Involvement of β adrenergic receptors in spasmolytic effect of caulerpine on guinea pig ileum

Abstract Previously, we demonstrated that caulerpine has spasmolytic effect on guinea pig ileum. The aim of this study was to investigate pathways of its spasmolytic action. We test caulerpine against phasic contractions induced by carbachol in the circular layer of guinea pig ileum and this alkaloid did not inhibit these contractions, indicating that caulerpine did not interfering with the mobilisation of Ca2+ from intracellular stores. Additionally, the spasmolytic effect of caulerpine did not involve K+ channels. Furthermore, we observed that α2-adrenergic receptors were not involved in the spasmolytic effect of caulerpine, since the relaxation curve induced by caulerpine was not shifted in the presence of yohimbine (α2-adrenergic antagonist). However, in the presence of propranolol (β-adrenergic antagonist), the relaxation curve induced by caulerpine was right-shifted, resulting in a fivefold increase in EC50. Thus, a possible mechanism for the spasmolytic action of caulerpine is the activation of β-adrenergic receptors.


Introduction
Our research group has worked with some marine natural products, particularly from marine algae and their secondary metabolites (de Souza et al. 2009;Brito da Matta et al. 2011;Cavalcante-Silva et al. 2014). Recently, we described the antinociceptive effect of caulerpine (Souza et al. 2009), a bisindole alkaloid, which has been isolated mainly from green algae of the genus Caulerpa (Aguilar-Santos 1970; Ornano et al. 2014). We also demonstrated that this alkaloid antagonised phasic contractions induced by carbachol, histamine and serotonin on guinea pig ileum, showing a non-selective spasmolytic effect (Cavalcante-Silva et al. 2013). In addition, caulerpine relaxed, in a significant and concentration-dependent manner, the ileum pre-contracted by KCl and carbachol. Additionally, we observed that this effect was in part by inhibition of Ca 2+ influx through voltage-gated calcium channels (Ca V ) (Cavalcante-Silva et al. 2013). Thus, the aim of this study was to investigate the mechanism of spasmolytic action.

Results and discussion
Intestinal smooth muscle has two muscle layers, longitudinal and circular, and despite that the increase in [Ca 2+ ] c is involved in the contractile mechanism in both layers, the sources of Ca 2+ which lead to this increase differ. The contraction of longitudinal muscle layer depends on Ca 2+ influx through Ca V . However, the blockade of Ca 2+ influx or absence of this ion in the extracellular medium does not affect the contraction of the circular muscle layer, which indicates that this one depends on the mobilisation of Ca 2+ from intracellular stores to initiate contraction (Grider & Makhlouf 1988). In a previous study, we hypothesised that caulerpine blocks Ca 2+ influx through Ca V (Cavalcante-Silva et al. 2013). Thus, in this study, we evaluated the effect of caulerpine on carbachol-induced phasic contractions in the circular muscle layer to demonstrate whether this alkaloid interferes with the mobilisation of Ca 2+ from intracellular stores. It was observed that caulerpine (10 −4 and 3 × 10 −4 M) did not antagonise phasic contractions induced by 10 −6 M carbachol (CCh) in the circular muscle layer from guinea pig ileum (Figures S1 and  S2). This finding supports the hypothesis that this alkaloid acts by blocking the influx of Ca 2+ , without interfering with the mobilisation of Ca 2+ from intracellular stores. Considering the fact that the Ca V may be negatively modulated indirectly by the opening of K + channels, we decided to investigate whether caulerpine would activate these channels, thereby indirectly blocking Ca V . Accordingly, CsCl was used as a pharmacological tool to block the K + channels non-specifically (Cecchi et al. 1987). If this hypothesis was true, the relaxation curve of caulerpine in the presence of 5 mM CsCl would be significantly rightshifted. However, this was not observed. The EC 50 of caulerpine in the presence of CsCl (EC 50 = 3.0 ± 0.7 × 10 −5 M) did not differ from that in its absence (EC 50 = 4.7 ± 0.7 × 10 −5 M), indicating that caulerpine did not activate K + channels to cause the indirect blockade of Ca V (Figures 1 and S3 (A,B)).
The gastrointestinal tract is innervated by fibres from the sympathetic nervous system, and catecholamines modulate a variety of digestive functions, such as smooth muscle relaxation, secretion of neurotransmitters and electrolyte absorption (de Ponti et al. 1996). Some of these functions are mediated by the α 2 -adrenergic receptors through Gi protein-adenylyl cyclase (Gi-AC) (Bülbring & Tomita 1987), which are located in the epithelial cells and intestinal smooth muscle cells (Bauer 1982).
Besides α 2 -adrenergic receptors, β-adrenergic receptors are involved in the relaxation of intestinal smooth muscle. There are three subtypes of these receptors, β1, β2 and β3, and all are expressed in intestinal smooth muscle (Bauer 1982;de Ponti et al. 1996). due to the importance of these receptors in smooth muscle relaxation, we decided to investigate whether caulerpine would act on β receptors to exert its spasmolytic effect. Accordingly, propranolol was used as a pharmacological tool to antagonise β receptors non-specifically (Bauer 1982). In the presence of 5 μM propranolol, the caulerpine-induced relaxation curve was right-shifted, resulting in a fivefold increase in EC 50 (2.2 ± 0.2 × 10 −4 M, p < 0.001), suggesting, at the functional level, the involvement of β-adrenergic receptors in the spasmolytic effect of caulerpine (Figures 3 and S3 (A,d)). Furthermore, Hou et al. (2012) also demonstrated that other indole alkaloids, akuamidina and Z-alstoscholarine, exert a spasmolytic effect through β adrenergic receptors activation.
The results of this study reveal for the first time that caulerpine has a spasmolytic effect that does not involve the activation of K + channels or α 2 receptors but rather the activation of β-adrenergic receptors (Table S1). These results provide a major contribution to the pharmacological study of marine organisms, especially for algae along the north-east coast of Brazil, and show the applicability of this alkaloid as a prototype candidate in the development of spasmolytic molecules that will be used to treat gastrointestinal diseases, since in such therapy, there are still no compounds that act on the β-adrenergic pathway to exert a therapeutic effect.

Conclusions
In summary, this work showed that the mechanism of spasmolytic action of caulerpine, at the functional level, involves the activation of β-adrenergic receptors.