Effects of Muscarinic Stimulation on Intracellular Calcium in the Rabbit Bladder: Comparison with Metabolic Response

Urinary bladder emptying is mediated primarily by a co-ordinated contraction of the bladder body in response to parasympathetic stimulation and muscarinic receptor activation. In a previous study using surface spectrophotometry to monitor the nicotinamide adenine dinucleotide (oxidized form)/nicotinamide adenine dinucleotide (reduced form) (NADH/NAD) ratio, we demonstrated that muscarinic stimulation results in a rapid decrease in this ratio which precedes the contractile response, and that the ED₅₀ for the NADH response is significantly lower than the ED₅₀ for contraction. The current study was designed to correlate changes in intracellular free calcium using FURA-2 fluorescence with both the contractile and metabolic response to muscarinic stimulation. Isolated strips of urinary bladder body were monitored in vitro for changes in intracellular free calcium, NADH/NAD ratio, and contraction. Intracellular free calcium was monitored by preincubation with FURA-2 AM and continuously measuring the fluorescence with an MB2 surface spectrofluorometer using excitation wavelengths of 340 and 380 nm, and an emission wavelength of 510 nm. The NADH/NAD ratio was monitored with the MB2 surface spectrophotometer using an excitation wavelength of 366 nm and an emission wavelength of 450 nm. Contraction was monitored using an isometric force transducer connected to a Grass model D polygraph. The” results can be summarized as follows: (1) Bethanechol stimulates a sharp decrease in the NADH/NAD ratio, a rapid increase in intracellular free calcium, and a slower increase in contractile force. (2) The ED₅₀ for NADH fluorescence was significantly less than the ED₅₀ for either contraction or calcium fluorescence which were equal to each other.