Inhibition Of Radioligand Binding Research Articles

Assessment of mazindane, a pro-drug form of mazindol, in assays used to define cocaine treatment agents

The current studies compared mazindane (5-(4-chlorophenyl)-2,3-dihydro-5H-imidazo [2,1a] isoindole) hydrogen sulfate, a water soluble pro-drug of mazindol (5-(4-chlorophenyl-2,3-dihydro-5H-imidazo [2,1-a] isoindol-5-ol), with mazindol in assays used to define cocaine treatment agents. Both compounds enhanced motor activity (LMA) in Swiss Webster mice with ED 50 values of 2.5 mg/kg i.p. for mazindane and 3.9 mg/kg i.p. for mazindol. At 25 mg/kg mazindane displayed toxic effects and death while mazindol was effect/death free at 50 mg/kg. In Sprague–Dawley rats trained to discriminate cocaine from saline both compounds fully substituted for cocaine with mazindane being fourfold more potent in the total session (0.33 vs. 1.3 mg/kg i.p.) and first reinforcer (0.29 vs. 1.2 mg/kg i.p). Complete substitution was observed in rhesus monkeys trained to discriminate cocaine from saline with ED 50 values for mazindane (0.134 mg/kg i.m.) and mazindol (0.119 mg/kg i.m.). Mazindol exhibited little or no activity at 10 −5 M in inhibiting radioligand binding at 14 neurotransmitter sites while mazindane gave weak activity at the histamine H 1 and 5-hydroxytryptamine 5-HT 3 sites. These results demonstrate that mazindane could be a useful alternative to mazindol as a pharmacological tool because of its similar profile of activity and enhanced water solubility.

Solid phase synthesis and evaluation of Tyr-Tic-Phe-Phe(p-NHCOCH(2)Br) ([Phe(p-bromoacetamide)(4)]TIPP), a potent affinity label for delta opioid receptors.

Derivatives of the delta opioid receptor selective peptide Tyr-Tic-Phe-Phe-OH (TIPP) containing a p-bromoacetamide moiety on the phenyl ring of Phe(3) or Phe(4) were prepared by solid phase synthesis. [Phe(p-NHCOCH(2)Br)(4)]TIPP exhibited high affinity for cloned delta receptors (IC(50) = 5.4 nM), and incubation with only 2.5 nM resulted in 85% wash resistant inhibition of radioligand binding to delta receptors. Therefore, this peptide is a potent affinity label for further study of delta opioid receptors.

Functional interaction of lithocholic acid conjugates with M3 muscarinic receptors on a human colon cancer cell line

Lithocholic acid (LA) conjugates interact with M3 receptors, the muscarinic receptor subtype that modulates colon cancer cell proliferation. This observation prompted us to examine the action of bile acids on two human colon cancer cell lines: H508, which expresses M3 receptors, and SNU-C4, which does not. Cellular proliferation was determined using a colorimetric assay. Interaction with muscarinic receptors was determined by measuring inhibition of muscarinic radioligand binding and changes in cellular inositol phosphate (IP) formation. Lithocholyltaurine (LCT) caused a dose-dependent increase in H508 cell proliferation that was not observed in SNU-C4 cells. After a 6-day incubation with 300 μM LCT, H508 cell proliferation increased by 200% compared to control. Moreover, in H508 cells, LCT caused a dose-dependent inhibition of radioligand binding and an increase in IP formation. LCT did not alter the rate of apoptosis in H508 or SNU-C4 cells. These data indicate that, at concentrations achievable in the gut, LA derivatives interact with M3 muscarinic receptors on H508 human colon cancer cells, thereby causing an increase in IP formation and cell proliferation. This suggests a mechanism whereby alterations in intestinal bile acids may affect the growth of colon cancer cells.

