P2 purinergic receptors for diadenosine polyphosphates in the nervous system

• 1.1. The actions of diadenosine polyphosphates, diadenosine tetraphosphate (Ap₄A), diadenosine pentaphosphate (Ap₅A) and diadenosine hexaphosphate (Ap₆A) in the nervous system have been reviewed.
• 2.2. In the peripheral nervous system, diadenosine polyphosphates bind to P_2Y-purinergic receptors such as the P_2Y in chromaffin cells and Torpedo synaptosomes, P_2X in vas deferens and urinary bladder and also Torpedo synaptosomes and P_2U in endothelial chromaffin cells.
• 3.3. In the central nervous system Ap_nA compounds can act through P_2X-purinoceptors opening cation channels in nodose ganglion neurones. Diadenosine polyphosphates bind to a P_2d-purinergic receptor in rat brain synaptic terminals and hippocampus, linked to protein kinase C (PKC) activation.
• 4.4. P₄-purinoceptors are specific receptors for diadenosine polyphosphates, coupled to the Ca²⁺ influx, in the central synapses. This purinoceptor is not activated by ATP and synthetic analogs. The P₄-purinoceptor could act as a positive modulator of the synaptic transmission, giving even more importance to diadenosine polyphosphates as neurotransmitters.

     

Cardiac effects of diinosine tetraphosphate,a putative dinucleotide receptor antagonist

The cardiac electrophysiological and vasomotor effects of diinosine tetraphosphate (Ip₄I, 10 nM and 1 μM) were examined in guinea pig isolated hearts together with its ability to antagonize the effects of diadenosine tetraphosphate (Ap₄A, 1 nM and 1 μM). Ip₄I had no effects on action potential duration or refractory period and attenuated the electrophysiological effects of Ap₄A at both concentrations. Although Ip₄I increased coronary perfusion pressure, the vasomotor effects of Ap₄A were resistant to antagonism by Ip₄I. These observations support data from neural tissues and indicate that diinosine polyphosphates may antagonize cardiac electrophysiological effects of diadenosine polyphosphates. Drug Dev. Res. 52:500–503, 2001. © 2001 Wiley‐Liss, Inc.     

Effects of adenosine 5’-triphosphate, uridine 5’-triphosphate, adenosine 5’-tetraphosphate and diadenosine polyphosphates in guinea-pig taenia caeci and rat colon muscularis mucosae

The functional effects of adenosine 5’-triphosphate (ATP), uridine 5’-triphosphate (UTP), adenosine 5’-tetraphosphate (AP₄) and the diadenosine polyphosphates P₁,P₃-diadenosine triphosphate (Ap₃A), P₁,P₄-diadenosine tetraphosphate (Ap₄A) and P₁,P₅-diadenosine pentaphosphate (Ap₅A) were studied in two isolated smooth muscle preparations thought to contain P_2Y (P2Y₁) receptors, the guinea-pig taenia caeci (which relaxes to ATP) and the rat colon muscularis mucosae (which contracts to ATP). In addition, the breakdown of these compounds by the rat colon muscularis mucosae was investigated by high pressure liquid chromatography. In the guinea-pig taenia caeci all the purine nucleotides caused relaxation with a potency order of Ap₃A=Ap₄A> ATP>AP₄=Ap₅A, and these relaxations were antagonised by suramin with apparent pA₂ values in the region of 5, consistent with activation of a P2Y₁ receptor. In the rat colon muscularis mucosae the nucleotides caused contraction with a potency order of Ap₃A = Ap₄A>ATP=AP₄ =Ap₅A >UTP. However, while suramin (100 μM) inhibited responses to ATP and UTP at all concentrations of agonist, it only inhibited contractions induced by the higher concentrations of AP₄, Ap₃A and Ap₄A and had little effect on contractions induced by Ap₅A. A higher concentration of suramin (1 mM) enhanced contractions induced by ATP but greatly inhibited those induced by UTP and had no effect on responses to the other agonists. The A₁ adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 10 nM) had no effect on responses to ATP or UTP but inhibited responses to Ap₃A, Ap₄A, Ap₅A and AP₄. A combination of suramin (1 mM) and DPCPX (10 nM) almost abolished responses to all the agonists. ATP and UTP were rapidly degraded by the rat colon muscularis mucosae while AP₄, Ap₃A, Ap₄A and Ap₅A were degraded more slowly, and the major product detected after breakdown of the purine nucleotides was inosine rather than adenosine. The breakdown of all the nucleotides was inhibited by suramin (1 mM), although this inhibition did not achieve statistical significance in the case of ATP. These results show that while the diadenosine polyphosphates appear to act as P2 agonists in the taenia caeci, in the rat colon muscularis mucosae their major action is via adenosine A₁ receptors rather than via P2 receptors. In addition, although they are more stable than ATP or UTP, their action in this tissue is clearly affected by their degradation which complicates the effects of suramin. Received: 23 March 1998 / Accepted: 29 June 1998     

