Biomedical subjects

V G Wilson

Publications and source records attributed to V G Wilson.

At least 37 records · Page 2Linked to original sources

PubMed1999

A study of NPY-mediated contractions of the porcine isolated ear artery.

Enhanced contractions of the porcine isolated ear artery by the alpha2-adrenoceptor agonist UK14304 are uncovered by pharmacological manipulation. As both neuropeptide Y (NPY) receptors and alpha2-adrenoceptors are negatively-coupled to adenylyl cyclase in this tissue, we determined whether NPY is also able to produce an enhanced contraction in the same tissue, under the same conditions. NPY (0.1 microM) produced a small contraction of porcine isolated ear arteries which was 5.1+/-0.8% of the response to 60 mM KCl (n = 14). An enhanced NPY response was uncovered if the tissue was pre-contracted with 0.1 microM U46619, and relaxed back to baseline with 1-2 microM forskolin before the addition of NPY (49.8+/-5.3%, n = 14). Forskolin (1 microM) stimulated cyclic AMP accumulation in porcine ear artery segments in the presence of 0.1 microM U46619 and 1 mM isobutylmethylxanthine (IBMX), NPY (0.1 microM) inhibited this response by 40%, but had no effect on basal levels of cyclic AMP. An enhanced response to 0.1 microM NPY was also obtained after pre-contraction with 0.1 microM U46619 and relaxation with either SNP (28.9+/-5.7%, n = 14), or dibutyryl cyclic AMP (21.2+/-4.6%, n = 14). This indicates that at least part of the enhanced response to NPY is independent of the agonist's ability to inhibit adenylyl cyclase. In conclusion, an enhanced contraction to NPY in the porcine isolated ear artery can be obtained by prior pharmacological manipulation. The enhanced responses are mediated through adenylyl cyclase-dependent and independent pathways similar to those reported for alpha2-adrenoceptors in this preparation.

Adenylyl Cyclases↗

PubMed1999

Molecular and pharmacological evidence for MT1 melatonin receptor subtype in the tail artery of juvenile Wistar rats.

1. In this study reverse transcriptase-polymerase chain reaction (RT-PCR) has been used to identify mt1 and MT2 receptor mRNA expression in the rat tail artery. The contributions of both receptors to the functional response to melatonin were examined with the putative selective MT2 receptor antagonists, 4-phenyl-2-propionamidotetraline (4-P-PDOT) and 2-benzyl-N-pentanoyltryptamine. In addition, the action of melatonin on the second messenger cyclic AMP was investigated. 2. Using RT-PCR, mt1 receptor mRNA was detected in the tail artery from seven rats. In contrast MT2 receptor mRNA was not detected even after nested PCR. 3. At low concentrations of the MT2 selective ligands, neither 10 nM 4-P-PDOT (pEC50=8.70+/-0.31 (control) vs 8.73+/-0.16, n=6) nor 60 nM 2-benzyl-NV-pentanoyltryptamine (pEC50= 8.53+/-0.20 (control) vs 8.83+/-0.38, n = 6) significantly altered the potency of melatonin in the rat tail artery. 4. At concentrations non-selective for mt1 and MT2 receptors. 4-P-PDOT (3 microM) and 2-benzyl-N-pentanoyltryptamine (5 microM) caused a significant rightward displacement of the vasoconstrictor effect of melatonin. In the case of 4-P-PDOT, the estimated pKB (6.17+/-0.16, n=8) is similar to the binding affinity for mt1 receptor. 5. Pre-incubation with 1 microM melatonin did not affect the conversion of [3H]-adenine to [3H]-cyclic AMP under basal condition (0.95+/-0.19% conversion (control) vs 0.92+/-0.19%, n=4) or following exposure to 30 microM forskolin (5.20+/-1.30% conversion (control) vs 5.35+/-0.90%, n=4). 6. Based on the above findings, we conclude that melatonin receptor on the tail artery belongs to the MT1 receptor subtype, and that this receptor is probably independent of the adenylyl cyclase pathway.

Animals↗

PubMed1999

The Pseudomonas aeruginosa quorum-sensing signal molecule, N-(3-oxododecanoyl)-L-homoserine lactone, inhibits porcine arterial smooth muscle contraction.

