Regular paperNociceptin and the micturition reflex
Introduction
A detailed survey of anatomic, physiological and pharmacological aspects of micturition is obviously beyond the aim of this article, therefore, the reader can retrieve more information about these topics in an appropriate monograph [49]; here we outline some concepts that could be useful for understanding the models used in the studies reviewed and the putative anatomic substrates that mediate the effects of nociceptin on the micturition reflex.
Micturition is regulated by a series of reflexes that act in coordinated sequence to subserve two distinct functional phases of the lower urinary tract (i.e. urinary bladder and urethra): the filling phase and the voiding phase of the micturition cycle. During the filling phase, the bladder accommodates increasing volumes of urine without a consistent increase of intravesical pressure. This process is favored by both the visco-elastic properties of the bladder wall (urothelium, submucosa, and smooth muscle) and the activity of the sympathetic inhibitory motor innervation (acting mainly through cathecolaminergic mechanisms), which inhibits the parasympathetic excitatory motor innervation, the genesis of sensory input (the transduction of bladder wall stretch and possibly of intravesical volume), and directly relaxes smooth muscle cells. The filling phase is also facilitated by the closure of the urethral sphincter that is also driven by sympathetic nerve activity [85]. The voiding phase starts when the intravesical pressure (and consequently the degree of stretching of the bladder wall) reaches to critical threshold values (pressure threshold). During the voiding phase the firing of sympathetic motor nerves is inhibited and replaced by parasympathetic nerve activity that triggers bladder smooth muscle contraction (through the release of acetylcholine and adenosin triphosphate) and the coordinated relaxation of the urethral outlet (that is mainly driven by nitric oxide production) [3], [33].
During the two phases of the micturition, the activity of the lower urinary tract is integrated and regulated at various levels along the neuraxis. In the rat and in most other mammalian species, brain nuclei are essential for the triggering of the micturition reflex. Although suprapontine structures (e.g. cortical, thalamic) can exert either inhibitory or/and excitatory modulation of this reflex, a pontine micturition center (Barrington nucleus), localized to the dorsolateral tegmentum (DLT) in the rat, is the most important nucleus where afferent inputs from the lower urinary tract converge to activate the motor output to the urinary bladder and urethra [17]. Recently, another important micturition center located in the caudal ventrolateral periaqueductal gray matter (PAG) of the midbrain has been also identified in the rat; this area seems to be cephalic extension of the Barrington nucleus [62]. The supraspinal excitatory and inhibitory bladder motor inputs are sent thereafter to preganglionic neurons in the autonomic regions of the lumbosacral spinal cord where they also receive sensory integration from the bladder, urethra, and dermatomes of somatic areas involved in the facilitation of storage and voiding processes [15]. In the rat, sympathetic preganglionic motor nerves (cholinergic) emerge from lumbar (L1-L2) segments of the spinal cord and project to the inferior mesenteric ganglion where cell bodies of cathecolaminergic postganglionic fibers are located: these fibers make up the hypogastric nerve and innervate the bladder (mainly the trigone), the urethra, and the pelvic ganglion. Some preganglionic sympathetic fibers also insert into the preganglionic trunk of the pelvic nerve after having traveled through the sympathetic chain of the paravertebral ganglia. Parasympathetic preganglionic neurons, which are localized to the sacral parasympathetic nuclus (SPN, L6-S1 segments in the rat), send their axons to postganglionic neurons located in the pelvic ganglia. In the rat, neurons of the pelvic ganglia are exclusively located outside the urinary bladder, between the dorsal lobe of the prostate and the colonic wall, whereas in other species (including humans) intramural vesical ganglia are also present. Besides the pelvic and the hypogastric inputs, the urethra receives a direct motor innervation from pudendal somatic motor nerves that arise from Onuf’s nucleus located in the ventral horns of the sacral spinal cord.
