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Aptamers are an attractive candidate for a molecular-targeted drug since they can show ability to precisely recognize proteins with a large affinity similar to antibodies. A drawback of aptamers is...

Intrarenal arterial infusion of norepinephrine (30 ng/kg per min) or of angiotensin II (4 ng/kg per min) reduced the glomerular filtration rate and urinary Na + excretion in denervated kidneys of anesthetized rabbits pretreated intrarenally with a nitric oxide (NO) synthase inhibitor N ω -nitro- l -arginine methyl ester (50 μ g/kg per min). Angiotensin II but not norepinephrine reduced fractional Na + excretion. Intrarenal administration of a spontaneous NO donor 1-hydroxy-2-oxo-3-( N -methyl-3-aminopropyl)-3-methyl-1-triazene (NOC 7, 30 ng/kg per min) in l -NAME pretreated kidneys did not affect basal values, but attenuated the reduction in urinary Na + excretion induced by these agonists without affecting the angiotensin II-induced reduction in glomerular filtration rate. The results suggest that NOC 7 can suppress the norepinephrine-induced hypofiltration and the angiotensin II-evoked tubular reabsorption and thereby attenuates the agonist-induced antinatriuresis in the denervated and endogenous NO-depleted rabbit kidney.

Multiple cholesterol stones are associated with more biliary complications and show more rapid cholesterol nucleation than solitary stones. Group II phospholipase A2 (PLA2-II) may play a critical role in the process of mucosal inflammation, which in turn may produce pronucleating agents. PLA2-II concentrations in gallbladders and gallbladder bile from patients with different types of gallstone disease were assayed to correlate PLA2-II with alterations in biliary composition.PLA2-II protein concentrations were assayed immunoradiometrically using monoclonal antibodies against human splenic PLA2-II.Immunoreactive PLA2-II levels in gallbladder bile were significantly higher in patients with multiple cholesterol stones (68.2 +/- 6.3 ng/dL, mean +/- SEM; n = 24) than in those with solitary stones (24.9 +/- 2.8; n = 20; P0.01), those with multiple pigment stones (24.2 +/- 3.7; n = 18; P0.01), or control subjects (13.4 +/- 1.7; n = 19; P0.01). Increased biliary immunoreactive PLA2-II levels in multiple cholesterol stones were associated with a concomitant increase in the lysophosphatidylcholine to phosphatidylcholine ratio; free arachidonate, protein, and hexosamine concentrations; and gallbladder bile viscosity. The gallbladders showed an increased PLA2-II protein mass and steady-state messenger RNA levels, which was associated with increased prostaglandin E2 levels.Increased biliary PLA2-II may be of pathogenetic importance in multiple cholesterol stones, probably through potentiating gallbladder mucosal inflammation with associated biliary alterations favoring cholesterol crystal formation.

1. The aim of this study was to investigate the renal vascular response to angiotensin II (3-8) (AIV). The effect of the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) or the cyclooxygenase inhibitor, indomethacin on the AIV-induced response was examined in anaesthetized spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). 2. Intrarenal infusion of AIV produced a biphasic vasoconstrictor response. The vasoconstriction developed rapidly to reach a maximum followed by a partial recovery to a sustained lesser level of vasoconstriction. The initial maximum response was enhanced by L-NAME but not affected by indomethacin treatment. The simultaneous administration of L-NAME and indomethacin prevented the partial recovery resulting in a greater sustained level of constriction. 3. A stable vasoconstriction of relatively constant magnitude was observed with angiotensin II (AII) infusion. The AII vasoconstriction was enhanced by L-NAME but not changed by indomethacin. The combination of these inhibitors further enhanced the AII-induced vasoconstriction in WKY, but not in SHR. 4. Pretreatment with the AII receptor antagonist, losartan, inhibited the vasoconstriction induced by AIV and AII. 5. These results suggest that nitric oxide and prostaglandins may modulate the renal vascular response to AIV.

