Ethanol Inhibits the Binding of Substance P to Rat Brain Cortex NK1 Receptors

The binding of ¹²⁵I-labeled substance P (SP) to rat brain cortex membranes has been studied Under control conditions and in the presence of ethanol. The binding of SP at low concentrations (20–1000 pM) gave two components, one with a K _D value of 80 pM and another one with a K _D of 500 pM. The higher-affinity component is due to NK₁ receptors, as confirmed by the inhibition Of the SP binding by the rodent NK1 specific agonist [Sar₉ Met(O₂)₁₁]SP. Ethanol (1.7 mM) added to the binding assays inhibited by more than 50% the specific binding at a very low SP concentration (20 pM); however, it had no effect at SP concentrations ranging from 50 to 120 pM. This suggests a decrease by ethanol of the affinity of SP to the NK₁ receptors involved in this binding component. The ethanol effect disappeared at [EtOH] 0.17 mM.     

Substance P

Substance P is a potent hypotensive peptide first noted in extracts of equine brain and intestinal tissue in 1931 that also stimulates the contraction of various intestinal tissues. Its isolation proved to be particularly difficult and was not accomplished until 1970 following the chance finding of a sialogogic factor in bovine hypothalamic extracts. Utilizing this biological property to monitor purification procedures, the active sialogogic peptide was obtained in pure form and identified as Substance P by multiple chemical and biological criteria. The amino acid sequence of Substance P is HArgProLysProGlnGlnPhePheGlyLeuMetNH₂. Synthetic Substance P has been prepared and a radioimmunoassay has been developed, thus facilitating well-defined research into its physiological roles. Much of the current research is centering on its possible role in sensory neurotransmission.     

Substance P Receptors on O-2A Progenitor Cells and Type-2 Astrocytes In Vitro

Abstract: Bradykinin- and substance P (SP)-stimulated second messenger studies in isolated subsets of neuroglia showed bradykinin-stimulated synthesis of phospho- inositides (PI) in type-1 astrocytes and oligodendrocytes. SP-stimulated PI accumulation was restricted to oligoden- drocyte/type-2 astrocyte progenitor cells and type-2 astrocytes. These data were confirmed by analysis of calcium transients in single cells. In a regional study, SP-stimulated PI accumulation in primary astrocyte cultures was restricted to white matter. We conclude that regional heterogeneity in the expression of peptide receptors in cultures of primary astrocytes arises from a restricted distribution on subsets of macroglia. SP receptors restricted on cells of the oligodendrocyte/type-2 astrocyte type-2 lineage in vitro, coupled with in vivo observations by others, suggests that SP receptor expression is conserved on subsets of macroglia in vitro and possibly reactive astrocytes in vivo.     

Functional Wild-Type and Carboxy-Terminal-Tagged Rat Substance P Receptors Expressed in Baculovirus-Infected Insect Sf9 Cells

Abstract: The rat substance P (SP) receptor (SPR) was expressed in insect Sf9 cells by infection with recombinant baculovirus. The receptor bound SP with high affinity ( K _D = 360 p M ) and had a rank order of affinity of SP > neurokinin A > neurokinin B. Ligand activation of the receptor resulted in an increase in both inositol lipid hydrolysis and intracellular Ca²⁺ concentration ([Ca²⁺]_i). However, high-level expression of the receptor, in the absence of ligand, was correlated with increased basal turnover of inositol lipids and an elevated rate of Ca²⁺ influx. These results demonstrate that the Sf9 cells provide a suitable environment for the high-level expression of a functionally active SPR. Two carboxy-terminal epitope-tagged receptors (SPR-KT3 = SPR-TPPPEPET, COOH; SPR-Glu = SPR-EEEEYMPME, COOH) were also expressed. The affinity of the KT3-tagged receptor for ligand was similar to that of the wild-type receptor ( K _D = 405 p M ), and that of the Glu-tagged receptor was slightly lower ( K _D = 1,082 p M ). The high-affinity SP binding site of all three receptors was sensitive to guanosine 5'- O -(3-thiotriphosphate) pretreatment. The maximal signal-transducing ability of the epitope-tagged receptors was comparable to that of the wild-type receptor ([Ca²⁺]_i rise as a percentage of wild-type: SPR-KT3, 80–100%; SPR-Glu, 88–100%). These data show that heterologous expression in the baculovirus system results in high expression of functional wild-type and tagged receptors.     

Characterization of Receptors for Substance P in Human Astrocytoma Cells: Radioligand Binding and Inositol Phosphate Formation

UC11 cells, derived from a human astrocytoma, have a high density of functional substance P receptors. Radioligand binding studies were conducted with the highly selective neurokinin-1 receptor ligand [3H][Sar9,Met(O2)11]-substance P. Kinetic binding experiments conducted at 4 degrees C yielded an association rate constant k1 of 1.86 x 10(7) M-1 min-1, a dissociation rate constant k-1 of 0.00478 min-1, and a calculated kinetic KD of 257 pM. Saturation binding experiments yielded average values of KD = 447 +/- 103 pM, Bmax = 862 +/- 93 fmol/mg of protein. This Bmax corresponds to more than 150,000 binding sites/cell. Competition binding experiments with unlabeled [Sar9,Met(O2)11]-substance P yielded average values of KD = 491 +/- 48 pM and Bmax = 912 +/- 67 fmol/mg of protein. In [3H]inositol-labeled cells, substance P induced a robust inositol phosphate formation. Inositol trisphosphate levels increased as much as 20-fold within approximately 15 s of addition of substance P. This inositol trisphosphate formation was transient and had returned to baseline within the first 60-120 s. Inositol monophosphate formation, however, was linear for at least 2 h. Structure activity data on binding and inositol monophosphate formation confirmed the presence of a neurokinin-1 receptor subtype in these cells. Thus, the UC11 cell should be a useful model cell for delineating the physiological role of substance P receptors in astrocytes.     

Synthesis of Substance P

SUBSTANCE P has been synthesized by the solid-phase procedure of Merrifield^1,2 according to the sequence H-Arg-Pro-LysPro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH₂ reported in the previous letter.     

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.     

Stress and Plasticity in the Limbic System

The adult nervous system is not static, but instead can change, can be reshaped by experience. Such plasticity has been demonstrated from the most reductive to the most integrated levels, and understanding the bases of this plasticity is a major challenge. It is apparent that stress can alter plasticity in the nervous system, particularly in the limbic system. This paper reviews that subject, concentrating on: a) the ability of severe and/or prolonged stress to impair hippocampal-dependent explicit learning and the plasticity that underlies it; b) the ability of mild and transient stress to facilitate such plasticity; c) the ability of a range of stressors to enhance implicit fear conditioning, and to enhance the amygdaloid plasticity that underlies it.     

Solid-Phase Radioimmunoassay for Substance P

The solid-phase immunoassay for quantification of substance P has been developed. The assay is based on the repartition of anti-substance P antibodies between the insoluble phase-immobilized substance P and the free peptide. The immobilized substance P-antibody complex is then quantified with 125I-protein A. The method allowed detection of 10 pg of substance P. The values of substance P concentration obtained by the present method in different regions of the rat brain were comparable to those obtained by standard radioimmunoassay with 125I-tyr-8-substance P as tracer. The described solid-phase radioimmunoassay is a simple, sensitive, and reliable technique for quantification of substance P-like immunoreactivity in biological samples.     

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.     

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 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.     

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.     

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.