PPT mRNA Research Articles

Inducible preprotachykinin mRNA expression in mucosal lymphoid organs following oral immunization with Salmonella

Using a sensitive and specific reverse transcribed-polymerase chain reaction (RT-PCR), it was possible to quantify relative increases in preprotachykinin (PPT) mRNA expression in vivo following oral administration of Salmonella. Despite the presence of constitutive levels of PPT mRNA expression in the Peyer's patches, expression of this mRNA increased within 20 h following oral administration of Salmonella. Increases in PPT mRNA expression were also detected in both the mesenteric lymph nodes and spleens following Salmonella administration despite the lack of constitutive PPT mRNA expression in these lymphoid organs. Furthermore, mononuclear leukocytes contributed to this increased expression of PPT mRNA, suggesting that the initial immune response against Salmonella occurs in the presence of increased tachykinin expression.

Neuropeptide gene expression and capsaicin-sensitive primary afferents: maintenance and spread of adjuvant arthritis in the rat.

1. Many experimental and clinical arthritides are characterized by their bilateral nature. There is strong evidence to suggest that this bilateral spread may be mediated by a neuronal mechanism. We have previously shown early and sustained induction of mRNAs encoding preprotachykinin (PPT) and calcitonin gene-related peptide (CGRP) in dorsal root ganglion (DRG) neurons innervating an inflamed, arthritic joint. We have now investigated the involvement of capsaicin-sensitive primary afferents and the expression of neuropeptide mRNAs in the maintenance and bilateral spread of mild adjuvant-induced arthritis in the rat. 2. Capsaicin was applied perineurally to either the left (Cap-L) or right (Cap-R) sciatic nerve of halothane-anaesthetized male Han Wistar rats. Two weeks after capsaicin lesioning, arthritis was induced by injection of Freund's complete adjuvant (FCA) around the left ankle at a dose that caused inflammation of the left ankle joint, and a delayed (14 days) contralateral (right) ankle arthritis. Arthritis was monitored for 15 days after injection, when animals were killed and the lumbar DRG dissected. PPT, CGRP, somatostatin (SS), and vasoactive intestinal polypeptide (VIP) mRNA expression was determined in L5 DRG using in situ hybridization. 3. Spread of inflammation/arthritis to the right limb was associated with bilateral rises in PPT and CGRP mRNA expression in L5 DRG. SS mRNA expression in right DRG was unaffected by spread of inflammation. FCA-L+Cap-L reduced left joint swelling and prevented spread of arthritis to the right joint when assessed by joint swelling. This inhibition of spread of arthritis was associated with significant reductions in all left L5 DRG neuropeptide mRNAs compared with controls, and the rise in right L5 DRG PPT mRNA expression seen in FCA-L-alone animals was blocked. FCA-L+Cap-R also reduced left joint swelling and prevented the spread of inflammation to the right ankle. This lesion prevented the rise in PPT and CGRP mRNA expression seen in right DRG with FCA-L alone. 4. These findings suggest a role for capsaicin-sensitive primary afferents and the primary afferent neuropeptides encoded by PPT and CGRP mRNA in the maintenance and spread of arthritis.

Endogenous Glucocorticoids and the Induction and Spread of Monoarthritis in the Rat

Using mono- and bilateral tarsal arthritic models in the rat, we have previously shown increases in the expression of mRNAs encoding substance P and calcitonin gene-related peptide (CGRP) in primary sensory neurons innervating inflamed joints. Dorsal root ganglion (DRG) neuropeptide content in rats is altered by glucocorticoids, and since glucocorticoids regulate the expression of preprotachykinin (PPT) gene, the substance P precursor in other tissues, these effects may be mediated at the level of transcription. Indeed adrenalectomy potentiates disease in polyarthritis although the relationship to joint disease itself is unclear. Secretion of corticosterone in both mono- and bilaterally inflamed rats showed a loss of the normal diurnal nadir with no elevation of evening values. However, there were no changes in glucocorticoid target organs (adrenal gland, thymus and spleen) suggesting the stress was intermittent. Adrenalectomy in mono- and bilaterally inflamed rats did not significantly alter either the severity of inflammation or its spread. Bilaterally inflamed animals did, however, show reduced weight gain. Adrenalectomy had no effect on the induction of PPT and CGRP mRNA expression in innervating DRG neurons in monoarthritis (14 days after adjuvant injection), the unilateral increase in both PPT and CGRP mRNA expression in ADX animals being similar to SHAM arthritic rats. (PPT: ADX 140 +/- 13 left; 99 +/- 6 right % control; SHAM 160 +/- 22 left, 100 +/- 5 right % control. CGRP: ADX 177 +/- 6 left, 97 +/- 3 right % control; SHAM 147 +/- 21 left, 100 +/- 5 right % control).(ABSTRACT TRUNCATED AT 250 WORDS)

