ABSTRACT
Morphine significantly stimulated invertebrate immunocyte intracellular calcium level increases in a concentration-dependent manner in cells preloaded with Fura 2/AM. Morphine's action was blocked by prior exposure of the cells to the opiate receptor antagonist naloxone. Various opioid peptides did not exhibit this ability, indicating a morphine-mu 3 mediated process. In comparing the sequence of events concerning morphine's action in stimulating both [Ca2+]i and NO production in these cells, we found that the first event precedes the second by 42 +/- 7 s. The opiate stimulation of [Ca2+]i- was attenuated in cells leached of calcium. strongly suggesting that intracellular calcium levels regulate cNOS activity in invertebrate immunocytes.
Subject(s)
Calcium/metabolism , Hemocytes/metabolism , Morphine/pharmacology , Nitric Oxide/metabolism , Animals , Bivalvia , Cells, Cultured , Enkephalin, Ala(2)-MePhe(4)-Gly(5)- , Enkephalins/pharmacology , Fura-2 , Hemocytes/drug effects , Kinetics , Naloxone/pharmacology , Narcotic Antagonists/pharmacology , Nitric Oxide Synthase/metabolism , Signal TransductionABSTRACT
Both morphine and anandamide significantly stimulated cultured endothelial intracellular calcium level increases in a concentration-dependent manner in cells pre-loaded with fura 2/AM. Morphine is more potent than anandamide (approximately 275 vs. 135 nM [Ca]i), and the [Ca]i for both ligands was blocked by prior exposure of the cells to their respective receptor antagonist, i.e., naloxone and SR 171416A. Various opioid peptides did not exhibit this ability, indicating a morphine-mu3-mediated process. In comparing the sequence of events concerning morphine's and anandamide's action in stimulating both [Ca]i and nitric oxide production in endothelial cells, we found that the first event precedes the second by 40+/-8 sec. The opiate and cannabinoid stimulation of [Ca]i was attenuated in cells leeched of calcium, strongly suggesting that intracellular calcium levels regulate cNOS activity.
Subject(s)
Analgesics, Opioid/pharmacology , Arachidonic Acids/pharmacology , Calcium Channel Blockers/pharmacology , Calcium/metabolism , Endothelium, Vascular/metabolism , Morphine/pharmacology , Nitric Oxide/metabolism , Cells, Cultured , Dose-Response Relationship, Drug , Endocannabinoids , Humans , Polyunsaturated Alkamides , Signal TransductionABSTRACT
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.