Pharmacological characterisation of the goldfish somatostatin sst 5 receptor

Somatostatin (somatotropin release inhibiting factor, SRIF), exerts its effects via specific G protein coupled receptors of which five subtypes have been cloned (sst 1–5). Recently, SRIF receptors have also been cloned from fish tissues. In this study, goldfish sst 5 receptors (gfsst 5) were expressed and characterised in the Chinese hamster lung fibroblast cell line, that harbours the luciferase reporter gene driven by the serum responsive element (CCL39-SRE-Luci). The agonist radioligands [ 125I]-LTT-SRIF-28 ([Leu 8, dTrp 22, 125I-Tyr 25]SRIF-28) and [ 125I][Tyr 10]cortistatin-14 labelled similar receptor densities with high affinity and in a saturable manner (p K d: 9.99–9.71; B max: 300–350 fmol mg −1). 5′-Guanylyl-imidodiphosphate inhibited radioligand binding to some degree (38.5–57.9%). In competition binding studies, the pharmacological profile of SRIF binding sites defined with [ 125I]LTT-SRIF-28 and [ 125I][Tyr 10]cortistatin-14 correlated significantly ( r 2=0.97, n=20). Pharmacological profiles of human and mouse sst 5 receptors expressed in CCL39 cells correlated markedly less with those of the gfsst 5 profile ( r 2=0.52–0.78, n≥16). Functional expression of the gfsst 5 receptor was examined by measurement of agonist-induced luciferase expression and stimulation of [ 35S]GTPγS ([ 35S]guanosine 5′- O-(3-thiotriphosphate) binding. Profiles were similar to those achieved in radioligand binding studies ( r 2=0.81–0.93, n=20), although relative potency (pEC 50) was reduced compared to p K d values. Relative efficacy profiles of luciferase expression and [ 35S]GTPγS binding, were rather divergent ( r 2=0.48, n=20) with peptides showing full agonism at one pathway and absence of agonism at the other. BIM 23056 ( d-Phe-Phe-Tyr- d-Trp-Lys-Val-Phe- d-Nal-NH 2) acted as an antagonist on the effects of SRIF-14 (p K B=6.74±0.23) on stimulation of [ 35S]GTPγS binding. Pertussis toxin abolished the effect of SRIF-14 on luciferase expression and [ 35S]GTPγS binding suggesting coupling of the receptor to G i/G o proteins. In summary, the present studies demonstrate that the gfsst 5 receptor has a similar pharmacological profile and transductional properties to mammalian sst 5 receptors. The difference in efficacy profiles defined using different functional assays suggests numerous, agonist specific, conformational receptor states, and/or ligand-dependent receptor trafficking.

Inhibition of μ and δ opioid receptor ligand binding by the peptide aldehyde protease inhibitor, leupeptin

We reported recently that the ubiquitin–proteasome pathway is involved in agonist-induced down regulation of μ and δ opioid receptors [J. Biol. Chem. 276 (2001) 12345]. While evaluating the effects of various protease inhibitors on agonist-induced opioid receptor down regulation, we observed that while the peptide aldehyde, leupeptin (acetyl- l-Leucyl- l-Leucyl- l-Arginal), did not affect agonist-induced down regulation, leupeptin at submillimolar concentrations directly inhibited radioligand binding to opioid receptors. In this study, the inhibitory activity of leupeptin on radioligand binding was characterized utilizing human embryonic kidney (HEK) 293 cell lines expressing transfected μ, δ, or κ opioid receptors. The rank order of potency for leupeptin inhibition of [ 3H]bremazocine binding to opioid receptors was μ>δ>κ. In contrast to the effect of leupeptin, the peptide aldehyde proteasome inhibitor, MG 132 (carbobenzoxy- l-Leucyl- l-Leucyl- l-Leucinal), had significantly less effect on bremazocine binding to μ, δ, or κ opioid receptors. We propose that leupeptin inhibits ligand binding by reacting reversibly with essential sulfhydryl groups that are necessary for high-affinity ligand/receptor interactions.

Interaction of anandamide with the M 1 and M 4 muscarinic acetylcholine receptors

