Cdc20 and securin overexpression predict short-term breast cancer survival.

BACKGROUND: Cdc20 is an essential component of cell division and responsible for anaphase initiation regulated by securin degradation. Cdc20 function is strongly regulated by the spindle assembly checkpoint to ensure the timely separation of sister chromatids and integrity of the genome. We present the first results on Cdc20 in a large clinical breast cancer material. METHODS: The study was based on 445 breast cancer patients with up to 20 years of follow-up (mean 10.0 years). DNA content was determined by image cytometry on cell imprints, and Cdc20 and securin immunohistochemistry on tissue microarrays of breast cancer tissue. RESULTS: In our results, high Cdc20 and securin expression was associated with aneuploid DNA content. In prognostic analyses, high Cdc20 immunoexpression alone and in combination with high securin immunoexpression indicated aggressive course of disease and up to 6.8-fold (P<0.001) risk of breast cancer death. Particularly, high Cdc20 and securin immunoexpression identified a patient subgroup with extremely short, on average 2.4 years, breast cancer survival and triple-negative breast cancer (TNBC) subtype. CONCLUSIONS: We report for the first time the association of high Cdc20 and securin immunoexpression with extremely poor outcome of breast cancer patients. Our experience indicates that Cdc20 and securin are promising candidates for clinical applications in breast cancer prognostication, especially in the challenging prognostic decisions of TNBC.

EGFR gene copy number assessment from areas with highest EGFR expression predicts response to anti-EGFR therapy in colorectal cancer.

BACKGROUND: Only 40-70% of metastatic colorectal cancers (mCRCs) with wild-type (WT) KRAS oncogene respond to anti-epidermal growth factor receptor (anti-EGFR) antibody treatment. EGFR amplification has been suggested as an additional marker to predict the response. However, improved methods for bringing the EGFR analysis into routine laboratory are needed. METHODS: The material consisted of 80 patients with mCRC, 54 of them receiving anti-EGFR therapy. EGFR gene copy number (GCN) was analysed by automated silver in situ hybridisation (SISH). Immunohistochemical EGFR protein analysis was used to guide SISH assessment. RESULTS: Clinical benefit was seen in 73% of high (≥ 4.0) EGFR GCN patients, in comparison with 59% of KRAS WT patients. Only 20% of low EGFR GCN patients responded to therapy. A high EGFR GCN number associated with longer progression-free survival (P<0.0001) and overall survival (P=0.004). Together with KRAS analysis, EGFR GCN identified the responsive patients to anti-EGFR therapy more accurately than either test alone. The clinical benefit rate of KRAS WT/high EGFR GCN tumours was 82%. CONCLUSION: Our results show that automated EGFR SISH, in combination with KRAS mutation analysis, can be a useful and easily applicable technique in routine diagnostic practise for selecting patients for anti-EGFR therapy.

Tumor-infiltrating lymphocytes and CD8+ T cells predict survival of triple-negative breast cancer.

PURPOSE: Tumor inflammatory response was evaluated as a prognostic feature in triple-negative breast cancer (TNBC) and compared with the clinical prognosticators of breast cancer and selected biomarkers of cancer cell proliferation. METHODS: TNBC patients (n = 179) with complete clinical data and up to 18-year follow-up were obtained from Auria biobank, Turku University Hospital, Turku, Finland. Tumor-infiltrating lymphocytes (TILs) and several subtypes of inflammatory cells detected with immunohistochemistry were evaluated in different tumor compartments in full tissue sections and tissue microarrays. RESULTS: Deficiency of stromal TILs and low number of CD8+ T cells independently predicted mortality in TNBC (HR 2.4, p 0.02 and HR 2.1, p 0.02, respectively). Each 10% decrease in stromal TILs resulted in 20% increased risk of mortality. An average of 13.2-year survival difference was observed between the majority (> 75%) of patients with low (< 14% of TILs) vs high (≥ 14% of TILs) frequency of CD8+ T cells. The prognostic value of TILs and CD8+ T cells varied when evaluated in different tumor compartments. TILs and CD8+ T cells were significantly associated with Securin and Separase, essential regulators of metaphase-anaphase transition of the cell cycle. DISCUSSION: TILs and CD8+ T cells provide additional prognostic value to the established clinical prognostic markers in TNBC. However, possible clinical applications would still benefit from systematic guidelines for evaluating tumor inflammatory response. Increasing understanding on the interactions between the regulation of cancer cell proliferation and inflammatory response may in future advance treatment of TNBC.

