077 Comparison of the Actomyosin ATPase Inhibitor Calponin and the Sphingosine-1-phosphate Cell Differentiation Pathway Regulatory Enzymes Sphingosine Kinase 1 and Sphingomyelin Synthase in Vaginal Wall Smooth Muscle of Women with and without Pelvic Organ Pro

Pelvic Organ Prolapse (POP) is the descent of the vaginal walls and uterus leading to herniation of nearby organs into the vagina. POP prevalence as reported by patient symptoms is ∼3%, but it has been reported as high as 50% upon physical examination. Many women with POP also present with voiding dysfunction, defecatory dysfunction or dyspareunia. Abnormalities in smooth muscle (SM) in the vagina and endopelvic structures play a role in POP pathogenesis. Calponin is a protein thought to regulate SM contraction by inhibiting both myosin ATPase and bundling of actin filaments via binding to alpha-SM actin. Sphingosine kinase-1 (SPHK1) and sphingomyelin synthase (SMS) regulate the sphingosine-1-phosphate (S1P) pathway of SM contraction and differentiation. 1) compare expression of calponin, alpha-SM actin, SPHK1 and SMS in vaginal wall of women with and without POP, 2) determine cellular localization of calponin and alpha-SM actin in vaginal SM tissue. Case-control study comparing vaginal tissue samples from five patients with POP and five without POP. Tissues were 1 x 1 x 1 cm sections of full-thickness anterior vaginal apical tissue. Exclusion criteria: history of pelvic radiation, previous pelvic reconstruction surgery or connective tissue disorders. Tissue samples were pulverized to a powder under liquid nitrogen using a freezer mill and then homogenized in extraction buffer. Western Blot was performed using a no-stain gel line. Fluorescently tagged antibodies were used to determine relative expressions of α-actin and calponin. Blots were imaged and quantified using image analysis software with normalization to total lane protein. Full-thickness sections were placed in buffered formalin and then embedded in paraffin blocks. 4 μm tissue sections were cut by microtome and placed on slides. One slide was stained with hematoxylin and eosin. Sequential slides were de-paraffinized in xylene, blocked in serum, and incubated with alpha-SM or calponin antibodies. Expression and cellular localization of proteins were observed using immunofluorescence (IF) probes and microscopy. Independent t-test was performed to compare SM protein expression in POP versus control specimens.