Endocannabinoid reactivity to acute stress: Investigation of the relationship between salivary and plasma levels
Introduction
The endogenous cannabinoid (eCB) system is an innate lipid signalling system made up of cannabinoid ligands arachidonoyl ethanolamine (AEA or anandamide) and 2-arachidonoyl glycerol (2-AG), as well as cannabinoid type 1 (CB1) and type 2 (CB2) receptors (Ligresti, De Petrocellis, & Di Marzo, 2016). Present in both the central and peripheral nervous systems, the eCB system is involved in a wide variety of functions, including immunity, pain responding, synaptic plasticity, appetite and mental health (Ligresti et al., 2016).
Recently, the role of the eCB system during acute stress responding has been extensively modelled in animal research, with both AEA and 2-AG involved in distinct roles during the stress response (Balsevich, Petrie, & Hill, 2017; Morena, Patel, Bains, & Hill, 2016). Specifically, AEA levels tonically gate the hypothalamic-pituitary-adrenal (HPA) stress response. Stress induction causes reduction in AEA in the hypothalamus and this reduction has been shown to be necessary for the production of cortisol (Hill & Tasker, 2012; Patel, Roelke, Rademacher, Cullinan, & Hillard, 2004). In contrast, 2-AG is reported to facilitate cortisol-induced negative feedback of the HPA response and thereby plays an important role in terminating the stress response (Hill & Tasker, 2012; Patel et al., 2004).
Understanding the involvement of the eCB system during stress is important because of the potential therapeutic use of cannabinoid drugs for stress-related disorders such as post-traumatic stress disorder (PTSD) (Hill, Campolongo, Yehuda, & Patel, 2018). Further, it is important to translate neurological processes identified in animal models to human studies for further clinical investigation. However, the few studies that have attempted to test this model in humans have been inconsistent, with reports of 2-AG either significantly increasing either immediately after stress (Crombie, Leitzelar, Brellenthin, Hillard, & Koltyn, 2019; Hill, Miller, Carrier, Gorzalka, & Hillard, 2009), not immediately but 45-minutes following stress (Schroeder et al., 2009), or decreasing in stress-resilient participants but not increasing in stress-prone participants (Chouker et al., 2010). Similarly, two studies found non-significant, small increases in AEA immediately following psychosocial stress (Hill et al., 2009; Mayo et al., 2020), one found decreases in AEA immediately following stress (Mayo et al., 2019) and two other studies found that AEA significantly increased immediately following stress (Crombie et al., 2019; Dlugos, Childs, Stuhr, Hillard, & de Wit, 2012). Notably, these studies used heterogeneous stress-inductions including social, physical and hemodynamic, which are not all relevant to PTSD. However, Crombie et al. (2019) recently reported a group*time interaction of 2-AG levels, with increased levels in control but not PTSD participants in response to both exercise and social stress. This suggests that peripheral eCB stress responses can differ between psychiatric populations.
Human studies of the eCB system are also generally limited by the necessity of blood sampling, with previous attempts at detecting eCBs in saliva being mostly unsuccessful (Battista et al., 2014). AEA and 2-AG are synthesised from the phospholipid N-arachidonoyl-phosphatidylethanolamine and diacylglycerols, respectively, through multiple pathways (Ligresti et al., 2016). There is good evidence that the majority of circulating eCBs do not originate from the central nervous system (Hill et al., 2008; Hillard, 2017; Jumpertz, Guijarro, Pratley, Piomelli, & Krakoff, 2011; Schmidt, Brune, & Hinz, 2006), suggesting that most eCBs measured in blood are synthesised in the peripheral system, such as in blood cells. There is also strong evidence that eCBs can be synthesised in the salivary glands, both with precursory compounds detectable in saliva (Fezza et al., 2003; Matias et al., 2012; Prestifilippo, Fernandez-Solari, Medina, Rettori, & Elverdin, 2009) and addition of arachidonic acid to saliva resulting in synthesis of AEA and 2-AG (Fezza et al., 2003). eCBs in blood are believed to mostly not be protein bound (Bojesen & Hansen, 2003) and therefore may possibly cross into salivary glands similar to free cortisol. However, whether salivary eCBs are predominately locally synthesised or originate from blood concentrations has not been tested to our knowledge.
Therefore, the current study formed part of a wider validation of a method to quantify eCBs in human saliva, with emphasis on the origin of salivary eCBs and the potential for salivary eCBs to display stress responsivity. Our goals were to determine whether salivary eCBs display stress reactivity, and whether salivary and plasma eCBs responded in a similar way to the existing animal model of eCB control of the HPA stress response (i.e., increase to 2-AG and decrease to AEA following stress). The study was divided into male and female arms, as limited resources restricted us from the ability to account for the impact of ovarian hormones in blood samples; however, the full cohort provided saliva. We hypothesised that, whilst salivary eCBs might show minimal stress reactivity, post-stress increases in plasma levels of 2-AG would be correlated with cortisol levels, whereas AEA levels would reduce rapidly following acute stress induction (Balsevich et al., 2017; Hill & Tasker, 2012; Morena et al., 2016). Based on existing evidence for sex differentiation of eCBs in various matrices (Fanelli et al., 2012; Mwanza et al., 2016; Ney, Matthews, Bruno, & Felmingham, 2018), we further hypothesised that female levels of AEA and 2-AG would be lower than male levels in saliva, and that male salivary eCB responding would be greater to stress than in females.
