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The developments of bio-analytical methods for analyzing bioactive peptides are of paramount importance. Neuropeptides and their bioactive fragments play a vital role in the regulation of many biological processes and diseases. This paper presents the use of matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) method for monitoring neuropeptides along with their degradation products in plasma samples from cancer patients. The neuropeptides focused in this study were beta-endrophin, substance P, and bradykinin. The method involves the enzyme digestion of the neuroactive peptides followed by MALDI-MS sample preparation and subsequent acquisition of the MS spectral data. The mass spectral profile identifies most of the C-terminal and N-terminal peptides, and the mass accuracy was in the range of -1.68 to 1.46 Da with the mass spectrometer utilised. Analysis of the neuropeptide degradation patterns from the cancer patients were compared with the controls showed similar results. The study reveals that this approach can be used to identify the enzymatic digestion products of protein.

We evaluated roles of kinins in allergen-induced nasal blockage and sneezing, and development of nasal hyperresponsiveness to leukotriene D4 in a Japanese cedar pollen-induced allergic rhinitis model of guinea pigs. Sensitised guinea pigs were repeatedly challenged by pollen inhalation once every week. Neither a bradykinin B1 receptor antagonist, des-Arg9-[Leu8]bradykinin nor a bradykinin B2 receptor antagonist, icatibant suppressed allergen-induced sneezing and nasal blockage. However, development of nasal hyperresponsiveness to leukotriene D4 was significantly suppressed by them. The amount of bradykinin in nasal cavity lavage fluid was immediately increased after the challenge. In non-sensitised animals, hyperresponsiveness to leukotriene D4 was developed by a bradykinin B2 receptor agonist, bradykinin, but not by a bradykinin B1 receptor agonist, des-Arg10-kallidin, while in the sensitised-challenged animal, both agonists developed hyperresponsiveness. In conclusion, the nasal hyperresponsiveness appeared to be induced by kinins produced in response to the antigen challenge through activation of not only bradykinin B2 but also B1 receptors.

The peptide hormone bradykinin is a mediator in many physiological and pathological processes. The generation and, to a limited extent, metabolism occur at the sites of action. The short half-life of bradykinin (approximately 15 s) renders measurements of its concentration in bodily fluids difficult. As a result, recent methods utilizing either ELISA or HPLC/mass spectrometry, have been developed for the measurement of the stable metabolic endproduct of bradykinin, i.e., the pentapeptide Arg-Pro-Pro-Gly-Phe (RPPGF; BK[1-5]). Both have been successfully used to quantitate levels of RPPGF in plasma, pulmonary and nasal exudates. In this study, we demonstrate the characterization and utility of a radioimmunoassay for the measurement of RPPGF, using a newly synthesized radioiodinated analogue of RPPGF, i.e., RPPG(125I)F. This radioimmunoassay shows an IC50 of 80.5 +/- 7.4 pg/tube (n = 23) with a linear range of detection between 10 and 500 pg/tube. The assay was used to demonstrate the time-dependent generation of RPPGF by cultured rat aortic vascular smooth muscle cells exposed to bradykinin (100 ng/ml). Peak generation of RPPGF was 74.5 +/- 9.7 pmol/well (n = 5) after 24 h of incubation. Captopril, an inhibitor of angiotensin-converting enzyme (kininase II), inhibited generation in a concentration-dependent manner. The results characterize a new radioimmunoassay for the stable metabolic endproduct of bradykinin, RPPGF.

