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Simple Summary: In vivo microdialysis applied to rat submandibular glands showed that an increase in the norepinephrine and serotonin levels in the dialysate was primarily dependent on their release from the nerve endings. Either infusion of imipramine into interstitial fluids in rat submandibular glands through a dialysis probe or intraperitoneal administration of imipramine significantly and dose-dependently increased both norepinephrine and serotonin concentrations in the interstitial fluids. Xerostomia induced by antidepressants such as imipramine has long been thought to be due to their anticholinergic effects. However, even antidepressants with low anticholinergic effects may have a high incidence of xerostomia. In salivary glands, norepinephrine activates alpha-adrenergic receptors in blood vessels and beta-adrenergic receptors in acinar cells, respectively, causing a decrease in the blood flow and an increase in the protein secretion, resulting in the secretion of viscous saliva with low water content and high protein content. A previous study demonstrated that perfusion of the submandibular glands of rats with serotonin significantly decreased saliva secretion. The results of the present study revealed the following: (1) that norepinephrine and serotonin, but not epinephrine nor dopamine, were detected in the interstitial fluids in rat submandibular glands; (2) that norepinephrine and serotonin concentrations in the dialysate was 4.3 ± 2.8 nM and 32.3 ± 19.6 nM at stable level, respectively; (3) that infusion with imipramine, a reuptake inhibitor of norepinephrine and serotonin, significantly and dose-dependently increased both norepinephrine and serotonin concentrations in the dialysate; and (4) that intraperitoneal administration of imipramine significantly increased both norepinephrine and serotonin concentrations in the dialysate. These results suggested that one of the mechanisms of xerostomia induced by reuptake inhibitors of norepinephrine and serotonin involves the activation of adrenergic and serotonin receptors in the salivary glands, respectively. [ABSTRACT FROM AUTHOR]

Simple Summary: Oral administration of d-serine, but not l-serine, increased its concentrations in the rat submandibular gland and pilocarpine-induced salivary secretion. Perfusion of the rat submandibular gland with d-serine via the submandibular gland artery increased carbachol-induced salivary secretion. The proportion of the d-form of serine in interstitial fluid was higher than that in plasma or saliva. Infusion of d-serine with l-glutamate through the microdialysis probe significantly increased acetylcholine levels in the submandibular glands in an N-methyl-d-aspartate receptor glycine site antagonist-sensitive manner. d-serine has been observed in submandibular gland tissue in rats, but its functions remain to be clarified. Oral administration of d-serine, but not l-serine, increased its concentrations in the submandibular gland and pilocarpine-induced salivary secretion. In vivo microdialysis was used to collect the d- and l-enantiomers of amino acids from local interstitial fluid in the rat submandibular gland. The proportion of the d-form of serine in interstitial fluid was higher than that in plasma or saliva. Perfusion of the rat submandibular gland with d-serine and l-glutamic acid via the submandibular gland artery resulted in a significant increase in salivary secretion after stimulation of muscarinic receptors with carbachol. In vivo microdialysis applied to the submandibular glands of rats showed that infusion of d-serine along with l-glutamate through the microdialysis probe significantly elevated acetylcholine levels in local interstitial fluids in the submandibular glands of anesthetized rats as compared to that with l-glutamate alone in an N-methyl-d-aspartate receptor glycine site antagonist-sensitive manner. These results indicate that d-serine augments salivary secretion by increasing acetylcholine release in the salivary glands. [ABSTRACT FROM AUTHOR]

