Novel Cytochrome P450 2C94 Functionally Metabolizes Diclofenac and Omeprazole in Dogs.

Cytochromes P450 (P450s or CYPs) are important drug-metabolizing enzymes. Because dogs are frequently used in drug metabolism studies, knowledge of dog CYP2C enzymes is essential because in humans these enzymes are abundant and play major roles in liver and intestine. The present study identified and characterized novel dog CYP2C94 along with previously identified dog CYP2C21 and CYP2C41. Dog CYP2C21, CYP2C41, and CYP2C94 cDNAs, respectively, contained open reading frames of 490, 489, and 496 amino acids and shared high-sequence identities (70%, 75%, and 58%) with human CYP2Cs. Dog CYP2C94 mRNA was preferentially expressed in liver, just as dog CYP2C21 and CYP2C41 mRNAs were. In dog liver, CYP2C21 mRNA was the most abundant, followed by CYP2C94 and CYP2C41 mRNAs. Moreover, the hepatic expressions of all three dog CYP2C mRNAs varied in four individual dogs, two of which did not express CYP2C41 mRNA. The three dog CYP2C genes had similar gene structures, and CYP2C94, although located on the same chromosome, was in a genomic region far from the gene cluster containing CYP2C21 and CYP2C41 Metabolic assays with recombinant proteins showed that dog CYP2C94, along with CYP2C21 and CYP2C41, efficiently catalyzed oxidations of diclofenac, warfarin, and/or omeprazole, indicating that dog CYP2C94 is a functional enzyme. Novel dog CYP2C94 is expressed abundantly in liver and encodes a functional enzyme that metabolizes human CYP2C substrates; it is, therefore, likely responsible for drug clearances in dogs. SIGNIFICANCE STATEMENT: Novel dog cytochrome P450 2C94 (CYP2C94) was identified and characterized along with dog CYP2C21 and CYP2C41. Dog CYP2C94, isolated from liver, had 58% sequence identity and a close phylogenetic relationship with its human homologs and was expressed in liver at the mRNA level. Dog CYP2C94 (and CYP2C21 and CYP2C41) catalyzed oxidations of diclofenac and omeprazole, human CYP2C9 and CYP2C19 substrates, respectively, but CYP2C41 also hydroxylated warfarin. CYP2C94 is therefore a functional drug-metabolizing enzyme likely responsible for drug clearances in dogs.

Intrathecally administered substance P activated the spinal defecation center and enhanced colorectal motility in anesthetized rats.

Noxious stimuli on the colorectum cause colorectal contractions through activation of descending monoaminergic pathways projecting from the supraspinal defecation center to the spinal defecation center. Since it is known that substance P is involved in the response to peripheral noxious stimuli in the spinal cord, we investigated the effects of intrathecally administered substance P at L6-S1 levels on colorectal motility in rats that were anesthetized with α-chloralose and ketamine. Intrathecally administered substance P enhanced colorectal motility, even after transection of the thoracic spinal cord at the T4 level. Severing the pelvic nerves, but not the colonic nerves, abolished substance P enhanced colorectal motility. In the spinal cord at L6-S1 levels, expression of mRNA coding neurokinin (NK) 1-3 receptors was detected by RT-PCR. Immunohistological experiments revealed that preganglionic neurons of the pelvic nerves express NK1 receptors, whereas expression of NK2 receptors was not found. In addition, substance P-containing fibers densely innervated around the preganglionic neurons expressing NK1 receptors. An intrathecally administered NK1 receptor antagonist (spantide) attenuated capsaicin-induced colorectal contractions. These results suggest that the colokinetic action of substance P is mediated by the NK1 receptor in the spinal defecation center. Our findings indicate that substance P may function as a neurotransmitter in the spinal defecation center.NEW & NOTEWORTHY We found that intrathecally administered substance P enhanced colorectal motility in anesthetized rats. Neurokinin (NK) 1 receptors, but not NK2 receptors, were detected in preganglionic neurons of the pelvic nerves. Blockade of NK1 receptors in the spinal cord attenuated the enhanced colorectal motility in response to intracolonic noxious stimuli. The findings indicate that substance P may function as a neurotransmitter in the spinal reflex pathway controlling defecation.

