The [Ca2+] i was elevated by high concentrations of extracellular Ca2+ and by prototypic agonists of the CaSR such as gentamicin. The action of gentamicin was antagonized by inhibitors of phospholipase C (PLC), inositol trisphosphate (IP3) receptors, NSCCs, and, importantly, by the CaSR antagonist, NPS2390. Furthermore, the action of gentamicin was potentiated by activators of PLC and protein kinase C (PKC). This confirmed the pathway components mediating Ca2+ responses to a known agonist of the CaSR. We then investigated whether nifedipine (an L-type Ca2+ channel blocker) stimulates CaSRs to elevate [Ca2+] i via a similar mechanism. Nifedipine Ca2+ responses were dose-dependently blocked by NPS2390 and by the same inhibitors of PLC, IP3 receptors, and NSCCs that disrupted the action of gentamicin. Calphostin C (a PKC inhibitor) and TMB-8 (an inhibitor of Ca2+ release from stores) also inhibited the nifedipine-induced [Ca2+] i elevation.At first, we confirmed the existence of CaSRs in these cells by showing that [Ca2+] i elevation in gingival fibroblasts.These findings suggest that CaSRs are involved in the nifedipine-induced [Ca Inhibitors of nonselective cation channels i increase caused by isradipine, a dihydropyridine-derived Ca2+ channel antagonist,[3) receptor.[2+ channel antagonists like isradipine may elevate [Ca2+]i in gingival fibroblasts via the activation of NSCCs and mobilization of Ca2+ from intracellular stores.[Gingival overgrowth is known to be a common side effect of calcium  have been shown to inhibit the secretion of parathyroid hormone (PTH) and thus to affect serum PTH levels.[2+]i in parathyroid adenoma cells.i.[CaH levels. This mayma cells. A key me [Ca2+]i. This mec [Ca2+]i.2+ signaling pathways downstream of isradipine  and the CaSR (in the parathyroid gland), we postulated that CaSRs may be involved in Ca2+ channel blocker-activated Ca2+ signaling in gingival cells. We show here that nifedipine, the archetypal dihydropyridine-derivative calcium channel inhibitor, indeed acts via a CaSR pathway to elevate [Ca2+]i in gingival fibroblasts.In addition to the parathyroid gland, CaSRs arn -(3-trifluoromethylphenyl)-2, 4, 6-trimethylbenzenesulfonamide (m-3M3FBS), and 1- -trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione (U-73122) were obtained from Calbiochem . All other chemicals were supplied by Nacalai Tesque . These chemicals were dissolved in dimethyl sulfoxide (Sigma) as stock solutions, and thereafter, added in the perfusate.Tissue culture reagents were purchased from Gibco BRL  and fura-2/AM and EGTA were obtained from Dojindo Laboratories . Nifedipine, gentamicin, neomycin, spermine, lanthanum chloride, verapamil hydrochloride, phorbol 12-myristate 13-acetate (PMA), and quinoxaline-2-carboxylic acid adamantan-1-ylamide (NPS2390) were purchased from Sigma . 1-[β-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF-96365), 3 per cm2) were plated on fibronectin-coated glass cover slips adhered to a flexiperm disc . The medium was supplemented with 10% fetal bovine serum in a humidified atmosphere of 95% air and 5% CO2 at 37°C. The medium also contained antibiotics , and was changed at intervals of 2–3 days.Normal human gingival fibroblast Gin-1 cells were obtained from Dainippon Pharmaceutical Co. Ltd. . The cells were cultured for 3–6 days in Dulbecco’s modified Eagle medium . The cells . The cells were kept in a buffer consisting of 135 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 20 mM HEPES–NaOH (pH 7.4), and loaded with the dye by incubation in 5 µM fura-2/AM for 45 min at 37°C. Cells were then washed to remove excess fura-2/AM and then incubated in a fresh buffer (without fura-2/AM) for 15 min postincubation to allow intracellular cleavage of the acetoxymethylester conjugate (and thus activation) of fura-2. Excitation light from a xenon lamp was passed through a 340- or 360-nm filter. The emission wavelength for analysis was 510 nm. Changes in the fluorescence intensity of fura-2 in the cells were recorded with a video-imaging analysis system . [Ca2+]i was determined as the ratio of the fluorescence stimulated by excitation at 340 nm/360 nm, compared with a standard calibration curve obtained using Calcium Calibration Buffer Kit I .[Ca2+]i in the range of 50–200 nM.To minimize the leakage of fura-2, cells were kept at 32°C during fluorescence measurement using a bath temperature controller . The cells were soaked in a flexiperm chamber containing 0.5 ml of saline and were perfused at 8.0 ml/min with a tubing pump system . Drugs were added to the perfusate at appropriate concentrations. The time of treatment with CaSR agonists was 1 min. To ensure that fura-2 fluorescence was maintained within the linear range , we only selected analysis cells with the basal [CaN). Statistical analyses of the data were performed by Student’s two-sided paired t-test. Differences between the mean values were considered significant if the probability of error (P) was less than 0.05.Data are represented as the mean value ± standard error of the mean (SEM) and the number of observations (2+]i changes following the administration of a range of concentrations (2–10 mM) of extracellular Ca2+. 2+ dose-dependently elevates [Ca2+]i in these cells.Though the study was initially planned to carry out the CaSRs expressed in the Gin-1 cells used in this experiment, later the protocol was modified to evaluate i. We further tested the concentration-dependence of the response to verapamil. Increasing concentrations of verapamil  induced progressively larger [Ca2+]i elevations above basal . This demonstrates that known CaSR agonists mediate intracellular Ca2+ signaling in normal human gingival fibroblast Gin-1 cells.As shown in 2+]i changes by promoting both the release of Ca2+ from stores and influx through NSCCs. We investigated the effects of inhibitors of these pathways on the [Ca2+]i changes elicited by the CaSR agonist, gentamicin. We used the PLC inhibitor, U73122 (10 µM);i elevation in normal human gingival fibroblast Gin-1 cells was evaluated [2+]i elevation.The effect of NPS2390, a CaSR antagonist, on the gentamicin-induced [Cavaluated . Pretreavaluated  showed s2+]i elevation induced by 10 µM of nifedipine [2+]i elevation. A similar effect was observed with calphostin C (a PKC inhibitor)[2+ store release inhibitor).[2+]i elevation [2+]i elevation, in a concentration-dependent manner . There was no significant difference between the first and second responses, demonstrating that the effect of NPS2390 was not an artifact of, for example, the store depletion.Nifedipine (10 µM) also stimulated CaSRs, and U73122 (10 µM), xestospongin C (2 µM), and SKF-96365 (10 µM) significantly inhibited the i elevation may be due to the direct stimulation of the CaSR by nifedipine. We conducted the present study to investigate this hypothesis.In addition to their function in the parathyroid gland, CaSRs are expressed in several other tissues where they are involved in various cellular processes such as proliferation, differentiation, and hormone secretion. The stim2+ concentration causes a concomitant elevation of [Ca2+]i,[2+]i in the Gin-1 cells used in this study. Importantly, the CaSR antagonist, NPS2390,i, an effec [Ca2+]i, neomycin [Ca2+]i, spermine [Ca2+]i, LaCl3,[2 [Ca2+]i, all of w2+ channel antagonist, and evokes gingival overgrowth. Therefore, the concentration-dependent changes in [Ca2+]i in response to verapamil are further evidence of the fact that calcium channel blockers are able to activate CaSRs in gingival fibroblasts.Verapamil is an interesting member of this group of compounds in that it is a structural member of the phenylalkylamine family of compounds, which were an important intermediate in the development of calcimimetic drugs. In addit2+]i increases that arise from two distinct mechanisms: (a) mobilization of Ca2+ from intracellular stores  and (b) influx of extracellular Ca2+ through a Ca2+ -permeable NSCC.i, while PKC, under the control of DAG,i by activating CaSRs, leading to vasoconstriction. The blockade of this response by U73122 lends further support to the conclusion that the PLC–IP3 pathway plays a major role in signaling the downstream of the CaSR.The agonist-induced activation of the CaSR causes i elevation is due to CaSR stimulation. The most direct evidence for this was the efficacy of NPS2390 in inhibiting the nifedipine-induced [Ca2+]i elevation. NPS2390 has been used previously to confirm the presence of CaSRs, most recently by Cortijo et al.[2+]i responses via CaSRs in A549 human airway epithelial cells. The nifedipine-induced [Ca2+]i elevation was also blocked by many of the same pathway inhibitors that disrupted signaling in response to gentamicin, a known agonist of the CaSR, suggesting a similar mechanism of action for these two agonists.Next, we tested whether the nifedipine-induced [Cajo et al. who obseHere we present evidence that the CaSR is present in our Gin-1 cells and that nifedipine acts through it to mediate intracellular signaling events similar to those seen during gentamicin treatment. It may be necessary to perform molecular biological and immunohistochemical experiments to further elucidate the detailed relationship between nifedipine and CaSRs. However, our findings represent an important step in our understanding of how such calcium channel blockers induce fibroblast proliferation in the gingiva, and, consequently, gingival outgrowth.


