Pituitary adenylate cyclase activating peptide (PACAP) in salivary glands of the rat: origin,and secretory and vascular effects

Pituitary adenylate cyclase activating peptide (PACAP)-38. injected Lv. to the anaesthetized rat. evoked secretion of saliva from the three major salivary glands. the submandibular glands responding with the greatest and the sublingual glands with the smallest volumes. The parotid saliva was rich in amylase and protein. In vitro. pieces of parotid and submandibular gland tissues released K⁺ and protein in response to PACAP-38. with atropine and adrenoceptor antagonists present. The blood flow in the submandibular gland increased in response to PACAP-38. despite a marked fall in mean aortic blood pressure. PACAP is a vasoactive intestinal peptide (VIP)-like neuropeptide. A comparison between the two peptides showed PACAP-38 to be more effective than VIP with respect to vascular responses and less or equi-effective with VIP with respect to the secretory responses. thus suggesting the involvement of PACAP type I and type II receptors. respectively PACAP-38 and -27 were present in the parotid gland as judged by radioimmunoassay. the concentration of the former being about twice that of the latter. Parasympathetic denervation. by cutting the auricula-temporal nerve. reduced the total parotid gland contents of PACAP-38 and -27 by 23 and 44%. respectively (compared with a previously demonstrated 95% reduction of VIP). Sympathetic de nervation. section of the facial nerve or treatment with the sensory neurotoxin capsaicin did not affect the content of PACAP. The difference in efficacy between PACAP and VIP in the vascular and secretory responses as well as the difference in localization suggest that the two peptides play different physiological roles in the salivary glands.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an upstream regulator of prodynorphin mRNA expression in neurons

Although dynorphins are widely involved in the control of not only nociceptive neurotransmission but also a variety of brain functions such as memory and emotion, no natural regulator for inducing the mRNA expression of prodynorphin (Pdyn), a precursor protein of dynorphins, is known. Using primary cultures of rat cortical neurons, we found that pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the vasoactive intestinal polypeptide (VIP)/secretin/glucagon neuropeptide family, markedly induces Pdyn mRNA expression. PACAP was much more effective than VIP, indicating a major role for PAC1 in the PACAP-induced Pdyn mRNA expression. The increase in Pdyn mRNA expression was independent of de novo protein synthesis. Administration of forskolin, an activator for adenylate cyclase/protein kinase A (PKA), but not TPA, an activator for protein kinase C (PKC), induced Pdyn mRNA expression, suggesting a major role for PKA. The involvement of PKA was supported by the inhibition of PACAP-induced Pdyn mRNA expression upon addition of H89, an inhibitor for PKA. The PACAP-induced potentiation of NMDA-R was involved in the mRNA expression of Bdnf or c-fos but not Pdyn. These results suggest PACAP to be an upstream regulator for inducing Pdyn mRNA expression through PKA.     

Expression of glucose-dependent insulinotropic polypeptide and its receptor in the rat major salivary glands

Glucose-dependent insulinotropic polypeptide receptors (GIPR) are expressed throughout the body. The expression of its ligand, glucose-dependent insulinotropic polypeptide (GIP) however, has only been reported in a limited numbers of organs. Although the rat submandibular salivary gland (SMG) has been found to express GIP, its biological role is still not understood. Moreover, nothing is known about the expression of GIP in other types of salivary glands, i.e. the parotid (PG) and sublingual (SLG) glands. We detected the expression of GIP mRNA in the rat PG, SMG and SLG. Immunohistochemical analyses revealed that GIP and GIPR were expressed only in the ductal area of all types of major salivary glands, and no immunostaining was found in the acini area. We also found GIP expression in the rat SMG to be age dependent, with 8-week-old rats showing 2–3-fold higher than those of 9- and 11-week-old rats, respectively. This is the first study to indicate both GIP and GIPR expression in the rat major salivary glands, as well as its variation in the rat SMG during the growth period. These findings are crucial for a better understanding of the physiological function of GIP in rat major salivary gland.     

