Substance P Fragments Research Articles

Substance P modulation of DAMGO binding in the brain of CXBK and Swiss-Webster mice

The effects of substance P (SP) on the binding of the selective μ opioid agonist [ 3H]DAMGO to brain membranes of CXBK and Swiss-Webster (SW) mice were compared. We have previously shown that subnanomolar concentrations of SP and N-terminal fragments of SP modulate DAMGO binding in SW brain membranes and hypothesized that modulation occurs via SP interaction with μ 1 sites. In the present study, binding assays using CXBK mice, a strain deficient in μ receptors including μ 1 sites, were performed to assess the effect of μ receptor deficiency on SP-induced modulation of DAMGO binding. Whereas the addition of 0.1 nM SP to the binding mixtures produced up to 30% increase in the values of K d and maximum binding capacity (R) for the SW strain, SP produced little or no change in the case of CXBK strain. Maximum binding capacity for DAMGO was 43% less in the brain of CXBK mice than in SW mice. No difference was observed in the estimated binding parameters of the spinal cord for the two strains. Whereas pretreatment of brain membranes of SW mice using β-funaltrexamine (β-FNA) increased from 2- to 10-fold the modulatory effect of SP, CXBK brain membranes pretreated with β-FNA remained nearly insensitive to modulation by SP. The effect of SP on the affinity of DAMGO binding in SW mice, but not in CXBK mice, was reversed by the addition of GTP. It is concluded that μ receptor deficiency can markedly influence SP-induced modulation of DAMGO binding.

Effects of N- and C-terminal fragments of substance P on ATPase and monoamine oxidase activities in rat brain

The in vitro influence of substance P (SP) C- and N-terminal fragments on the Na +,K +-ATPase and Ca 2+,Mg 2+-ATPase and monoamine oxidase (MAO) from synaptosomal membrane and extra-synaptosomal mitochondria were studied. The obtained results indicate: 1. C-terminal fragment of SP (SP 6–11) in 10 μM concentration stimulates the Ca 2+,Mg 2+-ATPase activities from cerebral cortex and hippocampus. Na +,K +-ATPase from cerebral cortex is hardly sensitive to the action of this fragment. 2. N-terminal fragment of SP (SP 1–5) in 10 μM concentration increases Na +,K +-ATPase activity from cerebral cortex and hippocampus. 3. N-terminal tetrapeptide (SP 1–4) exerts no influence on ATPases independently from their brain localization. 4. The activity of monoamine oxidase after use of C- and N-terminal fragments is unchanged.

Substance P enhancement of inhibitory avoidance learning: Mediation by the N-terminal sequence

Experiments were performed to investigate the effects of intraperitoneally administered undecapeptide substance P (SP), its N-terminal fragment SP(1–7) (SPN) and the C-terminal analog [pGlu 6]-SP(6–11) (SPC) on inhibitory avoidance learning, using a one-trial up-hill avoidance task. In Experiment 1 rats were injected with either SP (50 μg/kg), SPN (3.3, 33, 167, 333 μg/kg) or SPC (2.7, 27, 134, 268 μg/kg) immediately after the training trial. Controls received the diluent vehicles. When tested 24 hr later, rats injected with 50 μg/kg SP (37 nmol/kg) and 167 μg/kg SPN (185 nmol/kg) exhibited longer step-up latencies than vehicle-treated controls. None of the other doses of SPN nor of the C-terminal fragment influenced performance. In Experiment 2, 167 μg/kg SPN or vehicle was injected posttrial either immediately or 5 hr after the training trial. Retention latencies 24 hr later were longer for rats treated with 167 μg/kg SPN immediately after the training trial. Performance of the SPN 5-hr delay group did not differ from that of the vehicle-injected controls, ruling out proactive effects of SPN on recall.

