Substance P and antinociception

Substance P (SP)-induced antinociception is still a topic of controversy. Some investigators have failed to see an antinociceptive effect of SP, particularly following intraperitoneal administration. In the present experiments SP induced significant hot plate antinociception in male mice, following intraperitoneal administration. SP exhibited a bell-shaped dose response curve, and the antinociceptive effect was dependent on the pH of the vehicle. The antinociceptive effect of SP lasted for at least 1 hr and was naloxone-reversible. The antinociceptive effect of SP could be prevented by housing subjects collectively rather than individually during the experiment. In conclusion, the bell-shaped dose response curve, the solution pH and different testing procedures all influence the effects of SP on nociception. Given this complexity, it is not surprising that some experiments fail to demonstrate antinociception following SP administration.     

Amino-acid Sequence of Substance P

IN 1931 von Euler and Gaddum¹, studying the tissue distribution of acetylcholine, found that brain and intestine contained a substance that stimulated contraction of the isolated rabbit jejunum and caused transient hypotension when injected intravenously into anaesthetized rabbits. These effects could not be ascribed to acetylcholine, for they were not prevented by the previous administration of atropine. The initial studies were made on crude acid alcohol extracts of equine brain and intestine, dried in powder form. The active principle in the preparation was later referred to as substance P (P for powder) and this non-committal term subsequently achieved widespread acceptance in the literature, in the absence of any clearly definable biological role for the compound (or compounds) involved.     

Synthesis of peptides by the solid-phase method. V. Substance P and analogs

We have synthesized a series of 12 analogs of the undecapeptide substance P in order to perform a structure-activity study of this peptide. In the present work, each residue was substituted by L-alanine, and the C-terminal amide was replaced by the free carboxyl in order to pinpoint biologically important side chains and functional groups. The synthesis of the analogs was carried out by the automatic solid-phase method. Couplings were performed by the symmetrical anhydride procedure. After cleavage with liquid HF, the peptides were purified by gel filtration and ion-exchange chromatography. Their purity was assessed by thin-layer chromatography, paper electrophoresis, amino acid and elemental analyses, and high pressure liquid chromatography. They were tested for biological activity in vitro on the ileum of the guinea pig, the mesenteric vein of the rabbit, and the vas deferens of the rat, and in vivo by measuring their effect on the blood pressure of the rat.     

Substance P Receptor Expression and Cellular Responses to Substance P in Prenatal Rat Spinal Cord Cells

Abstract

Substance P receptors (SPRs) are expressed by prenatal rat spinal cord neurons and glial cells early in their differentiation, and SPRs may mediate developmental influences in the developing spinal cord. In order to understand better early SPR expression, we quantified SPR mRNA in the rat spinal cord during prenatal development using a cDNA probe for the rat SPR in nuclease protection assays. SPR mRNA was present in the rat spinal cord at E14, the earliest stage examined, and the presence of specific binding sites for radiolabeled SP suggested that SPRs were expressed at the protein level as well. Comparisons of samples from rats at different prenatal ages showed that the relative abundance of SPR mRNA declined by about 75% from E14 through the remainder of prenatal development. Assays of the hydrolysis of phosphatidyl inositol performed on prenatal spinal cord cells in culture revealed that SP caused a small but significant stimulation. These results show that expression of SPRs is an early molecular event in the development of the rat spinal cord in vivo and that SPRs on young spinal cord cells can mediate functional responses at early developmental stages.     

Substance P. New cyclic analogs

[Adpoc-Glu(N3)6, (Met-N3)11] substance P-(6-11)-peptide was reacted with diamines H2N(CH2) nNH2 (n = 3-10, 12) to give cyclopeptides. Subsequent careful cleavage of the Adpoc group leads to the formation of compounds of type cyclo-[H-Glu-Phe-Phe-Gly-Leu-Met-NH-(CH2) n-NH-] X HCl. The substances produce a specific two-phase myotropic effect in experiments on isolated guinea pig ileum. The compounds where n is 3, 7, 12 exhibit also a hypotensive activity when assayed on anaesthetized rats.     

Substance P and microcirculation during stress

The experiments have been made on rats with the use of animals' immobilization on the back during 1 or 24 h as a stressor. Intravital study of the microcirculation in the rat mesentery has shown that P substance in a concentration of 7 X 10(-8) M aggravated the disturbances caused by the immobilization, while antiserum to P substance with an activity of 150 ng SP/equiv. normalized the microcirculation. The action of P substance and antiserum was similar as regards disturbances of vascular permeability for colloid carbon particles. It is concluded that P substance participates in the mechanisms of microcirculatory and vascular permeability disturbances during stress.     

