preprotachykinin-A mRNA Research Articles

L-type calcium channel blockers and substance P induce angiogenesis of cortical vessels associated with beta-amyloid plaques in an Alzheimer mouse model

It is well established that L-type calcium channels (LTCCs) are expressed in astroglia. However, their functional role is still speculative, especially under pathologic conditions. We recently showed that the α1 subunit-like immunoreactivity of the CaV1.2 channel is strongly expressed in reactive astrocytes around beta-amyloid plaques in 11-month-old Alzheimer transgenic (tg) mice with the amyloid precursor protein London and Swedish mutations. The aim of the present study was to examine the cellular expression of all LTCC subunits around beta-amyloid plaques by in situ hybridization using 35S-labeled oligonucleotides. Our data show that messenger RNAs (mRNAs) of the LTCC CaV1.2 α1 subunit as well as all auxiliary β and α2δ subunits, except α2δ-4, were expressed in the hippocampus of age-matched wild-type mice. It was unexpected to see, that cells directly located in the plaque core in the cortex expressed mRNAs for CaV1.2 α1, β2, β4, and α2δ-1, whereas no expression was detected in the halo. Furthermore, cells in the plaque core also expressed preprotachykinin-A mRNA, the precursor for substance P. By means of confocal microscopy, we demonstrated that collagen-IV-stained brain vessels in the cortex were associated with the plaque core and were immunoreactive for substance P. In cortical organotypic brain slices of adult Alzheimer mice, we could demonstrate that LTCC blockers increased angiogenesis, which was further potentiated by substance P. In conclusion, our data show that brain vessels associated with beta-amyloid plaques express substance P and an LTCC and may play a role in angiogenesis.

Involvement of substance P in the development of cisplatin‐induced acute and delayed pica in rats

Although substance P (SP) and neurokinin NK1 receptors have been reported to be involved in cisplatin-induced acute and delayed emesis, their precise roles remain unclear. Pica, the consumption of non-nutrient materials such as kaolin in rats, can be used as a model of nausea in humans. We investigated the time-dependent changes in cisplatin-induced pica and the involvement of SP and NK1 receptors in this behaviour. Rats were administered cisplatin with or without a daily injection of a 5-HT3 receptor antagonist (granisetron) or an NK1 receptor antagonist (aprepitant), and kaolin intake was then monitored for 5 days. The effects of granisetron on the cisplatin-induced expression of preprotachykinin-A (PPT-A) mRNA, which encodes mainly for SP, and on SP release in the medulla, measured by in vivo brain microdialysis, were also investigated. Cisplatin induced pica within 8 h of its administration that continued for 5 days. Granisetron inhibited the acute phase (day 1), but not the delayed phase (days 2-5), of pica, whereas aprepitant abolished both phases. Within 24 h of the injection of cisplatin, PPT-A mRNA expression and SP release in the medulla were significantly increased; these findings lasted during the observation period and were inhibited by granisetron for up to 24 h. The profiles of cisplatin-induced pica in rats are similar to clinical findings for cisplatin-induced emesis in humans, and we showed that SP production in the medulla and activation of NK1 receptors are involved in this cisplatin-induced pica.

Dragon’s Blood Inhibits Chronic Inflammatory and Neuropathic Pain Responses by Blocking the Synthesis and Release of Substance P in Rats

As a traditional Chinese medicine, dragon’s blood (DB) is widely used in treating various pains for thousands of years due to its potent anti-inflammatory and analgesic effects. In the present study, we observed that intragastric administration of DB at dosages of 0.14, 0.56, and 1.12 g/kg potently inhibited paw edema, hyperalgesia, cyclooxygenase-2 (COX-2) protein expression, or preprotachykinin-A mRNA expression in carrageenan-inflamed or sciatic nerve–injured (chronic constriction injury) rats, respectively. A short-term (15 s or 10 min) pre-exposure of cultured rat dorsal root ganglion (DRG) neurons to DB (0.3, 3, and 30 μg/ml) or its component cochinchinenin B (CB; 0.1, 1, and 10 μM) blocked capsaicin-evoked increases in both the intracellular calcium ion concentration and the substance P release. Moreover, a long-term (180 min) exposure of cultured rat DRG neurons to DB or CB significantly attenuated bradykinin-induced substance P release. These findings indicate that DB exerts anti-inflammatory and analgesic effects by blocking the synthesis and release of substance P through inhibition of COX-2 protein induction and intracellular calcium ion concentration. Therefore, DB may serve as a promising potent therapeutic agent for treatment of chronic pain, and its effective component CB might partly contribute to anti-inflammatory and analgesic effects.

