title: Alpha 1-adrenergic stimulation and cytoplasmic free calcium concentration in cultured renal proximal tubular cells: evidence for compartmentalization of quin-2 and fura-2.
abstract: This study was designed to examine the role of changes in cytoplasmic free calcium concentration ([Ca2+]i) during the response to alpha 1-adrenergic agonists in cultured renal proximal tubular cells. Experiments were carried out on primary cultures of canine proximal tubular cells grown in defined culture medium on a solid support, on collagen-coated polycarbonate membranes, or on collagen-coated glass coverslips. Quin-2 and fura-2 were used to monitor [Ca2+]i. The basal level of [Ca2+]i was 101 nM, as measured with quin-2, and 122 nM, as determined using fura-2. Fluorescence flow cytometry revealed that about 85% of the population of proximal tubular cells responded to phenylephrine with an increase in [Ca2+]i. Phenylephrine (10(-5) M) caused an immediate actual increase in [Ca2+]i by 18 and 24%, as determined with quin-2 and fura-2, respectively, with the peak increase in [Ca2+]i averaging 22% and 44% over the basal level (180-300 sec). This effect did not require extracellular calcium. The effect of phenylephrine was abolished by prazosin and verapamil. Fluorescence microscopy of quin-2 or fura-2 loaded cells revealed punctate areas of fluorescence within the cytoplasm suggesting vesicular uptake of the dyes. Pinocytotic entrapment of the dyes was demonstrated by the transfer of cell-impermeant fura-2 across tubular cell monolayers mounted in Ussing chambers. The transfer of the dye was similar to that of a marker of fluid-phase pinocytosis, Lucifer Yellow (LY). This pinocytotic entrapment of Ca2+-indicators would lead to underestimation of the actual calcium transients. Microfluorometric study of single proximal tubular cells "scrape-loaded" with fura-2 revealed a four-fold increase in [Ca2+]i concentration following stimulation with phenylephrine.
mesh_terms: D000634:Aminoquinolines / Q000378:metabolism; D000818:Animals; D001572:Benzofurans / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002451:Cell Compartmentation; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D004285:Dogs; D005260:Female; D016257:Fura-2; D007546:Isoquinolines / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000187:drug effects*; D008297:Male; D010656:Phenylephrine / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D010873:Pinocytosis; D011224:Prazosin / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology
chemical_list: D000634:Aminoquinolines; D001572:Benzofurans; D007546:Isoquinolines; D010656:Phenylephrine; C017475:lucifer yellow; D014700:Verapamil; C035739:Quin2; D002118:Calcium; D016257:Fura-2; D011224:Prazosin