Upregulation of neurotrophins and transforming growth factor-ß expression in the bladder may lead to nerve hyperplasia and fibrosis in patients with severe ketamine-associated cystitis.

AIMS: To investigate the mechanism of bladder nerve hyperplasia and fibrosis in the patients with ketamine-associated cystitis (KC). METHODS: Sixteen patients with severe KC, six patients with mild KC, and five patients with localized invasive bladder cancer served as control patients. Bladder mucosa specimens were taken during the operations, and the specimens were stained for nerve growth factor (NGF) and S-100 to evaluated nerve hyperplasia. The quantitative Western blot analysis was performed for NGF, brain-derived neurotrophic factor (BDNF), growth-associated protein 43 (GAP-43), tropomyosin receptor kinase A and B (TrkA and TrkB), transforming growth factor-ß (TGF-ß), phosphorylated extracellular signal-regulated kinases (p-ERK), protein kinase B (p-Akt), and glycogen synthase kinase 3ß (p-GSK-3ß). RESULTS: The results demonstrated diffuse NGF expression in KC bladder epithelium, lamina propria, and muscle. The GAP-43, NGF, BDNF, TrkA, TGF-ß, p-ERK, P-AKT, and p-GSK-3ß expression in the bladder mucosa specimens of patients with severe KC was significantly higher than in patients with mild KC and control patients. Expression of neurotrophins was significantly correlated with bladder capacity and pain. NGF and BDNF expression were significantly higher in the KC bladder specimens with strongly positive S-100 staining. TGF-ß expression in the bladder specimens was significantly correlated with neurotrophins, p-ERK, P-AKT, and p-GSK-3ß levels. CONCLUSION: Our findings indicate upregulation of neurotrophins, TGF-ß, and activation of the cell proliferation kinases plays an important role in nerve hyperplasia and fibrosis mechanisms in severe KC bladders. The neurotrophins and TGF-ß interact as cause and effect, leading to bladder hypersensitivity and fibrosis in severe KC.

Honokiol blocks store operated calcium entry in CHO cells expressing the M3 muscarinic receptor: honokiol and muscarinic signaling.

BACKGROUND: Honokiol, a cell-permeable phenolic compound derived from the bark of magnolia trees and present in Asian herbal teas, has a unique array of pharmacological actions, including the inhibition of multiple autonomic responses. We determined the effects of honokiol on calcium signaling underlying transmission mediated by human M3 muscarinic receptors expressed in Chinese hamster ovary (CHO) cells. Receptor binding was determined in radiolabelled ligand binding assays; changes in intracellular calcium concentrations were determined using a fura-2 ratiometric imaging protocol; cytotoxicity was determined using a dye reduction assay. RESULTS: Honokiol had a potent (EC50 ≈ 5 µmol/l) inhibitory effect on store operated calcium entry (SOCE) that was induced by activation of the M3 receptors. This effect was specific, rapid and partially reversible, and was seen at concentrations not associated with cytotoxicity, inhibition of IP3 receptor-mediated calcium release, depletion of ER calcium stores, or disruption of M3 receptor binding. CONCLUSIONS: It is likely that an inhibition of SOCE contributes to honokiol disruption of parasympathetic motor functions, as well as many of its beneficial pharmacological properties.

Oxidative stress disruption of receptor-mediated calcium signaling mechanisms.

