Judith A. Cole

Parathyroid Hormone Regulation of Na+,K+-ATPase Requires the PDZ 1 Domain of Sodium Hydrogen Exchanger Regulatory Factor-1 in Opossum Kidney Cells

It was demonstrated that expression of murine sodium hydrogen exchanger regulatory factor (NHERF-1) lacking the ezrin-binding domain blocks parathyroid hormone (PTH) regulation of Na+,K+-ATPase in opossum kidney (OK) cells. The hypothesis that the NHERF-1 PDZ domains contribute to PTH regulation of Na+,K+-ATPase was tested by comparison of PTH regulation of Na+,K+-ATPase in wild-type OK (OK-WT) cells, NHERF-deficient OKH cells, OK-WT transfected with siRNA for NHERF (NHERF siRNA OK-WT), and OKH cells that were stably transfected with full-length NHERF-1 or constructs with mutated PDZ domains. OKH cells and NHERF siRNA OK-WT showed decreased expression of NHERF-1 but equivalent expression of ezrin and Na+,K+-ATPase alpha1 subunit when compared with OK-WT cells. PTH decreased Na+,K+-ATPase activity and stimulated phosphorylation of the Na+,K+-ATPase alpha1 in OK-WT cells but not in NHERF-deficient cells. Rubidium (86Rb) uptake was equivalent in OK-WT, OKH, and OKH cells that were transfected with all but the double PDZ domain mutants. PTH decreased 86Rb uptake significantly in OK-WT but not in OKH cells. PTH also significantly inhibited 86Rb uptake in OKH cells that were transfected with full-length NHERF-1 or NHERF-1 with mutated PDZ 2 but not in OKH cells that were transfected with mutated PDZ 1. Transfection with NHERF expressing both mutated PDZ domains resulted in diminished basal 86Rb uptake that was not inhibited further by PTH. PTH stimulated protein kinase Calpha activity and alpha1 subunit phosphorylation in OK-WT but not in NHERF-deficient cells. Transfection of OKH cells with NHERF constructs that contained an intact PDZ1 domain restored PTH-stimulated protein kinase Calpha activity and alpha1 subunit phosphorylation. These results demonstrate that NHERF-1 is necessary for PTH-mediated inhibition of Na+,K+-ATPase activity and that the inhibition is mediated through the PDZ1, not PDZ2, domain.

Novel regulatory function for NHERF-1 in Npt2a transcription

Several lines of evidence show that sodium/hydrogen exchanger regulatory factor 1 (NHERF-1) regulates the expression and activity of the type IIa sodium-dependent phosphate transporter (Npt2a) in renal proximal tubules. We have previously demonstrated that expression of a COOH-terminal ezrin binding domain-deficient NHERF-1 in opossum kidney (OK) cells decreased expression of Npt2a in apical membranes but did not affect responses to parathyroid hormone. We hypothesized that NHERF-1 regulates apical membrane expression of Npt2a in renal proximal tubule cells. To address this hypothesis, we compared regulation of Npt2a expression and function in NHERF-deficient OK cells (OK-H) and wild-type cells (OK-WT). In OK-H cells, phosphate uptake and expression of Npt2a protein in apical membranes were significantly lower than in OK-WT cells. Transient transfection of green fluorescent protein-tagged Npt2a cDNA into OK-H cells resulted in aberrant localization of an Npt2a fragment to the cytosol but not to the apical membrane. OK-H cells also exhibited a marked decrease in Npt2a mRNA expression. As demonstrated by luciferase assay, Npt2a promoter activity was significantly decreased in OK-H cells compared with that shown in OK-WT cells. Transfection of OK-H cells with human NHERF-1 restored Npt2a expression at both the protein and mRNA levels and regulation by parathyroid hormone. Expression of NHERF-1 constructs with mutations in the PDZ domains or the ezrin binding domain in OK-H cells suggested that the PDZ2 domain is critical for apical translocation of Npt2a and for expression at the mRNA level. Our data demonstrate for the first time that NHERF-1 regulates Npt2a transcription and membrane insertion.