The Pseudomonas aeruginosa quorum sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) has been shown to suppress cytokine production in macrophages. We have examined the effect of OdDHL and related compounds on constrictor tone of porcine blood vessels. OdDHL (1-30 microM) caused a concentration-dependent inhibition of U46619-induced contractions of the coronary artery through a largely endothelium-independent mechanism, but was markedly less effective in the pulmonary artery. Quantitively similar effects to those produced by OdDHL were observed with N-(3-oxododecanoyl)-L-homocysteine thiolactone, a thiolactone derivative, while N-3-oxododecanamide, a lactone-free acyl analogue, possessed 1/3rd the potency as a vasorelaxant. Neither N-butanoyl-L-homoserine lactone nor L-homoserine lactone (up to 30 microM) were active. Our findings indicate that OdDHL inhibits vasoconstrictor tone of both pulmonary and coronary blood vessels from the pig. The vasorelaxant action of OdDHL appears to be primarily determined by the N-acyl chain length, with a minor contribution by the homoserine lactone moiety.

4-Butyrolactone↗

PubMed1999

alpha(2)-adrenoceptor and NPY receptor-mediated contractions of porcine isolated blood vessels: evidence for involvement of the vascular endothelium.

1. Enhanced contractions to the alpha(2)-adrenoceptor agonist UK14304 and neuropeptide Y (NPY) in the porcine ear artery can be uncovered by pharmacological manipulation. The aim of this study was to determine whether similar pharmacological manipulation can uncover enhanced contractions in the porcine splenic artery, and to determine whether the endothelium modulates these responses. 2. UK14304 (0.3 microM) and NPY (0.1 microM) produced small contractions of the porcine splenic artery. After pre-contraction of the tissue with U46619, followed by relaxation with forskolin, the responses to both UK14304 and NPY were enhanced. Enhanced contractions to both UK14304 and NPY were also obtained after relaxation with SNP. These results demonstrate that, as in the porcine ear artery, alpha(2)-adrenoceptors and NPY receptors are able to produce enhanced contractile responses through both adenylyl cyclase-dependent and -independent signal transduction pathways. 3. Removal of the endothelium had no significant effect on responses to UK14304 either alone or in the presence of U46619 and forskolin in the porcine splenic artery. On the other hand, responses to UK14304 after relaxation with SNP were reduced after endothelium-denudation in both the porcine splenic artery and ear artery. Similar results were obtained with NPY in the porcine ear artery. 4. In conclusion, enhanced contractile responses to UK14304 and NPY in the porcine splenic artery can be uncovered using methods similar to those employed in the porcine ear artery. Under certain conditions the responses to both agents are modulated by the endothelium. These data highlight further the similarities in the signal transduction pathways used by both alpha(2)-adrenoceptors and NPY receptors to induce vasoconstriction.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1998

'Seeing through a glass darkly': casting light on imidazoline 'I' sites.

Although imidazoline sites have been the subject of research for several years, there is still controversy about their structure, diversity and physiology. The I1 site is thought to exist principally as a binding site and is widely purported to play a role in controlling systemic blood pressure, although this is still unclear. The majority of I2 sites are widely accepted as being allosteric sites on monoamine oxidase; however, even with selective ligands, their exact function remains to be determined. A putative I3 site modulates insulin secretion and could represent the first functional site to be pharmacologically defined with selective agonists and antagonists. The structure and relevance of the proposed endogenous ligand 'clonidine-displacing substance' remains elusive. A potential candidate for this substance is agmatine; however, although it is capable of displacing bound clonidine from imidazoline sites, it lacks the functionality ascribed to the clonidine-displacing substance. In this review, Richard M. Eglen and colleagues assess our knowledge of imidazoline sites in the light of recent data.

Adrenergic alpha-Agonists↗

PubMed1998

Selective potentiation by ouabain of naloxone-induced withdrawal contractions of isolated guinea-pig ileum following acute exposure to morphine.

