Neuroseience Letters. 143 (1992) 247 250 c¢" 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
247
NSL 08901
Decreased cerebrospinal fluid neuropeptide-converting enzyme activity in monoarthritic rats Stefan Persson ~, Claes Post ~'b, J e a n n e W e i l - F u g a z z a L S t e p h e n H. Butler a a n d F r e d N y b e r g ~ "Department g f Pharmaeolo,~y, University q[" Uppsala, Uppsala (Sweden), ~'Department q['Phurmucology, Astru Drueo A B, Ltmd (Sweden). ' l"nit~; de Recherehes de Physiopharmacologie du Svst&ne Nerveux, I N S E R M , U. 161. Paris ( Fram'e ; and 'lDepurtment o/ Anesthesioh~gy aml Pain ('enter. Universi O' qfl Wusho~gton, Seattle, WA 98195 I USA j (Received 12 February 1992; Revised version received 13 May 1992: Accepted 3 June 1992) Key wor~&
Dynorphin-converting enzyme; Dynorphin; Substance P endopeptidase; Substance P: Cerebrospinal tluid: Pain: Arthritis: Rat
The activity in rat cerebrospinal fluid (CSF) of dynorphin-converting enzyme (DCE) and substance P endopeptidase (SPE) was determined in control animals and in rats with monoarthritis. Enzymatic activities were measured with specific radioimmunoassays toward the N-terminal products Leu-enkephalin-Arg~and substance P~ 7, respectively. A monoarthritis stable during weeks 2 6 post-injection was induced by injection (0.05 ml) into one joint with Freund's adjuvant. Both SPE and DCE were significantly decreased 15 days after the intraarticular injection. Despite the degree of arthritis that was sustained equally at four weeks after inoculation, both DCE and SPE ,aere back to control levels at that time. It can therefore be concluded that arthritis from a single joint is sufficient to elicit changes in CSF convertase activities, and that these effects disappear between 2 and 4 weeks after injection, although the arthritis persists.
Peptidergic neurotransmission is not only terminated by peptidases [13], but may also be dynamically altered by conversion to other receptor agonists. This may be done by conversion of the neuropeptides by convertases (see [18] for review). In this way, dynorphin A is converted by dynorphin converting enzyme (DCE) to Leuenkephalin-Arg 6 [12] and substance P (SP) by substance P endopeptidase (SPE) to SP~ 7 [10], The fragments of both peptides have pharmacological profiles very different from those of the parent peptides. Awaiting the necessary isolation, purification and sequencing of the enzymes, both SPE and DCE have to be measured indirectly as enzyme activities and not as actual levels of the enzymes. We have recently demonstrated that rat cerebrospinal fluid (CSF) contains neuropeptide converting enzymes, whereof DCE and SPE have been studied in more detail [14, 15]. The enzyme activities can experimentally be altered by several procedures; subchronic administration of opiates causes an increased activity of both enzymes [14], whereas glucocorticoids decrease DCE and leave SPE unchanged (unpublished). We have also recently found that polyarthritic rats sensitized to Correspondenee: S. Persson, Department of Pharmocology, University of Uppsala, P.O. Box 591, S-751 24 Uppsala, Sweden. Fax: (46) 185599718.
