Submandibular Rat Research Articles

Structural and posttranslational analysis of human calcium-binding protein, spermatid-associated 1.

Recently, we detected a novel biomarker in human saliva called calcium-binding protein, spermatid-associated 1 (CABS1). CABS1 protein had previously been described only in testis, and little was known of its characteristics other than it was considered a structurally disordered protein. Levels of human CABS1 (hCABS1) in saliva correlate with stress, whereas smaller sized forms of hCABS1 in saliva are associated with resilience to stress. Interestingly, hCABS1 also has an anti-inflammatory peptide sequence near its carboxyl terminus, similar to that of a rat prohormone, submandibular rat 1. We performed phylogenetic and sequence analysis of hCABS1. We found that from 72 CABS1 sequences currently annotated in the National Center for Biotechnology Information protein database, only 14 contain the anti-inflammatory domain "TxIFELL," all of which are primates. We performed structural unfoldability analysis using PONDER and FoldIndex and discovered three domains that are highly disordered. Predictions of three-dimensional structure of hCABS1 using RaptorX, IonCom, and I-TASSER software agreed with these findings. Predicted neutrophil elastase cleavage density also correlated with hCABS1 regions of high structural disorder. Ligand binding prediction identified Ca2+ , Mg2+ , Zn2+ , leucine, and thiamine pyrophosphate, a pattern observed in enzymes associated with energy metabolism and mitochondrial localization. These new observations on hCABS1 raise intriguing questions about the interconnection between the autonomic nervous system, stress, and the immune system. However, the precise molecular mechanisms involved in the complex biology of hCABS1 remain unclear. We provide a detailed in silico analysis of relevant aspects of the structure and function of hCABS1 and postulate extracellular and intracellular roles.

Marked Differences in the Submandibular Salivary Proteome between Sardinian Alcohol-Preferring and Sardinian Alcohol-Non Preferring Rats Revealed by an Integrated Top-Down-Bottom-Up Proteomic Platform.

Sardinian alcohol-preferring (sP) and Sardinian alcohol-non preferring (sNP) rats have been selectively bred for opposite alcohol preference and consumption. Aiming to verify possible differences at the proteomics level between sP and sNP rats, we investigated the salivary proteome by a a liquid chromatography-mass spectrometry top-down-bottom-up integrated approach. For this purpose, submandibular saliva was collected from alcohol-naive sP and sNP rats under isoprenaline stimulation. A total of 200 peptides and proteins were detected and quantified in the two rat lines, 149 of which were characterized in their naturally occurring structure. The data are available via ProteomeXchange with identifier PXD006997. Surprisingly, sP rats exhibited marked quantitative and qualitative differences with respect to sNP rats, namely higher levels of proteoforms originating from submandibular gland protein C, and from submandibular rat protein 2, as well as those of several unidentified peptides and proteins. sP rats expressed some proteins not detectable in sNP rats such as the glutamine and glutamic acid-rich protein (GRP)-CB. The isoform GRP-B, detectable in both rat lines, was more abundant in sNP rats. The submandibular saliva of sNP rats was also characterized by very high levels of GRP-B proteolytic peptides and rat salivary protein 1. Whether these differences could contribute to the opposite alcohol preference and consumption of sP and sNP rats is currently unknown and requires further investigation.

A novel biomarker associated with distress in humans: calcium-binding protein, spermatid-specific 1 (CABS1).

Calcium-binding protein spermatid-specific 1 (CABS1) is expressed in the human submandibular gland and has an anti-inflammatory motif similar to that in submandibular rat 1 in rats. Here, we investigate CABS1 in human saliva and its association with psychological and physiological distress and inflammation in humans. Volunteers participated across three studies: 1) weekly baseline measures; 2) a psychosocial speech and mental arithmetic stressor under evaluative threat; and 3) during academic exam stress. Salivary samples were analyzed for CABS1 and cortisol. Additional measures included questionnaires of perceived stress and negative affect; exhaled nitric oxide; respiration and cardiac activity; lung function; and salivary and nasal inflammatory markers. We identified a CABS1 immunoreactive band at 27 kDa in all participants and additional molecular mass forms in some participants. One week temporal stability of the 27-kDa band was satisfactory (test-retest reliability estimate = 0.62-0.86). Acute stress increased intensity of 18, 27, and 55 kDa bands; 27-kDa increases were associated with more negative affect and lower heart rate, sympathetic activity, respiration rate, and minute ventilation. In both acute and academic stress, changes in 27 kDa were positively associated with salivary cortisol. The 27-kDa band was also positively associated with VEGF and salivary leukotriene B4 levels. Participants with low molecular weight CABS1 bands showed reduced habitual stress and negative affect in response to acute stress. CABS1 is readily detected in human saliva and is associated with psychological and physiological indicators of stress. The role of CABS1 in inflammatory processes, stress, and stress resilience requires careful study.