Recently, soluble(s) circulating isoforms of intercellular adhesion molecule-1 (sICAM-1) and sE-selectin (formerly endothelial leukocyte adhesion molecule-1) have been described in normal human serum. Elevated levels have been reported in acute and chronic inflammatory disorders, including allograft rejection. In this study, plasma levels of sICAM-1 and sE-selectin were determined in groups of tacrolimus (FK 506)-treated adult patients following either isolated small bowel (SB), liver, or combined SB plus liver (SB/L) transplantation. Each molecule was measured at 1, 2, 4, 6, 8, and 12 weeks (all patients) and at 6, 9, and 12 months after transplantation (SB and SB/L only) by enzyme linked immunosorbent assay. Levels were compared with those of soluble interleukin-2 receptor (sIL-2R ; a marker of lymphocyte activation) and soluble HLA class I (which has been reported to be elevated in liver transplant-related complications). Elevations above normal in mean plasma levels of sICAM-1 (2.4-fold), sE-selectin (1.8-fold), sIL-2R (10.6-fold), and sHLA class I (1.3-fold) were found in patients with stable isolated SB grafts during the first 12 weeks posttransplant. Except for sHLA class I, levels of each protein were subsequently reduced, up to 1 year posttransplant. However, further increases in sICAM-1 and in sIL-2R and sE-selectin levels were observed during episodes of SB rejection compared with stable grafts. Mean levels of all molecules were higher in patients with isolated SB grafts compared with those given liver or combined (SB/L) transplants, either during stable SB graft function (up to 12 weeks posttransplant) or rejection. The data demonstrate increased adhesion molecule production/shedding following SB transplantation and are suggestive of a reduced overall level of immune activation in liver and SB/L compared with isolated SB transplantation.

In order to clarify the mechanism of plasma gene transfection, the relationship between transfection efficiency and transferred molecular size was investigated. Molecules with low molecular mass (less than 50 kDa; dye or dye-labeled oligonucleotide) and high molecular mass (more than 1 MDa; plasmid DNA or fragment of plasmid DNA) were transferred to L-929 cells. It was found that the transfection efficiency decreases with increasing in transferred molecular size and also depends on the tertiary structure of transferred molecules. Moreover, it was suggested the transfection mechanism is different between the molecules with low (less than 50 kDa) and high molecular mass (higher than 1 MDa). For the amount of gene transfection after plasma irradiation, which is comparable to that during plasma irradiation, it is shown that H2O2 molecules are the main contributor. The transfection efficiency decreased to 0.40 ± 0.22 upon scavenging the H2O2 generated by plasma irradiation using the catalase. On the other hand, when the H2O2 solution is dropped into the cell suspension without plasma irradiation, the transfection efficiency is almost 0%. In these results, it is also suggested that there is a synergetic effect of H2O2 with electrical factors or other reactive species generated by plasma irradiation.

This study investigates the molecular mechanisms underlying the induction of and protection from T cell activation-associated hepatic injury. When BALB/c mice were given a single intravenous injection of concanavalin A (Con A) (> or = 0.3 mg/mouse), they developed acute hepatic injury as assessed by a striking increase in plasma transaminase levels within 24 h. Histopathologically, only the liver was injured while moderate infiltration of T cells and polymorphonuclear cells occurred in the portal areas and around the central veins. The induction of hepatic injury was dependent on the existence as well as the activation of T cells, as untreated BALB/c nu/nu mice or BALB/c mice pretreated with a T cell-specific immunosuppressive drug, FK506, failed to develop disease. Significant increases in the levels of various cytokines in the plasma were detected before an increase in plasma transaminase levels. Within 1 h after Con A injection, tumor necrosis factor (TNF) levels peaked, this being followed by production of two other inflammatory cytokines, interleukin 6 (IL-6) and IL-1. Passive immunization with anti-TNF but not with anti-IL-1 or anti-IL-6 antibody, conferred significant levels of protection. Moreover, administration of rIL-6 before Con A injection resulted in an IL-6 dose-dependent protection. A single administration of a given dose of rIL-6 completely inhibited the release of transaminases, whereas the same regimen induced only 40-50% inhibition of TNF production. More than 80% inhibition of TNF production required four consecutive rIL-6 injections. These results indicate that: (a) TNFs are critical cytokines for inducing T cell activation-associated (Con A-induced) hepatitis; (b) the induction of hepatitis is almost completely controlled by rIL-6; and (c) rIL-6 exerts its protective effect through multiple mechanisms including the reduction of TNF production.

