Arylazido Aminopropionyl ATP Research Articles

Comparative effects of adenosine‐5′‐triphosphate and related analogues on insulin secretion from the rat pancreas

Adenosine tri- and diphosphate (ATP and ADP) and their structural analogues stimulate insulin secretion from the isolated perfused rat pancreas, an effect mediated by P2Y-purinoceptor activation. Concerning the base moiety of the nucleotide, it was previously shown that purine but not pyrimidine nucleoside triphosphates were active and that substitution on purine C2 with the 2-methylthio group greatly enhanced the potency. In this study, we further analyze the consequences of ribose and polyphosphate chain modifications. Modifications in 2' and 3' position on the ribose led to a decrease in insulin response when bulky substitutions were made: indeed, 2'-deoxy ATP was similar in activity to ATP, whereas arylazido-aminopropionyl ATP (ANAPP3) was weakly effective and trinitrophenyl ATP (TNP-ATP) was inactive. Substitution on the gamma phosphorus of the triphosphate chain led to a decrease (gamma-anilide ATP) or no change (gamma-azido ATP) in potency; the replacement of the bridging oxygen between beta and gamma phosphorus by a peroxide group did not significantly change the activity, whereas substitution by a methylene group completely abolished stimulation of insulin secretion. As for the phosphorothioate analogues, adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) induced an insulin response similar to that produced by ATP, whereas adenosine-5'-O-(2-thiodiphosphate) (ADP beta S) was about 100-fold more potent than ATP, as previously shown. In conclusion, two structural features seem to have a strategic importance for increasing the insulin secretory activity of ATP analogues: substitution at the C2 position on the adenine ring of ATP and modifications of the polyphosphate chain at the level of the beta phosphorus.

Indirect evidence for separate vesicular neuronal origins of norepinephrine and ATP in the rabbit vas deferens

Various modulators of neurotransmission were examined for selective effects on the non-adrenergic or adrenergic components of neurotransmission in the vas deferens to test the hypothesis that ATP and norepinephrine are secreted from the same vesicles. The ATP receptor antagonist, arylazido aminopropionyl ATP (ANAPP), selectively depressed the non-adrenergic contraction and prazosin selectivity depressed the adrenergic contraction in response to electrical stimulation. These results are consistent with the presence of two neurotransmitters, ATP and norepinephrine, which mediate neurogenic contractions. Prostaglandin E 2 inhibited non-adrenergic, but enhanced adrenergic, electrically induced (10 Hz) contractions, presumably via a prejunctional mechanism. The adrenergic component of the neurogenic response was significantly more sensitive to treatment with guanethidine, guanabenz, and 6-hydroxydopamine. These results with a variety of agents are inconsistent with the hypothesis that the neurotransmitters, ATP and norepineprine, are released in tandem from the same neuronal granules.

Arylazido aminopropionyl ATP (ANAPP 3): Interaction with adenosine receptors in longitudinal smooth muscle of the guinea-pig ileum

Arylazido aminopropionyl ATP (ANAPP 3), an ATP-receptor antagonist containing a photosensitive arylazido moiety coupled to the 3′ hydroxyl of ATP, inhibited the twitch response of electrically stimulated ileal longitudinal muscle strips in a dose-dependent manner. These agonist responses to ANAPP 3 were attenuated by the enzyme adenosine deaminase and antagonized by the adenosine receptor antagonist 8-phenyltheophylline. Schild analysis yielded similar pA 2 values for ANAPP 3 and adenosine suggesting a common receptor site. Several 3′-ribose-modified adenosine analogs were tested for agonist activity and found to be inactive. Results suggest that ANAPP 3 interacts at the presynaptic adenosine receptor of the ileum following its metabolism to adenosine, which explains the lack of antagonism at adenosine receptors of ileal smooth muscle following photolysis of ANAPP 3.

Long-lasting blockade of P 2-receptors of the urinary bladder [formula omitted] following photolysis of arylazido aminopropionyl atp, a photoaffinity label

In the present study the possibility of provoking an irreversible blockade of P 2-receptors in vivo by photolyzing ANAPP 3 was investigated. ANAPP 3 administered as an intraarterial infusion produced a short-lived blockade of P 2-receptor mediated contractions of cat urinary bladders in situ . However, irradiation of the bladders with visible light during the infusion of ANAPP 3 resulted in a blockade of the response for at least 120 minutes. Thus, photoactivation of ANAPP 3 in vivo produced an essentially irreversible blockade of P 2-purinergic receptors.

