Molecular Regulation of Renal Phosphate Transport

Received: 18 April 1996/Revised: 26 June 1996     

Involvement of Intracellular or Extracellular Calcium in Activation of Tyrosine Hydroxylase Gene Expression in PC12 Cells

Abstract: The relationship between elevations in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) by different mechanisms and tyrosine hydroxylase (TH) gene expression was examined. Depolarization by an elevated K⁺ concentration triggered rapid and sustained increases in [Ca²⁺]_i from a basal level of ～50 to 110–150 nM and three- to fourfold elevations in TH mRNA levels, requiring extracellular calcium but not inositol 1,4,5-trisphosphate (IP₃). On the other hand, bradykinin or thapsigargin, both of which induce release of intracellular calcium stores via IP₃ or inhibition of Ca²⁺-ATPase, rapidly elevated [Ca²⁺]_i to >200 nM and increased TH gene expression (three-to fivefold). Confocal imaging showed that the elevations in [Ca²⁺]_i in each case occurred throughout the cyto- and nucleoplasm. The initial rise in [Ca²⁺]_i due to either bradykinin or thapsigargin, which did not require extracellular calcium, was sufficient to initiate the events leading to increased TH expression. Consistent with this, the effects of bradykinin on TH expression were inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid or 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester which chelates or inhibits the release of intracellular calcium, respectively. Bradykinin required a rise in [Ca²⁺]_i for <10 min, as opposed to 10–30 min for depolarization to increase TH mRNA levels. These results demonstrate that although each of these treatments increased TH gene expression by raising [Ca²⁺]_i, there are important differences among them in terms of the magnitude of elevated [Ca²⁺]_i, requirements for extracellular calcium or release of intracellular calcium stores, and duration of elevated [Ca²⁺]_i, indicating the involvement of different calcium signaling pathways leading to regulation of TH gene expression.     

Differential Inhibition of Secretagogue-Stimulated Sodium Uptake in Adrenal Chromaffin Cells by Activation of D4 and D5 Dopamine Receptors

Abstract: Recent studies have demonstrated that D1-selective and D2-selective dopamine receptor agonists inhibit catecholamine secretion and Ca²⁺ uptake into bovine adrenal chromaffin cells by receptor subtypes that we have identified by PCR as D5, a member of the D1-like dopamine receptor subfamily, and D4, a member of the D2-like dopamine receptor subfamily. The purpose of this study was to determine whether activation of D5 or D4 receptors inhibits influx of Na⁺, which could explain inhibition of secretion and Ca²⁺ uptake by dopamine agonists. D1-selective agonists preferentially inhibited both dimethylphenylpiperazinium- (DMPP) and veratridine-stimulated ²²Na⁺ influx into chromaffin cells. The D1-selective agonists chloro-APB hydrobromide (CI-APB; 100 µ M ) and SKF-38393 (100 µ M ) inhibited DMPP-stimulated Na⁺ uptake by 87.5 ± 2.3 and 59.7 ± 4.5%, respectively, whereas the D2-selective agonist bromocriptine (100 µ M ) inhibited Na⁺ uptake by only 22.9 ± 5.0%. Veratridine-stimulated Na⁺ uptake was inhibited 95.1 ± 3.2 and 25.7 ± 4.7% by 100 µ M CI-APB or bromocriptine, respectively. The effect of CI-APB was concentration dependent. A similar IC₅₀ (∼18 µ M ) for inhibition of both DMPP- and veratridine-stimulated Na⁺ uptake was obtained. The addition of 8-bromo-cyclic AMP (1 m M ) had no effect on either DMPP- or veratridine-stimulated Na⁺ uptake. These observations suggest that D1-selective agonists are inhibiting secretagogue-stimulated Na⁺ uptake in a cyclic AMP-independent manner.     

A Nonneuronal Isoform of Cell Adhesion Molecule L1: Tissue-Specific Expression and Functional Analysis

Abstract: The cell adhesion molecule L1 is a multifunctional protein in the nervous system characterizing cell adhesion, migration, and neurite outgrowth. In addition to full-length L1, we found an alternatively spliced variant lacking both the KGHHV sequence in the extracellular part and the RSLE sequence in the cytoplasmic part of L1. This L1 variant was expressed exclusively in nonneuronal cells such as Schwann cells, astrocytes, and oligodendrocytes, in contrast to the expression of the full-length L1 in neurons and cells of neuronal origin. To investigate the functions of the L1 variant, we established cell lines transfected with a cytoplasmic short L1 (L1cs) cDNA that lacks only the 12-bp segment encoding for the RSLE sequence. The promoting activities of homophilic cell adhesion, neurite outgrowth, and neuronal cell migration of L1cs-transfected cells (L4-2) were similar to those of full-length L1-transfected cells (L3-1), but the cell migratory activity of L4-2 itself was clearly lower than that of L3-1. In conclusion, the short form of L1 is a nonneuronal type, in contrast to the neuronal type of the full-length L1. Deletion of the four amino acids RSLE in the cytoplasmic region of L1 markedly reduced cell migratory activity, suggesting an importance of the RSLE sequence for the signaling events of neuronal migration mediated by L1.     

A Drosophila receptor-type tyrosine kinase (DFR1) acts as a fibroblast growth factor receptor in Xenopus embryos

Members of the fibroblast growth factor (FGF) family play important roles in various developmental processes in vertebrates. Since two genes closely related to the vertebrate FGF receptor (FGFR) genes DFR1 and DFR2/breathless have already been reported in Drosophila , the existence of a Drosophila FGF has been predicted. In the present study, we examined whether DFR1 is functionally interchangeable with a vertebrate FGFR in the Xenopus system. First, we found that the expression of DFR1 promoted Ca²⁺ efflux in response to human basic (b)FGF in Xenopus oocytes, whereas the coexpression of a dominant negative form of DFR1 (ΔDFR1) with a chick FGFR1/cek1 inhibited promotion of Ca²⁺ efflux induced by the expression of cek1 in the oocyte. Second, the expression of ΔDFR1 was observed to induce a defect in the posterior structure of the Xenopus embryo at stage 30, as observed with a dominant negative form of cek1 (Δcek1). Third, we found that the expression of ΔDFR1 inhibited the expression of FGF-regulated genes such as Xbra, Xnot , and Xshh in Xenopus embryos at stage 11, while the coexpression of DFR1 with ΔDFR1 could rescue the inhibited expression of FGF-regulated genes. These results indicate that DFR1 acts as an FGFR in Xenopus embryos and that an FGF is likely to exist in Drosophila .     

Nerve Growth Factor-Stimulated Nuclear S6 Kinase in PC12 Cells

Abstract: Previous work has shown that nerve growth factor (NGF) stimulates the phosphorylation of the ribosomal protein S6 in PC12 cells. In this study, we show that S6 kinase activity is also present in purified PC12 cell nuclei. This activity was increased by treatment of the cells with NGF and, to a lesser extent, by treatment with epidermal growth factor. The NGF-stimulated activity was obtained from nuclear extracts and some of its characteristics described. The increase in activity was prevented by treatment of the cells with rapamycin or with wortmannin, and the overall activity could be precipitated by antibodies directed against the p85^S6K. These data indicate that p85^S6K is the NGF-stimulated S6 kinase in PC12 cell nuclei. The presence of S6 protein in the nucleus of PC12 cells has been confirmed and evidence is presented that suggests that it is identical to a protein called SMP reported some years ago.