The M 1 and M 4 muscarinic acetylcholine receptors are the most abundant muscarinic receptor subtypes in the brain, and are involved in learning and memory. Because cannabinoid receptors are also abundantly expressed in similar brain regions and mediate opposite effects to acetylcholine on cognition, the present study investigated whether the endocannabinoid agonist, anandamide, and its metabolically stable derivative, methanandamide, directly modified the binding properties of the human M 1 and M 4 receptors individually expressed in CHO cell membranes. Experiments utilized the antagonists, [ 3H] N-methylscopolamine and [ 3H]quinuclidinyl benzilate. When acetylcholine was used as the inhibiting ligand, shallow, biphasic isotherms were observed at both receptors, characterised by similar apparent dissociation constants for high and low affinity binding at each receptor but with a greater proportion of high affinity sites at the M 4 (40–45%) than at the M 1 receptor (17–20%). In contrast, anandamide and methanandamide inhibited the binding of both radioligands over a narrow (low micromolar) concentration range, with monophasic isotherms characterized by Hill coefficients significantly greater than 1 at both receptors. These effects were not due to the vehicle used. Further saturation binding analyses found anandamide able to significantly reduce the apparent affinity and maximal density of binding sites labeled by [ 3H]quinuclidinyl benzilate. Interestingly, no significant inhibition of radioligand binding was noted using the synthetic cannabinoid agonist, WIN55212-2, or the cannabinoid CB 1 receptor antagonist, SR141716A. These data thus provide evidence for a direct role of anandamides in modulating muscarinic receptor binding properties through a non-competitive mechanism that is unrelated to their actions on cannabinoid receptors.

High level of alpha2-adrenoceptor in rat foetal liver and placenta is due to alpha2B-subtype expression in haematopoietic cells of the erythrocyte lineage.

1. Rat foetal liver contains large amounts of alpha2-adrenoceptors. The present work aimed to identify the receptor subtype and the cell type accounting for high expression and to clarify the mechanisms responsible for the sharp decrease in hepatic receptivity occurring during the late stage of foetal development. 2. Binding experiments indicated that the density of alpha2-adrenoceptors in the foetal liver (embryonic day 18; 615+/-155 fmol mg(-1) of protein) is 18 fold higher than in newborn or adult (35.2+/-4.3 fmol mg(-1)). A high amount of receptor is also found in the placenta (443+/-53 fmol mg(-1)). In both tissues, the rank order of antagonists to inhibit radioligand binding matched the pharmacological profile of the alpha2B-adrenoceptor and exclusively RNG transcripts were detected by RNase protection assays. 3. Isolation of cell fractions from foetal liver showed that alpha2B-adrenoceptor is primarily expressed by haematopoietic cells. Consistent with this view, the receptor is found to be abundant in foetal blood, carried by reticulocytes. The expression in blood gradually declines to zero at 3 weeks of age and it is not recovered following induction of reticulocytosis in adults. 4. In foetal reticulocytes, a low proportion of the receptor population is coupled to G-protein. The alpha2-agonist UK14304 has a marginal effect on cyclic AMP level but significantly increases arachidonic acid release. The function of the receptor remains to be elucidated. However, together with observations on alpha2B-knockout mice, the current finding strongly suggests a role for alpha2B-adrenoceptor during foetal haematopoiesis in rodents.

Differential cellular localization of estrogen receptor α in uterine and mammary cells

The classical α isoform of the estrogen receptor (ER) has been reported to localize almost exclusively in the nucleus. However, studies on non-genomic steroid effects have also suggested the existence of ERs residing at the cell surface. In this work, we present immunological data supporting extra-nuclear ERα localization in uterine (SHM) and mammary (MCF-7) cell lines. Immunocytological studies performed on SHM cells revealed that native ERs mainly localize as a perinuclear cytoplasmic ring. The receptors were rapidly translocated to the nucleus by 17β-estradiol. In addition to nuclear ERs, a peripheral reservoir of ERα immunoreactivity, most probably associated with the plasma membrane, was detected in MCF-7 cells. These results were confirmed by the detection of membrane estrogen binding sites using fluorescent estrogen-BSA derivatives and ligand binding assays to intact cells, where [ 3H]-estradiol could be partly displaced by impeded estrogen conjugates. Partial inhibition of radioligand binding by an antibody against the steroid binding domain of the ERα suggests that the isoform faces the extracellular media in MCF-7 cells. Moreover, ERα-like proteins (∼67 kDa) were found to be associated in isolated membrane subfractions from the cells. However, immunocytology of COS-1 (ER-negative) and SHM cells transfected with the complete cDNA coding for the cloned ERα and β isoforms showed exclusive nuclear localization of the overexpressed ERs. The non-classical distribution of native ERα-like proteins in each cell line, suggests an alternative mode of ERα cellular localization/function. Cell type-dependent processing may account for the differential localization shown by native and expressed receptors in the systems considered.

