Binding of an iodinated substance P analogue to cultured anterior pituitary prolactin- and luteinizing hormone-containing cells.

Several lines of anatomic, biochemical, and pharmacological evidence suggest that the neuropeptide substance P has a direct action on cells of the anterior pituitary lobe via a specific neurokinin-1 receptor. In the present study we confirmed this association by combining Bolton-Hunter iodinated substance P-receptor autoradiography with immunocytochemistry on cultured anterior pituitary cells. Radiolabeled substance P was bound to living cell cultures at 0 degrees C, and after a brief wash the cultures were fixed and processed immunocytochemically for prolactin and luteinizing hormone. A large proportion of cultured anterior pituitary cells possessed substance P binding sites. When receptor autoradiography was combined with immunocytochemistry, it was evident that both prolactin- and luteinizing hormone-immunoreactive cells were labeled with radiolabeled substance P. However, a small proportion of the radioligand-labeled cells were not stained by the immunocytochemical procedure, suggesting that additional cell types possess substance P receptors. The present study presents morphological evidence that substance P binds to prolactin- and luteinizing hormone-containing cells of the anterior pituitary lobe. Therefore, it is likely that substance P has a direct action on mammotrophs and gonadotrophs.     

Presence and Ontogeny of Enkephalin and Substance P in the Chick Ciliary Ganglion

The avian ciliary ganglion has been reported to contain both enkephalin and substance P in preganglionic terminals. However, extensive biochemical characterization of these antigens has not been completed. Using radioimmunoassays specific for Met5- and for Leu5-enkephalin and for substance P we identified immunoreactive substances in ganglionic extracts that comigrate on HPLC columns with standard Met5- and Leu5-enkephalin and with substance P. The ontogeny of Met5-enkephalin and substance P during embryogenesis was determined in ganglionic extracts and we found that the content of Met5-enkephalin in the ganglion reached a peak at embryonic stage 37 whereas the content of substance P in the ganglion reached its maximum in the adult.     

Purification of vanadium binding substance from the blood cells of the tunicate, Ascidia sydneiensis samea

Vanadium binding substance has been partially purified through chromatographies on Sephadex G-25 and SE-Cellulose at pH 2.3. The binding substance was colorless, relatively stable and maintained vanadium ion. The vanadium ion in the substance existed in vanadyl form (VO(IV)). Furthermore, the substance had an apparent affinity for exogenous vanadium ion(V) and contained a reducing sugar.     

The organization of capsaicin-sensitive visceral afferent systems

It has become clear that a number of neuropeptides are found in sensory nerves, some of which have been identified in visceral afferents. The best studied peptide is substance P, which has been localized in a population of capsaicin-sensitive visceral afferents. It has been established that there are a varied proportion of substance P-containing afferents in different visceral structures. In general, the peripheral termination of these nerves is around blood vessels. The central terminations of visceral afferents are in laminae I and V in the dorsal horn of the spinal cord. Substance P has been localized in these laminae and appears to be capsaicin-sensitive and therefore of sensory origin. Recently, substance K, which is derived from the same gene as substance P, has been found in visceral structures. Calcitonin gene-related peptide has been found in certain viscera to be contained in capsaicin-sensitive nerves. The contribution that other peptides make to visceral afferent innervation is not known.     

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).     

Lipid of Streptomyces sioyaensis

Three ninhydrin-positive lipids of Streptomyces sioyaensis were found. These lipids were called substance A, B and C, tentatively. Study on the distribution of these lipids in Actinomycetales has shown that substance A was common in all of the strains tested, and that substance B was found in the limited strains. The substance C was characteristic only in Streptomyces sioyaensis.     

Substance P: its pharmacology and physiological roles

The recent identification and synthesis of the endecapeptide substance P has renewed interest in this naturally occurring compound. The original substance P of Euler and Gaddum was a mixture of biologically active substances, some of which were peptides. The peptide component was responsible for gut-contracting, hypotensive and sialogogic properties attributed to substance P. For almost 40 years following its discovery, substance P resisted isolation and purification. Even the most highly active preparations were heterogeneous and there was a divergence of results from experiments using preparations which were partially purified by different techniques. Nevertheless much of the early work on the distribution and pharmacology of crude substances P has been confirmed using the synthetic endecapeptide and has stimulated research towards the elucidation of a possible functional role for substance P. Most proposed functions have been highly speculative but, at present, evidence is accumulating in support of a physiological role for substance P as a neurotransmitter in sensory pathways.     

