The advantages of haplotype analysis of the promoter region of the human apolipoprotein E gene.

Polymorphisms in the regulatory region of the human apolipoprotein E gene (gene, APOE; protein, apoE) have been implicated in Alzheimer's disease. Here we describe in detail the advantages of a simple method for haplotype analysis of this region (at -491 and -427 bases relative to the transcription start site of the gene). The promoter region of the APOE gene was amplified by polymerase chain reaction (PCR) and this fragment was then used as a template for PCR with "nested" primers to generate a 228-bp product incorporating both the -491 and the -427 loci. PCR products were then digested with DraI and AluI together and subjected to polyacrylamide gel electrophoresis. The distinct pattern of bands appearing on the gel was then used to ascribe [-491,-427] haplotypes to each subject, from which -491 and -427 genotypes were inferred. -491 and -427 genotypes were also confirmed by digestion with DraI alone or AluI alone. Haplotype analysis was successful in all 20 samples analyzed and was 100% consistent with genotyping. We suggest that this is a reliable, time-saving method that the will be useful in large-scale APOE promoter genotyping studies.

Effects of sevoflurane on dopamine, glutamate and aspartate release in an in vitro model of cerebral ischaemia.

Release of excitatory amino acids and dopamine plays a central role in neuronal damage after cerebral ischaemia. In the present study, we used an in vitro model of ischaemia to investigate the effects of sevoflurane on dopamine, glutamate and aspartate efflux from rat corticostriatal slices. Slices were superfused with artificial cerebrospinal fluid at 34 degrees C and episodes of 'ischaemia' were mimicked by removal of oxygen and reduction in glucose concentration from 4 to 2 mmol litre(-1) for < or = 30 min. Dopamine efflux was monitored in situ by voltammetry while glutamate and aspartate concentrations in samples of the superfusate were measured by HPLC with fluorescence detection. Neurotransmitter outflow from slices was measured in the absence or presence of sevoflurane (4%). After induction of ischaemia in control slices, there was a mean (SEM) delay of 166 (7) s (n = 5) before sudden efflux of dopamine which reached a maximum extracellular concentration of 77.0 (15.2) micromol litre(-1). Sevoflurane (4%) reduced the rate of dopamine efflux during ischaemia (6.90 (1.5) and 4.73 (1.76) micromol litre(-1) s(-1) in controls and sevoflurane-treated slices, respectively; P<0.05), without affecting its onset or magnitude. Excitatory amino acid efflux was much slower. lschaemia-induced glutamate efflux had not reached maximum after 30 min of ischaemia. Basal (pre-ischaemic) glutamate and aspartate efflux per slice was 94.8 (24.8) and 69.3 (31.5) nmol litre(-1) superfusate (n = 4) and was not significantly reduced by 4% sevoflurane. lschaemia greatly increased glutamate and aspartate efflux (to a maximum of 919 (244)% and 974 (489)% of control, respectively). However, ischaemia-induced efflux of both glutamate and aspartate was significantly reduced by 4% sevoflurane (P < 0.001 for glutamate, P < 0.01 for aspartate). In summary, sevoflurane may owe part of its reported neuroprotective effect to a reduction of ischaemia-induced efflux of excitatory amino acids and, to a lesser extent, dopamine.

Effects of chronic tramadol on pre- and post-synaptic measures of monoamine function.

