Enkephalin, substance P, and serotonin axonal input to c-fos-like immunoreactive neurons of the rat spinal cord

Mustard oil, which stimulates small diameter afferents, was used to evoke the expression of the oncogene c-fos in the lumbar spinal cord. C-fos-like immunoreactivity was concentrated in, but not limited to, neuronal nuclei of laminae I and II of the lumbar dorsal horn. Double-label immunocytochemistry was used to determine if neurons which expressed c-fos-like immunoreactivity received axonal input from enkephalin-, substance P- or serotonin-immunoreactive neurons. The analysis of vibratome and semithin plastic-embedded tissue sections demonstrated that the majority of c-fos-like immunoreactive neurons received input from enkephalin-, substance P- or serotonin-immunoreactive axonal varicosities.     

Excitatory responses of trigeminal neurons to substance P suggest involvement in sensory transmission

Responses to substance P application were studied with intracellular recording techniques in in vitro preparations of trigeminal root ganglion neurons of guinea pigs. Perfusion of substance P in micromolar concentrations markedly depolarized neurons and reduced their input conductances. Also, the threshold for spikes evoked by injections of depolarizing current pulses was decreased. Single electrode voltage-clamp recordings showed that substance P increased inward, and decreased outward currents evoked by hyperpolarizing voltage steps from holding potentials near rest. Depolarizing responses to substance P were attenuated in Na+-deficient solutions. The excitatory actions of this endogenous peptide on the perikarya of primary sensory neurons give rise to the possibility of physiological actions of substance P at multiple sites in the trigeminal system.     

Enkephalin, dynorphin and substance P in postmortem substantia nigra from normals and schizophrenic patients.

Three peptide neuromodulators that are found in high concentration in the substantia nigra: dynorphin A 1-8, met5-enkephalin-arg6-gly7-leu8 and substance P, were measured by specific radioimmunoassays in nigral tissue from normals and schizophrenics postmortem. Substance P and dynorphin were unchanged between the two groups. However, the proenkephalin-derived peptide was significantly elevated in the schizophrenic group. The immunoreactivity was identified as authentic met5-enkephalin-arg6-gly7-leu8 by high pressure liquid chromatography. The data suggest that a different set of regulatory controls exists for nigral enkephalin peptides as compared to dynorphin and substance P, and that the former system may be disordered in schizophrenia.     

Mass spectra of underivatized peptide amides related to substance P

Mass spectra of underivatized hexa- and heptapeptide amides related to Substance P have been obtained with a conventional electron ionization mass spectrometer using sample vaporization from a tungsten wire by the technique of rapid heating, proton transfer ionization using ammonia, and photoplate recording of spectra. These spectra exhibit little evidence of sample pyrolysis and are readily interpreted to yield amino acid sequences.     

Selective agonists for receptors of substance P and related neurokinins

Neurokinins and their receptors are a complex system consisting of at least three endogenous agents--substance P (SP), neurokinin A (NKA), and neurokinin B (NKB)--and their corresponding receptor types, respectively, NK-1, NK-2, and NK-3. Investigations on receptors have been made using sensitive and fairly selective pharmacological preparations (the dog carotid artery for the NK-1, the rabbit pulmonary artery devoid of endothelium for the NK-2, and the rat portal vein for the NK-3 receptor), and some natural peptides of mammalian and nonmammalian origin. Because of the nonselectivity of the natural peptides, analogues of the neurokinins have been found that act on one receptor only and show therefore high selectivity. The selective agonists [Sar9,Met(O2)11]SP, [Nle10]NKA (4-10), and [MePhe7]-NKB have been used successfully for (a) characterizing the three neurokinin receptors, (b) identifying isolated organs whose responses to neurokinins depend on the activation of a single (monoreceptor systems) or of more than one (multireceptor systems) receptor, and (c) elucidating some of the physiological function of the three receptor types. It is suggested that NK-1 mediate peripheral vasodilatation and exocrine secretions, NK-2 stimulate bronchial muscles and facilitate the release of catecholamines, and NK-3 promote the release of acetylcholine in peripheral organs.     

