Endomorphin-1 and endomorphin-2 differentially interact with specific binding sites for substance P (SP) aminoterminal SP1-7 in the rat spinal cord

Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) represent two opioid active tetrapeptides with high affinity and selectivity for the mu-opioid (MOP) receptor. Both EM-1 and EM-2 exhibit strong inhibition of pain signals in the central nervous system (CNS). In contrast to these compounds, the undecapeptide substance P (SP) facilitates pain influx in the CNS. SP has been implicated in a number of functions in the central nervous system, including pain processing and reward. Its aminoterminal fragment SP1-7 has been shown to modulate several actions of SP in the CNS, the nociceptive effect included. Although the actions of SP1-7 have been known for long no specific receptor for the SP fragment has yet been cloned. In this study, we demonstrate the presence of specific binding sites for the heptapeptide in the rat spinal cord. The binding affinity for unlabeled SP1-7 to the specific sites for the labeled heptapeptide highly exceeded those of SP and other C- or N-terminal fragments thereof. The NK-1, NK-2 and NK-3 receptor ligands [Sar9, Met(O2)11]SP, R396 and senktide, respectively, showed no or negligible binding. Moreover, both EM-1 and EM-2 were found to interact with SP1-7 binding. However, a significant difference in binding affinity between the two opioid active tetrapeptides was observed. As recorded from replacement curves the affinity of EM-2 was 10 times weaker than that for SP1-7 but about 100 times higher than that of EM-1. Among other Tyr-Pro-containing peptides Tyr-MIF-1 but not Tyr-W-MIF-1 exhibited affinity of similar potency as EM-2. These results strengthen the previously observed differences between EM-1 and EM-2 in various functional studies. Moreover, using a cell line (C6) expressing the MOP receptor it was shown that the labeled SP1-7 did not interact with binding to this receptor and no functional response was seen for the SP heptapeptide on the MOP receptor by means of stimulation in the GTPgammaS assay. This suggests that the identified SP1-7 binding sites, with high affinity also for EM-2, are not identical to the MOP receptor and apparently not to any of the known tachykinin receptors.     

A fragment of substance P with specific central activity: SP(1-7)

Amino-terminal fragments of substance P (SP), SP(1-7) and SP(1-8), were found to produce naloxone-reversible antinociception in the mouse similar to that produced by SP. Similar to SP, these peptides produce antinociception only within a narrow dose range. They have no activity on smooth muscle or blood pressure. These results suggest that contrary to peripheral effects of SP, which are mediated by receptors which recognize the carboxy-terminal part of the SP molecule, certain central actions of SP are mediated by receptors which recognize the amino-terminal part of the SP molecule. SP may be metabolized to this active fragment prior to its action at these receptors.     

ADP is the cognate ligand for the orphan G protein-coupled receptor SP1999

P2Y receptors are a class of G protein-coupled receptors activated primarily by ATP, UTP, and UDP. Five mammalian P2Y receptors have been cloned so far including P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11. P2Y1, P2Y2, and P2Y6 couple to the activation of phospholipase C, whereas P2Y4 and P2Y11 couple to the activation of both phospholipase C and the adenylyl cyclase pathways. Additional ADP receptors linked to Galpha(i) have been described but have not yet been cloned. SP1999 is an orphan G protein-coupled receptor, which is highly expressed in brain, spinal cord, and blood platelets. In the present study, we demonstrate that SP1999 is a Galpha(i)-coupled receptor that is potently activated by ADP. In an effort to identify ligands for SP1999, fractionated rat spinal cord extracts were assayed for Ca(2+) mobilization activity against Chinese hamster ovary cells transiently transfected with SP1999 and chimeric Galpha subunits (Galpha(q/i)). A substance that selectively activated SP1999-transfected cells was identified and purified through a series of chromatographic steps. Mass spectral analysis of the purified material definitively identified it as ADP. ADP was subsequently shown to inhibit forskolin-stimulated adenylyl cyclase activity through selective activation of SP1999 with an EC(50) of 60 nM. Other nucleotides were able to activate SP1999 with a rank order of potency 2-MeS-ATP = 2-MeS-ADP > ADP = adenosine 5'-O-2-(thio)diphosphate > 2-Cl-ATP > adenosine 5'-O-(thiotriphosphate). Thus, SP1999 is a novel, Galpha(i)-linked receptor for ADP.     

Design of chimeric peptide ligands to galanin receptors and substance P receptors.

