iCGRP Release Research Articles

Nerve growth factor treatment enhances release of immunoreactive calcitonin gene-related peptide but not substance P from spinal dorsal horn slices in rats

The effect of systemic nerve growth factor (NGF) on neuropeptide content and capsaicin-evoked release of neuropeptide from in vitro spinal cord dorsal horn slices was examined. Rats were injected subcutaneously every other day with murine NGF (mNGF) 1 mg/kg or saline for 7 days, or mNGF 0.1/kg, mNGF 1 mg/kg or saline for 13 days. Lumbar dorsal horn slices of the rat spinal cord from all groups showed a significant increase in immunoreactive calcitonin gene-related peptide (CGRP) release upon exposure to capsaicin. This release was enhanced in rats pretreated with mNGF 1 mg/kg for 7 days, but not after 13 days. No enhancement was seen after 7 or 13 days in any treatment group for immunoreactive substance P release. Upon examination of neuropeptide content in dorsal horn, no significant differences were noted between treatment groups. The increased iCGRP release from dorsal horn slices suggests a preferential release of CGRP and provides further evidence that NGF indirectly plays a role in the modulation of inflammation through the regulation of neuropeptide release.

Why cooling is beneficial: non-linear temperature-dependency of stimulated iCGRP release from isolated rat skin

The capsaicin receptor in nociceptive neurons is a target for the sensitizing actions of algogenic inflammatory mediators. Capsaicin and potential endogenous ligands are thought not to gate this heat-activated ion channel but to sensitize it so profoundly that even room temperature can open it. We investigated the temperature dependency of capsaicin-induced CGRP release from nociceptive nerve fibers in isolated rat skin over a range of ambient temperatures using different agonist concentrations (10 −7–10 −5 M) and KCl (60 mM) for control. Ambient temperature (4–40 °C) showed no significant influence on the basal iCGRP outflow. The supramaximal capsaicin concentration of 10 −6 M as a stimulus evoked a response that was not significantly diminished by temperatures decreasing from 40 to 24 °C but lost 65% of its amplitude between 24 and 14 °C (Q 10∼6.7). Such a collapse of the response occurred between 40 and 32 °C at lower capsaicin concentration (10 −7 M). The concentration-response curves showed a rightward shift upon cooling from 40 to 24 °C and a major loss of slope and maximum effect at 14 °C which formally describes a noncompetitive antagonism. KCl-induced iCGRP release showed a much more linear temperature dependency (Q 10∼2.4 between 24 and 14 °C). Significant capsaicin responses even at 8 °C suggest a contribution of noxious-cold sensitive neurons known to coexpress CGRP and the capsaicin receptor. The heat-activated ion channels (TRPV1-4) are thought to play a significant role in inflammatory pain which is effectively relieved by cooling. The present results contribute to understanding this phenomenon.

Effects of TRPV1 receptor antagonists on stimulated iCGRP release from isolated skin of rats and TRPV1 mutant mice

Capsaicin antagonists including ruthenium red, capsazepine and iodo-resiniferatoxin (I-RTX) have recently been shown to inhibit the activation by noxious heat of the capsaicin receptor (TRPV1) expressed in non-neuronal host cells, and natively, in cultured dorsal root ganglion cells. Noxious heat has been shown to release immunoreactive calcitonin gene-related peptide (iCGRP) from the isolated rat skin. In this model, ruthenium red, I-RTX as well as capsazepine 10 μM caused no alteration in iCGRP release at 32 °C by themselves whereas capsazepine 100 μM doubled it reversibly. In wild-type mice 100 μM capsazepine also stimulated iCGRP release while it was without effect in TRPV1 knockout littermates. In the rat skin, both ruthenium red and capsazepine (10/100 μM) reduced and abolished, respectively, capsaicin-induced iCGRP release while I-RTX (1/10 μM) was ineffective. Only ruthenium red 100 μM showed an unspecific effect inhibiting iCGRP release induced by KCl. Ruthenium red and capsazepine (10/100 μM) caused no significant alteration of iCGRP release induced by heat stimulation at 47 °C. Employing 45 °C stimulation intensity, capsazepine and I-RTX (in the higher concentrations) showed a significant facilitatory effect on the heat response suggesting a partial agonistic action of the compounds. It is concluded that noxious heat-induced iCGRP release in the isolated rat skin occurs through a mechanism that is not inhibited by TRPV1 antagonism reflecting a different pharmacological profile of noxious heat transduction in terminals of sensory neurons compared to that in cultured cell bodies and TRPV1-transfected host cells.

