMY-21950 inhibited sterol synthesis weakly in vitro with IC50 values 76- and 188-times higher than in hepatocytes; similar effects were seen for BMY-22089. However, the IC50 ratios (tissue/hepatocyte) for lovastatin and fluindostatin were near unity in these models. Thus, BMY-21950 and BMY-22089 are the first potent synthetic HMG-CoA reductase inhibitors that possess a very high degree of liver selectivity based upon differential inhibition sensitivities in tissues. This cellular uptake-based property of hepatic specificity of BMY-21950 and BMY-22089, also manifest in pravastatin, is biochemically distinct from the pharmacodynamic-based disposition of lovastatin, which along with fluindostatin exhibited potent inhibition in all tissues that were exposed to it.     

产毒性大肠杆菌毒素在豚鼠肠道定位的免疫组织化学研究

研究利用免疫组织化学方法对产毒性大肠杆菌（ETEC）感染豚鼠小肠组织中ETEC肠毒素的定位进行了研究,本研究结果表明,发病豚鼠从空肠到回肠明显充血,肿胀,但盲肠,结肠和直肠外观与对照组差别不明显,光镜下见到发病动物肠组织病变主要出现在空肠和回肠,以回肠最为严重。主要病理改变为水肿,炎症细胞浸润和充血,病变部位可以出现在粘膜层,粘膜下层,肌层和浆膜层,取回肠组织切片作免疫组织化学显示ETEC不耐热肠毒素（LT）和耐热肠毒素（ST）、可见回肠粘膜表层,粘膜肌层,肌层及浆膜层均呈LT和ST阳性反应,分布弥漫,空白对照和正常豚鼠回肠组织均呈阴性结果。本研究表明,ETEC主要作用于空肠和回肠,尤其是回肠;回肠组织各层都有病变,且与肠毒素的分布一致,证明毒素的作用并不仅限于肠粘膜细胞。     

Depolarizing agent-induced cyclic AMP accumulation in isolated rat spinal cord

The stimulation of cyclic adenosine 3',5'-monophosphate (cyclic AMP) accumulation by the depolarizing agents K+, ouabain and veratridine, was studied in rat and guinea pig spinal cord tissue slices. Significantly increased accumulation of cyclic AMP was produced by each of the agents in a concentration-dependent manner. Veratridine and ouabain were equipotent (EC50 = 5 x 10(-5)M) and approximately 500 fold more potent than K+ (EC50 = 10(-2)M). Depolarizing agent-induced cyclic AMP accumulation in slices from guinea pig spinal cord was approximately double the response in rat spinal cord. Maximum stimulation occurred within 2.5 min of incubation with these agents and lasted for at least 30 min. Regional studies demonstrated that the maximal accumulation of cyclic AMP occurred to a greater degree in tissue slices from the dorsal section of spinal cord from both rat and guinea pig. Whereas the ouabain and veratridine stimulatory responses are completely dependent on extracellular Ca++, the K+ response is only partially dependent. Stimulation due to ouabain and veratridine is dependent, and K+ is independent, of release of neurohumoral substances such as norepinephrine or adenosine from spinal neurons. These experiments indicate the possible modulatory role of depolarization-linked events in regulating the spinal cord cyclic AMP system.     

Characterization and tissue distribution of H3 histamine receptors in guinea pigs by N alpha-methylhistamine

We have used [3H]N alpha-methylhistamine to characterize H3-binding in the guinea pig brain and to study its tissue distribution. Kinetic and equilibrium binding experiments indicate a single class of high affinity sites in membranes isolated from guinea pig brain tissue (Kd = 0.4 nM, Bmax = 41 fmol/mg of protein). Competition binding experiments have confirmed that this ligand associates with H3-receptors and, under the conditions used in these experiments, does not bind to H1- or H2-receptors. Although there was some binding in the ileum and large intestine, H3-binding was found primarily in the central nervous system.     

Peptide YY induces nerve-mediated responses in the guinea pig intestine.

The actions of peptide YY (PYY) were studied in longitudinal organ-bath preparations of the guinea pig intestine. PYY induced concentration-dependent (10(-9)-5 x 10(-8) M) relaxations of tissue from the duodenum, jejunum, ileum, and colon. These responses were unaffected by adrenergic blockade and atropine treatment but could be prevented by tetrodotoxin. The pharmacology of PYY actions in segments of the small and large intestine indicated the involvement of intrinsic nonadrenergic, noncholinergic inhibitory neurones in the relaxation response to this peptide. All tissues could be made tachyphylactic to PYY without affecting their ability to respond to the direct acting muscle relaxants ATP or papaverine. Moreover, nicotinic ganglion stimulated relaxations and cholinergic nerve-mediated contractions were also unaffected. These results show applied PYY to have potent neurogenic actions in the guinea pig intestine with some similarities to PYY actions in the rat intestine.     

Comparison of capsaicin and substance P induced cyclic AMP accumulation in spinal cord tissue slices

Capsaicin (CAP) caused a time, concentration and Ca++ dependent increase in cyclic AMP accumulation in tissue slices from rat and guinea pig spinal cord. The CAP-induced increase occurred uniquely in slices from dorsal cord of both rat and guinea pig and the increase was significantly greater in dorsal cord slices from guinea pig vs rat spinal cord. CAP mediated release of substance P does not appear to mediate the CAP-induced increase in cyclic AMP accumulation since the increase in cyclic AMP is significantly less with substance P and the substance P antagonist [D-Pro2, D-Trp7,9]-substance P does not antagonize the CAP-induced increase. The CAP-induced increase in cyclic AMP accumulation appears to be a direct effect. Structural requirements for this effect are both the substituted aromatic and alkyl side chain portion of the CAP molecule. The present results suggest that CAP has the ability to interact with sites in dorsal spinal cord which are linked to the synthesis of cyclic AMP, which could modulate spinal processing of nociceptive information.