Endomorphins 1 and 2 reduce relaxant non-adrenergic,non-cholinergic neurotransmission in rat gastric fundus

It is now well established that opioids modulate cholinergic excitatory neurotransmission in the gastrointestinal tract. The aim of the present study was to characterize a possible effect of endomorphins on nonadrenergic, noncholinergic (NANC) relaxant neurotransmission in the rat gastric fundus in vitro. The drugs used in the experiments were the endogenous mu-opioid receptors (MORs) endomorphin 1 and 2 and the mu-opioid receptor antagonist CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2). CTAP left the basal tonus and the spontaneous activity of the preparation unchanged. Electrical field stimulation (EFS) under NANC conditions at frequencies ranging from 0.5 to 16 Hz caused a frequency-dependent relaxant response on the 5-hydoxytryptamine (5-HT) (10(-7) M) precontracted smooth-muscle strip. Both endomorphin 1 and endomorphin 2 significantly reduced this relaxation in a concentration-dependent manner. Endomorphin 1 proved to be more potent in reducing the relaxant responses. The endomorphin effects were significantly reversed by the MOR antagonist CTAP. CTAP itself did not influence the EFS-induced relaxation. In summary, these data provide evidence that the endogenous MOR agonists endomorphin 1 and 2 can reduce nonadrenergic, noncholinergic neurotransmission in the rat gastric fundus smooth muscle via a pathway involving MORs. The physiological relevance of these findings remains to be established, since the data presented suggest that the endomorphins act as neuromodulators within NANC relaxant neurotransmission.     

Effects of cannabinoids on endogenous K+ and Ca2+ currents in HEK293 cells

Effects of cannabinoids on endogenous potassium and calcium currents in HEK293 cells were studied using the whole-cell variant of the patch-clamp technique. The cannabinoid agonists WIN 55,212-2, methanandamide, and anandamide (1 microM) decreased the calcium current by 53.1 +/- 2.6, 47.5 +/- 1.2, and 38.8 +/- 3.1%, respectively, after transfection of human CB1 cannabinoid receptor (hCB1) cDNA into HEK293 cells. The delayed rectifier-like current was not changed after application of these agonists, but the inward rectifier was increased by 94.0 +/- 3.6, 83.7 +/- 5.1, and 63.0 +/- 2.5% after application of WIN 55,212-2, methanandamide, and anandamide, respectively. The effects of the cannabinoid antagonists (AM251, AM281, and AM630) on the inward rectifier and calcium currents were the opposite of those seen with cannabinoid agonists; thus, these compounds act as inverse agonists in this preparation. These results suggest that endogenous inward rectifier and calcium currents are modulated by cannabinoids in HEK293 cells, and that some expressed receptors may be constitutively active.     

Inhibition of peristaltic activity by cannabinoids in the isolated distal colon of mouse.

The effects of the cannabinoid receptor agonist Win 55,212-2 and of the competitive cannabinoid receptor antagonist SR 141716A on the electrically-evoked peristalsis of isolated distal colon of mouse were studied. Intraluminal pressure, longitudinal displacement, ejected fluid volume and changes in morphology of external intestinal wall were simultaneously recorded in the pre-drug period and in presence of Win 55,212-2 alone or in combination with SR 141716A. In the pre-drug period (control), peristaltic activity was characterised by regular, monophasic waves and the intraluminal content propelled towards anterograde (oro-aboral) direction with a propulsion velocity of 1.25 +/- 0.1 mm x s(-1). Pressure and shortening waves showed a peak amplitude of 2.44 +/- 0.32 kPa and 1.8 +/- 0.72 mm, respectively. The mean amount of fluid volume ejected during each contraction was 80 +/- 12.6 microl. The addition of Win 55,212-2 [10(-7)-10(-4) M] to the organ bath determined a dose-related attenuation of peristaltic activity consequent to the decrease of circular and longitudinal muscle strength. The decrease of contractile activity was followed by dose-dependent decrease of the amount of fluid ejected during peristalsis. The effects of Win 55,212-2 [10(-7)-10(-5) M] were prevented by SR 141716A, indicating the presence of cannabinoid CB1 receptors in the mouse distal colon. SR 141716A alone enhanced both tonic and phasic motor activities in the colonic longitudinal smooth muscle, suggesting that CB1 receptor antagonists could act either through antagonising the effect of endogenous CB1 receptor agonist or by an agonist effect on these receptors. The present results further support the hypothesis that cannabinoids perform a neuromodulatory role in various tracts of gastrointestinal system and first demonstrate their action also in the distal colon of rodents.     