Potentiation of evoked calcitonin gene-related peptide release from oral mucosa: a potential basis for the pro-inflammatory effects of nicotine.

Inflammation of the buccal mucosa, gingiva and periodontal tissues is a significant problem in users of nicotine-containing tobacco products; however, the potential role of nicotine in the development of this inflammation is unclear. In many tissues, nicotine, acting through nicotinic acetylcholine receptors (nAChRs), has been shown to increase the release of the pro-inflammatory mediator calcitonin gene-related peptide (CGRP) thereby potentially contributing to neurogenic inflammation. The purpose of the present studies was to determine the effects of nicotine and other nAChR agonists on capsaicin-evoked immunoreactive CGRP (iCGRP) release from rat buccal mucosa and to identify a potential cellular basis for these effects. Using a previously validated model of in vitro superfusion, we show that the nAChR agonists nicotine (EC50 557 micro m), epibatidine (EC50 317 pm) and cytisine (EC50 4.83 nm) potentiated capsaicin-evoked iCGRP release in a concentration-dependent manner by 123, 70 and 76%, respectively. The expression and distribution patterns of the mRNA transcripts encoding the alpha3, alpha4 and alpha6 nAChR subunits and their colocalization with CGRP and the capsaicin receptor VR1 were examined in rat trigeminal ganglion using combined in situ hybridization and immunohistofluorescence. Of all trigeminal neurons counted, mRNA encoding the alpha3, alpha4 and alpha6 subunits was found, respectively, in 14.45, 9.2 and 19.21% of neurons. The cell body diameter of most neurons containing any nAChR subunit was in the 30-40 micro m range with slightly fewer in the 20-30 micro m range. Co-localization of these alpha subunit transcripts with either CGRP or VR1 immunoreactivity ranged from approximately 5 to 7% for alpha4 and over 8% for alpha3 to 18% for alpha6. These data support the hypothesis that nicotinic agents, acting at nAChRs contained on primary sensory neurons, are capable of directly modulating the stimulated release of iCGRP. In the case of users of nicotine-containing tobacco products, this modulation could contribute to inflammatory processes within the oral cavity.

Intrinsic regulation of CGRP release by dental pulp sympathetic fibers.

Neurotransmission from sympathetic and peptidergic afferent fibers participates in the regulation of pulpal blood flow (PBF) via opposing effects. In this study, we directly tested the hypothesis that activation of pulpal sympathetic terminals inhibits exocytosis of immunoreactive calcitonin gene-related peptide (iCGRP) from peptidergic afferents innervating bovine dental pulp. The results demonstrate that norepinephrine inhibits capsaicin-evoked iCGRP release. The application of alpha-adrenergic antagonists (phentolamine or phenoxybenzamine) increased spontaneous release of iCGRP. Moreover, administration of agents that evoke the release of sympathetic neurotransmitters (guanethidine or reserpine) inhibited capsaicin-evoked iCGRP release. Collectively, these results indicate that sympathetic neurotransmission inhibits exocytosis from pulpal peptidergic afferent fibers. Analysis of these data supports the hypothesis that peripheral sympathetic vasomotor control may operate by a direct mechanism (vasoconstriction) as well as by an indirect mechanism (e.g., inhibition of exocytosis from afferent fibers). Since capsaicin-sensitive neurons are nociceptors, it is possible that certain sympathetic neurotransmission may modulate pain.