1. Ouabain, an inhibitor of Na+/K+ ATPase induces the release of acetylcholine from central and myenteric cholinergic neurones principally due to partial depolarization of the cell membrane. The effect of ouabain has been examined on neurogenic contractions in the guinea-pig ileum arising from either electrical field stimulation or from naloxone in morphine-exposed preparations. 2. Guinea-pig isolated ileum preparations were stimulated transmurally (0.1 Hz, 0.3 ms, 200 mA) to elicit contractions of the myenteric plexus-longitudinal smooth muscle. 3. Incubation with morphine (0.3 microM, 60 min) was followed by naloxone (1 microM) which produced withdrawal contractions in 16/26 preparations (median of 10.7 [2.2-40.0]% of a maximal contracture to KCl (60 mM)). 4. In parallel experiments, ouabain (1 microM) was added to the tissue before exposure to morphine (0.3 microM, 60 min). Naloxone (1 microM) subsequently displayed a withdrawal contraction in all 26/26 tissues (57.9 [30.5-151.7]% of a maximal contracture to KCl (60 mM). 5. Ouabain neither affected the concentration-dependent contractions of guinea-pig ileum produced by carbachol nor the inhibition of electrically-evoked contraction produced by morphine (0.3 microM). 6. The muscarinic antagonist atropine (0.1 microM) antagonized control naloxone withdrawal responses. The atropine resistant component, evident in ouabain-treated tissues, was blocked by SR140333((S)1-[2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenyla cetyl)piperidin-3-yl]ethyl]-4-phenyl-1-azoniabicyclo[2.2. 2]-octane, chloride), a substance P antagonist. 7. Clonidine (alpha2-adrenoceptor agonist) inhibited electrically-evoked contractions. Exposure to the alpha2-adrenoceptor antagonist RX811059 (2-(2-ethoxy-1,4-benzodioxan-2-yl)-2-imidazoline), resulted in a contracture which was not significantly enhanced by ouabain (1 microM). 8. Ouabain selectively potentiates the naloxone-induced withdrawal contraction following acute exposure to morphine the major components of which are mediated by both acetylcholine and substance P.

Adrenergic alpha-Agonists↗

PubMed1998

Alpha2-adrenoceptor-mediated contractions of the porcine isolated ear artery: evidence for a cyclic AMP-dependent and a cyclic AMP-independent mechanism.

1. The aim of this study was to determine the conditions under which the alpha2-adrenoceptor agonist UK14304 produces vasoconstriction in the porcine isolated ear artery. 2. UK14304 (0.3 microM) produced a small contraction of porcine isolated ear arteries which was 7.8+/-3.3% of the response to 60 mM KCl. Similar sized contractions were obtained after precontraction with either 30 nM angiotensin II, or 0.1 microM U46619 (8.2+/-1.8% and 10.2+/-2.6% of 60 mM KCl response, respectively). However, an enhanced alpha2-adrenoceptor response was uncovered if the tissue was precontracted with U46619, and relaxed back to baseline with 1-2 microM forskolin before the addition of UK14304 (46.9+/-9.6% of 60 mM KCl response). 3. The enhanced responses to UK14304 in the presence of U46619 and forskolin were not inhibited by the alpha1-adrenoceptor antagonist prazosin (0.1 microM), but were inhibited by the alpha2-adrenoceptor antagonist rauwolscine (1 microM), indicating that the enhanced responses were mediated via postjunctional alpha2-adrenoceptors. 4. In the presence of 0.1 microM U46619 and 1 mM isobutylmethylxanthine (IBMX), 1 microM forskolin produced an increase in [3H]-cyclic AMP levels in porcine isolated ear arteries. Addition of 0.3 microM UK14304 prevented this increase. 5. The enhanced UK14304 response was dependent upon the agent used to relax the tissue. After relaxation of ear arteries precontracted with 10 nM U46619 and relaxed with forskolin the UK14304 response was 46.9+/-9.6% of the 60 mM KCl response, and after relaxation with sodium nitroprusside (SNP) the response was 24.8+3.3%. However, after relaxation of the tissue with levcromakalim the UK14304 response was only 8.2+/-1.7%, which was not different from the control response in the same tissues (12.2+/-5.6%). An enhanced contraction was also obtained after relaxation of the tissue with the cyclic AMP analogue dibutyryl cyclic AMP (23.2+/-1.3%) indicating that at least part of the enhanced response to UK14304 is independent of the ability of the agonist to inhibit cyclic AMP production. 6. Relaxation of U46619 contracted ear arteries with SNP could be inhibited by the NO-sensitive guanylyl-cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) indicating that production of cyclic GMP is necessary for the relaxant effect of SNP. However, ODQ had no effect on the relaxation of tissue by forskolin, suggesting that this compound does not act via production of cyclic GMP. Biochemical studies showed that while forskolin increases the levels of cyclic AMP in the tissues, SNP had no effect on the levels of this cyclic nucleotide. 7. In conclusion, enhanced contractions to the alpha2-adrenoceptor agonist UK14304 can be uncovered in porcine isolated ear arteries by precontracting the tissue with U46619, followed by relaxation back to baseline with forskolin, SNP or dibutyryl cyclic AMP before addition of UK14304. There was a greater contractile response to UK14304 after relaxation with forskolin than with SNP or dibutyryl cyclic AMP, suggesting that cyclic AMP-dependent and- independent mechanisms are involved in the enhancement of the UK14304 response.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1998

Rilmenidine reveals differences in the pharmacological characteristics of prejunctional alpha2-adrenoceptors in the guinea-pig, rat and pig.

