A role for p53 in the regulation of lysosomal permeability by delta 9-tetrahydrocannabinol in rat cortical neurones: implications for neurodegeneration

The psychoactive ingredient of marijuana, Δ⁹-tetrahydrocannabinol (Δ⁹-THC), can evoke apoptosis in cultured cortical neurones. Whilst the intracellular mechanisms responsible for this apoptotic pathway remain to be fully elucidated, we have recently identified a role for the CB₁ type of cannabinoid (CB) receptor and the tumour suppressor protein, p53. In the current study, we demonstrate the Δ⁹-THC promotes a significant increase in lysosomal permeability in a dose- and time-dependent manner. The increase in lysosomal permeability was blocked by the CB₁ receptor antagonist, AM251. Δ⁹-THC increased the localization of phospho-p53Ser15 at the lysosome and stimulated the release of the lysosomal cathepsin enzyme, cathepsin-D, into the cytosol. The p53 inhibitor, pifithrin-α and small interfering RNA-mediated knockdown of p53 prevented the Δ⁹-THC-mediated increase in lysosomal permeability. Furthermore, the Δ⁹-THC -mediated induction of apoptosis was abrogated by a cell-permeable cathepsin-D inhibitor (10 μM). Thus, the study demonstrates that Δ⁹-THC impacts on the lysosomal system, via p53, to evoke lysosomal instability as an early event in the apoptotic cascade. This provides evidence for a novel link between the CB₁ receptor and the lysosomal branch of the apoptotic pathway which is crucial in regulating neuronal viability following exposure to Δ⁹-THC.     

CB1 knockout mice display impaired functionality of 5-HT1A and 5-HT2A/C receptors

Interaction between brain endocannabinoid (EC) and serotonin (5-HT) systems was investigated by examining 5-HT-dependent behavioral and biochemical responses in CB₁ receptor knockout mice. CB₁ knockout animals exhibited a significant reduction in the induction of head twitches and paw tremor by the 5-HT_2A/C receptor selective agonist (±) DOI, as well as a reduced hypothermic response following administration of the 5-HT_1A receptor agonist (±)-8-OH-DPAT. Additionally, exposure to the tail suspension test induced enhanced despair responses in CB₁ knockout mice. However, the tricyclic antidepressant imipramine and the 5-HT selective reuptake inhibitor fluoxetine induced similar decreases in the time of immobility in the tail suspension test in CB₁ receptor knockout and wild-type mice. No differences were found between both genotypes with regard to 5-HT_2A receptor and 5-HT_1A receptors levels, measured by autoradiography in different brain areas. However, a significant decrease in the ability of both, the 5-HT_1A receptor agonist (±)-8-OH-DPAT and the 5-HT_2A/C receptor agonist (−)DOI, to stimulate [³⁵S]GTPγS binding was detected in the hippocampal CA₁ area and fronto-parietal cortex of CB₁ receptor knockout mice, respectively. This study provides evidence that CB₁ receptors are involved in the regulation of serotonergic responses mediated by 5-HT_2A/C and 5-HT_1A receptors, and suggests that a reduced coupling of 5-HT_1A and 5-HT_2A receptors to G proteins might be involved in these effects.     

Endogenous cannabinoid anandamide inhibits nicotinic acetylcholine receptor function in mouse thalamic synaptosomes