Canine Limbic System Substance P Receptors

Abstract

A canine limbic system preparation is utilized as a source of substance P receptor(s) to screen gradient RP-HPLC fractions for the presence of receptoractive-substance P activity, and to quantify endogenous receptoractive-substance p in biological extracts such as human tooth pulp. The binding characteristics, K_D = 1.3 nM and B_max = 11 fmol mg^?1 protein, are similar to values obtained from receptors produced from other biological sources such as whole rat brain, minus cerebellum.     

Substance P and spinal neurones.

When applied by microiontophoresis, substance P (sP) had a strong, but slow and prolonged excitatory action on nearly half the neurones tested in the lumbar spinal cord of cats. Motoneuronal antidromic field potentials only occasionally showed a significant effect of sP. Cerebral cortical neurones in cats and rats were much less readily excited than spinal interneurones. Some unresponsive units showed evidence of a depressant effect of sP. Although sP may have a significant function in central afferent pathways, it is not likely to be a quickly-acting synaptic transmitter.     

Proteolysis Controls Endogenous Substance P Levels

Substance P (SP) is a prototypical neuropeptide with roles in pain and inflammation. Numerous mechanisms regulate endogenous SP levels, including the differential expression of SP mRNA and the controlled secretion of SP from neurons. Proteolysis has long been suspected to regulate extracellular SP concentrations but data in support of this hypothesis is scarce. Here, we provide evidence that proteolysis controls SP levels in the spinal cord. Using peptidomics to detect and quantify endogenous SP fragments, we identify the primary SP cleavage site as the C-terminal side of the ninth residue of SP. If blocking this pathway increases SP levels, then proteolysis controls SP concentration. We performed a targeted chemical screen using spinal cord lysates as a proxy for the endogenous metabolic environment and identified GM6001 (galardin, ilomastat) as a potent inhibitor of the SP _1–9-producing activity present in the tissue. Administration of GM6001 to mice results in a greater-than-three-fold increase in the spinal cord levels of SP, which validates the hypothesis that proteolysis controls physiological SP levels.     

Substance P Causes Seizures in Neurocysticercosis

Neurocysticercosis (NCC), a helminth infection of the brain, is a major cause of seizures. The mediators responsible for seizures in NCC are unknown, and their management remains controversial. Substance P (SP) is a neuropeptide produced by neurons, endothelial cells and immunocytes. The current studies examined the hypothesis that SP mediates seizures in NCC. We demonstrated by immunostaining that 5 of 5 brain biopsies from NCC patients contained substance P (SP)-positive (+) cells adjacent to but not distant from degenerating worms; no SP+ cells were detected in uninfected brains. In a rodent model of NCC, seizures were induced after intrahippocampal injection of SP alone or after injection of extracts of cysticercosis granuloma obtained from infected wild type (WT), but not from infected SP precursor-deficient mice. Seizure activity correlated with SP levels within WT granuloma extracts and was prevented by intrahippocampal pre-injection of SP receptor antagonist. Furthermore, extracts of granulomas from WT mice caused seizures when injected into the hippocampus of WT mice, but not when injected into SP receptor (NK1R) deficient mice. These findings indicate that SP causes seizures in NCC, and, suggests that seizures in NCC in humans may be prevented and/or treated with SP-receptor antagonists.     

Substance P innervation of the rat thymus

Immunohistochemistry for substance P (SP) in the rat thymus revealed fine varicose neural profiles in specific regions of the thymus. Thymic SP innervation was abundant within the capsule and interlobular septa. The majority of SP+ nerve fibers within the septa were free of vascular association, although some fibers were associated with the vasculature deep within the septa. SP+ nerve fibers entered the thymic cortex from the septa and distributed among cortical thymocytes and mast cells. Along the corticomedullary junction, SP+ nerve fibers were found in association with the vasculature. The medullary region of the thymus received only a sparse innervation of SP+ fibers. In addition, SP+ nerve fibers coursed adjacent to OX-8+ cells and mast cells in the extrathymic connective tissue surrounding the thymus. The present study provides evidence that SP is present in nerve fibers in the thymus, and may be available to interact with thymocytes, mast calls, and other cells in the thymus, and affect their development and function.     

