Modulation of articular chondrocyte proliferation and anionic glycoconjugate synthesis by glucosamine (GlcN), N-acetyl GlcN (GlcNAc) GlcN sulfate salt (GlcN.S) and covalent glucosamine sulfates (GlcN-SO4).

OBJECTIVES: To investigate, in chondrocyte cultures under conditions for maximizing responses in proliferation and proteoglycan (PG) synthesis, the effects of glucosamine hydrochloride (GlcN.HCl) and glucosamine sulfate (GlcN.S) salts, N-acetyl glucosamine (GlcNAc), and covalently substituted GlcN-X,Y,Z(SO(4))(n) (general formula). METHODS: Bovine articular chondrocytes (BAC) were studied under anchorage-independent (AI, alginate beads) and anchorage-dependent (AD, plastic surface) conditions. Differentiation markers were evaluated (e.g., cartilage-specific (V+C)(-) fibronectin). Varying concentrations of GlcN.HCl, GlcN.S, GlcNAc and GlcN sulfated at positions -2, -3, -6, (-2,3), (-3,6) and (-3,4,6), were tested. Cell proliferation, DNA synthesis and [(35)S]-sulfate incorporation into newly synthesized PG were determined. RESULTS: Increasing GlcN.HCl or GlcN.S concentrations gave decreasing net PG synthesis. Compounds showed more pronounced effects in AD cultures (expressing the V(-)C(+) fibronectin isoform) compared to AI cultures ((V+C)(-) isoform). Addition of GlcN.HCl or GlcN.S gave a concentration-dependent decrease in BAC proliferation, partially prevented by glucose (Glc). GlcNAc was not inhibitory. Addition of GlcN-2-SO(4) or GlcN-2,6-diSO(4) did not affect proliferation or DNA synthesis. The other GlcN-sulfates gave varying inhibitory effects, which for GlcN-3-SO(4) were reversed by inosine. CONCLUSIONS: The free amino group of GlcN seems responsible for inhibition of chondrocyte proliferation and PG synthesis. These effects were greater under higher concentrations of GlcN in AD vs AI conditions. GlcN.HCl behaves similarly to GlcN.S, but differential effects with GlcN-X,Y,Z(SO(4))(n) isomers were observed. Acetylation or sulfation of the GlcN amino group reverses or partially reverses, respectively, anti-proliferative effects of GlcN. Sulfation of GlcN, at positions 3 and 6 results in complex effects on AC proliferation and PG synthesis.

Endothelin receptor antagonism does not prevent the development of in vivo glyceryl trinitrate tolerance in the rat.

There is evidence that increased endothelial production of endothelin-1 (ET-1) may contribute to glyceryl trinitrate (GTN) tolerance. We used the competitive ET(A) receptor antagonist ZD2574 to determine whether chronic ET(A) receptor blockade affected the biochemical and functional responses to GTN during the development of GTN tolerance in vivo. Tolerance induced using transdermal GTN patches resulted in a 5.3 +/- 1.2-fold increase in the EC(50) value for GTN relaxation in isolated aorta from GTN-tolerant rats. Coadministration of ZD2574 (100 mg kg(-1) t.i.d. for 3 days) during tolerance induction had no effect on GTN-induced relaxation. This dose of ZD2574 markedly blunted the pressor response to ET-1, indicating effective blockade of ET(A) receptors, and also abolished the initial transient depressor response to ET-1, indicating that blockade of endothelial ET(B) receptors also occurred using this dosage regimen for ZD2574. Consistent with the relaxation data, coadministration of ZD2574 had no effect on the decrease in GTN-induced cGMP accumulation or on the decrease in GTN biotransformation that occurred in aortae from GTN-tolerant animals. Radioimmunoassay data indicated that the GTN tolerance induction protocol caused a 2.3 +/- 0.4-fold and a 2.2 +/- 0.5-fold increase in total tissue ET-1 levels in tolerant aorta and vena cava, respectively. These data suggest that chronic inhibition of ET receptors by ZD2574 was not sufficient to prevent or diminish the tolerance-inducing effects of GTN, and that the increase in ET-1 levels observed in tolerant tissues may occur as a consequence of the vascular changes that occur during chronic GTN exposure.

Aldehyde reductase: the role of C-terminal residues in defining substrate and cofactor specificities.

