Effects of Aconitum alkaloid kobusine and pseudokobusine derivatives on cutaneous blood flow in mice; II.

Aconitum alkaloids of the C20-diterpenoid type, kobusine (1) and pseudokobusine (2), their anisoyl, veratroyl, p-nitrobenzoyl, nicotinoyl or pivaloyl derivatives, and dehydrokobusine and N,6-seco-6-dehydropseudokobusine derivatives were examined for their peripheral vaso-activities by laser-flowmetrical measurement of the cutaneous blood flow in the hind foot of mice after intravenous administration. Kobusine 15-anisoate (4), 11-veratroate (5), 15-veratroate (6), 11-pivaloate (9) and 15-pivaloate (10) were significantly effective at a low dose of 0.5 or 0.05 mg/kg. Pseudokobusine derivatives were all active at 1, 0.5 or 0.05 mg/kg, and the effects of pseudokobusine 15-anisoate (13), 15-veratroate (16) and 15-p-nitrobenzoate (19) at 0.1 mg/kg were remarkable. Yesoline (26) and alkaloid (28) were significantly effective at a low dose of 1 mg/kg, whereas yesonine (25) and N-acetyl-N,6-seco-6-dehydropseudokobusine (27) were inactive. Dehydrokobusine derivatives (29, 30) were significantly effective at a low dose of 0.5 or 0.1 mg/kg. It is thought that the hydroxyl groups of alkaloids, especially a free OH group of 2 at C-6, are important for action on the peripheral vasculature leading to dilatation, and the results indicated that esterification of the hydroxyl group at C-15 with either anisoate, veratroate orp-nitrobenzoate may contribute to enhancement of the activity of the parent alkaloids.

Excitatory amino acid release in the locus coeruleus during naloxone-precipitated morphine withdrawal in adjuvant arthritic rats.

OBJECTIVE AND DESIGN: Excitatory amino acid levels in the locus coeruleus (LC) and the behavioral signs during naloxone-precipitated withdrawal in arthritic rats treated with chronic morphine were investigated by in vivo microdialysis. METHODS: Increases in glutamate (Glu) and aspartate (Asp) were noted after naloxone (48 nmol/5 microl, LC)-precipitated withdrawal from normal and adjuvant arthritic rats which had been intracerebroventricularly infused for 3 days with morphine (26 nmol/l microl/h). RESULTS: The increases in Glu and Asp levels on morphine withdrawal in normal rats were attenuated following naloxone challenge in the morphine-dependent arthritic rats. Moreover, behavioral signs during morphine withdrawal were detected following the naloxone challenge in both the morphine-dependent normal and adjuvant arthritic rats, but not in the saline-infused controls. CONCLUSIONS: These results show that the attenuation of Glu and Asp release from the LC in the adjuvant arthritic rats might explain the anti-inflammatory and analgesic effects of mu-opioids in adjuvant arthritic rats.

Anti-inflammatory effects of methotrexate on reversed passive Arthus reactions in rats.

The anti-inflammatory effects of methotrexate (MTX), an anti-rheumatic drug for treating rheumatoid arthritis, on acute inflammation were studied by using Arthus reactions induced in the pleural cavity and dorsal skin of rats. The effects were compared with those of dexamethasone (DEX), a synthetic analog of adrenocortical steroid, and of ketoprofen (KET), a nonsteroidal anti-inflammatory agent. In reversed passive Arthus (RPA) reactions induced in the pleural cavity by an anti-bovine-albumin serum, DEX significantly suppressed both neutrophil accumulation and plasma exudation at the sites of injection of an antibody, whereas MTX and KET had no effect. In the RPA reaction induced in the dorsal skin by an anti-ovalbumin serum, all three drugs inhibited exudation to the same level. However, DEX and MTX suppressed neutrophil accumulation, whereas KET did not. We found that the oral administration of MTX for 3 days significantly inhibited both neutrophil accumulation and exudation in the RPA reaction in the dorsal skin, suggesting that MTX is an effective anti-inflammatory agent. However, the manifestation of these anti-inflammatory effects might be restricted by differences in the inflammation models in rats.

