Stimulatory effect of Coca-Cola<Superscript>TM</Superscript> on gastroduodenal HCO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack> secretion in rats

We examined the effect of various carbonated beverages, especially Coca-Cola^TM, on the HCO₃⁻ secretion in the rat stomach and duodenum. Under urethane anaesthesia, a chambered stomach or a proximal duodenal loop was perfused with saline, and HCO₃⁻ secretion was measured at pH 7.0 using a pH-stat method and by adding 2 mM HCl. The amount of CO₂ contained in these beverages was about 4–7 g/mL. Coca-Cola^TM topically applied to the mucosa for 10 min significantly increased the HCO₃⁻ secretion in both the stomach and the duodenum. The HCO₃⁻ response in the duodenum was totally abolished by indomethacin and also partially inhibited by acetazolamide, an inhibitor of carbonic anhydrase. Likewise, the response in the stomach was also markedly inhibited by either acetazolamide or indomethacin. The mucosal application of Coca-Cola^TM increased the PGE₂ contents in both the stomach and the duodenum. Other carbonated beverages, such as sparkling water, Fanta Grape^TM or cider, also increased the HCO₃⁻ secretion in these tissues. These results suggest that Coca-Cola^TM induces HCO₃⁻ secretion in both the stomach and the duodenum, and these responses may be attributable to both the intracellular supply of HCO₃⁻ generated via carbonic anhydrase, and endogenous PGs, probably related to the acidic pH of the solution. Received 4 August 2006; accepted 10 November 2006     

β<Superscript>2</Superscript>/β<Superscript>3</Superscript>-di- and α/β<Superscript>3</Superscript>-tetrapeptide derivatives as potent agonists at somatostatin sst<Subscript>4</Subscript> receptors

Four linear beta(2)/beta(3)-di- and alpha/beta(3)-tetrapeptides (1-4) were investigated as somatostatin sst(4) receptor agonists on recombinant human and mouse somatostatin receptors. Human somatostatin receptor subtypes 1-5 (sst(1-5)), and mouse somatostatin receptor subtypes 1,3,4 and 5, were characterised using the agonist radioligands [(125)I]LTT-SRIF-28, [(125)I][Tyr(10)]CST(14) and [(125)I]CGP 23996 in stably transfected Chinese hamster lung fibroblast (CCL39) cells. The peptides bound selectively to sst(4) receptors with nanomolar affinity (pK(d)=5.4-7.8). The peptides were investigated on second messenger systems both as agonists, and as antagonists to SRIF-14-mediated effects in CCL39 cells expressing mouse sst(4 )receptors, via measurement of inhibition of forskolin-stimulated adenylate cyclase activity, and stimulation of luciferase expression. The peptides showed full agonism or pronounced partial agonism (40 to 100% relative intrinsic activity) in both inhibition of forskolin-stimulated adenylate cyclase activity (pEC(50)=5.5-6.8), and luciferase expression (pEC(50)=5.5-6.5). The agonist potential was confirmed since antagonism was very difficult to establish. The data show that beta(2)/beta(3)-di- and alpha/beta(3)-tetrapeptide derivatives have agonist potential at recombinant somatostatin sst(4) receptors. Therefore, they may be used to elucidate physiological and biochemical effects mediated by sst(4), and may also have potential as therapeutic agents.     

Molecular characterization of specific H<Subscript>1</Subscript>-receptor agonists histaprodifen and its N<Superscript>α</Superscript>-substituted analogues on bovine aortic H<Subscript>1</Subscript>-receptors

We determined the molecular properties of the selective and potent H(1)-receptor agonist histaprodifen and its N(alpha) substituted analogues: methyl-, dimethyl-, and imidazolylethyl-histaprodifen (suprahistaprodifen). All derivatives show high affinity for (3)H-mepyramine labeled bovine aortic H(1)-receptor binding sites with the following order of potency: suprahistaprodifen > dimethylhistaprodifen > methylhistaprodifen > histaprodifen > histamine. Suprahistaprodifen and dimethylhistaprodifen were the most potent displacers of (3)H-mepyramine binding (K(i)=4.3 and 4.9 nM, respectively). Histaprodifen, methylhistaprodifen and suprahistaprodifen binding was differentially influenced by GTP, whereas dimethylhistaprodifen was not affected. All drugs, except dimethylhistaprodifen, were activators of G-proteins. Their order of potency was suprahistaprodifen > histamine > histaprodifen > methylhistaprodifen. Their effect on G-protein activation was abolished by the addition of the H(1)-receptor antagonist triprolidine (10 microM), which given alone did not activate G-proteins. Our data suggest that histaprodifens are potent but heterogeneous H(1)-receptor ligands with diverse effects on the molecular level in our model system. While the histaprodifen, methylhistaprodifen and suprahistaprodifen data are in agreement with their agonistic nature, as shown in the functional studies performed on different species (rat and guinea pig H(1)-receptor), dimethylhistaprodifen behaved as an antagonist in our study.     

