[en] The distribution and effects of the two neuropeptides, vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine amide (PHI), on vascular and nonvascular smooth muscle in the urogenital tract of nonpregnant rabbit female, were investigated. Immunoreactive VIP and PHI were present in all regions except the ovary with the highest concentration in the uterin cervix. By using in vitro tension recordings of myometrial specimens, it was demonstrated that both peptides displayed a dose-dependent inhibition of the mechanical activity. The dose-response curves of VIP and PHI were superimposable with and ID₅₀ of 3 x 10^-8 mol/l, and their combined effect was additive. In addition, the influence of the two peptides on myometrial blood flow (MBF) was investigated by the xenon-133 washout technique. Both peptides were found to increase MBF with the same potency and efficacy. Their combined effect was additive. In conclusion VIP and PHI are present in the rabbit urogenital tract, and the two peptides are equipotent inhibitors of mechanical nonvascular and vascular smooth muscle activity in the uterus

[en] Pancreastatin, a 49-amino acid peptide with a COOH-terminal glycine amide, was originally isolated from porcine pancreas, but pancreastatin immunoreactivity has been found in several neuroendocrine tissues. There are strong indications that pancreastatin is derived from chromogranin A, since the amino acid sequence 240-288 in porcine chromogranin A corresponds to pancreastatin flanked by typical signals for proteolytic processing. The authors studied the effect of electric stimulation of the nervous supply to perfused porcine pancreas, antrum, nonantral stomach, and small intestine on the release of immunoreactive pancreastatin, and they have characterized the molecular nature of the secreted immunoreactivity by using a radioimmunoassay specific for the COOH-terminal glycine amide of porcine pancreastatin in combination with chromatography. In all tissues nerve stimulation significantly increased the release of immunoreactive pancreastatin. The secreted immunoreactive pancreastatin was heterogeneous, consisting of pancreastatin itself, a COOH-terminal pancreastatin fragment, and NH₂-terminally extended pancreastatin forms. Pancreastatin predominated in the perfusate from pancreas and antrum, whereas mainly NH₂-terminally extended molecular forms were secreted from the antrectomized stomach and small intestine. The different molecular forms of pancreastatin were secreted from the perfused organs in the same molar ratio as they occur in extracts of the corresponding tissues. Thus, pancreastatin and other chromogranin A-derived peptides in organ-specific proportions regularly accompany the secretion of the peptide hormones from the gastrointestinal tissues on appropriate stimulation. 40 refs., 5 figs

[en] A sensitive and specific radioimmunoassay for vasoactive intestinal polypeptide (VIP) has been developed, which can detect 3.3 pmol x L^-1 of the peptide in plasma. Antisera to highly purified porcine VIP coupled to albumin were raised in eight rabbits. The final dilution, the avidity, and the specificity of each antiserum were determined. ¹²⁵I-VIP served as label, and highly purified porcine VIP was used as standard. Separation of antibody-bound and free VIP was achieved by plasma-coated charcoal. Nonspecific interference with the assay system was excluded by extraction of plasm samples with ethanol. The reliability of the assay was investigated by recovery experiments, by serial dilution of plasma samples with high concentration of endogenous VIP, and by immunosorption. The within-and between assay reproducibility at a concentration of 18.3 pmol x L^-1 was 1.6 and 2.3 pmol x L^-1 (1 S.D.), respectively. Median fasting concentration of VIP in plasma from 74 normal subjects was 7.3 pmol x L^-1

[en] Synthetic porcine secretin was labelled by the conjugation-labelling method of Bolton and Hunter, the lactoperoxidase method, the gaseous diffusion method and the chloramine-T method. The chloramine-T technique was adapted as routine method. Ten μg (3.27 nmol) peptide was reacted with 5 mCi of Na¹²⁵I at a concentration of chloramine-T of 1.3 mmol/l. Synthetic secretin was suitable for labelling for at least eight months when stored as dry matter in nitrogen-filled glass ampoules. Purification and separation of labelled from unlabelled hormone was carried out by gel-permeation chromatography on Sephadex G-50 superfine. The labelled preparation had a specific radioactivity of 405π+33 μCi/nmol (mean π+S.E.M., n=9) and was usable for six weeks. 6-tyrosyl-secretin took up more iodine compared to porcine synthetic secretin but had lower immunoreactivity with all antisera tested. (Auth.)

[en] The chloramine-T method for radioiodination of neurotensin for radioimmunoassay was studied. As conventional procedures produced heterogeneous preparations, labelling was performed with a low amount of chloramine-T (1.8 nmol) in the presence of excess of peptide (6 nmol). Purification and complete separation of labelled from unlabelled peptide was obtained by ion-exchange chromatography on SP Sephadex C-25. Four labelled components were identified by isoelectric focusing, enzymatic cleavage and studies of immunoreactivity. The two components representing monoiodinated preparations labelled at Tyr 3 or Tyr 11 could be isolated. Depending on the binding site of the particular antiserum the appropriate tracer could be selected for use in the radioimmunoassay. The specific radioactivities were high (2303 (2137-2407) μCi/nmol and 1927 (1608-2307) μCi/nmol (median and range)) and the stability of the label and the reproducibility of the procedure was good. (author)

