Functional and proteomic analysis of submandibular saliva in rats exposed to chronic stress by immobilization or constant light.

OBJECTIVE: In this study, we have evaluated the effects of stress on functional and proteomic changes in submandibular saliva of rats. DESIGN: Male adult rats were divided in three groups: IMO (2 h/day of immobilization for 7 days), LL (constant light during 20 days), C (unstressed controls submitted to 14 h light-10h dark cycle). Body weight, food intake and the dry weight of submandibular gland were recorded. Saliva samples, collected under anaesthesia following i.p. administration of isoproterenol and pilocarpine (5 mg/kg), were assayed for total proteins (TP), amylase activity and SDS-PAGE electrophoresis. RESULTS: Body weight, food intake and the dry weight of submandibular gland of IMO rats were lower than those of C and LL groups. The salivary volumes secreted in IMO and LL rats, were significantly higher than in controls. The TP output (µg protein/µg saliva/mg of dry tissue) and amylase activity output (AU/µg of saliva/mg of dry tissue) in IMO were significantly higher than in C and LL animals. The electrophoretic pattern of saliva proteins of LL rats, revealed the absence of a protein band of approximately 25 kDa. This band was composed by the common salivary protein-1 and a prolactin-induced protein as identified by peptide mass fingerprinting. CONCLUSIONS: Differences in body weight and food intake between IMO and LL might be attributed to the sort and intensity of stressors stimuli. The changes in the volume of secreted saliva could be a compensatory mechanism in response to stressors. The increase of total protein in IMO rats and the absence of 25 kDa proteins in LL, would suggest that the submandibular glands respond to the sympathetic nervous system stimuli induced by the stress with an increase of activity of the sympathetic nerves in IMO and a reduction in LL rats.

Autonomic nervous system regulates secretion of anti-inflammatory prohormone SMR1 from rat salivary glands.

The autonomic nervous system regulates the secretion of bioactive proteins and peptides from salivary glands that can be important in systemic physiological responses. The prohormone submandibular rat-1, which is highly expressed in rat submandibular glands, can be cleaved to produce polypeptides with analgesic and anti-inflammatory activities. Human genes related to submandibular rat-1 have conserved biological functions and are potentially important in pain suppression, erectile function, and inflammation. In this study we describe the differential expression and posttranslational modification of submandibular rat-1 protein in salivary glands, the urogenital tract, lung, blood, and saliva in male Sprague-Dawley and Brown Norway rats. Submandibular rat-1 protein is secreted into saliva after the administration of beta-adrenergic or cholinergic agonists. Removal of the sympathetic ganglion that innervates the salivary glands results in increased levels of submandibular rat-1 protein in salivary glands. The secretion of submandibular rat-1 in response to physiological stress may provide a large pool of submandibular rat-1-derived peptide products that can promote analgesia and decrease inflammation locally and systemically. This pathway may be conserved among mammals and may constitute an important anti-inflammatory and analgesic response to stress.

The tripeptide phenylalanine-(D) glutamate-(D) glycine modulates leukocyte infiltration and oxidative damage in rat injured spinal cord.

The tripeptide, phenylalanine-glutamate-glycine (FEG) and its d-isomeric form phenylalanine-(D) glutamate-(D) glycine (feG), derived from submandibular gland peptide-T, significantly reduce the allergic inflammatory response and leukocyte trafficking and neutrophil migration into intestine, heart and lungs. Due to these actions, we hypothesized that feG would attenuate the early inflammatory response to spinal cord injury, reduce free radical production and improve neurological outcomes, like other leukocyte-limiting strategies we have used previously. We tested this using a clip compression model of spinal cord injury in rats. Following spinal cord injury at the 4th thoracic cord segment, we quantified leukocyte infiltration, free radical formation and oxidative damage at the lesion site after feG or control peptide phenylalanine-(D) aspartate-(D) glycine treatment. In rats treated with feG at 2 and 12 h, or 6 and 12 h after spinal cord injury, mean myeloperoxidase activity and ED-1 expression were significantly lower ( approximately 40%) than in controls at 24 h. Free radical formation generated in injured spinal cord was detected using 2',7'-dichlorofluorescin-diacetate as a fluorescent probe. Free radical production in the injured cord increased significantly after spinal cord injury and feG treatment significantly reduced this free radical production. Oxidative enzymes, lipid peroxidation and cell death were also significantly ( approximately 40%), gp91 ( approximately 30%), thiobarbituric acid reactive substance levels ( approximately 35%), 4-hydroxynonenal-bound protein ( approximately 35%) and caspase-3 ( approximately 32%). Early administration of feG decreases infiltration of inflammatory cells into the injured spinal cord and intraspinal free radical formation, thereby reducing oxidative damage and secondary cell death after spinal cord injury.