Aim. To identify risk factors for urticaria, to determine the relative proportion of the susceptibility to urticaria that is due to genetic factors in an adult clinical twin sample, and to further determine whether the genetic susceptibility to urticaria overlaps with the genetic susceptibility to atopic diseases. Methods. A total of 256 complete twin pairs and 63 single twins, who were selected from sibships with self-reported asthma via a questionnaire survey of 21,162 adult twins from the Danish Twin Registry, were clinically interviewed about a history of urticaria and examined for atopic diseases. Data were analysed with Cox proportional hazards regression and variance components models. Results. A total of 151 individuals (26%) had a history of urticaria, whereas 24 (4%) had had symptoms within the past year. Female sex, HR = 2.09 (1.46–2.99), P = 0.000; hay fever, HR = 1.92 (1.36–2.72), P = 0.000; and atopic dermatitis, HR = 1.44 (1.02–2.06), P = 0.041 were significant risk factors for urticaria. After adjustment for sex and age at onset of urticaria in the index twin, the risk of urticaria was increased in MZ cotwins relative to DZ cotwins, HR = 1.42 (0.63–3.18), P = 0.394. Genetic factors explained 45% (16–74%), P = 0.005, of the variation in susceptibility to urticaria. The genetic correlation between urticaria and hay fever was 0.45 (0.01–0.89), P = 0.040. Conclusions. Susceptibility to urticaria is partly determined by genetic factors. Urticaria is more common in women, and in subjects with hay fever and atopic dermatitis, and shares genetic variance with hay fever. Urticaria is a common complex disease characterised by disseminated or local eruption of migrating itching wheals sometimes complicated by swelling of mucosal membranes; angioedema. Urticaria carries a lifetime risk of about 20% and is a troublesome disease that significantly impacts on quality of life. The mainstay of treatment of urticaria is nonsedating antihistamines. However, in many patients treatment with immunosuppressive drugs such as corticosteroids, azathioprine, cyclosporine, and biological therapies is necessary in order to obtain symptomatic control , 2.εRI) on mast cells and basophils [ Urticaria can be divided into acute and chronic types arbitrarily based on whether symptoms have been present for more than six weeks. Acute urticaria is usually triggered by allergic reactions to foods, insect venoms, or drugs, or it can be triggered by infections. However, in many cases the cause remains unknown. Likewise, chronic urticaria can be classified into several clinical and aetiological subtypes of for example allergic, autoimmune, physical, or idiopathic origin. Particularly, chronic urticaria that has no detectable cause is termed chronic idiopathic urticaria, whereas chronic autoimmune urticaria is present in those with antibodies against the high-affinity IgE receptor , and mould (Alternaria iridis and Cladosporium herbarium) allergens . Reactions were read after 15 minutes. A positive result was defined as a positive reaction to at least one of the allergens, and a reaction was considered positive if the mean wheal diameter was at least 3 mm. Serum total IgE was measured with an enzyme-linked immunosorbent assay (ELISA) on Immulite 2500 . Results were expressed as KIU/L. Skin prick tests were performed using standard dilutions birch, grass, mugwort, horse, dog, cat, house dust mite  as the underlying time, and zygosity, sex, and age at onset of urticaria in the index twin as covariates. In this analysis an increased hazard ratio (HR) in MZ twins relative to DZ twins would signal a genetic susceptibility to urticaria. Regression analyses were performed in SPSS 16.0 . Using the classical twin method , the varA total of 575 subjects  underwent the clinical examination . Characteristics of the studied subjects are given in P = 0.000; hay fever, HR = 1.92 (1.36–2.72), P = 0.000; and atopic dermatitis, HR = 1.44 (1.02–2.06), P = 0.041 were retained as significant risk factors for urticaria  of the population; 43 (17.9%) of the men and 108 (32.2%) of the women had a history of urticaria, whereas 24 (4.2%); 6 (2.5%) of the men and 18 (5.4%) of the women had had symptoms within the past year. None of the subjects had urticaria at the day of examination. Among subjects with a history of urticaria, the median age at onset was 18.6 years . After prticaria .P = 0.394, indicating that the variation in the susceptibility to urticaria is of genetic origin , P = 0.000 of atopic dermatitis, 40% (24–56%), P = 0.000 of hay fever, and 25% (13–37%), P = 0.000 of asthma. The genetic and environmental correlations, respectively, between urticaria and atopic dermatitis were −0.06 (−0.58–0.46), P = ns and 0.23 (−0.32–0.78), P = ns; between urticaria and hay fever were 0.45 (0.01–0.89), P = 0.040 and −0.15 (−0.16–0.47), P = ns; and between urticaria and asthma were −0.07 (−0.62–0.49), P = ns and −0.22 (−0.78–0.34), P = ns, indicating that urticaria shares some genetic variance with hay fever, but not with atopic dermatitis and asthma. Variance components analysis showed that genetic factors explained 45% (16–74%), This study provides one among just a few available estimates of the heritability of urticaria and to our knowledge is the only clinical investigation of the heritability of the disease; genetic factors were found to explain about half of the variation in the susceptibility to urticaria, whereas the other half could be ascribed to random nongenetic variation. The lifetime prevalence of urticaria in the population was 26%, whereas the 12-month prevalence was 4%. The disease was about two times more common in women than in men and in subjects with hay fever and atopic dermatitis. Furthermore, urticaria was found to share genetic variance with hay fever, but not with atopic dermatitis or asthma.  One of the clear strengths of this study is the systematic selection of subjects from a nationwide population of twins, and the subsequent collection of detailed clinical interview data on 575 adults. Urticarial rash is transient and is therefore often not present at the day of examination. Therefore, the diagnosis relies chiefly on patient recollection and on the capability of the interviewing physician to obtain the correct information about past symptoms. Although we collected data on the lifetime history of urticaria, we did not obtain information on the duration and specific exposures relating to the occurrence of symptoms, which limits our ability to distinguish between different subtypes of disease. Particularly, we had no information on food allergy, allergy to medications, or other factors that could have aided in the aetiological workup. Also, we did not record whether urticaria episodes were accompanied by allergic reactions such as angioedema, acute gastroenteritis, airway symptoms, or anaphylaxis. Neither did we record whether the subjects had had several or just one episode of the disease. Common physical urticarias such as symptomatic dermographism, delayed pressure urticaria and cholinergic urticaria, and chronic spontaneous urticaria are entirely different dermatoses which all manifest as clinically different types of hives. The nature and importance of genetic factors will predictably differ in these diverse subtypes. Unfortunately the clinical interview did not allow discrimination between these different forms of chronic urticaria. However, a large proportion of the studied subjects had known type 1 allergy and associated atopic disorders, which makes it likely that their episodes of urticaria had an allergic basis. We therefore consider the main type of urticaria encountered in our population acute type 1 allergy-mediated urticaria and not chronic urticaria. This was also supported by our finding that the 12-month and current prevalence of the disease was much lower than the cumulative lifetime prevalence, and that the cumulative lifetime prevalence in this population with atopic predisposition was slightly higher than what could be expected in the general population. Our results may therefore only be applicable to an allergic population and not to the population as a whole, although we used statistical methods to correct for this skewed ascertainment of individuals.  Urticaria and hay fever were genetically correlated to a sizable extent; 0.45. Interestingly, in congruence with our finding, the genetic correlation between urticaria and hay fever was also exactly 0.45 among Swedish children . In contHowever, the Swedish study was based on questionnaire responses while our study was based on confirmed clinical examination. A recent finding among a Han Chinese population showed that polymorphisms in the high affinity receptor of IgE are associated with atopic dermatitis but not with urticaria, indicating that these diseases may have different genetic aetiologies, at least in respect to certain genetic polymorphisms . We conclude that the susceptibility to urticaria is determined partly by genetic factors. Notably, half of all variance of urticaria susceptibility is ascribable to differences between subjects on a genetic level. Moreover, urticaria is more common in women and in subjects with hay fever and atopic dermatitis and has overlapping genetic features with hay fever. Future genetic studies distinguishing different subphenotypes of urticaria  could contribute to further unravelling of the aetiology of the disease.