Purpose The N- and C-terminal regions of dynorphin (Dyn) A (1-17) activate opioid and N-methyl-D-aspartate receptors, respectively. Earlier studies demonstrated that Dyn-converting enzyme cleaved Dyn A (1-17) mainly at the [Arg.sup.6]-[Arg.sup.7] bond, resulting in the production of N- and C-terminal region peptide fragments, and that this enzyme was not inhibited by a mixture of the three peptidase inhibitors (PIs) amastatin (A), captopril (C), and phosphoramidon (P). The purpose of the present study was to evaluate antinociceptive potential and toxicity with intracerebroventricular administration of Dyn A (1-17) or (1-13) under pretreatment with a mixture of A, C, and P and/or Dyn-converting enzyme inhibitor (p-hydroxymercuribenzoate). Methods Peptide fragments from Dyn A (1-17) following incubation with membrane preparation under pretreatment with a mixture of the three PIs was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS). Infusion of drugs and peptides into the third ventricle in rats was performed via indwelling cannulae. Induction of antinociception and toxicity by Dyn A (1-17), Dyn A (1-13), Dyn A (1-6), or Dyn A (7-17) were determined by the tail-flick test and induction of barrel rotation, respectively. The effects of the PIs on antinociception and toxicity were evaluated by a dose-response study and a comparison of differences among various combinations of Dyn A (1-17) or Dyn A (1-13) and the three PIs and p-hydroxymercuribenzoate. Results MALDI-TOF-MS analysis identified Dyn A (1-6) and Dyn A (1-10) fragments as products following incubation of Dyn A (1-17) with membrane preparation of rat midbrain under pretreatment with a mixture of the three PIs. Pretreatment with a mixture of the three PIs produced an approximately 30-fold augmentation in antinociception induced by low-dose intracerebroventricular administration of Dyn A (1-17) or (1-13) in a [mu]-, [delta]- and [kappa]-opioid receptor antagonist-reversible manner, but without signs of toxicity such as barrel rotation in the rat. Dyn A (1-17)-induced antinociception and toxicity was greater than that of Dyn A (1-6), Dyn A (1-13), or Dyn A (7-17) at the same dose. Dyn A (1-17)-induced antinociception and toxicity under pretreatment with various combinations of the three PIs and p-hydroxymercuribenzoate was greater than that with a mixture of the three PIs alone. Conclusion These findings suggest that administration of a mixture of the three PIs increases Dyn A (1-17)- or (1-13)-induced antinociception under physiological conditions without toxicity. Keywords Dynorphin A * Peptidase * Dynorphin-converting enzyme * Antinociception * Toxicity, Introduction Dynorphin (Dyn) A (1-17) activates both opioid and nonopioid receptors, with its N- and C-terminal regions activating opioid and N-methyl-D-aspartate (NMDA) receptors, respectively. At physiological concentrations, Dyn A (1-17) [...]

Purpose Previous in vitro studies have shown that degradation of opioid peptides during incubation with cerebral membrane preparations is almost completely prevented by a mixture of three peptidase inhibitors (PIs), namely, amastatin, captopril, and phosphoramidon. In the present in vivo study, we evaluate the effects of intrathecal administration of these PIs on antinociception by [[Met.sup.5]]-enkephalin (ME) or PIs themselves. Methods Drugs were administered into the thoracolumbar level of the spinal cord in the intrathecal space in rat. Induction of antinociception was measured by the tail immersion assay, with 55 °C as the nociceptive stimulus. Effects of PIs on antinociception were evaluated by doseresponse study (ME, 1-20 nmol; PIs, 1-20 nmol each), by comparison of differences among two combinations of PIs (amastatin and captopril; captopril and phosphoramidon; amastatin and phosphoramidon) and three PIs (amastatin, captopril, and phosphoramidon), and by using opioid receptor selective antagonists. Results Intrathecal administration of ME with these three PIs or PIs alone significantly and dose dependently increased antinociception in a μ- and δ-opioid receptor antagonist-reversible manner; moreover, the degree of antinociception with a combination of any two of these was less than that with all three, indicating that any residual single peptidase could inactivate significant amounts of ME. Conclusion The present data, together with those of earlier studies, clearly demonstrate that amastatin-, captopril-, and phosphoramidon-sensitive enzymes play an important role in inactivation of opioid peptides at the spinal level. Keywords Antinociception * Peptidase-inhibitors * Methionine-enkephalin * Tail-flick response, Introduction Endogenous opioid peptide levels depend on the activity of opioid peptide-degrading enzymes, which terminate their action at the synaptic cleft. Enkephalins are hydrolyzed by five types of peptidase [1, [...]