[en] Vasoactive intestinal peptide (VIP) bound with high affinity (Kd 0.13 nmol/l) to receptors on the human glioma cell line U-343 MG Cl 2:6. The receptors bound the related peptides helodermin, PHM and secretin with 10, 400 and 5000 times lower affinity, respectively. Deamidated VIP (VIP-COOH) and [des-His1]VIP bound with 10 and 100 times lower affinity. The fragment VIP(7-28) displaced 25% of the receptor-bound ¹²⁵I-VIP whereas VIP(16-28) and VIP(1-22-NH2) were inactive. The binding of ¹²⁵I-VIP could be completely inhibited by 10 mumol/l of the antagonists [N-Ac-Tyr1,D-Phe2]GRF(1-29)-NH2, [pCl-D-Phe6,Leu17]VIP and VIP(10-28); in contrast, the antagonist L-8-K was inactive. Affinity labeling showed that VIP bound to proteins with Mr's of 75 kDa, 66 kDa and 50 kDa, respectively. Following binding, the peptide was rapidly internalized, and at steady-state only 20% of cell-associated ¹²⁵I-VIP was bound to receptors on the cell surface. The internalized ¹²⁵I-VIP was completely degraded to ¹²⁵I-tyrosine which was released from the cells. Degradation of internalized ¹²⁵I-VIP was significantly reduced by chloroquine phenanthroline and pepstatin-A. Surface binding and internalization of ¹²⁵I-VIP was increased 3 times by phenanthroline, and pepstatin-A caused a 5 times increase in surface binding. Chloroquine reduced surface-bound ¹²⁵I-VIP, but caused retention of internalized ¹²⁵I-VIP

[en] A sensitive and specific radioimmunoassay for the recently isolated neuropeptide with N-terminal histidine and C-terminal isoleucine amide (PHI) has been developed which can detect 3.0 pmol/l of the peptide in plasma. Labelling of PHI with ¹²⁵I was performed by the chloramine T method. Non-specific interference in the assay was excluded by extraction of plasma with ethanol to a mean recovery of 82.4%. Plasma samples diluted parallel to the standard curve. The intra-assay and inter-assay coefficient of variation (CV) values at a level of 24.0 pmol/l were 6.3% and 13.1%, respectively. In 75 normal adults, the fasting PHI concentration ranged from 3.5-30.0 pmol/l with a mean of 14.2 pmol/l. In 235 children, the PHI concentration varied with age. Ingestion of a meal caused a rapid and short-lived increase in the PHI concentration. (Auth.)

[en] In order to produce antibodies for radioimmunochemical analysis of secretin in plasma, 16 rabbits were immunized; two different immunization schemes were followed. All the rabbits produced detectable antibodies to secretin. The final dilution of the antisera necessary to bind 50% of 5 fmol [¹²⁵I]secretin varied from 1:5,000 to 1:2,300,000. The avidity of antisera, expressed by the equilibrium constant, ranged from 5 .10⁶ l . mol^-1 to 3 . 10¹¹ l . mol^-1. Six rabbits produced antisera that displayed avidity sufficient for measurement of the physiologic concentrations of secretin in plasma. The equation of Sips was used to evaluate the heterogeneity index of the antibodies. Seven of the 16 antibodies to secretin had a heterogeneity index of 0.90 or more. Cross-reactivity with structurally related peptide hormones (glucagon, vasoactive intestinal peptide, gastric inhibitory peptide) was found to be negligible for the six antisera examined (those with the highest avidity). Three of the high-avidity antisera were examined for reactivity with secretin fragments. They all bound the C-terminal secretin tricosa and tetradecapeptides almost as well as pure natural secretin, whereas the reactivity with the N-terminal tetradecapeptide was slightly lower. (Auth.)

[en] Highlights: ► Restricted Neuroglobin expression in the mouse retina. ► Antibody validation using Neuroglobin-null mice. ► Co-expression of Neuroglobin with Melanopsin and tyrosine hydroxylase. ► No effect of Neuroglobin deficiency on neuronal survival. -- Abstract: Neuroglobin (Ngb), a neuronal specific oxygen binding heme-globin, reported to be expressed at high levels in most layers of the murine retina. Ngb’s function is presently unknown, but based on its high expression level and oxygen binding capabilities Ngb was proposed to function as an oxygen reservoir facilitating oxygen metabolism in highly active neurons or to function as a neuroprotectant. In the present study, we re-examined the expression pattern of Ngb in the retina using a highly validated antibody. Furthermore, intactness of retino-hypothalamic projections and the retinal expression level of Melanopsin and Tyrosine Hydroxylase were investigated in Ngb-null mice. Ngb-immunoreactivity was found in a few neurons of the ganglion cell and inner nuclear layers co-expressing Melanopsin and Tyrosine Hydroxylase, respectively. Ngb deficiency neither affected the level of Melanopsin and Tyrosine Hydroxylase proteins nor the intactness of PACAP-positive retinohypothalamic projections in the suprachiasmatic nucleus. Based on the present results, it seems unlikely that Ngb could have a major role in retinal oxygen homeostasis and neuronal survival under normal conditions. The present study suggests that a number of previously published reports have relied on antibodies with dubious specificity.