Pre- and Postjunctional Effects of Diadenosine Polyphosphates in the Guinea-pig Vas Deferens

The pre- and postjunctional activities of a number of diadenosine polyphosphates were examined in the guinea-pig isolated vas deferens at the level of the membrane-potential, using a modified sucrose-gap technique. P¹,P³-Di(adenosine 5′)triphosphate (Ap₃A), P¹,P⁴-di(adenosine 5′)tetraphosphate (Ap₄A) and P¹,P⁵-di(adenosine 5′)pentaphosphate (Ap₅ A) all caused concentration-dependent depolarization of the smooth muscle membrane. The potency order was: Ap₅A > Ap₄A. Ap₃A. P¹, P²-Di(adenosine 5′)pyrophosphate (Ap₂A) did not evoke depolarization even at the highest concentration tested (1 mM). All the dinucleotides caused a reduction in the amplitude of evoked excitatory junction potentials (e.j.ps). The potency order was: Ap₅A = Ap₄A > Ap₃A > Ap₂A. The depolarizations evoked by the dinucleotides were markedly reduced by the selective P_2X-purinoceptor antagonist, pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS, 10 μM), as was the amplitude of the fully facilitated e.j.p. The inhibition of the e.j.p. evoked by Ap₃A and Ap₂A was reduced by the P₁-purinoceptor antagonist, 8-p-sulphophenyltheophylline (8-pSPT, 50 μM), but that evoked by Ap₅A and Ap₄A was not. Thus, Ap₃A, Ap₄A and Ap₅ A evoke depolarization of the guinea-pig vas deferens via P_2X-purinoceptors, and additionally Ap₂A and Ap₃A exert a prejunctional effect via P₁-purinoceptors. The prejunctional activity of Ap₄A and Ap₅A is mediated via an undefined purinoceptor, which is neither P₁ nor P_2X.     

Cardiac ATP-sensitive K+ channel: a target for diadenosine 5′,5″-P1,P5-pentaphosphate

In numerous studies the intracellular mononucleotide-dependent gating of ATP-sensitive K⁺ (K_ATP) channels has been demonstrated. However, it is not known whether dinucleotide polyphosphates, a family of endogenous compounds structurally-related to ATP, could also modulate this ion conductance. Therefore, in the present study we assessed the direct effect of diadenosine 5,5-P¹,P⁵-pentaphosphate (Ap₅A) on cardiac K_ATP channel activity using the inside-out configuration of the patch-clamp technique. Addition of Ap₅A (50 M) to the internal side of membrane patches, excised from guinea-pig ventricular cells, strongly inhibited K_ATP channel activity. The estimated NP_o (where N is the number of channels in the patch and P_o the open probability of each channel) was 4.16 ± 0.50 in the absence and 0.85 ± 0.30 in the presence of Ap₅A (50 M). This effect of Ap₅A was partially reversible, and the NP₀ was 2.26 ± 0.60 after washout of Ap₅A. Exposure of K_ATP channels to increasing concentrations of Ap₅A revealed that the Ap₅A-induced inhibition is concentration-dependent with the half-maximal effective concentration of 16 M (Hill coefficient: 1.6). On the basis of these results, we conclude that Ap₅A is a potent antagonist of the K_ATP channel activity. This represents a previously unrecognized property of Ap₅A, as well as the discovery of a potentially novel endogenous ligand of myocardial KATP channels.     