Inhibition of angiotensin converting enzyme by N-terminal fragments of substance P

A range of N-terminal fragments of substance P (SP) were evaluated for inhibitory activity against angiotensin converting enzyme (ACE) from rat lung and brain (striatum). SP inhibited the enzyme from both sources in a concentration dependent manner (IC 50 30 μM). The N-terminal fragments SP[1–7], SP[1–6], SP[1–4] and SP[3–4] were equipotent with SP for both sources of the enzyme. However, SP[1–3] showed a difference in its activity, being more active than SP (IC 50 10 μM) in inhibiting the brain enzyme, but inactive against lung ACE. These results suggest that the inhibitory action of SP on ACE resides in the N-terminus of the peptide. The difference in reactivity towards SP[1–3] lends support to the idea that lung and brain ACE are different isozymes.

Cardiovascular effects of substance P peptides in the nucleus of the solitary tract

Microinjection of the neuropeptide substance P (SP) into the baroreceptor portions of the nucleus of the solitary tract (NTS) caused a dose-dependent decrease in blood pressure (BP) and heart rate (HR), consistent with the putative role for SP as a transmitter in the baroreceptor reflex arc. In contrast, SP elevated BP and HR when microinjected into the adjacent area postrema. Structure-activity studies of effects of SP in the NTS revealed that an aminoterminal heptapeptide fragment of SP could fully reproduce the depressor and bradycardiac effects of SP. In contrast, a car☐yterminal hexapeptide fragment of SP significantly elevated both BP and HR. The structural requirements for aminoterminal fragment effects were quite specific in terms of peptide length and sensitivity to D-amino acid substitutions. These findings are consistent with a role for SP as a baroreceptor reflex transmitter and suggest, furthermore, that this action is mediated by the aminoterminal region of SP.

Substance P Receptors in Human Pituitary: A Potential Inhibitor of Luteinizing Hormone Secretion*

Specific saturable high affinity receptors for substance-P (SP) are present in human pituitaries obtained post-mortem. The human pituitary SP receptor bound SP and two of its analogs, [D-Tyr0]SP and [D-Tyr0,Nor,Leu11]SP, with similar affinities (Kd, 1.76 X 10(-8), 3.53 X 10(-8), and 1.93 X 10(-8) mol/L, respectively). The fragments SP-(3-11) and SP-(7-10) had lower affinities for the receptor than SP (Kd, 9.24 X 10(-8) and 2.86 X 10(-7) mol/L, respectively). SP-(5-8) and SP-(1-4) were poorly bound by the receptor (Kd, greater than 10(-6) mol/L). The progressive decrease in binding with the removal of amino acid residues from the N- or C-termini of the molecule is similar to that observed in the rat. GnRH and TRH did not compete for the SP-binding sites. SP was able to inhibit GnRH-stimulated LH secretion from cultured human pituitary cells in a dose-dependent manner, while having no effect on GnRH-stimulated FSH secretion. Basal release of TSH, PRL, GH, and ACTH was unaffected by SP. These findings suggest that SP may have a physiological role in the regulation of LH secretion in man.