S(+)-flurbiprofen but not 5-HT 1 agonists suppress basal and stimulated CGRP and PGE 2 release from isolated rat dura mater

Neurogenic inflammation of the meninges, expressed in plasma extravasation and vasodilatation, putatively contributes to certain types of headache. Both, non-steroidal antiinflammatory drugs (NSAIDs) and serotonin-1 (5-HT 1) receptor agonists are similarly effective antimigraine drugs but their mechanism of action is unclear. The clinical observation that sumatriptan lowered plasma levels of calcitonin gene-related peptide (CGRP), found increased during migraine attacks, drew attention to a possible inhibition of pro-inflammatory neuropeptide release from trigeminal afferents. An isolated preparation of fluid-filled rat skull cavities was used to study effects of NSAIDs and 5-HT 1B/D agonists on the dura stimulated by inflammatory mediators (bradykinin, histamine and serotonin, 10 −5 M each). The release of immunoreactive CGRP (iCGRP) and immunoreactive PGE 2 (iPGE 2) was measured in 5-min samples of superfusates using enzyme immunoassays. S(+)-flurbiprofen (10 −6 M) strongly reduced the basal and stimulated iCGRP release and abolished iPGE 2 release; R(−)-flurbiprofen showed much less effect on iPGE 2 liberation and did not influence iCGRP release. The 5-HT 1B/D agonists naratriptan and CP93,129 were ineffective on both iCGRP and iPGE 2 release. Inspite of its weak COX blocking effect, R(−)-flurbiprofen is reported to exert antinociceptive effects, although it has not been tested in migraine. Only the potent COX blocker S(+)-flurbiprofen also suppressed iCGRP release while the 5-HT 1B/D agonists were ineffective. Thus, inhibition of meningeal neuropeptide secretion is not a common action principle of the drugs that could be essential for their antimigraine effects.

Beta 2-Adrenoceptor regulation of CGRP release from capsaicin-sensitive neurons.

Previous studies have suggested that neurotransmitter substances from the sympatho-adrenomedullary system regulate pulpal blood flow (PBF), in part, by the inhibition of vasoactive neuropeptide release from pulpal sensory neurons. However, no study has evaluated the role of beta-adrenoceptors. We evaluated the hypothesis that activation of beta-adrenoceptors inhibits immunoreactive calcitonin gene-related peptide (iCGRP) release from capsaicin-sensitive nociceptive neurons via in vitro superfusion of bovine dental pulp. Either norepinephrine or epinephrine inhibited capsaicin-evoked iCGRP. The norepinephrine effect was blocked by the selective beta(2)-adrenoceptor antagonist, ICI 118,551, but not by pre-treatment with the selective beta(1)-adrenoceptor antagonist, atenolol. In addition, application of albuterol, a selective beta(2)-adrenoceptor agonist, significantly blocked capsaicin-evoked release of iCGRP. Collectively, these studies demonstrate that activation of beta(2)-adrenoceptors in dental pulp significantly reduces exocytosis of neuropeptides from capsaicin-sensitive nociceptors. This effect may have physiologic significance in regulating PBF. Moreover, since capsaicin selectively activates nociceptors, beta(2)-adrenoceptor agonists may have clinical utility as peripherally acting therapeutics for dental pain and inflammation.

