Treatment outcomes of midfacial segment pain: experience from the Liverpool multi-disciplinary team facial pain clinic.

OBJECTIVES: Midfacial segment pain (MSP) has the characteristics of tension-type headache which is confined to the midface cor- responding to the second division of the trigeminal nerve. This review presents treatment outcomes of MSP patients managed at the Multi-disciplinary Team (MDT) Facial Pain Clinic in Liverpool. METHODOLOGY: Prospective clinical outcome performed in a tertiary referral centre for complex facial pain syndromes. MAIN OUTCOME MEASURES: Sino-Nasal Outcome Test (SNOT). Clinical "success" was defined as an improvement in total SNOT score of >9 points and a reduction of the ear-facial symptoms sub-domain score by ≥50% from baseline. RESULTS: The average age of the cohort was 49 years, with an average follow-up of 12 months. The overall pre-treatment total SNOT-22 score was 59.5 which improved significantly to 42 at latest follow-up. Although the average scores of all sub-domains improved, only the ear-facial symptoms and psychological issues sub-domains achieved statistical significance. When the criterion for success was applied, nine patients fulfilled this definition at an average of 12 months follow-up. The baseline total SNOT score in this cohort improved from 60.6 to 19.7. Half of these patients achieved success within 18 months of commencing treatment and the probability of attaining success at long-term follow-up was high. CONCLUSIONS: Treatment of midfacial segment facial pain is complex and requires follow-up to achieve any meaningful clinical outcome.

The Utility of the Sino-Nasal Outcome Test (SNOT) as a Treatment Outcome Measure in Nonsinogenic Facial Pain Syndrome.

INTRODUCTION: The purpose of this study was to assess the strength of correlation between the Sino-Nasal Outcome Test (SNOT) subdomains to determine which bore the greatest disease burden so that it could be used as a suitable measure of clinical response. METHODS: Prospective clinical audit of patients attending the Liverpool Multi-Disciplinary Facial Pain Clinic. The Brief Pain Inventory-Facial (BPI-F) was used as a comparator and surrogate measure of quality of life. RESULTS: Fifty patients (38 female) were reviewed, of whom 66% had midfacial segment pain. The total SNOT score was highly correlated with the BPI-F. Both the sleep function and psychological issues were the most highly correlated SNOT subdomains with the BPI-F, followed by ear-facial symptoms. The rhinologic symptom subdomain had a weak correlation with the BPI-F. The total SNOT score was most significantly correlated with the sleep function and psychological issues subdomains. CONCLUSION: This study supports the utility of the SNOT questionnaire as a measure of clinical outcome for nonsinogenic facial pain. The total SNOT score is highly correlated with the BPI-F, which has been used as a surrogate measure of quality of life. In addition, the sleep function and psychological issues subdomains are suitable variables to measure response to treatment.

Understanding the role of the tumour vasculature in the transport of drugs to solid cancer tumours.

OBJECTIVES: The vasculature of tumours imposes certain barriers that transport of anti-cancer drugs must overcome. Here follows an account of development of a general computational model that describes the mechanisms of drug transport to a solid tumour, with an emphasis on modelling the vasculature using solute transport concepts. MATERIALS AND METHODS: Investigation into the biological parameters that enhance/prevent anticancer drug transport to the tumour provides a means to evaluate the effects of these parameters on the treatment process. Sensitivity analysis of these provides useful insights concerning anticancer drug transport mechanisms from the vasculature to the solid tumour for a non-specified drug and non-specified solid tumour by revealing the conditions that promote or prevent effective drug transport. The effect of the vasculature on transport efficiency is studied using a parametric analysis of some of the transport and biological parameters. Understanding the various transport mechanisms provides a basis to evaluate the effectiveness of the drug treatment a priori. RESULTS: It was found that increases in the capillary hydrostatic pressure, diffusive permeability coefficient and hydraulic conductivity all result in a decrease in tumour size. Similarly, decreases in the interstitium hydrostatic pressure and filtration constant result in a decrease in tumour size. Dependence of the change in the tumour size to changes in these parameters is non-linear. These results demonstrate the potential of the integrated computational model of the tumour and its vasculature to estimate efficacy of a particular treatment process. Regardless of the dependency of the outcome on the assumed model parameters and the assumed kinetics, mathematical models of this type can provide more explanation on the issues related to the transport barriers, the efficacy of the treatment, and the development of effective anticancer drugs. A case study also is presented to demonstrate the model's flexibility to accommodate a two-cell-glioma population.

