Ana Charrua

Anandamide-Evoked Activation of Vanilloid Receptor 1 Contributes to the Development of Bladder Hyperreflexia and Nociceptive Transmission to Spinal Dorsal Horn Neurons in Cystitis

The role of anandamide in the development of inflammatory hyperalgesia and visceral hyperreflexia was studied in the rat urinary bladder. Animals were given intraperitoneal cyclophosphamide injection, which evokes painful hemorrhagic cystitis accompanied by increased bladder reflex activity. The vanilloid receptor 1 [transient receptor potential vanilloid 1 (TRPV1)] antagonist capsazepine, applied onto the serosal surface of bladders, significantly reduced the hyperreflexia. Mass spectrometric analysis revealed that cyclophosphamide injection significantly and persistently increased the anandamide content of bladder tissues. The increase in the anandamide content paralleled the development of reflex hyperactivity. Anandamide (1-100 μm), applied onto the serosal surface of naive bladders, increased the reflex activity in a concentration-dependent manner. Repeated anandamide applications did not produce desensitization of the response. The anandamide-evoked effect was blocked by capsazepine or by instillation of resiniferatoxin, the ultrapotent TRPV1 agonist, into the bladders 24 hr before the anandamide challenge. The cannabinoid 1 receptor antagonist SR141716A [N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide] significantly increased the potency of anandamide in enhancing bladder reflex activity in naive but not in cyclophosphamide-injected animals. Application of the fatty acid amide hydrolyze inhibitor palmitoylisopropylamine onto the serosal surface of bladders also increased the reflex activity both in naive and cyclophosphamide-injected rats. This latter effect in naive animals was blocked by capsazepine and by resiniferatoxin pretreatment. Finally, intravesical instillation of anandamide (50 μm) increased c-fos expression in the spinal cord, which was reduced by capsazepine or by resiniferatoxin pretreatment. These results suggest that anandamide, through activating TRPV1, contributes to the development of hyperreflexia and hyperalgesia during cystitis.

GRC-6211, a new oral specific TRPV1 antagonist, decreases bladder overactivity and noxious bladder input in cystitis animal models.

PURPOSE We evaluated the effects of GRC-6211, an orally active TRPV1 antagonist, on the function and noxious input of naïve and inflamed bladders. MATERIALS AND METHODS In urethane (Sigma(R)) anesthetized rats 0.5 ml GRC-6211 (0.001, 0.01, 0.1 and 1 mg/kg weight) or its vehicle (0.5% methylcellulose) were administered through a duodenal catheter and cystometry was done during infusion of saline, 100 microM capsaicin or 0.5% acetic acid (Merck, Feltham, United Kingdom). Cystometry was also performed in WT and TRPV1 knockout mice treated with 1 mg/kg GRC-6211. Cystometry was done in rats inflamed with lipopolysaccharide after receiving 0.1 mg/kg GRC-6221 or vehicle. Spinal c-fos expression induced by 0.5% acetic acid was investigated after 0.1 mg/kg GRC-6211 or vehicle administration. TRPV1 immunoreactivity was evaluated in the bladder after GRC-6211 administration. RESULTS The reflex activity of rat and WT mice naïve bladders was unchanged by GRC-6211 up to a dose of 0.1 mg/kg. At 1 mg/kg contractions were transiently suppressed in naïve rats and WT mice but not in TRPV1 knockout mice. GRC-6211 (0.1 mg/kg) completely prevented capsaicin induced irritation, while the 0.001, 0.01 or 0.1 mg/kg dose decreased the mean +/- SD frequency of bladder contractions during acetic acid infusion from 1.5 +/- 0.3 to 1.35 +/- 0.35 (not significant), 0.9 +/- 0.2 (p <0.05) and 0.8 +/- 0.2 (p <0.05), respectively. Lipopolysaccharide inflamed rats had 1.4 +/- 0.4 and 0.8 +/- 0.1 contractions per minute after vehicle and GRC-6211, respectively (p <0.05). The c-fos expression induced by acetic acid was decreased by GRC-6211 (85.5 +/- 19.1 to 46.7 +/- 9.4, p <0.05). GRC-6211 did not change bladder TRPV1 immunoreactivity. CONCLUSIONS GRC-6211 counteracts the bladder hyperactivity and noxious input induced by cystitis. At high doses it suppresses normal bladder activity by a TRPV1 dependent mechanism. TRPV1 antagonists might be useful for cystitis.

