Inhibitory effects of amitriptyline on the stimulation-induced Ca2+ increase in parotid acini

The present study demonstrates the effects of the antidepressant, amitriptyline, and the acetylcholine antagonist, atropine, on the stimulation-induced rise in cytosolic, free Ca2+ (Cai2+). The changes in Cai2+ of collagenase-isolated rat parotid acini were measured by means of the Ca2(+)-sensitive dye, fura-2. It was found that stimulation by carbachol resulted in a maximal increase of 582 +/- 34 nM (mean +/- S.E.) in Cai2+ with a ks of 5.8 +/- 1.3 microM. Adrenaline caused a rise of 380 +/- 22 nM in Cai2+ with a ks of 0.5 +/- 0.2 microM. Amitriptyline and atropine were found to inhibit the carbachol-induced rise in Cai2+ with dissociation constants (kI) of 105 and 1.25 nM, respectively, in the absence of agonist. The adrenergic-induced rise in Cai2+ was inhibited by amitriptyline with a kI of 45 nM. Amitriptyline was found to inhibit both receptor classes by a competitive or mixed type of inhibition. Similarly, atropine exerted the same type of inhibition on the acetylcholine receptor. Amitriptyline and atropine were found to be mutually exclusive for competing for substrate binding on the receptor. These findings are consistent with a common binding site for amitriptyline and atropine on the acetylcholine receptor, possibly in close proximity with, but different from the substrate binding site. The stimulation-induced cell shrinkage evoked by the loss of electrolytes and water from the acini was measured by a 90 degree light scattering signal. It was found that this method makes possible the detection of autonomic side-effects of antidepressants on acini suspended in protein-containing media.