The effect of experimental occlusal interferences on nerve growth factor levels in periodontal tissues

Objective To test the hypothesis that experimental occlusal interferences increase the nerve growth factor (NGF) levels in periodontal tissues and cause an up-regulation of preprotachykinin-A (PPTA) mRNA. Background NGF is related to hyperalgesia and inflammation. PPTA mRNA, a primer of substance P, is a possible factor in the aetiology of pain. Methods Experimental interferences were created by placing inlays in the right maxillary molars of 15 dogs. The right side molars formed the experimental group. The left side molars served as controls. Three dogs with cavities prepared without changing the occlusion formed a sham group. The dogs in the first group were sacrificed, 3 at each time, after 3, 7, 14, 30, and 60 days. The sham group was sacrificed after 14 days. The levels of NGF in periodontal tissues and PPTA mRNA in the trigeminal ganglions were detected by ELISA and TR-PCR. Comparisons were made with paired t-tests and a multivariate MANOVA test. Results On all measurement days, there were higher levels of NGF mRNA, PPTA mRNA, and NGF on the experimental than on the control side in 14 of 15 comparisons and in the sham group. NGF production in periodontium was time-dependent. No differences in NGF protein levels were observed between the control and the sham groups. Conclusion The results which need confirmation in further tests are of clinical interest. They indicate that occlusal experimental interferences may be an etiologic factor in oral facial pain by increasing mRNA and NGF protein levels in the periodontal tissues.

Involvement of L-Selectin in Contact Hypersensitivity Responses Augmented by Auditory Stress

Stress affects the pathophysiology of cutaneous immune reactions, including contact hypersensitivity (CH) in individuals sensitized with sensitizing hapten, where local endothelial cell activation plays a critical role. To clarify the effects of stress in cutaneous immune reactions, we selected a CH model using annoying sound as a stress. Furthermore, we conducted the stress experiments by using selectin-deficient mice to determine the involvement of selectin molecules regarding local endothelial activation. Auditory stress augmented CH responses in the present study. Namely, ear thickness and mast cell numbers were significantly increased in stressed CH mice. mRNA expression of preprotachykinin-A, a precursor of substance-P; interferon-gamma; interleukin (IL)-4; IL-6; and tumor necrosis factor-alpha significantly increased in stressed CH mice. Furthermore, stressed L-selectin-deficient mice showed significant decreases in all parameters mentioned above relative to stressed wild-type mice in CH response. Meanwhile, treatment with anti-L-selectin Ab resulted in a significant decrease in ear thickness and mRNA levels of interferon-gamma, IL-4, IL-6, and tumor necrosis factor-alpha, but failed to significantly reduce preprotachykinin-A mRNA levels and mast cell numbers. Our results indicated that auditory stress enhances CH response and that the augmentation of this CH response might be mediated through L-selectin, but not through P- or E-selectin pathways.

Prenatal Exposure to Nicotine Affects Substance P and Preprotachykinin-A mRNA Levels in Newborn Rat

Prenatal nicotine exposure influences neuronal development including effects on several neurotransmitter systems. It also attenuates the ventilatory response to hypoxia, known to require a functional substance P-ergic system. Previous studies have shown that nicotine increases the risk for sudden infant death syndrome (SIDS) by 4-fold, and that SIDS-victims have elevated brainstem levels of substance P. We, therefore, studied the effect of prenatal nicotine exposure on the levels of substance P-like immunoreactivity by RIA in the brain in newborn rat pups. The expression of the substance P precursor preprotachykinin A mRNA was also determined by real-time reverse transcriptase-polymerase chain reaction in carotid body, in petrosal/jugular and trigeminal ganglia, in cervical and lumbar dorsal root ganglia, and in the brainstem. We found that prenatal nicotine exposure increased levels of substance P-like immunoreactivity in the brainstem without changing levels in other parts of the brain or in the adrenals. Furthermore, mRNA levels were increased in the carotid bodies and in the petrosal ganglia, in contrast to the decreased levels in the cervical dorsal root ganglia. We conclude that nicotine causes alterations in the substance P-ergic system in the brainstem, possibly linked to the increased risk for SIDS after prenatal nicotine exposure.