Altered tachykinin expression by dorsal root ganglion neurons in a rat model of neuropathic pain

The experiments described in the present study approached nerve injury from both a biochemical and anatomical perspective by monitoring changes in expression of preprotachykinin (PPT) mRNA encoding the prototypic tachykinin substance P and related peptide species in neurons of the rat dorsal root ganglia (DRG) following unilateral chronic constriction injury of the sciatic nerve. In situ hybridization histochemistry (ISHH) analyses in conjunction with computer-assisted image processing were employed to quantify levels of PPT mRNA distributed in DRG neurons. Injury-induced changes in PPT mRNA expression by affected DRG neurons included: 1. (1) at early postoperative times, generally increased levels of PPT mRNA associated with small and intermediate-size B cells exhibiting normal morphology, 2. (2) at late postoperative times, markedly decreased levels of PPT mRNA associated with degenerating B cells, and 3. (3) induction of PPT gene expression by large A cells which is highly correlated with degenerative morphological changes. The significant aspects of these changes are discussed with special emphasis on the contribution of altered transmitter expression by DRG neurons to the pathophysiology of causalgia. In particular, the induction of PPT gene expression by many of the large neurons undergoing degenerative changes may represent an important biochemical parameter which is associated with the development and persistence of experimental allodynia.

Fourth Isoform of Preprotachykinin Messenger RNA Encoding for Substance P in the Rat Intestine

Three different isoforms of preprotachykinin mRNA (PPT mRNA) encode for substance P and related neuropeptides (1). Here we report a fourth isoform of PPT mRNA which is generated by alternative exclusion of exon-4 and exon-6 from the PPT mRNA. It was present mainly in ileal smooth muscle and mucosa, colon, heart and brain and low level of this mRNA was detected in the jejunal smooth muscle and mucosa. This was not detected in the kidney or uterus. The level of this PPT mRNA was enhanced significantly by 60% during colitis in rat induced by trinitro benzene sulphonic acid.

Local anaesthesia prevents acute inflammatory changes in neuropeptide messenger RNA expression in rat dorsal root ganglia neurons

Sensory neuropeptides, synthesised in dorsal root ganglia (DRG), are implicated in neurogenic inflammation and nociception in arthritis. Adjuvant monoarthritis increases primary afferent activity and alters expression of neuropeptide genes in DRG We investigated the role of neural discharge in the early changes in neuropeptide gene expression. Adjuvant injection increased preprotachykinin (PPT) and calcitonin gene-related peptide (CGRP) messenger RNA (mRNA) after 8 h, whereas somatostatin mRNA expression remained unchanged, in innervating L5 DRG neurons. The changes in PPT mRNA expression were prevented by concurrent local anaesthesia of the sciatic nerve. Our results suggest that electrical activity mediates, in part, the changes in DRG gene expression in response to acute inflammation.

The expression of neuropeptides and their mRNAs in the trigeminal mesencephalic nucleus following masseteric nerve transection

By in situ hybridization and immunohistochemistry, we examined the expression of neuropeptides such as neuropeptide Y (NPY), galanin (Gal), substance P (SP), vasoactive intestinal polypeptide (VIP) and their mRNAs in the rat mesencephalic trigeminal nucleus (Mes5) following masseteric nerve transection. On the side contralateral to the nerve transection, none of the peptides examined were labeled in Mes5 cell bodies. However, on the side ipsilateral to the lesion, NPY, Gal and preprotachykinin (PPT) mRNAs appeared in Mes5 cell bodies. Double labeling for mRNAs by in situ hybridization and retrograde tracer fluorogold (FG) revealed that almost all (96–97%) the FG-labeled neurons which were cut expressed NPY and Gal mRNAs, whereas less neurons (87%) expressed PPT mRNA. NPY and Gal-like immunoreactivities were detected in Mes5 cell bodies ipsilateral to the axotomy. The results suggested that these neuropeptides play roles in adaptive processes after peripheral nerve injury in Mes5 neurons as they are thought to do so in dorsal root ganglion neurons.

