Kinji Kurihara

Characterization of a phosphorylation event resulting in upregulation of the salivary Na+-K+-2Cl−cotransporter

Previous studies from our laboratory have shown a close correlation between increased Na+-K+-2Cl-cotransporter activity and increased cotransporter phosphorylation after β-adrenergic stimulation of rat parotid acinar cells. We demonstrate here that these effects are paralleled by an increase in the number of high-affinity binding sites for the cotransporter inhibitor bumetanide in membranes prepared from stimulated acini. We also show that the sensitivity of cotransporter fluxes to inhibition by bumetanide is the same in both resting and isoproterenol-stimulated cells, consistent with the hypothesis that β-adrenergic stimulation and the accompanying phosphorylation result in the activation of previously quiescent transporters rather than in a change in the properties of already active proteins. In addition, we demonstrate that the increased phosphorylation on the cotransporter resulting from β-adrenergic stimulation is localized to a 30-kDa phosphopeptide obtained by cyanogen bromide digestion. Immunoprecipitation and Western blotting experiments demonstrate that this peptide is derived from the NH2-terminal cytosolic tail of the cotransporter, which surprisingly does not contain the sole protein kinase A consensus site on the molecule.

Immunohistochemical profile of basic fibroblast growth factor and heparan sulphate in adult rat mandibular condylar cartilage.

Basic fibroblast growth factor (bFGF) and heparan sulphate (HS) were detected immunohistochemically in mandibular condylar cartilage, and the findings compared with those on epiphyseal articular cartilage. In the condylar cartilage, both bFGF and HS were localized in chondrocytes throughout the various zones including the fibrous, proliferative, mature-cell and hypertrophic zones: bFGF immunostaining was most significant in the proliferative and mature-cell zones, while intense staining for HS was found mainly in the hypertrophic zone. Immunoreaction for bFGF was detected in the nuclei of chondrocytes, whereas HS staining was observed in the cytoplasm. In articular cartilage, only chondrocytes beneath the superficial zone (intermediate zone) demonstrated both bFGF and HS immunoreactivities. Chondrocytes in the deeper calcifying region of the articular cartilage did not immunoreact for either bFGF or HS. These findings suggest that, in contrast to the epiphyseal articular cartilage, a continuous bFGF-mediated remodelling of cells and matrix takes place in mandibular condylar cartilage during the process of endochondral ossification.

Effects of protein kinase inhibitors and protein phosphatase inhibitors on cyclic AMP-dependent down-regulation of vesicular monoamine transport in pheochromocytoma PC12 cells

Cyclic AMP down‐regulates vesicular monoamine transport in PC12 cells and thereby decreased catecholamine reuptake from the extracellular fluid. We examined the effects of protein kinase inhibitors and protein phosphatase inhibitors on this cAMP action. Treatment of cells with a protein kinase inhibitor, K252a, increased vesicular amine transport and cellular amine uptake, thereby antagonizing the regulatory action of cAMP. In contrast, a protein phosphatase inhibitor, okadaic acid, had the opposite effect on the amine transport, i.e. it enhanced the cAMP action. These results suggest the involvement of a protein phosphorylation process in the cAMP‐dependent modulation of vesicular monoamine transport.

Regulation of Na+,K+-ATPase in submandibular glands of hypophysectomized male mice by steroid and thyroid hormones.

The effects of thyroid hormone, androgen, glucocorticoid, and mineralocorticoid on Na+,K+-ATPase activity and on levels of its alpha-subunit protein (alpha 1 isoform) in mouse submandibular gland (SMG) were studied by enzyme assay for ouabain-sensitive ATP hydrolysis, by quantitative densitometric scanning of Western blots, and by immunohistochemistry. To define the specific regulatory effects of various pituitary-dependent hormones on expression of Na+,K+-ATPase in the SMG, we treated hypophysectomized (hypox) male mice with triiodo-L-thyronine (T3), 5 alpha-dihydrotestosterone (DHT), dexamethasone (Dex), and aldosterone (Ald), injected singly or in combination. Na+,K+-ATPase was confined to the duct system of the SMG. In intact mice there was a gender difference in SMG Na+,K+-ATPase, with levels of the enzymes activity and of its alpha 1-subunit being less in the glands of males. In males, hypophysectomy caused a rise in levels of Na+,K+-ATPase activity and in levels of the alpha 1-subunit protein of this enzyme, and in intensity of immunocytochemical staining for this subunit but there were no such changes in the SMG of hypox females. Changes caused by hormonal replacement to hypox males in Na+,K-ATPase activity, levels of its alpha 1-subunit, or the intensity of immunocytochemical staining for this subunit were complex. Ald had no effect. T3 or dexamethasone, given alone, induced Na+,K+-ATPase activity above control values (hypox males) and increased levels of its alpha 1-subunit protein and immunohistochemical staining for this subunit. By contrast, DHT did not cause a decline in any of these parameters. However, when treatment with T3 was combined with administration of Dex or DHT, enzymatic activity of Na+,K+-ATPase decreased but levels of the alpha 1-subunit protein and immunohistochemical staining for this subunit increased. Therefore, inductions of the alpha 1-subunit of this enzyme are not always correlated with increases in levels of activity of Na+,K+-ATPase, and we propose that both enzymatic and immunochemical analyses are essential for evaluation of hormonal regulation of Na+,K+-ATPase in salivary gland and in other tissues.

