Nitric oxide-mediated beta 2-adrenoceptor relaxation is impaired in mesenteric veins from portal-hypertensive rats

BACKGROUND & AIMS: beta-Adrenergic relaxation seems to be mediated by nitric oxide. The aim of this study was to evaluate changes induced by portal hypertension in beta 2-adrenergic vasorelaxation. METHODS: Isolated rat mesenteric veins were relaxed by salbutamol, and nerve-mediated vasocontractions were induced by electrical field stimulation. Responses were evaluated in the presence of NG-nitro-L-arginine methyl ester (L-NAME) or tetrodotoxin. Immunocytochemical techniques were used for localization of neuronal NO synthase. RESULTS: Salbutamol-induced relaxations were decreased in rings from portal-hypertensive animals. L-NAME reduced these relaxations, but its effects were more pronounced in sham-operated rats. Tetrodotoxin decreased the effect of salbutamol only in rings from sham-operated animals. Combination of L-NAME and tetrodotoxin did not exert a greater effect than either of these agents alone. Veins from portal-hypertensive animals were more sensitive to S-nitroso-N-acetyl penicillamine. L-NAME increased vasocontractions by electrical stimulation only in rings from sham-operated rats. Veins from portal-hypertensive animals exhibited a specific degeneration of NO-containing nerve endings. CONCLUSIONS: beta 2-Adrenergic relaxation is impaired in mesenteric veins from portal-hypertensive rats, possibly as a result of a defective neuronal release of NO.     

Agonist-induced down-regulation of the beta2-adrenoceptor and its mRNA in human mononuclear leukocytes

Agonist-mediated regulation of beta2-adrenoceptors in mononuclear leukocytes has been examined at the protein but not at the mRNA level. In the present study, incubation of mononuclear leukocytes with the beta-agonist (-)-isoproterenol (10(-6) M) for up to 42 hr led to a maximum decrease in both beta2-adrenoceptor mRNA concentration and total receptor number of ca. 56 and 70%, respectively. The decrease in the mRNA level, however, was slower than for the protein level. After 4 hr of incubation with the beta-agonist, the protein level decreased to a minimum of 65% of the initial amount, while an incubation of 8 hr was necessary to reach a similar decrease in the level of mRNA (69% of the initial level). Measurements of mRNA stability revealed a reduction in the half-life of beta2-adrenoceptor mRNA from 2.7 to 1.1 hr following 4 hr of incubation with (-)-isoproterenol. Our data clearly demonstrate that treatment of human mononuclear leukocytes with (-)-isoproterenol induces a beta2-adrenoceptor down-regulation together with a slower time course of mRNA down-regulation which is partly due to a reduction of mRNA stability.     

Polymorphism of the beta2-adrenoceptor and the response to long-term beta2-agonist therapy in asthma

DNA-based diagnosis of haemophilia A has previously been carried out by linkage analysis using two highly informative markers, Hind III RFLP and St14 VNTR, for affected Turkish families. In the present study the number and frequency of the microsatellite alleles at introns 13 and 22 in the factor VIII (FVIII) gene were analysed in order to increase the rate of informative females and accuracy of linkage analysis. Six alleles were observed at both loci. The two most frequent alleles of each locus were the same as the two common alleles found in Anglo-Americans. The comparison of heterozygosity of both microsatellite loci showed that the Turkish population is slightly less polymorphic than Anglo-Americans but more polymorphic than Chinese, Slavs and Uzbekians. The additional use of the two microsatellite repeat polymorphisms with the previously established informative markers has been accepted as the most effective strategy in DNA diagnosis by linkage analysis for the assessment of haemophilia A carriers and affected fetuses in the Turkish population. The modifications adopted in this study for the multiplex PCR analysis of the microsatellite repeat polymorphism eliminated the use of radioactivity and sequencing gels, reducing cost and labour.     

