ATP-Evoked Intracellular Ca²âº Signaling of Different Supporting Cells in the Hearing Mouse Hemicochlea.

Hearing and its protection is regulated by ATP-evoked Ca(2+) signaling in the supporting cells of the organ of Corti, however, the unique anatomy of the cochlea hampers observing these mechanisms. For the first time, we have performed functional ratiometric Ca(2+) imaging (fura-2) in three different supporting cell types in the hemicochlea preparation of hearing mice to measure purinergic receptor-mediated Ca(2+) signaling in pillar, Deiters' and Hensen's cells. Their resting [Ca(2+)]i was determined and compared in the same type of preparation. ATP evoked reversible, repeatable and dose-dependent Ca(2+) transients in all three cell types, showing desensitization. Inhibiting the Ca(2+) signaling of the ionotropic P2X (omission of extracellular Ca(2+)) and metabotropic P2Y purinergic receptors (depletion of intracellular Ca(2+) stores) revealed the involvement of both receptor types. Detection of P2X2,3,4,6,7 and P2Y1,2,6,12,14 receptor mRNAs by RT-PCR supported this finding and antagonism by PPADS suggested different functional purinergic receptor population in pillar versus Deiters' and Hensen's cells. The sum of the extra- and intracellular Ca(2+)-dependent components of the response was about equal with the control ATP response (linear additivity) in pillar cells, and showed supralinearity in Deiters' and Hensen's cells. Calcium-induced calcium release might explain this synergistic interaction. The more pronounced Ca(2+) leak from the endoplasmic reticulum in Deiters' and Hensen's cells, unmasked by cyclopiazonic acid, may also suggests the higher activity of the internal stores in Ca(2+) signaling in these cells. Differences in Ca(2+) homeostasis and ATP-induced Ca(2+) signaling might reflect the distinct roles these cells play in cochlear function and pathophysiology.

Immunolocalization of AQP5 in resting and stimulated normal labial glands and in Sjögren's syndrome.

OBJECTIVE: In our current work, in vivo examination of AQP5 distribution in labial salivary glands following stimulation of secretion has been carried out in normal individuals and in patients with Sjögren's syndrome. SUBJECTS AND METHODS: For this study, we selected five patients with primary Sjögren's syndrome (mean age 62.4 ± 10.6 s.d. years) diagnosed in accordance with the European Cooperative Community classification criteria. There were five patients (mean age 27 ± 2.5 s.d. years) in the control group. The subcellular distribution of AQP5 in human labial gland biopsies was determined with light and immunoelectron microscopy before and 30 min after administration of oral pilocarpine. RESULTS: In unstimulated control and Sjögren's labial glands, AQP5 is about 90% localized in the apical plasma membrane, with only rarely associated gold particles with intracellular membrane structures. We have found no evidence of pilocarpine-induced changes in localization of AQP5 in either healthy individuals or patients with Sjögren's syndrome. CONCLUSIONS: Our studies indicate that neither Sjögren's syndrome itself, nor muscarinic cholinergic stimulation in vivo caused any significant changes in the distribution of AQP5 in the labial salivary gland cells.

Protective effect of rasagiline in aminoglycoside ototoxicity.

