Young Wha Moon

Histamine H1 Receptor Induces Cytosolic Calcium Increase and Aquaporin Translocation in Human Salivary Gland Cells

One of the common side effects of antihistamine medicines is xerostomia (dry mouth). The current consensus is that antihistamine-induced xerostomia comes from an antimuscarinic effect. Although the effect of antihistamines on salivary secretion is both obvious and significant, the cellular mechanism whereby this happens is still unclear because of the lack of knowledge of histamine signaling in human salivary glands. Here, we have studied histamine receptors and the effect of antihistamines on human submandibular acinar cells. In primary cultured human submandibular gland and a HSG cell line, histamine increased the intracellular Ca2+ concentration. The histamine-induced cytosolic free Ca2+ concentration ([Ca2+]i) increase was inhibited by histamine H1 receptor-specific antagonists, and the expression of the functional histamine H1 receptor was confirmed by reverse transcription–polymerase chain reaction. Interestingly, histamine pretreatment did not inhibit a subsequent carbachol-induced [Ca2+]i rise without “heterologous desensitization.” Chlorpheniramine inhibited a carbachol-induced [Ca2+]i increase at a 100-fold greater concentration than histamine receptor antagonism, whereas astemizole and cetrizine showed more than 1000-fold difference, which in part explains the xerostomia-inducing potency among the antihistamines. Notably, histamine resulted in translocation of aquaporin-5 to the plasma membrane in human submandibular gland cells and green fluorescent protein-tagged aquaporin-5 expressing HSG cells. We found that histidine decarboxylase and the histamine H1 receptor are broadly distributed in submandibular gland cells, whereas choline acetyltransferase is localized only at the parasympathetic terminals. Our results suggest that human salivary gland cells express histamine H1 receptors and histamine-synthesizing enzymes, revealing the cellular mechanism of antihistamine-induced xerostomia.

Involvement of apoptosis and calcium mobilization in tetrahydrobiopterin-induced dopaminergic cell death☆

Parkinsons disease is a neurodegenerative disorder associated with selective loss of the dopaminergic neurons in the substantia nigra. We have previously shown that tetrahydrobiopterin (BH4), the obligatory cofactor for dopamine synthesis, exerts selective toxicity on dopamine-producing cells. In the present study we determined, both in vitro and in vivo, whether the cell death induced by this endogenous molecule involves apoptosis, resembling that which occurs in Parkinsons disease. Transmission electron microscopic analysis revealed that the dopamine-producing CATH.a cells underwent ultrastructural changes typical of apoptosis, such as cell shrinkage and chromatin condensation, upon exposure to BH4. The BH4 treatment also caused intranuclear DNA fragmentation as determined by TUNEL staining. A similar phenomenon also occurred in vivo, as the nigral cells became TUNEL-positive upon injection of BH4 into the substantia nigra. The BH4-induced CATH.a cell death seemed to involve macromolecule synthesis because cycloheximide and actinomycin D had protective effects. Concurrent treatment with the caspase inhibitor Z-VAD-FMK also suppressed cell death. BH4 treatment led to increases in the ratio of Bax/Bcl-x(L) mRNA and protein levels. Ca(2+) seemed to play a role in BH4-induced cell death, because BH4 caused an increase in Ca(2+) uptake and the intracellular Ca(2+) release blocker dantrolene, intracellular Ca(2+) chelator BAPTA/AM, and extracellular Ca(2+) chelator EGTA each attenuated the toxicity. These data provide evidence that the dopaminergic cell death induced by BH4 involves apoptosis and suggest relevance of this cell death to degeneration of the dopaminergic system in Parkinsons disease.

Enhanced Expression of the Sweet Taste Receptors and Alpha-gustducin in Reactive Astrocytes of the Rat Hippocampus Following Ischemic Injury

The heterodimeric sweet taste receptors, T1R2 and T1R3, have recently been proposed to be associated with the brain glucose sensor. To identify whether sweet taste signaling is regulated in response to an ischemic injury inducing acute impairment of glucose metabolism, we investigated the spatiotemporal expression of the sweet taste receptors and their associated taste-specific G-protein α-gustducin in the rat hippocampus after ischemia. The expression profiles of both receptor subunits and α-gustducin shared overlapping expression patterns in sham-operated and ischemic hippocampi. Constitutive expression of both receptors and α-gustducin was localized in neurons of the pyramidal cell and granule cell layers, but their upregulation was detected in reactive astrocytes in ischemic hippocampi. Immunoblot analysis confirmed the immmunohistochemically determined temporal patterns of sweet-taste signaling proteins. These results suggest that the expression of sweet taste signaling proteins in astrocytes might be regulated in response to altered extracellular levels of glucose following an ischemic insult.

