A comparison of the release of substance P and some synthetic analogues from micropipettes by microiontophoresis or pressure

The release of substance P (SP) and two analogues by iontophoresis or pressure from microelectrodes was compared. Substance P was released linearly by iontophoresis from electrodes while no release of the analogues was detected. [N-methylphenylalanine8, N-methylglycine9-] SP5-11 (DiMeC7) and [methyl-2-aminoethyl]11 SP (SP-DAE) were released from electrodes by pressure ejection with linear relationships in all cases between pressure and the amounts released. Under the tested experimental conditions, release of substance P by iontophoresis was between 2 and 3 orders of magnitude less than that by pressure over a given time. The release of substance P and the uncharged analogue DiMeC7 by pressure was very similar while release of SP-DAE was one order of magnitude less.     

The effects of depolarizing agents and neuropeptides on dopamine release from striatal synaptosomes

Synaptosomes (isolated nerve endings) from rat corpus striatum responded to several depolarizing agents by releasing dopamine. Among these agents were KCl, glutamic acid, ouabain, and veratrine. Substance P hexapeptide (SP6) also caused dopamine release, but the magnitude of this effect was small and variable. A number of other neuropeptides (cholecystokinin 1–8, des-Tyr-γ-endorphin, Leu⁵-β-endorphin and substance P) did not alter dopamine release. SP6-induced dopamine release may result from substance P receptor-induced depolarization; however, the lack of robustness of the response in this preparation makes it unsuitable for studying agonists and antagonists of substance P.     

Adenosine analogs do not inhibit the potassium-stimulated release of substance P from rat spinal cord slices

Summary Adenosine agonists produce antinociception when injected directly onto the spinal cord of rats and mice. One mechanism to account for this effect could be inhibition of neurotransmitter release from nociceptive sensory neurons. Consequently, we studied whether these agents could inhibit the potassium stimulated release of one such transmitter, substance P, from rat spinal cord slices. A 2 cm section of lumbar spinal cord was dissected from male Sprague-Dawley rats, chopped into 0.5 × 0.5 mm sections and perfused at 37°C with a modified Krebs bicarbonate buffer containing either 3.5 mM, 30 mM, or 50 mM KCl in the presence and absence of various adenosine analogs. Perfusates, collected every 2 min, were assayed for substance P by radioimmunoassay. Exposure of tissue to 50 mM KC1 produced an approximate three-fold increase in the release of substance P over basal release. This increase in release was calcium dependent. Perfusion of spinal cord tissues with either adenosine (10^–3 M), N⁶-cyclohexyladenosine (10^–5 M or 5 × 10^–5 M), 5-N-ethylcarboxamide adenosine (10^–5 M) or L-N⁶-phenylisopropyladenosine (10^–5 M) did not significantly alter basal or potassium-stimulated release of SP when compared to controls. In contrast to the adenosine agonists, exposure of the spinal cord tissue to 10^–5 M morphine significantly reduced the potassium-stimulated release of substance P. Pretreatment of the slices with 10^–5 M theophylline or 8-phenyltheophylline did not significantly attenuate the inhibition of substance P release produced by morphine. Theophylline alone (10^–5 M) had no significant effect on either basal or potassium-stimulated release of SP. These studies demonstrate that adenosine does not inhibit the release of SP from spinal cord slices and does not appear to mediate the morphine-induced inhibition of SP release. The results suggest that the mechanism of the antinociceptive effects of adenosine at the level of the spinal cord is not via inhibition of substance P release. Send offprint requests to M. R. Vasko at the above address     

Passive and Iontophoretic Transdermal Penetration of Chlorpromazine

The in vitro iontophoretic transdermal delivery of chlorpromazine (CPZ) across pig skin was investigated. Anodal iontophoresis considerably increased CPZ skin penetration and accumulation compared with the passive controls.

The effect of CPZ concentration in the donor solution was studied (1.4–8.2 mM). A higher penetration was observed with an increase of the concentration. In addition, the effect of NaCl concentration was also studied (154–200 mM). As expected, CPZ iontophoretic transport decreased with NaCl content. Finally, the influence of the current density (0.20–0.50 mA/cm²) was investigated. The iontophoretic transport of CPZ tends to increase with current density, although this effect was not statistically significant between 0.35 and 0.5 mA/cm². On the whole, this work shows that iontophoresis may be used to improve the transdermal delivery of CPZ for the treatment of chronic psychosis.     

Interaction of neurotensin with the substance P receptor mediating histamine release from rat mast cells and the flare in human skin.

