Neil S. Lamarre

A quantitative study to assess synergistic interactions between urotensin II and angiotensin II

Interaction between the vasoactive peptides, urotensin II and angiotensin II, could have important implications in various disease states. We examined this interaction using isolated rat aortic rings with intact adventitia and endothelium. The fixed-ratio combination we tested produced effect levels significantly greater than predicted by additivity. Thus, the interaction was synergistic, and this is illustrated in a response surface plot that shows the predicted additive effect for all possible combinations.

Bladder Reinnervation Using a Primarily Motor Donor Nerve (Femoral Nerve Branches) is Functionally Superior to Using a Primarily Sensory Donor Nerve (Genitofemoral Nerve)

PURPOSE We determined whether transfer of a primarily motor nerve (femoral) to the anterior vesicle branch of the pelvic nerve would allow for more effective bladder reinnervation than transfer of a primarily sensory nerve (genitofemoral). MATERIALS AND METHODS A total of 41 female mongrel dogs underwent bladder decentralization and then bilateral nerve transfer, or served as sham operated or unoperated controls. Decentralization was achieved by bilateral transection of all sacral roots that induced bladder contraction upon electrical stimulation. Retrograde neuronal labeling dye was injected in the bladder 3 weeks before sacrifice. RESULTS Increased detrusor pressure after direct stimulation of the transferred nerve, lumbar spinal cord or spinal root was observed in 12 of 17 dogs with genitofemoral nerve transfer and in 9 of 10 with femoral nerve transfer (mean ± SEM 7.6 ± 1.4 and 11.7 ± 3.1 cm H2O, respectively). Mean detrusor pressure after direct electrical stimulation of transferred femoral nerves was statistically significantly greater than after stimulation of transferred genitofemoral nerves. Retrograde labeled neurons from the bladder observed in upper lumbar cord segments after genitofemoral and femoral nerve transfer confirmed bladder reinnervation, as did labeled axons at the nerve transfer site. CONCLUSIONS While transfer of a mixed sensory and motor nerve (genitofemoral) or a primarily motor nerve (femoral) can reinnervate the bladder, using the primarily motor nerve provided greater return of nerve evoked detrusor contraction. This surgical approach may be useful to achieve bladder emptying in patients with lower motor spinal cord injury.

Cocaine synergism with alpha agonists in rat aorta: Computational analysis reveals an action beyond reuptake inhibition

BACKGROUND Cocaine has long been known to increase blood pressure, but the degree and mechanism of vasoconstricting action remain poorly understood. Here we examine the interaction between cocaine and alpha-adrenoceptor agonists, with the action of reuptake inhibition minimized. METHODS Cocaine was administered to isolated rings of rat thoracic aorta, alone and in combination with three different adrenoceptor agonists: phenylephrine, methoxamine, and norepinephrine. Synergy analysis begins with the predicted additive effect of the combination of two agonists, based upon dose equivalence theory. This case where one agonist (cocaine) has no effect when administered alone requires only a t-test to demonstrate that a departure from additivity has occurred. RESULTS At doses where cocaine alone produced no vasoconstriction, it potentiated the vasoconstriction produced by all three alpha agonists, a clear indication of synergism between cocaine and these agents. Higher doses of cocaine in combination with alpha adrenoceptor agents gave an inverted-U shaped (hormetic) dose-effect curve, i.e., dose-related relaxation at higher doses. The hormetic dose-effect relation was analyzed using computational methodology based on dose equivalence to derive the unknown second component of action that causes relaxation. CONCLUSIONS Cocaine exhibits both vasoconstricting and vasorelaxant effects. This relaxing component, possibly related to activation of myosin light chain phosphatase, was quantified as a dose-effect curve. Most important is the synergism between cocaine and alpha-adrenoceptor stimulation which cannot be explained as an action due to reuptake inhibition, and has not been previously described.

On the quantitation of an agonist with dual but opposing components of action: Application to vascular endothelial relaxation

In this communication we show that the same principle that underlies the use of the isobolograph for assessing agonist interactions also leads to a method for analyzing the opposing effects of a single agonist. This is the principle of dose equivalence whose application is illustrated here and applied to the endothelium-dependent relaxing component of two putative vasoconstrictor peptides. These studies, employing angiotensin II and endothelin-1, were conducted with isolated preparations of rat aorta that were measured for agonist-induced isometric tension development in both endothelial-denuded and -intact vessels. The dose-effect relation of the relaxing component of each agonist, which should not be calculated from simple effect subtraction, was derived by the method described here.