1. The alpha2A and alpha2D-adrenoceptor subtypes are thought to be species homologs most easily differentiated on the basis of the potency of antagonists. In the present study we have compared the effect of rilmenidine with two other selective alpha2-adrenoceptor agonists, UK-14304 (5-bromo-6- [2-imidazolin-2-ylamino]-quinoxaline) and clonidine, against electrically-evoked contractions in five isolated preparations from the rat, guinea-pig and pig, and, where possible, determined the receptor subtype involved. 2. UK-14034, clonidine and rilmenidine produced concentration-dependent inhibition of the electrically-evoked contractions of the rat isolated vas deferens and tail artery and the guinea-pig ileum. These inhibitory effects were reversed by the selective alpha2-adrenoceptor antagonist, RX-811058 (1 microM), except in the rat tail artery preparations where the remaining neurogenic response was inhibited; evidence for the involvement of 'innervated' alpha2-adrenoceptors. Both clonidine and UK-14304 produced concentration-dependent inhibition of responses in the porcine isolated tail artery and urinary bladder but clonidine was markedly less efficacious in these preparations. In contrast, rilmenidine failed to inhibit the neurogenic contractions in either preparation. 3. Although rilmenidine failed to elicit a detectable response in either the porcine isolated tail artery or urinary bladder, it (10 microM and 30 microM, respectively) competitively antagonised the inhibitory effects of UK-14304 with an estimated dissociation constant of (pK(B)) 5.82 and 5.93, respectively. 4. Prazosin (1 microM) failed to alter the effect of UK-14304 against neurogenic contractions in the porcine isolated urinary bladder, while rauwolscine (pK(B) 8.87) was 10 fold more potent than phentolamine (pK(B) 7.56). On the other hand, phentolamine (pK(B) 8.42) was only marginally more potent than rauwolscine (pK 8.05) against clonidine-induced inhibition of electrically-evoked contractions of the guinea-pig isolated ileum. This pharmacological evidence with antagonists supports the presence of alpha2D-adrenoceptors in the rat and guinea-pig and the alpha2A-adrenoceptors in the pig. 5. We have demonstrated that rilmenidine, unlike clonidine and UK-14304, is devoid of any agonist activity at prejunctional alpha2A-adrenoceptors in the pig, but is an efficacious agonist at alpha2D-adrenoceptors in the rat and guinea-pig.

Adrenergic alpha-2 Receptor Agonists↗

PubMed1998

Differential role of vasoactive prostanoids in porcine and human isolated pulmonary arteries in response to endothelium-dependent relaxants.

The pig is increasingly being used in medical research, both as a model of the human cardiovascular system, and as a possible source of organs for xenotransplantation. However, little is known about the comparative functions of the vascular endothelium between porcine and human arteries. We have therefore compared the effects of two endothelium-dependent vasorelaxants, acetylcholine (ACh) and the Ca2+-ATPase inhibitor, cyclopiazonic acid (CPA) on the porcine and human isolated pulmonary artery using isometric tension recording. ACh and CPA produced endothelium-dependent relaxations of both the human and porcine pulmonary arteries. In the porcine pulmonary artery, the cyclo-oxygenase inhibitor, flurbiprofen had no effect on relaxations to ACh (Emax: control 67.8+/-8.8% versus 72.4+/-9.5% (n=11)) or CPA (Emax: control 79.6+/-5.0% versus 94.0+/-10.6% (n=7)). The nitric oxide synthase inhibitor, L-NAME converted relaxations to both ACh and CPA into contractile responses (maximum response: ACh 30.0+/-11.1% (n = 10); CPA 80.4+/-26.2% (n = 8) of U46619-induced tone). These contractile responses in the presence of L-NAME were abolished by flurbiprofen. In the human pulmonary artery, L-NAME and flurbiprofen partly attenuated relaxations to ACh (Emax: control: 45.1+/-12.1%; flurbiprofen: 33.4+/-13.5%; L-NAME: 10.1+/-7.2%) and CPA (Emax: control: 78.1+/-5.5%; flurbiprofen: 69.6+/-7.2%; L-NAME 37.9+/-10.7% of U46619-induced tone). These responses were abolished by the combination of both inhibitors. We have demonstrated that while the release of nitric oxide is important in responses to endothelium-dependent vasorelaxants in both human and porcine pulmonary arteries, in the human arteries, there is an important role for vasorelaxant prostanoids whilst in the porcine arteries, vasoconstrictor prostanoids are released.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1998

Endothelium-dependent relaxation in response to ethanol in the porcine isolated pulmonary artery.

