Glutamate-stimulated ATP release from spinal cord astrocytes is potentiated by substance P

ATP has recently emerged as a key molecule mediating pathological pain. The aim of this study was to examine whether spinal cord astrocytes could be a source of ATP in response to the nociceptive neurotransmitters glutamate and substance P. Glutamate stimulated ATP release from these astrocytes and this release was greatly potentiated by substance P, even though substance P alone did not elicit ATP release. Substance P also potentiated glutamate-induced inward currents, but did not cause such currents alone. When glutamate was applied alone it acted exclusively through alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionate receptors to stimulate Ca(2+) influx-dependent ATP release. However, when substance P was co-applied with glutamate, ATP release could be elicited by activation of NMDA and metabotropic glutamate receptors. Activation of neurokinin receptor subtypes, protein kinase C and phospholipases A(2), C and D were needed for substance P to bring about its effects. These results suggest that astrocytes may be a major source of ATP in the spinal cord on activation of nerve fibres that release substance P and glutamate.     

Opioid modulation of capsaicin-evoked release of substance P from rat spinal cord in vivo

Capsaicin has been shown to evoke the release of substance P (SP) from small diameter primary afferent fibers. Using an in vivo perfusion of the rat spinal cord, this study examined the pharmacology of opioid receptor systems which modulate the capsaicin-evoked release of SP. The addition of capsaicin (200 μM) to the perfusate raised SP-like immunoreactivity (SP-LI) from resting levels of 31±5 to 74±14 pg/ml or an increase of 139% above the baseline. Using high pressure liquid chromatography (HPLC) the identity of the released SP-LI was determined to coelute primarily with authentic SP or the oxidized form of SP. Opioid receptor agonists were added to the perfusate and their ability to inhibit capsaicin-evoked release of SP-LI was assessed. Morphine (10–100 μM), DAGO (1–100 μM), DPLPE (10–100 μM), but not U50488H (100 μM) produced a dose-dependent reduction in the capsaicin-evoked release of SP-LI. Pretreatment with the opioid receptor antagonist naloxone (1 mg/kg, IP) had no effect on the basal or capsaicin-evoked release of SP-LI. Naloxone pretreatment was able to antagonize completely the opioid-produced inhibition of capsaicin-evoked SP-LI release. These data indicate that the release of SP from primary afferent fibers can be modulated by the activation of mu or delta but not kappa opioid receptors. Further, these data support the hypothesis that spinally administered mu and delta opioid agonists may produce their antinociceptive effect through the presynaptic inhibition of neuropeptide release from small diameter primary afferent fibers.     

Immunocytochemical study of substance P containing nerve terminals in rat spinal cord

Summary The subcellular localization of substance P (SP) in the dorsal horn of the rat spinal cord was studied using the unlabelled antibody procedure of Sternberger with different fixatives (4% paraformaldehyde alone or with varying amounts of glutaraldehyde), buffer systems for the immunohistochemical incubations, and the presence or absence of the detergent, Trition X-100. Hand-sliced tissues were compared with Vibratome sections, and showed adequate results which are described below. Labelled terminals of two types could be seen in all samples incubated with anti-SP sera. The two types of positive terminals can be described as those which contained mostly immunoreactive clear vesicles, and those which contained both immunoreactive clear and dense core vesicles. Brief fixation during pressure perfusion with increased concentrations of glutaraldehyde (up to 2%) improved the tissue preservation and, as a result, the intensity and definition of the SP immunoreaction products. The use of Tris or phosphate buffer for the immunohistochemical incubations maintained the intensity of staining in well-fixed tissues. However, Tris incubations contributed to a diffusion of immunoreaction products and increased the number of broken membranes in the labelled processes as well as those of myelin. These phenomena were not observed in phosphate buffer, which preserved the tissue better than Tris. Like Tris, pretreatment with the detergent Triton X-100 (TX) contributed further to the diffusion of the immunoreaction products, and increased the number of broken membranes. For example,without TX, the outer membrane and envelope of the mitochondria became intensely and clearly labelled when phosphate buffer was used for incubations;with TX pretreatment, the staining was far more diffuse, and the intensity of staining became reduced such that only the mitochondrial outer surface appeared somewhat immunopositive. Using phosphate buffer alone, we observed well-defined immunoreaction products around the microtubules of many-containing processes. This finding was less clear in other preparations, especially those pretreatedwith TX. We therefore submit that the conditions of tissue fixation and incubation may influence greatly the data amassed by the technique of immunocytochemistry. Results must be evaluated in view of the methods chosed for each immunocytochemical study. Optimal technical conditions encourage new morphological findings, as shown below concerning the circuitry of SP neurons in the dorsal horn.This work was initiated while Dr. K. Kakudo was a postdoctoral Fellow in Anatomic Pathology, Medical College of Georgia, from Osaka University Medical School, JapanPresently at the Department of Anatomy, Tulane Medical School, New Orleans, Louisiana, USAThe work, presented at the Annual Meeting of the 31st Annual Meeting of the Histochemical Society held in New Orleans, April 11–15, 1980, was partially supported by BRSG 10-16-04-3611-23, CCHD10-12-04-3600-00 (LLV) and the Department of Pathology (address reprint requests to LLV)     

