Muscarinic cholinergic components in the carp brain

The activity of the muscarinic cholinergic system (acetylcholine, ACh; acetylcholinesterase, AChE; choline acetyltransferase, ChAT; muscarinic acetylcholine receptors) was studied in the carp brain. The ACh content (13.9 ± 1.1 nmol/g wet tissue) was estimated by gas chromatography after microwave irradiation focused to the head. The AChE and ChAT activities were 153 ± 13 nmol/min/mg protein and 817 ± 50 pmol/min/mg protein, respectively. The characteristics of [³H](−)quinuclidinyl benzilate ([³H](−)QNB) and [³H]pirenzepine ([³H]PZ) binding were also studied in brain membranes. Their specific binding was linearly dependent on the protein content and they appeared to bind with high affinity to a single, saturable binding site. A dissociation constant (K_d) of 47 ± 6.3 pM and a maximum number of binding sites (B_max) of 627 ± 65 fmol/mg protein were obtained for [³H](−)QNB, with a K_d value of 3.85 ± 0.67 nM and a B_max value of 95.3 ± 6.25 fmol/mg protein for [³H]PZ binding. The [³H]PZ binding amounted to only 15% of the [³H](−)QNB-labeled sites, as estimated from the ratio of the B_max values of [³H](−)QNB and [³H]PZ, suggesting a low density of M₁ subtype. Atropine sulfate, atropine methylnitrate and PZ inhibited the binding of both radioligands with Hill slopes (n_H) close to unity. The n_H value of AF-DX 116 was close to 1 against [³H](−)QNB binding, while it was 0.75 against [³H]PZ binding. The displacement curves of oxotremorine and carbachol were shallow for the binding of both radioligands. The rank order of potency of muscarinic ligands against [³H](−)QNB binding (K_i nM) was atropine sulfate (0.55) > atropine methylnitrate (1.61) > PZ (61.19) > oxotremorine (156.3) > AF-DX 116 (307) > carbachol (1301), while in the case of [³H]PZ binding it was atropine sulfate (0.24) > atropine methylnitrate (0.34) > PZ (10.38) > AF-DX 116 (55.87) > oxotremorine (62.79) > carbachol (1696). The results indicate the presence of a well-developed muscarinic cholinergic system with predominantly M₂ receptors in the carp brain.     

Synthesis and preliminary evaluation of [3H]PSB-0413, a selective antagonist radioligand for platelet P2Y12 receptors

The selective antagonist radioligand [³H]2-propylthioadenosine-5′-adenylic acid (1,1-dichloro-1-phosphonomethyl-1-phosphonyl) anhydride ([³H]PSB-0413) was prepared by catalytic hydrogenation of its propargyl precursor with a high specific radioactivity of 74 Ci/mmol. In preliminary saturation binding studies, [³H]PSB-0413 showed high affinity for platelet P2Y₁₂ receptors with a K_D value of 4.57 nM. Human platelets had a high density of P2Y₁₂ receptors exhibiting a B_max value of 7.66 pmol/mg of protein.     

[H]lysergic acid diethylamide (LSD): differential agonist and antagonist binding properties at 5-HT receptor subtypes in rat brain

[³H]Lysergic acid diethylamide (LSD) in the presence of 40 nM ketanserin labeled the 5-HT_1A receptor subtype in rat hippocampal membranes. In the presence of guanosine triphosphate (GTP), the B_max and affinity of [³H]LSD binding to the 5-HT_1A binding site were significantly decreased. [³H]LSD in the presence of 40 nM WB4101 labeled the 5-HT₂ receptor subtype in homogenates of rat frontal cortex. In contrast to the effect on [³H]LSD binding to the 5-HT_1A binding site, GTP produced no significant effect on either the B_max or the K_D of [³H]LSD binding to the 5-HT₂ binding site. Competition of 5-HT for [³H]LSD binding to the 5-HT₂ binding site was best described by a computer-derived model assuming two binding sites. In the presence of GTP, the 5-HT competition curve was shifted significantly to the right with an approx. 3-fold increase in the IC₅₀. These binding characteristics are consistent with [³H]LSD acting as an antagonist at the 5-HT₂ receptor which has multiple affinity states for agonists and is coupled to a guanine nucleotide regulatory subunit. Thus, [³H]LSD has binding characteristics consistent with it acting as an agonist at the 5-HT_1A receptor subtype but as an antagonist at the 5-HT₂ receptor subtype in rat brain.     