The effects of the endogenous cannabinoid anandamide [arachidonylethanolamide (AEA)] on the function of nicotinic acetylcholine receptor (nAChR) were investigated using the ⁸⁶Rb⁺ efflux assay in thalamic synaptosomes. AEA reversibly inhibited ⁸⁶Rb⁺ efflux induced by 300 μM ACh with an IC₅₀ value of 0.9 ± 2 μM. Pre-treatment with the cannabinoid (CB₁) receptor antagonist SR141716A (1 μM), the CB₂ receptor antagonist SR144528 (1 μM), or pertussis toxin (0.2 mg/mL) did not alter the inhibitory effects of AEA, suggesting that known CB receptors are not involved in AEA inhibition of nAChRs. AEA inhibition of ⁸⁶Rb⁺ efflux was not reversed by increasing acetylcholine (ACh) concentrations. In radioligand binding studies, the specific binding of [³H]-nicotine was not altered in the presence of AEA, indicating that AEA inhibits the function of nAChR in a non-competitive manner. Neither the amidohydrolase inhibitor phenylmethylsulfonyl fluoride (0.2 mM) nor the cyclooxygenase inhibitor, indomethacin, (5 μM) affected AEA inhibition of nAChRs, suggesting that the effect of AEA is not mediated by its metabolic products. Importantly, the extent of AEA inhibition of ⁸⁶Rb⁺ efflux was significantly attenuated by the absence of 1% fatty acid free bovine serum albumin pre-treatment, supporting previous findings that fatty acid-like compounds modulate the activity of nAChRs. Collectively, the results indicate that AEA inhibits the function of nAChRs in thalamic synaptosomes via a CB-independent mechanism and that the background activity of these receptors is affected by fatty acids and AEA.     

NMDA-mediated modulation of dopamine release is modified in rat prefrontal cortex and nucleus accumbens after chronic nicotine treatment

In this study, we investigate the effects of chronic administration of (−)nicotine on the function of the NMDA-mediated modulation of [³H]dopamine (DA) release in rat prefrontal cortex (PFC) and nucleus accumbens (NAc). In the PFC synaptosomes NMDA in a concentration-dependent manner evoked [³H]DA release in rats chronically treated with vehicle (14 days) with an EC₅₀ of 13.1 ± 2.0 μM. The NMDA-evoked overflow of the [³H]DA in PFC nerve endings of rats treated with (−)nicotine was significantly lower (−43%) than in vehicle treated rats. The EC₅₀ was 9.0 ± 1.4 μM. Exposure of NAc synaptosomes of rats treated with vehicle to NMDA produced an increase in [³H]DA overflow with an EC₅₀ of 14.5 ± 5.5 μM. This effect was significantly enhanced in chronically treated animals. The EC₅₀ was 10.5 ± 0.5 μM. The K⁺-evoked release of [³H]DA was not modified by the (−)nicotine administration. Both the changes of the NMDA-evoked [³H]DA overflow in the NAc and PFC disappeared after 14 days withdrawal. The results show that chronic (−)nicotine differentially affects the NMDA-mediated [³H]DA release in the PFC and NAc of the rat.     

Neuropeptide Y inhibits [Ca2+]i changes in rat retinal neurons through NPY Y1, Y4, and Y5 receptors

Neuropeptide Y (NPY) and NPY receptors are widely distributed in the CNS, including the retina, but the role of NPY in the retina is largely unknown. The aim of this study was to investigate whether NPY modulates intracellular calcium concentration ([Ca²⁺]_i) changes in retinal neurons and identify the NPY receptors involved. As NPY decreased the [Ca²⁺]_i amplitudes evoked by 30 mM KCl in only 50% of neurons analyzed, we divided them in two populations: NPY-non-responsive neurons (Δ2/Δ1 ≥ 0.80) and NPY-responsive neurons (Δ2/Δ1 < 0.80), being the Δ2/Δ1 the ratio between the amplitude of [Ca²⁺]_i increase evoked by the second (Δ2) and the first (Δ1) stimuli of KCl. The NPY Y₁/Y₅, Y₄, and Y₅ receptor agonists (100 nM), but not the Y₂ receptor agonist (300 nM), inhibited the [Ca²⁺]_i increase induced by KCl. In addition, the inhibitory effect of NPY on evoked-[Ca²⁺]_i changes was reduced in the presence of the Y₁ or the Y₅ receptor antagonists. In conclusion, NPY inhibits KCl-evoked [Ca²⁺]_i increase in retinal neurons through the activation of NPY Y₁, Y₄, and Y₅ receptors. This effect may be viewed as a potential neuroprotective mechanism of NPY against retinal neurodegeneration.     