Capsaicin receptors are colocalized with sweet/bitter receptors in the taste sensing cells of circumvallate papillae.

We examined co-localization of vanilloid receptor (VR1) with sweet receptors T1R2, T1R3, or bitter receptor T2R6 in taste receptor cells of rat circumvallate papillae. Tissue sections of rat circumvallate papillae were doubly reacted with anti-VR1 antibodies and anti-T1R2, anti-T1R3 or anti-T2R6 antibodies, using double-immunofluorescence histochemistry technique. Localizations of VR1, T1Rs and T2R6 in the vallate taste cells containing α-gustducin were also examined. VR1 immunoreactivities (-ir) were observed in subsets of taste cells in the circumvallate papillae, and 96–99% of the vallate taste cells exhibiting T1R2-, T1R3- or T2R6-ir co-exhibited VR1-ir. Approximately half of T2R6-ir cells (~49%), and 50–58% of T1Rs-ir cells, co-exhibited α-gustducin-ir in the vallate taste buds. About 58% of VR1-ir cells in the vallate exhibited α-gustducin-ir as well. Results support the idea that capsaicin may interact with the transduction pathways of sweet and bitter taste stimuli, possibly in mediation of its receptor VR1 localized in taste receptor cells. Additionally, the partial co-localization of α-gustducin with VR1 suggests that a tentative modulatory function of capsaicin in sweet and bitter transductions in the rat circumvallate comprises of both α-gustducin-mediated and non-mediated transduction pathways.

Intra-oral pre-treatment with capsaicin increases consumption of sweet solutions in rats.

Abstract Sprague-Dawley rats received preference tests for sucrose or saccharin daily following oral treatment with 0.02% capsaicin. Consumed sweet solutions and preference scores increased in capsaicin-treated rats, compared to control rats on the second to fifth exposure period for sucrose and all exposure periods for saccharin. Chow intake was not affected by repeated treatment with capsaicin. Real-time RT-PCR analysis revealed decreased expression of sweet receptors T1R2 and T1R3 as well as capsaicin receptor VR1 in the circumvallate after this repeated oral exposure to capsaicin. VR1 immunoreactivities were also localized in the vallate taste cells by fluorescence immunohistochemistry. Results suggest that decreased expression of sweet receptors in the circumvallate may be related to increased sweet consumption in capsaicin-treated rats; any causal relationship should be further studied. Also, these data suggest that capsaicin may interact with a sweet transduction pathway in the mediation of its receptor VR1 that are located in the vallate taste cells.

Differential expression of the calcium-sensing receptor in the ischemic and border zones after transient focal cerebral ischemia in rats

G-protein-coupled calcium-sensing receptor (CaSR) has been recently recognized as an important modulator of diverse cellular functions, beyond the regulation of systemic calcium homeostasis. To identify whether CaSR is involved in the pathophysiology of stroke, we studied the spatiotemporal regulation of CaSR protein expression in rats undergoing transient focal cerebral ischemia, which was induced by middle cerebral artery occlusion. We observed very weak or negligible immunoreactivity for CaSR in the striatum of sham-operated rats, as well as in the contralateral striatum of ischemic rats after reperfusion. However, CaSR expression was induced in the ischemic and border zones of the lesion in ischemic rats. Six hours post-reperfusion there was an upregulation of CaSR in the ischemic zone, which seemed to decrease after seven days. This upregulation preferentially affected some neurons and cells associated with blood vessels, particularly endothelial cells and pericytes. In contrast, CaSR expression in the peri-infarct region was prominent three days after reperfusion, and with the exception of some neurons, it was mostly located in reactive astrocytes, up to day 14 after ischemia. On the other hand, activated microglia/macrophages in both the ischemic and border zones were devoid of specific labeling for CaSR at any time point after reperfusion, despite their massive infiltration in both regions. Our results show heterogeneity in CaSR-positive cells within the ischemic and border zones, suggesting that CaSR expression is regulated in response to the altered extracellular ionic environment caused by ischemic injury. Thus, CaSR may have a multifunctional role in the pathophysiology of ischemic stroke, possibly in vascular remodeling and astrogliosis.

