Mechanism of potentiation in substance p-stimulated amylase secretion in isolated rat parotid slices

Michalek, Roman (1988) Mechanism of potentiation in substance p-stimulated amylase secretion in isolated rat parotid slices. University of Southampton, Doctoral Thesis.

Abstract

Perifusion of rat parotid gland slices with isoprenaline (IPR) (100pM-100μM), phenylephrine (PHE) (3μM-300μM), acetylcholine (ACh) (10nM-10mM), carbachol (CCh) (300μM-1mM), substance P (SP) (100pM-100μM) and calcitonin gene-related peptide (CGRP) (10nM-10μM) for 15 seconds elicited a dose-dependent increase in amylase secretion, with IPR being the most potent and CGRP the least potent agonist. Combination of propranolol (1μM), phentolamine (1μM) and atropine (1μM) demonstrated that muscarinic cholinergic, substance P peptidergic, β- and α-adrenergic receptors are present in the rat parotid gland. CCh- and PHE-stimulated amylase secretion was greatly reduced after a 20 minutes depletion of cellular Ca^++, whereas IPR- and SP- stimulated amylase secretion was only slightly reduced. A subthreshold dose of IPR (2nM) potentiated the amylase response to submaximal doses of SP. In addition a subthreshold dose of forskolin (1μM), which in itself was a potent secretagogue, potentiated the SP-stimulated amylase secretion; whereas a subthreshold dose of CGRP (10nM) had no effect on SP-stimulated amylase secretion. IPR (100μM) stimulated tissue cAMP accumulation, whereas neither SP (10μM) nor a combination of SP (10μM) and IPR (2nM) had any effect on cAMP accumulation. Addition of either SP or IPR alone caused an increase in ^45Ca efflux which was dose dependent with a threshold at 1μM for SP and 100nM for IPR. However, when SP was applied simultaneously with a subthreshold dose of IPR (2nM) the threshold to SP was reduced to 10nM and the responses to larger doses of SP were potentiated. The calculated [Ca⁺⁺]; in unstimulated rat partoid acini was approximately 180nM. Both SP (30pM-10nM) and IPR (100nM-3μM) stimulated an increase in this value. Whereas SP (10nM) produced a rapid change in [Ca^++]; to twice the basal value,, both IPR (3μM) and CGRP (10nM) elicited relatively slow increases in [Ca⁺⁺]; to approximately 124% of basal. Addition of the active phorbol ester phorbol-12,13-dibutyrate (PDBu) (3μM-100μM), for 15 seconds, elicited a dose-dependent increase in amylase secretion. Subthreshold concentrations of PDBu (25nM) and a different phorbol ester, phorbol-12-myristate-13-acetate (PMA) (50nM), potentiated submaximal SP-stimulated amylase secretion. Similarly the submaximal IPR-stimulated amylase secretion was potentiated by PDBu (25mM). The C-kinase inhibitors 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) (50μM) and polymyxin B (PMB) (1215 Units/ml) inhibited both SP- and PDBu-stimulated amylase secretion, but were without effect on IPR- or forskolin-stimulated secretion. These findings suggest that the mechanism involved in IPR-potentiation of submaximal SP-stimulated amylase secretion may involve a synergistic interaction distal to the second messengers such as at the level of protein phosphorylation. (DX85715)

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