A new electrophoretic variant of α subunit of Na+/K+-ATPase from the submandibular gland of rats

Abstract The α catalytic subunits of Na + K + -ATPase were isolated from the kidney and brain of rats (α1 and α2, respectively). The antisera raised against these subunits were used as probes to analyze the isoform of catalytic subunits of Na + K + -ATPase in various tissues of rats. Of 27 rat tissues examined, most had a catalytic subunit identical to α1 but some, such as the nervous and muscle tissues, had both α1 and α2 isoforms as judged by their reactivities to antisera and their electrophoretic mobility. We found that the submandibular gland contained a new electrophoretic variant of immunoreactive α subunit (designated α(S) in this report) in addition to α1 identical to those found in kidney and brain. The new variant, α(S), strongly cross-reacted with anti-α1 antiserum, but to a lesser extent with anti-α2 antiserum. The α(S) had a molecular mass which was found to be slightly less (approx. 90 kDa) than brain and kidney α1. We examined whether or not the α(S) is formed by proteolytic cleavage of α subunits during preparation and concluded that this is not the case. The α(S) reacted with [γ-32P]ATP, resulting in the formation of radioactive α subunit which was stabilized by 2 mM ouabain but which was labile in the presence of 70 mM potassium chloride. Since N-terminal amino acid sequence of α(S) protein [G()DKY()PAAVS] corresponds exactly and uniquely with the sequence of the α1 chain between residues 1 and 11, it is very probable that α(S) protein originated from α1 protein following the post-translational processing.

Characterization of ecto-nucleoside triphosphatase on A-431 human epidermoidal carcinoma cells.

Hydrolysis of extracellular ATP and other nucleoside phosphates by A-431 human epidermoidal carcinoma cells was studied. The hydrolysis of extracellular ATP by these cells required either Mg2+ or Ca2+, and either cation could be replaced by Co2+, Fe2+, or Mn2+. Nucleoside triphosphates (ATP, GTP, CTP, UTP, and dTTP), but not nucleoside diphosphates, were hydrolyzed by the cells with Km and Vmax values similar to those for ATP (0.9-1.1 mmol/l and 6-10 nmol Pi formed/10(6) cells, respectively). The hydrolysis of ATP was inhibited strongly by ATP-gamma S and AMPPNP, and weakly by AMPCPP and ADP-beta S, but not by AMPCPP or AMPCP. Since the hydrolysis of [gamma-32P]ATP was inhibited by all these nucleoside triphosphates, the binding site for ATP is presumed to be the same as that for the other nucleoside triphosphates. All these results indicate that ecto-ATPase activity associated with A-431 cells is due to ecto-nucleoside triphosphatase. The nucleotide specificity shown in the present study indicates that ecto-nucleoside triphosphatase associated with A-431 cells is a molecule different from P2-purinergic receptors which can be stimulated specifically with nucleoside phosphates like ATP, ADP, UTP, UDP, and GTP, but not by other nucleotides.

Specific expression of an A-kinase anchoring protein subtype, AKAP-150, and specific regulatory mechanism for Na+,K+-ATPase via protein kinase A in the parotid gland among the three major salivary glands of the rat