Molecular and Functional Characterization of N-Acetyltransferases in Common Marmosets and Pigs.

Arylamine N-acetyltransferases (NATs) are drug-metabolizing enzymes that are essential for the metabolism of endogenous substrates and xenobiotics. The molecular characteristics of NATs have been extensively investigated in humans but remain to be investigated in common marmosets and pigs, animal species that are often used in drug metabolism studies. In this study, marmoset NAT1 and pig NAT1 cDNAs were isolated from liver samples and were characterized by molecular analyses and drug-metabolism assays. These NAT genes were intronless and formed gene clusters with one other NAT gene in the genome, just as human NAT genes do. Marmoset NAT1 and pig NAT1 amino acid sequences showed high sequence identities (94% and 85%, respectively) to human NAT1. Phylogenetic analysis indicated that marmoset NAT1 and pig NAT1 were more closely clustered with human NATs than with rat or mouse NATs. Marmoset NAT1 and pig NAT1 mRNAs were expressed in all the tissue types analyzed, with the expression levels being highest in the small intestine. Metabolic assays using recombinant proteins found that marmoset NAT1 and pig NAT1 metabolized human NAT substrates p-aminobenzoic acid, 2-aminofluorene, sulfamethazine, and isoniazid. Marmoset NAT1 and pig NAT1 substantially acetylated p-aminobenzoic acid and 2-aminofluorene relevant human NAT1, but their activities were lower toward sulfamethazine and isoniazid than those of the relevant human NAT2. Therefore, marmoset and pig NATs are functional enzymes with molecular similarities to human NAT1, but their substrate specificities, while similar to human NAT1, differ somewhat from human NAT2. SIGNIFICANCE STATEMENT: Marmoset N-acetyltransferase NAT1 and pig NAT1 were identified and showed high sequence identities to human NAT1. These NAT mRNAs were expressed in various tissues. Marmoset and pig NAT1s acetylated typical human NAT substrates, although their substrate specificities differed somewhat from human NAT2. Marmoset NAT1 and pig NAT1 have similarities with human NAT1 in terms of molecular and enzymatic characteristics.

Cytochrome P450 2J Genes Are Expressed in Dogs, Cats, and Pigs, and Encode Functional Drug-Metabolizing Enzymes.

Cytochrome P450s (P450s) have been identified and analyzed in dogs and pigs, species that are often used in preclinical drug studies. Moreover, P450s are clinically important for drug therapy not only in humans, but also in species under veterinary care, including dogs and cats. In the present study, seven P450s homologous to human CYP2J2, namely, dog CYP2J2; cat CYP2J2; and pig CYP2J33, CYP2J35, CYP2J91, and CYP2J93, were newly identified and characterized, along with pig CYP2J34 previously identified. The cDNAs of these CYP2Js contain open reading frames of 502 amino acids, except for CYP2J35 (498 amino acids), and share high sequence identity (77%-80%) with human CYP2J2. Phylogenetic analysis revealed that dog and cat CYP2J2 were closely related, whereas pig CYP2Js formed a cluster. All seven CYP2J genes contain nine coding exons and are located in corresponding genomic regions, with the pig CYP2J genes forming a gene cluster. These CYP2J2 mRNAs were predominantly expressed in the small intestine with additional expression in the kidney and brain for dog CYP2J2 and pig CYP2J91 mRNAs, respectively. All seven CYP2Js metabolized human CYP2J2 substrates terfenadine, ebastine, and astemizole, indicating that they are functional enzymes. Dog CYP2J2 and pig CYP2J34 and CYP2J35 efficiently catalyzed ebastine primary hydroxylation and secondary carebastine formation at low substrate concentrations, just as human CYP2J2 does. Velocity-versus-substate plots exhibited sigmoidal relationships for dog CYP2J2, cat CYP2J2, and pig CYP2J33, indicating allosteric interactions. These results suggest that dog, cat, and pig CYP2Js have similar functional characteristics to human CYP2J2, with slight differences in ebastine and astemizole oxidations. SIGNIFICANCE STATEMENT: Dog CYP2J2; cat CYP2J2; and pig CYP2J33, CYP2J34, CYP2J35, CYP2J91, and CYP2J93, homologous to human CYP2J2, were identified and characterized by sequence, phylogenetic, and genomic structure analyses. Intestinal expression patterns of CYP2J mRNAs were characteristic in dogs, cats, and pigs. Dog, cat, and pig CYP2Js likely play roles as drug-metabolizing enzymes in the small intestine, similar to human CYP2J2.

