a b s t r a c tIncreasing evidence from both clinical and experimental studies indicates that the insulin-releasing hormone, glucagon-like peptide-1 (GLP-1) may exert additional protective/reparative effects on the cardiovascular system. The aim of this study was to examine vasorelaxant effects of GLP-1(7-36)amide, three structurally-related peptides and a non-peptide GLP-1 agonist in rat aorta. Interestingly, all GLP-1 compounds, including the established GLP-1 receptor antagonist, exendin (9-39) caused concentrationdependent relaxation. Mechanistic studies employing hyperpolarising concentrations of potassium or glybenclamide revealed that these relaxant effects are mediated via specific activation of ATP-sensitive potassium channels. Further experiments using a specific membrane-permeable cyclic AMP (cAMP) antagonist, and demonstration of increased cAMP production in response to GLP-1 illustrated the critical importance of this pathway. These data significantly extend previous observations suggesting that GLP-1 may modulate vascular function, and indicate that this effect may be mediated by the GLP-1 receptor. However, further studies are required in order to establish whether GLP-1 related agents may confer additional cardiovascular benefits to diabetic patients.

transmitter(s) underlying nitric oxide synthase (NOS)-independent neural inhibition in the internal anal sphincter (IAS) is still uncertain. The present study investigated the role of purinergic transmission. Contractile and electrical responses to electrical field stimulation of nerves (0.1-5 Hz for 10 -60 s) were recorded in strips of mouse IAS. A single stimulus generated a 28-mV fast inhibitory junction potential (IJP) and relaxation. The NOS inhibitor N -nitro-L-arginine (L-NNA) reduced the fast IJP duration by 20%. Repetitive stimulation at 2.5-5 Hz caused a more sustained IJP and sustained relaxation. L-NNA reduced relaxation at 1 Hz and the sustained IJP at 2.5-5 Hz. All other experiments were carried out in the presence of NOS blockade. IJPs and relaxation were significantly reduced by the P2 receptor antagonists 4-[[4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzenedisulfonic acid (PPADS) (100 M), by desensitization of P2Y receptors with adenosine 5Ј-[␤-thio]diphosphate (ADP-␤S) (10 M), and by the selective P2Y1 receptor blocker 2Ј-deoxy-N 6 -methyl adenosine 3Ј,5Ј-diphosphate (MRS2179) (10 M). Relaxation and IJPs were also significantly reduced by the K ϩ channel blocker apamin (1 M). Removal of extracellular potassium (Ko) increased IJP amplitude to 205% of control, whereas return of Ko 30 min later hyperpolarized cells by 19 mV and reduced IJP amplitude to 50% of control. Exogenous ATP (3 mM) relaxed muscles in the presence of TTX (1 M) and hyperpolarized cells by 15 mV. In conclusion, these data suggest that purinergic transmission significantly contributes to NOS-independent neural inhibition in the mouse IAS. P2Y1 receptors, as well as at least one other P2 receptor subtype, contribute to this pathway. Purinergic receptors activate apaminsensitive K ϩ channels as well as other apamin-insensitive conductances leading to hyperpolarization and relaxation.gastrointestinal; enteric; motor inhibition; NANC transmission THE INTERNAL ANAL SPHINCTER (IAS) aids in maintaining fecal continence and permits evacuation of fecal contents during the rectoanal inhibitory reflex (RAIR). To fulfill these functions, the IAS usually exists in a state of contraction that can be relaxed by inhibitory nerves during the defecation reflex. In the present study we examined inhibitory motor innervation to the mouse IAS. The predominant neurotransmitters underlying inhibitory neural responses in the gastrointestinal tract include nitric oxide (NO), VIP, pituitary adenylate cyclase-activating polypeptide, and ATP (or a related compound). There is still controversy regarding the contribution of these neurotransmitters to neural inhibition in the IAS. Whereas some studies suggest that neural inhibition is almost exclusively due to NO in combination with VIP (40), earlier studies of the guinea pig (39), rat (14), and rabbit (32) IAS provided evidence that a purinergic neural pathway contributes as well.Membrane hyperpolarization is an important mechanism contributing to inhibitory motor responses. ...

