Apolipoprotein E is a putative autocrine regulator of the rat ovarian theca cell compartment.

Apolipoprotein (apo) E inhibits androgen production by ovarian theca cells. We found that apo E, as a synthetic peptide mimicked the full-size protein, induced theca and interstitial cell (TIC) apoptosis indicated by pyknotic cell morphology, increased DNA end-labeling (TUNEL), and DNA ladders. None of the low-density lipoprotein (LDL) receptor superfamily members were involved because the universal antagonist of these receptors, receptor-associated protein (RAP), did not block apo E-induced apoptosis. Furthermore, several apo E synthetic peptides that do not bind the LDL receptor did induce TIC apoptosis. Similar to apo E, apoptogenic agents such as ceramide and LY 294002, a phosphatidylinositol (PI) 3-kinase inhibitor, induced apoptosis and suppressed androstenedione production. However, apoptosis alone was not responsible for apo E suppression of androstenedione production because both insulin and IGF-I prevented apo E-induced apoptosis, but neither restored androstenedione production. Theca cells of atretic follicles express the greatest apo E mRNA, and here we show that cultured TIC produce apo E. When considered with the observation of TUNEL-positive theca cells in atretic follicles these results support our hypothesis that intraovarian apo E controls theca cell production of androgen as well as limiting the size of the theca cell compartment.

Effect of allelic polymorphism of the B(1) and B(2) receptor genes on the contractile responses of the human umbilical vein to kinins.

The human genes corresponding to the two receptor (R) subtypes for bradykinin (BK)-related peptides, the B(1)R and B(2)R, are known to be polymorphic. The human isolated umbilical vein responds by contractions to stimulation by kinins via constitutive B(2)Rs and inducible B(1)Rs. Vascular rings from 100 different umbilical cords were submitted to a standardized protocol where E(max) values were obtained at 2 and 6 h of incubation, and EC(50) values were estimated at 6 h for the B(1)R agonist Sar-¿D-Phe(8)des-Arg(9)-BK; E(max) and EC(50) values were also obtained for the B(2)R agonist BK at 4 h. The genotype of each tissue donor was determined for two polymorphic sites in the B(1)R gene and three such sites in the B(2)R gene. The (-/-) genotype of a frequent insertion/deletion polymorphism of the B(2)R exon 1 was associated with increased contractile efficiency of the B(1)R agonist, Sar-¿D-Phe(8)des-Arg(9)-BK, but had no effect on BK-induced contractility. A B(2)R exon 2 polymorphism (C(181) --> T) selectively influenced the potency of BK (EC(50) higher when the T allele was present). The other polymorphisms studied were not found to affect kinin-induced contractility. Although most of the frequent polymorphic alleles of the kinin receptor genes are functionally neutral or determine functional alterations that are not detectable using the method used here, two B(2)R polymorphic sites (exon 1, exon 2) modestly influence function. As the exon 1 B(2)R polymorphism predicts the response of the B(1)R agonist, it may be in linkage disequilibrium with an unknown, functionally important polymorphism of the neighboring B(1)R gene.

T-Cell epitopes and human leukocyte antigen restriction elements of an immunodominant antigen of Blastomyces dermatitidis.

