Substance P mRNA Research Articles

Regulatory effect of nerve growth factor on release of substance P in cultured dorsal root ganglion neurons of rat

To investigate the regulatory effects of nerve growth factor (NGF) on basal and capsaicin-induced release of neuropeptide substance P (SP) in primary cultured embryonic rat dorsal root ganglion (DRG) neurons. DRGs were dissected from 15-day-old embryonic Wistar rats. DRG neurons were dissociated and cultured, and then exposed to different concentrations of NGF (10 ng/mL, 30 ng/mL, or 100 ng/mL) for 72 h. The neurons cultured in media without NGF served as control. RT-PCR were used for detecting the mRNAs of SP and vanilloid receptor 1 (VR1) in the DRG neurons. The SP basal and capsaicin (100 nmol/L)-induced release in the culture were measured by radioimmunoassay (RIA). SP mRNA and VR1 mRNA expression increased in primary cultured DRG neurons in a dose-dependent manner of NGF. Both basal release and capsaicin-evoked release of SP increased in NGF-treated DRG neurons compared with in control group. The capsaicin-evoked release of SP also increased in a dose-dependent manner of NGF. NGF may promote both basal release and capsaicin-evoked release of SP. NGF might increase the sensitivity of nociceptors by increasing the SP mRNA or VR1 mRNA.

A lack of μ-opioid receptors modulates the expressions of neuropeptide Y and substance P mRNA

The present study was undertaken to investigate changes in the expressions of neuropeptide Y (NPY) and substance P (SP) in mice lacking μ-opioid receptors. In an in situ hybridization study, in which we compared wild type and μ-opioid receptor knockout mice, NPY mRNA levels were found to be lower in the caudate–putamen and nucleus accumbens of μ-opioid receptor knockout mice. In addition, SP mRNA levels were lower in the ventromedial hypothalamic nucleus of μ-opioid receptor knockout mice. Our findings suggest that a lack of μ-opioid receptors modulates basal NPY mRNA levels in striatal regions and SP mRNA levels in the ventromedial hypothalamic nucleus of the mouse, and that these changes are due to compensatory modulation in the brain.

Calcitonin gene-related peptide and substance P contribute to reduced blood pressure in sympathectomized rats

CGRP and substance P (SP) are produced in dorsal root ganglia (DRG) sensory neurons and modulate vascular tone. Sympathetic and sensory nerves compete for NGF, a potent stimulator of CGRP and SP, and it has been suggested that sympathetic hyperinnervation in spontaneously hypertensive rats may reduce the availability of NGF to sensory nerves, thus reducing CGRP and SP. The purpose of this study was to determine whether destruction of peripheral sympathetic nerves in normal rats would increase the availability of NGF for sensory neurons and enhance expression of CGRP and SP. Sympathectomy was produced in rats by guanethidine sulfate administration. Control rats received saline. Sympathectomized rats displayed reductions in blood pressure (BP) and atria norepinephrine levels, whereas NGF levels in the DRG, spleen, and ventricles were increased. Sympathectomy also enhanced CGRP and SP mRNA and peptide content in DRG. Administration of CGRP and SP receptor antagonists increased the BP in sympathectomized rats but not in the controls. Thus sympathectomy enhances sensory neuron CGRP and SP expression that contributes to the BP reduction.

Placental Expression of Substance P and Vasoactive Intestinal Peptide: Evidence for a Local Effect on Hormone Release

The present study evaluated vasoactive intestinal peptide (VIP) and substance P (SP) mRNA expressions and the localization of both peptides in first- and third-trimester human placentas. VIP and SP mRNAs were detected by slot blot analysis in first- and third-trimester placental tissues. By immunohistochemistry both neuropeptides were localized in the trophoblast (syncytium and cytotrophoblastic cells) of the chorionic villi. Because little information is available on the role of VIP and/or SP on the secretion of placental hormones, we investigated the effect of these neuropeptides on human chorionic gonadotropin (hCG) and progesterone release from primary cultured human trophoblastic and JEG-3 cells. The addition of increasing doses of VIP resulted in a dose-dependent stimulation of hCG release from cultured human trophoblast and JEG-3 cells. Increasing doses of VIP also dose-dependently stimulated progesterone secretion from primary cultured trophoblastic cells at all time points evaluated and from JEG-3 cells only after 3 h. SP did not affect hCG and progesterone secretion either in cultured human trophoblast or in JEG-3 cells. In conclusion, the present study demonstrates that VIP and SP are mainly expressed in human trophoblasts, and that VIP modulates the in vitro secretion of hCG and progesterone, suggesting a different role in trophoblastic function of the two peptides.

