Search

As (67)Ga is injected into the blood, (67)Ga is immediately bound to transferrin (Tf) and transported to various tissues and the Tf-(67)Ga complex binds to Tf receptor on various tissues. In partial hepatectomy (PH) a part of blood in circulation is lost together with removed liver tissues, consequently the amounts of blood cells and Tf in circulation decrease. In order to investigate the effect of those decreases on (67)Ga uptake by the liver, we compared the uptake in partially hepatectomized rats with that in venesectioned rats in which only a part of blood in circulation decreased. A two-thirds PH was performed. Two milliliters of blood was venesectioned. Each treated rat was intraveneously injected with (67)Ga. The changes of erythrocyte and reticulocyte contents after PH did not differ from those after venesection (VS) at all. But (67)Ga uptake by reticulocytes significantly increased after VS but did not after PH. On the other hand, (67)Ga uptake by the liver significantly increased after PH but did not after VS. These differences must be related to the different expression of Tf receptors on the liver after PH and VS.

We have recently reported that transferrin (Tf)-unbound gallium-67 (67Ga) may be taken up into the liver of carbon tetrachloride (CCl4)-treated rats. In the present study, we attempted to clarify detailed mechanism of Tf-unbound 67Ga uptake by hepatocytes treated with CCl4 using in vitro experimental system. Hepatotoxic damages by CCl4 are mostly attributed to radical formed by an action of cytochrome P450. P450 isozymes have a higher expression in the perivenous hepatocytes (PVH) more than periportal hepatocytes (PPH). Therefore, we thought that the uptake of 67Ga which had been used for the detection of liver damage might have a zonal difference. The results of ALT activities showed that the CCl4 exposure for 4 h strongly impaired PVH more than PPH. The uptake of 67Ga by PVH treated with CCl4 was also higher than that by PPH. Moreover, the uptake of 45Ca by PVH was higher than that by PPH. In order to investigate whether 67Ga passed through calcium channel of hepatocytes, we made use of calcium channel blocker and activator. The Ca2+-channel blocker, verapamil, significantly decreased the uptakes of 45Ca and 67Ga by PPH and PVH pretreated with CCl4. The addition of the Ca2+-channel activator, Bay K8644, significantly increased the uptake both of 45Ca and 67Ga by PPH pretreated with CCl4. In the present study, it was demonstrated that the uptake of Tf-unbound 67Ga preferentially occurred in CCl4-damaged PVH and 67Ga was taken up into the hepatocytes in part through calcium channel.

We have reported that transferrin (Tf)-unbound Gallium-67 ((67)Ga) was taken up into hepatocytes 1 d after carbon tetrachloride (CCl(4))-treatment in rats. It had been reported that the binding affinity of Iron-59 ((59)Fe) to Tf was greater than that of (67)Ga. In the present study, we investigated whether or not Tf was involved in (59)Fe uptake by hepatocytes of CCl(4)-damaged rats. The results showed that the uptake of (59)Fe by hepatocytes and the number of Tf-receptor decreased 1 d after CCl(4)-treatment and increased 2 d after the treatment. Our data demonstrated that the uptake of (59)Fe by hepatocytes differed from that of (67)Ga and Tf was involved in that of (59)Fe. We expect that these finding could serve to analyze the inflammatory stages, disorder stage at 1 d and regeneration stage at 2 d after CCl(4)-treatment.

We have recently demonstrated the inverse correlation between transglutaminase (TGase) activity and DNA synthesis in periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) at 1 d after partial hepatectomy. In order to elucidate a role of polyamines as substrates of TGase in the differential growth capacities between PPH and PVH from regenerating liver, we investigated the zonal differences in alteration of ornithine decarboxylase (ODC) activity and polyamines. In two subpopulations, the inverse correlation between DNA synthesis and epsilon-(gamma-glutamyl) lysine (Gln-Lys) cross-linking catalyzed by TGase was demonstrated at 1 d after partial hepatectomy. ODC activity in PPH significantly increased with a peak at 1 d after partial hepatectomy, whereas did not in PVH. Protein-binding SPD in PPH also transiently increased with a peak at 1 d after partial hepatectomy, but did not in PVH. These results suggest that at 1 d after partial hepatectomy, in PPH, the inhibition of Gln-Lys cross-linking by the formation of N-gamma-glutamyl SPD leads to the increase of DNA synthesis, whereas in PVH, enhanced formation of Gln-Lys cross-linking leads to the lower DNA synthesis.

