Transglutaminase-Synthesized γ-(Glutamyl5) Spermidine Derivative of Substance P Is a Selective Tool for Neurokinin-2 Receptors Characterization

Peptides, Vol. 19, No. 4, pp. 683– 690, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/98 $19.00 1 .00

PII S0196-9781(98)00014-X

Transglutaminase-Synthesized g-(Glutamyl5) Spermidine Derivative of Substance P Is a Selective Tool for Neurokinin-2 Receptors Characterization FRANCA MANCUSO,* CLAUDIA COSTA,†‡ ANTONIO CALIGNANO,* LOREDANA MARINIELLO,§ FRANCESCO ROSSI,‡ RAFFAELE PORTA§ AND CARLA ESPOSITO†1 Departments of *Experimental Pharmacology and §Food Science, University of Naples “Federico II” and †Department of Biochemistry and Biophysics and ‡Institute of Pharmacology and Toxicology, 2nd University of Naples, Naples, Italy Received 14 October 1997; Accepted 2 January 1998 MANCUSO, F., C. COSTA, A. CALIGNANO, L. MARINIELLO, F. ROSSI, R. PORTA AND C. ESPOSITO. Transglutaminase-synthesized g-(glutamyl5)spermidine derivative of substance P is a selective tool for neurokinin-2 receptors characterization. PEPTIDES 19(4) 683– 690, 1998.—The ability of transglutaminase-synthesized 1,3-diaminopropane, spermidine (Spd), spermine (Spm), and monodansylcadaverine g-(glutamyl5)derivatives of substance P (SP) to produce bronchoconstriction was investigated. In urethaneanaesthetized guinea pigs, intravenous injections of SP derivatives contracted differently bronchial smooth muscle and caused hypotension. The most effective bronchoconstrictor among SP analogs was the g-(glutamyl5)Spd derivative of SP (Spd-SP; EC50 5 5.3 nmol/kg), which was more potent than the native peptide (EC50 5 26.5 nmol/kg). In contrast, the g-(glutamyl5)Spm derivative of SP (Spm-SP) was found completely unable to cause bronchoconstriction and was significantly less effective than SP in determining hypotension. The contractile effect of Spd-SP and Spm-SP was investigated in vitro on rat isolated colon, a well-characterized preparation rich in NK2 receptors. In addition, Spd-SP was tested on the endothelium-denuded rabbit pulmonary artery (RPA) and the hamster isolated trachea (HT), both tissue preparations containing only a single functional receptor subtype (NK2A and NK2B, respectively). The results obtained showed that Spd-SP recognizes NK2 receptors occurring on rat isolated colon more effectively (EC50 5 11 nM) than the native peptide (EC50 5 45 nM). Conversely, Spm-SP evokes a contractile response less effective than that elicited by SP (EC50 5 312 nM). Furthermore, Spd-SP (0.1–10 mg kg21) produced a concentration-dependent contraction of both HT and RPA, exhibiting a potency respectively 12 and 30 times higher than SP in contracting HT and RPA. Our results indicate that the introduction of a Spd moiety at the level of glutamine-5 of SP gives rise to an analog that possesses a different capability to recognize NK2 receptors than the parent peptide. Moreover, since Spd-SP seems to contract more effectively RPA than HT, we conclude that it preferentially recognizes the NK2A receptor subtype. © 1998 Elsevier Science Inc. Substance P

Transglutaminase

Spermidine

Polyamines

Bronchoconstriction

NK2 receptor subtypes

system and non-neuronal tissues. Tachykinins are involved in multiple physiological responses, including pain transmission, neurogenic inflammation, vasodilation, smooth muscle contraction, intestinal motility, bronchoconstriction,

SUBSTANCE P (SP), neurokinin A (NKA) and neurokinin B (NKB) are members of a family of structurally related peptides (18,25). They represent the major tachykinins in mammals and are widely distributed in the central nervous 1

Requests for reprints should be addressed to Dr. Carla Esposito, Department of Biochemistry and Biophysics, via Costantinopoli 16, 80138 Naples, Italy. E-mail: [email protected]