SCH-202676: An Allosteric Modulator of Both Agonist and Antagonist Binding to G Protein-Coupled Receptors

A novel thiadiazole compound, SCH-202676 (N-(2,3-diphenyl-1,2, 4-thiadiazol-5-(2H)-ylidene)methanamine), has been identified as an inhibitor of both agonist and antagonist binding to G protein-coupled receptors (GPCRs). SCH-202676 inhibited radioligand binding to a number of structurally distinct, heterologously expressed GPCRs, including the human mu-, delta-, and kappa-opioid, alpha- and beta-adrenergic, muscarinic M1 and M2, and dopaminergic D1 and D2 receptors, but not to the tyrosine kinase epidermal growth factor receptor. SCH-202676 had no direct effect on G protein activity as assessed by [35S]guanosine-5'-O-(gamma-thio)triphosphate binding to purified recombinant G(oalpha)- or G(betagamma)-stimulated ADP-ribosylation of G(oalpha) by pertussis toxin. In addition, SCH-202676 inhibited antagonist binding to the beta2-adrenergic receptor expressed in Escherichia coli, a system devoid of classical heterotrimeric G proteins. SCH-202676 inhibited radiolabeled agonist and antagonist binding to the alpha2a-adrenergic receptor with an IC50 value of 0.5 microM, decreased the Bmax value of the binding sites with a slight increase in the KD value, and inhibited agonist-induced activation of the receptor. The effects of SCH-202676 were reversible. Incubation of plasma membranes with 10 microM SCH-202676 did not alter subsequent radioligand binding to the alpha2a-adrenergic receptor and the dopaminergic D1 receptor. Taken together, our data suggest that SCH-202676 has the unique ability to allosterically regulate agonist and antagonist binding to GPCRs in a manner that is both selective and reversible. The scope of the data presented suggests this occurs by direct interaction with a structural motif common to a large number of GPCRs or by activation/inhibition of an unidentified accessory protein that regulates GPCR function.

Synthesis and evaluation of isothiocyanate-containing derivatives of the delta-opioid receptor antagonist Tyr-Tic-Phe-Phe (TIPP) as potential affinity labels for delta-opioid receptors.

Derivatives of the delta-opioid receptor-selective peptide antagonist H-Tyr-Tic-Phe-Phe-OH (TIPP) containing an isothiocyanate moiety at the para position of either Phe(3) or Phe(4) were prepared as potential affinity labels for delta-opioid receptors. The synthesis was accomplished using a general solution-phase synthetic procedure which allows for introduction of affinity labeling groups late in the synthesis of a variety of small peptide substrates. The target peptides and their corresponding amines were then evaluated in radioligand binding experiments using Chinese hamster ovary (CHO) cells expressing delta- and mu-opioid receptors. The peptides [Phe(p-NCS)(3)]TIPP (2) and [Phe(p-NCS)(4)]TIPP (4) showed affinity for delta-receptors comparable to the parent compound TIPP (IC(50) = 12 and 5 nM, respectively, vs 6 nM for TIPP). Both peptides 2 and 4 were able to inhibit radioligand binding to delta-receptors in a wash-resistant manner at a concentration of 10 nM. Therefore, the peptides [Phe(p-NCS)(3)]TIPP (2) and [Phe(p-NCS)(4)]TIPP (4) represent two affinity labels that may prove useful in the study of delta-opioid receptors.

3α-(4-Substituted phenyl)nortropane-2β-carboxylic acid methyl esters show selective binding at the norepinephrine transporter

A series of 3α-(4-substituted)nortropane-2β-carboxylic acid methyl esters was synthesized and evaluated for the ability to inhibit radioligand binding at the dopamine, serotonin, and norepinephrine transporters. 3α-(4-Methylphenyl)nortropane-2β-carboxylic acid methyl ester ( 4c) was found to be selective and highly potent for the norepinephrine transporter (NET) relative to the dopamine and serotonin transporters.

Synthesis and evaluation of N,N-dialkyl enkephalin-based affinity labels for delta opioid receptors.

