Molecular Dynamics Simulation and NMR Investigation of the Association of the ß-Blockers Atenolol and Propranolol with a Chiral Molecular Micelle.

Molecular dynamics simulations and NMR spectroscopy were used to compare the binding of two ß-blocker drugs to the chiral molecular micelle poly-(sodium undecyl-(L)-leucine-valine). The molecular micelle is used as a chiral selector in capillary electrophoresis. This study is part of a larger effort to understand the mechanism of chiral recognition in capillary electrophoresis by characterizing the molecular micelle binding of chiral compounds with different geometries and charges. Propranolol and atenolol were chosen because their structures are similar, but their chiral interactions with the molecular micelle are different. Molecular dynamics simulations showed both propranolol enantiomers inserted their aromatic rings into the molecular micelle core and that (S)-propranolol associated more strongly with the molecular micelle than (R)-propranolol. This difference was attributed to stronger molecular micelle hydrogen bonding interactions experienced by (S)-propranolol. Atenolol enantiomers were found to bind near the molecular micelle surface and to have similar molecular micelle binding free energies.

A Molecular Dynamics Simulation Study of the Association of 1,1'-Binaphthyl-2,2'-diyl hydrogenphosphate Enantiomers with a Chiral Molecular Micelle.

Molecular dynamics (MD) simulations were used to investigate the binding of 1,1'-binaphthyl-2,2'-diyl hydrogenphosphate (BNP) enantiomers to the molecular micelle poly-(sodium undecyl-(L,L)-leucine-valine) (poly(SULV)). Poly(SULV) is used as a chiral selector in capillary electrophoresis separations. Four poly(SULV) binding pockets were identified and either (R)-BNP or (S)-BNP were docked into each pocket. MD simulations were then used to identify the preferred BNP binding site. Within the preferred site, both enantiomers formed hydrogen bonds with poly(SULV) and penetrated into the poly(SULV) core. Comparisons of BNP enantiomer binding to the preferred poly(SULV) pocket showed that (S)-BNP formed stronger hydrogen bonds, moved deeper into the binding site, and had a lower poly(SULV) binding free energy than the (R) enantiomer. Finally, MD simulation results were in agreement with capillary electrophoresis and NMR experiments. Each technique showed (S)-BNP interacted more strongly with poly(SULV) than (R)-BNP and that the site of chiral recognition was near the poly(SULV) leucine chiral center.

Granulocyte transfusions in fulminant invasive fungal sinusitis.

PURPOSE: Fulminant invasive fungal sinusitis (IFS) is an aggressive disease seen in patients who are severely neutropenic. The use of granulocyte transfusions to address neutropenia-associated IFS has been described for almost 2 decades. The objectives are to provide our experience using granulocyte transfusions in patients with IFS and to provide a current review of the literature. MATERIALS AND METHODS: A retrospective chart review was performed at the Medical College of Wisconsin to identify patients who received granulocyte transfusions for IFS. Data collected included age, original diagnosis, IFS pathogen, dates, transfusion number, reason for discontinuation, additional therapies, last known follow-up, and status at last known follow-up. A Medline search and manual review of citations within bibliographies was also performed. RESULTS: A total of 20 patients received granulocyte transfusions at the Medical College of Wisconsin between October 2003 and June 2009; 3 of these patients received granulocyte transfusions for fulminant IFS. A total of 22 reported cases of IFS treated with granulocyte transfusions exist in the current literature. CONCLUSIONS: Although limitations to the use of granulocyte transfusions exist, they still represent a viable treatment option in individuals who fail to respond to more conventional therapies.

Knowledge translation: Radiographers compared to other healthcare professionals.

