Simple and rapid determination of irinotecan and its metabolite SN-38 in plasma by high-performance liquid-chromatography: application to clinical pharmacokinetic studies

Irinotecan (CPT-11) is an anticancer agent widely employed in the treatment of colorectal carcinoma. A simple, rapid and sensitive high-performance liquid chromatographic method for the simultaneous determination of CPT-11 and its metabolite SN-38 in plasma, and their preliminary clinical pharmacokinetics are described. Both deproteinisation of plasma specimens (100 μl) and addition of the internal standard, camptothecin (CPT), are achieved by incorporating to samples 100 μl of a solution of CPT (1 μg/ml) in acetonitrile–1 mM orthophosphoric acid (90:10); 200 μl of this acidified acetonitrile solution, drug-free, is also added to accomplish complete deproteinisation: this procedure reduces sample preparation time to a minimum. After deproteinisation, samples are treated with potassium dihydrogenphosphate (0.1 M) and injected into a Nucleosil C₁₈ (5 μm, 250×4.0 mm) column. Mobile phase consists of potassium dihydrogenphosphate (0.1 M)–acetonitrile (67:33), at a flow-rate of 1 ml/min. CPT-11, SN-38 and CPT are detected by fluorescence with excitation wavelength set at 228 nm and emission wavelengths of CPT-11, SN-38 and CPT fixed, respectively, at 450, 543 and 433 nm. The limits of quantitation for CPT-11 and SN-38 are 1.0 and 0.5 ng/ml, respectively. This method shows good precision: the within day relative standard deviation (RSD) for CPT-11 (1–10 000 ng/ml) is 5.17% (range 2.15–8.27%) and for SN-38 (0.5–400 ng/ml) is 4.33% (1.32–7.78%); the between-day RSDs for CPT-11 and SN-38, in the previously described ranges, are 6.82% (5.03–10.8%) and 4.94% (2.09–9.30%), respectively. Using this assay, plasma pharmacokinetics of CPT-11, SN-38 and its glucuronidated form, SN-38G, have been determined in one patient receiving 200 mg/m² of CPT-11 as a 90 min intravenous infusion. The peak plasma concentration of CPT-11 at the end of the infusion is 3800 ng/ml. Plasma decay is biphasic with a terminal half-life of 11.6 h. The volume of distribution at steady state (V_ss) is 203 l/m², and the total body clearance (Cl) is 14.8 l/h·m². The maximum concentrations of SN-38 and SN-38G reach 28.9 and 151 ng/ml, respectively.     

Endogenous inhibitors (GALFs) of 11beta-hydroxysteroid dehydrogenase isoforms 1 and 2: derivatives of adrenally produced corticosterone and cortisol

Two isoforms of 11β-HSD exist; 11β-HSD1 is bi-directional (the reductase usually being predominant) and 11β-HSD2 functions as a dehydrogenase, conferring kidney mineralocorticoid specificity. We have previously described endogenous substances in human urine, “glycyrrhetinic acid-like factors (GALFs)”, which like licorice, inhibit the bi-directional 11β-HSD1 enzyme as well as the dehydrogenase reaction of 11β-HSD2.

Many of the more potent GALFs are derived from two major families of adrenal steroids, corticosterone and cortisol. For example, 35-tetrahydro-corticosterone, its derivative, 35-tetrahydro-11β-hydroxy-progesterone (produced by 21-deoxygenation of corticosterone in intestinal flora); 35-tetrahydro-11β-hydroxy-testosterone (produced by side chain cleavage of cortisol); are potent inhibitors of 11β-HSD1 and 11β-HSD2-dehydrogenase, with IC₅₀'s in range 0.26–3.0 μM, whereas their 11-keto-35-tetrahydro-derivatives inhibit 11β-HSD1 reductase, with IC₅₀'s in range 0.7–0.8 μM (their 35β-derivatives being completely inactive).

