Effects of Δ9-Tetrahydrocannabinol and the Aminoalkylindole K2/Spice Constituent JWH-073 on Cardiac Tissue and Mesenteric Vascular Reactivity.

Background: Although use of Cannabis sativa is not associated with serious adverse effects, recreational use of aminoalkylindole (AAI) cannabinoid receptor agonists found in K2/Spice herbal blends has been reported to cause adverse cardiovascular events, including angina, arrhythmia, changes in blood pressure, ischemic stroke, and myocardial infarction. Δ9-Tetrahydrocannabinol (Δ9-THC) is the primary CB1 agonist found in cannabis and JWH-073 is one of the AAI CB1 agonists found in K2/Spice brands sold to the public. Methods: This study used in vitro, in vivo, and ex vivo approaches to investigate potential differences on cardiac tissue and vascular effects betweenJWH-073 and Δ9-THC. Male C57BL/6 mice were treated with JWH-073 or Δ9-THC and cardiac injury was assessed by histology. Effects of JWH-073 and Δ9-THC on H9C2 cell viability and ex vivo mesenteric vascular reactivity were also determined. Results: JWH-073 or Δ9-THC induced typical cannabinoid effects of antinociception and hypothermia but did not promote death of cardiac myocytes. No differences in cell viability were observed in cultured H9C2 cardiac myocytes after 24 h of treatment. In isolated mesenteric arteries from drug-naive animals, JWH-073 produced significantly greater maximal relaxation (96%±2% vs. 73%±5%, p<0.05) and significantly greater inhibition of phenylephrine-mediated maximal contraction (Control 174%±11%KMAX) compared with Δ9-THC (50%±17% vs. 119%±16%KMAX, p<0.05). Discussion: These findings suggest that neither cannabinoid at the concentrations/dose studied caused cardiac cell death, but JWH-073 has the potential for greater vascular adverse events than Δ9-THC through an increased vasodilatory effect.

Differential Expression of CB1 Cannabinoid Receptor and Cannabinoid Receptor Interacting Protein 1a in Labor.

Background: The endocannabinoid system is present in multiple organ systems and is involved in smooth muscle regulation, immune function, neuroendocrine modulation, and metabolism of tissues. Limited data are available regarding the presence and role of this system in reproductive tissues. Components of the endocannabinoid system have been identified in myometrial and placental tissues. However, no study has investigated differential expression of the endocannabinoid system in labor. Objectives: The purpose of this study was to identify and quantify two components of the endocannabinoid system, the CB1 cannabinoid receptor and cannabinoid receptor interacting protein 1a (CRIP1a) in uterine and placental tissues, and to determine if there is differential expression in tissues exposed to labor. We hypothesized that CB1 cannabinoid receptor concentration would be altered in uterine and placental tissue exposed to labor compared with tissues not exposed to labor. Study Design: Uterine and placental tissue samples were collected in nine laboring and 11 nonlaboring women undergoing cesarean delivery. CB1 cannabinoid receptor and CRIP1a presence and quantification were evaluated using western blot, immunohistochemistry, and real-time quantitative polymerase chain reaction. Statistical comparisons of laboring and nonlaboring subjects were made for uterine and placental tissue using a Mann-Whitney test. Results: Immunohistochemistry demonstrated positive staining for CB1 cannabinoid receptors and CRIP1a in uterine tissue. The protein abundance of CB1 cannabinoid receptor in uterine tissue was significantly lower in tissues exposed to labor (p=0.01). The protein abundance of CRIP1a was lower in uterine tissue exposed to labor but did not reach statistical significance (p=0.06). mRNA expression of CB1 cannabinoid receptor (p=0.20) and CRIP1a (p=0.63) did not differ in labored compared with nonlabored uterine tissues. Conclusions: Our findings of diminished protein density of CB1 cannabinoid receptor in uterine tissue exposed to labor support the hypothesis that the endocannabinoid system plays a role in parturition. Our data add to the growing body of evidence indicating the endocannabinoid system is of importance for successful reproduction and support the need for additional research investigating this complex system as it pertains to labor. ClinicalTrials.gov ID: NCT03752021.