Simple Summary: Parotid, submandibular, and sublingual glands in rat were found to contain high concentrations of d-aspartic acid and low ones of d-serine and d-alanine. In addition to d-amino acid oxidase and d-aspartate oxidase, serine racemase was also detected in all three of these major salivary glands, as were N-methyl-d-aspartic acid receptor subunits NR1 and NR2D, but not NR2A, NR2B, or NR2C. Free d-amino acids, which are enantiomers of l-amino acids, are found in mammals, including humans, and play an important role in a range of physiological functions in the central nervous system and peripheral tissues. Several d-amino acids have been observed in saliva, but their origin and the enzymes involved in their metabolism and catabolism remain to be clarified. In the present study, large amounts of d-aspartic acid and small amounts of d-serine and d-alanine were detected in all three major salivary glands in rat. No other d-enantiomers were detected. Protein expression of d-amino acid oxidase and d-aspartate oxidase, the enzymes responsible for the oxidative deamination of neutral and dicarboxylic d-amino acids, respectively, were detected in all three types of salivary gland. Furthermore, protein expression of the d-serine metabolic enzyme, serine racemase, in parotid glands amounted to approximately 40% of that observed in the cerebral cortex. The N-methyl-d-aspartic acid subunit proteins NR1 and NR2D were detected in all three major salivary glands. The results of the present study suggest that d-amino acids play a physiological role in a range of endocrine and exocrine function in salivary glands. [ABSTRACT FROM AUTHOR]

Objectives: Ease of denture cleaning is of paramount importance in geriatric patients and those with limited dexterity. We have previously investigated methods of coating dentures with titanium dioxide (TiO2 ) and reported the effects (self-cleaning and antibacterial) of such treatments in in vitro studies. This study was to verify the biocompatibility of a TiO2 -coated acrylic resin produced by the new coating method with spray-coating technique.Methods: Specimens were prepared from denture base acrylic resin and polished up to grit #1000. The TiO2 -coating agent was sprayed onto the specimens using an airbrush gun. Specimens were then divided into 'polymethyl methacrylate (PMMA)', 'primer-coated PMMA' and 'TiO2 -coated PMMA' groups to be evaluated for biological safety using a hamster oral mucosa irritation test, a guinea pig skin sensitisation test and a rabbit intracutaneous test. The biological reaction was scored.Results: Reaction scores were considerably <1.0, the acceptable limit set by the ISO, in all three tests. Indeed, in most samples, there was no deleterious effect at all.Conclusion: These results tested on animals demonstrate that denture base resin coated with TiO2 by this method does not cause irritation or sensitisation of the oral mucosa, skin or intracutaneous tissue and is therefore good biocompatibility for use in close proximity to oral mucosa and skin. [ABSTRACT FROM AUTHOR]

1. This study examined the effect of diazepam (DZP) on phosphoinositide turnover, which plays an important role in the regulation of salivary secretion, in rat parotid acinar cells. 2. DZP (10(-9) M to 10(-5) M), a potent agonist of both central- and peripheral-type benzodiazepine receptors, dose-dependently decreased inositol 1,4,5-trisphosphate IP3 production stimulated by carbachol, a muscarinic receptor agonist, in the cells. 3. DZP produced a maximum inhibitory response at a concentration of 10(-5) M, with IP3 production decreased to 63% of maximal levels. The concentration inducing half maximal inhibition of IP3 production was approximately 3.5 x 10 (-8) M. 4. An inhibitory response to DZP was produced by a short-term pretreatment (<3 min) of the cells and prevented by antagonist and competing ligand for the central- and peripheral-type benzodiazepine receptors, flumazenil and PK 11195, respectively. 5. DZP showed a non-competitive inhibition of carbachol-stimulated IP3 production. It did not directly inhibit the activities of GTP-binding regulatory proteins and phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PLC) in the parotid gland membranes, though choline chloride inhibited PLC activity. 6. DZP (10(-5) M) attenuated the increase in the intracellular Ca2+ concentration ([Ca(2+)](i)) in the cells following stimulation of the muscarinic and alpha(1)-adrenoceptors. 7. These results suggest that in the parotid acinar cells, DZP inhibits muscarinic receptor-stimulated IP3 production through benzodiazepine receptors and that PLC activity which produces IP3 is inhibited by chloride. The decreases in IP3 and [Ca(2+)](i) in the cells may be connected with the suppression of salivary secretion induced by DZP. [ABSTRACT FROM AUTHOR]

