Effects of protease-activated receptors (PARs) on intracellular calcium dynamics of acinar cells in rat lacrimal glands.

Protease-activated receptors (PARs) represent a novel class of seven transmembrane domain G-protein coupled receptors, which are activated by proteolytic cleavage. PARs are present in a variety of cells and have been prominently implicated in the regulation of a number of vital functions. Here, lacrimal gland acinar cell responses to PAR activation were examined, with special reference to intracellular Ca(2+) concentration ([Ca(2+)]i) dynamics. In the present study, detection of acinar cell mRNA specific to known PAR subtypes was determined by reverse transcriptase polymerase chain reaction. Only PAR2 mRNA was detected in acinar cells of lacrimal glands. Both trypsin and a PAR2-activating peptide (PAR2-AP), SLIGRL-NH2, induced an increase in [Ca(2+)]i in acinar cells. The removal of extracellular Ca(2+) and the use of Ca(2+) channel blockers did not inhibit PAR2-AP-induced [Ca(2+)]i increases. Furthermore, U73122 and xestospongin C failed to inhibit PAR2-induced increases in [Ca(2+)]i. The origin of the calcium influx observed after activated PAR2-induced Ca(2+) release from intracellular Ca(2+) stores was also evaluated. The NO donor, GEA 3162, mimicked the effects of PAR2 in activating non-capacitative calcium entry (NCCE). However, both calyculin A (100 nM) and a low concentration of Gd(3+) (5 µM) did not completely block the PAR2-AP-induced increase in [Ca(2+)]i. These findings indicated that PAR2 activation resulted primarily in Ca(2+) mobilization from intracellular Ca(2+) stores and that PAR2-mediated [Ca(2+)]i changes were mainly independent of IP3. RT-PCR indicated that TRPC 1, 3 and 6, which play a role in CCE and NCCE, are expressed in acinar cells. We suggest that PAR2-AP differentially regulates both NCCE and CCE, predominantly NCCE. Finally, our results suggested that PAR2 may function as a key receptor in calcium-related cell homeostasis under pathophysiological conditions such as tissue injury or inflammation.

The effects of diuretics on intracellular Ca2+ dynamics of arteriole smooth muscles as revealed by laser confocal microscopy.

The regulation of cytosolic Ca(2+) homeostasis is essential for cells, including vascular smooth muscle cells. Arterial tone, which underlies the maintenance of peripheral resistance in the circulation, is a major contributor to the control of blood pressure. Diuretics may regulate intracellular Ca(2+) concentration ([Ca(2+)](i)) and have an effect on vascular tone. In order to investigate the influence of diuretics on peripheral resistance in circulation, we investigated the alteration of [Ca(2+)](i) in testicular arterioles with respect to several categories of diuretics using real-time confocal laser scanning microscopy. In this study, hydrochlorothiazide (100 microM) and furosemide (100 microM) had no effect on the [Ca(2+)](i) dynamics. However, when spironolactone (300 microM) was applied, the [Ca(2+)](i) of smooth muscles increased. The response was considerably inhibited under either extracellular Ca(2+)-free conditions, the presence of Gd(3+), or with a treatment of diltiazem. After the thapsigargin-induced depletion of internal Ca(2+) store, the spironolactone-induced [Ca(2+)](i) dynamics was slightly inhibited. Therefore, the spironolactone-induced dynamics of [Ca(2+)](i) can be caused by either a Ca(2+) influx from extracellular fluid or Ca(2+) mobilization from internal Ca(2+) store, with the former being dominant. As tetraethylammonium, an inhibitor of the K(+) channel, slightly inhibited the spironolactone-induced [Ca(2+)](i) dynamics, the K(+) channel might play a minor role in those dynamics. Tetrodotoxin, a neurotoxic Na(+) channel blocker, had no effect, therefore the spironolactone-induced dynamics is a direct effect to smooth muscles, rather than an indirect effect via vessel nerves.