The effect of thiorphan on release of sensory neuropeptides from guinea-pig cerebral venous sinuses

Pharmacological Research~ Vol. 23, No. 3, 1991 285 THE EFFECT OF THIORPHAN ON RELEASE OF SENSORY NEUROPEPTIDES FROM GUINEA-PIG CEREBRAL VENOUS SINUSES MANUELA TRAMONTANA*, ELENA DEL BIANCO*, MARINA ZICHEt, PAOLO SANTICIOLI~, CARLO A. MAGGPf and PIERANGELO GEPPETTI* *Institute of Internal Medicine IV, t Department of Preclinical and Clinical Pharmacology, University of Florence and $Department of Pharmacology, Menarini Pharmaceuticals, Florence, Italy Received in final form 22 June 1990 SUMMARY The effect of peptidase inhibitors on neuropeptide release from peripheral endings of capsaicin-sensitive sensory neurons was studied in cerebral superior sagittal and transverse sinuses of guinea-pig. Capsaicin (1 pM)-evoked release of substance Plike immunoreactivity (SP-H) was increased in a concentration-dependent manner by thiorphan (0.1-10pM). Captopril (10 ~M) or a mixture of bestatin (10 #M), leupeptin (10 btM) and bacitracin (i0 pM) did not affect the capsaiein-evoked SP-LI release. Thiorphan (10#M) increased also the capsaicin-evoked release of neurokinin A-like immunoreactivity (TK-LI) and calcitoniri gene-related peptidelike immunoreactivity (CGRP-LI) by 228% and 172%, respectively, while captopril (10 ~tM)was without effect. Thiorphan (10 pM), but not captopril (10 pM), enhanced by 239% CGRP-LI release induced by bradykinin (10 pM). In the cerebral venous vessels neutral endopeptidase (EC 3.4.24.11, NEP)-like activity was 58.8 + 6.1 pmol/mg protein/min, while angiotensin converting enzyme-like activity was below the detection limit of the assay. A thiorphan-sensitive mechanism, putatively attributable to NEP, plays a major role in the inactivation of peptides released from or acting on eapsaicin-sensitive sensory fibres of cerebral venous sinuses of guinea-pig. KEY WORDS:tachykinins, calcitonin gene-related peptide, thiorphan, neutral endopeptidase, cerebral venous sinuses. INTRODUCTION Stimulation of superior sagitta! sinus was found to be painful in humans [1, 2] and application of the algesic and proinflammatory peptide, bradykinin, to this vessel in Correspondence to: Dr P. Geppetti, Institute of Internal Medicine IV, V. le Pieraccini 18, 50139 Florence, Italy. 1043-6618/91/030285-10/S03.00/0 © 1991 The Italian Pharmacological Society 286 Pharmacological Research, Vol. 23, No. 3, 1991 the cat was shown to activate sensory pathways to the brain [3]. Vasodilatation and plasma extravasation produced by neuropeptides released from trigeminal perivascular fibres have been considered to play a role in the pathophysiology of migraine headache [4, 5]. Capsaicin, the pungent principle of hot red peppers, and bradykinin released substance P- and calcitonin gene-related peptide-like immunoreactivity (SP-LI and CGRP-LI, respectively) from superior sagittal and transverse sinuses (SSTS) of guinea-pig [6]. Neuropeptide release by both capsaicin and bradykinin was abolished by previous exposure of the tissue to capsaicin. Since capsaicin at ,UM concentrations acts selectively on a subpopulation of primary afferents producing in a time-related fashion, excitation followed by prolonged desensitization towards different stimuli (see for review [7, 8]), it was concluded that in the SSTS both SP-LI and CGRP-LI originated from terminals of capsaicinsensitive primary afferents [6]. Neutral endopeptidase (EC 3.4.24.11, NEP, also called enkephalinase) has shown to cleave efficiently various peptides, among which were tachykinins and bradykinin [9, 10]. Recently, the potentiating action by selective NEP inhibitors of tachykinin induced effects has been demonstrated in a variety of peripheral tissues [11-13I. Likewise, increase by thiorphan of SP-LI release from both central and peripheral endings of capsaicin-sensitive sensory neurons was demonstrated in guinea-pig dorsal spinal cord [14], gallbladder [12] and bronchi [11]. In the search for factors that could modulate release of sensory neuropeptides from SSTS, we have therefore addressed the question whether NEP might play a role in this respect. Here we show that a thiorphan-sensitive mechanism, putatively attributable to NEP, is present in cerebral venous sinuses and potently regulates neuropeptide release from capsaicin-sensitive sensory fibres. Since angiotensinconverting enzyme (ACE) inhibitors have been reported to potentiate certain tachykinin-induced actions [15], the effect of