| Summary: | 碩士 === 國立成功大學 === 生理學研究所 === 91 === After implantation, pregnant uteri undergo a series of spatial and temporal remodeling including extracellular matrix rearrangement and smooth muscle adaptation to accommodate growing embryos. With the processing of pregnancy, smooth muscle cells of the myometrium are slightly hypertrophic with mild edema in the interstitial compartment in early- gestation uteri. In late-gestation, smooth muscle cells are prominently hypertrophic. Expression of smooth muscle myosin heavy chain (SMMHC), one of the most specific smooth muscle differentiation markers, increased in the hypertrophied state. In rat aortic vascular smooth muscle cells (VSMC) culture system, the expression of SMMHC in cultured vascular smooth muscle cells is related to NO-PKG signaling pathway. Nitric oxide (NO) attenuates vascular remodeling by inhibiting smooth muscle proliferation. Even though it is known that hypertrophy and hyperplasia of vascular smooth muscles contribute to the increase of vascular wall in response to chronic pulmonary hypertension, it is not clear whether the increased NO suppresses muscle hypertrophy by lowering the expression of SMMHC. Since hypertrophy also contributes to vascular remodeling, we further explored whether the elevation of NO lowers smooth muscle hypertrophy by reducing the expression of contractile proteins. Therefore, we hypothesize nitric oxide decreased the expression of SMMHC mediate through NO-PKG dependent pathway.
In this regards, pregnant uteri were used as the experimental model. Three objectives were designed 1) to characterize the influence of pregnancy on uterine remodeling, 2) to determine the effect of a NO donor on the expression of SMMHC related to NO-cGMP dependent pathway, and 3) to examine the influence of pregnancy on the protein abundance of the molecules related to the NO. Western blot analysis was used to measure the protein abundance of SM-MHC, SM α-actin, eNOS and PKG. Immunohistochemistry was used to localize SM α-actin and eNOS. The level of NO production was measured by Sievers 280 nitric oxide analyzer. The immunoprecipitation was used to detect the level of PKG phosphorylation.
The morphological data showed that, the implanted regions in G7 uteri have thicker wall with increased endometrial thickness than non-implanted regions. In contrast, the implanted regions of G18 had much thinner wall with dramatic decrease of both endometrial and myometrial thickness than the non-implanted regions of G18 uteri.
In NO donor modulating effects, our data shown: 1) SMMHC was decreased in response to the highest concentration of SNP (10-4M). 2) SM α-actin and non SM-actin was not affected by increasing concentrations of SNP (10-7M-10-4M). 3) sGC was not affected by increasing concentrations of SNP (10-7M-10-4M).4) SNP at 10-7 and 10-4M significantly lowered PKG abundance. 5) PKG phosphorylation was increased in response to SNP (10-4M) after incubated 1 hr and 24 hr.
In NO production data indicated: 1) In whole tissue extracts, the production of NO in non-implantation regions of G7 uteri was greater than that in implantation regions of G7. However, non-implanted regions of G18 uteri generated a similar amount of NO as implanted regions of G18 uteri did. 2) Endometrial NO from non-implanted regions of G7 uteri had much greater production of NO than other regions tested but there was no difference in implanted regions between G7 and G18 uteri. 3) After incubated with SNP for 24-hr, medial NO was increased with increasing concentrations of SNP (10-7 — 10-4 M).
In the influence of pregnancy, Western blot analysis indicated: 1) When compare the temporal effect: SMMHC expressed higher in late-gestation (G18) than early-gestation (G7); SM α-actin and non SM-actin was not significantly different between G7 and G18; eNOS expressed higher in G7 than G18; PKG was not significantly different between G7 and G18. 2) When compare the spatial effect: SMMHC, non SM-actin and PKG were not significantly different between non-implanted and implanted regions; SM α-actin in implanted regions was slightly lower than in non-implanted regions; eNOS expressed higher in implanted regions than in non-implanted regions.
Immunostaining results shown: When compared with non-implanted regions of G7 uteri, implanted regions of G7 uteri had relatively intense staining for the immunoreactive eNOS in the inner decidual reaction zone of endometrium but had similar intensity of the staining in myometrium layers. When compared with non-implanted regions of G18 uteri, implanted regions of G18 uteri had relatively intense staining for the immunoreactive eNOS in myometrial layers.
Our data suggested, at high concentration, NO-decreased SMMHC expression may not be mediated by PKG activation. Implantation enhances local expression of eNOS. With the progression of pregnancy, the differential distribution of eNOS in the implantation region may be related to regulate the NO level mediated SMMHC expression in pregnant uteri.
|
|---|