Substance P Enhances Expression of Lipopolysaccharide-induced Inflammatory Factors in Dental Pulp Cells

To examine how substance P (SP) is related with dental pulp inflammation, we examined the effects of SP on expression of genes for inflammatory factors in human dental pulp cell cultures. Using reverse transcriptase-polymerase chain reaction, we found that Prevotella intermedia lipopolysaccharide (LPS) induced expression of SP and SP-receptor mRNAs, and that somatostatin inhibited the LPS-induced expression of SP mRNA. We also found that SP enhanced LPS-induced stimulation of NF-kappaB binding activity. In addition, SP induced expression of cyclooxygenase-2 and interleukin-10 receptor mRNAs. In contrast, SP inhibited expression of interferon-gamma receptor mRNA. These results suggest that SP may play a regulatory role in the immunological response of dental pulp tissue to pathogenic bacteria.

Substance P expression correlates with severity of diarrhea in cryptosporidiosis.

Cryptosporidiosis, caused by Cryptosporidium parvum, is self-limited in immunocompetent hosts but may cause chronic diarrhea in patients with acquired immunodeficiency syndrome (AIDS). Substance P (SP), a neuropeptide belonging to the tachykinin family, is expressed in gastrointestinal tract and can cause electrogenic chloride anion secretion. Therefore, we studied SP mRNA and protein expression in jejunal tissue samples of patients with AIDS with naturally occurring chronic cryptosporidiosis and healthy volunteers with mild cryptosporidiosis or asymptomatic infection after experimental C. parvum challenge. SP mRNA was associated with symptoms in cryptosporidiosis. SP protein levels were greater in symptomatic than asymptomatic volunteers. Similarly, greater expression of SP mRNA and protein were noted in patients with AIDS with chronic cryptosporidiosis versus immunocompetent volunteers with self-limited infection. This study demonstrates a direct correlation between SP levels and disease severity and may imply that SP plays a role in diarrhea mediation.

Substance P in the uterine cervix, dorsal root ganglia and spinal cord during pregnancy and the effect of estrogen on SP synthesis

Prior to parturition the non-pliable uterine cervix undergoes a ripening process (“softens” and dilates) to allow a timely passage of the fetus at term. The exact mechanism(s) triggering and involved in cervical ripening are unknown, though evidence for a role for sensory neurons and their contained neuropeptides is emerging. Moreover, an apparent increase in neuropeptide immunoreactive nerves occurs in the cervix during pregnancy, maternal serum estrogen levels rise at term and uterine cervix-related L6-S1 dorsal root ganglia (DRG) sensory neurons express estrogen receptor (ER) and neuropeptides. Thus, we sought to test the hypothesis that the neuropeptide substance P (SP) changes biosynthesis and release over pregnancy, that estrogen, acting via the ER pathway, increases synthesis of SP in DRG, and that SP is utilized in cervical ripening at late pregnancy. Using immunohistochemistry, in situ hybridization, reverse transcriptase–polymerase chain reaction (RT–PCR) and radioimmunoassay (RIA), we investigated coexpression of ER-α/β and SP; differential expression of ER-α and -β mRNA in DRG neurons; SP synthesis in DRG; and changes in SP concentration in the cervix, DRG and spinal cord over pregnancy. In addition, the effect of exogenous estrogen on SP synthesis in L6-S1 DRG of ovariectomized rats was examined. SP-immunoreactive neurons expressed ER-α and ER-β. SP synthesis (expressed as β-PPT mRNA label) was prominent in small DRG neurons. SP concentration increased in the L6-S1 DRG and spinal cord segments, with a peak at Day 20 of gestation, but decreased in the cervix during the first two trimesters, with a rise over the last trimester to Day 10 levels. SP and ER-α mRNA synthesis increased in DRG over pregnancy but ER-β mRNA levels were largely unchanged. When ovariectomized rats were treated with exogenous estrogen, SP mRNA synthesis in the DRG increased in a dose-related manner, an effect blocked by ER blocker ICI 182 780. From these results, we postulate that synthesis of SP in L6-S1 DRG and utilization in the cervix increase over pregnancy and this synthesis is under influence of the estrogen–ER system, most likely ER-α. We postulate that SP may play a role in cervical ripening and, consequently in the birth process.

