Lacunar-canalicular network in femoral cortical bone is reduced in aged women and is predominantly due to a loss of canalicular porosity.

The lacunar-canalicular network (LCN) of bone contains osteocytes and their dendritic extensions, which allow for intercellular communication, and are believed to serve as the mechanosensors that coordinate the processes of bone modeling and remodeling. Imbalances in remodeling, for example, are linked to bone disease, including fragility associated with aging. We have reported that there is a reduction in scale for one component of the LCN, osteocyte lacunar volume, across the human lifespan in females. In the present study, we explore the hypothesis that canalicular porosity also declines with age. To visualize the LCN and to determine how its components are altered with aging, we examined samples from young (age: 20-23 y; n = 5) and aged (age: 70-86 y; n = 6) healthy women donors utilizing a fluorescent labelling technique in combination with confocal laser scanning microscopy. A large cross-sectional area of cortical bone spanning the endosteal to periosteal surfaces from the anterior proximal femoral shaft was examined in order to account for potential trans-cortical variation in the LCN. Overall, we found that LCN areal fraction was reduced by 40.6% in the samples from aged women. This reduction was due, in part, to a reduction in lacunar density (21.4% decline in lacunae number per given area of bone), but much more so due to a 44.6% decline in canalicular areal fraction. While the areal fraction of larger vascular canals was higher in endosteal vs. periosteal regions for both age groups, no regional differences were observed in the areal fractions of the LCN and its components for either age group. Our data indicate that the LCN is diminished in aged women, and is largely due to a decline in the canalicular areal fraction, and that, unlike vascular canal porosity, this diminished LCN is uniform across the cortex.

The increased density of p38 mitogen-activated protein kinase-immunoreactive microglia in the sensorimotor cortex of aged TgCRND8 mice is associated predominantly with smaller dense-core amyloid plaques.

The role for phosphorylated p38 mitogen-activated protein kinase [p-p38(MAPK)] in ß-amyloid plaque deposition [a hallmark of Alzheimer's disease (AD) pathology] remains ambiguous. We combined immunohistochemistry and stereological sampling to quantify the distribution of plaques and p-p38(MAPK)-immunoreactive (IR) cells in the sensorimotor cortex of 3-, 6- and 10-month-old TgCRND8 mice. The aggressive nature of the AD-related human amyloid-ß protein precursor expressed in these mice was confirmed by the appearance of both dense-core (thioflavin-S-positive) and diffuse plaques, even in the youngest mice. p-p38(MAPK)-IR cells of the sensorimotor cortex were predominantly co-immunoreactive for the Macrophage-1 (CD11b/CD18) microglial marker. These p-p38(MAPK)-IR microglia were associated with both dense-core and diffuse plaques, but the expected age-dependent increase in the density of plaque-associated p-p38(MAPK)-IR microglia was restricted to dense-core plaques. Furthermore, the density of dense-core plaque-associated p-p38(MAPK)-IR microglia was inversely correlated with the size of the core within the given plaque, which supports a role for these microglia in restricting core growth. p-p38(MAPK)-IR microglia were also observed throughout wildtype and TgCRND8 mouse cortical parenchyma, but the density of these non-plaque-associated microglia remained constant, regardless of age or genotype. We conclude that the constitutive presence of p-p38(MAPK)-IR microglia in aging mouse brain is indicative of a longitudinal role for this kinase in normal brain physiology. We suggest that this fact, as well as the fact that a pool of p-p38(MAPK)-IR microglia appears to restrict ß-amyloid plaque core development, needs to be duly considered when ascribing functions for p38(MAPK) signalling in the AD brain.

The nuclear localization of 3'-phosphoinositide-dependent kinase-1 is dependent on its association with the protein tyrosine phosphatase SHP-1.