Hyperglycemia-Induced Changes in Na+/myo-Inositol Transport, Na+-K+-ATPase, and Protein Kinase C Activity in Proximal Tubule Cells

In many tissues, hyperglycemia alters the activities of the Na+-dependent myo-inositol (Na/MI) transporter, Na+-K+-ATPase, and protein kinase C (PKC). However, little is known concerning adaptive changes in renal proximal tubular function after acute or chronic hyperglycemia. We examined hyperglycemia-induced changes in Na/MI transport, Na+-K+-ATPase activity, and PKC activity using three proximal tubule–like cell lines (JTC12, LLC-PK1, and OK/E cells) and primary cultures of human proximal tubular epithelium (HK cells) cultured for varying periods in low- or high-glucose media, myo-Inositol (MI) transport was mediated by a high-affinity (Km ∼50 mumol/l) Na+-dependent saturable process in the four cell lines. Hyperglycemia produced a time-dependent and persistent increase in Na/MI transport in all cell lines. Chronic hyperglycemia increased the Km for MI transport in LLC-PK1 cells and increased the Vmax in both LLC-PK1 and JTC12 cells. Glucose competitively inhibited Na/MI transport in all low-glucose cells and in high-glucose HK, JTC12, and OK/E cells but had no effect on transport in high-glucose LLC-PK1 cells. Acute hyperglycemia also produced time-dependent increases in Na+-K+-ATPase activity in all cell lines, a change that persisted only in HK cells. A 24-h exposure to high glucose had no effect on PKC activity in any of the cell lines but increased Ca/phospholipid-dependent PKC activity in membrane fractions from chronically high-glucose LLC-PK1 and OK/E cells. These data suggest that hyperglycemia causes acute changes in proximal tubule function and long-lived adaptive responses in Na/MI transport and the PKC signaling pathway.

Down-Regulation of Protein Kinase C by Parathyroid Hormone and Mezerein Differentially Modulates cAMP Production and Phosphate Transport in Opossum Kidney Cells

We examined the effects of prolonged exposure to parathyroid hormone (PTH) and the protein kinase C (PKC) activator mezerein (MEZ) on cyclic adenosine monophosphate (cAMP) production, PKC activity, and Na+‐dependent phosphate (Na/Pi) transport in an opossum kidney cell line (OK/E). A 5 minute exposure to PTH stimulated, while a 6 h incubation reduced, cAMP production. Na/Pi transport was maximally inhibited under desensitizing conditions and was not affected by reintroduction of the hormone. MEZ pretreatment (6 h) enhanced PTH‐, cholera toxin (CTX)‐, and forskolin (FSK)‐stimulated cAMP production, suggesting enhanced Gsα coupling and increased adenylyl cyclase activity. However, PKA‐ and PKC‐dependent regulation of Na/Pi were blocked in MEZ‐treated cells. The PTH‐induced decrease in cAMP production was associated with a reduction in membrane‐associated PKC activity while MEZ‐induced increases in cAMP production were accompanied by decreases in membrane and cytosolic PKC activity. Enhanced cAMP production was not accompanied by significant changes in PTH/PTH related peptide (PTHrP) receptor affinity or number, nor was the loss of Na/Pi transport regulation associated with changes in PKA activity. The results indicate that down‐regulation of PKC by PTH or MEZ differentially modulates cAMP production and regulation of Na/Pi transport. The distinct effects of PTH and MEZ on PKC activity suggest that agonist‐specific activation and/or down‐regulation of PKC isozyme(s) may be involved in the observed changes in cAMP production and Na/Pi transport.

Autotransplantation of avian parathyroid glands: An animal model for studying parathyroid function

The parathyroid glands of chickens were autotransplanted and the return of parathyroid function following transplantation was determined. Parathyroidectomy (PTX) resulted in a marked hypocalcemia (5.2 +/- 0.2 mg/dl) 4 hr following PTX. Plasma calcium (PCa) had declined to 4.3 +/- 0.2 mg/dl 24 hr after PTX. Parathyroid glands were transplanted subcutaneously 24 hr after removal and 24 hr later, PCa had risen to 8.6 +/- 0.5 mg/dl. Seven days after PTX. PCa increased to 10.3 +/- 0.2 mg/dl and by 14 days was indistinguishable from control levels (10.8 +/- 0.2 mg/dl vs. 11.0 +/- 0.2 mg/dl, respectively). When chicks with transplanted glands were fed a low Ca (0.08%) diet for 2 weeks they were able to maintain plasma PCa at levels comparable to control birds. Removal of the transplanted glands resulted in marked decreases in PCa (from 9.7 +/- 0.3 to 5.6 +/- 0.8 mg/dl), in the fractional excretion of phosphate, in urine cAMP, and in renal 25OH-vitamin D3-1 alpha-hydroxylase activity. Stepwise reductions in PCa and 1 alpha-hydroxylase activity were produced in partially PTX and fully PTX chicks by removing part or all of the parathyroid tissue. These data suggest that the transplanted parathyroid tissue was the major source of circulating PTH and that it may be possible to produce different degrees of acute hypoparathyroidism by varying the amount of transplanted parathyroid tissue removed surgically. Chickens with transplanted parathyroid glands thus provide a convenient animal model in which to study parathyroid function in an avian species.