Anna P. Malykhina

Neural mechanisms of pelvic organ cross-sensitization

Clinical observations of viscerovisceral referred pain in patients with gastrointestinal and genitourinary disorders suggest an overlap of neurohumoral mechanisms underlying both bowel and urinary bladder dysfunctions. Close proximity of visceral organs within the abdominal cavity complicates identification of the exact source of chronic pelvic pain, where it originates, and how it relocates with time. Cross-sensitization among pelvic structures may contribute to chronic pelvic pain of unknown etiology and involves convergent neural pathways of noxious stimulus transmission from two or more organs. Convergence of sensory information from discrete pelvic structures occurs at different levels of nervous system hierarchy including dorsal root ganglia, the spinal cord and the brain. The cell bodies of sensory neurons projecting to the colon, urinary bladder and male/female reproductive organs express a wide range of membrane receptors and synthesize many neurotransmitters and regulatory peptides. These substances are released from nerve terminals following enhanced neuronal excitability and may lead to the occurrence of neurogenic inflammation in the pelvis. Multiple factors including inflammation, nerve injury, ischemia, peripheral hyperalgesia, metabolic disorders and other pathological conditions dramatically alter the function of directly affected pelvic structures as well as organs located next to a damaged domain. Defining precise mechanisms of viscerovisceral cross-sensitization would have implications for the development of effective pharmacological therapies for the treatment of functional disorders with chronic pelvic pain such as irritable bowel syndrome and painful bladder syndrome. The complexity of overlapping neural pathways and possible mechanisms underlying pelvic organ crosstalk are analyzed in this review at both systemic and cellular levels.

Hyperexcitability of convergent colon and bladder dorsal root ganglion neurons after colonic inflammation: mechanism for pelvic organ cross‐talk

Abstract  Clinical studies reveal concomitant occurrence of several gastrointestinal and urologic disorders, including irritable bowel syndrome and interstitial cystitis. The purpose of this study was to determine the mechanisms underlying cross‐organ sensitization at the level of dorsal root ganglion (DRG) after acute and subsided gastrointestinal inflammation. DiI (1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate) and Fast Blue were injected into the distal colon and urinary bladder of male rats, respectively. Convergent DRG neurons were found in L1‐L3 and L6‐S2 ganglia with an average distribution of 14% ± 2%. The resting membrane potential (RMP) of cells isolated from upper lumbar (UL) ganglia was −59.8 ± 2.7 mV, whereas lumbosacral (LS) neurons were more depolarized (RMP = −49.4 ± 2.1 mV, P ≤ 0.05) under control conditions. Acute trinitrobenzene sulfonic acid (TNBS) colitis (3 days) decreased voltage and current thresholds for action potential firing in LS but not UL convergent capsaicin‐sensitive neurons. This effect persisted for 30 days in the absence of overt colonic inflammation. The current threshold for action potential (AP) firing in UL cells was also decreased from 165.0 ± 24.5 pA (control) to 85.0 ± 19.1 pA at 30 days (P ≤ 0.05), indicating increased excitability. The presence of a subpopulation of colon‐bladder convergent DRG neurons and their persistent hyperexcitability after colonic inflammation provides a basis for pelvic organ cross‐sensitization.

Colonic inflammation increases Na+ currents in bladder sensory neurons.

The purpose of this study was to determine whether sensitization of pre-spinal afferents induced by colonic inflammation can affect the physiology of the urinary bladder. Tetrodotoxin-resistant (TTX-R) Na+ currents were examined in bladder sensory neurons after experimental colitis. Cell bodies of bladder dorsal root ganglia were retrogradely labeled and TTX-R Na+ currents were recorded in capsaicin-sensitive neurons. Colitis significantly enhanced response of bladder neurons to capsaicin by ∼60% and the peak amplitude of TTX-R Na+ current by 51%. These results suggest that colonic inflammation leads to increased excitability of nociceptive bladder neurons.

Experimental colitis triggers the release of substance P and calcitonin gene-related peptide in the urinary bladder via TRPV1 signaling pathways.