The effects of various protease inhibitors on naloxone-precipitated withdrawal jumping were examined in morphine-dependent mice. The doses of morphine were subcutaneously given twice daily for 2 days (day 1, 30 mg/kg; day 2, 60 mg/kg). On day 3, naloxone (8 mg/kg) was intraperitoneally administered 3h after final injection of morphine (60 mg/kg), and the number of jumping was immediately recorded for 20 min. Naloxone-precipitated withdrawal jumping was significantly suppressed by the intracerebroventricular administration of N-ethylmaleimide (0.5 nmol) and Boc-Tyr-Gly-NHO-Bz (0.4 nmol), inhibitors of cysteine proteases involved in dynorphin degradation, 5 min before each morphine treatment during the induction phase, with none given on the test day, as well as by dynorphin A (62.5 pmol) and dynorphin B (250 pmol). However, amastatin, an aminopeptidase inhibitor, phosphoramidon, an endopeptidase 24.11 inhibitor, and captopril, an angiotensin-converting enzyme inhibitor, caused no changes. The present results suggest that cysteine protease inhibitors suppress naloxone-precipitated withdrawal jumping in morphine-dependent mice, presumably through the inhibition of dynorphin degradation.

The effects of various protease inhibitors on the development of antinociceptive tolerance to morphine were examined in mice. Intrathecal (i.t.) administration of morphine (0.01–1nmol) produced a dose-dependent and significant antinociceptive effect in the 0.5% formalin test. When the doses of morphine (mg/kg, s.c. per injection) were given as pretreatment twice daily for two days [first day (30) and second day (60)], i.t. administration of morphine (0.1nmol) was inactive due to antinociceptive tolerance on the third day. Tolerance to i.t. morphine was significantly suppressed by the i.t. injection of N -ethylmaleimide or Boc-Tyr-Gly-NHO-Bz, inhibitors of cysteine proteases involved in dynorphin degradation, as well as by dynorphin A, dynorphin B and (−) U-50,488, a selective κ-opioid receptor agonist. On the other hand, amastatin, an aminopeptidase inhibitor, phosphoramidon, an endopeptidase 24.11 inhibitor, lisinopril, an angiotensin-converting enzyme inhibitor, and phenylmethanesulfonyl fluoride, a serine protease inhibitor, were inactive. These results suggest that cysteine protease inhibitors suppress the development of morphine tolerance presumably through the inhibition of dynorphin degradation.

The anti-inflammatory effect of propolis was compared with that of diclofenac, a non-steroidal anti-inflammatory drug, and L-N(G)-nitro arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, using carrageenin-induced mouse paw edema. When administered 10 min prior to carrageenin injection, propolis (1 : 1000, 1 : 100, p.o.), diclofenac (12.5, 50 mg/kg, p.o.) and L-NAME (10, 100 mg/kg, s.c.) showed a significant anti-inflammatory effect. The anti-inflammatory effects of propolis and L-NAME were significantly inhibited by L-arginine, a precursor of nitric oxide, but not by D-arginine. In contrast, the anti-inflammatory effect produced by diclofenac was not inhibited by either D-arginine or L-arginine. These results indicate that the anti-inflammatory effect of propolis on mouse paw edema acts via the inhibition of nitric oxide production, similar to that of L-NAME but not diclofenac.