NK-1 and NK-3 type tachykinin receptor mRNA expression in the rat spinal cord dorsal horn is increased during adjuvant or formalin- induced nociception

Substance P (SP) and other related tachykinins such as neurokinin B (NKB) have been studied widely as mediators of sensory information. The release of SP into the dorsal horn of the spinal cord is increased during nociception, and SP activates nociception-specific dorsal horn neurons. The tachykinin NKB has antinociceptive effects in the spinal cord and is contained in intrinsic spinal neurons; thus, NKB may also contribute to the processing of sensory information. Both neurokinin-1 (NK-1) and neurokinin-3 (NK-3) receptors have been localized in the superficial laminae of the dorsal horn. This study investigated changes in NK-1 and NK-3 receptor mRNA expression during nociception. Following injection of either formalin or complete Freund's adjuvant (CFA) into one hindpaw, the levels of expression of NK-1 and NK-3 mRNAs in the spinal cord dorsal horn and preprotachykinin (PPT) mRNA expression in the lumbar dorsal root ganglia (DRG) were quantitated using solution hybridization-nuclease protection assays. Peptide and receptor mRNA expression levels were normalized to beta-actin mRNA levels, which did not change during the treatments. Formalin (2 or 6 hr) or CFA (4 d) injection produced approximately a twofold increase in SP-encoding PPT mRNA expression in the ipsilateral lumbar DRG. Increased activity in primary afferent neurons containing SP may stimulate the production of SP precursors, providing substrate for increased SP production, release, and turnover in the dorsal horn and periphery.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain-derived neurotrophic factor enhances striatal neuropeptide expression in both the intact and the dopamine-depleted rat striatum.

In order to elucidate the importance of the striatal GABAergic neurones in mediating functional effects of exogenously applied brain-derived neurotrophic factor (BDNF) in the basal ganglia, we performed daily injections of BDNF or vehicle into the dopamine-depleted striatum of unilaterally 6-hydroxydopamine-lesioned rats for 1 week. In situ hybridization revealed that BDNF exacerbated the lesion-induced up-regulation of preproenkephalin (PPE) mRNA, and completely reversed the lesion-induced decrease of preprotachykinin (PPT) mRNA. In contrast, striatal levels of trkB mRNA were not significantly affected by BDNF administration. Up-regulation of PPE and PPT mRNA was also observed in unlesioned BDNF-injected animals. We conclude that exogenously applied BDNF increases neuropeptide mRNA expression in striatal neurones independently of the presence of a dopaminergic innervation.

PPT and CGRP mRNAs coexpress with trkA, but not, with trkB and trkC mRNAs in rat dorsal root ganglion neurons

PPT and CGRP mRNAs coexpress with trkA, but not, with trkB and trkC mRNAs in rat dorsal root ganglion neurons

Activity-dependent changes in GAD and preprotachykinin mRNAs in visual cortex of adult monkeys.

Tachykinin-immunoreactive neurons are a subgroup of the GABA neuronal population in layer IVC of monkey primary visual cortex. Following brief periods of monocular deprivation in adult monkeys, immunoreactivity for both GABA and tachykinins is dramatically reduced in layer IV cells that lie within the deprived ocular dominance columns of this cortical area. The present study shows that these activity-dependent changes are associated with changes in mRNA levels but over different time courses. Radioactive antisense riboprobes derived from monkey-specific cDNAs were used to localize glutamic acid decarboxylase (GAD) and beta-preprotachykinin (beta PPT) mRNAs by in situ hybridization histochemistry. GAD and beta PPT mRNAs decreased in deprived ocular dominance columns of adult monkeys when neural activity was abolished in one eye by intraocular injections of tetrodotoxin (TTX). beta PPT mRNA levels fell within 5 d of deprivation and thus appeared to parallel the fall in immunodetectable tachykinin levels. By contrast, reduced GAD mRNA levels were detectable only after 15 d of deprivation and long after the fall in immunoreactive GAD and GABA levels has maximized. These results suggest that tachykinin gene expression is regulated by transcriptional mechanisms as part of the first response to reduced neural activity whereas the initial downregulation of immunoreactive GAD and GABA depends on posttranscriptional mechanisms. Following a more prolonged period of deprivation, a secondary mechanism for GAD regulation appears to be engaged at the level of gene transcription or possibly by changes in mRNA stability.

