Endothelin-1 Elicits TRP-Mediated Pain in an Acid-Induced Oral Ulcer Model.

Oral ulcer is the most common oral disease and leads to pain during meals and speaking, reducing the quality of life of patients. Recent evidence using animal models suggests that oral ulcers induce cyclooxygenase-dependent spontaneous pain and cyclooxygenase-independent mechanical allodynia. Endothelin-1 is upregulated in oral mucosal inflammation, although it has not been shown to induce pain in oral ulcers. In the present study, we investigated the involvement of endothelin-1 signaling with oral ulcer-induced pain using our proprietary assay system in conscious rats. Endothelin-1 was significantly upregulated in oral ulcers experimentally induced by topical acetic acid treatment, while endothelin-1 production was suppressed by antibacterial pretreatment. Spontaneous nociceptive behavior in oral ulcer model rats was inhibited by swab applications of BQ-788 (ETB receptor antagonist), ONO-8711 (prostanoid receptor EP1 antagonist), and HC-030031 (TRPA1 antagonist). Prostaglandin E2 production in the ulcers was suppressed by BQ-788. Mechanical allodynia in the model was inhibited not only by BQ-788 and HC-030031 but also by BQ-123 (ETA receptor antagonist), SB-366791 (TRPV1 antagonist), and RN-1734 (TRPV4 antagonist). In naive rats, submucosal injection of endothelin-1 caused mechanical allodynia that was sensitive to HC-030031 and SB-366791 but not to RN-1734. These results suggest that endothelin-1 production following oral bacterial invasion via ulcerative regions elicits TRPA1-mediated spontaneous pain. This pain likely occurs through an indirect route that involves ETB receptor-accelerated prostanoid production. Endothelin-1 elicits directly TRPA1- and TRPV1-mediated mechanical allodynia via both ETA and ETB receptors on nociceptive fibers. The TRPV4-mediated allodynia component seems to be independent of endothelin signaling. These findings highlight the potential of endothelin signaling blockers as effective analgesic approaches for oral ulcer patients.

Endothelin receptor-mediated responses in trigeminal ganglion neurons.

Recent evidence implicates endothelin in nociception, but it is unclear how endothelin activates trigeminal ganglion (TRG) neurons. In the present study, we investigated the expression of the endothelin receptors ETA and ETB and endothelin-induced responses in rat TRG neurons. Double-immunofluorescence studies demonstrated that ETA and ETB were expressed in TRG neurons and that 26% of ETA- or ETB-expressing neurons expressed both receptors. During whole-cell patch-clamp recording, endothelin-1 enhanced an induced current in response to capsaicin, a TRPV1 agonist, in approximately 20% of dissociated neurons. The enhancement was blocked by the PKC inhibitor chelerythrine and by the ETA antagonist BQ-123, but not by the ETB antagonist BQ-788. Ca(2+)-imaging showed that endothelin-1 increased the intracellular Ca(2+) concentration in more than 20% of the dissociated neurons. Importantly, unlike the effect of endothelin-1 on capsaicin-induced current, the Ca(2+) response was largely suppressed by BQ-788 but not by BQ-123. These results suggest that ETA-mediated TRPV1 hyperactivation via PKC activation and ETB-mediated Ca(2+) mobilization occurs in different subsets of TRG neurons. These endothelin-induced responses may contribute to the induction of orofacial pain. The ETB-mediated function in TRG neurons is a special feature in the trigeminal system because of no ETB expression in dorsal root ganglion neurons.

Distinct effects of cevimeline and pilocarpine on salivary mechanisms, cardiovascular response and thirst sensation in rats.

