Tammy Sexton

Effects of anandamide on cannabinoid receptors in rat brain membranes.

Anandamide (arachidonylethanolamide) is a compound recently isolated from porcine brain as a putative endogenous ligand at cannabinoid receptors. The present studies examined the effects of anandamide on cannabinoid receptor binding sites and adenylyl cyclase in rat brain membranes. Receptor binding experiments, conducted at 25 degrees for 90 min, apparently resulted in significant degradation of anandamide, since anandamide (10 microM) had little effect on [3H]WIN 55212-2 binding in cerebellar membranes. Addition of the general serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) protected against this degradation, resulting in an IC50 value of 90 nM for anandamide versus [3H]WIN 55212-2 binding. Anandamide inhibited adenylyl cyclase in cerebellar membranes in a GTP-dependent manner, exhibiting a maximal inhibition level slightly less than that of WIN 55212-2 and CP-55,940, with an IC50 value of 1.9 microM. The effect of anandamide on adenylyl cyclase was region-specific, with maximal inhibition occurring in cerebellum and striatum. These results suggest that anandamide acts at G-protein-coupled cannabinoid receptors in brain with properties similar to those of exogenous cannabinoids.

Opioid and Cannabinoid Receptor Inhibition of Adenylyl Cyclase in Braina

Both opioids and cannabinoids bind to G-protein-coupled receptors to inhibit adenylyl cyclase in neurons. These reactions were assayed in brain membranes, where maximal inhibitory activity occurred in the following regions: mu-opioid inhibition in rat thalamus, delta-opioid inhibition in rat striatum, kappa-opioid inhibition in guinea pig cerebellum, and cannabinoid inhibition in cerebellum. The inhibition of adenylyl cyclase by both cannabinoid and opioid agonists was typical of G-protein-linked receptors: they required GTP, they were not supported by non-hydrolyzable GTP analogs, and they were abolished (in primary neuronal cell culture) by pertussis toxin treatment. The immediate targets of this system were determined by assaying protein phosphorylation in the presence of receptor agonists and App(NH)p, a substrate for adenylyl cyclase. In striatal membranes, opioid agonists inhibited the phosphorylation of at least two bands of MW 85 and 63 kDa, which may be synapsins I and II, respectively. Other experiments determined the long-term effects of this second messenger system. In primary neuronal cultures, opioid-inhibited adenylyl cyclase attenuated forskolin-stimulated pro-enkephalin mRNA levels, thus providing a feedback regulation of opioid synthesis. Finally, in cerebellar granule cells, both cannabinoid and opioid receptors may exist on the same cells. In these cells, agonists which bind to different receptor types may produce similar biological responses.

Novel 2-substituted cocaine analogs : binding properties at dopamine transport sites in rat striatum

A novel scheme utilizing vinylcarbenoid precursors has been developed for the synthesis of novel tropane analogs of cocaine. Using this method, 15 analogs were prepared and tested for activity in binding to dopamine transporters in rat striatal membranes using [125I]RTI-55. In all the analogs, the aryl group at the 3 position was directly bound to the tropane ring (as in WIN 35,428), and methyl or ethyl ketone moieties were present at the 2 position instead of the typical ester group. The most potent analog was a 2-naphthyl derivative (IC50 value of 0.2 nM, vs. 170 nM for cocaine), while replacement of the aryl with either ethyl or cyclohexyl drastically reduced potency (to > 50 microM and 5 microM, respectively).

Synthesis of 2β-acyl-3β-(substituted naphthyl)-8-azabicyclo[3.2.1]octanes and their binding affinities at dopamine and serotonin transport sites

A series of 3beta-naphthyltropane derivatives were synthesized and found to show high affinity at both the dopamine and serotonin transporter sites, leading to some of the most potent inhibitors known based on the tropane structure. Comparative molecular field analysis (CoMFA) models were developed for both dopamine and serotonin transporter binding data. These models provide insights into those factors that influence binding at the two transporters.