Experimental studies of the effects of abnormal venous valves on fluid flow.

The effects of variations in the venous valve anatomy are studied experimentally using an artificial system that mimics the bicuspid valves normally found in veins in the lower extremities. The artificial valves are constructed from thin-walled, latex tubing and polyurethane film. The experimental variables in the study are the gap width between the leaflet attachments at the vein wall and the ratio of the sinus depth to vein diameter. The results show that the antegrade mass flow rate is not affected to the same degree when compared to retrograde flow by the various valve configurations examined in this study. The results also indicate that increases in the gap width, which serve to increase the degree of imperfect wall attachment, have less effect on retrograde mass flow rate in valves with deeper sinuses.

4-Alkyl- and 4-cinnamylglutamic acid analogues are potent GluR5 kainate receptor agonists.

Enantiomerically pure (2S,4R)-4-substituted glutamic acids were prepared and tested for homomeric GluR5 and GluR6 kainate subtype receptor affinity. Some of the 4-cinnamyl analogues showed high selectivity and potency (K(i) < 25 nM) for the GluR5 receptors. The greatest selectivity and potency were achieved with the 3-(2-naphthyl)prop-2-enyl compound. This compound, LY339434, has negligible activity at the AMPA and kainate receptors GluR1, -2, -4 and -6. Although, LY339434 shows agonist activity at NMDA receptors in cultural hippocampal neurons (approximate EC(50) of 2.5 microM), we consider that LY339434 should be a useful pharmacological tool for the investigation of the functional role of GluR5 kainate receptors.

[3H]ATPA: a high affinity ligand for GluR5 kainate receptors.

The pharmacological properties of [3H]ATPA ((RS)-2-amino-3(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid) are described. ATPA is a tert-butyl analogue of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid) that has been shown to possess high affinity for the GluR5 subunit of kainate receptors. [3H]ATPA exhibits saturable, high affinity binding to membranes expressing human GluR5 (GluR5) kainate receptors (Kd approximately 13 nM). No specific binding was observed in membranes expressing GluR2 and GluR6 receptors. Several compounds known to interact with the GluR5 kainate receptor inhibited [3H]ATPA binding with potencies similar to those obtained for competition of [3H]kainate binding to GluR5. Saturable, high affinity [3H]ATPA binding (Kd approximately 4 nM) was also observed in DRG neuron (DRG) membranes isolated from neonatal rats. The rank order potency of compounds to inhibit [3H]ATPA binding in rat DRG and GluR5 membranes were in agreement. These finding demonstrate that [3H]ATPA can be used as a radioligand to examine the pharmacological properties of GluR5 containing kainate receptors.

Pharmacological characterization of a GluR6 kainate receptor in cultured hippocampal neurons.

