s-nitro-n-acetylpenicillamine and anandamide

In invertebrates, like Hydra and sea urchins, evidence for a functional cannabinoid system was described. The partial characterization of a putative CB1 cannabinoid receptor in the leech Hirudo medicinalis led us to investigate the presence of a complete endogenous cannabinoid system in this organism. By using gas chromatography-mass spectrometry, we demonstrate the presence of the endocannabinoids anandamide (N-arachidonoylethanolamine, 21.5+/-0.7 pmol/g) and 2-arachidonoyl-glycerol (147.4+/-42.7 pmol/g), and of the biosynthetic precursor of anandamide, N-arachidonylphosphatidyl-ethanolamine (16.5+/-3.3 pmol/g), in the leech central nervous system (CNS). Anandamide-related molecules such as N-palmitoylethanolamine (32.4+/-1.6 pmol/g) and N-linolenoylethanolamine (5.8 pmol/g) were also detected. We also found an anandamide amidase activity in the leech CNS cytosolic fraction with a maximal activity at pH 7 and little sensitivity to typical fatty acid amide hydrolase (FAAH) inhibitors. Using an antiserum directed against the amidase signature sequence, we focused on the identification and the localization of the leech amidase. Firstly, leech nervous system protein extract was subjected to Western blot analysis, which showed three immunoreactive bands at ca. approximately 42, approximately 46 and approximately 66 kDa. The former and latter bands were very faint and were also detected in whole homogenates from the coelenterate Hydra vulgaris, where the presence of CB1-like receptors, endocannabinoids and a FAAH-like activity was reported previously. Secondly, amidase immunocytochemical detection revealed numerous immunoreactive neurons in the CNS of three species of leeches. In addition, we observed that leech amidase-like immunoreactivity matches to a certain extent with CB1-like immunoreactivity. Finally, we also found that stimulation by anandamide of this receptor leads, as in mammals, to inhibition of cAMP formation, although this effect appeared to be occurring through the previously described anandamide-induced and CB1-mediated activation of nitric oxide release. Taken together, these results suggest the existence of a complete and functional cannabinoid system in leeches.

Topics: Adenylyl Cyclases; Amidohydrolases; Amino Acid Sequence; Animals; Antibodies; Arachidonic Acids; Calcium Channel Blockers; Cannabinoid Receptor Modulators; Central Nervous System; Colforsin; Endocannabinoids; Immunoenzyme Techniques; Leeches; Molecular Sequence Data; Nitric Oxide; Nitric Oxide Donors; Penicillamine; Polyunsaturated Alkamides; Receptors, Cannabinoid; Receptors, Drug; Signal Transduction

We demonstrate that immediate exposure to gp120 (5 min; 0.1 microg/ml) results in a significant shift of the macrophage population to an amoeboid and motile category (P<0.01; 91.7+/-5.5 vs. a control value of 42.4+/-4.2) and prior exposure with anti-gp120 antagonizes this shift. Acute exposure of the macrophages to morphine (10(-6) M) or anandamide (10(-6) M) resulted in the cells rounding up (shape factors of 0.84 and 0.87 respectively) and becoming non-motile. The action is blocked by prior treatment with the specific antagonists naloxone and SR 141716A. Chronic exposure (6 h) of the cells to all three agents resulted in a random migration pattern. Further, all agents blocked chemotaxis induced by DAMA and IL-1. Observation of the cells behavior during chronic exposure revealed a sporadic activity pattern with gp120 whereas morphine and anandamide first induced a period of inactivity which is followed by a period of activity (chemokinesis). Recent work from our laboratory has demonstrated that both morphine and anandamide acutely stimulate constitutive macrophage nitric oxide (NO) release, which then induces macrophage rounding and inactivity. It was therefore of interest to examine their behavior by exposing macrophages to the NO-donor SNAP. In a concentration dependent manner SNAP exhibited the same behavioral actions as both substances of abuse. Given this, we next determined if macrophages exposed to gp120 would release NO. We demonstrated that NO was released only when exposed to morphine and anandamide not gp120. Thus. the chemokinetic inducing activities of these agents may be the basis for excitotoxin liberation in neural tissues and/or a higher viral load in various organ systems since cellular adherence and random migration are stimulated.

Topics: Analysis of Variance; Arachidonic Acids; Cannabinoids; Cell Movement; Chemotaxis; Disease Susceptibility; Endocannabinoids; Enkephalin, Methionine; HIV Envelope Protein gp120; HIV Infections; Humans; In Vitro Techniques; Interleukin-1; Macrophages; Morphine; Narcotics; Nitric Oxide; Penicillamine; Polyunsaturated Alkamides