Effects of diadenosine triphosphate and diadenosine tetraphosphate on rat liver cells: Differences and similarities with ADP and ATP

Liver cells possess multiple types of purinoceptors that mediate the effects of extracellular nucleotides. Like ADP and ATP, the dinucleotides diadenosine triphosphate (Ap₃A) and diadenosine tetraphosphate (Ap₄A) fully activated glycogen phosphorylase, with ED₅₀ values of 0.31 μM and 1.3 μM, respectively. At variance with ATP, neither the dinucleotides nor ADP significantly increased the levels of IP₃. Ap₄A (and also ADP) moderately increased IP₃ (± 72%) whereas Ap₃A was completely ineffective. Like ATP, Ap₃A, Ap₄A, and ADP inhibited the cAMP increase after glucagon. Phorbol-12-myristate-13-acetate (PMA) pretreatment of the hepatocytes clearly inhibited the glycogenolytic potency of Ap₃A and ADP, but had only a minor effect on the potency of Ap₄A or ATP. It is concluded that, depending upon the effect studied (glycogenolytic effect with or without PMA, increasing IP₃ potency, or inhibition of cAMP increase), different analogies between the agonists studied emerged, indicating the complexity of the interaction of ATP and its analogues with liver purinoceptors and/or of the transduction mechanism(s) initiated by the different nucleotides.     

Ap4A and ADP-beta-S binding to P2 purinoceptors present on rat brain synaptic terminals.

1. Diadenosine tetraphosphate (Ap4A) a dinucleotide stored and released from rat brain synaptic terminals presents two types of affinity binding sites in synaptosomes. When [3H]-Ap4A was used for binding studies a Kd value of 0.10 +/- 0.014 nM and a Bmax value of 16.6 +/- 1.2 fmol mg-1 protein were obtained for the high affinity binding site from the Scatchard analysis. The second binding site, obtained by displacement studies, showed a Ki value of 0.57 +/- 0.09 microM. 2. Displacement of [3H]-Ap4A by non-labelled Ap4A and P2-purinoceptor ligands showed a displacement order of Ap4A > adenosine 5'-O-(2-thiodiphosphate) (ADP-beta-S) > 5'-adenylyl-imidodiphosphate (AMP-PNP) > alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-MeATP) in both sites revealed by the Ki values of 0.017 nM, 0.030 nM, 0.058 nM and 0.147 nM respectively for the high affinity binding site and values of 0.57 microM, 0.87 microM, 2.20 microM and 4.28 microM respectively for the second binding site. 3. Studies of the P2-purinoceptors present in synaptosomes were also performed with [35S]-ADP-beta-S. This radioligand showed two binding sites the first with Kd and Bmax values of 0.11 +/- 0.022 nM and 3.9 +/- 2.1 fmol mg-1 of protein respectively for the high affinity binding site obtained from the Scatchard plot. The second binding site showed a Ki of 0.018 +/- 0.0035 microM obtained from displacement curves. 4. Competition studies with diadenosine polyphosphates of [35S]-ADP-beta-S binding showed a displacement order of Ap4A > Ap5A > Ap6A in the high affinity binding site and Ki values of 0.023 nM, 0.081 nM and 5.72 nM respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of diadenosine tetraphosphate (Ap4A) binding sites in cultured chromaffin cells: evidence for a P2y site.