The expression and role of Fas ligand in intestinal inflammation.

Fas ligand (FasL) is involved in the pathogenesis of inflammatory diseases and immune privilege. We examined the expression of FasL in the enteric nervous system (ENS) in murine colitis and guinea-pig ileitis. We studied FasL immunoreactivity, functional integrity of the ENS, severity of colitis, and distribution of neutrophils in wild type and B6/gld mice that lack functional FasL. In ileitis, the distribution of FasL, CD4+ and CD8+ T cells was examined. FasL expression was increased in the ENS of wild type mice with colitis, but decreased labelling of nerve fibres was noted in B6/gld mice. Neutrophils were more abundant and widely distributed in B6/gld mice. Colitis was more severe and persistent in B6/gld mice 7 days after induction. Functional parameters of intestinal secretion and motility in B6/gld mice were the same as controls. In ileitis, FasL expression was increased in the guinea-pig ENS and returned to control levels following the resolution of inflammation. While T cells were not present in the ENS of controls, they were observed during inflammation, but were excluded from ganglia. The number of enteric neurons was unchanged over the course of inflammation. The expression of FasL is altered in intestinal inflammation and contributes to its resolution in experimental colitis.

Regulation of leukocyte adhesion to heart by the tripeptides feG and feG(NH2).

The role of the D-isomeric form of the salivary gland tripeptide FEG (feG) and its carboxyl-amidated derivative, feG(NH2), in regulating leukocyte adherence to nonfixed atrial slices from Sprague-Dawley rats was examined under static conditions. Optimal binding of the leukocytes was seen if the leukocytes were treated with platelet activating factor (PAF; 10(-9)M). The increased adherence of PAF-treated peripheral blood leukocytes was totally inhibited by both feG and feG(NH2) (10-9M), as well as by antibodies against CD18 and CD49d. In contrast, the binding of peritoneal leukocytes was blocked only by CD49d antibody. Circulating leukocytes obtained from lipopolysaccharide (LPS) treated (2 mg/kg ip) rats did not bind to atrial slices obtained from normal hearts, but readily bound to atrial slices obtained from LPS-treated rats. This leukocyte binding was inhibited by in vivo feG treatment (100 microg/kg ip, 24 h before harvest) or by treating the isolated cells with feG (10(-9)M). The amidated peptide feG(NH2) reduced neutrophil accumulation in the atrium elicited by ip injection of LPS, whereas feG was ineffective. The reduction in neutrophil infiltration into the myocardium by feG(NH2) and the prevention of leukocyte interaction with myocytes seen with both feG and feG(NH2) probably results in hindered leukocyte migration in the inflamed heart, resulting in less tissue damage. The inhibition by these tripeptides on neutrophil adhesion to myocytes suggests that salivary glands hormones regulate the severity of cardiac inflammation.

Inhibition of allergic inflammation by C-terminal peptides of the prohormone submandibular rat 1 (SMR-1).

BACKGROUND: The C-terminal of the prohormone submandibular rat 1 protein (SMR-1) contains several small peptides that reduce the severity of allergic inflammation and septic shock, and are part of the cervical sympathetic trunk-submandibular gland (SMG) axis of neuroendocrine immunology. These peptides include the heptapeptide, submandibular gland peptide-T and the tripeptide FEG. The D-isomeric form of this tripeptide, feG, which is active when administered orally, reduces LPS-provoked leukocyte rolling on mesenteric venules and influx of inflammatory cells into the peritoneum and intestinal muscle. METHODS: To investigate the mechanism of action of these peptides, the influx of inflammatory molecules into the airways, and several properties of human neutrophils were examined. RESULTS: Oral feG (1 mg/kg) inhibited the influx of inflammatory cells into the airways lumen of allergen challenged, sensitized Brown Norway rats. This inhibition occurred whether feG was given 30 min prior to 6 h post allergen challenge. Moreover, feG in picomolar to nanomolar concentrations inhibited PAF elicited chemotaxis by 30-40%, but the peptides did not affect superoxide production or phagocytosis by neutrophils. feG reduced PAF-stimulated expression of CD11b. CONCLUSIONS: feG may exert its anti-inflammatory effects by modulating the expression and functions of beta(2) integrins. The CST-SMG axis may be a major neuroendocrine pathway that modulates allergic asthma and other inflammatory responses.

