Charles A. Buffington

Protein kinase C contributes to abnormal capsaicin responses in DRG neurons from cats with feline interstitial cystitis

Interstitial cystitis (IC) is a painful disorder which affects urinary bladder function in cats and humans. We have used patch clamp techniques to examine the possibility that the properties of primary afferent neurons are changed in feline interstitial cystitis (FIC). We measured transient receptor potential vanilloid receptor 1 (TRPV1) responses to capsaicin (CAPS) in dorsal root ganglion (DRG) neurons (L4-S3) from normal cats and cats with FIC. We show that FIC neurons are increased in size and exhibit CAPS responses which are increased in amplitude and desensitize slowly. CAPS responses desensitized seven times slower in FIC neurons. Phorbol 12,13-dibutyrate (PDBu), an activator of PKC, slowed the desensitization of CAPS responses in normal cat bladder and non-bladder neurons, but had no effect in FIC neurons. Bisindolylmaleimide, an inhibitor of PKC, reversed the PDBu effects in normal cat neurons and normalized the desensitization of CAPS responses in FIC neurons. Our data suggest that FIC afferent neurons exhibit abnormal CAPS responses. The latter may be due to enhanced endogenous activities of PKC.

Beyond Neurons: Involvement of Urothelial and Glial Cells in Bladder Function

The urothelium, or epithelial lining of the lower urinary tract (LUT), is likely to play an important role in bladder function by actively communicating with bladder nerves, smooth muscle, and cells of the immune and inflammatory systems. Recent evidence supports the importance of non‐neuronal cells that may extend to both the peripheral and central processes of the neurons that transmit normal and nociceptive signals from the urinary bladder. Using cats diagnosed with a naturally occurring syndrome termed feline interstitial cystitis (FIC), we investigated whether changes in physiologic parameters occur within 3 cell types associated with sensory transduction in the urinary bladder: 1) the urothelium, 2) identified bladder dorsal root ganglion (DRG) neurons and 3) grey matter astrocytes in the lumbosacral (S1) spinal cord. As estrogen fluctuations may modulate the severity of many chronic pelvic pain syndromes, we also examined whether 17β‐estradiol (E2) alters cell signaling in rat urothelial cells.

Localization of P2X and P2Y Receptors in Dorsal Root Ganglia of the Cat

The distribution of P2X and P2Y receptor subtypes in upper lumbosacral cat dorsal root ganglia (DRG) has been investigated using immunohistochemistry. Intensity of immunoreactivity for six P2X receptors (P2X5 receptors were immuno-negative) and the three P2Y receptors examined in cat DRG was in the order of P2Y2 = P2Y4>P2X3>P2X2 = P2X7>P2X6>P2X1 = P2X4>P2Y1. P2X3, P2Y2, and P2Y4 receptor polyclonal antibodies stained 33.8%, 35.3%, and 47.6% of DRG neurons, respectively. Most P2Y2, P2X1, P2X3, P2X4, and P2X6 receptor staining was detected in small- and medium-diameter neurons. However, P2Y4, P2X2, and P2X7 staining was present in large- and small-diameter neurons. Double-labeling immunohistochemistry showed that 90.8%, 32.1%, and 2.4% of P2X3 receptor-positive neurons coexpressed IB4, CGRP, and NF200, respectively; whereas 67.4%, 41.3%, and 39.1% of P2Y4 receptor-positive neurons coexpressed IB4, CGRP, and NF200, respectively. A total of 18.8%, 16.6%, and 63.5% of P2Y2 receptor-positive neurons also stained for IB4, CGRP, and NF200, respectively. Only 30% of DRG neurons in cat were P2X3-immunoreactive compared with 90% in rat and in mouse. A further difference was the low expression of P2Y1 receptors in cat DRG neurons compared with more than 80% of the neurons in rat. Many small-diameter neurons were NF200-positive in cat, again differing from rat and mouse.