Inhibitors of 11β-HSD2 increase local cortisol levels, permitting it to act as a mineralocorticoid in kidney. Inhibitors of 11β-HSD1 dehydrogenase/11β-HSD1 reductase serve to adjust the set point of local deactivation/reactivation of cortisol in vascular and other glucocorticoid target tissues, including adipose, vascular, adrenal tissue, and the eye. These adrenally derived 11-oxygenated C₂₁- and C₁₉-steroidal substances may serve as 11β-HSD1- or 11β-HSD2-GALFs. We conclude that adrenally derived products are likely regulators of local cortisol bioactivity in humans.     

Rapid degradation of neurotensin by stimulated rat mast cells

A RIA towards neurotensin (NT) using C-terminal- and N-terminal-specific antisera was used to study degradation of this tridecapeptide by isolated rat mast cells. Incubation of NT (10 μM) with peritoneal or pleural mast cells resulted in a rapid loss of NT immunoreactivity (iNT), as measured by C-terminal-directed antiserum, with little effect on N-terminal iNT. The rate of the reaction was faster with pleural cells (T_1/2, 30 s) than with peritoneal cells (T_1/2, 180 s) and was > 10-fold slower in the presence of metabolic poisons. The enzyme(s) involved is most likely released from the cells during secretion, as NT was degraded by media conditioned by compound 48/80-stimulated mast cells 40–60 times faster than by media from unstimulated cells. This degradation by conditioned media was concentration dependent, pH dependent, and temperature sensitive. HPLC analyses indicated a near stoichiometric conversion of NT to NT(1–12) (66%) and NT(1–11) (34%) after incubation for 10–30 s with conditioned media. By 30 min only NT(1–11) and NT(1–10) were present. Phenanthroline (1 mM), an inhibitor of carboxypeptidase, prevented the loss of C-terminal iNT and the generation of NT(1–12) and NT(1–11). While NT(1–12) was effective in releasing histamine from mast cells in vitro and increasing vascular permeability in vivo, NT(1–11) was not. These results suggest that carboxypeptidase-like enzyme(s) could modulate the level and form of NT-related peptides in various states involving activation of mast cells.     

Functional expression of cDNAs for bovine 11 beta-hydroxylase-aldosterone synthases, P450(11 beta)-2 and -3 and their chimeras.

Two molecular species of bovine P450(11β), P450(11β)-2 and P450(11β)-3 have been identified, in which the amino acid differences were found at the 6th, 36th and 82nd positions from the NH₂-termini of the mature proteins. They catalyzed the 11β-, 18- and 19-hydroxylation and aldosterone formation from 11-deoxycorticosterone, and the rate of production of 18-hydroxycorticosterone and aldosterone by P450(11β)-3 was greater than that by P450(11β)-2 [Morohashi et al., J. Biochem. 107 (1990) 635–640].

In this study, chimeric clones were constructed whose 6th, 36th and 82nd amino acid residues were exchanged with each other. Two original clones and six chimeric clones were expressed in COS-7 cells, and their steroidogenic activities studied. The ratio of aldosterone or 18-hydroxycorticosterone production to corticosterone production by one clone was compared with that of the other. The ratios for the four clones having Gly36 [P450(11β)-3 type] were 0.08–0.22, whereas those for the clones having Ser36 [P450(11β)-2 type] were 0.03–0.05, suggesting that the Gly36 structure is important for aldosterone production.     

Biotransformation of 14-deacetoxyl sinenxan A by Ginkgo cell suspension cultures and the cytotoxic activity evaluation

14-Deacetoxyl sinenxan A [2,5,10β-triacetoxy-4(20),11-taxadiene, 1] was converted to two new products, 10β-hydroxy-2,5-diacetoxy-4(20),11-taxadiene (2) and 10β-butyloxy-2,5-diacetoxy-4(20),11-taxadiene (3) both about in 20% yields by Ginkgo cell suspension cultures. Their structures were identified on the basis of their chemical and spectroscopic data. The three compounds (1–3) were preliminarily evaluated for their in vitro cytotoxic activities against two solid tumor cell lines and their drug-resistant counterparts (KB and KB/V, MCF-7 and MCF-7/ADR), and the decreased activities were observed in the case of the two products. The results suggested that biotransformation might be a valuable approach to diversifying natural products and provide some useful information on the study on the structure–activity relationships of the type of compounds.     