The tripeptide feG reduces endotoxin-provoked perturbation of intestinal motility and inflammation.

Lipopolysaccharide (LPS)-induced intestinal endotoxaemia perturbs motility and causes activation and influx of inflammatory cells into the muscle tissue. Because rat submandibular gland peptide T (SGP-T; Thr-Asp-Ile-Phe-Glu-Gly-Gly), its carboxyl-terminal fragment tripeptide, FEG (Phe-Glu-Gly) and its D-isomeric analogue, feG, modulate intestinal anaphylactic reactions, we examined whether these peptides also modulate LPS-induced intestinal endotoxaemia in conscious rats. The disruption of the fasting pattern of intestinal MMCs (migrating motor complexes), induced by intravenous LPS (20 microg kg-1) injection, was prevented by all three peptides. The extravasation of leucocytes into the peritoneal cavity and increased expression of the activation marker CD18 on mesenteric tissue leucocytes (18 h after intraperitoneal injection of LPS) were reduced by orally administered feG, which also significantly decreased the number of intestinal tissue leucocytes expressing the integrin CD18. We conclude that feG attenuates both the immediate (intestinal motility) and late ( approximately 18 h) inflammatory reactions provoked by endotoxaemia.

The carboxamide feG(NH2) inhibits endotoxin perturbation of intestinal motility.

The submandibular gland rat-1 (SMR1) salivary gland prohormone contains several peptides, submandibular gland peptide-T (SGP-T) and the tripeptide, FEG, which possess anti-inflammatory activities. The D-isomeric form of FEG, feG, also is a potent anti-inflammatory peptide. In this study, we compared the inhibitory activity of feG and its carboxamide derivative, feG(NH2), on the perturbations of intestinal motility induced by intravenous lipopolysaccharide. feG(NH2) was 20-30 times more potent than feG in reducing the motility disturbances induced by lipopolysaccharide. feG may undergo square-amidation to yield a hormone that strongly down-regulates intestinal responsiveness to endotoxin.

Effects of repeated fluoxetine and citalopram administration on cytokine release in C57BL/6 mice.

This study examines the effects of repeated administration of the selective serotonin reuptake inhibitors (SSRIs), fluoxetine and citalopram (10 mg/kg, i.p.), on immunoreactivity in C57BL/6 mice. Immune functions were evaluated by the ability of splenocytes to reduce a tetrazolium salt to formazan (MTT test), to proliferate, and to produce cytokines, including interleukin (IL)-1, IL-2, IL-4, IL-6, IL-10 and interferon gamma (IFN gamma). Citalopram administered for 1, 2 and 4 weeks stimulates the proliferative activity of splenocytes and suppresses their ability to secrete the anti-inflammatory cytokine IL-4. Fluoxetine administration for 1 and 2 weeks, but not 4 weeks, stimulates the proliferative activity of splenocytes, whereas a 4-week administration of fluoxetine suppresses the secretion of IL-4. Four weeks of prolonged administration of citalopram and fluoxetine induces a significant increase in the production of IL-6 and IL-10, a cytokine with immunosuppressive and anti-inflammatory activities. The results show that, in C57BL/6 mice, the immunomodulatory effects of SSRIs depend on the SSRI used and the duration of administration.

The effect of repeated amitriptyline and desipramine administration on cytokine release in C57BL/6 mice.