Adenosine A2A receptors enable the synaptic effects of cannabinoid CB1 receptors in the rodent striatum

Adenosine A_2A, cannabinoid CB₁ and metabotropic glutamate 5 (mGlu₅) receptors are all highly expressed in the striatum. The aim of the present work was to investigate whether, and by which mechanisms, the above receptors interact in the regulation of striatal synaptic transmission. By extracellular field potentials (FPs) recordings in corticostriatal slices, we demonstrated that the ability of the selective type 1 cannabinoid receptor (CB₁R) agonist WIN55,212-2 to depress synaptic transmission was prevented by the pharmacological blockade or the genetic inactivation of A_2ARs. Such a permissive effect of A_2ARs towards CB₁Rs does not seem to occur pre-synaptically as the ability of WIN55,212-2 to increase the R2/R1 ratio under a protocol of paired-pulse stimulation was not modified by ZM241385. Furthermore, the effects of WIN55,212-2 were reduced in slices from mice lacking post-synaptic striatal A_2ARs. The selective mGlu₅R agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) potentiated the synaptic effects of WIN55,212-2, and such a potentiation was abolished by A_2AR blockade. Unlike the synaptic effects, the ability of WIN55,212-2 to prevent NMDA-induced toxicity was not influenced by ZM241385. Altogether, these results show that the state of activation of A_2ARs regulates the synaptic effects of CB₁Rs and that A_2ARs may control CB₁ effects also indirectly, namely through mGlu₅Rs.     

Serotonin-1A receptor function in the dorsal raphe nucleus following chronic administration of the selective serotonin reuptake inhibitor sertraline

Serotonin-1A (5-HT_1A) receptors in the dorsal raphe nucleus (DRN) function as somatodendritic autoreceptors, and therefore play a critical role in controlling serotonergic cell firing and serotonergic neurotransmission. We hypothesized that a decrease in the capacity of 5-HT_1A receptors to activate G proteins was a general mechanism by which 5-HT_1A receptors in the DRN are desensitized following chronic administration of selective serotonin reuptake inhibitors (SSRIs). Using in vivo microdialysis, we found that the ability of the 5-HT_1A receptor agonist 8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT) (0.025 mg/kg, s.c.) to decrease extracellular 5-HT levels in striatum was attenuated following chronic treatment of rats with the SSRIs sertraline or fluoxetine. This apparent desensitization of somatodendritic 5-HT_1A autoreceptor function was not accompanied by a decrease in 5-HT_1A receptor sites in the coupled, high-affinity agonist state as measured by the binding of [3H]8-OH-DPAT. In marked contrast to what was observed following chronic administration of fluoxetine, 5-HT_1A receptor-stimulated [³⁵S]GTPγS binding in the DRN was not altered following chronic sertraline treatment. Thus, desensitization of 5-HT_1A somatodendritic autoreceptor function following chronic sertraline administration appears not to be due to a decrease in the capacity 5-HT_1A receptors to activate G proteins in the DRN. Our findings suggest that the SSRIs may not be a homogeneous class of antidepressant drug with regard to the mechanism by which the function of somatodendritic 5-HT_1A autoreceptors is regulated.     

Imaging the Brain Marijuana Receptor: Development of a Radioligand that Binds to Cannabinoid CB1 Receptors In Vivo

Abstract: The major active ingredient of marijuana, (−)-Δ⁹-tetrahydrocannabinol, exerts its psychoactive effects via binding to cannabinoid CB1 receptors, which are widely distributed in the brain. Radionuclide imaging of CB1 receptors in living human subjects would help explore the presently unknown physiological roles of this receptor system, as well as the neurochemical consequences of marijuana dependence. Currently available cannabinoid receptor radioligands are exceedingly lipophilic and unsuitable for in vivo use. We report the development of a novel radioligand, [¹²³I]AM281{ N -(morpholin-4-yl)-5-(4-[¹²³I]iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H -pyrazole-3-carboxamide}, that is structurally related to the CB1-selective antagonist SR141716A [ N -(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H -pyrazole-3-carboxamide]. Baboon single photon emission computed tomography studies, mouse brain dissection studies, and ex vivo autoradiography in rat brain demonstrated rapid passage of [¹²³I]AM281 into the brain after intravenous injection, appropriate regional brain specificity of binding, and reduction of binding after treatment with SR141716A. AM281 has an affinity in the low nanomolar range for cerebellar binding sites labeled with [³H]SR141716A in vitro, and binding of [¹²³I]AM281 is inhibited by several structurally distinct cannabinoid receptor ligands. We conclude that [¹²³I]AM281 has appropriate properties for in vivo studies of cannabinoid CB1 receptors and is suitable for imaging these receptors in the living human brain.     

