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PubMed1995

Identification of alpha 1-adrenoceptor subtypes in the rabbit prostate.

1. The alpha 1-adrenoceptor subtypes of rabbit prostate were characterized in binding and functional experiments. 2. In saturation experiments, [3H]-prazosin bound to two distinct affinity sites in the rabbit prostate (pKD = 11.20 +/- 0.22 and 8.39 +/- 0.11, Bmax = 15.3 and 736 fmol mg protein-1). 3. In the displacement experiments, the binding was inhibited with shallow displacement curves by unlabelled prazosin, WB4101, and 5-methylurapidil, suggesting the presence of two distinct affinity sites for prazosin, WB4101, or 5-methylurapidil. On the other hand, HV723 displaced the [3H]-prazosin binding monophasically with a low affinity. From the results, the presence of two distinct alpha 1-adrenoceptor subtypes was suggested; presumably one is the classical alpha 1A (cloned alpha 1C) subtype with high affinity for prazosin, WB4101 and 5-methylurapidil but not for HV723 and the other corresponds to the alpha 1L subtype, which shows low affinity for the four antagonists. 4. In the functional experiments, prazosin, WB4101, HV723 and 5-methylurapidil competitively antagonized the contractile response to noradrenaline with low affinities close to those for the alpha 1L subtype determined in binding experiments. These results suggest that contractile response to noradrenaline in the rabbit prostate is predominantly mediated through the alpha 1L subtype.

Acetonitriles↗

PubMed1996

Drugs for treatment of benign prostatic hyperplasia: affinity comparison at cloned alpha 1-adrenoceptor subtypes and in human prostate.

1. We have previously shown that among alpha 1-adrenoceptor antagonists used or investigated for the treatment of benign prostatic hyperplasia, tamsulosin discriminates alpha 1-adrenoceptor subtypes in rat tissues whereas alfuzosin and naftopidil do not. We now expand these studies to additional drugs (doxazosin, terazosin) being used and/or investigated for this purpose, and have evaluated all of these drugs at cloned subtypes and in human prostate. 2. Competition binding studies were performed with [3H]-prazosin in membrane samples from rat spleen, kidney and cerebral cortex and human prostate and with cloned alpha 1-adrenoceptors expressed in COS cells. Doxazosin and terazosin did not discriminate alpha 1-adrenoceptor subtypes in rat kidney and cerebral cortex. In contrast, the subtypes present in the tissues were well discriminated by the alpha 1A-adrenoceptor-selective reference drug WB 4101. 3. Alfuzosin, doxazosin, naftopidil and terazosin did not discriminate cloned alpha 1-adrenoceptor subtypes transiently expressed in COS cells whereas tamsulosin and WB 4101 did. 4. In human prostate, alfuzosin, doxazosin, naftopidil and terazosin did not discriminate the alpha 1-adrenoceptor subtypes present in this tissue whereas tamsulosin and the alpha 1A-adrenoceptor-selective reference drugs WB 4101, phentolamine and 5-methylurapidil did. Based on data with the alpha 1A-adrenoceptor-selective drugs, human prostate contains alpha 1A- and alpha 1B-adrenoceptors in an approximate 70:30% ratio. 5. We conclude that tamsulosin, in common with WB 4101, but in contrast to alfuzosin, doxazosin, naftopidil, and terazosin is selective for alpha 1A-adrenoceptors which appear to dominate in the human prostate; the therapeutic relevance of this selectivity remains to be assessed in clinical studies.

Adrenergic alpha-Antagonists↗

PubMed2004

Evidence showing that beta-adrenoceptor subtype responsible for the relaxation induced by isoprenaline is principally beta 2 but not beta 1 in guinea-pig tracheal smooth muscle.