Human neuronal cells (NT2-N) express functional substance P and neurokinin-1 receptor coupled to MIP-1 beta expression.

Substance P (SP), the most extensively studied and potent member of the tachykinin family, is a major modulator of inflammation and immunomodulatory activities within the central and peripheral nervous systems. We have examined the gene expression of SP and its receptor in a human neuronal cell line (NT2-N). Using reverse transcribed polymerase chain reaction (RT-PCR), the four isoforms of preprotachykinin-A gene transcripts (alpha, beta, gamma, and delta) were detected in the NT2-N. We also identified the presence of mRNA for neurokinin-1 receptor (NK-1R), a primary receptor for SP, in the NT2-N cells. Concomitant with NT2 cell differentiation into neurons, SP and NK-1R mRNA expression increased consistently. Intracellular SP and cell membrane NK-1R immunoreactivity were all observed in NT2-N cells. Most importantly, we demonstrated that SP and NK-1R presented in NT2-N cells are functionally involved in the regulation of macrophage inflammatory protein 1 beta (MIP-1beta), an important beta-chemokine participating in the activation and directional migration of immune cells to sites of central nervous systems (CNS) inflammation. Thus, SP and its receptor may play an important role in modulation of neuronal functions related to regulation of immune activities within the CNS. The NT2-N cell line is well suited for in vitro investigations of the SP-NK-1R pathway in immune responses and inflammation in the CNS.

Sequential Expression of the Neuropeptides Substance P and Somatostatin in Granulomas Associated with Murine Cysticercosis

Neurocysticercosis, a parasitic infection of the human central nervous system caused by Taenia solium, is a leading cause of seizures. Seizures associated with neurocysticercosis are caused mainly by the host inflammatory responses to dying parasites in the brain parenchyma. We previously demonstrated sequential expression of Th1 cytokines in early-stage granulomas, followed by expression of Th2 cytokines in later-stage granulomas in murine cysticercosis. However, the mechanism leading to this shift in cytokine response in the granulomas is unknown. Neuropeptides modulate cytokine responses and granuloma formation in murine schistosomiasis. Substance P (SP) induces Th1 cytokine expression and granuloma formation, whereas somatostatin inhibits the granulomatous response. We hypothesized that neuropeptides might play a role in regulation of the granulomatous response in cysticercosis. To test this hypothesis, we compared expression of SP and expression of somatostatin in murine cysticercal granulomas by using in situ hybridization and immunohistochemistry. We also compared expression with granuloma stage. Expression of SP mRNA was more frequent in the early-stage granulomas than in the late-stage granulomas (34 of 35 early-stage granulomas versus 1 of 13 late-stage granulomas). By contrast, somatostatin was expressed primarily in later-stage granulomas (13 of 14 late-stage granulomas versus 2 of 35 early-stage granulomas). The median light microscope grade of SP mRNA expression in the early-stage granulomas was significantly higher than that in the late-stage granulomas (P = 0.008, as determined by the Wilcoxon signed rank test). By contrast, somatostatin mRNA expression was higher at later stages (P = 0.008, as determined by the Wilcoxon signed rank test). SP and somatostatin are therefore temporally expressed in granulomas associated with murine cysticercosis, which may be related to differential expression of Th1 and Th2 cytokines.

