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Results for “Nuclease Protection Assays”

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PubMed1997

Physical exercise induces FGF-2 and its mRNA in the hippocampus.

Clinical and experimental evidence indicate that physical activity has a positive impact on brain function; however, the molecular bases for how exercise affects the structure and function of the brain are largely unknown. We have investigated the influences of variable periods of voluntary wheel-running on the expression of basic fibroblast growth factor and its mRNA in various brain regions. Nuclease protection assays revealed that the hippocampus was the only region examined exhibiting changes in FGF-2 mRNA as a result of exercise. FGF-2 mRNA increased to reach a peak by the 4th night of wheel-running. FGF-2 immunoreactivity, normally located in the perinuclear area of astrocytes, following exercise became stronger and appeared to spread to the cytoplasm and processes of astrocytes. Quantification of the FGF-2-immunoreactive astrocytes showed an increase in density between 2 and 4 nights of running in discrete regions of the hippocampus. These results demonstrate that exercise regulates FGF-2 expression and suggest that growth factors are likely mediators of the positive effects of exercise on the brain.

Animals↗

PubMed2000

Tissue-specific expression of multiple mRNA variants of the mouse estrogen receptor alpha gene.

The isolation of five new mouse estrogen receptor alpha mRNA variants (mERalpha A, B, F1, F2 and H) is described. All of these transcripts are generated by alternative splicing and all encode the 66 kDa ERalpha protein that the previously identified mRNA C variant generates. However, these transcripts differ in their 5' untranslated regions. RT-PCR and S1 nuclease protection assays revealed a tissue- and sex-specific expression pattern of all variants. The C and F mRNA variants are the predominantly expressed mERalpha variants in mouse. The expression of mERalpha H mRNA is restricted to liver, although female mice produce around a five fold higher level of this transcript than males. Our results show that the mERalpha gene is a complex genomic unit in mice that exhibits alternative splicing which is regulated in a tissue-specific manner.

5' Untranslated Regions↗

PubMed1997

Measurement of relative quantities of different HLA-A and -B mRNAs in cells by reverse transcription-polymerase chain reaction and denaturing gradient gel electrophoresis.

In order to determine the relative quantities of different HLA-A and -B mRNAs in cells, we have developed a simple and reliable method by using reverse transcription-polymerase chain reaction (RT-PCR), denaturing gradient gel electrophoresis (DGGE) and phosphor imaging analysis. Cytoplasmic RNA from lymphoblastoid cell lines with well-characterized HLA phenotypes are reversely transcribed with a primer specific for all HLA-A and -B antigens. The first-strand cDNA is used as template for quantitative PCR. The primer pair used for quantitative PCR are specific for all class I HLA and one of the primers is labeled with [gamma-32P]ATP. The amplified sequences include parts of exon 2 and exon 3 which contain most polymorphic residues in class I HLA molecules. The RT-PCR products containing the amplified HLA-A and -B sequences are separated by DGGE. The radioactivities of different DNA bands separated in denaturing gradient polyacrylamide gels are measured by phosphor imaging and used to determine the relative amounts of HLA-A and -B mRNAs. This approach is validated by using samples containing known quantities of different HLA-A and -B mRNA transcripts and confirmed by S1 nuclease protection assay. The combined RT-PCR/DGGE approach therefore provides a simple and reliable method for quantitation of relative amounts of different HLA-A and -B mRNAs in cells. This method should also be useful for studying the expression of other highly conserved and duplicated genes.

Cell Line, Transformed↗

PubMed1999

Nociceptive regulation of GABA(B) receptor gene expression in rat spinal cord.