Substance P-induced contraction of rabbit airways: mechanism of action.

The mechanism by which substance P induces contraction of airway smooth muscle has been the subject of numerous reports. It has been suggested that in rabbit airways the action of substance P is indirect, via the release of endogenous acetylcholine, whereas this is not so in other species. The present detailed study investigated whether substance P-induced contraction in rabbit isolated bronchus and trachea is due to the release of endogenous acetylcholine or in bronchus is due to histamine release and whether substance P is metabolized by the enzymes enkephalinase and acetylcholinesterase. Isometric contraction to cumulative addition of substance P was measured in the presence of 10(-6) and 10(-4) M atropine, 10(-6) M pyrilamine, 10(-5) M phosphoramidon, or 3 x 10(-7) M neostigmine. Neither atropine nor pyrilamine had any effect on the substance P responses. Phosphoramidon, however, produced a 12-fold shift to the left in the response curve with a decrease in the 50% effective concentration from 7.0 x 10(-8) to 6.1 x 10(-9) M (n = 4 control and 5 treated; P less than 0.05). In contrast, neostigmine at a concentration that produced a sixfold shift to the left in the acetylcholine response curve had no effect on substance P responses. We conclude that, in rabbit airways in vitro, substance P-induced contraction is not mediated by release of endogenous acetylcholine or histamine. In addition, endogenous enkephalinase but not acetylcholinesterase may be involved in the degradation of substance P. Our results show that, in contrast to previous studies in rabbits, the mechanism of action of substance P may resemble that described in humans.     

Substance K: a novel mammalian tachykinin that differs from substance P in its pharmacological profile

The primary structure of one of the bovine brain substance P precursors has shown the existence of a second mammalian tachykinin sequence, named substance K, that is remarkably homologous to that of the amphibian peptide kassinin. In this study, three substance K sequences were chemically synthesized and were submitted to parallel bioassays with kassinin, substance P and physalaemin. The results show that the three substance K peptides all possess biological activities characteristic of the tachykinin family and that their biological activities more closely resemble those of kassinin than those of substance P or physalaemin. This suggests that substance K may have a physiological role which is related to but different from that of substance P in mammalian organisms.     

Characterization of substance P-like immunoreactivity in peripheral sensory nerves and enteric nerves by high pressure liquid chromatography and radioimmunoassay

Material exhibiting immunoreactivity for substance P in enteric nerves, obtained from the myenteric plexus of the guinea pig small intestine, and in the peripheral ends of sensory nerves of the ureter, atrium and superior mesenteric artery, was characterized by separation by high pressure liquid chromatography, and quantified by radioimmunoassay of fractions collected from the chromatograph. Capsaicin, which depletes substance P-like immunoreactivity from sensory, but not from other substance P-containing nerves, reduced the content of substance P-like immunoreactivity in ureter, atrium and superior mesenteric artery by more than 99.5%, whereas the reduction in immunoreactive material in the myenteric plexus was less than 10%. Separation of extracts of myenteric plexus, ureter and atrium on a reversed-phase column gave major peaks corresponding to authentic substance P and minor peaks that coeluted with oxidized substance P. If the extracts were oxidized with hydrogen peroxide before chromatography, all the immunoreactivity was found in the peak corresponding to oxidized substance P. In the superior mesenteric artery extracts, in addition to the components corresponding to substance P and its oxidized derivative, there was a small intermediate peak that has yet to be identified. Physalaemin, which has been suggested to be present in mammalian nerves, was not detectable in any of the extracts. It is concluded that both enteric nerves and the peripheral processes of sensory nerves which show immunoreactivity for substance P in this species contain the authentic peptide.     

Ostertagia ostertagi: excretory secretory chemotactic substance from infective larvae as cause of eosinophil locomotion

The infective (third stage) larva of Ostertagia ostertagi produced an excretory secretory substance that is chemotactic for bovine eosinophils. The eosinophil chemotactic substance was present in supernatants of larval cultures within 6 hr of incubation in Eagle's Minimum Essential Medium containing 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid. The substance responsible for eosinophil chemotaxis has activity in small amounts, a molecular weight (MW) greater than 2000, and is heat labile at 100 C. Bovine eosinophils appeared to have a receptor for the chemotactic excretory secretory substance which is either identical or structurally similar to the previously described eosinophil chemotactic substance present in soluble third stage larval extracts. The larval substance may cause eosinophil accumulation in the abomasal tissue of cattle with ostertagiasis.     

