Charles H.V. Hoyle

Suramin antagonizes responses to P2-purinoceptor agonists and purinergic nerve stimulation in the guinea-pig urinary bladder and taenia coli.

1 Suramin, an inhibitor of several types of ATPase, was investigated for its ability to antagonize responses mediated via P2X‐purinoceptors in the guinea‐pig urinary bladder and P2Y‐purinoceptors in the guinea‐pig taenia coli. 2 In isolated strips of bladder detrusor muscle, suramin (100 μm‐1 mm) caused a non‐competitive antagonism of responses to α,β‐methylene ATP with an estimated pA2 of approximately 4.7, and inhibited responses to stimulation of the intramural purinergic nerves, with a similar pA2 value. At a concentration of 10 μm, suramin had little effect, but at a concentration of 1 μm, suramin potentiated responses to α,β‐methylene ATP, and potentiated responses to electrical stimulation of intramural purinergic nerves. 3 In isolated strips of taenia coli, in which a standard tone had been induced by carbachol (100 nm), suramin at 100 μm and 1 mm significantly antagonized relaxant responses to ATP (at an EC50 concentration) with an estimated pA2 of 5.0 ± 0.82 and relaxant responses to electrical stimulation of the intramural non‐adrenergic, non‐cholinergic inhibitory nerves, either single pulses or trains of 8 Hz for 10 s, with estimated pA2 values of 4.9 ± 0.93 and 4.6 ± 1.01, respectively. Suramin had no significant effect at 1 or 10 μm. 4 Suramin, at any of the concentrations tested, did not affect contractile responses to histamine (10 μm) or carbachol (10 μm) in the bladder detrusor preparations. In the taenia coli, suramin did not affect either the relaxant responses to noradrenaline (at an EC50 concentration) or the contractile responses to carbachol (100 nm). 5 Thus, suramin at concentrations above 10 μm blocked actions mediated via P2X‐ and P2Y‐purinoceptors in the guinea‐pig urinary bladder and taenia coli respectively. Potentiation of purinoceptor‐mediated activity was seen only at a low concentration of suramin (1 μm) and only in the urinary bladder (P2X‐purinoceptor). For its antagonistic activity suramin did not discriminate between P2X‐ and P2Y‐purinoceptors, but it was selective for P2‐purinoceptor‐mediated activity rather than that mediated via cholinoceptors, adrenoceptors or histamine receptors.

Neuropeptide families and their receptors: evolutionary perspectives.

Examination of families of neuropeptides and their receptors can provide information about phyletic relationships and evolutionary processes. Within an individual a given signal molecule may serve many diverse functions, mediated via subtypes of the receptor which may be coupled to their transduction mechanisms in different ways. The rate of evolution of a peptide may reflect or be reflected in the rate of evolution of its receptor. For example, in the neuropeptide Y (NPY) family, pancreatic polypeptide (PP) shows significant structural diversity, while NPY is highly conserved. Molecular forms of a given subtype of NPY receptor that is selectively activated by NPY (Y1 or Y2 or Y5) are also highly conserved, but the subtype that is primarily activated by PP (Y4), shows remarkable diversity. Also, between receptor subtypes there can be remarkable diversity. This is evident in several neuropeptide families, where a neuropeptide sequence is highly conserved across a wide range of species but where the receptor homology of subtypes with species tends to be much lower than homology between species. For example, human and rat vasopressin are identical, but the human V(1)- or V(2)-vasopressin receptors are approximately 80% homologous with rat V(1)- or V(2)-receptors, but within humans or rats the V(1)-receptor is less than 50% homologous with the V(2)-receptor. Furthermore, duplication of an ancestral gene is thought to have led to the co-presence in eutherian mammals of oxytocin and vasopressin, which have maintained a close structural similarity, yet in many species the oxytocin receptor is only 30 to 50% homologous with vasopressin receptors. Thus it appears that there has been greater evolutionary pressure to conserve the signal molecule, than to conserve the structure of the receptor. Evaluation of the evolution of neuropeptides and their receptors may be useful in determining phyletic relationships. Traditional classification places the guinea pig as a hystricomorph rodent within the same order (Rodentia) as the muriform or myomorph rat and mouse. However, molecular analyses of polypeptides have led to the suggestion that guinea pigs belong to a distinct order. Analysis of several neuropeptide sequences and the Y4 receptor supports this view. In general terms for both neuropeptides and receptors, sequence homology reflects phylogeny and taxonomy as based on morphological features. Within the oxytocin/vasopressin family in which peptides and receptors have been characterised in invertebrate representatives as well as fish and amphibia in addition to mammals, the molecular diversity correlates well with evolutionary diversity.

