The association between prior statin use and long-term outcomes after critical care admission.

BACKGROUND: Statins may have immunomodulatory effects that benefit critically ill patients. Therefore, we retrospectively examined the association between survival and the prescription of statins prior to admission to an intensive care unit (ICU), or high dependency unit (HDU), as a result of major elective surgery or as an emergency with a presumed diagnosis of sepsis. METHODS: We retrospectively studied critical care patients (ICU or HDU) from a tertiary referral UK teaching hospital. Nottingham University Hospitals have more than 2200 beds, of which 39 are critical care beds. Over a 5-year period (2000-2005), 414 patients were identified with a presumed diagnosis of sepsis, and 672 patients were identified who had planned ICU/HDU admissions following elective major surgery. Patients prescribed statins prior to hospital admission were compared with those who were not. Demographics, medical history, drug history, and Acute Physiology and Chronic Health Evaluation II scores were examined. Univariate and multivariate analyses were applied using the primary end point of survival at 5 years after admission. RESULTS: Patients prescribed statins prior to critical care admission were, on average, older and had higher initial Acute Physiology and Chronic Health Evaluation II scores and more preexisting comorbidities. Statins were almost invariably stopped following admission to critical care. Statin use was not associated with significantly altered survival during hospital admission, or at 5 years, for either patients with sepsis (9% vs 15%, P=.121; 73% vs 84%, P=.503, respectively) or postoperative patients (55% vs 58%, P=.762; 57% vs 63%, P=.390). CONCLUSIONS: Prior statin use was not associated with improved outcomes in patients admitted to critical care after elective surgical cases or with a presumed diagnosis of sepsis.

Partite expression of the bovine papillomavirus E1 open reading frame in Escherichia coli.

Six recombinants were constructed which expressed portions of the bovine papillomavirus E1 open reading frame as OmpF/E1/beta-galactosidase tribrid fusion proteins in Escherichia coli. Rabbit sera containing E1-specific antibodies were generated against five of these six fusion proteins (which together constitute 74% of the full-length E1 open reading frame). The individual fusion proteins and their cognate antisera will be useful reagents for defining the structure and function of the BPV E1 protein(s).

Changes in DNA binding by purified simian RNA polymerase II under transcribing and nontranscribing conditions.

The interaction of RNA polymerase II from African Green Monkey liver tissue with SV40 DNA was examined by a DNAase protection technique. In the absence of nucleoside triphosphates, simian polymerase binds to nicked, linear SV40 DNA and protects 30 bp of binary complex DNA from DNAase I digestion. With the addition of nucleoside triphosphates to initiate transcription, polymerase protects 40 bp of the ternary complex DNA from DNAase I. Thus, a conformational change in either the polymerase, the DNA, or both occurs during the transition from binary to ternary complex, and this altered conformation allows a larger protection of template DNA. Similar results were seen with Escherichia coli RNA polymerase holoenzyme on SV40 DNA.

'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.

Angiotensin II causes vascular hypertrophy in part by a non-pressor mechanism.

Angiotensin II, when given in low doses, raises blood pressure slowly. When tested in vitro on vascular smooth muscle cells, it has mitogenic and trophic effects; it is not known if it has these effects in vivo. Our purpose was to determine whether vascular hypertrophy develops during slow pressor infusion of angiotensin II and, if so, whether it is pressure induced. Three experiments were done in rats infused subcutaneously with angiotensin II (200 ng/kg/min) by minipump for 10-12 days. Experiment 1: Angiotensin II gradually raised systolic blood pressure (measured in the tail) from 143 +/- 2 to 208 +/- 8 mm Hg (mean +/- SEM), significantly suppressing plasma renin and increasing threefold (NS) plasma angiotensin II. There was no loss of peptide in the pump infusate when tested at the end of the experiment. Experiment 2: In the perfused mesenteric circulation, vasoconstrictor responses to norepinephrine, vasopressin, and KCl were enhanced in rats given a slow pressor infusion of angiotensin II, but sensitivity of responses was not altered. This combination of changes suggests that vascular hypertrophy develops during slow pressor infusion of angiotensin II. Experiment 3: Vessel myography was done after angiotensin II infusion with and without a pressor response. Angiotensin II raised systolic blood pressure, increased heart weight, and produced myographic changes of vascular hypertrophy in the mesenteric circulation, increasing media width, media cross-sectional area, and media/lumen ratio. Hydralazine given with angiotensin II prevented the rise of pressure and the cardiac effect but not the vascular changes. Two-way analysis of variance showed that angiotensin II significantly increased media width, media cross-sectional area, and media/lumen ratio, all independent of hydralazine. Thus, although hydralazine inhibits the pressor and cardiac effects of angiotensin II, suggesting a pressor mechanism for the cardiac change, it does not inhibit structural vascular change, which suggests that at least part of the effect has a non-pressor mechanism.