In vitro three‐dimensional culture systems of salivary glands

Dry mouth can be caused by salivary gland hypofunction due to Sjögren's syndrome (SS) or radiation therapy for head and neck cancer, and it can also be a side effect of medications. The use of sialagogues effectively increases saliva secretion in patients with dry mouth. However, the application of sialagogues is not always satisfactory because of their side effects, such as sweating, nausea, runny nose and diarrhea. Two‐dimensional (2D) cell cultures have been used not only for drug screening and discovery but also to clarify disease mechanisms. However, three‐dimensional (3D) cell cultures are expected to be even more advantageous than 2D cell cultures. Therefore, we have tried to develop an in vitro cell culture system that can reconstitute 3D salivary glands. Sox9 and Foxc1 were identified as important genes that differentiate mouse embryonic stem cell‐derived oral ectoderm into salivary gland placode. Using these genes and organoid culture systems, we succeeded in generating salivary gland organoids that exhibited a morphology and gene expression profile that were similar to those of the embryonic rudiment from which salivary glands arise in normal mice. These organoids are expected to be a promising tool for disease modeling, drug discovery and regenerative medicine in salivary glands.     

Pituitary adenylate cyclase-activating polypeptide (PACAP)-like immunoreactive neuronal elements in rat hypothalamus and median eminence with special reference to morphological background of its effect on anterior pituitary — light and electron microscopic immunocytochemistry

Pituitary adenylate cyclase-activating polypeptide-like immunoreactive (PACAP-LI) neuronal elements in the rat hypothalamus including the median eminence (ME) were investigated by light and electron microscopic immunocytochemistry. PACAP-LI neuronal perikarya with well-developed cell organelles and dense granules were distributed mainly in the magnocellular portion of the paraventricular nucleus and throughout the entire supraoptic nucleus. In the ME, numerous PACAP-LI neuronal processes were found in the internal layer (IL), and immunoreactive terminals containing dense granules, vesicles and mitochondria were detected around portal capillaries which penetrated into the IL from the external layer. Thereafter, PACAP is released into the portal capillaries in the IL, transported to the anterior pituitary and plays a role in the stimulation of adenylate cyclase of anterior pituitary cells.     

Embryonic mouse submandibular salivary gland morphogenesis and the TNF/TNF‐R1 signal transduction pathway

TNF is a pleiotropic cytokine that modulates cell proliferation and apoptosis. The objective of the present study was to investigate the possible function(s) of the TNF/TNF‐R1 signaling pathway in embryonic mouse submandibular salivary gland (SMG) morphogenesis. After characterizing in vivo mRNA and protein expression of various constituents of this pathway, we utilized in vitro experiments to investigate the phenotypic outcomes of enhanced and deficient ligand. The results of these experiments indicate that the TNF/TNF‐R1 signal transduction pathway plays an important role in balancing cell proliferation and apoptosis during SMG duct and presumptive acini formation. Anat Rec 262:318–330, 2001. © 2001 Wiley‐Liss, Inc.     

Transient depletion of CD4+ CD25+ regulatory T cells results in multiple autoimmune diseases in wild‐type and B‐cell‐deficient NOD mice

Plasticity is a hallmark of macrophages, and in response to environmental signals these cells undergo different forms of polarized activation, the extremes of which are called classic (M1) and alternative (M2). Rapamycin (RAPA) is crucial for survival and functions of myeloid phagocytes, but its effects on macrophage polarization are not yet studied. To address this issue, human macrophages obtained from six normal blood donors were polarized to M1 or M2 in vitro by lipopolysaccharide plus interferon-γ or interleukin-4 (IL-4), respectively. The presence of RAPA (10 ng/ml) induced macrophage apoptosis in M2 but not in M1. Beyond the impact on survival in M2, RAPA reduced CXCR4, CD206 and CD209 expression and stem cell growth factor-β, CCL18 and CCL13 release. In contrast, in M1 RAPA increased CD86 and CCR7 expression and IL-6, tumour necrosis factor-α and IL-1β release but reduced CD206 and CD209 expression and IL-10, vascular endothelial growth factor and CCL18 release. In view of the in vitro data, we examined the in vivo effect of RAPA monotherapy (0·1 mg/kg/day) in 12 patients who were treated for at least 1 month before islet transplant. Cytokine release by Toll-like receptor 4-stimulated peripheral blood mononuclear cells showed a clear shift to an M1-like profile. Moreover, macrophage polarization 21 days after treatment showed a significant quantitative shift to M1. These results suggest a role of mammalian target of rapamycin (mTOR) into the molecular mechanisms of macrophage polarization and propose new therapeutic strategies for human M2-related diseases through mTOR inhibitor treatment.     