Binding of [<Superscript>3</Superscript>H]prazosin to α<Subscript>1A</Subscript>- and α<Subscript>1B</Subscript>-adrenoceptors,and to a cimetidine-sensitive non-α<Subscript>1</Subscript> binding site in rat kidney membranes

[(3)H]Prazosin bound to alpha(1A)- and alpha(1B)-adrenoceptors, as well as to a cimetidine-sensitive non-alpha(1)-adrenoceptor binding site in rat kidney membranes. An experimental design is presented where the alpha(1)-adrenoceptors are selectively exposed by blocking the non-alpha(1) binding site with 60 microM cimetidine. Conversely, the non-alpha(1) binding site can be selectively exposed by blocking the alpha(1)-adrenoceptors with 600 nM metitepine. The identity of the non-alpha(1) binding site for [(3)H]prazosin in the rat kidney, herein pharmacologically characterized by 33 competing substances, is still unknown.     

Chronic Δ<Superscript>9</Superscript>-Tetrahydrocannabinol Administration Reduces IgE<Superscript>+</Superscript>B Cells but Unlikely Enhances Pathogenic SIV<Subscript>mac251</Subscript> Infection in Male Rhesus Macaques of Chinese Origin

Delta9-tetrahydrocannabinol (Δ⁹-THC) is the major psychoactive component of the cannabis plant. Δ⁹-THC has been used in the active ingredient of Marinol as an appetite stimulant for AIDS patients. Its impact on progression of HIV-1 infection, however, remains debatable. Previous studies indicated that Δ⁹-THC administration enhanced HIV-1 infection in huPBL-SCID mice but seemingly decreased early mortality in simian immunodeficiency virus (SIV) infected male Indian-derived rhesus macaques. Here, we determine the chronic effect of Δ⁹-THC administration using 0.32 mg/kg or placebo (PBO), i.m., twice daily for 428 days on SIV_mac251 infected male Chinese-derived rhesus macaques. Sixteen animals were divided into four study groups: Δ⁹-THC⁺SIV⁺, Δ⁹-THC⁺SIV^?, PBO/SIV⁺ and PBO/SIV^? (n = 4/group). One-month after daily Δ⁹-THC or PBO administrations, macaques in groups one and three were challenged intravenously with pathogenic SIV_mac251/CNS, which was isolated from the brain of a Chinese macaque with end-staged neuroAIDS. No significant differences in peak and steady state plasma viral loads were seen between Δ⁹-THC⁺SIV⁺ and PBO/SIV⁺ macaques. Regardless of Δ⁹-THC, all infected macaques displayed significant drop of CD4/CD8 T cell ratio, loss of CD4⁺ T cells and higher persistent levels of Ki67⁺CD8⁺ T cells compared with uninfected animals. Moreover, long-term Δ⁹-THC treatment reduced significantly the frequency of circulating IgE⁺B cells. Only one Δ⁹-THC⁺SIV⁺ macaque died of simian AIDS with paralyzed limbs compared with two deaths in the PBO/SIV⁺ group during the study period. These findings indicate that chronic Δ⁹-THC administration resulted in reduction of IgE⁺B cells, yet it unlikely enhanced pathogenic SIV_mac251/CNS infection in male Rhesus macaques of Chinese origin.     