Cannabinoid receptor interacting protein 1a interacts with myristoylated Gαi N terminus via a unique gapped ß-barrel structure.

Cannabinoid receptor interacting protein 1a (CRIP1a) modulates CB1 cannabinoid receptor G-protein coupling in part by altering the selectivity for Gαi subtype activation, but the molecular basis for this function of CRIP1a is not known. We report herein the first structure of CRIP1a at a resolution of 1.55 Å. CRIP1a exhibits a 10-stranded and antiparallel ß-barrel with an interior comprised of conserved hydrophobic residues and loops at the bottom and a short helical cap at the top to exclude solvent. The ß-barrel has a gap between strands ß8 and ß10, which deviates from ß-sandwich fatty acid-binding proteins that carry endocannabinoid compounds and the Rho-guanine nucleotide dissociation inhibitor predicted by computational threading algorithms. The structural homology search program DALI identified CRIP1a as homologous to a family of lipidated-protein carriers that includes phosphodiesterase 6 delta subunit and Unc119. Comparison with these proteins suggests that CRIP1a may carry two possible types of cargo: either (i) like phosphodiesterase 6 delta subunit, cargo with a farnesyl moiety that enters from the top of the ß-barrel to occupy the hydrophobic interior or (ii) like Unc119, cargo with a palmitoyl or a myristoyl moiety that enters from the side where the missing ß-strand creates an opening to the hydrophobic pocket. Fluorescence polarization analysis demonstrated CRIP1a binding of an N-terminally myristoylated 9-mer peptide mimicking the Gαi N terminus. However, CRIP1a could not bind the nonmyristolyated Gαi peptide or cargo of homologs. Thus, binding of CRIP1a to Gαi proteins represents a novel mechanism to regulate cell signaling initiated by the CB1 receptor.

Cannabinoid receptor subtype influence on neuritogenesis in human SH-SY5Y cells.

Human SH-SY5Y neuroblastoma cells stably expressing exogenous CB1 (CB1XS) or CB2 (CB2XS) receptors were developed to investigate endocannabinoid signaling in the extension of neuronal projections. Expression of cannabinoid receptors did not alter proliferation rate, viability, or apoptosis relative to parental SH-SY5Y. Transcripts for endogenous cannabinoid system enzymes (diacylglycerol lipase, monoacylglycerol lipase, α/ß-hydrolase domain containing proteins 6 and 12, N-acyl phosphatidylethanolamine-phospholipase D, and fatty acid amide hydrolase) were not altered by CB1 or CB2 expression. Endocannabinoid ligands 2-arachidonoylglycerol (2-AG) and anandamide were quantitated in SH-SY5Y cells, and diacylglycerol lipase inhibitor tetrahydrolipstatin decreased 2-AG abundance by 90% but did not alter anandamide abundance. M3 muscarinic agonist oxotremorine M, and inhibitors of monoacylglycerol lipase and α/ß hydrolase domain containing proteins 6 &12 increased 2-AG abundance. CB1 receptor expression increased lengths of short (<30 µm) and long (>30 µm) projections, and this effect was significantly reduced by tetrahydrolipstatin, indicative of stimulation by endogenously produced 2-AG. Pertussis toxin, Gßγ inhibitor gallein, and ß-arrestin inhibitor barbadin did not significantly alter long projection length in CB1XS, but significantly reduced short projections, with gallein having the greatest inhibition. The rho kinase inhibitor Y27632 increased CB1 receptor-mediated long projection extension, indicative of actin cytoskeleton involvement. CB1 receptor expression increased GAP43 and ST8SIA2 mRNA and decreased ITGA1 mRNA, whereas CB2 receptor expression increased NCAM and SYT mRNA. We propose that basal endogenous production of 2-AG provides autocrine stimulation of CB1 receptor signaling through Gi/o, Gßγ, and ß-arrestin mechanisms to promote neuritogenesis, and rho kinase influences process extension.

Tpcn2 knockout mice have improved insulin sensitivity and are protected against high-fat diet-induced weight gain.

