31P nuclear magnetic resonance spectroscopic evidence that canine nucleus pulposus and anulus fibrosus cells differ in principal organophosphorus biomolecules.

STUDY DESIGN: 31P Nuclear magnetic resonance spectroscopy was used to compare amounts and types of principal organophosphorus biomolecules in canine intervertebral disc tissues ex vivo. OBJECTIVE: The goal of this study was to compare principal organophosphorus biomolecules in nucleus pulposus, anulus fibrosus, and in isolated nucleus pulposus and anulus fibrosus cells. SUMMARY OF BACKGROUND DATA: There is no published information on principal organophosphorus biomolecules in disc tissues for any animal. METHODS: Canine nucleus pulposus and anulus fibrosus were shown by hematoxylin-eosin staining to be healthy tissues characteristic of adult dogs. Viable cells liberated from these disc tissues by sequential protease digestion were directly visualized by light microscopy of wet mounts. 31P nuclear magnetic resonance spectra were recorded at room temperature for 12 hours according to conventional published methods. RESULTS: No resonances were detected for intact nucleus pulposus and anulus fibrosus tissue. 31P nuclear magnetic resonance spectra of viable chondrocyte-like cells from anulus fibrosus featured two distinctive resonance peaks consistent with phosphomonoesters and phosphodiesters. After undigested anulus fibrosus was removed, no resonances were detected. 31P nuclear magnetic resonance spectra of viable chondrocyte-like cells from nucleus pulposus before and after removal of undigested tissue featured only one resonance peak consistent with phosphodiesters. CONCLUSIONS: A phosphomonoester-containing biomolecule is present in healthy canine anulus fibrosus tissue but not in nucleus pulposus tissue, nor in nucleus pulposus cells or anulus fibrosus cells. A phosphodiester-containing biomolecule is present principally in nucleus pulposus cells. This study demonstrated that canine chondrocyte-like cells from nucleus pulposus and anulus fibrosus are phenotypically distinctive in principal organophosphorus biomolecules.

Osmotic regulation of taurine transport via system beta and novel processes in mouse preimplantation conceptuses.

Taurine was shown recently to increase the frequency at which 2-cell mouse conceptuses develop into blastocysts in vitro. For this reason and because taurine helps cells adapt to external stresses, we studied transport of this and related amino acids by preimplantation mouse conceptuses. The most conspicuous component of taurine transport in conceptuses at the 1-cell through blastocyst stages of development was both Na(+)- and Cl(-)-dependent. This Na(+)- and Cl(-)-dependent transport system interacted relatively strongly with beta- but not alpha-amino acids. By these criteria, transport system beta is responsible for Na(+)-dependent taurine transport in preimplantation mouse conceptuses. Moreover, detection of mRNA encoding the taurine transport protein (TAUT) in early conceptuses supports the theory that TAUT is a major component of system beta. Transport of taurine by system beta in 1-cell conceptuses was slower in hypotonic than in hypertonic media, whereas the reverse was true for system beta in blastocysts. In contrast, hypotonically stimulated Na(+)-independent taurine transport was, of course, more rapid in hypotonic than in hypertonic media in both 1-cell conceptuses and blastocysts. Transport via this hypotonically stimulated process also showed no sign of saturation by up to 10 mM taurine. Hypotonically stimulated taurine transport appeared transiently in 1-cell conceptuses under hypotonic conditions until they had recovered their initial volumes. Hence, we suggest that a decrease in taurine uptake via system beta and an increase in taurine exodus via the Na(+)-independent, nonsaturable transport process could contribute to the regulatory volume decrease in 1-cell conceptuses in hypotonic medium. Since taurine uptake by system beta in blastocysts is, however, higher in hypotonic than in hypertonic media, taurine uptake by system beta in blastocysts might intensify a tendency to increase cell volume in hypotonic medium. Such an increase in taurine uptake could further favor anabolic changes associated with cell swelling. In addition to contributing to regulation of cellular volume and perhaps metabolism, the hypotonically stimulated Na(+)-independent transport processes in early embryos have novel characteristics. Hypotonically stimulated Na(+)-independent taurine transport was inhibited by niflumate, N-ethylmaleimide and NaN3 but not by furosemide, iodoacetate, KCN, ouabain or alpha- or beta-amino acids. Furthermore, 4,4'-diisothiocyanostilbene-2,2'-disulfonate inhibited this transport in 1-cell conceptuses but not in blastocysts. Hence, different hypotonically stimulated Na(+)-independent taurine transport processes appear to be present in 1-cell conceptuses vs. blastocysts. The functions of these and other instances of developmental regulation of expression of transport processes in preimplantation conceptuses remain largely to be elucidated.(ABSTRACT TRUNCATED AT 400 WORDS)

The cation receptor subsite of the choline transporter in preimplantation mouse conceptuses resembles a cation receptor subsite of several amino acid transporters.

Mediated choline transport in preimplantation mouse conceptuses was inhibited competitively by Na+ and other cationic osmolites. Uptake of choline by conceptuses was also inhibited relatively strongly by ethanolamine, hemicholinium-3, harmaline, harmalol and harmine. The Ki values for inhibition of choline transport by most of the latter inhibitors were of the same order of magnitude as the Km value for choline transport (approximately 100 microM). To our knowledge, we are the first to show that mediated 'Na(+)-independent' choline transport is, nevertheless, inhibited strongly by the Na(+)-site inhibitor, harmaline. Inhibitions by harmaline, Na+ and other cations have been used to draw a parallel between the substrate receptor sites of amino acid transport systems y+ and bo.+. We suggest that the latter parallel should be extended to include the Na(+)-independent mammalian choline transporter. In addition, the choline transport activity in conceptuses increased by more than 100-fold between the 2-cell and blastocyst stages of development. Mouse blastocysts probably utilize choline for the synthesis of membrane phospholipids during cellular differentiation and when they begin to grow about ten hours prior to implantation. Since we show here that mouse conceptuses develop the capacity to transport choline prior to the onset of growth, some of the choline utilized for growth could come from an exogenous source.

Mediated Na(+)-independent transport of L-glutamate and L-cystine in 1- and 2-cell mouse conceptuses.

L-Glutamate and L-cystine appeared to compete for transport via a mediated Na(+)-independent transport process in 1- and 2-cell conceptuses. Not only did these substances competitively inhibit each others' uptake by conceptuses, but their Ki values for inhibition were about equal to their Km values for transport in 1-cell conceptuses. Moreover, the transport process interacted strongly with L-amino acids that had 3-6 atoms in a chain between their negatively charged groups, whereas it interacted weakly or not at all with amino acids that did not have these characteristics or that were N-methylated. Transport of anionic amino acids was not altered greatly by pH in the range 4.5-8.0, but transport of L-cystine was much faster at higher pH values. The slower cystine transport at lower pH values was due primarily to protonation of its second amino group. A small increase in the degree of deprotonation of cystine's carboxyl groups also probably contributed slightly to its faster transport at higher pH values. By all of these criteria, the transport process in conceptuses appears to be a form of amino acid transport system xc-. System xc- activity was several-fold higher in 1- than in 2-cell conceptuses. Similarly, L-glutamate uptake by unfertilized eggs was relatively rapid, whereas it was much slower in immature, fully-grown oocytes. System xc- activity in 1-cell conceptuses also appeared to increase in response to the oxidative stress of culture, whereas no such increase was observed for 2-cell conceptuses. We suggest that this transient increase in the activity of system xc- activity during development of 2-cell conceptuses from immature, fully-grown oocytes could help protect unfertilized and fertilized eggs from oxidative stresses in situ.