Using a continuous superfusion system of dog renal cortical slices, we studied the role of Ca(2+) in the intracellular control mechanism for renin release. The calcium ionophore A23187 (10 microM) produced a significant decrease in renin release. This effect was abolished in the absence of extracellular Ca(2+). Moreover, pretreatment with the calmodulin inhibitor W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, 20 microM) completely prevented the inhibitory effect of A23187 (10 microM). The beta-adrenoceptor agonist isoproterenol (1, 10 and 100 microM) produced a concentration-dependent increase in renin release. Pretreatment with W-7 (20 microM) potentiated the stimulation of renin release induced by isoproterenol (1 microM). These results suggest that A23187-induced inhibition of renin release is mediated by the activation of calmodulin via an increase in intracellular Ca(2+) and beta-adrenoceptor-stimulated renin release is modulated by intracellular Ca(2+) mobilization.

We examined modulation by nitric oxide (NO) of sympathetic neurotransmitter release and vasoconstriction in the isolated pump-perfused rat kidney. Electrical renal nerve stimulation (RNS; 1 and 2 Hz) increased renal perfusion pressure and renal norepinephrine (NE) efflux. Nonselective NO synthase (NOS) inhibitors [ Nω-nitro-l-arginine methyl ester (l-NAME) or Nω-nitro-l-arginine], but not a selective neuronal NO synthase inhibitor (7-nitroindazole sodium salt), suppressed the NE efflux response and enhanced the perfusion pressure response. Pretreatment with l-arginine prevented the effects of l-NAME on the RNS-induced responses. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), which eliminates NO by oxidizing it to NO2, suppressed the NE efflux response, whereas the perfusion pressure response was less susceptible to carboxy-PTIO. 8-Bromoguanosine cGMP suppressed and a guanylate cyclase inhibitor [4 H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one] enhanced the RNS-induced perfusion pressure response, but neither of these drugs affected the NE efflux response. These results suggest that endogenous NO facilitates the NE release through cGMP-independent mechanisms, NO metabolites formed after NO2rather than NO itself counteract the vasoconstriction, and neuronal NOS does not contribute to these modulatory mechanisms in the sympathetic nervous system of the rat kidney.

We studied the effect of oxatomide, an antiallergic drug, on the resistance of K562 cells to doxorubicin. Oxatomide synergistically potentiated the cytotoxicity of doxorubicin in doxorubicin-resistant K562 cells (K562/DXR) at a concentration of 1-10 microM, but had hardly any synergistic effect on the parental cell line (K562) at the same concentration. Oxatomide inhibit P-glycoprotein pump-efflux activity and the binding of [3H]-azidopine to the cell-surface protein P-glycoprotein, in a dose-related manner. These results indicate that oxatomide reverses the multidrug-resistance phenotype through direct interaction with P-glycoprotein.

We examined the effects of sarafotoxin 6c (S6c), an endothelin-B (ETB) receptor agonist, on adrenal catecholamine secretion in response to cholinergic stimuli in pentobarbital sodium-anesthetized dogs. Drugs were administered intra-arterially into the adrenal gland through the phrenicoabdominal artery. Infusion of S6c attenuated increases in adrenal catecholamine output induced by splanchnic nerve stimulation. The inhibitory effect of S6c on the catecholamine secretion response was suppressed with a selective ETB receptor antagonist N- cis2,6-dimethylpiperidinocarbonyl-l-γ-methylleucyl-d-1-methoxycarbonyltryptophanyl-d-norleucine (BQ-788), a nitric oxide synthase (NOS) inhibitor N ω-nitro-l-arginine methyl ester, and a neuronal NOS inhibitor 7-nitroindazole monosodium salt (7-NINA). Similar results were obtained with the catecholamine secretion response induced by injection of ACh. 7-NINA alone did not affect these catecholamine secretion responses. These results suggest that ETB receptors play an inhibitory role in adrenal catecholamine secretion by activating neuronal NOS, whereas neuronal NOS is unlikely to be involved in regulation of adrenal catecholamine secretion in the absence of simultaneous ETB receptor stimulation.

