Substance P-Evoked Calcium Mobilization and Ionic Current Activation in the Human Astrocytoma Cell Line U 373 MG: Pharmacological Characterization

Abstract— In the human astrocytoma cell line U 373 MG, application of substance P (SP) leads to a transient increase in cytosolic calcium concentration and to a biphasic current response in voltage-clamped cells. Using these two functional assays we have characterized pharmacologically the SP response in U 373 MG cells. SP and [l -Pro⁹]SP displayed high potencies in both assays with EC₅₀values of 2.5 ± 10^?9M and 1 ± 10^?9M on calcium responses and 110^?9M and 510^?9M on ion current responses, respectively. The high potency of SP and [l -Pro⁹]SP as well as the low potency of [Lys⁵,MeLeu⁹,N-Leu¹⁰]neurokinin A_(4-10) and the inactivity of senktide demonstrate the NK₁-type pharmacology of these responses. Furthermore, the NK₁ antagonists (±)-CP 96,345, its chloro analogue, (±)-cis-3-(2-chlorobenzylamino)-2-benz-hydrylquinuclidine, and RP 67580 were potent antagonists of both SP responses. For the calcium mobilization induced by SP (1 (10^?7M), the IC₅₀ values for the three antagonists were 4 ± 10^?10M, 4 ± 10^?9M, and 9 ± 10^?9M, respectively, whereas on the current response evoked by SP 10^?8M), the IC₅₀ values were 8 ± 10^?9M, 2.4 ± 10^?8M, and 1.2 10^?7M, respectively. Despite differences in the absolute IC₅₀ values obtained with both techniques, the relative potencies of the three antagonists correlate fairly well. The U 373 MG cell line provides a useful model system for studies of the pharmacology of the human NK₁receptor and its transduction mechanisms at the level of second messengers and modulation of ion currents.     

Synergistic Regulation of Cytosolic Ca2+ Concentration in Mouse Astrocytes by NK1 Tachykinin and Adenosine Agonists

The effects on cytosolic Ca2+ concentration of 2-chloroadenosine and [L-Pro9]-substance P, a selective agonist of NK1 receptors, were investigated on astrocytes from embryonic mice in primary culture. Cells responded to [L-Pro9]-substance P with a transitory increase in cytosolic Ca2+ which was of shorter duration when external Ca2+ was removed. A transient response to 2-chloroadenosine alone occurred. When simultaneously applied, [L-Pro9]-substance P and 2-chloroadenosine evoked a prolonged elevation of cytosolic Ca2+ (up to 30 min). This phenomenon was dependent on the presence of extracellular Ca2+, but insensitive to dihydropyridines, La3+, and Co2+, excluding the implication of voltage-operated Ca2+ channels. Arachidonic acid also induced a sustained elevation of cytosolic Ca2+, but did not increase further the response evoked by [L-Pro9]-substance P and 2-chloroadenosine. The activation of protein kinase C by a diacylglycerol analogue mimicked the effect of [L-Pro9]-substance P in potentiating the 2-chloroadenosine-evoked response. Like 2-chloroadenosine, pinacidil, which hyperpolarizes the cells by opening K+ channels, prolonged the elevation of cytosolic Ca2+ concentration induced by [L-Pro9]-substance P. Conversely, depolarization with 50 mM KCl canceled the effects of either pinacidil or 2-chloroadenosine applied with [L-Pro9]-substance P. Pertussis toxin pretreatment suppressed all the effects induced by 2-chloroadenosine.     

Colonic Mechanoreceptor Input to Neurons in Guinea Pig Caudal Mesenteric Ganglion

The background activity of pacemaker-like neurons (PLN) of the guinea-pig caudal mesenteric ganglion (CMG) and their reflex responses to colonic distention were studied on combined isolated preparations including the CMG and a colon segment connected with the lumbar colonic nerves. The results allow us to suggest that the spontaneous and reflex activity of PLN is of a peripheral origin. Synaptic transmission in the peripheral colonofugal nerve pathways is mediated by acetylcholine and substance P (SP). The SP-induced excitation of PLN probably involves activation of the tachykinin NK₃ receptors.     

Expression of tachykinin receptors in Xenopus oocytes injected with poly(A)~+RNA from cat dorsal root ganglion

ＴａｃｈｙｋｉｎｉｎｆａｍｉｌｙｉｓａｇｒｏｕｐｏｆｎｅｕｒｏｐｅｐｔｉｄｅｓｗｉｔｈｓｉｍｉｌａｒＣｔｅｒｍｉｎａｌｓｅｑｕｅｎｃｅｓａｎｄｒｅｌａｔｅｄｂｉｏａｃｔｉｖｉｔｉｅｓ.ＴｈｅｍａｊｏｒｔａｃｈｙｋｉｎｉｎｓｉｎｍａｍｍａｌｉａｎａｒｅｓｕｂｓｔａｎｃｅＰ(ＳＰ),ｎｅｕｒｏｋｉｎｉｎＡ(ＮＫＡ)ａｎｄｎｅｕｒｏｋｉｎｉｎＢ(ＮＫＢ).Ｃｏｒｒｅｓｐｏｎｄｉｎｇｔｏｔｈｅｓｅｐｅｐｔｉｄｅｓ,ｔｈｒｅｅｄｉｓｔｉｎｃｔｔａｃｈｙｋｉｎｉｎｒｅｃｅｐｔｏｒｓｗｅｒｅｄｉｓｃｏｖｅｒｅｄａｎｄｎ…     

Membrane‐induced structure of novel human tachykinin hemokinin‐1 (hHK1)

