Radiolabeling of the rat P2X4 purinoceptor: evidence for allosteric interactions of purinoceptor antagonists and monovalent cations with P2X purinoceptors

The rat recombinant P2X4 purinoceptor was expressed in CHO-K1 cells, and binding studies were performed using the radioligand [35S]adenosine-5'-O-(3-thio)triphosphate ([35S]ATPgammaS). In 50 mM Tris/1 mM EDTA assay buffer, pH 7.4 at 4 degrees, [35S]ATPgammaS bound with high affinity to the P2X4 purinoceptor (KD = 0.13 nM, Bmax = 151 pmol/mg of protein). The purinoceptor agonists ATP and 2-methylthioadenosine triphosphate possessed nanomolar affinity for the P2X4 purinoceptor, whereas the antagonist suramin possessed much lower affinity (IC50 = 0.5 mM). Cibacron blue was more potent than suramin but produced a biphasic competition curve, whereas d-tubocurarine potentiated binding at concentrations in excess of 10 microM. The complex effects of cibacron blue and d-tubocurarine seemed to be due to an allosteric interaction with the P2X4 purinoceptor because these compounds affected radioligand dissociation, measured after isotopic dilution with unlabeled ATPgammaS. Cibacron blue (1-100 microM) and d-tubocurarine (0.1-1 mM) produced rapid (10 sec to 5 min) decreases or increases, respectively, in the level of [35S]ATPgammaS binding measured immediately after initiation of the dissociation reaction. However, the subsequent rates of radioligand dissociation were not markedly different from those measured in their absence. Monovalent cations produced similar affects on the P2X4 purinoceptor and, like d-tubocurarine, increased [35S]ATPgammaS binding. The actions of d-tubocurarine and sodium were not additive. The findings from this study indicate that [35S]ATPgammaS can be used to label the P2X4 purinoceptor and suggest that this binding can be enhanced by monovalent cations and d-tubocurarine and may be subject to negative allosteric modulation to varying degrees by different purinoceptor antagonists.     

Coexpression of ligand-gated P2X and G protein-coupled P2Y receptors in smooth muscle. Preferential activation of P2Y receptors coupled to phospholipase C (PLC)-beta1 via Galphaq/11 and to PLC-beta3 via Gbetagammai3

P2 receptor subtypes and their signaling mechanisms were characterized in dispersed smooth muscle cells. UTP and ATP stimulated inositol 1,4,5-triphosphate formation, Ca2+ release, and contraction that were abolished by U-73122 and guanosine 5'-O-(3-thio)diphosphate, and partly inhibited (50-60%) by pertussis toxin (PTX). ATP analogs (adenosine 5'-(alpha, beta-methylene)triphosphate, adenosine 5'-(beta, gamma-methylene)triphosphate, and 2-methylthio-ATP) stimulated Ca2+ influx and contraction that were abolished by nifedipine and in Ca2+-free medium. Micromolar concentrations of ATP stimulated both Ca2+ influx and Ca2+ release. ATP and UTP activated Gq/11 and Gi3 in gastric and aortic smooth muscle and heart membranes, Gq/11 and Gi1 and/or Gi2 in liver membranes, and Go and Gi1-3 in brain membranes. Phosphoinositide hydrolysis stimulated by ATP and UTP was mediated concurrently by Galphaq/11-dependent activation of phospholipase (PL) C-beta1 and Gbetagammai3-dependent activation of PLC-beta3. Phosphoinositide hydrolysis was partially inhibited by PTX or by antibodies to Galphaq/11, Gbeta, PLC-beta1, or PLC-beta3, and completely inhibited by the following combinations (PLC-beta1 and PLC-beta3 antibodies; Galphaq/11 and Gbeta antibodies; PLC-beta1 and Gbeta antibodies; PTX with either PLC-beta1 or Galphaq/11 antibody). The pattern of responses implied that P2Y2 receptors in visceral, and probably vascular, smooth muscle are coupled to PLC-beta1 via Galphaq/11 and to PLC-beta3 via Gbetagammai3. These receptors co-exist with ligand-gated P2X1 receptors activated by ATP analogs and high levels of ATP.     