Characterization of the vasorelaxation to methanandamide in rat gastric arteries

In the present study, the relaxant effect of the cannabinoid methanandamide was explored in rat gastric arteries. Since in some vessels cannabinoids have been shown to release calcitonin gene-related peptide (CGRP) from perivascular nerves, the influence of methanandamide was compared with that of exogenous CGRP. Methanandamide and CGRP elicited concentration-dependent, endothelium-independent relaxations. Methanandamide-induced relaxations were unaffected by the CB1 receptor antagonist AM251, the CB2 receptor antagonists AM630 and SR144528, and combined pre-exposure to AM251 and SR144528. Pre-exposure to O-1918, an antagonist of a novel nonCB1/nonCB2 cannabinoid receptor, did not influence the relaxations to methanandamide. Capsaicin or capsazepine treatment slightly inhibited methanandamide-induced relaxations. Preincubation with 30 mmol/L extracellular K+ or 3 mmol/L TEA had no significant effect on the responses elicited by methanandamide, but reduced CGRP-induced relaxations. Relaxation to 10(-5) mol/L methanandamide was significantly blunted by Bay K8644 and by preincubation with nifedipine. Furthermore, 10(-5) mol/L methanandamide significantly inhibited CaCl2-induced contractions in norepinephrine-stimulated vessels previously depleted of intra- and extracellular Ca2+. Finally, preincubation with 10(-5) mol/L methanandamide almost completely abolished high K+-induced contractions. These findings suggest that the vasorelaxant action of methanandamide in rat gastric arteries is not mediated by stimulation of known cannabinoid receptors and only partly related to stimulation of TRPV1 receptors on perivascular nerves. At high concentrations, methanandamide might induce relaxation by reducing calcium entry into the smooth muscle cells.     

Cannabinoids Inhibit Acid-Sensing Ion Channel Currents in Rat Dorsal Root Ganglion Neurons

Local acidosis has been found in various pain-generating conditions such as inflammation and tissue injury. Cannabinoids exert a powerful inhibitory control over pain initiation via peripheral cognate receptors. However, the peripheral molecular targets responsible for the antinociceptive effects of cannabinoids are still poorly understood. Here, we have found that WIN55,212-2, a cannabinoid receptor agonist, inhibits the activity of native acid-sensing ion channels (ASICs) in rat dorsal root ganglion (DRG) neurons. WIN55,212-2 dose-dependently inhibited proton-gated currents mediated by ASICs. WIN55,212-2 shifted the proton concentration–response curve downwards, with an decrease of 48.6±3.7% in the maximum current response but with no significant change in the EC₅₀ value. The inhibition of proton-gated current induced by WIN55,212-2 was almost completely blocked by the selective CB1 receptor antagonist AM 281, but not by the CB2 receptor antagonist AM630. Pretreatment of forskolin, an AC activator, and the addition of cAMP also reversed the inhibition of WIN55,212-2. Moreover, WIN55,212-2 altered acid-evoked excitability of rat DRG neurons and decreased the number of action potentials induced by acid stimuli. Finally, WIN55,212-2 attenuated nociceptive responses to injection of acetic acid in rats. These results suggest that WIN55,212-2 inhibits the activity of ASICs via CB1 receptor and cAMP dependent pathway in rat primary sensory neurons. Thus, cannabinoids can exert their analgesic action by interaction with ASICs in the primary afferent neurons, which was novel analgesic mechanism of cannabinoids.     

