Intrathecal cholecystokinin interacts with morphine but not substance P in modulating the nociceptive flexion reflex in the rat

The effect of intrathecal (IT) cholecystokinin (CCK), substance P (SP) and morphine (MO) on spinal cord excitability was studied in decerebrate, spinalized rats. CCK had a weaker facilitatory effect on the nociceptive flexion reflex than SP. The possible functional significance of the coexistence of CCK and SP in neurons projecting to the spinal cord was tested by coadministration of the two peptides. At the doses tested no synergistic interaction on the reflex was found with CCK and SP. IT MO caused a brief enhancement followed by a prolonged depression of the reflex. A high dose of CCK injected prior to MO increased the facilitatory effect and decreased the depressive effect of the opiate on the reflex. The effect of desulfated (D) CCK was similar to CCK but at a higher dose. Naloxone (NAL) had a similar effect as CCK when administered prior to MO. The MO-induced depression of the reflex was readily reversed by NAL, but not by CCK. The results indicate that CCK may prevent the inhibitory effect of MO on spinal cord excitability to nociceptive stimulation, but does not reverse it. CCK may alter the balance of excitation-inhibition between various types of dorsal horn interneurons that are involved in the transmission of nociceptive information.     

The bradykinin B1 receptor antagonist B9858 inhibits a nociceptive spinal reflex in rabbits

There are two bradykinin receptor subtypes, designated B1 and B2. Whilst both have been implicated in nociception, it is believed that there is a low level of constitutive expression of B1 receptors and that their expression is induced by inflammation or tissue damage. The present study investigated the role of B1 receptors in spinal nociceptive processing using an in vivo electrophysiological assay in decerebrate, spinalized rabbits, a species that shares close B1 receptor homology with the human receptor. Inflammation was induced in the paw by an injection of complete Freund's adjuvant at least 1 h before recording single motor unit activity of the semitendinous/biceps femoris muscle in response to a noxious pinch of the foot. Control animals received an intraplantar injection of saline. The peptide B1 receptor antagonist B9858 was administered i.v. and caused dose-dependent and complete inhibition of the nociceptive spinal reflex (ID50 = 1 mg x kg(-1)). In control animals without paw inflammation, B9858 had no effect. These findings are consistent with other evidence that peptide B1 receptor antagonists inhibit spinal nociceptive reflexes only after induction of B1 receptors by inflammation and support the potential therapeutic utility of B1 receptor antagonists as analgesic and anti-inflammatory drugs.     

Differential inhibitory effects of mu-opioids on substance P- and capsaicin-induced nociceptive behavior in mice

The antinociceptive mechanisms of the selective mu-opioid receptor agonists [D-Ala2,NMePhe4,Gly(ol)5]enkephalin (DAMGO), H-Tyr-D-Arg-Phe-beta-Ala-OH (TAPA) or H-Tyr-D-Arg-Phe-beta-Ala-NH2 (TAPA-NH2) against substance P (SP)- or capsaicin-elicited nociceptive behaviors was investigated in mice. DAMGO, TAPA or TAPA-NH2 given intrathecally inhibited the nociceptive behaviors elicited by intrathecally administered SP or capsaicin, and these antinociceptive effects were completely eliminated by intrathecal co-administration with D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), a selective mu-opioid receptor antagonist. Pretreatment subcutaneously with naloxonazine, a selective mu1-opioid receptor antagonist, partially attenuated the antinociceptive effect of TAPA-NH2, but not DAMGO and TAPA, against SP. However, the antinociception induced by TAPA, but not DAMGO and TAPA-NH2, against capsaicin was significantly inhibited by naloxonazine. On the other hand, co-administration intrathecally with Tyr-D-Pro-Trp-Gly-NH2 (D-Pro2-Tyr-W-MIF-1), a selective mu2-opioid receptor antagonist, significantly attenuated the antinociceptive effects of DAMGO, but not TAPA and TAPA-NH2, against capsaicin, while the antinociceptions induced by three opioid peptides against SP were significantly inhibited by D-Pro2-Tyr-W-MIF-1. These results suggest that differential inhibitory mechanisms on pre- and postsynaptic sites in the spinal cord contribute to the antinociceptive effects of the three mu-opioid peptides.     