Neuromuscular Nicotinic Receptors Mediate Bladder Contractions Following Bladder Reinnervation with Somatic to Autonomic Nerve Transfer after Decentralization by Spinal Root Transection

PURPOSE We investigated whether the reinnervated neuronal pathway mediates contraction via the same neurotransmitter and receptor mechanisms as the original pathway. MATERIALS AND METHODS After decentralizing the bladder by transecting the sacral roots in dogs we performed peripheral nerve transfer, including bilateral genitofemoral to pelvic nerve transfer and unilateral left femoral nerve to bilateral pelvic nerve transfer. Reinnervation was assessed 7.5 months postoperatively by monitoring bladder pressure during electrical stimulation of the transferred nerves, spinal ventral roots and spinal cord. RESULTS Of the 17 dogs with genitofemoral to pelvic nerve transfer 14 (82%) demonstrated functional bladder reinnervation as evidenced by increased bladder pressure during stimulation of the transferred genitofemoral nerve, or L3 or L4 spinal ventral roots. Lumbar spinal cord stimulation caused increased bladder pressure in 9 of 10 dogs (90%) with unilateral left femoral nerve to bilateral pelvic nerve transfer. Succinylcholine virtually eliminated the bladder pressure increases induced by electrical stimulation of the transferred somatic nerves or of the lumbar spinal segments that contribute axons to these donor nerves. In unoperated or sham operated controls succinylcholine had no effect on nerve evoked bladder pressure increases but it substantially decreased the urethral and anal sphincter pressure induced by stimulating the lumbosacral spinal cord or the S2-S3 spinal ventral roots. The reinnervated detrusor muscles of dogs with genitofemoral to pelvic nerve transfer and unilateral left femoral nerve to bilateral pelvic nerve transfer also showed increased α1 nicotinic receptor subunit immunoreactivity in punctate dots on detrusor muscle fascicles and in neuronal cell bodies. This staining was not observed in controls. CONCLUSIONS Succinylcholine sensitive nicotinic receptors, which normally mediate only skeletal muscle neuromuscular junction neurotransmission, appeared in the new neuronal pathway after genitofemoral to pelvic and unilateral femoral nerve to bilateral pelvic nerve transfer. This suggests end organ neuroplasticity after reinnervation by somatic motor axons.

MP17-08 NEUROGENIC BLADDER DYSFUNCTION IN A MURINE MODEL OF MULTIPLE SCLEROSIS IS CAUSED BY CORONOVIRUS-INDUCED DEMYELINATION OF THE NERVOUS SYSTEM

INTRODUCTION AND OBJECTIVES: Neurogenic bladder dysfunction develops in patients with different neurodegenerative disorders including multiple sclerosis (MS). Lower urinary tract symptoms are reported in the majority of MS patients with the most common complaints of urinary urgency and frequency. The objective of this work was to evaluate neurogenic bladder dysfunction in a murine model of multiple sclerosis induced by a neurotrophic strain A59 of mouse hepatitis virus (MHV-A59). METHODS: Adult mice (C57BL/6J, 8 wks of age, N1⁄484) received single inoculation of MHV-A59 leading to the occurrence of coronavirus-induced encephalomyelitis (CIE). Animals were monitored daily for 4 wks for clinical signs of neurologic impairment. Clinical Symptom Score (CSS, 0-5) was assigned based on the level of tail tonicity, kyphosis and limb paresis/paralysis. Micturition patterns were assessed by filter paper assay and cystometric recordings in unrestrained mice. Contractile responses of the detrusor were evaluated in vitro by tension measurements in response to agonists and electric field stimulation (EFS). RESULTS: Inoculation with MHV-A59 virus induced a significant neural deficit with average CSS of 2.6 0.5 at 2 wks post-inoculation accompanied by 25 5 % of weight loss (p 0.001 to baseline). Histological analysis of spinal cord sections revealed multiple demyelinated lesions. Cystometric recordings confirmed neurogenic bladder overactivity at 4 wks post-inoculation including shortened inter-micturition interval, lower voided volume and elevated number of non-micturition contractions (p 0.05 to control group). In response to EFS, the cholinergic component of contraction was significantly reduced in mucosa intact CIE mouse bladders, while the atropine-resistant (purinergic) component was increased. Removal of the mucosa from CIE mouse bladders increased the cholinergic and decreased the purinergic components. CONCLUSIONS: Our results suggest that coronovirus-induced demyelination of the central nervous system causes the development of neurogenic bladder dysfunction that is similar to detrusor overactivity observed in MS patients. The underlying mechanisms may include an alteration in nerve-evoked contractions mediated by a urothelium dependent suppression of muscarinic and augmentation of purinergic mediated response in the bladder detrusor.