Clinical data provide evidence of high level of co-morbidity among genitourinary and gastrointestinal disorders characterized by chronic pelvic pain. The objective of this study was to test the hypothesis that colonic inflammation can impact the function of the urinary bladder via activation of TRPV1 signaling pathways followed by alterations in gene and protein expression of substance P (SP) and calcitonin gene-related peptide (CGRP) in sensory neurons and in the bladder. Inflammation was induced by intracolonic instillation of trinitrobenzene sulfonic acid (TNBS, 12.5mg/kg), and desensitization of TRPV1 receptors was evoked by intracolonic resiniferatoxin (RTX, 10(-)(7)M). mRNA and protein concentrations of CGRP and SP were measured at 3, 5 and 30 days. RTX instillation in the colon caused 3-fold up-regulation of SP mRNA in the urinary bladder at day 5 (n=7, p ≤ 0.05) followed by 35-fold increase at day 30 (n=5, p ≤ 0.05). Likewise, TNBS colitis triggered 15.8-fold up-regulation of SP mRNA 1 month after TNBS (n=5, p ≤ 0.05). Desensitization of colonic TRPV1 receptors prior to TNBS abolished SP increase in the urinary bladder. RTX led to 4.3-fold increase of CGRP mRNA at day 5 (n=7, p ≤ 0.05 to control) in the bladder followed by 28-fold increase at day 30 post-RTX (n=4, p ≤ 0.05). Colitis did not alter CGRP concentration during acute phase; however, at day 30 mRNA level was increased by 17.8 ± 6.9-fold (n=5, p ≤ 0.05) in parallel with 4-fold increase in CGRP protein (n=5, p ≤ 0.01) in the detrusor. Protein concentration of CGRP in the spinal cord was diminished by 45-65% (p ≤ 0.05) during colitis. RTX pretreatment did not affect CGRP concentration in the urinary bladder; however, it caused a reduction in CGRP release from lumbosacral DRG neurons during acute phase (3 and 5 days post-TNBS). Our results clearly demonstrate that colonic inflammation triggers the release of pro-inflammatory neuropeptides SP and CGRP in the urinary bladder via activation of TRPV1 signaling mechanisms enunciating the neurogenic nature of pelvic organ cross-sensitization.

The Effects of Sevoflurane and Propofol on Qt Interval and Heterologously Expressed Human Ether-a-go-go Related Gene Currents in xenopus Oocytes

Sevoflurane can induce prolongation of the cardiac QT interval by inhibiting the repolarization phase of the action potential. This may occur as a result of inhibition of the human ether-a-go-go related gene (HERG) channel. To clarify the mechanisms of anesthetics on HERG channels, we monitored the electrocardiogram and measured QT intervals in the guinea pig in the presence of sevoflurane and propofol. Sevoflurane (1%–4%) prolonged QTc dose-dependently (7.5%–21.2%), but propofol did not affect it. Furthermore, HERG channels were expressed in Xenopus oocytes and outward HERG currents were obtained on step depolarization from a holding potential of −70 mV. Repolarization to −70 mV from positive test potentials resulted in large outward tail currents. Sevoflurane (1%–4%), in a dose-dependent manner, inhibited the HERG outward tail currents (9.7%–26.6%), whereas steady-state currents were inhibited only at large concentrations. The time constant of the converging current was decreased in the presence of sevoflurane, but the inactivation and activation curves were not shifted. Propofol did not affect these currents within the clinically relevant concentration. In conclusion, compared with steady-state currents, sevoflurane was more potent in inhibiting the outward tail currents, suggesting that sevoflurane may modulate the HERG channel kinetics in its inactivated state.

Fenamate-induced enhancement of heterologously expressed HERG currents in Xenopus oocytes

The human ether-a-go-go related gene (HERG) product encodes for the pore-forming subunit of the rapid component of the delayed rectifier K(+) channel that mediates repolarization of cardiac action potential. HERG channels are also potential targets of a large variety of pharmacological agents most of which tend to block HERG currents. In this study, we examined the effects of the non-steroidal anti-inflammatory agents, flufenamic acid and niflumic acid, on heterologously expressed HERG channels in oocytes. The cRNA of HERG (30 ng) was injected into Xenopus oocytes and currents were recorded using two-electrode voltage clamp technique in a low Cl(-) solution. Flufenamic and niflumic acids (10(-4)-5 x 10 (-4) M) enhanced the amplitude of outward currents evoked by depolarizing pulses. At potentials positive to 0 mV, an initial transient component was also evident in the presence of fenamates. Fenamates accelerated the activation rate of HERG channels and decelerated their deactivation. Flufenamic acid (5 x 10 (-4) M) shifted the I(tail)-V relationship from -26.7+/-0.1 to -31.4+/-0.2 mV. Neither flufenamic acid or niflumic acid affected the kinetics of HERG channel inactivation. Using a voltage protocol that mimicked the cardiac action potential, both fenamates increased the outward current during the plateau and during the phase 3 repolarization of action potential. The effects of the fenamates were blocked by the HERG channel blocker, E-4031 and were also not observed in water-injected oocytes. Our data suggest that fenamates enhance HERG currents and affect the action potential duration in the heart.

Do the urinary bladder and large bowel interact, in sickness or in health? ICI-RS 2011.