To develop affinity labels for delta opioid receptors based on peptide antagonists, the Phe(4) residues of N,N-dibenzylleucine enkephalin and N,N-diallyl[Aib(2),Aib(3)]leucine enkephalin (ICI-174, 864) were substituted with either Phe(p-NCS) or Phe(p-NHCOCH(2)Br). A general synthetic method was developed for the conversion of small peptide substrates into potential affinity labels. The target peptides were synthesized using Phe(p-NH(2)) and a Boc/Fmoc orthogonal protection strategy which allowed for late functional group conversion of a p-amine group in the peptides to the desired affinity labeling moieties. A key step in the synthesis was the selective deprotection of a Boc group in the presence of a tert-butyl ester using trimethylsilyl trifluoromethanesulfonate (TMS-OTf). The target peptides were evaluated in radioligand binding experiments in Chinese hamster ovary (CHO) cells expressing delta or mu opioid receptors. The delta receptor affinities of the N, N-dibenzylleucine enkephalin analogues were 2.5-10-fold higher than those for the corresponding ICI-174,864 analogues. In general, substitution at the para position of Phe(4) decreased binding affinity at both delta and mu receptors in standard radioligand binding assays; the one exception was N, N-dibenzyl[Phe(p-NCS)(4)]leucine enkephalin (2) which exhibited a 2-fold increase in affinity for delta receptors (IC(50) = 34.9 nM) compared to N,N-dibenzylleucine enkephalin (IC(50) = 78.2 nM). The decreases in mu receptor affinities were greater than in delta receptor affinities so that all of the analogues tested exhibited significantly greater delta receptor selectivity than the unsubstituted parent peptides. Of the target peptides tested, only N, N-dibenzyl[Phe(p-NCS)(4)]leucine enkephalin (2) exhibited wash-resistant inhibition of radioligand binding to delta receptors. To our knowledge, 2 represents the first peptide-based affinity label to utilize an isothiocyanate group as the electrophilic affinity labeling moiety. As a result of this study, enkephalin analogue 2 emerges as a potential affinity label useful for the further study of delta opioid receptors.

Molecular Determinants of Tuberoinfundibular Peptide of 39 Residues (TIP39) Selectivity for the Parathyroid Hormone-2 (PTH2) Receptor: N-TERMINAL TRUNCATION OF TIP39 REVERSES PTH2 RECEPTOR/PTH1 RECEPTOR BINDING SELECTIVITY

Molecular Determinants of Tuberoinfundibular Peptide of 39 Residues (TIP39) Selectivity for the Parathyroid Hormone-2 (PTH2) Receptor: N-TERMINAL TRUNCATION OF TIP39 REVERSES PTH2 RECEPTOR/PTH1 RECEPTOR BINDING SELECTIVITY

Regulation of human D(1), d(2(long)), d(2(short)), D(3) and D(4) dopamine receptors by amiloride and amiloride analogues.

1. The modulatory effects of the allosteric effectors methylisobutylamiloride (MIA), benzamil and amiloride have been examined at human D(1), D(2), D(3) and D(4) dopamine receptors. The subtype selectivity and the mechanism of action of this allosteric regulation was examined. 2. In radioligand dissociation experiments each modulator accelerated dissociation from all four receptor subtypes indicating allosteric regulation. MIA displayed selectivity for the D(3) subtype for acceleration of radioligand dissociation. 3. In equilibrium binding (pseudo-competition) experiments the three compounds inhibited radioligand binding at the four receptor subtypes. Inhibition curves for D(1), D(2(short)), D(2(long)) and D(3) receptors were described by Hill coefficients exceeding unity and data were fitted best by a model that assumes binding of modulator to both the primary and allosteric binding sites of the receptor (the allosteric/competitive model). 4. At the D(4) subtype, Hill coefficients of unity described the binding data for amiloride and benzamil, consistent with competitive inhibition. The Hill coefficient for MIA at the D(4) subtype was less than unity and data could be fitted well by the allosteric/competitive model, but it was not possible to define unambiguously the modulatory mechanism. For this effect a better definition of the mechanism could be obtained by simultaneous analysis of data obtained in the presence of a range of concentrations of a purely competitive ligand. 5. MIA reduced the potency with which dopamine stimulated [(35)S]-GTPgammaS binding at the D(2) receptor. The effects of MIA could be described by the allosteric/competitive model with effects of MIA to inhibit the binding of dopamine but not its ability to induce a response.

Imidazoline receptor antisera-selected (IRAS) cDNA: cloning and characterization.