3'-Phosphoinositide-dependent protein kinase-1 (PDK1), the direct upstream kinase of Akt, can localize to the nucleus during specific signalling events. The mechanism used for its import into the nucleus, however, remains unresolved as it lacks a canonical nuclear localization signal (NLS). Expression of activated Src kinase in C6 glioblastoma cells promotes the association of tyrosylphosphorylated PDK1 with the NLS-containing tyrosine phosphatase SHP-1 as well as the nuclear localization of both proteins. A constitutive nucleo-cytoplasmic SHP-1:PDK1 shuttling complex is supported by several lines of evidence including (i) the distribution of both proteins to similar subcellular compartments following manipulation of the nuclear pore complex, (ii) the nuclear retention of SHP-1 upon overexpression of a PDK1 protein bearing a disrupted nuclear export signal (NES), and (iii) the exclusion of PDK1 from the nucleus upon overexpression of SHP-1 lacking the NLS or following siRNA-mediated knock-down of SHP-1. The latter case results in a perinuclear distribution of PDK1 that corresponds with the distribution of PIP3 (phosphatidylinositol 3,4,5-triphosphate), while a PDK1 protein bearing a mutated PH domain that abrogates PIP3-binding is excluded from the nucleus. Our data suggest that the SHP-1:PDK1 complex is recruited to the nuclear membrane by binding to perinuclear PIP3, whereupon SHP-1 (and its NLS) facilitates active import. Export from the nucleus relies on PDK1 (and its NES). The intact complex contributes to Src kinase-induced, Akt-sensitive podial formation in C6 cells.

Dephosphorylation of Akt in C6 cells grown in serum-free conditions corresponds with redistribution of p85/PI3K to the nucleus.

Withdrawal of serum from cell cultures constitutes a useful model for the study of mechanisms involved in the regulation of Akt function in vitro. However, there have been several reports of changes in Akt activity that are not fully explained by the current model of phosphatidylinositol 3'-kinase (PI3K)/Akt signaling. We demonstrate the expected loss of Akt phosphorylation in C6 glioma cells cultured in serum-free conditions, yet we also observed a paradoxical increase in PI3K-lipid kinase activity in the same cultures. These events corresponded with relocalization of p85, the regulatory subunit of PI3K, to the perinuclear region and a local increase in PI3K-lipid kinase products. Treatment with platelet-derived growth factor (PDGF) maintained the association between p85 and the PDGF receptor during serum withdrawal and restored PI3K-lipid production at the plasma membrane. Although this protected Akt from dephosphorylation, it only slightly reversed cell-cycle arrest. These effects were not sensitive to treatment with epidermal growth factor, thus precluding a generalized role for growth factors. Our data suggest that loss of growth factor signaling, including PDGF signaling, may disrupt recruitment and/or anchoring of an active p85(PI3K) complex at the plasma membrane during serum withdrawal, which could account for the concurrent loss of Akt function.

Haloperidol induces apoptosis via the sigma2 receptor system and Bcl-XS.

Toxicity of the typical antipsychotic haloperidol (HAL) comprises an apoptotic component that we link to pro-apoptotic Bcl-XS in PC12 preneuronal and N2a neuroblastoma cells. The mitochondrial translocation of Bcl-XS and its interaction with the pore-forming voltage-dependent anion channel (VDAC) correlates with the redistribution of cytochrome c and the cleavage of Poly(ADP-ribose) polymerase. Haloperidol-induced apoptosis is mediated by the sigma2 (sigma2) receptor system and does not involve the expected antagonism of the dopamine D(2) receptor, nor is it influenced by Vitamin E- or p53/Bax-mediated events. Pathological relevance is demonstrated by the cytotoxic synergism between HAL and the Alzheimer disease-related peptide beta-amyloid(1-40), which correlates with Bcl-XS expression and its interaction with VDAC, and with cytosolic cytochrome c translocation. These data provide for a unique apoptotic mechanism that could underscore the clinical risks associated with HAL, particularly following chronic regimens or in the elderly.

