Subcellular localization of neuronal nitric oxide synthase in rat small intestine.

The subcellular localization of neuronal nitric oxide synthase (NOS I, EC 1.14.13.39) was investigated in the longitudinal muscle/myenteric plexus (LM/MP) preparation of rat small intestine. The presence of NOS I, inducible nitric oxide synthase (NOS II), and endothelial nitric oxide synthase (NOS III) was assessed after homogenization and low-speed centrifugation in a postnuclear supernatant by immunological detection after PAGE and Western blotting. Only NOS I was clearly present, whereas NOS II and NOS III were below detection limits. After high-speed centrifugation of the postnuclear supernatant, soluble and particulate fractions were obtained, and the presence of NOS I in these fractions was investigated by measurement of NOS I immunoreactivity and enzyme activity. We found that 90 +/- 1% of NOS I immunoreactivity and 97 +/- 1% of NOS enzyme activity were confined to the soluble fraction of the tissue. Further immunological analysis demonstrated that washing the particulate fraction revealed detectable amounts of NOS I only after concentration of the washing supernatant. Most particulate NOS I remained in the pellet and therefore represents cell organelle-associated enzyme. No NOS I immunoreactivity could be detected as a soluble protein within organelles of the cell. Particulate NOS I could in part be solubilized by Triton X-100 treatment, and the detection of Triton X-100-soluble NOS I was dependent on the antibody used. In conclusion, our results indicate that NOS I in the LM/MP preparation of rat small intestine is mainly soluble and that the particulate NOS I is partly an intrinsic membrane protein and can partly be solubilized by detergent treatment.

Study of the muscarinic receptor subtypes in N1E 115 mouse neuroblastoma cells.

Muscarinic receptors in N1E 115 mouse neuroblastoma cells were characterized by competition binding experiments using three agonists and five antagonists, including 4-DAMP and AF-DX 116, and by studying the effect of agonist stimulation on the cellular cAMP and cGMP content. The results of the binding studies with the antagonists suggest that only one single homogeneous binding site of the M1 muscarinic receptor subtype is present. For the binding with the agonists, two binding sites were detected, one with high affinity for the ligand (between 53 and 77% of the total binding sites depending on the agonist) and one with low affinity. In contrast to the results obtained with the binding experiments using antagonists, the study of the cellular cyclic nucleotide response upon carbachol stimulation suggested the presence of both the M1 and M2 subtypes as there was an increase in cyclic GMP concentration while at the same time, the prostaglandin-stimulated synthesis of cyclic AMP was inhibited. Considering both binding and functional data we suggest that in N1E 115 cells a majority of M1 and a minority of M2 muscarinic receptors are present; there is no evidence for the presence of M3 muscarinic receptors.

Gradient distribution pattern of muscarinic receptors in N1E 115 mouse neuroblastoma cells.

The distribution pattern of muscarinic receptors in N1E 115 mouse neuroblastoma cells after linear and non-linear gradient centrifugation was investigated. In untreated cells, at least two forms of the receptors, with different densities, were found.

Glycan microheterogeneity of alpha 1-acid glycoprotein in sera and dialysates of patients on continuous ambulatory peritoneal dialysis.

Total concentration and concanavalin (Con A) dependent microheterogeneity of alpha 1-acid glycoprotein (AGP) were studied in sera of eight chronic renal failure patients before the start of continuous ambulatory peritoneal dialysis (CAPD), and in sera and dialysate fluids for up to 6 months of CAPD. The glycan heterogeneity of AGP in the samples was expressed as a reactivity coefficient, i.e. the ratio of the Con A-reactive AGP components to the Con A non-reactive AGP component. Concentrations of AGP in serum and reactivity coefficients were markedly elevated in non-dialysed uraemic patients. AGP concentrations in serum increased further during the first week on CAPD, and then gradually decreased to pre-dialysis values, which were reached after 1 to 6 mth. The reactivity coefficients did not change significantly during the CAPD treatment. Dialysate AGP concentrations were low in comparison to those in serum, and there was a good correlation between the reactivity coefficients in the dialysate fluids and those in the corresponding sera. The effect of peritonitis was evaluated in a separate group of eight CAPD patients. Serum and dialysate AGP concentrations were significantly higher during than after peritonitis while the corresponding reactivity coefficients were only slightly elevated.

Influence of differentiation on muscarinic receptors in N1E 115 neuroblastoma cells.

The effect of inducing morphological differentiation in N1E 115 mouse neuroblastoma cells on the number of muscarinic receptors and the ligand binding affinity was investigated using the lipophylic quinuclidinyl benzylate and the hydrophylic N-methylscopolamine as tritiated ligands. Induction of morphological differentiation was accompanied by a two- to three-fold increase of the number of receptors when assayed in a broken cell preparation; the ligand binding affinity was unaffected by differentiation. Using intact cells, this increase was not paralleled by a similar increase in binding sites accessible for N-methylscopolamine, which binds preferentially to extracellular sites.