Characterization of alpha1-adrenoceptor subtypes mediating contractions to phenylephrine in rat thoracic aorta, mesenteric artery and pulmonary artery

1. The subtype of alpha1-adrenoceptor mediating contractions to phenylephrine of the rat thoracic aorta, mesenteric artery and pulmonary artery were investigated by use of antagonists which show selectivity between the cloned alpha1-adrenoceptor subtypes in binding studies. 2. Cumulative concentration-contraction curves for phenylephrine were competitively antagonized in the rat thoracic aorta by prazosin (pA2 9.9), WB4101 (pA2 9.6), 5-methylurapidil (pA2 8.1), benoxathian (pA2 9.2) and indoramin (pA2 7.4). These compounds were also competitive antagonists in the mesenteric and pulmonary arteries (except for 5-methylurapidil in the pulmonary artery), (prazosin pA2 9.9 and 9.7; WB4101 pA2 9.8 and 9.6; 5-methylurapidil pA2 7.9 and pK(B) estimate 8.0; benoxathian pA2 8.8 and 9.3; indoramin pA2 7.2 and 7.5, respectively). 3. RS 17053 was not a competitive antagonist in any blood vessel as Schild plot slopes were greater than unity. The pK(B) estimates for RS 17053 were 7.1 in aorta, 7.0 in the mesenteric artery and 7.7 in the pulmonary artery. 4. The alpha1D-subtype selective antagonist BMY 7378 appeared to be non-competitive with shallow Schild plot slopes. The data were better fitted with two lines in all tissues, with Schild plot slopes that were no longer different from unity, except in the pulmonary artery. The higher affinity site for BMY 7378 in the aorta had a pA2 of 9.0, while it was 8.8 and 8.9 in the mesenteric and pulmonary arteries, respectively. 5. MDL73005EF acted in a non-competitive manner in all three blood vessels, with shallow Schild plot slopes. The pK(B) estimates for MDL73005EF were 8.4 in aorta, 7.5 in the mesenteric artery and 8.0 in the pulmonary artery. 6. In all three blood vessels the functionally determined antagonist affinity estimates correlated best with published pKi values for their displacement of [3H]-prazosin binding on membranes expressing cloned alpha1d-adrenoceptors compared with alpha1a- or alpha1b-adrenoceptors. The antagonist affinity estimates in the aorta, mesenteric and pulmonary arteries correlated highly with their previously published pA2 values in rat aorta (alpha1D) and less well with those for alpha1A- and alpha1B-adrenoceptors mediating contraction of the rat epididymal vas deferens and rat spleen, respectively. 7. The results of this study suggest that the contraction to phenylephrine of the rat thoracic aorta, mesenteric artery and pulmonary artery are mediated in part via the alpha1D-subtype of adrenoceptor. The data for both BMY 7378 and MDL73005EF in all three blood vessels are consistent with receptor heterogeneity. However, the identity of the second site is unclear.     

The role of beta 2-adrenoceptor on the pathogenesis of insulin resistance in essential hypertension

The role of beta 2-adrenoceptor on the pathogenesis of insulin resistance in essential hypertension (EH) was explored. After the measurement of blood pressure in 15 EH patients and 8 control subjects, EH patients were divided into two groups by the elevation of plasma NE (delta NE) 5 min after standing: 7 normoadrenergic EH (delta NE < 140 pg/ml) and 8 hyperadrenergic EH (delta NE > or = 140 pg/ml). On the morning after a 12-h overnight fast, regular insulin (0.1 U/kg) was injected intravenously, and glucose disappearance rate (GDR) was measured and used as an index of insulin sensitivity. On the following day, the test was reinvestigated following the administration of mabuterol, a beta 2 agonist. Plasma growth hormone (GH), cortisol, norepinephrine (NE) and epinephrine (Epi) were measured before and after the mabuterol administration. Although there were no significant differences of basal GDR among these three groups, mabuterol induced a considerable decrease in GDR in EH patients but not in control subjects. There was no significant difference in the decrease of GDR between normo- and hyperadrenergic EH. The decrease in GDR tended to correlate with the mean blood pressure at rest in EH but not in normal subjects. Plasma glucose and serum insulin in EH patients were increased more than in normal subjects. Plasma GH, cortisol and Epi were not elevated by mabuterol, but plasma NE increased in each group, significantly in hyperadrenergic EH. There was no correlationship between the increase in plasma NE and the decrease in GDR after mabuterol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Action potential shortening through the putative beta4-adrenoceptor in ferret ventricle: comparison with beta1- and beta2-adrenoceptor-mediated effects