1. The present study was carried out to pharmacologically identify the beta-adrenoceptor subtype that mediates isoprenaline-elicited relaxation in the isolated guinea-pig tracheal smooth muscle, to answer the question whether it is beta(1)- or beta(2)-subtype? 2. Isoprenaline as well as salbutamol, a well-known beta(2)-selective adrenoceptor agonist, produced a concentration-dependent relaxation with a pD(2) value of 8.12 vs. 7.54 for salbutamol. 3. Isoprenaline-elicited relaxation was not affected by beta(1)-selective antagonists, atenolol and CGP-20,712A, within the concentration ranges supposed to antagonize beta(1)-subtype: atenolol, < or =10(-6) M; CGP-20,712A, < or =10(-8) M. 4. By contrast, the concentration-response curves for isoprenaline as well as salbutamol were shifted rightwards in a competitive fashion by atenolol at the concentrations > or =3 x 10(-6) M. However, pA(2) values of atenolol against isoprenaline (5.86) and salbutamol (5.71) were consistent with the value corresponding to beta(2)- but not to beta(1)-subtype (around 7.00), and these values were not significantly different from each other. 5. Competitive antagonism of the relaxations to isoprenaline and salbutamol were also obtained with beta(2)-selective antagonists, butoxamine and ICI-118,551. Against isoprenaline and salbutamol, the pA(2) values of butoxamine (6.51 vs. 6.81) and ICI-118,551 (8.83 vs. 8.90) were substantially identical. Thus the primary mediation of beta(2)-receptor in the relaxations was strongly supported. 6. The present findings provide evidence that the beta-adrenoceptor which mediates isoprenaline-elicited relaxation of guinea-pig tracheal smooth muscle is essentially beta(2)- but not beta(1)-subtype. The present study also indicates the importance of using multiple receptor antagonists with different pA(2) values to pharmacologically identify the responsible receptor subtype in smooth muscle mechanical responses.

Adrenergic beta-1 Receptor Agonists↗

PubMed1991

Relation between adrenergic neurogenic contraction and alpha 1-adrenoceptor subtypes in dog mesenteric and carotid arteries and rabbit carotid arteries.

1. We examined the distribution of alpha 1-adrenoceptor subtypes and their relation to adrenergic neurogenic contraction induced by electrical transmural stimulation in the dog mesenteric and carotid arteries and the rabbit carotid artery. 2. In the dog mesenteric artery, contraction to noradrenaline was competitively inhibited by HV723 (pKB = 9.37) and prazosin (pKB = 8.40). Pretreatment with chlorethylclonidine (CEC) slightly attenuated only the contractions induced by low concentrations of noradrenaline. Contraction induced by electrical transmural stimulation was inhibited at lower concentrations of HV723 than of prazosin. 3. In the dog carotid artery, contraction to noradrenaline was inhibited with higher affinity by prazosin (pKB = 9.82) than by HV723 (pKB = 8.47). Prazosin was also more potent than HV723 in inhibiting the contraction to electrical stimulation. Pretreatment with CEC markedly attenuated or abolished contraction to noradrenaline and electrical stimulation. 4. In the rabbit carotid artery, prazosin inhibited noradrenaline-induced contraction biphasically (pKB = 9.91 and 8.60). After CEC pretreatment, contraction to noradrenaline was attenuated moderately and the high affinity site for prazosin was abolished. HV723 competitively inhibited the noradrenaline response with a similar pKB value (approximately 8.5) regardless of CEC treatment. Contraction to electrical stimulation was inhibited by prazosin more effectively than by HV723 in preparations not treated with CEC, while it was equipotently inhibited by both antagonists in CEC-treated preparations. 5. These results suggest that the contractions induced by endogenous and exogenous noradrenaline are mediated through the same subtypes of alpha,-adrenoceptor distributed in each artery; according to our recent subclassification: alpha 1N subtype in the dog mesenteric artery, alpha 1H subtype in the dog carotid artery and alpha lH and alpha 1L subtypes in the rabbit carotid artery. Different susceptibility to alpha l-adrenoceptor antagonists of sympathetic adrenergic responses in various blood vessels may be related to heterogeneous involvement of distinct alpha,-adrenoceptor subtypes in the sympathetic response.

Acetonitriles↗

PubMed1991

Delineation of three pharmacological subtypes of alpha 2-adrenoceptor in the rat kidney.

