Laboratory practical to study the differential innervation pathways of urinary tract smooth muscle

Abstract

In the mammalian lower urinary tract, there is a reciprocal relationship between the contractile state of the bladder and urethra. As the bladder fills with urine, it remains relaxed to accommodate increases in volume, while the urethra remains contracted to prevent leakage of urine from the bladder to the exterior. Disruptions to the normal contractile state of the bladder and urethra can lead to abnormal micturition patterns and urinary incontinence. While both the bladder and urethra are smooth-muscle organs, they are differentially contracted by input from cholinergic and sympathetic nerves, respectively. The laboratory practical described here provides an experiential approach to understanding the anatomy of the lower urinary tract. Several key factors in urinary tract physiology are outlined, e.g., the bladder is contracted by activation of the parasympathetic pathway via cholinergic stimulation on muscarinic receptors, whereas the urethra is contracted by activation of the sympathetic pathway via adrenergic stimulation on α₁-adrenoceptors. This is achieved by measuring the force generated by bladder and urethra smooth muscle to demonstrate that acetylcholine contracts the smooth muscle of the bladder, whereas adrenergic agonists contract the urethral smooth muscle. An inhibition of these effects is also demonstrated by application of the muscarinic receptor antagonist atropine and the α₁-adrenergic receptor blocker phentolamine. A list of suggested techniques and exam questions to evaluate student understanding on this topic is also provided.

Keywords: acetylcholine; bladder; detrusor; phenylephrine; urethra.

Fig. 1.

Preparation of detrusor muscle strips. A: diagrammatic representation of the LUT showing the bladder and urethra. During dissections, the urethra should be removed from the base of the bladder by cutting at the point of the dotted line. B: once the urethra has been removed, the bladder should be opened by making a longitudinal incision along the dotted line shown. C: once opened, the bladder should be pinned out flat on a Sylgard-bottomed dish, and the urothelium layer removed. Strips of DSM should then be taken by cutting the DSM along the dotted lines shown.

Fig. 2.

Preparation of the urethra tube. A: diagrammatic representation of the urethra tube after removal from the bladder. B: a suture should be passed through the opening in the urethral tube with the aid of a sewing needle. C: scissors should be used to cut the suture once it has passed through the urethral tube, resulting in two separate strings. D: these strings should be tied to create a loop above and below the urethral tube. The tissue is now ready for setup on the force transducer.

Fig. 3.

Organ bath used in experimentation. Diagrammatic representation is shown of the organ bath setup used in performing force measurements with the urethral tube set up to the force transducer, as detailed in the text. The looped sutures of the ring preparation and the strip preparation should be placed on the wires of the force transducer and the stable mount (A). The inflow of oxygen is indicated (B). Oxygen bubbles should be present in the bath in a manner that does not interfere with the tissue preparations.

Fig. 4.

Effect of CCh on DSM and USM contractions. A: representative contractile trace showing the effect of 1 µM CCh on DSM contractions. B: representative contractile trace showing the effect of 1 µM CCh on USM contractions. *Artifact resulting from washing of the tissue within the organ bath.

Fig. 5.

Effect of PE on DSM and USM contractions. A: representative contractile trace showing the effect of 10 µM PE on DSM contractions. B: representative contractile trace showing the effect of 10 µM PE on USM contractions. *Artifact resulting from washing of the tissue within the organ bath.

Fig. 6.

Effect of phentolamine on DSM contractions induced by CCh. Representative contractile trace shows the effect of 1 µM phentolamine on DSM contractions induced by CCh. *Artifact resulting from washing of the tissue within the organ bath. Double slash marks denote time period of 15 min.

Fig. 7.

Effect of phentolamine on USM contractions induced by PE. Representative contractile trace shows the effect of 1 µM phentolamine on USM contractions induced by PE. *Artifact resulting from washing of the tissue within the organ bath. Double slash marks denote time period of 15 min.

Fig. 8.

Effect of atropine on DSM contractions induced by CCh. Representative contractile trace shows the effect of 1 µM atropine on DSM contractions induced by CCh. *Artifact resulting from washing of the tissue within the organ bath. Double slash marks denote time period of 15 min.

Fig. 9.

Effect of atropine on USM contractions induced by PE. Representative contractile trace shows the effect of 1 µM atropine on USM contractions induced by PE. *Artifact resulting from washing of the tissue within the organ bath. Double slash marks denote time period of 15 min.