Isamu Yamaguchi

Prolongation of skin allograft survival in rats by a novel immunosuppressive agent, FK506

FK506, as immunosuppresant, was isolated from Streptomyces tsukubaensis. Intramuscular administration of FK506 (0.32 mg/kg or more) 5 days a week for two weeks after grafting prolonged the acdeptance time of F344 skin allograft to WKA rats. Similar results were obtained with cyclosporine at 32 mg/kg or more, but other immunosuppressives (i.e., prednisolone, azathioprine, and bredinin) gave only a marginal prolongation. The prolonging effect of FK506 was obtained in various donor-recipient combinations across a major or minor histocompatibility barrier. The agent also prolonged the acceptance time of mouse skin xenografts to rats. Furthermore, maintenance doses of 3.2 or 0.32 mg/kg twice a week after an initial 14-day treatment with the agent at 3.2 mg/kg gave graft survival as long as the treatment was continued for more than 120 days. Our findings show that FK506 has a potent immuno-suppressive effect in rate and suggest that the agent merits further investigation.

Smooth muscle cell migration induced by inflammatory cell products and its inhibition by a potent calcium antagonist, nilvadipine

The chemotactic activities of inflammatory cell products for rat aortic smooth muscle cells (SMC) were examined in modified Boyden chambers. A checker board analysis revealed that interleukin-1 (IL-1), leukotriene B4 (LTB4), platelet-derived growth factor (PDGF) and inflammatory exudate from zymosan-activated air pouches stimulated chemotaxis of SMC. The chemotaxis, irrespective of the attractants used, was strongly inhibited by nilvadipine, a potent calcium antagonist, and the IC50 values were around 1 x 10(-10) M. Removal of extracellular calcium abolished the chemotactic activities of the attractants. These results suggest that inflammatory cells such as macrophages and polymorphonuclear leukocytes (PMN) have an important role in the migration of SMC into the intima during atherogenesis, and that nilvadipine might be useful for preventing and treating atherosclerosis.

Inflammatory responses in cuff-induced atherosclerosis in rabbits

Cuff-treatment of the rabbit carotid artery produced a diffuse intimal thickening which resembled early lesions of atherosclerosis. A limited amount of focal endothelial damage occurred first (0.5 h), leukocytes infiltrated the subendothelium and extensive endothelial denudation occurred at 24 h. At 3 days, the regenerating endothelium covered the denuded area, and the media was edematous. At 7 days proliferation of intimal cells became visible. Maximum intimal thickening occurred at 3 weeks. Daily injection of dexamethasone (0.01-10 mg/kg i.m.) and ticlopidine (1-100 mg/kg i.m.) dose-dependently attenuated the intimal thickening. Indomethacin had little effect. Inflammatory exudate from zymosan-activated air pouch induced chemotaxis of rat smooth muscle cells (SMC) in vitro. Similar chemotactic activity was observed with leukotriene B4 (LTB4) but not with the other lipoxygenase products tested. The exudate contained reasonable amounts of LTB4, which would account for its chemotactic activity. Dexamethasone inhibited the chemotaxis by the exudate and proliferation of SMC. These results are discussed in relation to the mechanism of atherogenesis. It is concluded that leukocytes play a major role in cuff-induced intimal thickening, and that their products cause endothelial denudation and SMC chemotaxis. Involvement of platelet aggregation in atherogenesis is also suggested.

Antiatherogenic activity of FR34235 (Nilvadipine), a new potent calcium antagonist: Effect on cuff-induced intimal thickening of rabbit carotid artery

The antiatherogenic activity of FR34235 (Nilvadipine), a calcium antagonist, was examined in rabbits with carotid arteries sheathed with polyethylene cuffs, and compared with that of nifedipine, verapamil and diltiazem. The drugs were given intramuscularly in daily doses of 0.01-10 mg/kg for 3 weeks, starting on the day of cuff-placement. FR34235 dose-dependently inhibited the cuff-induced intimal thickening, and was more potent than the other calcium antagonists, whose order of potency was nifedipine, diltiazem and verapamil. In an in vitro experiment on inhibition of migration of rat aortic smooth muscle cells, using zymosan-activated air pouch exudate as a chemoattractant in modified Boyden chambers, FR34235 was also the most potent among the calcium antagonists tested. The IC50 values were 3.3 X 10(-11) M for FR34235, 1.7 X 10(-10) M for nifedipine, 6.0 X 10(-9) M for verapamil and 2.4 X 10(-7) M for diltiazem. Effects of these drugs on proliferation of rat aortic smooth muscle cells and rabbit platelet aggregation were also examined in vitro. At concentrations less than 10(-5) M, none of the drugs inhibited proliferation of the smooth muscle cells, and only verapamil inhibited collagen-induced platelet aggregation (IC50 = 9.0 X 10(-7) M). It is suggested that FR34235 should be useful for preventing and treating atherosclerosis. Inhibition of smooth muscle cell migration is thought to be its mechanism of antiatherogenic activity.