Humans infected with the dimorphic fungus Blastomyces dermatitidis develop strong T-lymphocyte responses to WI-1, an immunodominant antigen that has been shown to elicit protective immunity in mice. In the present study, the T-cell epitopes of WI-1 and human leukocyte antigen (HLA) restricting elements that display them were investigated. Peripheral blood mononuclear cells (PBMC) from 37 patients with a confirmed history of blastomycosis were tested for a response to WI-1 in primary proliferation assays; PBMC from 35 (95%) responded. Six patients whose PBMC proliferated strongly in response to WI-1 (defined as a stimulation index greater than 50) were tested further for responses to subcloned, recombinant fragments of the antigen. These patients responded chiefly to sequences within the N terminus and the 25-amino-acid tandem repeat. Cloned CD4(+) T cells from an infected individual were used to delineate more precisely the peptide epitopes in the fragments and HLA restricting elements that present them. A majority of the T-cell clones recognized an epitope spanning amino acids 149 to 172 within the N terminus, displayed by HLA-DR 15. A minority of the clones, which have been shown to perform a cytolytic function in vitro, recognized an epitope in the tandem repeat displayed by HLA-DPw4, an uncommon restricting element. Tandem repeat epitopes required display by the beta chain of DPw4 heterodimers. Thus, human T cells with different functions in vitro also recognize distinct regions of WI-1, raising the possibility that HLA restricting elements that present them could modulate immunity during blastomycosis by selection and display of WI-1 peptides.

Portacaval anastomosis results in more widespread alterations of cerebral metabolism in old versus young adult rats: implications for post-shunt encephalopathy.

Treatment of portal hypertension by portal decompressive surgery or transjugular intrahepatic portosystemic stent shunt (TIPS) results in new or worsening episodes of portal-systemic encephalopathy, particularly in older patients. As part of a series of studies to elucidate the pathophysiologic mechanisms responsible for the age-related increased portal-systemic encephalopathy following shunt surgery, local cerebral glucose utilization, a measure of regional brain functional activity, was assessed using the 14C-2-deoxyglucose autoradiographic technique in 2 month-old (young adult) and 24 month-old (old adult) rats following end-to-side portacaval anastomosis. Cerebral glucose utilization was decreased by 22% (p<0.05) in frontal cortex of 2 month-old rats following portacaval anastomosis. More widespread alterations of glucose utilization, involving frontal and frontoparietal cortices, as well as thalamic structures were observed in the brains of 24 month-old rats following portacaval anastomosis despite blood ammonia concentrations of a comparable magnitude. Decreased cerebral glucose utilization in frontal and frontoparietal cortex of old adult rats following portacaval anastomosis probably results from decreased cerebral energy requirements as a consequence of neurotransmitter-related dysfunction. The greater susceptibility of aging brain to the deleterious effects of portacaval anastomosis is consistent with the higher incidence of encephalopathy in older cirrhotic patients following portacaval anastomosis or TIPS.

Increased neuronal nitric oxide synthase expression in brain following portacaval anastomosis.

It has previously been suggested that increases of L-arginine uptake into brain following portacaval shunting may result in increased activities of constitutive neuronal nitric oxide synthase (nNOS). In order to further address this issue, nNOS protein and gene expression were studied by Western blot analysis using a monoclonal nNOS antibody and RT-PCR respectively in the brains of rats following portacaval shunting or sham operation. Portacaval shunting resulted in a 2-fold increase (P < 0.01) in nNOS protein and a concomitant 2.4-fold increase (P < 0.01) in nNOS mRNA. Increased nNOS activity in brain and the resulting increase in nitric oxide production could contribute to the increased cerebral blood flow and to the pathogenesis of hepatic encephalopathy in chronic liver disease.

The portacaval-shunted rat: a new model for the study of the mechanisms controlling voluntary ethanol consumption and ethanol preference?