Alterations in nerve-evoked bladder contractions in a coronavirus-induced mouse model of multiple sclerosis.

Background Patients with neurodegenerative diseases such as multiple sclerosis, Parkinson’s, and Alzheimer’s often present with lower urinary tract symptoms (LUTS, urinary frequency, urgency, nocturia and retention) resulting from damage to the peripheral and central nervous systems. These studies were designed to examine the changes in the function of the bladder that may underlie neurogenic bladder dysfunction using a mouse model of demyelination in the CNS. Methods Bladders from 12 week old male C57BL/6J mice with coronavirus-induced encephalomyelitis (CIE, a chronic, progressive demyelinating disease model of human MS), and age-matched controls, were cut into 5–7 strips and suspended in physiological muscle baths for tension measurement in response to agonists and electric field stimulation (EFS). Experiments were performed on intact and denuded (with mucosa removed) bladder strips. Results The maximum effect of EFS was not significantly different between CIE and control bladders. Nerve-evoked EFS contractions (tetrodotoxin-sensitive) were blocked by a combination of atropine (cholinergic antagonist) and α,β-methylene ATP (an ATP analog that desensitizes purinergic receptors). In response to EFS, the α,β-methylene ATP-resistant (cholinergic) component of contraction was significantly reduced, while the atropine-resistant (purinergic) component was significantly increased in CIE bladders. Removal of the mucosa in CIE bladders restored the cholinergic component. Bethanechol (muscarinic receptor agonist) potency was significantly increased in CIE bladders. Conclusions Our data demonstrate a deficit in the nerve-evoked cholinergic component of contraction that is not due to the ability of the smooth muscle to respond to acetylcholine. We conclude that neurodegenerative bladder dysfunction in this model of multiple sclerosis may be due, in part, to pathologic changes in the mucosa that causes suppression of muscarinic receptor-mediated contractile response and augmentation of purinergic response of the underlying muscle. Further studies utilizing CIE mice should help elucidate the pathological changes in the mucosa resulting from demyelination in the CNS.

'Null method' determination of drug biophase concentration.

ABSTRACTPK/PD modeling is enhanced by improvements in the accuracy of its metrics. For PK/PD modeling of drugs and biologics that interact with enzymes or receptors, the equilibrium constant of the interaction can provide critical insight. Methodologies such as radioliogand binding and isolated tissue preparations can provide estimates of the equilibrium constants (as the dissociation constant, K value) for drugs and endogenous ligands that interact with specific enzymes and receptors. However, an impediment to further precision for PK/PD modeling is that it remains a problem to convert the concentration of drug in bulk solution (A) into an estimate of receptor occupation, since A is not necessarily the concentration (C) of drug in the biophase that yields fractional binding from the law of mass action, viz., C/(C + K). In most experimental studies A is much larger than K, so the use of administered instead of biophase concentration gives fractional occupancies very close to unity. We here provide a simple way to obtain an estimate of the factor that converts the total drug concentration into the biophase concentration in isolated tissue preparation. Our approach is an extension of the now classic ‘null method’ introduced and applied by Furchgott to determination of drug-receptor dissociation constants.

