Evaluation of Short and Long Term Cold Stress Challenge of Nerve Grow Factor, Brain-Derived Neurotrophic Factor, Osteocalcin and Oxytocin mRNA Expression in BAT, Brain, Bone and Reproductive Tissue of Male Mice Using Real-Time PCR and Linear Correlation Analysis.

The correlation between the Ngf/p75ntr-Ntrk1 and Bdnf, Osteocalcin-Ost/Gprc6a and Oxytocin-Oxt/Oxtr genes, was challenged investigating their mRNA levels in 3 months-old mice after cold-stress (CS). Uncoupling protein-1 (Ucp-1) was used as positive control. Control mice were maintained at room temperature T = 25°C, CS mice were maintained at T = 4°C for 6 h and 5-days (N = 15 mice). RT-PCR experiments showed that Ucp-1 and Ngf genes were up-regulated after 6 h CS in brown adipose tissues (BAT), respectively, by 2 and 1.5-folds; Ucp-1 was upregulated also after 5-days, while Ngfr (p75ntr) and Ntrk1 genes were downregulated after 6 h and 5-days CS in BAT. NGF and P75NTR were upregulated in bone and testis following 5-days, and P75NTR in testis after 6 h CS. Bdnf was instead up-regulated in bone following 5-days CS and down-regulated in testis. OST was upregulated by 16 and 3-fold in bone and BAT, respectively, following 5-days CS. Gprc6a was upregulated after 6 h in brain, while Bglap (Ost) gene was downregulated. Oxt gene was upregulated by 5-fold following 5-days CS in bone. Oxtr was upregulated by 0.5 and 0.3-fold, respectively, following 6 h and 5-days CS in brain. Oxtr and Oxt were downregulated in testis and in BAT. The changes in the expression levels of control genes vs. genes following 6 h and 5-days CS were correlated in all tissues, but not in BAT. Correlation in BAT was improved eliminating Ngfr (p75ntr) data. The correlation in brain was lost eliminating Oxtr data. In sum, Ucp-1 potentiation in BAT after cold stress is associated with early Ngf-response in the same tissue and trophic action in bone and testis. In contrast, BDNF exerts bone and neuroprotective effects. Similarly to Ucp-1, Bglap (Ost) signaling is enhanced in bone and BAT while it may exert local neuroprotective effects thought its receptor. Ngfr (p75ntr) regulates the adaptation to CS through a feed-back loop in BAT. Oxtr regulates the gene-response to CS through a feed-forward loop in brain. Overall these results expand the understanding of the physiology of these molecules under metabolic thermogenesis.

Cannabinoid agonists induce relaxation in the bovine ophthalmic artery: evidences for CB1 receptors, nitric oxide and potassium channels.

Glaucoma pathophysiology appears to involve vascular deficits, which may contribute to initiation and progression of the disease. Anandamide, the endogenous cannabinoid ligand, and WIN55212-2, a synthetic cannabinoid agonist, are able to evoke concentration-dependent relaxations in bovine ophthalmic artery rings, precontracted with 5-hydroxytryptamine (5-HT) (1 microM). Endothelium removal reduces cannabinoid agonist potency and efficacy. The selective cannabinoid 1 (CB1) receptor antagonists SR141716A (100 nM) and AM251 (100 nM) cause a shift to the right in the concentration-response curves to anandamide and WIN55212-2 in arterial rings both in the presence and in the absence of endothelium. In endothelium-intact arteries, the nitric oxide synthase inhibitor, N(G)-monomethyl-L-arginine (L-NMMA, 300 microM), completely blocked the anandamide- and WIN55212-2-relaxant responses; by contrast, the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP, 100 microM) induced an increase in vasorelaxant responses to cannabinoid agonists. Relaxations to anandamide and WIN55212-2 were inhibited by iberiotoxin (IbTX, 200 nM), a blocker of large conductance, Ca2+-activated K+ channel (BK(Ca)), and by 4-aminopyridine (4-AP; 1 mM), a blocker of delayed rectifier K+ channel, whereas the blockade of K(ATP) channels by glibenclamide (5 microM) and of small conductance Ca2+-activated K+ channels (SK(Ca)) by apamin (100 nM) did not produce any effects. These data suggest that anandamide and WIN55212-2 relax the bovine ophthalmic artery by involving CB1 the cannabinoid receptor-sensitive pathway. In endothelium-intact arteries, relaxation occurs through activation of nitric oxide synthase cyclic GMP and Ca2+-activated K+ channels. They also cause endothelium-independent relaxation by involving potassium channel opening.