A comprehensive analysis of six forms of cytochrome P450 2C (CYP2C) in pigs.

Pigs are an important species used in drug metabolism studies; however, the cytochromes P450 (P450s or CYPs) have not been fully investigated in pigs.In this study, pig CYP2C32, CYP2C33, CYP2C34, CYP2C36, CYP2C42, and CYP2C49 cDNAs were isolated and found to contain open reading frames of 490 or 494 amino acids that shared 64-82% sequence identity with human CYP2C8/9/18/19.Pig CYP2C genes formed a gene cluster in a genomic region that corresponded to that of the human CYP2C cluster; an additional gene cluster was formed by pig CYP2C33a and CYP2C33b distant from the first cluster but located in the same chromosome.Among the tissues analysed, these pig CYP2C mRNAs were preferentially expressed in liver, small intestine, and/or kidney; pig CYP2C49, CYP2C32, CYP2C34, and CYP2C33 mRNAs were the most abundant CYP2C mRNAs in liver, jejunum, ileum, and kidney, respectively.Metabolic assays showed that pig CYP2C proteins (heterologously expressed in Escherichia coli) metabolised typical human CYP2C substrates diclofenac, warfarin, and/or omeprazole.The results suggest that these pig CYP2Cs are functional enzymes able to metabolise human CYP2C substrates in liver and small intestine, just as human CYP2Cs do.

Hypertension alters the endothelial-dependent biphasic response of bradykinin in isolated Microminipig basilar artery.

Angiotensin (Ang) II is known to promote vascular disease and hypertension, partly through its effect on vascular endothelium. Bradykinin (BK) is an endothelium-dependent agonist that induces relaxation followed by contraction of the porcine basilar artery through release of NO and PGF2α, respectively. In this study, we evaluated the effect of Ang II-induced hypertension on basilar artery responsiveness to BK in the Microminipig (MMPig). Ang II (200ng/kg/min) or vehicle was infused into MMPigs for 14days using an osmotic mini-pump and blood pressure was monitored regularly. The responsiveness of subsequently isolated basilar arteries was then measured using a micro organ bath system. MMPig basilar artery endothelial cells were cultured and stimulated with Ang II or vehicle for 48h. Mean blood pressure was significantly (P<0.05; n=5) higher in Ang II-infused MMPigs than in vehicle-infused MMPigs. In vitro, BK-induced endothelium-dependent dilation of isolated basilar artery specimens was abolished and BK-induced contraction was significantly increased (Emax: 15.85±2.42% and 56.54±2.71% of 60mM KCl in control and Ang II group respectively at 10-7M concentration of BK; P<0.01; n=5) in Ang II-infused MMPigs. Ang II stimulation of the endothelial cells significantly decreased (54.15% at 24h; P<0.05; n=three independent experiment performed in triplicate) the amount of BK-elicited NO and increased (44.27% at 24h; P<0.05; n=three independent experiment performed in triplicate) the amount of BK-elicited PGF2α. These results suggest that the decrease of NO and increase of PGF2α production from endothelial cells are responsible for cerebrovascular dysfunction in hypertension, possibly causing cerebrovascular contraction and thus increasing the risk of brain infarction.

Enzymatic synthesis and RNA interference of nucleosides incorporating stable isotopes into a base moiety.