Postischemic regulation of central histamine receptors.

This study characterizes changes occurring in the central histaminergic system associated with ischemia-reperfusion pathology in the rat. Specifically, after a postocclusion time period of 48 h, we have analyzed histamine H(1) receptor mRNA expression, histamine H(2) receptor protein amount and binding densities, and histamine H(3) receptor mRNA expression and binding densities in brain regions that have been suggested to be selectively vulnerable to transient global ischemia, i.e. hippocampus, thalamus, caudate-putamen, and cerebral cortex. We found an increase in H(1) receptor mRNA expression in the caudate-putamen: given that ischemia reduces glucose uptake and H(1) receptor activation has been shown to decrease this effect, an increase of expression levels may result in mitigating tissue damage due to energy failure observed in ischemia. A decrease in H(2) receptor binding densities in the caudate-putamen was also observed; the ischemia-induced decrease in H(2) receptor protein was also detectable by Western blot analysis. This phenomenon may underlie the previously reported ischemia induced striatal dopamine release. H(3) receptor mRNA expression was increased in the caudate putamen of the postischemic brain but was decreased in the globus pallidus and the thalamus; in association with this, H(3) receptor binding densities were increased in the cortex, caudate-putamen, globus pallidus, and hippocampus. The upregulation of H(3) receptor ligand binding may be involved in the previously reported continuous neuronal histamine release. Our data suggest that central histamine receptor expression and ligand binding are altered in brain ischemia in distinct areas, and may participate in neuroprotection and/or ischemia-associated neuronal damage.

Lack of histamine synthesis and down-regulation of H1 and H2 receptor mRNA levels by dexamethasone in cerebral endothelial cells.

The purpose of this work was to determine whether cerebral endothelial cells have the capacity to synthesize histamine or to express mRNA of receptors that specifically respond to available free histamine. The histamine concentrations and the expression of L-histidine decarboxylase (HDC) and histamine H1 and H2 receptor mRNA, both in adult rat brain and in cultured immortalized RBE4 cerebral endothelial cells, were investigated. In this study endothelial cells were devoid of any kind of detectable histamine production, both in vivo and in the immortalized RBE4 cells in culture. Both the immunostainings for histamine and the in situ hybridizations for HDC were negative, as well as histamine determinations by HPLC, indicating that endothelial cells do not possess the capacity to produce histamine. Also, glucocorticoid (dexamethasone) treatment failed to induce histamine production in the cultured cells. Although the cerebral endothelial cells lack histamine production, a nonsaturable uptake in RBE4 cells is demonstrated. The internalized histamine is detected both in the cytoplasm and in the nucleus, which could indicate a role for histamine as an intracellular messenger. Histamine H1 and H2 receptor mRNA was expressed in RBE4 cells, and glucocorticoid treatment down-regulated the mRNA levels of both H1 and H2 receptors. This mechanism may be involved in glucocorticoid-mediated effects on cerebrovascular permeability and brain edema.

Staurosporine induces a neuronal phenotype in SH-SY5Y human neuroblastoma cells that resembles that induced by the phorbol ester 12-O-tetradecanoyl phorbol-13 acetate (TPA).

Treatment of SH-SY5Y human neuroblastoma cells with the protein kinase inhibitor staurosporine, induced both morphological and functional differentiation in these cells. The effects of staurosporine were comparable to those induced by the protein kinase C (PKC) activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA), with respect to induction of neuronal differentiation, i.e. neurite outgrowth, inhibition of DNA synthesis, induction and down-regulation of c-myc protein expression, induction of mRNA for both neuropeptide Y (NPY) and growth associated protein 43 (GAP-43) and stimulation of tyrosine hydroxylase expression. Staurosporine failed to translocate PKC to the membrane fraction or to stimulate phosphorylation of the endogenous PKC substrate M(r) 80,000 (p80). Instead, staurosporine inhibited TPA-induced phosphorylation of p80.

In situ detection of H1-receptor mRNA and absence of apoptosis in the transient histamine system of the embryonic rat brain.