Portacaval anastomosis (PCA) is a surgical procedure whereby blood from the portal vein is shunted into the inferior vena cava. PCA in the rat results in a significant increase (from 0.77 +/- 0.26 to 3.51 +/- 0.37 g of ethanol/kg/day) in voluntary ethanol consumption in a free-choice paradigm between water and 5% ethanol solution. After PCA surgery, increased voluntary ethanol consumption starts abruptly at 6 to 7 days and is maintained for > 28 weeks. Voluntary ethanol consumption in rats after PCA results in blood ethanol levels up to 158 mg%. After PCA, the ethanol preference ratio (defined as the percentage of total fluid intake constituted by ethanol) increased from 19 +/- 2% to 78 +/- 2% (P < 0.001). Administration of the nonselective opioid receptor antagonist naloxone (5 mg/kg, sc) resulted in a significant 6-fold attenuation of voluntary ethanol consumption by rats with PCA, an effect that was not mediated by an effect on locomotor activity. These findings, together with previous reports of widespread alterations of the mu- and delta-opioid receptors in the brain after PCA, suggest that increased voluntary ethanol consumption and ethanol preference in PCA rats may result from activation of the endogenous opioid system. Preliminary studies suggest that rats with PCA manifest behavioral signs consistent with the development of dependence. The portacaval-shunted rat may provide a useful preparation for the study of mechanisms, in particular those involving the liver, implicated in the development of increased voluntary ethanol consumption and ethanol preference.

Portacaval shunting and hyperammonemia stimulate the uptake of L-[3H] arginine but not of L-[3H]nitroarginine into rat brain synaptosomes.

Elevated activities of nitric oxide synthase (NOS) have been reported previously in the brains of portacaval-shunted (PCS) rats, a model of chronic hepatic encephalopathy (HE). As L-arginine availability for nitric oxide synthesis depends on a specific uptake mechanism in neurons, we studied the kinetics of L-[3H]-arginine uptake into synaptosomes prepared from the brains of PCS rats. Results demonstrate that L-arginine uptake is significantly increased in cerebellum (60%; p < 0.01), cerebral cortex (42%; p < 0.01), hippocampus (56%; p < 0.01), and striatum (51%; p < 0.01) of PCS rats compared with sham-operated controls. Hyperammonemia in the absence of portacaval shunting also stimulated the transport of L-[3H]arginine; kinetic analysis revealed that the elevated uptake was due to increased uptake capacity (Vmax) without any change in affinity (Km). Incubation of cerebellar synaptosomes with ammonium acetate for 10 min caused a dose-dependent stimulation of L-[3H]arginine uptake. Neither portacaval shunting nor hyperammonemia had any significant effect on the synaptosomal uptake of NG-nitro-L-[3H]arginine. These studies demonstrate that increased NOS activity observed in experimental HE may result from increased availability of L-arginine resulting from a direct stimulatory effect of ammonia on L-arginine transport.

Cardiovascular effects of Sar-[D-Phe8]des-Arg9-bradykinin, a metabolically protected agonist of B1 receptor for kinins, in the anesthetized rabbit pretreated with a sublethal dose of bacterial lipopolysaccharide.

We investigated the mechanism of the hypotensive effect of Sar-[D-Phe8]des-Arg9-bradykinin (BK) in lipopolysaccharide-treated anesthetized rabbits. The study involved pharmacokinetic and hemodynamic measurements and tests of antagonism with various drugs. The rate of elimination of Sar-[D-Phe8]des-Arg9-BK from the rabbit plasma was slower than that of Lys-BK, a naturally occurring B1 agonist. The amplitude of the hypotensive effect of Sar-[D-Phe8]des-Arg9-BK was not affected by pretreatment with indomethacin, diclofenac, dazmegrel, NG-nitro-L-arginine, glibenclamide, MK-886, BN-50739, atropine or propranolol, but its duration was shortened by indomethacin and diclofenac. Sar-[D-Phe8]des-Arg9-BK-induced hypotension was associated with decreases of total peripheral resistance, cardiac output, carotid, mesenteric and femoral blood flow, transient reductions followed by secondary increases of vascular resistance in the carotid and femoral beds, reductions of central venous pressure, but no change of hematocrit. Animal pretreatment with diclofenac or hexamethonium abolished the secondary increases of carotid bed vascular resistance caused by the B1 agonist. These and other results suggest that peripheral vasodilation leading to a decrease of total peripheral resistance and a decrease of cardiac output may both contribute consecutively to the hypotensive effect of Sar-[D-Phe8]des-Arg9-BK in this animal model. Inappropriate compensatory responses to arterial hypotension, prostaglandin release, and slow rate of elimination of Sar-[D-Phe8]des-Arg9-BK from the rabbit plasma, may all be at the basis of the prolonged duration of the hypotension caused by the B1 agonist.