Correlation of substance P-induced desensitization with substance P amino terminal metabolites in the mouse spinal cord.

Intrathecal injection of mice with substance P (SP) or its C-terminal fragments results in a behavioral syndrome characterized by reciprocal caudally directed biting and scratching. Repeated injection of SP, but not SP C-terminal fragments, results in a decrease in the intensity of, or desensitization to, these SP-induced behaviors. Peptidase inhibitors, phosphoramidon (PH), bacitracin (BAC), diprotin A (DPA) and angiotensin converting enzyme inhibitor (ACEI OR SQ20881), together with [3H]SP, were used to investigate the possible accumulation of tritiated N-terminal metabolites in the mouse spinal cord in vivo during the development of desensitization to SP. SP N-terminal metabolites in the spinal cord were quantified by reverse-phase HPLC. The magnitude of SP-induced desensitization correlated well (r = .95) with total SP N-terminal metabolites recovered from the spinal cords of the same mice studied in vivo. The magnitude of SP-induced desensitization was also found to be negatively correlated (r = .95) with total recovered intact [3H]SP. The rank order of potency of the peptidase inhibitors in decreasing the magnitude of SP-induced desensitization was BAC = PH much greater than ACEI greater than DPA. The order of potency for in vitro inhibition of SP metabolism using synaptic membrane-derived peptidases was BAC greater than PH much greater than ACEI. These results support the hypothesis that desensitization to SP-induced behaviors depends, at least in part, on the concentration of SP N-terminal metabolites in the spinal cord.     

Myocardial ischemia induces the release of substance P from cardiac afferent neurons in rat thoracic spinal cord

Antibody-coated microprobes were inserted into the thoracic (T3-4) spinal cord in urethane-anesthetized Sprague-Dawley rats to detect the differences in the release of immunoreactive substance P-like (irSP) substances in response to differential activation of cardiac nociceptive sensory neurons (CNAN). CNAN were stimulated either by intrapericardial infusion of an inflammatory ischemic exudate solution (IES) containing algogenic substances (i.e., 10 mM each of adenosine, bradykinin, prostaglandin E2, and 5-hydroxytryptamine), or by transient occlusion of the left anterior descending coronary artery (CoAO). There was widespread basal release of irSP from the thoracic spinal cord. Stimulation of the CNAN by IES did not alter the pattern of release of irSP. Conversely, CoAO augmented the release of irSP from T3-4 spinal segments from laminae I-VII. This CoAO-induced irSP release was eliminated after thoracic dorsal rhizotomy. These results indicate that heterogeneous activation of cardiac afferents, as with focal coronary artery occlusion, represents an optimum input for activation of the cardiac neuronal hierarchy and for the resultant perception of angina. Excessive stimulation of cardiac nociceptive afferent neurons elicited during regional coronary artery occlusion involves the release of SP in the thoracic spinal cord and suggests that local spinal cord release of SP may be involved in the neural signaling of angina.     