Objectives: To determine the anatomic distribution of select neuropeptides (neurokinin A [NKA], substance P [SP], and bradykinin [BK]), of inflammatory cells (leukocytes and mast cells), and the histamine content in the normal swine ureter and compare the findings with regions of increased ureteral contractility., Methods: Ureters from 10 pigs were obtained and cut into eight segments, proximally to distally. A portion of each ureteral segment was suspended in Krebs buffer (37 degrees C) and attached to force displacement transducers, and spontaneous contractility was measured for 30 minutes. A second portion was assayed for histamine, NKA, SP, and BK using enzyme-linked immunosorbent assay. A third portion was fixed in 10% buffered formalin, stained with hematoxylin-eosin, and evaluated histologically., Results: Ureteral contractility was found to be highest in the most proximal and most distal regions of the ureter. Similarly, SP content was three times greater in the proximal ureter and two times greater in the distal ureter than in the midureter (P <0.05, n = 10). The total NKA and BK content were also higher in the proximal and distal ureter than in the midureter. Conversely, the histamine content was consistent throughout the ureter. Moreover, no significant difference in the distribution of inflammatory cells was identified throughout the ureter., Conclusions: The anatomic distribution of NKA, SP, and BK in the ureter corresponded to regions of increased spontaneous ureteral contractility, more specifically the proximal and distal ureter. Neuropeptides may play a significant role in ureteral contractility and may be a target for pharmacologic mediation during obstruction and stone passage.

We developed an enzyme immunoassay (EIA) specific for Arg1-Pro2-Pro3-Gly4-Phe5 ([1-5]-BK) for determination of the levels of this peptide in biological fluids. Previously developed EIAs for bradykinin (BK) and for des-Phe8-Arg9-BK ([1-7]BK) were also used. Incubation of rat plasma with glass powder resulted in the transient appearance of BK. A degradation product, [1-7]BK, could be detected in the incubation mixture for a longer period of time. When compared with BK and [1-7]BK, a larger amount of [1-5]BK was detectable even longer. In carrageenan-induced pleurisy in rats, which was associated with a peak rate of plasma exudation 5 hr after administration of carrageenan, BK was undetectable (< 160 pg/rat) in the pleural exudates. By contrast, [1-7]BK was detectable over the entire course of the inflammatory response. A larger amount of [1-5]BK was detectable. The peak level of [1-5]BK was 6050 +/- 1050 pg/rat, 5 hr after administration of carrageenan. Inhibition of the generation of BK by intrapleural administration of soy bean trypsin inhibitor (0.3 mg/rat) 30 min before collection of pleural fluid resulted in significant reductions in the levels of both [1-7]BK (by 51-65%) and [1-5]BK (by 63-79%) in the exudates 3, 7 and 19 hr after administration of carrageenan. Intraperitoneal administration of captopril (10 mg/kg) caused a marked reduction (by 98%) in levels of [1-5]BK in exudates 3 hr after administration of carrageenan. The reduction was accompanied by an increase in the level of BK up to 1250% of that in untreated rats. These results indicate that the newly developed EIA for [1-5]BK might be a useful tool for verifying the release of kinin in vivo.

Splenocytes from mice immunized either with bradykinin conjugated with carbodiimide to keyhole limpet hemocyanin or ovalbumin were fused using polyethylene glycol with the mouse myeloma cell line SP2/o. Nine monoclonal antibodies reactive with kinins were obtained from two fusions. All of the antibodies were of the IgG1k isotype, except for one, which was an IgG2ak. Based on their reactivities with biologically active kinins and biologically inactive degradation products, the antibodies were separated into three groups. The first group, which had the highest affinities for bradykinin, displayed about equal reactivities for bradykinin and des-Arg9-bradykinin, but little reactivities for the kinin fragments, des-Arg1-bradykinin and des-Phe8-Arg9-bradykinin, or for lysyl-bradykinin and methionyl-lysyl-bradykinin. The second group was similar to the first except that it showed about a 2.5- to 3.5-fold greater reactivity for des-Arg9-bradykinin than for bradykinin. The third group, which had the lowest affinities for bradykinin [50% inhibition of antibody binding to an enzyme-linked immunosorbent assay (ELISA) plate occurring with bradykinin concentrations ranging from about 8 to 39 nM], showed little reactivities with des-Arg1-bradykinin, des-Arg9-bradykinin and des-Phe8-Arg9-bradykinin, but 50-100% cross-reactivities with lysyl-bradykinin and methionyl-lysyl-bradykinin. The useful ranges for bradykinin detection (ng/well, 50 microL assay volume) using the highest affinity antibody in each group in ELISAs were: 0.01 to 0.5, 0.03 to 3, and 0.1 to 3 for groups 1, 2, and 3, respectively.