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salivary and airway secretion, as well as immune response (16,22). The physiological actions of tachykinins are exerted via three different receptors (NK1, NK2, and NK3) toward which SP, NKA, and NKB represent, respectively, the most potent natural ligands (10,30). The three receptor sites, all belonging to the superfamily of G-protein-coupled receptors (21), are expressed differently in the nervous system and peripheral tissues, one or more of them being responsible for distinct biologic responses (16,31). NK2 receptors are distributed in the central nervous system, where they appear involved in pain processing (32,34) and anxiety (33), in the periphery, predominantly in the airways (2,16), and in the genitourinary tract (23), where they are suggested to mediate smooth muscle contraction. Moreover, NK2 receptors occur on the smooth muscle of some blood vessels (e.g., rabbit pulmonary artery), where they mediate contraction (29). Finally, recent evidence suggests that NKA-induced vasodilatation in rat footpad skin is not associated with NK1 receptors and nitric oxide (28). Pharmacological studies to define the distribution and the physiological role of NK2 receptors have suggested the existence of NK2 receptor interspecies heterogeneity (14,24). Two putative receptor subtypes have been designated classic/ nonclassic (or NK2B/NK2A) NK2 receptors. These receptor subtypes have been described on the basis of a different rank order potency exhibited by some antagonists tested in several tissue preparations obtained from different animal species (16). By using the enzyme transglutaminase (EC 2.3.2.13), we have previously synthesized in vitro SP analogs displaying a different selectivity for NK1 and NK3 receptors (6,7,26). Transglutaminase catalyzes the covalent attachment of various primary amines to the g-carboxamide group of reactive protein-bound glutamine residues (1) and recognizes only the first of the two glutamine residue of SP (glutamine 5) as reactive site (27). The g-(glutamyl5)spermine derivative of SP (Spm-SP) was found to be completely ineffective in contracting the smooth muscle of different tissue preparations (stomach strips, duodenum, and ileal segments) isolated from both guinea pig and rat, and it was almost completely unable to induce edema formation when injected in the rat hind limb (6,26). In contrast, we demonstrated that Spm-SP retained several vascular NO-mediated biologic activities evoked by native SP, such as provoking rabbit aorta relaxation, to decrease rat arterial blood pressure and to inhibit collageninduced platelet aggregation (6). Further experiments performed with different g-(glutamyl5)derivatives of SP showed that the specific introduction of the spermine (Spm) moiety at the glutamine 5 level was essential in producing the observed effect. In fact, its substitution with amines of different molecular weight and physicochemical characteristics, as 1,3diaminopropane (Dap), spermidine (Spd), or monodansylcadaverine (Dns; Fig. 1), led to compounds possessing a pharmacological profile very similar to that exhibited by the

MANCUSO ET AL.

native neuropeptide (6). Moreover, we demonstrated that Spm-SP recognizes NK3 receptors occurring on the rat portal vein, showing a greater potency (EC50 5 588 nM) than native SP (EC50 5 1120 nM) and the other SP derivatives tested (7). The purpose of this study was to test the capability of transglutaminase-synthesized amine derivatives of SP to interact with NK2 receptors. EXPERIMENTAL PROCEDURES Drugs SP acetate, transglutaminase purified from guinea pig liver, Dap dihydrochloride, Spd trihydrochloride, Spm tetrahydrochloride, Dns, thiorphan, urethane, captopril, and bestatin were supplied by Sigma Chemical Co., (St. Louis, MO). [bAla8]NKA (4 –10) was supplied by Research Biochemicals International (Natik, MA), and pancuronium bromide (Pavulon) was supplied from Organon Teknika (Rome, Italy). Animals Male outbred Hartley guinea pigs (weighing 300 –350 g), male Charles River rats (weighing 250 –300 g), male Charles River hamsters (weighing 100 –150 g), and albino male New Zealand rabbits (weighing 2500 –3000 g) obtained from Charles River (Como, Italy), were fasted overnight and allowed free access to water before the experiments. The experimental protocol followed in this study was approved by the “Ministero della Sanita` Italiana” according to EEC rules. Transglutaminase-catalyzed Synthesis and Purification of SP Derivatives The SP analogs were obtained by incubating the native peptide SP with transglutaminase purified from guinea pig liver in the presence of calcium and Dap, Spd, Spm, or Dns as described previously by Esposito et al. (6). The identification of the adducts was performed by fast atom bombardment mass spectrometry (27). Measurement of Airway Responses Prior to the operation, the guinea pigs were anaesthetized with urethane (1.5 g kg21 intraperitoneally) and the trachea, carotid artery, and jugular vein were cannulated for measurement of airway obstruction, for systemic blood pressure, and for the introduction of drugs, respectively. Pancuronium bromide (4 mg kg21, IV) was injected to prevent spontaneous breathing. The animals were ventilated with a rodent ventilator (U. Basile, Milan, Italy) with room air supplemented with oxigen at 60 strokes min21 with a stroke volume of 3–7 ml. Airway obstruction was measured according to the method of Mooroka et al. (20) using a differential pressure transducer (U. Basile, Milan, Italy) connected by the side-arm of the tracheal cannula to a bronchospasm transducer and expressed as intrathoracic