1 Substance P induced histamine release from rat peritoneal mast cells in a dose-dependent manner over the concentration range 1 to 10 microM. 2 At concentrations in the range 2.5 to 1 0 microM, neurotensin produced only about 5% release of histamine, which was substantially less than the maximum effect obtained with substance P. 3 Neurotensin, 2.5 to 10 microM produced graded inhibition of histamine release induced by substance P. The inhibitory effect of neurotensin was not seen when histamine release was induced by an antigen-antibody effect of neurotensin was not seen when histamine release was induced by an antigen-antibody reaction or by the ionophore, A 23187. Some evidence was obtained to suggest that compound 48/80 may interact with the same receptor as substance P and neurotensin. 4 [D-Arg8]neurotensin, [D-Arg9]neurotensin, xenopsin and the C-terminal octapeptide of substance P (SP4-11) all inhibited histamine release by substance P, but physalaemin did not. 5 Neurotensin inhibited the wheal and flare reactions induced by substance P in human skin. 6 [D-Trp7,9]substance P released histamine from rat mast cells and was about 12 times more potent than substance P itself. [D-Trp7,9]SP1-11 also produced wheal and flare responses in human skin, being 1.8 times more potent than substance P in the production of flare.     

Modulatory effect of substance P on GABA-activated currents from rat dorsal root ganglion

INTRODUCTIONSP has been reported to serve as a pain transmit-ter or a modulator of noxious transmission in the dorsalhorn of the spinal cord[1,2]. Application of SP to neu-rons of the DRG produced a depolarizing response[3-5]and activated an inward current[6,7] associated with anincrease in neuronal excitability. g-Aminobutyric acid(GABA) is the primary inhibitory transmitter in the spi-nal cord, which causes a reduction of the release ofexcitatory transmitter from primary afferent n…     

Antagonist discrimination between subtypes of tachykinin receptors in the guinea-pig ileum

The effects of substance P and eledoisin on spontaneous and electrically-evoked release of [3H]acetylcholine, and on smooth muscle were studied in the guinea-pig myenteric plexus-longitudinal muscle preparation preloaded with [3H]choline. Substance P and eledoisin caused transient increases in spontaneous release of [3H]-acetylcholine and in longitudinal muscle tone. Both tachykinins were equipotent in contracting the muscle, but eledoisin was more potent than substance P in eliciting [3H]acetylcholine release. The release caused by substance P was enhanced in the presence of naloxone and scopolamine which suggests that the release is modulated through opioid and muscarinic receptors. Substance P and eledoisin inhibited the release of [3H]acetylcholine evoked by electrical stimulation at 0.1 Hz. The inhibition was not due to an activation of alpha-adrenoceptors, histamine or opioid receptors. The substance P antagonists (D-Pro2, D-Trp7,9)SP (10 and 30 microM) and (Arg5, D-Trp7,9, Nle11)SP5-11 (1 and 10 microM) competitively antagonized both the contractile effects of substance P and eledoisin, and the inhibition by the tachykinins of the electrically-evoked release of [3H]acetylcholine. The increase in spontaneous [3H]acetylcholine release elicited by substance P and eledoisin was not prevented by the substance P antagonists. The results suggest that the neuronal receptor whose activation causes inhibition of acetylcholine release and the smooth muscle receptor correspond to the SP-P type, whereas the neuronal receptor mediating an increase in spontaneous acetylcholine release is of the SP-E type. The two antagonists, (D-Pro2, D-Trp7,9)SP and (Arg5, D-Trp7,9, Nle11)SP5-11, selectively block only the SP-P receptor.     

Determination of the relative potency of two excitant amino acids

Recent reports have suggested that some cells in the CNS differ in their relative sensitivity to iontophoretically applied aspartate and glutamate. Attempts to investigate this phenomenon present difficulties since transport numbers for the same substance show considerable variation among different micropipette barrels. A method is described which allows corrections to be made for differences in efflux rates from different barrels. Efflux of tritiated glutamate and aspartate into saline for each pipette is measured by liquid scintillation counting before and after its use in vivo. Calibration curves of efflux (pmol min ^?1) against current (nA min^?1) are then constructed for each barrel containing amino acid. Using these calibrations, log dose-response curves for aspartate and glutamate can be plotted for each neurone by derivation from the apparent dose-response curves in which firing rate is plotted against log current (nA).When the method was tested on brainstem neurones it was found that the apparent relative sensitivity of most cells was changed following correction, and in some cases reversed.     

Effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on substance P-induced histamine release from rat peritoneal mast cells.