This study examines the effects of repeated amitriptyline and desipramine administration (10 mg/kg, IP) on the immunoreactivity of saline-injected C57BL/6 mice, as evaluated by the ability of splenocytes to reduce a tetrazolium salt to formazan (MTT test), to proliferate, and to produce cytokines, such as interleukin (IL)-1, IL-2, IL-4, IL-6, IL-10 and interferon gamma (IFN-gamma). Desipramine and amitriptyline administered for one or two weeks enhance the biochemical (estimated by MTT test) and proliferative activities of splenocytes. One and two weeks administration of desipramine significantly reduces the secretion of IL-4, an anti-inflammatory cytokine. Amitriptyline administration for four weeks stimulates the proliferative activity of splenocytes and enhances IL-2 bioactivity, whereas four weeks desipramine aministration does not change these parameters in comparison to saline treated control mice. Prolonged desipramine administration (seven and 28 days) significantly increased the bioactivity of IL-1. Four weeks of prolonged administration of amitriptyline and desipramine induces a significant increase in the secretion of IL-10, a cytokine with immunosuppressive and anti-inflammatory activities. The results show that the immunoregulatory effects of tricyclic antidepressants in C57BL/6 mice depend on the drugs used and on the duration of administration.

Rodent submandibular gland peptide hormones and other biologically active peptides.

The cervical sympathetic trunk-submandibular gland neuroendocrine axis plays an integral role in physiological adaptations and contributes to the maintenance of systemic homeostasis, particularly under the 'stress conditions' seen with tissue damage, inflammation, and aggressive behavior. The variety of polypeptides, whose release from acinar and ductal cells is under sympathetic nervous system control, offers coordinated and progressive levels of endocrine communication. Proteolytic enzymes (e.g. the kallikreins and furin maturases) are involved in the conversion of inactive precursors (e. g. Pro-EGF and SMR1) into biologically active molecules (e.g. EGF, SMR1-pentapeptide), which act on local or distant targets and thereby modulate the homeostatic process.

Submandibular gland peptide-T (SGP-T) modulates ventricular function in response to intravenous endotoxin.

A novel peptide hormone isolated from salivary glands, submandibular gland peptide-T (SGP-T), protects against the enhanced hypotensive response to intravenously administered lipopolysaccharide (LPS; 3.5 mg/kg; Salmonella typhosa) in rats with their submandibular glands removed (sialadenectomy). In this study, we examined the effects of SGP-T on LPS-provoked perturbations of heart function. Sialadenectomy did not alter basal heart function, although the sialadenectomized rats exhibited more pronounced reductions in ventricular peak systolic pressure (VPSP), ventricular pressure at max dP/dt (Pmax dP/dt), and maximum ventricular negative dP/dt (-dP/dt) relative to unoperated controls following LPS challenge. These changes were primarily due to changes in afterload (blood pressure). However, in sialadenectomized rats LPS-induced changes in Pmax -dP/dt (ventricular pressure at maximum -dP/dt) did not correlate with changes in VPSP, which suggests that sialadenectomy may alter the systolic component of the heart beat. The peptide SGP-T, at doses of 1 and 3.5 microg/kg, corrected Pmax -dP/dt and all other endotoxin-induced changes in heart function. Since Pmax -dP/dt is a measure of relaxation during diastole, the submandibular glands appear to play a role in protecting the heart against the adverse effects of acute endotoxin administration on ventricular relaxation. These effects of the submandibular glands may be mediated by the release of the peptide SGP-T.

Integration of neuro-endocrine immune responses in defense of mucosal surfaces.

Neuro-endocrine immunology, a field arising from curiosity about the mind-body connection, is evolving rapidly. From intriguing, but seemingly unexplainable observations with human infections and disease, experimental systems have been developed that provide a solid scientific basis for new understanding. There have been major efforts to understand influences of the nervous system on immune and inflammatory responses, e.g., innervation of the immune system, molecular communication pathways, and complex phenomena such as conditioning of immune responses and mechanisms of host defenses. In turn, the immune system communicates with the neuro-endocrine systems. Imbalances in the neuro-endocrine-immunologic circuitry are relevant in host defenses and in injury and repair. Examples of these themes in neuro-endocrine-immunology arise in several host-parasite models of neurogenic inflammation, immediate hypersensitivity responses, and granuloma formation. The hypothalamic-pituitary-adrenal axis and the cervical sympathetic trunk-submandibular gland axis provide important models to enhance understanding of this poorly known component of the host-parasite relationship.

