Influence of β2- and β3-adrenoceptor agonists on contractile activity of the porcine myometrium in the luteal phase and the first days of pregnancy

This study analysed the relaxant properties of salbutamol (β2-adrenoceptors agonist) and BRL 37344 (β3-adrenoceptors agonist) regarding the contractility of porcine myometrium on days 10-14 of the oestrous cycle (cyclic group; n = 10) and on days 3-5 of pregnancy (early pregnant group; n = 6). The activity of myometrial strips (tension, frequency and amplitude) was recorded under isometric conditions using force transducers. The contractility was assessed further following the administration of increasing concentrations of the agonists (10-9-10-4M), both with and without β-adrenoceptor antagonists (butaxamine - a selective β2- adrenoceptor antagonist, propranolol- a non-selective β1- and β2-adrenoceptor antagonist and bupranolol - a non-selective β1-, β2- and β3-adrenoceptor antagonist) at a concentration of 10-4M. Although neither salbutamol nor BRL 37344 caused changes in the tension, at the highest concentrations they decreased the frequency and amplitude of contractions. These changes were more evident after salbutamol treatment and in the early pregnant group. Antagonists given alone did not cause changes in the parameters examined but changed some activity of the agonists. Butoxamine reduced the decrease in frequency and amplitude induced by salbutamol and produced a decrease in the tension after BRL 37344 treatment in the early pregnant group. Propranolol reduced the decrease in frequency and amplitude induced by salbutamol in both examined groups and did not cause significant changes in BRL 37344 activity. The administration of bupranolol before salbutamol treatment caused an increase in the tension and reduced the decrease in the frequency in the cyclic group. Moreover, bupranolol eliminated a decrease in frequency and induced an increase in amplitude caused by BRL 37344 in both groups and these changes were more evident in the early pregnant group. The data indicates that both β2- and β3-adenoreceptors are involved in the regulation of the contractility in both groups, but the changes after agonists and antagonists treatment are more evident in the early pregnant myometrium.

sGCα1 mediates the negative inotropic effects of NO in cardiac myocytes independent of changes in calcium handling

In the heart, nitric oxide (NO) modulates contractile function; however, the mechanisms responsible for this effect are incompletely understood. NO can elicit effects via a variety of mechanisms including S-nitrosylation and stimulation of cGMP synthesis by soluble guanylate cyclase (sGC). sGC is a heterodimer comprised of a β1- and an α1- or α2-subunit. sGCα1β1 is the predominant isoform in the heart. To characterize the role of sGC in the regulation of cardiac contractile function by NO, we compared left ventricular cardiac myocytes (CM) isolated from adult mice deficient in the sGC α1-subunit (sGCα1−/−) and from wild-type (WT) mice. Sarcomere shortening under basal conditions was less in sGCα1−/− CM than in WT CM. To activate endogenous NO synthesis from NO synthase 3, CM were incubated with the β3-adrenergic receptor (β3-AR) agonist BRL 37344. BRL 37344 decreased cardiac contractility in WT CM but not in sGCα1−/− myocytes. Administration of spermine NONOate, an NO donor compound, did not affect sarcomeric shortening in CM of either genotype; however, in the presence of isoproterenol, addition of spermine NONOate reduced sarcomere shortening in WT but not in sGCα1−/− CM. Neither BRL 37344 nor spermine NONOate altered calcium handling in CM of either genotype. These findings suggest that sGCα1 exerts a positive inotropic effect under basal conditions, as well as mediates the negative inotropic effect of β3-AR signaling. Additionally, our work demonstrates that sGCα1β1 is required for NO to depress β1/β2-AR-stimulated cardiac contractility and that this modulation is independent of changes in calcium handling.

Progesterone decreases the relaxing effect of the β3-adrenergic receptor agonist BRL 37344 in the pregnant rat myometrium