Gallium-67 (67Ga) has been used as a tumor or inflammation-imaging agent in nuclear medicine. We recently reported that the peak of serum alanine aminotransferase (ALT) level was 1 d after carbon tetrachloride (CCl4) treatment in rats. However, the peak of 67Ga uptake by the liver tissue and hepatocytes was 2 d after the treatment. If 67Ga is taken in the hepatic disorder phase, the pattern of 67Ga uptake by the hepatocytes should be consistent with that of the ALT level. In order to answer why it was not, we carried out a detailed examination. The lipid peroxidation of hepatocytes from CCl4-treated mice was greatly increased 1 d after CCl4 treatment; serum ALT was also increased 1 d after the treatment. The uptake of 67Ga by the liver tissue reached a maximum 1 and 2 d after the treatment, while maximum by the hepatocytes was achieved after 2 d. The incorporation of bromodeoxyuridine into the hepatocytes also reached maximum 2 d after the treatment. These results suggest that the uptake of 67Ga by the hepatocytes is carried out during liver regeneration rather than during hepatic disorder by CCl4 treatment.

We investigated a relationship within zonal differences in DNA synthesis and in transglutaminase (TGase) activity between perivenous versus periportal regions of regenerating rat liver. Using the digitonin/collagenase perfusion technique, hepatocyte subpopulations were isolated from each region at various time points after partial hepatectomy. The amounts of DNA synthesis as well as the levels of TGase mRNA and activity in each subpopulation were measured. Although increased DNA synthesis was observed in both subpopulations with a peak at 24 h after partial hepatectomy, the amount of DNA synthesis in periportal hepatocytes (PPH) was twice as much as that in perivenous hepatocytes (PVH). In PVH, TGase activity peaked at 24 h after partial hepatectomy with a preceding increase in its mRNA expression at 12 h, whereas TGase activity in PPH at 24 h was one-half of that in PVH. As TGase is known to have a growth-arresting activity, our data indicate that relatively higher TGase activity in PVH at 24 h after partial hepatectomy might correlate with relatively lower DNA synthesis in this region compared to periportal region.

We recently suggested that the transferrin (Tf)-gallium-67 (67Ga) complex dissociated on the surface of the hepatocytes after partial hepatectomy and free 67Ga bound to heparan sulfate in the extracellular matrix. In the present study, we investigated whether the entering of indium-111 (111In) and iron-59 (59Fe) with high affinity to transferrin differed from the entering of 67Ga by the hepatocytes after partial hepatectomy. 111In was almost taken by the plasma and little taken by the red blood cell. On the other hand, the uptake of 59Fe by the red blood cell was higher than plasma. The uptake of 59Fe by the bone marrow was significantly higher than that of 111In. The uptake of 111In and 59Fe by the liver tissue was reached a maximum 2 d after partial hepatectomy but the uptake ratio of 111In was lower than that of 59Fe. We suspected that the uptake of 59Fe by the liver tissue was the highest because of the high binding affinity of Tf-59Fe to Tf-receptor. The entering of 111In and 59Fe into the hepatocytes was also reached a maximum 2 d after partial hepatectomy but the ratio of 59Fe was slightly lower than that of 111In. These results suggested that the binding affinity to Tf could have played a crucial role in the differences of the entering of 111In, 59Fe and 67Ga into the hepatocytes of partially hepatectomized rats.

We attempted to develop an experimental system for evaluation of stress effect on ethyl alcohol metabolism by the radiorespirometric analysis using [(14)C]ethyl alcohol. Each rat was immobilized in an adjustable restraint device for 3 h. The excretion of (14)CO(2) in the breath was significantly decreased by restraint stress. The liver alcohol dehydrogenase activity was also decreased by restraint stress. We think that radiorespirometric analysis may be useful for the evaluation of stress effect on real metabolism of ethyl alcohol in the liver.

We investigated the effect of transglutaminase (TGase) on in guinea pigs and rats. Serum alanine aminotransferase (ALT) level increased 1 d after CCl(4) treatment of both in guinea pigs and rats since TGaese activity was greatly higher in guinea pigs than rats. However, serum ALT level in guinea pigs was very much lower than that in rats. Liver TGase activities decreased after CCl(4) treatment in both guinea pigs and rats. However, TGase activities in the liver from guinea pigs were higher than that from rats. Decreased TGase activities by CCl(4) in the liver from guinea pigs and rats were significantly recovered by retinoic acid treatment that was reported to increase TGase. Degree of recovery of serum ALT level by retinoic acid in rats was larger than in guinea pigs. These results suggested that the distinction of the effect of retinoic acid on serum ALT level in CCl(4)-treated animals was due to the different TGase activity that increased membrane stability.