Simple Summary: Stimulation of the parasympathetic nervous system results in the secretion of saliva. Previous studies have demonstrated acetylcholine content in homogenate obtained from salivary glands. Acetylcholine in homogenate, however, includes that stored in the cells, as well as that released in the interstitial fluid. The activity of the parasympathetic nervous system is mainly determined by the amount of acetylcholine released. We established an in vivo microdialysis method for monitoring the acetylcholine released from nerve endings in the salivary glands in rats. The results of the present study demonstrated that acetylcholine levels in the dialysate reflect acetylcholine levels in the interstitial fluid of the submandibular gland, and that an increase in the acetylcholine level in the dialysate depends predominantly on the release of acetylcholine from the parasympathetic nerve endings. A microdialysis technique was used to monitor acetylcholine levels in the local interstitial fluid in rat submandibular glands, with the aim of determining parasympathetic nerve activity in vivo. The dialysis probe housed a 10 × 0.22 mm semipermeable membrane (molecular weight cutoffs: 50,000 Da). When the probe was perfused at 2 μL/min in vitro, the mean relative recovery of acetylcholine was 41.7% ± 2.5%. The dialysis probes were implanted in the submandibular glands of anesthetized rats and perfusion with Ringer's solution, at 2 μL/min, was performed. Acetylcholine concentrations in the dialysate were measured by high-performance liquid chromatography and electrochemical detection. The results revealed the following: (1) that mixing Eserine with Ringer's solution allowed acetylcholine in the salivary glands to be quantified; (2) that acetylcholine concentrations in the dialysate were highly variable and unstable over the first 120 min after probe implantation, but reached a nearly stable level (4.8 ± 2.7 nM) thereafter in the presence of 100 µM of Eserine; and (3) that electrical stimulation of the chorda tympani nerve, or perfusion with high potassium Ringer's solution, significantly increased acetylcholine concentrations in the dialysate. These results indicate that the present microdialysis technique offers a powerful tool for detecting changes in parasympathetic activity within the salivary glands. [ABSTRACT FROM AUTHOR]

Abstract: We have evaluated the effect of the systemic administration of MK-801 (0.4 mg/kg) on the gene expression of d-amino acid oxidase (DAO) in several brain areas of the rat. The levels of DAO mRNA in all the brain areas significantly increased and peaked at 4 h after the administration. The present results suggest that there is a link between the expression of DAO mRNA and the N-methyl-d-aspartate (NMDA) receptor activity. [Copyright &y& Elsevier]

We have used real-time quantitative PCR methods to evaluate the effect of the systemic administration of (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801; 0.4 mg/kg) on the gene expression of serine racemase in several brain areas of the rat. The levels of serine racemase mRNA in all the brain areas transiently increased after the administration. The present findings suggest that there is a link between the expression of serine racemase mRNA and the activity of the NMDA receptors. [Copyright &y& Elsevier]

Amyotrophic lateral sclerosis (ALS) is a motor neuron disease with adult onset, characterized by progressive loss of motor neurons. Prostaglandin E2 (PGE2), a lipid mediator, exerts its biological functions by binding to four subtypes of E-prostanoid (EP1-4). Among them, EP3 has been shown to have multiple isoforms, EP3α, EP3β, and EP3γ, produced by alternative splicing. Since PGE2 has been shown to have important pathophysiological roles in ALS, experiments were performed to identify EP3 receptor isoform(s) in spinal motor neurons of wild-type (WT) and ALS model (G93A) mice. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of adult mice demonstrated expression of EP3α and EP3γ mRNAs in the lumbar spinal cord, whereas EP3β mRNA was barely detectable. Laser capture microdissection was used to dissect out motor neurons from frozen samples of lumbar spinal cord in these mice for analysis by real-time PCR. We found that expression of EP3γ mRNA was predominant in these neurons, whereas EP3α and EP3β mRNAs were undetectable. At the early symptomatic stage, the mRNA expression profiles of these splice isoforms in G93A motor neurons were comparable to those in neurons from WT mice. These results suggest that the PGE2-to-EP3 signaling pathway is mediated mainly by the EP3γ isoform in the motor neurons of mice, and that modulation of the EP3γ isoform in motor neurons may be a promising new therapeutic approach for ALS.