The effect of arylazido aminopropionyl ATP (ANAPP 3) on inhibition of pelvic nerve evoked contractions of the cat urinary bladder

Previously reported work has provided evidence that arylazido aminopropionyl ATP (ANAPP 3), a P 2-receptor antagonist, blocks the effects of ATP and other purine analogs, in the urinary bladder of the cat. This antagonism appeared limited to those effects mediated by P 2-receptors, such as contraction of the urinary bladder. However, it was noted that ANAPP 3 did alter the effects of adenosine, mediated through P 1-receptors, in some preliminary experiments. A series of experiments was undertaken to determine if ANAPP 3 blocks the P 1-receptors and/or has effects on inhibition of bladder contractions. The urinary tract of anesthetized cats was exposed by a midline abdominal incision. The pelvic and hypogastric nerves were isolated and prepared for electrical stimulation. Bladder contractions were induced by stimulation of the pelvic nerves every 30s with square wave pulses. These contractions were inhibited by hypogastric nerves and the exogenous administration of ATP, noradrenaline, adenosine or β, γ-methylene ATP (APPCP), a hydrolysis resistant analog of ATP. ANAPP 3 (0.5 μmol, i.a.) was administered, but it did not antagonize the inhibition induced by any of the exogenously administered agents or hypogastric nerve stimulation. This study indicates that ANAPP 3 is specific for P 2-receptors and is not an effective antagonist of P 1-receptors, at least in the urinary tract of the cat.

Interaction of [ 3H]arylazido aminopropionyl ATP ([ 3H]ANAPP 3) with P 2-purinergic receptors in the smooth muscle of the isolated guinea-pig vas deferens

Following its photolysis in the presence of the isolated guinea-pig vas deferens, the ATP photoaffinity label ANAPP 3 produces a specific antagonism of adenine nucleotide-induced contractile responses which are mediated by P 2-purinergic receptors. To characterize the site of covalent photoincorporation of ANAPP 3, intact vasa deferentia were treated with [ 3H]ANAPP 3 and samples of homogenate, cytosol and a crude membrane fraction were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Photolysis of [ 3H]ANAPP 3 (10 −5 M; 3.0 μCi/ml) resulted in the incorporation of radioactivity into cellular components with apparent molecular weights of 54–66 and 43–57 kilodaltons. The photoincorporation of [ 3H]ANAPP 3 was associated with the crude membrane fraction and not the cytosol, was reduced in the presence of ATP in an ATP-concentration-dependent manner, was lessened following pretreatment of the tissues with photolyzed nonradiolabeled ANAPP 3, and was unaffected by the nucleoside transport inhibitor, dipyridamole. In tension studies on the same tissues the presence of ATP resulted in a concentration-dependent reduction in the initial contractile response to [ 3H]ANAPP 3 the response to 3H was antagonized in tissues which had been pretreated with nonradiolabeled ANAPP 3, and dipyridamole had no effect on the contractile response to [ 3H]ANAPP 3. According to several criteria these findings indicate that the antagonism by photolyzed ANAPP 3 of adenine nucleotide-induced responses is a direct result of the covalent insertion at or near the recognition site of cell-surface P 2-purinergic receptors.

Novel, selective purinoceptor antagonists: investigation of ATP as a neurotransmitter

Selective antagonists for ATP-mediated P 2-purinoceptors have recently become available and have proved useful tools in the study of ATP in synaptic transmission. Takeshi Katsuragi and Tatsuo Furukawa review the data on novel purinoceptor antagonists and discuss the implications of the observations that clonidine and the ATP analogs, arylazidoaminopropionyl ATP and α,β-methylene ATP, produce selective antagonism for P 1- and P 2-purinoceptors, respectively. Experiments utilizing these antagonists support the hypothesis that endogenous ATP and noradrenaline exist as co-transmitters in the neuromuscular junction of rodent vas deferens.