The only major structural difference between aldehyde reductase, a primarily NADPH-dependent aldo-keto reductase, and aldose reductase, a dually coenzyme-specific (NADPH/NADH) member of the same superfamily, is an additional eight amino acid residues in the substrate/inhibitor binding site (C-terminal region) of aldehyde reductase. On the premise that this segment defines the substrate specificity of the enzyme, a mutant of aldehyde reductase lacking residues 306-313 was constructed. In contrast to wild-type enzyme, the mutant enzyme reduced a narrower range of aldehydes and the new substrate specificity was not similar to aldose reductase as might have been predicted. A major change in coenzyme specificity was observed, however, the mutant enzyme being distinctly NADH preferring(Km, NADH = 35 microM, compared to <5 mM for wild-type and Km, NADPH = 670 microM, compared to 35 microM for wild type). Upon analyzing coordinates of aldehyde and aldose reductase, we found that deletion of residues 306-313 may have created a truncated enzyme that retained the three-dimensional structural features of the enzyme's C-terminal segment. The change in substrate specificity could be explained by the new alignment of amino acids. The reversal of coenzyme specificity appeared to be due to a significant backbone shift initiated by the formation of a strong hydrogen bond between Tyr319 and Val300. A similar bond exists in aldose reductase (Tyr309-Ala299). It appears, therefore, that as far as coenzyme specificity is concerned, deletion of residues 306-313 has converted aldehyde reductase into an aldose reductase-like enzyme.

Studies on the inhibitor-binding site of porcine aldehyde reductase: crystal structure of the holoenzyme-inhibitor ternary complex.

Aldehyde reductase is an enzyme capable of metabolizing a wide variety of aldehydes to their corresponding alcohols. The tertiary structures of aldehyde reductase and aldose reductase are similar and consist of an alpha/beta-barrel with the active site located at the carboxy terminus of the strands of the barrel. We have determined the X-ray crystal structure of porcine aldehyde reductase holoenzyme in complex with an aldose reductase inhibitor, tolrestat, at 2.4 A resolution to obtain a picture of the binding conformation of inhibitors to aldehyde reductase. Tolrestat binds in the active site pocket of aldehyde reductase and interacts through van der Waals contacts with Arg 312 and Asp 313. The carboxylate group of tolrestat is within hydrogen bonding distance with His 113 and Trp 114. Mutation of Arg 312 to alanine in porcine aldehyde reductase alters the potency of inhibition of the enzyme by aldose reductase inhibitors. Our results indicate that the structure of the inhibitor-binding site of aldehyde reductase differs from that of aldose reductase due to the participation of nonconserved residues in its formation. A major difference is the participation of Arg 312 and Asp 313 in lining the inhibitor-binding site in aldehyde reductase but not in aldose reductase.

Detection of metal binding to bovine inositol monophosphatase by changes in the near and far ultraviolet regions of the CD spectrum.

Mg2+ ions, essential for the catalytic activity of mammalian inositol monophosphatase, increase the ellipticity in the near-ultraviolet region of the CD spectrum of the enzyme. These spectral changes are not affected by the additional presence of substrate and are reversed if EDTA is added to the solution of enzyme and metal ions. Titration of the spectral perturbation at 275 nm shows that this binding occurs with a dissociation constant (Kd) around 275 microM, 292 microM and 302 microM for the wild-type, [Gln217]inositol monophosphatase and [Phe219]inositol monophosphatase enzymes respectively. The source of the spectroscopic change at 275 nm is not Trp219. The addition of Mg2+ also causes a decrease in ellipticity over most of the far-ultraviolet region of the spectrum (between 205-240 nm). The Kd values describing the binding of Mg2+ ions are 3.9 mM, 6.8 mM and 29.1 mM for the wild-type, [Gln217]inositol monophosphatase and [Phe219]inositol monophosphatase enzymes, respectively, each showing an approximate 12% change in ellipticity. In the additional presence of 10 mM Pi, there is a fourfold increase in the affinity of wild-type enzyme for Mg2+. It is concluded that CD spectral changes at wavelengths around 275 nm are indicative of metal ions interacting with a high-affinity metal-binding site (site 1). The spectral changes around 225 nm are associated with interactions at a lower-affinity site normally occupied by the Mg2+ ion which is reflected by the Km value for this metal ion. Other metal ions such as Ca2+ and Tb3+ (but not Mn2+ or Zn2+) also perturb the CD spectrum of the enzyme in both regions of the spectrum. The amplitudes of these signal changes are greater for Mg2+ or Tb3+ (25%) ions than for Ca2+ (8.5%), although two Ca2+-binding sites with Kd values of 20 microM and 100 microM have been identified. The uncompetitive inhibitor Li+ causes little change in the near-ultraviolet spectrum in the absence or presence of either substrate or Pi. However, in contrast to other metal ions, Li+ ions elicit a 10% increase in ellipticity at 220 nm with a Kd of 0.8 mM.

