Capsaicin-sensitive Afferents Research Articles

Expression of the Somatostatin Receptor Subtype 4 in Intact and Inflamed Pulmonary Tissues

Somatostatin released from capsaicin-sensitive sensory nerves of the lung during endotoxin-induced murine pneumonitis inhibits inflammation and hyperresponsiveness, presumably via somatostatin receptor subtype 4 (sst(4)). The goal of the present study was to identify sst(4) receptors in mouse and human lungs and to reveal its inflammation-induced alterations with real-time quantitative PCR, Western blot, and immunohistochemistry. In non-inflamed mouse and human lungs, mRNA expression and immunolocalization of sst(4) are very similar. They are present on bronchial epithelial, vascular endothelial, and smooth-muscle cells. The sst(4) receptor protein in the mouse lung significantly increases 24 hr after intranasal endotoxin administration as well as in response to 3 months of whole-body cigarette smoke exposure, owing to the infiltrating sst(4)-positive mononuclear cells and neutrophils. In the chronically inflamed human lung, the large number of activated macrophages markedly elevate sst(4) mRNA levels, although there is no change in acute purulent pneumonia, in which granulocytes accumulate. Despite mouse granulocytes, human neutrophils do not show sst(4) immunopositivity. We provide the first evidence for the expression, localization, and inflammation-induced alterations of sst(4) receptors in murine and human lungs. Inasmuch as tissue distribution of this receptor is highly similar, extrapolation of murine experimental results to human conditions might be possible.

Interdisciplinary review for correlation between the plant origin capsaicinoids, non-steroidal antiinflammatory drugs, gastrointestinal mucosal damage and prevention in animals and human beings

The plant origin capsaicinoids (capsaicin, dihydrocapsaicin, norcapsaicin, dihydrocapsaicin, homocapsaicin, homodihydrocapsaicin) are well known and used as nutritional additive agents in the every day nutritional practice from the last 9,500 years; however, we had have a very little scientifically based knowledge on their chemistry, physiology and pharmacology in animal observations, and in humans up to the mid-twentieth century. Our knowledge about their chemistry, physiology, pharmacology entered to be scientifically based evidence from the year 1980, dominantly in animal observations. The human observations with capsaicin (capsaicinoids), in terms of good clinical practice, have been started only in the last 10-year period (from 1997) in randomized, prospective, multiclinical studies. The name of "capsaicin" used only in the physiological and pharmacological research both in animal experiments and in human observation. The "capsaicin" (as a "chemically" used natural compound) modifies the so-called capsaicin-sensitive afferent nerves depending on their applied doses. The specific action of capsaicin (capsaicinoids) on sensory afferent nerves modifying gastrointestinal (GI) function (under very specific conditions) offers a possibility for the production of an orally applicable drug or for other drug combinations, which can be used in the human medical therapy. The production of new drug is based on the critical interdisciplinary review of the results obtained with capsaicinoids. This paper gives an interdisciplinary and critical overview on the chemical, physiological, pharmacological and toxicological actions of the natural origin capsaicinoids (from the point of drug production) under conditions of acute, subacute and chronic administration in animal experiments and human observations, toxicology, pharmacokinetics). This interdisciplinary review covers the following main chapters: (1) physiological and pharmacological research tool by capsaicin in the animals and human beings, (2) capsaicin research in animals (including the acute, subacute toxicology and chronic toxicology metabolism, genotoxicology), (3) capsaicin observation with capsaicin in human beings. (1) The capsaicin used in the physiological and pharmacological observations (in animals and human beings) chemically represents different chemical compounds, which can be obtained from the plants (paprika, chilli, etc.), (2) capsaicinoids are able to modify the capsaicin-sensitive afferent nerves, which have principle roles in the defence of different organs (including the gastrointestinal tract [against the different chemicals, heat, strech, chemical millieu-induced damage], (3) the application of capsaicin (capsaicinoids) can be repeated for the beneficial effects on the gastrointestinal tract as those in animal experiments. After this interdisciplinary and critical review, this paper demonstrates the well-planned research pathways of the discoveries of capsaicinoids from plant chemistry, via physiology, pharmacology and toxicology in animal experiments and human observations.