OBJECTIVES: This narrative review examines the current status of evidence-based practice and knowledge translation in diagnostic radiography. It explores knowledge translation efforts in the allied health professions aimed at systematically implementing evidence-based practice and suggests ways that these may be applied within diagnostic radiography. KEY FINDINGS: Knowledge translation in diagnostic radiography is in its infancy with numerous examples of key findings of rigorous studies not implemented in practice. Utilising frameworks, models and theories to systematically translate knowledge into evidence-based practice has been shown to be effective in other allied health professions. Whilst few studies in diagnostic radiography report utilising these systematic approaches to implementing evidence-based practice, those that do, show promising results. Attitudes towards evidence-based practice within diagnostic radiography are becoming more positive and it is important to use this positive shift in attitudes to create real evidence-based change in the profession. CONCLUSION: The potential benefits of systematically translating knowledge into evidence-based practice in diagnostic radiography are wide reaching with positive implications for our patients, the profession and wider community. Leaders at all levels of radiography must work towards implementing evidence-based practice in their daily work. IMPLICATIONS FOR PRACTICE: Systematic approaches to knowledge translation should be adopted and reported in diagnostic radiography in order to more effectively translate knowledge into evidence-based practice.

Investigation of Chiral Recognition by Molecular Micelles with Molecular Dynamics Simulations.

Molecular dynamics simulations were used to characterize the binding of the chiral drugs chlorthalidone and lorazepam to the molecular micelle poly-(sodium undecyl-(L)-leucine-valine). The project's goal was to characterize the nature of chiral recognition in capillary electrophoresis separations that use molecular micelles as the chiral selector. The shapes and charge distributions of the chiral molecules investigated, their orientations within the molecular micelle chiral binding pockets, and the formation of stereoselective intermolecular hydrogen bonds with the molecular micelle were all found to play key roles in determining where and how lorazepam and chlorthalidone enantiomers interacted with the molecular micelle.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) composite biomaterials for bone tissue regeneration: in vitro performance assessed by osteoblast proliferation, osteoclast adhesion and resorption, and macrophage proinflammatory response.

The efficacy of composite materials for bone tissue engineering is dependent on the materials' ability to support bone regeneration whilst inducing a minimal inflammatory response. In this study we examined the in vitro osteogenic and inflammatory properties of poly(3-hydroxybutyrate-co-3-valerate) (PHBV) with various calcium phosphate-reinforcing phases: nano-sized hydroxyapatite (HA); submicron-sized calcined hydroxyapatite (cHA); and submicron-sized beta-tricalcium phosphate (beta-TCP), using bioassays of cultured osteoblasts, osteoclasts, and macrophages. Our study showed that the addition of a nano-sized reinforcing phase to PHBV, whilst improving osteogenic properties, also reduces the proinflammatory response. Proinflammatory responses of RAW264.7/ELAM-eGFP macrophages to PHBV were shown to be markedly reduced by the introduction of a reinforcing phase, with HA/PHBV composites having the lowest inflammatory response. Osteoclasts, whilst able to attach to all the materials, failed to form functional actin rings or resorption pits on any of the materials under investigation. Cultures of osteoblasts (MC3T3-E1) readily attached and mineralised on all the materials, with HA/PHBV inducing the highest levels of mineralization. The improved biological performance of HA/PHBV composites when compared with cHA/PHBV and beta-TCP/PHBV composites is most likely a result of the nano-sized reinforcing phase of HA/PHBV and the greater surface presentation of mineral in these composites. Our results provide a new strategy for improving the suitability of PHBV-based materials for bone tissue regeneration.

New drug binding sites in Ca2+ channels.

New classes of drugs modifying Ca2+ channel activity have become available, this may enlarge the clinical utilities that have been associated with established Ca2+ channel antagonists such as the dihydropyridines (for example, nifedipine). Two such classes are reviewed by Michael Spedding, Barry Kenny and Pierre Chatelain. Fantofarone is a non-dihydropyridine with a novel site of action in the L-type Ca2+ channel that appears to yield a distinct cardiovascular profile. In contrast, fluspirilene and related Na+ and Ca2+ channel inhibitors have a distinct site of action in Ca2+ channels, which is not specific for one channel type. The utility of Na+ and Ca2+ channel inhibitors in ischaemic stroke is compared with new and more selective Na+ channel inhibitors.

The albino-deletion complex of the mouse: molecular mapping of deletion breakpoints that define regions necessary for development of the embryonic and extraembryonic ectoderm.