Immunosuppressive effect of FK506 on collagen-induced arthritis in rats

FK506, a new immunosuppressive agent, was given intramuscularly to rats for 12 days, starting on the day of type II collagen immunization. FK506 in doses of 0.32 mg/kg or more suppressed arthritis and also suppressed humoral and skin test response to type II collagen. FK506 suppressed arthritis only when given during the afferent limbs of immune response (0-4 days), whereas the drug was only marginally effective when treatment was started during the efferent limbs of immune response (7-11 days). FK506-induced immunosuppression continued and/or was maintained throughout the experiments (50 days). These rats immunized with type II collagen and treated with FK506 failed to develop arthritis even following a secondary immunization 50 days later but were fully capable of developing experimental allergic encephalomyelitis. This result suggest that FK506-treated rats develop specific unresponsiveness toward the type II collagen. It is concluded that FK506 is a strong immunosuppressive drug on collagen-induced arthritis.

Role of inflammatory responses in initiation of atherosclerosis: effects of anti-inflammatory drugs on cuff-induced leukocyte accumulation and intimal thickening of rabbit carotid artery

Immediately after a cuff-sheathing of rabbit carotid artery, a large number of leukocytes adhered to injured endothelium then infiltrated into the media. These inflammatory responses were followed by an atherosclerotic change, intimal thickening, of the artery. A simultaneous injection of dexamethasone (10 mg/kg i.m.) inhibited the leukocyte accumulation by 74% when evaluated 18 h thereafter. Similarly, 39% inhibition was obtained with the same dose of FR110302, a potent 5-lipoxygenase inhibitor. On the other hand, the same dose of indomethacin, a cyclooxygenase inhibitor, had little effect on the leukocyte accumulation. The intimal thickening which was evaluated 3 weeks after the cuff-treatment was attenuated by a daily dose (10 mg/kg i.m.) of dexamethasone or FR110302 but not by one of indomethacin. The inhibition by the two former drugs were 91 and 58%, respectively. In vitro, the three drugs in concentrations up to 10 microM hardly affected endothelial adhesion of PMN which was induced by LPS or IL-1. Though 10 microM of FR110302 and indomethacin significantly decreased PMN chemotaxis induced by LTB4, the decreases were less than that at 10 microM dexamethasone. These results confirm a possible linkage between inflammation and atherosclerosis, and suggest that 5-lipoxygenase products contribute to the initiation and development of atherosclerosis.

Immunosuppressive effect of FK506 on experimental allergic encephalomyelitis in rats

We investigated the effect of a new immunosuppressant, FK506, on the development of experimental allergic encephalomyelitis (EAE) in rats. EAE developed in 100% of rats immunized with myelin basic protein (MBP) in complete Freunds adjuvant. FK506 in doses of 1.0 mg/kg/day or more prevented the clinical signs of EAE for at least 50 days, when administered intramuscularly 5 days a week for 2 weeks starting on the day of immunization (days 0-4 and days 7-11), and a similar result was obtained, when the compound was given for 5 days (days 0-4). FK506, however, showed a significant but weak effectiveness when started from 7 days after immunization. Delayed-type hypersensitivity (DTH) to MBP developed before EAE, and anti-MBP antibody levels increased. Both humoral and cellular immune response to MBP were completely suppressed in rats treated with FK506. From these results, it is presumed that immunosuppression of cell-mediated immunity and/or humoral immunity by the treatment of FK506 actually causes the decreased incidence noted in the experiment for the development of EAE.

Meta-chlorophenylpiperazine attenuates formalin-induced nociceptive responses through 5-HT1/2 receptors in both normal and diabetic mice