Intravesical resiniferatoxin decreases spinal c‐fos expression and increases bladder volume to reflex micturition in rats with chronic inflamed urinary bladders

To evaluate the effect of intravesical resiniferatoxin on spinal c‐fos expression and bladder volume at reflex micturition in rats with chronic urinary bladder inflammation.

Functional Transient Receptor Potential Vanilloid 1 is Expressed in Human Urothelial Cells

PURPOSE We investigated the expression and functional status of TRPV1 receptor in human urothelial cells. MATERIAL AND METHODS Human urothelium was cultured and TRPV1 receptor expression was studied by immunocytochemistry and reverse transcriptase-polymerase chain reaction. The influence of inflammatory mediators on TRPV1 mRNA levels was also studied. Functional assays (cobalt uptake measurements and whole cell voltage clamp records) were used to study the response of urothelial cells to capsaicin, temperature, low pH and inflammatory mediators. Capsaicin induced adenosine triphosphate release from urothelial cells was assessed by bioluminescence. RESULTS TRPV1 protein and mRNA were detected in human urothelial cells and mRNA more than tripled in the presence of inflammatory mediators. Nerve growth factor treatment alone did not affect TRPV1 mRNA expression. Capsaicin (100 nM and 1 microM) and heat (41C and 45C) evoked cobalt uptake and inflammatory mediators lowered the temperature threshold for TRPV1 activation to 37C. Capsaicin (1 microM) induced TRPV1 desensitization to further applications of the agonist. In whole cell patch clamp experiments 1 microM capsaicin and a heat ramp from 37C to 50C caused inward currents. The same concentration of capsaicin induced the release of about 7 fmol adenosine triphosphate per mg. CONCLUSIONS TRPV1 receptors expressed by human urothelial cells respond to capsaicin and thermal stimuli. Capsaicin evoked release of adenosine triphosphate suggests that human urothelial TRPV1 is involved in the afferent branch of the micturition reflex. Inflammatory mediators decrease the TRPV1 thermal threshold of activation to body temperature and increase its expression. This finding may be relevant for symptoms associated with cystitis.

Insulin induces cobalt uptake in a subpopulation of rat cultured primary sensory neurons

Previous findings show that both the vanilloid receptor 1 and the insulin receptor are expressed on small primary sensory neurons. As insulin evokes activity in second messengers which could induce opening of the vanilloid receptor 1, we examined, by using the cobalt‐uptake technique, whether or not insulin can activate cultured rat primary sensory neurons through activating the vanilloid receptor 1. Capsaicin (50, 100 and 500 nm) induced concentration‐dependent labelling in primary sensory neurons. Preincubation of cells in insulin (10 µm) for 10 min followed by a 2‐min wash did not produce significant change in the capsaicin‐induced labelling. Coapplication of insulin (10 µm) with capsaicin, however, potentiated the 50 and 100 nm capsaicin‐evoked staining. Insulin itself also produced cobalt labelling in a concentration‐dependent manner. The size–frequency distributions of neurons showing capsaicin‐ or insulin‐induced cobalt accumulation were similar. The insulin‐induced cobalt labelling was significantly reduced by the tyrosine kinase inhibitor, tyrphostin AG1024, the vanilloid receptor 1 antagonists, ruthenium red and capsazepine, the protein kinase inhibitor, staurosporine and the phospholipase C inhibitor neomycin. Double immunostaining of cultured primary sensory neurons and sections from dorsal root ganglia revealed that about one‐third of the cells coexpress the insulin receptor and vanilloid receptor 1. These findings suggest that insulin activates a subpopulation of primary sensory neurons, probably through phosphorylation‐ and/or phosphatidylinositol(4,5)biphosphate hydrolysis‐evoked activation of the vanilloid receptor 1. Although the insulin‐induced activation of vanilloid receptor 1 seems to be a short‐lived effect in vitro, in vivo it might play a role in the development of burning pain sensation in hyperinsulinism.