Expression of preprotachykinin-A mRNA isoforms and substance P production in T lymphocytes of human healthy subjects

The influence of sex and age on the expression pattern of preprotachykinin-A (PPT-A) mRNA isoforms encoding substance P and other tackykinins such as neurokinin A (NKA), neuropeptide K (NPK) and neuropeptide gamma (NPgamma) in human immunocompetent cells and the role of this pattern on SP production are unknown. To investigate these questions, we assessed PPT-A isoform expression and SP production in CD3+ lymphocytes of normal healthy subjects. There were no significant differences in PPT-A isoforms in relation to sex or age. The most frequently expressed isoforms were beta and gamma: after lymphocyte stimulation with phytohemagglutinin (PHA), there was a significant increase in their frequency (p<0.0001). Significantly higher SP levels were found in subjects expressing beta and gamma PPT-A than in those with beta PPT-A only (p=0.001). These findings provide evidence of a heterogeneous expression of PPT-A isoforms in CD3+ lymphocytes of normal healthy subjects.

Preprotachykinin A mRNA is colocalized with tyrosine hydroxylase-immunoreactivity in bulbospinal neurons

Previous studies have generated controversy about the extent of co-localization between substance P- and catecholamine-containing neurons that project to the spinal cord. In earlier studies, estimates using immunofluorescence after colchicine have ranged from almost all, to almost none. We sought to resolve this issue by combining in situ hybridization and immunofluorescence. Catecholamine (A1 to A7, C1 to C3; tyrosine hydroxylase immunoreactive) neurons in the rat brainstem were examined to determine their content of mRNA for the preprotachykinin-A gene. In the A1 to A7 and the C1 to C3 cell groups, preprotachykinin-A mRNA was found only in substantial amounts in the C1–C3 cell groups. On average 20.9±0.9% (234/1120, n=3) of rostral C1 neurons contained preprotachykinin-A mRNA. Co-localization was also observed in C2 and C3 neurons to a similar extent. Retrograde tract-tracing with cholera toxin B subunit was used to identify bulbospinal neurons and 17.9±2.7% (96/529 cells) of the bulbospinal tyrosine hydroxylase-containing neurons of the rostral C1 cell group were found to contain preprotachykinin-A mRNA. In addition a new population of non-catecholaminergic bulbospinal preprotachykinin-A neurons is described in an area corresponding to the recently described caudal pressor area. To confirm that the preprotachykinin-A mRNA observed in cells in the medulla was converted to protein, dual immunofluorescence for fiber labeling at the confocal level was carried out. This confirmed colocalization of substance P and tyrosine hydroxylase in the intermediolateral cell column, but nowhere else, in a small number of cases. The results provide evidence for a much larger population of substance P/neurokinin A containing neurons in the brainstem than was previously suspected. Furthermore, many of these neurons are catecholaminergic and spinally projecting. The specific sympathetic outflow that these neurons influence remains to be determined.

Topical glucocorticoid augments scratching behaviour in dinitrofluorobenzene‐sensitized mice by the induction of substance P

Topical glucocorticoid (GC) is commonly applied in atopic dermatitis treatment. However, the chronic use of GC may be associated with significant side effects. In this study, we investigated whether long-term epicutaneous application of GC modulates scratching behaviour in dinitrofluorobenzene (DNFB) contact-sensitized mice. After challenge with DNFB, scratching behaviour was increased in DNFB-sensitized mice treated with GC in contrast to control mice. In addition, reverse transcriptase-polymerase chain reaction analysis demonstrated that the expression of preprotachykinin-A (PPT-A) mRNA, a precursor of substance P (SP), and inducible nitric oxide synthase (iNOS) mRNA in mice, to which GC was applied, was only observed. In order to evaluate the factors responsible for the augmented scratching behaviour, we injected various cytokines (interleukin-1alpha (IL-1alpha), IL-2, IL-3 and tumour necrosis factor-alpha (TNF-alpha)) subcutaneously into the ear of DNFB contact-sensitized mice before DNFB challenge. Among the cytokines, only IL-3 and TNF-alpha significantly increased scratching behaviour in DNFB contact dermatitis mice. Furthermore, PPT-A mRNA was only expressed in mice pre-injected with IL-3 before challenge, but not in those pre-injected with other cytokines. Taken together, our results suggest that topical GC may augment the itching sensation in DNFB-sensitized mice through modulation of iNOS and SP induced by IL-3.