Crystallinity and polysaccharide chains of beta-glucan in white sorghum, SK(5912).

The polysaccharide chains and the crystallinity of β-glucan in a white sorghum variety, SK₅₉₁₂ were investigated using chemical and enzymic studies. Mild periodate oxidation and methylation, coupled to descending paper chromatography of products revealed the presence of unresolved non-carbohydrate moiety, 2, 4-and 2, 3-di-O-methyl -glucose residues (molar ratio; 18:3) and 2, 4, 6-and 2, 3, 6-tri-O-methyl -glucose residues (molar ratio; 1:14). Paper chromatography of the total acid hydrolysate also revealed a non-carbohydrate spot, identified as protein on the basis of positive Biuret and ninhydrin tests. The O-methyl -glucose residues suggest two polysaccharide chains designated X and Y. Chain X is formed through linking of β- -glucopyranosyl residues by (1→3) linkages with 85–86% (1→6) bonds at branch points and constitute about 6–7% of the β-glucan sample. Chain Y, which is 93–94% of the β-glucan polysaccharide chains, constitutes β- -glucopyranosyl residues in (1→4) linkages and 4–5% (1→6) bonds at branch points. Of the 18 branch points on the X-chains in a given β-glucan sample, about 15 are the Y chains interlinked to the X-chains through their (Y-chains) reducing ends. Both acid and enzyme hydrolyses of the β-glucan suggest two structural organizations, a crystalline and less crystalline granules, based on two first order kinetics. This was correlated by the progress curves obtained during hydrolysis with two purified isoforms of β-glucanases from the sorghum malt. The short and highly branched polysaccharide chains, and longer but less branched polysaccharide chains found in this β-glucan are reminiscent of the structures of amylopectin and amylose, respectively. The K_ms of 0.30–0.32 and 0.42–0.50 mg β-glucan/ml for the β-glucanase isoforms also lay credence to both the crystalline forms and the highly polymerised nature of the β-glucan in white sorghum.     

Differential processing of substance P and neurokinin A by plasma dipeptidyl(amino)peptidase IV, aminopeptidase M and angiotensin converting enzyme

In addition to plasma metabolism of substance P (SP) by angiotensin converting enzyme (ACE; EC 3.4.15.1) (<1.0 nmol/min/ml), the majority of SP hydrolysis by rat and human plasma was due to dipeptidyl(amino)peptidase IV (DAP IV; EC 3.4.14.5) (3.15–5.91 nmol/min/ml), which sequentially converted SP to SP(3–11) and SP(5–11). In turn, the SP(5–11) metabolite was rapidly hydrolyzed by rat and human plasma aminopeptidase M (AmM; EC 3.4.11.2) (24.2–25.5 nmol/min/ml). The K_m values of SP for DAP IV and of SP(5–11) for AmM ranged from 32.7 to 123 μM. In contrast, neurokinin A (NKA) was resistant to both ACE and DAP IV but was subject to N-terminal hydrolysis by AmM (3.76–10.8 nmol/min/ml; K_m=90.7 μM. These data demonstrate differential processing of SP and NKA by specific peptidases in rat and human plasma.     

Determination of 1-methoxy-2-propanol and its metabolite 1,2-propanediol in rat and mouse plasma by gas chromatography

A method utilizing capillary GC and flame ionization detection was developed for the simultaneous determination of 1-methoxy-2-propanol (propylene glycol monomethyl ether; PGME) and its metabolite 1,2-propanediol (propylene glycol; PG) in rat and mouse plasma. The calibration graphs for rat and mouse plasma were linear with correlation coefficients at>0.997 over the range 2–700 μg/ml. The limit of quantification was ca. 2 μg/ml (2 ng on-column) for both compounds in plasma of each species. The ranges of the precision and accuracy for PGME were 2.8–8.8% and 3.2–13%, respectively, and for PG were 11–26% and 10–25%, respectively. The recovery of PGME from rat and mouse plasma was ca. 73% and for PG it was ca. 65 and 31% from rat and mouse plasma, respectively. The method was used to study the oral absorption and metabolism of PGME in mice. PGME was readily absorbed and metabolized to PG following oral gavage administration at 90 mg/kg. The maximum concentrations of PGME and PG in plasma were attained at 20 and 30 min following dosing, respectively.     