Normal functioning of the urinary bladder and the distal gut is an essential part of daily physiological activity coordinated by the peripheral and central nervous systems. Pathological changes in one of these organs may induce the development of cross‐organ sensitization in the pelvis and underlie clinical co‐morbidity of genitourinary and GI dysfunctions. Experimental human and animal data suggest that the bladder and distal colon interact under both normal and pathological conditions, however, the directions of these interactions can change dramatically depending on the nature and duration of the applied stimuli. This review article aimed to summarize the clinical data on colon–bladder cross‐reflexes in healthy individuals, as well as in patients with co‐morbid disorders. It also discusses currently used animal models, experimental approaches, and suggested mechanisms of colon–bladder cross‐talk. Additionally, it provides an overview of the potential pharmacological targets to develop treatment options for patients with co‐morbid disorders. Presented work resulted from the discussion of colon/bladder interactions during “Think Tank 9” presentations at the International Consultation on Incontinence Research Society meeting held in Bristol, UK, 2011. Neurourol. Urodynam. 31:352–358, 2012.

Nitrotyrosylation of Ca2+ Channels Prevents c-Src Kinase Regulation of Colonic Smooth Muscle Contractility in Experimental Colitis

Basal levels of c-Src kinase are known to regulate smooth muscle Ca2+ channels. Colonic inflammation results in attenuated Ca2+ currents and muscle contraction. Here, we examined the regulation of calcium influx-dependent contractility by c-Src kinase in experimental colitis. Ca2+-influx induced contractions were measured by isometric tension recordings of mouse colonic longitudinal muscle strips depolarized by high K+. The Emax to CaCl2 was significantly less in inflamed tissues (38.4 ± 7.6%) than controls, indicative of reduced Ca2+ influx. PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine], a selective Src kinase inhibitor, significantly reduced the contractile amplitude and shifted the pD2 from 3.88 to 2.44 in controls, whereas it was ineffective in inflamed tissues (3.66 versus 3.43). After pretreatment with a SIN-1 (3-morpholinosydnonimine)/peroxynitrite combination, the maximal contraction to CaCl2 was reduced by 46 ± 7% in controls but unaffected in inflamed tissues (13 ± 11%). Peroxynitrite also prevented the inhibitory effect of PP2 in control tissues. In colonic single smooth muscle cells, PP2 inhibited Ca2+ currents by 84.1 ± 3.9% in normal but only 36.2 ± 13% in inflamed tissues. Neither the Ca2+ channel Cav1.2b, gene expression, nor the c-Src kinase activity was altered by inflammation. Western blot analysis showed no change in the Ca2+ channel protein expression but increased nitrotyrosylated-Ca2+ channel proteins during inflammation. These data suggest that post-translational modification of Ca2+ channels during inflammation, possibly nitrotyrosylation, prevents c-Src kinase regulation resulting in decreased Ca2+ influx.

Spontaneous and evoked contractions are regulated by PKC-mediated signaling in detrusor smooth muscle: involvement of BK channels

Protein kinase C (PKC) and large conductance Ca(2+)-activated potassium channels (BK) are downregulated in the detrusor smooth muscle (DSM) in partial bladder outlet obstruction (PBOO). DSM from these bladders display increased spontaneous activity. This study examines the involvement of PKC in the regulation of spontaneous and evoked DSM contractions and whether pharmacologic inhibition of PKC in normal DSM contributes to increased detrusor excitability. Results indicate the PKC inhibitor bisindolylmaleimide 1 (Bim-1) prevented a decline in the amplitude of spontaneous DSM contractions over time in vitro, and these contractions persist in the presence of tetrodotoxin. Bim-1 also reduced the basal DSM tone, and the ability to maintain force in response to electrical field stimulation, but did not affect maximum contraction. The PKC activator phorbol-12,13-dibutyrate (PDBu) significantly reduced the amplitude and increased the frequency of spontaneous contractions at low concentrations (10 nM), while causing an increase in force at higher concentrations (1 μM). Preincubation of DSM strips with iberiotoxin prevented the inhibition of spontaneous contractions by PDBu. The BK channel openers isopimaric acid and NS1619 reduced the Bim-1-induced enhancement of spontaneous contractions in DSM strips. Our data suggest that PKC has a biphasic activation profile in the DSM and that it may play an important role in maintaining the quiescent state of the normal bladder during storage through the effects on BK channel, while helping to maintain force required for bladder emptying. The data also suggest that PKC dysfunction, as seen in PBOO, contributes to detrusor overactivity.

Colonic inflammation up‐regulates voltage‐gated sodium channels in bladder sensory neurons via activation of peripheral transient potential vanilloid 1 receptors

Background  Primary sensory neurons express several types of ion channels including transient receptor potential vanilloid 1 (TRPV1) and voltage‐gated Na+ channels. Our previous studies showed an increased excitability of bladder primary sensory and spinal neurons triggered by inflammation in the distal colon as a result of pelvic organ cross‐sensitization. The goal of this work was to determine the effects of TRPV1 receptor activation by potent agonists and/or colonic inflammation on voltage‐gated Na+ channels expressed in bladder sensory neurons.