Purification in quantity of the secreted form of WI-1: a major adhesin on Blastomyces dermatitidis yeasts.

WI-1, a 120-kDa adhesin on Blastomyces dermatitidis, binds the yeast to macrophages and is a major target antigen of immune recognition in acquired resistance to the fungus. In past studies, WI-1 has been purified by extracting the protein from the yeast cell wall, which yields microgram quantities for biological assays. We report a strategy for generating and purifying the secreted form of WI-1 in quantity. Yeasts of B. dermatitidis ATCC strain 60636 cultured in HMM medium were found to secrete 10 microg/ml of WI-1 into a supernate relatively free of other medium and yeast components. Using a two-step method of ion exchange and hydrophobic interaction chromatography, we achieved a 7.1-fold purification of WI-1. Purified WI-1 was sequenced at the N-terminus which revealed that the secreted protein exists in two different forms. In functional assays, purified WI-1 also retained its adhesivity for human macrophages, and its antigenicity in binding anti-WI-1 antibodies and stimulating T-cells to proliferate, but it lost some capacity to elicit delayed-type hypersensitivity in mice. These findings advance our understanding of the WI-1 adhesin/antigen and our ability to express and purify WI-1 in quantity and will permit a study of the relationship between three-dimensional structure and activity of the molecule.

Ammonium acetate challenge in experimental chronic hepatic encephalopathy induces a transient increase of brain 5-HT release in vivo.

Ammonia has been shown to cause release of neurotransmitters such as serotonin (5-hydroxytryptamine; 5-HT) from synaptosomal preparations in vitro. In the present study, frontal neocortical extracellular levels of 5-HT and its major metabolite, 5-hydroxyindole-3-acetic acid (5-HIAA), were determined in vivo by the use of microdialysis in portacaval shunted (PCS) rats, an experimental model of chronic hepatic encephalopathy (HE), prior to and after an acute coma-inducing administration of ammonium acetate (NH4Ac; 5.2 mmol/kg, i.p.). PCS rats displayed elevated (P < 0.01) 5-HIAA but unaltered 5-HT extracellular levels compared with controls, supporting the contention of an increased neocortical 5-HT metabolism but unaltered neuronal 5-HT output in chronic HE. However, a transient elevation of extracellular 5-HT levels was observed when PCS-NH4Ac rats were in coma. Increased brain ammonia may thus augment neuronal 5-HT release in chronic HE, which in turn could be a causative for precipitation of more severe stages of HE.

Mechanism of the contractile effect of diaspirin-cross-linked hemoglobin in rat isolated aorta strip denuded of endothelium as revealed using an oil-immersion procedure.