Modulation of 5-HT<Subscript>1A</Subscript> receptor-mediated Ca<Superscript>2+</Superscript> responses by co-expression with various recombinant G<Subscript>α</Subscript> proteins in CHO-K1 cells

The ability of the human 5-HT_1A receptor to activate different recombinant G proteins was investigated in CHO-K1 cells by monitoring 5-HT ligand-mediated Ca²⁺ responses upon co-expression with either G_q, G₁₅ or chimeric G_q/i3 proteins. Each G protein yielded a typical 5-HT-dependent Ca²⁺ response with different kinetic parameters both for the onset-time of maximal Ca²⁺ response (21 to 30 s) and time-dependent attenuation (43 to 73% of residual activity at 1 min upon peak Ca²⁺ response). Pertussis toxin-treatment fully abolished the Ca²⁺ responses mediated by both the endogenous G_i/o and the chimeric-PTX-sensitive G_q/i3 proteins. In contrast, Ca²⁺ responses driven by recombinant G_q and G₁₅ proteins were decreased by PTX, respectively by 52% and 35%, corresponding to the level of endogenous G protein activation. The pharmacology of the 5-HT ligand-mediated Ca²⁺ responses was highly affected by both the presence and nature of the co-expressed G protein. This influence was more pronounced for the partial agonists L 694247, 8-OH-DPAT, flesinoxan and buspirone in contrast to ipsapirone. The G protein rank order for apparent increase of ligands' intrinsic activity was: G_q <G_q/i3 <G₁₅ protein. Each of the 5-HT-mediated Ca²⁺ responses could be antagonised by WAY 100635, buspirone and methiothepin regardless of the absence or presence of a G_q, G_q/i3 or G₁₅ protein. In conclusion, these data reinforce that depending on the presence and nature of the G protein environment, 5-HT_1A ligands may display a large spectrum of activities.Abbreviations AFU Arbitrary fluorescence unit - 5-CT 5-Carboxamidotryptamine - 5-HT 5-Hydroxytryptamine (serotonin) - 8-OH-DPAT 8-(Hydroxy-2-(di-n-propylamino)tetralin - CHO Chinese hamster ovary - PLC Phospholipase C - WAY 100635 N-[2-[4-(2-methoxyphenyl)1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexanecarboxamide - PTX Bordetella pertussis toxin - wt Wild-type     

Pharmacological profile of the cyclic nociceptin/orphanin FQ analogues c[Cys<Superscript>10,14</Superscript>]N/OFQ(1–14)NH<Subscript>2</Subscript> and c[Nphe<Superscript>1</Superscript>,Cys<Superscript>10,14</Superscript>]N/OFQ(1–14)NH<Subscript>2</Subscript>

In this study we describe the activity of two cyclic nociceptin/orphanin FQ (N/OFQ) peptides; c[Cys^10,14]N/OFQ(1–14)NH₂ (c[Cys^10,14]) and its [Nphe¹] derivative c[Nphe¹,Cys^10,14]N/OFQ(1–14)NH₂ (c[Nphe¹,Cys^10,14]) in native rat and mouse and recombinant human N/OFQ receptors (NOP). Cyclisation may protect the peptide from metabolic degradation.In competition binding studies of rat, mouse and human NOP the following rank order pK_i was obtained: N/OFQ(1–13)NH₂(reference agonist)>N/OFQ=c[Cys^10,14]>>c[Nphe¹Cys^10,14]. In GTP³⁵S studies of Chinese hamster ovary cells expressing human NOP (CHO_hNOP) c[Cys^10,14] (pEC₅₀ 8.29) and N/OFQ(1–13)NH₂ (pEC₅₀ 8.57) were full agonists whilst c[Nphe¹Cys^10,14] alone was inactive. Following 30 min pre-incubation c[Nphe¹Cys^10,14] competitively antagonised the effects of N/OFQ(1–13)NH₂ with a pA₂ and slope factor of 6.92 and 1.01 respectively. In cAMP assays c[Cys^10,14] (pEC₅₀ 9.29, E_max 102% inhibition of the forskolin stimulated response), N/OFQ(1–13)NH₂ (pEC₅₀ 10.16, E_max 103% inhibition) and c[Nphe¹Cys^10,14] (~80% inhibition at 10 M) displayed agonist activity. In the mouse vas deferens c[Cys^10,14] (pEC₅₀ 6.82, E_max 89% inhibition of electrically evoked contractions) and N/OFQ(1–13)NH₂ (pEC₅₀ 7.47, E_max 93% inhibition) were full agonists whilst c[Nphe¹Cys^10,14] alone was inactive. c[Nphe¹Cys^10,14] (10 M) competitively antagonised the effects of N/OFQ(1–13)NH₂ with a pK_B of 5.66. In a crude attempt to assess metabolic stability, c[Cys^10,14] was incubated with rat brain membranes and then the supernatant assayed for remaining peptide. Following 60 min incubation 64% of the 1 nM added peptide was metabolised (compared with 54% for N/OFQ-NH₂).In summary, we report that c[Cys^10,14] is a full agonist with a small reduction in potency but no improvement in stability whilst c[Nphe¹Cys^10,14] displays tissue (antagonist in the vas deferens) and assay (antagonist in the GTP³⁵S assay and agonist in cAMP assay) dependent activity.Presented in part to The British Pharmacological Society at the Brighton, UK Meeting January 2003     