A non-peptide substance P antagonist down-regulates SP mRNA expression in human mononuclear phagocytes

Substance P (SP), a potent modulator of neuroimmunoregulation, exerts its activity by binding to the neurokinin-1 receptor (NK-1R). The SP–NK-1R interaction is important in inflammation and viral infections, including HIV infection of human immune cells. We recently demonstrated that SP modulates HIV replication and that a non-peptide SP antagonist CP-96,345 inhibits HIV replication in human monocyte-derived macrophages (MDM) by affecting the SP–NK-1R interaction. In order to examine the effect of the SP antagonist on SP mRNA expression, MDM was incubated with or without CP-96,345 in the presence or absence of HIV infection. SP mRNA expression in these cells was then determined by real-time PCR technology. The effect of CP-96,345 on chemokine gene expression was also investigated by using a cDNA array assay. CP-96,345 down-regulated SP mRNA expression and antagonized exogenous SP-enhanced SP expression at the mRNA level, suggesting that SP autocrine regulation was interrupted by CP-96,345. CP-96,345 inhibited HIV replication in MDM, associated with down-regulated SP mRNA expression in comparison to HIV infection controls. In parallel with down-regulated SP and CCR5 mRNA expression, cDNA array assays indicated that CP-96,345 treatment also inhibited IL-8 gene expression, while enhancing expression of fractalkine and monocyte chemotactic protein-3 (MCP-3). Since SP plays an important role in inflammation and viral infections, these studies may have potential applications for therapeutic intervention of inflammation and viral infection of immune cells.

Substance P Induced Preprotachykinin-A mRNA, Neutral Endopeptidase mRNA and Substance P in Cultured Normal Fibroblasts

In certain skin diseases, stress can modulate the induction and/or progression of cutaneous manifestations. However, little is known about the circuit in neuroendocrine and in the immune systems of the skin. To address this question, we have analyzed the regulatory mechanisms of autocrine induction of substance P (SP) by cultured normal human fibroblasts that compose the major population of the skin and might augment stress-induced skin inflammatory responses. In nonstimulated conditions, normal fibroblasts express a moderate amount of preprotachykinin-A (PPT-A), a precursor of SP mRNA, and exogenous SP significantly upregulated PPT-A mRNA expression. Maximum response of SP peptide and SP mRNA in fibroblasts was observed 1–3 h after stimulation with SP. In contrast, the expression of neutral endopeptidase (NEP), a cell surface peptide with hydrolyzing activity of SP, was increased in fibroblasts stimulated with SP after 24 h. The administration of NEP inhibitor (phosphoramidon) to the fibroblasts induced higher SP production. In addition, the neurokinin (NK) receptor antagonists (spantide, FK224 and FK888) and protein synthesis inhibitor (cycloheximide) inhibited SP production by 30–40% of control response. In immunostaining study, specific cytoplasmic staining of SP was observed in fibroblasts stimulated with SP. Finally, we confirmed that the nucleotide sequence of the PPT-A expressed in fibroblasts perfectly corresponded to the gene bank human PPT-A cDNA. This is the first report that SP mRNA, NEP mRNA and SP peptide can be induced by normal human skin fibroblasts in response to exogenous SP, and that fibroblast-derived SP might play an important role in the induction and acceleration of certain cutaneous diseases.

HIV enhances substance P expression in human immune cells.

Substance P (SP), a potent modulator of neuroimmunoregulation, is expressed in human immune cells. We observed elevated plasma SP levels in HIV-infected men compared with uninfected subjects. In the present study, we investigated the possible cellular source of the increased SP level caused by HIV infection. Using real-time reverse transcriptase-polymerase chain reaction, we demonstrated that monocyte-derived macrophages (MDM) and lymphocytes from both placental cord blood and adult peripheral blood expressed SP mRNA, which was significantly increased by HIV infection. HIV-induced SP expression was positively related to virus replication in the infected MDM. Purified recombinant HIV envelope glycoprotein 120 (gp120) derived from both the macrophage-tropic strain (MN) and the T lymphocyte-tropic strain (IIIB), when added to MDM cultures, enhanced SP mRNA expression. The gp120-induced SP expression was abrogated by pretreating the cells with soluble CD4. Furthermore, the activation of HIV in the latently infected promonocytic cell line (U1) and T-cell line (ACH-2) up-regulated SP mRNA expression. These data support the hypothesis that interaction of HIV and SP may have significant in vivo relevance to the immunopathogenesis of HIV infection and AIDS.