We have examined the expression of A-kinase anchoring protein (AKAP) in the three major salivary glands, i.e. the parotid gland (PG), submandibular gland (SMG), and sublingual gland (SLG), of the rat to elucidate the functional relevance between saliva secretion and Na(+),K(+)-ATPase regulation by protein kinase A (PKA)-dependent phosphorylation, since an AKAP subtype, AKAP-150, is known to be involved in the regulation of the ATPase in PG. Although AKAP-150 and its mRNA were clearly detected in the PG, they were hardly detectable in either the SMG or SLG. The membrane-bound form of the RII regulatory subunit of PKA, an index for the total amount of AKAP subtypes and therefore of the anchored PKA holoenzyme, was also undetectable in membranes from the SMG and SLG but was found in the PG; though a substantial and comparable amount of Na(+),K(+)-ATPase was present in all of these membrane preparations. Incubation with [gamma-32P]ATP revealed that Na(+),K(+)-ATPase in the PG membranes was quickly phosphorylated upon the addition of cAMP, whereas the ATPases in the membranes from SMG and SLG were not; though they were readily and equally phosphorylated by the exogenously added PKA catalytic subunit. AKAP-150 in the basolateral membranes of PG acinar cells was co-immunoprecipitated with RII by an anti-RII antiserum; and AKAP-150 and Na(+),K(+)-ATPase were immunohistochemically co-localized predominantly on the basolateral membranes, suggesting a possibility that the ATPase might directly interact with the AKAP to form an ATPase/AKAP/PKA complex or associate with the AKAP, such association being mediated via some scaffolding molecule. Expression of AKAP-150 and quick down-regulation of Na(+),K(+)-ATPase by AKAP-anchored PKA in response to cAMP elevation are characteristics specific to PG among the three major salivary glands, suggesting the presence of PG-specific regulatory mechanisms for saliva production/secretion.

Expression of Na+/K+-ATPase α subunit isoforms in rat salivary glands: Occurrence of sense and antisense RNAs of the α3 isoform in the sublingual gland

We examined the expression of Na(+)/K(+)-ATPase alpha-subunit isoforms in rat salivary glands by RT-PCR. Isoform alpha1 was expressed strongly in all three major salivary glands. The alpha2 isoform was expressed in both submandibular gland (SMG) and sublingual gland (SLG) but faintly in the parotid gland (PG). The alpha3 was detected only in the SLG, and the alpha3 mRNA in the SLG was 1/8 of its level in the brain. Na(+)/K(+)-ATPase alpha3 isoform in the SLG, was localized predominantly on the basolateral plasma membranes in serous cells by immunohistochemical method. We also found the presence of natural antisense RNA of the alpha3 isoform in rat SLG: the 1st-strand cDNA prepared with gene-specific forward primers targeted to the CDS region and to the promoter region of the alpha3 gene in place of oligo(dT) or gene-specific reverse primers produced reasonable PCR products corresponding to the alpha3 cDNA sequence by the subsequent PCR reaction. Synthesis of the 1st-strand cDNA with the gene-specific forward primers was prevented by RNase digestion of the total RNA preparation, indicating that the PCR products in the RT-PCR system were not due to the contaminated genomic DNA, if any. The alpha3 protein level in rat SLG increased with aging, and levels of both alpha3 mRNA (sense RNA) and alpha3 antisense RNA were higher in SLGs of aged rats than in those of young rats, respectively.

Increase in Tetrahydrobiopterin Release from PC 12 Cells under Hypotonic Culture Conditions is Inhibited by HgCl2

Abstract Tetrahydrobiopterin (BH4) was released from PC 12 cells to the extracellular fluid. We found that the BH4 outward flow from the cells placed in Hanks medium was increased when NaCl concentration of the medium was decreased. Increase in the BH4 out flow was not observed when NaCl in the Hanks medium was substituted with sodium glutamate (0.14 M), choline chloride (0.14 M) or sucrosc (0.25 M). HgCl2, an inhibitor of water channel, aquaporin, prevented the increase in BH4 out flow caused by the hypotonic medium. The results suggested the presence of a site in BH4 transport which was stimulated by osmotic pressure and/or water influx.

Postnatal changes in an alpha subunit isoform, alpha(S), of Na+,K(+)-ATPase in the submandibular gland of rats.

The alpha and alpha(+) isoforms of Na+,K(+)-ATPase were isolated from the kidney and brain of rats and purified. Their antisera were raised to analyze the alpha isoforms in rat tissues. We found that the submandibular gland (SMG) contains a new immunoreactive alpha subunit isoform, designated alpha(S) in this report, in addition to alpha identical with those found in the kidney or brain. The new alpha(S) strongly reacted with anti-alpha-antiserum but to a much lesser extent with anti-alpha(+)-antiserum. The alpha(S) had a slightly lower molecular weight (approximately 90,000) than the brain and kidney alpha isoforms. Various fractions of SMG tissues were added to the SMG microsomes and incubated in order to test whether or not the alpha(S) is formed artificially; no increase of alpha(S) was observed by these treatments, suggesting that the alpha(S) was not the product formed from alpha during the preparation of microsome sample, but was rather a protein originally present in the SMG. The alpha(S) protein was not detected in the SMG of 2- or 5-week-old rats, but it gradually increased in rats older than 8 weeks, reaching the maximum in 30-week-old animals. The Na+,K(+)-ATPase activity in the SMG increased concomitantly with the increase of alpha(S), indicating that Na+,K(+)-ATPase comprising alpha(S) also shows enzyme activity; it is speculated that alpha(S) may have some unique and unknown function(s) in older rats.