We have examined the pharmacology of kainate receptors in cultured hippocampal neurons (6-8 days in vitro (DIV)) from embryonic rats (E17). Cultured neurons were pre-treated with concanavalin A to remove kainate receptor desensitization and whole-cell voltage clamp electrophysiology employed to record inward currents in response to glutamatergic agonists and antagonists. N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor responses were blocked using MK801 (3 microM) and the 2,3-benzodiazepine, LY300168 (GYKI53655, 50 microM), respectively. Inward currents were recorded in hippocampal neurons upon application of kainate and the 2S,4R isomer of 4-methyl glutamic acid (SYM2081) with EC50 values of 3.4 +/- 0.4 microM and 1.6 +/- 0.5 microM, respectively (n = 6 cells). The GluR5 selective agonists, LY339434 (100 microM) and (RS)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl) propionic acid (ATPA) (100 microM), did not evoke detectable inward currents in any cell responding to kainate. LY293558 and the selective GluR5 antagonist, LY382884, had weak antagonist effects on responses evoked by either kainate or (2S,4R)-4-methyl glutamate (IC50 > 300 microM). The quinoxalinedione, 2,3-dihyro-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX), blocked both kainate and (2S,4R)-4-methyl glutamate-activated currents at much lower concentrations (IC50 approximately 10 microM). These results provide pharmacological evidence that ion channels comprised of GluR6 kainate receptor subunits mediate kainate receptor responses in hippocampal neurons cultured 6-8 DIV.

Decahydroisoquinolines: novel competitive AMPA/kainate antagonists with neuroprotective effects in global cerebral ischaemia.

In the present studies, we have evaluated the activity of a series of glutamate receptor antagonists from the decahydroisoquinoline group of compounds both in vitro and in vivo. Compound activity at alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate receptors was assessed using ligand binding to cloned iGluR2 and iGluR5 receptors and on responses evoked by AMPA and N-methyl-D-aspartate (NMDA) in the cortical wedge preparation. In vivo, compounds were examined for antagonist activity electrophysiologically in the rat spinal cord preparation and in the gerbil model of global cerebral ischaemia. Compounds tested were LY293558, which has been shown to protect in models of focal cerebral ischaemia, LY202157 (an NMDA antagonist), LY246492 (an NMDA and AMPA receptor antagonist), LY302679, LY292025, LY307190, LY280263, LY289178, LY289525, LY294486 (AMPA/kainate antagonists) and LY382884 (an iGluR5 selective antagonist). Results obtained support a role for AMPA receptors in cerebral ischemia. LY377770 (a mixed AMPA/iGluR5 antagonist and active isomer of LY294486) demonstrated good neuroprotection with a 2-h time window and may therefore be useful in the treatment of ischaemic conditions.

LY339434, a GluR5 kainate receptor agonist.

The activity of a gamma-substituted glutamate analogue, (2S, 4R, 6E)-2-amino-4-carboxy-7-(2-naphthyl)hept-6-enoic acid (LY339434) and (2S,4R)-4-methylglutamic acid at ionotropic glutamate receptors has been examined. Ligand binding studies were performed using [3H] AMPA binding to membranes expressing either homomeric recombinant GluR1, GluR2, GluR4 receptors, and [3H] kainate binding to GluR5 and GluR6 kainate receptors. LY339434 and (2S,4R)-4-methylglutamic acid showed selectivity in ligand binding studies for kainate receptors over AMPA receptors. Within the kainate class of glutamate receptors, LY339434 showed selectivity for GluR5 over GluR6 whereas (2S,4R)-4-methylglutamic acid showed high affinity for both GluR5 and GluR6 kainate receptors. Examination of the functional activity of LY339434 and (2S,4R)-4-methylglutamic acid showed that both compounds evoked inward currents in dorsal root ganglion neurons (DRG) with estimated EC50 values of 0.8 +/- 0.2 microM and 0.17 +/- 0.04 microM, respectively. In GluR5 expressing HEK 293 cells, LY339434 evoked inward currents with an estimated EC50 value of 2.5 +/- 0.9 microM but had little effect on GluR6 expressing cells at concentrations less than 100 microM. LY339434 was a weak AMPA receptor agonist (EC50 values > 300 microM) as determined by activity in acutely isolated cerebellar Purkinje neurons. LY339434 and (2S,4R)-4-methylglutamic acid had agonist activity at NMDA receptors studied in cultured hippocampal neurons with EC50s of 2.5 microM and 11.7 microM, respectively. These results indicate that both LY339434 and (2S,4R)-4-methyl glutamic acid may be useful pharmacological tools for the examination of kainate receptors.