Many drugs cannot be dissolved in distilled water and so other solvents such as ethanol, dimethylsulphoxide and methanol are used. Because very little is known about the direct effects of these three solvents on the cardiovascular system, we have examined their effects on isolated pulmonary and coronary arteries from the pig. Increasing concentrations of ethanol, dimethylsulphoxide and methanol induced relaxation in porcine pulmonary (at 1.2% v/v, 59.9+/-9.0% (n =9), 55.9+/-9.0% (n =6) and 12.3+/-6.4% (n = 8), respectively, of U46619-induced tone) and coronary arteries (at 1.2% v/v, 69.9+/-7.1% (n = 10), 78.9+/-6.1% (n = 7) and 12.9+/-8.2% (n = 6) respectively, of U46619-induced tone). In the pulmonary arteries the relaxation in response to ethanol was found to be endothelium-dependent whereas the responses to dimethylsulphoxide and methanol were unaffected by removal of the endothelium. In the coronary arteries the relaxation to all three solvents was independent of the presence of the endothelium. Comparison of the sensitivity of the tissues to the solvents showed that ethanol and dimethylsulphoxide produced comparative responses in both the pulmonary and coronary arteries, whereas methanol was much less potent. The endothelium-dependent response to ethanol in the porcine pulmonary artery (maximum response, Emax, 67.1+/-9.3% of U46619-induced tone, n = 7) was attenuated by the cyclooxygenase inhibitor, flurbiprofen (Emax 31.9 +/- 12.0%, n=7), the nitric oxide synthase inhibitor, L-NAME (NG-nitro-L-arginine methyl ester; Emax 23.5+/-10.2%, n = 7)) and the combination of both inhibitors (Emax 18.3+/-7.8%, n = 7). The residual relaxatory response to ethanol was abolished, and converted into a contractile response, both by removal of the endothelium (at 1.7% v/v ethanol 27.3+/-11.5% of U46619-induced tone, n=7) and by the addition of a low concentration of KC1 (49.9-/+10.3%, n=6), suggesting the release of a non-prostanoid, non-nitric oxide factor from the endothelium. This response, however, was not attenuated by the cannabinoid receptor-antagonist SR141716A (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide HCL; 52.5-/+4.3% relaxation, n =8), suggesting that the factor released in this preparation by ethanol is not a cannabinoid. The results of this study indicate that many solvents commonly used in pharmacological experiments have pronounced vasoactive properties. Methanol might be the vehicle of choice, because it was the least active solvent, whereas high concentrations of ethanol might influence vascular function at both the level of the smooth muscle and the endothelium, with the action on the endothelium involving the release of endothelium-derived relaxing factors.

Animals↗

PubMed1997

Reconstitution of a functional bovine papillomavirus type 1 origin of replication reveals a modular tripartite replicon with an essential AT-rich element.

A functional replication origin was reconstituted using oligonucleotide cassettes corresponding to three sequence subelements within the Bovine Papillomavirus Type 1 (BPV-1) replication origin: the 23-bp AT-rich region (ATR), the 18-bp binding site for the viral replication initiator protein E1 (E1BS), and a binding site for the viral transcriptional transactivator and replication enhancer protein E2 (E2BS). Replication of the reconstituted origin depended on heterologous expression of both the E1 and E2 proteins and on the presence of both the E1BS and E2BS, indicating that it is functionally analogous to the authentic BPV-1 origin. In addition, pairwise testing of subelement combinations revealed that the ATR was also essential and that a functional origin required at least one copy of all three subelements. While the E1BS and E2BS are sequence-specific elements, the function of the BPV-1 ATR could be at least partially substituted with heterologous AT-rich sequences, suggesting that the role of this element is primarily AT content-dependent rather than sequence-dependent. A stringent requirement for the ATR was also observed in the context of an authentic minimal origin sequence confirming that it is an intrinsic property of the BPV-1 origin and not simply an artifact of the reconstitution system. This study indicates that the minimal functional BPV-1 origin shares the tripartite modular organization characteristic of other simple eukaryotic replication origins. The reconstitution system described now provides a convenient approach to define the physical and functional interrelationships between the three subelements in a systematic fashion.