Inhibition of human and rat 11beta-hydroxysteroid dehydrogenase type 1 by 18beta-glycyrrhetinic acid derivatives

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) plays an important role in regulating the cortisol availability to bind to corticosteroid receptors within specific tissue. Recent advances in understanding the molecular mechanisms of metabolic syndrome indicate that elevation of cortisol levels within specific tissues through the action of 11β-HSD1 could contribute to the pathogenesis of this disease. Therefore, selective inhibitors of 11β-HSD1 have been investigated as potential treatments for metabolic diseases, such as diabetes mellitus type 2 or obesity. Here we report the discovery and synthesis of some 18β-glycyrrhetinic acid (18β-GA) derivatives (2–5) and their inhibitory activities against rat hepatic11β-HSD1 and rat renal 11β-HSD2. Once the selectivity over the rat type 2 enzyme was established, these compounds’ ability to inhibit human 11β-HSD1 was also evaluated using both radioimmunoassay (RIA) and homogeneous time resolved fluorescence (HTRF) methods. The 11-modified 18β-GA derivatives 2 and 3 with apparent selectivity for rat 11β-HSD1 showed a high percentage inhibition for human microsomal 11β-HSD1 at 10 μM and exhibited IC₅₀ values of 400 and 1100 nM, respectively. The side chain modified 18β-GA derivatives 4 and 5, although showing selectivity for rat 11β-HSD1 inhibited human microsomal 11β-HSD1 with IC₅₀ values in the low micromolar range.     

Effects of intraplantar injections of nociceptin and its N-terminal fragments on nociceptive and desensitized responses induced by capsaicin in mice

Injection of capsaicin into the hindpaw has been employed as a model of chemogenic nociception in mice. Intraplantar injection of nociceptin (30–240 pmol) produced a significant and dose-dependent antinociceptive activity in the capsaicin test. The nociceptin N-terminal fragments, (1–11) and (1–13), were also active with a potency higher than nociceptin and comparable to nociceptin, respectively. Intraplantar injection of the nociceptin (1–7) fragment had no effect on capsaicin-induced nociception. Antinociception induced by nociceptin or nociceptin (1–13) was reversed significantly by intraplantar co-injection of [Nphe¹]nociceptin (1–13)NH₂, an orphan opioid receptor-like 1 (ORL1) receptor antagonist, whereas local injection of the antagonist did not interfere with the action of nociceptin (1–11). Nociceptin (1–11) was approximately 2.0-fold more potent than naturally occurring peptide nociceptin, and 10-fold more active than intraplantar morphine. Nociceptive licking/biting response to intraplantar injection of capsaicin was desensitized by repeated injections of capsaicin at the interval of 15 min. Desensitization induced by capsaicin was attenuated significantly by co-injection of nociceptin at much lower doses than antinociceptive ED₅₀ for nociceptin. Capsaicin desensitization was also decreased by co-injection of nociceptin (1–11) and (1–13) to a similar extent. The present results indicate that not only nociceptin but also the N-terminal fragment (1–13) possesses a local peripheral antinociceptive action, which may be mediated by peripheral ORL1 receptors. In addition, the difference of the effective doses suggests that the antinociceptive action and inhibition of capsaicin-induced desenitization by nociceptin, nociceptin (1–11) and (1–13), may involve distinct mechanisms at the level of the peripheral nerve terminal.     