β-defensins are small, cationic peptides with broad-spectrum antimicrobial activity that are produced by mucosal epithelia. However, little is known about the expression of β-defensins in the major salivary glands. The purpose of this study was to characterize expression of rat β-defensin-1 (RBD-1) and -2 (RBD-2) mRNA within the major salivary glands together with the effect of injection of intraductal lipopolysaccharide (LPS) on that expression. β-defensin mRNA expression was quantitated by RT-PCR in salivary gland tissues and salivary acinar and striated duct cells collected by laser captured microdissection. RBD-1 and -2 were expressed in the parotid gland, the submandibular gland, and the sublingual gland. β-defensins were expressed in both the acinar and striated duct cells of the major salivary glands. Intraductal injection of LPS increased expression of RBD-1 and -2 mRNA, which peaked at 12 hrs. These results suggest that salivary cells (acinar and striated duct cells) have the potential to produce β-defensins.

Objective: Previous in vitro studies have shown that the degradation of [Leu5]enkephalin during incubation with cerebral membrane preparations is almost completely prevented by a mixture of three peptidase inhibitors such as amastatin, captopril, and phosphoramidon. The present in vivo study was performed to examine the antinociceptive effect of [Leu5]enkephalin administered intrathecally under pretreatment with these three peptidase inhibitors., Methods: A tail-flick test was used to determine the nociceptive threshold after administration of [Leu5]enkephalin. The time-course profiles of the latency to flick the tail and the area under the time response curve were evaluated for the antinociceptive action of the drug., Results: The antinociceptive effect of [Leu5]enkephalin administered intrathecally on the tail-flick test was increased more than 100-fold under i.t. pretreatment with three peptidase inhibitors. The antinociceptive effect produced by [Leu5]enkephalin in rats pretreated with any single peptidase inhibitor increased antinociception compared to that with saline. The antinociceptive potency of [Leu5]enkephalin under pretreatment with any combination of two peptidase inhibitors was smaller than that in rats pretreated with three peptidase inhibitors together. These results indicate that any residual single peptidase inactivates significant amounts of [Leu5]enkephalin., Conclusion: The present data, together with those of earlier studies, clearly demonstrate that amastatin-, captopril-, and phosphoramidon-sensitive enzymes play an important role in the inactivation of [Leu5]enkephalin administered intrathecally in rat.

The purpose of this study was to investigate activation of inhibitory regulation pathways by methamphetamine (METH)-withdrawal stress in rat salivary gland. Our previous study showed that METH-withdrawal stress activated steroid biosynthesis and that pregnenolone produced during the early stage of this process inhibited salivary secretion. However, how this type of stress inhibits salivary secretion and the activation pathway of steroid biosynthesis in salivary gland remain to be clarified. In the present study, using an in vivo cannulation method, METH-withdrawal stress decreased salivary secretion and increased expression of diazepam-binding inhibitor (DBI), an endogenous peripheral-type benzodiazepine receptor (PBR) agonist; Western blot and RT-PCR also showed increased expression of DBI mRNA in parotid, submandibular, and sublingual gland. In addition, METH-withdrawal stress also elicited an increase in pituitary adenylate cyclase-activating polypeptide (PACAP) and PBR mRNA, which is associated with DBI activity. These results suggest that METH-withdrawal stress activates a PACAP-DBI pathway in salivary gland, enhancing steroid genesis and inhibiting secretion.