Distribution of tachykinin- and opioid-expressing neurons in the hamster solitary nucleus: an immuno- and in situ hybridization histochemical study

In several sensory systems, tachykinin- and opioid-expressing neurons functionally interact and influence the processing of afferent information. To determine whether a similar relationship exists for the processing of general and special (gustatory) visceral afferent information, the present study mapped the distributions of these two neuronal phenotypes within the nucleus of the solitary tract (NST) of the hamster by employing a combination of immuno- and in situ hybridization histochemistry (ISHH). The hamster was chosen because it is frequently used as a model in taste studies, yet there is a relative dearth of data about peptide expression or the classical neurotransmitters in the brainstem of this animal. The immunohistochemical analyses employed 2 highly selective antisera directed towards the prototypical tachykinin and opioid peptides, i.e. substance P (SP) and methionine enkephalin (ENK), respectively. Intense staining of fibers and preterminal/terminal puncta was concentrated in the rostral pole or gustatory zone of the NST. SP-, but not ENK-like immunoreactivity was also observed in long courses of axon bundles traversing the brainstem enroute to the NST. Local application of colchicine engendered the appearance of a moderate number of SP-positive somata that were mostly clustered in the medial, central and intermediate subnuclei, as well as being scattered throughout the remainder of the NST, including the gustatory zone. A low number of isolated ENK-positive somata were also observed throughout the NST. The somal areas of the SP- and ENK-positive somata averaged 86.3 and 81.8 μm 2, respectively. The ISHH studies were performed using 2 selective oligodeoxynucleotide probes with complementary sequences to mRNAs encoding γ-preprotachykinin (PPT) and preproenkephalin (PPE) molecules. Overall, the cellular expression of PPT mRNA within the NST corresponded both in distribution and in number to those identified by immunohistochemical analyses using anti-SP serum. In contrast, ISHH analyses monitored a significantly greater number of PPE- expressing somata in the medial, central, intermediate and ventrolateral nuclei than were ENK immunoreactive. These findings indicate that tachykinin and opioid peptide phenotypes are represented in neurons throughout the hamster NST and suggest a functional role for PPT- and PPE-related peptide forms in the modulation of afferent general visceral and gustatory information.

Serotonin modulates the levels of mRNAs coding for thyrotropin-releasing hormone and preprotachykinin by different mechanisms in medullary raphe neurons

The serotonergic neurons of the medullary raphe also contain the peptide neurotransmitters substance P (SP) and thyrotropin releasing hormone (TRH). In this study we asked whether the manipulation of serotonin levels alters the levels of mRNA coding for pre-proTRH. Just like the mRNA coding for the precursor of SP (preprotachykinin, PPT), levels of TRH mRNA are increased when serotonin synthesis is inhibited by para-chlorophenylalanine (pCPA) and decreased when serotonin reuptake is blocked by zimelidine. However, subtle differences suggest that the mechanisms behind these changes are different. Levels of TRH mRNA are still decreased after 14 days of zimelidine treatment, a time when levels of PPT mRNA have returned to control values. In addition, the serotonin reuptake blocker fluoxetine lowers levels of TRH but not PPT mRNA. Finally, the 5-HT 1A receptor agonist 8-hydroxy-2-(di- n-propylamino)tetralin (8-OH-DPAT) induces a transient decrease in levels of PPT mRNA similar to that induced by zimelidine 34, but does not decrease levels of TRH mRNA even when 10-fold higher doses are administered. These results suggest that while some pharmacological manipulations appear to alter TRH and PPT mRNA levels coordinately, the mechanisms regulating the synthesis of these two colocalized neurotransmitters are different.

Tachykinin systems in the spinal cord and basal ganglia: influence of neonatal capsaicin treatment or dopaminergic intervention on levels of peptides, substance P-encoding mRNAs, and substance P receptor mRNA.