Several chimeric peptides were synthesized and found to be high-affinity ligands for both galanin and substance P receptors in membranes from the rat hypothalamus. The peptide galantide, composed of the N-terminal part of galanin and C-terminal part of substance P (SP), galanin-(1-12)-Pro-SP-(5-11) amide, which is the first galanin antagonist to be reported, recognizes two classes of galanin binding sites (KD(1) less than 0.1 nM and KD(2) approximately 6 nM) in the rat hypothalamus, while it appears to bind to a single population of SP receptors (KD approximately 40 nM). The chimeric peptide has higher affinity towards galanin receptors than the endogenous peptide galanin-(1-29) (KD approximately 1 nM) or its N-terminal fragment galanin-(1-13) (KD approximately 1 microM), which constitutes the N-terminus of the chimeric peptide. Galantide has also higher affinity for the SP receptors than the C-terminal SP fragment-(4-11) amide (KD = 0.4 microM), which constitutes its C-terminal portion. Substitution of amino acid residues, which is of importance for recognition of galanin by galanin receptors, such as [Trp2], in the galanin portion of the chimeric peptide or substitution of ([Phe7] or [Met11]-amide) in the SP portion of chimeric peptide both cause significant loss in affinity of the analogs of galantide for both the galanin- and the SP-receptors. These results suggest that the high affinity of the chimeric peptide, galantide, may in part be accounted for by simultaneous recognition/binding to both receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endomorphins interact with the substance P (SP) aminoterminal SP(1-7) binding in the ventral tegmental area of the rat brain

We have recently identified a specific binding site for the tachykinin peptide substance P (SP) fragment SP(1-7) in the rat spinal cord. This site appeared very specific for SP(1-7) as the binding affinity of this compound highly exceeded those of other SP fragments. We also observed that endomorphin-2 (EM-2) exhibited high potency in displacing SP(1-7) from this site. In the present work using a [(3)H]-labeled derivative of the heptapeptide we have identified and characterized [(3)H]-SP(1-7) binding in the rat ventral tegmental area (VTA). Similarly to the [(3)H]-SP(1-7) binding in the spinal cord the affinity of unlabeled SP(1-7) to the specific site in VTA was significantly higher than those of other SP fragments. Further, the tachykinin receptor NK-1, NK-2 and NK-3 ligands showed no or negligible binding to the identified site. However, the mu-opioid peptide (MOP) receptor agonists DAMGO, EM-1 and EM-2 did, and significant difference was observed in the binding affinity between the two endomorphins. As recorded from displacement curves the affinity of EM-2 for the SP(1-7) site was 4-5 times weaker than that for SP(1-7) but about 5 times higher than that of EM-1. The opioid receptor antagonists naloxone and naloxonazine showed weak or negligible binding. It was concluded that the specific site identified for SP(1-7) binding in the rat VTA is distinct from the MOP receptor although it exhibits high affinity for EM-2.     

Further delineation of the two binding sites (R*(n)) associated with tachykinin neurokinin-1 receptors using [3-Prolinomethionine(11)]SP analogues.

Two binding sites are associated with neurokinin-1 substance P receptors in both transfected cells and mammalian tissues. To further delineate the interactions between the crucial C-terminal methionine of substance P and these two binding sites, we have incorporated newly designed constrained methionines, i.e. (2S, 3S)- and (2S,3R)-prolinomethionines. The potencies of these C terminus-modified SP analogues to bind both sites and to activate phosphatidylinositol hydrolysis and cAMP formation have been measured, together with those of their corresponding sulfoxides and sulfones. The molecular nature of these two binding sites and their selective coupling to effector signaling pathways are discussed in the light of current models of receptor activation. The less abundant binding site is coupled to G(q/11) proteins, whereas the most abundant one interacts with G(s) proteins in Chinese hamster ovary cells transfected with human neurokinin-1 receptors. The specific orientation of the C-terminal methionine side chain imposed by these constraints shows that macroscopically chi(1) and chi(2) angles of this crucial C-terminal residue are similar in both binding sites. However, slight but significant variations in the rotation around the Cgamma-S bond yield different either stabilizing or destabilizing interactions in the two binding sites. These results highlight the need of such constrained amino acids to probe subtle interactions in ligand-receptor complexes.     

