Genistein, a soybean isoflavone, inhibits inward rectifier K(+) channels in rat osteoclasts.

Genistein, a soybean-derived isoflavone with an inhibitory effect on protein tyrosine kinases (PTKs), has been shown to suppress osteoclastic bone resorption. To clarify the mechanisms underlying this action, we investigated the effects of genistein on inward rectifier K(+) current (I(Kir)) in rat osteoclasts by using the whole-cell patch-clamp technique. Extracellularly applied genistein inhibited I(Kir) in a concentration-dependent manner. Physiologically attainable concentrations of genistein inhibited I(Kir). IC(50) values obtained 5 and 10 min after the application of genistein were 54 and 27 microM, respectively. The removal of genistein partially restored the current. Daidzein, an isoflavone without PTK-inhibiting activity, also showed a weak inhibitory effect on I(Kir), but genistin had no effect. Other PTK inhibitors, tyrphostin A25, tyrphostin B42, and tyrphostin B46, inhibited I(Kir), whereas herbimycin A and lavendustin A were without effect. The inactive tyrphostin, A1, showed a similar inhibitory effect as tyrphostin A25. The tyrosine phosphatase inhibitor, orthovanadate, did not affect the inhibitory potency of genistein on I(Kir). The inhibitory action of genistein was unaffected by changing intracellular Ca(2+) concentration ([Ca(2+)]i) or by pretreatment of the cell with GDPbetaS, Rp-cAMPS, okadaic acid, or staurosporine. Therefore the inhibition of I(Kir) by genistein does not depend on PTK inhibition, involvement of changes in [Ca(2+)]i, or secondary interaction with protein kinase A or protein kinase C. Genistein-induced inhibition of I(Kir) would cause membrane depolarization, elevation of [Ca(2+)]i, and inhibition of osteoclastic bone resorption.

Immunosuppressants decrease neutrophil chemoattractant and attenuate ischemia/reperfusion injury of the liver in rats.

BACKGROUND: Neutrophils may play an important role in the development of liver ischemia/reperfusion injury. We investigated the effects of the immunosuppressants azathioprine (AZA), cyclosporine A (CsA), tacrolimus (FK506), and rapamycin (RPM) on the expression of cytokine-induced neutrophil chemoattractant (CINC) after ischemia/reperfusion of the liver. METHODS: Liver ischemia was induced in male Wistar rats by occluding the portal vein with a microvascular clip for 30 minutes. Rats received two intramuscular injections of AZA (4 mg/kg), CsA (5 mg/kg), FK506 (0.5 mg/kg), or RPM (0.5 mg/kg) 3 and 24 hours before ischemia/reperfusion of the liver. RESULTS: Serum CINC concentrations in untreated animals increased, peaked 6 hours after reperfusion, and thereafter decreased gradually. Pretreatment with AZA, CsA, FK506, and RPM, however, inhibited the increase in serum CINC concentrations after reperfusion. CINC mRNA in liver tissue increased and peaked 3 hours after reperfusion, but was significantly lower in animals treated with AZA, CsA, FK506, and RPM. In vitro CINC production by Kupffer cells harvested from animals treated with AZA, CsA, FK506, or RPM 3 hours after reperfusion was also significantly lower than that observed in untreated animals. Both myeloperoxidase activity and the number of neutrophils accumulating in the liver 24 hours after reperfusion in animals treated with AZA, CsA, FK506, and RPM were significantly lower than in untreated animals. This correlated with lower serum aspartate transaminase, alanine transaminase, and lactate dehydrogenase levels in animals treated with AZA, CsA, FK506, and RPM 24 hours after reperfusion. CONCLUSION: The immunosuppressants AZA, CsA, FK506, and RPM reduce neutrophil accumulation and attenuate ischemia/reperfusion injury of the liver.

Indication of chemoembolization therapy without gelatin sponge for hepatocellular carcinoma.

This study evaluated the effect of chemoembolization (C-LIP) consisting of ethiodized oil (Lipiodol Ultra Fluid; André Guerbet, Aulnay-sous-Bois, France) and epirubicin, without gelatin sponge on hepatocellular carcinoma (HCC), administered by hepatic arterial infusion. We analyzed the cases from two points of view: the local recurrence rate for hypervascular solitary small HCC (tumor size: < or =3 cm in diameter) and the cumulative survival rate for advanced HCC (stage VI according to the criteria of Liver Cancer Group of Japan) following C-LIP therapy. The C-LIP also was compared with transcather arterial embolization (TAE; C-LIP followed by gelatin sponge) and percutaneous ethanol injection therapy (PEIT). In the small HCC cases, the recurrence rate at 1 year after C-LIP was 77% (10 of 13 patients), while the local recurrence rate was 46% (six of 13 patients) at 6 months and 61% (eight of 13 patients) at 1 year. The local recurrence rate at 1 year was 29% (four of 14 patients) after TAE and 20% (three of 15 patients) after PEIT. These results showed that the effect of local anticancer therapy by C-LIP was not as potent as that of TAE or PEIT. In advanced HCC cases, the cumulative survival rate for 13 patients treated by C-LIP was 72% at 6 months, 36% at 1 year, and 14% at 2 years. However, the survival rates for 13 patients at 6 months, 1 year, and 2 years after TAE were 46%, 23%, and 8%, respectively. There was no difference between the C-LIP patients and TAE patients with regard to the pretreatment liver function. Three patients died within 2 months after the initial TAE. These deaths were mainly due to damage to the noncancerous liver parenchyma. Therapy with C-LIP alone was not appropriate for hypervascular solitary small HCCs, and additional treatment was necessary. We think C-LIP therapy should be selected instead of TAE for advanced HCCs to avoid severe parenchymal damage.