12-O-tetradecanoylphorbol-13-acetate (TPA, 1 to 30 ng/ml) produced a dose-related inhibition of substance P (SP)-induced histamine release from rat peritoneal mast cells. TPA itself induced some histamine release over this concentration range (maximum release about 20% of total). Maximum inhibition of SP-induced release by TPA required preincubation with TPA for at least 10 min. The inhibitory action of TPA was observed in the absence as well as in the presence of extracellular calcium (0.4 mM). Inhibition of diacylglycerol kinase by R 59022 or of diacylglycerol lipase by RHC 80267 reduced SP-induced histamine release. Oleolylacetylglycerol (OAG, 50 microM) inhibited histamine release induced by SP but was less potent than TPA. It is concluded that protein kinase C activation in rat peritoneal mast cells is associated with inhibition of SP-induced histamine release.     

On the pharmacology of the glycine receptors on the cuneo-thalamic relay cells in the cat

1. In cats either decerebrated or anaesthetized with sodium pentobarbitone, strychnine released by iontophoresis from electrodes containing a 5 mM solution in 165 mM of NaCl, abolished the action of glycine and beta-alanine on cuneo-thalamic relay cells without disturbing their response to equally effective applications of gamma-aminobutyric acid (GABA) and beta-guanidino-propionic acid.2. The loss of glycine sensitivity appeared to increase as long as the strychnine release continued until even the largest currents tolerated by the electrode were unable to eject effective amounts of glycine. Parallel shifts of the glycine log-current response curves, equivalent to an equipotent dose-ratio of about 2.0 only occurred when the duration of the strychnine applications by current in excess of 28 nA was restricted to a few minutes.3. Without modifying either the frequency of the spike discharge or the amplitude or shape of the action potential, currents larger than 28 nA occasionally caused a loss of GABA sensitivity.4. Strychnine administered either intravenously (0.2 mg/kg) or applied topically to the surface of the cuneate (0.5 mM) blocked the response to glycine without any obvious effect on the response to GABA.5. It was concluded that the discovery of a strychnine-sensitive component of the inhibitory potentials recorded from the cuneate nucleus will reveal the physiological role of the glycine receptors on cuneo-thalamic relay cells.     

A comparison between different immobilised glucoseoxidase-based electrodes

Biosensors obtained by immobilising glucose oxidase ‘unentrapped’ and ‘entrapped in liposomes’, both with a classical H₂O₂ amperometric electrode and with screen-printed electrochemical sensor, were compared. Electrode response, linearity range and the influence of some parameters as phospholipid nature, temperature and measurement techniques were investigated. Experimental results showed that, while with the unentrapped enzyme the output current is linear only up to about 4 mM glucose concentration, the linearity range increases up to about 20 mM using enzyme-loaded liposomes; however the low permeability of the lipid bilayer decreases the electrode sensitivity to very low values (200 nA/M for palmitoylolelyl phosphatidylcholine liposomes). The approach with screen-printed sensors showed a better performance and gave biosensors with higher sensitivity (about 14 500 nA/mM). A mathematical model, useful to compare the behaviour of the different analytical systems and to design electrodes with the required properties, was also proposed.     

Correlation of the release of amines and antagonists with their effects

The microiontophoretic release of [¹⁴C]-noradrenaline into saline and brain tissue and the effect of a backing current of 15 nA on this release has been determined. It was found that the transport number of noradrenaline was similar for saline and tissue and that the backing current could markedly reduce the release of noradrenaline.The microiontophoretic release of [³⁵S]-chlorpromazine has been studied. The release was linearly related to the charge passed for only two out of the ten micropipettes tested and the transport number for these was very low.An attempt has been made to determine the range of diffusion after microiontophoretic release of α-methylnoradrenaline and noradrenaline using the fluorescence method for the demonstration of biogenic monoamines and soluble compound autoradiography. The results show that these substances can diffuse for considerable distances and therefore possibly affect more than one neurone.There appears to be a correlation between the effects of perfusing a range of concentrations of noradrenaline on the EEG and the amount of noradrenaline that is taken up by the tissue. The amount of noradrenaline that diffuses into the tissue under the conditions that produce phasic arousal can be released microiontophoretically by a relatively low charge. It may therefore be possible to use microiontophoresis to study the mechanisms underlying more gross physiological changes.     

The influence of substance P on the response of guinea-pig isolated ileum to periarterial nerve stimulation.