Use of substance P fragments to differentiate substance P receptors of different tissues

The C- and N-terminal fragments of substance P were compared to the parent molecule with respect to their ability to: (a) contract the isolated guinea pig ileum, (b) induce salivation in the rat, (c) excite single cat dorsal horn neurones, and (d) induce scratching by intracranial injections in mice. C-terminal fragments as small as the heptapeptide were potent SP agonists on all assay systems. C-terminal fragments containing five amino acids or less were, at most, only weakly active. The C-terminal hexapeptide was a potent SP receptor stimulant on the isolated guinea pig ileum and, when directly applied by microiontophoresis, on cat dorsal horn neurons. However, the same compound was only 2-5% as potent as substance P in eliciting salivation and scratching in vivo, an indication that this fragment may be especially labile to enzymatic degradation. N-terminal fragments were totally inactive on the isolated guinea pig ileum. On the rat salivation and central nervous system assays, however, N-terminal fragments were capable of weak SP-like activity. It is concluded that SP receptors exist in multiple forms which we have labelled SP1 and SP2 receptors for those insensitive or sensitive to N-terminal fragments, respectively.     

Substance P receptors in mammalian central nervous system.

1. Multiple distinct affinity states or sites of substance P (SP) receptors exist in freshly-prepared rat brain membranes. 2. Substance P receptors may couple with islet-activating protein (pertussis toxin) sensitive GTP-binding protein(s). 3. Substance P receptors may be regulated Mg2+ and Na+ in an opposite manner. 4. Some important factor(s), in addition to GTP-binding protein, appear to be involved in SP binding activity. 5. An apparent molecular weight of the SP binding site is approximately 46,000 Da.     

Distribution of NPY and SP and their effects on glucagon secretion from the in vitro normal and diabetic pancreatic tissues

Neuropeptides modulate the function of classic neurotransmitters in the regulation of body function. The role of neuropeptides in the regulation of endocrine secretion from the pancreas of diabetic rat is poorly understood. This study examined the pattern of distribution of neuropeptide-Y (NPY) and substance P (SP) in normal and diabetic rat pancreases. In addition to this, the effect of NPY and SP on glucagon secretion was also examined in the pancreases of normal and diabetic rats. Four weeks after the induction of diabetes, the pancreaseses of normal and diabetic rats were removed and processed for immunohistochemistry and glucagon secretion. The pattern of distribution of glucagon in the pancreas of diabetic rat was conspicuously deranged after the onset of diabetes. The pattern of distribution of NPY and SP was, however, similar in the pancreases of both normal and diabetic rats. Stimulation of normal rat pancreatic tissue with NPY (10(-12) and 10(-9) M) evoked large and significant (P < 0.001) increases in glucagon secretion compared to basal. In contrast to this, NPY inhibited glucagon secretion from the pancreas of diabetic rat. Treatment of pancreatic tissue fragments of normal rat with 10(-9) M SP resulted in significant (P < 0.03) increases in glucagon secretion. SP inhibited glucagon secretion from diabetic rat pancreas. In conclusion, NPY and SP stimulated glucagon secretion from the pancreas of normal rat. In contrast, NPY and SP inhibited glucagon secretion from diabetic rat pancreas.     

Tachykinin Receptors of the NK1 Type (Substance P) Coupled Positively to Phospholipase C on Cortical Astrocytes from the Newborn Mouse in Primary Culture

Specific 125I-Bolton-Hunter substance P (125I-BHSP) binding sites are present on intact cortical astrocytes of the newborn mouse in primary culture. Therefore, these cells were used to ascertain the existence of functional substance P (SP) receptors coupled positively to phospholipase C. SP stimulated phosphoinositide breakdown with an EC50 value (4.5 x 10(-10) M) similar to its IC50 value (3.8 x 10(-10) M) for inhibiting 125I-BHSP binding. The maximal response to (10(-6) M SP for 60 min) obtained was approximately 500% of control values. The rank order of potency of tachykinins was SP greater than neurokinin (NK) A greater than NKB. Long SP C-terminal fragments were more potent than shorter ones in stimulating the accumulation of 3H-inositol phosphates. SP free acid and SP N-terminal fragments were without effect. [L-Pro9]SP and SP methyl ester, two selective agonists of NK1 receptors, were almost as potent as SP. An excellent correlation was found when the abilities of tachykinins and their analogs for stimulating phosphoinositide breakdown and for inhibiting 125I-BHSP binding were compared. Finally, when used at a concentration of 3 x 10(-6) M, spantide [( D-Arg1, D-Trp7,9, Leu11]SP), an SP antagonist, competitively reduced the stimulatory effect of SP on accumulation of 3H-inositol phosphates. These results demonstrate the presence of functional SP receptors (NK1) on cortical astrocytes from the newborn mouse in primary culture.     

