Delineation on Therapeutic Significance of Transporters as Molecular Targets of Drugs

Transporters are membrane proteins mediating permeation of organic and inorganic solutes through the plasma membrane and membranes of intracellular organella. They play essential roles in the epithelial absorption and cellular uptake of nutrients as well as absorption, distribution, metabolism, and excretion of drugs. Because transporters contribute to determining the distribution of compounds in the body in concert with metabolic/synthetic enzymes, the drugs that affect the functions of transporters are expected to alter the distribution of compounds in the body and to ameliorate disrupted homeostasis. In this context, drugs targeting transporters have been used clinically. Such drugs include antidepressants targeting monoamine transporters, diuretics targeting inorganic ion transporters of renal tubules, and uricosuric agents targeting renal urate transporters. Now new transporter-targeting drugs designed based on post-genome drug development strategy have been in the process of clinical trials or basic/clinical researches. For example, the inhibitors of renal Na+/glucose cotransporter SGLT2 have been proved for their efficacy in the treatment of diabetes mellitus. The cancer L-type amino acid transporter 1 (LAT1) has been considered as a target of cancer diagnosis and therapeutics. The transporter-targeting drugs are expected to provide new rationale in the therapeutics of various diseases.     

PROTO-ONCOGENE EXPRESSION IN HUMAN GLOMERULAR DISEASES

The expression of the protein products and mRNA of c- fos , c- myc , p53, and c- raf was examined in normal renal tissues and biopsy specimens from 73 patients with various glomerular diseases. Immunofluorescent staining showed that there were cell nuclei stained for c-Fos, c-Myc, and p53, and cytoplasm positive for c-Raf, in the glomeruli of patients with proliferative types of glomerulonephritis, including IgA nephritis and lupus nephritis, and in patients with focal glomerular sclerosis. Glomerular expression of c- fos and c- myc mRNA was detected by in situ hybridization. The number of proto-oncogene-positive glomerular cells was significantly higher in lupus nephritis, IgA nephritis, and focal segmental sclerosis, as compared with minimal change nephrotic syndrome and normal specimens. In IgA nephritis, the population of glomerular cells positive for c-Fos and c-Myc and the grade of c-Raf immunoreactivity were significantly correlated with the proportion of proliferating cell nuclear antigen (PCNA)-positive glomerular cells, with histological grading of mesangial hypercellularity and matrix increase, and with the magnitude of proteinuria. These data indicate that proto-oncogene expression is associated with mesangial proliferation and matrix expansion in proliferative types of glomerulonephritis and in focal glomerular sclerosis.     

Application of Muscle Microdialysis to Evaluate the Concentrations of the Fluoroquinolones Pazufloxacin and Ofloxacin in the Tissue Interstitial Fluids of Rats

Muscle microdialysis has been used to determine the unbound concentrations of the fluoroquinolones, pazufloxacin and ofloxacin, in tissue interstitial fluids (C_{isf, u}) of rats under steady state conditions. C_{isf, u} was estimated from the concentration in dialysate and the in-vitro permeability rate constant by the extrapolation method based on the clearance concept. Paper disks were inserted under the abdominal skin of rats, and the drug concentrations in the fluids penetrating into the disks (C_disk) were measured and compared with C_{isf, u}. The C_{isf, u} of pazufloxacin and ofloxacin in muscle were close to their unbound concentrations in the venous plasma; these were 75.3% and 77.1%, respectively, of the total concentrations in plasma at the steady state. The C_disk of pazufloxacin and ofloxacin were also close to their C_{isf, u}. These results indicate that the unbound concentrations of the fluoroquinolones in the tissue interstitial fluids were the same as those in the venous plasma. The disk insertion technique seems to be useful for evaluating drug concentrations in tissue interstitial fluid.     

Effects of Thyrotrophin-releasing Hormone Tartrate and its Sustained Release Formulation on Cerebral Glucose Metabolism in Aged Rats

The effects of a sustained release formulation of thyrotrophin-releasing hormone (TRH) over two weeks (TRH-SR, 10 or 50 mg kg^? equivalent to 0.56 or 2.80 mg kg^? free TRH, respectively) and repeated treatment with TRH tartrate (TRH-T, 0.3, 1.0 or 3.0 mg kg^?, equivalent to 0.2, 0.7 or 2.0 mg kg^? free TRH, respectively) on the rate of local cerebral glucose utilization (LCGU) were investigated using the quantitative autoradiographic 2-deoxy-[¹⁴C]d -glucose method in various brain regions of aged rats. In aged rats (28 months old), the LCGU was significantly reduced as compared with young adult rats (3 months old), while treatment with TRH-SR ameliorated the reduction of the LCGU in a dose-dependent manner. The brain regions ameliorated by TRH-SR were the auditory cortex, septal nucleus, substantia nigra, cerebellar cortex and cerebellar nucleus. In contrast, once-daily repeated treatment over one week with TRH-T at a dose of 0.3 mg kg^? (equivalent to 50 mg kg^? of TRH-SR) had no effect on the reduced LCGU in various brain regions in aged rats (27 months old), whereas treatment with a higher dose of TRH-T (0.7 or 2.0 mg kg^? free TRH) significantly ameliorated the reduction. The comparison of the ameliorating potencies between TRH-T and TRH-SR indicated that TRH-SR had a potency about 7 times greater than TRH-T.     