Thymidine phosphorylase was used to catalyze the conversion of thymidine (or methyluridine) and uracil incorporating stable isotopes to deoxyuridine (or uridine) with the uracil base incorporating the stable isotope. These base-exchange reactions proceeded with high conversion rates (75-96%), and the isolated yields were also good (64-87%). The masses of all synthetic compounds incorporating stable isotopes were identical to the theoretical molecular weights via EIMS. (13)C NMR spectra showed spin-spin coupling between (13)C and (15)N in the synthetic compounds, and the signals were split, further proving incorporation of the isotopes into the compounds. The RNA interference effects of this siRNA with uridine incorporating stable isotopes were also investigated. A 25mer siRNA had a strong knockdown effect on the MARCKS protein. The insertion position and number of uridine moieties incorporating stable isotopes introduced into the siRNA had no influence on the silencing of the target protein. This incorporation of stable isotopes into RNA and DNA has the potential to function as a chemically benign tracer in cells.

Habu snakes (Protobothrops flavoviridis) show variation in thoracic aortic vasoreactivity between adjacent Japanese islands.

Habu snakes (Protobothrops flavoviridis) are pit vipers found in the geographically adjacent but ecologically divergent islands of Tokunoshima and Amami-Oshima in southwestern Japan. Abiotic factors can cause variation in animal populations between the two islands, and Habu snakes may show such intraspecific physiological variation. We therefore evaluated the vasoreactivity in aortas isolated from the Habu of both islands. Tokunoshima Habu showed significantly greater contractile responses to angiotensin (Ang) II, acetylcholine (ACh) and noradrenaline, and significantly higher affinities (pEC50) for Ang II and ACh, than Amami-Oshima Habu. ACh caused contractions in aortas from both populations, a finding previously unreported in snakes. Our findings indicate that vasoreactivity may differ between Tokunoshima and Amami-Oshima Habu.

Involvement of beta3-adrenergic receptors in relaxation mediated by nitric oxide in chicken basilar artery.

The response of basilar arteries to noradrenaline varies among many animal species, but remains little studied in poultry. Accordingly, we aimed to characterize the adrenergic receptor (AR) subtypes that modulate vascular response in basilar arteries in the chicken, with isometric recording of arterial ring tension using an organ bath. We demonstrated the presence of both alpha and beta (α and ß) receptor subtypes through evaluating the response to noradrenaline, with and without a range of ß-AR and α-AR antagonists. The concentration-dependent relaxations then induced by a range of ß-AR agonists indicated a potency ranking of isoproterenol > noradrenaline > adrenaline > procaterol. We then investigated the effects of ß-AR antagonists that attenuate the effect of isoproterenol (propranolol for ß1,2,3-ARs, atenolol for ß1-ARs, butoxamine for ß2-ARs, and SR 59230A for ß3-ARs), with Schild regression analysis, ascertaining multiple ß-AR subtypes, with neither the ß1-AR nor the ß2-AR as the dominant subtype. SR 59230A was the only antagonist to yield a pA2 value (7.52) close to the reported equivalent for the relevant receptor subtype. Furthermore, treatment with SR 58611 (a ß3-AR agonist) induced relaxation, which was inhibited (P < 0.01) by L-NNA and SR 59230A. Additionally, treating basilar arterial strips (containing endothelium) with SR 58611 induced nitric oxide (NO) production, which was inhibited (P < 0.01) by L-NNA and SR 59230A. Based on this first characterization of AR subtypes in chicken basilar arteries (to our knowledge), we suggest that α- and ß-ARs are involved in contraction and relaxation, and that ß3-ARs, especially those on the endothelium, may play an important role in vasodilation via NO release.

Relationship between Regional Distribution of Centenarians and Drinking Water Hardness in the Amami Islands, Kagoshima Prefecture, Japan.

