Simeon Todorov

Gastric antisecretory effects of synthetic cannabinoids after central or peripheral administration in the rat.

Previous studies have revealed that cannabinoid (CB)-receptor agonists inhibit gastric acid secretion stimulated by indirectly acting agents, but not by histamine. Aiming to investigate whether central or peripheral mechanisms are involved, the effects of the synthetic CB-receptor agonists WIN55,212-2 and HU-210, administered either intracerebroventricularly (i.c.v.) or intravenously (i.v.) to the anaesthetized rat with lumen-perfused stomach, against gastric acid secretion induced by pentagastrin were tested. Injected i.c.v., both WIN55,212-2 (50 and 100 microg/kg) and HU-210 (25, 50 and 100 microg/kg) were ineffective on either basal secretion or acid output induced by pentagastrin (7.7 microg/kg, i.v.). By contrast, i.v. injections of WIN55,212-2 (100 and 1000 microg/kg) or HU-210 (10-100 microg/kg) significantly inhibited pentagastrin-induced acid secretion, maximal reductions being 75.70 and 82.24% for WIN55,212-2 and HU-210, respectively. The gastric antisecretory effect of HU-210 was prevented by administration of the selective CB(1)-receptor antagonist SR141716A (1000 microg/kg, i.v.). These results show that CB(1)-receptors mediating inhibition of gastric acid secretion in the rat are mainly peripherally located.

Synthesis and biological activity of novel small peptides with aminophosphonates moiety as NOP receptor ligands

The aim of the present study was the synthesis and the biological screening of new analogs of Ac-RYYRWK-NH2, modified at the N-terminal with 1-[(methoxyphosphono)methylamino]cycloalkanecarboxylic acids. The four newly synthesized ligands for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) have been prepared by solid-phase peptide synthesis—Fmoc-strategy. These compounds were tested for agonistic activity in vitro on electrically stimulated smooth-muscle preparations isolated from vas deferens of Wistar rats. Our data showed that substitution of Arg at position 1 with aminophosphonates moiety decreased significantly the affinity of ligands to the NOP receptor. Furthermore, the enlargement of the cycle (with 5–8 carbon atoms) additionally diminished both the activity and the selectivity for NOP-receptor.

PRESYNAPTIC HISTAMINE H2 RECEPTORS MODULATE THE SYMPATHETIC NERVE TRANSMISSION IN THE ISOLATED RAT VAS DEFERENS ; NO ROLE FOR H3-RECEPTORS

The modulatory activity mediated by histamine receptors on the sympathetic nerve transmission was investigated in the rat vas deferens. Agonists and antagonists acting at the different histamine receptor subtypes (H1, H2 and H3) were tested on electrically-driven preparationsin vitro. Low-frequency stimulation (0.1 Hz) evoked muscle contractions almost completelysustained by ATP release, while at high-frequency stimulation (5–10 Hz) norepinephrine was mainly involved. The H1 receptor agonists, pyridilethylamine and 2-(2 aminoethyl)thiazole, enhanced the electrically evoked twitch responses, but not contractions induced by exogenously-applied norepinephrine and ATP. These effects were prevented by the H1-blocking drugs, mepyramine and phenyramine, but only at high concentrations (10 μmol/l). All these H1-antagonists strongly enhanced muscle response to electrical stimulation. The H2 receptor agonists, dimaprit, amthamine and impromidine, reduced the contractions evoked by field stimulation, but not by exogenously applied norepinephrine and ATP, the effect being antagonised by H2-blocking drugs, ranitidine and famotidine. The H3 receptor agonist,R(α)-methylhistamine, reduced the electrically evoked muscle contractions, the effect being not modified by the selective H3-blocking drug, thioperamide, but prevented by famotidine. These data suggest that rat vas deferens contains presynaptic histamine H2 receptors, able to mediate inhibitory effects on the sympathetic transmission, while histamine H3 receptors are apparently not involved. On the contrary, the role of H1 is still unclear, since both agonists and antagonists may have the same effects.

Prejunctional modulation of non-adrenergic non-cholinergic (NANC) inhibitory responses in the isolated guinea-pig gastric fundus.