Previous complementation analyses with five (c11DSD, c5FR60Hg, c2YPSj, c4FR60Hd, c6H) of the mouse albino deletions defined at least two genes on chromosome 7, known as eed and exed, which are necessary for development of the embryonic and extraembryonic ectoderm, respectively, of early postimplantation embryos. The region of chromosome 7 containing these two genes has now been accessed at the molecular level by cloning two of the deletion breakpoint-fusion fragments. The c2YPSj breakpoints were isolated by cloning an EcoRI fragment containing a copy of an albino region-specific repeat unique to c2YPSj DNA. Similarly, the c11DSD breakpoints were isolated by cloning a c11DSD EcoRI fragment detected by a unique-sequence probe mapping proximal to the albino-coat-color locus. By mapping the cloned breakpoints relative to the remaining three deletions, the c11DSD distal breakpoint was found to define the distal limit of the region containing eed, whereas the c2YPSj and c6H distal breakpoints were found to define the proximal and distal limits, respectively, of the region containing exed.

Antivascular therapy of oral tongue squamous cell carcinoma with PTK787.

OBJECTIVES/HYPOTHESIS: Vascular endothelial growth factor (VEGF) is an important mediator in tumor vascularization, growth, and metastasis. We investigated whether blockade of the VEGF receptor (VEGF-R) signaling pathway by the tyrosine kinase inhibitor PTK787 combined with CPT-11, a semisynthetic camptothecin analogue, can inhibit the tumor growth and angiogenesis of squamous cell carcinoma of the oral tongue in an orthotopic nude mouse model. METHODS: JMAR human oral squamous cell carcinoma cells were injected into the tongues of nude mice. Seven days later, the mice were randomized to receive a placebo, daily oral PTK787, weekly CPT-11 injection, or PTK787 plus CPT-11. After 4 weeks of treatment, the mice underwent necropsy, and the tongue tumors, cervical lymph nodes, and lungs were removed for immunohistochemical analyses. RESULTS: CPT-11, PTK787, and PTK787 plus CPT-11 significantly decreased tumor volumes and prolonged survival. The combination treatment group had the most significant decrease in volume and increase in survival. PTK787 alone or in combination with CPT-11 reduced the phosphorylation of VEGF-R in tumor cells and tumor-associated endothelial cells, was associated with decreased microvessel density, a decreased proliferative index, and an increased apoptotic index. PTK787 alone or the combination therapy resulted in apoptosis of both tumor cells and tumor-associated endothelial cells. CONCLUSIONS: These results suggest that targeting VEGF-R tyrosine kinase activity can be an effective therapeutic approach in squamous cell carcinoma of the oral tongue.

Symptom Burden and Functional Gains in a Cancer Rehabilitation Unit.

BACKGROUND/AIMS: To determine if there is a relationship between patient symptoms and functional improvement on inpatient rehabilitation. METHODS: Retrospective review of medical records at an American tertiary referral-based cancer center of all patients admitted to an inpatient rehabilitation unit between 3/1/2013-5/20/2013. Main outcome measures included the Edmonton Symptom and Assessment Scale (ESAS) and Functional Independence Measure (FIM). FINDINGS: The medical records for 71 unique cancer rehabilitation inpatients were analyzed. Statistical analysis of total admission ESAS on total FIM change found no significant relationships. The symptom burden of the patients was mild. Patients demonstrated statistically significant improvements in function and symptoms during inpatient rehabilitation. The mean change in total FIM and total ESAS were an increase of 19.20 and decrease of 7.41 respectively. Statistically significant changes occurred in fatigue, sleep, pain, and anxiety. CONCLUSION: Both symptom and functional scores improved significantly during inpatient rehabilitation. However, no significant relationships were found between symptoms at admission and improvement in FIM.

Implantation of human fetal ventral mesencephalon to the right caudate nucleus in advanced Parkinson's disease.