Capillary permeability induced by intravenous neurokinins

The effects on plasma extravasation of three increasing doses from 6.5 pmol to 650 nmol/kg of substance P (SP), SP fragments, neurokinin A (NKA), neurokinin B (NKB) and selective agonists for neurokinin receptors were assessed in three cutaneous tissues (skin of hind paws, dorsal skin and ears) by intravenous (i.v.) administration in the pentobarbitone anaesthetized rat. Dose-dependent increases in plasma extravasation were observed with the following rank orders of potency (SP greater than NKA greater than NKB) for neurokinins and (SP greater than [p-Glu6]SP(6-11) greater than SP(4-11) greater than [p-Glu5]SP(5-11) greater than SP(7-11] for C-terminal SP fragments. The metabolically stable SP analogue [p-Glu5, MePhe8, Sar9]SP(5-11) was slightly more potent than [p-Glu5]SP(5-11). The N-terminal fragments SP(1-4), SP(1-7) and SP(1-9) were inactive up to 650 nmol/kg. The NK-1 receptor selective agonists [Sar9, Met(O2)11]SP and [beta-Ala4, Sar9, Met (O2)11]SP(4-11) were more potent than the NK-2 [( Nle10]NKA(4-10] and NK-3 [( MePhe7]NKB and [beta-Asp4, MePhe7]NKB(4-10] receptor selective agonists. Plasma extravasation induced by SP (6.5 nmol/kg) was unchanged in the presence of atropine, methysergide, diphenhydramine or during the i.v. and intra-arterial (i.a.) infusion of D-Arg0[Hyp3.D-Phe7]BK, an antagonist of bradykinin. Plasma extravasation induced by SP and [Sar9, Met(O2)11]SP was significantly reduced by indomethacin while that induced by NKA, NKB, [beta-Ala4, Sar9, Met(O2)11]SP(4-11), SP(4-11) and [p-Glu6]SP(6-11) was unaffected by the cyclooxygenase inhibitor. Compound 48/80 (0.75 mg/kg), histamine (10 mg/kg) and 5-HT (10 mg/kg) caused an increase in plasma extravasation, only the effect of compound 48/80 was abolished by indomethacin. Pretreatment with compound 48/80 prevented its own action on plasma extravasation and significantly reduced that induced by 6.5 nmol/kg of SP. These results rule out the involvement of acetylcholine (muscarinic receptors), 5-HT (5-HT1 and 5-HT2 receptors), histamine (H1 receptors) and kinins (B2 receptors) in the response to SP and indicate that the two positively charged amino acids (Arg, Lys) at the N-terminal end of the SP molecule are essential to trigger the release of prostaglandins from mast cells. This mechanism is responsible for the indirect effect of SP and related peptides on capillary permeability and does not appear to be mediated by a selective SP receptor. In addition, neurokinins may increase capillary permeability by direct activation of a NK-1 receptor type on the vascular endothelium.

Degradation of substance P by neuronal and glial cells cultured from rat fetal brain and their membranes

By HPLC analysis, neuronal and glial cells cultured from rat fetal brain and their membrane preparations were shown to degrade substance P (SP) added exogenously. The degradation by neuronal cells and their membranes resulted in marked accumulation of SP fragments (1–4) and (1–6), and the accumulation, as well as the initial cleavage of SP, was strongly inhibited by metal chelators but not by phosphoramidon and captopril. Proposed cleavage sites by neuronal cells were almost identical to those by a substance P-degrading endopeptidase previously purified from rat brain by us (J. Biochem. 104: 999–1006 (1988)). On the other hand, the action of glial cells and their membranes on SP produced the fragments (1–6), (1–4), (10–11) and (8–9) in high amounts. The production, as well as the initial cleavage of SP, was inhibited not only by metal chelators and p-chloromercuribenzenesulfonic acid but also by phosphoramidon. Proposed cleavage sites by glial cells were almost identical to those attacked by endopeptidase-24.11. Thus, the proteases that degrade SP in neuronal cells and glial cells seem different.

The role of enzymatic processing in the biological actions of substance P

There is considerable evidence that substance P (SP) is a neurotransmitter in the CNS. Current findings suggest that the effects of synaptically released SP are terminated by enzymatic breakdown, primarily by endopeptidase 3.4.24.11 (endo 24.11). The products of cleavage by endo 24.11 include the amino-terminal fragment SP(1–7). Evidence suggests that SP is involved in mediating baroreceptor reflex activity in the nucleus of the solitary tract (NTS). Microinjection of SP into the NTS lowered blood pressure and heart rate. Microinjection of SP(1–7) into the NTS reproduced the effects of SP on both heart rate and blood pressure. Intra-NTS injection of phosphoramidon, an inhibitor of endo 24.11 activity, completely blocked the effects of a subsequent injection of SP. This blocking effect of phosphoramidon was unaltered by pretreatment with the opiate inhibitor naloxone. In contrast, phosphoramidon failed to block the depressor and bradycardic effects of SP(1–7). The implications of these findings regarding the role of endo 24.11 in the metabolism of SP are discussed.