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.

Regulation of preprotachykinin-A gene expression in an animal model of Alzheimer's disease.

A lzheimer’s disease is a neurodegenerative disorder of the CNS characterized by deposition of neuritic plaques and neurofibrillary tangles with consequent neuron death. Clinically it is characterized by a progressive loss of memory and impairment of learning capacity (Selkoe 1997; Seabrook et al. 1999). Substance P (SP) is a neurotransmitter related to memory functions and learning properties, highly expressed in several brain areas from forebrain to hindbrain (Rattan and Tejwani 1992; Alvarez et al. 1997). For example, the nucleus basalis of Meynert (NBM) is highly sensitive to memory enhancement effects of SP. SP also interacts with the cholinergic ascending system of the NBM that is implicated in memory functions. Patients with Alzheimer’s disease show a marked loss of cholinergic neurons from the NBM to the cortex and diminished brain SP immunoreactivity (Cleary et al. 1995; Nag et al. 1999). On the basis of these findings, the aim of this study was to verify the presence and the distribution of preprotachykinin-A (PPT-A) mRNA in discrete brain areas in an animal model of Alzheimer’s disease. Twenty male Albino Wistar rats weighing 340 g were implanted SC with miniosmotic infusion pumps (Alzet type) that deliver the mixed neurotoxins to the hippocampus via a catheterized cannula (Morimoto et al. 1998). Experimental animals included four groups (five animals each group). The first group (vehicle) was infused with a saline solution. The second group was infused with a solution of 0.075 g/ l of -amyloid (fragment 1–40) and 0.01875 g/ l of ibotenic acid (A dose). The third group was infused with a solution of 0.15 g/ l of -amyloid (fragment 1–40) and 0.0375 g/ l of ibotenic acid (B dose). The fourth group was infused with a solution of 0.224 g/ l of -amyloid (fragment 1–40) and 0.0562 g/ l of ibotenic acid (C dose). Finally, on Day 10, rats were sacrificed by decapitation and the brains were removed and then stored for in situ hybridization. Semiquantitative ISH histochemistry examined PPT-A mRNA levels in the bed nucleus of the stria terminalis (BNST) in the medial preoptic area (MPA). The infusion of the two neurotoxins -amyloid (fragment 1–40) and ibotenic acid induced a 24.29% statistically significant reduction in basal hybridizable PPT-A mRNA in the BNST brain area after administration with dose A, as well as a 19.26% and a 16.85% statistically significant decrease after administration with dose B and dose C compared to CON rats, respectively (Figure 1). The same figure shows a 30.7% statistically significant lower level of PPT-A mRNA in MPA after dose A, whereas PPT-A mRNA levels were shown to be sig-

In situ hybridization analysis of preprotachykinin-A mRNA levels in young and old rats.

Preprotachykinin-A (PPT-A) mRNA levels in discrete rat brain regions were examined. Analysis of silver grains revealed a 19.2% and 31.5% statistically significant decrease in PPT-A mRNA in the dorsal and ventral caudate putamen (d-CPu and v-CPu), respectively, a 30% lower expression of PPT-A mRNA in the bed nucleus of the stria terminalis (BNST), a 33.7% decrease in PPT-A mRNA in the habenula (Hb), and a 30% decrease of PPT-A mRNA levels in the posterodorsal part of the medial amygdala (MePD). Results show that aging of the CNS is associated with widespread changes in tachykinin gene expression, suggesting that alterations in the tachykinergic system may have implications in the physiopathology of the elderly.

Substance P modulates cocaine-evoked dopamine overflow in the striatum of the rat brain.