Chronic intoxication by methylmercury leads to oxidative damage and cell death in salivary glands of rats.

Methylmercury (MeHg) is one of the most toxic species of mercury, causing several systemic damages; however, its effect on the salivary glands has rarely been explored to date. This study was aimed at analyzing the mercury deposit, oxidative stress markers, and cell viability in parotid and submandibular rat salivary glands after chronic methylmercury intoxication. Herein, forty male Wistar rats (40 days old) were used in the experiment. The animals of the experimental group were intoxicated by intragastric gavage with MeHg at a dose of 0.04 mg per kg body weight per day for 35 days, whereas the control group received only corn oil, a diluent. After the period of intoxication, the glands were obtained for evaluation of total mercury deposit, cell viability, and the malondialdehyde (MDA) and the nitrite levels. Our results indicated mercury deposits in salivary glands, with a decrease in cell viability, higher levels of MDA in both glands of intoxicated animals, and a higher concentration of nitrite only in the submandibular gland of the mercury group. Thus, the intoxication by MeHg was able to generate deposits and oxidative stress in salivary glands that resulted in a decrease in cell viability in both types of glands.

Calcium-binding protein, spermatid-specific 1 is expressed in human salivary glands and contains an anti-inflammatory motif.

Salivary glands are involved in the production and exocrine and endocrine secretion of biologically active proteins, polypeptides, and hormones involved in growth and differentiation, homeostasis, and digestion. We have previously studied the prohormone submandibular rat 1 (SMR1), product of the Vcsa1 gene, which is highly expressed in the testes and salivary glands of rats, and can be cleaved to produce polypeptides with analgesic, erectile function, and anti-inflammatory activities. Humans lack the Vcsa1 gene, but homologous sequences and functions for analgesia and erectile function exist in the human genes Prol1, SMR3a, and SMR3b located on the human chromosomal region close to where Vcsa1 lies in the rat. Here we show the human protein calcium-binding protein spermatid-specific 1 (CABS1) contains a similar sequence to the anti-inflammatory sequence in rat SMR1, thus CABS1 may be another human gene with homologous function to Vcsa1. Using Western blot and PCR, we discovered that the human protein CABS1, previously thought to only be expressed in the testes, is also expressed in the salivary glands and lung, in a tissue-specific manner. Peptides derived from CABS1 were tested in an in vivo mouse model of lipopolysaccharide (LPS)-induced neutrophilia and an ex vivo rat model of antigen-induced intestinal anaphylaxis and significantly reduced both neutrophil accumulation in bronchoalveolar lavage fluid and antigen-induced ileal contractions, respectively. Thus human CABS1 has a peptide motif homologous to the anti-inflammatory peptide sequence of rat SMR1. Whether this similarity of CABS1 extends to the neuroendocrine regulation of the anti-inflammatory activity seen for SMR1 remains to be determined.

The sympathetic nervous system regulates the release of anti-inflammatory peptides from salivary glands (93.18)

Abstract Chronic and acute stress have profound effects on inflammation. In rats, allergic inflammation is regulated by the sympathetic nervous system acting on salivary glands. Human asthma is frequently accompanied by salivary gland inflammation. Salivary gland dysfunction in stressed individuals could enhance asthma severity. Salivary gland prohormone SMR1 (submandibular rat-1) is cleaved into two peptides that are anti-inflammatory in rats, mice, dogs, sheep, cats, and human cells in pulmonary inflammation, food allergy, septic shock, pancreatitis, and spinal cord injury. We hypothesized that modulation of the autonomic nervous system would change the expression, processing, and secretion of SMR1 and its peptides. Rats were injected with saline, isoproterenol, or pilocarpine, or the superior cervical ganglion was excised. Saliva, blood, and tissues were collected and analyzed for SMR1. Adrenergic stimulation caused the majority of SMR1 into be secreted into saliva in 60 min. Removal of the superior cervical ganglion that innervates the salivary glands changed SMR1 protein levels in the salivary glands. SMR1 secretion into saliva in response to acute stress may provide a large pool of SMR1-derived peptide products that mediate anti-inflammatory responses locally and systemically. This research is funded by AllerGen NCE.