Type 2 diabetes is a complex disorder affected by multiple genes and the environment. Our laboratory has shown that in response to a glucose challenge, two-pore channel 2 ( Tpcn2) knockout mice exhibit a decreased insulin response but normal glucose clearance, suggesting they have improved insulin sensitivity compared with wild-type mice. We tested the hypothesis that improved insulin sensitivity in Tpcn2 knockout mice would protect against the negative effects of a high fat diet. Male and female Tpcn2 knockout (KO), heterozygous (Het), and wild-type (WT) mice were fed a low-fat (LF) or high-fat (HF) diet for 24 wk. HF diet significantly increases body weight in WT mice relative to those on the LF diet; this HF diet-induced increase in body weight is blunted in the Het and KO mice. Despite the protection against diet-induced weight gain, however, Tpcn2 KO mice are not protected against HF-diet-induced changes in glucose or insulin area under the curve during glucose tolerance tests in female mice, while HF diet has no significant effect on glucose tolerance in the male mice, regardless of genotype. Glucose disappearance during an insulin tolerance test is augmented in male KO mice, consistent with our previous findings suggesting enhanced insulin sensitivity in these mice. Male KO mice exhibit increased fasting plasma total cholesterol and triglyceride concentrations relative to WT mice on the LF diet, but this difference disappears in HF diet-fed mice where there is increased cholesterol and triglycerides across all genotypes. These data demonstrate that knockout of Tpcn2 may increase insulin action in male, but not female, mice. In addition, both male and female KO mice are protected against diet-induced weight gain, but this protection is likely independent from glucose tolerance, insulin sensitivity, and plasma lipid levels.

Mouse Neuroblastoma CB1 Cannabinoid Receptor-Stimulated [35S]GTPÉ£S Binding: Total and Antibody-Targeted Gα Protein-Specific Scintillation Proximity Assays.

G protein-coupled receptors (GPCRs) are important regulators of cellular signaling functions and therefore are a major target for drug discovery. The CB1 cannabinoid receptor is among the most highly expressed GPCRs in neurons, where it regulates many differentiated neuronal functions. One model system for studying the biochemistry of neuronal responses is the use of neuroblastoma cells originating from the C1300 tumor in the A/J mouse, including cloned cell lines NS20, N2A, N18TG2, N4TG1, and N1E-115, and various immortalized hybrids of neurons with N18TG2 cells. GPCR signal transduction is mediated through interaction with multiple types and subtypes of G proteins that transduce the receptor stimulus to effectors. The [35S]GTPÉ£S assay provides a valuable pharmacological method to evaluate efficacy and potency in the first step in GPCR signaling. Here, we present detailed protocols for the [35S]GTPÉ£S-binding assay to measure the total G protein binding and the antibody-targeted scintillation proximity assay to measure specific Gα proteins in neuroblastoma cell membrane preparations. This chapter presents step-by-step methods from cell culture, membrane preparation, assay procedures, and data analysis.

Cannabinoid Receptor Interacting Protein 1a Competition with ß-Arrestin for CB1 Receptor Binding Sites.

Cannabinoid receptor interacting protein 1a (CRIP1a) is a CB1 receptor (CB1R) distal C-terminal-associated protein that alters CB1R interactions with G-proteins. We tested the hypothesis that CRIP1a is capable of also altering CB1R interactions with ß-arrestin proteins that interact with the CB1R at the C-terminus. Coimmunoprecipitation studies indicated that CB1R associates in complexes with either CRIP1a or ß-arrestin, but CRIP1a and ß-arrestin fail to coimmunoprecipitate with each other. This suggests a competition for CRIP1a and ß-arrestin binding to the CB1R, which we hypothesized could attenuate the action of ß-arrestin to mediate CB1R internalization. We determined that agonist-mediated reduction of the density of cell surface endogenously expressed CB1Rs was clathrin and dynamin dependent and could be modeled as agonist-induced aggregation of transiently expressed GFP-CB1R. CRIP1a overexpression attenuated CP55940-mediated GFP-CB1R as well as endogenous ß-arrestin redistribution to punctae, and conversely, CRIP1a knockdown augmented ß-arrestin redistribution to punctae. Peptides mimicking the CB1R C-terminus could bind to both CRIP1a in cell extracts as well as purified recombinant CRIP1a. Affinity pull-down studies revealed that phosphorylation at threonine-468 of a CB1R distal C-terminus 14-mer peptide reduced CB1R-CRIP1a association. Coimmunoprecipitation of CB1R protein complexes demonstrated that central or distal C-terminal peptides competed for the CB1R association with CRIP1a, but that a phosphorylated central C-terminal peptide competed for association with ß-arrestin 1, and phosphorylated central or distal C-terminal peptides competed for association with ß-arrestin 2. Thus, CRIP1a can compete with ß-arrestins for interaction with C-terminal CB1R domains that could affect agonist-driven, ß-arrestin-mediated internalization of the CB1R.

