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Electrochemical measurement of regulatory peptides in the neurointermediate lobe of brattleboro and control rats
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ELECTROCHEMICAL MEASUREMENT OF REGULATORY PEPTIDES IN THE NEUROINTERMEDIATE LOBE OF BRATTLEBORO AND CONTROL RATS L L Foyster, C A Marsden and G W Bennett, Department of Physiology and Pharmacology, Queen's Medical Centre, Clifton Boulevard, Nottingham. At present the only sensitive technique available for the measurement of regulatory peptide levels in tissue is by radioimmunoassay (RIA); in this study an alternative neuropeptide assay involving reverse-phase HPLC with on-line electrochemical detection (LCEC) has been developed. Measurement is made, using LCEC, of vasopressin and oxytocin levels in the neurointermediate lobe of both rats homozygous for diabetes insipidus (Brattleboro rats - BB) and the heterozygous control rats (Long Evans - LE). Reverse-phase HPLC was carried out on a Hypersil 5ODS column (25em x 5mm) using a 0.15M Na2HPO4/methanol (60:40v/v) mobile phase, at pB6.0~ flow rate 1.0ml/min. Electrochemical detection was with a BAS glassy carbon electrode, with an applied voltage of +I.00V. Single neurointermediate lobes were dissected out and extracted immediately by sonication in ].0ml 0.1M Acetic acid/methanol (50:50v/v), followed by centrifugation at 3,000 r.p.m, for 15 mins. The supernatant was dried down and the residue taken up in 1.0ml of 0.15M Na2HPO 4, pH6.O. 1O0~l of this was injected on the column. Vasopressin levels of 22.76±2.40 (n=6)nmol peptide/mg protein, were observed in LE rats~ whilst none was detected in BB rats, with a limit of detection of ipmol. Oxytocin levels in BB rats (10.41± 0.91 (n=6)nmol peptide/mg protein) were found to be higher than in LE rats (8.26±1.96nmol peptide/mg protein) although the difference was not significant. No difference was observed between BB and LE control rats for two other unidentified peaks detected by LCEC. The results indicate that an LCEC assay for neuropeptides could provide an alternative approach for peptide measurement to RIA, so avoiding problems of antibody specificity.
SUBSTANCE P IS A FUNCTIONAL TRANSMITTER AT A NEUROSECRETORY SYNAPSE IN RAT PAROTID GLAND. D. V. Gallacher. Dept. Physiol. University of Liverpool, Liverpool, U.K. The technique of electrical field stimulation (FS) was employed to stimulate the nerves of the isolated parotid gland preparation. The study revealed that a significant component of the FS response persisted in the presence of atropine, phentolamine and propranolol, i.e. due to release of a noncholinergic, non-adrenergic (NCNA) neurotransmi~ter. The NCNA transmitter evoked changes in membrane potential and conductance, potassium (86Rb)efflux and amylase release. Two biologically active peptides, substance P (8P) and vasoactive intestinal polypeptide (VIP) have been identified in nerve endings wlt • hin salivary glands, 2,3 . The NCNA FS eff e cts w ere d"ir ectly compared with those evoked by application of each of these pep±ides. The neurotoxin capsaicin, and the substance P antagonist, D-Pro 2, D-Trp 79 SP 4, were employed in the characterization of the NCNA neurotransmitter. It is concluded that substance P or a closely related peptide is the transmitter mediating the NCNA FS effects at a neurosecretory synapse in the rat parotid gland. i. Gallacher, D.V. an~ Petersen, O . H . J . Physiol. 3 0 5 : 4 3 - 5 7 (1980). 2. Hokfelt, T., Johansson, 0., Keller±h, J.O., Ljungdahl, A., Nilsson, G., Nygards, A., Pernow, B. In "Substance P" (Eds. von Euler, U.S., Pernow, B). Raven Press 1977. 3. Lundberg, J.M., Anggard, A., Fahrenkrug, J., Hokfelt, T. and Mutt, V. Proc. Nat. Acad. Sci. 7 7 : 1 6 5 1 - 1 6 5 5 (1980). 4. Engberg, G., Svensson, T.H., Rosell, S. & Folkers, K. Nature 2 9 3 : 2 2 2 - 2 2 3 (1981).