NK2 RECEPTOR RESPONSE TO SPD-SP

685

FIG. 1. Structure of the SP derivatives synthesized by transglutaminase.

pressure (ITP) in cmH2O. The intratracheal cannula was clipped before and after the experiment to obtain the maximal response. SP (1–20 nmol kg21), NKA (0.1–1 nmol kg21), Dap-SP (10 –20 nmol kg21), Spd-SP (1–20 nmol kg21), Spm-SP (2.5–20 nmol kg21), Dns-SP (5–20 nmol kg21), Dap (20 nmol kg21), Spd (20 nmol kg21), Spm (20 nmol kg21), and Dns (20 nmol kg21) dissolved in saline were injected alone or in combination via the jugular vein, and the % increase in ITP was evaluated at its peak. The arterial blood pressure was measured continuously with a pressure transducer connected to a recorder. Rat Colon Experimental Preparation Rats were stunned and killed by cervical dislocation. The colon was quickly removed, and the obtained muscle preparation (2 cm) was suspended under a load of 0.5 g in a 10-ml organ bath containing Krebs solution (millimolar composition: NaCl 118; KCl 4.7; KH2PO4 1.2; CaCl2 2.5; MgSO4 1.2; NaHCO3 25; glucose 11, pH 7.4), maintained at 37°C and gassed with 5% CO2 and 95% O2, and connected to an isotonic transducer (U. Basile, Milan, Italy). After an equilibration period of 30 min, NKA, SP, Spd-SP, Spm-SP, and [bAla8]NKA (4 –10) concentration–response curves (0.3–10 mM) were obtained. Experiments were performed

in a noncumulative way by consecutive injections of each stimulant at intervals of 20 –30 min to avoid desensitization of the tissues. All compounds were dissolved in Krebs’ solution. Only one agonist concentration–response curve was generated for each tissue. Hamster Trachea Experimental Preparation Hamsters were stunned and bled. The trachea was rapidly removed and placed in Krebs solution maintained at 37°C and gassed with 5% CO2 and 95% O2. Two rings were prepared from each trachea, which were mounted in a 5-ml organ bath to record tension isometrically by a transducer connected to a Unirecord (U. Basile, Milan, Italy). Each ring was suspended between two stainless steel plates under a tension of 0.5 g. The preparations were field-stimulated by two platinum electrodes placed at the top and the bottom of the organ bath. After an equilibration period of 1 h, NKA, SP, Spd-SP, and [bAla8]NKA (4 –10) concentration–response curves (0.3–10 mM) were obtained. Experiments were performed in a cumulative way, and only one agonist concentration–response curve was generated for each tissue. In some experiments, the concentration–response curves were obtained in the presence of a mixture of peptidase inhibitors (thiorphan, captopril, and bestatin, 1 mM each).