Localization of phosphatidylinositol 4‐phosphate 5‐kinase (PIP5K) α, β, γ in the three major salivary glands in situ of mice and their response to β‐adrenoceptor stimulation

Phosphatidylinositol 4‐phosphate 5‐kinase (PIP5K), which is composed of three isozymes (α, β and γ), catalyzes the production of phosphatidylinositol bisphosphate (PIP2). This phospholipid functions in membrane trafficking, as an anchor for actin cytoskeletons and as a regulator of intramembranous channels/transporters. It is also a precursor of such second messengers as diacylglycerol, inositol triphosphate and phosphatidylinositol (3,4,5)‐triphosphate. In the present study, the expression and localization of endogenous PIP5Ks were examined in the three major salivary glands of young adult mice in situ. In western blotting of normal control glands, immunoreactive bands for individual PIP5Ks were detectable, with the highest density in the parotid gland and the weakest density in the submandibular gland. In immuno‐light microscopy under non‐stimulated condition, weak immunoreactivity for PIP5Kα was confined to the apical plasmalemma in parotid, but not sublingual or submandibular, acinar cells. Immunoreactivity for PIP5Kβ was weak to moderate and confined to ductal cells but not acinar cells, whereas that for PIP5Kγ was selectively and intensely detected in myoepithelial cells but not acinar cells, and it was weak in ductal cells in the three glands. In western blot of the parotid gland stimulated by isoproterenol, a β‐adrenoceptor agonist, no changes were seen in the intensity of immunoreactive bands for any of the PIP5Ks. In contrast, in immuno‐light microscopy, the apical immunoreactivity for PIP5Kα in parotid acinar cells was transiently and distinctly increased after the stimulation. The increased immunoreactivity was ultrastructurally localized on most apical microvilli and along contiguous plasma membrane, where membranous invaginations of various shapes and small vesicles were frequently found. It was thus suggested that PIP5Kα is involved in post‐exocytotic membrane dynamics via microvillous membranes. The present finding further suggests that each of the three isoforms of PIP5K functions through its product PIP2 discretely in different cells of the glands to regulate saliva secretion.     

Aspiration biopsy of mammary analogue secretory carcinoma of accessory parotid gland: Another diagnostic dilemma in matrix‐containing tumors of the salivary glands

Mammary analogue secretory carcinoma (MASC) is a newly described rare salivary gland tumor, which shares morphologic features with acinic cell carcinoma, low‐grade cystadenocarcinoma, and secretory carcinoma of the breast. This is the first reported case of MASC of an accessory parotid gland detected by aspiration biopsy with radiologic and histologic correlation in a 34‐year‐old patient. Sonographically‐guided aspiration biopsy showed cytologic features mimicking those of low‐grade mucoepidermoid carcinoma, including sheets of bland epithelial cells, dissociated histiocytoid cells with intracytoplasmic mucinous material, and spindle cells lying in a web‐like matrix. Histologic sections showed a circumscribed tumor with microcystic spaces lined by bland uniform epithelial cells and containing secretory material. The tumor cells expressed mammaglobin and BRST‐2. The cytologic features, differential diagnosis, and pitfalls are discussed. The pathologic stage was pT1N0. The patient showed no evidence of disease at 1 year follow‐up. Diagn. Cytopathol. 2014;42:49–53. © 2012 Wiley Periodicals, Inc.     

Involvement of REG Iα protein in the regeneration of ductal epithelial cells in the minor salivary glands of patients with Sjögren's syndrome

The regenerating gene (Reg) was originally isolated from regenerating rat pancreatic islets and revealed recently to constitute a multi‐gene family in humans. REG Iα protein is known to be overexpressed not only in various human inflammatory diseases but also in various experimental models of inflammation in animal tissues. However, its involvement in pathophysiology of the minor salivary gland (MSG) is not clear. We investigated REG Iα expression in the MSG of patients with primary Sjögren's syndrome (SS) and assessed its role in ductal epithelial cell proliferation in such tissues. Lip biopsy specimens were obtained from 40 patients with primary SS and examined using immunohistochemistry for REG Iα protein, Ki67 and single‐strand DNA (ssDNA). The relationships among clinicopathological factors and expression of REG Iα protein, Ki67 and ssDNA in the MSG were then analysed. REG Iα protein was expressed rarely in ductal epithelial cells of the normal MSG but was apparently overexpressed in those of patients with SS. The labelling indices for both Ki67 and ssDNA in the ductal cells of the MSGs were significantly higher in SS patients than in controls. Moreover, these labelling indices were significantly higher in REG Iα‐positive than in negative SS patients. REG Iα protein may play a role in the regeneration of ductal epithelial cells in the MSGs of patients with SS.     

