Time course and localization of nerve growth factor expression and sensory nerve growth during progression of knee osteoarthritis in rats.

OBJECTIVES: Nerve growth factor (NGF) and sensory nerves are key factors in established osteoarthritis (OA) knee pain. We investigated the time course of NGF expression and sensory nerve growth across early and late stages of OA progression in rat knees. DESIGN: Knee OA was induced by medial meniscectomy in rats. OA histopathology, NGF expression, and calcitonin gene-related peptide immunoreactive (CGRP-IR) nerves were quantified pre-surgery and post-surgery at weeks 1, 2, 4 and 6. Pain-related behavior was evaluated using dynamic weight distribution and mechanical sensitivity of the hind paw. RESULTS: NGF expression in chondrocytes increased from week 1 and remained elevated until the advanced stage. In synovium, NGF expression increased only in early stages, whereas in osteochondral channels and bone marrow, NGF expression increased in the later stages of OA progression. CGRP-IR nerve density in suprapatellar pouch peaked at week 4 and decreased at week 6, whereas in osteochondral channels and bone marrow, CGRP-IR innervation increased through week 6. Percent ipsilateral weight-bearing decreased throughout the OA time course, whereas reduced paw withdrawal thresholds were observed only in later stages. CONCLUSION: During progression of knee OA, time-dependent alterations of NGF expression and CGRP-IR sensory innervation are knee tissue specific. NGF expression increased in early stages and decreased in advanced stage in the synovium but continued to increase in osteochondral channels and bone marrow. Increases in CGRP- IR sensory innervation followed increases in NGF expression, implicating that NGF is a key driver of articular nerve growth associated with OA pain.

Possible involvement of syntaxin 1A downregulation in the late phase of allodynia induced by peripheral nerve injury.

Syntaxin 1A is a membrane protein playing an integral role in exocytosis and membrane trafficking. The superficial dorsal horn (SDH) of the spinal cord, where nociceptive synaptic transmission is modulated, is rich in this protein. We recently reported that peripheral nerve ligation-induced nociceptive responses are considerably enhanced in syntaxin 1A-knockout mice [Takasusuki T, Fujiwara T, Yamaguchi S, Fukushima T, Akagawa K, Hori Y (2007) Eur J Neurosci 26:2179-2187]. On the basis of this earlier finding, we hypothesized that syntaxin 1A is involved in peripheral nerve injury-induced nociceptive plasticity. In this study, we examined this hypothesis by using nociceptive behavioral studies and tight-seal whole-cell recordings from neurons in the SDH of adult mouse spinal cord slices. Partial sciatic nerve ligation (PSNL) in adult male Institute of Cancer Research (ICR) mice increased the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs). The amplitude of the mEPSCs did not exhibit any changes, suggesting that peripheral nerve injury is associated with increased synaptic release of excitatory neurotransmitters. Western blot and real-time quantitative reverse transcription-polymerase chain reaction analyses revealed that PSNL gradually decreased the expression level of syntaxin 1A in the spinal SDH. This downregulation of syntaxin 1A took several days to develop, whereas behavioral allodynia developed within one day after PSNL. Syntaxin 1A knockdown by intrathecal injection of an antisense oligodeoxynucleotide against the syntaxin 1A gene led to the gradual development of allodynia. These results indicate a possible involvement of syntaxin 1A downregulation in the late maintenance phase of peripheral nerve injury-induced allodynia. In addition, syntaxin 1A knockdown by ribonucleic acid interference enhanced the axonal elongation and sprouting of spinal dorsal horn neurons in culture, suggesting that PSNL-induced syntaxin 1A downregulation may result in the rearrangement of the synaptic connections between neurons in the spinal dorsal horn. Taken together, it is possible to conclude that syntaxin 1A might be involved in spinal nociceptive plasticity induced by peripheral nerve injury.

Properties of a revertant of Escherichia coli viable in the presence of spermidine accumulation: increase in L-glycerol 3-phosphate.

Escherichia coli CAG2242 cells are deficient in the speG gene encoding spermidine acetyltransferase. When these cells were cultured in the presence of 0.5 to 4 mM spermidine, their viability was greatly decreased through the inhibition of protein synthesis by overaccumulation of spermidine. When the cells were cultured with a high concentration of spermidine (4 mM), a revertant strain was obtained. We found that a 55-kDa protein, glycerol kinase, was overexpressed in the revertant and that synthesis of a ribosome modulation factor and the RNA polymerase sigma(38) subunit, factors important for cell viability, was increased in the revertant. Levels of L-glycerol 3-phosphate also increased in the revertant. Transformation of glpFK, which encodes a glycerol diffusion facilitator (glpF) and glycerol kinase (glpK), to E. coli CAG2242 partially prevented the cell death caused by accumulation of spermidine. It was also found that L-glycerol 3-phosphate inhibited spermidine binding to ribosomes and attenuated the inhibition of protein synthesis caused by high concentrations of spermidine. These results indicate that L-glycerol 3-phosphate reduces the binding of excess amounts of spermidine to ribosomes so that protein synthesis is recovered.