Disaggregated ventral mesencephalic tissue from single aborted human fetuses of 11 to 18 weeks' gestation was implanted stereotaxically into a consistent striatal site in 12 patients with advanced Parkinson's disease. All were receiving optimum levodopa therapy and were examined preoperatively and at 3,6,9, and 12 months postoperatively. Immunosuppression was not used. There were significant sustained improvements at 12 months in three patients; motor fluctuations were absent in two. There were modest group improvements up to 6 months, with increased quality of "on" and "off" phases, quantity of on times, and specific improvements in contralateral upper limb bradykinesia. Preoperative levodopa requirements were reduced to a mean of 64% at 6 months and 61% at 12 months. Deterioration below baseline ratings occurred in three of nine patients who had consistent follow-up to 12 months. Grafting of midgestational human fetal tissue can lead to improvement in Parkinson's disease. Individual disease severity may be critical, and further trials are needed to identify host factors influencing outcome.

Haemolysin secretion from E coli.

Haemolysin (HlyA) secretion from E coli is directed by a specific C-terminal targeting signal, located within the last 27-50 amino acids, with quite novel characteristics. The HlyA molecule is secreted directly to the medium without a periplasmic intermediate or detectable proteolytic processing. The C-terminal domain of HlyA can also be used to promote the secretion of several other E coli and mammalian proteins. HlyD and HlyB are essential for translocation of HlyA to the medium and we propose that these proteins form a transenvelope complex which initially binds the HlyA signal followed by transport of HlyA to the medium. HlyB is a member of a family of membrane proteins engaged in ATP dependent secretion mechanisms conserved in many organisms including man (P-glycoprotein and the CF protein). In this review we discuss the structure, function and regulation of the secretion mechanism.

Identification of a novel calcium antagonist binding site in rat brain by SR 33557.

1. In K(+)-depolarized taenia preparations from guinea-pig caecum SR 33557 was a potent antagonist of Ca(2+)-induced contractions and antagonized the effect of the calcium channel activator Bay K 8644. 2. SR 33557 displayed high affinity (pKi 9.54 +/- 0.04, nH 1.01) for the [3H]-(+/-)-PN 200-110 binding site in rat cerebral cortex membranes. In the presence of 5 mM Ca2+ this affinity was reduced (pKi 8.82 +/- 0.01, nH 1.05) whilst the affinity of nitrendipine was unaffected by this concentration of Ca2+. 3. Saturation binding experiments in rat cerebral cortex carried out in the absence and presence of SR 33557 (0.1-1.0 nM) indicated an apparently competitive interaction at the dihydropyridine site, in that SR 33557 reduced the KD of [3H]-(+/-)-PN 200-110 binding without any effect on Bmax. In kinetic experiments, the rate of dissociation of [3H]-(+/-)-PN 200-110 from rat cerebral cortex was unchanged in the presence of SR 33557 (5 nM). 4. D-cis-diltiazem fully reversed the inhibition [3H]-nitrendipine binding to rat cerebral cortex produced by SR 33557 indicating the site of action of SR 33557 to be distinct from the dihydropyridine (DHP) binding site. 5. Saturation analysis indicated that [3H]-SR 33557 (0.01-0.8 nM) labelled a single class of binding sites in rat cerebral cortex membranes with high affinity (KD 0.12 +/- 0.01, Bmax 222 +/- 20 fmol mg-1 protein), although kinetic data indicated the existence of negative cooperativity between the binding sites. 6.In competition studies, a variety of different calcium antagonists displayed similar affinity for [3H]-SR 33557 and [3H]-(+/-)-PN 200-110 sites. The [3H]-SR 33557 site was sensitive to the inhibitory effect of divalent cations. The affinity of Cd2+ was 0.026 +/- 0.015 mM and the rank order of affinity was Cd2+ >Ca2+ >Mn2+ >Mg2+ >Na+.7. We propose that SR 33557 labels a distinct site in rat cerebral cortex. The coupling between the SR 33557 and DHP site appears to be very close, resulting in apparently competitive interactions in some experimental protocols but can be revealed as negatively allosteric in other circumstances.

Changes in [3H]-PK 11195 and [3H]-8-OH-DPAT binding following forebrain ischaemia in the gerbil.