We investigated the effects of restraint and footshock stress and norepinephrine treatment on gastric emptying. The gastric emptying was significantly inhibited by restraint stress. beta(3)-adrenergic antagonist canceled the inhibition of gastric emptying caused by restraint stress, while beta(1)-, beta(2)-, alpha(1)-, and alpha(2)-adrenergic antagonists did not affect the inhibition. Norepinephrine treatment also inhibited gastric emptying. The inhibition by norepinephrine treatment was canceled by beta(1)-, beta(2)-, and beta(3)-adrenergic antagonists, but not by alpha(1)- and alpha(2)-adrenergic antagonists. On the other hand, footshock stress did not affect gastric emptying. These results suggest that beta(3)-adrenoceptors play an important role in the inhibition of gastric emptying caused by restraint stress, while nonspecific beta-adrenoceptors are involved in that caused by norepinephrine treatment.

We attempted to identify the exchange mechanism of transferrin-67Ga complex to heparan sulfate-67Ga complex. The effect of phosphate on the binding ability of 67Ga to transferrin and heparan sulfate was studied by the dialysis method. The phosphate inhibited the binding ability of 67Ga to transferrin, while the phosphate enhanced the 67Ga binding ability to heparan sulfate. The results suggest that the phosphate is involved in the translocation of 67Ga from the transferrin to the heparan sulfate.

We have previously reported that restraint stress inhibits small intestinal motility in rats, and that the adrenergic beta3-antagonist SR59230A administration recovered the inhibition. In the present study, we compared the effects of restraint stress and norepinephrine on small intestinal motility using alpha- and beta-adrenergic antagonists. SR59230A did not recover the norepinephrine-induced inhibition of small intestinal motility. The norepinephrine-induced inhibition of small intestinal motility was recovered after administration of the alpha2-antagonist yohimbine, but not by alpha1-, beta1-, and beta2-antagonists. Considering these results, it is reasonable to assume that the mechanisms of inhibition of small intestinal motility due to restraint stress and norepinephrine treatment are different.

Ever since the first observation of 67Ga accumulation in tumors and inflammatory lesions, 67Ga has been used to detect various tumors and inflammations. The aims of this study were to clarify whether or not transferrin is involved in the uptake of 67Ga by the regenerating liver after partial hepatectomy. The uptake of 67Ga by the liver of rats reached a maximum 2 d after partial hepatectomy. In order to inhibit the binding of 67Ga to transferrin in the blood, FeCl3 was administered 5 min before the injection of 67Ga. The administration of FeCl3 decreased the uptake of 67Ga by the liver of the partially hepatectomized rats, suggesting that transferrin is involved in the uptake by the liver. However, 67Ga was taken up only slightly by hepatocytes obtained from the liver of these rats. We conclude that transferrin is involved in the uptake of 67Ga by the liver tissue of partially hepatectomized rats but is not involved in its entry into the hepatocytes. Only a slight amount of gallium-67 enters the hepatocytes, and may accumulate primarily in the extracellular matrix of the liver tissue of these rats.

We previously reported that small intestinal motility was significantly inhibited by restraint stress, but not by footshock stress. In the present study, we found that plasma beta-endorphin levels were more significantly elevated by footshock stress than restraint stress, and that preloading of footshock stimulus canceled the inhibition of small intestinal motility by restraint stress. Pretreatment with the mu-opioid receptor antagonist naltrexone significantly attenuated this canceling effect of footshock stimulus. These results suggest that footshock stimulus may cancel the inhibition of small intestinal motility by restraint stress via activation of mu-opioid receptors.

We have reported that acute restraint stress inhibits small intestinal motility in rats. In order to clarify this inhibitory mechanism, we examined the effects of alpha- and beta-adrenergic antagonists on the inhibition of small intestinal motility induced by restraint stress. This inhibition underwent recovery by propranolol (beta1/beta2-antagonist) or SR59230A (beta3-antagonist), but not by atenolol (beta1-antagonist), ICI-118,551 (beta2-antagonist), prazosin (alpha1-antagonist) or yohimbine (alpha2-antagonist). These results suggest that beta3-adrenoceptors play an important role in the inhibition of small intestinal motility caused by restraint stress.