Sensorineural hearing losses (SNHLs; e.g., ototoxicant- and noise-induced hearing loss or presbycusis) are among the most frequent sensory deficits, but they lack effective drug therapies. The majority of recent therapeutic approaches focused on the trials of antioxidants and reactive oxygen species (ROS) scavengers in SNHLs. The rationale for these studies was the prominent role of disturbed redox homeostasis and the consequent ROS elevation. Although the antioxidant therapies in several animal studies seemed to be promising, clinical trials have failed to fulfill expectations. We investigated the potential of rasagiline, an FDA-approved monomanine oxidase type B inhibitor (MAO-B) inhibitor type anti-parkinsonian drug, as an otoprotectant. We showed a dose-dependent alleviation of the kanamycin-induced threshold shifts measured by auditory brainstem response (ABR) in an ototoxicant aminoglycoside antibiotic-based hearing loss model in mice. This effect proved to be statistically significant at a 6-mg/kg (s.c.) dose. The most prominent effect appeared at 16kHz, which is the hearing sensitivity optimum for mice. The neuroprotective, antiapoptotic and antioxidant effects of rasagiline in animal models, all targeting a specific mechanism of aminoglycoside injury, may explain this otoprotection. The dopaminergic neurotransmission enhancer effect of rasagiline might also contribute to the protection. Dopamine (DA), released from lateral olivocochlear (LOC) fibers, was shown to exert a protective action against excitotoxicity, a pathological factor in the aminoglycoside-induced SNHL. We have shown that rasagiline enhanced the electric stimulation-evoked release of DA from an acute mouse cochlea preparation in a dose-dependent manner. Using inhibitors of voltage-gated Na(+)-, Ca(2+) channels and DA transporters, we revealed that rasagiline potentiated the action potential-evoked release of DA by inhibiting the reuptake. The complex, multifactorial pathomechanism of SNHLs most likely requires drugs acting on multiple targets for effective therapy. Rasagiline, with its multi-target action and favorable adverse effects profile, might be a good candidate for a clinical trial testing the otoprotective indication.

Malaria pigment crystals as magnetic micro-rotors: key for high-sensitivity diagnosis.

The need to develop new methods for the high-sensitivity diagnosis of malaria has initiated a global activity in medical and interdisciplinary sciences. Most of the diverse variety of emerging techniques are based on research-grade instruments, sophisticated reagent-based assays or rely on expertise. Here, we suggest an alternative optical methodology with an easy-to-use and cost-effective instrumentation based on unique properties of malaria pigment reported previously and determined quantitatively in the present study. Malaria pigment, also called hemozoin, is an insoluble microcrystalline form of heme. These crystallites show remarkable magnetic and optical anisotropy distinctly from any other components of blood. As a consequence, they can simultaneously act as magnetically driven micro-rotors and spinning polarizers in suspensions. These properties can gain importance not only in malaria diagnosis and therapies, where hemozoin is considered as drug target or immune modulator, but also in the magnetic manipulation of cells and tissues on the microscopic scale.

Morphological evidence of local reflex arc in the rat's tongue.

Lingual components of the autonomic nervous system are considered to be the most rostral portion of the enteric nervous system. Therefore our aim was to study the intrinsic nerve cell bodies and synapses using immunohisto-, immunocytochemical methods. Several small groups of ganglia with cell bodies immunoreactive (IR) for vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) and substance P (SP) were observed just below the gustatory epithelium. A few somatostatin and galanin IR nerve cell bodies were also found. Many IR cell bodies were also demonstrated in the glands and next to blood vessels. Some of these cell bodies were multipolar and some of them were small neurons with an ovoid shape having only one process. Cell bodies positive for calcitonin gene-related peptide (CGRP) were detected neither in the superficial nor in the deep portion. Electronmicroscopical analysis demonstrated different IR nerve fibres having axo-somatic and axo-dendritic synapses with other immunonegative cells. In a few cases VIP IR nerve processes were found to synaptize with other VIP positive nerve cell bodies. These results support the existance of intralingual reflex in the tongue, where the ganglia might have an integrative role of the different neuropeptide containing nerve fibres.

The role of N-methyl-D-aspartate receptors and nitric oxide in cochlear dopamine release.

Dopamine (DA) released from lateral olivocochlear (LOC) terminals may have a neuroprotective effect in the cochlea. To explore the role of N-methyl-d-aspartate (NMDA) receptors and nitric oxide (NO) in the modulation of a cochlear DA release, we measured the release of [3H]DA from isolated mouse cochlea in response to the application of NMDA. NMDA at 100 muM significantly increased the electrical-field stimulation-evoked and resting release of DA from the cochlea. The NO donor sodium nitroprusside enhanced the basal outflow of DA but failed to influence the evoked release. The administration of the nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) alone was ineffective, but it significantly inhibited the initial phase of the NMDA-induced elevation of DA outflow, which suggested the role of NO in the NMDA-induced DA release. The DA uptake inhibitor nomifensine increased the electrically evoked release of DA. Nomifensine failed to change the effect of NMDA on the resting or electrically-evoked DA release, which suggested that the uptake mechanism does not play a role in NMDA-evoked and NO-mediated DA release. In summary, we provide evidence that NO can modulate the release of DA from the cochlea following NMDA receptor activation, but does not affect the uptake of DA.