In the developing brain, histamine is one of the first neurotransmitters to appear. The concentration of histamine in the prenatal brain is fivefold that of adult levels. During the prenatal development a large transiently histamine-immunoreactive cell population distinct from the adult histaminergic system can be found within a subpopulation of the developing serotonergic raphe nuclei neurons. Also histamine-immunoreactive nerve fibers are widely distributed already during the prenatal development extending to the diencephalon, the thalamus, the cortex, and the spinal cord. Large numbers of histamine-containing mast cells also migrate into the brain during the late prenatal life. The wide distribution and high prenatal concentrations imply important functions for the histaminergic system during intrauterine development. However, little is known about the actual functions of histamine during development, and which of the histamine receptors are present in the prenatal rat brain is currently unknown. In the present study, we used in situ hybridization to study the distribution of H1-receptor (H1R) mRNA in the embryonic rat brain and spinal cord. H1R mRNA could be detected in rat brain and in spinal cord on embryonic day (E) 14, and the expression pattern seemed to partially localize in areas containing histamine-immunoreactive nerve fibers through E14-E20. H1R mRNA was also detected by reverse transcriptase polymerase chain reaction from embryonic brain samples and by Northern hybridization. The possible involvement of apoptosis in the disappearance of the developing transiently histaminergic system was studied by using apoptosis detection based on the terminal dUTP nick end labeling (TUNEL) technique and with c-Fos immunostaining. Although histamine immunoreactivity disappears dramatically from the developing raphe nuclei after E18, only occasional apoptotic nuclei could be seen in the histamine-immunoreactive cell bodies. The presence of H1R mRNA during the embryonic development renders it possible that histamine could exert an H1R-specific function at the time of the embryonic histamine peak.

The histaminergic system in the brain: structural characteristics and changes in hibernation.

Histaminergic neurons in adult vertebrate brain are confined to the posterior hypothalamic area, where they are comprised of scattered groups of neurons referred to as the tuberomammillary nucleus. Histamine regulates hormonal functions, sleep, food intake, thermoregulation and locomotor activity, for example. In the zebrafish, Danio rerio, histamine was detected only in the brain, where also the histamine synthesizing enzyme L-histidine decarboxylase (HDC) was expressed. It is possible that histamine has first evolved as a neurotransmitter in the central nervous system. We established sensitive quantitative in situ hybridization methods for histamine H(1) and H(2) receptors and HDC, to study the modulation of brain histaminergic system under pathophysiological conditions. A transient increase in H(1) receptor expression was seen in the dentate gyrus and striatum after a single injection of kainic acid, a glutamate analog. H(1) antagonists are known to increase duration of convulsions, and increased brain histamine is associated with reduced convulsions in animal models of epilepsy. No HDC mRNA was detected in brain vessels by in situ hybridization, which suggests lack of histamine synthesis by brain endothelial cells. This was verified by lack of HDC mRNA in a rat brain endothelial cell line, RBE4 cells. Both H(1) and H(2) receptor mRNA was found in this cell line, and the expression of both receptors was downregulated by dexamethasone. The findings are in agreement with the concept that histamine regulates blood-brain barrier permeability through H(1) and H(2) receptor mediated mechanisms. Hibernation is characterized by a drastic reduction of central functions. The activity of most transmitter systems is maintained at a very low level. Surprisingly, histamine levels and turnover were clearly elevated in hibernating ground squirrels, and the density of histamine-containing fibers was higher than in euthermic animals. It is possible that histamine actively maintains the low activity of other transmitters during the hibernation state.

Histamine is required for H3 receptor-mediated alcohol reward inhibition, but not for alcohol consumption or stimulation.