Mucosal Muscarinic Receptors Enhance Bladder Activity in Cats With Feline Interstitial Cystitis

PURPOSE Interstitial cystitis is a chronic pelvic pain syndrome of which the origin and mechanisms involved remain unclear. In this study Ca(2+) transients in the bladder wall of domestic cats diagnosed with naturally occurring feline interstitial cystitis were examined. MATERIALS AND METHODS Cross-sections of full-thickness bladder strips from normal cats and cats with feline interstitial cystitis were examined by optically mapping Ca(2+) transients and recording tension. Responses of Ca(2+) activity and detrusor contractions to pharmacological interventions were compared. In addition, pharmacological responses were compared in mucosa denuded preparations. RESULTS Optical mapping showed that feline interstitial cystitis bladders had significantly more spontaneous Ca(2+) transients in the mucosal layer than control bladders. Optical mapping also demonstrated that feline interstitial cystitis bladders were hypersensitive to a low dose (50 nM) of the muscarinic receptor agonist arecaidine when the mucosal layer was intact. This hypersensitivity was markedly decreased in mucosa denuded bladder strips. CONCLUSIONS In feline interstitial cystitis cat bladders there is increased Ca(2+) activity and sensitivity of muscarinic receptors in the mucosal layer, which can enhance smooth muscle spontaneous contractions.

Cystitis, Co-morbid disorders and associated epithelial dysfunction†‡

Introduction Interstitial cystitis (painful bladder syndrome / interstitial cystitis; PBS/IC) is a persistent pain syndrome affecting the urinary bladder with symptoms including urinary frequency, bladder pain and nocturia.(1–6) Various animal models have been studied, most of which mimic some aspect of the human condition of interest to the investigator(s). This review will provide examples of various animal models including those incorporating chronic stress, thought to produce features that share similarities to that of PBS/IC patients, whose symptoms are often exacerbated by various stressors. (7–12) This review also provides evidence that patients with PBS/IC exhibit abnormalities within the bladder epithelium (or urothelium), even though a consistent relationship of such changes with symptom severity has not been demonstrated. These changes include alterations in urothelial integrity, differentiation and/or proliferation as well as changes in ‘sensory’ function (altered expression or sensitivity of receptors and ion channels). Establishing a diagnostic ‘indicator’ with a high degree of correlation in this syndrome would be of value in terms of disease status, diagnosis and treatment. There have been reports of a number of factors/mediators altered in PBS/IC. However, the lack of a validated biomarker and a well-defined etiology for this syndrome introduces a number of complications, including diagnostic confidence, choice of appropriate animal models to study basic mechanism with the goal toward treatment, and rational therapies. It is also becoming increasingly apparent that patients with PBS/IC often overlap or share symptoms commonly associated with other persistent pain disorders. These include (but are not limited to) irritable bowel syndrome (IBS), non-cardiac chest pain, fibromyalgia and even overactive bladder syndrome (OAB).(13–18) Such types of changes are not limited to the urinary bladder, however, as reports of alterations in epithelial signaling/barrier function have been described in patients diagnosed with a wider variety of syndromes, including functional and inflammatory bowel disorders such as irritable bowel syndrome (IBS), gastrointestinal esophageal reflux disease (GERD) and asthma.(19–21) These and other findings suggest that changes within the epithelium (barrier as well as signaling functions) may be a common occurrence that may contribute to peripheral mechanisms of hypersensitivity in a number of disorders.

Alterations in the non-neuronal acetylcholine synthesis and release machinery in esophageal epithelium.

AIMS A non-neuronal cholinergic system has been described in epithelial cells including that of the urinary bladder (urothelium) and the upper gastrointestinal tract (esophagus). Epithelial dysfunction has been implicated in the pathophysiology of persistent pain conditions such as painful bladder syndrome as well as functional heartburn. For example, alterations in the ability to synthesize and release acetylcholine may contribute to changes in epithelial sensory and barrier function associated with a number of functional genitourinary and intestinal disorders. MAIN METHODS We examined using immunoblot, acetylcholine (ACh)-synthesis and release components in cat esophageal mucosa and whether elements of these components are altered in a naturally occurring model of chronic idiopathic cystitis termed feline interstitial cystitis (FIC). KEY FINDINGS We identified proteins involved in ACh synthesis and release (high affinity choline transporter, CHT1; ACh synthesizing enzyme choline acetyltransferase ChAT and carnitine acetyltransferase CarAT; vesicular ACh transporter VAChT and the organic cation transporter isoforms 1-3 or OCT-1-3) in cat esophageal mucosa. Significant alterations in CHT, ChAT, VAChT and OCT-1 were detected in the esophageal mucosa from FIC cats. Changes in the vesicular nucleotide transporter (VNUT) and the junctional protein pan-cadherin were also noted. SIGNIFICANCE Taken together, these findings suggest that changes in the non-neuronal cholinergic system may contribute to alterations in cell-cell contacts and possibly communication with underlying cells that may contribute to changes in sensory function and visceral hyperalgesia in functional esophageal pain.