OBJECTIVE: Cevimeline and pilocarpine (muscarinic receptor agonists) are used as sialogogues in xerostomia treatment. It is important to know the different effects on their salivary mechanisms and the side effects. The aim of the present study was to clarify and compare the comprehensive effects of cevimeline to pilocarpine on salivary, cardiovascular and central mechanisms in rats. DESIGN: Under anaesthesia, whole saliva secretion, parotid blood flow and blood pressure were measured following intra-peritoneal administrations of the sialogogues. In digested parotid cells, intracellular Ca(2+) concentrations were measured after the sialogogue application. In the conscious condition, changes in angiotensin II-induced water intake were observed after cevimeline administration. In the subfornical organ, which is a thirst-related central nucleus, the effect of cevimeline on the neuronal activity was electrophysiologically investigated. RESULTS: Cevimeline at 80µmolkg(-1) showed slowly increasing and lasting salivation, a similar blood flow increment in the parotid gland and higher pressor response when compared to pilocarpine at 4µmolkg(-1). In parotid cells, cevimeline increased the intracellular Ca(2+) concentration in a similar manner to pilocarpine, but at a higher concentration than pilocarpine. Cevimeline inhibited angiotensin II-induced water intake and neuronal activity in the subfornical organ, which is in contrast to reported effects of pilocarpine. CONCLUSIONS: Cevimeline activates common salivary mechanism with pilocarpine but has a slower onset of activation, longer duration of salivation and an increased pressor response at higher doses. The anti-dipsogenic effect of cevimeline is due to the inhibitory neuronal effect on the thirst-related central nuclei.

Central glial activation mediates cancer-induced pain in a rat facial cancer model.

Peripheral and central glial activation plays an important role in development of pain hypersensitivity induced by inflammation and nerve injury. However, the involvement of glial cells in cancer pain is not well understood. The present study evaluated the peripheral and central glial activation and the effect of an inhibitor of glial activation, propentofylline, on pain-related behaviors in a rat facial cancer model of the growth of Walker 256B cells in the unilateral vibrissal pad until days 3-4 post-inoculation. As compared with sham animals, the facial grooming period was prolonged, the withdrawal latency to radiant heat stimulation was shortened, and the withdrawal threshold by von Frey hair stimulation was decreased at the inoculated region, indicating the development of spontaneous pain, thermal hyperalgesia and mechanical allodynia. In immunostainings for Iba1 and glial fibrillary acidic protein (GFAP), although there were no morphological changes of GFAP-immunopositive satellite glial cells in the trigeminal ganglion, Iba1-immunopositive microglia and GFAP-immunopositive astrocytes in the medullary dorsal horn showed large somata with cell proliferation. After the daily i.p. administration of propentofylline beginning pre-inoculation, the central glial activation was attenuated, the prolonged facial grooming was partially suppressed, and the induced allodynia and hyperalgesia from day 2 were prevented, without a change in tumor size. These results suggest that glial activation in the CNS, but not in the peripheral nervous system, mediates the enhancement of spontaneous pain and the development of allodynia and hyperalgesia at an early stage in the facial cancer model.

Differences between orofacial inflammation and cancer pain.

Rat models of orofacial cancer exhibit both allodynia and hyperalgesia; however, it is unclear whether cancer-induced pain is secondary to cancer-induced inflammation. To address this question, we compared the effects of an anti-inflammatory drug, indomethacin, on pain and neurochemical changes in the medullary dorsal horn in orofacial inflammation and cancer models. Daily peripheral administration of indomethacin largely suppressed mechanical allodynia and thermal hyperalgesia in the inflammation model. The same procedure suppressed allodynia and hyperalgesia in the cancer model, but the suppression was weak when compared with that in the inflammation model. In the medullary dorsal horn, calcitonin gene-related peptide and substance P levels were significantly increased in the inflammation model, but did not change in the cancer model. These results suggest that pain in the orofacial cancer model is not significantly mediated by cancer-induced peripheral inflammation, although it may have some involvement.

Small salivary gland size in patients with xerostomia of unknown etiology.

OBJECTIVE: We have recently reported that flow rates of whole saliva in young healthy humans correlate positively with salivary gland sizes. The low rate of salivary secretion in xerostomia patients may be related to the small size of the salivary glands. To investigate this possibility, relationships between salivary secretions and salivary gland sizes were investigated in unknown-etiology xerostomia patients and healthy controls. DESIGN: The sizes of the three major salivary glands in seven xerostomia patients and seven age- and gender-matched healthy controls who have no previous disease history and prescription medication related to xerostomia, were measured by use of a magnetic resonance imaging technique. The salivary glands of all subjects failed to show any pathological aspects in magnetic resonance images. The flow rates of unstimulated and chewing-stimulated whole saliva were also measured. RESULTS: Flow rates of unstimulated and chewing-stimulated whole saliva and the sizes of the parotid and submandibular glands were significantly lower and smaller in xerostomia patients of unknown etiology when compared with healthy controls. In addition, salivary flow rates per size of the combined three major salivary glands were also significantly lower in the xerostomia patients of unknown etiology. CONCLUSIONS: The smaller salivary gland size in xerostomia patients of unknown etiology may be one of the causes of the reduced salivary secretion. The secretion rates as a function of gland sizes were also lower, and so it is likely that functional impairments of the salivary gland are also present in patients with xerostomia of unknown etiology.