Substance P in Subtotal Nephrectomy-Salt Hypertension

We have previously demonstrated that calcitonin gene-related peptide (CGRP) plays a counterregulatory role in subtotal nephrectomy-salt (SN-salt) hypertension through an increase in vascular responsiveness to the dilator activity of this neuropeptide. Substance P (SP) is often co-localized with CGRP in perivascular sensory nerves. To determine the role and mechanism of action of SP in SN-salt hypertension, we induced hypertension in 4- to 6-week-old male Sprague-Dawley rats (n=8) by subtotal nephrectomy and 1% saline drinking water. Sham-operated rats were given either tap water (n=9) or 1% saline to drink (n=9). Eleven to 13 days after each protocol, all rats had intravenous (for drug administration) and arterial (for continuous monitoring of mean arterial pressure [MAP]) catheters surgically implanted and were studied in the conscious and unrestrained state. Baseline MAP was significantly elevated in the SN-salt rats (157 +/- 6 mm Hg) compared with tap water--fed controls (128 +/- 3 mm Hg) and 1% saline--fed controls (132 +/- 5 mm Hg). Vehicle administration did not alter the MAP in any group. In contrast, administration of spantide-II (0.2 micromol/L in saline), an SP receptor antagonist, significantly elevated the MAP in SN-salt rats (13.9 +/- 0.8 mm Hg) compared with the tap water (1.7 +/- 1.7 mm Hg) and 1% saline controls (2.0 +/- 1.9 mm Hg). SP mRNA and peptide levels in dorsal root ganglia were not significantly different between the 3 groups. Administration of exogenous SP (12 and 24 nmol center dot L(-1) center dot kg(-1) intravenously) resulted in a significantly greater decrease in MAP in the SN-salt rats compared with both control groups. Taken together, these data suggest that in SN-salt hypertension, SP plays a counterregulatory role in the absence of an increase in its neuronal expression, thereby suggesting that one possible mechanism of this compensatory vasodilator response is enhanced vascular reactivity to SP.

Quantification of substance p mRNA in human immune cells by real-time reverse transcriptase PCR assay.

We have applied a newly developed real-time reverse transcriptase (RT) PCR (RT-PCR) assay for quantification of substance P (SP) mRNA expression (the SP real-time RT-PCR assay) in human blood monocyte-derived macrophages, peripheral blood lymphocytes, and microglia isolated from fetal brain. The SP real-time RT-PCR assay had a sensitivity of 60 mRNA copies, with a dynamic range of detection between 60 and 600,000 copies of the SP gene transcript per reaction mixture. The coefficient of variation of the threshold cycle number between the SP real-time RT-PCR assays was less than 1.16%. This assay with an SP-specific primer pair efficiently recognizes all four isoforms of preprotachykinin A (the SP precursor) gene transcripts. In order to use this assay to measure the levels of SP mRNA in the human immune cells quantitatively, we designed a specific probe (molecular beacon) derived from exon 3 of the SP gene. We demonstrated that the real-time RT-PCR quantitatively detected SP mRNA in the human immune cells, among which the microglia isolated from fetal brain had the highest levels of SP mRNA. The SP real-time PCR assay yielded reproducible data, as the intra-assay variation was less than 1%. Thus, it is feasible to apply the real-time RT-PCR assay for quantification of SP mRNA levels in human immune cells, as well as in other nonneuronal cells. Since SP is a major modulator of neuroimmunoregulation, this assay has the potential for widespread application for basic and clinical investigations.

