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Intestinal inflammation is a painful syndrome with multiple symptoms, including chronic pain. This study examined the possible role of sensory neurons and substance P in symptoms of an animal model of acute intestinal inflammation. The model was induced by injecting ethanol and zymosan into the colon of anesthetized male rats. Three hours later, sections of the colon were stained with hematoxylin and eosin. To determine the role of substance P, 5 mg/kg of the neurokinin-1 receptor (NK-1r) antagonist, CP-96,345, or 300 microg/kg of an antisense oligonucleotide targeted at NK-1r mRNA was administered. Spinal cord sections were examined for internalization of NK-1r, as an indicator of substance P release. Sections of colon revealed infiltration of inflammatory cells following ethanol and zymosan treatment. Plasma extravasation in rats given ethanol and zymosan was significantly greater than in controls given saline only (P<0.0001) or saline and ethanol (P<0.001). In ethanol- and zymosan-treated rats given CP-96,345, plasma extravasation was significantly less than in rats given ethanol and zymosan without the antagonist (P<0.0001). Administration of the antisense oligonucleotide also resulted in lower levels of plasma extravasation compared with controls (P<0.01). Internalization of the NK-1r was observed in neurons of lamina I in the T13-L2 and L6-S2 regions of the spinal cord, as well as in sympathetic preganglionic neurons at the L1 level. This internalization was observed in the absence of any other stimulus besides the inflammation itself. This study implicates substance P and its receptor, the NK-1r, in acute inflammation of the colon.

In view of the widespread use of non-steroidal anti-inflammatory drugs for treatment of inflammatory pain, we determined the effects of the non-steroidal anti-inflammatory drug, indomethacin, on dorsal horn neurons in the rat spinal cord in vivo. At 2.0-12.0 mg/kg (i.v.), indomethacin depressed the responses of spinal dorsal horn neurons to the effects of iontophoretic application of substance P, N-methyl-D-aspartate, quisqualate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. As indomethacin inhibits cyclo-oxygenase, these are the first data linking prostanoids and possibly arachidonic acid and other eicosanoids to the effects of substance P and glutamate in the spinal dorsal horn. As responses to iontophoretic application can be assumed to have been postsynaptic and as indomethacin had an effect generalized to all excitatory responses, we suggest a postsynaptic site for cyclo-oxygenase. We also suggest that elements in the cyclo-oxygenase signal transduction pathway may thus mediate at least some of the effects of substance P and glutamate receptor activation. Activation of the cyclo-oxygenase pathway in CNS neurons is Ca2- dependent, and activation of both N-methyl-D-aspartate and substance P receptors increases intracellular Ca2+. This led to the expectation that indomethacin would have a greater effect on responses to N-methyl-D-aspartate than to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate, but the reverse was observed. Thus, in addition to a mediator role, we hypothesize that an element(s) of the cyclo-oxygenase pathway may regulate the efficacy of excitation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors and perhaps other membrane-bound receptors. The cyclo-oxygenase signal transduction pathway thus appears to play at least two major roles in regulation of sensory processing in the spinal cord. Therefore, non-steroidal anti-inflammatory drugs, via cyclo-oxygenase inhibition, may have multiple actions in control of spinal sensory mechanisms.

The effects of novel substance P (NK-1) receptor antagonists LY303870 and LY306740, as well as LY306155, the enantiomer of LY303870, were tested on the responses of nociceptive spinal dorsal horn neurons to iontophoretically applied substance P and to peripheral noxious stimuli. The peripheral stimuli included noxious thermal and pinch stimuli applied to the cutaneous receptive field in the hind paw and stimulation of the superficial peroneal nerve with a train of high-intensity electrical stimuli. Extracellular recordings were obtained using multi-barrel electrodes from L4-L7 segments of the spinal cord in cats anaesthetized with alpha-chloralose and spinalized at the L1 level. The antagonists were given i.v. (0.5-4.0 mg/kg). Responses to substance P were inhibited by LY303870 and by LY306740 in a dose-related manner, but were not affected by LY306155. Responses to peripheral noxious thermal stimulation were inhibited in a dose-related manner by LY303870 and LY306740, and only at higher doses (2 mg/kg or more) by LY306155. Responses to pinch stimuli were inhibited by LY303870 and LY306740. LY306155 lacked consistent effects on pinch responses. LY303870 selectively inhibited the late component of the response to electrical stimulation of the superficial peroneal nerve. When these three drugs were tested against the responses of dorsal horn neurons to the excitatory amino acid, N-methyl-D-aspartate, the responses were unaffected. These data suggest that LY303870 and LY306740 pass from the circulation into the spinal cord where they antagonize dorsal horn neuronal responses to substance P and nociceptive inputs.

