Cloning and characterization of an electrogenic Na/HCO3− cotransporter from the squid giant fiber lobe

The squid giant axon is a classic model system for understanding both excitable membranes and ion transport. To date, a Na+-driven Cl-HCO3− exchanger, sqNDCBE—related to the SLC4 superfamily and cloned from giant fiber lobe cDNA—is the only HCO3−-transporting protein cloned and characterized from a squid. The goal of our study was to clone and characterize another SLC4-like cDNA. We used degenerate PCR to obtain a partial cDNA clone (squid fiber clone 3, SF3), which we extended in both the 5′ and 3′ directions to obtain the full-length open-reading frame. The predicted amino-acid sequence of SF3 is similar to sqNDCBE, and a phylogenetic analysis of the membrane domains indicates that SF3 clusters with electroneutral Na+-coupled SLC4 transporters. However, when we measure pHi and membrane potential—or use two-electrode voltage clamping to measure currents—on Xenopus oocytes expressing SF3, the oocytes exhibit the characteristics of an electrogenic Na/HCO3− cotransporter, NBCe. That is, exposure to extracellular CO2/HCO3− not only causes a fall in pHi, followed by a robust recovery, but also causes a rapid hyperpolarization. The current-voltage relationship is also characteristic of an electrogenic NBC. The pHi recovery and current require HCO3− and Na+, and are blocked by DIDS. Furthermore, neither K+ nor Li+ can fully replace Na+ in supporting the pHi recovery. Extracellular Cl− is not necessary for the transporter to operate. Therefore, SF3 is an NBCe, representing the first NBCe characterized from an invertebrate.

Changes in Phenylalanine Ammonia-lyase Activity and Gene Expression during Storage of Asparagus Spears

A cDNA clone coding phenylalanine ammonia-lyase (PAL) was isolated from a cDNA library prepared from asparagus spears (Asparagus officinalis L. cv. Welcome) using the reverse transcription-polymerase chain reaction (RT-PCR). The partial cDNA clone encoded an mRNA of 527 bp and the derived amino acid sequence was found highly homologous to PAL from rice, maize and barley. Northern blot analysis showed an increase of pAS-PAL mRNA until 24 h at 20 °C, which coincided well with PAL activity and fiber development, suggesting that the increase is a response to the wounding associated with harvest.

Cortisol alters carbonic anhydrase-mediated renal sulfate secretion

Active transepithelial sulfate secretion rate by winter flounder renal proximal tubule epithelium in primary culture (fPTC) is dependent on intracellular carbonic anhydrase (CA) and enhanced by cortisol. To further evaluate this relationship, a partial cDNA clone (327 bp) of carbonic anhydrase II (CAII) with high sequence similarity to CAII from numerous species including fish, chicken, and human was obtained from fPTCs. The majority of CA activity and CAII protein was present in the cytosol of fPTCs; however, significant amounts of both (in addition to SDS-resistant CA activity, i.e., CAIV-like isoform) were present in concentrated plasma membranes. CAII from concentrated membranes migrated differently than purified CAII on nondenaturing PAGE gels, suggesting that CAII associates with another membrane component. Treatment of fPTCs with the cell-soluble CA inhibitor methazolamide (100 μM) caused a 58% reduction in active transepithelial SO42- secretion. fPTCs that were previously cultured under high-cortisol concentrations, when subjected to 5 days of low physiological levels of cortisol, had decreased CA activity (28%), CAII protein abundance (65%), and net active SO42- secretion (28%), with no effect on epithelial differentiation. Methazolamide and low-cortisol treatment in combination inhibited net active SO42- secretion 56%, which was not different than the effect of methazolamide treatment alone. These data indicate that cortisol directly increases renal CA activity, CAII protein abundance, and CA-dependent SO42- secretion in the marine teleost renal proximal tubule.

Identification and Initial Characterization of the Murine Gammaherpesvirus 68 Gene M3, Encoding an Abundantly Secreted Protein

ABSTRACT Several viruses, including members of the gammaherpesvirus family, encode proteins that are secreted into the extracellular environment. We have identified an abundant 44-kDa secreted protein that is present in the supernatant of fibroblasts infected with murine gammaherpesvirus 68 (γHV68; also referred to as MHV-68) but not in that of uninfected fibroblasts. Sequence analysis of the amino terminus and of internal peptides revealed that this protein is encoded by the γHV68 M3 open reading frame (ORF). The amino-terminal sequence of the secreted protein starts at residue 25 of the M3 ORF, consistent with the first 24 residues functioning as a signal peptide. Northern blot analysis revealed a single abundant ∼1.4-kb early-late lytic transcript encoded by the M3 ORF. Analysis of a partial cDNA clone and subsequent analyses of products of rapid amplification of cDNA ends coupled with S1 nuclease protection assays demonstrate that the M3 protein is encoded by an unspliced, polyadenylated mRNA initiating at bp 7294 and terminating at bp 6007 of the γHV68 genome. The 3′ end of the M3 transcript maps 9 bp downstream of a consensus polyadenylation signal. Thus, the predicted M3 ORF is a functional gene that encodes an abundant secreted protein which is a candidate for interacting with host cellular receptors or cytokines.

Expression of the sodium-chloride cotransporter in osteoblast-like cells: effect of thiazide diuretics

The use of thiazide diuretics is associated with increased bone mineral density and, in some studies with reduced incidence of fractures, suggesting a potential role for these drugs in the treatment of osteoporosis. Our objective was to examine the effects of thiazides on osteoblast-like cells using the rat UMR-106 osteosarcoma cell line. Treatment of UMR-106 cells with chlorothiazide caused membrane depolarization and a rise of intracellular calcium but had no effect on adenosine 3,5'-cyclic monophosphate accumulation. The rise of intracellular calcium was partially inhibited by nifedipine and removal of extracellular calcium, indicating calcium uptake from the extracellular media, as well as by thapsigargin or dantrolene, indicating contributions from calcium release from intracellular stores. Reverse transcriptase-polymerase chain reaction was used to isolate a partial cDNA clone for the thiazide-sensitive sodium-chloride cotransporter from UMR-106 cells that hybridized to 5.0- and 11.0-kilobase mRNAs when Northern blot analysis was conducted. Antisense oligonucleotides to the sodium-chloride cotransporter specifically inhibited the chlorothiazide-induced depolarization and rise of intracellular calcium and reduced immunofluorescence staining for the sodium-chloride cotransporter protein in UMR-106 cells. We conclude that thiazide diuretics inhibit sodium-chloride cotransporter activity in UMR-106 cells, thereby altering intracellular calcium regulation. These results provide evidence for direct effects of thiazide diuretics on bone cells.