Increased Sensitivity to Cocaine Self-Administration in HIV-1 Transgenic Rats is Associated with Changes in Striatal Dopamine Transporter Binding

Cocaine abuse in HIV patients accelerates the progression and severity of neuropathology, motor impairment and cognitive dysfunction compared to non-drug using HIV patients. Cocaine and HIV interact with the dopamine transporter (DAT); however, the effect of their interaction on DAT binding remains understudied. The present study compared the dose–response functions for intravenous self-administration of cocaine and heroin between male HIV-1 transgenic (HIV-1 Tg) and Fischer 344 rats. The cocaine and heroin dose–response functions exhibit an inverted U-shape for both HIV-1 Tg and F344 rats. For cocaine, the number of infusions for each dose on the ascending limb was greater for HIV-1 Tg versus F344 rats. No significant changes in the heroin dose–response function were observed in HIV-1 Tg animals. Following the conclusion of self-administration experiments, DAT binding was assessed in striatal membranes. Saturation binding of the cocaine analog [125I] 3β-(4-iodophenyl)tropan-2β-carboxylic acid methyl ester ([125I]RTI-55) in rat striatal membranes resulted in binding curves that were best fit to a two-site binding model, allowing for calculation of dissociation constant (Kd) and binding density (Bmax) values that correspond to high- and low-affinity DAT binding sites. Control HIV-1 Tg rats exhibited a significantly greater affinity (i.e., decrease in Kd value) in the low-affinity DAT binding site compared to control F344 rats. Furthermore, cocaine self-administration in HIV-1 Tg rats increased low-affinity Kd (i.e., decreased affinity) compared to levels observed in control F344 rats. Cocaine also increased low-affinity Bmax in HIV-1 Tg rats as compared to controls, indicating an increase in the number of low-affinity DAT binding sites. F344 rats did not exhibit any change in high- or low-affinity Kd or Bmax values following cocaine or heroin self-administration. The increase in DAT affinity in cocaine HIV-1 Tg rats is consistent with the leftward shift of the ascending limb of the cocaine dose–response curve observed in HIV-1 Tg vs. F344 rats, and has major implications for the function of cocaine binding to DAT in HIV patients. The absence of HIV-related changes in heroin intake are likely due to less dopaminergic involvement in the mediation of heroin reward, further emphasizing the preferential influence of HIV on dopamine-related behaviors.

1-Naphthyl and 4-indolyl arylalkylamines as selective monoamine reuptake inhibitors

A series of enantiomerically pure 1-naphthyl and 4-indolyl arylalkylamines were prepared and evaluated for their binding affinities to the monoamine transporters. The two series of enantiomers displayed considerable differences in binding selectivity between the monoamine transporters, leading to the design of (S)-4-(3,4-dichlorophenyl)-4-(1H-indol-4-yl)-N-methylbutan-1-amine as a potent inhibitor for the dopamine and serotonin transporters.

Opioid‐Inhibited Adenylyl Cyclase in Rat Brain Membranes: Lack of Correlation with High‐Affinity Opioid Receptor Binding Sites

Abstract: Opioid agonists bind to GTP‐binding (G‐protein)‐coupled receptors to inhibit adenylyl cyclase. To explore the relationship between opioid receptor binding sites and opioid‐inhibited adenylyl cyclase, membranes from rat striatum were incubated with agents that block opioid receptor binding. These agents included irreversible opioid agonists (oxymorphone‐p‐nitrophenylhydrazone), irreversible antagonists [naloxonazine, β‐funaltrexamine, and β‐chlornaltrexamine (β‐CNA)], and phospholipase A2. After preincubation with these agents, the same membranes were assayed for high‐affinity opioid receptor binding [3H‐labeled D‐alanine‐4‐N‐methylphenylalanine‐5‐glycine‐ol‐enkephalin (μ), 3H‐labeled 2‐D‐serine‐5‐L‐leucine‐6‐L‐threonine enkephalin (δ), and [3H]ethylketocylazocine (EKC) sites] and opioid‐inhibited adenylyl cyclase. Although most agents produced persistent blockade in binding of ligands to high‐affinity μ, δ, and EKC sites, no change in opioid‐inhibited adenylyl cyclase was detected. In most treated membranes, both the IC50 and the maximal inhibition of adenylyl cyclase by opioid agonists were identical to values in untreated membranes. Only β‐CNA blocked opioid‐inhibited adenylyl cyclase by decreasing maximal inhibition and increasing the IC50 of opioid agonists. This effect of β‐CNA was not due to nonspecific interactions with Gi, Gs, or the catalytic unit of adenylyl cyclase, as neither guanylylimido‐diphosphate‐inhibited, NaF‐stimulated, nor forskolin‐stimulated activity was altered by β‐CNA pretreatment. Phospholipase A2 decreased opioid‐inhibited adenylyl cyclase only when the enzyme was incubated with brain membranes in the presence of NaCl and GTP. These results confirm that the receptors that inhibit adenylyl cyclase in brain do not correspond to the high‐affinity μ, δ, or EKC sites identified in brain by traditional binding studies.

