Edward C. Burgard

Modulation of BzATP and formalin induced nociception: attenuation by the P2X receptor antagonist, TNP‐ATP and enhancement by the P2X3 allosteric modulator, cibacron blue

Exogenous ATP produces acute and localized pain in humans, and P2X receptor agonists elicit acute nociceptive behaviours in rodents following intradermal administration to the hindpaw. The predominant localization of P2X3 mRNA in sensory neurones has led to the hypothesis that activation of P2X3 and/or P2X2/3 receptors contributes to nociception. The local administration of the P2X receptor agonist, BzATP (100 – 1000 nmol paw−1, s.c.) into the rat hindpaw produced an acute (<15 min) paw flinching response that was similar to that observed in the acute phase of the formalin (5%) test. The co‐administration of the potent P2X receptor antagonist, TNP‐ATP (30 – 300 nmol paw−1), but not an inactive analogue, TNP‐AMP, with BzATP into the rat hindpaw attenuated BzATP‐induced nociception. Similarly, co‐administration of TNP‐ATP, but not TNP‐AMP, with 5% formalin reduced both acute and persistent nociception in this test. Co‐administration of cibacron blue (30 and 100 nmol paw−1), a selective allosteric enhancer of P2X3 and P2X2/3 receptor activation, with BzATP (30 and 100 nmol paw−1) into the rat hindpaw produced significantly greater nociception as compared to the algogenic effects of BzATP alone. Intradermal co‐administration of cibacron blue (30 and 100 nmol paw−1) with formalin (1 and 2.5%) into the rat hindpaw also produced significantly greater nociceptive behaviour as compared to formalin alone. The ability of TNP‐ATP and cibacron blue to respectively attenuate and enhance nociceptive responses elicited by exogenous BzATP and formalin provide further support for the hypothesis that activation of peripheral P2X3 containing channels contributes specifically to both acute and persistent nociception in the rat.

Serotonergic modulation of bladder afferent pathways

Normal bladder function is based on activation and maintenance of a sophisticated reflex mechanism involving sympathetic, parasympathetic, and somatic control of the lower urinary tract. The spinal and supraspinal neuronal pathways involved can be modulated by activation or inhibition of neurons in the periphery, at the lumbosacral and thoracolumbar spinal levels, and at supraspinal regulatory sites. Activation of the primary afferent neurons that innervate the lower urinary tract is the first step on this reflex pathway. Under conditions in which bladder function is compromised, abnormal activity in these afferent neurons can induce changes in these circuits, resulting in bladder dysfunction. Control and modulation of afferent pathways is a recent focus for the development of novel treatments for lower urinary tract disorders. This review focuses on the central regulation of bladder function by central serotonergic modulation of sensory pathways. Modulation of this monoaminergic system has dramatic effects on bladder activity and can be a target for pharmacologic treatment of bladder disorders.

2', 3'-O-(2,4,6,trinitrophenyl)-ATP and A-317491 are competitive antagonists at a slowly desensitizing chimeric human P2X3 receptor.

Rapid desensitization of ligand‐gated ion channel receptors can alter the apparent activity of receptor modulators, as well as make detection of fast‐channel activation difficult. Investigation of the antagonist pharmacology of ATP‐sensitive homomeric P2X3 receptors is limited by agonist‐evoked fast‐desensitization kinetics. In the present studies, chimeric receptors were created using the coding sequence for the N‐terminus and the first transmembrane domain of either the nondesensitizing human P2X2a or fast‐desensitizing P2X3 receptor joined to the sequence encoding the extracellular loop, second transmembrane domain, and C‐terminus of the other receptor (designated P2X2–3 and P2X3–2, respectively). These clones were stably transfected into 1321N1 astrocytoma cells for biophysical and pharmacological experiments using both electrophysiological and calcium‐imaging methods. Chimeric P2X2–3 and P2X3–2 receptors were inwardly rectifying and agonist responses showed desensitization properties similar to the wild‐type human P2X2a and P2X3 receptors, respectively. The P2X2–3 chimera displayed an agonist pharmacological profile similar to the P2X3 wild‐type receptor being activated by low concentrations of both ATP and α,β‐meATP. In contrast, the P2X3–2 chimera had markedly reduced sensitivity to both agonists. The P2X3 receptor antagonists TNP‐ATP and A‐317491 were shown to be potent, competitive antagonists of the P2X2–3 chimera (Ki=2.2 and 52.1 nM, respectively), supporting the hypothesis that rapid receptor desensitization can mask the competitive antagonism of wild‐type homomeric P2X3 receptors.