Extracellular and intracellular studies were undertaken to test the effects of the non-peptide, substance P (NK-1) receptor antagonists CP-96,345 and CP-99,994, and of CP-96,344, the inactive enantiomer of CP-96,345, on the responses of spinal dorsal horn neurons to peripheral noxious and non-noxious cutaneous stimuli in spinalized cats anesthetized with alpha-chloralose. The effect of these agents on the response of dorsal horn neurons to iontophoretic application of substance P was also tested in extracellular studies. The substance P-induced slow, prolonged discharge of dorsal horn neurons was blocked by administration (0.5 mg/kg, i.v.) of CP-96,345 (n = 10) or CP-99,994 (n = 9), but was unaffected by CP-96,344 (n = 9). The response of substance P-sensitive neurons to noxious thermal stimulation of the cutaneous receptive field, especially the late afterdischarge phase, was also significantly inhibited by CP-96,345 (n = 10) and by CP-99,994 (n = 7). The response of such neurons to noxious pinch stimulation of the receptive field was also significantly inhibited by CP-96,345 (n = 7) and CP-99,994 (n = 8), but the response of three other substance P-sensitive neurons to pinch was unaffected by CP-96,345. CP-96,344 did not affect the response of any neuron tested to either of these noxious stimuli (noxious thermal, n = 7; pinch, n = 6). The response to hair afferent stimulation was unaffected by any of these compounds (CP-96,345, n = 16; CP-96,344, n = 5; CP-99,994, n = 6). In intracellular studies, the effect of these antagonists was tested on responses of dorsal horn neurons to noxious pinch stimulation or to a train of high intensity electrical stimulation of the superficial peroneal nerve. Both stimuli produced an initial fast depolarization followed by a slow and prolonged depolarization with corresponding discharge patterns. CP-96,345 (n = 3) and CP-99,994 (n = 6) selectively blocked the late, slow components of the stimulus-evoked response without affecting the early components. Responses to single electrical pulses of the same intensity and duration were not affected. CP-96,344 did not affect any of the responses tested (n = 5). The data indicate that nociceptive responses of a subset of spinal dorsal horn cells are selectively blocked by the NK-1 receptor antagonists, CP-96,345 and CP-99,994, thus confirming the involvement of NK-1 receptors in these responses.(ABSTRACT TRUNCATED AT 400 WORDS)

As excitatory amino acid receptors have been implicated in nociceptive sensory transmission, the principal objective of the present study was to investigate the effects of various excitatory amino acid antagonists on naturally evoked responses in spinal dorsal horn neurons. Extracellular single unit activity was recorded from functionally identified, spinal dorsal horn neurons in unanesthetized, decerebrated cats and in alpha-chloralose-anesthetized cats. The tests included iontophoretic application of the N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovaleric acid (APV), the non-N-methyl-D-aspartate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and kynurenate, and also the intravenous administration of the N-methyl-D-aspartate receptor antagonist, ketamine. In addition, attempts were made to determine the effects on these neurons of iontophoretic application of the excitatory amino acid agonists, L-glutamate, N-methyl-D-aspartate, quisqualate, (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and domoate. Marked differences were noted in the actions of agonists and antagonists between the responses observed in the unanesthetized, decerebrated and the anesthetized animals. In decerebrated cats, responses to hair afferent stimulation were blocked by kynurenate, 6-cyano-7-nitroquinoxaline-2,3-dione and 2-amino-5-phosphonovaleric acid. Responses to noxious thermal stimulation were attenuated by 2-amino-5-phosphonovaleric acid and in one unit also by ketamine. Neither 6-cyano-7-nitroquinoxaline-2,3-dione nor kynurenate affected the responses to noxious thermal stimulation. The proportion of cells responding to the agonists were: N-methyl-D-aspartate 24/27 (89%), quisqualate 12/13 (92%) and domoate 6/7 (86%). In chloralose-anesthetized cats, responses to hair afferent stimulation were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione and kynurenate but not by 2-amino-5-phosphonovaleric acid. Responses to noxious thermal stimulation were not affected by any of these antagonists, while the response to non-noxious thermal stimulation was blocked by 2-amino-5-phosphonovaleric acid, ketamine and kynurenate in the one neuron studied. The proportion of cells excited by the agonists differed from those observed in decerebrated cats: N-methyl-D-aspartate 9/32 (28%), quisqualate 50/54 (93%), (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate 19/23 (83%) and domoate 17/38 (45%). Application of the putative endogenous excitatory amino acid precursor N-acetyl-aspartyl-glutamate (NAAG) did not elicit a response in any of the neurons studied.(ABSTRACT TRUNCATED AT 400 WORDS)