Immunosuppressive acidic protein (IAP) level used to monitor adjuvant arthritis in rats.

To determine if immunosuppressive acidic protein (IAP) responds to the degree of inflammation in adjuvant arthritis in rats (AA), we measured the serum IAP level of the rats by using the single immunodiffusion method, and the level of concanavalin A (Con A) binding protein by using nepherometry. Pharmacologic treatment with 0.3mg/kg of methotrexate (MTX) was done from day 5 to 14 postimmunization in rats with AA. The measured serum IAP level on day 21 indicated the most severe inflamed phase. IAP levels reflected the degree of primary and secondary inflammatory phases in AA, whereas ConA binding protein (CBP) levels did not. Our results suggest that IAP can be used to monitor the symptoms of inflammation and the efficacy of anti-inflammatory drugs.

Renal carbonic anhydrase activity in DBA/2FG-pcy/pcy mice with inherited polycystic kidney disease.

DBA/2FG-pcy/pcy (D2-pcy) mice are a hereditary murine model of slowly progressive polycystic kidney disease (PKD) and characterized by the persistent excretion of acidic urine, in association with polyuria, after weaning. In this study, the activity of carbonic anhydrase (CA) and it histological distribution in the kidney of D2-pcy mice were investigated by immunohistochemistry. Significantly higher CA activity was detected in the cytosolic, but not membrane, fraction of kidney homogenates in 5-week-old D2-pcy mice than in age-matched, control DBA/2 (D2) mice, and a more rapid rate of urine acidification was noted in 11-week-old mice when acetazolamide, an inhibitor of the enzyme, was administered orally. By immunohistochemistry for the major renal CA isoenzyme (CA II), epithelial cells in the distal straight tubules and the cortical collecting ducts were stained intensely, whereas those of the proximal convoluted tubules had only weak and diffuse staining. The glomeruli, the proximal straight tubules and the ascending thin limb of Henle's loop were almost free from staining. In the cells lining cysts and/or dilated tubules, CA II activity was well preserved, although the staining intensity was considerably reduced in fully-flattened, lining cells of cysts, but no difference was found between D2-pcy and D2 mice in any segmental localization of renal CA II activity. From these results it seems that D2-pcy mice in the early stages of the cystic disease continue to secrete excess protons through the CA-mediated reaction that is stimulated for regulation of acid-base balance in the distal portion of the nephron and the collecting duct in kidney. It also suggests that monitoring urine pH may be useful in predicting the effects of early interventions on the progression of slowly developing renal cysts.

Effects of Aconitum alkaloid kobusine and pseudokobusine derivatives on cutaneous blood flow in mice.

Aconitum alkaloids of the C20-diterpenoid type, kobusine (1) and pseudokobusine (2), and their acetyl, benzoyl, propionyl or cinnamoyl derivatives are examined for their peripheral vaso-activities by laser-flowmetrical measurement of the cutaneous blood flow in the hind foot of mice after intravenous administration. A dose-relationship of maximally increased blood flow after the administration of either of the Aconitum alkaloids existed. Kobusine 15-acetate (4), 11-benzoate (6) and 15-benzoate (7) were significantly effective at a low dose of 1 mg/kg, whereas the other kobusine derivatives were all inactive. Alkaloid 2, alone, and the 11-acetate (14), 15-acetate (15), 15-propionate (22) and 15-cinnamoate (25) were all active at 1 mg/kg and the effect of 14 at 5 mg/kg was remarkable. The activity of 2 was abolished by esterification of the hydroxyl group at C-6. Alkaloid 15 at 5 mg/kg showed a pattern of time course of blood flow in which the increase was rapidly replaced with a decrease below the basal flow, probably suggesting the effect of excessive doses. Conclusively, it is considered that the hydroxyl groups of alkaloids, especially a free OH group of 2 at C-6, are important for action on the peripheral vasculature leading to dilatation, and these results indicated that esterification of the hydroxyl group at C-15 with either acetate or benzoate may contribute to enhancement of the activity of the parent alkaloids.