We examined the hypotensive effect of enalapril in relation to the local renin-angiotensin system of the kidney in spontaneously hypertensive rats (SHR). Oral administration of enalapril for 7 days decreased mean arterial blood pressure and renal tissue angiotensin II concentration without affecting plasma angiotensin II concentration in SHR. The enalapril treatment did not affect maximum binding of angiotensin II to renal tubules and glomeruli in SHR. In normotensive Wistar-Kyoto rats, no significant changes in mean arterial blood pressure, renal and plasma angiotensin levels were observed with enalapril treatment. Direct infusion of enalapril into the renal medullary interstitium decreased mean arterial blood pressure in association with the reduction of renal tissue angiotensin II concentration without changes in plasma angiotensin II concentration in SHR. These observations suggest that the inhibition of angiotensin conversion in the kidney is important for the hypotensive action of enalapril.

Effects of protein kinase C (PKC) activator and inhibitors on adrenal catecholamine release were examined in anesthetized dogs. Output of epinephrine (EPI) and norepinephrine (NE) was determined from adrenal venous blood by high-performance liquid chromatography (HPLC) with electrochemical detection. All drugs were infused intraarterially (i.a.) into the adrenal gland through the phrenic abdominal artery. Infusion of the PKC activator phorbol-12,13-dibutyrate (PDB 0.1 micrograms/min) increased EPI and NE output during basal state and enhanced increases in catecholamine output induced by splanchnic nerve stimulation (SNS 1 and 3 Hz). These effects of PDB were abolished by the PKC inhibitor staurosporine (SSP 0.3 microgram/min), when both drugs were infused simultaneously. Infusion of SSP (0.1, 0.3, and 1 micrograms/min) caused a dose-dependent inhibition of the SNS-induced increases in EPI and NE output. SNS-induced increases in catecholamine output were also inhibited by another PKC inhibitor polymyxin B (PMB 0.1, 0.3, and 1 micrograms/min) and by the phospholipase C (PLC) inhibitor neomycin (NM 0.3, 1, and 3 mg/min). SSP, PMB, and NM did not affect basal output of EPI and NE. These results suggest that activation of PKC promotes release of adrenal catecholamines and provide indirect evidence that activation of PKC and PLC may be involved in SNS-induced release of catecholamines from dog adrenal gland.

Human renal dipeptidase cDNA and genomic DNA were isolated from human kidney cDNA and genomic libraries, respectively. The human renal dipeptidase gene has a total length of approximately 6 kb and consists of ten exons and nine introns. The exons and cDNA each encode the 411 amino acid residues of the precursor protein, including 16 amino acid residues of signal sequence and a hydrophobic carboxyl terminal sequence for the attachment of a phosphatidylinositol glycan. Although the cDNA was slightly different from the cDNA reported by Adachi et al. (1990), the differences observed suggest, by comparison with human genomic DNA, that it may not represent an allelic variant but a cloning artifact. The recombinant human renal dipeptidase was produced on the surface of transfected L929 cells and had the same character as native renal dipeptidase. Northern blotting hybridization analysis showed that renal dipeptidase mRNA is only transcribed in kidney.

Mouse renal dipeptidase (mouseRDP, EC 3.4.13.11) was purified from the membrane fraction of kidney. The molecular mass of the enzyme was 115 kDa by size-exclusion HPLC and SDS-PAGE under non-reduced conditions and 58 kDa by SDS-PAGE under reduced conditions. The mouseRDP cDNA fragment was amplified from mouse kidney total RNA by reverse transcriptation-polymerase chain reaction (RT-PCR). The mouseRDP cDNA was isolated from a kidney cDNA library using the probe. The primary structure of mouseRDP deduced from the cDNA showed a high homology with renal dipeptidase from various mammals, except for the amino-terminal and carboxy-terminal domains. Recombinant mouseRDP obtained from transfected mouse L929 cells containing the expression plasmids has the same Km value and molecular mass as native mouse renal dipeptidase. From Northern blotting analysis, expression of the mouseRDP gene was recognized in both kidney and liver.