Alendronate distributed on bone surfaces inhibits osteoclastic bone resorption in vitro and in experimental hypercalcemia models.

Alendronate is an aminobisphosphonate that acts as a potent inhibitor of osteoclastic bone resorption. To understand the mechanism of action of alendronate in vivo, in this study we investigated the relationship between distribution of [14C]-alendronate in rat bone and its effects on bone resorption in vitro or in rat hypercalcemic models. A single IV dose of 0.05 approximately 1.25 mg/kg inhibited the increase in plasma calcium level induced by bovine PTH or 1 alpha(OH)D3. The minimal effective dose of pamidronate (1.25 mg/kg) and etidronate (over 31.25 mg/kg) were at least 5 times and 25 times, respectively, higher than the dose of alendronate in the rat hypercalcemic model prepared by 1 alpha(OH)D3. The relative potencies of compounds in the hypercalcemic rat models reflected those of inhibitory effects on bone resorption in vitro. We conducted the ivory-slice assay under two conditions: (a) addition of a given bisphosphonate after adherence of the osteoclasts; and (b) preincubation of the ivory slices with a given bisphosphonate. The inhibitory IC50 values of alendronate under condition (b) were similar to those under condition (a). To evaluate the interaction between osteoclasts and alendronate in bone, we investigated the localization of [14C]-alendronate in the tibia of growing rats (4-day-old rats). Alendronate did not distribute uniformly in the tibia. At 1 day after injection (0.05 mg SC), dense labeling was seen primarily under osteoclasts. We injected 0.05 mg/kg of [14C]-alendronate (single i.v.) into rats [14C]-alendronate was rapidly eliminated from plasma, and mainly distributed to the bone in rats. These data suggest that alendronate which distributed on bone surface mainly contributed to the antihypercalcemic action in vivo.

Improvement of multiple coronary risk factors in obese hypertensives by reduction of intra-abdominal visceral fat.

The association of obesity and hypertension is well documented, and the combination is important as a coronary risk factor, but its non-pharmacological management is very difficult. Japanese hypertensive obese subjects (HO, n = 95) selected from 321 non-medicated obese subjects with a body mass index > 25 kg/m2 were characterized by the clinical features of significant diaphragmatic elevation, higher heart rate (HR), fasting blood glucose (FBS), total cholesterol (Tch), uric acid and gamma GTP values and lower vital capacity (VC) compared to those of normotensive-obese subjects (NO, n = 226) (p < 0.01). During a diet therapy program (about 1,200 kcal/day) for HO (n = 55), 25 subjects were treated with a non-drug-dependent pulse-synchronized transpercutaneous electric abdominal muscle stimulator (PEM) (ca. 30,000 muscle contractions/day) for 4 weeks. These subjects showed significant improvement with reduction in body weight (9.4%, 7.4 kg), intra-abdominal visceral fat (VF) CT scan area (29%), abdominal subcutaneous area (10%) at the level of the umbilicus, blood pressure (BP), HR, FBS, gamma GTP, Tch, plasma norepinephrine, plasma renin activity and plasma insulin, an increase of VC and lowering of the diaphragm (p < 0.05). The reductions in weight, BP, FBS and Tch in the diet group (n = 30, 1,200 kcal/day for 4 weeks) were smaller than those in the PEM-diet group (p < 0.05). The Japanese hypertensive obese patients had complications of many other coronary risk factors, and the reduction in weight and VF with PEM-diet therapy seems to be effective for improving these risk factors.

Preventing denervation atrophy of a grafted muscle.

Electrodes were implanted in the grafted muscles of rabbits to generate continuous stimulation for the purpose of preventing denervation atrophy. Denervation atrophy could be prevented, to some extent, in dissected muscles with vascular pedicles not occluded during the experiment. Denervation atrophy was found to be promoted, rather than prevented, in grafted muscles with occluded vascular pedicles. The crucial conditions are varying stimulation and the long duration of occlusion (90 min). Overly strong stimulation of muscle contraction appears to result in "fatigue" of grafted muscles in which blood circulation has been blocked for more than the 90 min used in the reported experiment. Further investigations are necessary of the conditions of stimulation and the point in time at which stimulation should commence after grafting.