ELECTROCHEMICAL MEASUREMENT OF REGULATORY PEPTIDES IN THE NEUROINTERMEDIATE LOBE OF BRATTLEBORO AND CONTROL RATS L L Foyster, C A Marsden and G W Bennett, Department of Physiology and Pharmacology, Queen's Medical Centre, Clifton Boulevard, Nottingham. At present the only sensitive technique available for the measurement of regulatory peptide levels in tissue is by radioimmunoassay (RIA); in this study an alternative neuropeptide assay involving reverse-phase HPLC with on-line electrochemical detection (LCEC) has been developed. Measurement is made, using LCEC, of vasopressin and oxytocin levels in the neurointermediate lobe of both rats homozygous for diabetes insipidus (Brattleboro rats - BB) and the heterozygous control rats (Long Evans - LE). Reverse-phase HPLC was carried out on a Hypersil 5ODS column (25em x 5mm) using a 0.15M Na2HPO4/methanol (60:40v/v) mobile phase, at pB6.0~ flow rate 1.0ml/min. Electrochemical detection was with a BAS glassy carbon electrode, with an applied voltage of +I.00V. Single neurointermediate lobes were dissected out and extracted immediately by sonication in ].0ml 0.1M Acetic acid/methanol (50:50v/v), followed by centrifugation at 3,000 r.p.m, for 15 mins. The supernatant was dried down and the residue taken up in 1.0ml of 0.15M Na2HPO 4, pH6.O. 1O0~l of this was injected on the column. Vasopressin levels of 22.76±2.40 (n=6)nmol peptide/mg protein, were observed in LE rats~ whilst none was detected in BB rats, with a limit of detection of ipmol. Oxytocin levels in BB rats (10.41± 0.91 (n=6)nmol peptide/mg protein) were found to be higher than in LE rats (8.26±1.96nmol peptide/mg protein) although the difference was not significant. No difference was observed between BB and LE control rats for two other unidentified peaks detected by LCEC. The results indicate that an LCEC assay for neuropeptides could provide an alternative approach for peptide measurement to RIA, so avoiding problems of antibody specificity.
SUBSTANCE P IS A FUNCTIONAL TRANSMITTER AT A NEUROSECRETORY SYNAPSE IN RAT PAROTID GLAND. D. V. Gallacher. Dept. Physiol. University of Liverpool, Liverpool, U.K. The technique of electrical field stimulation (FS) was employed to stimulate the nerves of the isolated parotid gland preparation. The study revealed that a significant component of the FS response persisted in the presence of atropine, phentolamine and propranolol, i.e. due to release of a noncholinergic, non-adrenergic (NCNA) neurotransmi~ter. The NCNA transmitter evoked changes in membrane potential and conductance, potassium (86Rb)efflux and amylase release. Two biologically active peptides, substance P (8P) and vasoactive intestinal polypeptide (VIP) have been identified in nerve endings wlt • hin salivary glands, 2,3 . The NCNA FS eff e cts w ere d"ir ectly compared with those evoked by application of each of these pep±ides. The neurotoxin capsaicin, and the substance P antagonist, D-Pro 2, D-Trp 79 SP 4, were employed in the characterization of the NCNA neurotransmitter. It is concluded that substance P or a closely related peptide is the transmitter mediating the NCNA FS effects at a neurosecretory synapse in the rat parotid gland. i. Gallacher, D.V. an~ Petersen, O . H . J . Physiol. 3 0 5 : 4 3 - 5 7 (1980). 2. Hokfelt, T., Johansson, 0., Keller±h, J.O., Ljungdahl, A., Nilsson, G., Nygards, A., Pernow, B. In "Substance P" (Eds. von Euler, U.S., Pernow, B). Raven Press 1977. 3. Lundberg, J.M., Anggard, A., Fahrenkrug, J., Hokfelt, T. and Mutt, V. Proc. Nat. Acad. Sci. 7 7 : 1 6 5 1 - 1 6 5 5 (1980). 4. Engberg, G., Svensson, T.H., Rosell, S. & Folkers, K. Nature 2 9 3 : 2 2 2 - 2 2 3 (1981).