Inhibition of small conductance K+ -channels attenuated melatonin-induced relaxation of serotonin-contracted rat gastric fundus

The aim of this study was to investigate the effects of melatonin on rat gastric fundus smooth muscle. Melatonin (10(-4) to 10(-3) M) had no effect on the basal tone of gastric smooth muscle. After precontraction with carbachol (10(-6) M) or serotonin (10(-7) M), melatonin caused a concentration dependent inhibitory action. The half maximal effect on serotonin-induced contraction was found with 1.12 +/- 0.86 x 10(-5) M of melatonin. Increasing concentrations of melatonin (10(-5) to 10(-3) M) resulted in a right shift of the serotonin concentration response curve (10(-10) to 10(-5) M). This inhibitory effect of melatonin was partially blocked in the presence of apamin (10(-10) to 10(-7) M), a specific blocker of the small conductance calcium-dependent potassium channel, but not in the presence of other potassium channel blockers like charybdotoxin (10(-8) M), glibenclamide (l0(-5) M), or tetraethylammonium (ODQ, 10(-4) M). The inhibitory effect was not changed in the presence of the neuronal blocker tetrodotoxin (10(-6) M), the selective P2-receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (3 x 10(-5) M), the nitric-oxide synthase inhibitor N-nitro-L-arginine (3 x 10(-4) M), or the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one (10(-4) M), suggesting that neither the purinergic, nitrergic, nor guanylate cyclase pathways were involved. We further investigated inhibitory responses to electrical field stimulation (EFS) at different frequencies under non-adrenergic, non-cholinergic (NANC) conditions on a serotonin-induced contraction in the presence of melatonin (10)-5 to 10(-4) M). Melatonin significantly reduced these inhibitory NANC responses in higher (8-32 Hz), but not lower (05-4 Hz), frequencies (16 Hz without melatonin, 103 +/- 6.3%; melatonin 10(-5) M, 80.4 +/- 7.5%; melatonin 10(-4) M, 39.1 +/- 17.1%). Melatonin had no effect on contractile responses induced by EFS under basal tone. These results demonstrate that the inhibitory effect of melatonin in rat gastric fundus smooth muscle is apamin sensitive, but is not affected by other potassium channel blockers. This suggests that melatonin may be another transmitter candidate for the apamin sensitive responses within the gastrointestinal tract.     

Pharmacological characterization of receptor types mediating the dilator action of anandamide on blood vessels of the rat knee joint

This study investigates the actions of N-(2-hydroxyethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (anandamide) on blood flow of the rat knee joint. Topical bolus administration of anandamide (10-1000 nmol) onto the exposed knee joint capsules produced dose-dependent increases in the knee joint blood flow. Various antagonists were tested on the vasodilator response to 100 nmol anandamide. Capsazepine (N-[2-(4-chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide), an antagonist of the transient receptor potential vanilloid type 1 (TRPV1) receptor, given at 10 and 100 nmol, suppressed the response by a maximum of 71%. A cannabinoid CB(1) receptor antagonist AM281 (10 nmol) and a CB(2) receptor antagonist AM630 (10 nmol) shortened its duration from 15 min to 5 min. O-1918 (1 nmol), an antagonist of the putative endothelial anandamide/abnormal-cannabidiol receptor, on its own or combined with capsazepine and the two cannabinoid receptor antagonists produced 38% and 24% inhibition on the peak vasodilator response to anandamide, respectively. URB597 (1 nmol), an inhibitor of fatty acid amide hydrolase (FAAH) suppressed the response by 40%, and an anandamide transporter inhibitor [N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide] (AM404; 1 nmol) or a cyclo-oxygenase (COX) inhibitor flurbiprofen (20 nmol) abolished the response. These findings suggest the vasodilator action of anandamide in the rat knee joint involved hydrolysis of the compound by FAAH, production of COX-derived eicosanoid(s), activation of TRPV1 receptors, and a small component involved activation of endothelial anandamide/abnormal-cannabidiol receptors; a minor delayed dilator response was mediated by activation of conventional cannabinoid receptors.     