Kainate GluR5 receptor subtype mediates the nociceptive response to formalin in the rat.

In order to study the roles of the AMPA and kainate subtypes of non-NMDA glutamate receptors in the processing of persistent nociceptive information, compounds with varying activities at these receptors were examined for effects on the formalin-induced paw-licking behavior in rats. The selective AMPA antagonist, LY300164 and the mixed AMPA/kainate antagonist, NBQX, were compared for their effects on formalin-induced pain behavior. NBQX (3, 10, 20 mg/kg, i.p.), caused antinociception as well as ataxia whereas the selective AMPA antagonist, LY300164 (3,5,10 mg/kg, i.p.), did not cause antinociception at doses that did not produce ataxia. In view of the well documented distribution of kainate receptors on C fibres and of the kainate-preferring iGluR5 subtype on dorsal root ganglia (DRG), we tested a series of three decahydroisoquinolines with different profiles of activity between iGluR5 and AMPA receptors and all without activity on iGluR6, iGluR7 or KA2 subtypes. LY293558 (0.1, 1, 3, 5 mg/kg, i.p.), which had low micromolar affinity for both iGluR5 and 2 caused, like NBQX, both antinociceptive and ataxic effects. However, the selective iGluR5 antagonist LY382884 (5, 10, 30, 100 mg/kg, i.p.), exhibited antinociceptive actions without ataxia while the iGluR2 preferring antagonist LY302679 (5 mg/kg, i.p), caused ataxia but did not produce antinociceptive effects at that dose. These actions were stereoselective since the enantiomeric compounds, LY293559 and LY302680, were ineffective in these tests. The data strongly suggest an involvement of iGluR5 in the processing of nociceptive information.

Synthesis of willardiine and 6-azawillardiine analogs: pharmacological characterization on cloned homomeric human AMPA and kainate receptor subtypes.

Both willardiine and azawillardiine analogs (18-28) have been reported to be potent and selective agonists for either AMPA or kainate receptors. We report here the novel synthesis and pharmacological characterization of a range of willardiine (18-23) and 6-azawillardiine (24-28) analogs on cells individually expressing human homomeric hGluR1, hGluR2, hGluR4, or hGluR5 receptors. Reaction of the sodium salts of substituted uracils (7-12) or 6-azauracils (13-16) with (S)-3-[(tert-butoxycarbonyl)amino]oxetan-2-one (17) in dry DMF, subsequent deprotection in TFA, and purification by ion-exchange chromatography gave mainly the willardiine analog in which alkylation took place on N1 of the uracil ring. We have investigated the subtype selectivity of these compounds by examining their binding affinity for homomeric hGluR1, -2, -4, or -5 (and hGluR6 in the case of 5-iodowillardiine (22)). From this study we have demonstrated that 22 has high affinity for hGluR5 and, compared to kainate, displays excellent selectivity for this receptor over both the AMPA receptor subtypes and the homomeric kainate receptor, hGluR6. 5-Fluorowillardiine (19) has higher affinity than AMPA for both homomeric hGluR1 and hGluR2 and compared to AMPA displays greater selectivity for AMPA receptor subtypes over the kainate receptor, hGluR5. Some structural features required for optimal activity at homomeric AMPA or kainate receptor subtypes have also been identified. It would appear that quite large lipophilic substituents at the 5-position of the uracil ring not only are accommodated by hGluR5 receptors but also lead to enhanced affinity for these receptors. In contrast to this, for optimal binding affinity to hGluR1, -2, or -4, smaller, electron-withdrawing substituents are required. For optimal activity at hGluR4 receptors a 6-aza-substituted willardiine is favored. The subtype-selective compounds described here are likely to be useful tools to probe the distribution and the physiological roles of the various glutamate receptor subunits in the central nervous system.

A hippocampal GluR5 kainate receptor regulating inhibitory synaptic transmission.