SR142801 behaves as a tachykinin NK-3 receptor agonist on a spinal nociceptive reflex in the rat

Effects of two commonly used tachykinin NK-3 receptor antagonists (SR 142801 and R820) intrathecally (i.t.) administered were assessed in the rat tail-flick test. SR142801 and its (R)-enantiomer SR142806 (1.3, 6.5 and 65 nmol) were found as potent as senktide and [MePhe7]NKB (NK-3 selective agonists) to induce transient antinociceptive effects. Naloxone (10 microg) and R820 (6.5 nmol) blocked reversibly the responses to 6.5 nmol senktide, [MePhe7]NKB, SR142801 and SR142806 when administered i.t. 15 min earlier. However, the antinociceptive responses induced by SR142801 and SR142806 were not affected by i.t. pretreatments with NK-1 (6.5 nmol SR140333) and NK-2 (6.5 nmol SR48968) receptor antagonists. In control experiments, the NK-1 and NK-2 antagonists prevented the hyperalgesic effects to NK-1 ([Sar9,Met(O2)11]SP) and NK-2 ([beta-Ala8] NKA(4-10)) receptor agonists (6.5 nmol i.t.), respectively. R820 had no direct effect on nociceptive threshold and failed to alter angiotensin II-induced antinociception. The data suggest that the antinociceptive effect of SR142801 is due to an agonist effect at NK-3 receptor in the rat spinal cord that involves a local opioid mechanism. These results can be best explained by the existence of inter-species NK-3 receptor subtypes.     

Effect of galanin on substance P- and vasoactive intestinal polypeptide-induced nociceptive trigemino-hypoglossal reflex in rats.

Substance P (SP), vasoactive intestinal polypeptide (VIP) and galanin (GAL), present in primary sensory neurons, are involved in transmission of nociceptive signaling from the peripheral to central nervous system. In this study we investigated the effect of GAL on SP-induced or VIP-induced evoked tongue jerks (ETJ) in response to noxious tooth pulp stimulation during perfusion of the cerebral ventricles with SP or VIP solutions. The experiments were carried out on rats under chloralose anesthesia. It was shown that both, SP and VIP, perfused through the cerebral ventricles enhanced the ETJ amplitude as compared with control, but the effect produced by SP was stronger. The intracerebroventricular perfusion of GAL 5 minutes before SP caused a dose-dependent inhibition of SP-induced ETJ, whereas GAL perfused through the cerebral ventricles 5 minutes before VIP did not reduce the excitatory effect of VIP on ETJ. These results indicate that the antinociceptive effect of GAL perfused through the cerebral ventricles, tested on the trigemino-hypoglossal reflex in rats, is specifically mediated by the SP-ergic system.     

Substance P effects on spinal nociceptive neurones.

In decerebrated spinal cats, the effects of iontophoretically applied acetylcholine (ACh) and substance P were examined on the responses of dorsal horn neurones to noxious stimulation and touch of the skin. Both agents, in amounts that did not have a significant direct effect on the neuronal firing rate, prolonged the response of the cells to noxious stimulation but did not alter that to touch stimulation. The peptide and ACh potentiated the late, but not the early, responses of dorsal horn neurones to sural A_δ and C afferent stimulation. Substance P-induced potentiation of the above responses was observed even when the agent did not produce a significant depolarization of nociceptive cells. In greater amounts, the peptide depolarized the neurones, an effect that was not associated with a detectable change in the membrane resistance. These results indicate that substance P facilitates nociceptive pathways by potentiating the subliminal fringe and, in greater amounts, by depolarizing the cells. The failure by the peptide to potentiate touch-induced excitation of the nociceptive neurones appears not to be due to the selectivity of the drug effect but due to the absence of subliminal fringe.     