The inhibitory action of exo- and endocannabinoids on [³H]GABA release are mediated by both CB₁and CB₂receptors in the mouse hippocampus

Exogenous and endogenous cannabinoids play an important role in modulating the release of neurotransmitters in hippocampal excitatory and inhibitory networks, thus having profound effect on higher cognitive and emotional functions such as learning and memory. In this study we have studied the effect of cannabinoid agonists on the potassium depolarization-evoked [(3)H]GABA release from hippocampal synaptosomes in the wild-type (WT) and cannabinoid 1 receptor (CB(1)R)-null mutant mice. All tested cannabinoid agonists (WIN55,212-2, CP55,940, HU-210, 2-arachidonoyl-glycerol, 2-AG; delta-9-tetra-hydrocannabinol, THC) inhibited [(3)H]GABA release in WT mice with the following rank order of agonist potency: HU-210>CP55,490>WIN55,212-2>2-AG>THC. By contrast, 2-AG and THC displayed the greatest efficacy eliciting almost complete inhibition of evoked [(3)H]GABA efflux, whereas the maximal inhibition obtained by HU-210, CP55,490, and WIN55,212-2 were less, eliciting not more than 40% inhibition. The inhibitory effect of WIN55,212-2, THC and 2-AG on evoked [(3)H]GABA efflux was antagonized by the CB(1) receptor inverse agonist AM251 (0.5 μM) in the WT mice. In the CB(1)R knockout mice the inhibitory effects of all three agonists were attenuated. In these mice, AM251 did not antagonize, but further reduced the [(3)H]GABA release in the presence of the synthetic agonist WIN55,212-2. By contrast, the concentration-dependent inhibitory effects of THC and 2-AG were partially antagonized by AM251 in the absence of CB(1) receptors. Finally, the inhibition of evoked [(3)H]GABA efflux by THC and 2-AG was also partially attenuated by AM630 (1 μM), the CB(2) receptor-selective antagonist, both in WT and CB(1) knockout mice. Our data prove the involvement of CB(1) receptors in the effect of exo- and endocannabinoids on GABA efflux from hippocampal nerve terminals. In addition, in the effect of the exocannabinoid THC and the endocannabinoid 2-AG, non-CB(1), probably CB(2)-like receptors are also involved.     

Neural control of relaxation in cat airways smooth muscle

Functional innervation of cat airways smooth muscle was examined in isolated segments of trachea and bronchi using electrical field stimulation (EFS) techniques. Field stimulation caused contraction in tissues at resting tone and biphasic responses (contraction followed by relaxation) in tissues precontracted with 5-hydroxytryptamine (5-HT). Contractions were abolished by 10(-6) M atropine. Inhibitory responses were dependent on impulse voltage, duration, and frequency. At low voltages (less than or equal to 10 V) and pulse durations (less than or equal to 0.3 ms), EFS induced relaxations were abolished by 3 X 10(-6) M tetrodotoxin (TTX). Greater stimulus parameters elicited TTX-resistant relaxations. Pretreatment of the tissues with 10(-6) M propranolol and 10(-5) M guanethidine caused rightward shifts in relaxation frequency-response curves. These findings indicate that cat airways are innervated by excitatory cholinergic, inhibitory adrenergic, and inhibitory nonadrenergic noncholinergic (NANC) nerves. Pretreatment of the tissues with hexamethonium, cimetidine, indomethacin, or nordihydroguaiaretic acid did not affect NANC relaxation responses. It is concluded that NANC inhibitory responses in cat airway smooth muscle are mediated through intrinsic postganglionic nerve fibers and occur independently of histamine H2-receptor activation and without involvement of cyclooxygenase or lipoxygenase products of arachidonic acid metabolism.     

Functional characterisation of tachykinin receptors in the circular muscle layer of the mouse ileum

OBJECTIVES: Tachykinins are important mediators in neuromuscular signalling but have not been thoroughly characterised in the mouse gut. We investigated the participation of tachykinin receptors in contractility of circular muscle strips of the mouse ileum. RESULTS: Electrical field stimulation (EFS) of excitatory nonadrenergic noncholinergic (NANC) nerves induced frequency-dependent contractions which were mimicked by substance P (SP). Desensitisation of SP and NK(1), NK(2) or NK(3) receptors significantly reduced contractions to EFS. The NK(1) receptor blocker RP67580 significantly inhibited NANC contractions to EFS. The NK(2) and NK(3) receptor blockers nepadutant and SR142801 did not affect NANC contractions per se but increased the RP67580-induced inhibition of NANC contractions to EFS. Contractions to SP were significantly reduced by RP67580 but not affected by nepadutant or SR142801. The NK(1) and NK(2) receptor agonists, septide and [beta-ala(8)]-NKA 4-10 (beta-A-NKA), respectively, but not the NK(3) receptor agonist senktide-induced dose-dependent contractions. Atropine inhibited and l-NNA augmented contractions to septide. Contractions to beta-A-NKA were insensitive to atropine but augmented by l-NNA. CONCLUSIONS: Tachykinins mediate NANC contractions to EFS in the mouse small intestine. Endogenously released tachykinins activate mainly NK(1) receptors, located on cholinergic nerves and smooth muscle cells and, to a lesser degree, NK(2) and NK(3) receptors, most likely located presynaptically.     