Selective Pharmacological Blockade of the TRPV1 Receptor Suppresses Sensory Reflexes of the Rodent Bladder

We investigated the pharmacological effect of TRPV1 antagonists in anesthetized rodent models of bladder function. The TRPV1 antagonists JNJ17203212 and JYL1421 were evaluated in the anesthetized rat volume induced micturition reflex model. JNJ17203212 was further evaluated in this model in capsaicin (Sigma) desensitized rats, and in rat capsaicin and mouse citric acid models of irritant induced detrusor overactivity. Systemic JNJ17203212 and JYL1421 administration in the anesthetized rat volume induced micturition reflex model resulted in an increased micturition threshold volume. JNJ17203212 also decreased bladder contraction amplitude but JYL1421 had no effect. Capsaicin desensitization significantly increased baseline micturition threshold volume and decreased bladder contraction amplitude in the volume induced micturition reflex model compared to those in sham treated controls and JNJ17203212 produced no further effect after capsaicin desensitization. JNJ17203212 was also effective in 2 models of irritant induced detrusor overactivity, preventing the decrease in micturition threshold volume and the increase in bladder contraction amplitude observed with intravesical instillation of 10 microM capsaicin, and the decreased voiding interval induced by intravesical citric acid. The TRPV1 antagonists JNJ17203212 and JYL1421 increased the threshold for activation of the micturition reflex in the anesthetized rat volume induced micturition reflex model. This effect appeared to be mediated by capsaicin sensitive afferents. JNJ17203212 also inhibited detrusor overactivity induced by intravesical capsaicin and intravesical citric acid. These data extend our understanding of the role of TRPV1 in sensory modulation of the micturition reflex under nonirritant and inflammatory conditions.

Capsaicin-sensitive sensory nerve fibers contribute to the generation and maintenance of skeletal fracture pain

Although skeletal pain can have a marked impact on a patient's functional status and quality of life, relatively little is known about the specific populations of peripheral nerve fibers that drive non-malignant bone pain. In the present report, neonatal male Sprague–Dawley rats were treated with capsaicin or vehicle and femoral fracture was produced when the animals were young adults (15–16 weeks old). Capsaicin treatment, but not vehicle, resulted in a significant (>70%) depletion in the density of calcitonin-gene related peptide positive (CGRP+) sensory nerve fibers, but not 200 kDa neurofilament H positive (NF200+) sensory nerve fibers in the periosteum. The periosteum is a thin, cellular and fibrous tissue that tightly adheres to the outer surface of all but the articulated surface of bone and appears to play a pivotal role in driving fracture pain. In animals treated with capsaicin, but not vehicle, there was a 50% reduction in the severity, but no change in the time course, of fracture-induced skeletal pain–related behaviors as measured by spontaneous flinching, guarding and weight bearing. These results suggest that both capsaicin-sensitive (primarily CGRP+ C-fibers) and capsaicin-insensitive (primarily NF200+ A-delta fibers) sensory nerve fibers participate in driving skeletal fracture pain. Skeletal pain can be a significant impediment to functional recovery following trauma-induced fracture, osteoporosis-induced fracture and orthopedic surgery procedures such as knee and hip replacement. Understanding the specific populations of sensory nerve fibers that need to be targeted to inhibit the generation and maintenance of skeletal pain may allow the development of more specific mechanism-based therapies that can effectively attenuate acute and chronic skeletal pain.

S1736 EP4 Receptor Blockade Inhibits Epithelial Ion Secretion Induced By PGE2, TNFα and Il1β in Human Colon