The electrophysiological responses to (-)-CGP 12177 ((-)-4-(3-tertiarybutylamino-2-hydroxypropoxy) benzimidazol-2-one), an agonist for the putative beta4-adrenoceptor, were investigated on isolated perfused ferret hearts paced at 100 min(-1) and compared to those of (-)-noradrenaline and (-)-adrenaline, mediated through beta1- and beta2-adrenoceptors respectively. The three agonists decreased ventricular monophasic action potential duration but prolonged the action potential plateau; beta3-adrenoceptor-selective agonists had no effect. (-)-CGP 12177 was the most potent, but (-)-noradrenaline the most efficacious; both agonists caused ventricular extra-systoles. Because only (-)-noradrenaline but not (-)-CGP 12177 elicited shortening of the refractory period, the mechanism of arrhythmias mediated through beta1- and putative beta4-adrenoceptors may be different.     

Role of the N- and C-terminal domains of bovine beta 2-glycoprotein I in its interaction with cardiolipin

beta 2-Glycoprotein I (beta 2-GPI) is a cofactor in the recognition of the phospholipid antigen cardiolipin by anti-cardiolipin antibodies in autoimmune diseases such as systemic lupus erythematosus. We examined the interactions of various forms of bovine beta 2-GPI, such as its intact form, desialylated form (Asialo-beta 2-GPI), N-terminal domain (Domain I), and modified forms of beta 2-GPI and Asialo-beta 2-GPI with nicks in their C-terminal domains, with phospholipid liposomes under different conditions of pH and ionic strength. We found that at neutral pH and low ionic strength, beta 2-GPI became bound to liposome membranes containing cardiolipin, phosphatidylglycerol, phosphatidylserine, phosphatidylserine, phosphatidic acid, or phosphatidylinositol, but not phosphatidylcholine alone. The number of phospholipids involved in the binding seemed to depend on the head group structure of the negatively charged phospholipids, but the dissociation constant did not, being about 10(-8) M, except that for the interaction with phosphatidylinositol, which was one order of magnitude lower. We also found that Domain I and Asialo-beta 2-GPI bound to liposome membranes containing negatively charged phospholipids, and that in the interaction with cardiolipin, their dissociation constants were about 10(-6) and 10(-8) M, respectively. At neutral pH and both low and high ionic strengths, the affinities of the nicked forms of beta 2-GPI and Asialo-beta 2-GPI for cardiolipin were both lower than those of their intact forms but similar to that of Domain I.(ABSTRACT TRUNCATED AT 250 WORDS)     

The prognostic significance of beta human chorionic gonadotrophin and its metabolites in women with cervical carcinoma

AIMS: To examine long term survival of women with primary and recurrent cervical carcinoma in relation to (1) excretion of beta-core (a urinary metabolite of beta human chorionic gonadotrophin (beta hCG)) and (2) beta hCG immunostaining of the tumours, to determine the suitability of these markers for assessing prognosis. METHODS: This was a prospective observational study undertaken in a gynaecological oncology centre: 57 women with primary cervical cancer and 42 with recurrent disease were recruited between January 1990 and September 1992. Kaplan-Meier survival analysis with the log rank test was used to assess survival differences with survival rate given per year of follow up. RESULTS: In primary disease, the four year survival for the beta-core negative group was 79%, compared with 14% for the beta-core positive group (p = 0.001). This was still significant for early stage disease or squamous lesions alone. In recurrent disease, beta-core positivity was not prognostically significant. Immunohistochemistry was of no prognostic significance in either group. CONCLUSIONS: beta-core excretion appears to be useful in assessing prognosis of primary cervical cancer but not of recurrent disease. A large prospective study of urinary beta-core in early stage cervical cancer is needed to determine whether it can be used as an index for modifying treatment.     

