Proceedings of The Physiological Society
University of Cambridge (2004) J Physiol 555P, C19
The role of muscarinic receptors in the control of ovine submandibular function
G. Tobin and A.V. Edwards
* Department of Pharmacology, University of Goteborg, Medicinaregatan 15D, Goteborg 413 90, Sweden and Physiological Laboratory, University of Cambridge, Downing Street, Cambridge, CB2 3EG, UK
This study was undertaken to determine which muscarinic receptors mediate parasympathetic responses in the ovine submandibular gland. The effects of selective muscarinic blocking agents on secretory and vascular responses to electrical stimulation of the chorda-lingual nerve were determined and morphological correlates were assessed by Western blotting. Anaesthesia was induced and maintained with sodium pentobarbitone (Sagatal; 15-30 mg kg-1 I.V., then 0.1-0.3 mg min-1 kg-1 I.V.). The ipsilateral ascending cervical sympathetic nerve was cut to eliminate effects of sympathetic activity and the animals were eventually killed with a lethal dose of barbiturate.
Stimulation of the cut end of the parasympathetic chorda-lingual nerve continuously at 2 Hz, or in bursts of 1 s at 20 Hz every 10 s, for 10 min induced similar fluid responses (19 ± 5 vs. 21 ± 5 µl min-1 g gland-1; n = 10) although the protein output was about 50 % greater (P = 0.015; Student's t test) during stimulation in bursts. The vasodilator response was also significantly greater at 20 Hz 1:10 than at 2 Hz continuously as reflected by the fall in submandibular vascular resistance -53 ± 5 vs. -45 ± 4% P = 0.016; n = 10). Stimulation at 8 Hz induced a fluid response approximately three times as large (74 ± 11 µl/min g gland; n = 10) and a protein output 5 times greater than at 2 Hz; the reduction in vascular resistance was -75 ± 3 % (n = 10). Pirenzepine (40 µg/kg I.V.) significantly reduced the fluid response at all frequencies tested (P < 0.05-0.01), most conspicuously at 2 Hz where it was reduced by 85 %, but did not cause any significant change in the protein output or the vascular response. p-Fluoro-hexahydro-sila-diphenidol (4 µg/kg I.V.) in the presence of pirenzepine tended to further reduce the salivary secretion (reduced by 20 % at 8 Hz; n.s.), without causing any further change in the vascular or protein responses. Methoctramine (100 µg/kg IV), had no effect on either the fluid or the vascular response but significantly increased the protein output during the initial few min (2-4) of stimulation at 2 (+170% P < 0.007) and 20 Hz 1:10 (+125% P = 0.014), but not at 8 Hz. Western blot studies employing muscarinic receptor subtype-specific antibodies, revealed distinct bands corresponding to muscarinic M1, M3 and M5 receptors, with a faint band corresponding to the M4 type.
Thus, M1 receptors occur in the ovine submandibular gland and contribute to the secretory response to parasympathetic stimulation but have little effect on the vasodilatation. Inhibitory muscarinic receptors seem to be of the M4 subtype, and blockade by methoctramine of neuronal inhibitory receptors causing increases in transmitter release would explain the increase in protein output.
Where applicable, experiments conform with Society ethical requirements