AM630 is a competitive cannabinoid receptor antagonist in the guinea pig brain

AM 630 has been demonstrated to be a cannabinoid receptor antagonist in the mouse brain and vas deferens. Conversely, it was recently reported that AM630 acts as a cannabinoid agonist in the guinea pig ileum. This research was designed to determine whether the difference in the action of AM630 is species specific. Studies conducted in guinea pig brain reveal that AM630 antagonizes the stimulatory effect of the cannabinoid agonist WIN 55,212-2 on [³⁵S]GTPγS binding suggesting that difference in AM630 activity in different tissues is not due to species Variation.     

Nitric oxide involvement in the effect of acute lithium administration on the nonadrenergic noncholinergic-mediated relaxation of rat gastric fundus

INTRODUCTION: Lithium has largely met its initial promise as the first drug to be discovered in the modern era of psychopharmacology. However, the mechanism for its action remains an enigma. The aim of the present study was to verify the effect of acute lithium administration on the nonadrenergic noncholinergic (NANC)-mediated relaxation of rat isolated gastric fundus and to evaluate the role of nitric oxide pathway in this manner. MATERIALS AND METHODS: The isolated rat gastric fundus strips were precontracted with 0.5 microM serotonin and electrical field stimulation (EFS) was applied at 5 Hz frequency to obtain NANC-mediated relaxation in the presence or absence of lithium (0.1, 0.5, 1 and 5 mM). Also, effects of combining lithium (0.1 mM) with the NO synthase (NOS) inhibitor L-NAME (0.03 microM) or the guanylyl cyclase inhibitor ODQ (1 microM) on relaxant responses to EFS was investigated. Moreover, effects of combining lithium (1 mM) with 0.1 mM L-arginine (a precursor of NO) on neurogenic relaxation were assessed. Also, the effect of lithium (1 mM) on relaxation to sodium nitroprusside (SNP; 1 nM-0.1 mM) and glyceryltrinitrate (GTN; 0.1-10 microM) was investigated. RESULTS: The NANC-mediated relaxation was significantly (P<0.001) reduced by lithium in a dose- and time-dependent manner. Combination of lithium (0.1 mM) with L-NAME (0.03 microM), which separately had partial inhibitory effect on relaxations, significantly (P<0.001) reduced the NANC-mediated relaxation of gastric fundus. ODQ (1 microM) significantly inhibited the neurogenic relaxations in the presence or absence of lithium (0.1 and 1 mM). Although L-arginine at 0.1 mM had no effect on relaxation to EFS, it prevented the inhibition by lithium (1 mM) of relaxant responses to EFS. Also, SNP and GTN produced concentration-dependent relaxation in precontracted rat gastric fundus which was not altered by lithium incubation (1 mM). DISCUSSION: Our experiments indicated that lithium likely by interfering with L-arginine/NO pathway in nitrergic nerve can result in impairment of NANC-mediated relaxation of rat gastric fundus.     

Influence of relaxin on the neurally induced relaxant responses of the mouse gastric fundus