Effects of substance P and its fragments in physiological and pathological pain

It has been shown that substance P and its fragments can produce under certain conditions an analgetic effect on both physiological and pathological pain (i.e. on pain syndrome of spinal origin). The data obtained give evidence that prolonged hypoalgesia is caused by the injection of substance P and its fragments to nucleus raphe dorsal--a structure of the antinociceptive system. This analgetic effect can be initiated by the activation of the antinociceptive system influenced by substance P and or its fragments.     

Analgesic and cardiovascular effects of centrally administered substance P

Substance P (SP) injected intracerebroventricularly (ICV) into rabbits caused dose-related thermal analgesia with the maximum effect after 2 micrograms. The analgesia was measured by timing the withdrawal of the rabbit's ear from an infrared beam. Equimolar amounts of the related peptides physalaemin and eledoisin-related peptide also caused analgesia, but the SP N-terminal fragment (1-9) was inactive. This suggests that the analgesic message of SP resides within the C-terminal fragment. The analgesia caused by each peptide developed more rapidly but did not last as long as that after central injection of beta-endorphin. In separate experiments, 2 micrograms SP injected ICV increased blood pressure and decreased heart rate. The analgesic, bradycardic and pressor responses to central administration of SP were opposite to effects of peripherally administered SP, described previously. These results indicate that the effect induced by SP depends upon its specific neuroanatomical site of action.     

Neurobiology of trigeminal pain

The brainstem trigeminal complex integrates somatosensory inputs from orofacial areas and meninges. Recent studies have shown the existence of a double representation of pain within the brainstem, at the level of both caudalis and oralis subnuclei. Noxious messages are mainly conveyed by C-fibers that activate the subnucleus caudalis neurons. These neurons in turn activate the subnucleus oralis whose neurons share similar features with the deep spinal dorsal horn neurons. In contrast with the nearness of the laminar organization of the dorsal horn, the vertical organization of the trigeminal complex offers an easier access for the study of segmental mechanisms of nociceptive processing. This model allowed us to show the existence of subtle NMDA-related mechanisms of segmental nocious processing. The trigeminal complex conveys nociceptive messages to several brainstem and thalamic relays that activate a number of cortical areas responsible for pain sensations and reactions. Cortical processing is sustained by reciprocal interactions with thalamic areas and also by a direct modulation of their pre-thalamic relays. The dysfunction of these multiple modulatory mechanisms probably plays a key role in the pathophysiology of chronic trigeminal pain.     

Properties of substance P aggregates. Application to the synthesis and purification of substance P

By 1H-NMR spectroscopy it has been shown that Substance P is largely aggregated at basic and acid pH and in saline solutions. These SP polymers dissociate rapidly by addition of pyridine and acetonitrile and slowly by addition of methanol. The difficulties previously encountered in the purification of SP and SP analogs may be attributed to this aggregation and can be overcome under disaggregating conditions. As a first application of our study we propose a reliable method for obtaining SP with good yield.     

Use of substance P fragments to differentiate substance P receptors of different tissues

The C- and N-terminal fragments of substance P were compared to the parent molecule with respect to their ability to: (a) contract the isolated guinea pig ileum, (b) induce salivation in the rat, (c) excite single cat dorsal horn neurones, and (d) induce scratching by intracranial injections in mice. C-terminal fragments as small as the heptapeptide were potent SP agonists on all assay systems. C-terminal fragments containing five amino acids or less were, at most, only weakly active. The C-terminal hexapeptide was a potent SP receptor stimulant on the isolated guinea pig ileum and, when directly applied by microiontophoresis, on cat dorsal horn neurons. However, the same compound was only 2-5% as potent as substance P in eliciting salivation and scratching in vivo, an indication that this fragment may be especially labile to enzymatic degradation. N-terminal fragments were totally inactive on the isolated guinea pig ileum. On the rat salivation and central nervous system assays, however, N-terminal fragments were capable of weak SP-like activity. It is concluded that SP receptors exist in multiple forms which we have labelled SP1 and SP2 receptors for those insensitive or sensitive to N-terminal fragments, respectively.     