People who drink naturally hardened water may experience longevity-enhancing effects. In this study, we investigated water hardness and longevity from both geological and epidemiological perspectives in Japan's Amami islands, where drinking water is drawn from coralline or non-coralline bedrock. We investigated drinking water hardness, limestone bedrock occupancy, and the centenarian rate (number per 10,000 population) by municipality across four adjacent islands (Amami-Oshima (non-coralline), Tokunoshima, Okinoerabu, and Yoron (predominantly coralline)). Limestone was strongly correlated with water hardness (r = 0.99; p < 0.01), occupying more than 80% of the bedrock where the water was the hardest (Tokunoshima's Isen municipality: 86.5%; Yoron: 82.9%) and being scarcely detectable in Amami-Oshima (0.0 to 0.2%), where the water was the least hard. The centenarian rate was also strongly correlated with water hardness (r = 0.84, p < 0.01), with the highest figures in Yoron (29.7) and Isen (29.2), and the lowest in Amami-Oshima (0.0 to 12.2). Therefore, we hypothesize a potentially beneficial effect of hard water on longevity when that water is drawn from coralline limestone. Water hardness is determined by the water content of calcium and magnesium and may plausibly influence life expectancy through a preventative effect against cardiovascular disease. Our findings are of interest to current debates about future global access to drinking water and its quality.

Characterization of Vasoreactivity in a Semi-Arboreal Snake, the Tokara Habu (Protobothrops tokarensis).

Vasoreactivity is relatively well documented in terrestrial snakes but has previously been investigated in only one semi-arboreal snake species. Consequently, the extent to which vasoreactivity is common across snake taxa or varies by habitat is unclear. The Tokara habu (Protobothrops tokarensis) is a semi-arboreal snake endemic to only two small adjacent Japanese islands, and hence a useful species for further investigation of vasoreactivity. We evaluated responses to known vasoactive substances in thoracic aortas isolated from Tokara habu. Under resting tension, noradrenaline and angiotensin II induced concentration-dependent contraction, but acetylcholine, serotonin (5-hydroxytriptamine; 5-HT), and isoproterenol induced relaxation followed by contraction. Histamine and rattlesnake bradykinin had no effect. Experiments with receptor-specific antagonists suggest that M1 and M3 receptors are involved in the acetylcholine-induced response; 5-HT1, 5-HT2, and 5-HT7 receptors in the serotonin-induced response; and ß1 and ß2 adrenoceptors in isoproterenol-induced relaxation. This is the first report on such response patterns in snakes (including serotonin- and isoproterenol-induced relaxation). Nitric oxide may be involved in acetylcholine-induced relaxation but not in the responses to serotonin or isoproterenol. In contrast to the uniform vasoreactivity observed in terrestrial snakes, the vasoreactivity of semi-arboreal snakes may be governed by diverse regulatory mechanisms.

Reduced Nitric Oxide Synthase Involvement in Aigamo Duck Basilar Arterial Relaxation.

The basilar arterial endothelium mediates blood vessel relaxation partly through the release of nitric oxide (NO). Apoptosis of cerebrovascular endothelial cells is linked to a high mortality rate in chickens infected with the highly pathogenic avian influenza virus, but interestingly, ducks exhibit a greater resistance to this virus. In this study, we examined the responsiveness of duck basilar arteries (BAs) to various vasoactive substances, including 5-hydroxytryptamine (5-HT), histamine (His), angiotensin (Ang) II, noradrenaline (NA), acetylcholine (ACh), and avian bradykinin ornithokinin (OK), aiming to characterize the receptor subtypes involved and the role of endothelial NO in vitro. Our findings suggest that arterial contraction is mediated with 5-HT1 and H1 receptors, while relaxation is induced with ß3-adrenergic and M3 receptors. Additionally, OK elicited a biphasic response in duck BAs, and Ang II had no effect. Endothelial NO appears to be crucial in relaxation mediated with M3 and OK receptors but not ß3-adrenergic receptors in the duck BA. The reduced endothelial NO involvement in the receptor-mediated relaxation response in duck BAs represents a clear difference from the corresponding response reported in chicken BAs. This physiological difference may explain the differences in lethality between ducks and chickens when vascular endothelial cells are infected with the virus.

MARCKS dephosphorylation is involved in bradykinin-induced neurite outgrowth in neuroblastoma SH-SY5Y cells.

