Nitric oxide-induced signalling in rat lacrimal acinar cells.
The aim of the present study was to investigate the physiological role of nitric oxide (NO) in mediating secretory processes in rat lacrimal acinar cells. In addition, we wanted to determine whether the acinar cells possess endogenous nitric oxide synthase (NOS) activity by measuring NO production using the fluorescent NO indicator 4,5-diaminofluorescein (DAF-2). We initiated investigations by adding NO from an external source by means of the NO-donor, S-nitroso-N-acetyl-penicillamine (SNAP). Cellular concentrations of cyclic guanosine 5'-phosphate (cGMP) ([cGMP]) were measured by radioimmunoassay (RIA), and we found that SNAP induced a fast increase in the [cGMP], amounting to 350% of the [cGMP] in resting cells. Moreover, addition of SNAP and elevating [cGMP] in fura-2 loaded lacrimal acinar cells, resulted in a cGMP-dependent protein kinase-mediated release of Ca2+ from intracellular stores, leading to a rise in the intracellular free Ca2+ concentration ([Ca2+]i). The Mn2+ quenching studies revealed that the Ca2+ release was not accompanied by Ca2+ influx. Finally, we demonstrate that lacrimal acinar cells possess endogenous NOS activity, which is activated by beta-adrenergic stimulation and not by a rise in [Ca2+]i alone. We show that in rat lacrimal acinar cells, NO and cGMP induce Ca2+ release from intracellular stores via G kinase activation. However, the changes in [Ca2+]i are relatively small, suggesting that this pathway plays a modulatory role in Ca2+ signalling, thus not by itself causing fast transient increases in [Ca2+]i. In addition, we suggest that endogenously produced NO activated by beta-adrenergic receptor stimulation, plays an important role in signalling to the surrounding tissue.
Looms
D K
DK
Department of Medical Physiology, The Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 N, Denmark.
Tritsaris
K
K
Dissing
S
S
D000818:Animals; D002118:Calcium / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D019793:Fluorescein / Q000378:metabolism; D016257:Fura-2 / Q000494:pharmacology; D007202:Indicators and Reagents / Q000378:metabolism; D007765:Lacrimal Apparatus / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D009924:Organ Culture Techniques; D010396:Penicillamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D011863:Radioimmunoassay; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000502:physiology; D013997:Time Factors
C111205:4,5-diaminofluorescein; D007202:Indicators and Reagents; C110881:S-nitro-N-acetylpenicillamine; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D010396:Penicillamine; D006152:Cyclic GMP; D002118:Calcium; D019793:Fluorescein; D016257:Fura-2