Introduction: Increased levels of proand anti-inflammatory cytokines were observed in various segments of histologically intact small intestines in animal models of acute and chronic colitis. We recently demonstrated that these cytokines are produced de novo shortly after the induction of colitis. The trigger for this upregulation is not known. Aim: To study the possible neural involvement in the synthesis of inflammatory cytokines in remote areas from the ulcer site in rat models of colitis. Methods: Colitis was induced by rectal instillation of trinitrobenzenesulphonic acid (TNBS) or iodoacetamide in adult Sprague-Dawley rats. The capsaicin sensitive primary afferents (CSPA) were ablated using subcutaneous injections of capsaicin at time 0 (25mg/kg), 8 and 32 hours (50mg/kg). Using real time PCR, TNF-α and IL-10 mRNA expression was measured in mucosal scrapings of the duodenum, jejunum, ileum and colon at different time intervals after induction of colitis. Results: TNF-α mRNA expression increased by 3-40 times in the different intestinal segments (p<0.05) 48h after iodoacetamide induced colitis. CSPA ablation completely inhibited this upregulation in the small intestine but not in the colon. Similar results were obtained in TNBS induced colitis. Intestinal IL-10 mRNA expression increased by 6-43 times (p<0.01) 48h after iodoacetamide administration. This increase was abolished in rats subjected to CSPA ablation except in the colon where IL-10 further increased by 2 times (p<0.05). In TNBS group, there was respectively a 4-12 and 4-7 folds increase of small intestinal IL-10 mRNA expression at 1 and 21 days after colitis induction (both p<0.01). This increase was not observed in rats pretreated with capsaicin. Both capsaicin treated and untreated rats elicited a similar visual ulcer score after colitis induction. Conclusion: Intestinal CSPA fibers play a key role in the induction of a de novo synthesis of inflammatory cytokines in intestinal segments distant from the site of colitis. The study was supported by the medical practice plan (MPP) and university research board (URB), American University of Beirut-Lebanon.

S1733 The Role of the IL-6/STAT3/SOCS3 Pathway in Ulcerative Colitis Related Carcinogenesis

Introduction: Increased levels of proand anti-inflammatory cytokines were observed in various segments of histologically intact small intestines in animal models of acute and chronic colitis. We recently demonstrated that these cytokines are produced de novo shortly after the induction of colitis. The trigger for this upregulation is not known. Aim: To study the possible neural involvement in the synthesis of inflammatory cytokines in remote areas from the ulcer site in rat models of colitis. Methods: Colitis was induced by rectal instillation of trinitrobenzenesulphonic acid (TNBS) or iodoacetamide in adult Sprague-Dawley rats. The capsaicin sensitive primary afferents (CSPA) were ablated using subcutaneous injections of capsaicin at time 0 (25mg/kg), 8 and 32 hours (50mg/kg). Using real time PCR, TNF-α and IL-10 mRNA expression was measured in mucosal scrapings of the duodenum, jejunum, ileum and colon at different time intervals after induction of colitis. Results: TNF-α mRNA expression increased by 3-40 times in the different intestinal segments (p<0.05) 48h after iodoacetamide induced colitis. CSPA ablation completely inhibited this upregulation in the small intestine but not in the colon. Similar results were obtained in TNBS induced colitis. Intestinal IL-10 mRNA expression increased by 6-43 times (p<0.01) 48h after iodoacetamide administration. This increase was abolished in rats subjected to CSPA ablation except in the colon where IL-10 further increased by 2 times (p<0.05). In TNBS group, there was respectively a 4-12 and 4-7 folds increase of small intestinal IL-10 mRNA expression at 1 and 21 days after colitis induction (both p<0.01). This increase was not observed in rats pretreated with capsaicin. Both capsaicin treated and untreated rats elicited a similar visual ulcer score after colitis induction. Conclusion: Intestinal CSPA fibers play a key role in the induction of a de novo synthesis of inflammatory cytokines in intestinal segments distant from the site of colitis. The study was supported by the medical practice plan (MPP) and university research board (URB), American University of Beirut-Lebanon.

Involvement of capsaicin-sensitive afferent nerves in the proteinase-activated receptor 2–mediated vasodilatation in the rat dura mater