Molecular forms of dopamine beta-hydroxylase in rat superior cervical ganglion and adrenal gland.

Dopamine beta-hydroxylase (DBH) enzyme activity was associated in rat superior cervical ganglion with tetrameric DBH-A (294,000 D) and dimeric DBH-B (147,000 D) and in rat adrenal gland with DBH-A and a novel molecular form of DBH, defined as DBH-C, with a molecular weight of 125,000 D. Pretreatment of the rats with cycloheximide markedly reduced DBH activity without altering the molecular heterogeneity.

Influence of inflammation on serum concentration, molecular heterogeneity and drug binding properties of canine alpha-1-acid glycoprotein.

The concentration and the heterogeneity of alpha-1-acid glycoprotein (alpha-1-AGP) and oxprenolol binding were determined in serum of healthy dogs and dogs with inflammatory disease. In inflammation, an increase in the mean alpha-1-AGP concentration from 0.47 to 2.85 g/l was accompanied by a reduction in the mean free oxprenolol fraction from 25% to 6%. alpha-1-AGP concentration and oxprenolol binding were inversely correlated. The heterogeneity of canine alpha-1-AGP remained essentially unchanged in dogs with inflammation and, in both these dogs and the controls, between five and seven forms with different isoelectric points and one single concanavalin A-reactive form were detected. It is concluded that in dogs, as in humans, oxprenolol binds to serum alpha-1-AGP. Changes in serum binding of oxprenolol during inflammation result from a change in the serum concentration of alpha-1-AGP rather than a change of molecular heterogeneity.

Influence of continuous ambulatory peritoneal dialysis on serum alpha 1-acid glycoprotein concentration and drug binding.

The influence of continuous ambulatory peritoneal dialysis (CAPD) on the concentrations of alpha 1-acid glycoprotein in serum and dialysate and on the serum binding of oxprenolol, propranolol and phenytoin has been studied. Before starting CAPD treatment, the serum binding of oxprenolol and propranolol was higher and that of phenytoin lower than in healthy volunteers, and the serum alpha 1-AGP concentration was higher. During the first days to weeks after starting CAPD, the serum alpha 1-AGP concentration rose with a concomitant increase in the binding of oxprenolol and propranolol. Subsequently, the alpha 1-AGP level and the binding of oxprenolol and propranolol decreased to the values found before starting CAPD. The binding of phenytoin showed little change. The concentration of alpha 1-AGP in dialysate was 2 to 5% of that in serum.

A monoclonal antibody-based enzyme-linked immunosorbent assay of alpha 1-acid glycoprotein.

A "sandwich"-type enzyme-linked immunosorbent assay for determining concentrations of human alpha 1-acid glycoprotein (AGP) is described. Microtiter plates coated with a polyclonal rabbit antibody to human AGP were subsequently incubated with the antigen, with a specific murine monoclonal antibody, and with goat anti-mouse immunoglobulins conjugated to alkaline phosphatase. To evaluate the method for assay of AGP in human sera, we compared it with single radial immunodiffusion and "rocket" electroimmunoassay. The respective correlations were r = 0.988 (n = 45) and r = 0.973 (n = 47). Repeated assays of a human serum sample with an average AGP concentration of 859 mg/L yielded within-day and between-day CVs of 1.4% (n = 5) and 6.3% (n = 10), respectively. Because of its low detection limit (4.4 micrograms/L), this assay is also suitable for determination of AGP concentrations in other biological fluids, such as dialysates of patients being treated by continuous ambulatory peritoneal dialysis.

Dog alpha-1-acid glycoprotein: purification and biochemical characterization.

Dog alpha-1-acid glycoprotein was purified to homogeneity from dog serum in a three-step procedure involving precipitation with sulphosalicylic acid, isoelectric focusing and size exclusion chromatography. The molecular heterogeneity in the peptide part and in the carbohydrate part of the molecule was investigated with analytical isoelectric focusing in a narrow pH range and crossed immunoaffinity electrophoresis with concanavalin A (con A) in the first-dimension gel. Up to seven molecular forms with different isoelectric points were found, whereas only a single con A-dependent molecular form was detected.

A study of the heterogeneity of human alpha 1-acid glycoprotein with monoclonal antibodies.