Diaspirin-cross-linked hemoglobin (DCLHb) is a chemically modified hemoglobin (Hb) (i.e., alpha-subunits are cross-linked by a covalent bond) currently being tested as a potential oxygen-carrying blood substitute. It was examined for possible vasoactive properties, using the rat isolated aorta strip denuded of endothelium. In this experimental model, DCLHb (1.6-155 microM) was found to be inactive as a vasoconstrictor when added to the Krebs medium but to elicit contractile responses once the Krebs medium containing DCLHb was replaced by mineral oil, a procedure that favors the sequestration of a fixed amount of DCLHb within a substantially reduced volume of extracellular fluid. The contractile activity of DCLHb in our experimental model (i.e., prior exposure of tissues to drugs in the Krebs medium followed by replacement of the Krebs medium by mineral oil) was mimicked by methemoglobin and metmyoglobin, but not by cytochrome c, albumin, hemin, hematin, Fe2+, and a variety of hemorphins. It was abolished by indomethacin, SQ-29548 (prostaglandin H2-thromboxane A2 receptor antagonist), thiourea, or N-2-mercaptopropionylglycine (MCPG), reduced partially by verapamil, but not affected by dazmegrel, MK-886 (leukotriene biosynthesis inhibitor), dimethylsulfoxide, vitamin C or E, deferoxamine, NG-nitro-L-arginine, naloxone, and a variety of other drug receptor antagonists (e.g., prazosin) and protease inhibitors (e.g., pepstatin). Rat aorta strips denuded of endothelium exhibited contractile responses to arachidonic acid added in the Krebs medium (i.e., with no mineral oil added afterwards). Such contractile activity was reduced by SQ-29548, thiourea, or MCPG. Addition of U-46619 (prostaglandin H2-thromboxane A2 mimetic) to the Krebs medium also elicited contractile responses in rat aorta strips denuded of endothelium. Such contractile activity was reduced by SQ-29548, thiourea, or verapamil but not by MCPG. Within the limitations of our experimental approach, these results suggest that (1) the contractile activity of DCLHb in rat aorta strips denuded of endothelium following replacement of the Krebs medium by mineral oil involves the participation of a secondary mediator, which could be a vasoconstrictor metabolite of arachidonic acid; (2) the participation of reactive oxygen species, potential degradation products of DCLHb (e.g., heme, Fe2+, hemorphins), or other mediators in the contractile activity of DCLHb is unlikely; and (3) Ca2+ entry into target cells might be involved in the process by which DCLHb elicits its contractile activity in our experimental model.

Portacaval anastomosis induces region-selective alterations of the endogenous opioid system in the rat brain.

Portacaval anastomosis (PCA) in the rat results in a broad spectrum of neurological and neurobehavioral changes, including alterations of circadian rhythms, impaired locomotor activity, and reflexes, as well as decreased threshold to noxious stimuli. In addition, following portacaval shunting, rats drink significantly more ethanol in a free-choice drinking paradigm. Available evidence suggests that many of these behavioral changes may be modulated by the endogenous opioid system of the brain. To evaluate this possibility, the effects of PCA on circulating beta-endorphin (beta-EP), as well as beta-EP content in the pituitary and specific brain nuclei, was evaluated using a sensitive radioimmunoassay. Furthermore, the characteristics and regional densities of mu and delta opioid receptors in the brains of PCA and sham-operated control rats were studied using an in vitro technique, as well as quantitative receptor autoradiography and the specific ligands 125I [D-Ala2, MePhe4, Met(o)ol5]enkephalin (FK 33-824) and 125I [2-D-penicillamine, 5-D-penicillamine]-enkephalin (DPDPE) for micro and delta sites, respectively. PCA resulted in region-selective modifications of beta-EP in brain, but not in pituitary or blood. Autoradiographic studies revealed a generalized decrease in mu binding sites (up to 70% decreases compared with sham-operated controls) and region-selective alterations of delta receptor densities following PCA. Portacaval-shunted rats drank significantly more ethanol in a free-choice drinking paradigm, an effect that was significantly attenuated by the administration of the opiate antagonist naloxone. Increased ethanol preference thus appeared to result from modifications of the endogenous opioid system in nucleus accumbens of rats following PCA.

Selective alterations of extracellular brain amino acids in relation to function in experimental portal-systemic encephalopathy: results of an in vivo microdialysis study.