Characterization and distribution of somatostatin SS-1 and SRIF-1 binding sites in rat brain: identity with SSTR-2 receptors

Somatostatin (SRIF) SS-1 binding sites were initially defined in radioligand binding studies performed in rat brain cerebral cortex membranes using [125I]204-090 (a radiolabelled Tyr3 analogue of SMS 201-995, octreotide). SRIF-1 recognition sites were defined in binding studies performed with [125I]MK 678 (seglitide). Both SS-1 and SRIF-1 sites were characterized by their high affinity for SRIF-14, SRIF-28 and for cyclic peptides such as octreotide and seglitide, in marked contrast to SS-2 and SRIF-2 sites which have very low affinity for these synthetic SRIF analogues. In the present study, SS-1 and SRIF-1 radioligand binding studies were performed in rat cortex membranes and compared to results obtained in cloned Chinese hamster ovary cells expressing human SSTR-2 receptors using [125I]204-090 and/or [125I]MK-678. The rank orders of affinity of a variety of SRIF analogues and synthetic peptides for SS-1/SRIF-1 binding sites and recombinant SSTR-2 receptors were very similar and correlated highly significantly (r = 0.94-0.99); by contrast, correlation between SS-1 and SSTR-5 (r = 0.44) or SSTR-3 binding (r = 0.07) was not significant. Autoradiographic studies were performed in rat brain using both radioligands [125I]204-090 and [125I]MK-678 and compared with the distribution of SSTR-2 receptor mRNA determined using in situ hybridization. A clear overlap was observed between the distribution of SSTR-2 mRNA and binding sites labelled with both radioligands. SSTR-2 receptor-mediated inhibition of forskolin-stimulated adenylate cyclase in Chinese hamster ovary cells by a variety of SRIF analogues and short synthetic peptides displayed a rank order of potency highly similar to their rank order of affinity at SS-1/SRIF-1 binding sites. It is concluded that SS-1 and SRIF-1 binding sites respectively labelled with [125I]204-090 and [125I]MK 678, both display the pharmacological profile of SSTR-2 receptors, that the distribution of [125I]204-090 and [125I]MK-678 binding sites in rat brain is superimposable and largely comparable to that of SSTR-2 mRNA expression. It is also shown that neither [125I]204-090 nor [125I]MK-678 label SSTR-3 or SSTR-5 receptors in rat brain. Finally, it is demonstrated that SSTR-2 receptors can very efficiently couple to adenylate cyclase activity in an inhibitory manner.     

Neuropeptide Y Y1 receptors in the rat forebrain: autoradiographic demonstration of [125I][Leu31,Pro34]-NPY binding sites and neurons expressing Y1 receptor mRNA

Using the specific monoiodinated NPY analog [Leu31,Pro34]-NPY we have localized NPY binding sites of the Y1 type in forebrain areas of the rat. The resulting receptor autoradiograms were compared with the regional distribution and cellular localization of the mRNA encoding Y1 receptor as demonstrated by in situ hybridization histochemistry. High densities of Y1 binding sites were present in the cerebral cortex, the claustrum, the thalamus and the medical mammillary nucleus, while moderate densities of Y1 binding sites were observed in the amygdalahippocampal complex. Lower binding densities were observed in septal nuclei, most hypothalamic nuclei and the circumventricular organs. High levels of Y1 mRNA were observed in the granula cell layer of the hippocampal dentate gyrus, several thalamic nuclei and the hypothalamic arcuate nucleus, while moderate levels of Y1 mRNA were seen in the frontoparietal cortex, several thalamic nuclei, the hippocampal pyramidal layers, the subiculum, the olfactory tubercle, the claustrum and a number of hypothalamic nuclei. Using the hypothalamic arcuate nucleus as an example, the distribution of immunoreactive NPY, Y1 mRNA and Y1 binding sites was compared, and possible implications of Y1 mediated actions within this nucleus are discussed. The present study further enlightens the anatomical distribution of NPY binding sites of the Y1 type within the central nervous system of the rat, and extends the understanding of central actions of NPY mediated via this type of receptor.     

