Arecoline induces dual modulation of blood pressure in rat, including an initial downregulation and a subsequent upregulation

Purpose: To determine the role of arecoline in cardiovascular modulation in rats. Methods: After rats were anaesthetized with intraperitoneal urethane (1.4 g/kg body weight), saline or arecoline (at doses of 1.0, 3.0 and 10.0 mg/kg) was intraperitoneally administered, and blood pressure (BP) was continuously recorded using a physiological apparatus. Mean arterial pressure (MAP), maximum changes in MAP and reaction time due to arecoline stimulations were calculated and analyzed. Results: Arecoline induced biphasic modulation in BP, including an initial downregulation followed by a subsequent upregulation. The MAP and maximum change in MAP exhibited a concentration-dependent effect in the downregulation phase (p < 0.001 within each group), but not in the upregulation phase (p > 0.05 within each group), while BP reaction time showed a dose-dependent prolongation in both downregulation and upregulation phases (ps < 0.01 within each group). Remarkably, arecoline-induced BP downregulation more rapidly and drastically than upregulation in each arecoline group. Conclusion: These results indicate that arecoline exerts a complex effect in cardiovascular modulation that should be considered as side effects in the clinical use of arecoline and/or with the habitual chewing of areca nuts.


INTRODUCTION
nuts are the fruits of the Areca catechu palm tree, which are endemic to South and Southeast Asia (especially in China, India, Indonesia, Malaysia, Philippines, and New Guinea). Areca nuts are mainly used for chewing, which is reported to cause relaxation, evoke euphoria, and enhance postprandial satisfaction [1,2]. It is estimated that more than 600 million people frequently chew areca nuts, making it the fourth most widely-used psychoactive substance in the world, after alcohol, tobacco, and caffeine [1,3,4]. Many studies have demonstrated that areca nut chewing causes dependency syndrome and withdrawal syndrome, resulting in emotional fluctuations, agrypnia, anxiety and irritability [3]. Nevertheless, areca nuts are a popular herbal medicine that has long been used in clinical practice, such as the treatment of parasitic diseases, digestive disorders, and inflammation [1].
Arecoline is the main bioactive alkaloid in areca nuts, and it has several pharmacological effects that are parasympathetic in nature [5]. Arecoline has frequently been reported to exhibit beneficial effects in patients with Alzheimer dementia (AD), schizophrenia, and depression [6][7][8][9]. Because of the parasympathetic features, arecoline also causes cardiovascular modulations, but with contradictory effects. For example, many studies recorded that chewing areca nuts raised the risk of cardiovascular disorders [10][11][12][13], while other studies reported that arecoline decreased or increased blood pressure (BP) and heart rate [14][15][16]. Also, other studies found that arecoline failed to exert a significant effect altogether on BP [9].
The present study aimed to explore whether and how arecoline modulated BP, to provide a reference for clinical practice and a perspective for preclinical investigations on the pharmacological and toxicological characteristics of arecoline, or areca nuts in general.

EXPERIMENTAL Animals
Healthy adult male Sprague-Dawley rats (2 months old, 240 ± 20 g; n = 17) were purchased from Jinan Pengyue Experimental Animal Breeding Co. LTD (Jinan, China). The animals were housed in a temperature-controlled (25 ± 1 °C) environment with a 12 h light/12-h dark cycle and ad libitum feed and water. All animal experiments abided by the rules of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised in 1996), and were approved by the Academic and Ethics Committee of Lingnan Normal University.

Surgical procedures
Blood pressure recordings followed the procedure used in previous reports [17,18]. The rats were anesthetized with intraperitoneal urethane (1.4 g/kg body weight; Sinopharm Chemical Reagent Co. Ltd., Shanghai, China), followed by cervical surgery and tracheal intubation. Then, a catheter was inserted into the right carotid artery and connected to a physiological apparatus (BL-420F, Taimeng Technology Co. Ltd., Chengdu, China) for BP signal collecting and processing.