We have previously reported a vibration-induced, adenosine-mediated inhibition of nociceptive dorsal horn neurons in the cat spinal cord. The present study was conducted to investigate the mechanisms of this inhibition. In vivo intracellular recording was obtained from dorsal horn neurons in the lower lumbar segments of the anaesthetized cat. Vibration (80-250 Hz for 2-3 s every 15-20 s) was applied to the glabrous skin of the toes of the hind foot using a feedback-controlled mechanical stimulator. In 32 of 43 neurons tested, vibration produced a pronounced hyperpolarization of the membrane potential. This hyperpolarization peaked at -10 mV and decayed throughout the period of the application of vibration. It was associated with a decrease in membrane resistance, had a reversal potential negative to the resting membrane potential and was Cl(-)-independent, suggesting that it was due to an increase in a K+ conductance, properties typical of the response to adenosine. This inhibitory postsynaptic potential was unaffected by intravenous administration of bicuculline, strychnine and naloxone but was blocked by iontophoretic administration of 8-sulphophenyltheophylline, a P1-purinergic receptor antagonist. These results confirm our previous finding that vibration-induced inhibition of nociceptive dorsal horn neurons is mediated via the release of an endogenous purine compound and further suggests that this inhibition involves a postsynaptic inhibitory mechanism.

The mammalian tachykinins, substance P and neurokinin A, and the non-mammalian tachykinin, physalaemin, were tested on functionally identified dorsal horn neurones in vivo. The experiments were done on cats which were anaesthetized with sodium pentobarbital or were anaemically decerebrated. Extracellular single-unit recordings were made in the lumbar spinal cord and the tachykinins were applied by iontophoresis. Each neurone was classified functionally as wide dynamic range, non-nociceptive, nociceptive specific or proprioceptive. The response to tachykinin application was determined for each neurone. Application of each of the tachykinins evoked a characteristic excitatory response which was delayed in onset, slow in developing and prolonged: physalaemin excited 99/131 neurones tested, neurokinin A excited 45/63 neurones and substance P excited 32/49 neurones. With two neurones physalaemin evoked a depression of the rate of firing, which may have been caused indirectly by excitation of a neighbouring neurone. Such depression was not elicited by either substance P or by neurokinin A. Physalaemin had a preferential excitatory effect on nociceptive neurones evoking excitation of 76/94 nociceptive neurones compared with 12/23 non-nociceptive neurones (chi 2 = 7.9, 1 d.f., P = 0.005). Substance P also caused a preferential excitation, with 30/40 nociceptive neurones being excited while all of the non-nociceptive neurones (n = 7) were unaffected (chi 2 = 11.5, 1 d.f., P = 0.0007). In contrast, neurokinin A failed to have a preferential effect; 32/46 nociceptive and 9/10 non-nociceptive neurones were excited (chi 2 = 1.0, 1 d.f., P = 0.40). Comparing the proportions of nociceptive neurones excited by the different tachykinins indicated that this type of neurone was not differently sensitive to any of the three peptides (chi 2 = 3.2, 2 d.f., P = 0.20). On the other hand, non-nociceptive neurones were preferentially excited by neurokinin A and physalaemin compared with substance P (chi 2 = 13.4, 2 d.f., P = 0.001). With regard to the endogenous tachykinins the results of this study may be interpreted in the following ways. The differential excitatory effect of substance P on nociceptive neurones supports the proposed role for this peptide in the transmission specifically of nociceptive inputs at the first afferent synapse. On the other hand, as neurokinin A excited non-nociceptive as well as nociceptive neurones, there may be a functional role for neurokinin A distinct from that of substance P.