Activation of gamma-aminobutyric acid (GABA) neurotransmission evokes antinociceptive responses in laboratory animals. The recent cloning of GABA(B) receptor gene products makes it possible to examine the regulation of this receptor system as it relates to the mediation of pain-related sensory information. Inasmuch as acute and chronic pain alter the expression of a number of nociception-related receptors, and because such changes are important components in the regulation of pain, the present study was undertaken to determine whether GABA(B) receptor gene expression is altered in sensory systems following a peripheral nociceptive stimulus. Solution hybridization-nuclease protection assays conducted 24 h after formalin injection into the right hindpaw of the rat revealed a significant bilateral increase in GABA(B) R1 and R2 receptor expression in the dorsal lumbar spinal cord, and a significant increase in GABA(B) R1 receptor mRNA in the ipsilateral lumbar dorsal root ganglion. These findings indicate an activity-dependent, differential regulation of GABA(B) R1 and R2 receptor gene expression in spinal sensory systems in response to chemogenic nociceptive activation, suggesting that GABA(B) receptor plasticity may play an important role in regulating the mediation, and perception, of chronic pain.

Animals↗

PubMed1997

Overexpression of endothelin-1 mRNA and protein in portal hypertensive gastric mucosa of rats: a key to increased susceptibility to damage?

BACKGROUND: Portal hypertension predisposes gastric mucosa to increased injury by various noxious factors. Because endothelin-1 (ET-1) is a potent vasoconstrictor that enhances gastric mucosal injury, we examined ET-1 expression in the portal hypertensive (PHT) gastric mucosa and its possible role in increased mucosal susceptibility to damage. METHODS: In gastric specimens of PHT or sham-operated rats, ET-1 mRNA expression was studied by S1-nuclease protection assay and ET-1 protein by enzyme immunoassay and immunostaining. We also determined the extent of ethanol-induced gastric mucosal necrosis in PHT and sham-operated rats after administering either a placebo or FR 139317, a selective ETA receptor antagonist. RESULTS: In PHT stomachs ET-1 mRNA expression and protein concentration were significantly increased compared with sham-operated controls: mRNA expression (ET-1/glyceraldehyde-3-phosphate-dehydrogenase ratio), 0.54 +/- 0.18 versus 0.30 +/- 0.08; protein concentration, 7.36 +/- 2.21 pg/mg versus 3.93 +/- 0.40 pg/mg, respectively; both p < 0.01. Immunofluorescence signal of ET-1 protein was predominantly localized to endothelia of gastric mucosal and submucosal vessels. In PHT stomachs FR 139317 significantly reduced mucosal necrosis (percentage of necrotic area, from 24.9 +/- 5.9% to 10.8 +/- 4.0%; p < 0.01), although it had no effect on sham-operated controls. CONCLUSIONS: Portal hypertension activates the ET-1 gene with overexpression of ET-1 protein in the gastric mucosa. Protection of PHT gastric mucosa by ETA receptor antagonist against damage indicates that overexpression of ET-1 plays an important role in increased susceptibility of PHT gastric mucosa to injury.

Animals↗

PubMed1997

The translocating RecBCD enzyme stimulates recombination by directing RecA protein onto ssDNA in a chi-regulated manner.

Double-stranded DNA break repair and homologous recombination in E. coli are initiated by the RecBCD enzyme, which unwinds and simultaneously degrades DNA from a double-stranded DNA end. This process is stimulated by cis-acting DNA elements, known as chi sites. Using both in vitro pairing and nuclease protection assays, we demonstrate that the translocating RecBCD enzyme, which has been activated by chi, coordinates the preferential loading of the homologous pairing protein, RecA, onto the resultant single-stranded DNA downstream of chi. This facilitated loading of RecA protein results in a substantial increase in both the efficiency and rate of in vitro recombination reactions and offers an explanation for stimulation of recombination and repair in vivo by chi.

Base Sequence↗

PubMedDate not supplied

Expression of platelet-derived growth factor-A mRNA in human placenta: effect of magnesium infusion in pre-eclampsia.