1. Simultaneous computer modelling of plain and ARC 239- and guanoxabenz-masked [3H]-RX821002 saturation curves, plain ARC 239 and guanoxabenz competition curves as well as ARC 239-masked guanoxabenz competition curves revealed that the drugs bound to three alpha 2-adrenoceptor subtypes in the rat kidney with grossly differing selectivities. These alpha 2-adrenoceptor subtypes were termed alpha 2 A, alpha 2B1 and alpha 2B2. The order of affinities for [3H]-RX821002 for the adrenoceptor sites was alpha 2A greater than alpha 2B1 greater than alpha 2B2, the KdS being 0.62 +/- 0.05, 2.52 +/- 0.11 and 6.74 +/- 1.21 nM, respectively. The order of affinities for ARC 239 was alpha 2B1 greater than alpha 2B2 much greater than alpha 2A with KdS 4.78 +/- 1.04, 28.8 +/- 4.1 and 1460 +/- 270 nM, respectively. For guanoxabenz the order of affinities was alpha 2A greater than alpha 2B1 much greater than alpha 2B2 with KdS 99.7 +/- 15.1, 508 +/- 135 and 25,400 +/- 2400 nM, respectively. 2. Binding constants for 14 compounds for the three rat kidney alpha 2-adrenoceptor subtypes were determined by the simultaneous computer modelling of plain and ARC 239- and guanoxabenz-masked drug competition curves, plain ARC 239 and guanoxabenz competition curves as well as ARC 239-masked guanoxabenz competition curves. Of the 14 compounds tested, oxymetazoline and guanfacine were found to bind with low affinities to both of the alpha 2B1- and alpha 2B2-adrenoceptor but with high affinity to the alpha 2A-adrenoceptor. 3. (-)-Adrenaline and (-)-noradrenaline showed dissimilar order of affinities for the three alpha2-adrenoceptors. For (-)-adrenaline the order of affinities was alpha2Bl >- alpha2A> alpha2B2 and for (-)-noradrenaline alpha2B2 > alpha2Bl > alpha2A. All three alpha2-adrenoceptors showed the expected stereoselective binding for adrenaline enantiomers, the (+)-form being 7-10 fold less potent than the (-)form. 4. [3H]-yohimbine was also used as radioligand. The data with this ligand were fully compatible with the [3H]-RX821002 data. However, [3H]-yohimbine appeared to label only alpha2Bl- and alpha2B2-adrenoceptors presumably because it had too low an affinity for alpha2A-adrenoceptors. 5. We conclude that three pharmacological subtypes of alpha2-adrenoceptors are labelled by [3H]-RX821002 in the rat kidney. Guanoxabenz and ARC 239 may be used in competition studies to delineate between these three alpha2-adrenoceptor subtypes. Moreoever, we here present a method allowing the determination of binding constants for an arbitrary drug to the three alpha2-adrenoceptor subtypes.

Animals↗

PubMed1992

Alpha 4-2 beta 2 and other nicotinic acetylcholine receptor subtypes as targets of psychoactive and addictive drugs.

1. Xenopus oocytes were injected with various muscle and neuronal nicotinic acetylcholine receptor (ACh receptor, cholinoceptor) subunit RNA combinations and their pharmacological properties studied using two-electrode voltage clamp. The functional expression of one of these combinations, rat alpha 4-2 beta 2, has not been previously described. The alpha 4-2 mRNA is a splicing variant transcribed from the alpha 4 gene. In the experiments reported here, the alpha 4-2 beta 2 subtype was functionally indistinguishable from the alpha 4-1 beta 2 subtype. 2. For each subtype, the relative potency of nicotine compared with acetylcholine was obtained by estimating the relative concentration of nicotine which would elicit the same current response as 0.1 microM Ach. The ratios of these concentrations (nicotine: ACh) for the mouse muscle ACh receptor-(alpha 1 beta 1 gamma delta) was 96.1:1. In contrast, the ratios for the rat neuronal subtypes were: alpha 2 beta 2, 1.01:1; alpha 3 beta 2, 2.01:1; alpha 4 beta 2, 0.76:1 and alpha 4-2 beta 2, 0.76:1. The much greater relative nicotine sensitivity of the neuronal subtypes as compared with muscle receptors illustrates their potential to mediate the psychoactive and addictive effects of nicotine. However, it does not appear that the differences in relative nicotinic sensitivity among the neuronal receptors themselves can be used as a simple discriminative tool in neuronal tissue. 3. The slopes of the log dose-log response curves at low ACh concentrations were all greater than 1 but less than 2, suggesting that at least two agonist binding sites mediate the functional response of each hetero-oligomer. 4. The response of all the neuronal subtypes to ACh could be inhibited by the psychoactive drugs mecamylamine, amitriptyline, phencyclidine, trifluoperazine and promethazine. With the exception of the very potent antagonist, mecamylamine, the degree of block of the peak current to ACh produced by 10 microM concentrations of these drugs was remarkably similar (around 50%). 5. The degree of inhibition produced when the antipsychotic drug, trifluoperazine, was co-applied with ACh increased as the duration of application increased. Such an effect was not observed with promethazine, a related phenothiazine derivative which does not have antipsychotic actions.