1. Diadenosine tetraphosphate (Ap4A) a dinucleotide, which is stored in secretory granules, presents two types of high affinity binding sites in chromaffin cells. A Kd value of 8 +/- 0.65 x 10(-11) M and Bmax value of 5420 +/- 450 sites per cell were obtained for the high affinity binding site. A Kd value of 5.6 +/- 0.53 x 10(-9) M and a Bmax value close to 70,000 sites per cell were obtained for the second binding site with high affinity. 2. The diadenosine polyphosphates, Ap3A, Ap4A, Ap5A and Ap6A, displaced [3H]-Ap4A from the two binding sites, the Ki values being 1.0 nM, 0.013 nM, 0.013 nM and 0.013 nM for the very high affinity binding site and 0.5 microM, 0.13 microM, 0.062 microM and 0.75 microM for the second binding site. 3. The ATP analogues displaced [3H]-Ap4A with the potency order of the P2y receptors, adenosine 5'-O-(2 thiodiphosphate) (ADP-beta-S) greater than 5'-adenylyl imidodiphosphate (AMP-PNP) greater than alpha, beta-methylene ATP (alpha, beta-MeATP), in both binding sites. The Ki values were respectively 0.075 nM, 0.2 nM and 0.75 nM for the very high affinity binding site and 0.125 microM, 0.5 microM and 0.9 microM for the second binding site.     

Characterization of the P2 receptors on the human umbilical vein endothelial cell line ECV304

1. To characterize the P2 receptors present on the human umbilical vein endothelial-derived cell line, ECV304, cytosolic Ca²⁺, ([Ca²⁺]_c), responses were recorded in single cells and in cell suspensions to a series of nucleotides and nucleotide agonists.
2. Concentration response curves were obtained in fura-2-loaded ECV304 cell suspensions, with EC₅₀ values of 4.2 μM for ATP, 2.5 μM for UTP and 14 μM for adenosine-5′-O-(3-thio)triphosphate (ATPγS). EC₅₀ values for 2-methylthioATP, ADP, adenosine-5′-O-(2-thio)diphosphate (ADPβS) and AMP were 0.5 μM, 3.5 μM, 15 μM and 4.7 μM respectively, but maximal [Ca²⁺]_c responses were less than those produced by a maximal addition of ATP/UTP. ECV304 cells were unresponsive to UDP and β,γ,methyleneATP.
3. Cross-desensitization studies on ECV304 cells suggested that ATP and UTP recognized the same receptor. However, ADP recognized a receptor distinct from the UTP-sensitive receptor and AMP recognized a third distinct receptor.
4. ECV304 [Ca²⁺]_c responses to 2-methylthioATP were inhibited in the presence of 30 μM pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), whereas [Ca²⁺]_c responses to UTP were unaffected by this treatment.
5. ECV304 cells responded to the diadenosine polyphosphate Ap₃A with rises in [Ca²⁺]_c. Apparent responses to Ap₄A, Ap₅A and Ap₆A, were shown to be due to a minor nucleotide contaminant that could be removed by pre-treatment of the diadenosine samples with either alkaline phosphatase or apyrase.
6. ECV304 cells display a pharmacology consistent with the presence of at least two P2 receptors; a P2Y₂ receptor insensitive to the diadenosine polyphosphates and a P2Y₁ receptor sensitive to Ap₃A. In addition, ECV304 cells respond to AMP with increases in [Ca²⁺]_c via an as yet uncharacterized receptor.

     

Diadenosine polyphosphates in cultured vascular smooth-muscle cells and endothelium cells--their interaction with specific receptors and their degradation

The role of diadenosine polyphosphates (ApnA, where "A" denotes "adenosine" and "n" denotes the number of phosphate groups "p") as vasoconstrictors of smooth-muscle cells and as blood-pressure regulating and insulin-releasing compounds has been described. It was the aim of this study to investigate whether specific receptors for these compounds, mediating the above mentioned effects, occur in cultured vascular smooth-muscle cells (VSMC) and in endothelium cells, and whether these compounds are degraded during incubation. Saturable binding sites for diadenosine polyphosphate [3H]Ap4A with an extremely quick saturation equilibrium, even at low temperature (4 degrees C), are present in vascular smooth-muscle cells. Diadenosine polyphosphates at micromolar concentrations displaced [3H]Ap4A from binding sites; the ranking order was Ap4A > Ap3A > Ap5A approximately Ap6A. Compounds interacting with purinergic P2X receptors such as suramin, alpha,beta-methylene ATP and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), albeit at high concentrations, displaced [3H]Ap4A from its binding sites. Surprisingly, at low concentrations the compounds tested increased the binding of [3H]Ap4A, which might imply the occurrence of positive receptor cooperativity or inhibition of [3H]Ap4A degradation. By use of thin-layer chromatography it was observed that [3H]Ap4A was quickly degraded (half-life approx. 12 min) in the extracellular medium to (mainly) adenosine and inosine. [3H]Ap4A and its degradation products were quickly taken up by the cells. Degradation can be inhibited by Ap6A, alpha,beta-methylene ATP or PPADS. Rather similar degradation and uptake results were also obtained when endothelium cells were used. These data indicate that specific binding sites for [3H]Ap4A are present in vascular smooth-muscle cells and that diadenosine polyphosphates at physiological concentrations displace binding. The receptors involved might be distinct diadenosine polyphosphate receptors, although the involvement of others, such as P2X receptors, is also possible. Ap4A is quickly degraded in the extracellular space and compounds that inhibit degradation result in an increase in [3H]Ap4A binding. It should be remembered that when diadenosine polyphos-phates are being investigated in physiological and pathophysiological studies of their impact on smooth-muscle cell proliferation and on vasoconstriction (blood-pressure regulation), results obtained from long-term incubations might be critical.     