3H]MDL 100,907 labels 5-HT2A serotonin receptors selectively in primate brain

The selective antagonist for the 5-HT2A serotonin receptor MDL 100,907, recently characterized autoradiographically in rat brain, has been characterized as a radioligand for the visualization of this receptor in human and monkey brain. In both species [3H]MDL 100,907 binding to brain sections was saturable, had sub-nanomolar affinity (Kd = 0.14-0.19 nM in human brain; Kd= 0.16-0.19 nM in monkey brain) and presented a pharmacological profile consistent with its binding to 5-HT2A receptors (rank order of affinity for [3H]MDL 100,907-labeled receptors: MDL 100,907 > spiperone > ketanserin > mesulergine). The autoradiographical signal obtained with [3H]MDL 100,907 was compared to the signal obtained with [3H]ketanserin, [3H]RP62203 and [3H]mesulergine in both species, and to the distribution of 5-HT2A receptor mRNA as determined by in situ hybridization in monkey brain. At variance with the other radioligands, [3H]MDL 100,907 showed a single population of binding sites with extremely low levels of non-specific binding. As expected, mesulergine showed low affinity for [3H]MDL 100,907-labeled receptors and the autoradiographic pattern shown by [3H]mesulergine confirmed the lack of labeling of the 5-HT2A receptor by this radioligand in primate brain. The similarity of the distribution of [3H]MDL 100,907-labeled receptors and 5-HT2A mRNA in monkey brain, supports the selectivity of this radioligand for 5-HT2A receptors and suggests a somatodendritic localization of these receptors. The present results confirm [3H]MDL 100,907 as the radioligand of choice at present for the autoradiographic visualization of 5-HT2A receptors in mammalian brain including post-mortem human brain.     

Localization of HIV-1 co-receptors CCR5 and CXCR4 in the brain of children with AIDS

The neuropeptide Y Y2 receptor is one of six receptor subtypes mediating the multiform physiological actions of neuropeptide Y. The Y2 receptor has been demonstrated to be the most predominant receptor subtype in the human brain and appears to be involved in many neuropeptide Y actions, such as the regulation of locomotor activity, cardiovascular functions, memory processing, circadian rhythms and release of other neurotransmitters. We have recently demonstrated the widespread and abundant distribution of neuropeptide Y Y1 receptor messenger RNA in the human cerebral cortex (different laminar patterns within distinct cortical regions), hippocampal dentate gyrus and striatum. To assess a possible differential distribution of Y1 and Y2 receptor messenger RNAs, the regional expression of neuropeptide Y Y2 messenger RNA-containing cells in the human brain was analysed, in particular within the cerebral cortex and striatum. In situ hybridization experiments revealed the localization of the Y2 messenger RNA signal throughout all cortical regions, with the highest intensity per cell apparent in lamina IV, with the exception of the striate cortex, which showed an intense labelling primarily in layer VI. The striatum expressed low to undetectable levels of the Y2 receptor messenger RNA. The dentate gyrus and the CA2 region presented the highest hybridization signals, while a very weak Y2 messenger RNA expression was found in the CA1 region and subiculum. Positive Y2 messenger RNA hybridization signals were also detected in the lateral geniculate nucleus, amygdala, substantia nigra, hypothalamus, cerebellum and choroid plexus. These results demonstrate the widespread distribution of neuropeptide Y Y2 receptor messenger RNA in the human brain, with a pattern of expression distinct from the Y1 subtype, suggesting that these two receptor subtypes may mediate different neuropeptide Y functions in the human brain, mainly through actions on different neuronal systems.     

