Neuromodulation in a rat model of the bladder micturition reflex

A rat model of bladder reflex contraction (BRC) was used to determine the optimal frequency and intensity of spinal nerve (SN) stimulation to produce neuromodulation of bladder activity and to assess the therapeutic mechanisms of this neuromodulation. In anesthetized female rats (urethane 1.2 g/kg ip), a wire electrode was used to produce bilateral stimulation of the L6 SN. A cannula was placed into the bladder via the urethra, and the urethra was ligated to ensure an isovolumetric bladder. Saline infusion induced BRC. Electrical stimulation of the SN produced a frequency- and intensity-dependent attenuation of the frequency of bladder contractions. Ten-herz stimulation produced maximal inhibition; lower and higher stimulation frequency produced less attenuation of BRC. Attenuation of bladder contraction frequency was directly proportional to the current intensity. At 10 Hz, stimulation using motor threshold pulses (T(mot)) produced a delayed inhibition of the frequency of bladder contractions to 34 ± 11% of control. Maximal bladder inhibition appeared at 10 min poststimulation. High current intensity at 0.6 mA (～6 * T(mot)) abolished bladder contraction during stimulation, and the inhibition was sustained for 10 min poststimulation (prolonged inhibition). Furthermore, in rats pretreated with capsaicin (125 mg/kg sc), stimulation produced a stronger inhibition of BRC. The inhibitory effects on bladder contraction may be mediated by both afferent and efferent mechanisms. Lower intensities of stimulation may activate large, fast-conducting fibers and actions through the afferent limb of the micturition reflex arc in SN neuromodulation. Higher intensities may additionally act through the efferent limb.     

Activation and inhibition of the micturition reflex by penile afferents in the cat

Coordination of the urinary bladder and the external urethral sphincter is controlled by descending projections from the pons and is also subject to modulation by segmental afferents. We quantified the effects on the micturition reflex of sensory inputs from genital afferents traveling in the penile component of the somatic pudendal nerve by electrical stimulation of the dorsal nerve of the penis (DNP) in alpha-chloralose anesthetized male cats. Depending on the frequency of stimulation (range, 1-40 Hz), activation of penile afferents either inhibited contractions of the bladder and promoted urine storage or activated the bladder and produced micturition. Stimulation of the DNP at 5-10 Hz inhibited distension-evoked contractions and increased the maximum bladder capacity before incontinence. Conversely, stimulation at 33 and 40 Hz augmented distension-evoked contractions. When the bladder was filled above a threshold volume (70% of the volume necessary for distension-evoked contractions), stimulation at 20-40 Hz activated de novo the micturition reflex and elicited detrusor contractions that increased voiding efficiency compared with distension-evoked voiding. Electrical stimulation of the DNP with a cuff electrode or percutaneous wire electrode produced similar results. The ability to evoke detrusor contractions by activation of the DNP was preserved following acute spinal cord transection. These results demonstrate a clear role of genital afferents in modulating the micturition reflex and suggest the DNP as a potential target for functional restoration of bladder control using electrical stimulation.     

Tachykinins as modulators of the micturition reflex in the central and peripheral nervous system

In the normal urinary bladder, tachykinins (TKs) are expressed in a population of bladder nociceptors that is sensitive to the excitatory and desensitizing effects of capsaicin (i.e., capsaicin-sensitive primary afferent neurons (CSPANs)). Several endobiotics or xenobiotics excite CSPANs and release TKs and other mediators at both the peripheral and spinal cord level. The peripheral release of TKs determines a set of responses (known as neurogenic inflammation) that includes vasodilatation, plasma protein extravasation, smooth muscle contraction and stimulation of afferent nerves. Following chronic inflammation, both immune cells and capsaicin-resistant sensory neurons can de novo express TKs: whether these pools of TKs are releasable and contribute to inflammatory processes is presently unsettled. At the spinal cord level, the release of TKs contributes in determining an altered pattern of vesicourethral reflexes in response to nociceptive stimulation of the bladder by conveying: (a) the afferent transmission to supraspinal sites, and (b) descending or sensory inputs to the sacral parasympathetic nucleus (SPN). Recent evidence also attribute a synergetic role of TKs in the supraspinal modulation of the sensory arm of the micturition reflex.The overall available information suggests that TK receptor antagonists may affect bladder motility/reflexes which occur during different pathological states, while having little influence on the normal motor bladder function.     