Transient receptor potential channels in bladder function

The transient receptor potential (TRP) superfamily of cationic ion channels includes proteins involved in the transduction of several physical and chemical stimuli to finely tune physiological functions. In the urinary bladder, they are highly expressed in, but not restricted to, primary afferent neurons. The urothelium and some interstitial cells also express several TRP channels. In this review, we describe the expression and the known roles of some members of TRP subfamilies, namely TRPV, TRPM and TRPA, in the urinary bladder. The therapeutic interest of modulating the activity of TRP channels to treat bladder dysfunctions is also discussed.

Neurochemical characterization of insulin receptor-expressing primary sensory neurons in wild-type and vanilloid type 1 transient receptor potential receptor knockout mice

The insulin receptor (IR) is expressed by a subpopulation of primary sensory neurons (PSN), including a proportion of cells expressing the nociceptive transducer vanilloid type 1 transient receptor potential receptor (TRPV1). Recent data suggest functional links between the IR and other receptors, including TRPV1, which could be involved in the development of PSN malfunctions in pathological insulin secretion. Here we used combined immunohistochemical labelling on sections from L4–5 dorsal root ganglia of wild‐type (WT) and TRPV1 knockout (KO) mice to examine the neurochemical properties of IR‐expressing PSN and the possible effect of deletion of TRPV1 on those characteristics. We found that antibodies raised against the high‐molecular‐weight neurofilament (NF‐200) and the neurofilament protein peripherin distinguished between small and large neurons. We also found that the IR was expressed predominantly by the small peripherin‐immunopositive cells both in the WT and in the KO animals. IR expression, however, did not show any preference between the major subpopulations of the small cells, the calcitonin gene‐related peptide (CGRP)‐expressing and Bandeiraea simplicifolia isolectin B4 (IB4)‐binding neurons, either in the WT or in the KO mice. Nevertheless, a significant proportion of the IR‐expressing cells also expressed TRPV1. Comparison of the staining pattern of these markers showed no difference between WT and KO animals. These findings indicate that the majority of the IR‐expressing PSN are small neurons, which are considered as nociceptive cells. Furthermore, these data show that deletion of the TRPV1 gene does not induce any additional changes in neurochemical phenotype of nociceptive PSN. J. Comp. Neurol. 503:334–347, 2007.

Rat detrusor overactivity induced by chronic spinalization can be abolished by a transient receptor potential vanilloid 1 (TRPV1) antagonist

PURPOSE To evaluate the effect of a transient receptor potential vanilloid 1 (TRPV1) antagonist GRC 6211 on neurogenic detrusor overactivity (NDO) of spinal origin. MATERIALS AND METHODS Cystometries under urethane anaesthesia were obtained in 14 chronic spinalized rats to confirm NDO. Two groups were created. In the first one (n=10), GRC 6211 (0.01, 0.1 and 1mg/kg weight) was administered via the duodenum in cumulative doses and cystometries performed 150 min after the administration of each dose of the drug. In the second group (n=4), used as control, the animals were submitted to cystometries during 12 hours, without administration of GRC 6211. Frequency and amplitude of bladder contractions were recorded in both groups. RESULTS The mean (±SDev) bladder detrusor muscle contraction frequency of spinalized rats was 0.7±0.27 contractions/min. GRC 6211 produced a significant dose-dependent effect, with the frequency diminished to 0.53±0.23, 0.40±0.20 and 0.20±0.13 contractions/min, respectively. The mean (± SDev) amplitude of bladder contractions was 48.4±4.4 cmH(2)O. After administration of 0.01 mg/kg, 0.1mg/kg and 1mg/kg of GRC 6211, the amplitude decreased to 47.1±4.3, 45.6±5.6 and 40.2±4.1 cm H(2)O respectively. The effect was significant at 0.1 and 1mg/kg doses. Cystometries performed in the control group of spinalized rats showed no evidence of detrusor fatigue caused by the urethane anaesthesia and long duration of the experiment. CONCLUSION TRPV1 antagonists may be very effective in reducing NDO of spinal origin. This finding may have profound implications for the pathogenesis and future treatment options of patients with spinal NDO.