PILRalpha, a novel immunoreceptor tyrosine-based inhibitory motif-bearing protein, recruits SHP-1 upon tyrosine phosphorylation and is paired with the truncated counterpart PILRbeta.

SHP-1-mediated dephosphorylation of protein tyrosine residues is central to the regulation of several cell signaling pathways, the specificity of which is dictated by the intrinsic affinity of SH2 domains for the flanking sequences of phosphotyrosine residues. By using a modified yeast two-hybrid system and SHP-1 as bait, we have cloned a human cDNA, PILRalpha, encoding a 303-amino acid immunoglobulin-like transmembrane receptor bearing two cytoplasmic tyrosines positioned within an immunoreceptor tyrosine-based inhibitory motif. Substrate trapping in combination with pervanadate treatment of 293T cells confirms that PILRalpha associates with SHP-1 in vivo upon tyrosine phosphorylation. Mutation of the tyrosine residues in PILRalpha indicates the pivotal role of the Tyr-269 residue in recruiting SHP-1. Surface plasmon resonance analysis further suggests that the association between PILRalpha-Tyr-269 and SHP-1 is mediated primarily via the amino-terminal SH2 domain of the latter. Polymerase chain reaction amplification of cDNA in combination with genomic sequence analysis revealed a second gene, PILRbeta, coding for a putative activating receptor as suggested by a truncated cytoplasmic tail and a charged lysine residue in its transmembrane region. The PILRalpha and PILRbeta genes are localized to chromosome 7 which is in contrast with the mapping of known members of the inhibitory receptor superfamily.

Plasma and CSF benzodiazepine receptor ligand concentrations in cirrhotic patients with hepatic encephalopathy: relationship to severity of encephalopathy and to pharmaceutical benzodiazepine intake.

Increased plasma and CSF concentrations of substances which bind to brain benzodiazepine receptors have previously been reported in cirrhotic patients with hepatic encephalopathy (HE). However, their relationship to previous intake of pharmaceutical benzodiazepines has not been clearly established. In the present study, plasma levels of benzodiazepine receptor ligands (BZRLs) were measured using a sensitive radioreceptor assay in 12 control subjects with no evidence of hepatic, neurological or psychiatric illness, 11 cirrhotic patients without HE, 24 cirrhotic patients with moderate (grade I-II) HE and in 45 cirrhotic patients with severe (grade II-IV) HE. In addition, CSF concentrations of BZRLs were measured in 8 cirrhotic patients with HE and an equal number of age-matched controls. Recent intake (within 10 days) of pharmaceutical benzodiazepines was assessed by detailed review of medical files, and interviews with the patient, at least one family member as well as the pharmacist. Significantly increased plasma concentrations of BZRLs were observed in cirrhotic patients with severe encephalopathy (p < 0.02) compared to controls and to cirrhotic patients without (or with mild) neurological impairment. Increased plasma BZRLs could be accounted for by prior exposure to benzodiazepine medication in all cases. CSF concentrations of BZRLs in cirrhotic patients were not significantly different from control values. These findings do not support a role for "endogenous" benzodiazepines in the pathogenesis of HE in chronic liver disease but suggest that pharmaceutic benzodiazepines administered to cirrhotic patients as sedatives or as part of endoscopic work-up could have contributed to the neurological impairment in some patients.

Increased density of catalytic sites and expression of brain monoamine oxidase A in humans with hepatic encephalopathy.