The neurokinins, physalaemin, substance P, neurokinin A and bradykinin, were tested on the responses of single spinal neurons to the purines, adenosine 5'-triphosphate (ATP) and adenosine 5'-monophosphate and to GABA. Experiments were done on anaesthetized cats, recording extracellularly from functionally identified sensory neurons in the lumbar dorsal horn. All compounds were administered by iontophoresis. Neurokinins caused a slow, prolonged excitation which outlasted the period of application. Physalaemin was tested on responses to ATP in 24 units. In each case application of ATP caused either depression, excitation or a biphasic response when the application was not pre-conditioned by ejection of physalaemin. For 11 units, with ATP applications subthreshold to alter the on-going firing rate, such applications caused depression when they were preceded by administration of physalaemin. Three units were tested with ATP applications which caused the excitatory response; when the applications of ATP were preceded by ejection of physalaemin, there was then a depressant component in the response. In these 14 cases, the magnitude of the depression or of the depressant component of the response, was measured using currents which failed to produce depression in the absence of physalaemin ejection; the mean magnitude of this depression was 34.7 +/- 1.6% (+/- S.E.M.). With the 10 remaining units, responses to ATP were unaffected by application of physalaemin. The early components of the biphasic and excitatory responses were unaffected by physalaemin and hence it appeared to have a differential effect, enhancing only the depressant effects of ATP. The enhancement of depression was reversible, lasting up to 30 min following a single ejection. Neither control current nor glutamate mimicked the effect of physalaemin in the responses to application of ATP. The depressant response to adenosine 5'-monophosphate was also enhanced by physalaemin: ejections of adenosine 5'-monophosphate subthreshold to affect the on-going firing rate caused depression after physalaemin application in 3 of 8 units (average depression: 35.0 +/- 3.3%). On the other hand, depression induced by GABA was unaffected by physalaemin in every case (n = 8); in 4 of these cases GABA was tested on units for which purine-induced depression was enhanced by physalaemin. Thus, physalaemin preferentially affected depressant responses to ATP and to adenosine 5'-monophosphate.(ABSTRACT TRUNCATED AT 400 WORDS)

Recent studies have implicated glutamate and substance P in synaptic transmission in the nuclei tractus solitarii and in central regulation of cardiorespiratory functions. Consequently, in chloralose-anaesthetized cats that were artificially ventilated, we examined the effects of the microiontophoretic application of both chemicals (and the substance P homologue, eledoisin-related peptide) on single neurones of the nuclei tractus solitarii implicated in the control of respiration and respiratory tract reflexes. These neurones were functionally identified as either respiratory neurones or presumed reflex interneurones, and showed functional properties comparable to those previously documented for each of these two types. The iontophoretic application of glutamate produced an excitation of rapid onset in 23 or 25 reflex interneurones tested, but the respiratory neurones showed a differential sensitivity: one type (n = 32) was "glutamate-sensitive" and showed rapid excitation with glutamate applications of less than 30 nA, the other type of respiratory neurone (n = 26) was termed "glutamate-insensitive" since it either showed excitation only with applications of 60 nA or more or showed no response even with currents up to 94 nA. Each neurone studied was clearly of one type or the other. Glutamate could increase the number of spikes per rhythmic burst and the burst duration of respiratory neurones, it facilitated evoked activity in the reflex interneurones and in those respiratory neurones having a superior laryngeal nerve or vagus nerve afferent input, and the magnitude of the excitatory responses to glutamate varied directly with the amount of ejecting current. Substance P and eledoisin-related peptide also had excitatory effects on respiratory neurones and reflex interneurones, but compared with glutamate-induced effects the excitation was slower in onset and more prolonged in after-discharge. Both rhythmic and evoked activity could be facilitated, and the magnitude of the effect varied directly with the magnitude of the ejecting current. In showing that both glutamate and substance P (and its analogue, eledoisin-related peptide) have excitatory effects on the activity of respiratory neurones and reflex interneurones, this study provides evidence suggesting that these neurones have receptors for these neural chemicals, supportive of a role for each chemical in the regulation of respiration and respiratory tract reflexes.