Herbal Medicines, Sairei-to and Tokishakuyaku-san, Differently Modulate the Release of Cytokines from Decidual Versus Peripheral Blood Mononuclear Cells

PROBLEM AND METHOD OF STUDY: We have shown that Tokishakuyaku-san (Toki) and Sairei-to (Sai) enhance T helper-1 (Th1) cytokine release from peripheral blood mononuclear cells (PBMCs): thereby, they could be a therapeutic means in the treatment of autoimmunity related recurrent abortion in which T helper-2 (Th2) polarization is exaggerated, the condition purported to benefit from these herbal medicines. However, an open question is whether these medicines might enhance Th1 cytokine release in decidual tissues and thereby stimulate the killer activity, thus, working counterproductively by accelerating maternal alloimmune reactions toward fetal tissues. To address this, we examined the effects of these medicines on the release of cytokines from decidual mononuclear cells (DMCs) in comparison with PBMCs on the assumption that they might act differently on these cell types. The effects of these medicines were investigated as related to human leukocyte antigen (HLA)-G, a nonclassical HLA class I antigen expressed on trophoblasts and a putative crucial player involved in fetomaternal immune interplay. RESULTS: Regarding Th1 cytokines. Toki marginally increased the release of tumor necrosis factor (TNF)-alpha, but not interferon (IFN)-gamma from DMCs while Sai did not affect the release of both. Both Toki and Sai were without effect in modulating the release of interleukin (IL)-4, a member of Th2 cytokines. Interestingly, the presence of HLA-G reduced the release of Th1 cytokines from DMCs regardless of the addition of Toki, Sai or none. These findings are in sharp contrast with PBMCs on which these medicines seem to act so as to enhance Th1 polarization and attenuate Th2 polarization. CONCLUSION: Differential effects of Toki and Sai on the release of Th1/Th2 cytokines between DMCs and PBMCs may afford the rationale of these medicines in the treatment of autoimmunity-related recurrent abortion.     

Endotoxin affects the expansion modes of liver macrophages after partial hepatectomy

We studied the effect of increased endotoxin levels on the expansion modes of liver macrophages after two-thirds partial hepatectomy using anti-endotoxin polymyxin B. The expansion consists of dual modes: local proliferation and immigration. Local proliferation of mature cells was evaluated by the S-phase proportion measured by flow cytometric DNA analysis. This proportion (11.8 +/- 1.3% before hepatectomy) decreased to 6.0 +/- 1.1% at 12 h and reached a maximum of 21.9 +/- 2.7% on the 5th day after hepatectomy. In polymyxin B treated rats, the proportion reached a maximum of 21.1 +/- 1.6% at 48 h without any preceding decrease. Immigration of macrophage precursors was evaluated by the decreasing proportion of latex beads-containing cells that were marked as resident liver macrophages by injection of latex beads before hepatectomy. This proportion (98.7 +/- 0.2% before hepatectomy) was significantly decreased to 90.5 +/- 1.6% at 48 h. In polymyxin B treated rats, however, the proportion showed no significant decrease within 3 days. These results indicate that endogenous endotoxin, which suppresses the local proliferation and promotes extrahepatic recruitment, regulates the number of liver macrophages in regenerating rat liver.     

Immunohistochemistry of collagen types II and X, and enzyme-histochemistry of alkaline phosphatase in the developing condylar cartilage of the fetal mouse mandible

We investigated the immunohistochemical localisation of types II and X collagen as well as the cytochemical localisation of alkaline phosphatase in the developing condylar cartilage of the fetal mouse mandible on d 14–16 of pregnancy. On d 14 of pregnancy, although no immunostaining for types II and X collagen was observed, alkaline phosphatase activity was detected in all cells in the anlage of the future condylar process. On d 15 of pregnancy, immunostaining for both collagen types was simultaneously detected in the primarily formed condylar cartilage. Alkaline phosphatase activity was also detected in chondrocytes at this stage. By d 16 of pregnancy, the hypertrophic cell zone rapidly increased in size. These findings strongly support a periosteal origin for the condylar cartilage of the fetal mouse mandible, and show that progenitor cells for condylar cartilage rapidly or directly differentiate into hypertrophic chondrocytes.