Modulation of cardiac ischemia-sensitive afferent neuron signaling by preemptive C2 spinal cord stimulation: effect on substance P release from rat spinal cord

The upper cervical spinal region functions as an intraspinal controller of thoracic spinal reflexes and contributes to neuronal regulation of the ischemic myocardium. Our objective was to determine whether stimulation of the C2 cervical spinal cord (SCS) of rats modified the input signal at the thoracic spinal cord when cardiac ischemia-sensitive (sympathetic) afferents were activated by transient occlusion of the left anterior descending coronary artery (CoAO). Changes in c-Fos expression were used as an index of neuronal activation within the spinal cord and brain stem. The pattern of substance P (SP) release, a putative nociceptive transmitter, was measured using antibody-coated microprobes. Two SCS protocols were used: reactive SCS, applied concurrently with intermittent CoAO and preemptive, sustained SCS starting 15 min before and continuing during the repeated intermittent CoAO. CoAO increased SP release from laminae I and II in the T4 spinal cord above resting levels. Intermittent SCS with CoAO resulted in greater levels of SP release from deeper laminae IV-VII in T4 than CoAO alone. In contrast, SP release from laminae I and II was inhibited when CoAO was applied during preemptive, sustained SCS. Preemptive SCS likewise reduced c-Fos expression in the T4 spinal cord (laminae I-V) and nucleus tractus solitarius but increased expression in the intermediolateral cell column of T4 compared with CoAO alone. These results suggest that preemptive SCS from the high cervical region modulates sensory afferent signaling from the ischemic myocardium.     

Interactions of substance P antagonists with serotonin in the mouse spinal cord

Administered intrathecally (IT) to mice, the putative substance P antagonist [D-Pro2,D-Trp7,9-substance P (DPDT) blocked substance P- and serotonin-induced reciprocal hindlimb scratching with ID50 values of 4.6 (2.9-6.9) and 3.0 (1.9-4.8) micrograms, respectively. The duration of this antagonistic effect was 90-120 min. In contrast, DPDT did not block bombesin-, somatostatin-, glycine- or glutamate-induced scratching. These data indicate that DPDT is an effective antagonist of serotonin-induced behaviors in the mouse spinal cord. Phenoxybenzamine (IT) also blocked substance P- and serotonin-induced scratching. Its onset of action was more rapid for serotonin than for DPDT implying differences in agonist-induced receptor activation. Methysergide (IT) blocked serotonin-induced scratching [ID50 = 0.7 (0.3-1.5) micrograms], but not substance P-induced scratching. Similar to DPDT, [D-Arg1,D-Trp7,9,Leu11]-substance P, [des-Arg1,D-Pro2, D-Trp7,9]-substance P(2-11) and [D-Pro4,D-Trp7,9]-substance P(4-11) blocked substance P and serotonin-induced scratching. In contrast, [D-Pro2,D-Phe7,D-Trp9]-substance P and [D-Pro4,D-Trp7,9,10]-substance P(4-11) blocked only substance P-induced scratching. Thus, some, but not all putative substance P antagonists may also be behavioral antagonists of serotonin in the mouse spinal cord.     

Ultrastructural localization of substance P immunoreactivity in the ventral horn of the rat spinal cord

At the light microscope level, the minute concentrations of substance P (SP) in rat spinal ventral horn can be visualized best by amplification with the double bridge PAP method of Vacca et al. (1975; 1980) in 5 microns paraffin tissue sections. Morphologically, the immunoreactive sites resemble punctate bodies. They occur in close apposition with the large ventral horn cells and their associated neuronal processes. By the Sternberger PAP procedure, we now describe these punctate bodies at the electron microscope level. Ultrastructurally, they appear as tiny boutons (terminal and preterminal) and small unmyelinated processes. The boutons and processes typically contain one to several immunolabeled dense core vesicles among many immunolabeled clear vesicles. They range in size near the limit of resolution of the light microscope (LM), thereby justifying further the use of LM amplification staining by the double bridge method. The immunolabeled boutons often synapse with large smooth dendrites (which may originate from motoneurons) by asymmetrical or symmetrical synaptic densities. Their synaptic densities appear immunostained as well. The data support the view that the electrophysiological action of SP in the ventral horn occurs in part by synaptic action along the processes of the ventral horn cells. Other mechanisms of action are considered for the peptide as well. Additional types of membrane specializations (synaptoid junctions) and SP neural circuits are described below.     