Spinal mechanism of micturition reflex inhibition by naftopidil in rats

Aim

We investigated the spinal mechanism through which naftopidil inhibits the micturition reflex by comparing the effects of noradrenaline and naftopidil in rats.

Methods

The following were investigated: the influence of oral naftopidil on plasma monoamine and amino acid levels, the distribution of oral 14C-naftopidil, the effects of intravenous (IV) or intrathecal (IT) injection of noradrenaline or naftopidil on isovolumetric bladder contractions, amino acid levels in the lumbosacral spinal cord after IT noradrenaline or naftopidil, and the effects of IT naftopidil and strychnine and/or bicuculline on isovolumetric bladder contractions.

Key findings

Oral naftopidil decreased the plasma adrenaline level, while it increased the serotonin and glycine levels. After oral administration, 14C-naftopidil was detected in the spinal cord and cerebrum, as well as in plasma and the prostate gland. When the bladder volume was below the threshold for isovolumetric reflex contractions, IV (0.1 mg) or IT (0.1 μg) noradrenaline evoked bladder contractions, but IV (1 mg) or IT (0.01–1 μg) naftopidil did not. When the bladder volume was above the threshold for isovolumetric reflex contractions, IV or IT noradrenaline transiently abolished bladder contractions. IT noradrenaline decreased the levels of glycine and gamma-aminobutyric acid (GABA) in the lumbosacral cord, while IT naftopidil increased the GABA level. IT strychnine and/or bicuculline blocked the inhibitory effect of IT naftopidil on bladder contractions.

Significance

Naftopidil inhibits the micturition reflex by blocking α1 receptors, as well as by the activation of serotonergic, glycinergic, and GABAergic neurons in the central nervous system.     

Changes in galanin immunoreactivity in rat micturition reflex pathways after cyclophosphamide-induced cystitis

Alterations in the expression of the neuropeptide, galanin, were examined in micturition reflex pathways of rat after cyclophosphamide (CYP)-induced cystitis of variable duration: acute (4 h), intermediate (48 h), or chronic (10 days). In control animals, galanin expression was present in specific regions of the gray matter in the rostral lumbar and caudal lumbosacral spinal cord, including: (1) the dorsal commissure (DCM); (2) superficial dorsal horn; (3) the regions of the intermediolateral cell column (L1–L2) and the sacral parasympathetic nucleus (SPN, L6–S1); and (4) the lateral collateral pathway (LCP) in lumbosacral spinal segments. Densitometry analysis demonstrated significant decreases (P≤0.01) in galanin immunoreactivity (IR) in these regions of the L1–S1 spinal cord after acute or intermediate CYP-induced cystitis. In contrast, increases (P≤0.01) in galanin–IR were observed in the DCM, SPN, or LCP regions in the L6–S1 spinal segments in rats with chronic cystitis. No changes in the number of galanin–immunoreactive cells were observed in the L1–S1 dorsal root ganglia (DRG) after CYP-induced cystitis of any duration. A small percentage of bladder afferent cells (Fast-blue-labeled) in the DRG expressed galanin–IR in control rats; this was not altered with cystitis. Galanin–IR was observed encircling DRG cells after chronic cystitis. These changes may contribute to urinary bladder dysfunction, altered sensation, and referred somatic hyperalgesia after cystitis.This work was supported in part through NIH grants DK051369, DK060481, DK065989, and NS040796.     

Nociceptin in rVLM mediates electroacupuncture inhibition of cardiovascular reflex excitatory response in rats.