Evidence for a G protein-coupled diadenosine-5',5''-P1,P4-tetraphosphate (Ap4A) receptor binding site in lung membranes from rat

Nucleotide receptors are of considerable importance in the treatment of lung diseases, such as cystic fibrosis. Because diadenosine polyphosphates may also be of significance as signalling molecules in lung, as they are in a variety of tissues, in the present work we investigated the binding sites for [3H]diadenosine-5',5'-P1,P4-tetraphosphate (Ap4A) in plasma membranes from rat lung and studied their possible coupling to G proteins. We present evidence for a single high-affinity binding site for [3H]Ap4A with similar affinity for other diadenosine polyphosphates ApnA (n = 2 to 6). Displacement studies with different nucleotides revealed that the [3H]Ap4A binding site was different from P2X and P2Y2 receptor binding sites. Pretreatment of lung membranes with GTPgammaS or GTP in the presence of Mg2+ increased the Ki for Ap4A from 91 nM to 5.1 microM, which is indicative of G protein coupling. The putative coupling to G proteins was further confirmed by the enhancement of [35S]GTPgammaS binding (to Galpha proteins) to lung membranes by Ap4A (63% increase over basal) in a concentration-dependent manner. Therefore, our data for the first time provide evidence of a G protein-coupled Ap4A binding site in lung membranes.     

Synthetic,nondegradable diadenosine polyphosphates and diinosine polyphosphates: their effects on insulin-secreting cells and cultured vascular smooth muscle cells

Diadenosine polyphosphates show a dissimilarity between their effects in static and perifusion experiments with respect to insulin release that may be due to degradation of the compounds. The aim was to investigate two nondegradable compounds of bisphosphorothioates containing a methylene or chloromethylene group (namely, diadenosine 5',5' "-(P(1),P(4)-dithio-P(2),P(3)-methylene)tetraphosphate and diadenosine 5',5' "-(P(1),P(4)-dithio-P(2),P(3)-chloromethylene)tetraphosphate), as mixtures of three or four diastereomers. Owing to their modified structures, these compounds are resistant to degradation (ectophosphodiesterases, diphosphohydrolases, and phosphorylases). Both compounds tested were minimally degraded (2%) even after 16 h when incubated with insulin-secreting (INS-1) cells. Additionally, diinosine polyphosphates (Ip(5)I and Ip(6)I), putative antagonists of diadenosine polyphosphates, were tested. By use of [(3)H]Ap(4)A, saturable binding sites for both diadenosine polyphosphate analogues were found in INS-1 cells, 3T3 preadipocyte cells, and vascular smooth muscle cells (VSMC) and for both Ip(5)I and Ip(6)I in INS-1 cells. The synthesized diadenosine polyphosphate analogues have the same affinity as Ap(4)A, whereas Ip(5)I and Ip(6)I inhibit binding at higher concentrations (10-100 microM). Insulin release was investigated in static experiments over 90 min in INS-1 cells. Insulin release was inhibited dose-dependently by both of the diadenosine polyphosphate analogues to the same degree as by Ap(4)A. The glucose-induced insulin release curve was not shifted to the right. Both compounds inhibit insulin release only at high (insulin stimulatory) glucose concentrations, e.g., 5.6 mM glucose. Ip(5)I and Ip(6)I antagonized Ap(5)A-mediated inhibition of insulin release. [(3)H]Thymidine incorporation into VSMC was not influenced by either synthetic diadenosine polyphosphate analogue, indicating that Ap(4)A does not act by itself in this case but (active) degradation products mediate the effect. The data indicate the following. (1) Since nondegradable compounds inhibit insulin release as well as Ap(4)A, it is Ap(4)A itself and not any of its degradation products that induces this effect. (2) Diadenosine polyphosphate effects on cell proliferation are mediated via a degradation product in contrast to their effect on insulin release. (3) Ip(5)I and Ip(6)I act like antagonists. Both synthetic analogues and diinosine polyphosphates are valuable tools for diabetes research.     