Acetylcholine↗

PubMed1994

Heterogeneity of alpha 1-adrenoceptor subtypes involved in adrenergic contractions of dog blood vessels.

1. We determined the alpha 1-adrenoceptor subtypes involved in adrenergic contractions of eight different blood vessels isolated from the dog. 2. Noradrenaline produced concentration-dependent contractions in all the blood vessels tested, which were competitively inhibited by prazosin, WB4101, HV723 and 5-methylurapidil. However, there was considerable difference between the vessels with regard to the pKB values for all the antagonists. The alpha 1-adrenoceptors of dog vertebral and carotid arteries had high affinity for prazosin (pKB > 9.0) but low affinity for WB4101 (< 8.5), 5-methylurapidil (< 7.5) and HV723 (< or = 8.5). By contrast, HV723 had higher affinity (> 9.0) than prazosin (< 8.3), WB4101 (< 8.7) and 5-methylurapidil (< 8.2) in the portal vein, mesenteric artery and vein, and renal artery. In the femoral artery and vein, however, the four antagonists showed pKB values in the range 8.0-8.7. 3. Chloroethylclonidine (10 microM) produced a remarkable reduction of the contractile responses to noradrenaline in the vertebral and carotid arteries as compared with those in the other vessels. Nifedipine inhibited the responses to noradrenaline in all the tissues tested, and had marked effects in the portal vein. 4. Sympathetic adrenergic contractions induced by transmural electrical stimulation were also inhibited by prazosin and HV723 at different potencies among tissues. The relative potencies of both the antagonists paralleled the relationship in inhibiting the responses to exogenous noradrenaline in each vessel. 5. According to recent alpha l-adrenoceptor subclassification, the present results suggest that the contractions of blood vessels induced by endogenous and exogenous noradrenaline are mediated through different alpha l-adrenoceptor subtypes heterogeneously distributed in each vessel; presumably, the alpha 1 B subtype in the carotid and vertebral arteries, the alpha IN subtype in the visceral region and the alpha IL subtype in the femoral region. Regionally different expression of alpha 1-adrenoceptor subtypes may be in part associated with the regional heterogeneity of sympathetic responses in the blood vessels.

Acetonitriles↗

PubMed1996

Rat pineal alpha 1-adrenoceptor subtypes: studies using radioligand binding and reverse transcription-polymerase chain reaction analysis.

1. The pharmacological characteristics of alpha 1-adrenoceptor binding sites in rat pineal gland membranes, detected by use of a selective alpha 1-adrenoceptor antagonist ([125I]-iodo-2-[beta-(4-hydroxyphenyl) ethylaminomethyl]tetralone, [125I]-HEAT), were investigated with the alkylating agent, chloroethylclonidine (CEC), and in competition experiments with a number of adrenoceptor agonists and antagonists. 2. Chloroethylclonidine (CEC) treatment (10 microM, 10 min) of rat pineal membranes inactivated approximately 70% of specific [125I]-HEAT binding sites. Higher concentrations of CEC (up to 100 microM) or longer treatment periods (up to 40 min) were no more effective. 3. Adrenoceptor agonists and antagonists competitively inhibited [125I]-HEAT binding with Hill coefficients close to unity indicating a single alpha 1-adrenoceptor subtype is present. The affinity (Ki) of subtype selective agonists (oxymetazoline, SDZ NVI-085) and antagonists (5-methylurapidil, WB4101, benoxathian, phentolamine) was consistent with binding to an alpha 1B-adrenoceptor subtype. 4. The (-)- and (+)-enantiomers of niguldipine had an equal and low affinity for alpha 1-adrenoceptor binding sites both in untreated (log Ki-6.66 and -6.90 respectively) and CEC-treated membranes in which approximately 70% of sites had been inactivated (log Ki-6.41 and -6.86 respectively). This indicates that the small proportion of alpha 1-adrenoceptors insensitive to CEC are not alpha 1A-adrenoceptors. 5. mRNA was isolated from rat pinealocytes, cDNA was synthesized and then amplified by the polymerase chain reaction with alpha 1-adrenoceptor subtype specific primers. These experiments identified both alpha 1A- and alpha 1B-adrenoceptor mRNA, but not alpha 1D-mRNA in rat pinealocytes, although all three adrenoceptor subtypes were readily identified in rat brain cortex. 6. These data indicate that although both alpha 1A- and alpha 1B-adrenoceptor mRNAs are present in the pineal the major subtype of alpha 1-adrenoceptor expressed is the alpha 1B.