Multiple polyamine transport systems on the vacuolar membrane in yeast.

We recently identified a gene (TPO1, YLL028w) that encodes a polyamine transport protein on the vacuolar membrane in yeast [Tomitori, Kashiwagi, Sakata, Kakinuma and Igarashi (1999) J. Biol. Chem. 274, 3265-3267]. Because the existence of one or more other genes for a polyamine transport protein on the vacuolar membrane was expected, we searched sequence databases for homologues of the protein encoded by TPO1. Membrane proteins encoded by the open reading frames YGR138c (TPO2), YPR156c (TPO3) and YOR273c (TPO4) were postulated to be polyamine transporters and, indeed, were subsequently shown to be polyamine transport proteins on the vacuolar membrane. Cells overexpressing these genes were resistant to polyamine toxicity and showed an increase in polyamine uptake activity and polyamine content in vacuoles. Furthermore, cells in which these genes were disrupted showed an increased sensitivity to polyamine toxicity and a decrease in polyamine uptake activity and polyamine content in vacuoles. Resistance to polyamine toxicity in cells overexpressing the genes was overcome by bafilomycin A(1), an inhibitor of the vacuolar H(+)-ATPase. Among the four polyamine transporters, those encoded by TPO2 and TPO3 were specific for spermine, whereas those encoded by TPO1 and TPO4 recognized spermidine and spermine. These results suggest that polyamine content in the cytoplasm of yeast is elaborately regulated by several polyamine transport systems in vacuoles. Furthermore, it was shown that Glu-207, Glu-324 (or Glu-323) and Glu-574 of TPO1 protein were important for the transport activity.

Identification of the putrescine recognition site on polyamine transport protein PotE.

The PotE protein can catalyze both uptake and excretion of putrescine. The K(m) values of putrescine for uptake and excretion are 1.8 and 73 microm, respectively. Uptake of putrescine is dependent on the membrane potential, whereas excretion involves putrescine-ornithine antiporter activity. Amino acids involved in both activities were identified using mutated PotE proteins. It was found that Cys(62), Trp(201), Trp(292), and Tyr(425) were strongly involved in both activities, and that Tyr(92), Cys(210), Cys(285), and Cys(286) were moderately involved in the activities. Mutations of Tyr(78), Trp(90), and Trp(422) mainly affected uptake activity, and the K(m) values for putrescine uptake by these PotE mutants increased greatly, indicating that these amino acids are involved in the high affinity uptake of putrescine by PotE. Mutations of Lys(301) and Tyr(308) mainly affected excretion activity (putrescine-ornithine antiporter activity), and excretion by these mutants was not stimulated by ornithine, indicating that these amino acids are involved in the recognition of ornithine. It was found that the putrescine and ornithine recognition site on PotE is located at the cytoplasmic surface and the vestibule of the pore consisting of 12 transmembrane segments. Based on the results of competition experiments with various putrescine analogues and the disulfide cross-linking of PotE between cytoplasmic loops and the COOH terminus, a model of the putrescine recognition site on PotE consisting of the identified amino acids is presented.

Identification of a gene for a polyamine transport protein in yeast.

Properties of a membrane protein encoded by YLL028w were examined using yeast cells transformed with the gene. The transformed cells became resistant to polyamine toxicity, and the resistance was overcome by bafilomycin A1, an inhibitor of vacuolar H+-ATPase. Although spermine uptake activity of the transformed cells was almost the same as that of wild type cells, the uptake activity of vacuolar membrane vesicles from the transformed cells was higher than that from wild type cells. The transformed cells became resistant to MGBG (methylglyoxal bis(guanylhydrazone)) and paraquat, but not Ni2+ and Co2+, suggesting that the protein encoded by YLL028w is a transport protein specific for polyamines. When the YLL028w gene was disrupted by inserting the HIS3 gene, the cells became sensitive to polyamines, and spermine uptake activity of the vacuolar membrane vesicles decreased significantly. The accumulated spermine in YLL028w gene-disrupted cells decreased greatly compared with that in wild type cells. The results indicate that a membrane protein encoded by YLL028w (TPO1) is a polyamine transport protein on the vacuolar membrane.