captopril on sensory neuropeptide release from SSTS was also studied. MATERIALS AND METHODS Release experiments Albino guinea-pigs of both sexes (Dunkin-Hartley strain, Morini) weighing 250-350 g, were killed by decapitation and the internal surface of the cranium was exposed. Then the transverse and superior sagittal sinuses along with the surrounding dura mater were removed. Thick slices (0.3-0.5 ram) were prepared at 4°C using a tissue slicer (Stoelting, IL) and tissues (100-150 mg) were transferred to 1 ml per~sion chambers. The slices were superfused with an oxygenated (96% 02 and 4% CO2) Krebs solution containing 0.1% bovine serum albumin at 37°C. A 30 rain equilibration period was allowed to elapse during which slices were supeffused with either Krebs solution or Krebs solution containing the appropriate concentration of the peptidase inhibitor. Four ml fractions were collected every 7 min in tubes containing acetic acid (2 N final concentration) and freeze-dried. At the end of the experiments the tissues were blotted 2-3 times and weighed. Pharmacological Research, Vol. 23, No. 3, 1991 287 Radioimmunoassays and enzyme immunoassay SP-LI radioimmunoassay was performed as published previously [16], by using SP as standard, [1251]-Bolton and Hunter conjugated SP and 144 anti-SP antiserum which cross-reacted to 1% with neurokinin A (NKA), 0.5% with neurokinin B and less than 0.1% with physalaemin and eledoisin. CGRP-LI was measured as already reported [16], by using rat a-CGRP as standard, [J25I]CGRP and RAS-6012 antihuman CGRP-II, which cross-reacted to 100% with rat fl-CGRP and human CGRP-I. Bradykinin up to 0.1 mM did not cross-react with the antiserum. Immunoreactivity for neurokinin A (NKA) was measured with a newly developed [11] enzyme immunoassay that uses acetylcholinesterase as label of the antigen. This solid phase competitive assay performed in microtitre plates coated with specific second-antibody uses NKA as standard and the K-12 antiserum, which cross-reacted to 80% with kassinin, 60% with neuropeptide K, 33% with NKB, 30% with eledoisin and less than 0.1% with SP [17]. Enzyme activity assay For enzyme activity measurements tissues were homogenized in 50 mM pH 7.4 HEPES/NaOH buffer and assayed in triplicate. NEP-like activity was evaluated by the fluorimetric method described previously [18] that utilizes succinyl-alanylalanyl-phenylalanyl (7-amido-4-methyl) coumarin as substrate. ACE-like activity was measured as already reported [19] following the method of Yang and Neff [20], employing hippuryl-histidyl-leucine as a substrate. Protein concentration was estimated with the Pierce protein assay reagent (Pierce, IL) with bovine serum albuinin as standard. Statistical analysis All data in the text and figures are mean _+s.z of the mean. Statistical analysis was performed by using the analysis of variance and Dunnett's test when applicable. Total peptide-like immunoreactivity released during exposure to capsaicin or bradykinin was calculated by the sum of the values observed in presence of the stimulus, subtracted of the mean basal value. Drugs and reagents All peptides and RAS-6012 (Peninsula); capsaicin, thiorphan, bacitracin, leupeptin, bestatin and hippuryl-histidyl-leucine (Sigma); captopril (Squibb); succinyl-alanyl-alanyl-phenylalanyl (7-amido-4-methyl) coumarin (Bacheln); [~esI]Bolton and Hunter SP, [125IJCGRP(Amersham); 144 antiserum was a gift of Dr P. Pradelles, SPI-LERI, CEN-Saclay, Gyf-sur-Yvette; K-12 antiserum was a gift of Dr E. Theodorsson, Department of Clinical Chemistry, Karolinska Hospital, Stockholm. RESULTS Capsaicin-evoked neuropeptide release Basal SP-LI outflow from guinea-pig SSTS was below the detection limit of our assay in absence or in presence of peptidase inhibitors. Exposure to capsaicin Pharmacological Research, Vol. 23, No. 3, 1991 288 produced a rapid and sharp increase of the SP-LI outflow, the total evoked release being 2 20 + 66 f m o l / g / 3 5 min (n = 8). In the presence of thiorphan (0.1-10 ~M) the capsaicin-induced SP-LI outflow in the superfusate was enhanced in a Concentration-dependent manner (Table I and Fig. 1). In contrast, addition of 10 ktM captopril (Fig. 1) or a mixture of other peptidase inhibitors such as leupeptin (10/~M), bacitracin (10/2M) and bestatin (10/*M) did not affect the capsaicin-evoked SP-LI release (Table II). Table I Effect of thiorphan on SP-LI released by capsaicin (1/~M) from superior sagittal and transverse sinuses ¢ of guinea-pig