The effects of cAMP accumulation evoked by
acetylcholine (ACh) stimulation were studied in rat
submandibular acinar cells by observing the exocytotic
events, swelling of intercellular canaliculi (IC) and
intracellular Ca2+ concentration ([Ca2+]i), which were monitored using
an optical microscope. ACh stimulation evoked transient increases
followed by sustained increases in the frequency of exocytotic events
and IC swelling, while isoproterenol (isoprenaline; IPR) stimulation
evoked sustained increases in these parameters. BAPTA treatment
reduced the frequency of exocytotic events evoked by 5 µM ACh in the
absence of extracellular Ca2+, and further addition of Rp-cAMPS or
H-89 (protein kinase A (PKA) inhibitors) eliminated the remaining
ACh-evoked responses (50 %). Addition of PKA inhibitors in the
presence of extracellular Ca2+ reduced the frequency of exocytotic
events evoked by 500 µM ACh in non-BAPTA-loaded cells. However,
IC swelling evoked by 5 µM ACh was not affected by addition of PKA
inhibitors, and was eliminated in BAPTA-loaded cells perfused with
Ca2+-free solution. These results indicate that the IC swelling is
regulated by [Ca2+]i and the frequency of exocytotic events is regulated
by both [Ca2+]i and [cAMP]i during ACh stimulation. Addition of H-89
inhibited the capacitative Ca2+ entry into ACh-stimulated acinar cells.
Biochemical analysis revealed that ACh stimulation increased the
cAMP content in perfused submandibular glands. These results indicate
that ACh stimulates the accumulation of cAMP in submandibular
acinar cells and that this accumulation of cAMP modulates
Ca2+-regulated exocytosis.