Nociceptive neurons in the dorsal horn of the cat spinal cord are depressed by vibration applied to the ipsilateral hind limb. The present study investigated the pharmacological properties of this depression because of the possibility that it represents the neural basis at the spinal level for the analgesic effects of vibration in humans. Experiments were done in cats anesthetized with sodium pentobarbital and acutely spinalized at the first lumbar level. Extracellular recordings were made from nociceptive neurons in the lower lumbar segments. The depression of these neurons induced by vibration to the hindlimb was attenuated by administration of the P1-purinergic (adenosine) receptor antagonist, caffeine (20-60 mg/kg i.v.); the maximum attenuation was 100%. Effects of caffeine began within 2 min after the start of injection (1-3 min injection period), were greatest in the 10 min period after the end of injection and lasted for up to 2 hr. Importantly, another P1-purinergic receptor antagonist, which does not cross the blood-brain barrier, 8-sulphophenyltheophylline (8-16 mg/kg), had no effect on the depression when given intravenously (n = 5); however, when administered by iontophoresis 8-sulphophenyltheophylline blocked the depression in 2 of 6 units. Dipyridamole (1.0-2.0 mg/kg i.v.), an inhibitor of adenosine uptake, potentiated the depression in 2 of 5 cases. These results prompt us to suggest that depression induced by vibration may be mediated by adenosine via the activation of P1-purinergic receptors. On the other hand, the GABAA antagonist, bicuculline, failed to attenuate vibration-induced depression when administered either intravenously (0.2-0.4 mg/kg; n = 5) or by iontophoresis (n = 10) and the glycine antagonist, strychnine (0.2-0.6 mg/kg; n = 3) and the opiate antagonist, naloxone (0.1-0.4 mg/kg; n = 4) were similarly ineffective. These findings suggest that vibration-induced depression of these units occurs without involvement of bicuculline-sensitive GABA receptors, strychnine-sensitive glycine receptors and naloxone-sensitive opiate receptors. In view of the fact that vibration-induced depression is evoked synaptically, this study is the first to demonstrate in the central nervous system a synaptic response which is mediated by adenosine. In addition, we suggest that the analgesic effects of vibration in humans may be mediated at the spinal level by activation of P1-purinergic receptors.

We have shown previously that intrathecal administration of substance P to the lower thoracic vertebral level increases sympathetic output and increases the adrenal output of catecholamines. As opioid peptides are co-released with catecholamines from the adrenal medullae, experiments were done to determine whether the intrathecal administration of substance P to the eighth thoracic vertebral level would alter reaction time in the tail-flick test. Intrathecal administration of substance P (6.5 nmoles in artificial cerebrospinal fluid) in the awake restrained rat increased the reaction time at 1 and 6 min after injection to about 130-140% of the preadministration values; reaction time returned to preadministration values by 11 min. Similar administration of cerebrospinal fluid was without effect. Administration of 6.5 nmoles of thyrotropin-releasing hormone or oxytocin, peptides which also increase sympathetic output, failed to mimic the effects of substance P. The substance P-induced increase in reaction time was absent in rats which had been medullectomized and in rats pretreated with naloxone (10 mg/kg). Pretreatment with 10 mg/kg of either phentolamine or the quaternary opiate antagonists, nalorphine methochloride and naloxone methobromide, had no effect on the substance P-induced increase in reaction time. These results suggest that substance P given intrathecally to the eighth thoracic vertebral level may activate spinal sympathetic neurons to the adrenal medullae to cause the release of an opioid into the circulation. This circulating opioid may in turn play a role in the regulation of the tail-flick reflex by a centrally-mediated depression.