Neuropeptide families: evolutionary perspectives

Examination of neuropeptide families can provide information about phyletic relationships and evolutionary processes. In this article the oxytocin/vasopressin family, growth hormone releasing factor (GRF) superfamily and the substance P/tachykinin family have been considered in detail because they have been isolated from an extraordinarily diverse array of species from several vertebrate classes and invertebrate phyla. More important is that the nucleotide sequence of mRNA or cDNA encoding many of these peptides has been determined, which has allowed evolutionary distances to be estimated based on the DNA mutation rate. The origin of a given family lies in a primordial gene that arose many millions of years ago, and through time, exon duplication and insertion, gene duplication, point mutation and exon loss, the family developed into the forms that are now recognised. For example, in birds, GRF and pituitary adenylate cyclase activating peptide (PACAP) are encoded by the same gene, which probably arose as a result of exon duplication and tandem insertion of the ancestral GRF gene. In mammals GRF is the sole product on one gene, and PACAP is the product of a gene that also produces PACAP-related peptide (PRP), which is homologous to GRF. Thus it appears that between birds and mammals the GRF/PACAP gene duplicated: exon loss gave rise to the mammalian GRF gene, while mutation led to the formation of the mammalian PRP/PACAP gene. The neuropeptide Y superfamily is considered briefly, as is cionin, which is an invertebrate peptide that is closely related to the mammalian gastrin/cholecystokinin family.

PPADS selectively antagonizes P2X-purinoceptor-mediated responses in the rabbit urinary bladder.

1 Pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), an inhibitor of P2X‐purinoceptor‐mediated responses in rabbit vas deferens, was investigated for its ability to antagonize contractions evoked by α,β‐methylene ATP (α,β‐MeATP), carbachol and electrical field stimulation in the rabbit urinary bladder detrusor muscle. 2 PPADS (1–30 μm) caused concentration‐dependent inhibition of contractions to the stable P2X‐purinoceptor agonist, α,β‐MeATP, decreasing the maximum response to α,β‐MeATP (30 μm) at concentrations of 3–30 μm. The pD2 value for α,β‐MeATP in the absence of PPADS was 6.52 ± 0.10 (8). In the presence of PPADS at concentrations of 1, 3, 10 and 30 μm the negative log concentrations of α,β‐MeATP that cause the same contractile response as the pD2 value were significantly different from control, being respectively 6.17 ± 0.09 (8), 5.64 ± 0.12 (7), 5.15 ± 0.23 (7) and 4.78 ± 0.22 (5). 3 PPADS (1–30 μm) caused concentration‐dependent inhibition of contractions to stimulation of intramural purinergic nerves (1–32 Hz). There was a greater inhibition at lower frequencies (1–8 Hz) than at higher frequencies (16–32 Hz). PPADS, 30 μm, did not produce significantly greater antagonism than 10 μm. 4 PPADS (30 μm) had no significant influence on the contractile potency of carbachol: the pD2 values of carbachol in the absence and presence of PPADS were not significantly different being 6.42 ± 0.16 (5) and 6.33 ± 0.18 (5), respectively. However, PPADS caused a small, but significant, suppression of the maximal response of carbachol, reducing it by approximately 9%. 5 Radioligand binding studies carried out on rabbit bladder membranes with [3H]‐α,β‐methylene ATP ([3H]‐α,β‐MeATP) showed that PPADS concentration‐dependently inhibited the binding of [3H]‐α,β‐MeATP to P2X‐purinoceptors, while the binding of [3H]‐quinuclidinyl benzilate to muscarinic cholinoceptors was not affected. 6 Thus, PPADS (1–30 μm) antagonized responses mediated via P2X‐purinoceptors in the rabbit urinary bladder. It was selective for P2‐purinoceptor‐mediated contractions rather than those mediated via muscarinic receptors. Binding studies demonstrated that the antagonistic effect of PPADS is via a direct interaction with P2X‐purinoceptors.