1. A high density of [3H]-PK 11195 binding sites was present in gerbil cortical membranes (Bmax [3H]-PK 11195 1360 +/- 71 fmol mg-1 protein) in comparison to rat cortical membranes (254 +/- 21 fmol mg-1 protein). This effect was species-specific as similar findings were obtained with hippocampal membranes (Bmax 1430 +/- 111 fmol mg-1 protein in gerbil, compared to 196 +/- 31 in rat). 2. RO 5-4864, also a peripheral type benzodiazepine compound, displayed low affinity for the [3H]-PK 11195 site in the gerbil (pKi 6.57 +/- 0.02 and 6.70 +/- 0.12 in hippocampus and cortex respectively) compared to rat (pKi 8.16 +/- 0.07 and 8.48 +/- 0.02). Central benzodiazepine compounds, diazepam and flunitrazepam, also displayed this trend. 3. RO 5-4864 displaced [3H]-PK 11195 binding from gerbil and rat cortical membranes through a competitive interaction with Hill slopes close to unity. In both tissues, saturation isotherms of [3H]-PK 11195 binding indicated that the presence of RO 5-4864 caused changes in Kd without any effect on Bmax. In kinetic experiments, the presence of RO 5-4864 failed to modify the rate of dissociation of [3H]-PK 11195 from equilibrium in both rat and gerbil cortical membranes. 4. Forebrain ischaemia in the Mongolian gerbil (5 min bilateral carotid artery occlusion) with 7 days recovery caused a significant (P<0.05) decrease in the density of hippocampal 5-HTlA binding sites labelled by [3H]-8-OH-DPAT (Bmax control, 393 +/- 33 fmol mg-1 protein; ischaemic, 289 +/- 21 fmol mg protein)and an increase (P<0.01) in [3H]-PK 11195 binding sites (Bmax control, 1430 +/- 111 fmol mg-1 protein; ischaemic, 2160 +/- 170 fmol mg-1 protein). Ischaemia and recovery had no effect on the affinity of either ligand.5. Autoradiography experiments in gerbil brain sections revealed that the ischaemia-induced increase in[3H]-PK 11195 binding was consistent and significant in the CA, subfield on the hippocampus (control,152 +/- 42 fmol mg-1 tissue; ischaemic, 314 +/- 43 fmol mg-1 tissue).

Selective antagonism of calcium channel activators by fluspirilene.

1. Fluspirilene has been claimed to bind to a high affinity site in the calcium channel in skeletal muscle. We have investigated its calcium-antagonistic effects in smooth muscle and affinity for the channel in radioligand binding assays. 2. Fluspirilene was weakly active as an antagonist of Ca2(+)-induced contractions in K(+)-depolarized taenia preparations from the guinea-pig caecum, with threshold antagonism starting from concentrations of 30 nM. Nitrendipine, nicardipine and nimodipine were very potent antagonists in this model (threshold antagonism, greater than 1 nM). 3. In contrast, fluspirilene (10-1000 nM) was a potent non-competitive antagonist of the effects of Bay K 8644 (1-3000 nM) on Ca2(+)-induced contractions and, at 10 nM, selectively antagonised the effects of Bay K 8644, abolished the Ca2(+)-channel activator effects of CGP 28392, without changing the calcium antagonist effects of nitrendipine, or modifying the sensitivity of the tissues to Ca2+. In contrast, the dihydropyridines were more effective as antagonists of Ca2+ than of Bay K 8644. Fluspirilene therefore selectively antagonised the effects of dihydropyridine Ca2+ channel activators without affecting the antagonist potency. 4. In radioligand binding experiments, fluspirilene was a potent displacer of [3H]-PN-200-110 binding to rat cerebral cortical membranes (EC50 30 nM), albeit with a low Hill slope (0.66), and was more potent than other lipophilic diphenylalkylamines such as flunarizine and lidoflazine. Fluspirilene interacted non-competitively with [3H]-PN-200-110 and increased dissociation of the radioligand.

[3H]-RS-15385-197, a selective and high affinity radioligand for alpha 2-adrenoceptors: implications for receptor classification.