Portal-systemic encephalopathy (PSE) is characterized by neuropsychiatric symptoms progressing through stupor and coma. Previous studies in human autopsy tissue and in experimental animal models of PSE suggest that alterations in levels of brain amino acids may play a role in the pathogenesis of PSE. To assess this possibility, levels of amino acids were measured using in vivo cerebral microdialysis in frontal cortex of portacaval-shunted rats administered ammonium acetate (3.85 mmol/kg, i.p.) to precipitate severe PSE. Sham-operated rats served as controls. Portacaval shunting resulted in significant increases of levels of extracellular glutamine (threefold, p < 0.001), alanine (38%, p < 0.01), aspartate (44%, p < 0.05), phenylalanine (170%, p < 0.001), tyrosine (140%, p < 0.001), tryptophan (63%, p < 0.001), leucine (75%, p < 0.001), and serine (60%, p < 0.001). Administration of ammonium acetate to sham-operated animals led to a significant increase in extracellular glutamine and taurine content, but this response was absent in shunted rats. The lack of taurine release into extracellular fluid following ammonium acetate administration in portacaval-shunted rats could relate to the phenomenon of brain edema in these animals. Ammonium acetate administration resulted in significant increases in the extracellular concentrations of phenylalanine and tyrosine in both sham-operated and portacaval-shunted rats. Severe PSE was not accompanied by significant increases in extracellular fluid concentrations of glutamate, aspartate, GABA, tryptophan, leucine, or serine, suggesting that increased spontaneous release of these amino acids in cerebral cortex is not implicated in the pathogenesis of hepatic coma.

Na+,K(+)-ATPase activities are increased in brain in both congenital and acquired hyperammonemic syndromes.

Activities of Na+,K(+)-ATPase were measured in brain regions of experimental animals with either congenital or acquired hyperammonemia. In the sparse-fur (spf) mutant mouse, with a genetic X-linked deficiency of ornithine transcarbamylase, an animal model of congenital hyperammonemia, Na+,K(+)-ATPase was increased in frontal cortex (by 57%, P < 0.001), cerebellum (by 61%, P < 0.001), brainstem (by 71%, P < 0.001) and striatum (by 48%, P < 0.01). Four weeks following portacaval anastomosis in the rat, Na+,K(+)-ATPase activities were increased in cerebellum and striatum (by 19%, P < 0.01) and in brainstem (by 28%, P < 0.01). Stimulation of Na+,K(+)-ATPase and the subsequent alteration of neuronal excitability could contribute to the CNS dysfunction characteristic of chronic hyperammonemic syndromes.

Increased nitric oxide synthase activities and L-[3H]arginine uptake in brain following portacaval anastomosis.

Glutamatergic synaptic dysfunction has been proposed as a causal factor in portal-systemic encephalopathy. Increased in vitro and in vivo glutamate release and decreased glutamate binding to NMDA receptors were previously reported in the brains of portacaval-shunted rats. Such changes could lead to alterations in the second messenger systems coupled to glutamate receptors. As NMDA receptors have been shown to act via the nitric oxide/cyclic GMP second messenger system, we studied the activities of constitutive nitric oxide synthase (NOS) in the brains of rats following portacaval shunting. Results demonstrate that NOS activities are significantly increased in cerebellum (by 54%, p < 0.01), cerebral cortex (by 65%, p < 0.01), hippocampus (by 88%, p < 0.01), and striatum (by 64%, p < 0.01) of shunted rats compared with sham-operated controls. As L-arginine transport is a prerequisite for nitric oxide production, we also studied L-[3H]arginine transport into cerebellar and cerebral cortical synaptosomes prepared from the brains of portacaval-shunted and sham-operated rats. L-[3H]Arginine uptake was significantly increased (by approximately 50%, p < 0.01) in both cerebellum and cortex. Increased NOS activities of neuronal and/or astrocytic origin and the resultant increased production of nitric oxide in brain could be the consequence of increased NMDA receptor activation following portacaval shunting. Furthermore, increased nitric oxide production could contribute to the increased cerebral blood flow consistently observed following portacaval shunting.

Quantification of des-Arg9-bradykinin using a chemiluminescence enzyme immunoassay: application to its kinetic profile during plasma activation.

