Changes in calcium concentrations in subcellular compartments of rat submandibular gland acinar cells induced by cholinergic stimulation.

Abstract

The effects of cholinergic and alpha-adrenergic stimulation on calcium concentrations in rat submandibular gland acinar cells were evaluated at the subcellular level by electron probe X-ray microanalysis, and the subcellular distribution of receptors for inositol 1,4,5-trisphosphate (IP(3)Rs) was investigated by electron microscopic immunohistochemistry. For measurement of calcium concentrations, experiments were carried out both in vivo and in vitro. In in vivo experiments, fragments of glands were removed and slam-frozen 3 min after intraperitoneal injection of an agonist. In in vitro experiments, the gland tissue was enzymatically dispersed, treated with an agonist in the presence or absence of extracellular Ca(2+), and slam-frozen. The frozen specimens were cut into ultrathin cryosections, which were then freeze dried. Spectra were collected from secretory granules, the perinuclear cytoplasm containing endoplasmic reticulum (ER), and the nucleus of the acinar cells. A clear decrease in calcium content in secretory granules was observed after cholinergic stimulation both in vivo and in vitro in the presence or absence of extracellular Ca(2+). In the presence of extracellular Ca(2+), cholinergic stimulation following alpha-adrenergic stimulation reduced the calcium content in secretory granules to the same extent as cholinergic stimulation alone. No significant changes in the calcium content of the perinuclear cytoplasm and the nucleus were noticed after cholinergic stimulation. alpha-adrenergic stimulation did not significantly affect the calcium concentrations in any of the three compartments studied. IP(3)Rs were localized to ER lamellae, but not to secretory granule membranes or to nuclear membranes. Our findings suggest that: (1) in rat submandibular acini, Ca(2+) can be released from secretory granules by exposure to cholinergic, but not alpha-adrenergic, stimuli, (2) there are, however, no IP(3)Rs present in the granular membrane and the trigger for the Ca(2+) release remains under study, and (3) the response to either type of stimulus does not involve changes in the Ca(2+) content of the nucleus or of the perinuclear cytoplasm/ER, despite the presence of IP(3)Rs in the latter structure. This may be the result of limitation in the technique used, which cannot measure changes in the proper compartment of the ER.