Electroacupuncture (EA) at Neiguan-Jianshi acupoints through an opioid mechanism inhibits the cardiovascular pressor response induced by mechanical stimulation of the stomach. Because nociceptin also may regulate cardiovascular activity through its action in the brain stem, we hypothesized that this neuromodulator serves a role in the EA-related inhibitory effect. Blood pressure in ventilated male Sprague-Dawley rats (400-600 g) anesthetized by ketamine and alpha-chloralose was measured during balloon inflation of the stomach. Gastric distension with 6-8 ml of air induced consistent pressor reflexes of 26 +/- 1 mmHg that could be repeated every 10 min for 100 min. When nociceptin (10 nM) was microinjected into the rostral ventrolateral medulla (rVLM), the pressor response induced by gastric distension was inhibited by 68 +/- 6%. Thirty minutes of EA also decreased the reflex response by 75 +/- 11%; microinjection of saline into the rVLM did not alter the inhibitory effect of EA. In contrast, microinjection of a nociceptin receptor antagonist into the rVLM promptly reversed the EA response. Pretreatment with the opioid receptor antagonist naloxone did not influence the EA-like inhibitory effect of nociceptin on the distension-induced pressor reflex (22 +/- 1 to 8 +/- 2 mmHg). Furthermore, a mu-opioid receptor agonist microinjected into the rVLM after microinjection of a nociceptin receptor antagonist during EA promptly reversed the nociceptin receptor antagonist-related inhibition of the EA effect. Thus, in addition to the classical opioid system, nociceptin, through opioid receptor-like-1 receptor stimulation in the rVLM, participates in the modulatory influence of EA on reflex-induced increases in blood pressure.     

Evidence for the involvement of a capsaicin-sensitive innervation in the afferent branch of micturition reflex in rats

Functional evidence is presented indicating the presence in the rat urinary bladder of a capsaicin-sensitive innervation which is involved in regulating micturition threshold. Endogenous substance P and/or related tachykinins appear to be present in the capsaicin-sensitive nerve endings of the rat bladder and may play a neurotransmitter role in relaying information concerned with bladder volume from the detrusor muscle to the central nervous system. In addition, neurotransmitter release from the peripheral ending of the capsaicin-sensitive fibers may play an efferent role in certain motor and/vascular responses at bladder muscle level.     

Nociceptin and neurotransmitter release in the periphery

Nociceptin exerts a general modulatory effect on transmitter release from sympathetic, parasympathetic, NANC and sensory nerve endings in the peripheral nervous system in various species. This effect occurs at a prejunctional level and is independent from the activation of mu, delta and kappa opioid receptors. Despite the growing evidence describing the peripheral activity of nociceptin since its discovery in 1995, the lack of selective and potent antagonists does not allow us to draw conclusions on the putative physiological role of this peptide at this level.     

Solution Conformation of Nociceptin

Nociceptin, a novel heptadecapeptide, interacts with ORL₁a G protein-coupled receptor whose sequence is closely related to that of the κ opioid receptor but has no opioid activity. We have investigated the conformational preferences of Nociceptin also in comparison to Dynorphin A. The N-terminal part of Nociceptin has the same conformational preferences of the message of endogenous opioids but the C-terminal part of the sequence is more flexible than the corresponding address of Dynorphin A. [Tyr¹]-Nociceptin, while retaining nociceptive activity, has also an opioid activity comparable to that of enkephalins.     

Sprouting of substance P-expressing primary afferent central terminals and spinal micturition reflex NK1 receptor dependence after spinal cord injury

The primary afferent neurotransmitter triggering the spinal micturition reflex after complete spinal cord injury (SCI) in the rat is unknown. Substance P detected immunohistochemically in the sacral parasympathetic nucleus was significantly higher in 12 SCI rats than in 12 spinally intact rats (P = 0.008), suggesting substance P as a plausible candidate for the primary afferent neurotransmitter. The effects of the tachykinin NK1 receptor antagonist L-733060 on the spinal micturition reflex were then determined by performing conscious cystometry in an additional 14 intact rats and 14 SCI rats with L-733060 (0.1-100 microg) administered intrathecally at L6-S1. L-733060 was without effect in intact rats, but blocked the spinal micturition reflex in 10 of 14 SCI rats and increased the intermicturition interval in 2 of 4 others at doses ranging from 10 to 100 microg. Both phasic and nonphasic voiding contractions, differentiated according to the presence of phasic external urethral sphincter (EUS) activity, were present in most SCI rats. Both types of contractions were blocked by high doses of L-733060. Interestingly, there was a relative decline in phasic voiding contractions at high doses as well as a decline in contraction amplitude in nonphasic voiding contractions. In other respects, cystometric variables were largely unaffected in either spinally intact or SCI rats. L-733060 did not affect tonic EUS activity at any dose except when the spinal micturition reflex was blocked and tonic activity was consequently lost. These experiments show that tachykinin action at spinal NK1 receptors plays a major role in the spinal micturition reflex in SCI rats.     