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.

Properties of urethral rhabdosphincter motoneurons and their regulation by noradrenaline

The urethral rhabdosphincter (URS), commonly known as the external urethral sphincter, facilitates urinary continence by constricting the urethra. Striated muscle fibres in the urethral rhabdosphincter are innervated by Onufs nuclei motoneurons in the spinal cord. Although noradrenaline (NA) reuptake inhibitors are shown to increase URS tone preventing urinary leakage in incontinent patients, whether or how NA affects URS motoneurons is unknown. Properties of dye‐labelled URS motoneurons were investigated by whole‐cell patch‐clamp recordings in isolated spinal cord slices prepared from neonatal female rats. As previously shown for adult sphincter motoneurons, neonatal URS motoneurons are more depolarized and possess higher input resistance than other spinal α‐motoneurons. These distinct properties make URS motoneurons more excitable than other α‐motoneurons. Moreover, bath application of noradrenaline (NA) significantly depolarizes URS motoneurons and in many cases evokes action potentials. NA also significantly increases input resistance and reduces rheobase. These changes are reversed with wash, are largely blocked by the α1‐adrenoceptor‐selective antagonist prazosin, and are mimicked by the α1‐adrenoceptor‐selective agonist phenylephrine. In addition, NA significantly reduces the amplitude of the afterhyperpolarization and increases action potential frequency. Both the increase in action potential frequency and the reduction in afterhyperpolarization are occluded by apamin, a small‐conductance calcium‐activated potassium (SKCa) channel blocker. In conclusion, NA effectively increases the excitability of URS motoneurons through multiple mechanisms. The NA‐induced increase in excitability of urethral rhabdosphincter motoneurons could be a key mechanism by which NA reuptake inhibitors improve stress urinary incontinence.

Antagonism of P2X3-containing channels: commentary on Spelta et al.