Granulocytic sarcoma (GS) is a rare extramedullary solid tumor defined as an accumulation of myeloblasts or immature myeloid cells. It can cooccur with or precede the acute myeloid leukemia (AML) as well as following treated AML. The incidence of GS in AML patients is 3–8% but it significantly rises in M2 FAB subtype AML. This variety of AML harbors t in up to 20–25% of cases  and, at a molecular level, it is characterized by the generation of a fusion gene known as RUNX1-RUNX1T1. Approximately 10% of M2 AML patients will develop GS, as a consequence, the t and the relative transcript represent the most common cytogenetic and molecular abnormalities in GS. FLT3-ITD mutation was rarely described in AML patients presenting with GS. FLT3 ITD is generally strongly associated with poor prognosis in AML, and is rarely reported in patients with t. GS presentation is extremely variable depending on organs involved; in general, cranial bones and sinus are very rarely affected sites. We report a rare case of GS occurring as a recurrence of a previously treated t, FLT3-ITD positive AML, involving mastoid bones and paravertebral tissues. FLT3 internal tandem duplication (ITD) mutation in AML patients presenting with GS. FLT3 ITD is generally strongly associated with poor prognosis in AML and is rarely reported in patients with t [Granulocytic sarcoma (GS) is a rare extramedullary solid tumor defined according to the 2008 WHO classification as an accumulation of myeloblasts or immature myeloid cells. It can cooccur with acute myeloid leukemia (AML) or precede its diagnosis as well as following previously treated AML as extramedullary isolated manifestation of relapse , 2. The  t –7. GS is t . GS clin2, days 1–3), etoposide , and cytosine arabinoside . Upon full hematologic recovery after induction chemotherapy, a morphologic complete remission (CR) was documented by bone marrow aspirate. Consolidation therapy consisted in three courses of chemotherapy associating daunorubicin  and cytosine arabinoside . Serial bone marrow (BM) aspirates, performed after each consolidation course, confirmed the condition of morphologic CR; however, quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) demonstrated the persistence of RUNX1-RUNX1T1 fusion transcript either after induction or consolidation cycles. In April 2012, after 9 months of continuous CR, the patient was admitted to the emergency department for headache and left ear pains. A cranial magnetic resonance imaging (MRI) indicated the presence of a fluid component in the left mastoid, therefore a diagnosis of otomastoiditis was posed and a broad spectrum antibiotic therapy was instituted. At the same time, a BM reevaluation confirmed the morphological CR and the persistence RUNX1-RUNXT1 transcript. In spite of the administration of antibiotic therapy, symptoms did not resolve, and in May 2012, the patient was again admitted to the emergency department for a VII facial nerve palsy. On clinical examination, the patient had fever and showed left retroauricular swelling and pain, as well as a painful bulging of the upper and posterior walls of the external auditory canal. The eardrum appeared opaque and swollen at the otoscopy. According to the House-Brackmann scale [2 twice a day, days 1, 3, 5, and 7) associated with an intrathecal injection of cytosine arabinoside (50 mg flat dose) given before systemic therapy and following full hematopoietic resumption to normal blood cell count after chemotherapy. Since a PET/CT scan performed on day 26 after chemotherapy showed a substantial stability of the radiologic picture, we made a decision to carry out a biopsy of the lumbo-octurator mass and a hysterectomy, whereas uterus mass biopsy was excluded due to the very high risk of procedure-related bleeding. The histological examination was consistent with a diagnosis of paravertebral GS and uterine fibroma. Once again bone marrow aspiration confirmed the morphologic CR. Based on the prolonged hematologic CR with a persistent condition of extramedullary disease, the patient was addressed to a program of radiotherapy to irradiate the left mastoid and the spine mass. Radiotherapy was initiated after one month from the end of chemotherapy, and the patient received a total dose of 2200 cGy on the mastoid and of 2800 cGy on the spine mass. In September, before completing the radiotherapy program, a routine blood cell count showed a decrease of platelets count, therefore a BM aspirate was performed. At that stage, the morphologic picture was consistent with a full AML relapse. The patient was then addressed to an additional regimen of chemotherapy in the attempt to induce a second morphologic CR.In July 2011, a 42-year-old female was diagnosed with a M2 FAB subtype AML. Karyotypic and molecular studies revealed that this AML carried t and was FLT3-ITD positive. For this reason, the patient received a 3 drug-based induction regimen including daunorubicin (45 mg/mBurns described the first case of granulocytic sarcoma (GS)—also called chloroma—in 1823, while the first case of Acute Myeloid Leukemia (AML) associated GS was reported in 1903 by Turk who, therefore, suggested the same origin for both of the tumors . ExtrameThe peculiarity of our case consists in the unusual site of localization of the GS and its association with an AML carrying both t8;21) and FLT3 ITD mutation. In AML, FLT3-ITD mutations appear to be associated with an increased risk of relapse, short duration of disease free, and overall survival  and FLT3. The incIn this view, physicians should be aware that imaging diagnostic techniques, such as CT and MRI, might not be conclusive so that a definitive diagnosis could be hard to make outside a surgical scenario. Moreover, a complete as possible disease staging, also including PET and/or TB-CT, should always be attempted to identify additional, asymptomatic, extra-medullary localizations. Patients with GS are treated in the same way as patients with AML independently from bone marrow involvement. Infact extra-medullary disease should be considered a systemic disease and treated with a double therapeutic approach including systemic chemotherapy and additional intrathecal chemotherapy in case of SNC involvement or localized radiation therapy to increase the effectiveness on tumor masses. Chemotherapy is considered the best therapeutic option also for patients with pseudomastoiditis, facial palsy, and GS-related hearing loss. Surgery other than biopsy, should be avoided since surgical decompression of the facial nerve and handling of the middle ear ossicular chain offer a worse outcome than an appropriate chemotherapeutic. In our case, GS did not respond to high dose chemotherapy, therefore we made decision to irradiate the persistent mastoid and paravertebral masses considering that the chemotherapy failure might have been caused not only by chemoresistance but also by a problematic vascular access of drugs to the involved sites. In fact, even though chemotherapy plays an important role in the control of bone marrow disease, it could be inefficient to reach extra-medullary sites which are considered “sanctuaries”. In our case, radiotherapy would have also served as a bridge to allogeneic stem cells transplant to prevent bone marrow relapse. Shimizu et al.  analyzed


C. elegans, whereas GPR26 deficiency in the hypothalamus is associated with high genetic susceptibility to the onset of obesity in mice. However, the metabolic function of GPR26 in mammals remains elusive. Herein, we investigated a role of GPR26 in regulating energy homeostasis by generating mice with targeted deletion of the GPR26 gene. We show that GPR26 deficiency causes hyperphagia and hypometabolism, leading to early onset of diet-induced obesity. Accordingly, GPR26 deficiency also caused metabolic complications commonly associated with obesity, including glucose intolerance, hyperinsulinemia, and dyslipidemia. Moreover, consistent with hyperphagia in GPR26 null mice, GPR26 deficiency significantly increased hypothalamic activity of AMPK, a key signaling event that stimulates appetite. In further support of a regulatory role of GPR26 in satiety, GPR26 knockout mice also demonstrate hypersensitivity to treatment of rimonabant, an endocannabinoid receptor-1 antagonist commonly used to treat obesity by suppressing appetite in humans. Together, these findings identified a key role of GPR26 as a central regulator of energy homeostasis though modulation of hypothalamic AMPK activation.G-protein coupled receptor 26 (GPR26) is a brain-specific orphan GPCR with high expression in the brain region that controls satiety. Depletion of GPR26 has been shown to increase fat storage in  Obesity and its associated metabolic diseases represent the most common health risks in developed nations, and have emerged as a major health issue in many developed countries due to sedative life style and consumption of Western diet enriched with animal fat 5A and gastrin releasing hormone BB2 receptor, suggesting a possible role in regulating energy homeostasis. In support of this hypothesis, GPR26 is most abundantly expressed in ventromedial hypothalamic nucleus and cortex C. elegans resulted in increased body fat storage Although the etiology of obesity is poorly understood, it has