The expression of platelet-derived growth factor-A (PDGF-A) mRNA was examined in the cotyledons of normal human placentae and those from patients with pre-eclampsia. These patients exhibited pre-delivery blood pressure of 154+/-4/99+/-4 mmHg (mean+/-SEM) and met the criteria established for pre-eclampsia. During labour they received MgSO4 infusion for various time intervals (4-25 h). The PDGF-A message was quantitated to beta-actin by the solution hybridization nuclease protection assay. Since the two groups differed in two parameters (pre-eclampsia and MgSO4 treatment), the direct comparison was not feasible. An analysis of covariance revealed a significant difference in the message between the pre-eclamptic and control groups (P<0.01); the gestational age was not a significant covariate for either group but the time on MgSO4 in pre-eclampsia group was significant (P<0.002). A linear regression analysis of PDGF-A mRNA values for the pre-eclamptic group showed a time-dependent downregulation of the message by MgSO4 (P<0.01, r=- 0.796). These results show a uniform expression of PDGF-A mRNA in cotyledons of normal human placenta between 35 and 40 weeks of gestation. Furthermore, MgSO4 has an inhibitory effect on the expression of this message which may have aside from its anticonvulsive action beneficial effect on the function of pre-eclamptic placenta.

Actins↗

PubMed1997

Structure of the promoter for the rat Fas antigen gene.

The Fas antigen is a receptor protein transducing cell death signals. Binding of Fas ligands to Fas antigens provokes apoptosis in target cells. Here we report the structure of the promoter of the gene coding for rat Fas antigens. The major transcription start site, identified by the S1 nuclease protection assay, was situated 188 nucleotides upstream of the translational initiation site. The promoter activity was located in the region at the nucleotide position from -142 to -24. In this region we identified a consecutive sequence of NF-kappaB and NF-IL6 consensus sequences, spanning from -142 to -122.

Animals↗

PubMed1998

Selective expression of specific histone H4 genes reflects distinctions in transcription factor interactions with divergent H4 promoter elements.

Expression of many histone H4 genes is stringently controlled during the cell cycle to maintain a functional coupling of histone biosynthesis with DNA replication. The histone H4 multigene family provides a paradigm for understanding cell cycle control of gene transcription. All functional histone H4 gene copies are highly conserved in the mRNA coding region. However, the putative promoter regions of these H4 genes are divergent. We analyzed three representative mouse H4 genes to assess whether variation in H4 promoter sequences has functional consequences for the relative level and temporal control of expression of distinct H4 genes. Using S1 nuclease protection assays with gene-specific probes and RNA from synchronized cells, we show that the mRNA level of each H4 gene is temporally coupled to DNA synthesis. However, there are differences in the relative mRNA levels of these three H4 gene copies in several cell types. Based on gel shift assays, nucleotide variations in the promoters of these H4 genes preclude or reduce binding of several histone gene transcription factors, including IRF2, HiNF-D, SP-1 and/or YY1. Therefore, differential regulation of H4 genes is directly attributable to evolutionary divergence in H4 promoter organization which dictates the potential for regulatory interactions with cognate H4 transcription factors. This regulatory flexibility in H4 promoter organization may maximize options for transcriptional control of histone H4 gene expression in response to the onset of DNA synthesis and cell cycle progression in a broad spectrum of cell types and developmental stages.

3T3 Cells↗

PubMed1997

Prostaglandin E2 enhances type 2-bradykinin receptor expression in human glomerular podocytes.

We examined the effect of prostaglandin E2 (PGE2) on bradykinin (BK) binding, BK-dependent intracellular calcium and inositol phosphate (i.p.) concentrations and BK mRNA in human glomerular visceral epithelial cells (hGVEC). PGE2 (10 nM) produced a concentration-dependent increase in [3H]-BK specific binding after a lag time of 24 h with a threshold at 0.1 nM. This increase appeared to be mediated exclusively by an increase in BK receptor (BKR)-2 expression. Scatchard analysis of [3H]-BK saturation binding showed that PGE2 produced an increase in the receptor site density without a change in the apparent dissociation constant. PGE2 also markedly stimulated cAMP production. This effect was thought to mediate the increase in expression of BKR-2 as 8-bromo cAMP and various cAMP-stimulating agents acted similarly. PGE2 did not change the BK-dependent intracellular IP3 and cytosolic calcium increases. The overexpression of BKR-2 in the presence of PGE2 was associated with an increase in mRNA as shown by the nuclease protection assay without any change in mRNA half-life. Cycloheximide, an inhibitor of protein synthesis, enhanced BKR-2 mRNA expression. In conclusion, treatment with PGE2 stimulates the synthesis of BKR-2 in hGVEC, possibly by interfering with an inhibitory protein involved in BKR-2 transcription.