Effect of the blockade of μ<Subscript>1</Subscript>-opioid and 5HT<Subscript>2A</Subscript>-serotonergic/α<Subscript>1</Subscript>-noradrenergic receptors on sweet-substance-induced analgesia

Rationale Sweet-substance-induced analgesia has been widely studied, and the investigation of the neurotransmitters involved in this antinociceptive process is an important way for understanding the involvement of the neural system controlling this kind of antinociception.Objective The aim of this study was to investigate the involvement of opioid and monoaminergic systems in sweet-substance-induced analgesia.Methods The present work was carried out in an animal model with the aim of investigating whether acute (24 h) or chronic (14 days) intake of a sweet substance, such as sucrose (250 g/l), is followed by antinociception. Tail withdrawal latencies in the tail-flick test were measured before and immediately after this treatment. Immediately after the recording of baseline values, independent groups of rats were submitted to sucrose or tap-water intake and, after chronic treatment, they were pretreated with intraperitoneal administration of (1) naltrexone at 0.5, 1, 2 or 3 mg/kg; (2) naloxonazine at 5, 10, 20 or 30 mg/kg; (3) methysergide at 0.5, 1, 2 or 3 mg/kg; (4) ketanserin at 0.5, 1, 2 or 3 mg/kg; or (5) physiological saline.Results Naltrexone and methysergide at two major doses decreased sweet-substance-induced analgesia after chronic intake of a sweet substance. These effects were corroborated by peripheral administration of naloxonazine and ketanserin.Conclusions These data give further evidence for: (a) the involvement of endogenous opioids and a ₁-opioid receptor in the sweet-substance-induced antinociception; (b) the involvement of monoamines and 5HT_2A serotonergic/₁-noradrenergic receptors in the central regulation of the sweet-substance-produced analgesia.     

Discriminative stimulus effects of zolpidem in squirrel monkeys: role of GABA<Subscript>A</Subscript>/α<Subscript>1</Subscript> receptors

Abstract Rationale. The discriminative stimulus effects of zolpidem in squirrel monkeys trained at doses greater than or equal to 3.0 mg/kg differ from those of conventional benzodiazepines (BZs), but the extent to which these effects reflect the selectivity of zolpidem for GABA_A/α₁ receptors is not known. Objectives. The present study investigated the ability of GABA_A/α₁-preferring agonists to substitute for training doses of zolpidem greater than or equal to 3.0 mg/kg and the ability of GABA_A/α₁-preferring antagonists to block zolpidem's discriminative stimulus effects. Methods. Squirrel monkeys were trained to discriminate intravenous injections of zolpidem (3.0 or 5.6 mg/kg) from saline and tested with BZ agonists differing in selectivity and efficacy at GABA_A/α₁ receptors. Antagonism of the effects of zolpidem was studied using the GABA_A/α₁-preferring antagonists β-carboline-3-carboxylate-t-butyl ester (β-CCT) and 3-propyloxy-β-carboline (3-PBC). Results. Zolpidem and quazepam (GABA_A/α₁-preferring agonist) engendered full substitution for zolpidem, whereas CL 218,872 (GABA_A/α₁-preferring partial agonist) and the non-selective BZ agonists alprazolam and flunitrazepam engendered low and variable levels of zolpidem-lever responding (35–58%). Both β-CCT and 3-PBC antagonized the discriminative stimulus effects of zolpidem in a surmountable fashion. Conclusions. Our findings provide evidence for a key role of GABA_A/α₁ receptors in the discriminative stimulus effects of zolpidem at relatively high training doses, and suggest that selectivity and relatively high efficacy at GABA_A/α₁ receptors is required for BZ agonists to reproduce these discriminative stimulus effects. Electronic Publication     