Leptin blocks the fasting-induced increase of pERK1/2 in the paraventricular nucleus of rats

This study was conducted to define molecular mechanisms by which food deprivation increases phosphorylated extracellular signal-regulated protein kinase (pERK1/2) in the hypothalamic paraventricular nucleus of rats. pERK1/2 immunoreactivity (-ir) is markedly increased in the paraventricular nucleus by 48h of food deprivation. Treatment with RU486, glucocorticoid antagonists, during food deprivation did not affect the fasting-induced increase of pERK1/2-ir in the paraventricular nucleus, but intracerebroventricular (icv) leptin blocked the increase of pERK1/2-ir by food deprivation. Fasting-induced increases of neuropeptide Y (NPY) expression both in the arcuate nucleus and the paraventricular nucleus were also blunted by icv leptin; however, the icv NPY to satiated rats did not increase pERK1/2 in the paraventricular nucleus. These results suggest that the fasting-induced increase of pERK1/2 in the paraventricular nucleus may not be mediated either by plasma corticosterone or the hypothalamic NPY, but require leptin dis-inhibition.

5-hydroxy-L-tryptophan Suppressed Food Intake in Rats Despite an Increase in the Arcuate NPY Expression

This study was conducted to define the underlying mechanism of hypophagia induced by increased central serotonergic action. Rats received 3 daily injections of 5-hydroxy-L-tryptophan (5-HTP), a serotonin precursor, at a dose of 100 mg/kg/10 ml saline at 1 h before lights off. A significant suppression in food intake was observed shortly after the 5-HTP injection and persisted during 3 daily 5-HTP injections. Neuropeptide Y (NPY) expression in the arcuate nucleus increased after 3 days of 5-HTP treatment, as high as in the pair-fed group. Immunoreactivity of phosphorylated extracellular signal-regulated protein kinase (pERK1/2) in the hypothalamic paraventricular nucleus (PVN) was increased markedly by 3 days of 5-HTP treatment, but not by 3 days of pair-fed. mRNA expression levels of serotonin reuptake transporter (5-HTT) was increased in the dorsal raphe nucleus of the 5-HTP treated rats, but not in the pair-fed group. Results suggest that increased pERK1/2 in the PVN of 5-HTP injected rats may be a part of serotonergic anorectic signaling, perhaps blunting the orectic action of NPY; i.e., 5-HTP injected rats showed hypophagia despite of increased NPY expression in the arcuate nucleus.

Proteomic Analysis of Lithium-Induced Gene Expression in the Rat Hypothalamus

The hypothalamic proteomes were analyzed 1 and 6 hr after an intraperitoneal injection of lithium chloride or sodium chloride (0.15 M, 12 ml/kg). Results showed that expression of 14 and 32 proteomes was increased consistently by 1 hr and 6 hr of lithium treatment, respectively. Among them, tentative implications of glial fibrillary acidic protein, receptor-type protein tyrosine phosphatase, spectrin, and glutamate dehydrogenase in the lithium-induced activation of the hypothalamic-pituitary-adrenal axis, and conditioned taste aversion have been discussed. The proteomes listed in this study will provide, at least, a new insight to understand the molecular mechanism of lithiums action in the brain.

Cellular distribution of isozymes of protein kinase C in septal olfactory epithelium of mice

To determine the presence of protein kinase C (PKC) isozymes in the septal olfactory epithelium of mice (mSOE), western blotting and immunohistochemistry were performed using antibodies against PKC isozymes. With the exception of PKC-betaI, all of the PKC isozymes were detected in the whole lysate of septal tissue layer and apparent molecular weights for each isoform were found. PKC-alpha, PKC-gamma and PKC-epsilon were detected in the olfactory glandular cells of the lamina propria, and PKC-betaI and PKC-betaII were located in the microvillar cells. Neither novel PKC nor atypical PKC was detected in olfactory glandular cells or microvillar cells, except for PKC-epsilon. PKC-lambda was localized in the mucous layer of the mSOE. Meanwhile, PKC-delta and PKC-xi were distributed in the receptor cells in the mSOE. These data demonstrate the isoform-specific expression of PKC in mSOE and suggest a role for the novel and atypical types of PKC in olfactory transduction.