BACKGROUND AND PURPOSE: Conflicting data have been published on whether histamine is inhibitory to the rewarding effects of abused drugs. The purpose of this study was to clarify the role of neuronal histamine and, in particular, H3 receptors in alcohol dependence-related behaviours, which represent the addictive effects of alcohol. EXPERIMENTAL APPROACH: Alcohol-induced conditioned place preference (alcohol-CPP) was used to measure alcohol reward. Alcohol-induced locomotor stimulation, alcohol consumption and kinetics were also assessed. mRNA levels were quantified using radioactive in situ hybridization. KEY RESULTS: Low doses of H3 receptor antagonists, JNJ-10181457 and JNJ-39220675, inhibited alcohol reward in wild-type (WT) mice. However, these H3 receptor antagonists did not inhibit alcohol reward in histidine decarboxylase knock-out (HDC KO) mice and a lack of histamine did not alter alcohol consumption. Thus H3 receptor antagonists inhibited alcohol reward in a histamine-dependent manner. Furthermore, WT and HDC KO mice were similarly stimulated by alcohol. The expression levels of dopamine D1 and D2 receptors, STEP61 and DARPP-32 mRNA in striatal subregions were unaltered in HDC KO mice. No differences were seen in alcohol kinetics in HDC KO compared to WT control animals. In addition, JNJ-39220675 had no effect on alcohol kinetics in WT mice. CONCLUSIONS AND IMPLICATIONS: These data suggest that histamine is required for the H3 receptor-mediated inhibition of alcohol-CPP and support the hypothesis that the brain histaminergic system has an inhibitory role in alcohol reward. Increasing neuronal histamine release via H3 receptor blockade could therefore be a novel way of treating alcohol dependence.

Histamine in brain development and tumors.

Histamine is found in developing mammalian brain in both neurons and mast cells. Under normal conditions, histamine H1 and H2 receptors are found in neural, glial and endothelial cells, and H3 receptors at least on neurons. Experimental brain tumors display both H1 and H2 receptors, and histamine increases permeability in the tumors and in the neighboring areas. Many studies have addressed histaminergic signalling mechanisms in cell lines originating from brain tumors. However, the role of histamine in normal development of brain structures, proliferation and differentiation of neurons and glial cells, and growth of malignant tumors in situ is still poorly understood.

Protein kinase C-alpha but not protein kinase C-epsilon is differentially down-regulated by bryostatin 1 and tetradecanoyl phorbol 13-acetate in SH-SY5Y human neuroblastoma cells.

SH-SY5Y human neuroblastoma cells can be induced to differentiate by phorbol esters but not by bryostatins although both agents increase protein kinase C (PKC) activity in these cells to a similar extent. We examined whether this difference could be explained by differences in the responses of specific PKC isoenzymes. Both TPA and bryostatin 1 at 10 nM induced a rapid increase in membrane-associated PKC-alpha immunoreactivity which was sustained for 72 hours in TPA-treated cells, but was down-regulated within 24 hours in bryostatin-treated cells. TPA likewise induced a sustained phosphorylation of an 80 kDa PKC substrate whereas in bryostatin-treated cells the 80 kDa substrate was rapidly phosphorylated reaching a maximum at 6 hours followed by a decline to basal level within 48 hours. A higher concentration of TPA (300 nM), which results in a less differentiated phenotype, induced down-regulation of PKC-alpha within 24 hours. In contrast, both TPA and bryostatin 1 stimulated translocation and a partial down-regulation of PKC-epsilon with similar kinetics. These results suggest that the divergent actions of bryostatin 1 and TPA in SH-SY5Y cells are at least partially due to differential modulation of PKC-alpha but not PKC-epsilon by these two agents.

Intracellular PPi concentration is not directly dependent on amount of inorganic pyrophosphatase in Escherichia coli K-12 cells.

No correlation was observed between the level of inorganic pyrophosphatase (PPase) and the intracellular concentration of PPi in Escherichia coli cells. In exponentially growing cells the intracellular PPi concentration was in every case 1.5 nmol/mg (dry weight) or about 0.5 mM, even though the amount of PPase was varied from 15 to 2,600% of the control amount by mutation or by using a multicopy plasmid with an inserted gene (ppa) encoding PPase. The PPi concentration could, however, be increased or decreased from the control level under some stressful conditions.

Status epilepticus induces changes in the expression and localization of endogenous palmitoyl-protein thioesterase 1.

Kainic acid (KA)-induced experimental epilepsy, a model of excitotoxicity, leads to selective neuronal death and synaptic restructuring. We used this model to investigate the effects of neuronal hyperactivation on palmitoyl-protein thioesterase 1 (PPT1), the deficiency of which causes drastic neurodegeneration. Immunological stainings showed that epileptic seizures in adult rats led to a progressive and remarkable increase of PPT1 in limbic areas of the brain. Within 1 week, the maximal expression was observed in CA3 and CA1 pyramidal neurons of the hippocampus. In the surviving pyramidal neurons, PPT1 localized in vesicular structures in cell soma and neuritic extensions. After seizures, colocalization of PPT1 with synaptic membrane marker (NMDAR2B) was enhanced. Further, synaptic fractionation revealed that after seizures PPT1 was readily observed on the presynaptic side of synaptic junction. These data suggest that PPT1 may protect neurons from excitotoxicity and have a role in synaptic plasticity.