Effects of alkaloids from Aconitum yesoense var. macroyesoense on cutaneous blood flow in mice.

Nine alkaloid constituents in the root of Aconitum yesoense var. macroyesoense, as well as three acetylated derivatives, were examined for their peripheral vaso-activities by measuring laser-flowmetrically the cutaneous blood flow in the hind foot of mice after intravenous administration. The major constitutive delcosine (1), 14-acetyldelcosine (2) and lucidusculine (3), respectively, had little or very mild vaso-activity. Kobusine (4) and pseudokobusine (5) and three minor constituents, luciculine (6), 1-acetylluciculine (7) and dehydroluciculine (8), together exhibited a rapid increase in blood flow reaching a peak with a magnitude almost equal to that produced by hydralazine, when administered intravenously at the same dosage level of 20 mg/kg. Among them, 4 was characterized by successive reversal of the increase to a decrease in blood flow, while 7 produced a flow with a more delayed peak time. Dehydrolucidusculine (9) exhibited a transient decrease in blood flow prior to occurrence of the increase, as did papaverine. Consequently, it is assumed that the alkaloids, especially those of the C20-diterpenoid type, in the root of this Aconitum plant have peripherally vaso-dilating activities to varying degrees in mice, probably due to their direct action on the cutaneous microvasculature in a similar fashion to that shown by hydralazine. The laser blood flowmetric method would be useful as an in vivo means of qualitative as well as quantitative screening of chemically modified derivatives of peripherally vasoactive agents in mice.

Effects of H2-antihistamines in murine models of immediate hypersensitivity and chronic inflammation.

Effects of H2-antihistamines alone and in combination with H1-antihistamines on immediate hypersensitivity and chronic inflammation in mice and rats were investigated. The mouse PCA reaction was inhibited partially by a minimum effective dose of diphenhydramine and blocked completely when administered simultaneously with either famotidine, ranitidine or cimetidine. There was similar synergism between H1- and H2-antihistamines in inhibiting the PCA-like reaction in rat paw. Famotidine alone was able to inhibit those two reactions in a large oral dose of 100 mg/kg. A five-day course of therapy with H2-antihistamines alone or in combination with pyrilamine, suppressed effectively the chronic swelling induced in the hind paw of BUF rats. From these results, it is deduced that simultaneous blockage of both histamine H1- and H2-receptors may be necessary for sufficient inhibition of the microvascular permeability increase in some kinds of anaphylactic reactions, and that histamine, mainly interacting with H2-receptors, may take an important role for activation of a certain phase of chronic inflammation where bone destruction, blood capillary growth and mast cell degranulation involve.

Prostaglandins: a possible mediator to inhibit hepatic drug metabolism in adjuvant arthritic rats.

In the liver of adjuvant arthritic rats perfused with a hemoglobin-free buffer solution, the rate of metabolism of a model drug, 2,6-dichloro-4-nitroanisole, was approximately half that of the control, while the bile flow rate was normal. Granulation tissue extracts and arthritic rat serum had no effect on the activity of CNA metabolism in normal rat liver preparations. In the perfused normal rat liver, the rate of CNA metabolism was inhibited by addition of prostaglandin (PG) E1, PGE2, and PGF2 alpha, respectively, in a final concentration of 0.5 microM. The inhibition by PGE1 was increased in the concentration range from 0.1 to 2.5 microM. The bile flow rate was not affected by the added PGs. However, these PGs had no direct effect on the CNA demethylating activity of the isolated hepatocytes from normal rat liver in a high concentration of 10 microM. Serotonin stimulated slightly CNA metabolism and bile production in the perfused livers by the intermittent infusion, but was without effect in the isolated hepatocytes. Epinephrine and histamine had no significant effect on CNA metabolism in both liver preparations. A similar pattern of the inhibition of CNA metabolism by PGs was reproduced in the normal rat liver perfused with the medium containing the supernatant of the hepatic nonparenchymal cells incubated in the presence of PGE1. The involvement of liver sinusoidal cells as secretory cells in depression of hepatic drug metabolism has been discussed.