Binding of (−)-[<Superscript>3</Superscript>H]-CGP12177 at two sites in recombinant human β<Subscript>1</Subscript>-adrenoceptors and interaction with β-blockers

To verify the hypothesis that the non-conventional partial agonist (–)-CGP12177 binds at two ₁-adrenoceptor sites, human ₁-adrenoceptors, expressed in CHO cells, were labelled with (–)-[³H]-CGP12177. We compared the binding affinity and antagonist potency of 12 clinically used -blockers against the cyclic AMP-enhancing effects of (–)-isoprenaline and (–)-CGP12177.(–)-[³H]-CGP12177 bound to a high affinity site (H; K_H=0.47 nM) and low affinity site (L); K_L=235 nM). (–)-[³H]-CGP12177 dissociated from the ₁-adrenoceptors with a fast component (k_off=0.45 min^–1), consistent with the L-site, and a slow component (k_off=0.017–0.033 min^–1), consistent with the H-site. (–)-Isoprenaline and (–)-CGP12177 caused 96-fold and 12-fold maximal increases in cyclic AMP levels with –logEC₅₀M of 8.2 and 7.6. (–)-CGP12177 antagonised the effects of (–)-isoprenaline with a pK_B of 9.9. The -blockers antagonised the effects of (–)-isoprenaline more than the effects of (–)-CGP12177 with potency ratios: (–)-atenolol 1,000, (±)-metropolol 676, (–)-pindolol 631, (–)-timolol 589, (±)-carvedilol 204, (±)-oxprenolol 138, (±)-sotalol 132, (–)-propranolol 120, (±)-bisoprolol 95, (±)-alprenolol 81, (±)-nadolol 68 and (–)-bupranolol 56. In intact cells the binding constants of -blockers, estimated from competition with 3–5 nM (–)-[³H]-CGP12177 (binding to the H-site), correlated with the corresponding affinities estimated from antagonism of the (–)-isoprenaline effects.We conclude that (–)-[³H]-CGP12177 binds at two sites in the recombinant ₁-adrenoceptor. (–)-CGP12177 is an antagonist of catecholamine effects through the H-site and a non-conventional partial agonist through the L-site. -blockers are more potent antagonists through the H-site than the L-site.     

Antiproliferative and cytotoxic effects of some σ<Subscript>2</Subscript> agonists and σ<Subscript>1</Subscript> antagonists in tumour cell lines

To establish the activity of ligands at ₁ and ₂ receptor, we chose two tumour cell lines, the human SK-N-SH neuroblastoma and the rat C6 glioma lines, which express ₂ receptors at a high density and ₁ receptors in their high-affinity or low-affinity state. We tested the ₂ receptor agonist PB28 and the ₂ antagonist AC927, and (+)-pentazocine and NE100 as agonist and antagonist, respectively, at ₁ receptors, with regard to antiproliferative and cytotoxic effects. In addition, 1,3-di(2-tolyl)guanidine (DTG) and haloperidol were tested as reference compounds displaying nearly equipotent affinity (₂>₁ and ₁>₂, respectively). In both SK-N-SH and C6 cells, PB28 and NE100 displayed the most potent results both in antiproliferative and cytotoxic assay while AC927 and (+)-pentazocine were inactive in both assays. The cytotoxic and antiproliferative effects of DTG and haloperidol reflected their ₁ antagonist activity and ₂ agonist activity. Moreover, our results in the tumour cell lines correlated well with those for ₂ activity found previously in a functional assay in the guinea-pig bladder. These findings establish a new model for evaluating both ₂ and ₁ receptor activity of ligands, which could be useful for developing new ligands having mixed ₂ agonist/₁ antagonist activity as potential antineoplastic agents.     