A pharmacological investigation of the biphasic nature of the contractile response of rabbit and rat vas deferens to field stimulation

It has been demonstrated previously with the vas deferens of the guinea-pig that the first and second phases of the contractile response to motor nerve stimulation are preferentially antagonized by the P 2-purinoceptor antagonist arylazido aminopropionyl ATP (ANAPP 3), and the α 1-adrenoceptor antagonist prazosin, respectively. We have now investigated the effect of the two antagonists on the biphasic contraction in the vas deferens of two other species; rabbit and rat. ANAPP 3, in a concentration which antagonized responses to exogenously applied ATP but not those to exogenous norepinephrine, preferentially reduced the initial phasic response of the rabbit vas deferens to motor nerve stimulation without significantly reducing the secondary, tonic phase of the response. Prazosin had the opposite effect; antagonizing the response to norepinephrine but not to ATP and reducing the tonic response to motor nerve stimulation without significantly reducing the initial phasic response. Results obtained with the rat vas deferens were similar. The present results combined with previous findings suggest that ATP and norepinephrine act as cotransmitters in the vas deferens of several species.

Comparison of the effects of apamin, a Ca 2+-dependent K + channel blocker, and arylazido aminopropionyl ATP (ANAPP 3), a P 2-purinergic receptor antagonist, in the guinea-pig vas deferens

Apamin, which blocks Ca 2+-dependent increases in K + permeability, antagonizes ATP-induced relaxation of several smooth muscles. The ATP photoaffinity label arylazido aminopropionyl ATP (ANAPP 3), following its photolysis in the presence of the guinea-pig vas deferens, antagonizes contractile responses to ATP. This study was conducted to determine whether apamin antagonizes ATP-induced responses in the guinea-pig vas deferens, and also to evaluate whether ANAPP 3 antagonizes responses to ATP by interfering with Ca 2+-dependent K + permeability changes. Apamin (10 −6 M) potentiated ATP-induced contractions. This potentiation was nonspecific in that responses to norepinephrine, histamine and acetylcholine also were enhanced; responses to KCl were unaffected. To evaluate the possible interactions between the two agents at the same cellular site, the effect of apamin was examined in ANAPP 3-treated tissues. In such tissues apamin did not potentiate the residual responses to ATP; however, apamin was nevertheless able to potentiate responses of ANAPP 3-treated tissues to norepinephrine, histamine and acetylcholine, and responses to KCl remained unaffected. These studies provide additional support for the view that ANAPP 3 antagonizes ATP-induced responses of the guinea-pig vas deferens by blocking P 2-purinergic receptors. The antagonism by ANAPP 3 is not attributable to a blockade of Ca 2+-dependent K + permeability changes.

Pharmacological evidence that adenosine triphosphate and noradrenaline are co-transmitters in the guinea-pig vas deferens.

The contractile response of the guinea-pig vas deferens to tetanic nerve stimulation was biphasic. The first phase was mimicked by exogenously applied ATP. The second more tonic phase was mimicked by exogenously applied noradrenaline (NA). Intracellular micro-electrodes were used to record the electrical response of the vas deferens to nerve stimulation and to exogenously applied ATP and NA. Local application of ATP (10(-5) to 10(-3)M), by pressure ejection from a micropipette, produced a depolarization similar in magnitude and time course to the excitatory junction potential (e.j.p.). NA produced no such response. Superfusion of the vas deferens with ATP and NA (10(-6) to 10(-4)M) produced a depolarization. The depolarization produced by NA was more gradual than that produced by the same concentration of ATP. The ATP-receptor antagonist ANAPP3 (arylazido aminopropionyl-ATP) preferentially antagonized the first component of the neurogenic contractile response and also antagonized the e.j.p. The alpha-receptor antagonist prazosin preferentially antagonized the second phase of the neurogenic contractile response and enhanced the e.j.p. Similar results were obtained using the irreversible alpha-receptor antagonists phenoxybenzamine and dibenamine. Cocaine (10(-6) and 10(-5)M) enhanced the second phase of the contractile response to nerve stimulation, but reduced the first phase. Lidocaine (10(-5) and 10(-4)M) had no such effect. Cocaine (10(-6) and 10(-5)M) reduced the magnitude of e.j.p.s. at all stimulation frequencies from 1 to 8 Hz. In the presence of the selective alpha 2-receptor antagonist yohimbine (10(-7)M), both phases of the contractile response to nerve stimulation were enhanced to the same degree. This concentration of yohimbine also increased the magnitude of e.j.p.s. In the presence of 10(-7) M-yohimbine, cocaine (10(-6) and 10(-5)M) still enhanced the second phase of the contractile response, but no longer reduced the initial phase of the contraction or e.j.p.s to the same degree. In vas deferens from animals pre-treated with reserpine (2 mg/kg.day), the second phase of the contractile response to nerve stimulation was reduced but neither the first phase of the contraction nor the e.j.p.s was blocked. These results suggest that the first phase of the neurogenic contractile response of the vas deferens and the e.j.p. are mediated by ATP acting on P2-purinoreceptors, whereas NA mediates phase two, via alpha 1-adrenoceptors. The results also suggest that release of ATP and NA is influenced by a negative feed-back mechanism involving presynaptic alpha 2-adrenoceptors.