Adrenergic alpha-Antagonists↗

PubMed1996

Quantification and distribution of alpha 1-adrenoceptor subtype mRNAs in human prostate: comparison of benign hypertrophied tissue and non-hypertrophied tissue.

1. There are at least three alpha 1-adrenoceptor subtypes, alpha 1a, alpha 1b and alpha 1d, in human tissues. Using an RNase protection assay, we have now determined the amount of each subtype mRNA in human prostatic tissue, for both benign prostatic hypertrophy (BPH) and non-BPH. In all tissue samples examined, the predominant subtype mRNA was alpha 1a. The total abundance of alpha 1-adrenoceptor mRNA in BPH samples was over six times that in non-BPH samples. This increase was mostly accounted for by alpha 1a, which was almost nine times as abundant in BPH samples as in non-BPH samples. The abundance of alpha 1b was almost the same between BPH and non-BPH samples, and the abundance of alpha 1d in BPH samples was about three times that in non-BPH samples. The ratio of the numbers of the subtype mRNAs, alpha 1a: alpha 1b: alpha 1d, was 85:1:14 in BPH samples and 63:6:31 in non-BPH samples. 2. In situ hybridization studies showed no significant differences in the tissue localization of alpha 1-adrenoceptor subtype mRNAs between BPH and non-BPH samples. alpha 1a and alpha 1d were clearly detected in the interstitium of the prostate, where alpha 1a was stained more intensely than alpha 1d, and the positive sites were primarily smooth muscle cells. In contrast, alpha 1b staining was very faint. 3. This increase in mRNA abundance may be directly related to the contraction of prostatic tissue that leads to obstruction of the urinary tract in BPH patients. Specifically, our data suggest that increased expression of the alpha 1a subtype may be primarily responsible for the contraction of the prostate.

Aged↗

PubMed2006

Implications of clinical subtypes of migraine with aura.

OBJECTIVE: To compare the clinical characteristics of familial hemiplegic migraine (FHM), sporadic hemiplegic migraine (SHM), and nonhemiplegic migraine with aura (NHMA) and further, to compare subtypes of NHMA. BACKGROUND: To discover distinct underlying genetic and pathophysiological mechanisms it is crucial to drive clinical subdivision of migraine with aura as far as possible. The documentation of subtypes of migraine with aura depends on the clinical characteristics as the genetic mechanisms are unknown except for the dominantly inherited FHM. METHODS: Patients with FHM, SHM, or familial NHMA were recruited from specialist practice and diagnosed according to the International Classification of Headache Disorders (ICHD) in a validated interview by a physician. Patients with hemiplegic migraine had a physical and neurological examination. Patients with population-based NHMA from a previous Danish study were used for comparison. RESULTS: We recruited 147 patients with FHM, 105 with SHM, and 362 with familial NHMA. FHM and SHM had similar aura and headache characteristics. Patients with FHM and SHM were more likely to experience two or more aura symptoms (100% vs. 31%, P < .0001), they more often had prolonged aura symptoms, they almost always had a headache associated with the aura (93% vs. 58%, P < .0001), and they more frequently had basilar-type symptoms (69% vs. 10%, P < .0001) than patients with population-based NHMA. Patients with familial NHMA were more likely to experience two or more aura symptoms than patients with population-based NHMA (61% vs. 32%, P < .0001). Within the subtypes of NHMA, patients with typical aura with migraine headache had an earlier age at onset (20 +/- 10 vs. 23 +/- 13 years, P= .044) and a higher co-occurrence of migraine without aura (43% vs. 22%, P= .002) than patients with typical aura with nonmigraine headache. CONCLUSIONS: The present study proves that distinct subtypes of migraine with aura exist. It further underlines the phenotypic differences between the different subtypes of migraine with aura which likely are caused by different etiological mechanisms. In future studies FHM, SHM, and NHMA therefore should be analyzed as separate entities and patients with NHMA may be stratified by ICHD-2 subtype of NHMA.

Adolescent↗

PubMed2005

Bowel habit subtypes and temporal patterns in irritable bowel syndrome: systematic review.