The aim of the study was to test whether the synthesis of substance P (SP) and that of its receptor (also known as NK1 receptor) are coordinately regulated after chronic pharmacologic intervention in two neural systems, the spinal cord and basal ganglia. In one set of experiments, capsaicin was administered subcutaneously during the early postnatal period (day 3 after birth) to induce degeneration of afferent sensory neurons in the spinal cord. In the other set of experiments, interruption of dopaminergic transmission was achieved by two methods: (a) The neurotoxin 6-hydroxydopamine was used to denervate dopaminergic neurons during the early postnatal period, and (b) haloperidol was used in adult animals to block dopaminergic transmission by receptor blockade. The spinal cord, striatum, or both were used for the quantification of tachykinin [SP and neurokinin A (NKA)] and opioid peptides [[Met5]-enkephalin (ME) and dynorphin A (1-8) (DYN)] by radioimmunoassays. The abundance of total SP-encoding preprotachykinin (PPT) mRNA and SP receptor (SPR) mRNA in spinal cord (C5 to T1 segments), striatum, or microdissected substantia nigra was determined by northern blot or solution hybridization analysis. Amines and their acid metabolites were quantified by HPLC. Capsaicin administration (subcutaneously) during the early postnatal period increased latency in a hot-plate test, decreased SP and NKA levels, increased levels of PPT mRNAs, and did not affect SPR mRNA levels in the spinal cord. Intraspinal SP systems may attempt to compensate for the loss of afferent SP input, whereas spinal cord receptor mRNA levels do not appear to be altered.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased expression of preprotachykinin, calcitonin gene-related peptide, but not vasoactive intestinal peptide messenger RNA in dorsal root ganglia during the development of adjuvant monoarthritis in the rat

Neuropeptides in dorsal root ganglia (DRG) have been implicated in the pathogenesis of pain and neurogenic inflammation in experimental and clinical arthritis. Recently we demonstrated increased levels of substance P (SP) and calcitonin gene-related peptide (CGRP) confined to innervating DRG in adjuvant-mediated monoarthritis. We have now investigated whether changes in peptide content are reflected in altered neuropeptide gene expression and the time course involved. Using in situ hybridization we found marked increases in expression of β-preprotachykinin (PPT; 81±24% rise) and α-CGRP (44±6% rise)mRNAs in innervating (ipsilateral L 5) DRG neurones only. These a increases occured at the onset of acute inflammation (8 h) and persisted until chronic arthritis developed after 14 days. There were no changes in the proportion of DRG neurones expressing PPT or CGRP mRNAs. Messenger RNA encoding vasoactive intestinal polypeptide (VIP) was not induced. These data suggest that increased synthesis of PPT and CGRP peptides in DRG may play a role in the pathogenesis both of adjuvant-mediated acute inflammation and chronic arthritis.

Gene regulation in an ascending nociceptive pathway: inflammation- induced increase in preprotachykinin mRNA in rat lamina I spinal projection neurons

Tachykinin peptides are distributed widely in the nervous system and have been shown to play a prominent role in nociceptive pathways in the spinal cord and dorsal root ganglia. This study investigated the inflammation-induced response of dorsal horn projection neurons and local circuit neurons expressing preprotachykinin (PPT) mRNA using RNA blot analysis and in situ hybridization histochemistry. To identify projection neurons, fluorogold was injected into the parabrachial area of the brainstem. In laminae I, II and V/VI ipsilateral to inflammation, there was a differential increase in the number of neurons exhibiting PPT mRNA. In lamina I, the number of spinal projection neurons containing PPT mRNA showed a greater than 200% increase. The identification of spinal projection neurons with inflammation-induced increases in PPT mRNA suggests that tachykinin peptides may act as neurotransmitters in nociceptive CNS projection pathways.