BACKGROUND: Oxidative stress increases the cytosolic content of calcium in the cytoplasm through a combination of effects on calcium pumps, exchangers, channels and binding proteins. In this study, oxidative stress was produced by exposure to tert-butyl hydroperoxide (tBHP); cell viability was assessed using a dye reduction assay; receptor binding was characterized using [3H]N-methylscopolamine ([3H]MS); and cytosolic and luminal endoplasmic reticulum (ER) calcium concentrations ([Ca2+]i and [Ca2+]L, respectively) were measured by fluorescent imaging. RESULTS: Activation of M3 muscarinic receptors induced a biphasic increase in [Ca2+]i: an initial, inositol trisphosphate (IP3)-mediated release of Ca2+ from endoplasmic reticulum (ER) stores followed by a sustained phase of Ca2+ entry (i.e., store-operated calcium entry; SOCE). Under non-cytotoxic conditions, tBHP increased resting [Ca2+]i; a 90 minute exposure to tBHP (0.5-10 mM ) increased [Ca2+]i from 26 to up to 127 nM and decreased [Ca2+]L by 55%. The initial response to 10 µM carbamylcholine was depressed by tBHP in the absence, but not the presence, of extracellular calcium. SOCE, however, was depressed in both the presence and absence of extracellular calcium. Acute exposure to tBHP did not block calcium influx through open SOCE channels. Activation of SOCE following thapsigargin-induced depletion of ER calcium was depressed by tBHP exposure. In calcium-free media, tBHP depressed both SOCE and the extent of thapsigargin-induced release of Ca2+ from the ER. M3 receptor binding parameters (ligand affinity, guanine nucleotide sensitivity, allosteric modulation) were not affected by exposure to tBHP. CONCLUSIONS: Oxidative stress induced by tBHP affected several aspects of M3 receptor signaling pathway in CHO cells, including resting [Ca2+]i, [Ca2+]L, IP3 receptor mediated release of calcium from the ER, and calcium entry through the SOCE. tBHP had little effect on M3 receptor binding or G protein coupling. Thus, oxidative stress affects multiple aspects of calcium homeostasis and calcium dependent signaling.

Effects of different urodynamic characteristics on therapeutic outcomes of overactive bladder medication in a real-life clinical practice.

Objective: This study retrospectively investigated the influence of urodynamic parameters and patient characteristics on success rates among patients with overactive bladder (OAB) and urodynamic detrusor overactivity (DO). Materials and Methods: Consecutive patients with OAB and urodynamic DO initially received solifenacin, mirabegron, or combination of both for 1-3 months. If failed, patients were switched to another OAB medication subtype or provided additional OAB medication for a total of 6 months. A successful treatment was defined as an improvement in urgency severity and a global response assessment of ≥1. Success rates after initial or modulated OAB medication were analyzed based on patient and urodynamic characteristics. Results: A total of 453 patients were enrolled, among whom 144, 255, and 54 received solifenacin, mirabegron, and combined medications, respectively. Among the patients, 259 (57.2%) had OAB dry and 194 (42.8%) had OAB wet. Patients receiving mirabegron alone had a significantly higher initial medication success rate compared to that of others. Patients with a phasic DO (50.7%), bladder outlet obstruction (BOO, 52.5%), and no central nervous system (CNS) lesions (47.5%) exhibited higher success rates than those with a terminal DO (42.0%), no BOO (42.7%), and CNS lesions (31.6%), respectively. After switching or modulating the initial OAB medication following treatment failure, 115 (62.2%) of 185 patients still showed improvement in OAB symptoms, with an overall success rate of 70.2% after 6 months of treatment. Conclusion: Initial solifenacin or mirabegron treatment had a success rate of around 50%. In general, patients with a phasic DO, urodynamic BOO, and no CNS lesions have higher success rates than those with a terminal DO, no BOO, and CNS lesions, respectively. Success rates can further be improved by switching or modulating OAB medication.

Predictive Factors for a Satisfactory Treatment Outcome with Intravesical Botulinum Toxin A Injection in Patients with Interstitial Cystitis/Bladder Pain Syndrome.

A botulinum toxin A (BoNT-A) intravesical injection can improve the symptoms of interstitial cystitis/bladder pain syndrome (IC/BPS). Patients with IC/BPS have different clinical characteristics, urodynamic features, and cystoscopic findings. This study assessed the treatment outcomes of a BoNT-A intravesical injection and aimed to identify the predictive factors of a satisfactory outcome. This retrospective study included IC/BPS patients treated with 100 U BoNT-A. The treatment outcomes were assessed by global response assessment (GRA) at 6 months. We classified patients according to different clinical, urodynamic, and cystoscopic characteristics and evaluated the treatment outcomes and predictive factors. A total of 238 patients were included. Among these patients, 113 (47.5%) had a satisfactory outcome (GRA ≥ 2) and 125 (52.5%) had an unsatisfactory outcome. Improvements in the IC symptom score, IC problem score, O'Leary-Sant symptom score, and visual analog scale score for pain were significantly greater in patients with a satisfactory outcome than in patients with an unsatisfactory outcome (all p = 0.000). The IC disease duration and maximal bladder capacity (MBC) were significantly different between patients with and without a satisfactory outcome. Multivariate analysis revealed that only the MBC was a predictor for a satisfactory outcome. Patients with a MBC of ≥760 mL and glomerulations of 0/1 (58.7%) or glomerulations of 2/3 (75.0%) frequently had a satisfactory outcome. We found that BoNT-A intravesical injection can effectively improve symptoms among patients with IC/BPS, with a remarkable reduction in bladder pain. A MBC of ≥760 mL is a predictive factor for a satisfactory treatment outcome.

