Chapter 7 Neuropeptide converting and processing enzymes in the spinal cord and cerebrospinal fluid

Extracellular Biotransformation of β‐Endorphin in Rat Striatum and Cerebrospinal Fluid

“…Because immunoblotting experiments have demonstrated that IDE is present in human CSF (30), we explored the possibility that this enzyme is present and functional in rat CSF. Indeed, the observed fragments resulting from b-endorphin-r incubation with rat CSF, namely b-endorphin (1-17), (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18), (18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31) and (19-31) (Fig. 4A), are consistent with the known cleavage sites of b-endorphin-h by purified IDE (25).…”

“…Of the fragments resulting from b-endorphin detected in the present study, (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) and (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) are both predicted to bind to opioid receptors because they contain the initial enkephalinergic fragment YGGFM (21). Indeed, a number of studies have investigated the activity of (1-17) (c-endorphin) and associated fragments, showing this peptide to have opioidergic, as well as non-opioidergic, activity (10,11).…”

“…The protein A ⁄ G sepharose beads used to effect the immunoprecipitation have an apparent affinity for full-length b-endorphin-r, which was not observed in the mass spectra of the immunoprecipitated fraction (this was also the case in mock immunoprecipitated CSF samples, to which no monoclonal antibody was added; data not shown). It is noteworthy that synthetic b-endorphin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18), when added to mock immunoprecipitated CSF samples, did not adhere to the protein A ⁄ G sepharose beads, consistent with the presence of b-endorphin (1-18) and the absence of full-length b-endorphin-r in the immunoprecipitated fraction (bottom spectrum). The immunoprecipitation did not result in complete immunodepletion of the enzymatic activity (middle-spectrum, CSF supernatant).…”

“…The incubation fluid was used, rather than simply adding the peptide in the presence of the slices, to prevent the possibility of the peptide entering the cells of the tissue (whether by passive diffusion or by active transport) and being metabolised by intracellular enzymes, followed by the products in turn exiting the tissue into the sampled media. In the absence of protease inhibitors, 5 lM b-endorphin-r yielded the following fragments: (1-18), (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18), (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31) and (20)(21)…”

Extracellular Biotransformation of β‐Endorphin in Rat Striatum and Cerebrospinal Fluid

“…Because immunoblotting experiments have demonstrated that IDE is present in human CSF (30), we explored the possibility that this enzyme is present and functional in rat CSF. Indeed, the observed fragments resulting from b-endorphin-r incubation with rat CSF, namely b-endorphin (1-17), (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18), (18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31) and (19-31) (Fig. 4A), are consistent with the known cleavage sites of b-endorphin-h by purified IDE (25).…”

“…Of the fragments resulting from b-endorphin detected in the present study, (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) and (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) are both predicted to bind to opioid receptors because they contain the initial enkephalinergic fragment YGGFM (21). Indeed, a number of studies have investigated the activity of (1-17) (c-endorphin) and associated fragments, showing this peptide to have opioidergic, as well as non-opioidergic, activity (10,11).…”

“…The protein A ⁄ G sepharose beads used to effect the immunoprecipitation have an apparent affinity for full-length b-endorphin-r, which was not observed in the mass spectra of the immunoprecipitated fraction (this was also the case in mock immunoprecipitated CSF samples, to which no monoclonal antibody was added; data not shown). It is noteworthy that synthetic b-endorphin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18), when added to mock immunoprecipitated CSF samples, did not adhere to the protein A ⁄ G sepharose beads, consistent with the presence of b-endorphin (1-18) and the absence of full-length b-endorphin-r in the immunoprecipitated fraction (bottom spectrum). The immunoprecipitation did not result in complete immunodepletion of the enzymatic activity (middle-spectrum, CSF supernatant).…”

“…The incubation fluid was used, rather than simply adding the peptide in the presence of the slices, to prevent the possibility of the peptide entering the cells of the tissue (whether by passive diffusion or by active transport) and being metabolised by intracellular enzymes, followed by the products in turn exiting the tissue into the sampled media. In the absence of protease inhibitors, 5 lM b-endorphin-r yielded the following fragments: (1-18), (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18), (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31) and (20)(21)…”

N-terminal truncations of substance P1–7 amide affect its action on spinal cord injury-induced mechanical allodynia in rats

“…As with many other peptides, it thus appears that SP 1-7 may have both peripheral and central effects. In accordance with this, it appears that the SP 1-7 peptide fragment can be produced both centrally and peripherally, since enzymes capable of generating it are available in both the CNS and the periphery (Persson et al, 1995) as well as at the blood-brain barrier (Freed et al, 2002). in vitro studies have shown that SP and its C-and N-metabolites can pass this barrier (Freed et al, 2002).…”

Extracellular Biotransformation of β‐Endorphin in Rat Striatum and Cerebrospinal Fluid