ATP can enhance the proton-induced CGRP release through P2Y receptors and secondary PGE 2 release in isolated rat dura mater

Trigeminal afferent neurons express ionotropic P2X receptors for extracellular ATP which are known to be sensitive to low interstitial pH. Both conditions – ATP release and tissue acidosis – may occur in the dura following the ischemia phase of migraine attacks. Aim of this study was to investigate whether and how ATP and protons may cooperate in exciting meningeal afferents. After removal of the cerebral hemispheres hemisected scull cavities of adult Wistar rats were used as organ bath of their own lining, the dura mater. The dura was chemically stimulated and the amounts of immunoreactive calcitonin gene-related peptide (iCGRP) and prostaglandin E 2 (PGE 2) released into incubation fluid were measured using enzyme immunoassays. Stimulation with ATP (10 −4 and 10 −3 M) augmented iPGE 2 release dose-dependently whereas iCGRP secretion was minimally enhanced only if the dura had previously been depleted of extracellular ATP using hexokinase. Acid buffer solutions (pH 5.9 and 5.4) resulted in pH-dependent increase of iCGRP release but reduced iPGE 2 release. Purines (ATP 10 −3>UTP 10 −4 M>ATP 10 −4 M) and PGE 2 (10 −5 M) were found to facilitate the proton-induced increase in iCGRP release. The proton-reduction of PGE 2 release was overcome by adding ATP (10 −3 M). S(+)-flurbiprofen (10 −6 M) suppressed both the basal and stimulated iPGE 2 release and prevented the ATP(10 −4 M)-induced facilitation of the proton response. The facilitating effect of ATP was also blocked under suramin, a non-selective P2 antagonist, and under reactive blue, an non-selective P2Y-antagonist, but not under pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid, a P2X-antagonist. The present results provide evidence that ATP has poor, if at all, direct excitatory effects on CGRP-containing trigeminal nerve endings in the isolated dura and its facilitatory action seems to depend on G-protein coupled P2Y receptors and secondary PGE 2 release. The UTP effect and the antagonist profile is indicative for the P2Y 2 receptor subtype.

Capsaicin-evoked CGRP release from rat buccal mucosa: development of a model system for studying trigeminal mechanisms of neurogenic inflammation.

Many of the physiological hallmarks associated with neurogenic inflammatory processes in cutaneous tissues are similarly present within orofacial structures. Such attributes include the dependence upon capsaicin-sensitive sensory neurons and the involvement of certain inflammatory mediators derived therein, including calcitonin gene-related peptide (CGRP). However, there are also important differences between the trigeminal and spinal nervous systems, and the potential contributions of neurogenic processes to inflammatory disease within the trigeminal system have yet to be fully elucidated. We present here a model system that affords the ability to study mechanisms regulating the efferent functions of peptidergic terminals that may subserve neurogenic inflammation within the oral cavity. Freshly dissected buccal mucosa tissue from adult, male, Sprague-Dawley rats was placed into chambers and superfused with oxygenated, Krebs buffer. Serial aliquots of the egressing superfusate were acquired and analysed by radioimmunoassay for immunoreactive CGRP (iCGRP). Addition of the selective excitotoxin, capsaicin (10-300 microm), to the superfusion buffer resulted in a significant, concentration-dependent increase in superfusate levels of iCGRP. Similarly, release of iCGRP from the buccal mucosa could also be evoked by a depolarizing concentration of potassium chloride (50 mm) or by the calcium ionophore A23187 (1 microm). The specific, capsaicin receptor antagonist, capsazepine (300 microm), completely abolished the capsaicin-evoked release of iCGRP while having no effect whatsoever on the potassium-evoked release. Moreover, capsaicin-evoked release was dependent upon the presence of extracellular calcium ions and was significantly, though incompletely, attenuated by neonatal capsaicin denervation. Collectively, these data indicate that the evoked neurosecretion of iCGRP in response to capsaicin occurs via a vanilloid receptor-mediated, exocytotic mechanism. The model system described here should greatly facilitate future investigations designed to identify and characterize the stimuli that regulate the release of CGRP or other neurosecretory substances in isolated tissues. This system may also be used to elucidate the role of these mediators in the aetiology of inflammatory processes within the trigeminal field of innervation.