There is a renewed interest in the kininase I pathway of kinin metabolism, because des-Arg9-bradykinin (des-Arg9-BK) and des-Arg10-Lys-BK are selective and potent agonists of the B1 receptors, that are apparently upregulated by tissue injury. We have developed a polyclonal rabbit antiserum against des-Arg10-Lys-BK. In a radioimmunoassay for des-Arg10-Lys-BK, this antiserum exhibited high specificity. Notably, native kinins with the C-terminal Arg residue, bradykinin (BK) and Lys-BK, did not cross-react to a significant extent, whereas des-Arg9-BK and digoxigenin (DIG)-des-Arg9-BK exhibited a complete cross-reactivity. The antibodies were used to set up a sensitive chemiluminescence enzyme immunoassay (CLEIA) using the DIG-anti-DIG system as intermediate for the revelation of the immune complexes. The detection limit and the half-maximal saturation concentration for des-Arg9-BK were 27 and 1530 fmol/ml respectively. This assay, as well as another for BK quantification, have been applied in vitro to rabbit plasma activated by kaolin. The conversion of BK into des-Arg9-BK was generally efficient, and the persistence and concentration of both peptides were increased in the presence of enalaprilat an inhibitor of the angiotensin converting enzyme (ACEI). Rabbits treated with bacterial lipopolysaccharide exhibited an increase of plasma immunoreactive des-Arg9-BK that was potentiated in animals also treated with ACEI. This CLEIA for des-Arg9-BK is a new analytical tool applicable to analyze of the kininase I metabolites of kinins in vitro and in vivo. Measurements of des-Arg9-BK may be useful indicators of the kallikrein-kinin system activation.

Further analysis of the upregulation of bradykinin B1 receptors in isolated rabbit aorta by using metabolic inhibitors.

Kinins exert a contractile effect that develops as a function of the in vitro incubation time with isolated rabbit aorta. This response is mediated via receptors of the bradykinin B1 type and interleukin-1 amplifies this upregulation process. Tissues continuously treated with the protein synthesis inhibitor cycloheximide (71 microM) or with the protein trafficking inhibitor, brefeldin A (18 microM), failed to develop a contractile response to the bradykinin B1 receptor agonist, des-Arg9-bradykinin (1.7 microM) (72-100% inhibition of kinin response recorded at 3 or 6 h), whether or not they were exposed to interleukin-1 beta (290 pM). The protein glycosylation inhibitor tunicamycin exerted a selective and significant, but partial (50-76%), inhibition of des-Arg9-bradykinin-induced responses. The biochemical effect of the metabolic inhibitors on the tissue has been validated in assays involving incorporation of [3H]leucine and of [3H]mannose into protein or glycoprotein fractions, respectively. The modulatory effects of metabolic inhibitors on the responses to kinins of the isolated rabbit aorta support the idea that a de novo formation of membrane bradykinin B1 receptors is the molecular basis of both the spontaneous and the interleukin-1-stimulated upregulation phenomenon.

Recombinant human hemoglobin inhibits both constitutive and cytokine-induced nitric oxide-mediated relaxation of rabbit isolated aortic rings.

A genetically engineered recombinant human hemoglobin (rHb1.1) was recently developed for use as a blood substitute (Nature 1992;356:258-60). Like other mammalian hemoglobin (Hb) molecules, it might bind and antagonize the actions of nitric oxide (NO). We used an isolated rabbit aortic ring preparation to examine the ability of rHb1.1 to inhibit acetylcholine (ACh)- and interleukin-1 beta (IL-1 beta)-induced reductions of vasoconstrictor responses to the alpha-adrenoceptor agonist phenylephrine (PE). rHb1.1 (0.04-4.4 microM) rapidly and reversibly inhibited, in a concentration-dependent manner, both ACh- and IL-1 beta-induced decreases in PE contractile responses. These inhibitory effects of rHb1.1 were non-competitive and were equipotent to those of purified, cell-free human Hb (p.hHb). These two forms of soluble Hb were at least 10 times more potent than Hb in erythrocytes (red blood cells: RBC-Hb). Both NG-nitro-L-arginine (10 microM) a NO synthase inhibitor, and LY-83583 (10 microM), a guanylyl cyclase inhibitor, mimicked the effects of rHb1.1. The inhibitory effects of rHb1.1 were not shared by either human serum albumin (HSA 44 microM), which combines with but does not deactivate NO, or cytochrome C (44 microM), a heme-containing protein that does not bind NO; neither were they reversed by L-arginine (L-ARG) (1 mM), the presumed NO precursor. These and other results suggest that the chemical antagonism of NO is likely to be the mechanism by which rHb1.1 and other Hbs inhibit ACh- and IL-1 beta-induced decreases in the response to PE in rabbit aortic rings.