Rats
were maintained in the ventral decubitus position for BP recordings. Once the BP was stable, saline (0.9 % NaCl) or arecoline (at a dose of 1.0, 3.0 and 10.0 mg/kg/1.0 mL) was separately injected (~10 s) into the abdominal cavity. The effects of arecoline on BP regulations were regarded as substance-specific if they were reversible and reproducible.
Repeated injections were administered at intervals of 60 min to avoid the drug interference between administrations [19]. In general, each rat was given 4 -6 injections with different drugs, and those that did not return to the basal level (i.e., those that deviated over 10 % from the pretest levels) were excluded from further analyses.
Arecoline induced biphasic modulations in the BP (an initial downregulation, followed by a subsequent upregulation). The reaction times of BP responses to arecoline stimulations (including period 1 for the BP downregulation phase, and period 2 for the BP upregulation phase), mean arterial pressure (MAP) during period 1 and period 2, the maximum change in MAP, including MDMAP (maximum decreased MAP) and MIMAP (maximum increased MAP) during each arecoline trial were calculated. Since no alterations in BP were observed upon saline treatment, the MAPs in period 1 and period 2 for the saline treatment were calculated from two 60sec BP sequences beginning at 10 sec and 100 sec, respectively.

Statistical analysis
All data are presented as mean ± standard error of the mean (SEM). A one-way analysis of variance (ANOVA) followed by a Fisher's least significant difference post hoc test were conducted for statistical analyses. P < 0.05 was considered statistically significant.

RESULTS
Compared to the saline treatment group (Figure  1 A), arecoline treatment induced biphasic modulations in the BPs of the rats, i.e., an initial downregulation followed by a subsequent upregulation (Figure 1 B -D).  Figure 2).

Arecoline induced maximum changes in MAP
Post hoc analyses indicated that the MDMAP in period 1 were significantly different among different arecoline treatments (F2,25 = 13.302, p < 0.001; Figure 3). Remarkably, the MDMAP in the 10.0 mg/kg arecoline-treatment group increased by 101.27% compared to that of the 1.0 mg/kg treatment (p < 0.001; Figure 3). Post hoc analyses also revealed that MIMAP in in period 2 (F2,25 = 0.358, p = 0.703; Figure 3), was not significantly different among arecoline treatments.

Reaction time of arecoline-induced BP modulations
The BP reaction time due to arecoline treatment was also significantly different between groups in period 1 (F2,25 = 47.889, p < 0.001, Figure 4; in which the reaction time was prolonged by 123.15% in the 10.0 mg/kg arecoline-treatment group compared to that in the 1.0 mg/kg treatment group) and in period 2 (F2,25 = 6.375, p = 0.006, Figure 4; in which the reaction time was prolonged by 78.97% in the 10.0 mg/kg arecoline-treatment group compared to the 1.0 mg/kg treatment group).

BP downregulation and upregulation due to arecoline stimulations
The arecoline-induced BP downregulations were of higher magnitude (1-3 fold in MDMAP vs. MIMAP) than the upregulations. However, the durations of downregulation were longer (~5 fold in period 2 vs. period 1) than those of the upregulation (Table 1). Thus, those data indicated the intense, but transient downregulation relative to the upregulation in response to arecoline treatments.

DISCUSSION
Arecoline has long been known to exhibit multiple cholinomimetic effects that are predominantly parasympathetic in nature. Such effects have frequently been studied in animal models and observed in clinical settings, including effects on stress reduction, cardiovascular modulation, digestive promotion, and endocrine functions [5][6][7][8]20,21]. However, relatively little is known about how arecoline regulates BP. This study reported that arecoline evoked biphasic modulations on the BPs of rats, with an initial downregulation in BP, followed by a subsequent upregulation.
The BP changes induced by arecoline (1.0, 3.0 and 10.0 mg/kg) generally recovered within 800 sec (Figure 1), which was consistent with previous reports on arecoline action times in behavioral demonstrations [8] and metabolic examinations [4]. Arecoline-induced decreases in BP may be due to arecoline-mediated relaxation of the aorta endothelium and/or improvement of vasorelaxation [2]. However, arecoline-mediated BP upregulations cannot be explained merely by the cholinergic effect. Notably, several lines of experimental evidence suggest that arecoline is excitatory to the hypothalamic-pituitary-adrenal (HPA) axis and stimulates adrenocorticotropic hormone release [22]. Therefore, it is hypothesized that arecoline-mediated upregulation of BP may be due to the activation of the HPA axis. However, the precise mechanisms leading to the biphasic regulations in BPs require further investigations.

CONCLUSION
Given that arecoline or areca nuts are frequently used as therapeutic substances for various ailments, the findings of this study suggest that the cardiovascular effects of such treatments be taken into consideration during clinical use.

DECLARATIONS
institutions for access and distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/ 4.0) and the Budapest Open Access Initiative (http://www.budapestopenaccessinitiative.org/rea d), which permit unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.