Ultrastructural localization of substance P immunoreactivity in the ventral horn of the rat spinal cord

Summary At the light microscope level, the minute concentrations of substance P (SP) in rat spinal ventral horn can be visualized best by amplification with the double bridge PAP method of Vacca et al. (1975; 1980) in 5 m paraffin tissue sections. Morphologically, the immunoreactive sites resemble punctate bodies. They occur in close apposition with the large ventral horn cells and their associated neuronal processes. By the Sternberger PAP procedure, we now describe these punctate bodies at the electron microscope level. Ultrastructurally, they appear as tiny boutons (terminal and preterminal) and small unmyelinated processes. The boutons and processes typically contain one to several immunolabeled dense core vesicles among many immunolabeled clear vesicles. They range in size near the limit of resolution of the light microscope (LM), thereby justifying further the use of LM amplification staining by the double bridge method. The immunolabeled boutons often synapse with large smooth dendrites (which may originate from motoneurons) by asymmetrical or symmetrical synaptic densities. Their synaptic densities appear immunostained as well. The data support the view that the electrophysiological action of SP in the ventral horn occurs in part by synaptic action along the processes of the ventral horn cells. Other mechanisms of action are considered for the peptide as well. Additional types of membrane specializations (synaptoid junctions) and SP neural circuits are described below.The work, presented at the Histochemical Society's 29 Annual Meeting in Vancouver, B.C. April 1–2, 1978, was partially supported by CCHD 10-12-04-3600-67 (LLV)     

Left vagal stimulation induces dynorphin release and suppresses substance P release from the rat thoracic spinal cord during cardiac ischemia

Electrostimulatory forms of therapy can reduce angina that arises from activation of cardiac nociceptive afferent fibers during transient ischemia. This study sought to determine the effects of electrical stimulation of left thoracic vagal afferents (C(8)-T(1) level) on the release of putative nociceptive [substance P (SP)] and analgesic [dynorphin (Dyn)] peptides in the dorsal horn at the T(4) spinal level during coronary artery occlusion in urethane-anesthetized Sprague-Dawley rats. Release of Dyn and SP was measured by using antibody-coated microprobes. While Dyn and SP had a basal release, occlusion of the left anterior descending coronary artery only affected SP release, causing an increase from lamina I-VII. Left vagal stimulation increased Dyn release, inhibited basal SP release, and blunted the coronary artery occlusion-induced release of SP. Dyn release reflected activation of descending pathways in the thoracic spinal cord, because vagal afferent stimulation still increased the release of Dyn after bilateral dorsal rhizotomy of T(2)-T(5). These results indicate that electrostimulatory therapy, using vagal afferent excitation, may induce analgesia, in part, via inhibition of the release of SP in the spinal cord, possibly through a Dyn-mediated neuronal interaction.     

Substance P antagonist-induced spinal cord vasoconstriction: Effects of thyrotropin-releasing hormone and substance P agonists

Local spinal cord vasomotor effects of 3 substance P (SP) antagonists were studied in the rat following intrathecal (IT) administration. Each SP antagonist (3.3 nmol) increased spinal cord vascular resistance and reduced blood flow. A LH-RH antagonist analog (10 nmol) of similar molecular weight and which also contained multiple D-Trp residues did not cause spinal cord vasoconstriction. The vasoconstrictor action of the SP antagonist, [D-Arg¹, D-Pro², D-Trp^7,9, Leu¹¹]-SP ([D-Arg]-SP) was unaffected by pretreatment with a stable SP receptor agonist (5 nmol IT). Given evidence for a cerebral vasodilator action of TRH agonists, the effects of TRH (IV) and a stable TRH analog (MK-771, IT) on [D-Arg]-SP-induced vasoconstriction were also assessed. Neither TRH nor MK-771 prevented the [D-Arg]-SP-induced vasoconstriction. However, TRH (IV) but not MK-771 (IT) partially opposed [D-Arg]-SP-induced reduction in thoracic spinal cord blood flow. Thus, SP antagonists cause spinal cord vasoconstriction by a non-SP receptor mediated phenomenon. In addition, the attenuation of SP-antagonist-induced neuropathological changes previously reported with IV. TRH administration is likely due to less severe consequences of vasoconstriction in the presence of a higher initial baseline blood flow rather than direct prevention of the vasoconstriction.     