1 The effects of substance P (SP) on the responses of the guinea-pig isolated ileum to periarterial nerve stimulation were studied. Electrical stimulation (2-50 Hz) of mesenteric periarterial nerves resulted in contraction of preparations pretreated with guanethidine. The responses were abolished by atropine and morphine, but unaffected by hexamethonium. 2 SP in a high concentration (0.65 mumol) inhibited reversibly, by about 40%, the responses of the guinea-pig isolated ileum to periarterial nerve stimulation. 3 SP in a very low concentration (0.18 pmol) potentiated (by about 20%) the responses of the guinea-pig isolated ileum to periarterial nerve stimulation. 4 In the presence of low concentrations (0.06-0.32 pmol) of SP, morphine (2.65 mumol) produced less inhibition of the responses to periarterial nerve stimulation, and recovery from morphine inhibition of contraction was accelerated. 5 These results indicate that SP may act as a modulator on prejunctional acetylcholine release in the guinea-pig ileum.     

Morphine, but not sodium cromoglycate, modulates the release of substance P from capsaicin-sensitive neurones in the rat trachea in vitro.

1. Opioids have been shown to inhibit substance P (SP) release from primary afferent neurones (PAN). In addition, opioid receptors have been identified on PAN of the vagus nerves. Sodium cromoglycate (SCG) decreases the excitability of C-fibres in the lung of the dog in vivo. We have utilised a multi-superfusion system to investigate the effect of opioids and SCG on the release of SP from the rat trachea in vitro. 2. Pretreatment of newborn rats with capsaicin (50 mg kg-1 s.c. at day 1 and 2 of life) resulted in a 93.2 +/- 6.3% reduction in tracheal substance P-like immunoreactivity (SP-LI) content when determined by radioimmunoassay in the adult. 3. Exposure to isotonically elevated potassium concentrations (37-90 mM), capsaicin (100 nM-10 microM), and bradykinin (BK; 10nm-1 microM) but not des-Arg9-BK (1 microM) stimulated SP-LI release by a calcium-dependent mechanism. 4. SCG (1 microM and 100 microM) did not affect spontaneous, potassium (60 mM)- or BK (1 microM)-stimulated SP-LI release. 5. Morphine (0.1-100 microM) caused dose-related inhibition of potassium (60 mM)-stimulated SP-LI release with the greatest inhibition of 60.4 +/- 13.7% at 100 microM. The effect of morphine was not mimicked by the kappa-opioid receptor agonist, U50,488H (10 microM) or the delta-opioid receptor agonist, Tyr-(D-Pen)-Gly-Phe-(D-Pen) (DPDPE). 6. The effect of morphine was totally abolished by prior and concomitant exposure to naloxone (100 nM) which had no effect on control release values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of neuropeptide-induced histamine release from human dispersed skin mast cells.

1. Human skin mast cells, unlike other human mast cells so far studied, released histamine in a concentration-related manner in response to substance P, vasoactive intestinal peptide (VIP) and somatostatin (1 microM to 30 microM). In contrast, eledoisin, physalaemin, neurokinin A, neurokinin B, calcitonin gene-related peptide (CGRP), neurotensin, bradykinin and Lys-bradykinin induced negligible histamine release. 2. The low histamine releasing activity of physalaemin, eledoisin, neurokinin A and neurokinin B relative to substance P suggests that the human skin mast cell activation site is distinct from the tachykinin NK-1, NK-2 or NK-3 receptors described in smooth muscle. 3. The relative potencies of substance P and its fragments SP2-11, SP3-11, SP4-11 and SP1-4 in releasing histamine from human skin mast cells suggests that both the basic N-terminal amino acids and the lipophilic C-terminal portion of substance P are essential for activity. 4. Peptide-induced histamine release, like that induced by compound 48/80, morphine and poly-L-lysine, is rapid, reaching completion in 10-20 s, is largely independent of extracellular calcium but requires intact glycolysis and oxidative phosphorylation. 5. The substance P analogue, [D-Pro4,D-Trp7,9,10] SP4-11 (SPA), not only reduced substance P-induced histamine release in a concentration-related manner but also inhibited that induced by VIP, somatostatin, compound 48/80, poly-L-lysine and morphine but not anti-IgE. 6. The similar characteristics of histamine release induced by substance P, VIP, somatostatin, compound 48/80, poly-L-lysine and morphine suggest that they share a common pathway of activation-secretion coupling distinct from that of IgE-dependent activation. Furthermore, the ability of human skin mast cells to respond to basic non-immunological stimuli including neuropeptides may reflect a specialised function for these cells.     

