Impaired contractile G protein function in antrum and colon circular muscle from pregnant guinea pigs

Abstract

It has been previously shown that pregnancy has an inhibitory effect on the motility of gastrointestinal (GI) tract. We therefore examined the mechanisms responsible for this impaired GI motility in pregnant guinea pigs. Antrum and left colon were removed from adult third-trimester pregnant guinea pigs and nonpregnant female guinea pigs (control). Circular smooth muscle ceils were isolated by enzymatic digestion with collagenase and permeabilized by brief exposure to saponin as needed. Cell contraction was expressed as percent shortening with respect to control muscle cells. Contraction in response to CCK-8 (0.1 pM to 0.1 laM) was significantly reduced in both antrum and colon circular muscle from pregnant guinea pigs with a maximal shortening of only 14.2-+0.2% and 14.5 -+0.7% respectively when compared to 22.5 -+ 1.1% and 21.7 -+0.9% shortening in control animals (p<0.01, by ANOVA). Contraction in response to direct G protein activation with GTP,/S was also significantly reduced in antrum and colon circular muscle cells from pregnant guinea pigs. GTP)'S at 10 laM caused a maximal shortening of only 11.9 -+ 0.8% and 11.3 -+ 0.7% in antrum and colon circular muscle ceils from pregnant animals, respectively, as opposed to 20.1:1: 0.4% and 20.5 -+ 1.1% in controls. In contrast, the contraction of both antrum and colon circular muscle cells induced by a second messenger IP3 was similar between pregnant and control guinea pigs. Furthermore, the contractile responses to potassium chloride, which is a receptor-G protein independent agonist, were not significantly different from controls, These data suggested that pregnancy might affect the function of G proteins in the GI circular smooth muscle. To further confirm this hypothesis, the function of G proteins in antrum and colon circular muscle from both groups of animals was assessed by using [35S]GTP'¢S binding. Crude membranes derived from antrum and colon circular muscle were incubated with 30 nM [35S]GTPTS with or without agonist stimulation. CCK-8 at 1paM caused a significant increase of [35S]GTP)'S binding to Gq/1 let over the basal level (without CCK), but not to Gietl-2, Giet3, or Gsa. The stimulation of Gq/1 let binding was significantly reduced in antrum and colon circular muscle from pregnant guinea pig when compared to that from control animals (p<0.05, by t test). However, VIP at 1 taM caused a significant increase of [35S]GTPyS binding to Gset, but not to Gq/llet, Gietl-2 or Giot3. The stimulation of Gset binding induced by VIP was similar between these two groups. In conclusion, pregnancy appears to impair gastrointestinal muscle contractility by downregulating certain G proteins that mediate muscle contraction such as Gq/1 let protein. This research was supported by NIH grant R01-DK27389.