The atypical analgesic tramadol has strong structural similarities to the antidepressant venlafaxine and is a mixed noradrenaline (NA) and serotonin (5-HT) uptake inhibitor. Because tramadol has been found active in the forced swim test, a common predictor of antidepressant efficacy, we therefore examined the effects of chronic tramadol on various pre- and post-synaptic monoamine measures. Male Wistar rats (150-200 g) received tramadol (20 mg/kg i.p.) or vehicle for 21 days and were sacrificed 24 h after the last dose. Quantitative autoradiography revealed that specific frontocortical [3H]dihydroalprenolol and [3H]ketanserin binding was lower in the chronic tramadol group than controls (beta: 37+/-8 and 217+/-56 fmol/mg; 5-HT2A: 23+/-3 and 44+/-7 fmol/mg, respectively, p < 0.05). Chronic tramadol had no effect on the magnitude of electrically stimulated noradrenaline (NA) efflux or uptake in locus coeruleus (LC) slices. Although dexmedetomidine (10 nM) decreased LC NA efflux equally (by approximately 60%) in chronic tramadol and vehicle groups, desipramine (50 nM) increased LC NA efflux more in vehicle (to 164+/-7%) than tramadol-treated rats (144+/-6%; p < 0.05). Chronic tramadol had no effect on dorsal raphé (DRN) or median raphé (MRN) 5-HT efflux. However, 5-HT uptake in tramadol-treated rats was slower (p < 0.05) in MRN and nearly so (p = 0.055) in DRN. The selective 5-HT1A agonist 8-OH-DPAT reduced 5-HT efflux in both DRN and MRN. Its effect in DRN was greater in rats given chronic tramadol than in vehicle controls (54+/-2 versus 32+/-6% reduction in 5-HT efflux, respectively). In conclusion, we suggest that tramadol has many of the pre- and postsynaptic neurochemical features of a conventional antidepressant, as might be predicted from its pharmacology.

Comparison of ketamine stereoisomers on tissue metabolic activity in an in vitro model of global cerebral ischaemia.

Ketamine (2-o-chlorophenenyl-2-methylaminocyclohexanone hydrochloride) is a dissociative general anaesthetic with neuroprotective properties. Since ketamine is optically active, we compared the neuroprotective efficacy of the (+)- or (-)-enantiomers in global cerebral ischaemia. Rat corticostriatal slices superfused with, or incubated in, artificial CSF at 34 degrees C were subjected to a brief ischaemic insult. Dopamine efflux was measured using fast cyclic voltammetry. Tissue metabolism was determined with 2,3,5-triphenyltetrazolium chloride staining, a marker of mitochondrial enzyme activity. In control slices, ischaemia caused rapid striatal dopamine release (to 122 microM over 18 s) after an initial delay of 149s. Racemic ketamine (100 micromol/l) significantly delayed (by 24%, P<0.05), slowed (by 63%, P<0.01) and reduced (by 27%, P<0.05) ischaemia-induced dopamine release. Ischaemia (10 min) also caused significant decreases in striatal (25%, P<0.01) and cortical (31%, P<0.001) metabolic activity, manifested as a drop in mean TTC staining intensity. Racemic ketamine and its (+)- and (-)-enantiomers (each 100 microM) attenuated the loss of metabolic activity in the striatum. However, in the cortex, only (+)-ketamine (100 microM) was significantly neuroprotective. We conclude that neuroprotection by ketamine in cerebral ischaemia is both region- and isomer-dependent.

LY393615, a novel neuronal Ca(2+) and Na(+) channel blocker with neuroprotective effects in models of in vitro and in vivo cerebral ischemia.