The imidazoline-1 receptor (IR1) is considered a novel target for drug discovery. Toward cloning an IR1, a truncated cDNA clone was isolated from a human hippocampal lambda gt11 cDNA expression library by relying on the selectivity of two antisera directed against candidate IR proteins. Amplification reactions were performed to extend the 5' and 3' ends of this cDNA, followed by end-to-end PCR and conventional cloning. The resultant 5131-basepair molecule, designated imidazoline receptor-antisera-selected (IRAS) cDNA, was shown to encode a 1504-amino acid protein (IRAS-1). No relation exists between the amino acid sequence of IRAS-1 and proteins known to bind imidazolines (e.g., it is not an alpha2-adrenoceptor or monoamine oxidase subtype). However, certain sequences within IRAS-1 are consistent with signaling motifs found in cytokine receptors, as previously suggested for an IR1. An acidic region in IRAS-1 having an amino acid sequence nearly identical to that of ryanodine receptors led to the demonstration that ruthenium red, a dye that binds the acidic region in ryanodine receptors, also stained IRAS-1 as a 167-kD band on SDS gels and inhibited radioligand binding of native I1 sites in untransfected PC-12 cells (a source of authentic I1 binding sites). Two epitope-selective antisera were also generated against IRAS-1, and both reacted with the same 167-kD band on Western blots. In a host-cell-specific manner, transfection of IRAS cDNA into Chinese hamster ovary cells led to high-affinity I1 binding sites by criteria of nanomolar affinity for moxonidine and rilmenidine. Thus, IRAS-1 is the first protein discovered with characteristics of an IR1.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and PACAP-Receptor Type 1 Expression in Rat and Human Placenta

Pituitary adenylate cyclase-activating polypeptide (PACAP), the new hypophysiotropic factor member of the vasoactive intestinal peptide (VIP)/secretin/glucagon/GHRH family of neuropeptides, exerts its biological action by interacting with both PACAP-selective type I receptors (PAC1) and type II receptors (VPAC1), which bind both PACAP and VIP. The placenta is a site of production of hypophysiotropic factors that participate in the control of local hormone production, as well as the respective hypothalamic-pituitary neurohormones. In the present study, we show the expression of PACAP gene and irPACAP distribution within rat and human placental tissues, by means of RT-PCR and immunohystochemical experiments. In both rat and human placenta, we evaluated the expression of PAC1 gene by Northern hybridization analysis performed with a 32P-labeled 706 nt complementary DNA probe, derived from the full-length coding region of the rPAC1 complementary DNA. The results of these experiments demonstrate the presence, in both human and rat placenta, of a 7.5-kb transcript similar in size to those detected in the ovary, brain, and hypothalamus. Alternative splicing of two exons occurs in human and rat PAC1 gene generating splice variants with variable tissue-specific expression. To ascertain which of the splice variants were expressed in placental tissue we performed RT-nested PCR using primers flanking the insertion sequence termed hip/hop cassette in rat or SV1/SV2 box in human gene. Electrophoretic analysis of the PCR products showed a different pattern of expression of messenger RNA splicing variants in human and rat placenta. In particular, the rat placenta expresses the short PAC1 receptor (PAC1short), the rPAC1-hip or hop (which are indistinguishable with the primers used), and the rPAC1-hip-hop, whereas the human placenta expresses only the PAC1SV1 (or SV2) variant, structurally homologous to the rat PAC1 hip (or hop). Sequence analysis of the human PCR-amplified PAC1 variant was therefore carried out and revealed that human placenta only expresses the PAC1SV2 isoform. The presence and characterization of PACAP binding sites was then investigated in human placenta by radioligand binding studies performed on crude membrane preparation using [125I]PACAP27 as tracer. Scatchard analysis of the binding results revealed the presence of two binding sites, one with high affinity and low capacity (Kd 0.33 ± 0.04 nm; Bmax 36.9 ± 12.1 fmol/mg protein) and one with low affinity and high capacity (Kd 24 ± 6.9 nm, Bmax 9.3 ± 0.19 pmol/mg protein). The relative potencies of PACAP-related peptides for inhibition of radioligand binding were: PACAP27 ≥ PACAP38 > VIP, whereas GHRH and other unrelated peptides, such as CRH and β-endorphin, did not inhibit [125I]PACAP27 binding. In conclusion, in this study, we provide evidence for the expression of PACAP within rat and human placenta. We also demonstrate that both human and rat placenta express the PAC1 gene and that the human tissue has binding sites for PACAP. These findings may suggest a role for PACAP in the regulation of placental physiology through autocrine and/or paracrine mechanisms.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and PACAP-Receptor Type 1 Expression in Rat and Human Placenta1

