Hypothesis / aims of study
Misuse of ketamine, a non-competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor, has been strongly associated with lower urinary tract (LUT) dysfunction and the development of ketamine-induced cystitis (KIC) (1). However, the early functional alterations that precede and potentially drive subsequent structural bladder pathology remain poorly characterised (2).In this study, we examined the expression of NMDA receptors in the porcine urinary bladder and evaluated the acute effects of ketamine on bladder wall contractility, both in the presence and absence of NMDA receptor co-agonists (glycine and glutamate).
Study design, materials and methods
Female pig (Sus scrofa domestica, ~6 months) bladders were collected from a local abattoir and the dome dissected to prepare longitudinal strips: intact (I; detrusor + mucosa), denuded detrusor (D), and mucosa (M) alone. Tissues were mounted in Krebs solution (37°C, 2 g tension) for isometric recording of spontaneous contractions (SCs). Following baseline measurements, 500µM ketamine or vehicle was applied for 30 min. In separate experiments, tissues were pre-treated with 500µM glycine/glutamate before addition of ketamine (or vehicle). Contractile responses to 10µM carbachol (CCh) were also assessed in absence and presence of 500µM ketamine, with or without glycine/glutamate pre-treatment. Western blotting was performed on protein extracted from the same tissue regions (intact, denuded, mucosa) to assess NMDA receptor expression using NMDAR1 subunit-specific antibodies. Data are presented as mean ± SEM (n = number of preparations) and analysed using parametric or non-parametric t-tests.
Results
Western blotting confirmed NMDAR1 expression in porcine bladder tissue, with specific bands detected in experimental samples but absent in negative controls (Fig. 1A–B). In mucosal strips, 500µM ketamine significantly reduced the amplitude and frequency of SCs at 30 mins vs. baseline (Figures 2A, 2B). Paired mucosal control strips receiving Krebs showed no significant change over the same period. Ketamine did not have a significant effect on the SCs of intact or denuded preparations. Similarly, no significant changes in SC amplitude or frequency were observed in strips pre-treated with 500 µM glycine/glutamate prior to ketamine exposure (Figures 2C, 2D).
For agonist-evoked responses, 500µM ketamine significantly attenuated CCh-induced tonic contractions in mucosal, intact, and denuded preparations compared to control strips (Fig. 2E). Following glycine/glutamate pre-treatment, a significant reduction in CCh responses was observed only in intact strips (Fig. 2F).
Interpretation of results
Western blotting confirmed expression of the NMDAR1 subunit in porcine bladder tissue, indicating the presence of functional NMDA receptors, as this subunit is essential for receptor assembly and activity.
Ketamine selectively reduced spontaneous activity in mucosal strips, but not in intact preparations, suggesting a mucosa-specific contribution that is not apparent in whole tissue, likely due to masking by greater detrusor activity. The abolition of this effect by glycine/glutamate pre-treatment indicates involvement of a NMDA receptor–linked signalling mechanism in the mucosa. Ketamine significantly attenuated carbachol-evoked contractions across all tissue types, and this effect was only partially modified by co-agonists, indicating additional NMDA-independent mechanism.