The effect of arylazido aminopropionyl ATP on atropine resistant contractions of the cat urinary bladder

This work was designed to study neurogenic, atropine resistant contractions of the cat urinary bladder. Urinary bladder contractions were induced by pelvic nerve stimulation (PNS) and the administration of acetylcholine (Ach) and purine compounds. The results show that atropine (2 mg/kg) depresses the maintained portion (phase 2) of PNS-induced contractions and Ach-induced contractions while having no effect on the initial portion (phase 1) of the PNS-induced contractions or those contractions induced by the purines. Arylazido aminopropionyl ATP (ANAPP 3), an ATP antagonist, depressed phase 1 of the PNS contraction and the purine induced contractions. These results suggest that ATP is responsible for at least part of the atropine-resistant contractions of the cat urinary bladder.

Comparison of photoaffinity labeling og P 2-purinergic receptors of isolated guinea-pig vas deferens by arylazido aminopropionyl ATP and by arylazido aminobutyryl ATP

Two chemically related arylazido photoaffinity analogs of ATP (arylazido aminopropionyl ATP (ANAPP 3) and arylazido aminobutyryl ATP (ANABP 3)), which have been reported in the literature to differ in their ability to inhibit myosin ATPase, were compared for their ability to antagonize contractile responses of the isolated guinea-pig vas deferens to ATP. During photolysis in organ chambers the photoconversion (ΔA 260/Δt) of ANAPP 3 occured with greater than first order kinetics or was multiexponential and t 1 2 = 7.5 min , while ΔA 260/Δt for ANABP 3 was first order and t 1 2 = 2.25 min . After photolysis of these compounds in the presence of the guinea-pig vas deferens, using irradiation periods which caused 80% consumption of the compounds, ANABP 3 was 2–3 times more potent than ANAPP 3 in antagonizing contractions to ATP, which are mediated by P 2-purinergic receptors. A comparison of concentration-response curves obtained for nonphotolyzed ANAPP 3 and ANABP 3 used as agonists suggested that the greater antagonism produced by photolyzed ANABP 3 is not attributable to a greater potency. The results suggest that the longer 3′-hydroxyl-arylazide bridge length of ANABP 3 places the arylnitrene intermediate in a position at or near the P 2-receptor which is more favorable for covalent insertion.

Comparison of the effects of arylazido aminopropionyl ATP (ANAPP 3), an ATP antagonist, on responses of the smooth muscle of the guinea-pig vas deferens to ATP and related nucleotides