Cannabinoid agonists induce contractile responses through Gi/o-dependent activation of phospholipase C in the bovine ciliary muscle.

This study was undertaken to investigate the effect of some cannabinoid agonists on the bovine ciliary muscle. Both anandamide and CP 55,940 (cis-3-(2-hydroxy-4-(1,1-dimethyl heptyl) phenyl)-trans-4-(3-hydroxypropyl) cyclohexanol) produced a concentration-dependent contractile response in ciliary muscle. These responses were inhibited by SR 141716A (N-[piperidin-1-yl]-5-(4-cholophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) (0.1 and 1 microM) but not by SR 144528 (N-[1S)-endo-1,3,3-trimethyl bicyclo[2.2.1] heptan-2-yl] 5-(4-chloro-3-methylphenyl)-1-(4 methoxy benzyl)-pyrazole-3-carboxamide) (1 and 10 microM). A preincubation with G(i/o) protein inhibitor pertussis toxin (500 ng/ml) for 20 min inhibited the contractile action of anandamide and CP 55,940. In addition, the phospholipase C inhibitor U73122 (1[6-[[(17 beta)-3-methoxyestra-1,3,5(10)-trien-17-yl] amino] hexyl]-1H-pyrrole-2,5-dione) blocked the anandamide- and CP 55,940-induced contractions, whereas the protein kinase C activator, phorbol 12,13 dibutyrate (PDBu) significantly potentiated the contractions evoked by cannabinoid receptor agonists. We evaluated the binding of [(3)H]CP 55,940, which specifically labelled a single class of cannabinoid sites with affinity in low subnanomolar range (K(d)=0.6 nM) and the maximal number of binding sites of 1243 fmol/mg protein. Binding of [(3)H]CP 55,940 was inhibited by ligands having a major selectivity for cannabinoid (CB(1)) receptors. These findings provide strong evidence of the involvement of cannabinoid CB(1) receptors promoting contraction in the bovine ciliary muscle. Furthermore, the action of cannabinoid receptor agonists appears to be mediated via phospholipase C. These data also contribute to elucidate the cannabinoid CB(1) receptor pivotal role in the modulation of intraocular pressure and to show that cannabinoid receptor agonists may be regarded as potential antiglaucoma agents.

Pharmacological characterization of the 5-HT1A, 5-HT2 and 5-HT3 receptors in the bovine ciliary muscle.

We aimed to investigate the effects of serotonin (5-hydroxytryptamine, 5-HT) on the bovine ciliary muscle and subsequently to characterize and identify the subtypes of 5-HT receptors involved in the serotonin-evoked contractility muscle. The binding of [3H]ketanserin, [3H]granisetron and [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) was analyzed. All labelled compounds bound with high affinity to a single site in the membrane preparations studied. The affinity (K(d)) of the binding site was 7.5+/-1.2 nM for [3H]ketanserin, 6.9+/-0.8 nM for [3H]granisetron and 4.4+/-0.31 nM for [3H]8-OH-DPAT. The density of receptors (B(max)) was 1062+/-43.0 fmol/mg protein for [3H]ketanserin, 566+/-2.32 fmol/mg protein for [3H]granisetron and 205+/-4.63 fmol/mg protein for [3H]8-OH-DPAT. The serotonin-induced contraction appeared to be competitively antagonized by ketanserin (0.1, 1 and 10 microM) and ondansetron (0.1, 10 and 100 microM) which produced a pA(2) value of 8.5+/-0.12 and 8.0+/-0.19, respectively. 8-OH-DPAT and 5-carboxamidotryptamine (5-CT) proved to be completely ineffective. We conclude that serotonin induces bovine ciliary muscle contraction via 5-HT(2) and 5-HT(3) receptors while the 5-HT(1A) receptors, although present, do not mediate the contractile response.