Phage formation in Staphylococcus muscae cultures. VII. Partial purification of the protein factor necessary for virus synthesis

1. A substance is present in autolyzed pepsin solutions which stimulates the release of phage by some strains of S. muscae when added to Fildes' synthetic medium. 2. The substance is assayed by determining the quantity necessary to increase the phage yield to one-half the maximum value, using the one-step growth curve technique. 3. The substance has been concentrated and partially purified (500-fold) by heavy metal precipitation, butyl alcohol extraction, and absorption on norit. 4. No known amino acid or accessory growth substance tested could replace this substance.     

Prostaglandin in the saliva of the cattle tick Boophilus microplus.

Previous studies of saliva from engorged female cattle ticks revealed a component which induced contration of some isolated smooth muscles. Fractionation and further characterisation have shown that this substance is of the "slow-reacting" type, but that it is neither a bradykinin nor slow-reacting substance of anaphylaxis. The substance is deactivated by incubation with 15-hydroxprostaglandin dehydrogenase and its pharmacological properties also support its classification as a prostaglandin. A second pharmacologically-active component has now been found in the saliva but has not yet been characterised.     

Substance P enhances cation permeability of neuronal cell lines

Substance P stimulated the uptake of guanidinium in neuroblastoma X glioma hybrid cells and neuroblastoma cells but not in polyploid glioma cells. Guanidinium has previously been shown to pass the action potential Na+ channel in the two neuronal cell lines. Half-maximal stimulation was reached at 3 microM substance P and, with the hybrid cells, a saturation was seen above 10 microM. The analogue (D-Pro2,D-Trp7,9)-substance P, recently described as a substance P antagonist, caused a stimulation of guanidinium uptake comparable to that seen in the presence of substance P and did not inhibit the stimulation exerted by substance P. The pharmacological properties of the substance P-activated ion channel were investigated. Tubocurarine, phentolamine and propranolol blocked the substance P-stimulated guanidinium uptake with half-maximal inhibitory concentrations of 0.5, 5 and 50 microM. A similar characteristics has been found previously with the veratridine-activated Na+ channel in the cell lines investigated here. Peptides structurally related to substance P such as physalaemin and eledoisin, or others such as neurotensin, bradykinin, D-Ala2, Met5-enkephalinamide and ACTH(1-24) did not affect guanidinium uptake. In view of the high concentrations of substance P required for eliciting an effect in the cell lines, the involvement of specific receptors is questioned. A direct interaction of the peptide with the action potential Na+ channel is discussed.     

Features of intermediary steps around the 688-nm substance in the heme oxygenase reaction

The degradation of protoheme in the heme oxygenase reaction involves three oxidation steps: from protoheme to hydroxyheme, from hydroxyheme to a 688-nm substance, a protein-bound intermediate, and from the 688-nm substance to a biliverdin-iron complex. The 688-nm substance has a ferrous iron and it readily binds carbon monoxide to form a CO-complex, called the 638-nm substance (Yoshida, T., Noguchi, M., & Kikuchi, G. (1980) J. Biochem. 88, 557-563). The ferric 688-nm substance was prepared from the 638-nm substance by the addition of potassium ferricyanide together with aspiration to eliminate CO. The ferric 688-nm substance did not show any distinct absorption maximum in the red region of the absorption spectrum. The ferric 688-nm substance was readily reduced on the addition of the NADPH-cytochrome P-450 reductase system, but the ferric 688-nm substance could also be reduced spontaneously though at a very low rate. The ferrous 688-nm substance free from excess reducing agents was prepared by passing the 638-nm substance through a column of Sephadex G-25. The ferrous 688-nm substance was degraded to a biliverdin-iron complex much more rapidly in the presence of the NADPH-cytochrome P-450 reductase system than in its absence, indicating that a reducing equivalent is essential for the initiation of heme degradation even when starting from the ferrous 688-nm substance. Cyanide was found to bind to the ferrous 688-nm substance to form a stable compound; the cyanide compound formed could revert to neither the ferrous 688-nm substance nor the 638-nm substance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of substance P in post-tetanic potentiation in myenteric plexus-longitudinal muscle preparations; the effect of substance P-antagonist

A role of substance P in post-tetanic potentiation in the myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum was investigated by means of substance P-antagonist, (D-Pro2, D-Trp7,9)-substance P. After the addition of substance P-antagonist (10(-7)-10(-5) mol x l-1) a dose-dependent reduction of post-tetanic potentiation of neurogenic twitches was observed. Post-tetanic potentiation was actually abolished in the presence of 10(-5) mol x l-1 substance P-antagonist. It has been shown previously by our group that post-tetanic potentiation is reduced in preparations desensitized by substance P; this, together with our present results supported the hypothesis that the release of substance P-like compounds during tetanic stimulation played a key role in the mechanism of post-tetanic potentiation.     