Hypersensitivity to endotoxin hepatotoxicity in rats with inflammatory lesions.

The ratio of sinusoidal nonparenchymal cells to hepatocytes in rat liver was significantly increased following induction of inflammation, and decreased after subsequent exposure to endotoxin, particularly in the region around the terminal portal venules. Rats with inflammatory lesions were more sensitive to endotoxin hepatocytotoxicity than normal controls, as judged from the dose-dependent increase in activity of serum transaminases and from the extent of liver tissue injury. In addition, these animals, which were already in a state of depletion of hepatic glycogen, demonstrated marked hyperglycemia 24 h after endotoxin administration in small doses of less than 2 mg/kg.

Progressive foot swelling in BUF rats. A new animal model for screening of anti-inflammatory and anti-rheumatic drugs.

Buffalo rats of a low adjuvant-arthritis responder strain demonstrate a continually increasing swelling in the hind foot inoculated with mycobacterial adjuvant, characterized by extensive panostitis, new bone formation and long-delayed bony calcification. A rapid regression of foot swelling was produced by a three-day course of therapy with indomethacin (1 mg/kg) or with dexamethasone (0.1 mg/kg), but a definite rebound was followed soon after termination of the therapy. This recurrent episode occurred repeatedly in the second and third therapeutic trials with the anti-inflammatory drugs in the same animals. The 5 days treatment with either levamisole (5 mg/kg) or cyclophosphamide (5 mg/kg) prevented further increase in the swelling for a long time period after withdrawal of the drug, and enhanced bone mineralization.

Mouse paw anaphylaxis.

Anaphylactic swelling passively transferred to the hind paws of mice with homologous antibodies was investigated. The ascites fluid containing homocytotropic antibodies of the IgG1 class in a high concentration was obtained by repeated injections of bovine serum albumin in complete Freund's adjuvant into the peritoneal cavity of female ICR mice. The standard procedure for mouse paw anaphylaxis defined was as follows: female ICR mice received subcutaneously 25 microliters of a 1:20 diluted ascites in the hind footpad and 2 h later were given intravenously 1.0 mg of the antigen in saline, and subsequently at 5 min intervals paw thickness was assessed by means of a simple thickness gauge. Maximal paw edema occurred invariably 15 min after antigen challenge. Sex difference in response was not observed. Heating at 56 degrees C for 4 h resulted in a reduction of the activity of mouse ascites by approximately 40%. Histological examinations revealed that paw anaphylaxis was accompanied by mast cell degranulation and release of biologically active constituents from mast cell granules. Promethazine, pyrilamine, diphenhydramine, methysergide, cyproheptadine, ketotifen and phenoxybenzamine provided significant protection from the anaphylactic reaction in mice, while bromocriptine, dexamethasone and indomethacin were inactive. Relative drug effectiveness in paw anaphylaxis, passive cutaneous anaphylaxis and active and passive systemic anaphylaxis in mice was compared and discussed. Consequently, it was shown that mouse paw anaphylaxis was not only convenient for quantitative assessment of homocytotropic antibodies, but also useful in the routine work for screening of potential anti-allergic drugs.

Peroxidative status of isolated hepatocytes from adjuvant arthritic rats.