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.

Dual loci of DNA bending in the bovine papillomavirus upstream regulatory region.

Recombinant clones containing all or portions of the bovine papillomavirus (BPV) upstream regulatory region (URR) were constructed. When analyzed by polyacrylamide gel electrophoresis, the cloned URR sequences exhibited electrophoretic properties characteristic of DNA containing bend sites. Two distinct bend centers were identified, mapping to approximately 7477 and 7790 respectively on the BPV genome. No other loci of static DNA bending were detected in the URR region of the BPV genome.

Viral interaction with the host cell sumoylation system.

A novel host cell post-translational modification system termed sumoylation was discovered recently. Sumoylation is an enzymatic process that is biochemically analogous to, but functionally distinct from ubiquitinylation. As in ubiquitinylation, sumoylation involves the attachment of a small protein moiety, SUMO, to substrate proteins. Conjugation of SUMO does not typically lead to degradation of the substrate and instead causes functional alterations or changes in intracellular localization. While the majority of identified SUMO targets are cellular proteins, both herpesvirus and papillomavirus proteins have also been identified as authentic substrates for this modification. The exact effect of sumoylation on viral proteins appears to be substrate specific, but does have functional consequences that are likely to be important for the viral life cycle. In addition to viral proteins being targets for sumoylation, there is both direct and indirect evidence that viruses can alter the sumoylation status of host cell proteins. Such modulation of critical host proteins may be important for inhibiting cellular defense mechanisms or for promoting an intracellular state that is supportive of viral reproduction. This review highlights the enzymology of sumoylation and discusses the known examples of how viruses impact and are impacted by sumoylation.

The bovine papillomavirus type 1 genome contains multiple loci of static DNA bending, but bends are absent from the functional origin of replication.

Twenty-four overlapping restriction fragments spanning the entire bovine papillomavirus type 1 (BPV-1) genome were analyzed by electrophoresis to determine the extent of static DNA bending in the BPV-1 genome. Thirteen of 24 fragments contained static bends. Based on known locations of previously mapped bend loci and the overlapping pattern of these 13 fragments, we estimate that there are 8-11 distinct static bend loci in the BPV-1 genome. The bend loci were not uniformly distributed on the genome and with one exception, were clustered from nucleotides 5816 to 2621 on the BPV-1 map. This portion of the BPV-1 genome contains most of the transcriptional regulatory sequences as well as the origin of replication. The concordance between the genomic distribution of DNA bends and cis-active elements is consistent with the possibility that bent sequences may contribute to the function of at least some of these elements. However, unlike SV40, there was no static bend at that functional origin of replication for BPV-1. The nearest bends to the origin were approximately 120 bp to the 5' side and 300 bp to the 3' side. As both of these bends were outside of the sequences required for origin function, it is unlikely that static bending plays a critical role in BPV-1 replication.

Pharmacological characterization of noradrenaline-induced contractions of the porcine isolated palmar lateral vein and palmar common digital artery.