Expression of apoptosis-regulating genes in the rat prostate following botulinum toxin type a injection

BackgroundOnabotulinumtoxin A (OnabotA) injection has been investigated as a novel treatment for benign prostatic enlargement caused by benign prostatic hyperplasia. An OnabotA - induced volume reduction caused by sympathetic fibers impairment has been proposed as a potential mechanism of action. Our aim was to investigate the expression of apoptosis-regulating proteins in the rat prostate following OnabotA intraprostatic injection.MethodsAdult Wistar rats were injected in the ventral lobes of the prostate with 10 U of OnabotA or saline. A set of OnabotA-injected animals was further treated with 0.5 mg/kg of phenylephrine (PHE) subcutaneously daily. All animals were sacrificed after 1 week and had their prostates harvested. Immunohistochemical staining was performed for Bax, Bcl-xL and caspase-3 proteins and visualized by the avidin-biotin method. The optical density of the glandular cells was also determined, with measurement of differences between average optical densities for each group.ResultsSaline-treated animals showed intense epithelial staining for Bcl-xL and a faint labelling for both Bax and Caspase-3. OnabotA-treated rats showed a reduced epithelial staining of Bcl-xL and a consistently increased Bax and Caspase-3 staining when compared with saline-treated animals. PHE-treated animals showed a stronger Bcl-xL staining and reduced staining of both Bax and Caspase-3 when compared to the OnabotA group. Mean signal intensity measurements for each immunoreaction confirmed a significant decrease of the signal intensity for Bcl-xL and a significant increase of the signal intensity for Bax and Caspase 3 in OnabotA-injected animals when compared with the control group. In OnabotA+PHE treated animals mean signal intensity for Bcl-xL, Bax and Caspase 3 immunoreactions was identical to that of the control animals.ConclusionsThese results support the hypothesis that OnabotA activates apoptotic pathways in the rat prostate through a mechanism that involves sympathetic outflow impairment.

Brain-derived neurotrophic factor, acting at the spinal cord level, participates in bladder hyperactivity and referred pain during chronic bladder inflammation.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin (NT) known to participate in chronic somatic pain. A recent study has indicated that BDNF may participate in chronic cystitis at the peripheral level. However, the principal site of action for this NT is the central nervous system, most notably the spinal cord. The effects of centrally-acting BDNF on bladder function in normal animals and its central role during chronic cystitis are presently unknown. The present study was undertaken to clarify this issue. For that purpose, control non-inflamed animals were intrathecally injected with BDNF, after which bladder function was evaluated. This treatment caused short-lasting bladder hyperactivity; whereas chronic intrathecal administration of BDNF did not elicit this effect. Cutaneous sensitivity was assessed by mechanical allodynia as an internal control of BDNF action. To ascertain the role of BDNF in bladder inflammation, animals with cyclophosphamide-induced cystitis received intrathecal injections of either a general Trk receptor antagonist or a BDNF scavenger. Blockade of Trk receptors or BDNF sequestration notably improved bladder function. In addition, these treatments also reduced referred pain, typically observed in rats with chronic cystitis. Reduction of referred pain was accompanied by a decrease in the spinal levels of extracellular signal-regulated kinase (ERK) phosphorylation, a marker of increased sensory barrage in the lumbosacral spinal cord, and spinal BDNF expression. Results obtained here indicate that BDNF, acting at the spinal cord level, contributes to bladder hyperactivity and referred pain, important hallmarks of chronic cystitis. In addition, these data also support the development of BDNF modulators as putative therapeutic options for the treatment of chronic bladder inflammation.