The sensory branch of the micturition reflex arising from the bladder travels to the dorsal horns of the spinal cord passing through the pelvic ganglion and dividing thereafter into both pelvic and hypogastric nerves. Sensory fibers associated with the pelvic nerves are apparently more important for conveying afferent stimuli related to the motor activity and nociception arising from the urinary bladder than those associated with the hypogastric nerves [7]. The urethra is also innervated by both pelvic and hypogastric afferents that provide most of motor control; however, high frequency urethral obscillations, which typically conclude the voiding phase in the rat, are directly driven by somatic motoneurons of the Onuf’s nucleus. On the other hand, nociceptive stimuli arising from the urethra are largely transmitted to the central nervous system (CNS) through the sensory branch of the pudendal nerve [43], [63]. Bladder afferent projections from the pelvic nerve enter the spinal cord through the dorsal roots of L6 and S1 dorsal root ganglia and then insert into the Lissauer’s tract and their axon collaterals travel through the lamina I to make up two bundles that medially and laterally surround the dorsal horns. These bundles have been defined as the lateral collateral pathway and the medial collateral pathway: the former bundle (which is the most conspicuous) ends near the SPN, whereas the latter projects to the dorsal gray commissure (where pudendal afferents also converge). Another set of pelvic afferents make up a longitudinal bundle ventral to the central canal [16]. Second order neurons receiving inputs from the bladder have been identified by means of c-fos and pseudorabies virus immunohistochemistry [6], [69]. These neurons, which overlap with bladder central afferent terminals, are localized mainly in the lamina I, into the outer layer of the lamina II, lamina V, SPN and dorsal gray commissure. Therefore, the anatomic arrangement of sensory input from the bladder allows the facilitation of parasympathetic outflow at both spinal cord (spinal reflex, which is not sufficient to trigger the micturition in normal conditions) and supraspinal levels (supraspinal reflex, which is necessary for the physiological bladder voiding in adults).
Several classic low molecular weight neurotransmitters such as acetylcholine, cathecolamines, serotonin, excitatory and inhibitory aminoacids, in addition to neuropeptides are involved in the modulation of the micturition reflex in the CNS [17]. In the peripheral nervous system, a neurotransmitter function is exerted by acetylcholine (through nicotinic receptors) in both sympathetic and parasympathetic ganglia; on the other hand, acetylcholine (through muscarinic receptors) adenosine triphosphate and noradrenaline play a neuroeffector role being the most important mediators of the motor function at the bladder level.
Many neuropeptides can also participate in the modulation of the neurotransmission, the effector function, and sensory discharge of the lower urinary tract. It is worth mentioning that several neuropeptides are contained in sympathetic and parasympathetic (neuropeptide Y, vasoactive intestinal peptide, enkephalin and galanin) motor nerves, whereas some others (substance P, neurokinin A, calcitonin gene-related peptide, and pituitary adenylate cyclase-activating peptide), exclusively expressed in sensory nerves have been therefore defined as “sensory neuropeptides” [42], [51], [56]. Among this latter group, tachykininins such as substance P, neurokinin A, and calcitonin gene-related peptide exert a dual transmitter function: in the spinal cord as classic sensory neurotransmitters (afferent, or sensory function) and in the periphery as neuroeffectors (efferent, or motor function) [47], [50]. These sensory neuropeptides are contained in a subset of primary afferent neurons that are sensitive to the stimulant and to the desensitizing/neurotoxic effects of capsaicin.
Capsaicin, the pungent component of red pepper, has been instrumental in the discovery of those sensory neurons that exert the dual, afferent and motor, function [57]. Capsaicin acutely excites sensory neurons expressing the vanilloid receptor type 1, a recently cloned capsaicin receptor [10], by inducing Na+ and Ca2+ ions entry; this determines the generation of an orthodromic action potential (driven by Na+ ions) and the triggering of an antidromic neurosecretion (driven by Ca2+ ions). If the sensory neuron is exposed to large doses of capsaicin for a relatively long time, the increase of the intracellular concentration of calcium is so large that it exceeds the buffering capacities of the neuron, leading to neurotoxic effects (i.e. degeneration of peripheral nerve terminals and, eventually of the soma of sensory neurons). An additional mechanism accounting for the capsaicin-induced modification of physiological properties of sensory neurons involves an altered regulation of phenotype of the neuron: this phenomenon has been termed messenger plasticity [23].
Capsaicin-sensitive primary afferent neurons (CSPANs) represent an important fraction (about 50%) of the total number of bladder sensory neurons and, accordingly, the spinal projections of bladder CSPANs widely overlap the distribution of dye-labeled bladder sensory neurons [37]. Bladder CSPANs have been implicated in both the physiology and the pathophysiology of the micturition reflex [42]. In fact, although the micturition reflex is fundamentally resistant to the capsaicin pretreatment (at least when this is carried out on adult rats), some urodynamic alterations (i.e. increased bladder capacity and pressure threshold for triggering the micturition) are evident under urethane anesthesia in capsaicin-pretreated rats [59], [75]. On the other hand, the acute application of capsaicin onto the serosal or mucosal side of the urinary bladder induces an increased the micturition frequency during cystometries thus mimicking the urodynamic picture of cystitis [60]. It has been suggested that the mechanisms through which capsaicin induces bladder hyperactivity are important from a pathophysiological point of view, because bladder hyperactivity accompanying some model of cystitis, is prevented or reduced by pretreating the animals with large doses of capsaicin [42].