Thiorphan (#M) N SP-LI (frno//g/35 rain) % increase 0 0.01 0.1 1 10 5 5 5 4 4 196+_73 208 +_61 446 +_73* 807 +_97** 993 +_135"* -106% 227% 411% 506% *P< 0.05; ** P< 0.01; Dunnett's test. 1.5 -g_= E -6 ,z o_ q~ 02 I I Fig. 1. Effect of thiorphan (10~M) (dashed bar) or captopril (10#v) (dotted bar) on capsaicin (1 ktM)-induced release of substance P-like immunoreactivity (SP-LI) from slices of guinea-pig superior sagittal and transverse sinuses. Open bar indicates release in absence of peptidase inhibitors. Values are mean +-s~ of at least 5 experiments. *P< 0.01, Dunnett's test. Pharmacological Research, Vol. 23, No. 3, 1991 289 Table lI Effect of a mixture of various peptidase inhibitors on the capsaicin (1 #M)evoked SP-LI release from superior sagittal and transverse sinuses of guinea-pig Controls Bacitracin (10 ktM)+ bestatin (10 ktM)+ leupeptin (10 ktM) N SP-LI (froo//g/35 rain) 5 5 251 + 38 293 + 61 The mean basal outflow of TK-LI was 55 _+ 11 fmol/g/fraction, 62 + 9 fmol/g/ fraction or 64 + 8 fmol/g/fraction in control samples or in presence of thiorphan or captopril, respectively. The total evoked release of TK-LI induced by exposure to capsaicin was 476 _+93 fmol/g/35 min (n= 8). Capsaicin-induced increase of TKLI outflow was significantly enhanced by 228% in presence of thiorphan (P< 0.05, n=6), while captopril produced a slight insignificant increase (139%, n= 5)(Fig. 2). 1.5~ t* E to to -6 ilil Fig. 2. Effect of thiorphan (10 #M) or captopril (10 #M) on capsaicin (1 ktM)-evokedrelease of neurokinin A-like immunoreactivity (TK-LI) from slices of guinea-pig superior sagittal and transverse sinuses. For symbols see Fig. 1. Values are mean_+s~ of at least 5 experiments. *P< 0.01, Dunnett's test. The mean basal outflow of CGRP-LI averaged 102+4fmol/g/fraction, 110 + 9.2 fmol/g/fraction and 98 + 3 fmol/g/fraction when peptidase inhibitor present in the bath was none, thiorphan or captopril, respectively. Capsaicin (1 ktM) elicited a rapid and sustained increase of the CGRP-LI outflow (total evoked release reached 3 4 0 6 + 7 2 1 f m o l / g / 3 5 rain, n=6). The increase of CGRP-LI outflow produced by capsaicin in presence of thiorphan (10 ktM) was significantly Pharmacological Research, Vol. 23, No. 3, 1991 290 (P<0.05) enhanced by 172% (Fig. 3). Captopril (10/zM) did not affect the capsaicin-induced CGRP-LI release (Fig. 3). 7.5 "~ tO 5 -6 & z.s n-(D o Fig. 3. Effect of thiorphan ( ] 0 # M ) and captopril ( 1 0 # M ) o n capsaicin (1 #M)-evoked release of calcitonin gene-related peptide-immunoreactivity (CGRP-LI) from slices of guineapig superior sagittal and transverse sinuses. For symbols see Fig. 1. Values are mean + SEof at least 5 experiments. *P< 0.05, Dunnett's test. Bradykinin-evoked CGRP-LI release The mean basal outflow of CGRP-LI before administration of bradykinin was 96 + 3 fmol/g/fraction (n= 6). The presence of 10 #M thiorphan (n= 6) or 10 #M captopril (n = 6) did not affect the basal CGRP outflow, that was 103 +_5 fmol/g/ fraction and 93 +_4 fmol/g/fraction for the two compounds, respectively. Exposure to 10 #M bradykinin produced a delayed and sustained increase of the CGRP-LI outflow. The total evoked release in the absence of peptidase inhibitors was 675 +71 fmol/g/35 rain. Captopril (10 #M) did not significantly affect CGRP-LI release induced bradykinin (Fig. 4). On the contrary bradykinin-evoked CGRP-LI outflow was enhanced by 239% (P<0.01) when 10ktM thiorphan (10#M) was added in the perfusion chamber (Fig. 4). Enzyme activities NEP-like activity in the SSTS (n = 6) averaged 58.8 + 6.7 pmol/mg protein/rain. ACE-like activity in four out of five samples was under the sensitivity of our assay. In the remaining sample converting enzyme-like activity was 0.5 pmol/mg protein/ rain. DISCUSSION There is increasing evidence that tachykinins and CGRP released from peripheral terminals of capsaicin-sensitive primary afferents produce a large array of effects in Pharmacological Research, Vol. 23, No. 3, 1991 "E E 291 1.5 & or, t.D 0.5 Fig. 4. Effect of thiorphan (10/AM) o r captopril (10/~M) on bradykinm (10/~M)-induced release of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) from slices of guinea-pig superior sagittal and transverse sinus. For symbols see Fig. 1. Values are mean _+sE of at least 5 experiments. *P< 0.01, Dunnett's test. various tissues including the airways, genitourinary and gastrointestinal tract, eye and others [7, 8]. SP is considered the mediator of neurogenic plasma