Application of two-photon microscopy to the study of cellular pharmacology of central neurons.

Two-photon microscopy is an especially powerful tool for combining anatomical and physiological experiments in the central nervous system: the possibility of simultaneously studying physiological phenomena in well-defined anatomical compartments allows fluorescence imaging of neurons in deeper layers of the brain. In this review we summarize the most commonly used brain preparation techniques together with the methods of loading neurons with fluorescent indicators. We will focus primarily on issues of drug delivery specifically related to two-photon experiments highlighting the different ways of drug administration. Methods of chemical stimulation via caged neurotransmitters are also discussed. Finally a few specific areas of two-photon applications in drug research on neuronal tissue are highlighted.

Evaluation of palatal saliva flow rate and oral manifestations in patients with Sjögren's syndrome.

OBJECTIVE: The purpose of this study was to describe the oral properties of Sjögren's syndrome (SS), including the determination of palatal saliva (PS) flow rate. SUBJECTS AND METHODS: Forty-nine SS patients and 43 healthy controls participated. Subjective symptoms were recorded and clinical assessments of the oral mucosal, dental and periodontal status were made. Unstimulated whole saliva (WS) and PS flow rates, the number of decayed, missing and filled teeth (DMF-T number), the gingival bleeding index (GBI) and the periodontal probing depth (PPD) were determined. RESULTS: Despite the decrease in the flow rate of WS in SS patients, PS was not different from those of the controls (1.57 +/- 1.02 and 1.35 +/- 2.5 microl cm(-2) min(-1), respectively). GBI (20.0% vs. 10.5%, respectively), DMF-T (27.1 +/- 6.12 vs. 23.0 +/- 6.99, respectively) and PPD (2.28 +/- 1.09 mm vs. 1.82 +/- 0.73 mm, respectively) were higher in SS compared with the controls (P < 0.05). DMF-T and PPD showed a positive correlation with anti-SSA and/or anti-SSB antibody positivity in the serum (P < 0.05). CONCLUSIONS: Data of the present study suggest that the subjective feeling of xerostomia in SS patients is the result of a decrease in the volume of the whole saliva, and not of the viscous PS. Correlation of DMF-T and PPD with autoantibody positivity reveals that the oral health status of SS patients may be associated with the general autoimmune process.

D2 autoreceptor inhibition reveals oxygen-glucose deprivation-induced release of dopamine in guinea-pig cochlea.

Dopamine (DA), released from the lateral olivocochlear (LOC) efferent terminals, the efferent arm of the short-loop feedback in the cochlea, is considered as a protective factor in the inner ear since it inhibits auditory nerve dendrite firing in ischemia- or noise-induced excitotoxicity leading to sensorineural hearing loss (SNHL). In the present study we investigated the effect of oxygen-glucose deprivation (OGD), an in vitro ischemia model, on guinea-pig cochlear [(3)H]DA release in a microvolume superfusion system. We found that OGD alone failed to induce a detectable elevation of [(3)H]DA level, but in the presence of specific D(2) receptor antagonists, sulpiride and L-741,626, it evoked a significant increase in the extracellular concentration of [(3)H]DA. D(2) negative feedback receptors are involved not exclusively in the regulation of synthesis and vesicular release of DA, but also in the activation of its reuptake. Thus, D(2) receptor antagonism interferes with the powerful reuptake of DA from the extracellular space. To explore the underlying mechanism of this DA-releasing effect we applied nomifensine and found that the effect of OGD on cochlear DA release in the presence of D(2) antagonists could be inhibited by this selective DA uptake inhibitor. This finding indicates that the OGD-evoked DA release was mainly mediated through the reverse operation of the DA transporter. The two structurally different D(2) antagonists also augmented the electrical field stimulation-evoked release of DA proving the presence of D(2) autoreceptors on dopaminergic LOC terminals. Our results confirm the presence and role of D(2) DA autoreceptors in the regulation of DA release from LOC efferents, and suggest a protective local mechanism during ischemia which involves the direct transporter-mediated release of DA. Increasing the release of the protective transmitter DA locally in the inner ear may form the basis of future new therapeutic strategies in patients suffering from SNHL.