Persistent tissue kinetics and redistribution of nanoparticles, quantum dot 705, in mice: ICP-MS quantitative assessment.

BACKGROUND: Quantum dots (QDs) are autofluorescent semiconductor nanocrystals that can be used for in vivo biomedical imaging. However, we know little about their in vivo disposition and health consequences. OBJECTIVES: We assessed the tissue disposition and pharmacokinetics of QD705 in mice. METHODS: We determined quantitatively the blood and tissue kinetics of QD705 in mice after single intravenous (iv) injection at the dose of 40 pmol for up to 28 days. Inductively coupled plasma-mass spectrometry (ICP-MS) measurement of cadmium was the primary method of quantification of QD705. Fluorescence light microscopy revealed the localization of QD705 in tissues. RESULTS: Plasma half-life of QD705 in mice was short (18.5 hr), but ICP-MS analyses revealed QD705 persisted and even continued to increase in the spleen, liver, and kidney 28 days after an iv dose. Considerable time-dependent redistribution from body mass to liver and kidney was apparent between 1 and 28 days postdosing. The recoveries at both time points were near 100%; all QD705s reside in the body. Neither fecal nor urinary excretion of QD705 was detected appreciably in 28 days postdosing. Fluorescence microscopy demonstrated deposition of QD705 in the liver, spleen, and kidneys. CONCLUSION: Judging from the continued increase in the liver (29-42% of the administered dose), kidney (1.5-9.2%), and spleen (4.8-5.2%) between 1 and 28 days without any appreciable excretion, QD705 has a very long half-life, potentially weeks or even months, in the body and its health consequences deserve serious consideration.

Involvement of substance P and neurogenic inflammation in arsenic-induced early vascular dysfunction.

Vascular-related diseases, including Blackfoot Disease and atherosclerosis, are prominent clinical findings among populations residing in arseniasis areas. While oxidative stress provided a general but nonspecific mechanistic base for arsenic-induced endothelial cell damage in vitro, more specific mechanism is needed to explain the highly targeted vascular lesions induced by arsenic in vivo. Based on our previous studies, we hypothesized that arsenic exerted its action on blood vessels via the neurogenic inflammation process involving release of a neuropeptide (substance P) and activation of endothelial Neurokinin 1 (NK-1) receptor in vivo. Indeed, our present study demonstrated a significantly higher substance P levels in arsenic-treated tissues when compared to saline-treated controls indicating a rapid release of substance P under the influence of arsenic. Furthermore, the arsenic-induced vascular leakage could be significantly reduced when the neurogenic inflammation process was interrupted (via either disruption on the release of substance P, interference on the action of substance P, or blockage of endothelial NK-1 receptor) showing that the neurogenic inflammation process was indeed involved. Histamine release was not found to play a significant role in arsenic-induced vascular permeability change. Our present study affirmed a de novo concept that a pathophysiological mechanism involving the neurogenic release of substance P and activation of endothelial NK-1 receptor underlies the arsenic-induced vascular injury and dysfunction in vivo. This pathophysiological process constituted a two-tiered biological interaction between the nervous system and vascular system and therefore was not readily unveiled by traditional in vitro studies in the past. Our present finding unveiled an important de novo concept on arsenic vascular toxicity in vivo.

Inorganic arsenic exposure and its relation to metabolic syndrome in an industrial area of Taiwan.

