Nevertheless, pharmacological inhibition of anandamide amidohydrolase activity which can be achieved by using a number of covalent inhibitors with varying degrees of selectivity does not appear to affect the uptake of radioactive anandamide, at least when this is measured over brief time intervals . These findings indicate that, in the short-term, anandamide hydrolysis does not provide the driving force for anandamide transport. Importantly, however, they do not rule out an involvement of anandamide amidohydrolase in the long-term biodisposition of anandamide: it is conceivable, in fact, that anandamide transport into cells may be transiently driven by passive sequestration in lipid storage sites, as it may happen with free fatty acids , followed by a step of enzymatic hydrolysis. If this hypothesis is correct, prolonged inhibition of amidohydrolase activity would result in the intracellular accumulation of unmetabolized anandamide, and may eventually compromise its transmembrane transport and biodisposition. Thus, drugs that block anandamide amidohydrolase activity may cause long-term modififications of anandamide homeostasis, which might in turn be exploited therapeutically in an approach analogous to that used with monoamino oxidase inhibitors . To summarize, the biological actions of anandamide may be terminated by two temporally distinct, but functionally linked mechanisms. A high-affinity transporter protein may combine with extracellular anandamide and remove it from its sites of action by facilitated diffusion. Inside cells, anandamide may betemporarily collected in lipid stores to be then disposed of by enzymatic hydrolysis .Pharmacological experiments with cortical neurons in culture indicate that 2-AG formation is mediated by the PLC/DAG lipase pathway.

According to this hypothesis, illustrated in Fig. 3, PLC activity may catalyze the cleavage of a membrane phospholipid producing 1,2-diacylglycerol,indoor grow shelves which may be converted to 2-AG by DAG lipase activity. Thus 2-AG may be an intermediate component of a well known and ubiquitous enzymatic pathway that generates sequentially three distinct signaling molecules: 1,2-diacylglycerol, 2-AG, and arachidonic acid . Two of these molecules, 1,2-diacylglycerol and arachidonic acid, serve a host of regulatory functions, most of which have no apparent relationship with cannabinoid signaling. The question arises, therefore, of what biochemical checkpoints may be in place to control the traffic of these widely different messenger molecules. For several reasons, the consecutive reactions catalyzed by DAG lipase and monoacylglycerol lipase are likely to be essential. It is generally thought that the second messenger effects of 1,2-diacylglycerol, which include protein kinase C activation, are terminated by its conversion to phosphatidic acid, catalyzed by DAG kinase . This suggests that the presence of an active DAG lipase may be a critical factor in determining whether 2-AG will be formed under given circumstances, i.e., that a high DAG lipase/DAG kinase ratio may be characteristic of cells that have the need to produce substantial amounts of 2-AG from 1,2-diacylglycerol. Yet, the ability to generate 2-AG is not sufficient to conclude that a cell may use it as a signaling molecule. In platelets, for example, 2-AG is produced in large quantities but it is also immediately converted to arachidonic acid by monoacylglycerol lipase activity . This very short life span suggests that 2-AG may serve primarily as an intermediate in arachidonate release . In neurons, by contrast, 2-AG appears to escape immediate metabolism and to accumulate in response to Ca21 -mobilizing stimuli . Is such accumulation the consequence of a specific regulation of DAG lipase, DAG kinase, and monoacylglycerol lipase activities? The molecular characterization of neuronal DAG lipase and monoacylglycerol lipase should be instrumental to answer this question.