A twin study of antisocial and neurotic symptoms in childhood

1. 2,2'-Pyridylisatogen tosylate (PIT) has been reported to be an irreversible antagonist of responses to adenosine 5'-triphosphate (ATP) at metabotropic purinoceptors (of the P2Y family) in some smooth muscles. When a recombinant P2Y1 purinoceptor (derived from chick brain) is expressed in Xenopus oocytes, ATP and 2-methylthioATP (2-MeSATP) evoke calcium-activated chloride currents (ICl,Ca) in a concentration-dependent manner. The effects of PIT on these agonist responses were examined at this cloned P2Y purinoceptor. 2. PIT (0.1-100 microM) failed to stimulate P2Y1 purinoceptors directly but, over a narrow concentration range (0.1-3 microM), caused a time-dependent potentiation (2-5 fold) of responses to ATP. The potentiation of ATP-responses by PIT was not caused by inhibition of oocyte ecto-ATPase. At high concentrations (3-100 microM), PIT irreversibly inhibited responses to ATP with a IC50 value of 13 +/- 9 microM (pKB = 4.88 +/- 0.22; n = 3). PIT failed to potentiate inward currents evoked by 2-MeSATP and only inhibited the responses to this agonist in an irreversible manner. 3. Known P2 purinoceptor antagonists were tested for their ability to potentiate ATP-responses at the chick P2Y1 purinoceptor. Suramin (IC50 = 230 +/- 80 nM; n = 5) and Reactive blue-2 (IC50 = 580 +/- 130 nM; n = 6) reversibly inhibited but did not potentiate ATP-responses. Coomassie brilliant blue-G (0.1-3 microM) potentiated ATP-responses in three experiments, while higher concentrations (3-100 microM) irreversibly inhibited ATP-responses. The results indicated that potentiation and receptor antagonism were dissociable and not a feature common to all known P2 purinoceptor antagonists. 4. In radioligand binding assays, PIT showed a low affinity (pKi < 5) for a range of membrane receptors, including: alpha 1, alpha 2-adrenoceptors, 5-HT1A, 5-HT1B, 5-HT2, 5-HT3, D1, D2, muscarinic, central benzodiazepine, H1, mu-opioid, dihydropyridine and batrachotoxin receptors. PIT showed some affinity (pKi = 5.3) for an adenosine (A1) receptor. 5. In guinea-pig isolated taenia caeci, PIT (12.5-50 microM) irreversibly antagonized relaxations to ATP (3-1000 microM); PIT also directly relaxed the smooth muscle and histamine was used to restore tone. Relaxations to nicotine (10-100 microM), evoked by stimulating intrinsic NANC nerves of taenia caeci preparations in the presence of hyoscine (0.3 microM) and guanethidine (17 microM), were not affected by PIT (50 microM, for 25-60 min). 6. These experiments indicate that PIT causes an irreversible antagonism of ATP receptors but, for recombinant chick P2Y1 purinoceptors, this effect is preceded by potentiation of ATP agonism. The initial potentiation by PIT (and by Coomassie brilliant blue-G) of ATP-responses raises the possibility of designing a new class of modulatory drugs to enhance purinergic transmission at metabotropic purinoceptors.     

125I]Leu31, Pro34-PYY is a high affinity radioligand for rat PP1/Y4 and Y1 receptors: evidence for heterogeneity in pancreatic polypeptide receptors

Cloned receptors for the PP-fold peptides are subdivided into Y1, Y2, PP1/Y4, Y5 and Y6. NPY and PYY have similar affinity for Y1, Y2, Y5 and Y6 receptors while PP has highest affinity for PP1. Pro34-substituted analogs of NPY and PYY have selectivity for Y1 and Y1-like receptors over Y2 receptors. In the present study, we found the putative Y1-selective radioligand, [125I]Leu31, Pro34-PYY, also binds with high affinity to the rat PP1 receptor in cell lines expressing the receptor. However, in rat brain sections, [125I]Leu31, Pro34-PYY does not appear to bind to the interpeduncular nucleus, a brain region containing a high density of [125I]-bPP binding sites. Therefore, it appears there is additional heterogeneity in receptors recognizing PP.     