Modulation of [ 3H]DAGO binding by substance P (SP) and SP fragments in the mouse brain and spinal cord via MU 1 interactions

Binding of [ 3H]DAGO to fresh, frozen or β-funaltrexamine (β-FNA) pretreated membranes of mouse brain and spinal cord was extensively studied using substance P (SP) or SP fragments as potential competitors and/or modulators. The objective was to determine whether SP exerts its analgesic effect by interacting with μ opioid receptors. The affinity of DAGO was reduced and binding capacity was increased in the presence of SP or the N-terminal SP fragments SP(1–9) and SP(1–4) but not the C-terminal SP fragment SP(5–11). Because sub-nanomolar concentrations of SP or N-terminal SP fragments displaced [ 3H] DAGO binding to a minor but detectable degree, it is suggested that SP interacts with μ 1 sites through its N-terminus portion. The effect of SP on DAGO binding was less in the spinal cord compared to the rest of the brain. Modulation of DAGO binding by SP was enhanced in the brain after pretreatment of membranes with the narcotic antagonist β-FNA. These results suggest a novel mechanism for the analgesic action of SP.

Spinal action of neurokinins producing cardiovascular responses in the conscious freely moving rat: evidence for a NK-1 receptor mechanism

This study was initiated to characterize the receptors which mediate the cardiovascular responses elicited by the intrathecal (i.th.) administration of neurokinins (NK) in the conscious freely moving rat. The dose response profile for substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) was determined over 0.065-65 nmol doses of the peptides. After i.th. administration at the T8-T10 thoracic level, only SP elicited a dose dependent pressor response. However, all NK elicited a dose dependent increase in heart rate (HR), and the following rank order of potency was observed: SP greater than NKA greater than NKB. SP (6.5 nmol) produced cardiovascular responses markedly greater than an equimolar dose of any of the seven SP fragments which were studied. The C-terminal sequences SP (4-11), [pGlu5]SP (5-11), [pGlu6]SP (6-11), and SP (7-11), as a group were slightly more potent than the N-terminal fragments, SP (1-4), SP (1-7) and SP (1-9) which were almost inactive. The NK-1 receptor selective agonists [Pro9, Met(O2)11]SP and [beta-Ala4, Sar9, Met(O2)11]SP (4-11), produced pressor and positive chronotropic responses equal to or greater in intensity than SP. With up to 6.5 nmol of the NK-2 receptor selective agonist [Nle10]NKA (4-10), no dose dependent cardiovascular response was produced and the NK-3 receptor selective agonist senktide (succinyl-[Asp6, MePhe8]SP (6-11], produced neither a cardiac nor pressor response when 6.5 nmol was administered. These results are consistent with the hypothesis that, receptors of the NK-1 subtype mediate the cardiovascular responses evoked by the spinal action of NK.

Substance P(1–7) antagonizes substance P-induced aversive behaviour in mice

Substance P (SP) and its fragments were administered intrathecally into awake mice. SP and C-terminal fragments caused dose-dependent reciprocal hindlimb scratching responses. SP(5–11) was more potent than SP not only in inducing scratching response but also in inducing aversive behaviour including licking and biting. SP(1–7) induced no behavioural reactions. However, when low doses of SP(1–7) (1.0–4.0 pmol) were injected simultaneously with SP or SP(5–11) (0.1 nmol), aversive behaviours induced by SP or SP(5–11) were significantly reduced. These results indicate that SP(1–7) formed endogenously could modulate the actions of SP or SP(5–11) in the spinal cord.

Activation of human neutrophils by substance P. Effect on oxidative metabolism, exocytosis, cytosolic Ca2+ concentration and inositol phosphate formation.