Adaptive Plasticity in Tachykinin and Tachykinin Receptor Expression after Focal Cerebral Ischemia Is Differentially Linked to GABAergic and Glutamatergic Cerebrocortical Circuits and Cerebrovenular Endothelium

To test the hypothesis of an involvement of tachykinins in destabilization and hyperexcitation of neuronal circuits, gliosis, and neuroinflammation during cerebral ischemia, we investigated cell-specific expressional changes of the genes encoding substance P (SP), neurokinin B (NKB), and the tachykinin/neurokinin receptors (NK1, NK2, and NK3) after middle cerebral artery occlusion (MCAO) in the rat. Our analysis by quantitative in situ hybridization, immunohistochemistry, and confocal microscopy was concentrated on cerebrocortical areas that survive primary infarction but undergo secondary damage. Here, SP-encoding preprotachykinin-A and NK1 mRNA levels and SP-like immunoreactivity were transiently increased in GABAergic interneurons at 2 d after MCAO. Coincidently, MCAO caused a marked expression of SP and NK1 in a subpopulation of glutamatergic pyramidal cells, and in some neurons SP and NK1 mRNAs were coinduced. Elevated levels of the NKB-encoding preprotachykinin-B mRNA and of NKB-like immunoreactivity at 2 and 7 d after MCAO were confined to GABAergic interneurons. In parallel, the expression of NK3 was markedly downregulated in pyramidal neurons. MCAO caused transient NK1 expression in activated cerebrovenular endothelium within and adjacent to the infarct. NK1 expression was absent from activated astroglia or microglia. The differential ischemia-induced plasticity of the tachykinin system in distinct inhibitory and excitatory cerebrocortical circuits suggests that it may be involved in the balance of endogenous neuroprotection and neurotoxicity by enhancing GABAergic inhibitory circuits or by facilitating glutamate-mediated hyperexcitability. The transient induction of NK1 in cerebrovenular endothelium may contribute to ischemia-induced edema and leukocyte diapedesis. Brain tachykinin receptors are proposed as potential drug targets in stroke.

Human stem cells express substance P gene and its receptor.

We studied the expression of Substance P (SP) and its receptor in an established human stem cell line (TF-1) and primary stem cells derived from human placental cord blood (HPCB). Using reverse transcriptase polymerase chain reaction (RT-PCR) assay, SP mRNA is detected in both TF-1 cells and HPCB stem cells. Among the alpha, beta, gamma, and delta transcripts of the SP gene, only the beta, gamma, and delta transcripts are detectable in these cells. These RT-PCR-amplified transcripts are confirmed by Southern blot assay using a specific SP probe. Sequence analysis of the RT-PCR-amplified products transcribed from mRNA extracted from the HPCB stem cells also confirmed that these transcripts are identical to those found in human neurons. At the protein level, TF-1 cells produced endogenous SP as determined by an enzyme-linked immunosorbent assay (EIA). Capsaicin, a vanillyl fatty acid amide (ingredient of hot pepper), released SP from TF-1 cells. In addition, using RT nested-PCR analysis, we identified the presence of mRNA for neurokinin-1 receptor (NK-1R, the receptor for SP) in both TF-1 cells and HPCB stem cells, which was confirmed by Southern blot and DNA sequencing analysis. The demonstration that human stem cells express SP and its receptor support the notion that SP is biologically involved in the hematopoietic regulating network.

Selective activation of group I metabotropic glutamate receptors upregulates preprodynorphin, substance P, and preproenkephalin mRNA expression in rat dorsal striatum.