Abstract  The inhibitory neurotransmission of the stomach was investigated in isolated guinea‐pig gastric fundus. In preparations treated with guanethidine (1 μmol L−1) and p‐fluoro‐hexahydro‐sila‐difenidol (1 μmol L−1), electrical stimulation evoked neurogenic inhibitory responses not modified by hexamethonium (100 μmol L−1), suggesting that inhibitory postganglionic non‐adrenergic non‐cholinergic (NANC) nerve fibres are involved. The nitric oxide (NO)‐synthase inhibitor Nω‐nitro‐l‐argininine‐methyl‐ester hydrochloride (1–100 μmol L−1) and the soluble guanylyl cyclase inhibitor ODQ (0.1–3 μmol L−1) also abolished such relaxant response, suggesting the involvement of NO/Cyclic Guanosine 3′,5′ monophosphate (cGMP) system as the final mechanism of muscle relaxation. The α2‐adrenoceptor agonist, UK 14 304 (10 nmol L−1–10 μmol L−1) did not influence the electrical field stimulation (EFS)‐evoked NANC responses. These latter responses were also refractory to a variety of receptor agonists and antagonists, acting at Gamma Aminobutyric Acid (GABA), serotonin 5HT1a, opioid μ, δ and κ, muscarinic M1 and M2, histamine H2 and H3 and cannabinoid receptors. The NANC response was insensitive to the P/Q‐type Ca2+‐channel blocker ω‐agatoxin TK (1 nmol L−1–0.1 μ mol L−1), but partially inhibited by the N‐type Ca2+‐channel blocker ω‐conotoxin GVIA (0.1 nmol L−1−0.1 μmol L−1), and by the L‐type Ca2+‐channel blockers nifedipine and calcicludine (0.1 nmol L−1−0.1 μmol L−1). These data suggest that the NANC relaxation of the isolated guinea‐pig gastric fundus is mediated by NO as the final inhibitory (neuro)transmitter at the longitudinal smooth muscle cells. The mechanism(s) promoting NO production is/are Ca2+‐dependent, but apparently insensitive to presynaptic modulation. Both N‐ and L‐type channels seem to occur in nitrergic nerve endings, where they contribute to trigger NO diffusion at the synaptic cleft.

In vivo effects of N/OFQ(1–13)NH2 and its structural analogue [ORN9]N/OFQ(1–13)NH2 on carrageenan-induced inflammation: rat-paw oedema and antioxidant status

The effects of nociceptin(1–13)NH2 (N/OFQ(1–13)NH2) and its structural analogue [Orn9]N/OFQ(1–13)NH2 on acute carrageenan (CG)-induced peripheral inflammation and paw antioxidant status were studied. CG was injected intraplantarly in the right hind paw of rats and the volume of the inflamed paw was measured each 30 min for a period of 4h. When administered simultaneously with CG, N/OFQ(1–13)NH2 decreased the paw volume, whereas if injected 15 min before CG it had no effect. [Orn9]N/OFQ(1–13)NH2 produced the opposite effects at the same time-intervals of its administration. We also investigated whether these neuropeptides influence CG-induced changes in cell antioxidant system, especially at the 4th hour of CG administration. CG alone decreased the glutathione level and superoxide dismutase activity, as measured in post-nuclear homogenate of the inflamed paw. However, CG injection increased glutathione peroxidase and glucose-6-phospate dehydrogenase activities, while the activity of glutathione reductase was unchanged. The peptides themselves did not change all measured parameters. Moreover, neither N/OFQ(1–13)NH2 nor [Orn9]N/OFQ(1–13)NH2 modified CG-induced changes in the antioxidant status, regardless of the time of their injection (simultaneously or 15 min before CG). The present results suggest that N/OFQ(1–13)NH2 and [Orn9]N/OFQ(1–13)NH2 most likely affect the neuronal inflammation, rather than act as pro- or antioxidants.

Study of the cytotoxicity and antioxidant capacity of N/OFQ(1–13)NH2 and its structural analogues