Selective antagonism by PPADS at P2X‐purinoceptors in rabbit isolated blood vessels

1 Pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), a P2‐purinoceptor antagonist, was investigated for its ability to antagonize: (1) P2X‐purinoceptor‐mediated contractions of the rabbit central ear artery and saphenous artery evoked by either α,β‐methylene ATP (α,β‐MeATP) or electrical field stimulation (EFS); (2) P2Y‐purinoceptor‐mediated relaxations of the rabbit mesenteric artery; (3) endothelium‐dependent and endothelium‐independent, P2Y‐purinoceptor‐mediated relaxations of the rabbit aorta. 2 α,β‐MeATP (0.1–100 μm) caused concentration‐dependent contractions of the rabbit ear and saphenous arteries. The negative log[α,β‐MeATP] that produced a contraction equivalent to the EC25 for noradrenaline (ear artery) or histamine (saphenous artery) in the absence of PPADS was 6.60 ± 0.18 (9) and 6.18 ± 0.17 (9) in the ear artery and saphenous artery, respectively. These effects of exogenous α,β‐MeATP were concentration‐dependently inhibited by PPADS (1–30 μm). In the ear artery, the negative log[α,β‐MeATP] producing a contractile response equivalent to the EC25 of noradrenaline, in the presence of PPADS at 1, 3 and 10 μm was 6.16 ± 0.18 (8), 5.90 ± 0.18 (8) and 4.72 ± 0.36 (8), respectively (P < 0.01). In the saphenous artery, the negative log[α,β‐MeATP] values equivalent to the EC25 for histamine in the presence of PPADS at concentrations of 1, 3, 10 and 30 μm were 5.90 ± 0.19 (8), 5.73 ± 0.16 (8), 4.99 ± 0.14 (8) and 4.51 ± 0.13 (8), respectively (P < 0.01). 3 PPADS at a concentration of 1 μm had no effect on contractions of the ear artery evoked by EFS (4–64 Hz; 1 μm phentolamine present). At higher concentrations (3–30 μm) it caused concentration‐dependent inhibition of neurogenic contractions. In the saphenous artery, PPADS (1–30 μm) concentration‐dependently inhibited contractions evoked by EFS at frequencies of 4, 8 and 16 Hz. Contractions evoked by EFS at frequencies of 32 and 64 Hz were significantly inhibited by PPADS only at concentrations of 10 and 30 μm. 4 PPADS (30 μm) had no effect on relaxations to 2‐methylthio ATP (3 nm‐3 μm) in rabbit mesenteric artery and to ATP (1 μm − 1 mm) in rabbit aorta (with endothelium intact or removed). In addition, PPADS (30 μm) had no significant influence on the contractile potency of noradrenaline and histamine in rabbit ear and saphenous artery, respectively. 5 In conclusion, these results support the evidence that PPADS is a selective antagonist of P2X‐purinoceptor‐mediated responses.

Involvement of melatonin MT3 receptors in the regulation of intraocular pressure in rabbits

Melatonin, a neurohormone secreted by the pineal gland, can stimulate three subtypes of receptors, namely: mt(1), MT(2) and MT(3). We examined the ability of melatonin and the selective MT(3) receptor agonist, 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT), to modify intraocular pressure in rabbits. Both compounds significantly reduced intraocular pressure, maximally by 24% and 43%, respectively, with IC(50) values of 363+/-23.0 and 423+/-30.0 ng/10 microl (1.6+/-0.1 and 1.8+/-0.1 nmol, respectively). The non-specific melatonin receptor antagonist, luzindole, abolished the depressant effect of both compounds, thus confirming the involvement of melatonin receptors. Our results show, for the first time, a functional response that may be regulated by melatonin MT(3) receptors, and provide evidence that supports a role of melatonin in the circadian changes of intraocular pressure.