The peptide hormone relaxin has been reported to depress the amplitude of contractile responses in the mouse gastric fundus by upregulating nitric oxide (NO) biosynthesis at the neural level. In the present study, we investigated whether relaxin also influenced nonadrenergic, noncholinergic (NANC) gastric relaxant responses in mice. Female mice in proestrus or estrus were treated for 18 h with relaxin (1 microg s.c.) or vehicle (controls). Mechanical responses of gastric fundal strips were recorded via force-displacement transducers. In carbachol precontracted strips from control mice and in the presence of guanethidine, electrical field stimulation (EFS) elicited fast relaxant responses that may be followed by a sustained relaxation. All relaxant responses were abolished by tetrodotoxin. Relaxin increased the amplitude of the EFS-induced fast relaxation without affecting either the sustained one or the direct smooth muscle response to papaverine. In the presence of the NO synthesis inhibitor L-N(G)-nitro arginine (L-NNA), that abolished the EFS-induced fast relaxation without influencing the sustained one, relaxin was ineffective. In strips from relaxin-pretreated mice, EFS-induced fast relaxations were enhanced in amplitude with respect to the controls, while sustained ones as well as direct smooth muscle responses to papaverine were not changed. Further addition of relaxin to the bath medium did not influence neurally induced fast relaxant responses, whereas L-NNA did. In conclusion, in the mouse gastric fundus, relaxin enhances the neurally induced nitrergic relaxant responses acting at the neural level.     

Influence of Cannabinoids on Electrically Evoked Dopamine Release and Cyclic AMP Generation in the Rat Striatum

Abstract: Using the endogenous cannabinoid receptor agonist anandamide, the synthetic agonist CP 55940 {[1α,2β( R )5α]-(−)-5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]phenol}, and the specific antagonist SR 141716 [ N -(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H -pyrazole-3-carboxamide hydrochloride], second messenger activation of the central cannabinoid receptor (CB1) was examined in rat striatal and cortical slices. The effects of these cannabinoid ligands on electrically evoked dopamine (DA) release from [³H]dopamine-prelabelled striatal slices were also investigated. CP 55940 (1 µ M ) and anandamide (10 µ M ) caused significant reductions in forskolin-stimulated cyclic AMP accumulation in rat striatal slices, which were reversed in the presence of SR 141716 (1 µ M ). CP 55940 (1 µ M ) had no effect on either KCl- or neurotransmitter-stimulated ³H-inositol phosphate accumulation in rat cortical slices. CP 55940 and anandamide caused significant reductions in the release of dopamine after electrical stimulation of [³H]dopamine-prelabelled striatal slices, which were antagonised by SR 141716. SR 141716 alone had no effect on electrically evoked dopamine release from rat striatal slices. These data indicate that the CB1 receptors in rat striatum are negatively linked to adenylyl cyclase and dopamine release. That the CB1 receptor may influence dopamine release in the striatum suggests that cannabinoids play a modulatory role in dopaminergic neuronal pathways.     

Distribution and function of the cannabinoid-1 receptor in the modulation of ion transport in the guinea pig ileum: relationship to capsaicin-sensitive nerves

We investigated the distribution and function of cannabinoid (CB)(1) receptors in the submucosal plexus of the guinea pig ileum. CB(1) receptors were found on both types of submucosal secretomotor neurons, colocalizing with VIP and neuropeptide Y (NPY), the noncholinergic and cholinergic secretomotor neurons, respectively. CB(1) receptors colocalized with transient receptor potential vanilloid-1 receptors on paravascular nerves and fibers in the submucosal plexus. In the submucosal ganglia, these nerves were preferentially localized at the periphery of the ganglia. In denervated ileal segments, CB(1) receptor immunoreactivity in submucosal neurons was not modified, but paravascular and intraganglionic fiber staining was absent. Short-circuit current (I(sc)) was measured as an indicator of net electrogenic ion transport in Ussing chambers. In the ion-transport studies, I(sc) responses to capsaicin, which activates extrinsic primary afferents, and to electrical field stimulation (EFS) were reduced by pretreatment with the muscarinic antagonist atropine, abolished by tetrodotoxin, but were unaffected by VIP receptor desensitization, hexamethonium, alpha-amino-3-hydroxy-5-methlisoxazole-4-proprionic acid, or N-methyl-d-aspartate glutamate receptor antagonists. The responses to capsaicin and EFS were reduced by 47 +/- 12 and 30 +/- 14%, respectively, by the CB(1) receptor agonist WIN 55,212-2. This inhibitory effect was blocked by the CB(1) receptor antagonist, SR 141716A. I(sc) responses to forskolin or carbachol, which act directly on the epithelium, were not affected by WIN 55,212-2. The inhibitory effect of WIN 55,212-2 on EFS-evoked secretion was not observed in extrinsically denervated segments of ileum. Taken together, these data show cannabinoids act at CB(1) receptors on extrinsic primary afferent nerves, inhibiting the release of transmitters that act on cholinergic secretomotor pathways.     