Morphological basis of sensory neuropathy and neuroimmunomodulation in minor salivary glands of patients with Sjögren's syndrome.

OBJECTIVE: A predominance of sensory neuropathy was earlier described in Sjögren's syndrome (SS), which might precede the presence of sicca symptoms. The mechanism of sensory neuropathy in SS is unknown. Therefore, the aim of this study was to determine the quantitative changes of the different neuropeptide containing nerve terminals and the immunocompetent cells in labial salivary glands of primary SS. DESIGN: Immunohisto- and immunocytochemical methods were used for the detection of immunoreactive (IR) elements and the data were compared with the healthy controls. RESULTS: All of the investigated IR nerve fibres were found in different quantity and localisation in both of control and SS glands. The density of them was changed variously in SS. The number of the substance P (SP), neuropeptide Y (NPY) (P < 0.05), galanin (GAL) IR nerve terminals was decreased, however, the number of vasoactive intestinal polypeptide (VIP) and tyrosine beta-hydroxylase (TH) IR nerve fibres (P < 0.05) was increased compared to the control. There were no IR immunocompetent cells in the control materials, however, a large number of them showed IR for SP (46.2%) and NPY (34.4%) in the SS. The IR was demonstrated mainly in the mast cells, plasma cells and some of the lymphocytes. CONCLUSIONS: These neuropeptides might have a role in the sensory neuropathy; they might activate nociceptive and sympathetic pathways. Some neuropeptides (SP, NPY) are endogenous in the immune system and produced in certain conditions, e.g. inflammation and chronic autoimmune disorders such as SS, so they might participate in the neuroimmunomodulation and contribute to the atrophy, apoptosis and necrosis.

Expression of aquaporin isoforms during human and mouse tooth development.

Previously, we described the development of hyaluronan (HA) deposition in human tooth germ tissues that are consistent with water transport in different stages of tooth development. The aquaporins (AQP) constitute a family of membrane water channels that are expressed in many organs. However, there are no data available about the expression pattern of aquaporin water channels in dental structures. In the present study we have characterised the expression of six different aquaporin isoforms (AQP1-5, AQP-9) in developing human and mouse tooth germs by immunohistochemistry using isoform specific antibodies. In the "bell stage" AQP1 was expressed in endothelial cells of small vessels whereas no other structures of the tooth primordial were labeled. AQP2, AQP3 and AQP9 immunoreactivity was not observed in tooth germs, whereas strong AQP4 and AQP5 expression was observed in dental lamina, inner enamel epithelium, stratum intermedium, stellate reticulum and the outer enamel epithelium. Oral epithelium also exhibited AQP4 and AQP5 immunolabeling. During development of the matrices of the dental hard tissues AQP4 and AQP5 immunostaining was observed in the odontoblasts and their processes, as well as in the secretory ameloblast and their apical processes. Immunolabeling controls were negative. In conclusion, AQP4 and AQP5 are expressed in tooth germ tissues in early development in cells that previously have been shown to express HA and/or CD44, indicating that AQP water channels may play a role for ECM hydration during tooth development.

Immunohistochemical analysis of substance P containing nerve fibres and their contacts with mast cells in the diabetic rat's tongue.

Sensory neuropathy is common symptom of the diabetes mellitus and the prevalence of oral lesions is higher in diabetic patients. The distribution of substance P was studied immunohistochemically in streptozotocin induced diabetic rat's tongue. The morphological association of sensory nerves (substance P immunoreactive) with mast cells (nerve fibre-mast cell contact) was monitored. The substance P nerve fibre mast cell contacts were very scanty in control tongue. The number of substance P nerve terminals and mast cells was significantly increased (p < 0.05) in diabetes mellitus after 4 weeks of the treatment compared with the control tongue. The number of mast cell nerve contacts was even more significantly increased (p < 0.001) in diabetes. The distance between nerve fibres and mast cells was about 1 mm and very often less than 200 nm. In some instances, the mast cells were degranulated in the vicinity to nerve fibres. Increased number of mast cell nerve contacts in neurogenic inflammation might cause vasoconstriction and lesions of the oral mucosa, so some disorders such lichen planus, leukoplakia and cancer might frequently develop in diabetes mellitus.