Isolated human bladder: evidence for an adenine dinucleotide acting on P2x-purinoceptors and for purinergic transmission

In isolated strips of human urinary bladder detrusor muscle, ATP, alpha, beta-methylene ATP and P1,P6-diadenosine hexaphosphate caused concentration-dependent contractions. ATP was less potent than the two synthetic purine compounds and gave smaller maximum responses. Responses to ATP, P1,P6-diadenosine hexaphosphate and noncholinergic nerve stimulation were blocked following desensitization of P2X-purinoceptors by alpha,beta-methylene ATP. Thus, adenine dinucleotides can act on P2X-purinoceptors and there is an element of purinergic neuromuscular transmission in the human urinary bladder.

Ocular hypotensive effects of melatonin receptor agonists in the rabbit: further evidence for an MT3 receptor

Melatonin is involved in the control of intraocular pressure during the night and day photoperiod. We have investigated the receptor that regulates intraocular pressure in New Zealand white rabbits by means of agonists and antagonists of melatonin receptors. Melatonin and its analogues: 2‐Phe‐melatonin, 6‐Cl‐melatonin, 2‐I‐melatonin, 5‐ methoxycarbonylamino‐N‐acetyltryptamine (5‐MCA‐NAT) and N‐acetyltryptamine all produced a reduction in intraocular pressure. Dose‐response analysis for these compounds gave pD2 values of 9.3±0.24 for melatonin; 9.0±0.09 for 6‐Cl‐melatonin; 9.0±0.84 for 2‐I‐melatonin; 8.9±0.07 for 5‐MCA‐NAT; 8.7±0.18 for 2‐Phe‐melatonin and 9.4±0.30 for N‐acetyltryptamine (all n=8). At a dose of 0.5 nmol (in 10 μl) melatonin and the selective melatonin MT3 agonist 5‐MCA‐NAT, induced greater reductions of intraocular pressure (22.8±2.3% and 32.5±1.4%, respectively) than the other compounds. The melatonin‐receptor antagonists, prazosin, DH‐97 and 4‐P‐PDOT, reversed the effect of 5‐MCA‐NAT in a dose‐dependent manner, with pA2 values of 13.5±0.17 for prazosin, 10.6±0.16 for DH‐97 and 9.4±0.20 for 4‐P‐PDOT (n=8). Cholinoceptor antagonists (hexamethonium and atropine) and α2‐ and β2‐adrenoceptor antagonists (yohimbine and ICI 118,551) partially reversed the effects produced by melatonin and 5‐MCA‐NAT, suggesting the possible involvement of cholinergic and noradrenergic systems in the hypotensive actions mediated by melatonin agonists. The α1‐adrenoceptor antagonist, corynanthine, had no significant effect. The strong hypotensive effect of the MT3 agonist, 5‐MCA‐NAT, suggests that this compound may be a useful agent for treating those pathologies where intraocular pressure is abnormally elevated.

Pharmacological activity of adenine dinucleotides in the periphery: possible receptor classes and transmitter function.

1. The pharmacological actions of adenine dinucleotides, in particular beta-nicotinamide adenine dinucleotide (NAD), beta-nicotinamide adenine dinucleotide phosphate (NADP) and a homologous series of alpha,omega-adenine dinucleotide polyphosphates has been reviewed. 2. It is apparent that many actions of NAD can be explained in terms of activation of P1-purinoceptors, but actions of NADP cannot be explained in terms of activation of P1- or P2-purinoceptors. 3. Similarly, pharmacological activities of P1,P3-diadenosine triphosphate and P1,P4-diadenosine tetraphosphate are not in keeping with activation of P1- or P2-purinoceptors. 4. In the vas deferens and urinary bladder, P1,P4-diadenosine tetraphosphate, P1,P5-diadenosine pentaphosphate and P1,P6-diadenosine hexaphosphate act on P2x-purinoceptors and can cause desensitization of these receptors. 5. It is suggested that classes of receptors for adenine dinucleotides exist which are distinct from either P1- or P2-purinoceptors. 6. It is also suggested that in view of the finding of high concentrations of alpha,omega-adenine dinucleotide polyphosphates in adrenal medullary chromaffin cells, and of the involvement of the P2x-purinoceptor in the vas deferens and urinary bladder with purinergic neuromuscular transmission, that alpha,omega-adenine dinucleotide polyphosphates may yet be discovered in autonomic neurones and serve as neurotransmitters.