Nicotinic receptor subtypes in rat subfornical organ neurons and glial cells.

It is unclear which nicotinic acetylcholine receptor (nAChR) subtypes are involved in the nicotinic activation of cells in the subfornical organ (SFO). We investigated the nAChR subtype using molecular biological, electrophysiological, pharmacological and immunohistochemical techniques. The use of reverse transcription-polymerase chain reaction in rats demonstrated the presence of mRNAs for the alpha2, alpha3, alpha4, alpha6, alpha7, beta2 and beta4 subunits in the SFO. The characteristics and dose-dependency of nicotine-induced inward currents in many dissociated SFO neurons were similar to those induced by acetylcholine in the presence of atropine. The nicotine-induced currents were larger than those induced by cytisine in most responding cells, suggesting the predominance of the beta2- rather than the beta4-containing nAChR. NIC-induced currents were significantly inhibited by dihydro-beta-erythroidine (a relatively selective antagonist for alpha4-containing nAChRs, and a partial antagonist for alpha2 or alpha3) at 300 nM in all responding cells. Additionally, the currents were significantly inhibited by alpha-conotoxin MII at 10 nM (a selective antagonist for alpha3- and/or alpha6-containing nAChRs) in some but not all responding cells. Methyllycaconitine at 10 nM (a selective alpha7-nAChR antagonist) reduced the nicotine-induced current significantly, but to a lesser extent. Fluorescence-labeled alpha-bungarotoxin (a homomeric alpha7 subtype selective binding drug) binding and immunofluorescence for the alpha7 subunit showed that positive images almost overlapped with those immunopositive for an astrocyte marker. These results suggest that the alpha4beta2 subtype is the main functional receptor in SFO neurons while alpha2, alpha3, alpha6, and beta4 subunits have some effect, and homomeric the alpha7 subtype exists in SFO astrocytes.

Functional evaluations of the parotid and submandibular glands using dynamic magnetic resonance sialography.

PURPOSE: To evaluate the functional differences between the parotid and submandibular glands using dynamic MR sialography. METHODS: In 30 volunteers, the time-dependent changes (dynamic changes) in the maximum area of the detectable parotid and submandibular gland ducts on dynamic MR sialographic images were analysed. RESULTS: Dynamic changes in the parotid gland ducts were detectable on MR sialographic images in all volunteers, but images of the submandibular gland ducts were detectable in only 23 volunteers. In addition, the dynamic changes in the submandibular gland ducts in these 23 subjects were less than those seen in the parotid gland ducts. A relationship was found between the changing ratio of parotid (Pearson r=0.448, P=0.013) or submandibular gland ducts (Pearson r=0.418, P=0.047) and the salivary flow rate during the stimulation period. CONCLUSIONS: Dynamic MR sialography allows evaluation of rest and stimulated functioning and morphological evaluation of the parotid and submandibular glands. This technique appears to have many possible applications in the dental, medical and biological fields.

Galanin inhibits neural activity in the subfornical organ in rat slice preparation.

The activation of the subfornical organ (SFO), a circumventricular organ, induces water intake and vasopressin release. Since central administrations of galanin (GAL) suppress water intake and vasopressin release, GAL may inhibit the neural activity of SFO neurons. In the present study, we investigated effects of GAL on the SFO using molecular biological, electrophysiological and anatomical techniques. Reverse transcription-polymerase chain reaction analysis demonstrated the presence in the SFO of rats of the mRNAs for each of the three known GAL receptor subtypes (GalR1, GalR2 and GalR3). In extracellular recordings in SFO slice preparations, GAL dose-dependently inhibited the neural activity of cells from a number of recording sites. Many GAL-sensitive SFO neurons showed excitatory responses to angiotensin II (ANGII). The GalR1 agonist M617 inhibited the activity of SFO neurons, whereas the GalR2 and GalR3 agonist GAL(2-11) had almost no effect. In patch-clamp recordings, GAL induced an outward current in SFO neurons without influencing synaptic currents. An immunoelectron microscopic study revealed the existence of GAL-containing synaptic vesicles in the SFO. These results suggest that the SFO has neural inputs involving GAL. The response to GAL is inhibitory, mediated at least in part by GalR1 and provides a plausible explanation for the opposite effects of ANGII and GAL seen in vivo on water intake and vasopressin release.