Changes in dopamine transporter binding in nucleus accumbens following chronic self-administration cocaine: Heroin combinations

Concurrent use of cocaine and heroin (speedball) has been shown to exert synergistic effects on dopamine neurotransmission in the nucleus accumbens (NAc), as observed by significant increases in extracellular dopamine levels and compensatory elevations in the maximal reuptake rate of dopamine. The present studies were undertaken to determine whether chronic self‐administration of cocaine, heroin or a combination of cocaine:heroin led to compensatory changes in the abundance and/or affinity of high‐ and low‐affinity DAT binding sites. Saturation binding of the cocaine analog [125I] 3β‐(4‐iodophenyl)tropan‐2β‐carboxylic acid methyl ester ([125I]RTI‐55) in rat NAc membranes resulted in binding curves that were best fit to two‐site binding models, allowing calculation of dissociation constant (Kd) and binding density (Bmax) values corresponding to high‐ and low‐affinity DAT binding sites. Scatchard analysis of the saturation binding curves clearly demonstrate the presence of high‐ and low‐ affinity binding sites in the NAc, with low‐affinity sites comprising 85 to 94% of the binding sites. DAT binding analyses revealed that self‐administration of cocaine and a cocaine:heroin combination increased the affinity of the low‐affinity site for the cocaine congener RTI‐55 compared to saline. These results indicate that the alterations observed following chronic speedball self‐administration are likely due to the cocaine component alone; thus further studies are necessary to elaborate upon the synergistic effect of cocaine:heroin combinations on the dopamine system in the NAc. Synapse 68:437–444, 2014.

Chronic baclofen desensitizes GABAB-mediated G-protein activation and stimulates phosphorylation of kinases in mesocorticolimbic rat brain

The GABAB receptor is a therapeutic target for CNS and neuropathic disorders; however, few preclinical studies have explored effects of chronic stimulation. This study evaluated acute and chronic baclofen treatments on GABAB-activated G-proteins and signaling protein phosphorylation as indicators of GABAB signaling capacity. Brain sections from rats acutely administered baclofen (5 mg/kg, i.p.) showed no significant differences from controls in GABAB-stimulated GTPγS binding in any brain region, but displayed significantly greater phosphorylation/activation of focal adhesion kinase (pFAK(Tyr397)) in mesocorticolimbic regions (caudate putamen, cortex, hippocampus, thalamus) and elevated phosphorylated/activated glycogen synthase kinase 3-β (pGSK3β(Tyr216)) in the prefrontal cortex, cerebral cortex, caudate putamen, nucleus accumbens, thalamus, septum, and globus pallidus. In rats administered chronic baclofen (5 mg/kg, t.i.d. for five days), GABAB-stimulated GTPγS binding was significantly diminished in the prefrontal cortex, septum, amygdala, and parabrachial nucleus compared to controls. This effect was specific to GABAB receptors: there was no effect of chronic baclofen treatment on adenosine A1-stimulated GTPγS binding in any region. Chronically-treated rats also exhibited increases in pFAK(Tyr397) and pGSK3β(Tyr216) compared to controls, and displayed wide-spread elevations in phosphorylated dopamine- and cAMP-regulated phosphoprotein-32 (pDARPP-32(Thr34)) compared to acutely-treated or control rats. We postulate that those neuroadaptive effects of GABAB stimulation mediated by G-proteins and their sequelae correlate with tolerance to several of baclofens effects, whereas sustained signaling via kinase cascades points to cross-talk between GABAB receptors and alternative mechanisms that are resistant to desensitization. Both desensitized and sustained signaling pathways should be considered in the development of pharmacotherapies targeting the GABA system.

Synthesis and monoamine transporter affinity of 3β-(4-(2-pyrrolyl)phenyl)-8-azabicyclo[3.2.1]octanes and 3β-(5-indolyl)-8-azabicyclo[3.2.1]octanes

3β-(5-Indolyl)-8-azabicyclo[3.2.1]octanes display potent binding affinity for both the dopamine and serotonin transporters, while certain 3β-(4-(2-pyrrolyl)phenyl)-8-azabicyclo[3.2.1]octanes selectively bind to the serotonin transporter.