Capsaicin-evoked release of immunoreactive calcitonin gene-related peptide from rat trigeminal ganglion: evidence for intraganglionic neurotransmission

Chemically-mediated cross-excitation has been described between neurons within sensory ganglia. However, the identity and source of the chemical mediators is not known. Ca 2+-dependent release of neurotransmitters from cultured sensory neurons in vitro has been observed, although neurite outgrowth may confound the ability to extrapolate findings from culture systems to in vivo conditions. Thus, the present studies evaluate the hypothesis of capsaicin-sensitive intraganglionic neuropeptide release from freshly prepared slices of rat sensory ganglia. The ganglionic slice preparation provides an advantage over neuronal cultures, because release may be assessed within minutes after tissue collection (minimizing phenotypic changes) and while maintaining gross anatomical relationships. Trigeminal ganglia (TGG) were quickly removed from male, Sprague–Dawley rats (175–200 g), chopped into 200 μm slices and placed into chambers within 3 min of collection. Chambers were perfused with buffer, and superfusates were collected and assayed for immunoreactive calcitonin gene-related peptide (iCGRP) release via radioimmunoassay. After about 90 min of baseline collection, tissue was treated with capsaicin followed by a washout period. Capsaicin (1–100 μM) evoked concentration-dependent increases in iCGRP release. A competitive capsaicin receptor antagonist, capsazepine, significantly inhibited capsaicin-evoked release of iCGRP. In addition, capsaicin-evoked release of iCGRP was dependent on the presence of extracellular calcium. Furthermore, capsaicin-evoked release from TGG slices was significantly greater than that from slices of equivalent weights of adjacent trigeminal nerve shown histologically to be free of neuronal somata. These data support the hypothesis that Ca 2+-dependent exocytosis of neuropeptides may occur within the TGG in vivo and that the majority of this release derives from neuronal somata.

Prostaglandin E2 enhances bradykinin-evoked iCGRP release in bovine dental pulp.

Mediators produced during inflammation are responsible for hyperalgesia and expression of neurotransmitters and receptors in the nervous system. The production of bradykinin (BK) and the prostaglandins (PGs) may regulate initiation of pain. This study tested the hypothesis that BK and prostaglandin E2 (PGE2) have a positive interaction in evoking neurosecretion of immunoreactive calcitonin gene-related peptide (iCGRP). Bovine dental pulp was prepared and stimulated by the superfusion method with BK alone and in combination with PGE2. Kinin receptor antagonists to bradykinin-evoked release of iCGRP were also tested. Also tested was the hypothesis that dental pulp contains either the B1 or B2 or both BK receptors. Results showed that PGE2 enhanced BK-evoked iCGRP release by more than 50%. Western immunoblots revealed detectable B2 receptor protein with no detectable B1 receptor protein. We conclude that BK evokes iCGRP release from bovine dental pulp which is enhanced by a positive interaction with PGE2. Neurosecretion is evoked from isolated terminals of dental pulp fibers via the bradykinin B2 receptor-dependent mechanism.

Prostaglandin E2-mediated sensitization of rat sensory neurons is not altered by nerve growth factor

To ascertain whether chronic exposure to nerve growth factor (NGF) alters the responsiveness of sensory neurons to prostaglandin E2 (PGE2), sensory neurons taken from adult rats were grown in culture in the presence or absence of NGF for 7 days. Neurons then were exposed to PGE2 and release of immunoreactive calcitonin gene-related peptide (iCGRP) and production of immunoreactive cAMP (icAMP) were examined. Growing neurons in the presence of 250 ng/ml NGF increased the content and the release of iCGRP from sensory neurons. Independent of NGF treatment, exposure to 100 nM PGE2 augmented capsaicin- or potassium-stimulated release of iCGRP by 1.5-fold compared with cells not exposed to PGE2. In a similar manner, NGF treatment did not alter the ability of PGE2 to increase the content of icAMP. These data suggest that prostaglandin-induced sensitization of sensory neurons is not influenced by NGF.