collagen I1 have a markedly decreased activity of both SPE and DCE during the acute phase of the disease [15]. This may have been due to many factors, such as pain, weight loss, etc, that were reported in that study. The present study was undertaken to compare the CSF profile of DCE and SPE in animals with monoarthritis to the previously reported results from animals with polyarthritis. The monoarthritic rats have previously been shown to have a hyperalgesic arthritic joint, but otherwise appear normal and do not develop the systemic disease observed in polyarthritic rats [2]. Sprague-Dawley male rats from the Centre d'Elevage Charles Rivers, France, a weight range of 150 175 g were used. They were housed in groups of 6 per cage on sawdust bedding, given food and water ad libitum and kept in an animal house at a constant temperature of 22°C with a 12 hour alternating light-dark cycle. For the induction of monoarthritis complete adjuvant was prepared as follows: 60 mg of killed Mvcohacterium buo'ricum(Difco laboratories) was added to a mixture of parraffin oil (6 ml), NaC1 0.9% (4 ml), Tween 80 (1 ml), mixed thoroughly and then autoclaved for twenty minutes at 120°C to rupture the cell walls of the mycobacteria. Four groups o f t e n rats were used: groups one and two were injected with vehicle alone in the left tibio-tarsal
248 joint; groups three and four were injected with 50/al complete adjuvant prepared from killed mycobacterium butyricum. Injection of the left ankle joint was performed under brief halothane/N20/O 2 anesthesia as follows: the tarsial area of the hind paw was grasped and the fossa distal medial to the lateral malleolus of the fibula was palpated. A 26 gauge needle was introduced into the capsule of the tibio-tarsal joint percutaneously by directing it cephalad, mesiad and superiorly from the midpoint of the inframalleolar fossa, until a distinct loss of resistance was felt - approximately 4 ram. The complete adjuvant or vehicle was then injected. Groups one and three (day 15 post-inoculation (PI)) and groups two and four (day 30 PI) were anesthetized with pentobarbital (3.5 mg/kg body weight, i.p.). The atlanto-occipital membrane was exposed and a 30 gauge needle was inserted through the midline of the membrane. CSF was collected into a polyethylene tubing by gently aspirating with a syringe connected to the tubing. The fluid (around 100/11) was transferred to an Eppendorf tube and immediately frozen on dry ice and stored at -80°C until analyzed. In a previous investigation we found a correlation between extremely high enzymatic activities, e.g. DC and SPE, and hemoglobin concentration in CSF [15]. An unsuccessful penetration through the atlanto-occipital membrane or possibly small capillary bleeding into the subarachnoid space undoubtedly leads to leakage of blood constituents into the CSF. Therefore, in order to minimize contributions from plasma proteases, all CSF samples were examined for their hemoglobin content. The blood protein was analyzed by colourimetric procedure described elsewhere [7, 17]. Briefly, 10 ¢tl CSF was incubated in glass tubes at room temperature together with 2 ml 3,3',5,5'-tetramethylbenzidine (5 mg/ml) in 90% (v/v) glacial acetic acid and 2 ml 0.3% (v/v) hydrogen peroxide. Exactly l0 minutes after the addition of the hydrogen peroxide solution the absorbance was measured in a spectrophotometer at 600 nm. Rat hemoglobin dissolved and diluted in artificial CSF (Na" 149.9 mM; K + 2.6 mM; Ca 2+ 1.3 mM; Mg 2+ 0.9 mM; C1 134.6 mM; HCO3- 20.2 mM; HPO42 1.0 mM, 0.5% bovine serum albumin, adjusted to pH 7.4) was used as standard. The detection limit of the hemoglobin assay was estimated to 90 ltg/ml. All CSF samples which contained measurable amounts of hemoglobin were discarded from the study so that converting enzyme activities were evaluated in 5 respectively 7 control rats 15 days, 4 control rats 30 days, 8 monoarthritic rats 15 and 30 days PI, respectively. The DCE activity was determined by measuring the release of Leu-enkephalin-Arg~' from dynorphin A using a specific radioimmunoassay for the hexapeptide product. Incubations were performed in duplicates in Eppen-