RP 67580, a potent and selective substance P non-peptide antagonist]

The pharmacological properties of 7,7-Diphenyl-2 [1-imino-2 (2-methoxy-phenyl)-ethyl] perhydroisoindol-4-one (3 aR, 7 aR) or RP67580 are described. This compound, derived from a novel chemical family, is a potent and selective substance P (SP) antagonist, in vitro and in vivo. In vitro, it inhibited in a competitive manner (IC50 = 10 nM) 3H-SP binding in rat brain (NK1 receptors). It did not interact with the two other tachykinin receptor sites (NK2 and NK3) nor the other receptor sites tested. Moreover, RP67580 competitively antagonized the contractile activity of SP on guinea-pig ileum (pA2 = 7.16); in contrast, it was inactive in rabbit pulmonary artery and in rat portal vein tissues which contain NK2 and NK3 receptors, respectively. In vivo, in the rat, RP67580 inhibited the plasmatic extravasation induced by administration of SP (ED50 = 0.04 mg/kg i.v.) as well as that induced by antidromic stimulation of a peripheral sensory nerve (ED50 = 0.15 mg/kg i.v.). In mice and rats, RP67580, like morphine, potently blocked the nociceptive effects of phenylbenzoquinone and formalin; its antinociceptive effect does not involve opiate receptors since it was not reversed by naloxone. These results indicate that RP67580 is a particularly valuable tool for investigating the physiological and pathological role of SP.     

The effect of substance P (SP) and SP partial sequences on nerve fiber growth in tissue culture

Explants of the ganglion trigeminale (PNS) and of the telencephalon (CNS) from chick embryos were cultivated in MAXIMOW chambers in semisynthetic media in the presence of dipeptide fragments (Lys(Z)-Pro . HCl, Lys-Pro-2HBr, Arg-Pro-2HCl) and the heptapeptide (SP5-11) of substance P as well as the complete substance P (SP1-11). 1. Histological examination of the dipeptide-treated CNS explants indicates that the structure of outgrowth in vitro is changed. Fascicel were observed. A stimulation of nerve fibre extension did not take place. 2.1. In dipeptide-treated PNS cultures the index of areas covered by the explants increased. 2.2. The index of nerve fibre growth increased significantly. The stimulation was caused in multiplication of fibres. Only Lys(Z)-Pro . HCl presents a prolongation of neurites. 2.3. SP5-11 effects in no case the growth of nerve fibres. SP1-11 stimulated significantly the fibre regeneration. 3. The possible role of SP1-11 with different effects under in vitro conditions is discussed. Only the N-terminal dipeptides stimulate the growth of nerve fibres. The C-terminal SP5-11 is without effect. Finally it is stated that the best results in neuritic enlargement and neurogenesis can only be obtained by cultivation with SP1-11.     

Anti-substance P anti-idiotypic antibodies. Characterization and biological activities

Antibodies to substance P (SP) produced in rabbits have been characterized for their specificity toward SP and some 30 SP-related peptides. For each compound, we observed a close correlation between capacity of binding to anti-SP antibodies and biological activity, namely their spasmogenic effect on guinea pig ileum in vitro and their hypotensive effect in the rat in vivo, indicating that the combining sites of anti-SP and SP receptor(s) are structurally very similar. Further immunization of five rabbits with anti-SP immunoglobulins elicited in two allotype-matched animals the production of anti-SP anti-idiotypic antibodies. These latter antibodies were found to strongly inhibit the spasmogenic action of SP on the guinea pig ileum. In contrast, they specifically enhanced, like SP, phospholipid turnover in rat parotid gland cells, a physiological function mediated through an activation of SP receptors. Immunocytochemical studies actually revealed the presence of specific membranous binding sites for anti-idiotypic antibodies on the parotid gland-dissociated cells. The anti-idiotypic antibodies described here, which thus behave either as agonists or antagonists for SP depending on the biological test, might be used as original and powerful tools not only in studies of the receptor stereospecificity but also in attempts to purify the membranous SP receptors.     

Further Demonstration that [Pro9]-Substance P Is a Potent and Selective Ligand of NK-1 Tachykinin Receptors