Contractile responses of the smooth muscle of the guinea-pig vas deferens to ATP and related analogs have been suggested previously to involve stimulation of cell-surface P 2-purinergic receptors and obligatory hydrolysis, depending on the concentration and structure of the nucleotide. This hypothesis was further tested by evaluating the antagonistic effect of arylazido aminopropionyl ATP (ANAPP 3), a specific P 2-purinergic receptor antagonist, on concentration-response relationships and on the profiles of individual responses to ATP and analogs containing phosphate-chain, ribose and adenine modifications. The concentration-response curves for ATP, β, γ-methylene ATP (APPCP), β, γ-imido ATP (APPNP) and adenosine 5′-O-(3-thiotriphosphate) (ATPγS) were bimodal and suggested the existence of two receptors, one with high and one with low affinity for adenine nucleotides. The type of antagonism observed was nucleotide-specific, and concentration-response curves were monosigmoidal after ANAPP 3 treatment. For ATP, antagonism was greatest at low concentrations (< 3 × 10 −5 M) and did not affect the maximum response; for APPCP and APPNP, it was greatest at high concentrations (> 3 × 10 −5 M) and resulted in a reduction in maximum response; for adenosine tetraphosphate (APPPP) the antagonism was pronounced at low and high concentrations; for ATPγS the antagonism was moderate at all concentrations. The concentration-response curves for a second group of less potent nucleotides was monosigmoidal. Included in this group was ADP, α, β-methylene ADP (APCP), 8-bromo ATP (8-BrATP) and 2-deoxy ATP (2d-ATP). Treatment with ANAPP 3 shifted the concentration-response curves for these analogs to the right but did not reduce maximum responses. The characteristics of individual response profiles were structurally related. For most analogs responses to low concentrations of nucleotides were phasic and spike-like. Discernible tonic phases appeared with higher concentrations of ATP, ADP, ATPγS, APPPP and 2d-ATP. ANAPP 3 antagonized the initial component of responses but did not affect the tonic phase. The initial phasic component of response has been related to receptor-mediated stimulation of the preparations, while the tonic component, characteristic of nucleotides without 5′-anhydride bridge substitutions, appears to be initiated by hydrolysis of the compounds, a process which is insensitive to ANAPP 3.

Direct evidence against a role of ATP as the nonadrenergic, noncholinergic inhibitory neurotransmitter in guinea pig tenia coli.

Electrical field stimulation of the isolated guinea pig tenia coli in the presence of a muscarinic receptor antagonist (atropine) and an adrenergic neuron blocker (guanethidine) produces relaxation. A large amount of indirect evidence has suggested that the neurotransmitter that is released from these nonadrenergic, noncholinergic inhibitory neurons is ATP or a related nucleotide, and the nerves have been termed "purinergic." A photoaffinity analog of ATP, arylazido aminopropionyl ATP, which produces a specific pharmacological antagonism of P2 purinergic receptors in isolated guinea pig vas deferens and urinary bladder, was utilized in the present study to evaluate directly whether ATP is the nonadrenergic, noncholinergic inhibitory neurotransmitter in tenia coli. By blocking postjunctional P2 receptors, arylazido aminopropionyl ATP produced a pronounced antagonism of relaxations induced by exogenously added ATP. Responses produced by ADP, AMP, and adenosine also were antagonized by arylazido aminopropionyl ATP, but to a lesser extent. Inhibitory responses to isoproterenol were not antagonized. Under these conditions of established, specific P2-receptor blockade of responses to exogenously added ATP, relaxations induced by field stimulation of intrinsic inhibitory nerves in the presence of atropine (1 microM) and guanethidine (1 microM) were not antagonized. Though these results provide no indication of the actual substance involved, they suggest strongly that the nonadrenergic, noncholinergic inhibitory neurotransmitter in the guinea pig tenia coli is not ATP.

Mechanism of photoaffinity labeling of P 2-purinergic receptors by arylazido aminopropionyl ATP in isolated guinea-pig vas deferens

Following photolysis in the presence of the isolated guinea-pig vas deferens, arylazido aminopropionyl ATP (ANAPP 3), a photoaffinity label analog of ATP, produces an irreversible and specific pharmacological antagonism of contractile responses to adenine nucleotides. Experiments were performed to determine whether the antagonism follows the photolysis-dependent formation of nitrene intermediates at occupied receptors (true photoaffinity labeling) or if the reactive intermediate is photogenerated in solution prior to diffusion to the receptor and formation of covalent bonds (pseudo-photoaffinity labeling). When present during photolysis, para-aminobenzoic acid (PABA; 10 mM), a scavenger for nitrenes generated in solution, did not prevent the antagonism of ATP-induced responses by ANAPP 3. However, the absorption spectrum of ANAPP 3 photolyzed in the presence of PABA was different from that obtained when PABA was not present. This evidence for the formation of additional photolysis products suggests that ANAPP 3 had inserted into PABA during photolysis. Thus, covalent bonds arise from true photoaffinity labeling of the receptor. An analysis of the pharmacological effects of PABA indicated that responses to ATP, KCl and acetylcholine were unaffected either in the presence of, or after a 23 min incubation, with 10 mM PABA. In contrast, PABA produced a substantial, but reversible, antagonism of histamine- and norepinephrine-induced contractions. Irradiation of tissues in the presence of 10 mM PABA produced a slight potentiation of responses to ATP. Thus, information on the mechanisms of photoaffinity labeling may be obtained from functional studies on intact tissues. However, the pharmacological effects of agents used to define these mechanisms should be evaluated as well.