1. The venom of the solitary scoliid wasp Colpa interrupta (F.) shows a kinin-activity, when tested on a cascade of mammalian smooth muscle preparations, and, in addition, a contraction of the rat colon. 2. The venom also irreversibly blocks the nicotinic synaptic transmission from the cercal nerve to a giant interneuron in the sixth abdominal ganglion of the cockroach, Periplaneta americana. 3. The same activities have been found within one HPLC fraction. 4. However, rechromatography of this fraction resulted in four subfractions being active on smooth muscles. 5. One fraction caused contraction of the colon, three other fractions contained kinin-activity. 6. Only the most active kinin fraction blocked synaptic transmission in the insect CNS. 7. This fraction contained threonine-bradykinin. 8. Synthetic Thr-bradykinin causes irreversible presynaptic activation-induced block of transmission in the insect CNS.

1. The presence of smooth muscle contracting substances in venom preparations made from six species of sphecid wasps was investigated using a number of standard pharmacological techniques. 2. In addition, acetylcholine was identified by enzymatic degradation as well as by mass fragmentography, while histamine was determined using a radio-enzymatic method. 3. The venom reservoir of P. triangulum contains about 400 ng acetylcholine, but no histamine-, 5-HT- or bradykinin-like activity. 4. The venoms of the five other species contain histamine in amounts varying from 20 to 2000 ng per venom reservoir, but no acetylcholine-, 5-HT- or bradykinin-like activity.

Extracts prepared from dried or fresh skins of more than 200 American amphibian species were subjected to biological screening in order to determine occurrence and contents of peptides active on smooth muscle preparations, systemic blood pressure and, subordinately, external secretions, anterior pituitary and the central nervous system. The peptide families identified in skin extracts were as follows: caruleins (caerulein, phyllocaerulein), tachykinins (physalaemin, phyllomedusin), bombesins (phyllolitorin, [Leu8]phyllolitorin, rohdeilitorin), bradykinins (phyllokinin and others), sauvagine, dermorphins (dermorphin, [Hyp6]dermorphin), tryptophyllins (numerous peptides) and, finally, miscellaneous peptides. None of the above peptide families showed a widespread distribution, but all were restricted to particular amphibian genera or stocks. The hylid frogs of the Phyllomedusinae family occupy a unique position, as their skin displayed the greatest variety and abundance of active peptides ever found in any amphibian is destined to increase because numerous other peptide molecules await isolation, elucidation of structure and definition of possible biological activities.

In the present investigation the hemolytic properties of citral were examined. Tests with different concentrations of citral showed that hemolysis of rat erythrocytes commenced after a lag period the length of which depended on the concentration of the hemolysin and tended to 100% hemolysis. Comparison of the characteristics of the hemolysis induced by high and low citral concentrations, indicated that two mechanisms are involved--a non specific steroid--terpenoid or glutathione depletion mechanism dominating at high citral concentrations and a free radical mechanism dominating at low citral concentrations. Experiments performed with various free radical scavengers indicate that 1O2 might be involved.

To determine the effective time of indomethacin (Id) action in disrupting early pregnancy, groups of rabbits were treated at various stages after mating with Id and/or replacement doses of PGF2 alpha. Effect of Id on the secretion of uterine proteins was also investigated. The highest rate of embryonic death occurred when Id was injected twice daily during days 5-7 of pregnancy, and days 5-6 appear to be the most critical. Persisting levels of Id during this critical stage is required to induce the antifertility effect. The results also indicate that the adverse effect of Id is not mediated through an effect on uterine proteins, and is mainly due to the inhibition of PG's synthesis. PG's are apparently required for normal implantation and early development.