The possible role of L-serine as an allosteric modulator of anandamide (AEA) action on cannabinoid receptors has been investigated along with the studying the actions of AEA metabolite, ethanolamine.  Dorsal root ganglia (DRG) neurons from neonate Sprague Dawley rats were used in primary culture. L-serine was found to modulate anandamide actions on overall neuronal excitability, voltage-activated K⁺ and Ca²⁺ currents and intracellular Ca²⁺ flux.  These modulations were suggested to be mediated via allosteric modulation of AEA actions at CB1 receptors.  These observations strengthen previous data obtained from binding studies of L-serine at CB1 receptors.  Furthermore, these modulations were abolished by CB1 antagonist, SR141716A, while L-serine alone failed to activate CB1 receptors.  We found that L-serine modulations were AEA-dependent and CB1 mediated, while no modulatory effects for L-serine were reported on TRPV1 or GPR55 receptors.  Similar modulations were reported from the CB1 allosteric modulator, Org-27569. Ethanolamine was fond to enhance the intracellular Ca²⁺ level via influencing thapsigargicin- and caffeine-sensitive calcium stores.  Ethanolamine actions were not abolished in PTX-treated DRG neurones or in the presence of CB1 antagonist, SR141716A indicating that these actions were conducted independently from CB1 receptors and inhibitory G-proteins. Additionally, ethanolamine modulated the voltage- activated potassium currents independently from its effect on intracellular Ca²⁺ level. In conclusion CB1 receptor modulation by ligands acting at an allosteric site may provide a novel approach to endocannabinoids-mediated therapies.

Thesis (M.A.)--California State University, Chico. / Includes abstract. "Located in the Chico Digital Repository." Includes bibliographical references (p. 31-44).

Bioactive compounds from Cannabis sativa have been used for millennia to alleviate the symptoms of a range of diseases. The physiological basis of effects such as analgesia, stimulation of hunger and reduction of inflammation was established in the late 20th century with the discovery of cannabinoid receptors but efforts to synthesise safe and potent drugs targeting these proteins have so far failed. The major barrier to research in this area is the instability of the receptors outside of biological settings, rendering elucidation of the binding sites by traditional means difficult. Certain small molecules can interact with the cannabinoid type 1 receptor (CB1) at locations distinct to the primary ligand docking site. Such allosteric modulation of the endocannabinoid system offers significant advantages over using orthosteric drugs and in this research a range of indole based structures were synthesised and tested in an attempt to improve the activity and drug-like nature of a lead compound. A partial structure-activity relationship was established, including the description of the most potent allosteric enhancer of CB1 so far reported. Efforts were also undertaken to investigate the allosteric binding environments using photoactivatable ligands based on a CB1 inhibitor. In combination with mutation studies and computer modelling this technique could allow the rational design of allosteric modulators, a task which is not trivial at present. Two photoactivatable compounds were synthesised and shown to interact with the receptor, with a method for isolating covalently labelled peptide fragments from other biomolecules demonstrated using “click chemistry” and a modified Wang resin. This work may find application in future investigations aiming to produce allosteric pharmaceuticals targeting CB1. Furthermore, the techniques described may be applied to study the binding site of a recently described allosteric endocannabinoid or could potentially be adapted to look at secondary binding domains in other G protein-coupled receptors.

Bone is a dynamic living organ that differentiates vertebrates from other animals. Calcification makes the tissue dense, stiff, strong and careful control of the material and geometrical properties of bone attempts to match their properties to the prevailing mechanical environment. Bone homeostasis involves an array of factors, only a few of which are known. The cells that produce bone, osteoblasts, share a common origin with adipocytes in mesenchymal stem cells (MSC) and there is mounting evidence of dysregulation of MSC differentiation in bone disorders such as osteoarthritis and osteoporosis. The cannabinoid system has recently been shown to be involved in bone homeostasis and has been proposed as a potential therapeutic target for treatment of bone diseases. However studies published to date look almost exclusively at its effect on trabecular bone. A full understanding of any regulatory factor can come only by considering both cortical and trabecular bone. This thesis describes experiments to characterize the mechanical, material and geometrical properties of cortical and trabecular bone from mice in which cannabinoid, CB1, CB2, or putative cannabinoid, GPR55, receptors have been deleted. Further studies started to explore the biological basis underlying the role of GPR55 and one of its antagonists, cannabidiol (CBD). The hypothesis was that endogenous cannabinoids can regulate bone properties and the aim Abstract ii was to resolve discrepancies found in previous studies and begin to explore the effect of cannabidiol acting through the GPR55 receptor on MSC differentiation. CB1 -/- mice had inferior mechanical properties to wild type mice due to reduced geometrical properties while the material properties remained unchanged. No change was found in trabecular bone volume in males but female CB1 -/- mice showed a high bone mass phenotype. CB2 -/- mice had mechanically superior bones to wild type animals due to larger geometrical properties. These superior characteristics were also seen in trabecular bone, where the female KOs had a high bone mass phenotype. The effect of knocking out GPR55 showed an increase in the amount of both cortical and trabecular bone. The knockout mice were fatter and were protected against age related bone loss. In addition, CBD enhanced adipogenesis in both humans and mice, while no effects were seen on osteogenesis. Enhanced adipogenesis was also found in GPR55-/- cell cultures. These results suggest that blocking GPR55 with small molecules such as CBD may be beneficial in bone diseases such as osteoporosis but not osteoarthritis.