Group I metabotropic glutamate receptors (mGluRs) are positively coupled to phosphoinositide hydrolysis through G-proteins and are densely expressed in the medium-sized spiny neurons of striatum. Activation of this group of mGluRs in the striatum produces long-lasting stimulation of behavioral activity. In this study, the role of group I mGluRs in the modulation of neuropeptide mRNA expression in striatal neurons was investigated using a Group I-selective agonist, 3,5-dihydroxyphenylglycine (DHPG) in chronically cannulated rats. Unilateral injections of DHPG into the dorsal striatum (caudoputamen) at behaviorally active doses of 20, 40, and 80 nmol elevated basal levels of preprodynorphin (PPD), substance P (SP), and preproenkephalin (PPE) mRNAs in the injected dorsal striatum as revealed by quantitative in situ hybridization. The elevation of all three mRNAs was dose-dependent and the responsiveness of opioid peptide mRNAs (PPD and PPE) to acute injection of DHPG at each dose surveyed was greater than that of SP mRNA. Induction of the mRNAs was delayed and prolonged as increases in hybridization signal became evident at 2 (SP and PPE) or 3 (PPD) h, reached a peak between 3 and 6 h, and returned to normal levels 24 h after DHPG injection. Coadministration of a Group I-selective antagonist, n-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carbo xamide (PHCCC, 10 nmol), with DHPG markedly attenuated DHPG-stimulated PPD, PPE, and, to a lesser extent, SP expression. Administration of PHCCC alone had no significant effect on basal levels of three mRNA expression in the striatum. This study provides a detailed description of the dose- and time-related alterations in striatonigral PPD/SP and striatopallidal PPE mRNA expression in response to a single injection of the Group I agonist DHPG. Data obtained demonstrate a facilitatory, dynamic regulation of constitutive expression of PPD, SP, and PPE mRNAs by local enhancement of glutamatergic tone on DHPG- and PHCCC-sensitive Group I mGluRs.

Hyperthyroidism decreases thyrotropin-releasing hormone gene expression in the caudal raphe nuclei and the parapyramidal regions in rats

Altered thyroid statuses are associated with autonomic disorders. Medullary thyrotropin-releasing hormone (TRH) and substance P (SP) regulate autonomic nervous activity. The influences of thyroid statuses on TRH and SP gene expressions in the caudal raphe nuclei and the parapyramidal regions were studied using quantitative in situ hybridization histochemistry. In male rats thyroidectomized (Tx) for 30 days, the serum T 4 levels decreased by 64% and the medullary pro-TRH mRNA signals (silver grains per neuron) significantly increased by 32–45%. These changes were prevented by daily i.p. T 4 (2 μg/100 g) injection in Tx rats. In sham operated/T 4 (20 μg/100 g, daily) injected rats, T 4 levels significantly increased by 88% and the silver grains decreased by 38–40%. Medullary SP mRNA signals were not significantly changed by altering thyroid status. These results support the concept that thyroid hormone regulates medullary TRH gene expression by negative feedback.