Peptide antagonist of delta-opioid receptor attenuates inhibition of spinal nociceptive reflex induced by stimulation of arcuate nucleus of the hypothalamus

In lightly pentobarbital-anesthetized and acutely prepared rats, electrical stimulation within the arcuate nucleus of the hypothalamus (ARH) consistently inhibited the tail-flick responses to noxious heating of the tail. The peptide ICI174864, a delta-opioid receptor antagonist applied intrathecally at the lumbar level at a dose of 1 nmol, markedly attenuated this inhibition without affecting the baseline nociceptive threshold. Normal saline injected by the same approach had no effect on the ARH inhibitory modulation. This is the first report showing an involvement of the delta-opioid receptor in the descending inhibition of spinal nociceptive reflex resulting from ARH stimulation.     

失血性休克大鼠离体淋巴管对P物质的反应性

目的:应用离体淋巴管灌流技术,观察失血性休克(HS)发展进程中淋巴管对P物质(SP)的反应性。方法:Wistar雄性大鼠随机分为对照组(仅麻醉与手术)和HS组(通过股静脉放血至平均动脉血压为40 mmHg,复制HS模型,分为休克0h、0.5 h、1 h、2h、3 h五个亚组)。各组在相应时间点分离胸导管,制备淋巴管,3 cmH2O跨壁压下行离体灌流,分别给予从低到高浓度的SP,测量淋巴管收缩末期口径、舒张末期口径、收缩频率(CF)和被动管径,计算收缩幅度(CA)、泵流分数(FPF)和紧张指数(TI),以给予SP前后淋巴管的CF、TI、CA、FPF的差值△CF、△TI、△CA、△FPF作为评价淋巴管对SP反应性的指标。结果:Shock 0 h与shock 0.5 h大鼠淋巴管对多个或一个SP浓度的△CF、△TI、△CA、△FPF显著高于对照组,shock 2 h淋巴管对SP的△CF(3×10-7mol/L)、△TI(1×10-7mol/L)以及shock 3 h淋巴管对SP的△CF(1×10-7mol/L、3×10-7mol/L)、△TI(1×10-7mol/L)、△CA(1×10-7mol/L)均显著低于对照组。结论:休克淋巴管对SP反应性呈双相变化,即早期升高,晚期降低。     

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)     

Dopamine antagonist haloperidol decreases substance P, substance K, and preprotachykinin mRNAs in rat striatonigral neurons

Rat genomic clones were used to quantitate preprotachykinin mRNAs in the rat basal ganglia, while the tachykinin peptide products substance P and substance K were measured by radioimmunoassay. Administration of the dopamine antagonist (antipsychotic) drug haloperidol significantly decreased substance P, substance K, and both alpha (substance P encoding) and beta (substance P/substance K encoding) preprotachykinin mRNAs, suggesting a drug-induced decrease in striatonigral tachykinin biosynthesis. The time course for decreased preprotachykinin mRNAs and tachykinins apparently parallels the period of maximum risk for the development of certain antipsychotic drug-induced extrapyramidal side effects seen clinically. Tachykinin interaction with dopamine neurons may play an important role in the modulation of basal ganglia function.     

Effect of monoamines on motoneurons of the isolated rat spinal cord

Superfusion of isolated hemisected spinal cords of 9-13-day old rats with noradrenalin (NA) solution depolarized or hyperpolarized the motoneurons depending on the NA concentration. Both effects were the result of the direct action of NA on the motoneurons, for they were given in medium containing an excess of Mg and deficiency of Ca ions.a-Adrenoblockers depressed both the depolarizing and hyperpolarizing effects of NA. The depolarizing effect of dopamine on motoneurons was abolished in medium containing excess of Mg ions. Its direct hyperpolarizing action of motoneurons was suppressed by haloperidol but unchanged by phentolamine. The depolarizing effect of serotonin and its metabolites — mexamine, kynurenine, and 3-hydroxy-anthranilic acid — persisted in the presence of an excess of Mg and deficiency of Ca ions, but it was suppressed by deseryl (methysergide) and the benzyl analog of serotonin. The hyperpolarizing effect of serotonin at high concentrations (10^–4–10^–3 M), revealed in some experiments, was abolished in medium containing excess of magnesium ions in the presence of morphine.A. M. Gorkii Donetsk State Medical Institute. Translated from Neirofiziologiya, Vol. 12, No. 4, pp. 391–396, July–August, 1980.     