Cannabinoid receptor interacting protein suppresses agonist-driven CB1 receptor internalization and regulates receptor replenishment in an agonist-biased manner.

Cannabinoid receptor interacting protein 1a (CRIP1a) is a CB1 receptor (CB1 R) distal C-terminus-associated protein that modulates CB1 R signaling via G proteins, and CB1 R down-regulation but not desensitization (Blume et al. [2015] Cell Signal., 27, 716-726; Smith et al. [2015] Mol. Pharmacol., 87, 747-765). In this study, we determined the involvement of CRIP1a in CB1 R plasma membrane trafficking. To follow the effects of agonists and antagonists on cell surface CB1 Rs, we utilized the genetically homogeneous cloned neuronal cell line N18TG2, which endogenously expresses both CB1 R and CRIP1a, and exhibits a well-characterized endocannabinoid signaling system. We developed stable CRIP1a-over-expressing and CRIP1a-siRNA-silenced knockdown clones to investigate gene dose effects of CRIP1a on CB1 R plasma membrane expression. Results indicate that CP55940 or WIN55212-2 (10 nM, 5 min) reduced cell surface CB1 R by a dynamin- and clathrin-dependent process, and this was attenuated by CRIP1a over-expression. CP55940-mediated cell surface CB1 R loss was followed by a cycloheximide-sensitive recovery of surface receptors (30-120 min), suggesting the requirement for new protein synthesis. In contrast, WIN55212-2-mediated cell surface CB1 Rs recovered only in CRIP1a knockdown cells. Changes in CRIP1a expression levels did not affect a transient rimonabant (10 nM)-mediated increase in cell surface CB1 Rs, which is postulated to be as a result of rimonabant effects on 'non-agonist-driven' internalization. These studies demonstrate a novel role for CRIP1a in agonist-driven CB1 R cell surface regulation postulated to occur by two mechanisms: 1) attenuating internalization that is agonist-mediated, but not that in the absence of exogenous agonists, and 2) biased agonist-dependent trafficking of de novo synthesized receptor to the cell surface.

CB1 cannabinoid receptor-mediated increases in cyclic AMP accumulation are correlated with reduced Gi/o function.

BACKGROUND: CB1 cannabinoid receptors (CB1Rs) stimulate Gi/o-dependent signaling pathways. CB1R-mediated cAMP increases were proposed to result from Gs activation, but CB1R-stimulated GTPγS binding to Gs has not heretofore been investigated. METHODS: Three models of CB1R-stimulated cAMP production were tested: pertussis toxin disruption of Gi/o in N18TG2 cells; L341A/A342L-CB1R expressed in Chinese hamster ovary (CHO) cells; and CB1 and D2 dopamine receptors endogenously co-expressed in MN9D cells. cAMP was assayed by [3H]cAMP binding competition. G protein activation was assayed by the antibody-targeted scintillation proximity assay. RESULTS: In L341A/A342L-CB1-CHO cells, cannabinoid agonists significantly stimulated cAMP accumulation over vehicle; (-)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxyl propyl] cyclohexan-1-ol (CP55940)-stimulated [35S]GTPγS binding to Gi1/2/3 was reversed, whereas binding to Gs was not different from CB1R. In MN9D cells, CB1 agonist HU210 or D2 agonist quinpirole alone inhibited forskolin-activated cAMP accumulation, whereas HU210 plus quinpirole increased cAMP accumulation above basal. HU210 alone stimulated [35S]GTPγS binding to Gi1/2/3, whereas co-stimulation with quinpirole reversed HU210-stimulated [35S]GTPγS binding to Gi1/2/3. CONCLUSIONS: CB1R couples to Gs but with low efficacy compared to Gi/o. The L341A/A342L mutation in CB1R reversed CP55940 activation of Gi to an inhibition, but had no effect on Gs. Combined CB1 plus D2 agonists in MN9D cells converted the CB1 agonist-mediated activation of Gi to inhibition of Gi. In these models, the CB1 agonist response was converted to an inverse agonist response at Gi activation. Cannabinoid agonist-stimulated cAMP accumulation can be best explained as reduced activation of Gi, thereby attenuating the tonic inhibitory influence of Gi on the major isoforms of adenylyl cyclase.