Altered biogenic amine metabolism and function are believed to underlie certain of the neuropsychiatric symptoms, e.g., depression, mania, and anxiety, encountered in clinical hepatic encephalopathy (HE). We therefore investigated the activity of the degradative enzyme monoamine oxidase (MAO) and its binding parameters using [3H]Ro 41-1049 (defining MAO-A) and [3H]Ro 19-6327 (Lazabemide; defining MAO-B) in autopsied brain tissue from male cirrhotic patients with HE. The MAO-B parameters in HE patient tissue were not significantly different from those determined for control tissue. In contrast, increases in MAO-A activities in HE patient frontocortical (by approximately 50%) and cerebellar (by approximately 145%) tissues were observed, confirming our previous findings using comparable tissues. Increases in the abundance of the active MAO-A protein were of the same order of magnitude, e.g., in frontal cortex by approximately 85% and in cerebellum by approximately 225%. Reverse transcriptase-polymerase chain reaction indicated an increase in the level of gene expression (by approximately 155%) and thus offers some of the first evidence of a transcriptional event potentially mediating MAO-A function in human brain tissue. The levels of biogenic amine acid metabolites were increased as expected. As HE patients are most often treated for their hepatic symptoms rather than their neuropsychiatric manifestations, they represent an important "untreated" psychiatric population. The present findings are therefore not only important for our understanding of the pathophysiology of HE but also extremely relevant to our understanding of the pharmacotherapy of other neuropsychiatric disorders in which biogenic amine and MAO-A dysfunction is indicated.

Vesicular dysfunction during experimental thiamine deficiency is indicated by alterations in dopamine metabolism.

Experimental and clinical studies indicate that catecholamines play an important role in the neurobehavioural symptomatology of thiamine deficiency. Given the cerebral region-selective vulnerability and the behavioural impairment commonly encountered in thiamine deficiency, we undertook to investigate regional catecholamine metabolism in the brains of pyrithiamine-induced thiamine-deficient rats. Dopamine metabolism was unaffected in the striatum. In contrast, other regions also known to be involved in sensory processing and intellectual function (e.g., frontal cortex, hypothalamus, thalamus), but having a greater noradrenergic input, had increased levels of 3,4-dihydroxyphenylacetic acid (DOPAC) and decreased levels of other dopaminergic metabolites including noradrenaline. In these regions levels of the vesicular amine transporter, defined by tetrabenazine-sensitive [3H]ketanserin binding, were also decreased. Our data suggest a region-selective vesicular dysfunction resulting in intraneuronal release, and subsequent degradation, of dopamine. These disruptions of dopamine and consequently noradrenaline metabolism may account for certain neurobehavioural deficits commonly encountered in thiamine deficiency.

Alterations in serotonin parameters in brain of thiamine-deficient rats are evident prior to the appearance of neurological symptoms.

Biochemical alterations of serotoninergic parameters have been demonstrated in experimental thiamine deficiency. In addition, hypophagia and hypothermia, two physiological processes associated with changes in the serotonin [5-hydroxytryptamine (5-HT)] system, are manifest early during the progression of thiamine deficiency. The binding of selected 5-HT radioligands was therefore investigated in discrete brain regions of pyrithiamine-induced thiamine-deficient rats. Using quantitative receptor autoradiography, the binding of 8-hydroxy-2-(di-n-[3H]propylamino) tetralin, a ligand used to label the somatodendritic 5-HT1A autoreceptor of the dorsal raphe nucleus, was found to be unaffected in this region, suggesting that the structural integrity of the 5-HT cell bodies is maintained throughout the course of pyrithiamine treatment. Increased binding of [3H]-ketanserin was observed in regions considered vulnerable as well as in some considered to be nonvulnerable during the course of thiamine deficiency. These binding changes, which appear to represent changes in the density of the postsynaptic 5-HT2A receptor population rather than the "tetrabenazine-sensitive" vesicular monoamine transporter, are evident before the appearance of histopathologic lesions and coincide with altered tissue concentrations of 5-HT. These data suggest that 5-HT neurons, although structurally intact, are functionally affected early during the progression of thiamine deficiency. These alterations, which are likely a part of adaptive neuronal change consequent to thiamine dysfunction, may be important in the physiological manifestations and the learning deficits commonly encountered in experimental thiamine deficiency.

Na+,K(+)-ATPase activity is selectively increased in thalamus in thiamine deficiency prior to the appearance of neurological symptoms.