1. The aim of this study was to examine the pharmacological characteristics of alpha-adrenoceptor-mediated contractions in two porcine isolated blood vessels, the palmar lateral vein (PLV) and the palmar common digital artery (PCDA). This was carried out with noradrenaline used as the agonist throughout, and either phentolamine (non-selective alpha-adrenoceptor antagonist), prazosin and YM-12617 (selective alpha 1-adrenoceptor antagonists) or rauwolscine and CH-38083 (selective alpha 2-adrenoceptor antagonists). 2. Noradrenaline (0.003-10 microM) produced concentration-dependent contractions in both vessels, with the PCDA (pD2 = 6.33 +/- 0.07, n = 10) being approximately 10 fold less sensitive to noradrenaline compared to the PLV (pD2 = 7.39 +/- 0.09, n = 8). Also, the maximal response to noradrenaline was greater in the PCDA compared to the PLV. Phentolamine (0.03-30 microM) produced parallel rightward shifts in the CRC to noradrenaline in both tissue preparations. The pA2 values were similar and slopes of the Schild plots were not significantly different from unity, indicating an interaction between phentolamine and a single receptor in each preparation. 3. In the PCDA the alpha 1-adrenoceptor antagonists, prazosin (0.01-1 microM) and YM-12617 (0.01-1 microM) produced non-parallel rightwards shifts in the CRC to noradrenaline, with the lower 10-15% of the CRC exhibiting greater resistance to the effects of these antagonists compared to the upper part. In contrast, rauwolscine (1-10 microM) and CH-38083 (10 microM) produced parallel displacement of the CRC to noradrenaline. In the PLV, low concentrations of either alpha l- (0.01 microM) or alpha2-adrenoceptor antagonists(0.1-1 microM) produced a large shift in the CRC, but subsequent higher concentrations had only small additional effects. Based upon pKB values estimated from the effects of the lower concentrations of antagonists, the results are consistent with a large population of alpha1-adrenoceptors in the PCDA and a mixture of alpha l- and alpha2-adrenoceptors in the PLV.4. In both tissues, when an ac,- and an a2-adrenoceptor antagonist were used in combination the effect produced was greater than that with either agent alone. In contrast, the combination of the alpha1-adrenoceptor antagonists (prazosin and YM-12617 together) or the alpha2-adrenoceptor antagonists (CH-38083 and rauwolscine together) were no more effective than that produced by the individual antagonists. These findings suggest the presence of functional alpha l- and alpha2-adrenoceptors in the PLV andPCDA.5. Phenoxybenzamine (0.3-3 microM, 60min exposure) produced a concentration-dependent reduction in the maximal response to noradrenaline which was more pronounced in the PCDA than the PLV. After a 60 min exposure to a combination of phenoxybenzamine (1 microM) and rauwolscine (1 microM), the remaining NA-induced contraction after washout was resistant to prazosin (0.1 microM) and sensitive to rauwolscine(1 microM) in both tissue preparations, indicating the existence of functional alpha2-adrenoceptors in both vessels.6. Evidence suggests that post-junctional alpha l- and alpha2-adrenoceptors contribute to noradrenaline-induced contractions in the PCDA and PLV, with the latter possessing a larger population of functional alpha2-adrenoceptors.

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.

Influence of angiotensin II on the alpha-adrenoceptors involved in mediating the response to sympathetic nerve stimulation in the rabbit isolated distal saphenous artery.

Under normal experimental conditions, sympathetic nerve-mediated responses to electrical field stimulation in the isolated distal saphenous artery of the rabbit are sensitive to prazosin (0.1 microM) and so, by definition, are mediated by alpha 1-adrenoceptors. In the presence of angiotensin II (A II, 0.05 microM) however, a component of the response to nerve stimulation became resistant to prazosin. This 'uncovered' response was virtually abolished by the selective alpha 2-adrenoceptor antagonist rauwolscine (1 microM), a concentration that in the absence of A II had enhanced nerve-mediated responses. Exposure to A II therefore, allows the clear demonstration of a role for postjunctional alpha 2-adrenoceptors in mediating the contractile response to sympathetic nerve stimulation in this arterial preparation.

Evidence that the population of postjunctional-adrenoceptors mediating contraction of smooth muscle in the rabbit isolated ear vein is predominantly alpha 2.