Effect of electroacupuncture on the activity of neurons in the central gray matter of the midbrain

The effect of electroacupuncture in locally-segment and general analgetic points on background impulse activity of central gray substance neurons and their activity caused by nociceptive stimulation of the dental pulp, infraorbital nerve and forearm skin surface was studied in acute experiments on cats. It has been established that general analgetic points are better represented in the central gray substance, as compared to locally-segment points. Different degree involvement of central gray substance in the realization of acupuncture analgetic effect in different points is postulated. The role of dorsal and ventral compartments of the central gray substance in acupunctural analgesia is discussed.     

Epithelium removal alters responsiveness of guinea pig trachea to substance P

Removal of epithelium from mammalian tracheae has been shown to enhance responsiveness to a variety of contractile and relaxant agents. One of the most dramatic shifts reported has been for guinea pig tracheal tissue denuded of epithelium and treated with substance P. We investigated whether this shift in responsiveness was because of 1) removal of an epithelium-associated enzyme, neutral endopeptidase, which degrades substance P and 2) loss of an epithelium-derived noncyclooxygenase relaxant factor. Using a muscle bath preparation we performed concentration-response curves with substance P and acetylcholine on indomethacin-treated tissues with and without intact epithelium and with and without pretreatment with the neutral endopeptidase inhibitor, phosphoramidon. Epithelium removal potentiated the mean agonist concentration calculated to causes 30% of the maximal contractile response by 148-fold for substance P and by 7-fold for acetylcholine. Phosphoramidon potentiated the contractile response to substance P, but not to acetylcholine, by both the epithelium-intact and denuded tissues (P less than 0.05). However, the degree of enhancement by phosphoramidon was much greater in the intact tissues. With phosphoramidon treatment, therefore, the difference in responsiveness to substance P between the intact and denuded tissues was reduced from 148-fold to 18-fold. This effect of phosphoramidon suggests that the hyperresponsiveness to substance P of epithelium-denuded airway tissue is largely because of removal of neutral endopeptidase. Because all tissues were treated with indomethacin, the leftward shifts in substance P and in acetylcholine responsiveness induced by epithelium removal further suggest that an epithelium-derived noncyclooxygenase factor other than neutral endopeptidase also modulates the contractile response to substance P and to acetylcholine.     

Radioimmunoassay of substance P and its stability in tissue

A sensitive and specific radioimmunoassay for substance P has been developed and its full characterisation is described. The assay was used to investigate the stability of substance P in tissue in order to establish the optimal conditions of tissue storage for maximal recovery of the peptide. The results indicate unexpected post-mortem changes in tissue substance P content and suggests its existence in at least two separate pools, which differ in their susceptibility to degradation. These results highlight the need for careful consideration of both extraction technique and assay characteristics in radioimmunological investigations of substance P.     

Modulation of endopeptidase activity by calcitonin gene related peptide: a mechanism affecting substance P action?

Peptides with hormonal or neuronal activity are derived by enzymatic processing from pro-hormones, which by themselves are biologically inert. Processing and other enzymatic conversions may occur step-wise, leading to the formation of a cascade of biologically active (or inactive) peptides. The neurokin in substance P is known to be metabolically transformed both by amino- and endopeptidases. More N-terminal substance (1-7) has been found than C-terminal (2-11 to 5-11) fragments in various CNS areas. The substance P (1-7) fragment also shows biological activity e.g., providing analgesia, lowering blood pressure, inhibiting aggressive behavior and (in contrast to substance P) inhibiting grooming behavior. An endopeptidase generating substance P (1-7) and to a lesser extent, substance (1-8), has been isolated and characterized from human cerebrospinal fluid (CSF) and bovine spinal cord, as a metalloenzyme with essential SH-groups. Substance P co-exists with calcitonin gene related peptide (CGRP) in a large population of non-myelinated primary afferent ('pain') fibers. Intrathecal injection of substance P causes behavioral and physiological responses which are potentiated and prolonged by CGRP. It was found that CGRP competes with substance P for the endopeptidase. It is suggested that the main action of CGRP in the spinal cord is to inhibit substance P degradation.