Degradation of substance P by human plasma

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ORGAN SPECIFIC METABOLISM OF NEUROTENSIN A. Shulkes, D.R. Fletcher, K.J. Hardy, Department of Surgery, University of Melbourne, Victoria, Australia. Heurotensin is a 14 amino acid intestinal peptide which is thought to function as a circulating hormone. However, little is known of its metabolism and the organs of clearance have not been defined. The aims of this study were to determine the metabolic clearance rate, disappearance half rate and the organs involved in the metabolism of circulating neurotensin. Percentage destruction of neurotensin across the lung, gut, liver, kidney and brain was determined by taking blood samples from nine ~onscious sheep with cannulae in the aorta, pulmonary artery, portal vein, hepatic vein, renal vein and left and right internal jugular veins. Neurotensin was infused for 1 hour at 200 pmol/kg/hr via a jugular cannula. Plasma neurotensin was measured by radioimmunoassay using an amino terminal directed antibody. Metabolic clearance rate was 14±l ml/kg/min (mean±SEM) and t½ 6.3±0.5 minutes. The kidney was the major organ involved in the disappearance of immunoreactive neurotensin with 17±2% destruction (determined by difference between levels in aorta and renal vein). There was lO±l% destruction across the brain and 6±2% across the gut but no Jisappearance across the lung or liver. Gel chromatography of the plasma from the aorta and subsequent assay with. C and N terminal directed neurotensin antibodies revealed that the neurotensin was being metabolised to a smaller amino terminal fragment which comprised at least half the circulating immunoreactive neurotensin after 30 minutes infusion. The relatively long half life, the low clearance and the fact that neurotensin immunoreactivity was not affected by the liver or lung suggests that neurotensin functions as a circulating hormone. However, further studies are needed to determine where the amino terminal neurotensin is produced and whether it is biologically active.

DEGRADATION OF SUBSTANCE P BY HUMAN PLASMA J.M.Conlon and C.S.Hethrington. Pain Relief Foundation, Walton Hospital, Liverpool, U.K. and Department of Clinical Biochemistry, University of Newcastle upon Tyne, U.K. The degradation of Substance P (SP) by human plasma was studied because (a) release of neuropeptides into plasma may represent a physiologically important mechanism of clearance and (b) tracer degradation by plasma peptidases interferes with radioin%munoassay. I~gradation of SP in the plasma of healthy fasted subjects was determined by radioimmunoassay using • . a carboxy-t erminally directed antibody and of ,~s l-(Tyr 8 ) SP by blndlng to talc. The SP-degrading activity of blood was associated with the plasma fraction (>90%) and rates of degradation were at least 20 times greater in plasma than in equal volumes of cerebrospinal fluid. Plasma SPdegrading activity exhibited a pH optimum of 7.5, was strongly inhibited by 1,10 phenanthroline (lmM) but not by Trasylol at concetrations up to 5000 KIU/ml. Rates of degradation were not significantly affected by incubation with lO00-fold molar excesses of TRH, LHRH, pentagastrin, somatostatin, glucagon or insulin. SP-degrading activity was partially purified by ammonium sulphate fractionation, ion-exchange chromatography and gel filtration, and was eluted from a Sephacryl S-300 column as a major peak with Kay=0.32, indicating an approximate mol.wt, of 150,000. The lability of the tracer and the presence in plasma of high mol.wt. components that non-specifically inhibit binding of tracer to antibody render invalid measurements of SP in unextracted plasma. These problems have been overcome by development of a radioimmunoassay in which SP is quantitatively extracted from plasma and CSF using octadecasilyl-silica at low pH and subsequently recovered using 80% methanol. The rapid and

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selective inactivation of SP in plasma suggests a physiological role for the plasma degrading enzyme(s) in regulation of the concentration of SP reaching peripheral sites.

INSULIN AND CCK REGULATE PANCREATIC CCK RECEPTORS AND SECRETORY RESPONSES John A. Williams and Makoto Otsuki. Cell Biology Research Laboratory, Mount Zion Hospital and Medical Center and Departments of Physiology and Medicine, University of California, San Francisco, California, USA. The present study was designed to evaluate the hormonal regulation of pancreatic CCK receptors and subsequent CCK action. Male Sprague Dawley rats were injected with either streptozotocin to induce hypoinsulinemia (diabetes) or CCK 8 at a dose which induces pancreatic hypertrophy. Isolated acini were then prepared and zymogen release in response to secretagogues was measured. The characteristi~5of CCK binding to its receptors were studied using biologically active -I-labeled porcine CCKRR. In the first group of experiments with diabetes, Scatchard plots of CCK binding to normal and diabetic acini were concave, consistent with the presence of two orders of binding sites; both high and low affinity receptors showed a 2-3 fold decreased affinity in diabetic as compared to normal acini. Moreover, diabetic rats showed a selective decrease in the sensitivity to CCK as the dose-response curves for amylase, ribonuclease and --Ca-- release were shifted 3 fold towards higher concentrations of hormone; maximal enzyme release from diabetic acini, required 300 pM CCK 8 as compared to 100 pM for acini from control rats. In contrast to CCK, there was no change in the dose-response curve for the cholinergic analog carbachol which acts on a different receptor. After 7 days treatment of diabetic rats with insulin (6-10 U/day), the secretory dose response curve and the affinity of CCK receptors returned to normal. In the second group of experiments, chronic stimulation with CCK injections (5 ~g/Kg twice daily) caused pancreatic hypertrophy as evidenced by an increased pancreatic content of protein, DNA, and amylase. Acini prepared from these pancreases showed an increased amount of amylase released per cell, but a 3i0 fold decreased sensitivity to stimulation by both CCK and carbachol. In contrast to insulin injections, there was no effect on the CCK receptor. In conclusion, both insulin and CCK have long term regulatory effects on the ability of acinar cells to respond to CCK. Insulin increases sensitivity whereas CCK decreases it. The effect of insulin is selective to CCK action and may be due to changes in the CCK receptor. In contrast, the effect of CCK is nonselective and is most likely due to changes in postreceptor mechanisms.

EFFECT OF VIP, PHI AND SECRETIN ON HEARTAND LUNGMEMBRANESIN MAN AND RAT. P. Robberecht, P. Chatelain, M. Delhaye, G. Taton, M. Waelbroeck, K. Tatemoto and J. Christophe. Department of Biochemistry, Medical School, Universit~ Libre de Bruxelles, Brussels, Belgium. VIP-, PHI- and secretin-receptors were investigated on human heart and lung membranes by their a b i l i t y to stimulate adenylate cyclase activity. Human heart specimens consisted in right atrium tissue collected during the establishment of extracorporeal circulation for cardiac surgery, in ventricular material obtained during resection of cardiac aneurysm and in one foetal heart obtained during a medical abortion. Humanlung specimens were obtained during the resection of bronchopulmonary tumors. The results obtained with human tissues were compared to those obtained with heart and lung membranes from rat tested under the same conditions. In