Neurogenic inflammation of the dura mater encephali has been suggested to contribute to the mechanisms of meningeal nociception and blood flow regulation. Recent findings demonstrated that the rat dura mater is innervated by trigeminal capsaicin-sensitive peptidergic nociceptive afferent nerves which mediate meningeal vascular responses through activation of the transient receptor potential vanilloid type 1 (TRPV1) receptor. The present work explored the functional significance of the capsaicin-sensitive subpopulation of dural afferent nerves via their contribution to the meningeal vascular responses evoked through activation of the proteinase-activated receptor 2 (PAR-2). The vascular responses of the dura mater were studied by laser Doppler flowmetry in a rat open cranial window preparation. Topical applications of trypsin, a PAR-2-activator, or Ser–Leu–Ile–Gly–Arg–Leu–amide (SLIGRL-NH 2), a selective PAR-2 agonist peptide, resulted in dose-dependent increases in meningeal blood flow. The SLIGRL-NH 2-induced vasodilatation was significantly reduced following capsaicin-sensitive afferent nerve defunctionalization by prior systemic capsaicin treatment and by pretreatment of the dura mater with the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP 8-37. Nω-nitro- l-arginine methyl ester hydrochloride ( l-NAME) an unspecific inhibitor of nitric oxide (NO) production, but not 1-(2-trifluoromethylphenyl) imidazole (TRIM), a neuronal NO synthase inhibitor, also inhibited the vasodilator response to SLIGRL-NH 2. The vasodilator responses elicited by very low concentrations of capsaicin (10 nM) were significantly enhanced by prior application of SLIGRL-NH 2. The present findings demonstrate that activation of the PAR-2 localized on capsaicin-sensitive trigeminal nociceptive afferent nerves induces vasodilatation in the dural vascular bed by mechanisms involving NO and CGRP release. The results indicate that the PAR-2-mediated activation and sensitization of meningeal capsaicin-sensitive C-fiber nociceptors may be significantly implicated in the pathophysiology of headaches.

Involvement of capsaicin-sensitive afferents and the Transient Receptor Potential Vanilloid 1 Receptor in xylene-induced nocifensive behaviour and inflammation in the mouse

The inflammatory actions of xylene, an aromatic irritant and sensitizing agent, were described to be predominantly neurogenic in the rat, but the mechanism and the role of the Transient Receptor Potential Vanilloid 1 (TRPV1) capsaicin receptor localized on a subpopulation of sensory nerves has not been elucidated. This paper characterizes the involvement of capsaicin-sensitive afferents and the TRPV1 receptor in nociceptive and acute inflammatory effects of xylene in the mouse. Topical application of xylene on the paw induced a short, intensive nocifensive behaviour characterized by paw liftings and shakings, which was more intensive in Balb/c than in C57Bl/6 mice. Genetic deletion of the TRPV1 receptor as well as destroying capsaicin-sensitive nerve terminals with resiniferatoxin (RTX) pretreatment markedly reduced, but did not abolish nocifensive behaviours. In respect to the xylene-induced plasma protein extravasation detected by Evans blue leakage, significant difference was neither observed between the Balb/c and C57Bl/6 strains, nor the ear and the dorsal paw skin. These inflammatory responses were diminished in the RTX pretreated group, but not in the TRPV1 gene-deleted one. Injection of the antioxidant N-acetylcysteine 15 min prior to xylene smearing significantly reduced plasma protein extravasation at both sites. These results demonstrate that xylene-induced acute nocifensive behaviour is mediated by capsaicin-sensitive afferents via TRPV1 receptor activation in mice. Neurogenic inflammatory components play an important role in xylene-induced plasma protein extravasation, but independently of the TRPV1 ion channel. Reactive oxygen or carbonyl species participate in this process presumably via stimulation of the TRPA1 channel.

Cannabinoid Modulation of Perivascular Sympathetic and Sensory Neurotransmission

Cannabinoids are signalling molecules which elicit their vascular effects mainly via G protein-coupled CB(1) receptors and transient receptor potential (TRP) channels (chiefly vanilloid TRPV1 receptors). Cannabinoids can act at prejunctional CB(1) receptors to inhibit perivascular sympathetic neurotransmission. The effects of cannabinoids on perivascular capsaicin-sensitive sensory nerves are more complex. Certain cannabinoids can activate sensory nerves through actions on TRPV1 receptors and other TRP channels, which leads to sensory neurotransmitter release (mainly calcitonin gene-related peptide) and vasorelaxation. However, activation of TRP/TRPV1 channels can also lead to desensitization and loss of sensory nerve activity. Concentration and time of exposure may be critical in determining which of these opposite effects of cannabinoids prevails. In addition, there is evidence for the expression of CB(1) receptors on perivascular capsaicin-sensitive sensory nerves, coupled to inhibition of neurotransmitter release. There is evidence that the archetypal cannabinoid anandamide is released as a neurotransmitter in the central nervous system, and from sensory ganglia, but a release of cannabinoids from perivascular nerves has yet to be clearly demonstrated. Hence, with regard to perivascular nerves, cannabinoids appear to act principally as prejunctional modulators of neurotransmission. The diverse prejunctional effects of cannabinoids may be novel targets for therapies designed to treat vascular disease.