Dual Effects of Anandamide on NMDA Receptor-Mediated Responses and Neurotransmission

Abstract: Anandamide is an endogenous ligand of cannabinoid receptors that induces pharmacological responses in animals similar to those of cannabinoids such as Δ⁹-tetrahydrocannabinol (THC). Typical pharmacological effects of cannabinoids include disruption of pain, memory formation, and motor coordination, systems that all depend on NMDA receptor mediated neurotransmission. We investigated whether anandamide can influence NMDA receptor activity by examining NMDA-induced calcium flux (ΔCa²⁺_NMDA) in rat brain slices. The presence of anandamide reduced ΔCa²⁺_NMDA and the inhibition was disrupted by cannabinoid receptor antagonist, pertussis toxin treatment, and agatoxin (a calcium channel inhibitor). Whereas these treatments prevented anandamide inhibiting ΔCa²⁺_NMDA, they also revealed another, underlying mechanism by which anandamide influences ΔCa²⁺_NMDA. In the presence of cannabinoid receptor antagonist, anandamide potentiated ΔCa²⁺_NMDA in cortical, cerebellar, and hippocampal slices. Anandamide (but not THC) also augmented NMDA-stimulated currents in Xenopus oocytes expressing cloned NMDA receptors, suggesting a capacity to directly modulate NMDA receptor activity. In a similar manner, anandamide enhanced neurotransmission across NMDA receptor-dependent synapses in hippocampus in a manner that was not mimicked by THC and was unaffected by cannabinoid receptor antagonist. These data demonstrate that anandamide can modulate NMDA receptor activity in addition to its role as a cannabinoid receptor ligand.     

Manipulation of fatty acid amide hydrolase functional activity alters sensitivity and dependence to ethanol

The aim of this study was to examine the role of fatty acid amide hydrolase (FAAH) on ethanol sensitivity, preference, and dependence. The deletion of FAAH gene or the inhibition of FAAH by carbamoyl-biphenyl-3-yl-cyclohexylcarbamate (URB597) (0.1 mg/kg) markedly increased the preference for ethanol. The study further reveals that URB597 specifically acts through FAAH and that cannabinoid-1 (CB₁) receptor is critical for N -arachidonoyl ethanolamide (AEA) mediated ethanol-reinforced behavior as revealed by lack of URB597 effect in both FAAH and CB₁−/− mice compared with vehicle-treated −/− mice. The FAAH −/− mice displayed a lower sensitivity to hypothermic and sedative effects to acute ethanol challenge. The FAAH −/− mice also exhibited a reduction in the severity of handling-induced convulsions following withdrawal from chronic ethanol exposure. The CB₁ receptor and proenkephalin gene expressions, and CB₁ receptor and μ-opioid (MO) receptor-mediated G-protein activation were found to be significantly lower in the caudate-putamen, nucleus accumbens core and shell of FAAH −/− than +/+ mice. Interestingly, the MO receptor-stimulated G-protein signaling was greater in the striatum of FAAH −/− than +/+ mice following voluntary ethanol consumption. These findings suggest that an elevation in the AEA content and its action on the limbic CB₁ receptor and MO receptor might contribute to ethanol-reinforced behavior. Treatment with drugs that decrease AEA tone might prove useful in reducing excessive ethanol consumption.     

A 10-kDa acyl-CoA-binding protein (ACBP) from Brassica napus enhances acyl exchange between acyl-CoA and phosphatidylcholine