Capsaicin-evoked release of immunoreactive calcitonin gene-related peptide from the spinal cord is mediated by nitric oxide but not by cyclic GMP

Recent data support a role for nitric oxide (NO) in pain processing at the level of the spinal cord, possibly via regulation of neuropeptide release. The goal of this study was to determine whether capsaicin, which selectively activates primary afferent neurons and evokes neuropeptide release, acts in an NO-dependent manner. Our results indicate that capsaicin (1 μM)-evoked release of immunoreactive calcitonin gene-related peptide (iCGRP) is significantly reduced in the presence of the NO synthase inhibitor, l-NAME (10–400 nM; F 3,45=68.38; P<0.001) and, the selective nNOS inhibitor, 3-bromo-7-nitroindazole (170–680 nM; F 5,48=56.2; P<0.01). d-NAME (200 nM) had no effect on capsaicin-evoked iCGRP release. Hemoglobin (an extracellular scavenger of NO; 3 mg/ml) significantly reduced the effect of capsaicin on the release of iCGRP ( F 1,8=9.12; P<0.05). The NOS substrate, l-arginine, effectively reversed the inhibitory effect of 3-bromo-7-nitroindazole on capsaicin-evoked iCGRP release. To determine whether the NO-mediated release was NMDA-driven, we superfused spinal cord slices with competitive and non-competitive NMDA antagonists in the presence and absence of capsaicin. MK-801 (0.1–10 μM; F 4,33=8.49; P<0.0001) and AP-5 (0.01–10 μM; F 4,38=3.34; P<0.05) reduced capsaicin-evoked iCGRP release. CNQX, an AMPA/kainate antagonist (10 nM–10 μM), significantly decreased capsaicin-evoked release of iCGRP ( F 6,42=8.76; P<0.01) in a dose-dependent fashion. Additionally, our results demonstrate that while capsaicin-evoked release is significantly reduced in the presence of LY-83583 (10 μM; F 2,18=3.46; P<0.01; a cyclic GMP lowering agent), there is no effect of ODQ (a potent and selective inhibitor of guanylate cyclase). Moreover, the application of a cell permeable analog of cyclic GMP (8-bromo-cGMP; 0.01–1000 μM) is without effect on both basal and evoked iCGRP release. Finally, we observed no colocalization of immunoreactive neuronal NOS (nNOS) with CGRP in the dorsal horn. In summary, these data indicate that capsaicin evokes the release of iCGRP, in part, via the production of NO which enters the extracellular space prior to having an effect. Moreover, iCGRP and nNOS are produced in distinct populations of neurons within the dorsal horn. We conclude that capsaicin-evoked release involves the activation of the NMDA receptor but is also modified by the activation of AMPA or kainate receptors. Finally, these data suggest that while capsaicin-evoked iCGRP release is modified by NO, this release does not require the activation of guanylate cyclase and subsequent production of cyclic GMP.

OR 54 Tissue cooling reduces BK/PGE 2 release of iCGRP in rat dental pulp

OR 54 Tissue cooling reduces BK/PGE 2 release of iCGRP in rat dental pulp

Activation of excitatory amino acid receptors in bovine dental pulp evokes the release of iCGRP.

The activation of excitatory amino acid (EAA) receptors within the central nervous system is associated with numerous centrally mediated phenomena, including hyperalgesia. However, relatively little is known about the peripheral mechanisms which these receptors may regulate when activated. This research evaluated the hypothesis that EAA receptors in bovine dental pulp activate a population of peptidergic sensory neurons as measured by the release of immunoreactive calcitonin gene-related peptide (iCGRP), a neuropeptide associated with neurogenic inflammation. In vitro superfusion of bovine dental pulp was used to evaluate the regulation of iCGRP secretion by the EAA receptor agonists AMPA, kainate, NMDA, and L-glutamate. Both AMPA and kainate stimulated the release of iCGRP in a concentration-dependent manner (AMPA EC50 = 0.27 +/- 3.3 nM; kainate EC50 = 3.2 +/- 1.1 microM). Pre-treatment and co-administration of the AMPA/kainate receptor antagonist CNQX significantly reduced the iCGRP release evoked by either of these agonists. In contrast, neither NMDA nor L-glutamate induced any consistent changes in iCGRP release. These results suggest that the activation of AMPA and kainate receptors in dental pulp may contribute to peripheral release of vasoactive neuropeptides which mediate a neurogenic component of inflammation.