Purinoceptors: are there families of P2X and P2Y purinoceptors?

There has been an exponential growth in interest in purinoceptors since the potent effects of purines were first reported in 1929 and purinoceptors defined in 1978. A distinction between P1 (adenosine) and P2 (ATP/ADP) purinoceptors was recognized at that time and later, A1 and A2, as well as P2x and P2y subclasses of P1 and P2 purinoceptors were also defined. However, in recent years, many new subclasses have been claimed, particularly for the receptors to nucleotides, including P2t, P2z, P2u(n) and P2D, and there is some confusion now about how to incorporate additional discoveries concerning the responses of different tissues to purines. The studies beginning to appear defining the molecular structure of P2-purinoceptor subtypes are clearly going to be important in resolving this problem, as well as the introduction of new compounds that can discriminate pharmacologically between subtypes. Thus, in this review, on the basis of this new data and after a detailed analysis of the literature, we propose that: (1) P2X(ligand-gated) and P2Y(G-protein-coupled) purinoceptor families are established; (2) four subclasses of P2X-purinoceptor can be identified (P2X1-P2X4) to date; (3) the variously named P2-purinoceptors that are G-protein-coupled should be incorporated into numbered subclasses of the P2Y family. Thus: P2Y1 represents the recently cloned P2Y receptor (clone 803) from chick brain; P2Y2 represents the recently cloned P2u (or P2n) receptor from neuroblastoma, human epithelial and rat heart cells; P2Y3 represents the recently cloned P2Y receptor (clone 103) from chick brain that resembles the former P2t receptor; P2Y4-P2Y6 represent subclasses based on agonist potencies of newly synthesised analogues; P2Y7 represents the former P2D receptor for dinucleotides. This new framework for P2 purinoceptors would be fully consistent with what is emerging for the receptors to other major transmitters, such as acetylcholine, gamma-aminobutyric acid, glutamate and serotonin, where two main receptor families have been recognised, one mediating fast receptor responses directly linked to an ion channel, the other mediating slower responses through G-proteins. We fully expect discussion on the numbering of the different receptor subtypes within the P2X and P2Y families, but believe that this new way of defining receptors for nucleotides, based on agonist potency order, transduction mechanisms and molecular structure, will give a more ordered and logical approach to accommodating new findings. Moreover, based on the extensive literature analysis that led to this proposal, we suggest that the development of selective antagonists for the different P2-purinoceptor subtypes is now highly desirable, particularly for therapeutic purposes.     

Selectivity and activity of adenine dinucleotides at recombinant P2X2 and P2Y1 purinoceptors

1. Adenine dinucleotides (Ap3A, x = 2-6) are naturally-occurring polyphosphated nucleotidic substances which are found in the CNS and are known to be released in a calcium-dependent manner from storage vesicles in brain synaptosomes. The selectivity and activity of adenine dinucleotides for neuronally-derived recombinant P2 purinoceptors were studied using P2X2 and P2Y1 subtypes expressed in Xenopus oocytes. 2. For the P2Y1 subtype derived from chick brain, Ap3A was equipotent and as active as ATP (EC50 values: 375 +/- 86 nM and 334 +/- 25 nM, respectively). Ap4A was a weak partial agonist and other dinucleotides were inactive as agonists. None of the inactive dinucleotides were antagonists nor modulated the activity of Ap3A and ATP. 3. For the P2X2 subtype derived from rat PC12 cells, Ap4A was as active as ATP but less potent (EC50 values: 15.2 +/- 1 microM and 3.7 +/- 0.7 microM, respectively). Other adenosine dinucleotides were inactive as either agonists or antagonists. 4. Ap5A (1-100 nM) potentiated ATP-responses at the P2X2 subtype, showing an EC50 of 2.95 +/- 0.7 nM for this modulatory effect. Ap5A (10 nM) shifted the concentration-response curves for ATP to the left by one-half log10 unit but did not alter the Hill co-efficient for ATP (nH = 2.1 +/- 0.1). Ap5A (10 nM) failed to potentiate Ap4A-responses but did enhance the efficacy of the P2 purinoceptor antagonist, suramin, by 12 fold at the P2X2 subtype. 5. In conclusion, the results show that ionotropic (P2X2) and metabotropic (P2Y1) ATP receptors which occur in the CNS are activated selectively by naturally-occurring adenine dinucleotides which are known to be released with nucleotides from storage vesicles. The observed potentiation of P2X2-responses by Ap5A, where co-released with ATP by brain synaptosomes, may have a functional bearing in purinergic signalling in the CNS.     