686

MANCUSO ET AL. TABLE 1 EFFECT OF THE g(GLUTAMYL )AMINE DERIVATIVES OF SP ON THE INTRATHORACIC PRESSURE (ITP) AND ON THE MEAN ARTERIAL BLOOD PRESSURE (MABP) IN ANAESTHETIZED GUINEA PIGS 5

ITP Drugs SP

NKA

Dap-SP

Spd-SP

Spm-SP

Dns-SP

MABP

nmol/kg

% Increase

EC50

D mmHg

EC50

1.0 2.5 5.0 10.0 20.0 60.0 0.05 0.1 0.25 0.5 1.0 1.0 2.5 5.0 10.0 20.0 60.0 1.0 2.5 5.0 10.0 20.0 60.0 2.5 5.0 10.0 20.0 60.0 5.0 10.0 20.0 60.0

2.0 6 0.5 3.5 6 0.7 9.5 6 5.2 21.3 6 1.7 42.2 6 2.8 85.0 6 3.0 5.1 6 1.7 15.2 6 1.2 44.2 6 1.8 78.3 6 2.1 95.1 6 3.0* 1.3 6 0.3 3.2 6 1.0 9.0 6 1.2 21.5 6 0.7 38.2 6 1.7 72.8 6 5.0 21.7 6 2.4* 35.2 6 1.7* 40.2 6 0.7* 70.1 6 1.1* 77.3 6 2.2* 97.0 6 2.7 1.6 6 0.9** 2.7 6 2.0** 4.1 6 1.9* 6.3 6 4.1* 8.4 6 3.0 2.1 6 1.1** 12.5 6 1.0** 22.2 6 1.4** 65.6 6 3.1*

26.5 6 1.5

15.2 6 2.5 38.5 6 4.6 42.0 6 5.0 47.3 6 4.7 51.1 6 5.2 75.8 6 4.9 21.1 6 1.7 42.3 6 3.1 53.1 6 2.0 75.6 6 2.0 78.4 6 2.1* 12.2 6 1.3 35.4 6 1.0 42.3 6 1.6 45.2 6 3.0 49.6 6 2.7 79.8 6 3.0 25.1 6 2.5 42.2 6 1.9 55.6 6 2.0** 64.5 6 1.7** 74.2 6 1.3** 80.1 6 1.0 30.6 6 2.4** 30.2 6 3.7* 33.8 6 3.9* 38.1 6 2.5* 41.5 6 1.5 15.6 6 1.7** 39.2 6 1.7** 42.1 6 1.3** 67.8 6 4.5

11.5 6 2.1

0.27 6 0.06

27.8 6 2.1

5.3 6 1.9

———

42.1 6 1.8

0.16 6 0.07

10.9 6 2.5

4.16 6 0.9

———

21.3 6 2.1

Data are mean 6 SEM values. Six animals were used in each group. EC50 values are nmol/kg. Further experimental details are given in the text. *p , 0.01; **p , 0.05 versus SP (Student’s t test).

Rabbit Pulmonary Artery Experimental Preparation Rabbits were stunned and bled. The rings, prepared according to D’Orleans–Juste et al. (5), were suspended under a load of 1.0 g in a 5-ml organ bath containing Krebs solution, maintained at 37°C, gassed with 5% CO2 and 95% O2, and connected to an isometric transducer (U. Basile, Milan, Italy). After an equilibration period of 90 min, NKA, SP, Spd-SP, and [bAla8]NKA (4 –10) concentration–response curves (0.3–10 mM) were obtained. Experiments were performed in a cumulative way, and only one agonist concentration–response curve was generated for each tissue. In some experiments, the concentration–response curves were obtained in the presence of a mixture of peptidase inhibitors (thiorphan, captopril, and bestatin, 1 mM each). Statistical Analysis All values in the text, figure, and tables are means 6 SEM. Statistical analysis was performed by means Student’s t test for paired or unpaired data. Regression analysis was per-

formed by means of the least squares method. EC50 and 95% confidence limits (CL) were calculated accordingly. The EC50 of each agonist was calculated as the concentration of the agonist needed to produce 50% of the maximal response. RESULTS Effect of g-(glutamyl5)Amine Derivatives of SP on Bronchoconstriction in Guinea Pigs The IV administration to urethane-anaesthetized guinea pig of NKA, SP, or its derivatives Dap-SP, Spd-SP, and Dns-SP caused a dose-dependent increase in intrathoracica pressure and a concurrent reduction in the blood pressure (Table 1). In this respect, NKA was the most potent bronchoconstrictor agent, being 48-fold more potent than SP at 1.0 nmol kg21. The analog Spd-SP (1.0 nmol kg21) was 10-fold more potent than SP in increasing ITP. Among the transglutaminase-synthesized derivatives of SP, Dap-SP produced effects very similar to those observed following the injection