Antinociceptive and Met-enkephalin releasing effects of tachykinins and substance P fragments

Substance P (SP), physalaemin, SP4-11, SP5-11 and the SP5-11 analog DiMe-C7 induce an antinociceptive effect in rats after intraventricular administration. Other tachykinins and the N-terminal fragments of SP are inactive. All antinociceptive peptides increase the Met-enkephalin efflux from slices of rat periaqueductal gray matter and their antinociceptive potency is correlated with their capacity to release Met-enkephalin. The results, discussed in the light of current theories on different tachykinin receptors, suggest that the SP-P receptor subtype may be involved in the control of noxious stimulation elicited by SP at supraspinal levels.     

Postsynaptic effects of substance P in frog neuromuscular junction

We have studied the effect of substance P on the end-plate currents (EPC) and the miniature EPC (MEPC) after acetylcholine esterase (ACE) inhibition in the cut neuromuscular preparation of the frog sartorius muscle using the voltage-clamp technique. At concentrations of 5·10^–7–1·10^–6 moles/liter substance P had no effect on the amplitude and the time characteristics of single EPC and MEPC but promoted prolongation of EPC decay on repetitive stimulation of the nerve with a frequency of 10/sec, indicating intensification of postsynaptic potentiation. Elevation of the concentration of the given peptide to 5·10^–6 moles/liter led to the shortening of the decay of single EPC and a more marked depression of the EPC amplitude in the trains as compared to the control, reflecting a decrease in the sensitivity of the postsynaptic membrane to the mediator, i.e., development of desensitization.S. V. Kurashov State Medical Institute, Kazan. I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Leningrad. Translated from Neirofiziologiya, Vol. 23, No. 4, pp. 436–441, July–August, 1991.     

Nitric oxide modulates renal sensory nerve fibers by mechanisms related to substance P receptor activation

Nerve terminals containing neuronal nitric oxide synthase (nNOS) are localized in the renal pelvic wall where the sensory nerves containing substance P and calcitonin gene-related peptide (CGRP) are found. We examined whether nNOS is colocalized with substance P and CGRP. All renal pelvic nerve fibers that contained nNOS-like immunoreactivity (-LI) also contained substance P-LI and CGRP-LI. In anesthetized rats, renal pelvic perfusion with the nNOS inhibitor S-methyl-L-thiocitrulline (L-SMTC, 20 microM) prolonged the afferent renal nerve activity (ARNA) response to a 3-min period of increased renal pelvic pressure from 5 +/- 0.4 to 21 +/- 2 min (P < 0.01, n = 14). The magnitude of the ARNA response was unaffected by L-SMTC. Similar effects were produced by N(omega)-nitro-L-arginine methyl ester (L-NAME) but not D-NAME. Increasing renal pelvic pressure produced similar increases in renal pelvic release of substance P before and during L-SMTC, from 5.9 +/- 1.4 to 13.6 +/- 4.2 pg/min before and from 4.9 +/- to 12.6 +/- 2.7 pg/min during L-SMTC. L-SMTC also prolonged the ARNA response to renal pelvic perfusion with substance P (3 microM) from 1.2 +/- 0.2 to 5.6 +/- 1.1 min (P < 0.01, n = 9) without affecting the magnitude of the ARNA response. In conclusion: activation of NO may function as an inhibitory neurotransmitter regulating the activation of renal mechanosensory nerve fibers by mechanisms related to activation of substance P receptors.     

Loss of spinal substance P pain transmission under the condition of LPA1 receptor-mediated neuropathic pain