The effect of side-chain shortening of N/OFQ(1-13)NH(2) at position 9 ([Orn(9)]N/OFQ(1-13)NH(2), [Dab(9)]N/OFQ(1-13)NH(2), [Dap(9)]N/OFQ(1-13)NH(2)) was studied regarding potential toxicity and antioxidant capacity. Staurosporine- and H2O2-induced damage of SH-SY5Y neuroblastoma cells was not changed in the presence of N/OFQ(1-13)NH(2) and [Orn(9)]N/OFQ(1-13)NH(2), but was strongly enhanced in the presence of [Dab(9)]N/OFQ(1-13)NH(2) and [Dap(9)]N/OFQ(1-13)NH(2). Moreover, treatment of cells with the latter two analogues alone led to cell injury. Neuropeptide-dependent differences in the viability of SH-SY5Y cells were also observed, i.e., a cytoprotective effect was observed only in the presence of N/OFQ(1-13)NH(2) and [Orn(9)]N/OFQ(1-13)NH(2). Compared to [Dab(9)]N/OFQ(1-13)NH(2) and [Dap(9)]N/OFQ(1-13)NH(2), the effects of N/OFQ(1-13)NH(2) and [Orn(9)]N/OFQ(1-13)NH(2) were more beneficial in systems generating free oxygen radicals (O(2)(-) and .OH), as well as on the antioxidant status of rat brain and liver. Taken together, our findings show that N/OFQ(1-13)NH(2) and its structural analogue [Orn(9)]N/OFQ(1-13)NH(2) possess more favorable profiles than the other two nociceptin (N/OFQ) analogues. The present results suggest that shortening of the side-chain of N/OFQ(1-13)NH(2) might increase cell damage and reduce the viability of SH-SY5Y neuroblastoma cells. Moreover, such alterations may lead to changes in free-oxygen generating systems and in antioxidant status in animal tissues.

Role of histamine H3 receptors in control of mouse intestinal motility in vivo and in vitro: comparison with alpha2-adrenoceptors.

We tested drugs acting at histamine H3 receptors in mice on the gastrointestinal transit of a charcoal meal in vivo and on neurogenic contractions of isolated ileal preparations. The agonist (R)-α-methylhistamine (100 μmol/kg) caused a maximum 25% reduction of gastrointestinal transit, an effect mimicked by immepip (100 μmol/kg) and antagonized by thioperamide (20 μmol/kg) or clobenpropit (20 μmol/kg). In the isolated ileum, (R)-α-methylhistamine (10–100 μM) caused a slight, thioperamide-insensitive, reduction (maximum 15%) of electrically evoked cholinergic contractions. In comparison, the α2-adrenoceptor agonist clonidine (0.1 μmol/kg) caused a 35.2% inhibition of the gastrointestinal transit and almost completely reduced (maximum 82% at 1 μM) the cholinergic contraction of the isolated ileum, both effects being antagonized by idazoxan (0.4 μmol/kg and 1 μM, respectively). These results suggest that histamine H3 receptors, located outside the myenteric plexus, mediate an inhibition of the gastrointestinal transit in vivo. Conversely, the presence of α2-adrenoceptors in the cholinergic nerve endings and their inhibitory role in the control of gastrointestinal propulsion is confirmed.

In-vivo effects of nociceptin and its structural analogue [Orn9] nociceptin on the antioxidant status of rat blood and liver after carrageenan-induced paw inflammation

The production of reactive oxygen species (ROS) in cells is well balanced with their elimination by the antioxidant defence system. This balance is essential for maintenance of physiological conditions, and its disturbance (oxidative stress) has been suggested as a potential pathogenic mechanism in a variety of diseases, accompanied by inflammation. In this study, the in-vivo effects of nociceptin (N/OFQ(1–13)NH2) and its structure analogue [Orn9]N/OFQ(1–13)NH2 were studied on markers of oxidative stress in erythrocytes and liver of rats 4 hours after subplantar administration of carrageenan (CG) (1%, 100 µl) in the right hind paw. A considerable inflammatory oedema of the paw was observed. CG did not change blood haemoglobin content, hematocrit value, glutathione level and antioxidant enzyme activities in the erythrocytes, but there was an increase in lipid peroxidation. In liver, CG-induced imbalance was manifested by an increase in lipid peroxidation and a decrease in glutathione level. Both peptides (20 µg, i.p.), when administered alone, had no effect on all parameters tested. When either [Orn9]N/OFQ(1–13)NH2 or N/OFQ(1–13)NH2 was injected simultaneously with CG or 15 minutes before it, they did not affect the CG-induced changes in the antioxidant status of the erythrocytes and liver. Our results suggest that the peptides tested did not play a role in the free radical processes that accompany CG-induced paw inflammation.

Receptor subtypes involved in the modulatory action of histamine on the contractility of the rat vas deferens

The data in the literature concerning the effects of histamine (HIST) on the rat vas deferens and the mechanisms involved in its modulatory action are rather scarce and contradictory. The presence of different receptor subtypes has been suggested: postsynaptic excitatory H1and presynaptic inhibitory H 2receptors [1]; inhibitory H2-receptors [2]; inhibitory H1and Hz-receptors [3]; presynaptic H3-receptors [4]. The aim of the present study was to study further the receptor subtypes involved in the modulatory effects of HIST on contractions of the rat vas deferens evoked by low-frequency electrical stimulation (ES).