Effects of progesterone on motility and prostaglandin levels in the distal guinea pig colon

Abstract

Progesterone (P4) inhibits the gastrointestinal muscle contraction by downregulating Galpha(q/11) proteins that mediate contraction, by upregulating Galpha(s) proteins that mediate relaxation, and by altering the pattern of cyclooxygenase (COX) enzymes and prostaglandins. We aimed to examine whether P4 treatment of guinea pigs in vivo affects basal colon motility [basal motility index (MI)] by altering the levels and actions of PGF(2alpha) and PGE(2). Guinea pigs were treated with intramuscular (IM) P4 for 4 days. The BASAL MI, the PGF(2alpha)-induced contraction, and PGE(2)-induced inhibition of contraction were examined in muscle strips and cells. The levels of PGF(2alpha) and PGE(2) were measured by radioimmunoassay. Treatment with P4 reduced the basal MI, the levels of PGF(2alpha), and PGF(2alpha)-induced contraction. P4 increased PGE(2) levels, and PGE(2) induced relaxation. Pretreatment with IM RU-486 (10 mg/kg per day), a P4 receptor antagonist, 1 h before P4 blocked the actions of P4. The PGF(2alpha) antagonist Al-1180 abolished basal MI and PGF(2alpha)-induced contraction. N-ethylmaleimide, which blocks unoccupied membrane receptors, blocked Ach and VIP actions but had no effect on PGF(2alpha) and PGE(2) effects. A COX-1 inhibitor decreased and a COX-2 inhibitor increased PGF(2alpha) levels; GTPgammaS increased and GDPbetaS decreased the levels of PGF(2alpha). Galpha(q/11) protein antibodies (Abs) reduced PGF(2alpha) levels, and Galpha(i3) Abs blocked its motor actions. Galphas Abs increased PGF(2alpha) but decreased PGE(2) levels. We concluded that P4 decreases basal MI by reducing PGF(2alpha) levels caused by downregulation of Galpha(q/11) and that PGF(2alpha)-induced contraction was blocked by downregulating Galpha(i3). P4 also decreased the basal MI by increasing PGE(2) levels, and PGE(2) induced relaxation by upregulating Galpha(s) proteins.

Fig. 1.

The basal motility index (MI) of colon muscle strips treated with progesterone (P4) (2 mg/kg per day) or saline intramuscular (IM) for 4 days. A: basal motility tracings of muscle strips from guinea pigs treated with saline (control) or P4. B: P4 decreased the basal MI of muscle strips compared with saline treatment (*P < 0.01 by ANOVA). Values are means ± SE of basal MI (n = 4).

Fig. 2.

A: P4 blocked the contraction (Δ MI) of ACh (10⁻⁶ M) and PGF₂α (10⁻⁶ M) (*P < 0.001, **P < 0.005 by ANOVA). B: in contrast, P4 increased the percent inhibition of contraction induced by PGE₂ (10⁻⁶ M) (#P < 0.05 by ANOVA). C: there were no differences in the percent inhibition of contraction of muscle cells induced by 8-bromo c′AMP (8B-cAMP) between P4- and saline-treated muscle cells. Values are means ± SE of n = 3.

Fig. 3.

A: treatment with P4 in vivo decreased the levels of PGF₂α (*P < 0.001 by ANOVA) and was blocked by pretreatment with RU-486 (RU) (#P < 0.001 by ANOVA, mg/kg body wt). B: P4 increased the levels of PGE₂ (**P < 0.01 by ANOVA) and was blocked by pretreatment with RU-486 (##P < 0.01 by ANOVA). Values are means ± SE of n = 4.

Fig. 4.

Effects of selective and nonselective (indomethacin) inhibitors on basal MI (A) and on PGF_2α levels (B) in cells from normal animals. SC560, a cyclooxygenase (COX)-1 inhibitor (10⁻⁵ M), decreased the basal MI (*P < 0.01 by ANOVA) and the levels of PGF_2α (#P < 0.01 by ANOVA). In contrast, NS398 (10⁻⁵ M), a COX-2 inhibitor, increased the basal MI (**P < 0.01 by ANOVA) and the levels of PGF_2α (##P < 0.05 by ANOVA). Indomethacin (10⁻⁵ M) lowered the basal MI and the PGF_2α levels (&P < 0.01 by ANOVA). Values are means ± SE of n = 4.