Differentiation of neuronal from non-neuronal Substance P

Substance P (SP) originally found as a neuropeptide in capsaicin-sensitive sensory neurons, had more recently been identified in non-neuronal cells, especially under pathological conditions. Neuronal and non-neuronal SP may perform distinct functions. A simple technique to differentiate different SP sources is currently unavailable. Herein, we describe a two-step sequential acetic acid extraction to differentiate SP source. The efficiency of this two-step extraction in differentiating SP in capsaicin-sensitive neurons was verified by using capsaicin as a tool to deplete SP in sensory neurons. Specifically, Balb-c mice were treated with high dose capsaicin (200 mg/kg). Skin was removed two weeks after treatment. In a separate experiment, lung and skin tissues from control animals (untreated) were incubated in-vitro with capsaicin, and sequential acetic acid extraction was performed. Following capsaicin treatment, both in-vivo and in-vitro, SP recovered in first extraction decreased significantly in lung and skin. Lastly, presence of capsaicin solvent (10% methanol and 10% Tween 80) or protease inhibitor cocktail in solution altered SP EIA test, yielding false positive results. These results demonstrated that SP in capsaicin sensitive sensory neurons was extracted in initial extraction of 15 min while non-neuronal SP was present in second extraction. Because SP in non-neuronal tissues may possibly be more important in pathological conditions, this technique could be useful in determining effects of various treatments on neuronal and non-neuronal SP levels and their consequences.     

Substance P Hydrolysis by Human Serum Cholinesterase

Highly purified human serum cholinesterase (EC 3.1.1.8, also known as pseudocholinesterase and butyrylcholinesterase) had peptidase activity toward substance P. Digestion of substance P was monitored by high performance liquid chromatography, which separated three product peptides. The cleavages occurred sequentially. The first peptide to appear as Arg1-Pro2. The Km for this hydrolysis was 0.3 mM; maximum activity was 7.9 nmol min-1 mg-1 of protein, which corresponded to a turnover number of 0.6 min-1. A second cleavage yielded Lys3-Pro4. A third cleavage occurred at the C-terminal, where the amide was removed from Met11 to yield a peptide containing residues 5-11. Both the peptidase and esterase activities of the enzyme were completely inhibited by the anticholinesterase agent, diisopropylfluorophosphate. Substance P inhibited the hydrolysis of benzoylcholine (a good ester substrate) with a KI of 0.17 mM, indicating that substance P interacted with cholinesterase rather than with a trace contaminant. Peptidase and amidase activities for serum cholinesterase are novel activities for this enzyme. It was demonstrated previously that the related enzyme acetylcholinesterase (EC 3.1.1.7) catalyzed the hydrolysis of substance P, but at entirely different cleavage sites from those reported in the present work. Since butyrylcholinesterase is present in brain and muscle, as well as in serum, it may be involved in the physiological regulation of substance P.     

Measurement of Substance P Metabolites in Rat CNS

A procedure based on ion-exchange chromatography for chemical separation and radioimmunoassays for quantitation of substance P (SP), the SP(1-7), and C-terminal fragments, respectively, has been developed. The procedure allows the determination of these fragments in the presence of large (i.e., 50- to 100-fold) excess of parent compound. The chemical identity of isolated SP and fragments was studied with preparative electrophoresis on dilute agarose gel and with HPLC. The activity identified as SP(1-7) comigrated with the authentic standard whereas practically all activity isolated as C-terminal fragments comigrated with SP(5-11). The levels of C-terminal fragments in rat brain areas rich in SP and in spinal cord were 1-2% of those of parent compound. The levels of SP(1-7) were always higher, in the spinal cord markedly higher (three to five times). Postmortem storage of samples from brain and spinal cord indicated that SP(1-7) levels fell more rapidly than those of SP or C-terminal fragments.     

Preincubation with Substance P Induces Substance P-Stimulated Phosphatidylinositol Turnover in Cultured Cerebellar Astrocytes

In this study we have investigated the effects of preincubation of cultured astrocytes with substance P (SP) on subsequent 125I-Bolton-Hunter conjugated SP (125I-BHSP) receptor binding, and SP-stimulated phosphatidyl-inositol (PI) accumulation. Spinal cord astrocytes preincubated for up to 96 h with SP (0.001-1,000 nM) suffered a dose-dependent decrease in both subsequent 125I-BHSP and SP-stimulated PI turnover. In contrast, preincubation of cerebellar astrocytes with SP resulted in an increase in SP-stimulated PI turnover, with no change in 125I-BHSP receptor binding. SP-induced PI turnover in cerebellar astrocytes was maximal after 72 h of preincubation with 0.1 nM SP. These data suggest that increased coupling between receptor and second messenger occurs in response to chronic exposure to SP.     