The gene encoding a 10-kDa acyl-CoA-binding protein (ACBP) from Brassica napus was over-expressed in developing seeds of Arabidopsis thaliana . Biochemical analysis of T₂ and T₃ A. thaliana seeds revealed a significant increase in polyunsaturated fatty acids (FAs) (18:2 ^{cis Δ9,12} and 18:3 ^{cis Δ9,12,15}) at the expense of very long monounsaturated FA (20:1 ^{cis Δ11}) and saturated FAs. In vitro assays demonstrated that recombinant B. napus ACBP (rBnACBP) strongly increases the formation of phosphatidylcholine (PC) in the absence of added lysophosphatidylcholine in microsomes from ΔYOR175c yeast expressing A. thaliana lysophosphatidylcholine acyltransferase ( AthLPCAT ) cDNA or in microsomes from microspore-derived cell suspension cultures of B. napus L. cv. Jet Neuf. rBnACBP or bovine serum albumin (BSA) were also shown to be crucial for AthLPCAT to catalyse the transfer of acyl group from PC into acyl-CoA in vitro . These data suggest that the cytosolic 10-kDa ACBP has an effect on the equilibrium between metabolically active acyl pools (acyl-CoA and phospholipid pools) involved in FA modifications and triacylglycerol bioassembly in plants. Over-expression of ACBP during seed development may represent a useful biotechnological approach for altering the FA composition of seed oil.     

Lack of CB1 receptor activity impairs serotonergic negative feedback

Serotonergic and endocannabinoid systems are important substrates for the control of emotional behaviour and growing evidence show an involvement in the pathophysiology of mood disorders. In the present study, the absence of the activity of the CB₁ cannabinoid receptor impaired serotonergic negative feedback in mice. Thus, in vivo microdialysis experiments revealed increased basal 5-HT extracellular levels and attenuated fluoxetine-induced increase of 5-HT extracellular levels in the prefrontal cortex of CB₁ knockout compared with wild-type mice. These observations could be related to the significant reduction in the 5-HT transporter binding site density detected in frontal cortex and hippocampus of CB₁ knockout mice. The lack of CB₁ receptor also altered some 5-HT receptors related to the 5-HT feedback. Extracellular recordings in the dorsal raphe nucleus (DRN) revealed that the genetic and pharmacological blockade of CB₁ receptor induced a 5-HT_1A autoreceptor functional desensitization. In situ hybridization studies showed a reduction in the expression of the 5-HT_2C receptor within several brain areas related to the control of the emotional responses, such as the DRN, the nucleus accumbens and the paraventricular nucleus of the hypothalamus, whereas an over-expression was observed in the CA3 area of the ventral hippocampus. These results reveal that the lack of CB₁ receptor induces a facilitation of the activity of serotonergic neurons in the DRN by altering different components of the 5-HT feedback as well as an increase in 5-HT extracellular levels in the prefrontal cortex in mice.     

Acid potentiation of the capsaicin receptor determined by a key extracellular site

The endocannabinoid anandamide (AEA) and some of its oxidative metabolites are putative ligands for the vanilloid receptor (VR₁). AEA affords protection against excitotoxicity induced in vivo by ouabain, a Na⁺/K⁺-ATPase inhibitor. This effect is only partly dependent of the cannabinoid CB₁ receptor. Here, we assessed whether VR₁ is involved in neuroprotection by AEA and arvanil, which is a hydrolysis-stable ligand for both VR₁ and CB₁. Using magnetic resonance imaging we show that: (i) modulation of VR₁, by the agonists arvanil and capsaicin and the antagonist capsazepine, leads to neuroprotective effects in the late but not acute phase after i.c. ouabain-injection; (ii) arvanil is a potent neuroprotectant, acting at both CB₁ and VR₁; and (iii) the neuroprotective effects of AEA are mediated by CB₁ but not by lipoxygenase metabolites or VR₁.     

Long-term treatment with fluoxetine induces desensitization of 5-HT4 receptor-dependent signalling and functionality in rat brain

The mode of action of antidepressant drugs may be related to mechanisms of monoamines receptor adaptation, including serotonin 5-HT₄ receptor subtypes. Here we investigated the effects of repeated treatment with the selective serotonin reuptake inhibitor fluoxetine for 21 days (5 and 10 mg/kg, p.o., once daily) on the sensitivity of 5-HT₄ receptors by using receptor autoradiography, adenylate cyclase assays and extracellular recording techniques in rat brain. Fluoxetine treatment decreased the density of 5-HT₄ receptor binding in the CA1 field of hippocampus as well as in several areas of the striatum over the doses of 5–10 mg/kg. In a similar way, we found a significant lower response to zacopride-stimulated adenylate cyclase activity in the fluoxetine 10 mg/kg/day treated group. Furthermore, post-synaptic 5-HT₄ receptor activity in hippocampus-measured as the excitatory action of zacopride in the pyramidal cells of CA1 evoked by Schaffer collateral stimulation was attenuated in rats treated with both doses of fluoxetine. Taken together, these results support the concept that a net decrease in the signalization pathway of 5-HT₄ receptors occurs after chronic selective serotonin reuptake inhibitor treatment: this effect may underlie the therapeutic efficacy of these drugs.     