Stimulatory effect of substance P on the spontaneous release of newly synthesized [3H]dopamine from rat striatal slices: a tetrodotoxin-sensitive process

Striatal slices from the rat were continuously superfused with [3H]tyrosine in order to estimate the release of newly synthesized [3H]dopamine [( 3H]DA). Substance P (SP) and neuromedin K (NKB) stimulated the spontaneous release of [3H]DA when used in concentrations (from 10(-9) M to 10(-7) M). The stimulatory effect of substance P (10(-8) M) was prevented completely when slices were superfused with tetrodotoxin (10(-7) M) or the substance P antagonist (D-Arg1,D-Pro2,D-Trp7,9,Leu11) substance P (10(-5 M) indicating that the response evoked by substance P was mediated by substance P receptors but that these were not located on DA nerve terminals. In addition, substance P did not modify the stimulatory effect of acetylcholine (ACh) (10(-5) M) on the spontaneous release of [3H]DA since the effects of substance P (10(-7) M) and ACh (10(-5) M) were additive.     

NMDA receptor activation modulates evoked release of substance P from rat spinal cord

The possible modulation exerted by glutamate on substance P (SP) release from the rat spinal cord has been investigated. The N-methyl-D-aspartate (NMDA) receptor agonist, NMDA (1 μM), increased SP basal outflow by 46.5±10.9% (n=3, P<0.01) without changing the evoked release of the peptide. Conversely, NMDA antagonists but not 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) inhibited both electrically-evoked and capsaicin-induced release of SP. In particular, D-2-amino-5-phosphonopentanoate (D-AP5; 50 μM) inhibited electrically-evoked and capsaicin-induced release of SP by 93±2.4% and 93.2±3.8% (n=12, P<0.01), respectively. Functional pharmacological evidence is provided for glutamate exerting a positive feedback on SP release evoked by C fibre stimulation via NMDA receptor activation.     

Release of histamine by neuropeptides from the perfused rat hindquarter

The release of histamine and serotonin by neuropeptides and capsaicin was measured in the isolated perfused rat hindquarter preparation. Substance P and two antagonistic peptides, [D-Pro2, D-Phe7, D-Trp9]-SP and [D-Pro2, D-Trp7,9)]-SP, release histamine, the SP(4-11) and SP(6-11) analogues did not. VIP and somatostatin released histamine and also serotonin. No amines were released by bombesin. Thus, all amine releasing peptides possessed at least two basic charges. However, the histamine releasing activity of the neuropeptides tested did not correlate with their reported ability to cause vasodilation and plasma extravasation. The SP(4-11) and SP(6-11) analogues which did not release histamine caused plasma extravasation. It is concluded that SP causes plasma extravasation by a direct action on blood vessels. Capsaicin released only serotonin but no histamine either in untreated rats and such desensitized with capsaicin as neonates. In rats desensitized with capsaicin 4 days prior to the experiment the substance P induced histamine release was as high as in untreated controls; it was, however, absent in rats desensitized with capsaicin as neonates. It is assumed that the sensitivity of mast cells to substance P is lost after degeneration of substance P containing primary sensory fibers.     

Transport and interaction of drugs in leukocytes

The accumulation of selected CNS drugs by rat leukocytes was previously reported. This paper presents evidence for the transport into leukocytes of additional drugs. Also studied was the inhibition of the latter processes by various structurally related compounds. The markedly rapid and sodium-independent uptakes into rat leukocytes of amphetamine, codeine, methadone and naloxone fulfilled the basic criteria for active transport. The uptake of morphine was apparently accomplished by more than one process. The affinities of the high capacity transport systems (approximate V_max: 100 nmoles/g cells/5sec) varied considerably as reflected by the two extreme K_m values obtained for methadone (20 μM) and morphine (1.8 mM). A variety of amines inhibited the cellular transport of the drugs. Most potent inhibitors were quinacrine (K_i: 0.5 to 3 μM), desipramine (K_i: 6–20 μM) and methadone (K_i: 18–25 μM). Morphine and tryptamine exhibited inhibition constants higher than 1 mM. The cellular transport processes newly described in rat leukocytes apparently represent a novel addition to the heterogenous biological transport of basic amines. The structural specificity of amine transport in various tissues is discussed.     

The effect of substance P on the mechanical and electrical responses of frog, chick and rat skeletal muscle

The effect of substance P (SP) on the electrical and contractile responses of skeletal muscles of frog, chick and rat were studied using electrophysiological techniques. In low concentrations, SP increased the amplitude of twitch and tetanic contractions in response to motor nerve stimulation of frog, chick and rat preparations, increased the amplitude and duration of frog sciatic nerve compound action potential and reduced the contracture responses produced by acetylcholine (ACh) or tetraethylammonium (TEA) in the chick skeletal muscle. In all these actions, the effect of SP was calcium-dependent. The results provide evidence that SP had a dual action; a prejunctional facilitatory effect by causing a further release of ACh and/or Ca2+ entry or release, and postjunctional effect by reducing the sensitivity of the postjunctional membrane to depolarizing drugs.