CGRP antagonists and capsaicin on celiac ganglia partly prevent postoperative gastric ileus

The role of capsaicin-sensitive pathways and CGRP in postoperative gastric ileus was investigated. Abdominal surgery was performed under enflurane anesthesia, and 5 min later, the 20-min rate of gastric emptying was measured by the phenol red method in conscious rats. Surgery inhibited gastric emptying by 76–83% compared with rats receiving anesthesia alone. Capsaicin on the celiac/mesenteric ganglia (10–21 days before) reduced gastric ileus by 33 ± 8%, whereas perivagal capsaicin had no effect. The IV CGRP-induced inhibition of gastric emptying was completely reversed by the CGRP antagonist, CGRP(8–37) (30 μg, IV); CGRP(8–37) (15, 30, or 60 μg) or CGRP monoclonal antibody #4901 (2 mg protein) decreased the inhibition of gastric emptying by 11 ± 7%, 51 ± 13%, 47 ± 3%, and 45 ± 17%, respectively. These results indicate that CGRP and splanchnic capsaicin-sensitive afferents are involved in mediating part of the gastric ileus observed immediately after abdominal surgery.     

Therapy of Cushing''s syndrome with steroid biosynthesis inhibitors

Several substances with different inhibitory effects on adrenal steroid biosynthesis were investigated in patients with Cushing's syndrome. It has been shown that trilostane, a 3β-hydroxysteroid-dehydrogenase inhibitor, is not potent enough to block cortisol biosynthesis in patients with hypercortisolism. Aminoglutethimide inhibits side chain cleavage of cortisol synthesis, but it has been demonstrated that the blocking effect on cortisol secretion is not strong enough to normalize urinary cortisol excretion in patients with Cushing's disease. For metyrapone, an inhibitor of adrenal 11β-hydroxylase, promising results were reported for the treatment of Cushing's syndrome. However, the drug has several side effects and depending on the definition of the desired reduction of cortisol secretion a true remission was only found in a minority of patients. The antifungal drug ketoconazole in vitro predominantly blocks 17,20-desmolase (IC₅₀ 1 μM) and to a lesser extent 17-hydroxylase (IC₅₀ 10 μM) and 11β-hydroxylase (IC₅₀ 15–40 μM). Therefore, ketoconazole in vivo most potently suppresses androgen secretion and only to a lesser extent cortisol biosynthesis. Several therapeutic trials with ketoconazole treatment in patients with pituitary Cushing's disease showed various remission rates between 30 and 90%. In contrast, in almost all patients with benign, primary adrenal Cushing's syndrome cortisol levels were normalized. In patients with ectopic ACTH syndrome ketoconazole was effective in about 50% of all reported cases, while cortisol hypersecretion due to adrenocortical carcinoma was only rarely inhibited by ketoconazole. The main side effect of ketoconazole treatment was liver toxicity which occurred in 12% of all treated patients. In contrast to ketoconazole, the narcotic drug etomidate shows a strong inhibitory effect on 11β-hydroxylase (IC₅₀ 0.03–0.15 μM) but only a weak inhibition of 17,20 desmolase (IC₅₀ 380 μM). This correlates with in vivo studies where even low, non-hypnotic doses of etomidate induced a pronounced fall in serum cortisol levels in normals and in patients with Cushing's syndrome. However, its clinical use is limited by its mandatory intravenous application and its sedative effects. In conclusion, ketoconazole remains the only available steroid-inhibitory drug for a therapeutic trial in patients with Cushing's syndrome who cannot be treated definitively by surgery.     

Novel non-steroidal inhibitors of human 11beta-hydroxysteroid dehydrogenase type 1

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) regulates glucocorticoid action at the pre-receptor stage by converting cortisone to cortisol. 11β-HSD1 is selectively expressed in many tissues including the liver and adipose tissue where metabolic events are important. Metabolic syndrome relates to a number of metabolic abnormalities and currently has a prevalence of >20% in adult Americans. 11β-HSD1 inhibitors are being investigated by many major pharmaceutical companies for type 2 diabetes and other abnormalities associated with metabolic syndrome. In this area of intense interest a number of structural types of 11β-HSD1 inhibitor have been identified. It is important to have an array of structural types as the physicochemical properties of the compounds will determine tissue distribution, HPA effects, and ultimately clinical utility. Here we report the discovery and synthesis of three structurally different series of novel 11β-HSD1 inhibitors that inhibit human 11β-HSD1 in the low micromolar range. Docking studies with 1–3 into the crystal structure of human 11β-HSD1 reveal how the molecules may interact with the enzyme and cofactor and give further scope for structure based drug design in the optimisation of these series.     

