Expression of functional nicotinic acetylcholine receptors in rat urinary bladder epithelial cells

Abstract

Although nicotinic acetylcholine receptors in both the central and peripheral nervous systems play a prominent role in the control of urinary bladder function, little is known regarding expression or function of nicotinic receptors in the bladder epithelium, or urothelium. Nicotinic receptors have been described in epithelial cells lining the upper gastrointestinal tract, respiratory tract, and the skin. Thus the present study examined the expression and functionality of nicotinic receptors in the urothelium, as well as the effects of stimulation of nicotinic receptors on the micturition reflex. mRNA for the alpha3, alpha5, alpha7, beta3, and beta4 nicotinic subunits was identified in rat urothelial cells using RT-PCR. Western blotting also confirmed urothelial expression of the alpha3- and alpha7-subunits. Application of nicotine (50 nM) to cultured rat urothelial cells elicited an increase in intracellular Ca2+ concentration, indicating that at least some of the subunits form functional channels. These effects were blocked by the application of the nicotinic antagonist hexamethonium. During in vivo bladder cystometrograms in urethane-anesthetized rats, intravesical administration of nicotine, choline, or the antagonists methyllycaconitine citrate and hexamethonium elicited changes in voiding parameters. Intravesical nicotine (50 nM, 1 microM) increased the intercontraction interval. Intravesical choline (1-100 microM) also affected bladder reflexes similarly, suggesting that alpha7 nicotinic receptors mediate this effect. Intravesical administration of hexamethonium (1-100 microM) potentiated the nicotine-induced changes in bladder reflexes. Methyllycaconitine citrate, a specific alpha7-receptor antagonist, prevented nicotine-, choline-, and hexamethonium-induced bladder inhibition. These results are the first indication that stimulation of nonneuronal nicotinic receptors in the bladder can affect micturition.

Fig. 1

A: RT-PCR detects presence of nicotinic acetylcholine receptor (nAChR) subunits. Notice positive results in α₃, α₅, α₇, β₃, and β₄ lanes; 1.2% agarose gel in 1× TBE buffer stained with ethidium bromide. Results are typical of cells cultured from 3 rat bladders or urothelial tissue surgically removed from the bladder. B: Western blot of cultured urothelial cells detects the presence of the α₃ and α₇ nAChR subunits. Results are typical for blots performed with cultured cells, surgically stripped urothelium, or dorsal root ganglion cells used as a control. Visualization of bands is blocked by the addition of an antigen to each antibody.

Fig. 2

Application of nicotine to urothelial cells causes an increase in the fura 2 signal, indicating an increase in intracellular calcium. A: representative trace of fura 2 ratio. B: application of nicotine (NIC; 50 nM, n = 34 cells from 5 independent cultures) causes a 2.5-fold increase in the fura 2 ratio, which is blocked by the pretreatment of the nicotinic antagonist hexamethonium (20 μM, C6, n = 18, from 3 separate cultures).

Fig. 3

Effects of intravesical nicotine on voiding function in the rat. A: 3 representative tracings of CMG recordings. All tracings were recorded from the same animal. Intravesical administration of NIC (50 nM and 1 μM, n = 6 each) increases the interval between voiding bladder contractions (intercontraction interval or ICI). B: effect is reversed by saline washout. *P < 0.05.

Fig. 4

Effect of hexamethonium (C6) on voiding function in the rat. A: representative traces of CMG recordings during intravesical administration of hexamethonium (C6). B: intercontraction interval changes following instillation of hexamethonium (C6) intravesically, n = 8 for each concentration. C: effects on ICI of nicotine infusion alone (50 nM and 1 μM) compared with simultaneous infusion of C6 (20 μM) and NIC; n = 6 for each. Bars 1 and 3 are taken from different rats than bars 2 and 4. All results are shown as a percent change from saline-infused controls. *P < 0.05 compared with control.

Fig. 5

Effect of methyllycaconitine citrate (MLA) on voiding function in the rat. A: representative traces of CMG recordings during intravesical administration of MLA. B: intercontraction interval following instillation of MLA intravesically, n = 8 for each MLA concentration. C: effects on ICI of nicotine infusion alone (50 nM and 1 μM, columns 1 and 3) compared with simultaneous infusion of MLA (10 μM) and NIC (columns 2 and 4), n = 6 for each, columns 1 and 3 are taken from different rats than columns 2 and 4. All results are shown as a percent change from saline-infused controls. NS, not statistically significant compared with control.

Fig. 6

Effect of simultaneous infusion of methyllycaconitine citrate and hexamethonium on micturition. Comparison of the percent change in ICI from saline control following instillation of hexamethonium (C6) alone or concurrent instillation of C6 and MLA, n = 6 for each. *P < 0.05. NS, not significant.

Fig. 7

Effect of the α₇-specific agonist, choline, on ICI in the rat. Choline (1, 10, 100 μM) was infused intravesically for 1 h each and ICI was measured. A: representative tracings of CMG recordings during intravesical administration of choline. B: graph shows change in ICI as a percent change from saline-infused controls. *P < 0.05 compared with control, n = 8 for each concentration.

Fig. 8

Effect of permeabilization of the urothelial barrier by protamine sulfate (PS) on nicotine-induced changes in bladder reflexes. A: representative tracings of CMG recordings taken during a control period of protamine sulfate (10 mg/ml) infusion and PS infusion with simultaneous NIC (1 μM) infusion. B: graph depicting changes in ICI during PS infusion or simultaneous PS and nicotine infusion as a change from saline-infused controls, n = 6 for each column. *P < 0.05 compared with control.

Fig. 9

A: effect of the potent nicotinic agonist, epibatidine, on bladder function. B: control recordings after 250 nM epibatidine instillation into the bladder (continuous infusion, 0.04 ml/min). C: after 1 h of continuous instillation of epibatidine. D: cystometrogram of bladder pressure following systemic administration of epibatidine (0.01 μg ip). A–C were recorded from the same animal, D from a separate animal.

Fig. 10

Hypothetical model of nicotinic cholinergic signaling in the urothelium. ACh, acetylcholine; ATP, adenosine triphosphate; C6, hexamethonium; NO, nitric oxide; UTC, urothelial cells (urothelium).