In the present studies we have examined the effects of a new calcium channel blocker, LY393615 ((N-Butyl-[5,5-bis-(4-fluorophenyl)tetrahydrofuran-2-yl]methylamine hydrochloride, NCC1048) in a model of hypoxia-hypoglycaemia in vitro and in a gerbil model of global and in two rat models of focal cerebral ischaemia in vivo. Results indicated that LY393615 protected against hypoxia-hypoglycaemic insults in brain slices and also provided significant protection against ischaemia-induced hippocampal damage in gerbil global cerebral ischaemia when dosed at 10, 12.5 (P<0.05) or 15 mg/kg i.p. (P<0.01) 30 min before and 2 h 30 min after occlusion. The compound penetrated the brain well after a 15 mg/kg i.p. dose and had a half-life of 2.5 h. In further studies LY393615 was protective 1 h post-occlusion when administered at 15 mg/kg i.p. followed by 2 doses of 5 mg/kg i.p. 2 and 3 h later. LY393615 dosed at 15 mg/kg i.p. followed by 2 further doses of 5 mg/kg i.p. (2 and 3 h later) also produced a significant reduction in the infarct volume following Endothelin-1 (Et-1) middle cerebral artery occlusion in the rat when administration was initiated immediately (P<0.01) or 1 h (P<0.05) after occlusion. The compound was also evaluated in the intraluminal monofilament model of focal ischaemia. The animals had the middle cerebral artery occluded for 2 h, and 15 min after reperfusion LY393615 was administered at 15 mg/kg i.p. followed by 2 mg/kg/h i.v. infusion for 6 h. There was no reduction in infarct volume using this dosing protocol. In conclusion, in the present studies we have reported that a novel calcium channel blocker, LY393615, with good bioavailability protects against neuronal damage caused by hypoxia-hypoglycaemia in vitro and both global and focal cerebral ischaemia in vivo. The compound is neuroprotective when administered post-occlusion and may therefore be a useful anti-ischaemic agent.

Rapid quantification of ischaemic injury and cerebroprotection in brain slices using densitometric assessment of 2,3,5-triphenyltetrazolium chloride staining.

2,3,5-Triphenyltetrazolium chloride (TTC), a marker of mitochondrial enzyme activity, is widely used to assess the effects of cerebral ischaemia in vivo. In the present study, we characterised its utility as a simple rapid macrohistological measure of ischaemic damage in brain slices. Coronal rat corticostriatal slices were incubated in oxygenated artificial cerebrospinal fluid (aCSF) until subjected to 'ischaemia' (deoxygenated, hypoglycaemic aCSF) for up to 12 min. After a further 30 min to 16 h of reincubation in oxygenated aCSF, slices were stained with TTC, fixed with formalin and transferred to cover slips. The slices were scanned in 8-bit greyscale using a standard desktop scanner and the staining analysed by densitometry of the acquired images. Control slices stained a rich pink/red. Ischaemia (10 min) reduced both the area and intensity of staining. Both measures of striatal staining were negatively correlated with the duration of ischaemia (0-12 min). Furthermore, staining in the striatum correlated significantly with cortical TTC staining. The effects of TTC concentration (0.063-0.5% w/v) and post-ischaemic interval (30 min to 16 h) were examined upon the intensity of TTC staining. (+)-MK 801 prevented the ischaemia-induced reduction in TTC staining, consistent with cerebroprotection. These data suggest that TTC staining of brain slices may be used to quantify ischaemic injury and cerebroprotection.

Control of dorsal raphé 5-HT function by multiple 5-HT(1) autoreceptors: parallel purposes or pointless plurality?

The serotonergic cells of the dorsal raphé nucleus innervate much of the forebrain and are thought to be involved in the mechanism of action of antidepressants. Dysfunction of these cells might be involved in the neural mechanisms underlying depression and suicide. The traffic in pathways emanating from the dorsal raphé nucleus is controlled by 5-HT(1) autoreceptors. Until recently it was thought that the autoreceptors in the dorsal raphé nucleus were solely of the 5-HT(1A) subtype. In this article, we discuss evidence that the situation is more complex and that multiple 5-HT(1) subtypes govern different aspects of 5-HT function in the dorsal raphé nucleus presenting new therapeutic opportunities.

Brain dopamine transporter messenger RNA and binding sites in cocaine users: a postmortem study.