Neurotransmitter signalling via NMDA receptors leads to decreased T helper type 1‐like and enhanced T helper type 2‐like immune balance in humans

Given the pivotal roles that CD4⁺ T cell imbalance plays in human immune disorders, much interest centres on better understanding influences that regulate human helper T‐cell subset dominance in vivo. Here, using primary CD4⁺ T cells and short‐term T helper type 1 (Th1) and Th2‐like lines, we investigated roles and mechanisms by which neurotransmitter receptors may influence human type 1 versus type 2 immunity. We hypothesized that N‐methyl‐d ‐aspartate receptors (NMDA‐R), which play key roles in memory and learning, can also regulate human CD4⁺ T cell function through induction of excitotoxicity. Fresh primary CD4⁺ T cells from healthy donors express functional NMDA‐R that are strongly up‐regulated upon T cell receptor (TCR) mediated activation. Synthetic and physiological NMDA‐R agonists elicited Ca²⁺ flux and led to marked inhibition of type 1 but not type 2 or interleukin‐10 cytokine responses. Among CD4⁺ lines, NMDA and quinolinic acid preferentially reduced cytokine production, Ca²⁺ flux, proliferation and survival of Th1‐like cells through increased induction of cell death whereas Th2‐like cells were largely spared. Collectively, the findings demonstrate that (i) NMDA‐R is rapidly up‐regulated upon CD4⁺ T cell activation in humans and (ii) Th1 versus Th2 cell functions such as proliferation, cytokine production and cell survival are differentially affected by NMDA‐R agonists. Differential cytokine production and proliferative capacity of Th1 versus Th2 cells is attributable in part to increased physiological cell death among fully committed Th1 versus Th2 cells, leading to increased Th2‐like dominance. Hence, excitotoxicity, beyond its roles in neuronal plasticity, may contribute to ongoing modulation of human T cell responses.     

Deficiency of prolactin‐inducible protein leads to impaired Th1 immune response and susceptibility to Leishmania major in mice

Although the strategic production of prolactin‐inducible protein (PIP) at several ports of pathogen entry into the body suggests it might play a role in host defense, no study has directly implicated it in immunity against any infectious agent. Here, we show for the first time that PIP deficiency is associated with reduced numbers of CD4⁺ T cells in peripheral lymphoid tissues and impaired CD4⁺ Th1‐cell differentiation in vitro. In vivo, CD4⁺ T cells from OVA‐immunized, PIP‐deficient mice showed significantly impaired proliferation and IFN‐γ production following in vitro restimulation. Furthermore, PIP‐deficient mice were highly susceptible to Leishmani major infection and failed to control lesion progression and parasite proliferation. This susceptibility was associated with impaired NO production and leishmanicidal activity of PIP KO macrophages following IFN‐γ and LPS stimulation. Collectively, our findings implicate PIP as an important regulator of CD4⁺ Th1‐cell‐mediated immunity.     

Dendritic‐cell expression of Ship1 regulates Th2 immunity to helminth infection in mice

In mouse models of infection with the gastrointestinal parasite Trichuris muris, appropriate dendritic‐cell (DC) Ag sampling, migration, and presentation to T cells are necessary to mount a protective Th2‐polarized adaptive immune response, which is needed to clear infection. SH2‐containing inositol 5′‐phosphatase 1 (SHIP‐1) has been shown to be an important regulator of DC function in vitro through the negative regulation of the phosphoinositide 3‐kinase (PI3K) pathway, but its role in vivo is relatively unexplored. In the current work, mice with a specific deletion of SHIP‐1 in DCs (Ship1^ΔDC) were infected with the parasite T. muris. Ship1^ΔDC mice were susceptible to infection due to ineffective priming of Th2‐polarized responses. This is likely due to an increased production of interleukin (IL) 12p40 by SHIP‐1‐deficient DCs, as in vivo antibody blockade of IL‐12p40 was able to facilitate the clearing of infection in Ship1^ΔDC mice. Our results describe a critical role for SHIP‐1 in regulating the ability of DCs to efficiently prime Th2‐type responses.