Autoradiographic visualization of group III metabotropic glutamate receptors using [3H]-L-2-amino-4-phosphonobutyrate

1. In vitro receptor autoradiography using [3H]-L-2-amino-4-phosphonobutyrate ([3H]-L-AP4) binding to sections of rat brain has been characterized and shown to most likely represent labelling of group III metabotropic glutamate receptors. 2. Specific [3H]-L-AP4 binding to rat brain sections was observed at high densities in the molecular layer of the cerebellar cortex and the outer layer of the superior colliculus. Moderate levels were observed throughout the cerebral cortex, in the molecular layer of the hippocampal dentate gyrus, in thalamus, striatum, substantia nigra and in the medial geniculate nucleus. Low levels of [3H]-L-AP4 binding were found in other regions of the hippocampal formation, in the entorhinal cortex and the granule cell layer of cerebellum. 3. Inhibitors of sodium- or calcium/chloride-dependent glutamate uptake did not displace [3H]-L-AP4 binding to rat brain sections indicating that the observed binding does not represent [3H]-L-AP4 uptake via these carriers. Furthermore, in contrast to [3H]-L-AP4 uptake into cerebellar membranes, [3H]-L-AP4 binding to brain sections was sensitive to guanosine-5'-O-(3-thio)trisphosphate-gamma-S. 4. In the molecular layer of the cerebellar cortex, [3H]-L-AP4 binding showed a maximal binding density (Bmax) of 0.52+/-0.06 pmol mg(-1) tissue and an affinity (Kd) of 346 nM. The rank order of affinity for displacement of [3H]-L-AP4 binding to rat brain sections was: L-AP4 > L-serine-O-phosphate > glutamate > (L)-2-aminomethyl-4-phosphonobutanoate > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate which is in agreement with a group III metabotropic glutamate receptor pharmacology.     

Modelling the P2Y purinoceptor using rhodopsin as template

The P2Y1 purinoceptor cloned from chick brain (Webb, T. et al (1993) FEBS Lett., 324, 219-225) is a 362 amino acid, 41 kDa protein. To locate residues tentatively involved in ligand recognition a molecular model of the P2Y purinoceptor has been constructed. The model was based on the primary sequence and structural homology with the G-protein coupled photoreceptor rhodopsin, in analogy to the method proposed by Ballesteros and Weinstein ((1995) Meth. Neurosci. 25, 366-428). Transmembrane helices were constructed from the amino acid sequence, minimized individually, and positioned in a helical bundle. The helical bundle was then minimized using the Amber forcefield in Discover (BIOSYM Technologies) to obtain the final model. Several residues that have been shown to be critical in ligand binding in other GPCRs are conserved in the P2Y1 purinoceptor. According to our model the side chains of these conserved residues are facing the internal cleft in which ligand binding likely occurs. The model also suggests four basic residues (H121 in TM3, H266 and K269 in TM6 and R299 in TM7) near the extracellular surface that might be involved in ligand binding. These basic residues might be essential in coordinating the triphosphate chain of the endogenous ligand adenosine 5'-triphosphate (ATP). Potential binding sites for agonists have been explored by docking several derivatives (including newly synthesized N6-derivatives) into the model. The N6-phenylethyl substituent is tolerated pharmacologically, and in our model this substituent occupies a region predominantly defined by aromatic residues such as F51 (TM1), Y100 (TM2) and F120 (TM3). The dimethylated analogue of ATP, N6,N6-dimethyl-adenosine 5'-triphosphate, is less well tolerated pharmacologically, and our model predicts that the attenuated activity is due to interference with hydrogen bonding capacity to Q296 (TM7).     