Bleb-like protrusions frequently appear in the plasma membrane of the hepatocytes freshly isolated from adjuvant arthritic rats by the collagenase-perfusion method. These hepatocytes show reduced overall synthesis of phospholipids and triglycerides, decreased percentages of arachidonic acid present in the plasma membrane, microsomal membrane and bile canalicular membrane, decreased levels of soluble thiol compounds, and increased lipid peroxidation. The content of thiobarbituric acid reactive substances in liver tissue homogenate and the level of lipid peroxides in blood are significantly higher in adjuvant arthritic rats than in normal controls. These observations suggest that inflammation might cause enhanced membrane fragility and altered membrane-bound enzyme function in rat hepatocytes, probably due to an increasing tendency to lipid peroxidation.

Reduced drug metabolism in isolated hepatocytes from adjuvant arthritic rats.

Viable hepatocytes were isolated from the livers of rats with adjuvant arthritis by the collagenase-perfusion method and measured for activities of drug-metabolizing enzymes. These cells produced radioactive metabolites from 14C-aminopyrine and 14C-aniline to a much lesser extent than the control hepatocytes that were derived from pair-fed normal rats. On the other hand, 14C-aminopyrine was scarcely metabolized by non-parenchymal cells other than hepatocytes, even when incubated with those from control rats. Although there were no significant differences in cell yield, viability and oxygen consumption, the cellular uptake of indocyanine green was significantly slower in the arthritic hepatocytes than the control hepatocytes. Morphologically, the freshly isolated arthritic hepatocytes demonstrated the disappearance of the microvilli, the appearance of bleb-like protrusions in the plasma membrane and the widespread distribution of the rough endoplasmic reticulum associated with a relatively decreased area of the smooth endoplasmic reticulum in the cytoplasma. Biochemically, these cells showed a significantly higher RNA/DNA ratio and an ability to incorporate 14C-leucine into proteins more rapidly, as compared to the control hepatocytes. A possible relationship between the reduction of the drug metabolizing activity and the production of the acute phase proteins in rat hepatocytes after an inflammatory stimulus was discussed.

Secretin-like bioactivity in the duodenal mucosa in patients with peptic ulcer and chronic pancreatitis.

Biopsy specimens of the duodenal mucosa were assayed to determine their secretin-like activity in 9 controls, 9 patients with gastric ulcer, 19 patients with duodenal ulcer, 4 patients with gastric and duodenal ulcer, and 13 patients with chronic pancreatitis. The bioassay of secretin was done on the pancreatic secretion in anesthetized rats. The sensitivity was in the orcer of 0.0625 CHR unit/rat (4 ng/rat). In the range between 0.0625 and 0.5 CHR units a satisfactory dose dependency was recognized. The following results were obtained. 1) The level of duodenal mucosal secretin-like activity in patients with gastric ulcer was the same as that in the controls, but was elevated in 32% of the patients with duodenal ulcer, 50% of those with gastric and duodenal ulcer, and 8% of those with chronic pancreatitis. 2) The high level of secretin-like activity noted in patients with duodenal ulcer was suspected to be related to the hypersecretion of gastric acid which is characteristic of this disease, but there was no correlation between gastric acid secretion and secretin-like activity in the duodenal mucosa.

Reserve capacity of bicarbonate secretion in chronic pancreatitis.

When secretin was given by continuous intravenous infusion in the control subjects, the dose of secretin inducing maximal bicarbonate output was found to be around 6.0 CHR U/kg/hr. Then the pancreatic exocrine secretory response to sequential standard (1.2 CHR U/kg/hr) and augmented (6.0 CHR U/kg/hr) dose of secretin was studied in the controls, in patients with chronic pancreatitis and in its suspected cases. This new method for exocrine pancreatic function did not offer advantage for the diagnosis of well established chronic pancreatitis. But from the results obtained in suspected chronic pancreatitis it was supposed that the decline of increasing rate of bicarbonate output with augmentation of dose and the decrease of response to augmented dose of secretin might be one of functional disorders occurred in the early stage of chronic pancreatitis.