been realized that central in the pathogenesis of obesity is a chronic positive energy balance resulted from increased caloric intake or/and decreased energy expenditure. The neuro-endocrine system plays a pivotal role in regulation of energy homeostasis, in which G protein-coupled receptor (GPCR) pathways are increasingly discovered to be an important modulator GPR26 gene. We show that mice with GPR26 deficiency exhibit hyperphagia and decreased energy expenditure, leading to high propensity to diet-induced obesity and its related metabolic complications. Consistent with the findings, GPR26 deficiency significantly increased phosphorylation of AMPK at ser172, a major activation site that is implicated in hyperphgia and onset of obesity. Our findings identified for the first time a key role of GPR26 in energy homeostasis, suggesting that targeting GPR26 with chemical compounds may provide a novel treatment for obesity thought modulation of appetite.In the study, we investigate a possible role of GPR26 in energy homeostasis by generating mice with targeted deletion of the in vivo, we generated mice deficient in GPR26 expression by homologous recombination in ES cells. We engineered a targeting vector to replace the first exon of the coding sequence of GPR26 with a neomycin resistance cassette . However, after feeding on the high-fat diet which contains 40% calories from animal fat for 12 consecutive weeks, the weight gain in GPR26−/− mice (KO) was significantly higher in female . As shown by −/− mice during the 12 weeks of high fat feeding when compared with the wild type controls, implicating a role of GPR26 in regulating appetite. In contrast, there were no significant differences in accumulated food intake between the male GPR26−/− mice and the same gender wild type controls  equipped with an open circuit calorimetry system (Oxymax) −/− mice.Hyperphagia and increased adiposity in GPR26−/− mice on a high-fat diet. As shown in −/− mice, suggesting hyperinsulinemia in GPR26 knockout mice. Ghrelin is a gastric peptide that stimulates food intake, and circulating ghrelin levels are decreased by the onset of human obesity −/− mice , and an animal protocol (protocol number: #2006-053) was approved by the Institutional Animal Care and Use Committee (IACUC) of Penn State University College of Medicine for this study.All experiments were performed in F2 (C57BL/6 background) mice. Mice were maintained on a 12-hour light and 12-hour dark cycle. The animals were individually housed at 7 weeks of age and received standard mouse chow  or high fat-chow . Body weight and food consumption were measured once a week. Fat and lean body masses were analyzed by quantitative nuclear magnetic resonance (QMR) using an Echo System instrument , as previously described EcoR V fragment that contains the 5′ promoter region and a 9.2-kb EcoR I fragment that contains genomic sequences downstream from the first exon of GPR26  and neo6R (catagccgaatagcctctcc) for targeting at the 5′ end. A PCR product of ∼3.8 kb was generated in heterozygous and knockout mice. The primer pair neo1746F (cctgccatagcctcaggttactc) and 52595r (ctgacccagagctcagtggagcagc) was used to check for targeting at the 3′ end. A PCR product of ∼9.2 kb was generated in heterozygous and knockout mice. The positive founder mice were further confirmed by Southern blot using a 3′ external probe on EcoR I digested DNA, which yielded a predicated 10.5 kb band for wild type and 12.5 kb band for the mutant allele.A targeting vector was engineered by inserting a 3.2-kb of GPR26 . The tarMice on high-fat diet with same gender, age, and genetic background (wild type or GPR26 knockout) were randomized into treatment and control groups respectively. The animals were daily administrated by oral gavage with 10 mg of rimonabant, also known as SR141716A, a CB1 antagonist, per kg body weight formulated in 100 µl sodium carboxymethylcellulose (NaCMC). The same volume of NaCMC was utilized as the vehicle control. Body weight and food intake were monitored daily for 20 consecutive days.OGTT was initiated by orally administrating 2.0 g of glucose per kg of body weight. Mouse tail blood samples were collected at the time points of 0, 30, 60, 120 minutes and analyzed for blood glucose levels by using a ACCU-CHEK Blood Glucose Meter .Body fat and lean body mass were measured using LF90 TD-NMR (Bruker Optics). Measurements of food intake, energy expenditure, and respiratory exchange ratio were performed using metabolic cages equipped with a comprehensive lab animal monitoring system (CLAMS) . Constant airflow (0.4 l/min) was drawn through the chamber and monitored by a mass-sensitive flow meter. The concentrations of oxygen and carbon dioxide were monitored at the inlet and outlet of the sealed chambers to calculate oxygen consumption and respiratory quotient (RQ). Each chamber was measured for 1 min at 15 min intervals.Mice were fasted overnight and heart blood was drawn into both heparinized and EDTA tubes on ice. Following centrifugation at 2000 rpm for 5 minutes to obtain plasma, the concentrations of blood triglyceride and cholesterol were determined using the Roche/Hitach 912 automatic analyzer system. Insulin, leptin, ghrelin and adiponectin were measured by Linco Research .The mouse hypothalamus and liver tissues were rinsed twice in PBS and homogenized in a lysis buffer containing 1% Triton X-100 and protease inhibitors. The protein content was determined using a bicinchoninic acid protein assay kit . Proteins (20 µg) were separated by SDS-PAGE on 10% polyacrylamide gels and then transferred to nitrocellulose membrane. Following blocking of nonspecific binding by 5% nonfat milk, the membranes were probed with a phosphor (p)-AMPK (Thr 172) antibody  at 4°C for 12 hours. The membranes were then washed and incubated with HRP-conjugated, goat anti-Rabbit IgG  and developed using western blot chemiluminescence reagent . Following demonstration of p-AMPK, the nitrocellulose membranes were stripped by treatment with a stripping buffer (Pierce) for 30 min and total AMPK level was detected in the same membranes using a rabbit anti-AMPK antibody . Densitometry was performed for standardization of the phosphorylated AMPK isoform relative to the total AMPK.t-test to determine difference between GPR26−/− and wild type mice. Analysis of covariance (ANCOVA) was also used to assess differences between GPR26−/− and wild type mice in metabolic parameters relative to total body weight and lean mass. Values are presented as means ± SEM; p<0.05 was considered significantly different.Statistical comparisons were done using two-tailed non-paired


This current undergoes marked changes in expression during chicken hair cell development. Although the properties of IA are not normally classified as Ca2+-dependent, we demonstrate that throughout the development of chicken hair cells, IA is greatly reduced by acute alterations of intracellular Ca2+. As determinants of spike timing and firing frequency, intracellular Ca2+ buffers shift the activation and inactivation properties of the current to more positive potentials. Our findings provide evidence to demonstrate that the kinetics and functional expression of IA are tightly regulated by intracellular Ca2+. Such feedback mechanism between the functional expression of IA and intracellular Ca2+ may shape the activity of spontaneous action potentials, thus potentially sculpting synaptic connections in an activity-dependent manner in the developing cochlea.Spontaneous action potentials have been described in developing sensory systems. These rhythmic activities may have instructional roles for the functional development of synaptic connections. The importance of spontaneous action potentials in the developing auditory system is underpinned by the stark correlation between the time of auditory system functional maturity, and the cessation of spontaneous action potentials. A prominent K Structured spontaneous action potentials (SAPs) play an instructive role in the survival and refinement of neuronal connections in developing sensory systems 2+-dependent 2+ (Ca2+i) 2+i mediate the expression of K+ channels that modulate SAPs, or influence the gene expression that may refine neuronal connections + currents (IA) regulate spike timing and firing frequency in neurons A is not known to be Ca2+-sensitive, in neurons and myocytes, the kinetics and expression have been associated with changes in Ca2+i via channel protein interactions with Ca2+-sensitive proteins 2+ homeostasis and IA expression are unknown but the correlation of the feedback between Ca2+i and Ca2+-sensitive processes, such as the expression of IA, can have important ramifications in Ca2+ oscillations and wave propagation in developing HCs Spontaneous activity in developing HCs is CaA and its possible sensitivity to Ca2+i handling. Chelation of Ca2+i shifted the activation properties of the current to more positive potentials and reduced the expression of IA. Our findings provided direct evidence to demonstrate that IA is tightly regulated by Ca2+i, a feedback mechanism that may shape the patterning of SAPs and ultimately sculpt synaptic connections in the developing cochlea.In order to better understand the mechanisms of these spontaneously generated action