In a recent patent, GPR55 emerged as a novel target for cannabinoid modulation, and was found to be activated by a number of synthetic and endogenous cannabinoids, including anandamide, virodhamine, and AM251.  There is mounting evidence to suggest that cannabinoids play a role in tumour progression.  The potential role of GPR55 in cancer, however, remains to be explored. This study used qPCR to determine GPR55, CB₁ and CB₂ expression levels in a number of native breast cancer cell lines, and found that GPR55 was expressed in highly metastatic cells, including MDA-MB-231 and B02-GFP cell lines. Studies into the modulation of GPR55 by its suggested ligands showed that agonist L-α-lysophosphatidylinositol enhanced the migration of metastatic breast cancer cells, and augmented certain behaviours associated with a more aggressive phenotype, including enhancing the ability of these cells to orientate and polarise in response to grooved substratum.  Meanwhile the GPR55 antagonist CBD inhibited the migration and invasion of these cells.  Using over-expression and siRNA knockdown of GPR55, we have implicated this receptor in both the LPI- and CBD-mediated effects on migration/invasion.  Furthermore, we found that LPI significantly stimulated both RhoA activation, and ERK1/2 phosphorylation in hGPR55-expressing HEK293 cells, but not in untransfected HEK293 cells.  These studies highlight an important role for GPR55 as a new biomarker in cancer, and suggest a possible therapeutic role for cannabinoids in the treatment of metastasis.

The novel compound VSN16R was characterised and its functional effect in the central nervous system and periphery was investigated.  VSN16R behaved as an agonist in the mouse isolated vas deferens assay.  The functional effect of VSN16R appeared to involve GPR55 and GPR18 activation. The possibility that in addition to the CB₁ receptor; the CB₂ receptor, GPR55 and GPR18 contribute towards the effects of certain cannabinoid ligands in the nervous system (both central and peripheral) was investigated.  Results suggest that the cannabinoid CB₂ receptor and also GPR55 and GPR18 are functional in the mouse vas deferens. The results imply that in addition to the CB₁ receptor, the CB₂ receptor can mediate the effects of certain cannabinoids, including CP55940, AEA and Δ⁹-THC, but not JWH015 in the [³⁵S]GTPγS assay with brain region membranes.  GPR55 can mediate the effects of certain cannabinoids, including CP55940 and JWH015 in the [³⁵S]GTPγS assay with brain region membranes.  GPR55 was also shown to mediate the effect of JWH015, but  not O-1602 in the mouse isolated vas deferens assay, indicating an additional target for O-1602 in this tissue.  Data obtained suggest that CP55940 and AEA, but not Δ⁹-THC, O-1602 or JWH015 can activate GPR18. The effect of cannabinoid ligands and VSN16R on human neutrophil migration was investigated. The functional effect of a range of phytocannabinoids and the ligand N-arachichidonoyl glycine (NAGly) at the receptor GPR902 was investigated.  The findings implied that CBD, CBG and Δ⁹-THC acid may be able to interact with GPR92 to antagonise the GPR92 agonist lysophosphatidic acid.

Thesis (Ph.D.)--University of Hawaii at Manoa, 1994. / Includes bibliographical references. / Microfiche. / xv, 329 leaves, bound ill. 29 cm

On t.p. "[delta]" appears as the Greek symbol. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1994. / Includes bibliographical references (leaves 257-262). / Microfiche. / xiv, 262 leaves, bound ill. 29 cm

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2007. / Vita. Includes bibliographical references.

Diss. (sammanfattning) Umeå : Umeå universitet, 2006. / Härtill 4 uppsatser.