Cytoplasmic calcium in individual proximal tubular cells in culture

1986 ◽  
Vol 251 (5) ◽  
pp. F938-F944 ◽  
Author(s):  
M. S. Goligorsky ◽  
D. J. Loftus ◽  
K. A. Hruska
Keyword(s):  
Parathyroid Hormone ◽  
Calcium Concentration ◽  
Complete Recovery ◽  
Calcium Transients ◽  
Cytoplasmic Calcium ◽  
Free Medium ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Proximal Tubular ◽  
Detailed Technique

The development of high quantal yield Ca2+-sensitive fluorescent indicators has made microspectrofluorometric monitoring of cytoplasmic calcium feasible. The detailed technique to monitor cytoplasmic calcium concentration in individual proximal tubular cells grown on glass cover slips is described. Manipulations of cytoplasmic calcium concentration by means of a Ca2+ ionophore or Ca2+-free medium resulted in corresponding changes of fura-2 fluorescence. Parathyroid hormone elicited a fivefold increase in cytoplasmic calcium concentration, with the subsequent complete recovery of this parameter in 3 min. This effect of parathyroid hormone was abolished by perfusion of the cells with Ca2+-free medium. Repeated pulses of parathyroid hormone spaced at an interval of 20 min, caused refractoriness of adenosine 3',5'-cyclic monophosphate response, whereas cytoplasmic calcium transients remained unaltered. When the frequency of the sequential pulses with parathyroid hormone was increased (5 min intervals), the amplitude of calcium transients was diminished, and the recovery of basal level of cytoplasmic calcium was incomplete and protracted. These observations may have application to disordered renal cell calcium metabolism in hyperparathyroid states.

Parathyroid hormone-dependent endocytosis of renal type IIc Na-Pi cotransporter

2007 ◽  
Vol 292 (1) ◽  
pp. F395-F403 ◽  
Author(s):  
Hiroko Segawa ◽  
Setsuko Yamanaka ◽  
Akemi Onitsuka ◽  
Yuka Tomoe ◽  
Masashi Kuwahata ◽  
...  
Keyword(s):  
Parathyroid Hormone ◽  
Apical Membrane ◽  
Hypophosphatemic Rickets ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Transporter Protein ◽  
Protein Levels ◽  
Proximal Tubular ◽  
Renal Proximal Tubular Cells ◽  
Renal Proximal Tubular

Hereditary hypophosphatemic rickets with hypercalciuria results from mutations of the renal type IIc Na-Pi cotransporter gene, suggesting that the type IIc transporter plays a prominent role in renal phosphate handling. The goal of the present study was to investigate the regulation of the type IIc Na-Pi cotransporter by parathyroid hormone (PTH). Type IIc Na-Pi cotransporter levels were markedly increased in thyroparathyroidectomized (TPTX) rats. Four hours after administration of PTH, type IIc transporter protein levels were markedly decreased in the apical membrane fraction but recovered to baseline levels at 24 h. Immunohistochemical analyses demonstrated the presence of the type IIc transporter in the apical membrane and subapical compartments in the proximal tubular cells in TPTX animals. After administration of PTH, the intensity of immunoreactive signals in apical and subapical type IIc transporter decreased in the renal proximal tubular cells in TPTX rats. Colchicine completely blocked the internalization of the type IIc transporter. In addition, leupeptin prevented the PTH-mediated degradation of the type IIa transporter in lysosomes but had no effect on PTH-mediated degradation of the lysosomal type IIc transporter. In PTH-treated TPTX rats, the internalization of the type IIc transporter occurred after administration of PTH(1–34) (PKA and PKC activator) or PTH(3–34) (PKC activator). Thus the present study demonstrated that PTH is a major hormonal regulator of the type IIc Na-Pi cotransporter in renal proximal tubules.

scholarly journals Parathyroid hormone transport effects and hormonal processing in primary cultured rat proximal tubular cells

1993 ◽  
Vol 293 (2) ◽  
pp. 377-380 ◽  
Author(s):  
R M O'Donovan ◽  
C C Widnell ◽  
T C Chen ◽  
J B Puschett
Keyword(s):  
Parathyroid Hormone ◽  
Specific Binding ◽  
Primary Cultures ◽  
Subcellular Fractionation ◽  
Phosphate Transport ◽  
Surface Binding ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Culture Models ◽  
Proximal Tubular

The development of satisfactory cell culture models for the study of parathyroid hormone (PTH)-induced inhibition of Pi transport has proven difficult. Using subcellular fractionation techniques we investigated the response of primary cultures of rat proximal tubular cells to PTH-(1-34). Specific binding of 125I-bPTH-(1-34) occurred at 2 degrees C. After 5 min of rewarming, trypsin-releasable radioactivity decreased from 90 to 50%, indicating internalization of the ligand. Cell disruption, followed by density centrifugation with 17% Percoll either directly after binding at 2 degrees C or post-rewarming for 20 min, showed a shift of 125I label from the plasma membrane (5′-nucleotidase) to lysosomal fractions (beta-D-glucosaminidase), confirming the sequential occurrence of cell surface binding, internalization and transport to lysosomes of 125I-bPTH-(1-34). Reculture at 37 degrees C revealed steady accumulation of trichloroacetic acid-soluble radioactivity in the medium, indicating degradation of 125I-bPTH-(1-34). Phosphate transport in the absence of sodium was minimal. Incubation of the cells with bPTH-(1-34) resulted in up to 50% inhibition of sodium-dependent phosphate transport. Prior phosphate depletion abrogated the response to PTH.

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