potentials, we investigated the functional expression of I2, 2 MgCl2, 5 Hepes, and 3 glucose, pH 7.4. The tegmentum vasculosum and the tectorial membrane were removed without any prior enzymatic treatment using a fine minutia needle. Chicken basilar papillae were stored in a 37°C incubator in Minimum Essential Medium (Invitrogen) before recording from HCs in situ. All experiments were performed at room temperature (21–23°C) within 5–45 mins of isolation. All reagents were obtained from Sigma Chemicals, unless specified otherwise.The present investigation was approved in accordance with the guidelines of the Institutional Animal Care and Use Committee of University of California, Davis. The protocol number was 15544 under the institutional authorization code A3433-01. This study included chickens at different stages of embryonic development ranging from E6–E21 as well as post-hatched chickens. Fertilized eggs were incubated at 37°C in a Marsh automatic incubator (Lyon Electric). Before experiments, chicken embryos were killed and staged according to the following: from E8–E12: based on visceral arches, feather gems and eyelids: and after E12 based on the length of the beak + currents were recorded in a whole-cell voltage-clamp configuration, using 2–3 MΩ resistance pipettes. Currents were amplified with an Axopatch 200B amplifier  and filtered at a frequency of 2–5 kHz through a low-pass Bessel filter. The data was digitized at 5–500 kHz using an analog-to-digital converter . The sampling frequency was determined by the protocols used. No online leak current subtraction was made, and only recordings with holding currents less than 50 pA were accepted for analyses. The liquid junction potentials were measured  and corrected online K2 1, CaCl2 0–2, D-glucose 10, Hepes 10, pH 7.3. For whole-cell recordings of action potentials we used pipette solution containing (in mM): KCl 130, Hepes 10, D-glucose, 5 KATP, 2–10, EGTA or BAPTA. Regarding perforated patch experiments, the tips of the pipettes were filled with the internal solution containing (in mM): KCl 150, Hepes 10, D-glucose 10, pH 7.3. The pipettes were front-filled with the internal solution and back-filled with the same solution containing 250-µg/ml amphotericin. The stock solutions of all toxins were made either in ddH2O or DMSO and stored at −20°C.Action potentials were amplified (100×) with an Axopatch 200B amplifier (Axon Instruments) and filtered at 2–5 kHz through a low-pass Bessel filter. The data were digitized at 5–500 kHz using an analog-to-digital converter . The sampling frequency was determined by the protocols used. Action potentials were recorded using extracellular solution containing (in mM) NaCl 145, KCl 6, MgClA, we eliminated Ca2+ from the bath and used 20 mM TEA and 300 nM apamin to block K+ currents, such as the delayed rectifier and Ca2+-activated K+ currents (IK(Ca)). Extracellular solution contained (in mM) NaCl 125, KCl 6, CaCl2 0, 20 TEA, D-glucose 10, MgCl2 1, HEPES 10, pH 7.3, 310 mOsm. Intracellular solution contained (in mM) KCl 120, Na2ATP 5, MgCl2 2, HEPES 10, EGTA 1–10, or BAPTA 1–10, D-glucose 10, pH 7.3, 300 mOsm.To record I0 is the initial current magnitude, τ1, τ2…τn are the time constants, and A1, A2…An, are the proportionality constants. Voltage-dependence of activation was examined from peak amplitude (measured at ∼3–5 ms from the onset of voltage step) of tail currents at different developmental stages  day-six to postnatal (P) day-two (E6-P2), then normalized steady-state curves were fitted with the Boltzmann distribution. Additionally, the steady-state inactivation curve was generated from normalized currents measured at a test potential following several conditioning pre-pulses. Pooled data were presented as mean ± SD. Significant differences between groups were tested using the Student's t test, with p<0.05 or 0.01 indicating statistical differences.The number of cells (n) is given for each data set. Data were analyzed using pClamp8 (Molecular Devices), Origin7.0  and Excel (Microsoft). Time constants (τs) were obtained from fits using Origin software. Time constants were obtained by fitting multiple exponential terms to the activation and decay of the current. The equation was of the form:A on SAPs in developing HCs, we employed pharmacological strategies +-current (IA) blocker, to spontaneously active HCs from the developing basilar papilla. Shown in p<0.01; n = 7). Similarly, basal HCs at E12 fire APs at the rate of ∼0.5 Hz . Moreover, after application of 4-AP, the frequency of firing increased to ∼2 Hz . These experiments were performed using 2 mM BAPTA in the pipette solution to mimic physiological buffering capacity 2+-dependent 2+ (Ca2+i) in 4-AP-mediated changes in AP firing rates, we used 10 mM pipette-BAPTA. Surprisingly, the typical phasic and burst pattern seen in E12 apical HCs (versus pipette-BAPTA concentrations are shown (A) may be sensitive to Ca2+I availability. Additionally, the data hinted that IA regulates the firing pattern of developing HCs in a Ca2+I-dependent manner. The effects of BAPTA and 4-AP on individual action potentials are outlined in To examine the roles of Iical HCs  was replical HCs . Even more shown . The corA during development, and tonotopic expression of the current could, in part, be responsible for diverse spiking activity  of inactivation was ∼−56 mV for apical and ∼−59 mV for basal cells. By E18, the transient outward current in basal HCs inactivated at more negative potentials than apical HCs  ∼0 and 23 using (in mM) 2 and 10 BAPTA, respectively. Moreover the V1/2s of the steady-state inactivation were ∼−56 mV and −48 mV using (in mM) 2 and 10 BAPTA, respectively. Additionally, we examined the time-dependence of the development of inactivation after varying durations at −50 mV and −90 mV. The time constants of inactivation (τI) were compared at different stages of development and with respect to different concentrations of Ca2+I buffer  of IA were ∼5 ms and 75 ms, using 2 mM BAPTA. Using 10 mM BAPTA the τs were ∼75 ms and 300 ms  in the developing chicken HC was identified functionally by Murrow (1994) and Griguer and Fuchs (1996) A in developing chicken HC is unknown, despite the currents purported role in spike timing in SAPs. Also important, roles of IA and its regulation and the ensuing effects on the structure of SAPs in developing HCs are unclear. This study fills in major gaps in our understanding of the patterning of SAPs in HCs. Our findings include; 1) IA contributes towards the patterning of SAPs. Since the current dictates the spike timing and firing pattern, it is conceivable to suggest that it may regulate the release of neurotransmitters in the developing cochlea to sculpt synapse formation. 2) Expression of IA is regulated developmentally and tonotopically. At the apical aspects of the basilar papilla the expression of IA is prominent only in early development. By contrast, IA persists in HCs at the basal aspects of the matured basilar papilla. 3) Alterations of Ca2+I by Ca2+ buffers produced marked effects on the functional expression, voltage-dependence and kinetics of IA. Increased concentrations of the Ca2+I buffer, BAPTA shifted the voltage-dependence of activation and inactivation to more positive potentials and reduced the number of functional channels expressed. These findings reaffirm the tight interplay that occurs between Ca2+I, and K+ currents.The transient outward KA visibly alters spike timing and frequency, transforming phasic action potential burst into tonic firing in spontaneously active chicken HCs. The results are reminiscent of the effects of a transient outward current in the developing mouse HC 2+ buffer, not only did the phasic patterning of spikes appear tonic, but also suppression of IA had no effect on the firing pattern, raising the possibility that reduced Ca2+I could stifle the availability of IA. The deduced amino acid sequence of the α-subunits of Kv1.2, Kv1.3 and Kv1.5 and the auxiliary subunit, Kvβ1 that confer A-type transient current reveals multiple putative phosphorylation sites at the N- and C-termini 2+-dependent. For example, there is a Src tyrosine kinase proline-rich binding site at the N-terminal of Kv1.5 that confers Ca2+-dependent modulation of the kinetics and voltage-dependent activation of transient K+ currents in Schwann cells 2+-dependent regulatory sites at the C-terminus of the channel and other interacting partners that form the channel complex could affect the localization, expression and specific functional properties of the native K+ conductance, as observed in cardiac myocytes and neurons Suppression of I2+ handling in the developing chicken basilar papilla may undergo marked plasticity to account for varying demands for the expression of Ca2+-dependent processes and increased Ca2+ influx through increased transduction channel numbers 2+ buffer, calbindin, is regulated during development and along the tonotopic axis of the cochlear in accordance with the demands' of Ca2+ regulation 2+ regulation can be seen in the apico-basal gradient of the basilar papilla 2+ buffers during development dovetails well with the expression pattern of Ca2+ buffers 2+ buffers in HCs of the developing basilar papilla and our findings, the kinetics and voltage-dependence of IA are expected to be altered. Voltage dependence of the activation and inactivation of IA are shifted to more positive potentials with increased Ca2+ buffers. Throughout development, basal HCs express IA of faster kinetics of inactivation than at the apical aspects of the basilar papilla. Moreover, with maturation this difference is magnified as apical HCs lose the transient component of K+ currents. At the basal aspects, the ratio of IA to the total outward K+ current is diminished with maturation. Whereas these findings are in accordance with changes in Ca2+ handling in the chicken basilar papilla, they are in stark contrast to reports on the expression of 4-AP sensitive current, presumably IA, in the developing mouse HC, where the expression of the current increases with development and persists in mature HCs 2+I availability has profound effects not only on the kinetics of IA and the number of channels expressed in HCs, but also on the ensuing SAP, which is sculpted by the current.Ca


