Studies of the effect of parasympathetic denervation on the muscarinic cholinergic receptors of the rat parotid gland

Adham, Nika (1986) Studies of the effect of parasympathetic denervation on the muscarinic cholinergic receptors of the rat parotid gland. University of Southampton, Doctoral Thesis.

Abstract

High affinity, specific [³H] quinuclidinyl benzilate, ([³H] QNB) binding sites were identified in the rat salivary gland membranes. The binding process exhibited Kd and B_max of 0.57 ± 0.16 nM and 52 ± 8.02 fmol/mg protein for the parotid gland respectively and 0.3 ± 0.025 nM and 101 ± 9.11 fmol/mg protein for the submaxillary gland respectively. Parasympathetic denervation (Px) of the rat parotid gland by means of avulsion of the auriculo-temporal nerve was found to be specific to the parotid gland and did not affect the submaxillary gland. (Px) caused a maximum reduction of ∼ 30% in the parotid gland wet weight and a maximum decrease of ∼ 80% in choline acetyl transferase (ChAT) activity, 3-6 weeks following surgery. When expressed as fmol/gland B_max of [³H] QNB was decreased by a maximum of ∼ 30% . This loss was probably due to the removal of the presynaptic nerve terminals. Muscarinic receptors were further studied using [^3H] N-methylscopolamine ([^3H] NMS) which, due to its hydrophilic nature is a more favourable ligand than [^3H] QNB. [^3H] NMS binding was of high affinity and specificity with a Kd of 0.4 ± 0.05 nM and B_max of 138 ± 9.56 fmol/mg protein. (Px) caused a decrease of ∼ 40% in the B_max of [³H] NMS expressed as fmol/gland, 3 weeks following surgery. (Px) also caused an increase in the affinity of carbachol as determined from displacement of [³H] NMS binding. The results suggested that denervation causes an increase in the affinity of M₁ receptor subtypes. The physiological responsiveness of the rat parotid gland was measured by monitoring K⁺ transport, measured by ⁸⁶Rb efflux. Denervation caused a shift to the left of the ⁸⁶Rb efflux dose-response curve to carbachol and phenylephrine (3.75 and 3.37 fold respectively). Further (Px) increased the intrinsic activity of the partial agonist pilocarpine and converted it to a full agonist. When surface receptors were by-passed using the Ca^{+ +} ionophore A-23187 to elicit ⁸⁶Rb efflux, control and denervated parotid gland slices were equally responsive. These findings suggest that the observed supersensitivity of receptors on the rat parotid gland caused by (Px) is not due to an increase in receptor number but may be associated with an increase in the affinity of agonists for the receptor subtype M₁. Further the possibility of an altered receptor-signal transduction mechanism between the receptor and phospholipid turnover/Ca^{+ +} mobilization requires to be fully investigated. (D72311/87)

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