Early changes in adenosine A1 receptors in cerebral cortex slices submitted to in vitro ischemia.

The effects of brain ischemia on the maximum binding capacity (B_max) and affinity (K_d) of A₁ receptors were studied in the rat cerebral cortex, with an in vitro approach. The results were correlated with changes in ³H-adenosine release, studied under identical experimental conditions. Fifteen minutes of in vitro ‘ischemia’ (hypoxic, glucose-free medium) induced a significant increase in both B_max (2398±132 fmol/mg protein, 151% of the control, P<0.05) and in K_d (2.43±0.12 nM, 161% of the control, P<0.01). At the same time, an increase in tritium efflux from [³H]-adenosine labeled cerebral cortex slices to 324% of the control was observed. A trend toward normalization was evident 5–15 min after ‘reoxygenation’ (restoring normal medium), but the binding parameters were still altered after 60 min (B_max 2110±82 fmol/mg protein, K_d 2.26±0.14 nM, P<0.01 vs the corresponding control) as was adenosine release (196% of the control). These findings suggest that the increased availability of adenosine and its receptors may be a defense mechanism against ischemic injury, while the reduced affinity of A₁ receptors, possibly due to desensitization, may be a sign of ischemia-induced cellular damage.     

The sodium channel in membranes of electroplax. Binding of batrachotoxinin-a [H]benzoate to particulate preparations from electric eel (electrophorus)

Batrachotoxinin-A [³H]benzoate ([³H]BTX-B) binds specifically and with high affinity (K_D 48 nM) to sites (B_max 2.1 pmol/mg protein) associated with voltage-dependent sodium channels in rodent brain vesicular preparations. High affinity binding requires the presence of scorpion (Leiurus) venom and a membrane potential. Local anesthetics antagonize the binding. Nonspecific binding is defined in the presence of veratridine. In particulate preparations from electroplax of the eel Electrophorus electricus, [³H]BTX-B binds with a K_D of about 140 nM and a B_max of 2.5 pmol/mg protein in the presence of scorpion venom. Higher concentrations of scorpion venom are required to enhance binding in Electrophorus preparations than in brain preparations. Local anesthetics antagonize binding in Electrophorus preparations with potencies similar to those in brain preparations. Veratridine and batrachotoxin are less potent in blocking binding in Electrophorus than in brain preparations. It appears likely that binding in Electrophorus preparations is primarily to membrane fragments rather than vesicular entities as in brain. Binding of [³H]BTX-B to particulate preparations from electroplax of the ray Torpedo californica and the catfish Malapterurus electricus is mainly nonspecific. Scorpion venom does not enhance total binding and local anesthetics are not effective in antagonizing binding.     

Choline acetyltransferase activity and muscarinic binding in brain regions of aging fischer-344 rats

Muscarinic receptor binding and choline acetyltransferase (EC 2.3.1.6.) activity were assayed in three brain regions of 4-, 12- and 24-month-old Fischer-344 rats. Statistically significant age differences in cholinergic parameters were observed in each region. The affinity for [³H]quinuclidinyl benzilate increased in the cortex (24 vs 12 and 4 months), but B_max decreased in the cortex (24 vs 12 vs 4 months), striatum (24 vs 12 vs 4 months) and hippocampus (24 vs 12 and 24 vs 4). Assays of carbamylcholine inhibition of [³H]quinuclidinyl benzilate binding in the hippocampus showed that high affinity agonist binding increased with age (24 vs 12 and 4 months), and the percentage of muscarinic binding to high affinity agonist sites decreased (24 vs 12 vs 4 months). In addition, the affinity of the agonist oxotremorine for muscarinic binding sites also increased in the hippocampus (12 and 24 vs 4 months). Although the K_m of choline acetyltransferase for choline chloride did not change in any region tested, the K_m for acetyl coenzyme A decreased in the hippocampus (24 vs 12 months), but increased (4 vs 12 months) and then decreased (12 vs 24 months) in the striatum. Statistically significant age-related declines in V_max for choline acetyltransferase were noted in the striatum (24 < 12 < 4 months), but no age differences in this parameter were observed in the cortex or the hippocampus. Statistically significant positive correlations between V_max for choline acetyltransferase and B_max for [³H]quinuclidinyl benzilate binding were observed in each of the brain regions of 4-, 12- and 24-month-old rats.