Past arsenic exposure was found associated with increased incidence of type 2 diabetes. However, the mechanisms remain unclear. Metabolic syndrome has been shown as a strong predictor for diabetes occurrence. We aimed at examining the association of inorganic arsenic exposure and the prevalence of metabolic syndrome. The authors recruited 660 age and gender stratified random population of residents in central Taiwan during 2002-2003. They received home interviews and health examinations at local health care units, where blood and hair specimens were collected. Hair arsenic (H-As) concentrations were determined by inductively coupled plasma-mass spectrometry. Metabolic syndrome was defined as the presence of three or more of the following risk factors: elevated levels of blood pressure, plasma glucose, and triglycerides, also the body mass index, and reduced high-density lipoprotein. Prevalence of metabolic syndrome increased from the 2nd tertile (0.034 ug/g) of H-As levels (odds ratio=2.54, 95% confidence interval: 1.20-5.39, p=0.015) after the adjustment for age, gender, occupation and life styles including cigarette smoking. We further found linear relation between H-As concentrations and increased levels of plasma glucose and lipids, and blood pressures. This first report may help identify modifiable factors associated with diabetogenesis and cardiovascular disease progression and thus be worth following for community health.

Survey of urinary nickel in residents of areas with a high density of electroplating factories.

The soil metal contamination arising from the discharge of the high density of electroplating factories in the geographic center of Taiwan has prompted concern about human exposure to harmful metals. This study aimed to determine the levels of nickel (Ni) in urine of residents living in the high vs. low factory-density areas, and to examine how these levels relate to gender and age. A total of 660 subjects, resident in the area for the last five years, were sampled according to the stratified random sampling approach, at ages 35-44, 45-54, and 55-64years for both genders. Metals in urine samples were analyzed by inductively coupled plasma mass spectrometry (ICPMS). The geometric mean (95% confidence interval (CI)) of urinary Ni was 6.30 (5.99-6.62)mug/l. The 0.95 parametric reference interval (90% CI) of urinary Ni was estimated to be 1.74 (1.62-1.88) to 22.73 (21.14-24.44)mug/l. Subjects in the areas with a high density of electroplating factories had significantly higher urinary Ni levels than those in the low-density areas, but both types of areas had obviously higher urinary Ni levels when compared to the non-occupationally exposed population from western countries. The health significance of elevated urinary Ni and its causative factors remain to be determined.

A mouse model for the study of vascular permeability changes induced by arsenic.

The primary objective of our present research was to develop an animal model for the investigation of arsenic-induced vasculopathy. Epidemiological evidence indicated that, aside from cancer, cardiovascular-related diseases were probably the most prominent health concerns in arseniasis areas. Although there were many investigations on the effect of arsenic on endothelial cells, most of these studies were conducted under in vitro conditions. A good animal model for studying the effects of arsenic on vascular integrity in vivo is very much needed. We have previously developed a rat model that could be used to demonstrate vascular changes induced by arsenic in vivo. In the present report, we are introducing a new model that is even more sensitive, economical, and effective than our original rat model. Taking advantage of the characteristics of mouse ears (thin, delicate, and with easily visible blood vessels), we demonstrated the pattern and extent of vascular leakage induced by arsenic clearly, quantitatively and convincingly. With this model, we demonstrated a time-dependent increase in vascular permeability induced by arsenic (a fourfold increase in vascular leakage was observed between 10-60 minutes). With this model, we were also able to demonstrate that small caliber vessels were more vulnerable than vessels of larger caliber. Thus, this animal model can provide dynamic information (from toxic effects, pathology, and functional alterations to cellular/molecular mechanisms) that cell culture techniques are unable to provide. The introduction of this model hopefully will stimulate and inspire many other new in vivo investigations on vasculopathy induced by arsenic or other chemicals in the future.

Vascular permeability alterations induced by arsenic.

The impact of arsenic on the integrity of blood vessels in vivo via in situ exposure (local injection) of arsenic was investigated. Vascular permeability changes were evaluated by means of the Evans blue assay and the India ink tracer techniques. Rats were intravenously injected with Evans blue followed by intradermal injections of various doses of sodium arsenite on the back skins of the animals. Evans blue at different time points was extracted and assayed as indices of vascular leakage. Skin at various time point injection sites was sampled for arsenic measurement via graphite furnace atomic absorption spectroscopy. Our time course study with Evans blue technique demonstrated a biphasic pattern of vascular permeability change: an early phase of permeability reduction and a later phase of permeability promotion at all dose levels tested. The India ink tracer technique also demonstrated a time-correlated increase in vascular labelling in the tissues examined, signifying an increase in vascular leakage with time. Moreover, we found that despite an early increase in tissue arsenic content at time of injection, tissue arsenic declined rapidly and returned to near control levels after 30-60 min. Thus, an inverse correlation between tissue arsenic content and the extent of vascular permeability was apparent. This study provides the first demonstration that in situ exposure to arsenic will produce vascular dysfunction (vascular leakage) in vivo.