Attenuation of intestinal and cardiovascular anaphylaxis by the salivary gland tripeptide FEG and its D-isomeric analog feG.

The effects of the submandibular gland peptide-T (SGP-T; Thr-Asp-Ile-Phe-Gly-Gly; TDIFEGG), its carboxy-terminal fragment (the tripeptide FEG; Phe-Glu-Gly), and the D-isomeric analog (feG) on intestinal and cardiovascular anaphylactic reactions were studied. The tripeptides, FEG and feG, when administered intravenously or orally to egg albumin-sensitized Hooded Lister or Sprague-Dawley rats 30 min prior to challenge with the antigen, totally prevented the disruption of intestinal motility and the development of anaphylaxis provoked diarrhea and inhibited anaphylactic hypotension by 66%. Submandibular gland peptides participate in the regulation of systemic inflammatory reactions, and the D-amino acid tripeptide, feG, is a potent, orally active anti-anaphylactic agent.

Modulation of neutrophil activity by submandibular gland peptide-T (SGP-T).

The heptapeptide submandibular gland peptide-T (SGP-T; sequence = Thr-Asp-Ile-Phe-Glu-Gly-Gly) was isolated from rat submandibular glands based on its ability to reduce endotoxic hypotension. Since these glands also modulate neutrophil function, the effects of SGP-T on neutrophil function were investigated. I.v. SGP-T (35 and 100 micrograms/kg), when administered prior to and after the s.c. implantation of a carrageenan soaked sponge into rats, significantly reduced the accumulation of neutrophils in the sponge. Neutrophils retrieved from saline soaked sponges generated substantial amounts of superoxide anion in response to both phorbol myristate acetate (PMA) and N-fornyl-methionyl-leucyl-phenylalanine (fMLP), whereas those obtained from carrageenan impregnated sponges were refractory to these oxidative stimuli. Treatment with SGP-T promoted a dose-dependent recovery in the ability of neutrophils obtained from carrageenan soaked sponges to generate superoxide anion. This study, by identifying SGP-T as a regulator of neutrophil function, supports the concept that salivary glands are involved in the regulation of inflammatory responses.

Submandibular gland peptide-T (SGP-T) inhibits intestinal anaphylaxis.

A novel peptide, submandibular gland peptide-T (SGP-T), which reduces allergen-induced hypotension, was examined for effects on intestinal anaphylaxis. Hooded-Lister rats were sensitized to egg albumin and prepared for the measurement of in vivo myoelectric activity of the jejunum. The disruption of migrating myoelectric complexes (MMCs) that occurs upon intraluminal, duodenal challenge with antigen of sensitized rats was inhibited by 75% upon intravenous treatment with 100 micrograms/kg of SGP-T. In addition, SGP-T reduced the number of rats experiencing anaphylactic diarrhea and disrupted MMCs, but the peptide did not alter antigen-provoked release of rat mast cell protease II. The mechanism of action of SGP-T remains to be determined, but it apparently does not act directly on mast cells to exert its antianaphylactic action. These results emphasize that modulation of immediate hypersensitivity reactions is only one of several gastrointestinal activities that are affected by growth factors and peptides released from salivary glands.

A novel submandibular gland peptide protects against endotoxic and anaphylactic shock.

Submandibular glands release peptides and proteins that, through exocrine and endocrine actions, facilitate tissue repair in the oral cavity, gastrointestinal tract, and more distal sites such as liver. It has been shown that salivary gland factors also modulate inflammatory responses, because we found that removal of the submandibular glands increases the hypotensive responses to endotoxin. From this observation we proposed that these glands contain a factor that regulates cardiovascular response to shock. With the use of classical peptide isolation procedures, a heptapeptide (TDIFEGG) called submandibular gland peptide T was identified in rat submandibular glands. A synthetic form of this peptide reduced endotoxic shock in sialadenectomized rats by 50% at doses as low as 1 microgram/kg and prevented allergen-induced hypotension by 90% in rats with intact salivary glands at a dose of 100 micrograms/kg. This novel peptide is probably generated from a prohormone, submandibular gland rat 1 protein, a product of the VCSA1 gene. These data indicate that submandibular glands participate in the regulation of systemic homeostasis.