Bradykinin (BK) plays a major role in producing peripheral sensitization in response to peripheral inflammation and in pain transmission in the central nerve system (CNS). Because BK activates protein kinase C (PKC) through phospholipase C (PLC)-ß and myristoylated alanine-rich C kinase substrate (MARCKS) has been found to be a substrate of PKC, we explored the possibility that BK could induce MARCKS phosphorylation and regulate its function. BK stimulation induced transient MARCKS phosphorylation on Ser159 with a peak at 1 min in human neuroblastoma SH-SY5Y cells. By contrast, PKC activation by the phorbol ester phorbol 12,13-dibutyrate (PDBu) elicited MARCKS phosphorylation which lasted more than 10 min. Western blotting analyses and glutathione S-transferase (GST) pull-down analyses showed that the phosphorylation by BK was the result of activation of the PKC-dependent RhoA/Rho-associated coiled-coil kinase (ROCK) pathway. Protein phosphatase (PP) 2A inhibitors calyculin A and fostriecin inhibited the dephosphorylation of MARCKS after BK-induced phosphorylation. Moreover, immunoprecipitation analyses showed that PP2A interacts with MARCKS. These results indicated that PP2A is the dominant PP of MARCKS after BK stimulation. We established SH-SY5Y cell lines expressing wild-type MARCKS and unphosphorylatable MARCKS, and cell morphology changes after cell stimulation were studied. PDBu induced lamellipodia formation on the neuroblastoma cell line SH-SY5Y and the morphology was sustained, whereas BK induced neurite outgrowth of the cells via lamellipodia-like actin accumulation that depended on transient MARCKS phosphorylation. Thus these findings show a novel BK signal cascade-that is, BK promotes neurite outgrowth through transient MARCKS phosphorylation involving the PKC-dependent RhoA/ROCK pathway and PP2A in a neuroblastoma cell line.

Direct evidence of nitric oxide production induced by lactoferrin and its enhancement by magnesium ions in cultured endothelial cells.

Bovine lactoferrin (BLF) reportedly lowers blood pressure and induces vasorelaxation, but its effect on nitric oxide (NO) production has not been established. Accordingly, we aimed to determine whether BLF induces NO production in bovine aortic endothelial cells, and the effects of extracellular free magnesium (Mg) ion concentrations on this NO production. BLF induced NO production time-dependently. NO production was markedly inhibited by the NO synthase inhibitor, NG-nitro-L-arginine methyl ester, in an effect abolished by L-arginine, but not D-arginine. NO production was suppressed at low concentrations, and enhanced at high concentrations, of Mg ions in culture medium. These results suggest that BLF has an important role in hypotensive effects. Mg ions may affect BLF-induced NO production.

Measurement of canine blood microparticles by flow cytometry: effect of anticoagulants and staining reagents.

Microparticles (MPs) are released from budding plasma membranes into body fluids. The use of flow cytometry for the measurement of MP in canines has not been standardized. In this fundamental study, we compared the effect of anticoagulant agents, such as acid-citrate-dextrose (ACD) and heparin on the measurement of canine MPs in platelet-free plasma (PFP) using flow cytometry. In addition, we used annexin V, carboxyfluorescein succinimidyl ester (CFSE), or calcein tetraacetoxymethyl ester (calcein-AM), and explored the characteristics of the staining reagents in MP detection using flow cytometry. We were able to measure canine MPs in PFP prepared from ACD-anticoagulated blood using flow cytometry, in which the highest positive rate for fluorescent staining was observed when CFSE was used.

α-MSH-induced activation of spinal MC1R but not MC4R enhances colorectal motility in anaesthetised rats.

The central nervous system is involved in regulation of defaecation. It is generally considered that supraspinal regions control the spinal defaecation centre. However, signal transmission from supraspinal regions to the spinal defaecation centre is still unclear. In this study, we investigated the regulatory role of an anorexigenic neuropeptide, α-MSH, in the spinal defaecation centre in rats. Intrathecal administration of α-MSH to the L6-S1 spinal cord enhanced colorectal motility. The prokinetic effect of α-MSH was abolished by severing the pelvic nerves. In contrast, severing the colonic nerves or thoracic cord transection at the T4 level had no impact on the effect of α-MSH. RT-PCR analysis revealed MC1R mRNA and MC4R mRNA expression in the L6-S1 spinal cord. Intrathecally administered MC1R agonists, BMS470539 and SHU9119, mimicked the α-MSH effect, but a MC4R agonist, THIQ, had no effect. These results demonstrate that α-MSH binds to MC1R in the spinal defaecation centre and activates pelvic nerves, leading to enhancement of colorectal motility. This is, to our knowledge, the first report showing the functional role of α-MSH in the spinal cord. In conclusion, our findings suggest that α-MSH is a candidate for a neurotransmitter from supraspinal regions to the spinal defaecation centre.