Immunolocalization of electroneutral Na(+)-HCO cotransporters in human and rat salivary glands.

Patterns of salivary HCO secretion vary widely among species and among individual glands. In particular, virtually nothing is known about the molecular identity of the HCO transporters involved in human salivary secretion. We have therefore examined the distribution of several known members of the Na(+)-HCO cotransporter (NBC) family in the parotid and submandibular glands. By use of a combination of RT-PCR and immunoblotting analyses, the electroneutral cotransporters NBC3 and NBCn1 mRNA and protein expression were detected in both human and rat tissues. Immunohistochemistry demonstrated that NBC3 was present at the apical membranes of acinar and duct cells in both human and rat parotid and submandibular glands. NBCn1 was strongly expressed at the basolateral membrane of striated duct cells but not in the acinar cells in the human salivary glands, whereas little or no NBCn1 labeling was observed in the rat salivary glands. The presence of NBCn1 at the basolateral membrane of human striated duct cells suggests that it may contribute to ductal HCO secretion. In contrast, the expression of NBC3 at the apical membranes of acinar and duct cells in both human and rat salivary glands indicates a possible role of this isoform in HCO salvage under resting conditions.

The nootropic drug vinpocetine inhibits veratridine-induced [Ca2+]i increase in rat hippocampal CA1 pyramidal cells.

The alkaloid derivative vinpocetine (14-ethoxycarbonyl-(3alpha,16alpha-ethyl)-14,15-eburnamine; Cavinton) has a well known beneficial effect on brain function in hypoxic and ischemic conditions. While it increases CNS blood flow and improves cellular metabolism, relatively little is known about vinpocetine's underlying molecular mechanisms on the single cell level. Since apoptotic and necrotic cell damage is always preceded by an increase in [Ca2+]i, this study investigated the effect of vinpocetine on [Ca2+]i increases in acute brain slices. Sodium influx is an early event in the biochemical cascade that takes place during ischemia. The alkaloid veratridine can activate this Na+ influx, causing depolarization and increasing [Ca2+]i in the cells. Therefore, it can be used to simulate an ischemic attack in brain cells. Using a cooled CCD camera-based ratio imaging system and cell loading with fura 2/AM, the effect of vinpocetine on [Ca2+]i changes in single pyramidal neurons in the vulnerable CA1 region of rat hippocampal slices was investigated. Preperfusion and continuous administration of vinpocetine (10 microM) significantly inhibited the elevation in [Ca2+]i induced by veratridine (10 microM). When the drug was administered after veratridine, it could accelerate the recovery of cellular calcium levels. Piracetam, another nootropic used in clinical practice, could attenuate the elevation of [Ca2+]i only at a high, 1 mM, concentration. We have concluded that vinpocetine, at a pharmacologically relevant concentration, can decrease pathologically high [Ca2+]i levels in individual rat hippocampal CA1 pyramidal neurons; this effect might contribute to the neuroprotective property of the drug.

Functional and immunocytochemical evidence that galanin is a physiological regulator of human jejunal motility.