Cell Line, Transformed↗

PubMed2003

Function of Corynebacterium glutamicum promoters in Escherichia coli, Streptomyces lividans, and Bacillus subtilis.

The function of seven promoters from Corynebacterium glutamicum, P-hom, P-leuA, P-per, P-aes1, P-aes2, P-45, and P-104, was analyzed in a heterologous background. DNA fragments carrying the promoters were cloned into shuttle promoter-probe vectors replicating in Escherichia coli and C. glutamicum (pET2), Streptomyces lividans (pGL7011) and Bacillus subtilis (pRB394). With the exception of P-hom, P-leuA and P-104 in B. subtilis, all promoters were found to be active in all species. Non-radioactive methods of primer-extension analysis and of S1-nuclease protection assay using automatic sequencer were developed to determine the respective transcriptional start points (TSPs). All TSPs were determined by primer extension and in two promoters (P-45 and P-hom) the main TSPs were confirmed by S1-mapping. While the main TSPs were identical in all four species, utilization of multiple TSPs varied among the species and additional TSPs were detected in S. lividans. Knowledge of the efficiency of promoters and of exact respective TSPs may be of practical value for the construction of expression systems in a heterologous background.

Bacillus subtilis↗

PubMed2002

Transcriptional and translational expression kinetics of the bovine herpesvirus 1 UL51 homologue gene.

We characterized the expression kinetics of the transcript and protein generated from the bovine herpesvirus 1 (BHV1) homologue of the herpes simplex virus 1 (HSV1) UL51 gene. The BHV1 UL51 ORF, located at positions 7236-->7967 of the viral genome, generated a major 1.05 kb transcript accumulating at very low abundance as soon as 3 h post-infection (p.i.), after which its levels increased to reach a plateau from 6 to 12 h p.i., and then slowly decreased up to 24 h p.i. As determined by S1 nuclease protection assays, UL51 transcription initiated at two distinct sites located at 191 and 196 bases upstream from the initiation codon, corresponding to positions 7045 and 7040 of the viral genome, respectively. Western blotting of BHV1-infected protein cell lysates, using a BHV1-specific antiserum generated against a recombinant protein expressed in Escherichia coli, detected a 28 kDa protein of the expected size (24985 Da) whose expression kinetics followed that of its transcript. As evidenced by in situ immunofluorescence assays, the protein mainly localized to the cytoplasm and the perinuclear region of infected cells. In contrast to HSV1 UL51 which is classified as a gamma2 gene, BHV1 UL51 belongs to viral genes of the gamma1 class as expression of its transcript is partially dependent on viral DNA synthesis.

Animals↗

PubMed1999

NMDA receptor subunit gene expression in the rat brain: a quantitative analysis of endogenous mRNA levels of NR1Com, NR2A, NR2B, NR2C, NR2D and NR3A.