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.

Transcription of G-Protein Coupled Receptors in Corporeal Smooth Muscle is Regulated by the Endogenous Neutral Endopeptidase Inhibitor Sialorphin

Several reports suggest that the rat Vcsa1 gene is down-regulated in models of erectile dysfunction. The Vcsa protein product sialorphin is an endogenous neutral endopeptidase inhibitor and its down-regulation could result in prolonged activation of G-protein activated signaling pathways by their peptide agonists. We investigated whether Vcsa1 down-regulation could result in an adaptive change in GPCR (G-protein coupled receptor) expression. Gene expression in cultured rat corporeal smooth muscle cells following treatment with siRNA directed against Vcsa1 or the neutral endopeptidase gene was analyzed using microarray and quantitative reverse transcriptase-polymerase chain reaction. In rats Vcsa1 is one of the most down-regulated genes following bilateral transection of the cavernous nerves. In that animal model we also investigated whether Vcsa1 down-regulation was accompanied by similar changes in gene expression in corporeal smooth muscle cells in which Vcsa1 was knocked down in vitro. Microarray analysis and quantitative reverse transcriptase-polymerase chain reaction demonstrated that corporeal smooth muscle cells treated in vitro with siRNA against Vcsa1 resulted in GPCR up-regulation as a functional group. In contrast, treatment of corporeal smooth muscle cells that lowered neutral endopeptidase activity resulted in decreased GPCR expression. These results suggest that the peptide product of Vcsa1, sialorphin, can effect GPCR expression by acting on neutral endopeptidase. In animals with bilaterally transected cavernous nerves the decreased Vcsa1 expression is accompanied by increased GPCR expression in cavernous tissue. These experiments suggest that the mechanism by which Vcsa1 modulates erectile function is partly mediated through changes in GPCR expression.

Vcsa1 Gene Peptides for the Treatment of Inflammatory and Allergic Reactions

The recently emerged Vcsa1 gene is one member of the variable coding sequence (VCS) multigene family of Rattus norvegicus. This gene encodes the precursor prohormone SMR1 (submandibular rat-1), which on enzymatic processing gives rise to several 5 to 11 amino acid peptides that modulate a variety of physiological functions. The analgesic pentapeptide sialorphin and anti-inflammatory heptapeptide submandibular gland peptide-T (TDIFEGG) are the most intensively studied. Although the Vcsa1 gene and its protein product are unique to rats, TDIFEGG or a derivative acts on all species examined to date, including human cells, in functions related to allergic reactions and inflammation. In this review, the patent and academic literature on SMR1 and its natural peptides and their derivatives are reviewed for consideration of biological targets and relevance to the development of novel therapeutic agents. The VCS gene family is discussed and we speculate on possible human homologs of these potent anti-inflammatory rat-derived peptides. The biologically active peptide products of SMR1 are considered and the mechanism of action and structure-activity relationships of the anti-inflammatory submandibular gland peptide-T and its derivatives are discussed.

Frontline: Inhibition of allergen‐induced pulmonary inflammation by the tripeptide feG: a mimetic of a neuro‐endocrine pathway