NK2 RECEPTOR RESPONSE TO SPD-SP

687

TABLE 2 CONTRACTILE RESPONSE OF THE RAT ISOLATED COLON TO SPD-SP AND SPM-SP Peptides

EC50 (nM)

95% CL (nM)

RP

NKA SP Spd-SP Spm-SP [bAla8]NKA(4-10)

7.5 45.0 11.0 312 6.0

5.4–10 15–102 8–21 96–927 4.1–5

6 1 4 0.1 7.5

Each value is the mean of four to six experiments. RP, relative potency. EC50 and Emax for producing contraction of the rat isolated colon. CL, 95% confidence limits.

of the same concentration of the parent peptide whereas Dns-SP exerted a lower contractile effect at the same doses. Conversely, Spm-SP was found almost completely unable to cause bronchoconstriction and significantly less effective than SP in determining hypotension, even when it was used at doses much higher. The different potency of SP derivatives is indicated by EC50 values reported in Table 1. Finally, it is worthy to note that free amines, when administered alone or when injected together with SP, did not increase intrathoracic pressure and did not decrease blood pressure (data not shown). Effect of g-(glutamyl5)Spermidine and g-(glutamyl5)Spermine Derivatives of SP on Rat Isolated Colon Data in Table 2 show the apparent affinity as EC50 and the relative potency of both natural tachykinins and of the SP derivatives Spd-SP and Spm-SP in producing contraction of the rat colon. This preparation strongly responded to NKA

and to [bAla8]-NKA (4 –10), a selective NK2 receptor agonist, which displayed the most potent activity (EC50 5 7.5 nM and 6.0 nM, respectively). Spd-SP contracts isolated rat colon (EC50 5 11 nM) better than the native SP (EC50 5 45 nM) whereas Spm-SP was tenfold less potent than SP (EC50 5 312 nM). Effect of g-(glutamyl5)Spermidine derivative of SP on Endothelium-denuded Rabbit Pulmonary Artery (RPA) and Hamster Isolated Trachea (HT) The dose-dependent contractile response of the HT and RPA to Spd-SP, as well as to selective tachykinin agonists SP, NKA, and [bAla8]NKA (4 –10) is shown in Figure 2. The plateau of response was achieved in about 2 min, with no appreciable difference between the two tissues (Fig. 2). The same results were obtained when the time course of serial dose–response curves was evaluated in the presence of a mixture of peptidase inhibitors such as thiorphan, captopril, and bestatin (1 mM each). These results, as reported previously (14), indicate that Spd-SP effect is not influenced by peptide degradation. In Table 3, EC50 values are shown and the relative potency of Spd-SP, NKA, and the NK2 agonist [bAla8]NKA (4 –10) in the HT and RPA. For each compound both in the HT and RPA, the EC50 values did not differ from those obtained in the absence of enzyme inhibitors (data not shown). Both [bAla8]NKA (4 –10) and the natural NK2 receptor ligand NKA were more potent than SP in HT and RPA, respectively. The newly developed SP analog, Spd-SP, displayed strong affinity in both tissues and was more potent than SP mostly in RPA. These results indicate that NK2 receptor(s) are more sensitive to Spd-SP than SP, particularly the NK2A receptor subtype occurring on the RPA, which seems to recognize Spd-SP better than NK2B subtype that occurring on the HT.

FIG. 2. Serial dose-dependent contractile response to Spd-SP and to NK2 receptor agonists in the hamster trachea (A) and in the endothelium-denuded rabbit pulmonary artery (B) in presence of [bAla 8]NKA (4 –10) (f), NKA (M), Spd-SP (F), and SP (E). Each point is the mean 6 SE of three to six replications.