Electrophysiological recordings have shown NMDA receptors to be heterogenous structures capable of responding to selected antagonists and agonists in multiple ways. This diversity in functional response has led investigators to conclude that these channels are comprised of unique combinations of receptor subunits which determine a cell's functional NMDA-signature [H. Meguro, H. Mori, K. Araki, E. Kushiya, T. Kutsuwada, M. Yamazaki, T. Kumanishi, M. Arakawa, K. Sakimura, M. Mishina, Functional characterization of a heteromeric NMDA receptor channel expressed from cloned cDNAs, Nature (London) 357 (1992) 70-74; T. Ishii, K. Moriyoshi, H. Sugihara, K. Sakurada, H. Kadotani, M. Yokoi, C. Akazawa, R. Shigemoto, N. Mizuno, S. Nakanishi, Molecular characterization of the family of the N-methyl-d-aspartate receptor subunits, J. Biol. Chem. 268 (1993) 2836-2843; K.A. Wafford, C.J. Bain, B. Le Bourdelles, P.J. Whiting, J.A. Kemp, Preferential co-assembly of recombinant NMDA receptors composed of three different subunits, NeuroReport 4 (1993) 1347-1349; T. Priestley, P. Laughton, J. Myers, B. Le Bourdelles, J. Kerby, P.J. Whiting, Pharmacological properties of recombinant human N-methyl-d-aspartate receptors comprising NR1a/NR2A and NR1a/NR2B subunit assemblies expressed in permanently transfected mouse fiberblast cells, Mol. Pharmacol. 48 (1995) 841-848; P.H. Seeburg, N. Burnashev, G. Kohr, T. Kuner, R. Sprengel, H. Monyer, The NMDA receptor channel: molecular design of a coincidence detector, Recent Prog. Horm. Res. 50 (1995) 19-34; A.L. Buller, D.T. Monagahan, Pharmacological heterogeneity of NMDA receptors: characterization of NR1a/NR2D heteromers expressed in Xenopus oocytes, Eur. J. Pharmacol. 320 (1997) 87-94]. In situ hybridization and immunocytochemical studies have shown that there is a spatio-temporal level of expression throughout the brain for each of the receptor subunits with some regions showing a strong preference for a particular subunit. Although these studies collectively show that there are regional differences with respect to NMDA receptor subunit expression in the brain, it has not been determined at what level(s) these genes are expressed or whether each region displays a unique NMDA-subunit signature. The present study was undertaken to examine the level of gene expression for the NR1, NR2A, NR2B, NR2C, NR2D and NR3A receptor subunits in isolated regions of rat brain using the nuclease protection assay. Results show that each of the brain regions examined expresses all six NMDA receptor subunits. The level of message expression for NR1 greatly exceeded that of the other subunits combined, with values ranging from 67-88% of the total subunit gene expression. The relative proportions of the other subunits (NR2A-D and NR3A) varied widely, suggesting that NMDA receptor composition is unique to each region of the brain.

Animals↗

PubMed2002

Sensory nerves and neuropeptides in uterine cervical ripening.

At the time of parturition (fetal delivery) the uterine cervix must "ripen," becoming soft, pliable, and dilated to accommodate the fetus' delivery. The fundamental processes underlying cervical ripening remain poorly understood. Knowledge that abundant autonomic and sensory nerves supply the uterine cervix, that transection of afferent nerves supplying the cervix blocks parturition, and that some of the changes in the cervix resemble those seen in inflammatory reactions suggests nerves may have a role in the cervical ripening changes. The present study utilized immunohistochemistry, plasma extravasation, and solution hybridization-nuclease protection assay to elucidate the complement of primary afferent nerves and some receptors in the rat cervix during pregnancy, and to determine if they may have roles in the ripening process at term. This study revealed an abundance of nerves associated with the cervical vasculature and myometrial smooth muscle containing immunoreactivity for substance P, calcitonin gene-related peptide, secretoneurin, and nitric oxide synthase throughout pregnancy. Many of these are small unmyelinated capsaicin-sensitive C-fibers. Substance P- (NK1-) and calcitonin gene-related peptide receptors were apparent on uterine cervix vasculature from pregnant, parturient, and postpartum rats. NK1 receptor mRNA was maximal at 20 days of pregnancy. Plasma extravasation of i.v. administered Evans Blue or Monastral Blue was most pronounced at parturition (shortly after NK1 mRNA is maximal); this was similar to plasma extravasation evoked by i.v. administration of substance P or capsaicin-treatment. This study revealed new data about the nervous system of the rat uterine cervix and that these nerves and their transmitters could very well be part of a neurogenic inflammatory process involved in cervical ripening.