Characterization of the Substance P (NK-1) Receptor in Tunicamycin-Treated Transfected Cells Using a Photoaffinity Analogue of Substance P

Abstract: Chinese hamster ovary cells expressing the N -glycosylated substance P (NK-1) receptor were treated with the glycosylation inhibitor tunicamycin and photolabeled with ¹²⁵I-Bolton-Hunter- p -benzoyl- l -phenylalanine⁸-substance P. Two radioactive proteins of M_r 80,000 and 46,000, representing the glycosylated and nonglycosylated substance P (NK-1) receptor, respectively, were observed. The IC₅₀ for the inhibition of photolabeling of both receptor forms was 0.3 ± 0.1 n M for substance P and 30 ± 5 n M for neurokinin A (substance K). Thus, glycosylation of the substance P (NK-1) receptor has no detectable effect on the affinity of the substance P (NK-1) receptor for substance P or neurokinin A (substance K).     

Substance P analogues act as broad-spectrum neuropeptide antagonists

Summary Neuropeptides including bombesin, vasopressin and bradykinin are increasingly implicated in the control of cell proliferation. There is now considerable evidence that the growth of certain common cancers including small cell lung cancer (SCLC) can be stimulated by multiple neuropeptides which act in an autocrine/paracrine fashion. Consequently, the development of broad spectrum neuropeptide, antagonists could be of therapeutic interest. Indeed, certain substance P (SP) analogues including (DArg¹, DPhe⁵, DTrp^7,9, Leu¹¹) SP and (Arg⁶, DTrp^7,9, MePhe⁸)SP (6–11) inhibit the actions of multiple neuropeptides and block the growth of SCLC cells in vitro and in vivo. Moreover, one of these compounds is now in a phase I clinical study and so an understanding of the mechanism of action of these SP analogues is both of fundamental as well as clinical interest. We have found that the SP analogues coordinately and reversibly inhibit the downstream signals which emanate from neuropeptide receptors and competitively block the binding of neuropeptides to their respective receptors. These and other results using novel SP analogues which are reviewed here, suggest that the SP analogues act directly on the neuropeptide receptors to block neuropeptide action.     

Substance P effects on spinal nociceptive neurones.

In decerebrated spinal cats, the effects of iontophoretically applied acetylcholine (ACh) and substance P were examined on the responses of dorsal horn neurones to noxious stimulation and touch of the skin. Both agents, in amounts that did not have a significant direct effect on the neuronal firing rate, prolonged the response of the cells to noxious stimulation but did not alter that to touch stimulation. The peptide and ACh potentiated the late, but not the early, responses of dorsal horn neurones to sural A_δ and C afferent stimulation. Substance P-induced potentiation of the above responses was observed even when the agent did not produce a significant depolarization of nociceptive cells. In greater amounts, the peptide depolarized the neurones, an effect that was not associated with a detectable change in the membrane resistance. These results indicate that substance P facilitates nociceptive pathways by potentiating the subliminal fringe and, in greater amounts, by depolarizing the cells. The failure by the peptide to potentiate touch-induced excitation of the nociceptive neurones appears not to be due to the selectivity of the drug effect but due to the absence of subliminal fringe.     

Substance P immunoreactivity in rat von Ebner's gland

Summary The present immunohistochemical study revealed substance P-immunoreactive neuronal elements in the von Ebner's gland of rats. Immunoreactive ganglion cells were observed as single cells or groups of several immunoreactive ganglion cells among intra-lingual muscles, at the base of the vallate papillae and near the von Ebner's gland. Very numerous substance P-immunoreactive varicose nerve fibres ran closely associated with the serous cells and excretory duct cells, and were seen to run along blood vessels in the gland. Since substance P-immunoreactive ganglion cells were present near the glands, the immunoreactive varicose nerve fibres in the von Ebner's gland may be partly derived from the intra-lingual ganglion cells. These substance P-immunoreactive varicose nerve fibres may have an effect on the secretory activity of the serous cells and duct cells, and on the vasodilation of blood vessels of the von Ebner's gland. Actin immunoreactivity was seen in numerous myoepithelial cells embracing serous cells and duct cells, and in the smooth muscle cells of blood vessels of the gland. By using a double immunolabelling technique with anti-substance P and anti-actin sera, substance P-immunoreactive varicose nerve fibres were found to be in close contact with myoepithelial cells.