Effect of azapetine phosphate on the isolated rat ureter preparation.

Azapetine phosphate produced a stimulant effect on the spontaneously contracting and quiescent isolated rat ureter preparations.     

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.     

Tonic enhancement of the sensitivity of baroreceptor reflex response by endogenous substance P in the rat.

Modulation of baroreceptor reflex (BRR) by endogenous substance P (SP) in the brain was investigated in rats anesthetized with pentobarbital sodium. Intracerebroventricular administration of the undecapeptide (15 or 30 nmol) and its antagonist, (D-Pro2, D-Trp7,9)-SP (30 or 60 nmol) or SP antiserum (1:20), respectively, promoted a significant increase and decrease in the sensitivity of BRR response. Prolonging the endogenous activity of SP with the aminopeptidase blocker, bestatin (200 nmol) or with the endopeptidase-24.11 inhibitor, phosphoramidon (200 nmol) significantly augmented the same reflex. Combining the undecapeptide with either peptidase blocker, moreover, promoted additional potentiation of the BRR response. On the other hand, simultaneous administration of bestatin and (D-Pro2, D-Trp7,9)-SP produced a reduction of the augmented effect of bestatin on the sensitivity of BRR response. Bilateral microinjection of SP (600 pmol) or an antiserum against SP (1:20) into the nucleus tractus solitarius (NTS) elicited respectively an enhancement of and reduction in the BRR response. These data suggest that neurons that contain SP may participate in central cardiovascular control by tonically enhancing the sensitivity of the BRR response, possibly via an action on the NTS.     

Effect of substance P on thyrotropin secretion from the pituitary gland in the rat

The role of substance P (SP) on thyrotropin (TSH) secretion was investigated in ovariectomized (OVX) female, estrogen-primed OVX, and normal male rats. Third ventricular administration of SP induced a significant increase in plasma TSH levels when compared to control animals in E-primed OVX rats (p less than 0.001). The plasma TSH levels increased in a dose-related manner and reached maximum levels at 10 min after injection. In contrast, intraventricularly injected SP failed to alter plasma TSH levels in both OVX rats and normal male rats. Intravenous administration of SP dramatically stimulated TSH release in E-primed OVX rats (p less than 0.001), whereas SP had no effect on the release of TSH when injected in OVX rats and normal male rats. To investigate any direct action of SP on TSH release from the anterior pituitary gland, synthetic SP was incubated with dispersed anterior pituitary cells harvested from E-primed OVX rats and normal male rats. SP, in the dose range between 10(-8) M and 10(-6) M, failed to alter the release of TSH into the culture medium in vitro. These findings indicate that SP has a stimulatory role in the control of TSH release by an action on the hypothalamus but only in estrogen-primed rats.     

Effect of substance P on neonatally axotomized noradrenaline neurons in rat brain

Transection (axotomy) of the dorsal tegmental noradrenaline bundle in the neonatal stage leads to a permanent degeneration of noradrenaline nerve terminal projections distal to the lesion (e.g. in the neocortex), while projections proximal to the lesion increase their nerve terminal density (e.g. in the cerebellum). These structural changes are reflected by marked reductions and elevations respectively of the endogenous noradrenaline levels, [3H]-noradrenaline uptake in vitro and nerve density as demonstrated by fluorescence histochemistry. Intracisternal administration of substance P after the transection did not alter these noradrenaline parameters in the neocortex, whereas dose-dependent and significant increases were found in the cerebellum and pons-medulla. The results indicate that substance P may have a growth-stimulatory effect on damaged locus coeruleus noradrenaline neurons in the CNS during ontogeny.