Vasomotor effects of noradrenaline, 5-hydroxytryptamine, angiotensin II, bradykinin, histamine, and acetylcholine on the bat (Rhinolophus ferrumequinum) basilar artery.

The responsiveness of the basilar artery to intrinsic vasoactive substances is species-specific and can be a unique characteristic. We investigated the responsiveness of the bat (Rhinolophus ferrumequinum) basilar artery to noradrenaline (NA), 5-hydroxytryptamine (5-HT), angiotensin (Ang) II, bradykinin (BK), histamine (His), and acetylcholine (ACh). NA, 5-HT, Ang II, and BK induced contraction, whereas His and ACh induced relaxation, in a concentration-dependent manner. The NA cumulative concentration-response curve was shifted to the right in parallel with phentolamine (an α-antagonist). However, propranolol, a ß-antagonist, had no significant effect. The 5-HT curve was shifted to the right in parallel by ketanserin (a 5-HT2 antagonist) and methiothepin (a 5-HT1 and 5-HT2 antagonist). Losartan (an AT1 antagonist) shifted the Ang II curve to the right, whereas PD123319 (an AT2 antagonist) had no significant effect. L-NA, indomethacin, and des-Arg9-[Leu8]-BK (a B1 antagonist) did not significantly affect BK-induced contractions. HOE140 (a B2 antagonist) shifted the BK concentration-response curve to the right. The His curve was shifted to the right weakly by diphenhydramine (an H1 antagonist) and strongly by cimetidine (a H2 antagonist). ACh-induced relaxation was significantly inhibited by L-NA, atropine, and pFHHSiD (a muscarinic M3 antagonist), whereas pirenzepine and methoctramine (muscarinic M1 and M2 antagonists, respectively) showed no significant effects. At a resting vascular tone, L-NA-induced contraction and indomethacin induced relaxation. These results suggest that α-adrenergic, 5-HT1, 5-HT2, AT1, and B2 receptors might be important in arterial contraction, whereas M3 and H2 (>H1) receptors might modify these contractions, inducing relaxation.

Modulation of rabbit platelet aggregation and calcium mobilization by platelet cholesterol content.

Hypercholesterolemia is one of the major contributing factors in atherosclerosis and the development of cardiovascular disease. Platelets from hypercholesterolemic rabbit have an increased cholesterol content and a hypersensitivity to endogenous aggregating agonists. Although rabbit has been widely used in studies of hypercholesterolemia, the precise role of platelet cholesterol in rabbit platelet activation has not been studied. In the present study, to determine the direct role of cholesterol on rabbit platelet activation, we examined the effect of in vitro modulation of cholesterol content on platelet activation. Cholesterol-depleted rabbit platelets by the treatment with methyl-beta-cyclodextrin showed decreased platelet aggregation by physiological agonists such as thrombin, adenosine diphosphate, and collagen. The inhibition of thrombin-induced aggregation in cholesterol-depleted platelets was restored by cholesterol repletion in platelets. The cholesterol depletion also inhibited Ca(2+) mobilization, which plays a pivotal role in the platelet activation induced by physiological agonists. We showed that the Ca(2+) influx pathway is strongly suppressed by cholesterol depletion more than Ca(2+) release from intracellular Ca(2+) stores in platelets stimulated with thrombin. Furthermore, platelet aggregation induced by PMA, a potent protein kinase C activator, was also depressed by cholesterol depletion. On the other hand, cholesterol enrichment in platelets augmented thrombin-induced aggregation and Ca(2+) mobilization. These findings suggest that cholesterol plays a critical role in regulating rabbit platelet activation, and provides fundamental information regarding hypercholesterolemia-mediated effects on cells in the rabbit model.