Taxonomic characterization of two marine peritrichous ciliates, Epicarchesium corlissi n. sp. and Pseudovorticella jiangi n. sp. (Ciliophora: Peritrichia), from northern China

Two new marine peritrich ciliates, Epicarchesium corlissi n. sp. and Pseudovorticella jiangi n. sp., were discovered in mariculture waters on the coast of northern China near Qingdao. Their morphology, infraciliature and silverline system were investigated based on both living and silver-impregnated specimens. E. corlissi is characterized as follows: marine Epicarchesium with dichotomously branched stalk; zooids elongate, approximately 60–70×25–35 μm in vivo; peristomial collar double-folded; macronucleus J-shaped; single, small contractile vacuole ventrally positioned; more than 60 striations between peristome and aboral trochal band, 13–18 from aboral trochal band to scopula; abstomal end of row 1 of infundibular polykinety 3 terminating at same level as rows 2 and 3 of infundibular polykinety 3; rows 2 and 3 of infundibular polykinety 3 much longer than row 1 and converging adstomally with infundibular polykinety 1. The new species P. jiangi is diagnosed as follows: marine Pseudovorticella; zooid inverted bell-shaped, approximately 80×60 μm in vivo and with a broad, flat, thin peristomial collar that measures approximately 90 μm across; pellicle with transparent cortical vesicles; macronucleus J-shaped; number of silverlines between peristome and aboral trochal band 20–24, from aboral trochal band to scopula 9–11; abstomal end of row 1 of infundibular polykinety 3 diverges from the other two rows of this polykinety and ends alongside row 3 of infundibular polykinety 2.     

Pregnancy diagnosis based on the fecal progesterone concentration in beef and dairy heifers and beef cows

The present study was undertaken to examine whether pregnancy diagnosis was possible by measuring fecal progesterone concentrations in beef and dairy heifers and beef cows. Rectal fecal samples collected on days 18–24 after insemination or days 11–17 after embryo transfer were mixed with methanol and shaken for preparation of a fecal solution. After centrifugation, the supernatant was extracted with petroleum ether followed by an enzyme immunoassay for progesterone. All pregnant animals showed fecal progesterone concentrations greater than 50 ng/g of fecal material on days 18–24 after AI or estrus. In non-pregnant animals, however, the fecal progesterone concentrations ranged widely from 5 to 180 ng/g of fecal material. In non-pregnant cattle, the percentage of cattle with <50 ng progesterone/g of fecal material compared with the total number was 37–60% on days 18–20, whereas the percentages increased more than 70% to a maximum of 78.1% on day 23. When 50 ng/g was considered as the cut-off value, the sensitivity and specificity of positive pregnancy tests were less than 70% on days 21–24, and 100% for negative pregnancy tests on days 18–24. There were significant differences in the mean fecal progesterone concentrations between pregnant and non-pregnant cattle on days 19–24. These results suggest that feces can be utilized to substitute for plasma and milk to measure progesterone for the purpose of pregnancy diagnosis in heifers and cows.     

N-Acetylated endorphins in ovine anterior pituitary and neuro-intermediate lobe

We have used an antiserum for immunohistochemistry and RIA/RP-HPLC which recognizes all fragments of N-acetylated endorphin (NacEP). In the rat neurointermediate lobe (N-IL), in addition to the N-acetylated forms of immunoreactive-β-endorphin (ir-βEP) already reported, we have demonstrated NacβEP_1–17 as a minor component. In the sheep pituitary processing of βEP is markedly different. In the anterior pituitary (AP), staining was indistinguishable with βEP and NacEP antisera, in contrast with the rat where many fewer AP cells stained with the NacEP antiserum. Secondly, as in the rat, all N-IL cells stained with both antisera; on RP-HPLC, however, the major forms of NacEP in the sheep N-IL were NacβEP_1–17 (40%), NacβEP_1–27 (25%) and NacβEP_1–16 (20%), with NacβEP_1–31 (2%) as a minor component. A similar profile was seen on RP-HPLC of sheep AP. These data suggest that (1) patterns of processing in sheep AP are similar to those in N-IL, though the extent of acetylation is less and (2) in the sheep pituitary low molecular weight acetylated fragments predominate, in contrast with the rat.     