Gender difference in unstimulated whole saliva flow rate and salivary gland sizes.

OBJECTIVE: A gender difference in the unstimulated whole saliva flow rate (UWSFR) may be due to a difference in the sizes of the salivary glands. In this study, we investigated the relationships among the UWSFR, gland sizes and body sizes of healthy young adult males and females. DESIGN: Unstimulated whole saliva was collected for 5 min by the spitting method in 50 healthy young adults, and the flow rate of the saliva was measured. Heights and weights were measured, and body mass indices (BMI) were calculated. The sizes of the salivary glands were measured by use of a magnetic resonance imaging technique. RESULTS: Parotid and submandibular gland sizes and flow rates in females were significantly smaller than those in males, as were also the weights, heights and BMI. In both males and females, there were significant positive correlations between gland sizes and the flow rates, weights and BMI. The variations of the flow rates were reduced by standardizing them with gland sizes, weights and BMI. CONCLUSIONS: These results suggest that the lower UWSFR in females as compared with males is due to the smaller gland sizes due to the smaller body sizes.

Dynamic magnetic resonance sialography as a new diagnostic technique for patients with Sjögren's syndrome.

OBJECTIVE: To evaluate the clinical utility of dynamic magnetic resonance (MR) sialographic images as a diagnostic tool for patients with Sjögren's syndrome. METHODS: The morphological findings and various kinds of functional parameters in volunteers on dynamic MR sialographic images were compared with those in five patients with definite Sjögren's syndrome. RESULTS: On the MR sialographs of all five patients with Sjögren's syndrome, the so-called 'apple-tree appearance' was seen. The difference in two functional parameters using the dynamic MR sialographic data was elucidated between the two groups. The maximum area of the detectable ducts in the group of patients was significantly smaller (P < 0.001) than that in the group of volunteers. The ratio of change in the detectable ducts in the group of patients was significantly lower (P = 0.011) than that in the group of volunteers. CONCLUSIONS: Our study suggests that dynamic MR sialographic data in addition to MR sialographic images might be useful for the diagnosis of Sjögren's syndrome.

Pilocarpine-induced salivation and thirst in conscious rats.

The muscarinic receptor agonist pilocarpine is widely used as a sialogogue. It has been well-established that it also induces water intake in animals. However, the mechanisms underlying the relationships between these events are unknown. To address this problem, we examined water intake and parotid salivary secretion in conscious rats. Intraperitoneally injected pilocarpine increased both water intake and salivary secretion. Intracerebroventricularly injected pilocarpine also induced water intake, but not salivary secretion. Intracerebroventricularly applied atropine, a muscarinic receptor antagonist, suppressed the water intake produced by pilocarpine applied intraperitoneally and intracerebroventricularly. However, it did not affect the salivary secretion induced by pilocarpine applied peripherally. We conclude that peripherally applied pilocarpine affects the parotid glands and the thirst center in the central nervous system, while it may induce salivary secretion mainly via peripheral responses, but water intake mainly via the central nervous system.

DAMGO suppresses both excitatory and inhibitory synaptic transmission in supraoptic neurones of mouse hypothalamic slice preparations.