Tissue-specific alterations of binding sites for peripheral-type benzodiazepine receptor ligand [3H]PK11195 in rats following portacaval anastomosis.

Kinetics of binding of [3H]PK11195, an antagonist ligand with high selectivity for the peripheral-type (mitochondrial) benzodiazepine receptor (PTBR), was studied in homogenates of cerebral cortex, kidney, heart, and testis of portacaval shunted rats and sham-operated controls. Portacaval anastomosis resulted in a significant two- to threefold increase in the number of [3H]PK11195 binding sites in cerebral cortex and kidney. A reduction in the number of [3H]PK11195 binding sites was observed in testis preparations, while the number of binding sites in the heart remained unaltered. These differences in the response of PTBRs to portacaval anastomosis, in different organs suggest that the physiological function of these receptors and the factors regulating them are modulated by distinct mechanisms. The finding of increased densities of [3H]PK11195 binding sites in brain and kidney following portacaval anastomosis parallels the cellular hypertrophy in these tissues and, together with previous observations of similar increases of these binding sites in brain and kidney in congenital hyperammonemia, suggest a pathophysiologic role for ammonia in these changes. In contrast, the significant loss of [3H]PK11195 binding sites in testicular preparations following portacaval anastomosis together with the known effects of steroid hormones on these sites suggests a role for PTBRs in the pathogenesis of testicular atrophy in chronic liver disease.

Development and in vivo evaluation of metabolically resistant antagonists of B1 receptors for kinins.

The B1 receptors for kinins are selectively stimulated by bradykinin (BK) and Lys-BK metabolites that do not have the C-terminal arginine, des-Arg9-BK and Lys-des-Arg9-BK, respectively. B1 receptors mediate a definite subset of the cardiovascular effects of kinins in normal and septic animals. We have studied the metabolism of the best selective B1 antagonist, Lys[Leu8]des-Arg9-BK, in order to support the rational design of new antagonists that have increased metabolic stability. The affinity of the new compounds was evaluated using the pA2 scale and was based on the antagonism of the contractile effect of kinins in the rabbit isolated aortic preparation. Acetylation of the alpha-amino group of the N-terminal Lys residue provided an excellent protection from the degradation by rabbit blood plasma. This and the inhibitory effect of amastatin on the metabolism of Lys[Leu8]des-Arg9-BK indicated that aminopeptidase M (AmM) is the major route of inactivation for this class of peptides in plasma. Various other modifications afforded a more or less complete resistance to purified angiotensin I converting enzyme (ACE). One analog, Ac-Lys[MeAla6, Leu8]des-Arg9-BK, was found resistant to the above-mentioned enzymes and to neutral endopeptidase-24.11 extracted from rabbit kidney. This antagonist, although 100 times less potent than the parent peptide Lys[Leu8]des-Arg9-BK on the rabbit aortic preparation, was equipotent in vivo against the hypotensive effect of a B1 agonist in lipopolysaccharide-treated rabbits, and unlike the original compound, its effect was persistent after the end of the infusion. Ac-Lys[MeAla6, Leu8]des-Arg9-BK does not antagonize B2 receptors either in vitro or in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)