Section snippets
Participants
Forty-five healthy females aged 18–34 completed the female arm of the study. Participants were excluded if they had current psychiatric diagnoses (measured using the Kessler Psychological Distress Scale; K10, Kessler et al., 2002), were using medications (other than hormonal contraceptives), had serious neurological or physical illness, were pregnant, had used illicit drugs in the previous six months or reported a lifetime use of more than 15 times, were using tobacco, displayed problematic
Demographics
Twenty-three females and 16 males participated in the MAST condition, whereas 22 females and 16 males were randomised to the pMAST condition. Demographics between sexes and overall are reported in Table 1. Male participants were significantly older than female participants (p = .002). There were no significant differences in age between MAST and pMAST conditions (p=.640), and no Sex*Condition interaction for age (p = .834). There were no significant differences between conditions or sex on K10
Discussion
In this study we measured changes in salivary and plasma eCBs and cortisol 5-, 30- and 45-minutes following acute psychosocial stress induction. We report evidence for stress reactivity in male and female salivary 2-AG levels, with a significant increase 5-minutes following stress induction compared to the control condition. Women in our sample reported significantly higher subjective ratings of distress to the stress task and had significantly higher overall salivary cortisol responses than
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgements
This work was supported by an NHMRC Program grant to KLF (APP1073041).
References (52)
- et al.
Interpreting scores on the Kessler Psychological Distress Scale (K10)
Australian and New Zealand Journal of Public Health
(2001) - et al.
Endocannabinoids: Effectors of glucocorticoid signaling
Frontiers in Neuroendocrinology
(2017) - et al.
Binding of anandamide to bovine serum albumin
Journal of Lipid Research
(2003) - et al.
Sex differences in cannabinoid pharmacology: a reflection of differences in the endocannabinoid system?
Life Sciences
(2013) - et al.
Loss of exercise- and stress-induced increases in circulating 2-arachidonoylglycerol concentrations in adults with chronic PTSD
Biological Psychology
(2019) - et al.
Estimation of reference intervals of five endocannabinoids and endocannabinoid related compounds in human plasma by two dimensional-LC/MS/MS
Journal of Lipid Research
(2012) - et al.
Endocannabinoids and related fatty acid amides, and their regulation, in the salivary glands of the lone star tick
Biochimica et Biophysica Acta
(2003) - et al.
Endocannabinoid signaling, glucocorticoid-mediated negative feedback, and regulation of the hypothalamic-pituitary-adrenal axis
Neuroscience
(2012) - et al.
Circulating endocannabinoids and N-acyl ethanolamines are differentially regulated in major depression and following exposure to social stress
Psychoneuroendocrinology
(2009) - et al.
Sex differences in HPA axis responses to stress: A review
Biological Psychology
(2005)
Low estradiol levels: A vulnerability factor for the development of posttraumatic stress disorder
Biological Psychiatry
Why are women so vulnerable to anxiety, trauma-related and stress-related disorders? The potential role of sex hormones
The Lancet Psychiatry
A solid-phase method for the extraction and measurement of anandamide from multiple human biomatrices
Analytical Biochemistry
Simultaneous HPLC-APCI-MS/MS quantification of endogenous cannabinoids and glucocorticoids in hair
Journal of Chromatography B Analytical Technologies in the Biomedical and Life Sciences
Modulation of the endocannabinoid system by sex hormones: Implications for Posttraumatic Stress Disorder
Neuroscience and Biobehavioral Reviews
An alternative theory for hormone effects on sex differences in PTSD: The role of heightened sex hormones during trauma
Psychoneuroendocrinology
Cannabinoid interventions for PTSD: Where to next?
Progress in Neuro-psychopharmacology & Biological Psychiatry
Endocannabinoids mediate hyposalivation induced by inflammogens in the submandibular glands and hypothalamus
Archives of Oral Biology
Regulation of salivary gland function by autonomic nerves
Autonomic Neuroscience
Introducing the Maastricht Acute Stress Test (MAST): A quick and non-invasive approach to elicit robust autonomic and glucocorticoid stress responses
Psychoneuroendocrinology
Analytical approaches for the determination of phytocannabinoids and endocannabinoids in human matrices
Drug Testing and Analysis
Motion sickness, stress and the endocannabinoid system
PloS One
Concurrent validity of the alcohol use disorders identification test (AUDIT)
Alcohol and Alcoholism
Acute stress increases circulating anandamide and other N-acylethanolamines in healthy humans
Neuropsychopharmacology
Sex differences in schizophrenia, bipolar disorder and PTSD: Are gonadal hormones the link?
British Journal of Pharmacology
Integrating endocannabinoid signaling and cannabinoids into the biology and treatment of posttraumatic stress disorder
Neuropsychopharmacology
Cited by (18)
Trauma film viewing and intrusive memories: Relationship between salivary alpha amylase, endocannabinoids, and cortisol
2024, PsychoneuroendocrinologyRegular cannabis use is associated with history of childhood and lifetime trauma in a non-clinical community sample
2023, Journal of Psychiatric ResearchThe endocannabinoid system and posttraumatic stress disorder (PTSD): A new narrative
2023, Neurobiology and Physiology of the Endocannabinoid System