Nociceptin Induces Hypophagia in the Perifornical and Lateral Hypothalamic Area

Nociceptin/orphanin FQ (N/OFQ) is known to induce food intake when administered into the lateral ventricle or certain brain areas. This is somewhat contradictory to its reward-suppressing role, as food is a strong rewarding stimulus. This discrepancy may be due to the functional diversity of N/OFQ’s target brain areas. N/OFQ has been shown to inhibit orexin and melanin-concentrating hormone (MCH) neurons, both of which are appetite-inducing cells. As the expression of these neurons is largely confined to the lateral hypothalamus/perifornical area (LH/PFA), we hypothesized that N/OFQ inhibits food intake by acting in this area. To test this hypothesis, we examined the effect of local N/OFQ infusion within the LH/PFA on food intake in the rat and found that N/OFQ decreased sugar pellet as well as chow intake. This effect was not seen when the injection site was outside of the LH/PFA, suggesting a site-specific effect. Next, to determine a possible cellular mechanism of N/OFQ action on food intake, whole cell patch clamp recordings were performed on rat orexin neurons. As previously reported in mice, N/OFQ induced a strong and long lasting hyperpolarization. Pharmacological study indicated that N/OFQ directly inhibited orexin neurons by activating ATP-sensitive potassium (KATP) channels. This effect was partially but significantly attenuated by the inhibitors of PI3K, PKC and PKA, suggesting that the N/OFQ signaling is mediated by these protein kinases. In summary, our results demonstrate a KATP channel-dependent N/OFQ signaling and that N/OFQ is a site-specific anorexic peptide.     

Nociceptin/orphanin FQ receptor ligands

Nociceptin (NC), alias Orphanin FQ (OFQ) is a heptadecapeptide structurally related to opioid peptides, especially Dynorphin A, which, however, does not interact with classic opioid receptors. NC selectively activates its own receptor (OP(4)), which has been shown to be insensitive to the naturally occurring opioid peptides as well as to a large number of non-peptide opioid receptor ligands, including naloxone. Thus, the NC/OP(4) system represents a new peptide-based signaling pathway, which is pharmacologically distinct from the opioid systems. The pharmacological tools available for investigating NC actions are at present rather limited and include: 1) peptide ligands obtained from structure activity studies performed using NC(1-13)NH(2) as a template or discovered by screening peptide combinatorial libraries; 2) nonpeptide ligands that are either molecules already known to interact with classic opioid receptors or novel molecules designed and synthesized as selective ligands of the OP(4) receptor. In the present paper the functional data obtained from both in vitro and in vivo studies with each relevant OP(4) receptor ligand will be analyzed and discussed comparing the advantages and disadvantages of each molecule. We hope that the present work will aid investigators, working in the NC/OP(4) field, in the choice of the pharmacological tools suitable for their experiments.     

Nociceptin stimulates thyrotropin secretion in rats

Effects of nociceptin on thyrotropin (TSH) and thyrotropin-releasing hormone (TRH) secretion in rats were studied. Nociceptin (150 microgram/kg) was injected intravenously and rats were serially decapitated after the injection. The effects of nociceptin on TRH release from the hypothalamus and TSH release from the anterior pituitary in vitro were also investigated. TRH and thyroid hormones were measured by individual radioimmunoassays. TSH was determined by enzyme immunoassay. TRH contents in the hypothalamus decreased significantly after nociceptin injection, whereas plasma TRH concentrations showed no changes. Plasma TSH concentrations increased significantly in a dose-related manner. The TRH release from the hypothalamus was enhanced significantly in a dose-related manner with the addition of nociceptin. The TSH release from the anterior pituitary in vitro was not affected by the addition of nociceptin. The plasma thyroxine and 3,3',5-triiodothyronine levels did not change significantly after nociceptin administration. The inactivation of TRH by plasma or hypothalamus in vitro after nociceptin injection did not differ from that of controls. The findings suggest that nociceptin acts on the hypothalamus to stimulate TRH and TSH secretion.     