α2-Adrenergic receptor activation inhibits calcitonin gene-related peptide expression in cultured dorsal root ganglia neurons

Calcitonin gene-related peptide (CGRP), a potent vasodilator, is produced in dorsal root ganglia (DRG) neurons which extend nerves peripherally to blood vessels and centrally to the spinal cord. We previously reported that neuronal CGRP expression is significantly reduced in the spontaneously hypertensive rat (SHR) which could contribute to the elevated BP. Other studies suggest that the enhanced activity of the sympathetic nervous system in the SHR may mediate, at least in part, this reduction in neuronal CGRP expression via activation of α 2-adrenoreceptors ( α 2-AR) on DRG neurons. To test this hypothesis in vitro we employed primary cultures of adult rat DRG neurons. Neuronal cultures were initially exposed (24 h) to either the α 2-AR agonist UK 14,304 (10 −6 M) or vehicle; however, no changes in CGRP mRNA content or immunoreactive CGRP (iCGRP) release were observed. Using the rationale that in vivo DRG neurons receive a continuous supply of target tissue derived nerve growth factor (NGF), which stimulates CGRP synthesis, the cultured neurons were treated (24 h) with either vehicle, NGF (25 ng/ml) alone, or NGF plus UK. NGF treatment increased CGRP mRNA accumulation 5.5±0.9-fold ( p<0.001) and iCGRP release 2.9±0.4-fold ( p<0.001) over control levels. The stimulatory effects of NGF were markedly attenuated, but not abolished, by UK (NGF+UK vs. control, CGRP mRNA, 2.9±0.4-fold, p<0.05; iCGRP, 1.7±0.2-fold, p<0.05). These values were also significant ( p<0.05) when compared to NGF treatment alone. Experiments performed using the α 2-antagonist yohimbine confirmed that the effects of UK were mediated by the α 2-AR. These results, therefore, demonstrate that α 2-AR activation attenuates the stimulatory effects of NGF on CGRP expression in DRG neurons.

Characterization of iCGRP release from adrenal capsule primary afferent neurons

The rat adrenal capsule and cortex are innervated by primary afferent neurons that contain neuropeptides, including calcitonin gene-related peptide (CGRP; 1). Furthermore, CGRP has been implicated in modulating adrenal cortical function (2–5). These results suggest that factors influencing the release of CGRP from these fibers may represent an additional regulatory system for modulation of adrenal cortical function in vivo. In the present study, we used an in vitro superfusion technique to pharmacologically characterize the release of immunoreactive CGRP (iCGRP) from adrenal capsular sensory fibers. These studies employed capsaicin, the pungent principle of hot peppers, which selectively activates a subset of nociceptive primary afferent neurons (6). In addition, the effects of capsaicin on aldosterone biosynthesis by adrenal capsules were assessed.Briefly, adrenal glands were quickly removed from male, Sprague-Dawley rats (250-275g) and decapsulated. The adrenal capsules were then placed into superfusion chambers and perfused with buffer. After a period of baseline collection, tissue was treated with either high K+ (50 mM) buffer, which results in non-specific activation of neurons, or capsaicin (100 μM), followed by a washout period. Superfusates were collected and assayed for iCGRP via RIA. In some experiments, superfusates were also assayed for aldosterone via RIA.