Formation of a complex containing ATP, Mg2+, and spermine. Structural evidence and biological significance.

The conformation of ATP in the presence of Mg2+ and/or spermine was studied by 31P and 1H NMR, to clarify how polyamines interact with ATP. Spermine predominantly interacted with the beta- and gamma-phosphates of ATP in the presence of Mg2+. A conformational change of the beta- and gamma-phosphate of ATP with spermine could not be observed in the absence of Mg2+ by 31P NMR. It was found by 1H NMR that the conformation of adenosine moiety of ATP was not influenced significantly by spermine. The binding of Mg2+ to ATP was slightly inhibited by spermine and vice versa. The results indicate that the binding sites of Mg2+ and spermine on ATP only partially overlap. The PotA protein, an ATP-dependent enzyme, was used as a model system to study the biological role of the ATP-Mg2+-spermine complex. The ATPase activity of PotA was greatly enhanced by spermine. Double reciprocal plots at several concentrations of spermine as an activator indicate that spermine interacts with ATP, but not with PotA. The activity of protein kinase A was also stimulated about 2-fold by spermine. The results suggest that a ternary complex of ATP-Mg2+-spermine may play an important role in some ATP-dependent reactions in vivo and in the physiological effects of endogenous polyamines.

Crystallization and preliminary X-ray analysis of the periplasmic receptor (PotF) of the putrescine transport system in Escherichia coli.

The primary receptor (PotF) of the putrescine transport system in E. coli has been crystallized by the hanging-drop vapor-diffusion technique. The crystals belong to the space group P21212 with unit-cell dimensions a = 269.4, b = 82.33 and c = 93.74 A. The crystals diffract beyond 2.2 A with a rotating-anode X-ray source. A complete data set from the native crystals has been collected and processed at 2.3 A resolution. Two heavy-atom derivatives have been prepared from the same Pt compound at 293 and 277 K. The difference Patterson maps revealed completely different major heavy-atom sites between these two derivatives.

Crystal structure and mutational analysis of the Escherichia coli putrescine receptor. Structural basis for substrate specificity.

PotF protein is a periplasmic substrate-binding protein of the putrescine transport system in Escherichia coli. We have determined the crystal structure of PotF protein in complex with the substrate at 2.3-A resolution. The PotF molecule has dimensions of 54 x 42 x 30 A and consists of two similar globular domains. The PotF structure is reminiscent of other periplasmic receptors with a highest structural homology to another polyamine-binding protein, PotD. Putrescine is tightly bound in the deep cleft between the two domains of PotF through 12 hydrogen bonds and 36 van der Waals interactions. The comparison of the PotF structure with that of PotD provides the insight into the differences in the specificity between the two proteins. The PotF structure, in combination with the mutational analysis, revealed the residues crucial for putrescine binding (Trp-37, Ser-85, Glu-185, Trp-244, Asp-247, and Asp-278) and the importance of water molecules for putrescine recognition.

Excretion and uptake of putrescine by the PotE protein in Escherichia coli.

The structure and function of the polyamine transport protein PotE was studied. Uptake of putrescine by PotE was dependent on the membrane potential. In contrast, the putrescine-ornithine antiporter activity of PotE studied with inside-out membrane vesicles was not dependent on the membrane potential (Kashiwagi, K., Miyamoto, S., Suzuki, F., Kobayashi, H., and Igarashi, K. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4529-4533). The Km values for putrescine uptake and for putrescine-ornithine antiporter activity were 1.8 and 73 microM, respectively. Uptake of putrescine was inhibited by high concentrations of ornithine. This effect of ornithine appears to be due to putrescine-ornithine antiporter activity because it occurs only after accumulation of putrescine within cells and because ornithine causes excretion of putrescine. Thus, PotE can function not only as a putrescine-ornithine antiporter to excrete putrescine but also as a putrescine uptake protein. Both the NH2 and COOH termini of PotE were located in the cytoplasm, as determined by the activation of alkaline phosphatase and beta-galactosidase by various PotE-fusion proteins. The activities of putrescine uptake and excretion were studied using mutated PotE proteins. It was found that glutamic acid 207 was essential for both the uptake and excretion of putrescine by the PotE protein and that glutamic acids 77 and 433 were also involved in both activities. These three glutamic acids are located on the cytoplasmic side of PotE, and the function of these three residues could not be replaced by other amino acids. Putrescine transport activities did not change significantly with mutations at the other 13 glutamic acid or aspartic acid residues in PotE.