Expression of glutathione S-transferases in fetal lung and liver tissue from parental strains and F1 crosses between C57BL/6 and BALB/c F1 mice following in utero exposure to 3-methylcholanthrene.

GST isoforms have been extensively studied in adult tissues but little is known about the composition and levels of these enzymes in fetal tissues. As part of our ongoing studies to determine the potential role of metabolic enzymes in mediating the differential susceptibility of different strains of mice to lung tumorigenesis following in utero exposure to 3-methylcholanthrene (MC), we screened for GST enzyme activity and for expression of the individual GSTalpha, pi, mu, and theta isoforms in murine fetal lung and liver tissues isolated from the parental strains and F1 crosses between C57BL/6 (B6) and BALB/c (C) mice. Using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate, we found that treatment with MC had no effect on the levels of GST enzyme activity in either the fetal lung or liver in either of the two parental strains or their F1 crosses. Low levels of expression of each of the four enzymes were detected by Western blotting in both fetal lung and liver tissues in all four strains. A statistically significant 3.5-fold induction was observed only for GSTmu in the fetal lung of the parental strain of BALB/c mice 48 h after exposure to MC. None of the other enzymes showed any significant differences in the levels of expression following exposure to MC. Although strain-specific differences in the expression of the GSTs that were independent of MC treatment were observed, they could not account for the differences previously observed in either the Ki-ras mutational spectrum or lung tumor incidence in the different strains of mice. Similar results were obtained when the maternal metabolism of MC was assayed in liver microsomal preparations. The results are consistent with previous studies showing low levels and poor inducibility of phase II enzymes during gestation, and demonstrate for the first time that all four of the major GST enzymes are expressed in fetal tissues. While the high inducibility of activating enzymes, such as Cyp1a1, and low, uninducible levels of phase II conjugating enzymes probably account for the high susceptibility of the fetus to transplacentally induced tumor formation, the results also suggest that factors other than metabolism may account for the strain-specific differences in susceptibility to carcinogen-mediated lung tumor induction following in utero exposure to chemical carcinogens.

Oxathiolene oxides: a novel family of compounds that induce ferritin, glutathione S-transferase, and other proteins of the phase II response.

Compounds that induce the synthesis of cytoprotective phase II enzymes have shown promise as cancer chemopreventive agents. Although chemically diverse, phase II enzyme inducers are capable of participating in Michael reaction chemistry. We have synthesized a novel class of organosulfur compounds, termed oxathiolene oxides (OTEOs). Based on their chemical properties, we hypothesized that these compounds could function as phase II enzyme inducers. Northern blot analysis showed that oxathiolene oxides induce the phase II enzymes glutathione S-transferase (GST), NAD(P)H:quinone oxidoreductase 1 (NQO1), and ferritin H and L mRNA in a concentration-dependent fashion in a normal embryonic mouse liver cell line, BNLCL.2. OTEO-562 (3-cyclohexenyl-4-methyl-1,2-oxathiol-3-ene-2-oxide) was the strongest inducer. Western blot analysis demonstrated that GST-alpha and ferritin H protein levels were also induced in cells treated with OTEO-562, as was total GST and NQO1 enzyme activity. Further, induction of NQO1 activity by OTEO-562 was equivalent in aromatic hydrocarbon (Ah) receptor wild-type and Ah receptor mutant cell lines, suggesting that oxathiolene oxides activate phase II enzymes by an Ah receptor-independent mechanism. Consistent with this observation, OTEO-562 failed to induce cytochrome P450 1A1 mRNA. These results suggest that oxathiolene oxides may merit further investigation as candidate chemopreventive agents.