Lowering [Ca2+]o paradoxically enhances ADP-evoked aggregation, an effect that has been attributed to enhanced thromboxane A2 production. This study examined the role of ectonucleotidases in the [Ca2+]o-dependence of platelet activation. Reducing [Ca2+]o from millimolar to micromolar levels converted ADP (10 μmol/l)-evoked platelet aggregation from a transient to a sustained response in both platelet-rich plasma and washed suspensions. Blocking thromboxane A2 production with aspirin had no effect on this [Ca2+]o-dependence. Prevention of ADP degradation abolished the differences between low and physiological [Ca2+]o resulting in a robust and sustained aggregation in both conditions. Measurements of extracellular ADP revealed reduced degradation in both plasma and apyrase-containing saline at micromolar compared to millimolar [Ca2+]o. As reported previously, thromboxane A2 generation was enhanced at low [Ca2+]o, however this was independent of ectonucleotidase activity. P2Y receptor antagonists cangrelor and MRS2179 demonstrated the necessity of P2Y12 receptors for sustained ADP-evoked aggregation, with a minor role for P2Y1. In conclusion, Ca2+-dependent ectonucleotidase activity is a major factor determining the extent of platelet aggregation to ADP and must be controlled for in studies of P2Y receptor activation.ADP is considered a weak platelet agonist due to the limited aggregation responses it induces  However, ADP is the only platelet agonist that stimulates Gαi signalling at physiological concentrations and is therefore an essential co-stimulus to achieve full functional responses for all known platelet agonists , ectonucleotide pyrophosphatase/phosphodiesterases (E-NPPs), alkaline phosphatases and 5′ nucleotidase . ADP der2+-dependent ectonucleotidases is an important factor in determining the amplitude and duration of platelet aggregation. The results have consequences for understanding the effectiveness of ADP as a platelet agonist, and in the selection of experimental conditions to explore P2Y receptor activation.In this study we have investigated further the mechanism(s) underlying the differential responses to ADP at physiological compared to low (micromolar) extracellular calcium concentrations. We demonstrate that degradation of ADP by Ca2 (TXB2) assay kits were purchased from Cambridge Bioscience LTD . Cangrelor (ARC-69931MX) was a kind gift from AstraZeneca . ADP, apyrase (gradeVII), aspirin, phosphoenolpyruvate and all other chemicals were purchased from Sigma .CHRONO-LUME was purchased from Labmedics , Pyruvate kinase was obtained from Roche Diagnostics Limited , GF109203X and MRS2179 were purchased from Tocris . Thromboxane Bv/v. Platelet-rich plasma (PRP) was obtained by centrifugation at 700 g for 5 min. When re-calcified, 20 mmol/l CaCl2  to the micromolar range  within approximately 2 min (2+]o-dependent aggregation response showed that production of TXA2 was enhanced at micromolar compared to millimolar [Ca2+]o levels and concluded that secondary stimulation of TXA2 receptors is responsible for the reversible nature of the ADP-evoked aggregation (2+]o on aggregation when TXA2 synthesis was blocked by aspirin (2+ levels respectively). In platelets resuspended in a physiological saline with apyrase, 10 μmol/l ADP evoked a transient aggregation response in the presence of 2 mmol/l [Ca2+]o, which was also converted to a sustained response by omission of CaCl2 from the saline (2+]o (2+ levels respectively). To ensure that TXA2 generation was completely inhibited, experiments were repeated in the presence of 1 mmol/l aspirin, which also had no significant effect on the ability of reduced [Ca2+]o to enhance platelet aggregation . Together, these data suggest that factor(s) other than altered TXA2 production must contribute to the ability of reduced [Ca2+]o to enhance ADP-evoked aggregation.ADP (10 μmol/l) evoked a sustained aggregation of platelets in plasma anti-coagulated with citrate that reduced the extracellular Caly 2 min . Previou2+ levels. Aggregation evoked by the hydrolysis-resistant analogue ADPβS was not significantly different in the presence or absence of extracellular Ca2+ . Moreover, when platelets were resuspended in the absence of apyrase, and experiments performed rapidly to limit the effects of desensitization, ADP-evoked aggregation was also sustained in the presence of 2 mmol/l extracellular Ca2+ (2+ respectively). Finally, the [Ca2+]o-dependence of ADP-evoked aggregation responses in plasma was abolished when platelets were resuspended in autologous heat-treated plasma  to destroy enzymatic activity (versus micromolar [Ca2+]o respectively). Together, these observations are consistent with a role for Ca2+-dependent nucleotidase activity in the [Ca2+]o-dependence to ADP-evoked sustained aggregation responses.Given that our washed platelet preparation contained apyrase (E-NTPDase1 isolated from potato to prevent P2Y receptor desensitization) and PRP has been reported to contain endogenous ectonucleotidases, we considered whether degradation of ADP contributed to the transient nature of the ADP-evoked aggregation at millimolar Caversus micromolar [Ca2+]o concentrations, ADP (10 μmol/l) was added to apyrase-treated saline or platelet-free plasma and the ADP concentration after 2 min measured by luminescence following conversion to ATP (see Methods). The concentration of ADP remaining in nominally Ca2+-free saline was 1·61 ± 0·06 μmol/l, which was significantly reduced to 0·079 ± 0·03 μmol/l (P < 0·001) in the presence of 2 mmol/l Ca2+, indicating accelerated nucleotidase activity by physiological [Ca2+]o (P < 0·001)  . A signinly 10 s . This suresponse . Togetheαs-coupled adenosine A2a receptors. In citrated PRP, adenosine (10 μmol/l) inhibited ADP (10 μmol/l)-evoked responses, resulting in aggregation responses similar to those observed with ADP in physiological calcium concentrations -stimulated platelets was measured in response to apyrase (0·32 u/ml), the P2Y12 antagonist cangrelor (1 μmol/l) or the P2Y1 antagonist MRS2179 (10 μmol/l)  . Cangrel μmol/l) . In cont μmol/l) . Thus, r2 generation previously reported by others  at micromolar and millimolar [Ca2+]o was examined on TXB2 production from washed platelets 3 min after stimulation with 10 μmol/l ADP , respectively, 3 min after stimulation  remains sustained for a considerable time near sites of vascular injury  , results from this study demonstrate the impact of variable ectonucleotidase activity on platelet function, which may have profound implications in certain clinical conditions. It has previously been reported that in blood from patients with elevated leucocyte counts, degradation of ADP is accelerated and aggregation in response to ADP is reduced due to increased NTPDase levels (2+ levels. The sustained inside-out activation of fibrinogen receptors that occurs in response to ADP at low [Ca2+]o is likely to be more representative of the potential contribution of ADP to a developing thrombus in vivo, where a constant supply of this P2Y receptor agonist from activated platelets can override enzymatic clearance in the vicinity of a developing thrombus.In conclusion, the present study shows that reduced degradation of ADP by ectonucleotidases contributes to the paradoxical amplification of ADP-evoked aggregation at micromolar compared to millimolar extracellular Ca