To study the role of the neuropeptide substance P in modulating some of the effects of cocaine in the striatum, we administered cocaine to rats and measured preprotachykinin-A (PPT-A) messenger RNA and substance P peptide in the nigrostriatal pathway. We also measured the effect of a neurokinin-1 (NK-1) receptor antagonist on striatal cocaine-evoked dopamine overflow by in vivo microdialysis in freely moving animals. Acute administration of cocaine to naive rats (15 mg/kg of body weight) increased preprotachykinin-A mRNA levels in the dorsal and ventral aspects of the caudate putamen 4 hours after the intraperitoneal injection of cocaine. Concomitantly, in a separate group of animals, substance P peptide levels were decreased in the ventral caudate putamen and substantia nigra (38% below controls). In a separate experiment, infusion through the microdialysis probe of the neurokinin-1 receptor antagonist L-733,060 significantly decreased cocaine-evoked striatal dopamine overflow (approximately 50% inhibition at 30 minutes after cocaine administration). Taken together, these results suggest a direct role for substance P in the modulation of some of the actions of cocaine in the striatum of the rat brain.

Ciliary neurotrophic factor induces preprotachykinin A gene expression in the rat carotid body

Substance P (SP), a translational product of preprotachykinin-A (PPT-A) mRNA plays an important role in hypoxic chemotransmission in the rat carotid body. Although hypoxic exposure has been associated with an increase in SP content in the carotid body, factors that cause induction, regulation and release of PPT-A and SP in the carotid body remain to be elucidated. The purpose of this study was to investigate whether ciliary neurotrophic factor (CNTF), a factor that has been shown to regulate neurotransmitter phenotype in tissue from neural crest origin, could induce PPT-A gene expression in the rat carotid body. We used in situ hybridization histochemistry with radioactive ribonucleotide probes to investigate the effect of CNTF on PPT-A gene induction in the carotid body. Exposure of the rat superior cervical ganglia and carotid body to increasing concentrations of CNTF in culture resulted in up-regulation and induction of PPT-A mRNA, respectively.

Identification of the α isoform of preprotachykinin-A mRNA in synovial fibroblasts isolated from patients with rheumatoid arthritis

Previous study showed that synovial fibroblasts derived from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) express β-preprotachykinin (PPT)-A mRNA and release substance P (SP). In this study, we examined the presence of an isoform of PPT-A mRNA in synovial and normal skin fibroblasts by reverse transcription-polymerase chain reaction (RT-PCR) analysis. RA synovial fibroblasts, but not OA synovial and skin fibroblasts, expressed α-isoform PPT-A mRNA together with β-PPT-A mRNA. However, expression of γ- and δ-PPT mRNAs was not observed in synovial and skin fibroblasts. Levels of both α- and β-PPT-A mRNAs were increased in RA fibroblasts treated with transforming growth factor (TGF)-β and basic fibroblast growth factor (bFGF). Treatment with TGF-β and bFGF strongly increased the expression of α- and β-PPT-A mRNAs compared to the individual treatment. Peak levels of α-PPT-A mRNA were reached at 8h whereas those of β-PPT-A mRNA were maximal at 12h after treatment with TGF-β and bFGF. These findings suggest that SP production by synovial fibroblasts in RA is regulated through signaling pathways that lead to the generation of both α- and β-PPT-A mRNAs.

Patterns of status epilepticus-induced substance P expression during development

Substance P, which modulates synaptic excitability, can be induced by a variety of stimuli. We studied the expression of hippocampal substance P in rats in using lithium-pilocarpine model of status epilepticus during development. Status epilepticus resulted in an age-specific manner of substance P expression that was anatomically distinctive in hippocampal subfields. Maximal induction of substance P immunoreactivity was seen in the CA1 region of the two-week-old rats, and progressively decreased in the three-, four-week-old rats and adults. Meanwhile, the number of substance P-immunoreactive neurons in the CA3 region and dentate granule cell layer was minimal in the two-week-old animals, but approximated the adult level in the three- and four-week-old rats. No substance P-immunoreactive axon terminals were seen in the strata pyramidale and lucidum in the CA3 region of the two-week-old rats, but they were found to progressively increase in the three-, four-week-old rats and adults. To confirm substance P expression after status epilepticus, we studied the expression of preprotachykinin-A mRNA in the hippocampus of the three-week-old rats by in situ hybridization. Two hours following injection of lithium-pilocarpine, preprotachykinin-A mRNA dramatically increased in the granule cells, as well as in the CA3 and CA1 pyramidal cell layers of the hippocampus. To evaluate the relationship between behavioral seizures and substance P induction, we used the NMDA receptor antagonist MK-801. Injection of MK-801 completely blocked lithium-pilocarpine-induced behavioral seizures and SP induction in the two-week-old rats. These results indicate that seizure activity selectively evokes age-dependent and region-selective expression of substance P.