Although the published results regarding the function of the β3-adrenergic receptors (β3-ARs) in the regulation of smooth muscle activity are very promising, the question of the mechanism of β3-ARs' action in the pregnant myometrium cannot be fully answered by human investigations. To assess whether it possesses an essential role in the regulation of uterine contractility in pregnant rats, as in humans, we performed functional, western blotting and molecular biology experiments on the late-pregnant rat myometrium. The influence of progesterone on the function of the β3-ARs was also investigated. We demonstrated the presence and the functional activity of the β3-ARs in the late-pregnant rat myometrium. The maximum dose-dependent uterus-relaxing effect of the selective β3-agonist BRL 37344 was recorded at the end of pregnancy in rats, similarly as in humans. The extent of its relaxing action was regarded as moderate. The expression of β3-AR protein and mRNA remained unchanged during the investigated period. The administration of progesterone had no effect on the β3-AR mRNA and protein expression or the maximum relaxation effect of BRL 37344, but shifted the dose–response curve to the right and decreased the synthesis of the second messenger, cAMP. It can be concluded that the β3-ARs play an additional role in the regulation of the contractile activity of the pregnant rat uterus. The inhibitory effect of progesterone on the functional activity of the β3-ARs may have important consequences in the case of human application if this effect is also demonstrated in pregnant human myometrial tissue.

NO production and eNOS phosphorylation induced by epinephrine through the activation of β-adrenoceptors

Epinephrine plays a key role in the control of vasomotor tone; however, the participation of the NO/cGMP pathway in response to β-adrenoceptor activation remains controversial. To evaluate the involvement of the endothelium in the vascular response to epinephrine, we assessed NO production, endothelial NO synthase phosphorylation, and tissue accumulation of cGMP in the perfused arterial mesenteric bed of rat. Epinephrine elicited a concentration-dependent increase in NO (EC50 of 45.7 pM), which was coupled to cGMP tissue accumulation. Both NO and cGMP production were blocked by either endothelium removal (saponin) or NO synthase inhibition ( Nω-nitro-l-arginine). Blockade of β1- and β2-adrenoceptors with 1 μM propranolol or β3-adrenoceptor with 10 nM SR 59230A displaced rightward the concentration-NO production curve evoked by epinephrine. Selective stimulation of β1-, β2-, or β3-adrenoceptors also resulted in NO and cGMP production. Propranolol (1 μM) inhibited the rise in NO induced by isoproterenol or the β2-adrenoceptor agonists salbutamol, terbutaline, or fenoterol. Likewise, 10 nM SR 59230A reduced the effects of the β3-adrenoceptor agonists BRL 37344, CGP 12177, SR 595611A, or pindolol. The NO production induced by epinephrine and BRL 37344 was associated with the activation of the phosphatidylinositol 3-kinase/Akt pathway and phosphorylation of eNOS in serine 1177. In addition, in anaesthetized rats, bolus administration of isoproterenol, salbutamol, or BRL 37344 produced NO-dependent reductions in systolic blood pressure. These findings indicate that β1-, β2-, and β3-adrenoceptors are coupled to the NO/cGMP pathway, highlighting the role of the endothelium in the vasomotor action elicited by epinephrine and related β-adrenoceptor agonists.

Stimulation of β3-adrenoceptors relaxes rat urinary bladder smooth muscle via activation of the large-conductance Ca2+-activated K+ channels

We investigated the role of large-conductance Ca2+-activated K+ (BK) channels in β3-adrenoceptor (β3-AR)-induced relaxation in rat urinary bladder smooth muscle (UBSM). BRL 37344, a specific β3-AR agonist, inhibits spontaneous contractions of isolated UBSM strips. SR59230A, a specific β3-AR antagonist, and H89, a PKA inhibitor, reduced the inhibitory effect of BRL 37344. Iberiotoxin, a specific BK channel inhibitor, shifts the BRL 37344 concentration response curves for contraction amplitude, net muscle force, and tone to the right. Freshly dispersed UBSM cells and the perforated mode of the patch-clamp technique were used to determine further the role of β3-AR stimulation by BRL 37344 on BK channel activity. BRL 37344 increased spontaneous, transient, outward BK current (STOC) frequency by 46.0 ± 20.1%. In whole cell mode at a holding potential of Vh = 0 mV, the single BK channel amplitude was 5.17 ± 0.28 pA, whereas in the presence of BRL 37344, it was 5.55 ± 0.41 pA. The BK channel open probability was also unchanged. In the presence of ryanodine and nifedipine, the current-voltage relationship in response to depolarization steps in the presence and absence of BRL 37344 was identical. In current-clamp mode, BRL 37344 caused membrane potential hyperpolarization from −26.1 ± 2.1 mV (control) to −29.0 ± 2.2 mV. The BRL 37344-induced hyperpolarization was eliminated by application of iberiotoxin, tetraethylammonium or ryanodine. The data indicate that stimulation of β3-AR relaxes rat UBSM by increasing the BK channel STOC frequency, which causes membrane hyperpolarization and thus relaxation.