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Identification of Mg2+/Ca2+-sensing system in mouse distal convoluted tubule cells and characterization of its role in hormone-stimulated magnesium transport

University of British Columbia

Distal convoluted tubule cells of the kidney play a crucial role in the determination of the final urinary magnesium excretion. In addition to responding to changesin local divalent cation concentrations, magnesium absorption in these cells is also under the influence from a number of hormones. Immortalized mouse distal convoluted tubule cells (MDCT) retain a number of the properties ofin vivo distal convoluted tubule cells. This study was performed to identify polyvalent-cation sensing mechanisms in MDCT cells and to describe the co-ordinated response to hormones that control magnesium reabsorption. The presence of Ca²⁺-sensing receptor (Ca²⁺-SR) was confirmed by sequencing a MDCT RT-PCR product that had greater than 92% identity with the published rat clone. Western blot analysis demonstrated the presence of the Ca²⁺-SR protein. Microfluorescence studies and analysis of hormone-induced cAMP generation, were used to determine changes in the cell's physiological response to stimulation of the polyvalent cation-sensing receptor. MDCT cells respond to extracellular Mg²⁺, Ca²⁺, Gd³⁺, and neomycin with a transient increase in [Ca²⁺] j . With respect to Mg²⁺ and Ca²⁺, MDCT cells can differentiate between small concentration changes in Mg²⁺ and Ca²⁺, even in the presence of an excess of the other cation. Evidence is provided for a separate mechanism that responds to extracellular Mg²⁺. Parathyroid hormone (PTH), arginine vasopressin (AVP), calcitonin (CT), glucagon, and prostaglandin E2 , stimulate cAMP generation and increased the Mg²⁺ uptake rate in MDCT cells. Aldosterone potentiates this hormonal stimulation. Hormone-stimulated Mg2"1' uptake and cAMP generation are inhibited with stimulation of the Mg²⁺/Ca²⁺-SR. Pre-treatment of MDCT cells with l,25,(OH)₂D₃ (16 hr, 10-7 M) increased Mg²⁺ uptake by 78 ±4%. Stimulation of the cation-sensing mechanism did not inhibit l,25,(OH)₂D₃ stimulated Mg²⁺ transport. Accordingly, the polyvalent cation-sensing receptor present in MDCT cells has differential effects on hormone mediated transport. We conclude further still that a specific Mg2+-sensing mechanism is present in MDCT cells and that this mechanism inhibits hormone mediated Mg²⁺ uptake in these cells. We infer from these studies that a cation-sensing mechanism is located in the distal convoluted tubule and plays a role in the control of distal Mg²⁺ absorption.

Thesis/Dissertation

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2009-06-29

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http://hdl.handle.net/2429/9829

Experimental Medicine

University of British Columbia

UBCV

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