688

MANCUSO ET AL. TABLE 3 CONTRACTILE RESPONSE OF THE HAMSTER TRACHEA AND OF THE ENDOTHELIUM-DENUDED RABBIT PULMONARY ARTERY TO SPD-SP Hamster Trachea

Rabbit Pulmonary Artery

Peptides

EC50 (nM)

95% (CL) (nM)

RP

EC50 (nM)

95% (CL) (nM)

RP

NKA SP Spd-SP [bAla8]NKA(4–10)

22 6689 539 18

15–35 520–9018 203–1725 8–65

304 1 12 372

4.1 718 24 4.0

3.9–5.4 642–903 16–35 3.5–5.7

175 1 30 179

Each value is the mean of four to six experiments. RP, relative potency. EC50 and Emax for producing contraction of the hamster isolated trachea and the endothelium-denuded rabbit pulmonary artery. CL, 95% confidence limits.

DISCUSSION The post-translational modification of SP in vitro by the use of the enzyme transglutaminase and polyamines gives rise two analogs of SP (Spd-SP and Spm-SP), which display a pharmacological profile quite different from that exhibited by the native neuropeptide (Table 4). In particular, the introduction of the polyamine Spm at the glutamine-5 level gives rise to a derivative of SP, which could be a useful tool in differentiating NK1 receptors. In fact, Spm-SP retains several vascular NO-mediated biologic activities evoked by native SP and, in contrast, is completely ineffective to contract the smooth muscle occurring in various tissues isolated from different species (6,26). Further experiments performed with different SP g(glutamyl5)derivatives showed that the substitution of Spm with Spd, a polyamine lacking only in a propylamine moiety in comparison with Spm (Fig. 1), led to a SP analog possessing contractile

activity on guinea pig ileal segments very similar to that exhibited by the native neuropeptide (6). The effects of the transglutaminase-synthesized derivatives of SP have been further characterized by testing their ability to contract in vitro the rat portal vein, a pharmacological preparation particularly rich in NK3 receptors (19). The results of the latter experiments demonstrated that Spm-SP recognizes NK3 receptors, showing a greater potency than both native SP and the other SP-derivatives tested (7). In the attempt to elucidate whether the transglutaminasecatalyzed polyamine attachment to SP influences the capability of the neuropeptide to recognize NK2 receptors, we first tested in vivo the effect of Dap-, Spd-, Spm-, and Dns-SP derivatives to induce bronchoconstriction in guinea pigs. It is well known that NK2 receptors widely occur in animal and human airway smooth muscle tissues and are believed to be involved in the bronchospastic action of the

TABLE 4 BIOLOGICAL ACTIONS OF THE TRANSGLUTAMINASE-SYNTHESIZED SPD-SP AND SPM-SP IN DIFFERENT EXPERIMENTAL MODELS Biological Action Smooth muscle contraction

Experimental Model Rabbit pulmonary artery Rat portal vein

Spd-SP @ SP Spm-SP . SP 5 Spd-SP

Rat stomach

Spm-SP no active Spd-SP 5 SP

Rat duodenum

Spm-SP no active Spd-SP 5 SP Spd-SP . SP @ Spm-SP Spm-SP no active Spd-SP 5 SP Spd-SP . SP . Spm-SP Spd-SP @ SP Spd-SP 5 SP 5 Spm-SP SP 5 Spm-SP SP 5 Spm-SP SP . Spm-SP

Rat colon Guinea pig ileum

Vasodilatation Blood pressure decrease Platelet aggregation Plasma protein extravasation

Order of Potency

Guinea pig bronchus Hamster trachea Rabbit aorta Rat Rat Rat paw edema

Reference present paper (7) (26) unpublished data (26) unpublished data present paper (6) (6) present paper present paper (6) (6) (6) (6)

