The role of TRPM8 in the Guinea-pig bladder-cooling reflex investigated using a novel TRPM8 antagonist.

Patients with overactive bladder often exhibit abnormal bladder contractions in response to intravesical cold saline (positive ice-water test). The molecular entity involved in cold sensation within the urinary bladder is unknown, but a potential candidate is the ion channel, transient receptor potential (melastatin)-8 (TRPM8). The objective of the present study was to investigate the role of TRPM8 in a bladder-cooling reflex evoked in anaesthetised guinea-pigs that is comparable to the positive ice-water test seen in patients. Guinea-pig TRPM8 was cloned from L6 dorsal root ganglia (DRG) and expressed in HEK293 cells. Functional agonist- and cold-induced Ca2+ influx and electrophysiology assays were performed in these cells, and for comparison in HEK293 cells expressing human TRPM8, using a novel TRPM8 antagonist, the S-enantiomer of 1-phenylethyl 4-(benzyloxy)-3-methoxybenzyl (2-aminoethyl) carbamate hydrochloride (PBMC). Potency data from these assays was used to calculate intravenous infusion protocols for targeted plasma concentrations of PBMC in studies on micturition reflexes evoked by intravesical infusion of menthol or cold saline in anaesthetised guinea-pigs. Tissue expression of TRPM8 in guinea-pig bladder, urethra and in dorsal root ganglia neurones traced from the bladder was also investigated. TRPM8 mRNA and protein were detected in L6 dorsal root ganglia, bladder urothelium and smooth muscle. PBMC antagonised in vitro activation of human and guinea-pig TRPM8 and reversed menthol and cold-induced facilitation of the micturition reflex at plasma concentrations consistent with in vitro potencies. The present data suggest that the bladder-cooling reflex in the guinea-pig involves TRPM8. The potential significance of TRPM8 in bladder disease states deserves future investigation.

Reversibility of hyperglycaemia and islet abnormalities in the high fat-fed female ZDF rat model of type 2 diabetes.

INTRODUCTION: We characterised the development of Type 2 diabetes and associated changes in islet appearance in female ZDF rats and explored its suitability for studies with novel therapeutic agents. METHODS: Female ZDF rats were either chow or high fat (60%) fed for up to 36 days and blood glucose and plasma insulin concentration measured. Additionally, we restored two groups of rats back to chow diet after ten and nineteen days of high fat feeding to determine the reversibility. Finally, two other groups of high fat-fed animals were dosed either orally with drug vehicle or had a minipump implanted subcutaneously to determine the effect of dosing method upon the progression of this disease model. The beta cell mass and morphology were assessed by immunohistochemistry for insulin. RESULTS: High fat feeding elevated blood glucose compared to chow-fed controls which peaked by 15 days, and maintained throughout the study. Plasma insulin reached a maximum after 8 days, but declined over the remaining 4 weeks. Assessment of islets revealed marked disruption, dispersion and weaker insulin staining. The area and percentage ß-cells were higher in high fat-fed animals. High fat diet treatment reversal when animals were moderately hyperglycaemic, when plasma insulin was still elevated, reversed the hyperglycaemia and maintained islet morphology similar to that of chow-fed animals. In contrast, dietary reversal when plasma insulin was declining, did not prevent continual decline in plasma insulin, ß-cell mass or islet disruption. Oral dosing tended to increase blood glucose and decrease plasma insulin whereas administration by minipump lowered blood glucose. DISCUSSION: The obese female ZDF rat offers the opportunity for preclinical evaluation of novel therapies directed towards improving pancreatic function, provided treatment is initiated prior to the precipitous decline in insulin production. Caution should be exercised in comparison of compounds administered by different dosing routes however.