Secretory organelles in ECL cells of the rat stomach: an immunohistochemical and electron-microscopic study.

ECL cells are numerous in the rat stomach. They produce and store histamine and chromogranin-A (CGA)-derived peptides such as pancreastatin and respond to gastrin with secretion of these products. Numerous electron-lucent vesicles of varying size and a few small, dense-cored granules are found in the cytoplasm. Using confocal and electron microscopy, we examined these organelles and their metamorphosis as they underwent intracellular transport from the Golgi area to the cell periphery. ECL-cell histamine was found to occur in both cytosol and secretory vesicles. Histidine decarboxylase, the histamine-forming enzyme, was in the cytosol, while pancreastatin (and possibly other peptide products) was confined to the dense cores of granules and secretory vesicles. Dense-cored granules and small, clear microvesicles were more numerous in the Golgi area than in the docking zone, i.e. close to the plasma membrane. Secretory vesicles were numerous in both Golgi area and docking zone, where they were sometimes seen to be attached to the plasma membrane. Upon acute gastrin stimulation, histamine was mobilized and the compartment size (volume density) of secretory vesicles in the docking zone was decreased, while the compartment size of microvesicles was increased. Based on these findings, we propose the following life cycle of secretory organelles in ECL cells: small, electron-lucent microvesicles (pro-granules) bud off the trans Golgi network, carrying proteins and secretory peptide precursors (such as CGA and an anticipated prohormone). They are transformed into dense-cored granules (approximate profile diameter 100 nm) while still in the trans Golgi area. Pro-granules and granules accumulate histamine, which leads to their metamorphosis into dense-cored secretory vesicles. In the Golgi area the secretory vesicles have an approximate profile diameter of 150 nm. By the time they reach their destination in the docking zone, their profile diameter is between 200 and 500 nm. Exocytosis is coupled with endocytosis (membrane retrieval), and microvesicles in the docking zone are likely to represent membrane retrieval vesicles (endocytotic vesicles).

Postnatal expression of H1-receptor mRNA in the rat brain: correlation to L-histidine decarboxylase expression and local upregulation in limbic seizures.

Histamine is implicated in the regulation of brain functions through three distinct receptors. Endogenous histamine in the brain is derived from mast cells and neurons, but the importance of these two pools during early postnatal development is still unknown. The expression of histamine H1-receptor in the rat brain was examined using in situ hybridization during postnatal development and in adults. For comparison, the expression of L-histidine decarboxylase (HDC) in the two pools was revealed. H1-receptor was evenly expressed throughout the brain on the first postnatal days, but resembled the adult, uneven pattern already on postnatal day 5 (P5). HDC was expressed in both mast cells and tuberomammillary neurons from birth until P5, after which the mast cell expression was no more detectable. In adult rat brain, high or moderate levels of H1-receptor expression were found in the hippocampus, zona incerta, medial amygdaloid nucleus and reticular thalamic nucleus. In most areas of the adult brain the expression of H1-receptor mRNA correlates well with binding data and histaminergic innervation. A notable exception is the hypothalamus, with high fibre density but moderate or low H1-receptor expression. Systemic kainic acid administration induced increased expression of H1-receptor mRNA in the caudate-putamen and dentate gyrus, whereas no change was seen in the hippocampal subfields CA1-CA3 or in the entorhinal cortex 6 h after kainic acid injections. This significant increase supports the concept that histaminergic transmission, through H1-receptor, is involved in the regulation of seizure activity in the brain.

Effects of reserpine on ECL-cell ultrastructure and histamine compartmentalization in the rat stomach.