Aim The mechanisms underlying detection and transmission of sensory signals arising from visceral organs, such as the urethra, are poorly understood. Recently, specialized ACh-expressing cells embedded in the urethral epithelium have been proposed as chemosensory sentinels for detection of bacterial infection. Here, we examined the morphology and potential role in sensory signalling of a different class of specialized cells that express serotonin (5-HT), termed paraneurones. Methods Urethrae, dorsal root ganglia neurones and spinal cords were isolated from adult female mice and used for immunohistochemistry and calcium imaging. Visceromotor reflexes (VMRs) were recorded in vivo. Results We identified two morphologically distinct groups of 5-HT+ cells with distinct regional locations: bipolar-like cells predominant in the mid-urethra and multipolar-like cells predominant in the proximal and distal urethra. Sensory nerve fibres positive for calcitonin gene-related peptide, substance P, and TRPV1 were found in close proximity to 5-HT+ paraneurones. In vitro 5-HT (1 µm) stimulation of urethral primary afferent neurones, mimicking 5-HT release from paraneurones, elicited changes in the intracellular calcium concentration ([Ca2+]i) mediated by 5-HT2 and 5-HT3 receptors. Approximately 50% of 5-HT responding cells also responded to capsaicin with changes in the [Ca2+]i. In vivo intra-urethral 5-HT application increased VMRs induced by urethral distention and activated pERK in lumbosacral spinal cord neurones. Conclusion These morphological and functional findings provide insights into a putative paraneurone-neural network within the urethra that utilizes 5-HT signalling, presumably from paraneurones, to modulate primary sensory pathways carrying nociceptive and non-nociceptive (mechano-sensitive) information to the central nervous system.

Transient receptor potential cation channel subfamily M member 4 (TRPM4) has been shown to play a key role in detrusor contractility under physiological conditions. In this study, we investigated the potential role of TRPM4 in detrusor overactivity following spinal cord transection (SCT) in mice. TRPM4 expression and function were evaluated in bladder tissue with or without the mucosa from spinal intact (SI) and SCT female mice (T8-T9 vertebra; 1-28 days post SCT) using PCR, western blot, immunohistochemistry, and muscle strip contractility techniques. TRPM4 was expressed in the urothelium (UT) and detrusor smooth muscle (DSM) and was upregulated after SCT. Expression levels peaked 3-7 days post SCT in both the UT and DSM. Pharmacological block of TRPM4 with the antagonist, 9-Phenanthrol (30 μM) greatly reduced spontaneous phasic activity that developed after SCT, regardless of the presence or absence of the mucosa. Detrusor overactivity following spinal cord injury leads to incontinence and/or renal impairment and represents a major health problem for which current treatments are not satisfactory. Augmented TRPM4 expression in the bladder after chronic SCT supports the hypothesis that TRPM4 channels play a role in DSM overactivity following SCT. Inhibition of TRPM4 may be beneficial for improving detrusor overactivity in SCI.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a debilitating chronic disease of unknown etiology. A naturally occurring disease termed feline interstitial cystitis (FIC) reproduces many features of IC/BPS patients. To gain insights into mechanisms underlying IC/BPS, we investigated pathological changes in the lamina propria (LP) of the bladder and proximal urethra in cats with FIC, using histological and molecular methods. Compared to control cat tissue, we found an increased number of de-granulated mast cells, accumulation of leukocytes, increased cyclooxygenase (COX)-1 expression in the bladder LP, and increased COX-2 expression in the urethra LP from cats with FIC. We also found increased suburothelial proliferation, evidenced by mucosal von Brunn’s nests, neovascularization and alterations in elastin content. Scanning electron microscopy revealed normal appearance of the superficial urethral epithelium, including the neuroendocrine cells (termed paraneurons), in FIC urethrae. Together, these histological findings suggest the presence of chronic inflammation of unknown origin leading to tissue remodeling. Since the mucosa functions as part of a “sensory network” and urothelial cells, nerves and other cells in the LP are influenced by the composition of the underlying tissues including the vasculature, the changes observed in the present study may alter the communication of sensory information between different cellular components. This type of mucosal signaling can also extend to the urethra, where recent evidence has revealed that the urethral epithelium is likely to be part of a signaling system involving paraneurons and sensory nerves. Taken together, our data suggest a more prominent role for chronic inflammation and tissue remodeling than previously thought, which may result in alterations in mucosal signaling within the urinary bladder and proximal urethra that may contribute to altered sensations and pain in cats and humans with this syndrome.