Modulation of nitrergic relaxant responses by peptides in the mouse gastric fundus

The effects of pituitary adenylate cyclase-activating peptide (PACAP-38) and vasoactive intestinal polypeptide (VIP) were investigated in the gastric fundus strips of the mouse. In carbachol (CCh) precontracted strips, in the presence of guanethidine, electrical field stimulation (EFS) elicited a fast inhibitory response that may be followed, at the highest stimulation frequencies employed, by a sustained relaxation. The fast response was abolished by the nitric oxide (NO) synthesis inhibitor L-N(G)-nitro arginine (L-NNA) or by the guanylate cyclase inhibitor (ODQ), the sustained one by alpha-chymotrypsin. alpha-Chymotrypsin also increased the amplitude of the EFS-induced fast relaxation. PACAP-38 and VIP caused tetrodotoxin-insensitive sustained relaxant responses that were both abolished by alpha-chymotrypsin. Apamin did not influence relaxant responses to EFS nor relaxation to both peptides. PACAP 6-38 abolished EFS-induced sustained relaxations, increased the amplitude of the fast ones and antagonized the smooth muscle relaxation to both PACAP-38 and VIP. VIP 10-28 and [D-p-Cl-Phe6,Leu17]-VIP did not influence the amplitude of both the fast or the sustained response to EFS nor influenced the relaxation to VIP and PACAP-38. The results indicate that in strips from mouse gastric fundus peptides, other than being responsible for EFS-induced sustained relaxation, also exerts a modulatory action on the release of the neurotransmitter responsible for the fast relaxant response, that appears to be NO.     

Effect of a synthetic cannabinoid agonist on the proliferation and invasion of gastric cancer cells

Although cannabinoids are associated with antineoplastic activity in a number of cancer cell types, the effect in gastric cancer cells has not been clarified. In the present study, we investigated the effects of a cannabinoid agonist on gastric cancer cell proliferation and invasion. The cannabinoid agonist WIN 55,212‐2 inhibited the proliferation of human gastric cancer cells in a dose‐dependent manner and that this effect was mediated partially by the CB₁ receptor. We also found that WIN 55,212‐2 induced apoptosis and down‐regulation of the phospho‐AKT expression in human gastric cancer cells. Furthermore, WIN 55,212‐2 treatment inhibited the invasion of gastric cancer cells, and down‐regulated the expression of MMP‐2 and VEGF‐A through the cannabinoid receptors. Our results open the possibilities in using cannabinoids as a new gastric cancer therapy. J. Cell. Biochem. 110: 321–332, 2010. © 2010 Wiley‐Liss, Inc.     

Gastrointestinal motility during pregnancy: role of nitrergic component of NANC nerves

This study evaluated whether increased release of nitric oxide (NO) from the nitrergic component of the nonadrenergic, noncholinergic (NANC) nerves may be partly responsible for the decrease in gastrointestinal motility observed during pregnancy. Segments of fundal strip, ileum, and colon were obtained from nonpregnant rats, rats in midpregnancy (days 9-11), and rats in late pregnancy (days 18-20). NANC activity was studied by assessing changes in tone after application of electric field stimulation (EFS). The role of NO was determined by observing the effects of EFS in the presence and absence of the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) and the reversibility of the effects of L-NAME by L-arginine. The magnitude of change in cGMP levels in the tissues after application of EFS was also assessed. Our studies indicate that there was increased magnitude of relaxation of isolated strips of rat gastric fundus and rat colon, after application of EFS to tissues obtained only from animals in late pregnancy. These results paralleled the changes in cGMP levels in tissues. NOS activity in the gastric fundus was significantly increased in animals in late pregnancy compared with nonpregnant controls. Our studies suggest that the delay in gastric emptying and increase in colonic transit time observed in rats during pregnancy may be caused in part by increased activity of the nitrergic component of the NANC nerves innervating these organs.     