The histamine-storing ECL cells in the stomach play a key role in the control of acid secretion. They contain granules, secretory vesicles and microvesicles, and sustained gastrin stimulation results in the additional formation of vacuoles and lipofuscin bodies. The cells are rich in the vesicle monoamine transporter type-2 (VMAT-2), which can be inhibited by reserpine. The present study examines the effect of reserpine on ECL-cell ultrastructure and histamine compartmentalization. Rats received reserpine and/or gastrin. Reserpine was given twice by the intraperitoneal route (25 mg/kg once daily). Gastrin-17 was given by subcutaneous infusion (5 nmol/kg/h), starting at the time of the first reserpine injection and continuing for 4 days when the rats were killed. At this stage, histamine in the oxyntic mucosa was unaffected by reserpine but elevated by gastrin. Immunocytochemical analysis (confocal microscopy) showed ECL-cell histamine in control and gastrin-treated rats to be localized in cytoplasmic organelles (e.g., secretory vesicles). After treatment with reserpine alone or reserpine+gastrin, ECL-cell histamine occurred mainly in the cytosol. Planimetric analysis (electron microscopy) of ECL cells showed reserpine to increase the number, size and volume density of the granules and to reduce the size and volume density of the secretory vesicles. Gastrin reduced the number and volume density of granules and secretory vesicles, increased the number and volume density of microvesicles and caused vacuoles and lipofuscin bodies to appear. Reserpine+gastrin increased the number, volume density and size of the granules. Reserpine prevented the effects of gastrin on secretory vesicles, vacuoles and microvesicles, but did not prevent the development of lipofuscin. Our findings are in line with the views: (1) that preformed cytosolic histamine is taken up by granules/secretory vesicles via VMAT-2, that histamine is instrumental in the transformation of granules into secretory vesicles and in their consequent enlargement and (2) that vacuoles are formed by the fusion of large secretory vesicles.

Histamine in the chick pineal gland: origin, metabolism, and effects on the pineal function.

The chick pineal gland contains histamine and tele-methylhistamine. The levels of both substances are elevated after treatment of chicks with the amino acid precursor of histamine, L-histidine (1 g/kg, ip). In control and L-histidine-loaded animals the pineal levels of histamine and tele-methylhistamine are higher in light-exposed than in dark-adapted animals (measured at the end of the light phase and in the middle of the dark phase of 12 hr light, 12 hr dark illumination cycle, respectively). The chick pineal gland contains histamine-immunofluorescent cells displaying mast cell morphology; they are seen in the vicinity of the capsule and in the parenchyma. Enzymatic studies showed the presence of the activity of histamine synthesizing and inactivating enzyme, i.e., L-histidine decarboxylase (HDC) and histamine-methyltransferase (HMT). The detected enzyme activities were sensitive to specific inhibitors of HDC (alpha-fluoromethylhistidine and alpha-hydrazinohistidine) and HMT (quinacrine and metoprine); inhibitors of aromatic amino acid decarboxylase alpha-methyl-DOPA and NSD-1015 were inactive on HDC. Exogenous histamine added to organ-cultured chick pineals strongly stimulated endogenous cyclic AMP accumulation and moderately increased melatonin secretion. The data, considered collectively, suggest that in avians histamine, probably originating from the pineal mast cell compartment, may function as a regulator of pineal gland activity.

Gene for pain modulatory neuropeptide NPFF: induction in spinal cord by noxious stimuli.

Neuropeptides FF (NPFF), AF (NPAF), and SF (NPSF) are homologous amidated peptides that were originally identified on the basis of similarity to the molluscan neuropeptide FMRF-amide. They have been hypothesized to have wide-ranging functions in the mammalian central nervous system, including pain modulation, opiate function, cardiovascular regulation, and neuroendocrine function. We have cloned the NPFF gene from human, bovine, rat, and mouse, and show that the precursor mRNA encodes for all three of the biochemically identified peptides (NPFF, NPAF, and NPSF). We demonstrate that NPFF precursor mRNA expression by Northern analysis and map sites of expression by in situ hybridization. We confirm the validity of the in situ hybridization by showing that its distribution in the brain and spinal cord matches the distribution of NPFF and NPSF immunoreactivity. We go on to show that the mRNA levels (as measured by in situ hybridization) in the spinal cord can be up-regulated by a model for inflammatory pain (carrageenan injection), but not by a model for neuropathic pain (lumbar nerve ligation). Our results confirm the evolutionary conservation of NPFF, NPAF, and NPSF neuropeptide expression in mammalian brain. They also provide a context for the interpretation of the pain-sensitizing effects of injections of these peptides that have been previously reported. Our results support a model for the role of these peptides in pain regulation at the level of the spinal cord.