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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-Arg 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 μ-, δ- and κ-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. [ABSTRACT FROM AUTHOR]

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]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 dose-response 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. [ABSTRACT FROM AUTHOR]

Transient receptor potential (TRP) cation channels are unique cellular sensors involved in multiple cellular functions. Their role in salivary secretion remains to be elucidated. The expression and localization of temperature-sensitive TRP channels in salivary (submandibular, sublingual and parotid) glands were analyzed by immunohistochemistry and quantitative real-time reverse transcription plus the polymerase chain reaction (RT-PCR). The effects of various TRP channel agonists on carbachol (CCh)-induced salivary secretion in the submandibular gland and on the intracellular Ca concentration ([Ca]) in a submandibular epithelial cell line were also investigated. Immunohistochemistry revealed the expression of TRP-melastatin subfamily member 8 (TRPM8) and TRP-ankyrin subfamily member 1 (TRPA1) in myoepithelial, acinar and ductal cells in the sublingual, submandibular and parotid glands. In addition, TRP-vanilloid subfamily member 1 (TRPV1), TRPV3 and TRPV4 were also expressed in myoepithelial, acinar and ductal cells in all three types of gland. Quantitative real-time RT-PCR results demonstrated the mRNA expression of TRPV1, TRPV3, TRPV4, TRPM8 and TRPA1 in acinar and ductal cells in these salivary glands. Perfusion of the entire submandibular gland with the TRPV1 agonist capsaicin (1 μM) via the submandibular artery significantly increased CCh-induced salivation, whereas perfusion with TRPM8 and TRPA1 agonists (0.5 μM WS12 and 100 μM allyl isothiocyanate) decreased it. Application of agonists for each of the thermosensitive TRP channels increased [Ca] in a submandibular epithelial cell line. These results indicate that temperature-sensitive TRP channels are localized and distributed in acinar, ductal and myoepithelial cells in salivary glands and that they play a functional role in the regulation and/or modulation of salivary secretion. [ABSTRACT FROM AUTHOR]