The principal excitatory neurotransmitter in the vertebrate central nervous system, L-glutamate, acts on three classes of ionotripic glutamate receptors, named after the agonists AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxalole-4-propionic acid), NMDA (N-methyl-D-aspartate) and kainate. The development of selective pharmacological agents has led to a detailed understanding of the physiological and pathological roles of AMPA and NMDA receptors. In contrast, the lack of selective kainate receptor ligands has greatly hindered progress in understanding the roles of kainate receptors. Here we describe the effects of a potent and selective agonist, ATPA ((RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid) and a selective antagonist, LY294486 ((3SR, 4aRS, 6SR, 8aRS)-6-((((1H-tetrazol-5-yl) methyl)oxy)methyl)-1, 2, 3, 4, 4a, 5, 6, 7, 8, 8a-decahydroisoquinoline-3-carboxylic acid), of the GluR5 subtype of kainate receptor. We have used these agents to show that kainate receptors, comprised of or containing GluR5 subunits, regulate synaptic inhibition in the hippocampus, an action that could contribute to the epileptogenic effects of kainate.

Synthesis of a series of aryl kainic acid analogs and evaluation in cells stably expressing the kainate receptor humGluR6.

The synthesis and pharmacological characterization of a novel series of 4-aryl-substituted kainic acid analogs are described. Receptor affinities were determined on recombinantly expressed humGluR6 kainate receptors and on [3H]kainate binding to rat forebrain kainate receptors. Functional agonist potencies were assessed using whole cell voltage clamp recordings in cells expressing humGluR6 receptors. Substitution of phenyl for the methyl at the C-4 position of kainic acid produced 11 which has high affinity and agonist potency at the GluR6 receptor. Substitution on phenyl led to a series of compounds with varying affinity for this kainate receptor. Agonist potency correlated with receptor affinity and with no derivative could antagonist activity be identified. Affinities for the humGluR6 kainate receptor were approximately 10-50 less than the observed affinities at rat forebrain kainate receptors. Furthermore, within the series of 4-aryl-substituted kainic acid analogs, there was a high degree of correlation between binding affinities for humGluR6 receptors and competition with kainate binding to rat forebrain kainate receptors.

Temporal image database design for outcome analysis of lung nodule.

This paper presents the design of a temporal image database system and its application in thoracic imaging. The design of this information system is based on the client/server architecture. The system consists of a chest imaging database server, a library of image processing modules, a link to the picture archiving and communication system (PACS) archive, and a low end client workstation with motif-based graphic user interface (GUI). The database system can be used to aid the radiologists in quantitating solitary or multiple long nodules and in assessing effectiveness of therapeutic procedures for these lung cancers. The GUI allows a user to retrieve any patient study from PACS. After a nodule is visually identified, it will be segmented automatically to obtain relevant features, such as the center of mass, volume, and surface area. Such 3D nodule information, together with the patient textual information, is subsequently organized in the chest imaging database to facilitate outcome analysis.

Cloning, expression and pharmacological characterization of a human glutamate receptor: hGluR4.

A member of the ionotropic family of glutamate receptors, hGluR4, was isolated from a human cDNA library and characterized following expression in mammalian cell lines. Human GluR4 possessed a 99% amino acid and 92% nucleotide homology to that of its rat counterpart with sequence differences restricted to the carboxy and amino terminal regions of the molecule. Transfection of simian kidney cells (COS-1) with an hGluR4 expression plasmid resulted in the transient formation of a membrane protein that possessed high specific binding for [3H](RS)-alpha-amino- 3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA) but not [3H]kainate. Competition studies yielded a displacement profile of AMPA = quisqualate > glutamate > domoate > kainate >> N-methyl-D-aspartate (NMDA) or dihydrokainate. Whole-cell, voltage-clamp recordings from a human embryonic kidney cell line (HEK 293) stably expressing hGluR4 confirmed the presence of constitutively active, ligand-gated ion channels activated by AMPA, glutamate and kainate but not N-methyl-D-aspartate. Kainate-evoked currents were reversibly attenuated by 6-cyano-7-nitro- quinoxaline-2,3-dione (CNQX) but not DL-2-amino-5- phosphonovalerate (DL-AP5). Agonist-evoked currents exhibited inward rectification and ion substitution experiments indicated that hGluR4 receptor-linked ion channels in their homomeric state are permeable to both CA2+ and Na+ ions. In the same cell line antibody to rat GluR4 immunoprecipitated a major protein band at approximately 108 kDa and a minor one at approximately 340 kDa. The immunoblot analysis of membranes chemically crosslinked with dithiobis(succinimidylpropionate) showed a broad band at 550-600 kDa suggesting that the GluR4 receptor forms a pentamer in situ. This is the first report of the cloning of hGluR4 receptor and its stable expression in a human cell line.