The dependence of anandamide and 2-AG formation on neural activity suggests that endogenous cannabinoids may be released from neurons in vivo and may contribute to the control of behavior. This possibility has not been confirmed directly yet, but finds support in pharmacological experiments with the CB1 antagonist SR141716A. The administration of SR141716A to naı¨ve animals causes a series of effects that may be accounted for by the reversal of an endogenous cannabinoid tone. For example, SR141716A produces hyperalgesia , arousal , memory improvement , anxiety-like responses , and increased acetylcholine release . Unfortunately, the inverse agonist properties of SR141716A and the current lack of biochemical information on the modalities of endogenous cannabinoid release in vivo make these pharmacological data difficult to interpret. One notable exception may be the pronociceptive effect of SR141716A administration in skin. The high levels of anandamide found in this tissue suggest that local CB1 receptors may be activated by the native ligand at least under certain conditions . An alternative experimental approach may be provided by the anandamide uptake inhibitor AM404. If this drug acts effectively in vivo, as initial studies appear to suggest, it may cause the accumulation of endogenous anandamide at its sites of action, and produce a selective ‘‘hypercannabinoid state’’ that should be reversed by CB1 receptor antagonists.Research has found persistent variation in SUD risk by sex. In the general population, men experience single and co-occurring SUDs at higher levels than women . Among SMs, however, sex differences are typically reduced or even reversed, with greater sexual orientation disparities among adult women compared to men, and especially elevated rates among bisexual women . Nonetheless, studies have rarely tested whether sex modifies relationships between sexual orientation and SUDs by including interaction terms in statistical models. Prevalence of SUDs tends to peak around age 25 and declines with age . Research examining SUDs among SMs, however, suggests a slower agenormative decline .

Rarely have researchers compared sexual orientation or gender identity disparities in SUDs among individuals older than 25 years with those in younger age groups. Knowledge of how the magnitude of sexual orientation and gender identity differences in SUDs vary by birth sex and age can help identify subgroups in need of interventions. This study analyzed data from the longitudinal Growing Up Today Study when participants were aged 20-35 to estimate sexual orientation and gender identity differences in probable SUDs. Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition criteria were used to assess past 12-month nicotine dependence, alcohol abuse/dependence, drug abuse/dependence, any SUD, and co-occurring multiple SUDs . Because research demonstrates sex differences in associations between sexual orientation and substance outcomes , we estimated statistical interactions between 1) sexual orientation and birth sex, and 2) gender identity and birth sex, and present birth-sex-stratified estimates. We hypothesized that SGMs would be more likely than non-SGMs of their same birth sex to meet criteria for SUDs, and that sexual orientation differences would be larger among participants assigned female at birth compared to those assigned male. Additionally, we estimated statistical interactions between 1) sexual orientation and age, and 2) gender identity and age. We hypothesized that sexual orientation and gender identity differences in SUD risk would be larger in older versus younger periods. Among participants meeting criteria for a past 12-month drug use disorder, we examined associations of sexual orientation and gender identity with past 12-month specific drug use.Sexual orientation.Past 12-month nicotine dependence, alcohol abuse/dependence, and drug abuse/dependence were assessed in 2010 and 2015-2017 with questions adapted from the National Survey on Drug Use and Health corresponding to DSM-IV criteria for SUDs . We coded responses as evidencing or not evidencing symptoms of dependence and abuse , classifying participants as having probable substance dependence if they endorsed 3 or more of 7 dependence symptoms and as having probable abuse if they endorsed at least 1 of 4 abuse symptoms. We then created 4 SUD variables: nicotine dependence , alcohol use disorder , drug use disorder ,indoor garden table and co-occurring multiple SUDs . GUTS questionnaires cover multiple health-related topics. Thus, to reduce participant burden, questions assessing drug use disorder for marijuana and other drugs were combined into a single set of questions. Drug use. Past 12-month use of marijuana, cocaine, heroin, MDMA/ecstasy, LSD/mushrooms, methamphetamine, amphetamines, and nonmedical use of prescription benzodiazepines, painkillers, sleeping pills, and stimulants were assessed in 2010 and 2015- 2017. Inhalants was assessed in 2010. Covariates. Race/ethnicity , region of residence , cohort , and age at the time of SUD assessment were included in analyses as potential confounders.Analyses were stratified by birth sex . Unadjusted prevalences of past 12-month SUD outcomes were examined for each sexual orientation and gender identity subgroup. We estimated multi-variable associations of SGM statuses with SUDs using generalized estimating equations with exchangeable correlations structure to account for non-independence of sibling clusters and repeated measures among individuals . When exchangeable correlation structure did not yield convergence for three models estimating drug type , we used independence correlation structure. For nicotine dependence, binary logistic regression estimated adjusted odd ratios . For alcohol use disorders, drug use disorders, and co-occurring SUDs, multi-nomial logistic regression estimated AOR. To test whether birth sex modified relationships between sexual orientation and SUDs and gender identity and SUDs, we included sexual-orientation-bysex and gender-identity-by-sex interaction terms.