BACKGROUND: Results of recent radioligand binding experiments suggest that chronic cocaine exposure increases dopamine transporter (DAT) synthesis throughout the striatum of humans. However, detection of cocaine binding site increases in animals and humans has varied depending on the radioligand used. The present experiment tested the hypothesis in cocaine-using humans that synthesis of midbrain DAT messenger RNA increases parallel with increased striatal DAT binding sites. METHODS: Striatal and midbrain samples were collected during autopsy examination from human cocaine users (n = 34) and from age-, sex-, and race-matched control subjects (n = 36). Levels of DAT messenger RNA were quantified in the medial and lateral midbrain regions using in situ hybridization, and striatal DAT binding sites were assessed by quantitative autoradiography using the DAT-specific radioligand [3H]WIN 35428. RESULTS: Striatal DAT binding sites were markedly increased in cocaine users, but, paradoxically, medial DAT messenger RNA levels were decreased. CONCLUSION: Cocaine exposure has a marked effect on DAT function, but the mechanisms involved may be complex.

Cocaine, ethanol, and genotype effects on human midbrain serotonin transporter binding sites and mRNA levels.

OBJECTIVE: Earlier platelet and postmortem brain studies have found alterations in serotonin transporter function in ethanol-abusing human subjects. The present investigation tested the hypothesis that brain serotonin transporter function is altered in chronic users of ethanol and cocaine, which might be related to a common serotonin transporter promoter polymorphism. METHOD: Serotonin transporter binding sites, serotonin transporter mRNA levels, and serotonin transporter promoter variants were quantified in postmortem samples from a group of human subjects who had been ethanol users or cocaine users and then compared to those of a matched group of comparison subjects. Quantitative autoradiographic and in situ hybridization assays were performed in midbrain samples that contained the dorsal and median raphe nuclei (the location of serotonin cell bodies that innervate the forebrain). RESULTS: There was a significant overall cocaine-by-ethanol-by-genotype interaction. Dorsal raphe [125I]CIT binding to the serotonin transporter was lower in cocaine users than in comparison subjects. In addition, serotonin transporter binding and serotonin transporter mRNA levels varied significantly by genotype. It was also found that serotonin transporter binding in subjects with either the short or heterozygote genotype was significantly higher in the ethanol-user subjects. CONCLUSIONS: Serotonin transporter binding sites were regulated in a region-specific and substance-specific pattern, which was not simply a local response to functional blockade. Also, a reciprocal relationship appeared to exist between cocaine and ethanol effects in the dorsal raphe, which may have interesting clinical implications for dual-diagnosis patients. It is possible that serotonin transporter promoter genotype may play a complex role in chronic ethanol dependence.

Expression of serotonin transporter mRNA in human brainstem raphe nuclei.

The distribution of the messenger RNA (mRNA) encoding the serotonin transporter was investigated in the entire extent of the ascending raphe systems of the human brainstem, using a sensitive cRNA probe. This mRNA is abundant in the neurons of the raphe nuclei, especially in the dorsal and median raphe. The subregional distribution of this mRNA closely corresponds to that described for tryptophan hydroxylase in immunocytochemical studies. It is expressed at the highest levels in ventral and ventrolateral subregions of the dorsal raphe, with lower levels of expression in the median raphe, oral pontine reticular nuclei, and the supralemniscal cell groups. These results provide a description of the distribution of this mRNA in normal subjects and will serve as the basis for future studies of the subregional distribution of this mRNA in the brains of depressed patients and those who have committed suicide.

Effects of catecholamines on isolated human colonic smooth muscle.