1. RS-15385-197 is the most potent and selective alpha 2-adrenoceptor antagonist available. We have used [3H]-RS-15385-197 to define alpha 2-adrenoceptor subtypes. The binding of [3H]-RS-15385-197 to membranes of rat cerebral cortex, rat neonatal lung and human platelets was reversible, saturable and of high affinity. Saturation experiments indicated that [3H]-RS-15385-197 bound to a single population of sites in all 3 tissues with high affinity (0.08-0.14 nM). The density of sites labelled by [3H]-RS-15385-197 was greater in the cortex (275 fmol mg-1 protein) than in the neonate lung (174 fmol mg-1 protein) and human platelet (170 fmol mg-1 protein). The density of sites labelled with [3H]-RS-15385-197 in the cortex was significantly greater than that labelled with [3H]-yohimbine (121 fmol mg-1 protein). 2. The selective alpha 2-adrenoceptor antagonists, idazoxan, yohimbine, rauwolscine and WY 26703 displaced [3H]-RS-15385-197 binding to rat cerebral cortex in a simple manner with Hill slopes close to unity. The affinities derived for these antagonists against [3H]-RS-15385-197 were similar to the values obtained for the displacement of [3H]-yohimbine indicating the alpha 2-adrenoceptor nature of the binding site. 3. alpha 2A-Adrenoceptor selective compounds, oxymetazoline and BRL 44409, showed high affinity for [3H]-RS-15385-197 binding in the human platelet and lower affinity in the neonate lung, while the alpha 2B-selective compounds, prazosin and imiloxan, showed high affinity for [3H]-RS-15385-197 binding in the neonate lung.This suggests that [3H]-RS-15385-197 labels both alpha2A- and alpha2B-adrenoceptor subtypes.4. Prazosin and methysergide inhibited the binding of [3H]-RS-15385-197 in the rat cerebral cortex in a simple manner consistent with an interaction at a single site. Although oxymetazoline inhibited [H]-RS- 15385-197 with a Hill slope significantly different from unity, the slope was increased to unity in the presence of Gpp(NH)p, suggesting an agonist-like interaction.5. The site labelled by [3H]-RS-15385-197 in the rat cortex shows high affinity for oxymetazoline and low affinity for prazosin which could be taken as evidence for classifying the site as an alpha2A-subtype.However, the affinities of yohimbine, rauwolscine and oxymetazoline at this site do not correspond to the population of sites in the human platelet. Yohimbine and rauwolscine were 20 fold selective for the platelet alph2A-subtype, whereas phentolamine was 2 fold and imiloxan was 10 fold selective for the cortex subtype. Indeed although the site showed some similarities with the alpha2A-subtype, the highest degree of homology was observed between this site and the rat submaxillary gland and the RG20 clone,tentatively called the alpha2D-adrenoceptor subtype. We propose that the alpha2-adrenoceptor in the rat cortex may therefore correspond to the putative alpha2D-subtype of the adrenoceptor.

Muscarinic activity of McN-A-343 and its value in muscarinic receptor classification.

The affinity and potency of McN-A-343 (4-(m-chlorophenyl-carbamoyloxy) -2-butynyltrimethylammonium chloride) has been assessed at a range of M1 and M2 muscarinic receptors. McN-A-343 was shown to act as a full agonist at M2 receptors present in the guinea-pig isolated taenia caeci (-log EC50 = 5.14). McN-A-343 exhibited no agonist action in the guinea-pig ileum, atria, bladder or trachea. McN-A-343 was not selective in terms of affinity since its dissociation constants at M1 and M2 binding sites in the rat cerebral cortex and myocardium respectively, were very similar (cortical pPKi = 5.05; myocardial pKi = 5.22). The selectivity previously reported for the compound may be due to differences in intrinsic efficacy and/or tissue receptor reserve. Based on differential antagonist affinities, the muscarinic receptor profile of the taenia caeci, trachea and bladder was similar to that observed in the ileum, but dissimiliar to that observed in the atria.

Characterization of an alpha 1D-adrenoceptor mediating the contractile response of rat aorta to noradrenaline.