Pituitary adenylate cyclase-activating polypeptide (PACAP), the new hypophysiotropic factor member of the vasoactive intestinal peptide (VIP)/secretin/glucagon/GHRH family of neuropeptides, exerts its biological action by interacting with both PACAP-selective type I receptors (PAC1) and type II receptors (VPAC1), which bind both PACAP and VIP. The placenta is a site of production of hypophysiotropic factors that participate in the control of local hormone production, as well as the respective hypothalamic-pituitary neurohormones. In the present study, we show the expression of PACAP gene and irPACAP distribution within rat and human placental tissues, by means of RT-PCR and immunohystochemical experiments. In both rat and human placenta, we evaluated the expression of PAC1 gene by Northern hybridization analysis performed with a 32P-labeled 706 nt complementary DNA probe, derived from the full-length coding region of the rPAC1 complementary DNA. The results of these experiments demonstrate the presence, in both human and rat placenta, of a 7.5-kb transcript similar in size to those detected in the ovary, brain, and hypothalamus. Alternative splicing of two exons occurs in human and rat PAC1 gene generating splice variants with variable tissue-specific expression. To ascertain which of the splice variants were expressed in placental tissue we performed RT-nested PCR using primers flanking the insertion sequence termed hip/hop cassette in rat or SV1/SV2 box in human gene. Electrophoretic analysis of the PCR products showed a different pattern of expression of messenger RNA splicing variants in human and rat placenta. In particular, the rat placenta expresses the short PAC1 receptor (PAC1short), the rPAC1-hip or hop (which are indistinguishable with the primers used), and the rPAC1-hip-hop, whereas the human placenta expresses only the PAC1SV1 (or SV2) variant, structurally homologous to the rat PAC1 hip (or hop). Sequence analysis of the human PCR-amplified PAC1 variant was therefore carried out and revealed that human placenta only expresses the PAC1SV2 isoform. The presence and characterization of PACAP binding sites was then investigated in human placenta by radioligand binding studies performed on crude membrane preparation using [125I]PACAP27 as tracer. Scatchard analysis of the binding results revealed the presence of two binding sites, one with high affinity and low capacity (Kd 0.33+/-0.04 nM; Bmax 36.9+/-12.1 fmol/mg protein) and one with low affinity and high capacity (Kd 24+/-6.9 nM, Bmax 9.3+/-0.19 pmol/mg protein). The relative potencies of PACAP-related peptides for inhibition ofradioligand binding were: PACAP27 > or = PACAP38 > VIP, whereas GHRH and other unrelated peptides, such as CRH and beta-endorphin, did not inhibit [125I]PACAP27 binding. In conclusion, in this study, we provide evidence for the expression of PACAP within rat and human placenta. We also demonstrate that both human and rat placenta express the PAC1 gene and that the human tissue has binding sites for PACAP. These findings may suggest a role for PACAP in the regulation of placental physiology through autocrine and/or paracrine mechanisms.

Molecular Cloning and Pharmacological Characterization of a Somatostatin Receptor Subtype in the Gymnotiform Fish Apteronotus albifrons