UNLABELLED: Irritable bowel syndrome (IBS) is a heterogeneous condition characterized by the presence of abdominal discomfort or pain and bowel habit alterations: constipation (C-IBS), diarrhea (D-IBS), or alternating C and D (A-IBS). Its clinical course is poorly known. OBJECTIVES: (i) To compare bowel habit subtypes distribution in IBS according to sample origin and diagnosis criteria; (ii) To evaluate IBS temporal patterns based on follow-up studies. METHODS: A literature search (1966-2003) was conducted in the MEDLINE and EMBASE databases. A total of 72 studies were found and 22 were finally selected. RESULTS: Population-based studies from the United States (Manning) found similar distribution among C-IBS, D-IBS, and A-IBS, while European studies (Rome I, Rome II, or self-reporting) showed either C-IBS or A-IBS as the most prevalent subtypes. Primary care office-based studies (Rome I or Rome II) showed A-IBS as the most prevalent group. Gastroenterology specialized office-based studies found either C-IBS or D-IBS as the most frequently reported subtype. Prospective follow-up investigations showed that the most frequent IBS temporal pattern profile consists of mild to moderate symptoms appearing in cluster in an intermittent way, about once a week, and lasting 2-5 days on average. CONCLUSION: IBS clinical subtypes distribution differs depending on the population evaluated, the geographical location, and the criteria employed to define IBS and bowel habit subtypes. In most cases, clinical course is characterized by the presence of mild-to-moderate symptoms appearing sequentially. Prospective studies, using clear and stable diagnostic criteria and subtype definitions, and based on daily data collection should further characterize IBS clinical course.

Abdominal Pain↗

PubMed1994

Genomic analysis of the F subtypes of human complement factor B.

Factor B of human complement is encoded within the Major Histocompatibility Complex (MHC) and is polymorphic, with up to 30 alleles defined by electrophoretic mobility. One of the most common alleles, BF*F, is subdivided into the FA and FB subtypes, which differ at the gene level by non-synonymous base substitutions in the seventh codon. We have found at this position a new restriction site polymorphism, as a Bsl I site absent from the FB allele. Using this restriction polymorphism, we have developed a method for BF F subtype determination, based on amplification by polymerase chain reaction of the 5' end of the BF gene, and digestion with Bsl I. This new method has been applied to a panel of 29 selected BF F individuals. A single strand DNA conformation analysis of the same region of the gene allowed us to confirm the above DNA-based BF F subtyping. During this study, two BF*F1 alleles showed discrepancies between protein and DNA typing, which were confirmed by our sequencing data. These were identical, in the 5' region, to BF*S and BF*FB genes, respectively. In a comparison with two protein subtyping methods, identical results were found for only one third of the selected samples. The conflicting results may arise, in part, from previously undescribed molecular heterogeneity within BF F subtypes, or from the presence of a null allele. Our new method allows BF*F subtyping to be used with confidence in the definition of disease-associated MHC haplotypes.

Alleles↗

PubMed2005

P2X purinoceptor subtypes on paraventricular nucleus neurones projecting to the rostral ventrolateral medulla in the rat.

The rostral ventrolateral medulla (RVLM) is essential for the generation of sympathetic nerve activity. The RVLM receives a substantial innervation from the hypothalamic paraventricular nucleus (PVN). Activation of P2X purinoceptors via ATP has been shown to mediate fast excitatory synaptic neurotransmission. There is mounting evidence to suggest the presence of P2X purinoceptors in hypothalamic nuclei, including the PVN. In this study, we determined whether P2X1-P2X6 purinoceptor subtypes were present on PVN neurones that projected to the RVLM. Injection of the retrogradely transported tracer, rhodamine-tagged microspheres, into the pressor region of the RVLM was used to identify the neurones in the PVN that innervated the RVLM. P2X1-P2X6 purinoceptors were detected by immunohistochemistry. Double-labelled neurones were quantified and expressed as a proportion of the retrogradely labelled neurones. The proportions of double-labelled neurones for each of the P2X purinoceptor subtypes varied, on average, from 14 to 29%. The P2X3 purinoceptor subtype was found to be the dominant purinoceptor subtype present on PVN neurones projecting to the RVLM. Additionally it was apparent that more than one P2X purinoceptor subtype was present on the PVN neurones projecting to the RVLM, since the sum of the average percentages of double-labelled neurones for each P2X purinoceptor subtype exceeded 100%. These findings highlight the presence of the P2X1-P2X6 purinoceptors on PVN neurones projecting to the RVLM. The results suggest a potential role for ATP in the PVN in the regulation of sympathetic nerve activity.