Positive allosteric modulatory effects of ajulemic acid at strychnine-sensitive glycine α<Subscript>1</Subscript>- and α<Subscript>1</Subscript>β-receptors

The synthetic cannabinoid ajulemic acid (CT-3) is a potent cannabinoid receptor agonist which was found to reduce pain scores in neuropathic pain patients in the absence of cannabis-like psychotropic adverse effects. The reduced psychotropic activity of ajulemic acid has been attributed to a greater contribution of peripheral CB receptors to its mechanism of action as well as to non-CB receptor mechanisms. Loss of inhibitory synaptic transmission within the dorsal horn of the spinal cord plays a key role in the development of chronic pain following inflammation or nerve injury. Inhibitory postsynaptic transmission in the adult spinal cord involves mainly glycine. As we hypothesised that additional non-CB receptor mechanisms of ajulemic acid might contribute to its effect in neuropathic pain, we investigated the interaction of ajulemic acid with strychnine-sensitive α₁- and α₁β-glycine receptors by using the whole-cell patch clamp technique. Ajulemic acid showed a positive allosteric modulating effect in a concentration range which can be considered close to clinically relevant concentrations (EC₅₀ values: α₁ = 9.7 ± 2.6 μM and α₁β = 12.4 ± 3.4 μM). Direct activation of glycine receptors was observed at higher concentrations above 100 μM (EC₅₀ values: α₁ = 140.9 ± 21.5 μM and α₁β = 154.3 ± 32.1 μM). These in vitro results demonstrate that ajulemic acid modulates strychnine-sensitive glycine receptors in clinically relevant concentrations.     

Molecular structure of the rabbit α<Subscript>2A</Subscript>-adrenoceptor: a contribution to the α<Subscript>2A</Subscript>-adrenoceptor versus I<Subscript>1</Subscript> imidazoline receptor controversy

In view of the high structural and pharmacological similarities between the alpha(2A)-adrenoceptors of humans and other mammalian species, it has been concluded, in particular, from experiments in rabbits that the (2A)-adrenoceptor is the exclusive site of action of central antihypertensive drugs, although the amino acid sequence of the alpha(2A)-adrenoceptor of just this species was unknown. Therefore, the aim of the present investigation was to determine the complete nucleotide sequence of the coding region of the rabbit alpha(2A)-adrenoceptor gene. Degenerate oligonucleotides corresponding to regions of the alpha(2A)-adrenoceptor conserved between rat and man were used in a polymerase chain reaction with genomic DNA prepared from rabbit. A 1,356-base pair product with an open reading frame of 1,353 base pairs was obtained that encodes a protein of 451 amino acids which is similar to the alpha(2A)-adrenoceptors of other mammals (man, pig, rat, mouse, guinea-pig and cattle) but not to their alpha(2B)- and alpha(2C)-adrenoceptor subtypes suggesting its classification as an alpha(2A)-adrenoceptor. However, the degree of amino acid sequence identity is, at best, only 80% and, thus, about 10% less than between the other mammalian species. Compared with the human sequence there are 81 substantial changes of amino acids. In conclusion, rabbit and human alpha(2A)-adrenoceptors substantially differ in their amino acid sequence which may explain the opposite pharmacodynamic properties of the central antihypertensive drug rilmenidine (alpha(2)-adrenoceptor agonism and antagonism, respectively) reported in the literature. Hence, the present study supports the view that experiments with central antihypertensive drugs in rabbits are not reliably predictive for the site of action of such drugs in man.     

Calcium channel function and regulation in β<Subscript>1</Subscript>- and β<Subscript>2</Subscript>-adrenoceptor transgenic mice

Cardiac effects of catecholamines on the L-type calcium channel depend on -adrenoceptor subtype (₁- vs. ₂-adrenoceptor). Chronic overexpression of these receptors leads to hypertrophy and early death at moderate (₁) or excessive (₂) levels of overexpression respectively. In order to examine the role of L-type calcium channels in altered cardiomyocyte calcium homeostasis found with ₁-adrenoceptor overexpression, and to understand the quantitative differences between -adrenoceptor subtypes regarding calcium channel regulation, we examined single channels in myocytes obtained from ₁- and ₂-adrenoceptor transgenic mice. The effects of the agonist isoproterenol were investigated and compared with acute receptor stimulation in the respective non-transgenic littermates.Channels from ₁-adrenoceptor transgenic mice have normal baseline activity, and channel number is not reduced. This contrasts to previous findings with ₂-adrenoceptor transgenic mice, where channel activity is depressed. Isoproterenol is unable to stimulate channel activity in both transgenic models.In conclusion, the L-type calcium channel is not likely to be involved in alterations of calcium handling of ₁-adrenoceptor transgenic myocytes. Furthermore, chronic ₁-adrenoceptor overexpression does not depress channel activity, giving another example of the difference between ₁- and ₂-adrenoceptor signal transduction.K.F. and T.K. equally contributed to this work     

