Inhibition of calcium‐calmodulin kinase restores nitric oxide production and signaling in submandibular glands of a mouse model of salivary dysfunction

Abstract

1 Nitric oxide is an intracellular and diffusible messenger of neurotransmitters involved in salivary secretion, as well as an inflammatory mediator in salivary gland diseases. It is synthesized by three different isoforms of nitric oxide synthase (NOS), each subject to a fine transcriptional, posttranscriptional and/or post-translational regulation. Our purpose was to study the possible mechanisms leading to NOS downregulation in submandibular glands of normal mice and in the nonobese diabetic (NOD) mouse model of salivary dysfunction with lower NOS activity. 2 NOS activity and cGMP accumulation were determined by radioassays in submandibular glands of both mice in the presence of the protein kinase inhibitors KN-93 and bisindolylmaleimide. NOS I mRNA and protein expression and localization were assessed by RT-PCR, Western blot and immunohistochemistry. 3 A downregulatory effect of calcium-calmodulin kinase II (CaMK II) on NOS activity in submandibular glands of both NOD and BALB/c mice was observed. Our results are consistent with a physiological regulation of NOS activity by this kinase but not by PKC in normal BALB/c mice. They are also supportive of a role for CaMK II in the lack of detectable NOS activity in submandibular glands of NOD mice. KN-93 also restored cGMP accumulation in NOD submandibular glands. 4 The downregulation of NOS in NOD mice seems to be mainly mediated by this kinase rather than the result of a lower expression or different cellular localization of the enzyme. It was not related to different substrate or cofactors availability either.

Vasoactive intestinal peptide/vasoactive intestinal peptide receptor relative expression in salivary glands as one endogenous modulator of acinar cell apoptosis in a murine model of Sjögren's syndrome

“…The localization of this enzyme is normally confined to neural fibres in close proximity to gland epithelial cells where NO contributes to salivary flow. Consistent with this, NOD mice submandibular glands showed a reduced NOS activation through VIP receptors that coincided with the reduction in salivary flow [15]. While VIP can induce NOS in peripheral and central neurones, VIP expression is regulated by neural NOS activity and knock-out mice for neural NOS isoform express lower neuronal VIP levels [29].…”

“…In keeping with this, early neurotransmitter receptor-signalling alterations have been reported in NOD females' submandibular glands unrelated to the onset of the autoimmune response [12][13][14]. Among them, a progressive loss of activity of the neural isoform of nitric oxide synthase (NOS 1) in NOD exocrine glands at the Sjögren's syndromelike period has been described [12,15]. The lower levels of NOS activity were found in glands of 16-week-old NOD mice that presented increased apoptosis of acinar cells and increased levels of tumour necrosis factor (TNF)-a, among other T helper type 1 (Th1) cytokines in the serum [15,16].…”

“…Among them, a progressive loss of activity of the neural isoform of nitric oxide synthase (NOS 1) in NOD exocrine glands at the Sjögren's syndromelike period has been described [12,15]. The lower levels of NOS activity were found in glands of 16-week-old NOD mice that presented increased apoptosis of acinar cells and increased levels of tumour necrosis factor (TNF)-a, among other T helper type 1 (Th1) cytokines in the serum [15,16]. Vasoactive intestinal peptide (VIP), described initially as a vasodilator and prosecretory neuropeptide, has trophic effects on acini [17,18] and strong anti-inflammatory properties in several models of chronic inflammatory diseases [19][20][21].…”

NOD Mice Exocrinopathy: Towards a Neuroimmune Link

“…It has been proposed that the initial trigger of autoimmune exocrinopathy in NOD mice may reside in a defect in salivary gland homeostasis [8] . In keeping with this, defects in metalloproteinase expression [9] and autonomic receptor activation [10] were reported in NOD glands and we described a loss of nitric oxide synthase (NOS) activity as well as a reduced salivary secretion and signaling upon vasoactive intestinal peptide (VIP) stimulation [7,11] . A differential regulation of the neural isoform of NOS by CaMK II [11] occurred prior to the appearance of detectable levels of cytokines or infiltrating cells in NOD glands [7] .…”

“…Total RNA isolation and reverse transcription were performed using Ready-to-Go (Amersham) [11,18] . PCR amplification cycles (94 ° C for 20 s, primer annealing at 62 ° C for 30 s, extension at 72 ° C for 40 s) and 30 cycles (94 ° C for 45 s, 60 ° C for 30 s, and 72 ° C for 45 s) with initial incubation at 94 ° C for 5 min and final incubation at 72 ° C for 10 min for Bax 2 and for the standard GAPDH were performed.…”