Opioid effects on synaptic transmission in the mouse supraoptic nucleus (SON) were investigated using whole-cell, patch-clamp techniques. The mu-opioid receptor agonist, [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) decreased the amplitude of both evoked excitatory postsynaptic currents (eEPSCs) and inhibitory postsynaptic currents (eIPSCs), and also decreased the frequency of both miniature EPSCs and IPSCs without effect on the amplitude. The selective mu-opioid receptor antagonist, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2), and the nonselective antagonist naloxone, antagonized these inhibitory effects. The application of DAMGO suppressed the amplitude of both the first and second evoked postsynaptic currents with a paired-pulse stimulus protocol, but increased the paired-pulse ratios (second ePSC/first ePSC). DAMGO induced neither inward nor outward currents, and had no significant changes in either glutamate- or GABA-induced currents. When compared with the relatively selective kappa- and delta-opioid receptor agonists dynorphin and [D-Pen(2), D-Pen(5)]-enkephalin, DAMGO showed the most potent inhibitory effects on evoked and miniature postsynaptic currents. Taken together, these results imply that DAMGO strongly suppresses the release of glutamate and GABA via mu-opioid receptors in the mouse SON, and support the involvement of presynaptic regulation by opioids in the control of magnocellular neurosecretory neurones.

Activation of muscarinic receptors in rat subfornical organ neurones.

Cholinergic muscarinic inputs to subfornical organ (SFO) neurones in rats were studied using histochemical, molecular-biological and electrophysiological techniques. Neurones in the medial septum and the diagonal band (MS-DBB) were retrogradely labelled by a tracer wheat germ agglutinin-conjugated horseradish peroxidase-colloidal gold complex injected into the SFO. Some in the MS-DBB were double-labelled by choline acetyltransferase (ChAT) antibody. Many ChAT-immunoreactive fibres were observed in the SFO. M3 muscarinic receptor subtype-like immunoreactivity, detected using a polyclonal antiserum, was observed in the SFO. In slice preparations, muscarine induced inward currents in a dose-related manner. The inward currents were suppressed by the relatively M3 muscarinic receptor selective antagonist 4-diphenylacetoxy-N-methylpiredine methiodide. In the whole-cell current mode, muscarine depolarized the membrane with increased frequency of action potentials. Reverse transcriptase-polymerase chain reaction showed the presence of M2-M5 receptor mRNA in the SFO tissues. These results suggest that the SFO receives cholinergic muscarinic synaptic inputs from the MS-DBB. Acetylcholine postsynaptically activates and depolarizes neurones in the SFO partly through specific muscarinic receptors, including M3 receptor subtypes.

Spontaneously active GABAergic interneurons in the subfornical organ of rat slice preparations.

Inhibitory postsynaptic currents (IPSCs) were recorded from subfornical organ (SFO) neurons in slice preparations of rats, using whole-cell voltage clamp techniques. Some SFO neurons showed bimodal distributions in amplitude with the large and small IPSCs. The large IPSCs vanished in the tetrodotoxin perfusion medium, but the small did not. Both sizes of the IPSCs were completely abolished by application of bicuculline and picrotoxin. Further subpopulation of SFO neurons with the bimodal distributions showed intermittent bursts of the large IPSCs. Immunohistochemical approach revealed existence of gamma-aminobutyric acid (GABA)-immunoreactive neurons and axons in the SFO. These suggest that spontaneously-active and intermittently-burst-firing GABA interneurons affect other SFO neurons in slice preparations of rats.

Angiotensin II induces inward currents in subfornical organ neurones of rats.

The action of angiotensin II on subfornical organ (SFO) neurones was studied using whole-cell current and voltage-clamp recordings in rat slice preparations. In the current-clamp mode, membrane depolarization in response to angiotensin II was accompanied by an increased frequency of action potentials and an increased membrane conductance. In the voltage-clamp mode, angiotensin II elicited inward currents in a dose-dependent manner. The net angiotensin II-induced inward currents were voltage-independent, with a mean reversal potential of -29.8 +/- 6.2 mV. Amplitudes of the angiotensin II-induced inward currents were decreased during perfusion with a low sodium medium. The angiotensin II-induced inward currents were blocked by the AT1 antagonist losartan, and were partially blocked by the AT2 antagonist PD-123319. Neurones which were sensitive to angiotensin II were found in the peripheral region of the SFO, whereas neurones in the central region were less sensitive to angiotensin II. These results suggest that angiotensin II induces inward currents, with opening of nonselective cation channels through mainly AT1 receptors in a subpopulation of SFO neurones of rats.

Muscarinic modulation of GABAergic transmission to neurons in the rat subfornical organ.