1. The affinities of a number of alpha 1-adrenoceptor antagonists were determined by displacement of [3H]-prazosin binding from cloned human alpha 1A-adrenoceptors (previously designated cloned alpha 1c subtype), alpha 1B alpha 1D and rat alpha 1D-adrenoceptors, stably expressed in rat-1 fibroblasts. Functional affinity estimates for these compounds were also determined from noradrenaline-mediated contractions of rat aorta. 2. BMY 7378 displayed high affinity for cloned human alpha 1D-adrenoceptors (pKi = 8.2 +/- 0.10) and was selective over alpha 1A (pKi = 6.2 +/- 0.10) and alpha 1B subtypes (6.7 +/- 0.11). WB 4101, benoxathian and phentolamine displayed high affinity for alpha 1A and alpha 1D adrenoceptors compared to the alpha 1B subtype. Spiperone displayed high affinity and selectivity for alpha 1B adrenoceptors (pKi 8.8 +/- 0.16). 5-Methyl-urapidil was selective for cloned alpha 1A adrenoceptors. 3. Comparative binding affinities (pKi) for compounds at cloned human and rat1D adrenoceptors were almost identical (r = 0.99, slope = 1.08). 4. Prazosin, doxazosin and 5-methyl-urapidil were potent, competitive antagonists of noradrenaline-mediated contractions of rat aorta (pA2 values of 9.8, 8.8 and 7.8 respectively). The selective alpha 1D antagonist BMY 7378 was also a potent antagonist on rat aorta (pKB = 8.3 +/- 0.1) but the interaction of this compound was not consistent with competitive antagonism at a single population of receptors. 5. Functional affinities for compounds determined against noradrenaline-mediated contractions of rat aorta correlated well with binding affinities at cloned alpha 1D-adrenoceptors (r = 0.96), but not with alpha 1A (r = 0.61) or alpha 1B (r = 0.46) subtypes. 6. Noradrenaline-mediated contractions of rat aorta were sensitive to the alkylating effects of chlorethylclonidine (CEC). CEC (10 microM) caused a small rightward shift in the noradrenaline concentration-response curve. CEC at 100 microM caused a further shift and suppression of the maximum response to noradrenaline.7. The results of this study suggest that noradrenaline predominantly, but not exclusively, mediates contraction of rat aorta through the activation of an alphalD-adrenoceptor.

Neuroprotective properties of lifarizine compared with those of other agents in a mouse model of focal cerebral ischaemia.

1. Changes in the peripheral type benzodiazepine binding site density following middle cerebral artery occlusion in the mouse, have been used as a marker of neuronal damage. These sites can be identified using the selective ligand [3H]-PK 11195 located on non neuronal cells, macrophages and astroglia, within the CNS. Glial cell proliferation and macrophage invasion is an unvoidable sequelae to cerebral ischaemic injury, secondary to neuronal loss. Following occlusion of the left middle cerebral artery (left MCA) a reproducible lesion was found in the parietal cortex within 7 days which gave rise to a significant increase in [3H]-PK 11195 binding. 2. Treatment of animals with the sodium channel blocker, lifarizine, significantly reduced the ischaemia-induced increase in [3H]-PK 11195 binding when given either 30 min pre-ischaemia and three times daily for 7 days at 0.5 mg kg-1, i.p. (P < 0.01) or delayed until 15 min post-ischaemia and three times daily for 7 days at 0.5 mg kg-1, i.p. (P < 0.001). Lifarizine was an effective neuroprotective agent in this model of focal ischaemia in the mouse. 3. Lifarizine also showed a dose-related protection against the ischaemia-induced increase in [3H]-PK 11195 binding with significant protection at doses of 0.1 mg kg-1, i.p. (P < 0.05), 0.25 mg kg-1, i.p. (P < 0.01) or 0.5 mg kg-1, i.p. (P < 0.01) 15 min post-ischaemia and b.i.d. for 7 days. No significant change is seen in the Kd for [3H]-PK 11195. The first dose could be delayed for up to 4 h after cerebralartery cauterization and protection was maintained.4. Phenytoin (28 mg kg-1, i.v. 15 min and 24 h post-ischaemia) was also neuroprotective in this model(P<0.01). This agent is thought to interact with voltage-dependent sodium channels to effect its anticonvulsantactions and this mechanism may also underlie its neuroprotective actions in focal cerebralischaemia.5. Agents with other mechanisms of action were also shown to have significant neuroprotection in this model. The non-competitive NMDA antagonist, MK 801, showed significant neuroprotection in the model when given at 0.5 mg kg-1, i.p. 30 min pre-ischaemia with t.i.d. dosing for 7 days (P< 0.001). The dihydropyridine calcium antagonist, nimodipine was not protective when given using the same dosing protocol as MK 801, 0.5 mg kg-1 30 min pre-occlusion and three times daily for 7 days but showed significant protection when given at 0.05 mg kg-1 15 min post-ischaemia and three times daily for 7days. The lipid peroxidation inhibitor, tirilazad (single dose 1 mg kg-1, i.v.) showed significant neuroprotection when given 5 min post-ischaemia but not when the first dose was delayed for 4 h.