The moderate affinity of clozapine at H3 receptors is not shared by its two major metabolites and by structurally related and unrelated atypical neuroleptics

We determined the affinity and/or potency of two metabolites of clozapine (clozapine-N-oxide and N-desmethylclozapine) and of five atypical neuroleptics, chemically related (olanzapine) or unrelated to clozapine (remoxipride, risperidone, thioridazine, zotepine), at H3 receptors. The specific binding of 3H-N alpha-methylhistamine to rat brain cortex homogenates was inhibited by the seven compounds; the pKi values were: N-desmethylclozapine (5.33); clozapine-N-oxide (4.18); olanzapine (5.45); thioridazine (4.91); zotepine (4.75); remoxipride (4.51) and risperidone (4.43). Three compounds were examined in a functional H3 receptor model as well. The electrically evoked tritium overflow from superfused mouse brain cortex slices, which represents quasi-physiological noradrenaline release, was not affected by N-desmethylclozapine (3.2 and 10 microM), clozapine-N-oxide (3.2-100 microM) and olanzapine (3.2-32 microM). On the other hand, the three compounds shifted to the right the concentration-response curve of histamine for its inhibitory effect on the evoked overflow; the apparent pA2 values were 5.84, 4.21 and 5.80, respectively. The present study shows that five atypical neuroleptics of different chemical classes and the two major metabolites of clozapine possess a lower affinity and/or antagonistic potency at H3 receptors than clozapine itself (pKi 6.15, pA2 6.33; Kathmann M, Schlicker E, G?thert M (1994). Psychopharmacology 116: 464-468).     

Genomic cloning and species-specific properties of the recombinant canine beta3-adrenoceptor

A molecular clone encoding a beta3-adrenoceptor was isolated from a canine genomic library. The cloned receptor exhibited a pharmacological profile similar to that of other species: in particular, high efficiency of the two selective beta3-adrenoceptor agonists, CL 316,243 (disodium(R,R)-5[2[[2-(chlorophenyl)-2hydroxyethyl]-amino]propyl]- 1,3-benzodioxole-2,2-dicarboxylate) and ICI 201651 ((R)4-(2-hydroxy-3-phenoxypropylaminoethoxy)-N-(2-methoxyethyl)phe noxy acetic acid) and a low affinity for the radioligand (-)-[3-(125)I]-iodocyanopindolol. Interestingly, CGP 12177A ((+/-)-4-(3-t-butylamino-2-hydroxypropoxy)benzimidazol-2-one), which is described as a partial agonist for the human receptor, was a full agonist for the canine receptor. After expression and stimulation of the canine beta3-adrenoceptor in stably transfected Chinese hamster ovary cells there was a very low accumulation of cAMP, suggesting weak coupling to Gs-protein and adenylyl cyclase. However, the response was much better in human embryonal kidney cells transfected with the canine beta3-adrenoceptor gene. The cloning of the canine beta3-adrenoceptor and the insights gained from its pharmacological characterization may allow the development of selective compounds for use in the treatment of obese dogs.     

The actions of some cannabinoid receptor ligands in the rat isolated mesenteric artery

Premature ovarian failure (POF) is a defect of ovarian development and is characterized by primary or secondary amenorrhea, with elevated levels of serum gonadotropins, or by early menopause. The disorder has been attributed to various causes, including rearrangements of a large "critical region" in the long arm of the X chromosome. Here we report identification, in a family with POF, of a gene that is disrupted by a breakpoint. The gene is the human homologue of the Drosophila melanogaster diaphanous gene; mutated alleles of this gene affect spermatogenesis or oogenesis and lead to sterility. The protein (DIA) encoded by the human gene (DIA) is the first human member of the growing FH1/FH2 protein family. Members of this protein family affect cytokinesis and other actin-mediated morphogenetic processes that are required in early steps of development. We propose that the human DIA gene is one of the genes responsible for POF and that it affects the cell divisions that lead to ovarian follicle formation.     