Adenovirus E2 Proteins↗

PubMed1997

Studies on the vasoconstrictor action of melatonin and putative melatonin receptor ligands in the tail artery of juvenile Wistar rats.

1. In this study we compared the vasoconstrictor activity of melatonin in rat isolated tail artery using two different recording systems, the Halpern pressure myograph and the Halpern-Mulvany wire myograph, with the view to determining a reliable method for obtaining pharmacological data on vascular melatonin receptors. In addition, we characterized the melatonin receptor in this preparation, using analogues of melatonin, and examined the activity of various naphthalenic derivatives with biological activity in non-vascular models of melatonin receptors. 2. Using the Halpern pressure myograph, cumulative addition of melatonin (0.1 nM to 1 microM) produced direct vasoconstriction (19.3+/-6.4% reduction in lumen diameter, n=5) in five of 11 pressurized segments, with pEC50 of 9.14+/-0.17. Similarly, non-cumulative application of melatonin caused vasoconstriction (19.7+/-4.6% reduction in lumen diameter, n=7) in seven of 20 preparations examined with pEC50 of 8.74+/-0.26. The selective alpha2-adrenoceptor agonist, UK-14304 (5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline bitartrate), produced vasoconstriction in all 'melatonin-insensitive' preparations. 3. Melatonin (0.1 nM to 1 microM) failed to elicit isometric contractions of tail artery segments in the Halpern wire myograph, but produced concentration-dependent potentiation of electrically-evoked, isometric contractions (maximum effect of 150-200% enhancement) when applied either noncumulatively (seven of seven preparations) or cumulatively (four of seven preparations). The pEC50 value of melatonin (non-cumulative) was 8.50+/-0.10 (n=7) which was not different from that obtained in the pressure myograph. All further experiments were conducted using a non-cumulative protocol against electrically-evoked, isometric contractions. 4. Based on the pEC50 values for the melatonin analogues examined, the pharmacological profile for the enhancement of electrically-evoked contractions was 2-iodomelatonin > 6-chloromelatonin > or = (-)-AMMTC > or = S21634 > or = melatonin > or = S20098 > S20242 > or = S20304 > 6-hydroxymelatonin > S20932 > (+)-AMMTC > N-acetyl-5-HT. Our data suggests the vascular receptor belongs to the MEL1-like subtype. All the indole-based analogues of melatonin, 2-iodomelatonin, (-)-AMMTC, (+)-AMMTC, S20932, 6-chloromelatonin, 6-hydroxymelatonin and N-acetyl-5-HT, behaved as full agonists. All the naphthalenic derivatives examined, S21634, S20098, S20242 and S20304 behaved as partial agonists relative to melatonin. 5. The naphthalenic-based antagonists, S20928 and S20929, did not modify electrically-evoked, isometric contractions of the tail artery, but produced a parallel, rightward displacement of the melatonin concentration-response curve. Based upon the effect of 1 microM S20928 and S20929, the estimated pK(B) values for these antagonists were 7.18+/-0.25 (n=4) and 7.17+/-0.25 (n=5), respectively. 6. We demonstrated that enhancement of electrically-evoked, isometric contractions of the rat isolated tail artery (using the Halpern-Mulvany wire myograph) is a simple and reproducible model for assessing the activity of putative agonists, partial agonists and antagonists at vascular melatonin receptors. Pharmacological characterization of the receptor suggests the presence of a MEL1-like subtype.

Animals↗

PubMed1997

Casein kinase II phosphorylates bovine papillomavirus type 1 E1 in vitro at a conserved motif.

The E1 protein of bovine papillomavirus type 1 (BPV-1) is a phosphoprotein which specifically binds and unwinds the virus replication origin by ATP-dependent helicase activity. The El protein has been shown to be multiply phosphorylated in vivo, although the sites of modification are incompletely mapped. Examination of the predicted amino acid sequence of all available E1 proteins revealed strong conservation between amino acids 25 and 60 of a motif consisting of a serine residue followed by a stretch of acidic residues. This conserved motif resembled a phosphorylation consensus site for the ubiquitous cellular kinase casein kinase II (CKII). Biochemical and mutational analysis demonstrated that the BPV- 1 E1 protein is an in vitro substrate for CKII at the serine within this conserved motif.