The effect of two different stress stimuli, restraint stress and footshock stress, on small intestinal motility was evaluated by a more reliable method with improvement of the previous method using a radionuclide, 51Cr. The small intestinal transit was significantly inhibited by restraint stress, but not by footshock stress, although plasma corticosterone levels were significantly elevated to the same extent by restraint stress and footshock stress. These results suggest that restraint stress and footshock stress stimuli influence small intestinal motility via different mechanisms, but the reason for the difference is unclear. This experimental system using 51Cr seems to be useful for the elucidation of mechanisms for restraint stress-induced dysfunction of small intestinal motility because of its excellent quantitative evaluation of small intestinal transit.

We investigated the effect of extracellular Ca2+ on the uptake of immunoglobulin G (IgG)-coated normal sheep red blood cells (SRBC) by the mouse peritoneal macrophages. The uptake of SRBC was dose dependently enhanced by Ca2+ concentration. We also investigated the effect of ryanodine on the uptake of SRBC. Ryanodine showed approximately 15% inhibition of the uptake. The activity of transglutaminase (TGase) in the cytosolic fraction of macrophages was enhanced as the Ca2+ concentration increased. The uptake of SRBC decreased by the addition of histamine, a TGase inhibitor. These results suggest that TGase is involved in the receptor-mediated endocytosis system of mouse peritoneal macrophages.

We attempted to develop an experimental system for evaluation of the influence of stress on small intestinal motility using a radionuclide, 51Cr, and an acute restraint stress model. Each rat was immobilized in an adjustable restraint device for 1 to 5 h. The rat was given 51Cr immediately after the end of stress loading through a catheter inserted into the duodenum, and sacrificed 20 min after administration. The small intestine was removed and the 51Cr radioactivity was continuously monitored with an NaI-scintillation survey meter. The small intestinal motility was estimated by the distance of the radioactive peak from the top of the duodenum. The small intestinal motility was significantly inhibited by 3 h-restraint stress. This experimental system is easy, rapid, and simple for the evaluation of the effect of stress on small intestinal motility.

The effect of inescapable footshock stress on open-field activity, as measured by the number of ambulations, was studied in male mice. Ambulations significantly increased after footshock stress, the most significant effect appeared after 20 min-stimulation and the effect decreased as footshock time lengthened. The footshock stress-induced enhancement of ambulation was inhibited by haloperidol (0.2, 0.5 and 1 mg/kg), phentolamine (5 and 10 mg/kg), mianserin (20 mg/kg), atropine (0.5, 1 and 2 mg/kg), naltrexone (10 mg/kg) and MK-801 (0.05, 0.1 and 0.2 mg/kg), but was not influenced by propranolol (5, 10 and 20 mg/kg) or diazepam (1, 2 and 5 mg/kg). Haloperidol (0.5 and 1 mg/kg) and mianserin (5, 10 and 20 mg/kg) also exerted an inhibitory effect on non-stressed normal mice. These results suggest that dopaminergic, alpha-adrenergic, cholinergic, opioidergic and N-methyl-D-aspartate (NMDA) receptor-mediated neurotransmission systems are involved in the footshock stress-induced ambulatory activation.

Effects of two types of stress, restraint and footshock, on plasma glucose level, insulin secretion, and glucose uptake by the liver, heart and femoral muscle were investigated in mice. Plasma glucose level gradually increased and reached maximum at 60 min after the onset of restraint stress, while footshock stress slightly increased plasma glucose level only 15 min after the onset of the stress, and this subsequently decreased significantly. The basal plasma insulin level and glucose-induced insulin secretion significantly decreased during both the stress stimuli. Glucose uptake by the femoral muscle was slightly increased during restraint stress, and was significantly increased during footshock stress. These results suggest that the transient increase in plasma glucose level during the early period of both stress stimuli might be caused by the inhibition of insulin secretion, and subsequent decrease in plasma glucose level during the latter phase of both stress stimuli was probably due to the increase in glucose uptake by the skeletal muscle. The present study confirmed that there was a difference in stress-induced glucoregulatory changes between restraint stress and footshock stress. This difference might be due to the degree of muscle movement during the stress stimuli.