1. Noradrenaline (NA), phenylephrine and UK-14304 elicited concentration-dependent contractions of the rabbit isolated ear vein of similar maximal magnitude. The rank order of potency, UK-14304 greater than noradrenaline greater than phenylephrine, is consistent with that of an effect mediated through an alpha 2-subtype. 2. The potent and highly selective alpha 1-adrenoceptor antagonists prazosin and YM-12617, at concentrations as high as 1 microM, produced less than a 4 fold rightward displacement of the NA concentration-response curve. 3. The selective alpha 2-adrenoceptor antagonists rauwolscine, Wy-26703 and CH-38083 antagonized responses to noradrenaline in a competitive manner. For all three antagonists, the pA2 values were consistent with an effect at alpha 2-adrenoceptors. However, 0.1 microM YM-12617 increased the potency of rauwolscine 2 fold indicating the presence of a small population of postjunctional alpha 1-adrenoceptors. 4. The relative antagonist potency of the yohimbine diastereoisomers rauwolscine and corynanthine against noradrenaline (rauwolscine 30 fold greater than corynanthine) is also consistent with an effect at alpha 2-adrenoceptors. 5. Contractions elicited by noradrenaline in the rabbit isolated ear vein appear to be mediated predominantly by postjunctional alpha 2-adrenoceptors.

Pharmacological analysis of postjunctional alpha-adrenoceptors mediating contractions to (-)-noradrenaline in the rabbit isolated lateral saphenous vein can be explained by interacting responses to simultaneous activation of alpha 1- and alpha 2-adrenoceptors.

1. The pharmacological characteristics of the alpha-adrenoceptor population in the rabbit isolated saphenous vein has been examined with (-)-noradrenaline (NA), as principal agonist, and a number of antagonists with selectivity for either alpha 1- or alpha 2-adrenoceptors. 2. The rank order of potency of various agonists is consistent with a population of alpha 2-adrenoceptors; UK-14304 greater than (-)-noradrenaline = (-)-adrenaline greater than B-HT 920 = cirazoline greater than phenylephrine greater than amidephrine, but the rank order of pA2 values for the antagonists against (-)-noradrenaline: BDF-6143 greater than rauwolscine = prazosin greater than CH-38083 = YM-12617 greater than Wy-26703 = phentolamine greater than corynanthine, is indicative of a mixed population of alpha 1- and alpha 2-adrenoceptors or, alternatively, a new subtype with characteristics of both the alpha 1- and alpha 2-subtypes. 3. Further evidence for two discrete populations of alpha-adrenoceptors is provided by, (a) the potent but non-competitive effect of prazosin against (-)-noradrenaline, (b) the presence of a component of the contractions elicited by NA and phenylephrine which is resistant to the selective alpha 2-adrenoceptor antagonists rauwolscine and CH-38083: these responses were inhibited by the selective alpha 1-adrenoceptor antagonists prazosin and YM-12617, but not by the selective alpha 2-adrenoceptor antagonist BDF-6143 and, (c) the relative potency of the yohimbine diastereoisomers rauwolscine and corynanthine against NA, phenylephrine and UK-14304. 4. In spite of the overwhelming evidence for a population of postjunctional alpha 2-adrenoceptors, prazosin was similarly effective against all agonists and failed to discriminate between those with putative selectivity for alpha 1- and alpha 2-adrenoceptors. This suggests an interaction of the effects of agonists at the two alpha-adrenoceptor subtypes. 5. An attempt has been made to reconcile a number of paradoxical observations with regard to the identification of postjunctional alpha 2-adrenoceptors in vitro, and it is suggested that in many of the isolated blood vessels presently available for examination both subtypes reside on the same smooth muscle cell. The pharmacological consequences of multiple subtypes of receptors mediating the same response is considered.

An examination of the postjunctional alpha-adrenoceptor subtypes for (-)-noradrenaline in several isolated blood vessels from the rabbit.