The relationship between progression of neurological status and the activities of both Na+,K(+)- and Mg(2+)-dependent-ATPase (adenosine 5'-triphosphate phosphohydrolase) was investigated in brain regions of pyrithiamine-induced thiamine deficient rats. Thalamic Na+,K(+)-ATPase activity was selectively increased by 200% (P < 0.01) prior to the appearance of symptoms of thiamine deficiency and normalized in symptomatic rats. This selective transitory activation precludes a mediation by brain soluble fraction Na+,K(+)-ATPase modifiers as does the unaltered distribution in regional high-affinity [3H]ouabain binding densities observed throughout the time-course used in these experiments. Na+,K(+)-ATPase maintains cellular ionic gradients and has been implicated in neurotransmitter uptake and release mechanisms. The fact that the increased thalamic Na+,K(+)-ATPase activity coincides with the early alterations in serotonin metabolism observed in similarly treated animals and the concomitantly early increase in glucose utilization previously observed in the thalamus of thiamine-deficient rats is discussed.

Nitric oxide synthase activities are selectively decreased in vulnerable brain regions in thiamine deficiency.

Pyrithiamine-induced thiamine deficiency in the rat exhibits many neuropathological and biochemical similarities to Wernicke's Encephalopathy in human. Activities of constitutive nitric oxide synthase (NOS) were measured in vulnerable (thalamus and cerebellum) and non-vulnerable (hippocampus and striatum) brain regions of pyrithiamine-induced thiamine-deficient rats. NOS activities were significantly decreased in the thalamus (by 26%, P < 0.05) of presymptomatic thiamine-deficient rats compared to pair-fed controls. Following onset of symptoms, in addition to thalamus (-38%, P < 0.01), cerebellum (-50%, P < 0.01) also manifested significantly decreased activities of NOS. Hippocampal and striatal activities of NOS were unchanged at both presymptomatic and symptomatic stages of thiamine deficiency. Selectively decreased activities of neuronal NOS in the thalamus and the cerebellum extends the previous observations of region-selective metabolic changes and, ultimately, neuronal cell loss observed in thiamine deficiency.

A quantitative autoradiographic study of muscarinic cholinergic receptor subtypes in the brains of pyrithiamine-treated rats.

Previous studies describe decreased acetylcholine synthesis in brain as well as neurobehavioral evidence for a central muscarinic cholinergic deficit in pyrithiamine-induced thiamine-deficient rats. In order to further evaluate this possibility, quantitative autoradiographic procedures using [3H]quinuclidinyl benzilate (for total muscarinic binding sites), [3H]pirenzepine (for muscarinic M1 sites) and [3H]AF-DX 384 (for muscarinic M2 sites) were performed at early (presymptomatic) and late (symptomatic) stages of thiamine deficiency induced in rats by administration of the central thiamine antagonist, pyrithiamine. No significant alterations in densities of M1, M2 or total muscarinic binding sites were observed in any brain structure evaluated at either early or late stages of thiamine deficiency. These findings do not support a major role for modifications of muscarinic cholinergic function in the pathogenesis of the neurological symptoms of thiamine deficiency.

Trace amines in hepatic encephalopathy.

In 1971 Fischer and Baldessarini proposed the hypothesis that hepatic encephalopathy (HE), a neuropsychiatric syndrome associated with hepatic dysfunction, could result from the direct decarboxylation of amino acids leading to trace amines such as tyramine and octopamine which could then act as false neurotransmitters. This was supported by the observation that the clinical symptoms of HE appeared to improve following treatment with L-Dopa, which cannot be metabolized to either of these trace amines. In addition to serum and urine levels of octopamine correlating roughly with the grade of clinical HE, levels of octopamine were also significantly increased in rat brain following coma induced by hepatic devascularization and in portacaval-shunted rats fed high aromatic amino acid content diets. This hypothesis was questioned, however, given the lack of observable adverse behavioural effects following treatments with octopamine. Finally, the equivocal results of a limited number of clinical trials (using L-Dopa) argued against a direct intervention by catecholamine-like trace amines in HE. An alternative hypothesis was advanced by Sourkes in 1978 implicating increased tryptophan metabolism as a factor in the etiology of HE. Hepatic dysfunction in humans alters CNS concentrations of tryptophan which correlate well with levels of the tryptamine metabolite indoleacetic acid (IAA). Furthermore, regional densities of [3H]tryptamine receptors in HE patient brain tissue are significantly decreased. These data support a pathophysiologic role for tryptophan and its neuroactive trace amine metabolite tryptamine in HE.