Characterization of C-terminal-truncated urinary metabolites of a recombinant hirudin in rats

Although it has been reported that hirudin was excreted in urine mainly as its nonmetabolized form in humans, dogs, and rabbits, no report has been published about the molecular nature of urinary metabolites in rats. We found that nonmetabolized hirudin could not be detected in rat urine after its i.v. administration and that urinary metabolites of recombinant hirudin CX-397 consisted of at least the following six C-terminal-truncated peptides: CX-397^1–49, CX-397^1–50, CX-397^1–51, CX-397^1–52, CX-397^1–54, and CX-397^1–55, in the ratio of roughly 11, 51, 3, 11, 19, and 5%, respectively. In conclusion, the urinary metabolism of recombinant hirudin in rats is different from that in humans, dogs, and rabbits, suggesting that the handling of hirudin in rat kidney is unique among them.     

Interaction of ovokinin(2-7) with vascular bradykinin 2 receptors

Intravenous administration of ovokinin(2–7), a cleavage peptide derived from ovalbumin, dose-dependently (0.1–5 mg/kg) lowered the mean arterial pressure (MAP) that was not accompanied by a significant change in the heart rate (HR) of urethane-anesthetized rats. The hypotensive effects of ovokinin(2–7) were five orders of magnitude lower compared to that of bradykinin and were largely prevented by pretreatment with the bradykinin B₂ receptor antagonist HOE140 (81.6±18.4%) and moderately affected by the B₁ receptor antagonist [des-Arg¹⁰]-HOE140 (26.3±15.5%). Intracellular Ca²⁺ levels, as measured by Fur 2-AM, were significantly elevated in cultured aorta smooth muscle cells by ovokinin(2–7). The increases were abolished by HOE140 and unaffected by [des-Arg¹⁰]-HOE140. The elevation of intracellular Ca²⁺ by ovokinin(2–7) was dependent on Ca²⁺ entry from extracellular space as it was reduced in a Ca²⁺-free solution. Pretreatment of the cells with the phospholipase C inhibitor U73122 (2 μM) eliminated the Ca²⁺ increase by the peptide. PA phosphohydrolase and phospholipase A₂ inhibitors significantly reduced the responses as well. Our results show that ovokinin(2–7) modulates cardiovascular activity by interacting with B₂ bradykinin receptors.     

Arginine 56 mutation in the beta subunit of nitrile hydratase: importance of hydrogen bonding to the non-heme iron center

Arginine 56 in the β subunit (βArg56) of the iron-containing nitrile hydratase (NHase), one of the strongly conserved residues within the NHase family, is known to form hydrogen bonds to the sulfinyl (–SO₂H) and sulfenyl (–SOH) groups of the post-translationally modified cysteine residues in the catalytic center. βArg56 was substituted by tyrosine, glutamate or lysine, respectively, and the respective mutant enzymes generated by reconstitution were characterized. The βR56K mutant complex exhibited about 1% of the enzymatic activity of native NHase, while the others were totally inactive. The kinetic analysis of the βR56K mutant complex exhibited a drastic decrease in turnover number and decreases in kinetic constants for substrate and inhibitors as compared to the native NHase. Changes in UV–visible absorption and light-induced Fourier transform infrared difference spectra suggest that βArg56 is involved in the positioning of the –SO₂H and –SOH groups of the modified Cys residues in the catalytic center so as to fine tune the electronic state of the iron center suitable for catalysis. Thus, βArg56 is essential for catalysis.