Static magnetic field-induced anti-nociceptive effect and the involvement of capsaicin-sensitive sensory nerves in this mechanism

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Dietary Resistant Starch Increases Hypothalamic POMC Expression in Rats

Resistant starch (RS) is fermentable dietary fiber. Inclusion of RS in the diet causes decreased body fat accumulation and altered gut hormone profile. This study investigates the effect of feeding RS on the neuropeptide messenger RNA (mRNA) expressions in the arcuate nucleus (ARC) of the hypothalamus and whether vagal afferent nerves are involved. The rats were injected intraperitoneally with capsaicin to destroy unmyelinated small vagal afferent nerve fibers. The cholecystokinin (CCK) food suppression test was performed to validate the effectiveness of the capsaicin treatment. Then, capsaicin-treated rats and vehicle-treated rats were subdivided into a control diet or a RS diet group, and fed the corresponding diet for 65 days. At the end of study, body fat, food intake, plasma peptide YY (PYY) and glucagon-like peptide 1 (GLP-1), and hypothalamic pro-opiomelanocortin (POMC), neuropeptide Y (NPY), agouti-related peptide (AgRP) gene expressions were measured. RS-fed rats had decreased body fat, increased POMC expression in the hypothalamic ARC, and elevated plasma PYY and GLP-1 in both the capsaicin and vehicle-treated rats. Hypothalamic NPY and AgRP gene expressions were not changed by RS or capsaicin. Therefore, destruction of the capsaicin-sensitive afferent nerves did not alter the response to RS in rats. These findings suggest that dietary RS might reduce body fat through increasing the hypothalamic POMC expression and vagal afferent nerves are not involved in this process. This is the first study to show that dietary RS can alter hypothalamic POMC expression.

Transient Receptor Potential Ankyrin 1 Mediates Toluene Diisocyanate–Evoked Respiratory Irritation

Toluene diisocyanate (TDI), a reactive, hazardous irritant, causes respiratory symptoms such as cough, rhinitis, dyspnea, and chest tightness in exposed workers. Although previous animal studies have shown that TDI causes respiratory reflexes that are abolished by desensitization of capsaicin-sensitive sensory nerves, the specific molecular identity of the transducer(s) responsible for sensing this noxious stimulus has, to date, remained elusive. Recent studies have demonstrated that transient receptor potential ankyrin 1 (TRPA1), an ion channel largely restricted to a subset of capsaicin-sensitive sensory nerves, functions as a transducer capable of initiating reflex responses to many reactive chemical stimuli. We therefore hypothesized that TRPA1 is the primary molecular transducer through which TDI causes sensory nerve activation and respiratory reflexes. Consistent with this hypothesis, TDI activated TRPA1, but not the capsaicin-sensitive transient receptor potential vanilloid 1 channel, in heterologous expression systems. TDI also activated a subset of dissociated trigeminal sensory neurons from wild-type but not TRPA1-deficient mice. In vivo, TDI mimicked known TRPA1 agonists by causing a pronounced decrease in breathing rate, indicative of respiratory sensory irritation, and this reflex was abolished in TRPA1-deficient mice. Together, our data suggest that TDI causes sensory nerve activation and airway sensory irritation via the activation of the ion channel, TRPA1.

Nitroglycerin Protects Small Intestine from Ischemia–Reperfusion Injury via NO–cGMP Pathway and Upregulation of α-CGRP

IntroductionNitroglycerin (NTG) has been reported to possess preconditioning-like (PCL) protections on heart and other tissues. Our previous studies showed that NTG has acute PCL effects on rat small intestine. The present studies were designed to study whether NTG has delayed PCL protection on rat small intestine and to explore its mechanism(s). MethodsThe intestine lesions were evaluated by histologic examination and serum lactate dehydrogenase (LDH) measurement. The effects of nitric oxide (NO), cGMP, and α-calcitonin gene-related peptide (CGRP) synthesis on the effects of NTG were analyzed. ResultsPretreatment with NTG (0.12 mg/kg i.v.) 24 h before ischemia–reperfusion (I/R) of super mesenteric artery significantly reduced histologic lesions and serum LDH with elevated blood levels of NO and CGRP. Inhibition of guanylate cyclase by methylene blue (30 mg/kg i.p.) or specific depletion of transmitters in capsaicin-sensitive sensory nerve by capsaicin (50 mg/kg s.c.) abrogated the protection conferred by NTG. Reverse-transcription polymerase chain reaction analysis showed that NTG upregulates the expression of α-CGRP messenger RNA (mRNA), but not β-CGRP mRNA in lumbar dorsal root ganglia. ConclusionIn conclusion, NTG prevents rat small intestine from I/R injury by delayed PCL effects 24 h after administration. The protective effects are mediated by NO–cGMP pathway and α-CGRP upregulation.

