Functional activity of cannabinoid receptors in RIN-M5F insulinoma beta-cells: interactions with bombesin and vanilloid receptors

Pietro Marini1, Luciano De Petrocellis2, Maura Palmery1, Luigia Cristino2, Aniello Schiano Moriello2, Santosh Nigam4, Katarzyna Starowicz3, Vincenzo Di Marzo3. 1Department of Human Physiology and Pharmacology, University of Rome “La Sapienza”, Rome, Italy, 2Institute of Cybernetics National Research Council, Pozzuoli, Naples, Italy, 3Institute of Biomolecular Chemistry National Research Council, Pozzuoli, Naples, Italy, 4Eicosanoid & Lipid Research Division, and Centre for Experimental Gynecology & Breast Research, Univ.-Klinikum Benjamin Franklin, Free University Berlin, Berlin, Germany.

Cannabinoid CB1 receptors were found in RIN-m5F rat insulinoma β cells and shown to modulate insulin release (Matias et al. 2006). Transient receptor potential vanilloid type 1 (TRPV1) channels (Akiba et al., 2004) and bombesin receptors are also expressed in insulinoma cells and stimulate insulin release via enhancement of intracellular Ca2+ ([Ca2+]I) (Anubhuti, S.A, 2006) (Matsumoto et al., 2003).

Here we investigated in these cells the effect on [Ca2+]I of activation of cannabinoid, bombesin and TRPV1 receptors. The effect on [Ca2+]I without extracellular calcium was determined by using Fura-2-AM and the loaded cells transferred in a quartz cuvette for fluorescence detection (Perkin-Elmer LS50B) under continuous stirring. The dye was excited at 340 and 380 nm to indicate relative [Ca2+]I changes by the F340/F380 ratio, and the emission was at 540 nm[Ca2+]I was measured as the maximum increase in F340/F380 ratio upon addition of the drugs. The effect of the substances on [Ca2+]I in the presence of extracellular Ca2+ was determined by using Fluo-4-AM. Experiments were carried out by measuring cell fluorescence at 25°C (lambaEX=488 nm, lambaEM=516 nm) before and after the addition of the test compounds at various concentrations. The efficacy of the agonists was determined by comparing it to the maximal effect on [Ca2+]I observed with 4 μM ionomycin.

Bombesin (pEC50 7.78±0.15), the CB1 agonist arachidonoyl-chloroethanolamide (ACEA) (pEC50 5.72±0.12), and the CB2 agonist JWH133 (pEC50 6.31±0.31), elevated [Ca2+]I in a way sensitive to the inhibitor of phosphoinositide-specific phospholipase C (PI-PLC), U73122, but not to pertussis toxin or forskolin. PI-PLC-dependent Ca2+ mobilization by ACEA and bombesin was entirely accounted for by activation of inositol-1,3,5-triphosphate (IP3) receptors on the endoplasmic reticulum (ER), whereas the effect of JWH133 was not sensitive to all tested inhibitors of IP3 and ryanodine receptors. ACEA, but not JWH133, significantly inhibited the effect of bombesin on [Ca2+]I, and bombesin slightly reduced the effect of ACEA. The “hybrid” CB1/TRPV1 agonists, anandamide (AEA: pEC50 5.69±0.04) and N-arachidonoyldopamine (NADA: pEC50 6.25±0.06), but not CB1 (ACEA pEC50 5.84±0.34) or TRPV1 (RTX: pEC50 6.09±0.43) selective agonists, exhibited higher potency at elevating [Ca2+]I in the presence of extracellular Ca2+, in a way sensitive to both CB1 and TRPV1 antagonists and to protein kinase C and phosphoinositide-3-kinase inhibitors.

These results suggest that, in RIN-m5F cells, PI-PLC-coupled CB1 receptors: 1) inhibit bombesin signalling, thereby possibly leading to inhibition of bombesin-induced insulin release; 2) facilitate TRPV1-induced effects on [Ca2+]I, possibly via inhibition of TRPV1 desensitization

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