The findings have implications for use of the Fischer-344 male rat as an animal model of aging and age-related disorders of the human brain, including dementia of the Alzheimer type.     

Characterization of muscarinic acetylcholine receptors on the rat pancreatic gastrin-producing cell line B6 RIN

The mechanisms of cholinergic stimulation of gastrin cells were studied in the rat pancreatic cell line B6 RIN. Carbachol induced an increase in intracellular Ca²⁺ and stimulated gastrin release in a dose-dependent manner over the range 10⁻⁵-10⁻³ M. These effects were completely abolished by atropine, suggesting the implication of muscarinic cholinergic receptors. The binding properties of these receptors were investigated. [N-Methyl-³H]scopolamine ([³h]nms) binding on cell homogenates was time-dependent, saturable and consistent with a single high-affinity binding class (K_d = 39.5 pM, and B_max = 7.9 fmol/mg DNA). Carbachol competitively inhibited [³H]NMS binding. The potency of inhibition of [³H]NMS binding by subtype selective antagonists was hexahydrodifenidol> pirenzepine> AF-DX 116. These results suggest the M₃, muscarinic receptors may be involved in the carbachol-induced gastrin release from B6 RIN cells.     

Temperature-dependent regulation of d-cis-[H]diltiazem binding to Ca channels by 1,4-dihydropyridine channel agonists and antagonists

The binding of the Ca²⁺-channel blocker d-cis-[³H]diltiazem to guinea pig skeletal muscle microsomes is temperature-dependent. At 2°C the K_D is 39 nM and B_max is 11 pmol/mg protein. The binding is fully reversible (K₋₁ = 0.02 min⁻¹). The binding sites discriminate between the diastereoisomers 1- and d-cis-diltiazem, recognize verapamil, gallopamil and tiapamil, and are sensitive to La³⁺-inhibition. At 30°C the K_D is 37 nM and the B_max is 2.9 pmol/mg protein. D-cis-diltiazem-labelling is regulated by the 1,4-dihydropyridine Ca²⁺-channel blockers and a novel Ca²⁺-channel activator in a temperature-dependent manner. At 30°C an enhancement of d-cis-diltiazem binding by the channel blockers is observed. This is attributed to a B_max increase. EC₅₀-values for enhancement and the maximal enhancement differ for the individual 1,4-dihydropyridines. At 2°C 1,4-dihydropyridines inhibit d-cis-[³H]diltiazem binding. This is attributed to a B_max decrease. We have directly labelled one of the drug receptor sites within the Ca²⁺-channel which can allosterically interact with the 1,4-dihydropyridine binding sites.     

Mechanism of modulation of [H]raclopride binding to dopaminergic receptors in rat striatal membranes by sodium ions

The mechanism of modulation of [³H]raclopride binding to dopaminergic receptors in rat brain striatal membranes by sodium ions was studied by means of equilibrium and kinetic measurements. Among different mono- and divalent cations studied, only sodium and lithium ions significantly enhanced [³H]raclopride binding to rat striatal membranes, but the effect of lithium was considerably smaller if compared with that of sodium. The equilibrium binding studies revealed that the increase in Na⁺ concentration from 0.5 to 150 mM increased both the radioligand affinity and the number of binding sites. The meaning of these changes was established by kinetic studies, which yielded hyperbolic plots of [³H]raclopride binding rate constants over the radioligand concentration. These plots correspond to the two-step ligand binding reaction mechanism, involving fast binding equilibrium followed by a slow isomerization of the receptor-antagonist complex. Sodium ions did not influence the antagonist affinity for the receptor sites in the first step of the binding process, nor the rate of isomerization of the receptor-ligand complex, but slowed down the rate of deisomerization. This led to a change in the value of the receptor-ligand dissociation constant K_d determined under equilibrium conditions. The same change in deisomerization rate was also sufficient to alter the receptor density (B_max), measured by the conventional ligand binding procedure.     