1 This study was designed to investigate the effect of meta‐chlorophenylpiperazine (m‐CPP; a 5‐hydroxytryptamine (5‐HT) receptor agonist) on the formalin‐induced nociceptive responses in normal, insulin‐dependent streptozotocin (STZ) diabetic and non‐insulin dependent genetically diabetic (db/db) mice. 2 A subcutaneous injection of diluted formalin (1% formaldehyde in 0.9% saline, 10 μm) under the plantar surface of the left hindpaw induced biphasic nociceptive responses, the first and second phases considered to represent acute and chronic pain, respectively. The former response in db/db mice was significantly lower than those in normal mice, and the latter responses in STZ and db/db mice were significantly lower than those in normal mice. 3 In normal mice, m‐CPP (0.32‐3.2 mg ml−1, p.o.) exhibited potent antinociceptive activity, dose‐dependently attenuating the frst and second phase; the ID50 value of the second phase was 0.4 mg kg−1. m‐CPP (0.32‐3.2 mg kg−1, p.o.) also dose‐dependently attenuated the formalin‐induced nociceptive responses in STZ‐induced diabetic mice and genetically diabetic db/db mice, and the activities were comparable to those in normal mice. 4 The antinociceptive activities of m‐CPP (1 mg kg−1, p.o.) were significantly inhibited by pretreatment with pindolol (a 5‐HT1‐receptor antagonist, 1 mg kg−1, i.p.) or ketanserin (a 5‐HT2 receptor antagonist, 1mg kg−1, i.p.) but were hardly affected by ICS205‐930 (a 5‐HT3 receptor antagonist, 1 mg kg−1, i.p.). 5 These results suggest that m‐CPP inhibits not only acute but also chronic pain transmission through 5‐HT1 and 5‐HT2 receptors, and that the 5‐hydroxytryptaminergic antinociceptive pathways are little affected by diabetes.

Osteopenia in genetically diabetic db/db mice and effects of 1α-hydroxyvitamin D3 on the osteopenia

Abstract To explore the pathogenesis of non-insulin-dependent diabetes mellitus associated osteopenia, we examined age-related changes of the femur metaphyseal bone mineral density in genetically diabetic (db/db) mice and non-diabetic (+/+) mice of the same strain using single photon absorptiometry and characterized the osteopenia pharmacologically and biochemically. Bone mineral density increased with age in the +/+ mice from 5 to 16 weeks of age, but reached a plateau in the db/db mice at 8 weeks of age, and significant differences between the two groups were observed after 12 weeks of age. Ash weight (A) and dry weight (D) of the femur and A/D ratio were significant lower in the db/db mice than in the +/+ mice after 8 weeks of age. Significant elevations of serum calcium and parathyroid hormone (PTH) were observed after 8 weeks and 12 weeks of age, respectively. Serum 1α,25-dihydroxyvitamin D levels were significantly decreased in the db/db mice compared to the +/+ mice. Daily oral treatment with 1α-hydroxy vitamin D3 (1α-(OH)D3) for 4 weeks starting from 8 weeks of age significantly attenuated the bone loss in the db/db mice. These results suggest that an impaired bone mineralization probably by insufficient vitamin D activity and high PTH levels are involved in the osteopenia in the db/db mice. 1α-(OH)D3 exerted beneficial effects on the bone loss.

Antinociceptive Effects of Morphine Were Different Between Experimental and Genetic Diabetes

This study was designed to investigate the effect of morphine on formalin-induced nociceptive responses in streptozotocin (STZ) induced-diabetic mice, noninsulin-dependent genetically diabetic db/db mice and their respective controls (ddY and (+/+)). In nondiabetic (ddY and (+/+)) mice, morphine (1-10 mg/kg, PO) dose dependently attenuated the biphasic nociceptive responses induced by s.c. injection of formalin to the hindpaw, demonstrating equipotency on both the first and second phases. Para-chlorophenylalanine (800 mg/kg x 2, PO) and pindolol (1 mg/kg, i.p.) reduced the effect of morphine on the first phase, sulpiride (10 mg/kg, i.p.) abolished the effect on both phases, while ketanserin (1 mg/kg, i.p.) had no effect. In STZ (200 mg/kg, i.p.)-diabetic mice, morphine weakly attenuated the nociception in comparison to control ddY mice, whereas it had comparable effects in both the first and second phases of control (+/+) mice and db/db mice. The serotonergic agonist, meta-chlorophenylpiperazine (0.32-3.2 mg/kg, PO), dose dependently attenuated the biphasic nociceptive responses to formalin in both phases of diabetic mice; however, FR64822, a dopaminergic compound (0.1-10 mg/kg, PO), had little effect. We speculate that activation of both dopaminergic (DA)- and serotonergic-mediated mechanisms are potentially responsible for the effect of morphine on the first phase, while the DA-mediated effect is involved in the second phase. The DA-mediated mechanism, but not the serotonin-mediated one, appears to be altered in both STZ-diabetic and db/db mice. These results suggest that the attenuated effects of morphine might be due to a dopaminergic dysfunction in STZ mice, and that there might be other mechanisms compensating for this attenuation of dopaminergic function in db/db mice.