A novel receptor for diadenosine polyphosphates coupled to calcium increase in rat midbrain synaptosomes.

1. Diadenosine polyphosphates, Ap4A and Ap5A, as well as ATP, alpha,beta-MeATP and ADP-beta-S, were able to elicit variable intrasynaptosomal Ca2+ increases in rat midbrain synaptic terminals. The origin of the Ca2+ increment was the extrasynaptosomal space since the elimination of extracellular Ca2+ abolished the effect of all the agonists. 2. The P2-purinoceptor antagonist, suramin, did not affect the Ca(2+)-increase evoked by diadenosine polyphosphates but dramatically blocked the Ca2+ entry induced by ATP and its synthetic analogues. 3. The actions of Ap5A and ATP on the intrasynaptosomal Ca2+ increase did not cross-desensitize. 4. Concentration-response studies for diadenosine polyphosphates showed pD2 values of 54.5 +/- 4.2 microM and 55.6 +/- 3.8 microM for Ap4A and Ap5A, respectively. 5. The entry of calcium induced by diadenosine polyphosphates could be separated into two components. The first represented a selective voltage-independent Ca2+ entry; the second, a sustained phase which was voltage-dependent. 6. Studies on the voltage-dependent Ca(2+)-channels involved in the effects of the diadenosine polyphosphates, demonstrated that omega-conotoxin G-VI-A inhibited the sustained Ca(2+)-entry, suggesting the participation of an N-type Ca(2+)-channel. This toxin was unable to abolish the initial cation entry induced by Ap4A or Ap5A. omega-Agatoxin IV-A, tetrodotoxin, or nifedipine did not inhibit the effects of the diadenosine polyphosphates. 7. The effect of ATP on Ca(2+)-entry was abolished by nifedipine and omega-conotoxin G-VI-A, suggesting the participation of L- and N-type Ca(2+)-channels in the response to ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potent desensitization of human P2X3 receptors by diadenosine polyphosphates

In this study, the receptor desensitizing effects of diadenosine polyphosphates at recombinant human P2X3 (hP2X3) receptors were examined. Administration of Ap3A, Ap4A, Ap5A or Ap6A inhibited the hP2X3 receptor-mediated response to a subsequent application of 3 muM alphabeta-methyleneATP (alphabeta-meATP), in a concentration-dependent manner, with IC50 values 2707, 42, 59 and 46 nM, respectively. These agonists did not desensitize alphabeta-meATP responses mediated by the slowly desensitizing heteromeric human P2X2/3 receptor. hP2X3 receptor desensitization was reversible and was not observed following the increase in intracellular Ca2+ levels produced by carbachol. A similar pattern of desensitization evoked by Ap5A was also observed using electrophysiological recordings of Xenopus oocytes expressing hP2X3 receptors. These data demonstrate that diadenosine polyphosphates, found endogenously in the central nervous system, can readily desensitize hP2X3 receptors at nanomolar concentrations that are 10-fold lower than are required to produce agonist-induced receptor activation. Thus, P2X3 receptor desensitization by diadenosine polyphosphates may provide an important modulatory mechanism of P2X3 receptor activation in vivo.     