NK2 RECEPTOR RESPONSE TO SPD-SP

689

endogenous tachykinins (2,16). The results of the experiments reported in the present paper indicate that only Spd-SP was a bronchoconstrictor more potent than SP among the transglutaminase-synthesized derivatives tested. Dap-SP and Dns-SP (the latter of which at higher doses) were able to elicit a contractile response similar to that evoked by native SP. In contrast, Spm-SP was found completely unable to cause bronchoconstriction and was significantly less effective than SP in determining hypotension. Maggi et al. (15) demonstrated by using guinea pig isolated bronchi that the contractile response to neurokinins is essentially mediated by both NK2A subtype and NK1 receptors. In addition, Floch et al. (8) reported that NK1 receptors in guinea pig bronchopulmonary system are pharmacologically distinct from those present in the rat vascular system. A similar conclusion has been drawn by Burcher and Zeng (4) for the epithelium-deprived guinea pig main bronchus and by Boni et al. (3), who obtained a corresponding in vivo evidence in anaesthetized guinea pigs. Therefore, Spm-SP, which is able to elicit NO-releasing effects (6), was shown to not recognize NK1 receptor also on guinea pig bronchi in vivo, confirming our previous observations in vitro (6,26). The potent effect of Spd-SP on the anaesthetized guinea pig ITP was confirmed by its selective contractile effect on the isolated rat colon, a well-characterized NK2 receptorcontaining preparation (11). Since binding and functional experiments carried out with selective antagonists have demonstrated the existence of NK2 subtype receptors (14), we have performed further experiments with different tissue preparations to elucidate whether Spd-SP is able to differently recognize NK2A and NK2B receptors. By using RPA and HT, tissue preparations containing NK2A and NK2B receptor subtypes, respectively (16), we showed that the insertion of Spd moiety in Position 5 on the backbone of SP yielded an adduct of the neuropeptide that apparently recognizes NK2A better than NK2B receptors. One possible explanation of the observed effect is that the transglutaminase-catalyzed structural modification

of SP delays or prevents the degradation of the neuropeptide by the proteolytic enzymes without affecting its ability to bind to the receptor. Although preliminary results (data not shown) suggest that the Spd moiety added into SP produces a protease-resistant analog of the neuropeptide, tissue peptidases seem to be not responsible for the different potency of the agonists tested. In fact, in the used tissue preparations, the RPA and HT EC50 value obtained in the presence of enzyme inhibitors did not differ from those obtained in their absence. Extensive mutational analysis of both NK2 and NK1 receptor proteins (9,12,13) have identified many common residues involved in the ligand binding. Moreover, these studies showed that differential peptide binding affinity for NK1 receptor include “conformational factors,” determined by divergent residues in the sequence, rather than differential interactions with specific residues (e.g., N-terminal sequence) of tachykinins. Therefore, as hypothesized by Maggi et al. (17), the existence of different molecular determinants could be responsible for species-related heterogeneity in NK2 receptor subtypes. In this regard, the chemical nature of the Spd moiety may confer to the adduct a favorable conformation for interaction with a specific NK2 subtype receptor. In conclusion, our results demonstrate that the specific introduction of a large size hydrophilic compound, like Spd, at the glutamine-5 level 1) gives rise to an analog of SP showing a different capability to recognize NK2 receptors and 2) provides further evidence for the occurrence of a species-related heterogeneity of the NK2 receptors. Therefore, Spd-SP could be considered an additional tool in differentiating tachykinin receptors and in ascertaining the molecular determinants of their species-related pharmacological heterogeneity. ACKNOWLEDGEMENT This work was supported from Grant 95.02923.CT14 (to R. P.) from the Consiglio Nazionale delle Ricerche, Roma, Italy.

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ent receptors are involved in the endothelium-mediated relaxation and the smooth muscle contraction of the rabbit pulmonary artery in response to substance P and related neurokinins. Eur. J. Pharmacol. 125:37– 44; 1986. 6. Esposito, C.; Mancuso, F.; Calignano, A.; Di Pierro, P.; Pucci, P.; Porta, R. Neurokinin receptors could be differentiated by their capacity to respond to the transglutaminase-synthesized g-(glutamyl5)spermine derivative of substance P. J. Neurochem. 65:420 – 426; 1995. 7. Filippelli, A.; Esposito, C.; Falciani, M.; Costa, C.; Cozzolino, A.; Rossi, F.; Porta, R. Transglutaminase-synthesized spermine derivative of substance P recognizes rat portal vein neurokinin-3 receptors. Life Sci. 60:403– 411; 1997. 8. Floch, A.; Fardin, V.; Cavero, I. Characterization of NK1 and NK2 tachykinin receptors in guinea pig and rat bronchopul-

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