Characterization of proctolin binding sites on locust oviduct membranes

The binding of [³H]proctolin to oviduct membranes has been analyzed to investigate the nature of proctolin binding sites in the oviduct. Proctolin binding was found to be time dependent, proportional to concentration of membrane protein, saturable, specific and reversible. Two apparent proctolin binding sites were recognized. The first had a K_d of 400 ± 82 nM and a B_max of 23.7 ± 6.7 pmol/mg protein. The second had a K_d of 2.4 ± 0.2 μM and a B_max of 96.3 ± 16.7 pmo/mg protein.

Binding was specific in thatcompetition experiments with a wide range of peptides showed that only Arg-Tyr-Leu-Pro-Ala was an effective competitor at μM concentrations. All other peptides examined weekly reduced proctolin binding at concentrations above 50 μM. Certain peptides were found to potentiate [³]pproctolin binding at low μM concentrations (1–10 μM) and to inhibit proctolin binding at higher concentrations. The significance of these findings is discussed.     

Comparison of histamine-H1 receptors in bovine intracerebral microvessels with cerebral grey matter by [H]mepyramine binding

Small intracerebral blood vessels (microvessels) of bovine brain are known to contain the vasoactive amine histamine, and the presence of histamine-H₁ receptors in microvessels was examined using the radioligand, [³H]mepyramine. Microvessels were isolated from cerebral cortex grey matter, striatum and hippocampus by a sieving technique and membranes prepared for binding studies. [³H]Mepyramine bound to a single, high affinity site, which displayed stereoselectivity for (+) chlorpheniramine relative to its (−) isomer and was consistent with binding to H₁-receptors. The density of binding sites (B_max), in microvessel membranes from cortical grey matter, was approximately one-third of that seen in membranes prepared from cortical grey matter. Microvessels isolated from striata and hippocampi had a similar density of H₁-receptor sites to that seen in cortical microvessels.

These results demonstrate that bovine intracerebral microvessels contain significant numbers of histamine-H₁ receptors and strengthen the hypothesis that histamine could regulate the calibre of intracerebral blood vessels.     

Short-term effects of 3,4-methylen-dioxy-metamphetamine (MDMA) on 5-HT(1A) receptors in the rat hippocampus

The first effects of 3,4-methylen-dioxy-metamphetamine (MDMA, “ecstasy”), on serotonin 1A (5-HT_1A) receptors in rat hippocampus were determined by means of [³H]-8-hydroxy-dipropylamino-tetralin ([³H]-8-OH-DPAT) and 5′guanosine-(γ-[³⁵S]-thio)triphosphate ([³⁵S]-GTPγS) binding as well as inhibition of forskolin (FK)-stimulated adenylyl cyclase (AC) activity. The study was completed by [³⁵S]-GTPγS functional autoradiography experiments carried out in frontal sections of rat brain, including the hippocampal region. Results showed that MDMA was either able to displace [³H]-8-OH-DPAT binding (K_i  500 nM) or to reduce the number of specific sites (B_max) without affecting K_d. The drug also failed to change the [³⁵S]-GTPγS binding or to inhibit AC velocity, underlying its behavior as a non-competitive 5-HT_1A receptor antagonist. Further, MDMA (1 or 100 μM), partially antagonized either [³⁵S]-GTPγS binding stimulation of the agonists 5CT and 8-OH-DPAT or the AC inhibition induced by 5CT and DP-5CT. However, in contrast to binding studies, in AC assays the amphetamine displayed an effect also on EC₅₀, always being less potent than the reference antagonist WAY100,635. In functional autoradiography, MDMA behaved either as a partial 5-HT_1A antagonist in limbic areas or, added alone, as an agonist, increasing the coupling signal presumably through 5-HT release from synapses. Interestingly, the selective 5-HT re-uptake inhibitor (SSRI) fluoxetine had no effect on MDMA [³⁵S]-GTPγS binding activation. This latter finding indicates that the amphetamine can release 5-HT via alternative mechanisms to 5-HT transporter binding, probably via membrane synaptic receptors or vesicular transporters. The release of other transmitters is not excluded. Therefore, our results encourage at extending the study of MDMA biochemical profiles, in the attempt to elucidate those amphetamine-induced pathways with a potential for neurotoxicity or psycho-stimulant activity.     