The neuropeptide substance P (SP), which has been suggested to mediate neurogenic inflammation, induces in human neutrophils the activation of the respiratory burst measured as O2 consumption and H2O2 production, and a cytochalasin B-dependent secretion of specific and azurophilic granules. The SP(4-11) fragment is much more stimulant than the entire molecule, whereas the SP(1-4) fragment is inactive. The respiratory and secretory response to SP are associated with an activation of phosphoinositide turnover, of Ca2+ influx and release from intracellular stores. Pertussis toxin inhibits 70% of the respiratory response and the residual 30% activity remains, even increasing 10-fold the concentration of the toxin. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine, a putative inhibitor of protein kinase C, does not modify the respiratory response to SP. Cytochalasin B significantly depresses the activation of the respiration by SP, whereas it moderately enhances the activation of phosphoinositide turnover and potentiates the increase of intracellular Ca2+ concentration. The results are discussed in relation to the receptor apparatus involved in SP activity, the signal transduction sequence activated by SP for the stimulation of NADPH oxidase, and the role of cell response to SP in the inflammatory process.

Effects of substance P and neurokinin A (substance K) on motor behavior: unique effect of substance P attributable to its amino-terminal sequence

The effects of intraventricular injections of the neuropeptides substance P (SP) and neurokinin A (NK-A; also called substance K) on spontaneous motor behavior were examined in mice. SP and NK-A were essentially equipotent at enhancing grooming and scratching behavior, and at reducing sniffing behavior. However, SP significantly enhanced hindlimb rearing behavior, while NK-A reduced this behavior. The effects of 3 other tachykinins, physalaemin, eledoisin and kassinin, were comparable to those of NK-A, including the reduction in rearing. Thus, SP is unique among tachykinins in its potentiation of rearing behavior. It was further demonstrated that carboxy-terminal SP fragments with tachykinin activity on smooth muscle resemble NK-A, and not SP, in their effects on motor behavior. In contrast, amino-terminal SP fragments, devoid of tachykinin-like activity, reproduced the one motor effect unique to SP, enhanced rearing, while lacking those actions common to all tachykinins. The structural requirements for enhanced rearing behavior by amino-terminal fragments were quite specific, in terms of chain length and sensitivity to d-amino acid substitutions, with the natural amino-terminal hexa- and heptapeptides being most active. The implications of these findings are discussed in light of recent observations that these same amino-terminal SP fragments are produced in vivo as metabolites of SP.

Relationship of Substance P to Catecholamines, Stress, and Hypertension

This paper presents results on the function of the adrenal medulla, especially the influence of substance P (SP) on the cholinergic-adrenergic interaction. Interconnections between the function of SP and its role in the development and maintenance of hypertension as well as its role in stress and adaption are investigated. Substance P acts only in situations with an increased nervous activity, for example, after stress-induced activation of the pituitary-adrenal and sympathetic-adrenal axis. Substance P normalizes stress-induced disorders by maintaining homeostasis in the catecholamine system. The basis of the antistress effect is the modulation of both biosynthesis and release of catecholamines in the adrenals. The homeostasis effect of SP, as well as the antistress effect do not require the complete sequence of the molecule. The N-terminal SP fragment is fully active and has no side effects.

Modulation of blood pressure by substance P: Opposite effects of N- and C- terminal fragments on anesthetized rats

Considerable evidence suggests that substance P (SP) may be a transmitter mediating the depressor effects of baroreceptor reflex activation within the brainstem, yet intracerebroventicular administration of SP is reported to result in a pronounced pressor effect. In this study, SP injected into the 4th cerebral ventricle produced a biphasic effect; a brief decrease in blood pressure followed by a lengthy increase. Similar injections of a carboxyterminal fragment of SP produced only a pressor effect of long duration. Injection of an amino-terminal SP fragment produced only a brief, rapid depressor effect. These results suggest that the amino-terminal sequence of SP may be involved in mediating the depressor effects of baroreceptor activation.