1. The effects of catecholamines and some adrenoceptor agonists and antagonists on isolated preparations of human colonic smooth muscle obtained from surgical resections were examined. 2. Strips of circular smooth muscle displayed rhythmic myogenic spontaneous contractions which were inhibited by catecholamines with an order of potency of isoprenaline (1.0) > noradrenaline (0.32) > adrenaline (0.2). Phentolamine (0.7 microM) significantly shifted the noradrenaline concentration-response curve (CRC) to the right but had no significant effect on isoprenaline or adrenaline. Propranolol (1 microM) significantly shifted the isoprenaline to the right but had no significant effect on noradrenaline or adrenaline. 3. Salbutamol (30 microM) had no inhibitory effect on the spontaneous activity and ICI 118,551 (1 microM) had no effect on inhibitory responses to isoprenaline. Betaxolol (1 microM) significantly shifted the CRC to isoprenaline to the right. BRL 37344 had no effect on spontaneous activity. 4. Responsiveness of circular strips to catecholamines was not affected by age of the patient and no consistent differences between males and females were shown. 5. Strips of taenia coli exhibited little or no spontaneous phasic activity. Noradrenaline and isoprenaline relaxed KCl-induced tone. The effects of noradrenaline and isoprenaline were antagonized by propranolol but not by phentolamine. BRL 37344 had no effect on KCl-induced tone. 6. In conclusion, catecholamines relaxed spontaneous activity of human colon circular smooth muscle through an action on both alpha- and beta-adrenoceptors. The alpha-adrenoceptors were of the alpha 1-subtype. The beta-adrenoceptor-mediated relaxation appeared to be primarily beta 1. In taenia coli, catecholamines relaxed KCl-induced tone via beta-adrenoceptors only.

Adrenoceptors mediating relaxation to catecholamines in rat isolated jejunum.

1. The characteristics of adrenoceptors mediating relaxation to catecholamines in rat isolated jejunum were investigated. 2. Catecholamines and BRL 37344 produced relaxation of the KCl-contracted strips with an order of potency of isoprenaline (1.0) > BRL 37344 (0.63) > noradrenaline (0.1) > adrenaline (0.04). 3. In the presence of both prazosin (1 microM) and propranolol (1 microM) only small dextral shifts of the concentration-response curves to agonists were observed and an order of potency of BRL 37344 (2.5) > isoprenaline (1.0) > noradrenaline (0.2) > adrenaline (0.1) was obtained. 4. In the presence of prazosin (1 microM) and propranolol (1 microM), cyanopindolol (0.1-10 microM) produced a concentration-dependent rightward shift of the concentration-response curve to adrenaline with a Schild slope not significantly different from unity and a mean pA2 value of 7.01. 5. The resistance of relaxant responses to propranolol, the relatively high potency of BRL 37344 compared to catecholamines and the competitive antagonism of relaxant responses to adrenaline by cyanopindolol suggest that beta-adrenoceptors in rat small intestine are mainly atypical in nature.

Hemodynamic and metabolic correlates of dipyridamole-induced myocardial thallium-201 perfusion abnormalities in multivessel coronary artery disease.

The mechanisms responsible for the development of reversible thallium-201 (TI-201) defects with dipyridamole stress in patients with coronary artery disease (CAD) is not well understood. Previous experimental animal studies have demonstrated coronary steal characterized by an absolute decrease in subendocardial flow distal to a stenosis in response to dipyridamole infusion. Accordingly, the purpose of this study was to determine if reversible TI-201 defects in response to dipyridamole infusion are reflective of myocardial ischemia or secondary to regional differences in flow reserve. Dipyridamole (0.56 mg/kg) TI-201 imaging was performed in 23 patients in whom serial electrocardiographic, hemodynamic, aortic and coronary sinus lactate, and coronary sinus adenosine measurements were obtained. All patients with CAD had TI-201 redistribution (3.8 +/- 2.0 defects/patient), and all patients without CAD had normal scans. Mean aortic pressure was similar in both groups and did not change in response to dipyridamole (non-CAD 103 +/- 11 vs CAD 99 +/- 15 mm Hg, p = NS). Pulmonary capillary wedge pressure was similar at baseline (non-CAD 11 +/- 4 vs CAD 13 +/- 5 mm Hg, p = NS) and did not change in response to the drug (non-CAD 14 +/- 3 vs CAD 15 +/- 7 mm Hg, p = NS). Lactate extraction fraction was similar at baseline (non-CAD 0.22 +/- 0.09 vs CAD 0.17 +/- 0.14, p = NS) and decreased similarly in both groups (non-CAD 0.08 +/- 0.06 vs CAD 0.05 +/- 0.12, p = NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Physiological assessment of coronary artery disease and myocardial viability in ischemic syndromes using adenosine echocardiography.