dorf tubes at 37°C in a final volume oi" 40 ~tl buffered at pH 7.4 with 50 mM Tris-HC1. The reaction mixture contained 0.1/~g (=36 pmol) of dynorphin A, undiluted CSF (20 #1) and a cocktail of various protease inhibitors (amastatin, captopril and GEMSA). The inhibitors were included in order to prevent further degradation of the generated product and were preincubated with the CSF sample for 15 min at 37°C betbre the addition of substrate. Amastatin (20/~M) was added to inhibit aminopeptidases, whereas captopril (20/aMt and GEMSA (1 mM) were added in order to inhibit angiotensin converting enzyme (ACE or EC 3.4.15.1) or enkephalin convertase (carboxypeptidase H or EC 3.4.17.10), respectively. After completed incubation (2 hours) the reaction was terminated by the addition of 500/~1 ice-cold methanol, followed by centrifugation for 1 min in a Beckman Microfuge u. The supernantant was evaporated in a Savant Vac centrifuge (Hicksville. N.Y., U.S.A.) prior to RIA analysis. SPE activity was determined by a similar procedure. Thus, the formation of substance Pl 7 from the synthetic undecapeptide was recorded by a RIA specific for this fragment. The only inhibitor used in this case was amastatin, which was added to yield a final concentration of 20/~M. A volume of 10/,tl CSF was preincubated for 15 rain at 37°C together with the inhibitor. Following the addition of 0.1/,tg (74 pmol) of substance P the incubation was performed in duplicate in Eppendorf tubes at 37°C for 1 hour in a final volume of 40/al buffered at pH 7.4 with 50 mM Tris-HCl. After completed incubation, samples were treated in accordance with those in the D e E assay described above. The release of the reaction products Leu-enkepalin-Arg6 and substance P~ 7 have previously been verified by HPLC (unpublished). The radioimmunoassays for Leu-enkephalin-Arg6 and substance P~ 7 were based on the charcoal adsorption technique and were conducted as described previously [8,
ll]. Statistical analyses of the recorded enzymatic activities were carried out by means of duplicate determinations using two factor analysis of variance (ANOVA) with factor interaction. Data were tested for normality and were found to be approximately log-normally distributed, which is a common feature of enzymes and hormones [5]. Hence, the ANOVA were calculated on log-transformed enzymatic activities. The neuropeptide-converting enzyme activities were measured 15 and 30 days alter inoculation. Both DCE and SPE activities were observed to decrease at day 15, whereas the activity for these two enzymes returned to control level at day 30. In Fig. I DCE activity at day 15 and 30 in control and monoarthritic rats is depicted. Two factor ANOVA, using log-transformed data, revealed a
249 100
day 15 p.i.
90
~
~
day 30 p.i.
.~ 70
~ 6o ~ ©
i
50
~ 4o ~ 2o
10 Control n=5
Monoanhritis n=8
Control n=4
Monoarthritis n=8
Fig. 1. Dynorphin-converting enzyme (DCE) activity in CSF from rats injected with incomplete adjuvant into the tibio-tarsal joint (control; open bar) and rats injected intraarticular with complete adjuvant (monoarthritis: shaded bar). CSF was sampled day 15 and day 30 postinoculation (p.i.). Enzymatic activity was measured as the rate of formarion of Leu-enk-Arg ~ from dynorphin A. Data presented as mean + S.E.M. (2-factor ANOVA; FL>-7.836, P
247
NSL 08901
Decreased cerebrospinal fluid neuropeptide-converting enzyme activity in monoarthritic rats Stefan Persson ~, Claes Post ~'b, J e a n n e W e i l - F u g a z z a L S t e p h e n H. Butler a a n d F r e d N y b e r g ~ "Department g f Pharmaeolo,~y, University q[" Uppsala, Uppsala (Sweden), ~'Department q['Phurmucology, Astru Drueo A B, Ltmd (Sweden). ' l"nit~; de Recherehes de Physiopharmacologie du Svst&ne Nerveux, I N S E R M , U. 161. Paris ( Fram'e ; and 'lDepurtment o/ Anesthesioh~gy aml Pain ('enter. Universi O' qfl Wusho~gton, Seattle, WA 98195 I USA j (Received 12 February 1992; Revised version received 13 May 1992: Accepted 3 June 1992) Key wor~&
Dynorphin-converting enzyme; Dynorphin; Substance P endopeptidase; Substance P: Cerebrospinal tluid: Pain: Arthritis: Rat
The activity in rat cerebrospinal fluid (CSF) of dynorphin-converting enzyme (DCE) and substance P endopeptidase (SPE) was determined in control animals and in rats with monoarthritis. Enzymatic activities were measured with specific radioimmunoassays toward the N-terminal products Leu-enkephalin-Arg~and substance P~ 7, respectively. A monoarthritis stable during weeks 2 6 post-injection was induced by injection (0.05 ml) into one joint with Freund's adjuvant. Both SPE and DCE were significantly decreased 15 days after the intraarticular injection. Despite the degree of arthritis that was sustained equally at four weeks after inoculation, both DCE and SPE ,aere back to control levels at that time. It can therefore be concluded that arthritis from a single joint is sufficient to elicit changes in CSF convertase activities, and that these effects disappear between 2 and 4 weeks after injection, although the arthritis persists.