Sudden unexpected deaths in epilepsy--where are we now?

AIM: To study the effects of menidipine (Men) on the affinity and density of dihydropyridines (DHP) binding sites in the cell membranes of left ventricle (LV) and brain in elderly renovascular hypertensive rats (RVHR) with LV hypertrophy. METHODS: Renovascular hypertension was produced by clipping the left renal artery in 20-month-old rats. The affinity and density of DHP binding sites in the cell membranes of LV and brain were assessed by radioligand assay. RESULTS: Men (20 mg.kg-1.d-1 ig for 9 wk) decreased markedly the systolic blood pressure and the LV weight (P < 0.01). Though not affecting the density of DHP receptor (Bmax), Men markedly decreased the total number of DHP binding sites in hypertrophied LV (from 5.95 +/- 0.62 to 4.0 +/- 1.1 pmol.LV). Men also reduced Bmax of DHP binding sites in the thalamus (from 522 +/- 27 to 371 +/- 24 pmol/g protein) and hippocampus (from 498 +/- 26 to 332 +/- 32 pmol/g protein). CONCLUSION: Men reversed the LV hypertrophy from renovascular hypertension accompanied with reduced total number of DHP binding sites in the cell membranes of LV and Bmax of the cell membranes of thalamus and hippocampus from elderly LV hypertrophied rats.     

Glycosyltransferase catalyzed assemblage of sialyl-Lewis(a)-saccharopeptides

P1,P4-Diadenosine 5'-tetraphosphate (Ap4A) acts as an extracellular modulator through its interaction with purinoceptors. Our laboratory has demonstrated the presence of an Ap4A receptor in cardiac tissue [1,2]. Due to the rapid hydrolysis of ATP by cardiac membranes the relationship of ATP and Ap4A binding to purinoceptors on cardiac membranes has not been characterized. In this communication we used two approaches to determine the relationship of ATP to the Ap4A receptor. Radioligand binding carried out with [alpha-32P]Ap4A and adenosine 5'-O-?3-thiotriphosphate? ([gamma-35S]ATPgammaS) demonstrates the presence of a single high affinity binding site for Ap4A and the presence of two binding sites for ATPgammaS. The second approach utilized immunoaffinity purified Ap4A receptor that was shown to be free of ATPase and Ap4Aase activities. Non-radiolabeled Ap4A and ATPgammaS effectively inhibited photocrosslinking of [alpha-32P]8-N3Ap4A to the receptor polypeptide while ATP was a much less effective inhibitor. Furthermore, on plasma membranes [alpha-32P]8-N3Ap4A photocrosslinked to only a 50 kDa polypeptide. These data are consistent with Ap4A interacting with a homogeneous population of receptors on cardiac plasma membranes but with ATP having a low affinity for the receptor.     

Detailed mapping of serotonin 5-HT1B and 5-HT1D receptor messenger RNA and ligand binding sites in guinea-pig brain and trigeminal ganglion: clues for function