Pharmacological properties of the cloned alpha 1A/D-adrenoceptor subtype are consistent with the alpha 1A-adrenoceptor characterized in rat cerebral cortex and vas deferens.

1. The pharmacological characteristics of cloned mammalian alpha 1A/D-, alpha 1B- and alpha 1C-adrenoceptor subtypes expressed in rat 1 fibroblasts were determined in comparison to the binding and functional properties of these subtypes in rat tissues. 2. Analysis of [3H]-prazosin binding to membrane homogenates from rat 1 fibroblast cells expressing each of the alpha 1-subtypes indicated high affinity binding to a single population of binding sites. Binding affinities were similar for alpha 1A/D-, alpha 1B- and alpha 1C-subtypes (Kds: 0.13, 0.10 and 0.15 nM respectively) although a higher density of alpha 1B- and alpha 1C-receptors (Bmax: 4068 and 10,323 fmol mg-1 protein respectively) were expressed in comparison to alpha 1A/D (838 fmol mg-1). 3. Displacement of [3H]-prazosin from membranes expressing cloned alpha 1-adrenoceptor subtypes revealed that 5-methyl-urapidil, WB 4101, benoxathian and phentolamine displayed high affinity and selectivity for alpha 1A/D- over alpha 1B-subtypes. These compounds also had high affinity and selectivity for alpha 1C- over alpha 1B-subtypes. 5-Methyl-urapidil showed selectivity for alpha 1C (Ki 0.60 +/- 0.16 nM) over both alpha 1A/D (Ki, 9.8 +/- 2.8 nM) and alpha 1B (Ki 57.2 +/- 12 nM) subtypes. Prazosin and doxazosin were not subtype selective. 4. In comparison to [3H]-prazosin a similar pharmacological profile was obtained with [125I]-HEAT using cloned alpha 1A/D-, alpha 1B- and alpha 1C-adrenoceptors expressed in rat 1 fibroblasts. 5. The affinities of prazosin, WB 4101, 5-methyl-urapidil, phentolamine and benoxathian at cloned alpha 1A/D-receptors were consistent with alpha 1A affinities determined with chlorethylclonidine-treated rat cortical membranes. Affinities at cloned XIB-receptors were consistent with alpha 1B affinities determined with rat liver membranes.6. Using the epididymal rat vas deferens as a functional measure of alpha 1A affinity, prazosin (pA29.23 +/- 0.28), WB 4101 (pA2 9.58 +/- 0.12), phentolamine (pKB 7.90 +/- 0.16), benoxathian (pKB 9.21 +/- 0.21)and 5-methyl-urapadil (pKB 8.51 +/-0.16) were potent antagonists of noradrenaline-induced contractions.7. At present, evidence from cloning studies suggests the existence of at least three alpha 1-adrenoceptor subtypes. In contrast to the recent proposal for alpha l-adrenoceptor classification, the pharmacology of the cloned alpha 1A/D (or alpha lD)-adrenoceptor is more consistent with that of an alpha 1A-adrenoceptor characterized in rat cerebral cortex and vas deferens.