Anti-beta 2-glycoprotein I autoantibodies from patients with the "antiphospholipid" syndrome bind to beta 2-glycoprotein I with low affinity: dimerization of beta 2-glycoprotein I induces a significant increase in anti-beta 2-glycoprotein I antibody affinity

"Antiphospholipid" autoantibodies are associated with arterial and venous thrombosis, recurrent fetal loss, and thrombocytopenia. At present, the best-characterized antigenic target for these autoantibodies (or Abs) is the phospholipid-binding protein beta2-glycoprotein I (beta2GPI). These Abs bind beta2GPI only in the presence of negatively charged phospholipids or microtiter polystyrene plates that have been specially treated to give the surface a negative charge. To determine whether the binding of these Abs to beta2GPI on negatively charged surfaces is dependent on increased density or neo-epitopes formed as a consequence of a conformational change on beta2GPI, we generated mutants of beta2GPI by site-directed mutagenesis and assessed the binding characteristics of anti-beta2GPI Abs to these mutants. Our results demonstrate that mutant F307*, which spontaneously forms significant dimerization, is bound best by all the anti-beta2GPI Abs in an anti-beta2GPI ELISA using irradiated polystyrene microtiter plates. In addition, these Abs bound mutant F307* coated onto standard polystyrene microtiter wells in the absence of phospholipid, whereas there was minimal binding with wild-type and mutant F307*/C288A, which formed minimal dimerization. Affinity-purified anti-beta2GPI Abs from patients with the antiphospholipid syndrome demonstrated significantly higher binding affinity for mutant F307* in fluid phase than for wild-type or mutant F307*/C288A of beta2GPI. These results demonstrate that autoantibody binding to beta2GPI is intrinsically of low affinity and that the binding is dependent on the density of the Ag and not on neo-epitope formation.     

Characteristics of cyanopindolol analogues active at the beta 3-adrenoceptor in rat ileum

1. Cyanopindolol (CYP) is a potent antagonist at the beta 3-adrenoceptor in rat ileum. Several analogues of CYP and pindolol were synthesized that also produced antagonist effects at the beta 3-adrenoceptor. However, at high concentrations, these compounds appear to act as "partial agonists'. This study was conducted to determine the structural requirements of CYP analogues necessary for antagonist activity and to examine the possibility that the agonist effects of CYP and its analogues may occur through a mechanism independent of beta-adrenoceptor activation. 2. Analogues of CYP and pindolol were tested for antagonist activity in rat ileum in which the beta 1- and beta 2-adrenoceptors were blocked. Fourteen compounds were tested against (-)-isoprenaline, and four of the more potent analogues were then tested against BRL 37344. The two most potent antagonists were CYP and iodocyanopindolol. The pKb values (negative log of equilibrium dissociation constant) obtained against (-)-isoprenaline were significantly higher than those obtained against BRL 37344, but the cause of this difference is not known. 3. Several structural requirements were determined for antagonist activity. Modification at the carbon atom alpha to the secondary amine caused the antagonist potency to fall as the level of saturation was reduced. Thus, a quaternary carbon group, such as t-butyl, produced the most potent antagonist. Substitution with a large moiety such as a cyclohexyl or benzyl group reduced antagonist activity, probably due to steric hindrance. Inclusion of an electron-withdrawing group, such as a cyano or ethylester moiety, alpha to the indole nitrogen, also increased the potency. Iodination of CYP and ethylesterpindolol at the 3-position of the indole ring did not increase antagonist potency. In contrast, iodination of the almost inactive analogues produced a significant increase in potency, suggesting that a beneficial electronic effect on the indole ring imparted by the iodo moiety may be able to offset partially the negative effects caused by either the steric hindrance, of lack of a quaternary carbon alpha to the secondary amine. 4. Values for pseudo-pD2 were also determined by conducting cumulative concentration-response studies up to the limit of drug solubility. For nine of the compounds tested, the pKb was significantly higher than the pseudo-pD2 value. 5. The discrepancy between the pKb and pseudo-pD2 values was examined further. The agonist effects of iodocyanopindolol, the agonist with the highest potency, were not antagonized by CYP which was the most potent antagonist of (-)-isoprenaline and BRL 37344 at the beta 3-adrenoceptor. This suggests that the agonist effects of iodoCYP were produced through a different mechanism: either via another receptor, another isoform of the rat beta 3-adrenoceptor, or through a non-receptor-mediated effect. Pseudo-pD2 values did not correlate with log P values for these compounds, indicating that their relaxant effects were not simply a function of their lipid solubility. 6. This study has highlighted several structural requirements for antagonist binding potency at the rat ileum beta 3-adrenoceptor and should assist in the development of potent selective antagonists for this receptor.     