The neuropeptide galanin has species-dependent effects on intestinal motility. It has a contractile effect on rat jejunal muscle while it relaxes guinea-pig ileum by inhibiting cholinergic transmission. Its effect on human gut motility has been unknown. Extensive work led to the discovery of selective galanin analogues such as M15 [galanin(1-12)-Pro-substance-P(5-11)], M35 [galanin(1-12)-Pro-bradykinin(2-9)-amide] that competitively inhibit various actions of galanin in the central nervous system. The present study was designed to examine the effect of galanin, M15 and M35 on longitudinal jejunal smooth muscle strips isolated from humans and rats, and to localize galanin-immunoreactivity in human jejunum. Galanin and ACh were equally effective in stimulating contractions of the isolated jejunal muscle: sigmoid curve fitting showed that maximal contractile response to galanin and ACh were 25.7+/-11.1 mN and 23.7+/-9.7 in humans, while 8.0+/-0.6 and 8.1+/-0.3 mN in rats, respectively. These effects of galanin were not inhibited by either atropine (5 x 10(-6) M) or tetrodotoxin (3 x 10(-6) M). The potency of galanin inducing the contractile actions were similar in humans and rats. Interestingly, neither M15 nor M35 (up to 10(-7) M) were able to inhibit the responses of the smooth muscle to galanin. However, both putative galanin receptor antagonists showed agonist effects in our experimental models. In accordance with the functional studies, both the longitudinal and the circular muscle layers were abundant in nerve fibers and varicosities showing galanin immunoreactivity. Our data suggest that galanin is a potent physiological regulator of jejunal contractions in humans. Its action on the jejunum, however, is mediated by galanin receptors that are different from those located in the central nervous system.

Changes of salivary amylase in serum and parotid gland during pharmacological and physiological stimulation.

Although serum amylase level is an important diagnostic factor in certain salivary and pancreatic diseases, little information is available regarding the mechanism by which parotid amylase reaches the circulatory system. The present study was carried out to investigate the relationship between parotid isoamylase concentrations in blood serum and in parotid tissue in response to various stimuli. Wistar rats were fed with standard laboratory rodent chow; water was supplied ad libitum. In the first experiment, after a 16-h fasting, rats received either 5 mg/kg pilocarpine or saline (control). In the second study, after fasting, half of the rats were fed for 1 h, the other half received no food. In the third experiment, the changes in serum and tissue enzyme levels were monitored in freely fed animals during the peak-food intake phase, the first 2 h of the dark period. Amylase concentration was determined by using starch as a substrate. Pancreatic and parotid isoamylase levels in serum were separated by gel-electrophoresis utilizing differences in ionic properties of the isoenzymes. As expected, pilocarpine strongly stimulated tissue amylase discharge and serum amylase elevation. Similar, but less pronounced changes were observed not only during refeeding of fasted animals, but also in nonfasted rats during their peak-feeding period. Our data suggest that pharmacological stimulation, such as with pilocarpine or feeding in fasted state, as well as a mild stimulation of parotid function by spontaneous food intake during nonfasted state results in a decrease in parotid tissue amylase activity and a proportional increase in serum levels of parotid isoamylase.

Effects of putative galanin antagonists M35 and C7 on rat exocrine pancreas.

Galanin is a neuropeptide having a wide range of biological actions. Recently selective galanin receptor antagonists such as M35 [galanin(1-12)-Pro-bradykinin(2-9)-amide] and C7 [galanin(1-12)-Pro-spantide-amide] have been described. These antagonists have blocked the actions of galanin on flexor reflex, glucose-induced insulin secretion, and acetyicholine release from hippocampus. Our present aim was to investigate whether M35 and C7 can affect galanin-induced inhibition of pancreatic enzyme secretion in rats. Pancreatic enzyme secretion was studied in urethane-anesthetized rats supplied with jugular vein catheter and pancreatic cannula. Amylase secretion evoked by submaximal CCK-8 stimulation was inhibited dose-dependently by galanin in anesthetized rats. Surprisingly, neither M35 nor C7 was able to inhibit the responses of the exocrine pancreas to galanin. However, both putative galanin receptor antagonists behaved as agonists in our experimental models. Our data suggest that the effects of galanin on pancreatic enzyme secretion are not mediated by M35- or C7-sensitive galanin receptors. Therefore, these galanin receptors are different from those described in the central nervous system.

Reduced oral wound healing in the NOD mouse model for type 1 autoimmune diabetes and its reversal by epidermal growth factor supplementation.