Regulation of adenohypophyseal messenger RNAs in female rats by age, hypothyroidism, estradiol and neonatal androgenization

Hormonal regulation of adenohypophyseal messenger ribonucleic acids (mRNAs) encoding preprotachykinin (PPT), prolactin (PRL) and thyrotropin β subunit (TSHβ) was examined in juvenile and pubertal female rats. Hypothyroidism, initiated on day 2 (d2) or 22 (d22) of life, increased PPT and TSHβ mRNAs but decreased PRL mRNA 17 days later. Exogenous estradiol given for 3 days reduced PPT mRNA in pubertal (d38) but not juvenile (d18) euthyroid females; conversely, estradiol increased PRL mRNA on d18 but not d38. In hypothyroid females however, estradiol decreased PPT and TSHβ mRNAs at both ages and increased PRL mRNA in pubertal but not juvenile females. Thus, regulation of adenohypophyseal mRNAs by estradiol varies with age and thyroid status. In previous studies, adenohypophyseal tachykinins increased in male, but not female rats at puberty. This sex difference was not reproduced here by neonatal androgenization of females, suggesting that it is not mediated by hypothalamic sexual differentiation. However, PRL mRNA increased in androgenized females; this increase was prevented by ovariectomy, suggesting its mediation by estradiol.

D 2 dopaminergic regulation of striatal preproenkephalin mRNA levels is mediated at least in part through cholinergic interneurons

The effect of administration of the muscarinic antagonist scopolamine on the increase in striatal preproenkephalin (PPE) mRNA following a 6-hydroxydopamine (6-OHDA) lesion or chronic D 2 dopamine (DA) antagonist treatment was examined by dot-blot hybridization. Administration of scopolamine dose-dependently attenuated the 6-OHDA lesion-induced increase in striatal PPE mRNA. Administration of the D 2 DA antagonist eticlopride to naive rats increased striatal PPE mRNA in a dose- and time-dependent fashion. Chronic coadministration of scopolamine attenuated the eticlopride-induced increase in striatal PPE mRNA. Chronic administration of scopolamine alone did not alter striatal PPE mRNA levels. In contrast, chronic administration of eticlopride, scopolamine or the two combined decreased striatal preprotachykinin (PPT) mRNA to the same extent, suggesting that there was no direct interaction between D 2 dopaminergic and cholinergic mechanisms in the regulation of striatal PPT mRNA. These data indicate that DA differentially regulates striatal PPE and PPT mRNA and suggest that dopaminergic regulation of striatal PPE mRNA is mediated in part through D 2 DA effects on striatal cholinergic neurons.

Preprotachykinin gene expression in the human basal ganglia: characterization of mRNAs and pre-mRNAs produced by alternate RNA splicing

The nature and distribution of preprotachykinin (PPT, i.e. substance P/neurokinin A-encoding) gene expression in human basal ganglia was determined. Northern blot analysis visualized a single band of approximately 1300 bases, confirming the postmortem stability of PPT mRNA. Gross anatomical analysis indicated that PPT gene expression was relatively evenly distributed throughout the human caudate and putamen, but absent in the globus pallidus and substantia nigra. Nuclease protection analysis of these tissues established that human PPT mRNA consisted of approximately 80–85% β-PPT (exon 1–7 derived) mRNA and 15–20% γ-PPT (minus exon 4), with no α-PPT (minus exon 6) mRNA detected; these data contrast with the proportion of PPT mRNAs seen in non-human species. The incompletely spliced PPT RNA species detected in basal ganglia accounted for approximately 8% of total human PPT RNA and suggested a fixed order of exon splicing. Since various PPT mRNAs encode different combinations of tachykinin peptides with distinct biological activities, the markedly different proportions of PPT mRNAs seen in human basal ganglia compared to non-human tissues may be of physiological significance.

Identification of Nerve Growth Factor-Responsive Sequences Within the 5′ Region of the Bovine Preprotachykinin Gene

The production of substance P and the mRNA encoding its precursor (preprotachykinin, PPT) is regulated by nerve growth factor (NGF) in dorsal root ganglion (drg) neurons. To explore the mechanism by which NGF regulates the production of PPT mRNA, we have transfected PC12 cells and F11 cells with plasmids containing the bovine PPT promoter linked to the reporter gene chloramphenicol acetyltransferase (CAT). We have identified (i) functional elements within the PPT promoter which are necessary for expression in the absence of NGF and (ii) two separate regions, each of approximately 250 bp, which confer NGF responsiveness. Both regions contained a sequence element, similar to a known transcription factor binding site, which is present in several other NGF-regulated genes.