Previous studies have indicated that [Pro9]-substance P ([Pro9]-SP) possesses very good affinity for NK-1 binding sites and that, in contrast to substance P, it interacts selectively with these sites. Therefore, [3H][Pro9]-SP (75 Ci/mmol) was synthesized in order to study its binding to membranes of the rat brain. Specific binding of [3H][Pro9]-SP (75% of total binding) was temperature-dependent, saturable, and reversible. Scatchard analysis and Hill plots revealed the existence of a single population of noninteracting binding sites (KD and Bmax values: 1.48 nM and 29.7 fmol/mg of protein, respectively). Competition studies with several tachykinins and analogues indicated that the pharmacological profile of [3H][Pro9]-SP binding sites is identical to that of NK-1 binding sites. Rat brain sections labeled with either [3H][Pro9]-SP or [3H]SP, revealed a close similarity in the topographical distribution of [3H][Pro9]-SP and [3H]SP binding sites. Biochemical, pharmacological, and autoradiographic data obtained with [3H][Pro9]-SP did not provide any evidence for the existence of subtypes of NK-1 binding sites. [Pro9]-SP had neither agonist nor antagonist properties on NK-2 and NK-3 receptors. Indeed, it did not stimulate phosphoinositide turnover on the hamster urinary bladder (NK-2 assay) and was devoid of activity on the contraction of the rabbit pulmonary artery (NK-2 assay) and of the rat portal vein (NK-3 assay). As a result of its high selectivity, [Pro9]-SP thus appears an excellent tool for investigating the functional properties of NK-1 receptors.     

Tachykinins Induce Secretion of Prolactin from Perifused Rat Anterior Pituitary Cells by Interactions with Two Different Binding Sites

Abstract

Substance P and the two other mammalian tachykinins, neurokinin A and B, are accepted to have direct regulating effects at the anterior pituitary level. We have examined the effects of substance P (SP) and neurokinin B (NKB), alone and in combination, on prolactin release from cultured anterior pituitary cells grown on collagen-coated micro beads and placed in a perfusion system. Prolactin (Prl) secretion was observed within 25 s after exposure to either secretagogue and reached a maximum within 60-80 s. Furthermore, the prolactin response induced by SP and NKB was dose-dependent. Prl secretion remained constant for up to 4 h when SP or NKB were perifused and then fell gradually towards basal levels. Simultaneous addition of submaximal concentrations of SP and NKB resulted in an additive response compared with the responses of either secretagogue alone. Continuous (8 h) perifusion with SP did not prevent a normal prolactin response by NKB or TRH. These results indicate that the tachykinins, substance P and neurokinin B, release Prl from perifused female rat anterior pituitary cells by interaction with two different receptors, possibly the NK1 and NK3 tachykinin receptor subtypes.     

Characterization of the PL-I-related SP2 protein from Xenopus

The complete cDNA sequence of Xenopus laevis sperm specific proteins SP1 and SP2 has been determined. This information when taken together with N-terminal sequencing and mass spectroscopy data indicates that these two proteins share a product precursor relationship in which SP2 results from cleavage of a short N-terminal peptide of SP1. The secondary and tertiary structures of SP2 have been characterized using circular dichroism and three dimension structure prediction. These structural analyses have conclusively shown that SP1/SP2 proteins are related to proteins of the histone H1 family, particularly to vertebrate histone H1x. Hence, they can be considered bona fide members of the protamine-like- I (PL-I) group of sperm nuclear basic proteins (SNBPs) that have been described in other vertebrate and invertebrate groups. SP2 binds to nucleosomal DNA in a way that is very similar to that of histone H1. However, its interaction with circular DNA does not exhibit an enhanced preference for the supercoiled conformation, and it appears to be mainly driven by ionic interactions.     

The NK-1 Receptor and a Calcium-Phospholipid Pathway: Inositol Trisphosphate Production and Calcium Movements Induced by Selective Agonists of Neurokinin Receptors in Rat Parotid Glands

In the rat parotid gland, substance P has been shown to induce a phosphatidylinositol bisphosphate breakdown resulting in an inositol trisphosphate production. These data suggested that substance P activated a phospholipase C and thus mediated its effects through the calcium-phospholipid pathway. To determine which neurokinin (NK) receptor was involved in the substance P response, we have used selective agonists of the different NK receptors and examined their effects on both inositol trisphosphate production and calcium movements. A selective NK-1 receptor agonist, [Sar9Met(O2)11]-substance P, evoked an [3H]inositol trisphosphate production and a rapid and transient 45Ca2+ efflux. On the other hand, selective NK-2 and NK-3 receptor agonists, [beta-Ala8]-NKA(4-10) and [MePhe7]-NKB, respectively, were without effect. We conclude that, in the rat parotid glands, only the NK-1 receptors are coupled to the calcium-phospholipid pathway. The C-terminal part of substance P appeared to be sufficient to stimulate this route because the C-terminal octapeptide, substance P(4-11), mimicked substance P effects on both inositol trisphosphate production and calcium movements. The NK-2 and NK-3 receptors, if present in the rat parotid glands, are not associated with the calcium-phospholipid pathway.