Clarification of the Innervation of the Bladder, External Urethral Sphincter and Clitoris: A Neuronal Tracing Study in Female Mongrel Hound Dogs: GENITOURINARY STRUCTURE INNERVATION IN DOGS

Many studies examining the innervation of genitourinary structures focus on either afferent or efferent inputs, or on only one structure of the system. We aimed to clarify innervation of the bladder, external urethral sphincter (EUS) and clitoris. Retrograde dyes were injected into each end organ in female dogs. Spinal cord, mid‐bladder, and spinal, caudal mesenteric, sympathetic trunk and pelvic plexus ganglia were examined for retrograde dye‐labeled neurons. Neurons retrogradely labeled from the bladder were found primarily in L7‐S2 spinal ganglia, spinal cord lateral zona intermedia at S1‐S3 levels, caudal mesenteric ganglia, T11‐L2 and L6‐S2 sympathetic trunk ganglia, and pelvic plexus ganglia. The mid‐bladder wall contained many intramural ganglia neurons labeled anterogradely from the pelvic nerve, and intramural ganglia retrogradely labeled from dye labeling sites surrounding ureteral orifices. Neurons retrogradely labeled from the clitoris were found only in L7 and S1 spinal ganglia, L7‐S3 spinal cord lateral zona intermedia, and S1 sympathetic trunk ganglia, and caudal mesenteric ganglia. Neurons retrogradely labeled from the EUS were found in primarily at S1 and S2 spinal ganglia, spinal cord lamina IX at S1‐S3, caudal mesenteric ganglia, and S1‐S2 sympathetic trunk ganglia. Thus, direct inputs from the spinal cord to each end organ were identified, as well as multisynaptic circuits involving several ganglia, including intramural ganglia in the bladder wall. Knowledge of this complex circuitry of afferent and efferent inputs to genitourinary structures is necessary to understand and treat genitourinary dysfunction. Anat Rec, 2018.

MP42-16 INNERVATION OF GENITOURINARY STRUCTURES: A NEURONAL TRACING STUDY IN FEMALE DOGS

identification of novel pathological regulators in this tissue. Oxidative stress is a fundamental pathological mediator; ROS generating enzyme NADPH oxidase (Nox enzyme) has attracted intense interest recently as it is the only enzyme that produces ROS as its sole function and can be targeted without compromising normal biochemical oxidation. Our recent pilot study provided initial evidence for the presence of such system in bladder urothelium and its potential functional significance. This study aimed firstly to define the importance of urothelial superoxide production in the body and secondly to dissect the enzymatic sources of superoxide production in the bladder. METHODS: C57BL/6J mice were euthanized. Bladder and other types of tissue were isolated. Lucigenin-enhanced chemiluminescence quantified superoxide production in live tissue. Western blot determined Nox subtype expressions. RESULTS: Superoxide production in bladder mucosa (RLU/mg tissue: 536.8 104.8, mean SEM) was many folds as high as those in detrusor muscle (21.8 3.8, n1⁄415, p<0.01), aorta (67.2 26.6, n1⁄47, p<0.05), brain (9.4 1.7, n1⁄46, p<0.01), kidney (84.3 23.0, n1⁄46, p<0.05), ventricle (21.8 3.8, n1⁄46, p<0.01) and liver (80.8 12.9, n1⁄47, p<0.05). NADPH oxidase inhibitor diphenyleneiodonium (DPI, 20mM) reduced superoxide production to 10.8 3.4 % of control (n1⁄46, p<0.01) in bladder mucosa and to 30.8 8.4% of control (n1⁄46, p<0.01) in detrusor. Mitochondria de-coupler FCCP (10mM) suppressed superoxide production to 51.8 10.5 % of control (n1⁄46; p<0.01) in bladder mucosa and to 59.8 10.4 % of control (n1⁄46, p<0.05) in detrusor. Xanthine oxidase inhibitor oxypurinol (100mM) produced no significant effect in bladder mucosa (87.9 17.4 % of control, n1⁄46, p>0.05) but a small inhibition in detrusor (78.0 6.6% of control). Western blot showed specific bands for Nox1, Nox2 and Nox4 but no Nox3 expression in bladder mucosa and detrusor with significantly higher expression in bladder mucosa (p<0.05, n1⁄44-6). CONCLUSIONS: These data demonstrate for the first time that the urothelium is the most active tissue for superoxide production in the body. Nox enzymes are the main enzymatic source for superoxide in bladder. The main Nox subtypes are Nox1, Nox2 and Nox4, mainly located in the urothelium. Exceptionally high levels of Nox-driven superoxide explain why bladder urothelium is prone to oxidative stress, inflammation and sensory dysfunction.