CO-LOCALIZATION OF SUBSTANCE P RECEPTOR (NK1) AND PREPROTACHYKININ (PPT) MRNA IN RAT DORSAL HORN NEURONS

S358 INTRODUCTION: Substance P (SP) and related tachykinins modulate pain processing in the dorsal horn of the spinal cord via release of SP from primary afferents terminating in superficial laminae. SP also is synthesized by intrinsic dorsal horn neurons located in superficial laminae (primarily lamina I) and in deeper laminae (primarily lamina V), and many of these are spinothalamic neurons [1]. The effects of SP on nociception are likely mediated by the NK1 tachykinin receptor, since NK1 antagonists block behavioral nociceptive sensitization resulting from tissue injury [2]. Dorsal horn neurons containing NK1 receptor are located in superficial laminae (lamina I) and in deeper laminae (laminae III-V), and many of these are spinothalamic neurons [3]. Release of SP from terminals in the dorsal horn is modulated by opioid peptides which produce their actions, in part, through mu opioid receptors (MOR1), which are expressed both by primary afferents and by intrinsic dorsal horn neurons. In order to determine if SP is synthesized by dorsal horn neurons expressing NK1 or MOR1 receptors, we performed double-label in situ hybridization (ISH) on sections of lumbar spinal cord. METHODS: Fresh frozen spinal cord tissue from male Sprague Dawley rats was cryostat sectioned at 12 um and processed for ISH. RNA probes antisense for preprotachykinin (PPT) were transcribed using digoxigenin-UTP and probes antisense for NK1 or MOR1 mRNA were transcribed using33 P-UTP, and the PPT probe was combined with either the NK1 or the MOR1 probe and spinal cord sections were hybridized with this mixture overnight, washed, processed for immunohistochemical detection of digoxigenin labeled cells (Boehringer Mannheim), and then processed for autoradiographic detection of (33) P-UTP labeled cells. Spinal cord sections were examined under brightfield and darkfield illumination for the presence of blue-black reaction product associated with digoxigenin labeling (PPT cells) or the presence of silver grains associated with33 P labeling (NK1 or MOR1 cells), and for colocalization of digoxigenin and33 P labeling in the same cell. RESULTS: In the dorsal horn PPT positive cells were primarily located in lamina I and in deeper lamina V, which partially overlapped the distribution of NK1 positive cells, which were densely distributed in lamina I and more loosely distributed in laminae III-V. MOR1 positive cells were primarily located in lamina II and in laminae III-V. In lamina I approximately 70% of NK1 positive cells were also positive for PPT, but in deeper laminae only 20% of NK1 cells expressed PPT. No MOR1 positive cells contained PPT mRNA, either in superficial or deep laminae. DISCUSSION: These results describe co-localization of mRNA coding for the SP receptor NK1 and for the neuropeptide SP in dorsal horn neurons. In contrast, dorsal horn neurons containing mRNA coding for the mu opioid receptor do not co-localize with SP mRNA. Since spinothalamic neurons are located in both superficial (I-II) and deep (III-V) laminae of the dorsal horn and approximately 75% of laminae I-II spinothalamic neurons and 35% of laminae III-V spinothalamic neurons contain NK1 receptors [3], these data suggest that most lamina I cells and a significant percentage of lamina V cells co-localizing NK1 and SP project directly to thalamic pain nuclei. Together these data suggest a disynaptic SPergic pathway transmitting nociceptive information from the periphery to the thalamus. Although it is well established that mu opioid receptors modulate SP transmission in primary sensory terminals at the first synapse in laminae I-II by presynaptic mechanisms [4], the absence of co-localization of SP mRNA with MOR1 mRNA in dorsal horn neurons suggests that opioid modulation of SP transmission in second order sensory neurons projecting to the thalamus is mediated postsynaptically by MOR1 expressing dorsal horn interneurons.

Lack of induction of substance P gene expression by hypoxia and absence of neurokinin 1-receptor mRNAs in the rat carotid body

Peripheral chemoreceptors are commonly thought to respond to hypoxia by releasing neurotransmitters from the type 1 cells of the carotid body; these molecules then bind to post-synaptic receptors on the carotid sinus nerve. The tachykinin substance P (SP) may act as an important neurotransmitter/neuromodulator in hypoxic chemotransmission in peripheral arterial chemoreceptors. In order to elucidate the role of SP in modulating hypoxic chemotransmission, we have used quantitative in situ hybridization histochemistry, to determine the effect of hypoxia on SP gene induction, and the localization of neurokinin 1 (NK-1) receptor mRNA in the carotid body and petrosal ganglia complex in rats at 21 days post-natal age. For comparison, we also determined: (1) the effect of hypoxia on tyrosine hydroxylase (TH) gene induction and (2) the localization of the mRNA encoding the D 2-dopamine receptor. SP mRNA was not detected in the rat carotid body during normoxia and its expression was not induced after a 1 h of exposure to hypoxia (10% O 2/90% N 2), a stimulus that was sufficient to cause a significant increase ( P<0.01) in TH mRNA levels in the carotid body. Both SP and TH mRNAs were abundantly expressed in multiple cells in the petrosal and the jugular ganglia. However, these mRNAs were not co-localized and SP and TH mRNA levels were not affected by hypoxia in these ganglia. Although D 2-dopamine receptor mRNA was abundantly expressed in the rat carotid body, we found no evidence of NK-1 receptor mRNA in the carotid body. In contrast, both NK-1 receptor mRNA and D 2-dopamine receptor mRNA were present in petrosal ganglion cells. In the rat, SP does not appear to modulate hypoxic chemotransmission by being made in and released from type 1 cells in the carotid body, and neither does SP modulate the activity of type 1 cells by binding to NK-1 receptors on these cells.