1. Postjunctional alpha-adrenoceptors in several isolated blood vessels from the rabbit have been characterized on the basis of the relative potency of the agonists noradrenaline (NA, non-selective), phenylephrine (alpha 1-selective) and UK-14304 (alpha 2-selective), and the potency of antagonists rauwolscine (alpha 2-selective) and corynanthine (alpha 1-selective) against contractions elicited by NA. In addition, the potency of prazosin against NA was also assessed in the venous preparations. 2. The thoracic aorta, ear artery and left renal vein appear to possess alpha 1-adrenoceptors since the agonist potency order was NA greater than phenylephrine greater than UK-14304, while corynanthine was 3-10 fold more potent than rauwolscine. 3. The ear vein appears to possess alpha 2-adrenoceptors. The rank order of agonist potency was UK-14304 greater than NA much greater than phenylephrine and all three agonists elicited responses of similar magnitude. Furthermore, rauwolscine was 30 fold more potent than corynanthine while prazosin failed to produce a concentration-dependent inhibition. 4. The saphenous vein and the plantaris vein appear to possess a mixture of both subtypes since the rank order of agonist potency was UK-14304 greater than NA much greater than phenylephrine, while responses elicited by UK-14304 were smaller than those to the other agonists. However, although rauwolscine was 20 to 100 fold more potent than corynanthine in both preparations, suggestive of predominantly alpha 2-adrenoceptors, prazosin was either potent (saphenous vein) or relatively inactive (plantaris vein). 5. The characteristics of postjunctional alpha 1- and alpha 2-adrenoceptors on isolated blood vessels from the rabbit are discussed in relation to the value of both the agonists, particularly NA, and the antagonists used in this study.

Evidence for functional dissociation of dependence and tolerance in guinea-pig isolated ileal segments following 20 hour exposure to morphine in vitro.

1. In the present study we have examined the relationship between tolerance and dependence in isolated ileal segments from the guinea-pig under three different conditions: fresh preparations not previously exposed to morphine (fresh/morphine naive); preparations stored overnight at 4 degrees C in modified Krebs-Henseleit saline (overnight-stored/morphine-naive); preparations stored overnight at 4 degrees C in Krebs-Henseleit saline containing 10 microM morphine and extensively washed with modified Krebs-Henseleit saline to remove residual morphine (overnight-stored/morphine-exposed). 2. Morphine produced a concentration-dependent inhibition of the response of ileal segment to 0.1 Hz, 1 ms and 10 V transmural field stimulation in fresh/morphine-naive, overnight-stored/morphine-naive and overnight-stored/morphine-exposed preparations. The maximum effect observed was similar in all three preparations-approximately 80% inhibition. Although, morphine was significantly more potent in the fresh/morphine-naive preparations (pD2 6.72 +/- 0.05, n = 8) than either the overnight-stored/morphine-native (pD2 6.42 +/- 0.11, n = 8) or the overnight-stored/morphine-exposed (pD2 6.44 +/- 0.14, n = 8), there was no significant difference between the overnight exposure to ileal segments to 10 microM morphine at 4 degrees C failed to induce tolerance to morphine. 3. The mu opiate receptor antagonist, naloxone (10 microM), produced contractions in both fresh/morphine-naive and overnight-stored/morphine-naive ileal segments following acute exposure to 10 microM morphine. Naloxone (10 microM) also produced contractions in 2/9 fresh/morphine-naive, 1/9 overnight-stored/morphine-naive and 7/9 overnight-stored/morphine-exposed preparations in the absence of morphine. The greater incidence of naloxone-induced contractions in overnight-stored/morphine-exposed preparations,suggests that dependence in this model is the product of adaptive changes that outlive the presence of morphine.4. The selective alpha2-adrenoceptor agonists, clonidine (0.3 microM) and 5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline bitartrate (UK-14304, 1 microM), inhibited naloxone-induced contractions in overnight-stored/morphine-exposed preparations of ileal segments (n = 4 preparations for each agonist), suggesting that the response is due to transmitter release from the myenteric plexus.5. The findings in the present study indicate that tolerance and dependence to morphine in ileal segments of the guinea-pig can be functionally dissociated by overnight exposure to morphine at 4 degrees C.The development of tolerance to morphine, unlike dependence, appears to be a temperature-dependent process. This also raises the possibility that naloxone possesses intrinsic negative agonism at morphine sensitive receptors, which is manifested as a functional response only after adaptive changes in the myenteric plexus following exposure to morphine.

Species-selective binding of [3H]-idazoxan to alpha 2-adrenoceptors and non-adrenoceptor, imidazoline binding sites in the central nervous system.