To test whether age modified relationships between sexual orientation and SUDs and gender identity and SUDs, we included sexualorientation-by-age and gender-identity-by-age interaction terms stratified by birth sex. To estimate sexual orientation and gender identity differences in past 12-month use of specific drugs, we used binary logistic regression. In these analyses, we combined LGB participants into one category due to small sample sizes. In all models, CH and cisgender participants were referent groups. Corresponding 95% confidence intervals and p-values were estimated. Multi-variable models adjusted for age, race/ethnicity, cohort, region of residence, and birth sex . All analyses were performed with SAS software, version 9.4, with a significance level of 0.05.Table 3 presents the multi-variable associations of sexual orientation, gender identity, and other covariates with SUDs among participants assigned female at birth. The odds of evidencing each SUD and co-occurring multiple SUDs were greater among all SM groups compared to CHs.Table 4 presents the multivariable associations of sexual orientation, gender identity, and other covariates with SUDs among participants assigned male at birth. All SM groups had elevated odds for nicotine dependence and one SUD compared to CHs. MHs also had elevated odds of alcohol dependence, drug abuse and dependence, and having 2 or more SUDs. Gay men also evidenced elevated odds for alcohol abuse and dependence, drug dependence, and having 2 or more SUDs compared to CHs. GMs had significantly higher odds of alcohol dependence than their cisgender peers. Like patterns observed among participants assigned female, associations between gender identity and SUDs were frequently smaller than associations of sexual orientation with SUDs.Table 5 presents the prevalence and multi-variable associations of sexual orientation and gender identity with past 12-month drug use among participants evidencing a drug use disorder, stratified by birth sex. Regardless of sexual orientation and gender identity, marijuana was the most prevalent drug reported. Among participants assigned female, LGBs were more likely than CHs to report using MDMA/ecstasy and LSD/mushrooms and GMs were more likely than their cisgender counterparts to report using LSD/mushrooms. Among those assigned male, LGBs were more likely than CHs to report using methamphetamine and inhalant and GMs were more likely than cisgender peers to report using heroin, amphetamines, inhalants, and non-medical use of prescription painkillers. No differences were found between MH and CH in the prevalence of types of drugs used.Our study quantified sexual orientation and gender identity differences in SUD risk during young adulthood, when SUD prevalence in the general U.S. population is high . We examined SUDs based on DSM-IV criteria including nicotine dependence, alcohol abuse and dependence, drug abuse and dependence, and multiple co-occurring SUDs. Aligning with previous literature , we found that SM status was associated with greater odds of past 12-month SUDs among young adults assigned female, and to a lesser extent among those assigned male. Co-occurrence of 2 or more SUDs in the past 12-months was also more common among SMs compared CHs, aligning with previous studies of lifetime SUD co-occurrence . Contrary to our hypothesis, age-related declines in SUD prevalence were largely similar across sexual orientation and gender identity groups. This finding may be due, in part, to our sample age range and age periods compared in analysis . Previous studies have shown differential age-related declines in alcohol problems between SMs and heterosexuals and noted the largest sexual orientation differences in ages 40 or older . An analysis of representative U.S. data showed declines in the prevalence of tobacco and alcohol disorders among SMs between ages 26-35 but increases in prevalence between the mid-30s to mid-40s .