PPT‐C encoded hemokinin‐1(hHK‐1) of Homo sapiens (TGKASQFFGLM) is a structurally distinct neuropeptide among the tachykinin family that participate in the NK‐1 receptor downstream signaling processes. Subsequently, signal transduction leads to execution of various effector functions which includes aging, immunological, and central nervous system (CNS) regulatory actions. However the conformational pattern of ligand receptor binding is unclear. The three‐dimensional structure of the hemokinin‐1 in aqueous and micellar environment has been studied by one and two‐dimensional proton nuclear magnetic resonance (2D 1H‐NMR spectroscopy) and distance geometry calculations. Data shows that hemokinin‐1 was unstructured in aqueous environment; anionic detergent SDS induces α‐helix formation. Proton NMR assignments have been carried out with the aid of correlation spectroscopy (gradient‐COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The inter proton distances and dihedral angle constraints obtained from the NMR data have been used in torsion angle dynamics algorithm for NMR applications (CYANA) to generate a family of structures, which have been refined using restrained energy minimization and dynamics. Helical conformation is observed from residue K3‐M11. The conformational range of the peptide revealed by NMR studies has been analyzed in terms of characteristic secondary features. Observed conformational features have been compared to that of Substance P potent NK1 agonist. Thus the report provides a structural insight to study hHK‐1‐NK1 interaction that is essential for hHK1 based signaling events. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 702–710, 2015.     

Substitution of conserved glycine residue by alanine in natural and synthetic neuropeptide ligands causes partial agonism at the stomoxytachykinin receptor

A few naturally occurring insect tachykinin-related peptides, such as stomoxytachykinin (Stc-TK), contain an Ala-residue instead of the highly conserved Gly-residue that is present in most other members of this peptide family. Stc-TK is a potent, partial agonist of the stable fly (Stomoxys calcitrans) tachykinin receptor, STKR. By means of synthetic analogues, the Gly/Ala exchange, representing the addition of a single methyl group in the active core region of these peptides, was shown to be fully responsible for the generation of this partial agonism, which was also accompanied by an increase in agonistic potency. Surprisingly, this Ala-dependent reduction in maximal response levels was only observed for the agonist-induced cellular calcium rise. Stomoxytachykinin, Stc-TK, did not display partial agonism for the STKR-mediated cyclic AMP response. A possible explanation for this differential partial agonism is that the Gly-containing and Ala-replaced peptides recognize and stabilize active receptor conformations that differ in their functional coupling efficacies towards these response pathways. Drosotachykinins, Drm-TK, tachykinin-like peptides encoded in the fruit fly genome, were shown to be STKR-agonists. Interestingly, one of these peptides, which contains an Ala-residue instead of the conserved Gly-residue, also proved to be a potent, partial agonist for STKR.     

Nuclear Localization of SP, CGRP, and NK 1 R in a Subpopulation of Dorsal Root Ganglia Subpopulation Cells in Rats

Signals generated by renal pelvic afferent nerves in response to stimulation are transmitted from peripheral processes of dorsal root ganglia neurons to their central terminals in the dorsal horn of the spinal cord to cause the release of neuropeptides, including SP and CGRP. All of the cellular activities of SP are considered to be mediated through interaction with NK₁R located on the cell surface. We have investigated the colocalization and subcellular distribution of NK₁R, SP, and CGRP in different subpopulations of neurons that innervate renal tissue. Our findings therefore provide the first evidence for the presence of NK₁R, SP, and CGRP in the nuclei of DGR neural cells. The physiological significance of this localization remains unknown. One possibility is that pelvic sensory neurons may regulate their responses to different stimuli by modulating the ratio of CGRP and SP release and/or nuclear NK₁R expression.     

Fruit fly behavior in response to chemosensory signals

An important question in contemporary sensory neuroscience is how animals perceive their environment and make appropriate behavioral choices based on chemical perceptions. The fruit fly Drosophila melanogaster exhibits robust tastant and odor-evoked behaviors. Understanding how the gustatory and olfactory systems support the perception of these contact and volatile chemicals and translate them into appropriate attraction or avoidance behaviors has made an unprecedented contribution to our knowledge of the organization of chemosensory systems. In this review, I begin by describing the receptors and signaling mechanisms of the Drosophila gustatory and olfactory systems and then highlight their involvement in the control of simple and complex behaviors. The topics addressed include feeding behavior, learning and memory, navigation behavior, neuropeptide modulation of chemosensory behavior, and I conclude with a discussion of recent work that provides insight into pheromone signaling pathways.     

The study of the conformation and interaction of two tachykinin peptides in membrane mimicking systems by NMR spectroscopy and pulsed field gradient diffusion

Pulsed‐field gradient diffusion has been used to study the binding of two tachykinin peptides, [Tyr⁸]‐substance P (SP) and [Tyr⁰]‐neurokinin A (NKA) to two membrane‐mimicking micelles, dodecylphosphocholine, and sodium dodecylsulfate. The structure of these peptides bound to the micelles have also been studied by using two‐dimensional nmr and restrained simulated annealing calculations. No major difference in the structures of each peptide in the two micellar media was found. The difference between the micelle‐bound structure of [Tyr⁸]SP and that of SP was also minor. The longer helical conformation on the C‐terminus for [Tyr⁰]NKA was observed, compared with that for NKA. The relationship between the difference in the biological potencies of [Tyr⁸]SP and SP and the differences in their structure, especially the interaction of the side chains of the two aromatic residues, and the difference in their binding affinities to membrane was discussed. In addition, differences between the result of restrained molecular dynamics simulations of [Tyr⁸]SP in the presence of an explicit micelle and the present results were observed and discussed. © 1999 John Wiley & Sons, Inc. Biopoly 50: 555–568, 1999