Amino Acid Sequence↗

PubMed1997

Isolation of an amino-terminal region of bovine papillomavirus type 1 E1 protein that retains origin binding and E2 interaction capacity.

In vitro DNA binding results from a series of E1 proteins containing amino-terminal or carboxy-terminal truncations indicated that sequences between amino acids 121 and 284 were critical for origin binding. Additional binding experiments with E1 proteins containing internal, in-frame insertions or deletions confirmed the importance of the region defined by truncated E1 proteins and also demonstrated that downstream sequences were not required for binding activity in the context of the full-length E1 protein. On the basis of mapping results from the E1 mutants, a clone (pE1(121-311)) was constructed that expressed E1 amino acids within the approximate boundaries of the critical sequences for DNA binding. The E1(121-311) protein retained origin-specific DNA binding, confirming that this region was not only necessary but was also sufficient for origin recognition. In addition to origin binding, E1(121-311) bound E2 protein in a cold-sensitive manner. Therefore, DNA binding and E2 binding activities colocalize to a 191-amino-acid functional domain derived from the amino-terminal half of the E1 protein. Finally, three E1 proteins with mutations in this region all lacked DNA binding activity and were all defective for in vivo replication. Two of these E1 mutants retained E2 binding capability, demonstrating that origin recognition by E1 is critical for replication and cannot necessarily be rescued by an interaction with E2 protein.

Animals↗

PubMed1997

Bovine papillomavirus type 1 DNA replication: the transcriptional activator E2 acts in vitro as a specificity factor.

We previously devised cell-free conditions supporting efficient replication of bovine papillomavirus type 1 (BPV1) DNA (C. Bonne-Andréa, S. Santucci, and P. Clertant, J. Virol. 69:3201-3205, 1995): the use of highly active preparations of viral initiator protein E1, together with extract from a particular cell source, allowed the synthesis of complete DNA circles through successive rounds of replication; this occurred in the absence of the viral transcriptional activator E2, required in vivo for the replication of viral genomes. We now report that adding E2 to cell-free assays produced only slight effects both on the yield of E1-dependent DNA synthesis and on the quality of newly made DNA molecules when a template carrying a wild-type BPV1 replication origin (ori) was used. The performance of mouse cell extracts, unable to sustain efficient BPV1 DNA replication in the presence of E1 only, was likewise not improved by the addition of E2. In a proper in vitro environment, E1 is thus fully capable of efficiently initiating viral DNA synthesis by itself, an activity which is not enhanced by interaction with E2. An important effect, however, was detected: E2 totally suppressed the nonspecific replication of ori-defective DNA templates, otherwise observed in high E1 concentrations. We examined the requirements for building a minimal DNA sequence behaving in vitro as a specific ori sequence under stringent recognition conditions, i.e., in the presence of both E1 and E2. Only two elements, the 18-bp E1 binding palindrome and an AT-rich sequence, were required in cis to allow specific cell-free DNA replication; there seemed to be no need for an E2 binding site to ensure discrimination between specific ori templates and other DNA molecules, even in the presence of E2. This suggests that during the initiation of BPV1 DNA replication, at least in vitro, E2 acts as a specificity factor restricting the action of E1 to a defined ori sequence; this function, likely not demanding the direct binding of E2 to cognate DNA sites, might primarily involve protein-protein interactions.

Animals↗

PubMed1996

Perturbation of the host cell cycle and DNA replication by the bovine papillomavirus replication protein E1.

A stable cell line expressing the bovine papillomavirus E1 protein (C2E1) was compared with an E1 minus control line (CNEO) to study the effects of E1 protein on host cell growth. C2E1 and CNEO cells were synchronized either at mitosis or at the G1/S boundary by the cell cycle inhibitors nocodazole and mimosine, respectively. After release from the drug-induced cell cycle block, the progression through the succeeding stages of the cell cycle was temporally monitored using flow cytometry. In addition, incorporation of bromodeoxyuridine (BrdUrd) was used to determine precisely the time of initiation of DNA synthesis in C2E1 and CNEO cells after release from drug-induced cell cycle arrest. Expression of E1 protein decreased the duration of G1 phase and increased S and G2 phase durations without affecting the overall cell doubling time. In conjunction with the increase in G2 phase duration, histone H1 kinase activity was prolonged during the G2 to M phase transition in C2E1 cells, which suggested that E1 protein may affect the mechanisms which ensure proper timing of kinase inactivation. During the G1 to S phase transition in C2E1 cells, the timing of appearance and abundance of cyclin D1 were altered compared to CNEO cells, while cyclin E levels were unaffected. Consequently, E1 protein may affect G1 phase duration through a cyclin D1-dependent pathway. Finally, a subpopulation of cells with a greater than G2 DNA content (>G2 DNA), and which was still capable of incorporating BrdUrd, was shown to exist only in the E1-expressing cell line. These combined results demonstrate that the viral replication protein E1 has the potential to influence the host cell environment significantly, which may contribute to pathogenesis and viral persistence.