Excitatory and inhibitory urinary bladder reflexes induced by stimulation of cervicovaginal capsaicin-sensitive sensory fibers in rats

We have investigated the effect of intravaginal application of capsaicin on micturition reflex in female rats. Urinary bladder contractility was measured by transurethral pressure recording at isovolumetric and subthreshold conditions in anaesthetized rats. The intravaginal application of capsaicin (15 microg/50 microl rat) induced reproducible bladder phasic contractions, without desensitization upon repeated applications, that were blocked by intravenous atropine (1 mg/kg) or hexamethonium (5 mg/kg) and prevented by removal of paracervical ganglia or systemic capsaicin pretreatment (125 mg/kg, s.c.). The inhibition of sympathetic transmission by guanethidine (30 mg/kg, s.c.) produced significant increase of the bladder reflex contractions activated by intravaginal capsaicin. Intravenous administration of the TRPV1 antagonist, capsazepine (3 mg/kg), significantly reduced the excitatory reflex response to capsaicin. Intravaginal administration of capsaicin (15 microg/50 microl), during distension-induced reflex bladder contractions, produced a transient block of reflexes, unaffected by guanethidine pretreatment. In conclusion, the stimulation of capsaicin-sensitive sensory nerve endings in the rat cervix-vagina induced a dual excitatory or inhibitory bladder response in anaesthetized female rats depending on the degree of bladder distension.

Alteration of cholinergic, purinergic and sensory neurotransmission in the mouse colon of food allergy model

It is well known that intestinal anaphylaxis results in a disturbed intestinal motility. It is hypothesized that the chronic intestinal anaphylaxis-induced changes in the enteric neuronal circuitry cause intestinal motor malfunctions. However, detailed mechanisms largely remain unclear. The aim of this study was to investigate the pathophysiological role of ATP, which acts as a non-cholinergic neurotransmitter and a neuroimmune modulator, in a disturbed intestinal motility of food allergy (FA). The FA mice developed allergic diarrhea accompanied with chronic inflammation and mast cell hyperplasia in the colon. The excised proximal colons (PCs) were suspended in the longitudinal direction in organ baths. In the PCs precontracted by KCl (50 mM), contractile responses to exogenous ATP (1 mM) were significantly ( P < 0.01) higher in FA mice (34.2% of KCl-induced precontractions) as compared to control mice (17.2%). Pretreatment with P2 purinoceptor antagonists [suramin and PPADs] significantly ( P < 0.01) reduced the ATP-evoked contractions to 7.7% and 1.5% in FA and control PCs, respectively. Furthermore, in the presence of inhibitors of cholinergic nerves and capsaicin-sensitive sensory nerves the electrical field stimulation (EFS; 10 Hz)-evoked contractions were significantly ( P < 0.05) higher in FA mice (65.8% of EFS-evoked maximum contractions, n = 6) than those in control mice (47.9%, n = 6). In addition, cumulative application of suramin and PPADs further inhibited EFS-induced contractions by 21.7% in FA mice ( n = 6, P < 0.01) and 8.7% in control mice ( n = 6, P < 0.05). Thus, the present study suggests that the sustained alteration in cholinergic, purinergic and sensory neurotransmission contribute to the disturbed motility during the chronic intestinal anaphylaxis.