Molecular cloning and expression of a human phosphodiesterase 4C.

A cDNA coding for a human phosphodiesterase 4C (PDE4C2) was isolated from the mRNA prepared from the glioblastoma cell line, U87. The cDNA contained an ORF of 1818 bp corresponding to a 605 amino acid polypeptide. The sequence differed at the 5' end from the human PDE4C previously reported (Engels, P. et al, 1995 FEBs Letters 358, 305-310) indicating that it represents a novel splice variant of the human PDE4C gene. Evidence was also obtained for a third 5' splice variant. The PDE4C2 cDNA was transfected into both COS 1 cells and yeast cells, and shown to direct the expression of an 80 kD polypeptide by Western blotting using a PDE4C specific antiserum. The activity of cell lysates was typical of PDE4 being specific for cAMP and inhibitable by the selective inhibitor, rolipram. However, the Km for cAMP of the enzyme produced in COS cells was 0.6 microM compared to 2.6 microM for the yeast 4C activity. In addition the COS cell PDE4 activity was much more sensitive to R rolipram than the yeast PDE4 enzyme (IC50 of 23 nM compared to 1648 nM). This difference in rolipram sensitivity was associated with the detection of a high affinity [3H] R rolipram binding site on the COS cell 4C enzyme but not on the yeast expressed enzyme. The results indicate that the enzyme can adopt more than one active conformation, which are distinguished by their interaction with rolipram.

Reversible denaturation of myo-inositol monophosphatase. The stability of the metal-binding loop.

The unfolding of bovine brain myo-inositol monophosphatase by guanidine. HCl (Gdn. HCl) has been investigated. The recovery of circular dichroism, emission spectra, and catalytic activity after dilution of Gdn.HCl-treated samples indicate that the overall process is reversible. The steepness of the spectroscopic changes between 3 M and 5 M Gdn.HCl, and the lack of any discernible plateau suggest that unfolding of the protein is a cooperative process. The sensitized luminescence of bound Tb(III) was used as a probe of conformational changes of the metal-binding loop. Denaturation of the enzyme by Gdn.HCl does not abolish sensitized luminescence. A 50% decrease in sensitized luminescence was observed in 5 M Gdn.HCl. Under this set of experimental conditions, the protein binds terbium with an association constant of 1 x 10(6)M-1. It is suggested that a residual structure of denatured myo-inositol monophosphatase is responsible for the binding of terbium ions. The kinetics of unfolding and refolding as a function of Gdn.HCl concentration were monitored by protein fluorescence in a stopped-flow instrument. The monophosphatase unfolded in a single kinetic phase with rate constants in the range 80-65 s-1 at 25 degrees C. The refolding kinetics fit monoexponential functions with rate constants in the range 120-65 s-1 depending on the Gdn.HCl concentration. Substantial refolding of the protein occurs within the dead time of mixing.

Bovine inositol monophosphatase. The identification of a histidine residue reactive to diethylpyrocarbonate.

The inositol monophosphatase from bovine brain is inactivated by the histidine-specific reagent diethylpyrocarbonate. Using 4 mM reagent at pH 6.5, the reaction results in the modification of 3 equivalents of histidine per polypeptide chain. The loss of activity occurs at the same rate as the slowest reacting of these residues. Site directed mutagenesis studies have been used to generate a mutated enzyme species bearing a His-217-->Gln replacement and have shown that it is the modification of histidine 217 which results in the inactivation of the enzyme.

Bovine inositol monophosphatase: proteolysis and structural studies.

Bovine brain inositol monophosphatase is inactivated when trypsin catalyses the cleavage of a single peptide bond between Lys-36 and Ser-37. This proteolysis is closely followed by cleavage at two other sites in the protein between Lys-78 and Ser-79 and between Lys-156 and Ser-157 suggesting that all of these sites are exposed in the native conformation of the protein. All of these residues are predicted to lie at the ends of alpha helices. The most susceptible bond (Lys-36--Ser-37) is predicted to lie in a highly flexible region of the protein. Circular dichroism studies suggest that approximately 40% of the secondary structure of this protein is helical which is similar to that predicted by the algorithm of Garnier et al. [(1978) J. Mol. Biol. 120, 97-120].