Using the NOD mouse, a model for type 1 diabetes, we examined how reduced concentrations of epidermal growth factor (EGF) in the saliva, after onset of type 1 diabetes, affect oral wound healing. Diabetic NOD/LtJ mice on insulin therapy, prediabetic NOD/LtJ, and age- and sex-matched BALB/cJ mice were given a cutaneous tongue punch and allowed to undergo normal healing. With diabetes onset and a reduction in saliva-derived growth factor levels, the rate of tongue wound healing was reduced compared with nondiabetic NOD/LtJ and healthy BALB/cJ mice. Addition of exogenous EGF to the drinking water did not accelerate the rate of healing in BALB/cJ or prediabetic NOD/LtJ; however, diabetic NOD/LtJ mice exhibited accelerated wound healing similar to healthy mice. These results demonstrate that loss of growth factors from saliva is associated with profoundly reduced oral wound healing, suggesting that therapeutic treatment with topical delivery may be beneficial to patients with type 1 diabetes and oral wound complications.

Analysis of high intracellular [Na+]-induced release of [3H]noradrenaline in rat hippocampal slices.

Our aim was to investigate the mechanisms involved in the high intracellular sodium-induced transmitter release in the CNS through the characterisation of the veratridine-evoked (40 microM) noradrenaline release from rat hippocampal slices. The response to veratridine was completely inhibited by tetrodotoxin (1 microM), indicating that the effect is due to the activation of sodium channels. Omission of Ca2+ from the superfusion fluid inhibited the veratridine-evoked release by 72%, showing that the majority of release results from external Ca2+-dependent exocytosis. The residual Ca2+-independent release was not blocked by the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester (100 microM) suggesting that intracellular Ca2+ stores are not involved in this component of veratridine effect. The noradrenaline uptake blockers, desipramine (10 microM) and nisoxetine (10 microM), inhibited the external Ca2+-independent release by 50 and 46%, respectively, indicating that the release partly originates from the reversal of transporters (carrier-mediated release). In contrast to uptake blockers, lowering the temperature, another possibility to inhibit transporter function, completely inhibited the effect of veratridine in the absence of Ca2+. Further experiments revealed that low temperature (20 and 12 degrees C) reduces the veratridine-induced increase of intracellular sodium concentration ([Na+]i) in rat cortical synaptosomes (68 and 78% inhibition, respectively). The clinical relevance of our data is that during ischemia a massive release of transmitters occurs mainly due to the elevation of [Na+]i, which contributes to the development of ischemic brain injury. Our results show that low temperature may be a better therapeutic approach to the treatment of ischemia because it has a dual action on this process. Firstly, it inhibits the function of uptake transporters and hence reduces the carrier-mediated outflow of transmitters. Secondly, it inhibits the sodium influx and therefore prevents the unwanted elevation of [Na+]i. Our data also suggest that veratridine stimulation can be a suitable model for ischemic conditions.

Identification and localization of aquaporin water channels in human salivary glands.

Aquaporin (AQP) water channels are expressed in a variety of fluid-transporting epithelia and are likely to play a significant role in salivary secretion. Our aim was to identify and localize the aquaporins expressed in human salivary glands. Total RNA was extracted from human parotid, submandibular, sublingual, and labial glands and from human brain. Expression of aquaporin mRNA was assessed by RT-PCR using specific primers for human AQP1, AQP3, AQP4, and AQP5. All four aquaporins were detected by RT-PCR in all of the glands, and the sequences were confirmed after further amplification with nested primers. Cleaned PCR products were then used as (32)P-labeled cDNA probes in a semiquantitative Northern blot analysis using glyceraldehyde-3-phosphate dehydrogenase as reference. Only AQP1, AQP3, and AQP5 mRNAs were present at significant levels. AQP localization was determined by immunohistochemistry on paraffin sections using affinity-purified primary antibodies and peroxidase-linked secondary antibodies. Each salivary gland type showed a broadly similar staining pattern: AQP1 was localized to the capillary endothelium and myoepithelial cells; AQP3 was present in the basolateral membranes of both mucous and serous acinar cells; AQP4 was not detected; and AQP5 was expressed in the luminal and canalicular membranes of both types of acinar cell. We conclude that AQP3 and AQP5 together may provide a pathway for transcellular osmotic water flow in the formation of the primary saliva.