1. We have used the imidazoline derivative [3H]-idazoxan to define alpha 2-adrenoceptors and non-adrenoceptor, imidazoline binding sites in cerebral cortex membranes of calf, mouse, rat, guinea-pig and man. 2. Competition experiments using the selective alpha-adrenoceptor drugs, rauwolscine and corynanthine, indicated that [3H]-idazoxan bound to a single population of sites in the calf and mouse membranes. However, [3H]-idazoxan also labelled non-adrenoceptor, imidazoline binding sites in the rat (15%), guinea-pig (30%) and human (40%) cerebral cortex membranes. 3. Competition experiments with adrenaline and cirazoline in the guinea-pig cortex, verified [3H]-idazoxan binding to both alpha 2-adrenoceptors and to non-adrenoceptor, imidazoline binding sites. 4. It has been postulated by several groups that [3H]-idazoxan may possess partial agonist activity. To investigate this further, saturation experiments were performed in the cerebral cortex membranes of all five species in the absence and presence of 300 microM guanosine triphosphate (GTP). GTP had no effect on [3H]-idazoxan binding in guinea-pig cerebral cortex; in both rat and mouse membranes 300 microM GTP increased the dissociation constant for [3H]-idazoxan by 2-3 fold without significantly affecting the Bmax. GTP reduced the Bmax by approximately 30% and 60% in calf and human cerebral cortex membranes, respectively, without significantly altering the Kd. 5. Saturation experiments were performed in the calf cerebral cortex membranes in the absence and presence of 300 microM GTP with the selective alpha 2-adrenoceptor agonist [3H]-clonidine and the selective muscarinic antagonist [3H]-quinuclidinyl benzilate (QNB). GTP reduced the Bmax for [3H]-clonidine without altering the Kd, but failed to affect either the Bmax or the Kd for [3H]-QNB.6. Saturation experiments were performed in human cerebral cortex membranes in the presence of alpha2-adrenoceptor blockade with and without GTP. GTP 300 microM reduced the Bmax for [3H]-idazoxan at the non-adrenoceptor, imidazoline binding sites, without affecting the Kd. GTP did not affect [3H]-QNB binding to muscarinic sites.7. Thus, there is a need to investigate further the pharmacological actions of [3H]-idazoxan in view of its ability to recognise both alpha2-adrenoceptors and non-adrenoceptor, imidazoline binding sites and because it might possess agonist activity at some of these sites.

Expression of functional postjunctional alpha 2-adrenoceptors in rabbit isolated distal saphenous artery--a permissive role for angiotensin II?

In the rabbit isolated distal saphenous artery, the population of postjunctional adrenoceptors is of the alpha 1 variety under normal in vitro experimental conditions, based on the potency order of selective agonists and on the effects of the antagonists prazosin and rauwolscine against responses to UK-14304. Angiotensin II (A II, 0.05 microM) however, without affecting resting baseline tension, markedly enhanced responses to UK-14304, particularly at low concentrations. This previously unseen component of the response to UK-14304 was resistant to prazosin (0.1 microM) but susceptible to rauwolscine (1 microM). A II would therefore appear to have a permissive role for the expression of a quiescent population of postjunctional alpha 2-adrenoceptors in the rabbit distal saphenous artery.

Effects of basal and acetylcholine-induced release of endothelium-derived relaxing factor on contraction to alpha-adrenoceptor agonists in a rabbit artery and corresponding veins.