Cholinergic muscarinic receptors of bovine adrenal medulla

Muscarinic receptor binding sites were identified on bovine adrenal medulla membranes, using [³H]-quinuclidinyl benzylate. A Scatchard analysis revealed a k_d of 0.07 nM and a Bmax of 3 fmol/mg prot. Muscarinic receptors of the medulla may, in part, be responsible for the release of catecholamines and the modulation of the cyclic nucleotide content of chromaffin cells.     

Localization of sites for 125I-labelled botulinum neurotoxin at murine neuromuscular junction and its binding to rat brain synaptosomes

Botulinum neurotoxin, purified to homogeneity from Clostridium botulinum (Type A), was found to be highly neurotoxic (>8 x 10⁷ mouse ld₅₀/mg protein). Labelling of this pure neurotoxin with ¹²⁵I-iodine to high specific radioactivity was achieved without appreciable loss of biological activity. This was used to demonstrate saturable binding sites for this toxin at the neuromuscular junction, following in vivo administration into mice. A demonstrable inhibitory effect of the neurotoxin on release of acetylcholine from rat cerebrocortical synaptosomes indicates that it affects synapses in the central nervous system. Kinetic studies on the binding of ¹²⁵I-labelled neurotoxin to brain synaptosomes yielded an association rate constant of 2.3 x 10⁵M^?1s^?1; dissociation plots were biphasic and the predominant species showed a rate constant of 1.2 x 10^?4s^?1. The saturable binding component is heatsensitive and inactivated by trypsin. Preliminary studies showed that botulinum neurotoxin associates with plasma membrane fractions of synaptosomes and that binding does not result in any gross structural changes, at least in the majority of the toxin molecules.     

Enlightened protein: Fhit tumor suppressor protein structure and function and its role in the toxicity of protoporphyrin IX-mediated photodynamic reaction

The Fhit tumor suppressor protein possesses Ap₃A (diadenosine triphosphate — ApppA) hydrolytic activity in vitro and its gene is found inactive in many pre-malignant states due to gene inactivation. For several years Fhit has been a widely investigated protein as its cellular function still remains largely unsolved. Fhit was shown to act as a molecular ‘switch’ of cell death via cascade operating on the influence of ATR–Chk1 pathway but also through the mitochondrial apoptotic pathway. Notably, Fhit was reported by our group to enhance the overall eradication effect of porphyrin-mediated photodynamic treatment (PDT). In this review the up-to-date findings on Fhit protein as a tumor suppressor and its role in PDT are presented.     

Interaction of calcium with3H-morphine binding to synaptosomes of the guinea-pig ileum

Summary Specific binding sites for³H-morphine were assayed in subcellular fractions of a crude mitochondrial P2 pellet from the guinea-pig ileum longitudinal muscle-myenteric plexus, prepared by discontinuous sucrose-gradient fractionation. The highest specific binding (ca. 100 fmol/mg protein) was obtained in the fraction containing synaptosomes, as examined by electron microscopy. A synaptosomal fraction prepared from guinea-pig brain had comparable specific binding (ca. 130 fmol/mg) to that of the ileal synaptosomal fraction. Addition of calcium (10 mM) to the binding assay medium resulted in a marked decrease in particular of the specific³H-morphine binding. Detailed analysis of the specific³H-morphine binding in the synaptosomal fraction of the ileum revealed that 1) a saturable component of specific opiate binding was present between 0.34 and 21.74 nM of³H-morphine; 2) in the presence of 3 and 10 nM of calcium similar decreases of specific³H-morphine binding were obtained, indicating that this binding was maximally inhibited already by 3 mM of calcium; 3) both in the absence and presence of calcium theK _D of specific³H-morphine binding was about 38 nM, indicating a non-competitive nature of the calcium inhibition; 4) addition of magnesium exhibited a similar effect as that of calcium, although magnesium appeared to be less potent than calcium in this respect. The data are discussed in the context of previously observed calcium effects on opioid actions in the electrically stimulated guinea-pig ileum bioassay and may contribute to the evidence that the interaction of calcium and opioids on the stimulus-release coupling mechanism at the neuromuscular junction of the guinea-pig ileum is occurring beyond opioid receptor activation as well.     