Structure-activity relationships of adenosines with heterocyclic N6-substituents

Two series of N⁶-substituted adenosines with monocyclic and bicyclic N⁶ substituents containing a heteroatom were synthesized in good yields. These derivatives were assessed for their affinity ([³H]CPX), potency, and intrinsic activity (cAMP accumulation) at the A₁ adenosine receptor in DDT₁ MF-2 cells. In the monocyclic series, the N⁶-tetrahydrofuran-3-yl and thiolan-3-yl adenosines (1 and 26, respectively) were found to possess similar activities, whereas the corresponding selenium analogue 27 was found to be more potent. A series of nitrogen containing analogues showed varying properties, N⁶-((3R)-1-benzyloxycarbonylpyrrolidin-3-yl)adenosine (30) was the most potent at the A₁AR; IC₅₀ = 3.2 nM. In the bicyclic series, the effect of a 7-azabicyclo[2.2.1]heptan-2-yl substituent in the N⁶-position was explored. N⁶-(7-Azabicyclo[2.2.1]heptan-2-yl)adenosine (38) proved to be a reasonably potent A₁ agonist (K_i = 51 nM, IC₅₀ = 35 nM) while further substitution on the 7″-nitrogen with tert-butoxycarbonyl (31, IC₅₀ = 2.5 nM) and 2-bromobenzyloxycarbonyl (34, IC₅₀ = 9.0 nM) gave highly potent A₁AR agonists.     

Rat brain guanosine binding site. Biological studies and pseudo-receptor construction

Rat brain guanosine binding sites were studied by (i) a pharmacological approach to confirm the hypothesis of the existence of specific G-coupled receptors for guanosine (1) and, for the first time, delineate a structure–activity relationship for a series of guanosine derivatives; (ii) a molecular modelling approach to design a pseudo-receptor construction. GTP and its non-hydrolysable analogue Gpp[NH]p decreased [³H]-guanosine binding to rat brain membranes. Gpp[NH]p 30 and 100 μM induced a dose-dependent decrease in [³H]-guanosine affinity and PTX pretreatment of rat brain membranes caused a 50% reduction in binding. In slices from rat brain cortex, guanosine induced a dose-dependent increase in intracellular cAMP. This increase is specific for guanosine, since neither the pretreatment with adenosine deaminase nor the A₁ and A₂ adenosine receptor antagonists were able to modify the guanosine-induced cAMP accumulation. The structure–activity relationship showed that the potency order of the best substances able to displace 50 nM [³H]-guanosine was guanosine (1)=6-thioguanosine (3)>8-bromoguanosine (4)>inosine (10)>7-methylguanosine (6)=3′-deoxyguanosine (9)>2′-deoxyguanosine (8)=guanine (11)=6-thioguanine (12)>>N²-methylguanosine (5). The competition studies confirmed that [³H]-guanosine site was distinct from the well characterized ATP and adenosine binding sites. The present results are rationalized in terms of a putative pseudo-receptor construct which includes all the relevant physicochemical interaction between guanosine analogues and their putative binding sites. This construct will be useful for the in silico screening of compound libraries in search for new potent and structurally diverse pharmacological tools.     

Identification of a novel 5-HT1 binding site in rat spinal cord

High affinity, specific [³H]5-hydroxytryptamine (5-HT) binding to spinal cord synaptosomes was examined to identify the 5-HT receptor subtypes present. Computer nonlinear regression analysis of competition studies employing 8-OH-DPAT indicated that this 5-HT_1A selective agonist demonstrated high affinity competition (K_{i = 1.3 nM}) for 24.6 ± 0.7% of the total [³H]5-HT binding sites. Competition studies employing the 5-HT_1B selective agonist RU24969, in the presence of 100 nM 8-OH-DPAT, indicated that RU24969 demonstrated high affinity (K_{i = 1.1 nM}) competitive inhibition for 26.2 ± 1.4% of all [³H]5-HT binding sites. Neither 5-HT_1C, 5-HT_1D, 5-HT₂ nor 5-HT₃ selective compounds demonstrated any high affinity competition for the residual 49% of specific [³H]5-HT binding. Therefore, three major classes of [³H]5-HT binding sites could be demonstrated in spinal cord synaptosomes: 5-HT_1A, 5-HT_1B and a novel [³H]5-HT binding site which respectively represented 25, 26 and 49% of spinal cord synaptosomal [³H]5-HT binding. Further studies focusing on the function of the latter binding site are needed to determine if the presently identified novel binding site is the major 5-HT₁ receptor subtype present in spinal cord.     