Vasomotor effects of 5-hydroxytryptamine, histamine, angiotensin II, acetylcholine, noradrenaline, and bradykinin on the cerebral artery of bottlenose dolphin (Tursiops truncatus).

From an evolutionary aspect, dolphins share a very close phylogenetic relationship with pigs. Previously, we characterized porcine cerebral artery responsiveness to intrinsic vasoactive substances. Therefore, here, we investigated dolphin (Tursiops truncatus) cerebral artery responsiveness to 5-hydroxytryptamine (5-HT), histamine (His), angiotensin (Ang) II, acetylcholine (ACh), noradrenaline (NA), and bradykinin (BK) to characterize their related receptor subtypes. We also compared dolphin cerebral artery responsiveness with porcine cerebral artery responsiveness. We found that 5-HT and His induced concentration-dependent contraction of the dolphin cerebral artery. Ketanserin (a 5-HT2 antagonist) and methiothepin (a 5-HT1 and 5-HT2 antagonist) shifted the concentration-response curve for 5-HT to the right. Although diphenhydramine (an H1 antagonist) shifted the concentration-response curve for His to the right, cimetidine (an H2 antagonist) had no such effect. Ang II and ACh did not produce any vasomotor actions. NA induced concentration-dependent relaxation. Propranolol (a ß antagonist) shifted the concentration-response curve for NA to the right, whereas phentolamine (an α antagonist) had no significant effect. BK induced relaxation followed by contraction in pre-contracted arteries with intact endothelium. HOE140 (a B2 antagonist) shifted the concentration-response curve for BK to the right, whereas des-Arg9-[Leu8]-BK (a B1 antagonist) had no significant effect. These results suggest that 5-HT1, 5-HT2, and H1 receptor subtypes are important in arterial contraction and that ß and B2 receptor subtypes modify these contractions to relaxations. The responsiveness of the dolphin cerebral artery is very similar to that of porcine cerebral artery, supporting their evolutionary linkage.

MARCKS regulates lamellipodia formation induced by IGF-I via association with PIP2 and beta-actin at membrane microdomains.

Myristoylated alanine-rich C kinase substrate (MARCKS) is considered to participate in formation of F-actin-based lamellipodia, which represents the first stage of neurite formation. However, the mechanism of how MARCKS is involved in lamellipodia formation is not precisely unknown. Using SH-SY5Y cells, we demonstrated here that MARCKS was translocated from cytosol to detergent-resistant membrane microdomains, known as lipid rafts, within 30 min after insulin-like growth factor-I (IGF-I) stimulation, which was accompanied by MARCKS dephosphorylation, beta-actin accumulation in lipid rafts, and lamellipodia formation. The protein kinase C inhibitor, Ro-31-8220, and Rho-kinase inhibitors, HA1077 and Y27632, themselves decreased basal phosphorylation levels of MARCKS and coincidently elicited translocation of MARCKS to lipid rafts. On the other hand, the phosphoinositide 3-kinase inhibitor, LY294002, abolished IGF-I-induced dephosphorylation, translocation of MARCKS to lipid rafts, and lamellipodia formation. Treatment of cells with neomycin, a PIP2-masking reagent, attenuated the translocation of MARCKS to lipid rafts and the lamellipodia formation induced by IGF-I, although dephosphorylation of MARCKS was not affected. Immunocytochemical and immunoprecipitation analysis indicated that IGF-I stimulation induced the translocation of MARCKS to lipid rafts in the edge of lamellipodia and formation of the complex with PIP2. Moreover, we demonstrated that knockdown of endogenous MARCKS resulted in significant attenuation of IGF-I-induced beta-actin accumulation in the lipid rafts and lamellipodia formation. These results suggest a novel role for MARCKS in lamellipodia formation induced by IGF-I via the translocation of MARCKS, association with PIP2, and accumulation of beta-actin in the membrane microdomains.