ISSN:
1432-2013
Schlagwort(e):
Key words Osmoregulation
;
Regulatory volume decrease
;
Cytosolic calcium
;
Calcium stores
;
IP3
;
Arachidonic acid
;
Collecting duct
Quelle:
Springer Online Journal Archives 1860-2000
Thema:
Medizin
Notizen:
Abstract In rat inner medullary collecting duct (IMCD) cells in primary culture, hypotonic stress induces Ca2+ transients consisting of an early peak phase caused by a Ca2+ release from intracellular stores and a subsequent plateau phase that involves Ca2+ entry from the extracellular milieu. In the present study, the mechanisms by which cell swelling is transduced into the Ca2+ release were investigated. The free intracellular Ca2+ concentration ([Ca2+]i) was measured using the fluorescent dye fura-2 and cell volume using a confocal laser scanning microscope. In control experiments, after reduction of extracellular osmolarity from 600 to 300 mosmol/l, by omission of sucrose, [Ca2+]i rapidly increased from 106 ± 9 nmol/l to a peak value of 405 ± 22 nmol/l (P≤ 0.05) and thereafter reached a steady-state of 230 ± 23 nmol/l. In low-Ca2+ conditions (10 nmol/l), the reduction of osmolarity evoked only a transient increase of [Ca2+]i by 182 ± 11 nmol/l (P≤ 0.05), which reflected Ca2+ release from intracellular stores. Hyposmotic stress had no effect on inositol 1,4,5-triphosphate (IP3) production measured by a [3H]IP3 radioreceptor assay. Preincubation with 100 μmol/l ETYA (a non-metabolisible derivative of arachidonic acid) reduced the Ca2+ response to hyposmotic stress under high and low Ca2+ conditions (87 and 85% inhibition respectively) as well as the regulatory volume decrease (RVD). Extracellular application of arachidonic acid in isotonic medium led to an increase in [Ca2+]i under high and low Ca2+ conditions. Pretreatment of IMCD cells with 50 μg/ml D609 (a phosphatidylcholine-directed phospholipase C inhibitor) or with 200 μmol/l propranolol (a phosphatidate phosphohydrolase inhibitor) reduced the hypotonic Ca2+ response more strongly than pretreatment with 5 μmol/l BPhB (a phospholipase A2 inhibitor). The Ca2+ response was also suppressed after preincubation with 200 μmol/l RHC 80267 (a diacylglycerol lipase inhibitor). Preincubation with 50 ng/ml pertussis toxin (a G-protein inhibitor) reduced the transient component of the Ca2+ response partially. We conclude that G-proteins, phosphatidylcholine-directed phospholipase C, phospholipase A2, diacylglycerol lipase and arachidonic acid, but not IP3, are involved in the mechanisms by which Ca2+ is released from the intracellular stores during RVD in IMCD cells.
Materialart:
Digitale Medien
URL:
http://dx.doi.org/10.1007/s004240050274
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