The similar pharmacology of the 5-HT1B and 5-HT1D receptors, and the lack of selective compounds sufficiently distinguishing between the two receptor subtypes, have hampered functional studies on these receptors. In order to provide clues for differential functional roles of the two subtypes, we performed a parallel localization study throughout the guinea-pig brain and the trigeminal ganglia by means of quantitative in situ hybridization histochemistry (using [35S]-labelled riboprobes probes for receptor messenger RNA) and receptor autoradiography (using a new radioligand [3H]alniditan). The anatomical patterns of 5-HT1B and 5-HT1D receptor messenger RNA were quite different. While 5-HT1B receptor messenger RNA was abundant throughout the brain (with highest levels in the striatum, nucleus accumbens, olfactory tubercle, cortex, hypothalamus, hippocampal formation, amygdala, thalamus, dorsal raphe and cerebellum), 5-HT1D receptor messenger RNA exhibited a more restricted pattern; it was found mainly in the olfactory tubercle, entorhinal cortex, dorsal raphe, cerebellum, mesencephalic trigeminal nucleus and in the trigeminal ganglion. The density of 5-HT(1B/1D) binding sites (combined) obtained with [3H]alniditan autoradiography was high in the substantia nigra, superior colliculus and globus pallidus, whereas lower levels were detected in the caudate-putamen, hypothalamus, hippocampal formation, amygdala, thalamus and central gray. This distribution pattern was indistinguishable from specific 5-HT1B receptor labelling in the presence of ketanserin under conditions to occlude 5-HT1D receptor labelling; hence the latter were below detection level. Relationships between the regional distributions of the receptor messenger RNAs and binding sites and particular neuroanatomical pathways are discussed with respect to possible functional roles of the 5-HT1B and 5-HT1D receptors.     

Binding of the non-classical cannabinoid CP 55,940, and the diarylpyrazole AM251 to rodent brain cannabinoid receptors

The binding of [123I]AM251 (a radioiodinated analog of the cannabinoid CB1 receptor antagonist SR141716A) was compared to that of [3H]CP 55,940 in mouse and rat brain preparations. Scatchard analysis of the binding of [123I]AM251 and [3H]CP 55,940 to membranes prepared from mouse cerebellum, striatum and hippocampus yielded similar Bmax values (15-41 pmol/g wet wt tissue). Kd values were lower for [123I]AM251 (0.23-0.62 nM) than for [3H]CP 55,940 (1.3-4 nM). CP 55,940 and SR141716A increased dissociation of [123I]AM251 from binding sites in mouse cerebellar homogenates to a similar extent. The structurally dissimilar cannabinoid receptor ligands THC, methanandamide, WIN 55, 212-2, CP 55,940 and SR141716A were each able to fully compete with binding of both [123I]AM251 and [3H]CP 55,940 in mouse cerebellum. In vitro autoradiography demonstrated that the distribution of binding sites for [123I]AM251 in rat brain was very similar to published distributions of binding sites for [3H]CP 55,940. Together, these observations suggest that AM251 binds to the same site (the cannabinoid CB1 receptor) in rodent brains as CP 55,940. However, the binding site domains which interact with AM251 and CP 55,940 may not be identical, since IC50 values for cannabinoid receptor ligands depended on whether [123I]AM251 or [3H]CP 55,940 was used as radioligand.     

Autoradiographic mapping of 5-HT1B- and 5-HT1D receptors in human brain using [3H]alniditan, a new radioligand

[3H]alniditan, a new potent non-indole serotonin 5-HT1B/1D agonist, was used as a radioligand to characterize 5-HT1B and 5-HT1D receptor (previously termed 5-HT1D beta and 5-HT1D alpha) in various regions of the human brain. Quantitative receptor autoradiography was applied for high anatomical resolution and sensitivity. Highest densities of 5-HT1B/1D receptors were found in the substantia nigra and in the globus pallidus. High to moderate densities were measured in the caudate nucleus, putamen, nucleus accumbens, central gray and hippocampal formation. Very low densities were detected in various cortical regions. In the cerebellum no [3H]alniditan binding was detected. Selective 5-HT1B receptor labeling was achieved using [3H]alniditan in the presence of 300 nM of ketanserin (sufficient to block 5-HT1D receptor labeling). The identity of the 5-HT1B binding sites under these conditions was corroborated by the pIC50 of sumatriptan, which corresponded to its affinity for cloned human 5-HT1B receptors expressed in cells. Surprisingly, the distribution of selective 5-HT1B receptor labeling was completely identical to the distribution of labeling of 5-HT1B + 5-HT1D receptors. The present data indicate that [3H]alniditan is a suitable radioligand for measuring 5-HT1B/1D receptor in the human brain and that the 5-HT1B binding sites are predominant in the presently investigated regions of the human brain.