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.     

A modified peroxidase--antiperoxidase procedure for improved localization of tissue antigens: localization of substance P in rat spinal cord

A procedure is presented which modifies the Sternberger peroxidase--antiperoxidase (PAP) technique in order to visualize additional amounts of immunodeposits representing the antigen substance (SP) in 5-micrometer paraffin tissue sections of rat spinal cord. For increased sensitivity, the new procedure utilizes a "double bridge" and diaminobenzidine in low pH buffer. The modifications have made possible the visualization of immunoreactive beaded processes and punctate bodies, which were then traced to determine patterns of SP circuitry. Using the modified PAP procedure, the greatest number of immunoreactive processes appeared in the dorsal horn, where some punctate bodies and varicose processes could be seen adjacent to the myelinated afferent fiber bundles that penetrate the substantia gelatinosa as dorsal root collaterals. Additional immunoreactive processes and punctate bodies coursed through the myelinated afferent fiber bundles that penetrate the dorsolateral white matter, and extend into the intermediolateral gray region. Substance P was also identified within immunoreactive processes found in Rexed's laminae V and VI, as well as the central canal region, the dorsal gray commissure, and the ventral gray and white commissures. Since the modifications improved the visualization of SP-containing processes in sparsely populated regions of the spinal cord, especially the ventral horn, they may be useful in demonstrating other antigens that normally occur in small quantities within tissues.     

Distribution and origin of bombesin,substance P and somatostatin in cat spinal cord

Bombesin (BN), substance P-(SP) and somatostatin (SRIF) were measured in individual laminae of the cervical, thoracic and lumbar (L) spinal cord of control cats, and in the L6 segment of cats receiving a spinal hemisection (L2) or deafferentation via dorsal rhizotomy at L6, 7, S1. The interlaminar distribution of BN, SP, and SRIF was remarkably similar. Highest concentrations were found in the superficial dorsal horn, and progressively less was found proceeding ventrally. Some intersegmental variations in peptide concentration within a single lamina were found. Dorsal rhizotomy caused a significant decline in BN, SP and SRIF in lamina I-III, therefore all three peptides appear to be contained in dorsal root ganglion cells. Evidence is presented for the existence of ascending BN and SP projections originating in lamina I-III and VII, for a descending SRIF pathway terminating in lamina VIII, and for an ascending BN path in lamina VIII. Dorsal root afferents to lamina VIII influence levels of BN, SP and SRIF.     

Ultrastructural localization of immunolabeled substance P and methionine-enkephalin-octapeptide in the surface layer of the dorsal horn of rat spinal cord

Summary A preembedding dual immunolabeling technique and electron microscopy were utilized to demonstrate the localization of immunoreactive substance P and methionine-enkephalin-octapeptide (Enk-8) in ultrathin sections of the surface layer (laminae I and II) of rat spinal dorsal horn. The immunoreaction of Enk-8 was visualized as goldtoned silver particles and that of substance P as diaminobenzidine reaction products. Axonal terminals with immunoreactive substance P, and also unlabeled axonal terminals, formed synaptic junctions with the perikarya and dendritic processes of Enk-8-containing neurons. Dendritic profiles immunolabeled for substance P were synaptically linked with unlabeled axons but not with Enk-8-positive ones. Furthermore, it was found that Enk-8 axons and substance P axons terminated synaptically in juxtaposition to one another on the same immunonegative dendrites. Among the Enk-8-containing neurons axonal profiles also appeared to be synaptically associated with immunoreactive Enk-8 dendritic processes.     

Substance P release from spinal cord slices by capsaicin

The release of substance P (SP) from slices of hypothalamus, substantia nigra and spinal cord of the rat was studied. In contrast to 47 mM potassium, capsaicin induced SP release from spinal cord slices only. The SP release was not reduced by 10^?6 M tetrodotoxin but was abolished by a Ca²⁺ free medium containing 3 mM ethylene-glycol-bis- (β-aminoethyl-ether) N,N-tetraacetic acid (EGTA). Capsaicin induced SP release was dose-dependent and exhibited tachyphylaxis. The ability of capsaicin to release SP supports the hypothesis that SP is involved in pain transmission and in neurogenic plasma extravasation.