A genomic DNA for human renal dipeptidase was isolated from a human genomic library using probes for human renal dipeptidase cDNA. The human renal dipeptidase gene, containing ten exons and nine introns, had a total length of approx. 6 kbp. The DNA sequence of these exons was slightly different from that of the human renal dipeptidase cDNA reported by Adachi et al. [1]. From the results of a comparison of the deduced amino acid sequence of each exon with various mammalian renal dipeptidases, the fourth exon was found to be highly conserved (90%).

A genomic DNA for rat platelet phospholipase A2 was isolated by screening a rat genomic library with oligonucleotide probes based on its published amino acid sequence. The rat platelet phospholipase A2 gene had a total length of about 2.5 kb and contained five exons and four introns. The intron-exon structure of the rate gene was similar to that of human non-pancreatic phospholipase A2.

We studied the effect of E-64, a new thiol protease inhibitor derived from Aspergillus japonicus TPR-64, and of its synthesized analogue, E-64-d, on dystrophic mice ( C57BL 6J -dy ). The locomotor activity of normal mice increased markedly, reaching a plateau at 8 weeks of age. In dystrophic mice, it increased until they were 7 weeks old, thereafter, it decreased gradually. This decrease reflected the degradation of the hind legs. Serum activities of creatine phosphokinase were significantly greater in dystrophic than in normal mice. When 3-week-old dystrophic mice were injected with E-64 (1 mg/kg, i.p.) or E-64-d (40 to 60 mg/kg, p.o.), the decrease in their locomotor activity was retarded and their serum enzyme activities decreased significantly. In addition, the survival time of treated dystrophic mice was prolonged. The locomotor activity of normal mice and their serum enzyme levels were not affected by the administration of E-64-d. We posit that the new thiol protease inhibitors we tested retard the porgression of dystrophy in mice.

We studied the effect of mitochondrial protein synthesis inhibitors on the neurotrophic action of the nerve stump in mice. The sciatic nerve was cut as close to, or as far from the extensor digitorum longus muscles as possible. At 2 and 3 days after denervation, depolarization in the resting membrane potential of muscles with long nerve stumps (14-16 mm) was significantly smaller than in muscles with very short (less than 2 mm) nerve stumps. Chloramphenicol (100 mg/kg, p.o.), erythromycin (4 micrograms/kg, i.p.), ethidium bromide, (10 micrograms/kg, i.p.) or acridine (10 micrograms/kg, i.p.), administered for 2 or 3 days after denervation, increased depolarization in the resting membrane potential of muscles with long nerve stumps; muscles with very short nerve stumps were not affected by these drugs. The administration of chloramphenicol and erythromycin resulted in an earlier increase in the depolarization than ethidium bromide and acridine. Activities of cathepsin B and L of lysosomal proteases in the nerve stumps were enhanced by nerve degeneration; they were not affected by the administration of chloramphenicol or ethidium bromide. We suggest that proteins synthesized in mitochondria of the nerve may participate in trophic actions.

The present study was designed to investigate the effect of bradykinin on renin release in relation to prostaglandins by use of indomethacin, a cyclooxygenase inhibitor, in pentobarbital-anesthetized rats. A nonhypotensive dose of bradykinin (1 microgram/kg X min), which was infused intrarenally, significantly increased plasma renin activity approximately 1.8-fold as compared to the control period. In indomethacin (5 mg/kg, i.v.)-pretreated rats, bradykinin did not significantly increase plasma renin activity. These results suggest that bradykinin does not directly increase plasma renin activity and that prostaglandins may participate in the bradykinin-induced renin release under these experimental conditions.