In the present study, we first report an in vivo characterization of flexor responses induced by three distinct sine-wave stimuli in the electrical stimulation-induced paw flexion (EPF) test in mice. The fixed sine-wave electric stimulations of 5 Hz (C-fiber), 250 Hz (Aδ-fiber) and 2000 Hz (Aβ-fiber) to the hind paw of mice induced a paw-flexion response and vocalization. The average threshold for paw flexor responses by sine-wave stimulations was much lower than that for vocalization. Neonatally (P3) pretreatment with capsaicin to degenerate polymodal substance P-ergic C-fiber neurons increased the threshold to 5 Hz (C-fiber) stimuli, but not to 250 Hz (Aδ-fiber) and 2000 Hz (Aβ-fiber). The flexor responses to 5 Hz stimuli were significantly blocked by intrathecal (i.t.) pretreatment with both CP-99994 and MK-801, an NK1 and NMDA receptor antagonist, respectively, but not by CNQX, an AMPA/kainate receptor antagonist. On the other hand, the flexor responses induced by 250 Hz stimuli were blocked by MK-801 (i.t.) but not by CP-99994 or CNQX. In contrast, flexor responses induced by 2000 Hz stimuli were only blocked by CNQX treatment. These data suggest that we have identified three pharmacologically different categories of responses mediated through different primary afferent fibers. Furthermore, we also carried out characterization of the in vivo functional sensitivity of each of the sensory fiber types in nerve-injured mice using the EPF test, and found that the threshold to both 250 Hz and 2000 Hz stimulations were markedly decreased, whereas the threshold to 5 Hz stimulations was significantly increased. Thus we found opposing effects on specific sensory fiber-mediated responses as a result of nerve injury in mice. These results also suggest that the EPF analysis is useful for the evaluation of plasticity in sensory functions in animal disease models.     

Vasoactive effects of substance P on isolated rabbit pulmonary artery

The vasoactive properties of substance P (SP) were studied in isolated rabbit pulmonary artery (PA) segments in vitro. In the absence of active base-line tone, noncumulative administration of SP (10(-11) to 10(-4) M) produced dose-dependent increases in PA tension. The peak isometric tension (Tmax) with SP was similar to the Tmax response to epinephrine; however, the doses of the agonist producing a threshold contraction and 25% of Tmax (ED25) were significantly lower for SP. In the presence of active base-line tone, induced by epinephrine or 5-hydroxytryptamine, SP produced transient PA relaxation which was directly related to the magnitude of the precontracted PA tension. Blockade of neurotransmission with tetrodotoxin (1 microgram/ml) and antagonists to alpha 1-adrenergic and histamine receptor binding had no effect on the contractile response to SP. On the other hand, PA contraction to an ED50 dose of SP was 1) inhibited by a mean of 33 +/- 10% (SE) following pretreatment with the cholinesterase inhibitor, neostigmine (10(-6) M) and 2) augmented by 52 +/- 21% with the cholinergic antagonist, atropine (10(-4) M). The latter also completely blocked the relaxation response to SP in precontracted PA. Similarly, removal of the PA endothelium also abolished the relaxation response to SP. In contrast, SP-induced contraction was markedly inhibited by the cyclooxygenase inhibitor, meclofenamate (1 microgram/ml), as well as the SP antagonist, D-Pro2, D-Trp7,9-SP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoradiographic localization of substance P receptors using I substance P

This paper describes a method for localization of substance P receptors in the rat central nervous system using ¹²⁵I labeled substance P in an autoradiographic procedure. Particularly high densities of substance P receptors were observed in the olfactory bulb, dentate gyrus, amygdala, superior colliculus, and locus coeruleus. Surprisingly low densities of substance P receptors were found in the substantia nigra pars reticulata, a region which contains high concentrations of substance P.     

Behavioral alterations induced by substance P, bombesin, and related peptides in mice

Bombesin, substance P and several structurally related peptides cause excessive grooming behavior after intracerebroventricular injection in mice. The present study describes the behavioral characteristics of these effects after acute administration. Substance P caused an elevation of grooming behavior which was short-lasting (less than 15 minutes), while bombesin induced both grooming and scratching behavior with a duration of action of about 2.5 hours. After repeated injections of high doses of either bombesin or a metabolically stable substance P analog, no tolerance-formation to these peptide-induced effects could be observed. Morphine partially antagonized bombesin-induced behaviors at a dose of 7.5 mg/kg subcutaneously while the same dose did not attentuate substance P-induced grooming. These results suggest that the behavioral changes induced by substance P and bombesin are mediated by distinct mechanisms. The lack of tolerance formation, together with the partial antagonism by morphine, suggests that the bombesin-induced behaviors may be related to a stimulation of nociceptive mechanisms.