Nociceptin or orphanin FQ, a 17 aminoacid neuropeptide, has been recently identified as the endogenous ligand of opioid-like receptors-1 (ORL1), a G-protein-coupled receptor sharing sequence analogies with mu, delta, and kappa opioid receptors but displaying very low affinity for classic opioid ligands [65], [70]. The expression of the nociceptin gene is abundant in the CNS but scarce in the periphery where its distribution seems to be restricted to ovary and immune cells [66]. ORL1 receptor mRNA has been localized in several peripheral organs such as intestine, skeletal muscle, vas deferens, and spleen [86]. In the CNS, nociceptin messenger RNA, nociceptin-like immunoreactivity and nociceptin-stimulated [35S]GTPγS binding (an index of the presence of ORL1-coupled G protein receptors) have been identified in brain (DLT and PAG) and spinal cord (SPN), areas directly involved in the control of the micturition reflex [8], [31], [68], [77], [79]. Therefore, it was our interest to review the evidence collected for the interaction occurring between nociceptin and the micturition reflex, with special emphasis on the effect of nociceptin on bladder CSPANs.
Two different models of intravesical pressure recordings have been used to study the effect of nociceptin on the micturition reflex in urethane-anesthetized rats: 1) transurethral, i.e. bladder motility was recorded in isovolumetric conditions (ureters were tied) through a cannula positioned into the proximal urethra [54]; and 2) transvesical, i.e. continuos cystometries were performed through a double lumen catheter (one lumen was used for the intravesical infusion of saline, the other one for intravesical pressure recording) inserted through the bladder dome, suitable to reproduce multiple filling-voiding cycles Fig. 1, Fig. 2 [55]. Both models were studied in two different experimental conditions. The transurethral model was performed with the bladder filled with 1) saline volumes (0.8–1.6 ml) over the threshold for the activation of distension-induced reflex (>15 mmHg) bladder contractions; 2) subthreshold volumes of saline (0.1–0.5 ml), so that the bladder is quiescent, but the reflex can be activated by stimuli that excite bladder afferents (chemonociceptive micturition reflex). In the transvesical model, intravesical saline infusion can be performed at: 1) low, physiological-like filling rate (slow-infusion cystometry, 50 μl/min), or 2) high filling rate (fast-infusion cystometry 250 μl/min). These latter experimental conditions differ from each other because of their differential susceptibility to capsaicin pretreatment (50 mg/kg, subcutaneously (s.c.), 5–7 days before); in capsaicin-pretreated rats the micturition frequency is decreased and the pressure threshold for the activation of micturition reflex is increased in the slow-infusion cystometry but not in the fast-infusion cystometry, with respect to controls [59]. Instead, the amplitude of micturition contractions is not modified by capsaicin pretreatment in any model; these results give experimental support to the intuitive concept that the mean amplitude of reflex bladder contractions is strictly linked to the activity of the efferent branch (capsaicin-resistant) of the micturition reflex, whereas micturition frequency and pressure threshold are urodynamic parameters related to its sensory branch (capsaicin-sensitive and -resistant) [75]. Therefore, the micturition reflex in the fast-infusion cystometry is completely mediated by capsaicin-resistant afferent pathways.
Section snippets
Inhibitory effects
The i.v. administration of nociceptin (3–100 nmol/kg) in urethane-anesthetized rats produced a naloxone-resistant suppression of distension-induced reflex contractions (transurethral model) and a concomitant drop in arterial blood pressure and heart rate; both the duration of distension-induced reflex contractions suppression and the cardiovascular changes were dose-dependent. The minimal effective dose for both cardiovascular and urinary effects was 10 nmol/kg [27], [29]. At the highest dose
Supraspinal sites
At the supraspinal level, nociceptin seems to be involved in many CNS functions, including the regulation of the autonomic nervous system [39], [13], [64], [68]. Among the effects that could indirectly affect the micturition reflex, it has been reported that the injection of nociceptin in the ventromedial nucleus of the hypothalamus facilitates sexual behavior in female rats [80], and it is known that sexual activity may have inhibitory influences on the micturition reflex. Another indirect
Therapeutical perspectives
It is obvious that the definition of the physiological role of nociceptin in the regulation of the micturition reflex awaits a potent, selective, and silent (devoid of intrinsic agonist activity) ORL1 receptor antagonist. So far, only the effect of [F/G]nociceptin(1–13)NH2 (30–300 nmol/kg, i.v.) has been tested on distension-induced reflex contractions; although the antagonist significantly inhibited nociceptin-induced cardiovascular changes [6], it inhibited distension-induced reflex
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