Role of receptor internalization in the agonist-induced desensitization of cannabinoid type 1 receptors

Agonist-induced internalization of G protein-coupled receptors (GPCRs) is an important mechanism for regulating signaling transduction of functional receptors at the plasma membrane. We demonstrate here that both caveolae/lipid-rafts- and clathrin-coated-pits-mediated pathways were involved in agonist-induced endocytosis of the cannabinoid type 1 receptor (CB1R) in stably transfected human embryonic kidney (HEK) 293 cells and that the internalized receptors were predominantly sorted into recycling pathway for reactivation. The treatment of CB1 receptors with the low endocytotic agonist Δ⁹-THC induced a faster receptor desensitization and slower resensitization than the high endocytotic agonist WIN 55,212-2. In addition, the blockade of receptor endocytosis or recycling pathway markedly enhanced agonist-induced CB1 receptor desensitization. Furthermore, co-expression of phospholipase D2, an enhancer of receptor endocytosis, reduced CB1 receptor desensitization, whereas co-expression of a phospholipase D2 negative mutant significantly increased the desensitization after WIN 55,212-2 treatment. These findings provide evidences for the importance of receptor endocytosis in counteracting CB1 receptor desensitization by facilitating receptor reactivation. Moreover, in primary cultured neurons, the low endocytotic agonist Δ⁹-THC or anandamide exhibited a greater desensitization of endogenous CB1 receptors than the high endocytotic agonist WIN 55,212-2, CP 55940 or 2-arachidonoyl glycerol, indicating that cannabinoids with high endocytotic efficacy might cause reduced development of cannabinoid tolerance to some kind cannabinoid-mediated effects.     

Characterization of [3H]CP-96,501 as a Selective Radioligand for the Serotonin 5-HT1B Receptor: Binding Studies in Rat Brain Membranes

Abstract: 3-(1,2,5,6-Tetrahydro-4-pyridyl)-5- n -propoxyindole (CP-96,501) was found to be a more selective ligand at the serotonin 5-HT_1B receptor than the commonly used 5-HT_1B agonist, 3-(1,2,5,6-tetrahydro-4-pyridyl)-5-methoxyindole (RU 24969). In rat brain membranes, the tritiated derivative, [³H]CP-96,501, was found to bind with a high affinity ( K _D, 0.21 n M ) to a single binding site ( n _H, 1.0). The receptor density of this site ( B _max, 72 fmol/mg of protein) matched that of the 5-HT_1B receptor determined with [³H]5-HT. Competition curves of 16 serotonergic compounds in [³H]CP-96,501 binding also indicated a single binding site. The rank order of their binding affinities with this new radioligand showed a high degree of correlation with their affinities at the 5-HT_1B receptor determined with [³H]5-HT or [¹²⁵I]iodocyanopindolol. Serotonergic compounds displayed competitive inhibition of [³H]CP-96,501 binding. In the presence of 5'-guanylylimidodiphosphate [Gpp(NH)p], [³H]CP-96,501 binding was reduced, while the potency of CP-96,501 to displace [¹²⁵I]iodocyanopindolol binding was also decreased. These findings are consistent with the agonist nature of CP-96,501. The results of this study suggest that [³H]CP-96,501 is a useful agonist radioligand for the 5-HT_1B receptor.     