Decrease in the synthesis and release of calcitonin gene-related peptide in dorsal root ganglia of spontaneously hypertensive rat: Role of nitric oxide synthase inhibitors

Calcitonin gene-related peptide (CGRP), the major transmitter in capsaicin-sensitive sensory nerves, and asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, participate in the regulation of blood pressure. In the present study, we tested the relationship between CGRP and ADMA in spontaneously hypertensive rats (SHRs). For the in vivo study, SHRs were treated with or without l-arginine for 2 weeks, and Wistar Kyoto (WKY) rats were used as controls. Systolic arterial pressure was measured, and the levels of CGRP, ADMA, and NO were analyzed. For the in vitro study, neural cells from dorsal root ganglia were treated with NO inhibitor or donor. Synthesis and release of CGRP were measured. Compared with WKY rats, serum concentration of ADMA in SHRs increased while CGRP and NO level decreased. Treatment with l-arginine significantly decreased blood pressure, concomitantly with an increase in the level of NO and the synthesis and release of CGRP in SHRs, but it did not affect ADMA levels. In cultured neural cells, ADMA reduced the level of NO and inhibited the synthesis and release of CGRP in a concentration-dependent manner. The effects of ADMA were reversed by l-arginine. Treatment with NOC-18, a donor of NO, increased the release and synthesis of CGRP in neural cells in a concentration-dependent manner. Decreased synthesis and release of CGRP is related to a reduction in NO levels, and corresponds to the increased concentrations of ADMA in spontaneously hypertensive rats.

Association of CALCA genetic polymorphism with essential hypertension

Calcitonin gene-related peptide (CGRP) is the predominant neurotransmitter in capsaicin-sensitive sensory nerves. Participation of CGRP in hypertension is one of the most extensively studied topics in the field. There is growing evidence to the effect that CGRP is associated with essential hypertension (EH). The aims of this study were to pinpoint whether single nucleotide polymorphisms (SNPs) in the genes coding for CALCA were associated with EH susceptibility in a Hunan Han population. A total of 293 subjects with EH and 208 controls were enrolled in the study. Genomic DNA was extracted from peripheral blood leucocytes by a phenol-chloroform method. The CALCA T-692C was genotyped using a restriction fragment length polymorphism method. A statistically significant difference in CALCA T-692C genotypic distribution was observed between cases and controls (P=0.001). Moreover, the frequencies of the C allele were 14.85% in the EH group and 7.45% in the control group, prevalence of C alleles in EH subjects and controls was significantly incomparable (P<0.001). Furthermore, the results of Logistic regression analysis showed that the carriers of C allele (TC+CC genotypes) were associated with increased EH risk (OR=2.093, 95% CI: 1.317-3.326, P<0.01). CALCA genetic polymorphism is associated with EH susceptibility. Carriers of at least one C allele at the polymorphic site CALCA T-692C showed increased risk for EH.

Agonistic Behavior of PACAP6-38 on Sensory Nerve Terminals and Cytotrophoblast Cells

The effects of pituitary adenylate cyclase activating polypeptide (PACAP) are mediated through G-protein-coupled receptors, the specific PAC1 receptor and VPAC1 and VPAC2 receptors which bind vasoactive intestinal peptide with similar affinity. Based on binding affinity studies, PACAP6-38 was discovered as a potent antagonist of PAC1 and it has been used by hundreds of studies as a PACAP antagonist. Recently, we have found that in certain cells/tissues, PACAP6-38 does not antagonize PACAP-induced effects, but surprisingly, it exerts similar actions to PACAP1-38, behaving as an agonist. In the present study, we report on the agonistic behavior of PACAP6-38 on neuropeptide release from sensory nerves of the isolated rat trachea and on the MAPK signaling pathways in cytotrophoblast cells. In isolated rat tracheae, PACAP6-38, similarly to PACAP1-38, induced significant inhibitory effects on the release of three simultaneously measured sensory neuropeptides, substance P, calcitonin gene-related peptide, and somatostatin evoked by both chemical excitation and electrical field stimulation of capsaicin-sensitive afferents. Effects of PACAP6-38 were the same as those of PACAP1-38 on MAPK signaling in human cytotrophoblast cells. Western blot analysis showed that both peptide forms stimulated ERK1/2 and JNK phosphorylation, while they both inhibited p38 MAPK phosphorylation. The most pronounced effects were observed when both peptides were present. In summary, our results show that PACAP6-38, which is a PACAP receptor antagonist in most cells/tissues, can behave as an agonist in other systems. The increasing interest in the effects of PACAP requires further studies on the pharmacological properties of the peptide and its analogues.