A high-affinity [3H]tryptamine binding site in human brain.

In vitro filtration binding revealed high-affinity specific [3H]tryptamine binding sites in human brain. These binding sites are heterogeneously distributed throughout brain, ranging from 280 fmol/mg protein in hippocampus and thalamus to approximately 90 fmol/mg protein in medulla oblongata and cerebellum. Preliminary autoradiographic studies indicate a heterogeneous distribution within layers of the frontal cortex. The observed stereoselectivity of the site, the interaction of the site with a G protein and the observed region-selective downregulation of the site in a human pathological condition, i.e. hepatic encephalopathy (Mousseau et al., 1994), suggests that this binding site is a functional [3H]tryptamine receptor. A similarity in kinetics and distribution of the [3H]tryptamine receptor in human and rat brain indicates that these two entities represent homologous structures, although the difference in pharmacological profiles suggests species variants. One cannot exclude the possibility that the rat and human [3H]tryptamine receptors do represent distinct subtypes. Finally, the suggested role for tryptamine in neuropsychiatric disorders as originally suggested by Dewhurst (1968) is supported by the present series of experiments.

The [3H]tryptamine receptor in human brain: kinetics, distribution, and pharmacologic profile.

The kinetics and distribution of [3H]tryptamine binding sites in human brain were investigated. Specific [3H]tryptamine binding in frontal cortex was of nanomolar affinity, reversible, saturable, and best fit to a single-site model. A heterogeneous distribution for this binding site was demonstrated, with the highest density observed in hippocampus, thalamus >> caudate nucleus, frontal cortex, pons, temporal cortex > globus pallidus/putamen, cerebellum. The similarities in kinetics and distribution of the [3H]tryptamine binding site in human and rat brain indicate that these two binding sites represent homologous structures. However, the present displacement studies using various ligands (indoleamines and other tryptophan metabolites, phenylethylamines, and miscellaneous drugs) and salts (Na+, K+, Ca2+, Mg2+, Cu2+) indicate stereospecific displacement as well as a rank-order potency profile that is different from that reported for the rat [3H]tryptamine binding site. This suggests the presence of distinct species-dependent [3H]tryptamine binding site subtypes. Taken together with the documented electrophysiological and behavioral evidence of tryptamine-mediated effects in the rat and the recent report of a significant loss of these binding sites in human portal systemic encephalopathy, as well as the present demonstration of an effect of guanine nucleotides on [3H]-tryptamine binding affinity, these findings suggest that these binding sites might be functional receptors. The implied role of tryptamine in neuropsychiatric disorders is supported by this demonstration of a receptor for [3H]-tryptamine in human brain.

Substance P analogs displace sigma binding differentially in the brain and spinal cord of the adult mouse.