Peripheral CGRP release as a marker for neurogenic inflammation: a model system for the study of neuropeptide secretion in rat paw skin

The local release of pro-inflammatory neuropeptides in the periphery has been associated with the development of neurogenic inflammation. However, there is an increasing number of reports demonstrating tissue-dependent differences regarding the mechanisms engaged by these neuropeptides to initiate and maintain the inflammatory response in the target tissue. Since skin is often involved in tissue injury, the present studies were designed to develop a model for assessing cutaneous peptide secretion as a marker for neurogenic inflammation in skin tissue. Calcitonin gene-related peptide (CGRP), as one of several neuropeptides known to be involved in neurogenic inflammation, was chosen to study capsaicin-induced effects on peripheral neurosecretion. The corial surface of the hairy skin of a rat hindlimb was superfused in vitro, and the basal and capsaicin-evoked peripheral release of immunoreactive CGRP (iCGRP) was measured using a radioimmunoassay. The main objectives of these studies were to characterize the various properties of this release including dose-dependency, exocytosis and receptor-mediation as well as the effects of acute and long-term capsaicin desensitization. Capsaicin significantly and dose-dependently increased the release of iCGRP at concentrations ranging from 3 to 300 μM. Omission of calcium ions or treatment with the competitive capsaicin receptor antagonist capsazepine completely inhibited the capsaicin-induced iCGRP release. Superfusion of the skin with 100 μM capsaicin following a conditioning stimulation with capsaicin at concentrations ranging from 0.3 to 100 μM led to an acute, dose-dependent desensitization of the CGRP response. In addition, chronic desensitization following the neonatal injection of capsaicin completely abolished the acute iCGRP response to capsaicin. The method described here should prove to be a valuable tool for the evaluation of the processes regulating the peripheral, cutaneous release of pro-inflammatory neuropeptides. This strategy, therefore, may lead to a better understanding of the mechanisms involved in the development and maintenance of neurogenic inflammation, particularly in the skin.

OR 5 Prostaglandin-bradykinin stimulated release of iCGRP in bovine dental pulp

OR 5 Prostaglandin-bradykinin stimulated release of iCGRP in bovine dental pulp

Differential regulation of evoked peptide release by voltage-sensitive calcium channels in rat sensory neurons

To determine whether the sensitizing actions of prostaglandins on sensory neurons are due to modulation of voltage-sensitive calcium channels (VSCC) we examined the effects of inhibiting these channels on PGE,-induced enhancement of evoked peptide release from isolated dorsal root ganglion neurons. The inhibitory effects of the VSCC blockers on stimulated release were dependent upon the type of chemical agent used to evoke the release. Bradykinin-stimulated release of immunoreactive substance P( iSP) and calcitonin gene-related peptide ( iCGRP) was attenuated by the N-type VSCC blocker, ω-conotoxin GVIA (100 nM), but was unaffected by blockade of L-type (1 μM nifedipine) or P-type (200 nM ω-agatoxin IVA) VSCC. In contrast, potassium-stimulated release of peptides was inhibited by nifedipine, but not by co-conotoxin GVIA or ω-agatoxin IVA. None of the VSCC blockers tested attenuated capsaicin-stimulated release of iSP and iCGRP. The combination of 1 μM nifedipine and 100 nM ω-conotoxin GVIA reduced the whole cell calcium current 89% ± 1.7%. Administration of 100 nM PGE 2 potentiated bradykinin- and capsaicin-evoked peptide release by 2–3-fold. Neither nifedipine nor ω-conotoxin GVIA attenuated the PGE 2-mediated potentiation of bradykinin-evoked release, and neither ω-conotoxin GVIA nor w-agatoxin IVA blocked the potentiation of capsaicin-evoked release induced by PGE 2. These results indicate that the sensitizing actions of PGE 2 as measured by enhanced peptide release, are not mediated by L-, N-, or P-type VSCC.