The ATP-activated P2X3-containing receptors (homomeric P2X3 and heteromeric P2X2/3) have been implicated in the processing of nociceptive information from evidence that these receptors are highly localized on small diameter primary sensory afferent neurons (nociceptors), that their expression is altered in pain states, and that pharmacological blockade reduces nociception in animal models (see references in Spelta et al., 2002; Jarvis & Kowaluk, 2001). The lack of truly potent and receptor-selective antagonists has hampered the pharmacological evaluation of P2X3-containing receptors. Early efforts to develop structure activity relationships (SAR) for P2X receptor antagonists have focused on micromolar affinity nonselective compounds like suramin and pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (Kim et al., 2001). As the present data by Spelta et al. (2002) clearly demonstrate, these two antagonists, as well as the nucleotide containing P2X receptor antagonist, trinitrophenyl-ATP (TNP-ATP), block P2X3-containing receptors with very different kinetic properties and affinities. But what determines the affinity of an antagonist? In the simplest sense, the Law of Mass Action states that affinity is defined by the dissociation constant (KD), or the ratio of dissociation/association rate constants. A common misconception among many biologists is that antagonist association rates are often relatively similar, and that the major determinant of affinity is the dissociation rate of the antagonist. After all, if an antagonist stays on the receptor longer, should it not display a higher affinity for that receptor? Studies with specific antagonists for a number of ligand-gated ion channels have suggested either that the dissociation rate is a primary determinant (Jones et al., 2001), or that a combination of association and dissociation rates (Benveniste & Mayer, 1991; Wenningmann & Dilger, 2001) governs antagonist affinity. In the present study by Spelta et al. (2002) fast drug application techniques were used to measure real-time antagonist kinetics at P2X receptors. By comparing the association and dissociation rates of three antagonists that display different affinities for P2X2/3 receptors, they have concluded that the antagonist association rate is a critical component of P2X receptor antagonist affinity. In this study, TNP – ATP shows >100 fold higher affinity for the P2X2/3 receptor than does suramin. The authors offer evidence that although the TNP – ATP dissociation rate is slightly slower, it is the 50 fold faster association rate that determines the increased affinity of TNP – ATP for the receptor. Likewise, although PPADS is shown to have an extremely slow dissociation rate, it exhibits only moderate affinity due to a correspondingly slow association rate. From these data, it is clear that antagonist association rates must be considered when addressing questions of relative antagonist affinity. What are the practical implications of association rate differences between antagonists? One obvious consideration should be careful control of antagonist pre-incubation times in in vitro experiments. For a slowly associating antagonist, a pre-application of several minutes may be required to achieve steady-state binding interactions, as previously shown for NF279, a suramin analogue P2X1 receptor antagonist (Rettinger et al., 2000). The work of Spelta et al. (2002) also illustrates the necessity for caution in the interpretation of agonist/antagonist interactions at the P2X2/3 receptor. Using a bicistronic expression system, these authors obtained an approximate 50 : 50 ratio of rat P2X2/3 to P2X2 receptor expression that complicated any Schild analysis of receptor antagonism since the P2X3 receptor-selective agonist α,β-meATP activates homomeric P2X2 receptors at high ⩾100 μM concentrations. Thus, for the most potent antagonist, TNP – ATP, these investigators note that P2X2/3 receptor block was consistent with competitive antagonism, yet a Schild analysis for P2X2/3 receptor block was not feasible. While not conclusively demonstrated, this interpretation is in agreement with our earlier work that characterized the apparent competitiveness of TNP – ATP to potently block human P2X2/3 receptors (Burgard et al., 2000). The competitive nature of TNP – ATP block of human P2X2/3 receptors has been recently supported using a non-desensitizing chimeric human P2X2/P2X3 receptor that showed P2X3-like pharmacology, but P2X2-like desensitization (Uchic et al., 2001). Spelta et al. (2002) have also demonstrated kinetic differences between the traditional lower affinity P2X receptor antagonists, suramin and PPADS. However, like TNP – ATP, the question of competitive versus non-competitive antagonism by these compounds has been debated, due to both low potency and nonspecific block of P2X receptor subtypes (Kim et al., 2001; Jacobson & Knutsen, 2001). Novel high affinity and selective P2X receptor antagonists are clearly needed in this field, and their kinetic profiles will require careful characterization. Although structurally diverse P2X receptor antagonists with enhanced pharmacological selectivity have been slow to emerge and the evolution of P2X receptor SAR is currently a more empirical than rational exercise, Spelta et al. (2002) have made a significant contribution to our understanding of the kinetics of the currently available P2X3 receptor antagonists.

[Lys5,MeLeu9,Nle10]-NKA(4–10) Elicits NK2 Receptor–Mediated Micturition and Defecation, and NK1 Receptor–Mediated Emesis and Hypotension, in Conscious Dogs