Nociceptin/orphanin FQ and drugs of abuse

Nociceptin/orphanin FQ (NC) binds with high affinity to the opioid receptor-like1 (ORL1) receptor. NC has been reported to block opioid-induced supraspinal analgesia, and it has been proposed that it may represent a functional antiopioid peptide in the control of brain nociceptive processes. The wide distribution of NC and of its receptors in the central nervous system suggests, however, that it may be involved in the control of a variety of biologic functions. Increasing evidence indicates that it may influence the rewarding and reinforcing properties of drugs of abuse. NC has been shown to abolish the rewarding properties of ethanol and morphine in the place conditioning paradigm, to reduce ethanol consumption in alcohol-preferring rats and to inhibit stress-induced alcohol-seeking behavior. These findings suggest that drugs directed at central NC receptors may represent an interesting approach to the treatment of ethanol and opiate abuse.     

Nociceptin/orphanin FQ metabolism and bioactive metabolites

The endogenous ligand for the orphan NOR receptor (earlier named ORL1) was recently discovered. This ligand, nociceptin/orphanin FQ is involved in a number of pharmacological actions in the CNS, including modulation of pain and cognition. However, its specific physiological role remains to be determined. Two major pathways of metabolism have been identified; the action of aminopeptidase(s) that prominently occurs in plasma, and endopeptidase activity that successively generates the N-terminal 1-13 and 1-9 fragments. Both pathways result in fragments that are inactive at the NOR receptor. However, short N-terminal fragments appear to be active in blocking the release of substance P from primary afferent C-fiber terminals in the dorsal spinal cord. The same endopeptidase(s) may also be involved in the fragmentation of dynorphin A since the inhibitor profile is similar. Enzyme activity is upregulated by morphine using either peptide as substrate that may lead to pharmacological interactions.     

Novel Nociceptin Analogues: Synthesis and Biological Activity

Syntheses are described of the nociceptin (1–13) amide [NC(1–13)-NH₂] and of several analogues in which either one or both the phenylalanine residues (positions 1 and 4), the arginine residues (positions 8 and 12) and the alanine residues (positions 7 and 11) have been replaced by N-benzyl-glycine, N-(3-guanidino-propyl)-glycine and β-alanine, respectively. The preparation is also described of NC(1–13)-NH₂ analogues in which either galactose or N-acetyl-galactosamine are β-O-glycosidically linked to Thr⁵ and/or to Ser¹⁰. Preliminary pharmacological experiments on mouse vas deferens preparations showed that Phe⁴, Thr⁵, Ala⁷ and Arg⁸ are crucial residues for OP₄ receptor activation. Manipulation of Phe¹ yielded peptides endowed with antagonist activity but [Nphe¹] NC(1–13)-NH₂ acted as an antagonist still possessing weak agonist activity. Introduction of the βAla residue either in position 7 or 11 of the [Nphe¹] NC(1–13)-NH₂ sequence, abolished any residual agonist activity and [Nphe¹, βAla⁷] NC(1–13)-NH₂ and [Nphe¹, βAla¹¹] NC(1–13)-NH₂ acted as competitive antagonists only. Modification of both Ala⁷ and Ala¹¹ abolished the antagonist activity of [Nphe¹]NC(1–13)-NH₂ probably by hindering receptor binding. Changes at positions 10 and 11 gave analogues still possessing agonist activity. [Ser(βGal)¹⁰] NC(1–13)-NH₂ displayed an activity comparable with that of NC(1–13)-NH₂, [Ser(βGalNAc)¹⁰] NC(1–13)-NH₂ and [βAla¹¹] NC(1–13)-NH₂ were five and 10 times less active, respectively.The α-amino acid residues are of the l-configuration. Standard abbreviations for amino acid derivatives and peptides are according to the suggestions of the IUPAC-IUB Commission on Biochemical Nomeclature (1984), Eur. J. Biochem. 138, 9–37. Abbreviations listed in the guide published in (2003), J. Peptide Sci. 9, 1–8 are used without explanation.