Intracellular Ca2+ signaling induced by vasopressin, ATP, and epidermal growth factor in epithelial LLC-PK1 cells

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Abstract
The free calcium concentrations in nucleus ([Ca2+]n) and in cytoplasm ([Ca2+]c) of cultured renal LLC-PK1 epithelial cells were estimated by confocal laser microscopy. No difference between the resting mean [Ca2+]n and [Ca2+]c was found. During stimulation with maximal effective concentrations of arginine vasopressin (AVP) or the purinergic agonist ATP, the transient Ca2+ rise was followed mostly by a decline to basal levels. A differential rise could be observed when the increase in [Ca2+]n attained higher values than [Ca2+]c. In 50-60% of the cells, epidermal growth factor (EGF) also induced a transient Ca2+ rise, and a differential increase ([Ca2+]n > [Ca2+]c) was found. The G protein-linked stimuli AVP and ATP were however quantitatively much more efficacious at stimulating the [Ca2+]n and [Ca2+]c increases than was EGF. To investigate whether AVP, ATP, and EGF released Ca2+ from distinct or overlapping stores, the agonists were sequentially added. AVP and ATP applied after EGF in Ca(2+)-free medium elicited an increase in [Ca2+]n and [Ca2+]c that was not significantly lower than the release of Ca2+ in control cells without EGF prestimulation. Similarly, the amplitude of the Ca2+ responses attained by EGF in cells prestimulated by ATP or AVP was comparable to the response in naive cells. Neither EGF, ATP, nor AVP evoked a Ca2+ signal after thapsigargin treatment, indicating that the intracellular Ca2+ pools stimulated by all these agonists are part of the thapsigargin-sensitive Ca2+ pools. In contrast, when ATP was applied after AVP in Ca(2+)-containing as well as in Ca(2+)-free solutions, the Ca2+ transients were lower as compared with the response without preincubation. No differential rise could be found in Ca(2+)-free conditions. An explanation could be the use of different phospholipase C isozymes by the different receptor types, which possibly gives rise to the mobilization of different Ca2+ pools.