Disruption of the integrity and function of brain microvascular endothelial cells in culture by exposure to diesel engine exhaust particles.

Diesel exhaust particles (DEPs), a by-product of diesel engine exhaust (DEE), are known to produce pro-oxidative and pro-inflammatory effects, thereby leading to oxidative stress-induced damage. Given the key role of DEPs in inducing oxidative stress, we investigated the role of DEPs in disrupting the integrity and function of immortalized human brain microvascular endothelial cells (HBMVEC). To study this, HBMVEC cells were exposed to media containing three different concentrations of DEPs or plain media for 24h. Those exposed to DEPs showed significantly higher oxidative stress than the untreated group, as indicated by the glutathione (GSH) and malondialdehyde (MDA) levels, and the glutathione peroxidase and glutathione reductase activities. DEPs also induced oxidative stress-related disruption of the HBMVEC cells monolayer, as measured by trans-epithelial electrical resistance. Taken together, these data suggest that DEPs induce cell death and disrupt the function and integrity of HBMVEC cells, indicating a potential role of DEPs in neurotoxicities.

Zinc oxide nanoparticle disruption of store-operated calcium entry in a muscarinic receptor signaling pathway.

The influences of ZnO nanoparticles on cellular responses to activation of muscarinic receptors were studied in Chinese hamster ovary cells expressing the human M3 muscarinic acetylcholine receptor. ZnO particles (20 nm) induced cytotoxicity in a time and concentration-dependent manner: following a 24h exposure, toxicity was minimal at concentrations below 20 µg/ml but virtually complete at concentrations above 28 µg/ml. ZnO particles did not affect antagonist binding to M3 receptors or allosteric ligand effects, but increased agonist binding affinity while eliminating guanine nucleotide sensitivity. At a noncytotoxic concentration (10 µg/ml), ZnO increased resting [Ca(2+)](i) from 40 to 130 nM without compromising calcium homeostatic mechanisms. ZnO particles had minimal effects on IP3- or thapsigargin-mediated release of intracellular calcium from the endoplasmic reticulum, but strongly inhibited store-operated calcium entry (capacitive calcium entry). The latter effect was seen as (1) a decrease in the plateau phase of the response and (2) a decrease in Ca(2+) entry upon introduction of calcium to the extracellular medium following thapsigargin-induced depletion of calcium from the endoplasmic reticulum (EC50's ≈ 2 µg/ml). Thus, ZnO nanoparticles interfere with two specific aspects of the M3 signaling pathway, agonist binding and store-operated calcium entry.

Computational and ultrastructural toxicology of a nanoparticle, Quantum Dot 705, in mice.

We conducted pharmacokinetic and toxicology studies on Quantum Dot 705 (QD705) in male ICR mice for up to 6 months after a single intravenous dose. Time-course sacrifices were carried out at 1, 4, and 24 h; 3, 7, 14, and 28 days; and 6 months on groups of six mice per time point. Mass balance studies were also carried out at 24 h, 28 days, and 6 months. Using inductively coupled plasma mass spectrometry, various tissues, urine, and feces were analyzed for cadmium (Cd111), which is a major (46%) component of QD705. On the basis of these experimental studies, a physiologically based pharmacokinetic computer simulation model was developed with excellent predictive capability for the time-dependent kinetic and distributional changes of QD705 in tissues. QD705 persisted and accumulated in the spleen, liver, and kidneys for at least 28 days with little or no disposition but was gradually and partially eliminated by 6 months. Although histological alterations of the spleen, liver, and kidney by light microscopy are unremarkable, investigation using electron microscopy on numerous renal samples revealed definitive mitochondrial alterations in renal tubular epithelial cells at 28 days and 6 months postdosing. Health implications and potential beneficial applications of QD705 are suggested.