“…Because immunoblotting experiments have demonstrated that IDE is present in human CSF (30), we explored the possibility that this enzyme is present and functional in rat CSF. Indeed, the observed fragments resulting from b-endorphin-r incubation with rat CSF, namely b-endorphin (1-17), (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18), (18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31) and (19-31) (Fig. 4A), are consistent with the known cleavage sites of b-endorphin-h by purified IDE (25).…”

“…Of the fragments resulting from b-endorphin detected in the present study, (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) and (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) are both predicted to bind to opioid receptors because they contain the initial enkephalinergic fragment YGGFM (21). Indeed, a number of studies have investigated the activity of (1-17) (c-endorphin) and associated fragments, showing this peptide to have opioidergic, as well as non-opioidergic, activity (10,11).…”

“…The protein A ⁄ G sepharose beads used to effect the immunoprecipitation have an apparent affinity for full-length b-endorphin-r, which was not observed in the mass spectra of the immunoprecipitated fraction (this was also the case in mock immunoprecipitated CSF samples, to which no monoclonal antibody was added; data not shown). It is noteworthy that synthetic b-endorphin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18), when added to mock immunoprecipitated CSF samples, did not adhere to the protein A ⁄ G sepharose beads, consistent with the presence of b-endorphin (1-18) and the absence of full-length b-endorphin-r in the immunoprecipitated fraction (bottom spectrum). The immunoprecipitation did not result in complete immunodepletion of the enzymatic activity (middle-spectrum, CSF supernatant).…”

“…The incubation fluid was used, rather than simply adding the peptide in the presence of the slices, to prevent the possibility of the peptide entering the cells of the tissue (whether by passive diffusion or by active transport) and being metabolised by intracellular enzymes, followed by the products in turn exiting the tissue into the sampled media. In the absence of protease inhibitors, 5 lM b-endorphin-r yielded the following fragments: (1-18), (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18), (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31) and (20)(21)…”

N-terminal truncations of substance P1–7 amide affect its action on spinal cord injury-induced mechanical allodynia in rats

“…As with many other peptides, it thus appears that SP 1-7 may have both peripheral and central effects. In accordance with this, it appears that the SP 1-7 peptide fragment can be produced both centrally and peripherally, since enzymes capable of generating it are available in both the CNS and the periphery (Persson et al, 1995) as well as at the blood-brain barrier (Freed et al, 2002). in vitro studies have shown that SP and its C-and N-metabolites can pass this barrier (Freed et al, 2002).…”

Extracellular Biotransformation of β‐Endorphin in Rat Striatum and Cerebrospinal Fluid

“…Because immunoblotting experiments have demonstrated that IDE is present in human CSF (30), we explored the possibility that this enzyme is present and functional in rat CSF. Indeed, the observed fragments resulting from b-endorphin-r incubation with rat CSF, namely b-endorphin (1-17), (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18), (18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31) and (19-31) (Fig. 4A), are consistent with the known cleavage sites of b-endorphin-h by purified IDE (25).…”

“…Of the fragments resulting from b-endorphin detected in the present study, (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) and (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) are both predicted to bind to opioid receptors because they contain the initial enkephalinergic fragment YGGFM (21). Indeed, a number of studies have investigated the activity of (1-17) (c-endorphin) and associated fragments, showing this peptide to have opioidergic, as well as non-opioidergic, activity (10,11).…”

“…The protein A ⁄ G sepharose beads used to effect the immunoprecipitation have an apparent affinity for full-length b-endorphin-r, which was not observed in the mass spectra of the immunoprecipitated fraction (this was also the case in mock immunoprecipitated CSF samples, to which no monoclonal antibody was added; data not shown). It is noteworthy that synthetic b-endorphin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18), when added to mock immunoprecipitated CSF samples, did not adhere to the protein A ⁄ G sepharose beads, consistent with the presence of b-endorphin (1-18) and the absence of full-length b-endorphin-r in the immunoprecipitated fraction (bottom spectrum). The immunoprecipitation did not result in complete immunodepletion of the enzymatic activity (middle-spectrum, CSF supernatant).…”

“…The incubation fluid was used, rather than simply adding the peptide in the presence of the slices, to prevent the possibility of the peptide entering the cells of the tissue (whether by passive diffusion or by active transport) and being metabolised by intracellular enzymes, followed by the products in turn exiting the tissue into the sampled media. In the absence of protease inhibitors, 5 lM b-endorphin-r yielded the following fragments: (1-18), (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18), (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31) and (20)(21)…”

N-terminal truncations of substance P1–7 amide affect its action on spinal cord injury-induced mechanical allodynia in rats

“…As with many other peptides, it thus appears that SP 1-7 may have both peripheral and central effects. In accordance with this, it appears that the SP 1-7 peptide fragment can be produced both centrally and peripherally, since enzymes capable of generating it are available in both the CNS and the periphery (Persson et al, 1995) as well as at the blood-brain barrier (Freed et al, 2002). in vitro studies have shown that SP and its C-and N-metabolites can pass this barrier (Freed et al, 2002).…”