The combined activities of cathepsin B and cathepsin L were studied in the forelimb and hind limb muscles of dystrophic mice. The activities of these proteases in the forelimb and hind limb muscles of young and adult dystrophic mice were significantly higher than those in normal mice. However, clinical involvement of dystrophy appeared in the hind limbs but not in the forelimbs. We therefore suggest that the increase in protease activity begins at a very early age and that the clinical involvement is not linked with the increase in cathepsins B and L.

the effect of captopril (D-3-mercapto-2-methylpropanoyl-L-proline), a converting enzyme inhibitor, on the systemic blood pressure in pentobarbital anesthetized rats was investigated. Experiments were performed in four groups of rats: untreated, Na-restricted, furosemide treated and bilaterally nephrectomized, respectively. Intravenous injection of 1 mg/kg of captopril caused a significant hypotensive effect in the three groups of rats but not in the nephrectomized group. Individual values for the hypotensive effect produced by captopril and plasma renin activity obtained just before injection of captopril showed a highly significant correlation.

In pentobarbital-anesthetized dogs, the increase in the renal norepinephrine secretion rate elicited by renal nerve stimulation (1 Hz) during infusion of angiotensin I (15 ng/kg/min) was partially but significantly inhibited (by 21-37%) after dosing with cilazaprilat (0.1 mg/kg), an angiotensin converting enzyme inhibitor, accompanied by a decrease in the renal venous plasma norepinephrine concentration. These results may suggest that cilazaprilat exerts at least a part of its hypotensive effect through decreasing the facilitatory action of endogenous angiotensin II on adrenergic transmission.

Angiotensin II markedly potentiated both PGE 2 and PGI 2 productions in the isolated dog renal arteries. This angiotensin II-induced response was significantly reduced by the treatments of EGTA and calcium antagonists such as verapamil, nifedipine and 8-(N,N′-diethylamino)-octyl-3,4,5,-trimethoxybenzoate (TMB-8). Calmodulin inhibitors, trifluoperazine and W-7 also inhibited the angiotensin II-induced PG productions while an inactive analogue of W-7, W-5 did not have any effect. The results suggest that angiotensin II may enhance the intracellular Ca 2+ level through the influx of extracellular Ca 2+ and then, calmodulin activated with Ca 2+ will stimulate both PGE 2 and PGI 2 productions via its activation of phospholipase A 2 in the dog renal arteries.

Anti-warfarin antiserum was prepared in rabbits by immunization with a synthesized warfarin antigen, 4′-azo-warfarin human serum albumin, which possesses two enantiomorphic haptenic sites of warfarin on the molecule. The antiserum recognized both R- and S-warfarin to the same degree, 50% cross reactivities of racemic warfarin, respectively. One of the warfarin metabolites, racemic warfarin alcohol, showed 1% cross reactivity, and R- or S-warfarin alcohol have half the reactivity of the racemic alcohol. Rabbit plasma warfarin levels were determined by radioimmunoassay using this antiserum and racemic [14C]warfarin and by fluorometric assay after isolation by thin layer chromatography. After a single administration of warfarin (2 mg kg−1 orally or 500 μg kg−1 i.v.), the plasma levels determined by both assay methods showed a good correlation (r = 0.97, P < 0.001, Y = 1.04–0.09). The results show that the radioimmunoassay can determine total plasma warfarin without interference of plasma metabolite. The applicability and limitation of the radioimmunoassay for pharmacokinetic study are discussed.

We studied the effect of chymostatin on dystrophic mice ( C57BL 6J -dy ). The locomotor activity of normal mice increased markedly, attaining a plateau at 8 weeks of age, whereas in dystrophic mice, it increased until the age of 7 weeks, and thereafter decreased gradually. Serum levels of creatine phosphokinase were significantly higher in dystrophic mice compared with normal mice, and dystrophic mice had a reduced muscle protein content. When 3-week-old dystrophic mice received chymostatin (1 mg/kg, i.p.), the decrease in locomotor activity was retarded, serum enzyme levels decreased significantly, and muscle protein content increased significantly. In addition, the survival time of treated dystrophic mice was prolonged. The locomotor activity, serum enzyme levels, and muscle protein content of normal mice were not affected by chymostatin. Therefore, we posit that chymostatin retarded the progression of dystrophy in mice.