Involvement of Rho/Rho-kinase signalling in the contractile activity and acetylcholine release in the mouse gastric fundus

In this study effects of Rho kinase inhibitors have been examined on the mouse gastric fundal smooth muscle reactivity and neurotransmitter (acetylcholine) release. Two Rho-kinase inhibitors, Y-27632 and fasudil (HA-1077), conspicuously suppressed the contractile responses to carbachol (CCh) and KCl as well as electrical field stimulation (EFS, 40 V, 0.5 ms, and 20 s). pEC(50) value for CCh and EC(50) value for KCl were 6.68+/-0.15 M and 10.4+/-2.8 mM, respectively. EFS induced reproducible contraction (38.3+/-4.75 mN/g tissue) which was almost abolished and potentiated in the presence of atropine (10(-6)M) and eserine (10(-6)M), respectively. The Rho-kinase inhibitors relaxed the fundic strips preconstricted by submaximal concentration of CCh or KCl in a concentration dependent manner. With CCh-elicited contraction, the pEC(50) values of Y-27632 and fasudil were 5.45+/-0.14 and 5.11+/-0.14 M, respectively (p>0.05). However, the pEC(50) values for Y-27632 and fasudil on KCl-induced tone were 6.09+/-0.1 and 5.35+/-0.06 M (p<0.001), respectively. Moreover, [3H]acetylcholine ([3H]ACh) release upon EFS from the gastric fundus was measured and it was found that Y-27632 (10(-4)M) significantly impaired the release. At 3 Hz the radioactivity ratio obtained after and before EFS (S(2)/S(1) ratio) was 0.88+/-0.03 in control but 0.63+/-0.08 in the presence of 10(-4)M Y-27632 (p<0.05). These results suggest that Rho kinase inhibitors can not only relax the gastric fundus but also modulate CCh, cholinergic nerve stimulation, and KCl-induced contraction. Furthermore, Rho/Rho kinase signalling may play a role in the neurotransmitter (ACh) release in the mouse gastric fundus.     

Receptor-independent depression of DA and 5-HT uptake by cannabinoids in rat neocortex--involvement of Na(+)/K(+)-ATPase

There is evidence that cannabinoids modulate the reuptake of some neurotransmitters in the central nervous system. In this study, we investigated the effects of the synthetic cannabinoid receptor agonist WIN55212-2, the endocannabinoid anandamide and the chemically related arachidonic acid on serotonin (5-HT) and dopamine (DA) uptake into rat neocortical synaptosomes. At micromolar concentrations, anandamide and arachidonic acid produced steep inhibition curves with Hill coefficients above unity. WIN55212-2 inhibited both DA and 5-HT uptake with Hill coefficients near unity, also within the micromolar range. The effect of WIN55212-2 was not mediated by cannabinoid receptors, since the CB1 receptor antagonist AM251 failed to diminish uptake inhibition by WIN55212-2 and since the Ki estimates of WIN55212-2 were outside the range of the dissociation constants of WIN55212-2 at both CB1 and CB2 receptors. A 100-fold higher concentration of DA, respectively 5-HT, did not induce a shift to the right of the WIN55212-2 concentration-inhibition curves, suggesting a carrier-independent mechanism. The Na(+)/K(+)-ATPase inhibitor ouabain concentration dependently inhibited 5-HT uptake. Possible drug effects on commercial Na(+)/K(+)-ATPase and synaptosomal ATP consumption were investigated using an ATP bioluminescence assay. Ouabain inhibited both commercial and synaptosomal Na(+)/K(+)-ATPase. WIN55212-2 had no effect on commercial Na(+)/K(+)-ATPase, but inhibited synaptosomal ATP consumption. Anandamide produced a sharp decrease in the activity of commercial Na(+)/K(+)-ATPase and on synaptosomal ATP consumption. Presence of ouabain significantly reduced the inhibitory effect of anandamide on synaptosomal ATP consumption, whereas the effect of WIN55212-2 remained unchanged. Our results show that cannabinoids and arachidonic acid inhibit DA and 5-HT uptake into rat neocortical synaptosomes. This effect is neither cannabinoid receptor-mediated nor due to competitive inhibition of membrane transporters, but is partly effected by a decreased Na(+)/K(+)-ATPase activity.