Autoradiography of CCK receptors in the rat brain using [H]Boc[Nle]CCK27–33 and [I]bolton-hunter CCK8. Functional significance of subregional distributions

[³H]Boc[Nle^28,31]CCK₂₇–₃₃ ([³H]BDNL-CCK₇) is a new ligand for cholecystokinin (CCK) receptors, endowed with a high specific activity (100 Ci/mmol). Binding sites for this ligand were visualized in the rat brain by autoradiography [³H]BDNL-CCK₇ binds specifically to an apparent single class of CCK receptors on rat striatum sections with a K_d of 1.76 nM and a B_max of 57 fmol/mg protein. Unsulfated CCK₈ was two times less potent than sulfated CCK₈ to displace binding of [³H]BDNL-CCK₇. Binding sites for [³H]BDNL-CCK₇ were present in many brain regions, the highest concentrations occurring in cortex, olfactory bulbs, nucleus accumbens, and medium to high concentrations in striatum, hippocampus, and several nuclei of thalamus, hypothalamus and amygdala. In the same experimental conditions, the binding sites for [¹²⁵I]BH-CCK₈ showed similar specificity and localization. We thus used both ligands to investigate the subregional distributions of CCK receptors in nucleus accumbens and hippocampus, where a highly organized topography of action of CCK has been reported. In nucleus accumbens, the CCK binding sites were concentrated in the anterior portion of the nucleus, whereas very low densities were observed within medial posterior nucleus accumbens, where injection of CCK has been shown to potentiate dopamine-induced hyperlocomotion. p]In hippocampus, CCK receptors were concentrated in the polymorphic zone of the hilus of the dentate gyrus and in stratum lacunosum moleculare of Ammon's horn. Very few receptors were observed in other regions of hippocampus, including stratum pyramidale and stratum moleculare. This is in contrast with the presence of numerous CCK terminals and the potent effect of CCK in these areas. The distributions of CCK receptors reported here in both nucleus accumbens and hippocampus were discussed in correlation with the distribution of CCK neurons and terminals, the related anatomical pathways, and the pharmacological profiles of the effects of CCK in these regions.     

Insect nervous system [H]saxitoxin binding sites: characterization and target size

Saxitoxin (STX) has proved useful in the isolation and characterization of vertebrate and invertebrate voltage-operated sodium channels. Membrane extracts from the nervous system of the cockroach Periplaneta americana contain a saturable component of specific [³H]STX binding. Scatchard analysis yields a K_D of 0.84 nM, similar to that (3.0 nM) determined in electrophysiological studies on axons in the same tissue (Sattelle et al., 1979). The maximum number of binding sites, B_max (8.25 pmol/mg protein), was higher than previously observed. The specific binding component was blocked by STX and tetrodotoxin (TTX), but not by scorpion (Leiurus quinquestriatus) venom, aconitine, veratridine, sea anemone toxin and deltamethrin, which act at different sites on the channel molecule. Unlabelled STX samples prepared from different sources (Mytilus, Saxidomus and Gonyaulax) were all effective as inhibitors of [³H]STX binding. Radiation inactivation was employed to determine the molecular target size of the [³H]STX binding molecule in membranes prepared from the cockroach nervous system. By this means M_r = 171,400 ± 25,000 was estimated for the insect sodium channel.     

