Immunoreactive tachykinins in 24-h collections of urine from patients with carcinoid tumours: characterization and correlation with plasma concentrations.

Tachykinins are a family of peptides that may be present in and secreted from carcinoid tumours of mid-gut origin. They are likely to play a role in the pathogenesis of, e.g. the flush, dyspnoea and valvular heart disease seen in the carcinoid syndrome. Since tachykinins are secreted from the tumour into the circulation in bursts, coinciding with flushing attacks, and have short half-lives, we anticipated that analysis of 24-h urine excretion of immunoreactive tachykinin metabolites might prove to be a more sensitive and stable parameter for monitoring than tachykinin-like immunoreactivity in plasma. The study included 48 patients hospitalized for treatment of advanced carcinoid tumours and 32 healthy controls. The urine excretion of tachykinin-like immunoreactive metabolites in the carcinoid patients (median 27.5 pmol 24 h-1, interquartile range (IQR) 8.5-51.0 pmol 24 h-1) was significantly (p<0.001) higher than that in the 32 healthy subjects (median 3.0 pmol 24 h-1, IQR 0.9-4.20 pmol 24 h-1). Of the patients, 38 (79%) had elevated 24-h urine excretion of tachykinin-like immunoreactive metabolites while 31 (64%) had elevated plasma concentrations of tachykinin-like immunoreactive metabolites. Of the patients, 27 (56%) had elevated concentrations of tachykinin-like immunoreactive metabolites both in plasma and urine, 12 (25%) had elevated concentrations only in urine excretion, 3 (6%) had elevated concentrations of only plasma tachykinin-like immunoreactive metabolites and 7 (14%) had elevation of neither plasma nor urine concentrations. Analysis by means of different column chromatographic techniques indicated that the immunoreactive material was heterogeneous, with some components co-eluting with oxidized neurokinin A (NKA) and neuropeptide K (NPK). The urine tachykinin-like immunoreactivity correlates well with that of plasma, but is a slightly more sensitive indicator of elevated tachykinin-like immunoreactivity, probably since levels of urine tachykinin-like immunoreactive metabolites reflect the overall amount of the latter secreted into the circulation during 24 h.

Occurrence, release, and effects of multiple tachykinins in cat colonic tissues and nerves.

Neurokinin A (NKA)-immunoreactivities and substance P (SP)-immunoreactivities were found in picomolar amounts in colonic tissues and almost an order of magnitude higher amounts in vagal, pelvic, splanchnic, and lumbar colonic nerves of the cat. Continuous electric stimulation of the pelvic nerve at 4 Hz or intermittent electric burst stimulation of the pelvic nerve at 40 Hz during 1 second with 10-second rest periods produced a marked release of NKA-like immunoreactivity (NKA-LI) and SP-LI from the colon to blood (P less than 0.001). Reflex activation of the pelvic nerve by mechanical stimulation of the anus or rectal distension produced a less pronounced release of NKA-LI and SP-LI from the colon to blood (P less than 0.01). There was a simultaneous colonic contraction and vasodilation during each nerve stimulation. Reverse-phase high performance liquid chromatography showed presence of NKA, NKA oxide, NKA (3-10)/NKA (4-10), and neuropeptide K (NPK) in colonic tissues and release of all these molecular forms except NPK on nerve stimulation. Substance P and SP oxide were present both in colonic tissue extracts and in released material. Close intraarterial infusions of NKA, neurokinin B, SP, NPK, eledoisin, and physalaemin at doses of 0.1-100 pmol/min induced dose-dependent contractions of the proximal and distal colon (P less than 0.001) and vasodilatation (P less than 0.001), NKA being the most potent. The effects of the tachykinins were reduced after tetrodotoxin (P less than 0.05) and atropine (P less than 0.05) but unchanged after treatment with hexamethonium. Our findings indicate that tachykinins are released from the pelvic nerve to induce a nonadrenergic noncholinergic contraction and vasodilatation of the colon in the cat.

Enzyme immunoassay for tachykinin-like immunoreactivity in the guinea pig spinal cord.

A solid-phase enzyme immunoassay for quantitation of tachykinin-like immunoreactivity (TK-LI) is presented. Because the antiserum K-12 recognizes various tachykinins, such as neurokinin A (100%), kassinin (103%), eledoisin (51%), neurokinin B (18%), physalaemin (0.7%), and substance P (0.7%), the immunoreactivity detected in this enzyme immunoassay has been termed TK-LI. The assay was performed on 96-well microtiter plates coated with a mouse monoclonal second antibody. After preincubation of soluble neurokinin A or samples and K-12 antiserum for 3 h at room temperature, acetylcholinesterase-labelled neurokinin A was allowed to react overnight at 4 degrees C. Samples were finally incubated with Ellman's reagent for 2 h and the absorbance was measured at 414 nm. The threshold for detection of TK-LI was 2 fmol/well. TK-LI release from guinea pig dorsal spinal cord slices was evoked by capsaicin or high K+ medium. The capsaicin-evoked TK-LI release was increased in the presence of thiorphan, but not in that of captopril.

Facile analysis and purification of deblocked N-terminal pyroglutamyl peptides with a strong cation-exchange sulfoethyl aspartamide column.

A high-performance strong cation-exchange Sulfoethyl Aspartamide column was used to analyze and purify five N-terminal pyroglutamyl peptides after treatment with Pyroglutamate Aminopeptidase. The resulting deblocked N-1 peptides possess an increased positive charge and are therefore retained to a greater extent by the column. Salt gradient elution in a pH 3 mobile phase was then used to recover the desired peptides and the purified deblocked peptides were directly subjected to N-terminal sequence analysis. The same digests were also chromatographed on a C18 reversed-phase column using standard trifluoroacetic acid-acetonitrile gradient elution. The elution order for the parent peptide and the N-1 peptide on the reversed-phase column was reversed from that on the Sulfoethyl Aspartamide column and the resolution of the two peptides obtained on the reversed-phase column was less than that observed on the cation-exchange column. In addition, the Sulfoethyl Aspartamide column was shown to be useful to monitor the extent of N-terminal glutamine cyclization formed during peptide purification and storage.

Capsaicin-sensitive tachykinin-like immunoreactivity in the thymus of rats and guinea-pigs.

Capsaicin-sensitive, substance P-like immunoreactivity (SP-LI) has been detected recently in rat thymus. Other tachykinins are frequently present with SP. In the present study, tachykinin-like immunoreactivity (TK-LI) was measured in guinea-pig, rat, mouse and hamster thymus with the amount detectable being greatest in guinea-pig, less in rat and least in mouse; it was not detectable in hamsters. In guinea-pig and rat thymus, but not in mouse, TK-LI was markedly reduced by pretreatment with capsaicin. TK-LI levels correlated significantly with those of SP-LI in both guinea-pig and rat thymus. High-performance liquid chromatography (HPLC) fractions considered to represent neurokinin A, eledoisin and neuropeptide K were present in guinea-pig thymus but only the first two were present in rat thymus.