cDNA cloning and functional properties of human glutamate receptor EAA3 (GluR5) in homomeric and heteromeric configuration.

We have isolated a new member of the human glutamate receptor family from a fetal brain cDNA library. This cDNA clone, designated EAA3a, shares a 90% nucleotide identity with the previously reported rat GluR5-2b cDNA splice variant and differed from human GluR5-1d in the amino and carboxy terminal regions. Cell lines stably expressing EAA3a protein formed homomeric ligand-gated ion channels responsive, in order of decreasing affinity to domoate, kainate, L-glutamate and (RS)-alpha-amino-3-hydroxy-5- methylisoxazole-propionate (AMPA). Kainate-evoked currents showed partial desensitization that was reduced on incubation with concanavalin A (conA) but not cyclothiazide and were attenuated by the non-N-methyl-D-aspartate (NMDA) receptor antagonist CNQX (6-cyano-7-nitro-quinoxalinedione). Coexpression of EAA3a and human EAA1 cDNAs in HEK 293 cells formed a heteromeric channel with unique properties. Kainate and AMPA activated the heteromeric channel with significantly higher affinities than observed for EAA3a alone. Ligand binding studies with the recombinant EAA3a receptor expressed in mammalian cells indicated a high affinity kainate binding site (Kd = 120 +/- 15.0 nM). The relative potency of compounds in displacing [3H]-kainate binding to EAA3a receptor was: domoate > kainate > L-glutamate = quisqualate > 6,7-dinitroquinoxaline-2,3-dione (DNQX) = CNQX > AMPA > dihydrokainate > NMDA.

Functional expression and pharmacological characterization of the human EAA4 (GluR6) glutamate receptor: a kainate selective channel subunit.

A cDNA encoding an ionotropic glutamate receptor subunit protein humEAA4 (GluR6), has been cloned from a human fetal brain library. This cDNA when expressed in COS or HEK-293 cells is associated with high-affinity kainate receptor binding and ion channel formation. We have successfully established cell lines stably expressing humEAA4 in HEK-293 cells This is the first report of the establishment of stable cell lines expressing a glutamate receptor channel. The relative potency of compounds for displacing [3H]-kainate binding to humEAA4 receptors expressed in COS or HEK-293 cells is domoate > kainate > quisqualate > 6-cyano-7-nitroquinoxaline-2,3-dione > L-glutamate = 6,7- dinitroquinoxaline-2,3-dione > dihydrokainate. Applications of kainate, glutamate, and domoate but not AMPA evoked rapidly desensitizing currents in cells expressing homo-oligomeric humEAA4 in a concentration dependent manner. The order of potency was: domoate > kainate > L-glutamate. Although AMPA did not itself activate humEAA4 receptors it did reduce, to a limited extent, kainate-evoked responses. AMPA may therefore be a weak partial agonist for this receptor. To date this effect has not been demonstrated with rat GluR6. It is possible that subtle species differences may exist in the nature of agonist receptor interaction. Kainate evoked currents were attenuated by the quinoxalinediones CNQX and DNQX but not by DAP5. The receptor desensitization was attenuated on application of concanavalin A. Ion-permeability studies indicated that the receptor-linked ion channel is permeable to both Na+ and Ca2+ ions.