Sparklets were virtually abolished by the removal of extracellular Ca2+ . Co-loading of smooth muscle strips with the slow Ca2+ chelator EGTA-AM (10 mM) confirmed that Ca2+ sparklets are restricted to the cell membrane. Ca2+ sparklets were inhibited by the calcium channel inhibitors R-(+)-Bay K 8644 (1 μM) , and diltiazem (10 μM) . Ca2+ sparklets were unaffected by inhibition of P2X1 receptors α,β-meATP (10 μM) whilst sparklet frequencies were significantly reduced by atropine (1 μM). Ca2+ sparklet frequency was significantly reduced by PKC inhibition with Gö6976 (100 nM) , demonstrating that Ca2+ sparklets are PKC dependant. In the presence of CPA (10 μM), there was no apparent change in the overall frequency of Ca2+ sparklets, although the sparklet frequencies of each UBSM became statistically independent of each other , implying that Ca2+ store mediated signals regulate Ca2+ sparklets. Under control conditions, inhibition of store operated Ca2+ entry using ML-9 (100 μM) had no significant effect. Amplitudes of Ca2+ sparklets were unaffected by any agonists or antagonists, suggesting that these signals are quantal events arising from activation of a single channel, or complex of channels. The effects of CPA and ML-9 suggest that Ca2+ sparklets regulate events in the cell membrane, and contribute to cytosolic and sarcoplasmic Ca2+ concentrations.Calcium is a ubiquitous second messenger in urinary bladder smooth muscle (UBSM). In this study, small discrete elevations of intracellular Ca During UBSM cell depolarization, several VGCCs are simultaneously activated in different areas of the cell membrane, resulting in Ca2+ entry, followed by Ca2+-induced Ca2+ release (CICR) and a whole-cell Ca2+ transient (WCT), which propagates rapidly through gap junctions, allowing micturition to take place In smooth muscle, intracellular Ca2+ plays in many cellular signalling processes, the maintenance of Ca2+ homeostasis in both the cytosol (Ca2+cyt) and the sarcoplasmic reticulum (Ca2+sr) is of significant importance. Following a WCT, Ca2+cyt is rapidly reduced to resting levels by a combination of plasma membrane (PMCA) and sarcolemmal (SERCA) Ca2+-ATPase activities +/Ca2+ exchange 2+ uptake Due to the vital role that intracellular Ca2+ signalling in UBSM cells, a range of signals can occur independently of VGCC activation, e.g. Ca2+ sparks, puffs and waves, which in UBSM are generated by Ca2+ release from the sarcoplasmic reticulum (SR) 2+ signals would imply that Ca2+sr can become depleted independently of Ca2+cyt, thus indicating a need for Ca2+ entry that promotes store refilling without necessarily activating smooth muscle contraction, an effect originally described as “capacitative Ca2+ entry” 2+ entry” (SOCE) Such is the ubiquitous role of Ca2+ sparklets are of significant importance to both local and global intracellular Ca2+ concentrations 2+srThe relatively recent, more widespread use of total internal reflection fluorescence (TIRF) microscopy in cellular imaging has revealed the presence of small VGCC-mediated events that are restricted to the membrane of isolated vascular smooth muscle cells 2+ sparklets in smooth muscle strips isolated from mouse urinary bladder, using an adapted TIRF microscopy approach. The relationship between Ca2+sr and Ca2+ sparklets was also investigated.The aim of this research was to investigate the presence of CaMale C57BL/6 mice between 6 and 10 weeks of age were killed by cervical dislocation. Efforts were made to minimise the suffering of experimental animals used in this study. All animal experiments were approved by the animal care and use committee of Saga University .3 25.0, NaH2PO4 1.13, KCl 4.7, glucose 11.1, CaCl2 1.8, and MgCl2 1.3. To ensure adequate oxygenation and to maintain pH between 7.3–7.4, solutions were bubbled with a mixture of 95% O2 and 5% CO2 gas. The ventral wall of the urinary bladder was opened longitudinally from the urinary bladder neck (posterior) to the top of the dome (anterior), and pinned to a Sylgard-coated surface. Urothelium was carefully removed from each individual strip. Urinary bladder strips (4–6 mm width and 10–15 mm length) were cut along the craniocaudal axis of the DSM, ensuring that several intact smooth muscle bundles were present in each strip.Urinary bladders were removed from the mice following cervical fracture. Isolated urinary bladders were sustained in an oxygenated Krebs solution, consisting of (in mM): NaCl 118.4, NaHCO2+ indicator Oregon Green BAPTA-1 AM (10 μM), dissolved in 1% DMSO –0.2% pluronic acid solution in oxygenated Krebs solution for 70 min at 35°C. Following indicator loading, the urinary bladder strip was placed, serosal side facing downwards, on the coverslip of a TIRF microscope  equipped with a 488 nm excitation laser and a CFI Plan Apo 60x/1.49na TIRF microscopy objective . The UBSM was perfused with oxygenated Krebs solution at 25°C, and held in place using a small plastic-coated weight of approximately 1.6–1.7 g. Using the weight ensured that a signal could be detected within the TIRF zone (i.e. the evanescent field), whilst maintaining the position of the UBSM strip inside the perfusion chamber . In order to minimize photobleaching, a maximum of 4 image sequences were recorded per smooth muscle strip, and the recordings were separated by a minimum period of 5 min. Where pharmacological treatments were applied, a minimum exposure period of 15 prior to imaging was used. Ca2+ signals could be detected more than 1 h after placement of the strip on the coverslip, implying that the setup was not detrimental to the survival of the UBSM strips. Example recordings of UBSM Ca2+ sparklets can be found in The smooth muscle strips were allowed to equilibrate for 10 min prior to imaging. Regions of the UBSM strip were selected for imaging, based upon; defined smooth muscle morphology; the occurrence of at least one Ca2+ image analysis plugin for imageJ was used . In order to be classified as a Ca2+ sparklet, an event had to be a minimum of 3 frames in duration, be 2.5 standard deviations above the baseline, and have a peak signal of at least 0.05 F/F0. Events that occurred simultaneously (or near simultaneously) in separate ROIs were excluded from the analysis, as detection in more than one ROI implies an event that is too large to be considered a sparklet. The occurrences of Ca2+ sparklets in the regions suggested by automated analysis were confirmed by visual inspection. Sparklet frequency data for each individual cell were corrected for cell membrane area because of the significant variations in the area of the cell membrane present within the TIRF zone.Image sequences were recorded using NIS-Elements imaging software , saved as AVI files, and transferred to imageJ (version 1.47q) for offline analysis. Arbitrary selection of individual regions of interest (ROIs) for analysis can be time consuming, and heavily prone to investigator bias; therefore, the “LC pro” CaAtropine, α,β-methylene-adenosine 5′-triphosphate  and diltiazem were dissolved in Krebs solution and stored as either 1 or 10 mM stock solutions at −20°C until dilution on the day of use. R-(+)-Bay K 8644, cyclopiazonic acid (CPA), EGTA-AM and ML-9 were all dissolved in DMSO and stored as either 1 or 10 mM stock solutions at −20°C, until dilution as required. All drugs were purchased from Sigma-Aldrich Japan  except for Oregon Green 488 BAPTA-1 AM, EGTA-AM  and R-(+)-Bay K 8644 .t-test was used on the basis that cells with no sparklets (and therefore a sparklet amplitude of 0) were removed from the analysis. In the case of EGTA-AM, sparklet frequencies were compared using a Mann-Whitney test, and amplitudes were compared using an unpaired Student's t-test. P values of less than 0.05 were considered statistically significant. For testing correlations between different paired datasets, the Spearman's rank correlation coefficient was used. P values of less than 0.05 were taken to indicate that sparklet frequencies were statistically dependent.Data were presented and analysed using Graphpad 6.0 statistical analysis software or Origin 6.0 statistical analysis software. For comparisons of sparklet frequency, Wilcoxon's matched-pairs signed rank test was used on the basis that the data were paired and not normally distributed around the mean value. For comparisons of sparklet amplitudes, an unpaired Student's 2+ imaging of UBSM strips using an adapted TIRF microscopy approach revealed a population of small transient Ca2+ signals that occurred on or near the cell membrane of UBSM cells  of preparations, and within the active preparations, in 72% (184/255) of cells. The detection of these transient Ca2+ signals (henceforth referred to as Ca2+ sparklets) in a relatively low percentage of preparations most likely reflected technical difficulties, e.g. excess connective tissues blocking access to the UBSM cell membranes, rather than an absence of activity. Over the course of the investigation, these signals were detected at minimum amplitude of 0.05 F/F0 and maximum amplitude of 0.27 F/F0 . The mean amplitude of the sparklets observed in the absence of extracellular Ca2+ was not significantly different from that under control conditions . In the presence of 10 mM Ca2+, sparklet frequency was not significantly different from control conditions . Surprisingly, in spite of an increased extracellular Ca2+ gradient, the mean sparklet amplitude was unaffected by the presence of 10 mM Ca2+ .The occurrence of Ca2+ sparklets occurred at the cell membrane and were entirely independent of CICR, UBSM strips were co-loaded with the Ca2+ chelator EGTA, in the acetoxymethyl ester (AM) form, in order to chelate intracellular Ca2+. As a relatively slow Ca2+ chelator 2+ events that originate from the SR, leaving rapid Ca2+ entry from outside the cell unaffected 2+ sparklets were detected in the presence of EGTA (10 mM) at a similar frequency to that in unmatched controls . No significant change in mean sparklet amplitude was detected in the presence of EGTA-AM , compared with controls. In the presence of EGTA-AM, there was a significant reduction in sparklet area, compared with controls  thus demonstrating the