The uptake of 45Ca by inside-out erythrocyte membrane vesicles (IOV) was dose-dependently inhibited by spermine. A cationic substance, Mg2+, did not affect this inhibitory activity of spermine. Ca(2+)-ATPase activity, however, was not inhibited by spermine at a concentration that could inhibit the uptake of 45Ca by the IOV. The release of 45Ca from IOV was significantly accelerated by spermine. Spermine bound to the surface of the IOV and was removed by treatment with acetic acid. These results suggest that spermine stimulates the release of Ca2+ from the IOV (or uptake of Ca2+ by erythrocytes) via an effect on the outer surface of the IOV (or the inner surface of erythrocytes).

We demonstrated the invagination of transferrin into reticulocyte plasma membrane to learn whether membrane-bound transglutaminase (TGase, a Ca(2+)-dependent enzyme) is involved in this invagination. The invagination was assessed by acid-resistance assay and antibody-inaccessibility assay. The invagination was blocked in the absence of ATP. [14C]Putrescine, a substrate for TGase, was incorporated into the membrane during the invagination. This incorporation was decreased in the absence of ATP or transferrin and was completely blocked in the presence of monodansylcadaverine or EGTA. The TGase inhibitor and EGTA also decreased the invagination. In the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, the labeling of 43 kDa membrane protein with [14C]putrescine and the increase in aggregation of proteins were observed in a transferrin-, ATP- and Ca(2+)-dependent manner. These results provide the first evidence for modification of protein by TGase accompanying the invagination of transferrin into the membrane, and suggest that membrane-bound TGase is involved in the invagination step of endocytosis.

Concomitant increase in putrescine incorporation with transferrin uptake into rat reticulocytes was found. [14C]Putrescine incorporation occurred in the presence of transferrin at 37 degrees C. The subcellular distribution of incorporated [14C]putrescine showed that the incorporation in the plasma membrane time dependently increased. Both 125I-labeled transferrin uptake and [14C]putrescine incorporation decreased when ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid was added, while it increased with the addition of A23187.

We have demonstrated the internalization of epidermal growth factor (EGF) using membrane isolated from rat liver. The isolated membrane exhibited a saturation curve of the binding of 125I-EGF. Furthermore, competition between the binding of 125I-EGF and unlabeled EGF to the isolated membrane was observed. These results were similar to those obtained from whole hepatocytes. In order to confirm whether or not the present experimental system using the isolated membrane can be used for the study of receptor-mediated endocytosis, the internalization of pre-bound EGF in the isolated membrane was assessed by two different methods. First, acid-insensitive 125I-EGF time-dependently increased following incubation at 37 degrees C. Secondly, EGF became inaccessible to the exogenous 125I-anti-EGF antibody when under the same condition. These processes were dependent on adenosine triphosphate, but independent of Ca2+ and stimulated by guanosine triphosphate. These results demonstrate that receptor-mediated endocytosis occurred in the isolated membrane.

Five [D-Ala2, D-Leu55]enkephalin (DADLE) analogs containing fluorinated Tyr1 or Phe4 residue, that is, [Phe(2F)4] (I), [Phe(3F)4] (II), [Phe(4F)4] (III), [Tyr(3F)1] (IV) and [Tyr(2F)1] (V), were synthesized by the solid phase method and their opioid receptor affinities were examined. Affinity profiles of five derivatives for the mu- and delta-receptor were similar to those of DADLE, and the affinity for kappa-receptor was zero or negligible.

Nine analogs based on a structure of Tyr-D-Arg-Phe-X (X = alkylamides or alkylhydrazide containing electron-withdrawing atoms or groups) were newly synthesized and their biological properties were examined by the opioid receptor binding properties of mu-, delta- and kappa-receptors, guinea-pig ileum (GPI) assay and analgesic activity in the tail pinch test after subcutaneous administration in mice. Analogs with X = NHCF2CF3, Sar-ol, or NH(CH2)2CN showed potent activities in the GPI and analgesic assays and high affinity for mu-receptor. An analog with X = taurinamide was found to possess 4-fold higher mu-receptor selectivity than that of [D-Ala2, MePhe4, Gly-ol5]enkephalin (DAGO). The receptor binding properties of previously reported analogs [Chem. Pharm. Bull., 33, 1528 (1985); ibid., 33, 4865 (1985); ibid., 36, 4834 (1988)] were also examined for overall discussion of the structure-activity relationships of this series of tripeptide amides.