The present investigation was prompted by previous studies in our laboratory which have indicated that tachykinins enhance depressant effects of purines and that the purine adenosine mediates a vibration-induced depression of nociceptive dorsal horn neurons. Extracellular recordings were made from single nociceptive neurons in the lower lumbar segments of anaesthetized cats. Vibration (80 Hz; 2.5-3.5 s every 20-25 s) was applied to the hindlimb using a feedback-controlled mechanical stimulator. The tachykinins physalaemin, substance P and neurokinin A were administered by iontophoresis. Physalaemin was tested on vibration-induced responses of 29 neurons; each neuron was excited by this tachykinin. To control for possible changes in the response to vibration caused by the excitation per se, statistical comparisons were made of the vibration-induced responses during excitation by tachykinins and during excitation by glutamate. In 16 cases the magnitude of the vibration-induced depression was significantly greater during the excitation caused by physalaemin. With the remaining neurons the response to vibration failed to differ during the excitation by physalaemin compared with the excitation by glutamate. In four of the 16 cases subthreshold applications of vibration caused depression after administration of physalaemin. The P1-purinergic (adenosine) antagonist, caffeine, was administered in three cases where vibration caused depression only with application of physalaemin. In each of these cases the depression was reversibly blocked by caffeine (10 or 40 mg kg-1 i.v.). The magnitude of vibration-induced depression was significantly increased during excitation by neurokinin A (5/14 neurons) or by substance P (1/9 neurons). From the results of the present study we suggest that tachykinins enhance the vibration-induced depression. This enhancement may be due to enhanced depression by adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration of 10 micrograms of substance P intrathecally to the spinal T9 level of the adult rat, anaesthetized with urethane, provoked an increase in free catecholamines in plasma taken from the inferior vena cava. Adrenaline levels at 1 min after administration were 154.8 +/- 10.8% (mean +/- SE; n = 11) of preadministration levels and noradrenaline levels were 153.5 +/- 11.8% of preadministration levels. Differences between the values of free catecholamines in animals given substance P vs those given vehicle only were statistically significant at 1 and 10 min postinjection, but not at 30 min. Administration of a substance P analogue with central antagonistic properties 15 min before substance P was given prevented expression of the effects of substance P. These results suggest that substance P may be an excitatory chemical mediator of synaptic transmission in spinal pathways controlling adrenal medullary output. Thus dysfunction of substance P mechanisms may underlie some animal models of hypertension and may be involved in some cases of essential hypertension in man as well as in autonomic dysfunction associated with some neurological entities.

A study was done of the effects of iontophoretic application of adenosine 5'-monophosphate (AMP) and adenosine 5'-triphosphate (ATP) on functionally identified neurones in the spinal dorsal horn of the cat. AMP depressed nearly two-thirds of the 32 neurones tested regardless of functional type; the remainder were unaffected. ATP, on the other hand, had three types of effect: depression, excitation and a biphasic effect which consisted of excitation followed by depression. A significant difference was found when a comparison was made of the frequency of occurrence of each of these three types of effect in the samples of non-nociceptive (n = 18) and of wide dynamic range neurones (n = 42): of non-nociceptive neurones 61% were excited, 11% were depressed, 6% had a biphasic response and 22% were unaffected; of wide dynamic range neurones 45% had a biphasic response, 19% were depressed, 14% were excited and 21% were unaffected (chi 2 = 16.2, P less than 0.005). The depressant effects of both AMP and ATP and the depressant phase of the biphasic effect of ATP seem to be mediated through activation of P1-purinergic receptors because these effects were blocked by theophylline, a P1-purinergic antagonist [Burnstock (1978) In Cell Membrane Receptors for Drugs and Hormones: A Multidisciplinary Approach, pp. 107-118]. Thus the biphasic effect appears to consist of excitatory and depressant responses in the same neurone. The differential effects of ATP on non-nociceptive vs wide dynamic-range neurones are similar to the differential effects on these neurones observed during activation of low-threshold primary afferents. This similarity, together with evidence that ATP can be released from primary afferent neurones [Holton and Holton (1954) J. Physiol., Lond. 126, 124-140; Holton (1959) J. Physiol., Lond. 145, 494-504], prompts us to suggest that ATP may be a chemical mediator of effects of low-threshold primary afferent inputs in the spinal dorsal horn.