Modulatory role of nitric oxide in the muscarinic receptor-induced vasodilation and secretion in the parotid gland of diabetic rabbit

Abstract

The aim of this study was to assess, on the experimental model of rabbit, the effect of type 1 diabetes mellitus (induced by a single intravenous injection of 100 mg kg−1 of alloxan) on vascular tonus of parotid artery, feeding artery for parotid gland, and secretory activity of this gland, induced by stimulation of cholinergic muscarinic receptors (by acetylcholine -ACh and carbachol, respectively). Vasorelaxant effect was investigated by isometric force measurements in parotid artery rings while salivary flow was registered collecting the saliva after iv application of carbachol. Quantification of inducible nitric oxide synthase (iNOS) mRNA expression was made in rabbit parotid artery and gland by Real-time RT-PCR. Also, basal tissue concentrations of cGMP in parotid artery and gland were determined by ELISA. Acetylcholine induced concentration- and endothelium-dependent vasorelaxation that was significantly decreased in parotid artery rings from diabetic rabbit compared to controls. Schild analysis of the ACh vasorelaxant effect, in the presence of selective muscarinic receptor antagonists, revealed involvement of the M3 receptor subtype in parotid artery rings from both control and diabetic rabbits, with no change in antagonist affinity constants. The inhibitory effects of indomethacin, a non-selective inhibitor of cyclooxygenase, and of high potassium, an inhibitor of hyperpolarization, on ACh vasorelaxation were increased. The effect of L-NOARG, a non-selective inhibitor of NOS, was decreased in diabetic parotid artery while SMT, a selective inhibitor of iNOS, significantly reduced ACh vasorelaxation only in parotid artery rings from diabetic rabbits. Carbachol induced dose-dependent increase in parotid gland secretion, being significantly reduced in the diabetic rabbits. Functional studies with the selective muscarinic receptor antagonists revealed involvement of the solely M3 receptor subtype in carbachol-induced parotid gland secretion from both control and diabetic rabbits. Inhibitory effects of Nω, a selective inhibitor of nNOS, as well as of L-NOARG and SMT on carbachol-induced parotid secretion were increased in diabetes. Also, up-regulation of iNOS mRNA expression and basal cGMP concentration were detected in parotid artery rings and glands from diabetic rabbits. These results suggest that decreased vasorelaxant effect of ACh in parotid artery and sialogouge effect of carbachol in parotid gland of diabetic rabbits are the consequence of impaired signal transduction mechanisms related to activation of cholinergic M3 receptors: disturbed balance between different NOS isoforms, but not a consequence of altered affinity and number of M3 receptors.

Cilj ovog rada je bio da se na eksperimentalnom modelu kunića ispita uticaj dijabetes melitusa tipa 1, izazvanog jednom intravenskom injekcijom aloksana (100mg/kg), na vaskularni tonus parotidne arterije, koja ishranjuje parotidnu žlezdu i sekretornu aktivnost ove žlezde, izazvane stimulacijom holinergičkih muskarinskih receptora (acetilholinom –ACh, odnosno karbaholom). Vazorelaksantni efekt je registrovan izometrijskim promenama u tonusu arterijskih prstenova, a protok pljuvačke je registrovan sakupljanjem pljuvačke posle intravenske primene karbahola. Takođe, u tkivu parotidne arterije i žlezde kvantifikovana je: ekspresija mRNK iNOS, metodom Real-time RT-PCR, i bazalna koncentracija cGMP, imunoesejskom metodom (ELISA). Dobijeni rezultati pokazuju da ACh izaziva koncentracijski i endotelno-zavisnu relaksaciju parotidne arterije koja je značajno manja na arterijama kunića sa dijabetesom u odnosu na kontrolnu grupu životinja. Šildova analiza efekta ACh, u prisustvu selektivnih muskarinskih antagonista, pokazuje da su samo M3 receptori uključeni u ovaj efekt ACh, i to kako kod kontrolnih tako i kod kunića sa dijabetesom. Inhibitorni efekti indometacina, neselektivnog inhibitora COX, i visokih koncentracija kalijuma na vazorelaksantnom dejstvu ACh rastu, a efekt L-NOARG, neselektivnog inhibitora NOS, se smanjuje na parotidnim arterijama kunića sa dijabetesom. Takođe, primena SMT, selektivnog inhibitora iNOS, značajno smanjuje vazorelaksantni efekt ACh samo na parotidnim arterijama kunića sa dijabetesom. Karbahol prouzrokuje dozno-zavisno povećanje sekrecije parotidne pljuvačke, koja je značajno smanjena u dijabetesu. Funkcionalna ispitivanja primenom selektivnih muskarinskih antagonista pokazuje učešće samo M3 receptora u parotidnoj sekreciji, izazvanoj karbaholom, i to kako kod kontrolnih tako i kod kunića sa dijabetesom. Takođe, u tkivu parotidne arterije i žlezde kunića sa dijabetesom značajno se povećava ekspresija mRNK iNOS kao i bazalne koncentracije cGMP. Na osnovu ovih rezultata može se zaključiti da smanjenje vazorelaksantnog efekta ACh na parotidnoj arteriji, i sekretornog efekta karbahola na parotidnoj žlezdi kunića sa dijabetesom su posledica poremećaja u signalnim transdukcionim mehanizmima vezanim za aktivaciju holinergičkih M3 receptora: poremećene ravnoteže između aktivnosti pojedinih NOS izoformi, a nisu posledica promena na nivou afiniteta i broja M3 muskarinskih receptora.