Animals↗

PubMed2002

Nicotinic ACh receptor subtypes on gastrointestinally projecting neurones in the dorsal motor vagal nucleus of the rat.

To determine the predominant nicotinic ACh receptor (nAChR) located on neurones in the dorsal motor nucleus of the vagus (DMV) that project to the gastrointestinal tract, we used the rat brainstem slice preparation and whole-cell recordings of DMV neurones identified by retrograde DiI tracing to pharmacologically characterize nAChRs. Pressure ejection of acetylcholine (ACh, 250 microM for 200 ms) from a patch pipette placed approximately 10-20 microm from the surface of the recorded cell produced an inward current in most DMV neurones sampled. The average currents for neurones projecting to the fundus, antrum and caecum were 149 +/- 38 (n = 25), 115 +/- 18 (n = 29) and 117 +/- 23 pA (n = 6), respectively. Blockade of the alpha7 subtype of nAChR with either alpha-bungarotoxin (alpha-BGT) or methyllycaconitine (MLA) counteracted 60-75 % of the ACh-evoked current in DMV neurones projecting to the fundus, antrum and caecum. In neurones projecting to the fundus and the antrum, currents resistant to alpha-BGT were significantly blocked by dihydro-beta-erythroidine (10-20 nM), an antagonist of the alpha4beta2 subtype of nAChR. In neurones projecting to the caecum, currents resistant to alpha-BGT were significantly depressed by a low concentration of mecamylamine (1 microM). Cytisine (100 microM), an agonist of nAChRs that contain the alpha7 or the beta4 subunit, evoked significant currents in caecum-projecting neurones that were previously exposed to alpha-BGT. In contrast, cytisine had no effect on DMV neurones previously exposed to alpha-BGT that project to the fundus or antrum. Our data indicate that the prevailing nAChR subtype in DMV neurones projecting to the GI tract is the alpha7 subtype. In addition, we obtained evidence for the co-expression of the alpha4beta2 nAChR subtype on DMV neurones projecting to the fundus and antrum, and the alpha3beta4 nAChR subtype on DMV neurones projecting to the caecum.

Acetylcholine↗

PubMed2004

The alpha 1 and alpha 6 subunit subtypes of the mammalian GABA(A) receptor confer distinct channel gating kinetics.

The GABA(A) receptors show a large degree of structural heterogeneity, with seven different subunit families, and 16 different subtypes in mammalian species. The alpha family is the largest, with six different subtypes. The alpha1 and alpha6 subtypes are among the most diverse within this family and confer distinct pharmacological properties to recombinant and neuronal receptors. To determine whether different single channel and macroscopic kinetic properties were also associated with these subtypes, the alpha1 or alpha6 subunit was expressed in mammalian cells along with beta3 and gamma2L subunits and the kinetic properties examined with outside-out patch recordings. The alpha1 beta3 gamma2L receptors responded to GABA with long-duration openings organized into multi-opening bursts. In contrast, channel openings of the alpha6 beta3 gamma2L receptors were predominately short in duration and occurred as isolated, single openings. The subunit subtype also affected the deactivation rate of the receptor, which was almost 2-fold slower for alpha6 beta3 gamma2L, compared with the alpha1 beta3 gamma2L isoform. Onset of fast desensitization did not differ between the isoforms. To determine the structural domains responsible for these differences in kinetic properties, we constructed six chimeric subunits, combining different regions of the alpha1 and alpha6 subunits. The properties of the chimeric subunits indicated that structures within the third transmembrane domain (TM3) and the TM3-TM4 intracellular loop conferred differences in single channel gating kinetics that subsequently affected the deactivation rate and GABA EC50. The effect of agonist concentration on the rise time of the current showed that the extracellular N-terminal domain was largely responsible for binding characteristics, while the transmembrane domains determined the activation rate at saturating GABA concentrations. This suggests that subunit structures outside of the agonist binding and pore-lining domains are responsible for the kinetic differences conferred by the alpha1 and alpha6 subtypes. Structural heterogeneity within these transmembrane and intracellular regions can therefore influence the characteristics of the postsynaptic response of GABA(A) receptors with different subunit composition.

Animals↗

PubMed2006

GABAA receptor subtype specific enhancement of inhibition in human motor cortex.