Effects of Gibberellic Acid on Primary Terpenoids and Δ9-Tetrahydrocannabinol in Cannabis sativa at Flowering Stage

Plants synthesize an astonishing diversity of isoprenoids, some of which play essential roles in photosynthesis, respiration, and the regulation of growth and development. Two independent pathways for the biosynthesis of isoprenoid precursors coexist within the plant cell: the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP) pathway. However, little is known about the effects of plant hormones on the regulation of these pathways. In the present study we investigated the effect of gibberellic acid (GA₃) on changes in the amounts of many produced terpenoids and the activity of the key enzymes, 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), in these pathways. Our results showed GA₃ caused a decrease in DXS activity in both sexes that it was accompanied by a decrease in chlorophylls, carotenoids and Δ⁹-tetrahydrocannabinol (THC) contents and an increase in α-tocopherol content. The treated plants with GA₃ showed an increase in HMGR activity. This increase in HMGR activity was followed by accumulation of stigmasterol and β-sitosterol in male and female plants and campestrol in male plants. The pattern of the changes in the amounts of sterols was exactly similar to the changes in the HMGR activity. These data suggest that GA₃ can probably influence the MEP and MVA pathways oppositely, with stimulatory and inhibitory effects on the produced primary terpenoids in MVA and DXS pathways, respectively.     

Adenosine A2a Receptor-Mediated Modulation of Striatal [3H]GABA and [3H]Acetylcholine Release

Abstract: The ability of adenosine agonists to modulate K⁺-evoked 4D†-[³H]aminobutyric acid ([³H]GABA) and acetylcholine (ACh) release from rat striatal synaptosomes was investigated. The A_2a receptor-selective agonist CGS 21680 inhibited Ca²⁺-dependent [³H]GABA release evoked by 15 m M KCI with a maximal inhibition of 29 ± 4% (IC₅₀ of ∼4 ± 10 ⁻¹² M ). The relative order of potency of three agonists was CGS 21680 ± 5'- N -ethylcarboxamidoadenosine > R-phenylisopropyladenosine (R-PIA), with the inhibition being blocked by A_2a receptor-selective antagonists (CP 66,713 and CGS 15943A) but not by the A₁-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). When release of [³H]GABA was evoked by 30 mM KCI, no significant inhibition was observed. In contrast, CGS 21680 stimulated the release of [³H]ACh evoked by 30 m M KCI, with a maximal stimulation of 26 ± 5% (IC₅₀ of ∼10⁻¹¹ M ). This effect was blocked by CP 66,713 but not by DPCPX. The A₁ agonist R -PIA inhibited [³H]ACh release, an effect blocked by DPCPX. It is concluded that adenosine A_2a receptors are present on both GABAergic and cholinergic striatal nerve terminals where they inhibit and stimulate transmitter release, respectively. Key Words : GABA—Acetylcholine—Adenosine receptors—Striatum.     

Chronic Exposure to Adenosine Receptor Agonists and Antagonists Reciprocally Regulates the A1 Adenosine Receptor-Adenylyl Cyclase System in Cerebellar Granule Cells

Abstract: Chronic treatment with the adenosine receptor antagonist caffeine evokes an up-regulation of A₁ adenosine receptors and increased coupling of the receptor to G proteins in rat brain membranes. However, chronic agonist exposure has not been explored. Primary cultures of cerebellar granule cells were exposed chronically to A₁ adenosine receptor agonists and antagonists. Exposure to the A₁ adenosine receptor agonist N ⁶-cyclopentyladenosine resulted in (1) a time- and concentration-dependent reduction in the density of receptors labeled by 1,3-[³H]dipropyl-8-cyclopentylxanthine, (2) an enhanced ability of guanyl nucleotides to decrease the fraction of A₁ adenosine receptor sites displaying high affinity for 2-chloroadenosine, and (3) a functional uncoupling of receptors from adenylyl cyclase (EC 4.6.1.1). The adenosine antagonists caffeine and 8- p -sulfophenyltheophylline produced alterations in A₁ adenosine receptor homeostasis that were antipodal to those associated with agonist treatment. Antagonist exposure (1) increased the density of A₁ adenosine receptors in cerebellar granule cell membranes, (2) blunted the effect of guanyl nucleotides on receptor coupling to G proteins, and (3) increased the functional coupling of receptors to adenylyl cyclase inhibition. Forskolin treatment of cerebellar granule cells did not affect receptor density, suggesting that cyclic AMP is not involved in the regulation of A₁ adenosine receptor expression.