Comparison of the subjective effects of Δ<Superscript>9</Superscript>-tetrahydrocannabinol and marijuana in humans

RATIONALE: There has been controversy about whether the subjective, behavioral or therapeutic effects of whole plant marijuana differ from the effects of its primary active ingredient, Delta(9)-tetrahydrocannabinol (THC). However, few studies have directly compared the effects of marijuana and THC using matched doses administered either by the smoked or the oral form.OBJECTIVE: Two studies were conducted to compare the subjective effects of pure THC to whole-plant marijuana containing an equivalent amount of THC in normal healthy volunteers. In one study the drugs were administered orally and in the other they were administered by smoking.METHODS: In each study, marijuana users (oral study: n=12, smoking study: n=13) participated in a double-blind, crossover design with five experimental conditions: a low and a high dose of THC-only, a low and a high dose of whole-plant marijuana, and placebo. In the oral study, the drugs were administered in brownies, in the smoking study the drugs were smoked. Dependent measures included the Addiction Research Center Inventory, the Profile of Mood States, visual analog items, vital signs, and plasma levels of THC and 11-nor-9-carboxy-THC.RESULTS: In both studies, the active drug conditions resulted in dose-dependent increases in plasma THC levels, and the levels of THC were similar in THC-only and marijuana conditions (except that at the higher oral dose THC-only produced slightly higher levels than marijuana). In both the oral study and the smoking study, THC-only and whole plant marijuana produced similar subjective effects, with only minor differences.CONCLUSION: These results support the idea that the psychoactive effects of marijuana in healthy volunteers are due primarily to THC.     

Comparison of smoked marijuana and oral Δ<Superscript>9</Superscript>-tetrahydrocannabinol in humans

Abstract Rationale. Although smoked marijuana contains at least 60 cannabinoids, Δ⁹-tetrahydrocannabinol (Δ⁹-THC) is presumed to be the cannabinoid primarily responsible for many marijuana-related effects, including increased food intake and subjective effects. Yet, there has been no systematic comparison of repeated doses of oral Δ⁹-THC with repeated doses of smoked marijuana in the same individuals. Objective. To compare the effects of oral Δ⁹-THC and smoked marijuana in humans under controlled laboratory conditions. Methods. Eleven healthy research volunteers, who reported smoking an average of six marijuana cigarettes per day, completed an 18-day residential study. Marijuana cigarettes (3.1% Δ⁹-THC, q.i.d.) were smoked or Δ⁹-THC (20 mg, q.i.d.) was taken orally using a staggered, double-blind, double-dummy procedure for three consecutive days. Four days of placebo administration separated each active drug condition. Psychomotor task performance, subjective effects, and food intake were measured throughout the day. Results. Relative to placebo baseline, oral Δ⁹-THC and smoked marijuana produced similar subjective-effect ratings (e.g., "high" and "mellow"), although some effects of smoked marijuana were more pronounced and less prone to the development of tolerance. Additionally, participants reported "negative" subjective effects (e.g., "irritable" and "miserable") during the days after smoking marijuana but not after oral Δ⁹-THC. Both drugs increased food intake for 3 days of drug administration, but had little effect on psychomotor performance. Conclusion. These results indicate that the behavioral profile of effects of smoked marijuana (3.1% Δ⁹-THC) is similar to the effects of oral Δ⁹-THC (20 mg), with some subtle differences. Electronic Publication     

Generation and Disease Model Relevance of a Manganese Enhanced Magnetic Resonance Imaging-Based NOD/scid-IL-2R<Emphasis Type="Italic">γ</Emphasis><Subscript>c</Subscript><Superscript><Emphasis Type="Italic">null</Emphasis></Superscript> Mouse Brain Atlas