Peptidergic neurotransmission is not only terminated by peptidases [13], but may also be dynamically altered by conversion to other receptor agonists. This may be done by conversion of the neuropeptides by convertases (see [18] for review). In this way, dynorphin A is converted by dynorphin converting enzyme (DCE) to Leuenkephalin-Arg 6 [12] and substance P (SP) by substance P endopeptidase (SPE) to SP~ 7 [10], The fragments of both peptides have pharmacological profiles very different from those of the parent peptides. Awaiting the necessary isolation, purification and sequencing of the enzymes, both SPE and DCE have to be measured indirectly as enzyme activities and not as actual levels of the enzymes. We have recently demonstrated that rat cerebrospinal fluid (CSF) contains neuropeptide converting enzymes, whereof DCE and SPE have been studied in more detail [14, 15]. The enzyme activities can experimentally be altered by several procedures; subchronic administration of opiates causes an increased activity of both enzymes [14], whereas glucocorticoids decrease DCE and leave SPE unchanged (unpublished). We have also recently found that polyarthritic rats sensitized to Correspondenee: S. Persson, Department of Pharmocology, University of Uppsala, P.O. Box 591, S-751 24 Uppsala, Sweden. Fax: (46) 185599718.
collagen I1 have a markedly decreased activity of both SPE and DCE during the acute phase of the disease [15]. This may have been due to many factors, such as pain, weight loss, etc, that were reported in that study. The present study was undertaken to compare the CSF profile of DCE and SPE in animals with monoarthritis to the previously reported results from animals with polyarthritis. The monoarthritic rats have previously been shown to have a hyperalgesic arthritic joint, but otherwise appear normal and do not develop the systemic disease observed in polyarthritic rats [2]. Sprague-Dawley male rats from the Centre d'Elevage Charles Rivers, France, a weight range of 150 175 g were used. They were housed in groups of 6 per cage on sawdust bedding, given food and water ad libitum and kept in an animal house at a constant temperature of 22°C with a 12 hour alternating light-dark cycle. For the induction of monoarthritis complete adjuvant was prepared as follows: 60 mg of killed Mvcohacterium buo'ricum(Difco laboratories) was added to a mixture of parraffin oil (6 ml), NaC1 0.9% (4 ml), Tween 80 (1 ml), mixed thoroughly and then autoclaved for twenty minutes at 120°C to rupture the cell walls of the mycobacteria. Four groups o f t e n rats were used: groups one and two were injected with vehicle alone in the left tibio-tarsal
248 joint; groups three and four were injected with 50/al complete adjuvant prepared from killed mycobacterium butyricum. Injection of the left ankle joint was performed under brief halothane/N20/O 2 anesthesia as follows: the tarsial area of the hind paw was grasped and the fossa distal medial to the lateral malleolus of the fibula was palpated. A 26 gauge needle was introduced into the capsule of the tibio-tarsal joint percutaneously by directing it cephalad, mesiad and superiorly from the midpoint of the inframalleolar fossa, until a distinct loss of resistance was felt - approximately 4 ram. The complete adjuvant or vehicle was then injected. Groups one and three (day 15 post-inoculation (PI)) and groups two and four (day 30 PI) were anesthetized with pentobarbital (3.5 mg/kg body weight, i.p.). The atlanto-occipital membrane was exposed and a 30 gauge needle was inserted through the midline of the membrane. CSF was collected into a polyethylene tubing by gently aspirating with a syringe connected to the tubing. The fluid (around 100/11) was transferred to an Eppendorf tube and immediately frozen on dry ice and stored at -80°C until analyzed. In a previous investigation we found a correlation between extremely high enzymatic activities, e.g. DC and SPE, and hemoglobin concentration in CSF [15]. An unsuccessful penetration through the atlanto-occipital membrane or possibly small capillary bleeding into the subarachnoid space undoubtedly leads to leakage of blood constituents into the CSF. Therefore, in order to minimize contributions from plasma proteases, all CSF samples were examined for their hemoglobin content. The blood protein was analyzed by colourimetric procedure described elsewhere [7, 17]. Briefly, 10 ¢tl CSF was incubated in glass tubes at room temperature together with 2 ml 3,3',5,5'-tetramethylbenzidine (5 mg/ml) in 90% (v/v) glacial acetic acid and 2 ml 0.3% (v/v) hydrogen peroxide. Exactly l0 minutes after the addition of the hydrogen peroxide solution the absorbance was measured in a spectrophotometer at 600 nm. Rat hemoglobin dissolved and diluted in artificial CSF (Na" 149.9 mM; K + 2.6 mM; Ca 2+ 1.3 mM; Mg 2+ 0.9 mM; C1 134.6 mM; HCO3- 20.2 mM; HPO42 1.0 mM, 0.5% bovine serum albumin, adjusted to pH 7.4) was used as standard. The detection limit of the hemoglobin assay was estimated to 90 ltg/ml. All CSF samples which contained measurable amounts of hemoglobin were discarded from the study so that converting enzyme activities were evaluated in 5 respectively 7 control rats 15 days, 4 control rats 30 days, 8 monoarthritic rats 15 and 30 days PI, respectively. The DCE activity was determined by measuring the release of Leu-enkephalin-Arg~' from dynorphin A using a specific radioimmunoassay for the hexapeptide product. Incubations were performed in duplicates in Eppen-
dorf tubes at 37°C in a final volume oi" 40 ~tl buffered at pH 7.4 with 50 mM Tris-HC1. The reaction mixture contained 0.1/~g (=36 pmol) of dynorphin A, undiluted CSF (20 #1) and a cocktail of various protease inhibitors (amastatin, captopril and GEMSA). The inhibitors were included in order to prevent further degradation of the generated product and were preincubated with the CSF sample for 15 min at 37°C betbre the addition of substrate. Amastatin (20/~M) was added to inhibit aminopeptidases, whereas captopril (20/aMt and GEMSA (1 mM) were added in order to inhibit angiotensin converting enzyme (ACE or EC 3.4.15.1) or enkephalin convertase (carboxypeptidase H or EC 3.4.17.10), respectively. After completed incubation (2 hours) the reaction was terminated by the addition of 500/~1 ice-cold methanol, followed by centrifugation for 1 min in a Beckman Microfuge u. The supernantant was evaporated in a Savant Vac centrifuge (Hicksville. N.Y., U.S.A.) prior to RIA analysis. SPE activity was determined by a similar procedure. Thus, the formation of substance Pl 7 from the synthetic undecapeptide was recorded by a RIA specific for this fragment. The only inhibitor used in this case was amastatin, which was added to yield a final concentration of 20/~M. A volume of 10/,tl CSF was preincubated for 15 rain at 37°C together with the inhibitor. Following the addition of 0.1/,tg (74 pmol) of substance P the incubation was performed in duplicate in Eppendorf tubes at 37°C for 1 hour in a final volume of 40/al buffered at pH 7.4 with 50 mM Tris-HCl. After completed incubation, samples were treated in accordance with those in the D e E assay described above. The release of the reaction products Leu-enkepalin-Arg6 and substance P~ 7 have previously been verified by HPLC (unpublished). The radioimmunoassays for Leu-enkephalin-Arg6 and substance P~ 7 were based on the charcoal adsorption technique and were conducted as described previously [8,
ll]. Statistical analyses of the recorded enzymatic activities were carried out by means of duplicate determinations using two factor analysis of variance (ANOVA) with factor interaction. Data were tested for normality and were found to be approximately log-normally distributed, which is a common feature of enzymes and hormones [5]. Hence, the ANOVA were calculated on log-transformed enzymatic activities. The neuropeptide-converting enzyme activities were measured 15 and 30 days alter inoculation. Both DCE and SPE activities were observed to decrease at day 15, whereas the activity for these two enzymes returned to control level at day 30. In Fig. I DCE activity at day 15 and 30 in control and monoarthritic rats is depicted. Two factor ANOVA, using log-transformed data, revealed a
249 100
day 15 p.i.
90
~
~
day 30 p.i.
.~ 70
~ 6o ~ ©
i
50
~ 4o ~ 2o
10 Control n=5
Monoanhritis n=8
Control n=4
Monoarthritis n=8
Fig. 1. Dynorphin-converting enzyme (DCE) activity in CSF from rats injected with incomplete adjuvant into the tibio-tarsal joint (control; open bar) and rats injected intraarticular with complete adjuvant (monoarthritis: shaded bar). CSF was sampled day 15 and day 30 postinoculation (p.i.). Enzymatic activity was measured as the rate of formarion of Leu-enk-Arg ~ from dynorphin A. Data presented as mean + S.E.M. (2-factor ANOVA; FL>-7.836, P