Cholinergic actions on subfornical organ (SFO) neurons in rat slice preparations were studied by using whole cell voltage- and current-clamp recordings. In the voltage-clamp recordings, carbachol and muscarine decreased the frequency of GABAergic inhibitory postsynaptic currents (IPSCs) in a dose-dependent manner, with no effect on the amplitudes or the time constants of miniature IPSCs. Meanwhile, carbachol did not influence the amplitude of the outward currents induced by GABA. Furthermore, carbachol and muscarine also elicited inward currents in a TTX-containing solution. From the current-voltage relationship, the reversal potential was estimated to be -7.1 mV. These carbachol-induced responses were antagonized by atropine. In the current-clamp recordings, carbachol depolarized the membrane with increased frequency of action potentials. These observations suggest that acetylcholine suppresses GABA release through muscarinic receptors located on the presynaptic terminals. Acetylcholine also directly affects the postsynaptic membrane through muscarinic receptors, by opening nonselective cation channels. A combination of these presynaptic and postsynaptic actions may enhance activation of SFO neurons by acetylcholine.

The effect of food consistency and dehydration on reflex parotid and submandibular salivary secretion in conscious rats.

Changes in salivary secretion with different consistency of diet and dehydration were studied in male Wistar rats under unrestricted conditions. To measure the salivary secretion, a stop-flow method was used. There was little unstimulated salivary secretion from the parotid and submandibular glands, but eating solid, powdered, and liquid diets induced parotid and submandibular saliva. There was no significant change in the volume and flow rate of saliva in bilateral parotid glands during the eating of solid diets. The solid and powdered diets induced significantly more salivary secretion from the parotid gland than did the liquid. The salivary flow rate with solid diets was significantly greater from the parotid gland than from the submandibular. On the other hand, the salivary flow rate with the liquid diet was significantly smaller from the parotid gland than from the submandibular. Appreciable amounts of submandibular saliva, but little parotid saliva were secreted during grooming. Clearly, parotid and submandibular saliva have different roles in the rat. When injected intraperitoneally with 1.5 M NaCl solution or water-deprived for 24 h, rats took longer to eat the solid diets, and had increased salivary volume and decreased flow rate from the parotid gland. These results indicate that the moisture content of the diet and the dryness of the mouth alters the volume of parotid saliva secreted in rats and show that parotid saliva plays an important part in mastication and swallowing.

Glutamatergic synaptic inputs activate neurons in the subfornical organ through non-NMDA receptors.

The subfornical organ (SFO) plays an important role in central regulation of the autonomic nervous system. The synaptic transmission properties of neurons in the SFO were studied with intracellular and whole-cell patch clamp recordings in the rat slice preparations. Both the spontaneous and evoked excitatory postsynaptic potentials (EPSPs) and currents (EPSCs) were almost completely suppressed by the glutamate receptor antagonist kynurenic acid and the non-NMDA (N-methyl-D-aspartic acid) antagonist CNQX. The non-NMDA agonist kainic acid depolarized the membrane most potently, compared with NMDA and quisqualic acid. These suggest that glutamate is a main excitatory neurotransmitter in the SFO and that its action is at least partly mediated through non-NMDA receptors.

Disorder of salivary secretion in inbred polydipsic mouse.

To find mechanisms of an extreme polydipsia in an inbred strain of mice, STR/N, this study was undertaken using Institute of Cancer Research (ICR) mice as a control. During food deprivation, daily water intake of both strains decreased. The decrement in the STR/N mice was larger than that in the ICR mice. During dehydration, daily food intake of the STR/N mice was smaller than that of the ICR mice. These data indicate that prandial drinking was more severely affected for the STR/N mice. Under anesthesia, the stimulated salivary secretion by pilocarpine of the STR/N mice was significantly smaller than that of the ICR mice. The submandibular gland of the STR/N mice was lighter and harder than that of the ICR mice. After desalivation from the major three salivary glands, the ICR mice drank as much as the STR/N mice. Young STR/N mice with undeveloped polydipsia did not show different salivary secretion stimulated by pilocarpine from the young ICR mice. These findings indicate a dysfunction with age in the salivary glands of the STR/N mice, and they suggest that the decreased saliva induces thirst and triggers extraordinary drinking in the polydipsic mice.