1. The effects of an endothelium-dependent (acetylcholine) and an endothelium-independent (sodium nitroprusside) relaxant against noradrenaline-induced contractions were compared in three isolated superficial blood vessels of the rabbit, the lateral saphenous vein, plantaris vein and distal saphenous artery. Both produced concentration-related relaxations of all three vessels and were more effective against submaximal than maximal contractions to noradrenaline. Transient contractions to high concentration of acetylcholine occurred only in endothelium-intact preparations of saphenous vein and were inhibited by flurbiprofen. 2. In endothelium-denuded preparations sodium nitroprusside was 3 times more effective than in endothelium-intact preparations, while acetylcholine (less than 3 microM) was inactive. Sensitivity was similar for each relaxant: lateral saphenous vein greater than or equal to plantaris vein greater than distal saphenous artery. The similar profile of sodium nitroprusside and acetylcholine suggests that differences in susceptibility to endothelium-derived relaxing factor (EDRF) are caused by inter-vessel variations in the excitation-coupling process for noradrenaline. 3. Haemoglobin inhibited acetylcholine-induced relaxations in the endothelium-intact preparation of the lateral saphenous vein and distal saphenous artery, which suggests a similar EDRF in each preparation and the likelihood that this is a single substance, presumably nitric oxide. 4. The influence of basal, spontaneously released EDRF on alpha-adrenoceptor function was tested either by mechanical disruption of the endothelium or by adding haemoglobin to endothelium-intact segments. Endothelial disruption slightly reduced contractions to noradrenaline (NA) in distal saphenous artery but increased response size of lateral saphenous and plantaris veins, in the latter also increasing sensitivity to NA: haemoglobin mimicked endothelial disruption. Thus, basal release of EDRF like acetylcholine and nitroprusside was more effective in the veins than in the corresponding artery. 5. In lateral saphenous vein responses to phenylephrine were enhanced by endothelial disruption, but without change in sensitivity: responses to UK-14304, B-HT 920 and cirazoline, which had a relatively slow speed of onset of contraction were not affected. There was no correlation between enhancement and alpha-adrenoceptor sub-type although the agonists which were enhanced all activate alpha 1-adrenoceptors. Competitive antagonists failed to reveal an alpha-adrenoceptor subtype enhanced by endothelial disruption. However, effects of phenoxybenzamine suggest that alpha 1-adrenoceptors are necessary for the influence of basal EDRF.

Postjunctional alpha-adrenoceptors in the rabbit isolated distal saphenous artery: indirect sensitivity to prazosin of responses to noradrenaline mediated via postjunctional alpha 2-adrenoceptors.

1. Under normal experimental conditions, the rabbit isolated distal saphenous artery appears to contain a homogeneous population of postjunctional alpha 1-adrenoceptors. Prazosin competitively antagonized responses to noradrenaline (NA) with a pA2 value of 8.6, while a relatively high concentration of rauwolscine (1 microM), produced only a 2 fold rightward displacement of the NA cumulative concentration-response curve (CCRC). 2. Despite the fact that angiotensin II (AII) was without effect on responses to NA or phenylephrine in this preparation, this peptide made responses to NA less susceptible to the antagonistic action of prazosin. This was particularly evident on the lower portion of the CCRC for NA. These results suggest that in the presence of AII, NA produces contractile responses by an action mediated through a prazosin-resistant adrenoceptor. 3. An attempt was made to isolate a homogeneous population of postjunctional alpha 2-adrenoceptors by use of a receptor protection procedure involving the combination of rauwolscine and phenoxybenzamine. After the protection protocol no responses were observed to the alpha-adrenoceptor agonists NA, phenylephrine or UK-14304. In the presence of angiotensin II however, concentration-dependent contractions were observed to each of these agonists. Under these conditions the rank order of potency, UK-14304 greater than NA greater than phenylephrine, is consistent with that of an effect mediated through postjunctional alpha 2-adrenoceptors. 4. The responses to NA, after the protection protocol, in the presence of AII, were susceptible to the selective alpha 2-adrenoceptor antagonist, rauwolscine (1 microM), but resistant to the selective alpha 2-adrenoceptor antagonist prazosin (0.1 microM). Furthermore, the combination of rauwolscine (1 microM) and prazosin (0.1 I microM) was no more effective in blocking responses to NA than was rauwolscine (1 microM) alone. These results are consistent with the presence of a homogeneous population of postjunctional alpha 2-adrenoceptors. 5. Inducing a small degree of tone with a low concentration of the selective alpha 1-adrenoceptor agonist, phenylephrine, markedly increased the threshold sensitivity to the selective alpha 2-adrenoceptor agonist UK- 14304, in a manner analogous to that seen with All. 6. The results in the present study indicate that responses mediated via postjunctional alpha 2-adrenoceptors in the rabbit isolated distal saphenous artery are dependent upon a degree of vascular smooth muscle stimulation by some other receptor system. It is hypothesized that under normal experimental conditions, this function is fulfilled by stimulation of alpha l-adrenoceptors, while after alpha 1-adrenoceptor blockade the necessary positive influence can be provided by stimulation of All receptors. The implications for such an interaction between postjunctional alpha-adrenoceptor subtypes in demonstrating prazosin-resistant, rauwolscine- or yohimbine-sensitive responses in isolated blood vessels is discussed.