Interactions between the neuro-endocrine system and immune system help maintain health. One interaction involves the superior cervical ganglia (SCG), which regulate the prohormone submandibular rat 1 (SMR1) produced by the submandibular gland (SMG). A peptide derived from SMR1, feG, has anti-inflammatory activity, and modification to D-isomer feG enhances bioactivity. We tested feG as a therapeutic agent for airways inflammation, using rats sensitized by OVA or Nippostrongylus brasiliensis (Nb). Treatment with feG but not fdG down-regulated OVA-challenge-induced increases in bronchoalveolar lavage (BAL)-derived macrophages, eosinophils and PMN (neutrophils) by 44%, 69% and 67%, respectively, at 24 h. We found that feG also reduced ICAM-1 on BAL-derived macrophages and eosinophils by 27% and 65%, and L-selectin on PMN by 55% following OVA challenge. Furthermore, feG but not fdG reduced the OVA-induced TNF increase in BAL fluid. We showed that feG also down-regulated both hyper-responsiveness to methacholine (by 27%) and microgranulomata formation in the lung parenchyma. In Nb-challenged rats, feG treatment inhibited ex vivo allergen-induced contraction of tracheal smooth muscle by up to 73%. In conclusion, feG, which is a mimetic of a peptide derived from a rat salivary gland prohormone, has anti-inflammatory properties in allergic airways inflammation in Brown-Norway rats. The role of the SCG-SMG neuro-endocrine pathway in allergic asthma and other inflammatory diseases requires additional study.

Microarray Analysis and Description of Smr1 Gene in Rat Penis in a Post-Radical Prostatectomy Model of Erectile Dysfunction

We focused on the post-radical prostatectomy model to advance the understanding of neurogenic erectile dysfunction. We attempted to identify previously undescribed molecular changes via gene discovery methods using GeneChip (Affymetrix, Santa Clara, California) microarray technology. Five male adult 120-day-old Sprague-Dawley rats underwent bilateral cavernous nerve neurectomy. Five age matched controls were prepared simultaneously. The penises were harvested on postoperative day 2 and snap frozen in liquid nitrogen. RNA was prepared and pooled into cut and uncut groups. Synthesis of cRNA was performed according to the GeneChip technical manual. Microarray analysis was performed on a U34A Rat Array (Affymetrix). This array has approximately 8,800 gene probe sets, approximately 6,600 known genes and approximately 2,200 estimated sequence transcripts. Dramatic results were found during GeneChip microarray expression analysis. A total of 126 candidate genes were noted to be altered based on the magnitude of expression change using rigorous statistical criteria, including 47 that were down-regulated and 79 that were up-regulated. Among the many significant changes seen 1 dominant class of genes was the submandibular rat genes. Submandibular rat 1 (SMR1) was down-regulated 82.5 fold. Other genes in this family were down-regulated 226 and 90 times. This result was confirmed by reverse transcriptase-polymerase chain reaction and Western blot analyses. These assays verified decreases in SMR1 at multiple time points after surgery. Impressive and previously unrecognized genetic changes are being intensely investigated as they are being unmasked by GeneChip technology. We have identified and begun the investigation of 1 interesting family of genes, namely submandibular gland proteins. The role of SMR as a clinically relevant change in penile and/or urethral function following cavernous nerve injury is speculative.

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 β₂ integrins. The CST-SMG axis may be a major neuroendocrine pathway that modulates allergic asthma and other inflammatory responses.

Novel genes and hormones in salivary glands: from the gene for the submandibular rat 1 protein (SMR1) precursor to receptor sites for SMR1 mature peptides

Submandibular rat 1 protein (SMR1) preprohormone and its maturation peptides constitute the novel characterized submandibular gland (SMG)-specific factors of the cervical sympathetic trunk (CST)-SMG (CST-SMG) axis. As is generally observed for major polypeptide hormones of the endocrine system, SMR1 peptides, including SMR1- imdecapeptide, -hexapeptide and -pentapeptide, are selectively matured from the precursor by cleavage at pairs of basic residues, and differentially accumulated and locally as well as systemically released under multifactorial neuroendocrine control. In turn, the final SMRl mature pentapeptide, at hormonal circulating concentrations, is selectively taken up by peripheral targets through specific binding sites. Localization of the target cells suggests that the SMRl-derived pentapeptide might be involved in modulating mineral ion balance in vivo. Furthermore, associated with male rat specific behavioral characteristics, one can propose that the androgen-regulated SMR1-pentapeptide is a SMG hormonal factor, which under stressful circumstances, is acutely secreted to counterregulate the mineral homeostatic responses to stress. The gene VCSA1, which encodes SMR1, belongs to a new multigene family, essentially expressed in the salivary glands of mammals. This family, whose several members also display putative sites of processing by convertases, has an unusual evolution characterized by multiple gene duplications and an accelerated divergence of coding sequences. Biomedical Reviews 1998; 9: 17-32.