We hypothesized that it would be feasible and safe to use adenosine echocardiography to assess the physiological significance of coronary stenoses, detect ischemic myocardium, and assess myocardial viability in a high risk group of patients with coronary artery disease (CAD). Therefore, in 40 patients with either unstable angina, non-Q myocardial infarction, or myocardial infarction treated with thrombolytic therapy, we performed adenosine echocardiography (140 mug/kg per min for 5 mins with a 16 segment model for analysis) and compared the findings with quantitative planar thallium-201 scintigraphy, and (in 26 patients) coronary angiography. The technique was safe, and there were no serious complications. Adenosine resulted in a significant increase in heart rate and decrease in blood pressure. The sensitivity of adenosine echocardiography and thallium scintigraphy were 96% and 88%, respectively, for detecting greater than 75% stenosis. The change in echo score from baseline during adenosine infusion was significantly higher with more severe coronary disease (single vessel right coronary artery {RCA} or left circumflex {LCX} disease = 0.125 +/- 0.15, proximal left anterior descending coronary artery {LAD} disease = 0.23 +/- 0.15, RCA and LCX disease = 0.30 +/- 0.14, LAD and RCA and/or LCX disease = 0.62 +/- 0.13). Likewise, the echo score during adenosine infusion was significantly higher in patients with high risk thallium scans (low risk = 1.29 +/- 0.26, medium risk = 1.74 +/- 0.22, and high risk = 2.21 +/- 0.37). In 13 patients receiving thrombolytic therapy, adenosine echocardiography identified 12 with viable myocardium as defined by quantitative thallium criteria. Furthermore, the wall-motion response of the viable segment was indicative of the degree of stenosis of the artery subtending the segment. Regional function deteriorated in patients with high grade (95 +/- 2%) stenoses and improved in those with nonflow limiting stenoses (66 +/- 25%, P = 0.03). Therefore, we conclude that adenosine echocardiography can detect significant coronary stenoses, has a high degree of concordance with thallium in detecting cardiac perfusion abnormalities, and can assess myocardial viability following thrombolytic therapy.

Expression of alternatively-spliced glutamate receptors in human hippocampus.

Rat glutamate receptors have been shown to be expressed as two developmentally regulated, alternatively spliced isoforms. We have investigated the expression of these isoforms of GluRA and GluRB in the human hippocampus. The expression pattern of the mRNAs coding for these subunits does not correspond to that in the rat hippocampus, both isoforms being preferentially expressed in the dentate gyrus and CA1 regions, with lower expression in CA3, with the exception of GluRB flop, where hybridization in CA3 is only lower than in dentate gyrus. Cloning of cDNA from human frontal cortex has also revealed that the two isoforms of human GluRB have virtual nucleotide sequence identity with the alternative exons in the rat, confirming the usefulness of oligonucleotides complementary to the rat cDNAs as probes for these receptor subunits in human neuropsychiatric disorders.

Characterization of catecholamine-mediated relaxations in rat isolated gastric fundus: evidence for an atypical beta-adrenoceptor.

