Effect of progesterone on nitric oxide/cyclic guanosine monophosphate signaling and contraction in gastric smooth muscle cells

Abstract

Previous studies have shown that progesterone could inhibit muscle contraction in various sites of the gastrointestinal tract. The underlying mechanisms responsible for these inhibitory effects of progesterone are not fully known. The aim of the current study was to investigate the effect of progesterone on the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway and muscle contraction in the stomach. Single gastric smooth muscle cells from female Sprague-Dawley rats were used. The expression of progesterone receptor (PR) mRNA was analyzed by reverse transcription polymerase chain reaction. NO and cGMP levels were measured via specific ELISAs. Acetylcholine (ACh)-induced contraction of single gastric muscle cells preincubated with progesterone was measured via scanning micrometry in the presence or absence of the NO synthase inhibitor, Nω-Nitro-L-arginine (L-NNA), or guanylyl cyclase inhibitor, 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and expressed as percent shortening from resting cell length. PR expression was detected in the stomach muscle cells. Progesterone inhibited ACh-induced gastric muscle cell contraction. Furthermore, progesterone increased NO and cGMP levels in single gastric muscle cells. Most notably, pre-incubation of muscle cells with either L-NNA or ODQ abolished the inhibitory action of progesterone on muscle contraction. These present observations suggest that progesterone promotes muscle cell relaxation in the stomach potentially via the NO/cGMP pathway.

Keywords: contraction; nitic oxide/cyclic guanosine monophosphate pathway; progesterone; smooth muscle; stomach.

Figure 1.

Expression of PR A+B mRNA in rat GSMCs. Primers aligning to a common interior sequence of PRA and PRB mRNA amplified a 102 bp product (PR A+B) in polymerase chain reaction of rat GSMC RNA. The identity and integrity of the product was confirmed by electrophoresis in agarose gel in the presence of ethidium bromide. PR A+B, progesterone receptor A and B isoforms; GSMC, gastric smooth muscle cell.

Figure 2.

Effect of progesterone on NO level in single GSMCs. Total NO metabolites (NO₂⁻ and NO₃⁻) were measured as indicators of NO levels. Treatment of GSMCs with progesterone (1 µM) for 10 min significantly increased NO levels in GSMCs. Values shown are representative of at least four independent experiments performed in triplicate. Samples were collected from 10 female rats. *P<0.05 vs. basal. NO, nitric oxide; NO₂⁻, nitrite; NO₃⁻, nitrate; P, progesterone; GSMC, gastric smooth muscle cell.

Figure 3.

Effect of progesterone on cGMP formation in single GSMCs. Incubation with progesterone (1 µM) for 10 min significantly increased cGMP levels in GSMCs. Values shown are representative of at least four independent experiments performed in triplicate. Samples were collected from 10 female rats. *P<0.05 vs. basal. cGMP, cyclic guanosine monophosphate; P, progesterone; GSMC, gastric smooth muscle cell.

Figure 4.

Effect of progesterone on ACh-induced gastric muscle contraction. Treatment of GSMCs with progesterone (1 µM) for 10 min significantly reduced ACh-induced contraction. n=50 cells from 10 different rats.*P<0.05 vs. ACh. ACh, acetylcholine; GSMC, cyclic guanosine monophosphate.

Figure 5.

Effect of the blockade of nitric oxide synthase on progesterone-induced relaxation. Treatment of gastric smooth muscle cells with progesterone (1 µM) for 10 min significantly reduced ACh-induced contraction. Relaxation induced by progesterone was significantly inhibited in muscle cells pre-incubated with L-NNA (1 µM). n=50 cells from 10 different rats. *P<0.05 vs. ACh. **P<0.05 vs. ACh+P. ACh, acetylcholine; P, progesterone; L-NNA, Nω-Nitro-L-arginine.

Figure 6.

Effect of the blockade of soluble guanylyl cyclase on progesterone-induced relaxation. Treatment of gastric smooth muscle cells with progesterone (1 µM) for 10 min significantly reduced ACh-induced contraction. Relaxation induced by progesterone was inhibited in muscle cells pre-incubated with ODQ (1 µM). n=50 cells from 10 different rats. *P<0.05 vs. ACh. **P<0.05 vs. ACh+P. ACh, acetylcholine; P, progesterone; ODQ, 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one.