Modulation of Capsaicin-Sensitive Nerve Activation by Low pH Solutions in Guinea-Pig Lung

We have studied the stimulation of airways sensory nerves by low pH solutions and concomitantly induced bronchoconstriction. The effect of low pH buffer and lactic acid solutions at the same pH (5 and 6) were compared and the influence of low pH on the capsaicin effect was recorded. We have used the isolated guinea-pig perfused lung model taking the insufflation pressure as an indicator of bronchial smooth muscle tone while the calcitonin gene-related peptide-like immunoreactivity measured in the lung perfusate represented sensory nerves activation. Low pH buffer and lactic acid solution (3 and 4.1 mM) at the same pH of 5 and 6 induced pH-dependent bronchoconstriction and peptides release which were completely abolished after systemic pretreatment with capsaicin. Both responses were significantly inhibited after Ca2+-free infusion. Capsazepine (10(-6) M), a selective capsaicin antagonist, significantly reduced the calcitonin gene-related peptide-like immunoreactivity overflow evoked by all the solutions studied. Diclofenac (10(-5) M), a cyclooxygenase blocker, inhibited pH 5, pH 6 and lactic acid 3 mM (pH 6)-evoked peptide release, but not lactic acid 4.1 mM (pH 5). The functional response was not significantly modified after diclofenac while only the lactic acid 3 mM response was significantly reduced by capsazepine. There was a synergistic interaction between capsaicin and low pH buffer on calcitonin gene-related peptide-like immunoreactivity release and an additive effect on bronchoconstriction. It is concluded that in the isolated perfused guinea-pig lung, lactic acid and low pH buffer induced calcitonin gene-related peptide-like immunoreactivity release and bronchoconstriction by stimulation of capsaicin-sensitive C fibres via a pathway partly dependent of extracellular Ca2+. The mechanism of calcitonin gene-related peptide-like immunoreactivity release seems to be the same at pH 6, while differences are evident at pH 5 between low pH buffer and lactic acid. Our results also suggest that proton activity could exert a modulatory role on the capsaicin-sensitive sensory nerves by a mechanism which remains to be clarified.

61 Visceral Hypersensitivity and Altered 5-HT Dynamics in the Intestine of Rats with a Loss of Function Mutation in the Serotonin Transporter

capsaicin-sensitive afferents was clearly demonstrated, as capsaicin pretreatment prevented the development of cross-sensitization and the associated bladder mastocytosis. Furthermore, acute bladder afferent sensitization was noted at 1 hr, while sub-acutely, the degree of bladder mast cell migration was directly and temporally associated with increases in bladder permeability and decreases in bladder voiding intervals (irritative voiding). A causal role of mast cells could not be established. We hypothesize that pelvic organ mast cell infiltration, mediator release, and consequential afferent modulation are the primary mechanisms responsible for chronic pelvic organ sensitization. Methods: Intra-colonic TNBS (or vehicle) was administered to mast cell deficient mice (W/Wv) and wild type (WT) littermates. Eight days later, colonic and bladder mast cells were quantified, and under urethane anesthesia, bladder voiding intervals and permeability were measured using saline cystometry and fluorescein, respectively. Animal weights were recorded daily throughout the experiment. Results: Animal weights were decreased following intra-colonic TNBS beginning as early as day 2 and peaking at 15-20% on day 4 (p<0.05). By day 6, weights had normalized and were no different from untreated controls. Histologic colitis paralleled decreases in weight and was no different in the c-kit deficient mice as compared to WT whose mast cell counts were elevated three-fold (p<0.05). Colonic mast cell counts in W/Wv mice were significantly less at baseline compared to WT mice, and there was no change following TNBS. In the bladder, mast cell responses paralleled those of the colon despite the lack of any acute inflammatory changes. Voiding intervals were decreased by 33% in TNBS-treated WT mice (p<0.05) (irritative voiding) but were unchanged inW/Wvmice. Likewise, bladder permeability was increased 4-fold (p<0.05) in TNBS-treated WT mice but unchanged in W/Wv mice. Conclusion: Mast cells and afferent nerves both play important roles in the development and maintenance of pelvic organ sensitization. Although afferent nerves and neuropeptide release are likely response for the acute effects, it would appear that the mast cells and their effects play an important role in maintaining pelvic organ sensitization.