Molecular characterization of the human EAA5 (GluR7) receptor: a high-affinity kainate receptor with novel potential RNA editing sites.

Several cDNA clones encoding EAA5 receptor polypeptides were isolated from a human fetal brain library. The EAA5 cDNAs demonstrated an 88.7-90.1% nucleotide identity with rat GluR7 cDNAs. The nucleotide sequence of EAA5 would encode a 919-amino acid protein, that has a 97.7-98.9% identity with the rat GluR7 receptor. Two variation of the EAA5 cDNA were identified which result in amino acid substitutions in the predicted extracellular amino-terminal region; Ser310-->Ala and Arg352-->Gln. These variations can be attributed to RNA editing involving T-->G and G-->A substitutions. Both the location (with respect to glutamate receptors), and the nucleotides involved, in this putative RNA editing are novel and may therefore involve novel mechanisms. Ligand binding studies with membranes of transfected COS-1 cells expressing EAA5 polypeptides demonstrate a rank order of ligand affinity similar to that observed with the rat GluR7 receptor, and a dissociation constant for kainate (2.72 +/- 0.12 nM (n = 3)) that is approximately 20- to 30-fold higher than that observed for the rat GluR7 receptor. All of the ligands tested had a higher affinity for the human EAA5 receptor as compared to the rat GluR7 receptor. This report provides another example of pharmacological differences for similar receptors across species.

Investigation of alpha 1-adrenoceptor subtypes in canine aorta, using alkylating agents.

The ability of putative selective irreversible ligands SZL-49 (1-(4-amino-6,7-dimethoxy-2,5-diene-2-carbonyl)) and CEC (chlorethylclonidine), for alpha 1A and alpha 1B adrenoceptor subtypes, respectively, to affect alpha 1-adrenoceptors of canine aorta microsomal membranes was investigated. These membranes contain an apparently homogeneous population of [3H]prazosin binding sites. SZL-49, like phenoxybenzamine, abolished all binding of [3H]prazosin. CEC abolished 75% of the prazosin binding sites under the most stringent conditions we applied. However, the remaining 25% of binding sites was identical in affinity for prazosin with control membranes, and competition studies of other subtype-selective ligands revealed unchanged ability to complete against CEC-sensitive and -insensitive sites. We concluded that SZL-49 and CEC are not alpha 1A- and alpha 1B-adrenoceptor selective under in vitro conditions. Our data led to the hypothesis that canine aortic membranes contain exclusively alpha 1B-adrenoceptors but that current tools for identifying alpha 1-adrenoceptor subtypes proved inadequate in vitro in this study.

Alpha-adrenoceptors in vascular smooth muscle: all is not well.

Studies of binding interactions and contractile responses of vascular muscles at alpha 1- and alpha 2-adrenoceptors revealed the following. (1) Agonists at alpha 1- and alpha 2-adrenoceptors may achieve selectivity by virtue of different efficacies despite similar affinities at the two receptors as well as by differing affinities. This implies that their potencies in binding studies may not correlate with potencies in response and that an agonist may produce positive or negative interactions by occupying both alpha 1- and alpha 2-receptors. (2) Agonists at alpha 2-adrenoceptors have the ability in some vascular muscles to release internal Ca2+ (implying an inositol triphosphate mechanism) as well as open Ca2+ channels. However, their contractile abilities are not closely related to function of Na+/H+ or Na+/Ca2+ exchange sites. Amiloride derivatives probably inhibit contractile effects of alpha-agonists and K+ elevation by an action at sites distal to the receptor or Ca channels. (3) The failure of alpha 2-agonists to contract arteries in vitro is not related to the absence of these receptors but most likely to their uncoupling from contractile responses, possibly owing to changes related to the in vitro condition (loss of modulating endogenous substances present in vivo such as angiotensin II or endothelins or to changed physical conditions such as may alter function of stretch-activated channels).