Tachykinin neurokinin 2 (NK2) receptor agonists may have potential to alleviate clinical conditions associated with bladder and gastrointestinal underactivity by stimulating contraction of visceral smooth muscle. The ability of [Lys5,MeLeu9,Nle10]-neurokinin A(4–10) (LMN-NKA) to elicit micturition and defecation was examined after repeated administration in groups of 2–10 conscious dogs. Administration of 10–100 μg/kg, i.v., four times daily for six consecutive days, reliably elicited micturition after ≥90% of doses and defecation after ≥50% of doses. Voiding occurred <4 minutes after dosing and was short lasting (<10 minutes). LMN-NKA was well tolerated, with emesis after ∼25% of doses at 100 μg/kg, i.v. Hypotension was induced by 100 μg/kg, i.v., of LMN-NKA but not by lower doses. Administration of 30–300 μg/kg, s.c., twice daily for seven consecutive days, reliably elicited both urination and defecation after 88%–100% of doses, and was accompanied by a high rate of emesis (50%–100%). The onset of voiding was rapid (<7 minutes) but was more prolonged than after intravenous administration (30–60 minutes). Emesis induced by 30 or 300 μg/kg, s.c., of LMN-NKA was significantly reduced (from 58% to 8% and from 96% to 54%, respectively) by a 30-minute pretreatment with the neurokinin 1 (NK1) receptor antagonist, (2S,3S)-N-(2-methoxybenzyl)-2-phenylpiperidin-3-amine (CP-99,994; 1 mg/kg, s.c.). The ability of selective NK2 receptor agonists to elicit on-demand voiding could potentially address a major unmet need in people lacking voluntary control of micturition and/or defecation. LMN-NKA unexpectedly activated NK1 receptors at doses that stimulated voiding, causing emesis and hypotension that may limit the clinical utility of nonselective NK2 receptor agonists.

Affinity, potency, efficacy, and selectivity of neurokinin A analogs at human recombinant NK2 and NK1 receptors

A series of peptide NK2 receptor agonists was evaluated for affinity, potency, efficacy, and selectivity at human recombinant NK2 and NK1 receptors expressed in CHO cells to identify compounds with the greatest separation between NK2 and NK1 receptor agonist activity. Binding studies were performed using displacement of [125I]-NKA binding to NK2 receptors and displacement of [3H]-Septide binding to NK1 receptors expressed in CHO cells. Functional studies examining the increase in intracellular calcium levels and cyclic AMP stimulation were performed using the same cell lines. A correlation was demonstrated between binding affinities (Ki) and potency to increase intracellular calcium (EC50) for NK2 and NK1 receptors. Ranking compounds by their relative affinity (Ki) or potency (EC50) at NK2 or NK1 receptors indicated that the most selective NK2 agonists tested were [Lys5,MeLeu9,Nle10]-NKA(4-10) (NK1/NK2 Ki ratio = 674; NK1/NK2 EC50 ratio = 105) and [Arg5,MeLeu9,Nle10]-NKA(4-10) (NK1/NK2 Ki ratio = 561; NK1/NK2 EC50 ratio = 70). The endogenous peptide, NKA, lacked selectivity with an NK1/NK2 Ki ratio = 20 and NK1/NK2 EC50 ratio = 1. Of the compounds selected for evaluation in cyclic AMP stimulation assays, [β-Ala8]-NKA(4–10) had the greatest selectivity for activation of NK2 over NK1 receptors (NK1/NK2 EC50 ratio = 244), followed by [Lys5,MeLeu9,Nle10]-NKA(4-10) (ratio = 74), and NKA exhibited marginal selectivity (ratio = 2.8).

Chapter 6. Urinary incontinence: Neuropharmacological approaches

Publisher Summary This chapter discusses neuropharmacological approaches to urinary incontinence (UI). UI occurs when the lower urinary tract does not store urine properly and there is an involuntary loss of urine. There are three types of UI: urge, stress, and mixed. Urge is considered to be due to an overactive bladder, while stress UI is considered to be due to decreased urethral outlet resistance. In urge incontinence, the reformulations of oxybutynin and other muscarinic cholinergic receptor antagonists continue to dominate the market and late stage clinical development programs. Enterprising clinicians have taken matters into their own hands and applied off label-use of various toxins as they try to find remedies for their patients, but safe, effective, and convenient therapy is needed to compliment the anticholinergics. The chapter describes the neural reflex pathways that control urine storage and micturition and describes pharmacological targets and drug discovery strategies within the context of these reflexes. It also elaborates the function and dysfunction of the lower urinary tract and discusses the reflex control of the lower urinary tract, along with the sites of drug action for the inhibition of micturition reflexes.