In situ hybridization analysis of preprotachykinin-A mRNA levels in young and old rats

The present study examined the effects of ageing on preprotachykinin-A (PPT-A) mRNA levels in discrete regions of the rat brain. Semiquantitative analysis of silver grains revealed a 16% statistically significant decrease in PPT-A mRNA in the shell of the nucleus accumbens (AcbSh), a 27.6% statistically significant lower level of PPT-A mRNA in the olfactory tubercle (Tu), a 19.2% and 31.5% statistically significant decrease in PPT-A mRNA in the dorsal and ventral caudate–putamen (d-CPu) (v-CPu), respectively, a 30% statistically significant lower expression of PPT-A mRNA in the bed nucleus of the stria terminalis (BNST), a 33.7% statistically significant decrease in PPT-A mRNA in the habenula (Hb) and a 30% statistically significant decrease of PPT-A mRNA levels in the postero-dorsal part of the medial amygdala (MePD). No changes in PPT-A mRNA levels were found in the nucleus accumbens, core (AcbC), in the islands of Calleja (Icj), and in the medial preoptic area (mPOA). These results show that ageing of the central nervous system (CNS) is associated with widespread changes in tachykinin gene expression, suggesting that alteration in the tachykinergic system may have implications in the physio-pathology of the elderly.

A tachykinin NK 1 receptor antagonist, CP-122,721-1, attenuates kainic acid-induced seizure activity

Substance P (SP) can play an important role in neuronal survival. To analyze the role of SP in excitotoxicity, kainic acid (KA) was administered to rats and in situ hybridization was used to analyze the levels of the SP encoding preprotachykinin-A (PPT-A) mRNA in striatal and hippocampal subregions 1, 4, and 24 h and 7 days after KA. In striatum and piriform cortex, PPT-A mRNA peaked 4 h after KA while in hippocampus, levels peaked after 24 h. KA caused seizures and neuronal toxicity as indicated by a reduction of the number of neurons in the hippocampal CA1 subregion after 7 days. KA was later administered alone or following pretreatment with the tachykinin NK 1 receptor antagonist CP-122,721-1 (0.3 mg/kg). The pretreatment decreased seizure activity and a negative correlation was found between seizure activity and survival of CA1 neurons. Conclusively, treatment with CP-122,721-1 has a seizure inhibiting property and may possibly counteract KA-induced nerve cell death in CA1.

Human lymphocytes express substance P and its receptor

We present data demonstrating the gene expression of substance P (SP) and its receptor in human peripheral blood-isolated lymphocytes. Using reverse transcribed polymerase chain reaction (RT-PCR) assay, preprotachykinin-A (substance-P) mRNA is detected in human peripheral blood-isolated lymphocytes. Among the α, β and γ transcripts of the SP gene, only the β and γ transcripts are detectable in these cells. These RT-PCR amplified transcripts are recognized by Southern blot assay using a specific SP probe. Direct DNA sequence analysis of the RT-PCR products from lymphocytes also confirmed the structure of these transcripts which are identical to those found in human neuronal cells. At the protein level, human lymphocytes produced endogenous SP as determined by an enzyme immunoassay. Capsaicin, a vanillyl fatty acid amide (ingredient of hot pepper), released preformed SP from lymphocytes. In addition, using RT/nested-PCR analysis, we identified the presence of mRNA for neurokinin-1 receptor (the receptor for SP) in human peripheral blood-isolated lymphocytes, which was confirmed by Southern blot and DNA sequencing analysis. The demonstration that human lymphocytes express SP and its receptor support the notion that SP is biologically involved in regulating the functions of these cells in an autocrine fashion.

Preprotachykinin-A gene expression in the forebrain of Sardinian alcohol-preferring and -nonpreferring rats

Increasing evidence suggests that TKergic mechanisms might play a role in ethanol intake control. Preprotachykinin-A (PPT-A) mRNA brain levels were measured in Sardinian alcohol-preferring (sP) and Sardinian alcohol-nonpreferring (sNP) rats. PPT-A mRNAs were about 50% lower in sP than in sNP rats in the bed nucleus of the stria terminalis (BNST), whereas levels in the olfactory tubercle (Tu) were about 30% higher in sP than in sNP rats. Our findings suggest that altered PPT-A gene expression might contribute to the different ethanol preference and intake of sP opposite to sNP rats.