Effect of arylazido aminopropionyl ATP (ANAPP 3), a putative ATP antagonist, on ATP responses of isolated guinea pig smooth muscle

Arylazido aminopropionyl ATP (ANAPP 3), a photoaffinity analogue of adenosine 5′-triphosphate, photoactivated with visible light (+hv), specifically and irreversibly antagonized ATP contractions of the guinea pig vas deferens. ANAPP 3 (30 μM) antagonized responses to exogenously added ATP in untreated, and in tissues pretreated with indomethacin (2.9 μM) and 6-(2-hydroxy-5-nitrobenzyl)-thio guanosine (10 μM). It was of interest to see if this pharmacological antagonist of ATP could be used to assess the validity of the purinergic nerve hypothesis by allowing a differentiation between, or proof of the identity of, responses to ATP and the non-adrenergic inhibitory transmitter in guinea pig stomach fundus. After photoactivation (+hv) in the organ bath and subsequent washout, ANAPP 3 (30 and 100 μM) failed to antagonize relaxant responses to ATP (1.0 – 1000 μM) in fundic strips. In addition ANAPP 3 failed to antagonize ATP-induced inhibition of the twitch response in electrically stimulated guinea pig ileum longitudinal muscle strips. We conclude that ANAPP 3 does not antagonize all actions of ATP, which may limit its usefulness in assessing the above hypothesis. Results with this compound suggest that ATP excitatory receptors may differ from those mediating relaxation and other ATP actions.

Contribution by purines to the neurogenic response of the vas deferens of the guinea pig

The specific purine-receptor antagonist, arylazido aminopropionyl ATP (ANAPP 3), was used to determine if purines released from nerves of the guinea-pig vas deferens contribute to the neurogenic response. ANAPP 3, which is a photoaffinity label, antagonized contractile responses of the in vitro vas deferens to transmural stimulation, reversibly in the presence of the compound and irreversibly after its photoactivation in the presence of the tissues. The antagonism by ANAPP 3 was augmented by depletion of norepinephrine produced by reserpine pretreatment. Responses of untreated tissues were only slightly antagonized by the α-adrenoceptor blocker prazosin. However, neurogenic responses were markedly reduced in the combined presence of ANAPP 3 and prazosin. ANAPP 3 did not affect the release of tritium from tissues prelabeled with [ 3H]norepinephrine. The initial phasic component of the neurogenic response was preferentially antagonized by ANAPP 3 whereas the secondary more tonic component of the response was preferentially antagonized by prazosin and reserpine pretreatment. Small, residual responses remaining after chemical sympathectomy produced by 6-hydroxydopamine pretreatment were potentiated, rather than inhibited, by ANAPP 3 and were, unlike untreated tissues, sensitive to atropine. These and previous findings indicate that ATP or a related purine, originating from adrenergic neurons, acts as a co-transmitter with norepinephrine in this tissue.

Arylazido aminopropionyl ATP, an active site directed photoaffinity reagent for mitochondrial adenosine triphosphatase

The photoaffinity label, arylazido aminopropionyl ATP ∗ ∗ Abbreviations: arylazido aminopropionyl ATP; (3′-0-{3-[N-(4-azido-2-nitrophenyl)amino]propionyl}adenosine-5′-triphosphate). , brings about a photodependent inhibition of mitochondrial F 1-ATPase and an associated specific covalent labeling of the soluble enzyme. In addition the ATP analog can act as a substrate when incubated with F 1-ATPase in the dark.