Strain specific mouse brain magnetic resonance imaging (MRI) atlases provide coordinate space linked anatomical registration. This allows longitudinal quantitative analyses of neuroanatomical volumes and imaging metrics for assessing the role played by aging and disease to the central nervous system. As NOD/scid-IL-2Rγ _c ^null (NSG) mice allow human cell transplantation to study human disease, these animals are used to assess brain morphology. Manganese enhanced MRI (MEMRI) improves contrasts amongst brain components and as such can greatly help identifying a broad number of structures on MRI. To this end, NSG adult mouse brains were imaged in vivo on a 7.0 Tesla MR scanner at an isotropic resolution of 100 μm. A population averaged brain of 19 mice was generated using an iterative alignment algorithm. MEMRI provided sufficient contrast permitting 41 brain structures to be manually labeled. Volumes of 7 humanized mice brain structures were measured by atlas-based segmentation and compared against non-humanized controls. The humanized NSG mice brain volumes were smaller than controls (p?<?0.001). Many brain structures of humanized mice were significantly smaller than controls. We posit that the irradiation and cell grafting involved in the creation of humanized mice were responsible for the morphological differences. Six NSG mice without MnCl₂ administration were scanned with high resolution T₂-weighted MRI and segmented to test broad utility of the atlas.     

The effects of hydrocortisone on rat heart muscarinic and adrenergic α<Subscript>1</Subscript>, β<Subscript>1</Subscript> and β<Subscript>2</Subscript> receptors,propranolol-resistant binding sites and on some subsequent steps in intracellular signalling

Glucocorticoids affect the expression and density of neurotransmitter receptors in many tissues but data concerning the heart are contradictory and incomplete. We injected rats with hydrocortisone for 1–12 days and measured the densities of cardiac muscarinic receptors, ₁-, ₁- and ₂-adrenoceptors and propranolol-resistant binding sites (formerly assumed to be the putative ₄-adrenoceptor). Some aspects of intracellular signalling were also evaluated: we measured adenylyl cyclase activity (basal, isoprenaline- and forskolin-stimulated and carbachol-inhibited), the coupling between muscarinic receptors and G proteins and basal and isoprenaline-stimulated heart rate. The density of cardiac muscarinic receptors increased (in both the atria and the ventricles). The density of ₁-adrenoceptors increased in the atria and was little changed in the ventricles. The density of ₂-adrenoceptors increased in both the atria and the ventricles. The number of ₁-adrenoceptors decreased initially, followed by a transient increase in the atria and did not change in the ventricles. The density of propranolol-resistant binding sites first increased and then diminished in the atria and did not change in the ventricles. Although there were noticeable changes in receptor densities, the stimulatory and inhibitory effects on adenylyl cyclase, basal and isoprenaline-stimulated heart rate and the coupling between muscarinic receptors and G proteins were not significantly altered. This may indicate that changes in receptor densities might be one of the mechanisms maintaining stable functional output. Deceased     

Role of β<Subscript>3</Subscript>-adrenoceptors in regulation of retinal vascular tone in rats

The aim of this study was to determine the role of β₃-adrenoceptors in the action of endogenous catecholamines (adrenaline and noradrenaline) on rat retinal arterioles in vivo. Using an original high-resolution digital fundus camera, the rat ocular fundus images were captured. The diameter of retinal arterioles contained in the images was measured. Both systemic blood pressure and heart rate were recorded continuously. Adrenaline (0.3–5.0 μg/kg/min, i.v.) increased the diameter of retinal arterioles, mean blood pressure and heart rate in a dose-dependent manner. Under blockade of β₁/β₂-adrenoceptors with propranolol (2 mg/kg, i.v. bolus followed by 100 μg/kg/min infusion), adrenaline decreased the diameter of retinal arterioles. Similar observation was made under treatment with the β₃-adrenoceptor antagonist L-748337 (50 μg/kg, i.v.). The pressor response to adrenaline was enhanced by propranolol, but not by L-748337. The positive chronotropic action of adrenaline was markedly prevented by propranolol, whereas it was unaffected by L-748337. Noradrenaline (0.03–1.0 μg/kg/min, i.v.) decreased the diameter of retinal arterioles but increased the mean blood pressure and heart rate. The effects of noradrenaline on retinal arteriolar diameter and blood pressure were unaffected by propranolol or L-748337. The positive chronotropic action of noradrenaline was almost completely abolished by propranolol. These results suggest that β₃-adrenoceptors play crucial roles in vasodilator responses to adrenaline of retinal arterioles but have minor or no effect on noradrenaline-induced responses. The results also indicate that the functional role of β₃-adrenoceptors may be more important than that in peripheral resistance vessels.