Animals↗

PubMed1998

Somatostatin decreases somatostatin messenger ribonucleic acid levels in the rat periventricular nucleus.

Growth hormone (GH) secretion from the pituitary is known to be under the dual control of GH-releasing factor (GRF) and somatostatin (SRIF). Hypothalamic SRIF, the major inhibitor of pituitary growth hormone secretion, inhibits its own release by a negative ultrashort-loop feedback mechanism. However, it is not known whether this negative regulation is mediated by inhibition of SRIF mRNA production. GRF may also inhibit its own release, thereby modifying pituitary GH secretion, possibly through an ultrashort-loop feedback mechanism. Thus, SRIF production and GRF release are both regulated by SRIF. Periventricular nucleus (PeN) and mediobasal hypothalamus (MBH) from adult male rats were incubated for 6 h in Waymouth's medium with either SRIF or the SRIF agonist analog RC 160 (10(-9) to 10(-6) M). Levels of SRIF mRNA were determined by an S1 nuclease protection assay using a 32[P]-labeled rat SRIF riboprobe. SRIF (10(-7) M) and RC 160 (10(-8), 10(-7) M) significantly (p< or =0.01) decreased SRIF mRNA levels in the PeN. The levels of SRIF mRNA in the MBH were not modified by either SRIF or RC 160. SRIF (10(-7) and 10(-6) M) significantly (p < or = 0.01 and p < or = 0.001, respectively) inhibited the release of GRF at 30 min in the MBH. Likewise, the release of GRF was slightly decreased by 10(-7) M RC 160, and significantly inhibited by 10(-6) M (p < or = 0.001) at 30 min. At 6 h, the levels of GRF were significantly reduced by 10(-7) M SRIF (p < or = 0.05) and by RC 160 (10(-7), 10(-6) M; p < or = 0.001 and p < or = 0.05, respectively). In contrast with these results, the SRIF analog was unable to alter SRIF release at 30 min. At 6 h incubation, RC 160 (10(-7) M) significantly (p < or = 0.001) reduced SRIF release from MBH fragments. These results demonstrate that SRIF and a SRIF analog decrease SRIF mRNA levels in the PeN and inhibit the release of SRIF from the nerve terminals of the MBH. Thus, SRIF appears to regulate its own gene expression by negative ultrashort-loop feedback. Therefore, when SRIF is secreted from these neurons in response to GRF, it down-regulates the preceding stimulatory input as well as its own secretion.

Animals↗

PubMed1999

Visual input regulates the expression of basic fibroblast growth factor and its receptor.

Emerging evidence indicates that the expression of trophic factors in the brain is regulated in an activity-dependent manner, which suggests an involvement of trophic factors in events controlled by input activity. We have investigated the possibility that visual sensory input impacts the expression of basic fibroblast growth factor and its receptor in the brain. Rats were maintained for seven days in darkness and then re-exposed to normal illumination for 0, 1, 3 or 6 h. We assessed relative levels of basic fibroblast growth factor and fibroblast growth factor receptor messenger RNAs using nuclease protection assays, and examined possible changes in the phenotypic expression of basic fibroblast growth factor and its receptor using immunohistochemistry. There was a significant decrease in levels of basic fibroblast growth factor and fibroblast growth factor receptor messenger RNAs as a result of dark rearing, and levels of messenger RNAs increased progressively with light re-exposure. Changes in messenger RNAs were observed primarily in the cerebral cortex (caudal portion) and were accompanied by alterations in the staining intensity and density of cells exhibiting basic fibroblast growth factor and fibroblast growth factor receptor phenotypes. Regulation of the basic fibroblast growth factor system by sensory input suggests that basic fibroblast growth factor, and perhaps other trophic factors, are mediators of the effects of experience on the structure and function of the CNS.

Animals↗

PubMed1999

Developmental expression of alpha-synuclein in rat hippocampus and cerebral cortex.