Fig. 5.

A: Al-8810 (10⁻⁶ M) blocked the contraction of muscle cells induced by increasing concentrations of PGF₂α (10⁻¹⁰–10⁻⁶ M) (P < 0.001, by ANOVA). B: Al-8810 (10⁻⁶ M) abolished the basal MI of colon muscle strips (*P < 0.001 by ANOVA). Values are means ± SE of n = 4.

Fig. 6.

Effect of N-ethylmaleimide (NEM) (10⁻⁴ M) on the basal MI of colon muscle strips and on the contraction induced by exogenous agonists. Left: NEM 10⁻⁴ M had no effect on the basal MI. Middle: in muscle strips, NEM blocked the contraction induced by ACh (10⁻⁶ M) (*P < 0.001 by ANOVA) but had no effect on the contraction induced by PGF₂α (10⁻⁶ M). Right: NEM blocked the contraction induced by ACh (10⁻⁶ M) in dissociated muscle cells (**P < 0.001 by ANOVA) but also had no effect on PGF₂α-induced contraction. Values are means ± SE of n = 4.

Fig. 7.

Effects of NEM (10⁻⁴ M) on the inhibition of contraction induced by agonists. NEM blocked the effect of the exogenous inhibitor VIP (10⁻⁶ M) (#P < 0.001 by ANOVA) but had no effect on the inhibition of contraction induced by PGE₂ (10⁻⁶ M) (an endogenous autacoid) and by 8B-cAMP (10⁻⁵ M) (receptor- and G protein-independent agonist). Values are means ± SE of n = 3.

Fig. 8.

A: GTPγS (10⁻⁴ M) increased the levels of PGF₂α (*P < 0.02 by ANOVA). This increase was blocked by pretreating muscle cells with P4 (10⁻⁵ M) (**P < 0.02 by ANOVA). B: GDPβS (10⁻⁴ M) reduced the levels of PGF₂α (#P < 0.01 by ANOVA). P4 (10⁻⁵ M) alone also reduced the levels of PGF₂α (##P < 0.01 by ANOVA). However, the addition of GDPβS (10⁻⁴ M) to muscle cells pretreated with P4 had no additional effects (10⁻⁵ M). Values are means ± SE of n = 4.

Fig. 9.

The effects of G protein antibodies (Abs) on levels of PGF₂α and PGE₂ in permeable colon muscle cells from normal animals. Left: Abs against Gα_q/11 decreased (*P < 0.01 by ANOVA) and Abs against Gα_s increased the basal levels of PGF₂α (**P < 0.01 by ANOVA). Right: Abs against G_αs lowered (#P < 0.005 by ANOVA) and Gα_q/11 Abs increased (##P < 0.01 by ANOVA) the levels of PGE₂. Values are means ± SE of n = 3.

Fig. 10.

A: effect of G protein Abs on contraction induced by PGF₂α (10⁻⁶ M) in normal permeable colon muscle cells. Abs against Gα_i3 blocked the contraction induced by PGF₂α (10⁻⁶ M) (*P < 0.01 by ANOVA), whereas Abs against Gα_q/11, Gα_i1/2, and Gα_s had no effect. Values are means ± SE of n = 4. B: Gα_s protein Ab treatment in normal permeable cells almost completely abolished the inhibition of contraction induced by PGE₂ (10⁻⁶ M) compared with that induced in control muscle cells (#P < 0.005 by ANOVA). Values are means ± SE of n = 3.

Fig. 11.

Effect of pertussis toxin (PTX) (100 μg/ml) on muscle contraction induced by PGF₂α (10⁻⁶ M) and basal levels of PGF₂α in intact colon muscle cells. A: PTX reduced the basal MI (*P < 0.01 by ANOVA). B: it also decreased the contraction induced by PGF₂α (10⁻⁶ M) (**P < 0.01 by ANOVA). C: PTX had no effect on the PGF₂α levels. Values are means ± SE of n = 4.