Identification of muscarinic receptor subtypes involved in catecholamine secretion in adrenal medullary chromaffin cells by genetic deletion

Background and Purpose

Activation of muscarinic receptors results in catecholamine secretion in adrenal chromaffin cells in many mammals, and muscarinic receptors partly mediate synaptic transmission from the splanchnic nerve, at least in guinea pigs. To elucidate the physiological functions of muscarinic receptors in chromaffin cells, it is necessary to identify the muscarinic receptor subtypes involved in excitation.

Experimental Approach

To identify muscarinic receptors, pharmacological tools and strains of mice where one or several muscarinic receptor subtypes were genetically deleted were used. Cellular responses to muscarinic stimulation in isolated chromaffin cells were studied with the patch clamp technique and amperometry.

Key Results

Muscarinic M₁, M₄ and M₅ receptors were immunologically detected in mouse chromaffin cells, and these receptors disappeared after the appropriate gene deletion. Mouse cells secreted catecholamines in response to muscarinic agonists, angiotensin II and a decrease in external pH. Genetic deletion of M₁, but not M₃, M₄ or M₅, receptors in mice abolished secretion in response to muscarine, but not to other stimuli. The muscarine-induced secretion was suppressed by MT7, a snake peptide toxin specific for M₁ receptors. Similarly, muscarine failed to induce an inward current in the presence of MT7 in mouse and rat chromaffin cells. The binding affinity of VU0255035 for the inhibition of muscarine-induced currents agreed with that for the M₁ receptor.

Conclusions and Implications

Based upon the effects of genetic deletion of muscarinic receptors and MT7, it is concluded that the M₁ receptor alone is responsible for muscarine-induced catecholamine secretion.     

Systematic evaluation of a panel of 30 synthetic cannabinoid receptor agonists structurally related to MMB-4en-PICA,MDMB-4en-PINACA,ADB-4en-PINACA,and MMB-4CN-BUTINACA using a combination of binding and different CB1 receptor activation assays: Part I—Synthesis,analytical characterization,and binding affinity for human CB1 receptors

Synthetic cannabinoid receptor agonists (SCRAs) are one of the largest and most structurally diverse classes of new psychoactive substances (NPS). Despite this, pharmacological data are often lacking following the identification of a new SCRA in drug markets. In this first of a three-part series, we describe the synthesis, analytical characterization, and binding affinity of a proactively generated, systematic library of 30 indole, indazole, and 7-azaindole SCRAs related to MMB-4en-PICA, MDMB-4en-PINACA, ADB-4en-PINACA, and MMB-4CN-BUTINACA featuring a 4-pentenyl (4en-P), butyl (B/BUT), or 4-cyanobutyl (4CN-B/BUT) tail and a methyl l -valinate (MMB), methyl l -tert-leucinate (MDMB), methyl l -phenylalaninate (MPP), l -valinamide (AB), l -tert-leucinamide (ADB), l -phenylalaninamide (APP), adamantyl (A), or cumyl head group. Competitive radioligand binding assays demonstrated that the indazole core conferred the highest CB₁ binding affinity (K_i = 0.17–39 nM), followed by indole- (K_i = 0.95–160 nM) and then 7-azaindole-derived SCRAs (K_i = 5.4–271 nM). Variation of the head group had the greatest effect on binding, with tert-leucine amides and methyl esters (K_i = 0.17–14 nM) generally showing the greatest affinities, followed by valine derivatives (K_i = 0.72–180 nM), and then phenylalanine derivatives (K_i = 2.5–271 nM). Adamantyl head groups (K_i = 8.8–59 nM) were suboptimal for binding, whereas the cumyl analogues consistently conferred high affinity (K_i = 0.62–36 nM). Finally, both butyl (K_i = 3.1–163 nM) and 4-cyanobutyl (K_i = 5.5–44 nM) tail groups were less favorable for CB₁ binding than their corresponding 4-pentenyl counterparts (K_i = 0.72–25 nM).