Catecholamines are not linked to myometrial phospholipase C and uterine contraction in late pregnant and parturient mouse

Abstract

1. We investigated whether catecholamines through activation of alpha(1)-adrenergic receptors (alpha(1)-AR) are involved in mouse uterine contraction at parturition. Myometrial phospholipase C (PLC) activity and uterine contraction were measured in response to noradrenaline (NA), the specific alpha(1)-AR agonist phenylephrine (Phe) and oxytocin (OT). 2. Using the reverse transcription-polymerase chain reaction RT-PCR, we detected the alpha(1a)-AR subtype in late pregnant mouse myometrium. We also detected, by immunoblotting studies, PLCbeta(1), PLCbeta(3) and different alpha-subunits of pertussis toxin-insensitive (Galpha(q/11)) and -sensitive G proteins (Galpha(o/i3), Galpha(i1/2)). 3. Phenylephrine and NA did not alter the myometrial inositol phosphate (InsP) production of late pregnant or parturient mouse. In similar conditions, OT increased InsP production in a dose-dependent manner. Consistent with these results, only OT (10 microM) recruited PLCbeta(1) and PLCbeta(3) to myometrial plasma membranes. The OT-induced InsP response was not altered by pertussis toxin (300 ng ml(-1), 2 h pretreatment), suggesting the involvement of a member of the Galpha(q) family. 4. Noradrenaline and Phe failed to increase uterine contraction at late pregnancy and at parturition. In contrast, OT induced uterine contraction in a dose-dependent manner with maximal increase (400 %) at a concentration of 1 microM. 5. The results indicate that OT receptors (OTR) but not alpha(1)-AR are linked to myometrial PLC activation and uterine contraction in late pregnant and parturient mouse. This discrepancy between mouse and other mammals could be attributed to the alpha(1)-AR subtype expressed in myometrium at this time.

Figure 1. RT-PCR for α₁-AR subtypes from late pregnant mouse and rat myometria

Sequences of α_1a- (450 bp), α_1b- (470 bp) and α_1d-AR (650 bp) were amplified from cDNAs derived from late pregnant mouse myometrium (m.M), late pregnant rat myometrium (r.M) and rat brain (r.B). The DNA size markers at 50 bp increments are shown on the left.

Figure 2. Immunodetection of PLCβ isoforms and Gα_i/o and Gα_q/11 proteins in plasma membranes of late pregnant mouse myometrium

Representative immunoblots for PLCβ isoforms (A) and Gα_i/o and Gα_q/11 proteins (B) with rabbit polyclonal antibodies in late pregnant mouse myometrium (m.M) and positive controls (r.B, r.M: rat brain, pregnant rat myometrium, respectively). A, PLCβ₁ and PLCβ₃ (150 kDa) but not PLCβ₄ (130 kDa) are expressed in late pregnant mouse myometrium. B, Gα_o/i3 (40 kDa), Gα_i1/2 (39 kDa) and Gα_q/11 (42 kDa) are all present in late pregnant mouse myometrium.

Figure 3. Effects of phenylephrine and oxytocin on PLCβ₁ and PLCβ₃ translocation towards the plasma membrane

Strips from late pregnant mouse myometrium were incubated in the absence (basal) or presence of 10 μm phenylephrine (Phe) or oxytocin (OT). Plasma membranes were then prepared and studied for PLCβ₁ (A) or PLCβ₃ (B) expression using specific rabbit polyclonal antibodies. Responses were expressed as a percentage of the basal plasma membrane PLCβ₁ or PLCβ₃ amounts. Results are the mean of three independent experiments. **P < 0.01; ***P < 0.001versus basal; ns, not significant.

Figure 4. Inositol phophate production in response to phenylephrine and oxytocin and effect of pertussis toxin on oxytocin effect in late pregnant and parturient mouse myometrium

A, myometrial inositol phosphate (InsP) production of strips from late pregnant mice in response to increasing concentrations of phenylephrine (Phe) or oxytocin (OT) (0^.1 nm to 10 μm). B, myometrial InsP production in response to increasing concentrations of OT in the absence or presence of pertussis toxin (PTX) (300 ng ml⁻¹, 2 h pretreatment) at late pregnancy. Mean basal InsP production was 170 ± 15 c.p.m. (mg tissue)⁻¹ in the absence or presence of PTX. C, myometrial InsP production in response to increasing concentrations of OT, Phe and NA at parturition. Mean basal InsP production was 260 c.p.m. (mg tissue)⁻¹. Responses were expressed as a percentage of basal InsP production and results are the means of four independent experiments (A and B) and two independent experiments (C). *P < 0.05; **P < 0.01; ***P < 0.001versus basal.

Figure 5. Contractile responses of uterine strips to phenylephrine, noradrenaline and oxytocin in late pregnant mouse

A, representative recordings of uterine tissue tension in the presence of oxytocin (OT), phenylephrine (Phe) and noradrenaline (NA). OT increased both the frequency and amplitude of uterine tension in a dose-dependent manner whereas Phe and NA induced relaxation at high concentrations. B, average results from four independent experiments done in triplicate. Data are represented as a percentage of spontaneous contraction in the absence of drugs. *P < 0.05; **P < 0.01; ***P < 0.001versus spontaneous contraction.

Figure 6. Effects of propranolol pretreatment on phenylephrine- and noradrenaline-induced uterine relaxation

Uterine strips were pre-incubated in the absence or presence of the β-AR antagonist propranolol (10 min, 10 μm) before being challenged with increasing concentrations of phenylephrine (Phe) in A and noradrenaline (NA) in B. Data are represented as a percentage of spontaneous contraction and are the means of four independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001versus spontaneous contraction. Curves obtained for Phe and NA in the absence of propranolol are shown in Fig. 5B.