The actions of the various forms of somatostatin (SRIF), including those of the tetradecapeptide SRIF14, are mediated by specific receptors. In mammals, five subtypes of SRIF receptors, termed sst1–5, have been cloned. Using a combination of reverse transcriptase-polymerase chain reaction and genomic library screening in the gymnotiform fish Apteronotus albifrons, a gene encoding the first-known nonmammalian SRIF receptor has been isolated. The deduced amino acid sequence displays 59% identity with the human sst3 receptor protein; hence, the gene is termed “Apteronotus sst3.” The predicted protein consists of 494 amino acid residues exhibiting a putative seven-transmembrane domain topology typical of G protein-coupled receptors. A signal corresponding to the Apteronotus sst3 receptor was detected in brain after amplification of poly(A)+-RNA by reverse transcriptase-polymerase chain reaction, but not by Northern blot analysis or in situ hybridization, suggesting a low level of expression. Membranes prepared from CCL39 cells stably expressing the Apteronotus sst3 receptor gene bound [125I][Leu8,d-Trp22,125 I-Tyr25]SRIF28 with high affinity and in a saturable manner (Bmax=4470 fmol/mg protein; pKD=10.5). SRIF14 and various synthetic SRIF receptor agonists produced a dose-dependent inhibition of radioligand binding, with the following rank order of potency: SRIF14 ≈ SRIF28 > BIM 23052 > octreotide > BIM 23056. Under low stringency conditions, an Apteronotus sst3 probe hybridized to multiple DNA fragments in HindIII or EcoRI digests of A. albifrons DNA, indicating that the Apteronotus sst3 receptor is a member of a larger family of Apteronotus SRIF receptors.

The pharmacological properties of the novel selective 5-HT3 receptor antagonist, alosetron, and its effects on normal and perturbed small intestinal transit in the fasted rat.

The purpose of this study was to investigate the pharmacological properties of the novel, selective 5-HT3 receptor antagonist, alosetron, and its effects on transit time in both the normal and perturbed small intestine of the rat. Alosetron concentration-dependently inhibited radioligand binding in membranes containing rat and human 5-HT3 receptors with estimated pKi values of 9.8 (n = 3) and 9.4 (n = 6), respectively. In selectivity studies alosetron had little or no significant affinity for any of the many other receptors and ion channels studied. Alosetron potently antagonized the depolarization produced by 5-HT in the rat vagus nerve (estimated pKB value of 9.8, n = 25). In anaesthetized rats, i. v. administration of alosetron inhibited 2-methyl-5-HT induced bradycardia (Bezold Jarisch index) at 1 and 3 microg kg-1, with an agonist dose ratio of approximately 3.0 at 1.0 microg kg-1, = 3-5). Alosetron administered via the duodenum also inhibited this reflex, with duration of action that was significantly longer than that seen with ondansetron (120-60 min, respectively, n = 6). Alosetron had no significant effect on normal small intestinal propulsion in the rat, but fully reversed the increase in intestinal propulsion (96%, n = 3) produced by egg albumin challenge. Alosetron is a highly selective 5-HT3 antagonist which normalizes perturbed small intestinal propulsion. Previous clinical data in IBS patients together with the transit data provide a good rationale for further studies with alosetron in IBS patients.

Saw palmetto extracts potently and noncompetitively inhibit human α1‐adrenoceptors in vitro

BACKGROUND We wanted to test whether phytotherapeutic agents used in the treatment of lower urinary tract symptoms have α1-adrenoceptor antagonistic properties in vitro. METHODS Preparations of β-sitosterol and extracts of stinging nettle, medicinal pumpkin, and saw palmetto were obtained from several pharmaceutical companies. They were tested for their ability to inhibit [3H]tamsulosin binding to human prostatic α1-adrenoceptors and [3H]prazosin binding to cloned human α1A- and α1B-adrenoceptors. Inhibition of phenylephrine-stimulated [3H]inositol phosphate formation by cloned receptors was also investigated. RESULTS Up to the highest concentration which could be tested, preparations of β-sitosterol, stinging nettle, and medicinal pumpkin were without consistent inhibitory effect in all assays. In contrast, all tested saw palmetto extracts inhibited radioligand binding to human α1-adrenoceptors and agonist-induced [3H]inositol phosphate formation. Saturation binding experiments in the presence of a single saw palmetto extract concentration indicated a noncompetitive antagonism. The relationship between active concentrations in vitro and recommended therapeutic doses for the saw palmetto extracts was slightly lower than that for several chemically defined α1-adrenoceptor antagonists. CONCLUSIONS Saw palmetto extracts have α1-adrenoceptor-inhibitory properties. If bioavailability and other pharmacokinetic properties of these ingredients are similar to those of the chemically defined α1-adrenoceptor antagonists, α1-adrenoceptor antagonism might be involved in the therapeutic effects of these extracts in patients with lower urinary tract symptoms suggestive of benign prostatic obstruction. Prostate 38:208–215, 1999. © 1999 Wiley-Liss, Inc.