Inhibitory action of PPADS on relaxant responses to adenine nucleotides or electrical field stimulation in guinea-pig taenia coli and rat duodenum.

1 The effect of pyridoxarphosphate‐6‐azophenyl‐2′, 4′‐disulphonic acid (PPADS) on the relaxant response to adenine nucleotides was examined in the carbachol‐contracted guinea‐pig taenia coli and rat duodenum, two tissues possessing P2y‐purinoceptors. In addition, in the taenia coli PPADS was investigated for its effect on relaxations evoked by adenosine, noradrenaline and electrical field stimulation. In order to assess the selectivity of PPADS between P2‐purinoceptor blockade and ecto‐ nucleotidase activity, its influence on ATP degradation was studied in guinea‐pig taenia coli. 2 The resulting rank order of potency for the adenine nucleotides in guinea‐pig taenia coli was: 2‐methylthio ATP≫ ATP>>α, β‐methylene ATP with the respective pD2‐values 7.96 ±0.08 (n = 23), 6.27 ±0.12 (n = 21) and 5.88 ±0.04 (n = 24). 3 In guinea‐pig taenia coli, PPADS (10–100 μm) caused a consistent dextral shift of the concentration‐response curve (CRC) of 2‐methylthio ATP and ATP resulting in a biphasic Schild plot. A substantial shift was only observed at 100 μm PPADS, the respective pA2‐values at this particular concentration were 5.26 ±0.16 (n = 5) and 5.15 ±0.13 (n = 6). Lower concentrations of PPADS (3–30 μm) antagonized the relaxant effects to α, β‐methylene ATP in a surmountable manner. An extensive shift of the CRC was produced only by 30 μm PPADS (pA2 = 5.97 ± 0.08, n = 6), and the Schild plot was again biphasic. 4 The relaxant responses to electrical field stimulation (80 V, 0.3 ms, 5 s, 0.5–16 Hz) in guinea‐pig taenia coli were concentration‐dependently inhibited by PPADS (10–100 μm). 5 In guinea‐pig taenia coli, the potency of ATP in inducing relaxation appeared to be independent of its rate of degradation by ecto‐nucleotidases, since the Km‐value (366 μm) obtained in the enzyme assay was much higher than the functional EC50‐value (0.45 μm) of ATP. PPADS (3–100 μm) was only weakly active in inhibiting ecto‐nucleotidase activity leaving a residual activity of 81.8 ±5.1% at 100 μm. Enzyme inhibition by PPADS was concentration‐independent and non‐competitive. 6 In rat duodenum, the rank order of potency was: 2‐methylthio ATP > ATP≫α, β‐methylene ATP, the respective pD2‐values being 6.98 ±0.04 (n = 76), 6.26 ±0.02 (n = 6) and 4.83 ±0.02 (n = 6). Among these agonists, 2‐methylthio ATP displayed the lowest apparent efficacy. 7 The CRC of 2‐methylthio ATP in rat duodenum was shifted to the right by PPADS (10–100 μm) in a concentration‐dependent manner, and Schild analysis gave a pA2‐value of 5.09 ±0.06 (slope =1.02, n=14). 8 PPADS was without any effect on the carbachol‐induced contraction in guinea‐pig taenia coli or rat duodenum and on the relaxation to noradrenaline or adenosine in guinea‐pig taenia coli. 9 In conclusion, the antagonistic properties of PPADS at the taenia coli and rat duodenum P2y‐ purinoceptors were different from those recently described at the P2x‐subtype: inhibition of P2y‐ purinoceptor‐mediated responses was observed at higher concentrations (3–100 μm vs. 1–10 (30) μm). Furthermore, we conclude that in addition to the classical P2y‐subtype, which is largely PPADS‐resistant, the guinea‐pig taenia coli may be endowed with a distinct relaxation‐mediating P2‐purinoceptor subtype which is sensitive to PPADS.