Targets for SMR1-pentapeptide suggest a link between the circulating peptide and mineral transport.

The submandibular rat 1 protein (SMR1) is selectively processed at pairs of basic amino acid residues in a tissue- and sex-specific manner. We have mapped peripheral targets for the final secretory maturation product of SMR1, the pentapeptide QHNPR, by examining in vivo the tissue distribution of the radiolabeled peptide using beta-radio imager whole body autoradiography. The characteristics of tissue uptake allowed specific binding sites at physiological peptide concentrations to be identified within the renal outer medulla, bone and dental tissue, glandular gastric mucosa, and pancreatic lobules. Direct evidence that pentapeptide binding sites are localized in selective portions of the male rat nephron, within the S3, S2, and S1 segments of the proximal tubules, was obtained. In bone tissue the pentapeptide exclusively accumulates within the trabecular bone remodeling unit, and in dental tissue it concentrates within the tubules of the dentinal rat incisor. In relation to male rat-specific behavioral characteristics, our data suggest that the circulating androgen-regulated SMR1-derived pentapeptide is primarily involved in the modulation of mineral balance between at least four systems: kidney, bone, tooth, and circulation.

Selective processing of submandibular rat 1 protein at dibasic cleavage sites

The amino acid sequence of submandibular rat 1 (SMR1) protein, deduced from its cDNA sequence, led to the prediction that the SMR1 gene encodes a hormone-like precursor [Rosinski-Chupin, I., Tronik, D. & Rougeon, F. (1988) Proc. Natl Acad. Sci. USA 85, 8553-8557]. SMR1 contains an N-terminal putative secretory signal sequence and a tetrapeptide (QHNP), located between dibasic amino acids which constitute the most common signal for prohormone processing. We have isolated and characterized from the male rat submandibular gland and its secretions three structurally related peptides, namely an undecapeptide (VRGPRRQHNPR), a hexapeptide (RQHNPR) and a pentapeptide (QHNPR) generated from SMR1 by selective proteolytic cleavages at pairs of arginine residues. The biosynthesis of these peptides is subjected to distinct regulatory pathways depending on the organ, sex and age of the rat. Furthermore, the peptides are differentially distributed in the submandibular gland and in resting or epinephrine-elicited submandibular salivary secretions, suggesting distinct proteolytic pathways for their maturation. The undecapeptide is generated in the gland of both male and female rats, but under basal conditions it is only released into the saliva in male animals. The hexapeptide is produced in large amounts in the gland of adult male rats and released into the saliva in both resting and stimulated conditions. The pentapeptide appears only in the male saliva and is present mostly under stimulated conditions. In addition, administration of epinephrine induces the release of the hexapeptide from the submandibular gland into the bloodstream. The evidence indicates that the rat submandibular gland can function as a dual exocrine and endocrine organ for the SMR1-derived hexapeptide, as has been reported for nerve growth factor, epidermal growth factor, renin and kallikrein. Although the biological activities of the SMR1-derived peptides are not yet known, their high production and adrenergic-induced release only into the saliva and bloodstream of adult male rats, suggest a physiological involvement in some male-specific processes.

Purification and characterization of a low and a high molecular weight form of epidermal growth factor from rat urine

Two forms of epidermal growth factor (EGF) have been purified to homogeneity from rat urine by immunoaffinity chromatography and gel filtration. For one of the purified peptides the molecular mass has been determined to be 5891 by mass spectrometry. This peptide consists of 51 amino acid residues. The sequence of the first 48 amino acid residues is identical to the previously published sequence for submandibular rat EGF. The C-terminal three residues (49–51) are Trp-Trp-Lys. The other purified peptide has a molecular mass of 45 kDa as determined by SDS-polyacrylamide gel electrophoresis. The N-terminal sequence is Asn-Tyr-Lys-Asp-(Cys)-Gly-Pro-Gly-Gly-(Cys)-Gly-Ser-His-Ala. Both the high and the low molecular mass form of urinary rat EGF are able to bind to the human placenta receptor for EGF.