chelating effects of EGTA. No Ca2+sr mediated events  could be detected in the presence of EGTA-AM (unquantified observation).In order to confirm that Ca2+ sparklets are cell membrane events, they were investigated pharmacologically to identify potential channels that may be involved in their generation. Ca2+ sparklet frequency, was significantly reduced by the L-type Ca2+ channel blockers, diltiazem  and R-(+)-Bay K 8644 -Bay K 8644 (1 μM): 0.034±0.021 Hz/mm2, nc = 22 np = 5; P<0.05, Wilcoxon matched pairs signed rank test). The amplitudes of the sparklets remaining in the presence of both blockers were not significantly different from those of the controls -Bay K 8644 -Bay K 8644, np = 5, P≥0.05, unpaired t-test).Having demonstrated that Ca2+ sparklets, α,β-meATP or atropine were applied to UBSM strips. In the presence of atropine (1 μM), there was a significant decrease in sparklet frequency  but not amplitude , compared with the controls. In the presence of 10 μM α,β-meATP, there was no statistically significant difference in sparklet frequency  or amplitude , compared with the controls.UBSM strips undergo small spontaneous depolarizations that have been shown to be largely purinergic in origin, and can lead to spontaneous contractions through activation of VGCCs atropine  μM, ther2+ sparklets require PKC-mediated phosphorylation of VGCCs. When UBSM strips were incubated in the presence of Gö6976 (100 nM), a PKC inhibitor, there was a significant decrease in sparklet frequency  but not amplitude .Several investigations in vascular smooth muscle indicate that Ca2+ sparklets, CPA (10 μM) was used to deplete SR Ca2+ stores, active depletion of SR Ca2+ stores favours the occurrence of SOCE. In the presence of CPA, there was no statistically significant difference in the overall frequency  or amplitude  of Ca2+ sparklets, compared with the controls. Although no significant change in mean sparklet frequency was detected following the addition of 10 μM CPA, the statistical correlation of events before and after the addition of CPA differed significantly to that observed for the other pharmacological interventions. Cells with a higher sparklet frequency under control conditions, generally showed a reduction in sparklet frequency in the presence of 10 μM CPA, whilst other cells saw an increase in sparklet frequency, typically from a very low frequency under control conditions (P≥0.05), indicating that a high sparklet frequency for any given cell under control conditions is in no way correlated with a high sparklet frequency in the presence of 10 μM CPA. In all other datasets using other pharmacological interventions, sparklet frequencies before and after the intervention were found to be statistically dependent on each other; this demonstrates a statistically significant change in the locations of Ca2+ sparklets, but not in the overall mean sparklet frequency -Bay K 8644+10 μM CPA: 0.15 ± 0.03 Hz/mm2; nc = 26, np = 5, P<0.05, Wilcoxon matched-pairs signed rank test) but not amplitude -Bay K 8644+10 μM CPA: 0.07 ± 0.003 F/F0, nc = 21 for control, 22 for 1 μM R-(+)-Bay K 8644+10 μM CPA, np = 5, P≥0.05, unpaired t-test), compared with the controls. When correlations between datasets were compared, the data were found not to be statistically dependent (P≥0.05). In order to confirm that Ca2+ sparklets in the absence of CPA were not mediated by stromal interacting molecule-1 (STIM1), and therefore probably caused by passive depletion of Ca2+sr, ML-9 (100 μM), an inhibitor of STIM1 plasma membrane interactions, was applied to UBSM strips. In the presence of 100 μM ML-9, there was no statistically significant change in overall sparklet frequency  or amplitude , compared with the controls.In order to investigate the role of the SR in the generation of Canditions . Correlarequency . CPA-ind2+ elevations detected in this investigation were limited to the cell membranes of UBSM cells. There are several pieces of evidence that support this claim: Firstly, signals of this nature cannot be routinely observed with high magnification epifluorescence microscopy or confocal laser scanning microscopy, implying that the superior resolution, and restricted excitation zone of a TIRF microscope was required to image the observed events. Secondly, the removal of extracellular Ca2+ significantly reduced the frequency of the Ca2+ sparklets, indicating that the source of Ca2+ for these events is extracellular .The small, discrete Cacellular . The mosmplitude . Because2+ sparklets -Bay K 8644 or diltiazem, there was a significant reduction in the frequency of Caparklets , indicat12+ sparklet frequency but not amplitude  the amplitude steps should be largely unaffected by the concentration or nature of the agonist used; (5) the durations of the events should be exponentially distributed; and (6) the channels should be highly Ca2+ permeable, and have an identified single channel conductance.In order to determine whether these events arise as a result of openings of a single ion channel/complex of ion channels or the near synchronous opening of several channels, a defined set of criteria need to be considered 2+ electrochemical gradient (criterion 3), to the point that increasing the extracellular Ca2+ concentration to 10 mM did not result in a statistically significant increase in the amplitude of the events . It was also not possible to plot a multiple Gaussian distribution of event amplitude (criterion 2), and the size of the events does not appear to be dependent upon the Cae events . In the In a physiological context, it may be very difficult to satisfy criterion 2 by plotting a multiple Gaussian function, due to interference from criterion 3. Smooth muscle cells in a poorly coupled electrical syncytium rarely maintain an entirely constant voltage in the same way as voltage-clamped isolated myocytes. Indeed, the resting membrane potential (RMP) can reportedly vary between −35 and −60 mV 2+ conductance in isolated vascular smooth muscle cells (criterion 6) Distinct amplitude steps are difficult to identify in the model used. However, there was no statistically significant change in the mean amplitudes of the events in response to any of the pharmacological treatments used in the present investigation  can occur through largely the same pathways as SOCE 2+ channel 2+ sparklets under control conditions. Three key pieces of evidence indicate that the former, rather than the latter, situation is occurring.This finding suggests that inhibition of SERCA, and the resultant depletion of intracellular Ca2+ sparklets, indicating that CPA-induced events are VGCC-dependent  membrane areas of the same cells in both the absence and presence of CPA, often at very different frequencies -Bay K 8644, there was no significant activation of SOCE by CPA; indeed, there was a statistically significant reduction in the frequency of Caependent . Second,ependent  and 7. Tquencies . This wo2+ sparklets are directly coupled with ryanodine receptors on the SR, and activate excitation-contraction coupling (ECC) through local activation of 4–6 ryanodine receptors that can lead to a WCT 2+cyt and indirectly Ca2+sr2+ sparklets can be detected using TIRF microscopy. These events can be affected by depletion of Ca2+sr, but can also occur independently of any signals from the SR, suggesting an additional role in local regulation of cell membrane processes.In cardiac muscle, VGCC Ca2+ entry into smooth muscle 22+ sparklets, although the clustered nature of VGCC distribution suggests that these events are due to activation of either single channels or complexes of channels.Other reports indicate that voltage dependant VGCC activity is the major conduit for Ca2+, it seems plausible that the use of BAPTA-1-based Ca2+ indicators for the monitoring of VGCC sparklets may produce a passive depletion of intracellular Ca2+ stores, and resultant SOCE 2+ indicator, it seems reasonable to expect a significant increase in sparklet frequency in the presence of a second chelator, in this case EGTA-AM 2+ sparklets are unrelated to Ca2+sr. The AM Ca2+ indicator loading protocol used in this investigation may have caused significant variations in the indicator concentration, resulting in variable concentrations of Ca2+sr and Ca2+cyt, although in many cases SR Ca2+ stores were clearly not depleted, as evidenced by the occurrence of intracellular Ca2+ events, sometimes in the same cells as VGCC sparklets Click here for additional data file.Video S12+ sparklets from a DSM strip loaded with Ca2+ indicator.Recording of spontaneous Ca Scale bar, 10 μm.(AVI)Click here for additional data file.Video S22+ sparklets in the presence of the VGCC antagonist, R-(+)-Bay K 8644 (1 μM).Recording of spontaneous Ca This recording is paired with the recording shown in video S1. Scale bar, 10 μm.(AVI)Click here for additional data file.Video S32+ indicator, showing various Ca2+ sparklets in addition to a WCT  and a Ca2+ puff .A DSM strip loaded with Ca Scale bar, 10 μm.(AVI)Click here for additional data file.Video S42+ indicator, showing various intracellular Ca2+ waves.A DSM strip loaded with Ca The poorly defined edges and propagating nature of many of the signals suggest that an intracellular Ca2+ signal is occurring.(AVI)Click here for additional data file.