1. Experiments were carried out in order to characterize the receptors mediating relaxant responses to catecholamines in the rat gastric fundus. The effects of noradrenaline, isoprenaline and the 'atypical' or beta 3-adrenoceptor agonist, BRL 37344, on methacholine-induced tone were measured. Prazosin, propranolol and cyanopindolol were used as antagonists. 2. Relaxant responses to noradrenaline, in the presence of propranolol (1 microM) were antagonized in a concentration-dependent manner by prazosin (0.01 to 1 microM), although this antagonism was weak and non-competitive in nature. Relaxant responses to isoprenaline, in the presence of prazosin (0.1 microM), were antagonized only by the highest concentration of propranolol (1 microM) giving a pKB of 6.3 BRL 37344 also relaxed the rat gastric fundus in the presence of prazosin (0.1 microM), and the responses to BRL 37344 were unaffected by propranolol (1 microM). 3. Tachyphylaxis to BRL 37344 was observed, a second concentration-response curve being significantly shifted to the right. Exposure of tissues to BRL 37344 (1 microM) between concentration-response curves also caused an 11 fold rightward shift in the response to isoprenaline. 4. In the presence of prazosin (0.1 microM) and propranolol (1 microM), the rank order of potency of the agonists was: (-)-isoprenaline (1.0) greater than (-)-noradrenaline (0.39) greater than BRL 37344 (0.10). 5. Responses to BRL 37344 in the presence of prazosin (0.1 microM) and propranolol (1 microM) were antagonized by (+/-)-cyanopindolol (1 microM), with a pKB of 6.56. Responses to isoprenaline, under the same conditions, were antagonized in a competitive manner by (+/-)-cyanopindolol (0.1-1 microM), with the slope of a Schild plot close to unity and a pA2 value of 7.44. 6. The resistance to blockade by prazosin and propranolol and the antagonism by cyanopindolol of the responses mediated by isoprenaline and BRL 37344 suggest that atypical beta-adrenoceptors similar to 'atypical',beta-adrenoceptors in rat adipocytes and other tissues are present in the rat gastric fundus.

Evidence for the existence of 'atypical' beta-adrenoceptors (beta 3-adrenoceptors) mediating relaxation in the rat distal colon in vitro.

1. Experiments were carried out to characterize the adrenoceptors mediating relaxant responses in the rat distal colon. Three agonists were used: noradrenaline, isoprenaline and the beta 3-adrenoceptor agonist BRL 37344. Phentolamine, propranolol and (+/- )-cyanopindolol were tested as antagonists. Tone in the rat distal colon was induced with KCl (30-40 mM) as a spasmogen, and relaxations of this KCl-induced tone produced by the agonists were measured. 2. Relaxant responses to noradrenaline that were obtained in the presence of propranolol (1 microM) were not antagonized by phentolamine (0.01 to 1 microM). Relaxant responses to isoprenaline that were obtained in the presence of phentolamine (1 microM) were antagonized in a concentration-dependent manner by propranolol (0.01 to 3 microM), although this antagonism was weak and non-competitive. Relaxant responses to BRL 37344 that were obtained in the presence of phentolamine (1 microM) were only weakly antagonized by high (1 microM) concentrations of propranolol. 3. Tachyphylaxis to BRL 37344 was observed, a second concentration-response curve being shifted to the right by 15 fold. Exposure of the tissues to BRL 37344 (1 microM) between concentration-response curves also caused rightward shifts in the responses to noradrenaline (18 fold) and isoprenaline (19 fold) but not to papaverine. 4. In the presence of phentolamine (1 microM) and propranolol (1 microM), the rank order of potency of the agonists was: (-)-isoprenaline (1.0) greater than or equal to BRL 37344 (0.93) greater than (-)-noradrenaline (0.3). 5. Responses to BRL 37344 in the presence of phentolamine (1 microM) and propranolol (1 microM) were antagonized by (+/-)-cyanopindolol (I microM), with an apparent pA2 value of 6.67. Responses to isoprenaline, under the same conditions, were antagonized in a competitive manner by (+/-)-cyanopindolol (0.1 to 10 microM), with the slope of the Schild plot close to unity and a pA2 value of 7.12. 6. The resistance of the relaxant responses to antagonism by phentolamine and propranolol, along with the relatively high potency of the beta 3-adrenoceptor agonist BRL 37344 and the antagonism of 'resistant' responses by (+/-)-cyanopindolol would suggest that 'atypical' beta 6-adrenoceptors, similar to the beta 3-adrenoceptors of rat adipocytes and other tissues, exist in the rat distal colon.