extravasation [21], while CGRP exerts potent Vasodilatatory effects [22]. The vascular actions produced by sensory neuropeptides have been also observed in the intracranial circulation, where they mediate neurogenic plasma extravasation in the dura mater [191, and modulate the tone of arteries and veins [23, 24]. The discovery that NEP inhibitors potentiated the actions produced by tachykinins in various tissue preparations [12, 13] led to the hypothesis that this enzyme might play a pivotal role in the termination of the action of endogenous tachykinins. Therefore investigation has been directed to the question whether peptidase inhibitors might affect the recovery of tachykinins released from neuronal sources. Indeed, SP-LI release from capsaicin-sensitive fibres of guineapig spinal cord [14], gallbladder [12] or bronchus [11] was increased by selective NEP inhibitor. We have previously demonstrated that SSTS contain neuropeptides, specifically SP and CGRP, that are released from terminals of capsaicin-sensitive sensory neurons [6]. The present results show that thiorphan produced a great enhancement in the recovery of SP-LI released by capsa!cin from the SSTS. Although the present findings do not allow.firm conclusions to be drawn on the identity of the thiorphan-sensitive SP-degrading activity found in SSTS, various considerations suggest that it might be attributable to NEE First, high amount of NEP-like activity was found in the SSTS, while ACE-like activity was practically undetectable. Second, potentiation of the capsaicin-evoked SP-LI release by thiorphml was dependent on the inhibitor concentration and was evident in presence of a concentration as low as 0.1 ¢tM. Third, other peptidase inhibitors, including captopril, up to 10 pM, failed to increase the recovery of SP-LI released by capsaicin. 292 Pharmacological Research, Vol. 23, No. 3, 1991 NKA coexists with SP in capsaicin-sensitive sensory neurons [251, and is efficiently cleaved in vitro by purified NEP preparation [26]; it is reasonable to hypothesize that thiorphan-sensitive metabolic activity might be involved in the inactivation of NKA released from perivascular sensory fibres. The lower increase of capsaicin-evoked TK-LI (228%) release as compared to SP-LI release (497%) observed in the presence of thiorphan could be due to the relative non-specificity of the K-12 antiserum, which recognizes peptides other than NKA, such as neuropeptide K and eledoisin-like material that could be less sensitive than NKA to the degrading actiyity inhibited by thiorphan. Addition of thiorphan to the medium enhanced the capsaicin-evoked release of CGRP-LI from SSTS. Little indication [27] has been reported so far on the possible existence of enzyme(s) able to cleave CGRP, which itself appears remarkably stable in blood [22]. To our knowledge, these results provide the first evidence that, at least in certain tissues, a thiorphan-sensitive degrading activity might be involved in the cleavage of endogenous CGRP. Thiorphan did not significantly increase the capsaicin-evoked release of CGRP from guinea-pig dorsal spinal cord [14]. The apparent discrepancy might be explained by the higher NEP-like activity found in the SSTS than in the dorsal spinal cord [14]. Clearly, further studies are needed to assess which mechanism(s) terminates the action of CGRP and whether the process(es) might occur in the organ- or tissue-specific manner for a given species. NEP have been shown to cleave bradykinin [10], and selective NEP inhibitors potentiated the actions of bradykinin in the ferret trachea [28] and in the guinea-pig urinary bladder [29]. The present observation of the potentiation by thiorphan of the bradykinin-evoked CGRP-LI release might therefore depend on the protection afforded toward the degradation of the applied stimulus, thereby allowing a higher bradykinin concentration to act on sensory fibres. However, since thiorphan also significantly enhanced the capsaicin-induced release of CGRP, we can not exclude th~at at least part of the increase in the bradykinin-evoked CGRP-LI outflow might be due to decreased degradation of the released sensory neuropeptide. The present findings strongly suggest that a thiorphan-sensitive enzymic activity, putatively ascribed to NEP, has an important role in regulating the amount of peptides either released from or acting on perivascular terminals of capsaicinsensitive sensory neurons. 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