Maureen Basha ¹, Ed LaBelle ¹, Tanchun Wang ², Robert S. Moreland ², Alan J. Wein, M ¹, Samuel K.Chacko ¹

1) Division of Urology, University of Pennsylvania 2) Department of Pharmacology and Physiology, Drexel University College of Medicine.

Introduction and Objectives: Muscarinic receptor stimulation of the inositol phospholipid signaling cascade is a major pathway of parasympathetic regulation of smooth muscle contraction in many tissues. Although the vaginal wall receives parasympathetic innervation, several investigators have reported that the contractile responses of vaginal smooth muscle to carbachol are weak or absent. Further studies are required to clarify the presence, or lack thereof, of muscarinic receptor mediated contraction in the vaginal muscularis. We have previously shown that the proximal and distal vagina differ in both molecular and functional characteristics and have shown that the proximal vagina is a phasic muscle molecularly designed for generating force quickly. Therefore, the objectives of this study were:1) to determine the expression of muscarinic receptors in the proximal and distal vaginal wall and 2) test the functional significance of receptor expression via measurement of both force and inositol phospholipid production in response to carbachol stimulation. We hypothesize that the proximal and distal vagina will also differ in their response to carbachol, a muscarinic agonist.

Methods: Adult female Sprague-Dawley rats were sacrificed on the day of estrus and the vagina was dissected into a proximal (~upper 2/3^rd) and distal (~lower 1/3^rd) segment.

Isolated RNA from proximal and distal segments underwent RT-PCR using primer pairs to amplify muscarinic receptor subtype 2 (M₂) and 3 (M₃) expression (n=3). Isometric force measurements were obtained using longitudinal tissue strips from the proximal and distal vagina mounted to a force transducer and equilibrated in physiological salt solution for 90 minutes (n=3). Strips were repeatedly stretched and contracted with KCl (110 mM) until the optimal length for maximal active contraction (L_o) was achieved. Strips were then activated with either 10 or 50 µM carbachol. Inositol triphosphate (IP₃) production of tissue strips exposed to 50 µM carbachol for 0,2,15 and 20 minutes was measured using [³H] labeled inositol and Dowex column chromatography (n=3).

Results Obtained: We detected both M₂ and M₃ receptors at the message level in the proximal and distal vagina. Muscle bath studies indicated a resting tension at L_o of 0.55 ± .05 g and 0.26 ± 0.02 g in proximal and distal strips, respectively. Peak force generation in response to 10 µM carbachol (normalized to maximal KCl contractions) indicated a robust yet similar response of proximal (98.76 ± 6.40 %) and distal vaginal (97.37± 7.72%) strips. However, force generation of proximal strips in response to 50µM carbachol increased to 145 ± 9.53 % whereas force generation of distal strips remained virtually unchanged. Accordingly, 50 µM carbachol stimulated greater IP₃ production of proximal vaginal strips compared to distal strips at all time points following agonist addition.

Conclusions: We demonstrate for the first time a functional significance of muscarinic receptors in the vaginal muscularis through measurement of force generation and IP₃ production in response to carbachol stimulation. We also show a regional difference in the muscarinic receptor mediated contractile pathway. These results strongly suggest that vaginal contraction is under parasympathetic control and raise the possibility of targeting muscarinic receptors in order to regulate vaginal contractility.

UroToday.com Coverage of SUFU 2007

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