Relationship of Substance P to Catecholamines, Stress, and Hypertension

Summary: This paper presents results on the function of the adrenal medulla, especially the influence of substance P (SP) on the cholinergic-adrenergic interaction. Interconnections between the function of SP and its role in the development and maintenance of hypertension as well as its role in stress and adaption are investigated. Substance P acts only in situations with an increased nervous activity, for example, after stress-induced activation of the pituitary-adrenal and sympathetic-adrenal axis. Substance P normalizes stress-induced disorders by maintaining homeostasis in the catecholamine system. The basis of the antistress effect is the modulation of both biosynthesis and release of catecholamines in the adrenals. The homeostasis effect of SP, as well as the antistress effect do not require the complete sequence of the molecule. The N-terminal SP fragment is fully active and has no side effects.

Pharmacological characterization and autoradiographic localization of substance P receptors in guinea pig brain

[ 3H]Substance P ([ 3H]SP) was used to characterize substance P (SP) receptor binding sites in guinea pig brain using membrane preparations and in vitro receptor autoradiography. Curvilinear Scatchard analysis shows that [ 3H]SP binds to a high affinity site ( K d =0.5 nM ) with a B max of 16.4 fmol/mg protein and a low affinity site ( K d =29.6 nM ) with a B max of 189.1 fmol/mg protein. Monovalent cations generally inhibit [ 3H]SP binding while divalent cations substantially increased it. The ligand selectivity pattern is generally similar to the one observed in rat brain membrane preparation with SP being more potent than SP fragments and other tachykinins. However, the potency of various nucleotides is different with GMP-PNP > GDP > GTP. The autoradiographic distribution of [ 3H]SP binding sites shows that high amounts of sites are present in the hippocampus, striatum, olfactory bulb, central nucleus of the amygdala, certain thalamic nuclei and superior colliculus. The cortex is moderately enriched in [ 3H]SP binding sites while the substantia nigra contains only very low amounts of sites. Thus, the autoradiographic distribution of SP binding sites is fairly similar in both rat and guinea pig brain.

Relationship of Substance P to Catecholamines, Stress, and Hypertension

This paper presents results on the function of the adrenal medulla, especially the influence of substance P (SP) on the cholinergic-adrenergic interaction. Interconnections between the function of SP and its role in the development and maintenance of hypertension as well as its role in stress and adaptation are investigated. Substance P acts only in situations with an increased nervous activity, for example, after stress-induced activation of the pituitary-adrenal and sympathetic-adrenal axis. Substance P normalizes stress-induced disorders by maintaining homeostasis in the catecholamine system. The basis of the antistress effect is the modulation of both biosynthesis and release of catecholamines in the adrenals. The homeostasis effect of SP, as well as the antistress effect do not require the complete sequence of the molecule. The N-terminal SP fragment is fully active and has no side effects.

The effect of substance P and its fragments on passive avoidance retention and brain monoamine activity

It has been shown that substance P(SP), as well as its carboxy and amino terminal fragments, affects a wide range of behaviors. In order to test the CNS activity of these fragments, we measured their effects on passive avoidance learning and monoamine activity. Following one-trial passive avoidance training, mice were injected intraventricularly with either a carboxy or amino terminal SP fragment (SP-C or SP-N), SP itself or phosphate-buffered saline (PBS). SP-N enhanced avoidance retention, which was tested 24 h after training. In a second experiment, monoamine activity was measured one hour after intraventricular injection of SP, PBS or SP fragments. SP-C decreased both nigral 5-hydroxyindoleacetic acid/5-hydroxytryptamine (5-HIAA/5-HT) and, to a lesser extent, 3,4-dihydroxyphenylacetic acid/dopamine, while SP-N increased nigral 5-HIAA/5-HT. It was concluded that SP-N and SP-C can exert behavioral and neurochemical effects that may be independent of the parent SP molecule.