Inhibition is of fundamental importance to regulate activity in cortical circuits. Inhibition is mediated through a diversity of different interneurones and gamma-aminobutyric acid A receptor (GABA(A)R) subtypes. Here we employed paired-pulse transcranial magnetic stimulation (TMS) to measure short interval intracortical inhibition (SICI), a GABA(A)R-mediated inhibition in human motor cortex, to address the question of which GABA(A)R subtype is responsible for this form of inhibition. It has been shown that classical benzodiazepines (diazepam and lorazepam) have a non-selective affinity profile at different alpha-subunit-bearing subtypes of the GABA(A)R while zolpidem has a 10-fold greater affinity to the alpha1-subunit-bearing GABA(A)R compared with those bearing the alpha2- or alpha3-subunit. We found that, in seven healthy subjects, a single oral dose of 20 mg of diazepam or 2.5 mg of lorazepam significantly increased SICI, whereas 10 mg of zolpidem did not change SICI. This dissociation occurred despite equal sedation by all three drugs, an alpha1-subunit GABA(A)R-mediated effect. The findings strongly suggest that SICI is not mediated by the alpha1-subunit-bearing subtype of the GABA(A)R but by those bearing either the alpha2- or alpha3-subunit. This study represents an attempt by means of TMS to identify GABA(A)R subtype-specific action at the systems level of human cortex, a highly relevant issue because the different alpha-subunit-bearing subtypes of the GABA(A)R are differently involved in benzodiazepine-mediated effects such as sedation, amnesia or anxiolysis, in developmental cortical plasticity, and in neurological disorders such as epilepsy.

Adult↗

PubMed2005

Nicotinic cholinergic receptors in the rat retina: simple and mixed heteromeric subtypes.

Neuronal nicotinic acetylcholine receptors (nAChRs) were measured in the rat retina to determine the heteromeric subtypes. We detected seven nicotinic receptor subunit mRNA transcripts, alpha2-alpha4, alpha6, and beta2-beta4, with RNase protection assays. The density of heteromeric nAChR binding sites is approximately 3 times higher in the retina than in the cerebral cortex. Moreover, the density of the sites in the retina measured with [3H]epibatidine ([3H]EB) is approximately 30% higher than with 125I-3-(2(S)-azetidinylmethoxy)pyridine (A-85380) and more than twice that measured with [3H]cytisine or [3H](-)nicotine. These data suggest that the retina expresses multiple subtypes of nAChRs, including a large fraction of receptors containing the beta2 subunit and a smaller fraction containing the beta4 subunit. Consistent with this, in binding competition studies, nicotinic ligands fit a model for two affinity classes of binding sites, with the higher affinity sites representing 70 to 80% of the nAChRs in the retina. To determine the specific subtypes of nAChRs in the rat retina, we used subunit-specific antibodies in immunoprecipitation assays. Immunoprecipitation of [3H]EB-labeled nAChRs with antibodies specific to the beta2 and beta4 subunits indicated that approximately 80% of the receptors contained beta2 subunits and approximately 25% contained beta4 receptors, consistent with the binding pharmacology results. Sequential immunoprecipitation assays indicated that the rat retina contains multiple subtypes of nAChRs. The majority of the receptors measured seemed to be simple heteromeric subtypes, composed of a single type of alpha and a single type of beta subunit; but a significant fraction are mixed heteromeric subtypes, composed of two or more alpha and/or beta subunits.

Animals↗

PubMed2003

In vitro hypersusceptibility of human immunodeficiency virus type 1 subtype C protease to lopinavir.

In order to characterize the impact of genetic polymorphisms on the susceptibility of subtype C strains of human immunodeficiency virus type 1 to protease inhibitors (PIs), a subtype B protease that originated from an infectious clone was modified through site-directed mutagenesis to include the amino acid residue signatures of subtype C viruses (I15V, M36I, R41K, H69K, L89 M) with (clone C6) or without (clone C5) an I93L polymorphism present as a molecular signature of the worldwide subtype C protease. Their susceptibilities to commercially available PIs were measured by a recombinant virus phenotyping assay. We could not detect any differences in the 50% inhibitory concentration (IC(50)s) of amprenavir, indinavir, ritonavir, saquinavir, and nelfinavir for the clones analyzed. However, we did observe hypersusceptibility to lopinavir solely in clone C6, which includes the I93L substitution (a 2.6-fold decrease in the IC(50) compared to that for the subtype B reference strain). The same phenotypic behavior was observed for 11 Brazilian and South African clinical isolates tested, in which only subtype C isolates carrying the I93L mutation presented significant hypersusceptibility to lopinavir.

Amino Acid Sequence↗