Animals↗

PubMed1996

The effect of forskolin on 5-HT1-like and angiotensin II-induced vasoconstriction and cyclic AMP content of the rabbit isolated femoral artery.

1. A characteristic feature of vasoconstrictor 5-HT1-like receptors in vitro is that responses mediated by these receptors are enhanced by other vasoconstrictor agents. In the present study, we have examined the influence of cellular cyclic AMP on vasoconstrictor responses to activation of 5-HT1-like receptors in isolated ring segments of the rabbit femoral artery (RbFA), and determined whether modulation of this second messenger underlies the ability of angiotensin II, an endogenous vasoconstrictor, to enhance 5-HT1-like responses. 2. In the presence of 0.1 microM ketanserin (to antagonize 5-HT2-receptors) and 0.3 microM prazosin (to antagonize alpha 1-adrenoceptors), 5-HT produced a concentration-related contraction, which was significantly augmented by pre-contraction of the vessel with 0.1-0.45 nM ([A30]) angiotensin II. Responses to 5-HT in the presence of angiotensin II were inhibited by the 5-HT1-like/5-HT2 antagonist, metergoline (1 microM). 3. The directly-acting adenylyl cyclase activator, forskolin (1 microM), abolished responses to angiotensin II and caused a rightward shift and concomitant depression of the 5-HT concentration-effect (E/[A]) curve. Higher concentrations of forskolin (> 10 microM) abolished responses to 5-HT and 1 microM sodium nitroprusside abolished responses to 5-HT and angiotensin II (n = 7). 4. In the presence of angiotensin II (0.1-0.45 nM), however, 1 microM forskolin failed to inhibit 5-HT-induced contractions; the E/A curve for 5-HT (in the presence of forskolin and angiotensin II) was not significantly different from that produced in the presence of angiotensin II alone. Similarly, the presence of angiotensin II (0.1-0.45 nM) was also able to overcome partially the inhibitory effect of 1 microM sodium nitroprusside against 5-HT-induced contractions (n = 7). In marked contrast, 5-HT failed to elicit a contraction in the presence of angiotensin II and 10 microM forskolin (n = 5). 5. 5-HT (1 microM) significantly reduced basal cyclic AMP accumulation by 35%, whereas angiotensin II (0.45 nM) was without effect. The combination of angiotensin II and 5-HT failed to alter significantly the reduction in cyclic AMP produced by 5-HT alone. Forskolin (1 microM) increased cyclic AMP levels 7 fold above basal, but neither 1 microM 5-HT nor a combination of 1 microM 5-HT and 0.45 nM angiotensin II produced a significant decrease in cyclic AMP content. 6. Whilst moderate concentrations of forskolin can inhibit the responses to either agent, simultaneous activation of angiotensin II and 5-HT1-like receptors can overcome the inhibitory effect of elevated levels of cyclic AMP. Since the potentiating effect of angiotensin II, in either the presence or absence of forskolin, occurs without significant alteration of cellular cyclic AMP, it seems likely that a cyclic AMP-independent pathway is implicated in the synergistic interaction between angiotensin II and vasoconstrictor 5-HT1-like receptors.

Angiotensin II↗

PubMed1996

Simple procedure for creation of in-frame deletion mutations throughout an open reading frame.

A general method is presented for randomly mutagenizing open reading frames (ORF) to generate in-frame deletions and insertions. The protocol requires expression of the ORF of interest as a hybrid ORF-beta-galactosidase fusion protein. This allows colorimetric screening for beta-galactosidase activity during subsequent mutagenesis steps. Consequently, proteins with no suitable phenotypic selection or screening properties can be readily screened for mutations that disrupt and subsequently restore the reading frame of the hybrid protein. In addition, this system provides gene expression for subsequent biochemical analysis of the mutant proteins. The bovine papillomavirus type 1 (BPV-1) EI ORF has been mutagenized using this method as an example.

Base Sequence↗