Monoclonal antibodies (MABS) were raised against human alpha 1-acid glycoprotein (AGP) and their reaction with the polymorphic forms of this plasma protein were evaluated. Spleen cells of BALB/c mice, immunized with native or desialylated human AGP, were fused with NSO mouse myeloma cells. The hybridoma products were screened with a direct ELISA test, in which the immunoplates were coated with a mixture of native and desialylated AGP. In this test, 14 anti-AGP antibody producing clones were retained. Coating the wells with either native or desialylated AGP showed that eleven clones reacted with both types of AGP ('Type I' MABS), while three MABS reacted specifically with the desialylated form ('Type II' MABS). Precoating the immunoplates with polyclonal anti-human AGP followed by incubation with native or desialylated AGP before the addition of hybridoma supernatant (indirect ELISA), confirmed the specificities observed in the direct ELISA. The molecular heterogeneity of both native AGP and desialylated AGP, based on Concanavalin A reactivity and isoelectric point, was not reflected by any specificity in the antibody reactions. Thus 'Type I' MABS reacted with all molecular forms present in native and desialylated AGP while 'Type II' MABS reacted with all molecular forms present in desialylated AGP.

Release of dopamine, noradrenaline and dopamine B-hydroxylase from mouse neuroblastoma.

The murine C1300 neuroblastoma tumor was found to secrete dopamine, noradrenaline and dopamine B-hydroxylase into the circulation of tumor-bearing A/J mice. The plasma levels of dopamine, noradrenaline and dopamine B-hydroxylase increased with the size of the tumor, and the increase in noradrenaline paralleled the increase in dopamine B-hydroxylase (r = 0.86). The vesicular storage of dopamine and noradrenaline in the tumor was evidenced by a decrease of the tissue content of dopamine and noradrenaline 24 hours after the administration of reserpine (5 micrograms/g) respectively to 17.6% and 7.8% of control values. A similar observation could be made for the levels of dopamine and noradrenaline in the plasma of reserpinized C1300 mice. The total activity of dopamine B-hydroxylase in the tumor and in plasma was unaffected by the reserpine treatment. Chronic administration of 6-hydroxydopamine (100 micrograms/g for 8 days) had no effect on the tissue contents of dopamine, noradrenaline or dopamine B-hydroxylase. The release of catecholamines and dopamine B-hydroxylase from the C1300 neuroblastoma was studied in vitro on superfused tumor slices. Stimulation of these slices with 56 mM KC1 or with 5.10(-5) M tyramine failed to induce the release of endogenous dopamine, noradrenaline or dopamine B-hydroxylase above the basal outflow levels. These results are suggestive for a non-exocytotic release of catecholamines and dopamine B-hydroxylase from the neuroblastoma tumor.

Multiple molecular forms of dopamine beta-hydroxylase in the C1300 mouse neuroblastoma tumor and in the serum of tumor-bearing mice.

The distribution of the enzymatic activity of dopamine beta-hydroxylase (DBH) in linear sucrose gradients was studied for a soluble fraction of the C1300 mouse neuroblastoma tumor, for the serum of tumor-bearing A/J mice, and for adrenal tissue and serum of control mice. In controls (adrenal gland and serum of A/J mice), about 75% of the DBH activity was associated with a high-molecular-weight form, denoted as DBHA, with an apparent sedimentation coefficient of 11.3 S. About 25% of the DBH activity was attributable to a slower-sedimenting species (7.1 S), denoted as DBHB. In tumor supernatants and in the serum of tumor-bearing mice, about 55% of the DBH activity was present as the 7.1 S species (DBHB), while only 35% was recovered as the high-molecular-weight form (DBHA). Approximately 5% of the activity could be attributed to a separate form, with a sedimentation coefficient of about 4.5 S. This form is designated DBHC. The ratio DBHB/DBHA is significantly higher in tumor tissue and in serum of tumor-bearing mice than in controls. The three enzymically active forms of DBH in the C1300 tumor are considered to represent the tetrameric (DBHA), dimeric (DBHB), and monomeric (DBHC) forms of the enzyme.

Immunohistochemical evidence for a plasma membrane localization of dopamine-beta-hydroxylase in the mouse neuroblastoma.

Dopamine-beta-hydroxylase (D beta H), a glycoprotein enzyme which converts dopamine into noradrenaline, was purified from C1300 mouse neuroblastoma and used to raise antibodies in rabbits. Using an indirect immunofluorescence technique the cellular localization of D beta H in C1300 mouse neuroblastoma was compared with that of the superior cervical ganglion. C1300 neuroblastoma D beta H was found to be predominantly localized in the plasma membrane, in contrast to its intracellular localization in the superior cervical ganglion of A/J mice. At least part of the enzyme was found to be associated with the external side of the plasma membrane.

Subcellular localization of noradrenaline and ATP in C1300 mouse neuroblastoma.

A density and velocity gradient centrifugation study of C1300 mouse neuroblastoma showed that ATP is nearly absent from noradrenaline-containing granules and is mainly localized in mitochondria, suggesting that in this tissue ATP is not involved in the storage of noradrenaline.