We investigated changes in the protein profile of submandibular gland (SMG) with inflammation induced by exposure to lipopolysaccharide (LPS) with the aim of identifying potential molecular markers of injured gland. Lipopolysaccharide (2.5µg) was directly administered into rat SMG unilaterally by retrograde ductal injection. At 12hr after treatment, the gland was excised and the proteins identified by two-dimensional difference gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Many proteins in the LPS-treated gland showed a marked change compared to those in the contralateral gland. Of particular note were increases in ubiquitin, a highly-conserved small regulatory protein and in calgranulin B, which has an immunological function in inflammation. Proteins related to apoptosis and stress also showed change in the inflamed gland. The results of this study suggest that the ubiquitin system of protein modification is involved in LPS-induced inflammation in salivary gland, and that a number of specific proteins might be applicable as molecular markers in the monitoring of inflamed or injured gland.

We studied the viability of high-performance liquid chromatography and mass spectrometry (LC/MS) as a selective and sensitive analytical method for measuring blood concentrations of the local anesthetic ropivacaine. Ropivacaine was effectively separated using a reverse-phase column and monitored at 275 m/z ion. The LC/MS method allowed measurement of concentrations of ropivacaine of lower than 75 ng/mL. The standard curve was linear and in the range of <1.5 microg/mL. Recovery of ropivacaine in plasma samples was over 90% after precipitation of plasma protein with trichloroacetic acid. The method was tested on the pharmacokinetics of plasma ropivacaine after single intravenous or subcutaneous administration in rabbits. The pharmacokinetic parameters showed a one-compartment model and a mean elimination half-life of 0.54+/-0.05 h and 2.83+/-0.51 h after administration at doses of 0.4 mg/kg, i.v. and 5 mg/kg, s.c., respectively. These values were in approximate agreement with previously obtained results in dogs. The results of the present study demonstrated that the LC/MS method was highly selective and sensitive for the measurement of ropivacaine, indicating that it offers a useful tool for monitoring the therapeutic effects and determining the pharmacokinetic parameters of this drug in blood.

Chronic administration of methamphetamine (MAP) up-regulated the mRNA expression of diazepam binding inhibitor (DBI) in rat brain, possibly leading to anxiety. Acute effects of MAP on anxiety associated with DBI, however, are not clear. In this study, we examined the effects of acute administration of MAP on behavior related to anxiety and the expression level of DBI mRNA and pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA, calibrated with the glyceraldehydes 3-phosphate dehydrogenase mRNA as the internal control in rat brain. The elevated plus-maze test was applied to the analysis of the possible anxiety-related profile of MAP. Acute administration of MAP (5 mg/kg, intraperitoneal administration) significantly increased spent time in the open-space arms at 4 h after the administration compared with a saline-treated group. The expression of DBI mRNA in a large number of regions of rat brain significantly decreased 2, 4, 8 and 16 h after acute administration of MAP. In contrast, the expression of PACAP mRNA in a large number of regions of rat brain significantly increased 4 and 8 h after the administration of MAP. These results suggest that MAP, at this dose, has an anxiolytic effect, based on the reduction of the putative anxiogenic peptides, DBI.

Anxiety is one of the common features of withdrawal syndrome caused by abuse-inducing drugs such as methamphetamine (MAP). The neural pathways associated with anxiety are established within the network sustained by diencephalon, cerebral cortex, cerebellum and hippocampus. Diazepam binding inhibitor (DBI), a peptide consisting of 87 amino acids, serves as an inverse agonist for the type A receptor of the gamma-aminobutyric acid (GABAA receptor) with endogenous anxiogenic potential. We examined the effect of chronic administration of MAP on the mRNA expression of DBI and DBI-related proteins, such as alpha 2 subunit of GABAA receptor (GABA-α2), peripheral-type benzodiazepine receptor (PBR), and pituitary adenylate cyclase-activating polypeptide (PACAP) in seven regions (diencephalon, cerebral cortex, cerebellum, striatum, hippocampus, midbrain, and pons-medulla) of the rat brain. The mRNA expression of DBI increased significantly in all areas of the brain, especially diencephalon, after chronic administration of MAP. The mRNA expression of PBR, GABA-α2 and PACAP increased significantly in all areas of the brain, especially cerebral cortex, after chronic administration of MAP. These results suggest that anxiety is associated with the mRNA expression of DBI as well as DBI-related genes.