We have previously observed similarities in the behavioral effects produced by the NH2-terminus of the undecapeptide substance P (SP) and by 1,3-di(2-tolyl)-guanidine (DTG) in the adult mouse. The present series of experiments indicate differences in the rank-order of potency of sigma ligands [DTG; haloperidol (HAL)], SP analogs [SP; SP(1-7); SP(5-11); [D-Pro2, D-Phe7]-SP(1-7) (D-SP(1-7))] and miscellaneous compounds [morphine (MOR), naloxone (NAL)] at competing for [3H]-DTG binding sites in the mouse brain and spinal cord in vitro: Brain; DTG = HAL >> SP = MOR = NAL >> SP(1-7) >> D-SP(1-7) >> SP(5-11): Spinal cord; DTG = HAL >> SP(1-7) = MOR = NAL >> SP >> D-SP(1-7) = SP(5-11). The observed difference in the rank-order potencies of the displacing ligands at these same binding sites supports the notion of two distinct populations of sigma binding sites in these tissues in the adult mouse. Given the low (micromolar) potency of SP analogs at displacing [3H]-DTG binding in the present series of experiments, it is unlikely that the similar behavioral effects we have previously observed elicited by SP(1-7) and DTG in the adult mouse are a result of a direct action of SP(1-7) at the sigma binding site.

Current theories on the pathogenesis of hepatic encephalopathy.

Hepatic encephalopathy (HE) is a serious neuropsychiatric complication of both acute and chronic liver disease. Several hypotheses have emerged following the development of appropriate animal models of HE and following studies using postmortem brain tissue from HE patients. It was originally suggested that primary energy failure was responsible for HE; however, there is now mounting evidence that the pathogenetic defect involves neurotransmission failure. Specific neurotransmitter systems implicated in the pathogenesis of portal-systemic encephalopathy (PSE) include the excitatory amino acid glutamate as well as neuroactive and/or neurotoxic biogenic amine metabolites. Although it has been proposed that alterations in the gamma-aminobutyric acid (GABA) system may play a pathogenic role in HE associated with both chronic and acute liver failure, there is now overwhelming evidence to the contrary. On the other hand, there is evidence to suggest that a subgroup of patients with HE have increased blood and CSF concentrations of substances that bind to GABA-related benzodiazepine receptors in brain. Alterations of both the glutamatergic and serotoninergic neurotransmitter systems in PSE likely result from the metabolic consequences of chronic exposure of brain to toxic levels of ammonia. In addition to its effects on glutamatergic and serotoninergic systems during chronic liver disease, ammonia has been intimately associated with the brain edema invariably observed in acute liver failure. It is evident that, regardless of the type of liver failure, effective reductions of ammonia levels remains the strategy of choice in the prevention of encephalopathy. The further elucidation of neurotransmitter alterations in HE could result in novel "downstream" neuropharmacologic approaches to its prevention and treatment.

An antinociceptive effect of capsaicin in the adult mouse mediated by the NH2-terminus of substance P.

Capsaicin in the adult animal is believed to evoke a massive release of substance P (SP) and a subsequent loss of primary afferent C-fiber activity. Given the antinociceptive effect of SP N-terminal metabolites, the present experiments were designed to compare behavioral and nociceptive responses after treatments with capsaicin or the SP NH2-terminal fragment, SP(1-7), and to determine whether they share a common mechanism of action. When adult mice were tested using the hot-plate assay, 24 hr after intrathecal injections of either capsaicin (0.3-2.6 nmol) or SP(1-7) (22.5 pmol-10 nmol), a dose-related antinociception was observed. The caudally directed biting and scratching behaviors induced by 1 nmol of capsaicin injected intrathecally were also greatly reduced 24 hr after either 2.6 nmol of capsaicin or 10 nmol of SP(1-7). Pretreatment with an antagonist of the NH2-terminus of SP, [D-Pro2,D-Phe7]-SP(1-7), prevented the long-term effects of capsaicin and of SP(1-7) on capsaicin-induced behaviors in our paradigm at doses that the COOH-terminal antagonist of neurokinin activity, [D-Pro2,D-Trp7,9]-SP, did not. The antinociceptive effect of capsaicin in the adult animal is, therefore, mimicked by SP(1-7) and attenuated by [D-Pro2,D-Phe7]-SP(1-7), suggesting that the NH2-terminus of SP and its NH2-terminal metabolites, released in response to capsaicin, may contribute to the mediation of capsaicin's antinociceptive effect.