The pharmacological characterization of 5-HT3 receptor binding sites in rabbit ileum: Comparison with those in rat ileum and rat brain

We have further characterized the 5-HT₃ receptors in rat and rabbit tissues by evaluating the binding of the 5-HT₃ receptor ligand, [³H]GR67330 to homogenates of rabbit ileum, rat ileum and rat brain (entorhinal cortex). In each tissue specific [³H]GR67330 binding represented a single saturable, high affinity site (K_d = 0.14, 0.18, 0.076 nM in rabbit ileum, rat ileum and rat brain respectively). The densities of sites present in rabbit and rat ileum were similar to that present in rat brain (B_max = 63, 47, 72 fmol/mg protein in rabbit ileum, rat ileum and rat brain respectively).

In each tissue, 5-HT₃ receptor agonists and antagonists potently competed for [³H]GR67330 binding. Derived inhibition constants were similar in rat ileum and brain. However marked differences in IC₅₀s were apparent for rabbit ileum compared with rat brain or ileum. These were most apparent with agonists. Thus, mCPBG [1-(meta-chlorophenylbiguanide)], phenylbiguanide, 5-HT and 2-methyl 5-HT were at least 5 times less potent to inhibit [³H]GR67330 binding in rabbit ileum than rat brain. The most pronounced differences were evident with phenylbiguanide and mCPBG which were 70 and 300 times less potent in the rabbit ileum respectively compared with the rat tissues. These differences were unlikely to be due to depletion effects because tissue combination experiments (rabbit ileum and rat brain) yielded biphasic inhibition curves for phenylbiguanide with affinities for each component similar to those in the individual tissues. Antagonist affinities also varied between the rabbit and rat tissues, although less markedly. Amongst the antagonists, the most marked differences were apparent with SDZ 206–830 and quipazine each being 10 times less potent to inhibit binding to rabbit than rat tissue.

Hill coefficients for inhibition of binding varied with tissue. In rat brain, as previously described for [³H]GR67330, Hill coefficients for agonist (and quipazine) inhibition of binding were greater than unity. This was less marked in rat and rabbit ileum tissues.

The present studies provide further evidence for species variation in 5-HT₃ receptors.     

Identification of vasoactive intestinal polypeptide (VIP) binding protein in bovine pineal gland

Vasoactive intestinal polypeptide (VIP) containing nerves are present in close proximity to epithelial, endocrine, and vascular smooth muscle cells. The pineal gland, known also as a “neuroendocrine transducer organ” contains a high content of VIP which prompted us to characterize the binding sites for VIP in this organ. [Tyr¹⁰−¹²⁵I]VIP was bound selectively and specifically to pineal membrane preparations in a time-dependent fashion. Scatchard analysis demonstrated a single class of high affinity binding sites with a dissociation constant (K_d) value of 5.7 ± 0.52 nmol/1 and a receptor density (B_max) value of 440 ± 35 fmol/mg protein. A Hill Plot with a slope of 1.013 indicated the absence of cooperativity. Covalent crosslinking with [Tyr¹⁰−¹²⁵I]VIP followed by SDS electrophoresis and autoradiography, revealed that the VIP binding protein exhibited a molecular weight of 51.8 ± 0.5 kDa. The precise function of pineal VIP binding protein remains to be delineated.     

Synthesis, radiolabeling and receptor binding of [3H][(1S,2R)ACPC2]endomorphin-2

Previously, we have shown that substitution of Pro² for cis-2-aminocyclopentanecarboxylic acid, ACPC in endomorphin-2 results in an analogue with greatly augmented proteolytic stability, high μ-opioid receptor affinity and selectivity. We now report the synthesis and biochemical characterization of [³H][(1S,2R)ACPC²]endomorphin-2 with a specific activity of 1.41 TBq/mmol (38.17 Ci/mmol). Specific binding of [³H][(1S,2R)ACPC²]endomorphin-2 was saturable and of high affinity with an equilibrium dissociation constant, K_d = 1.80 ± 0.21 nM and receptor density, B_max = 345 ± 27 fmol × mg protein⁻¹ at 25 °C in rat brain membranes. Similar affinity values were obtained in kinetic and displacement assays. Both Na⁺ and Gpp(NH)p decreased the affinity proving the agonist character of the radioligand. [³H][(1S,2R)ACPC²]endomorphin-2 retained the μ-specificity of the parent peptide. The new radioligand will be a useful tool to map the topographical requirements of μ-opioid peptide binding due to its high affinity, selectivity and enzymatic stability.