The effect of human beta2-microglobulin on major histocompatibility complex I peptide loading and the engineering of a high affinity variant. Implications for peptide-based vaccines

The ability to directly load cell surface major histocompatibility complex (MHC) class I molecules with peptides provides a potentially powerful approach toward the development of vaccines to generate cell-mediated immunity. We demonstrate that exogenous beta2-microglobulin (beta2m) stabilizes human cell surface MHC I molecules and facilitates their loading with exogenous peptides. Additionally, using three-dimensional crystal structures and known interaction sites between MHC I heavy chains and beta2m, we engineered variants of human beta2m (hbeta2m) with a single serine substitution at residue 55. This alteration was predicted to promote hydrophobic interactions at the MHC I heavy chain/beta2m interface and displace an ordered water molecule. Compared with hbeta2m, the serine to valine substitution at residue 55 had improved ability to bind to cell surface HLA-A1, HLA-A2, and HLA-A3 molecules, facilitate exogenous peptide loading, and promote recognition by peptide-specific T cells. The inclusion of hbeta2m or higher affinity variants when pulsing cells with MHC-restricted peptides increases the efficiency of peptide loading 50-80-fold. Therefore, the inclusion of hbeta2m in peptide-based vaccines may increase cell surface antigen densities above thresholds that allow recognition of peptide antigens by the immune system, particularly for cryptic, subdominant, or marginally antigenic peptides.     

Effects of glucagon on superior mesenteric artery and femoral artery haemodynamics in humans

OBJECTIVE: It remains unclear whether glucagon is a localized splanchnic arterial vasodilator in humans. This study examined this issue by assessing the haemodynamic effect of exogenous glucagon on splanchnic and extrasplanchnic arteries. METHODS: After an overnight fast, flow velocity of superior mesenteric artery and femoral artery was recorded by means of echo-Doppler in 10 controls and 10 patients with cirrhosis. Mean arterial pressure, heart rate and plasma glucagon level were also determined. These measurements were repeated after intramuscular injection of glucagon (1 mg) at 15 min and 30 min. RESULTS: Patients with cirrhosis had much higher glucagon levels than controls (P < 0.01). Plasma glucagon level rose following glucagon administration in controls (P < 0.01) and patients with cirrhosis (P < 0.01). Glucagon administration had no effect on mean arterial pressure, heart rate and femoral artery velocity in controls and patients with cirrhosis. In contrast, superior mesenteric artery velocity significantly increased after glucagon administration in both groups (P < 0.01, P < 0.01), although the effect was less pronounced in patients with cirrhosis than in controls (P < 0.05). CONCLUSION: These data suggest that glucagon might be a localized splanchnic arterial vasodilator. Thus, glucagon may be one of the factors contributing to the pathogenesis of the splanchnic hyperdynamic circulation seen in patients with cirrhosis.