Alpha-synuclein is an evolutionary highly conserved neuronal protein localized in presynaptic nerve terminals. The protein has been suggested to be involved in the pathogenesis of neurodegenerative diseases, but little is known about the physiological function of the protein. In the present study we used newborn, three, 14, 93 and 710-day-old rats to examine the expression of alpha-synuclein messenger RNA and protein during development of the hippocampus and cerebral cortex. Using in situ hybridization and an S1 nuclease protection assay, we found a high expression of alpha-synuclein messenger RNA during early postnatal development, followed by a marked decrease between postnatal days 14 and 93. In contrast, the amount of alpha-synuclein protein, as determined by immunoblotting, continued to increase throughout development and remained at a high level for at least two years. The persistent high expression of alpha-synuclein protein throughout development suggests that the protein is involved in maintaining synaptic function. Furthermore, the discrepancy between the levels of alpha-synuclein messenger RNA and protein after postnatal day 14 indicates that the amount of alpha-synuclein is determined by post-transcriptional regulation, and not by messenger RNA expression alone. To estimate the changes of alpha-synuclein expression per synapse, we compared the developmental expression of alpha-synuclein with synaptophysin, a well-established synaptic marker. The alpha-synuclein/synaptophysin messenger RNA and protein ratio was high during early development, but low in adult (postnatal day 93) and old (postnatal day 710) rats. This could indicate a higher expression of alpha-synuclein per synapse during early development.

Aging↗

PubMed1999

Central and peripheral expression of neurokinin-1 and neurokinin-3 receptor and substance P-encoding messenger RNAs: peripheral regulation during formalin-induced inflammation and lack of neurokinin receptor expression in primary afferent sensory neurons.

The neurokinin-1 receptor and its tachykinin neuropeptide ligand substance P are associated with the mediation of nociception. Substance P released from primary afferent sensory neurons activates neurokinin receptors on both central and peripheral targets that mediate specific aspects of central sensitization and inflammatory function; however, an autoreceptor function for the neurokinin-1 receptor remains highly controversial. Activation of the neurokinin-1 receptor by substance P during chronic nociception increases neurokinin-1 receptor gene expression in the spinal cord. Similarly, neurokinin-3 receptors on peripheral or target tissues or neurons could play an important role in the sensitization of sensory neurons. Therefore, this study (i) mapped the steady-state levels of substance P-encoding preprotachykinin, neurokinin-1 and neurokinin-3 receptor messenger RNAs in central and peripheral tissues including sensory ganglia, and (ii) investigated whether formalin-evoked nociception altered the quantity or location of neurokinin-1 or neurokinin-3 receptor messenger RNAs in the sensory ganglia or inflamed peripheral targets for substance P. Solution hybridization-nuclease protection assays quantified neurokinin receptor messenger RNA levels in central and peripheral tissues from normal and formalin-inflamed rats. High concentrations of the neurokinin-1 receptor were found in whole brain, spinal cord, and peripheral target organs innervated by substance P-containing neurons. Measurable levels of neurokinin-3 receptor messenger RNA were found only in brain, spinal cord and urinary bladder. Results also show that neither neurokinin-1 nor neurokinin-3 receptor messenger RNAs were detectable in primary afferent sensory neurons in the dorsal root ganglia of normal or formalin-inflamed rats. Neurokinin-1 receptor messenger RNA levels were, however, significantly increased in hindpaw tissues inflamed by formalin for 6 h. These results indicate that the plasticity of neurokinin-1 receptor gene expression in non-neuronal peripheral cells could regulate sensitivity to substance P in a manner similar to that in the spinal cord dorsal horn. Altered neurokinin-1 receptor gene expression provides a useful marker of long-term nociceptive activation and may mediate peripheral mechanisms of hyperalgesia and cellular sensitization during inflammation. Importantly, inflammation does not induce a phenotypic change in afferent sensory neurons providing neurokinin receptor targets for the direct sensitization of these neurons by substance P.

Animals↗