Dimitrios E. Kouzoukas

Intracellular calcium plays a critical role in the alcohol-mediated death of cerebellar granule neurons

Alcohol is a potent neuroteratogen that can trigger neuronal death in the developing brain. However, the mechanism underlying this alcohol‐induced neuronal death is not fully understood. Utilizing primary cultures of cerebellar granule neurons (CGN), we tested the hypothesis that the alcohol‐induced increase in intracellular calcium [Ca2+]i causes the death of CGN. Alcohol induced a dose‐dependent (200–800 mg/dL) neuronal death within 24 h. Ratiometric Ca2+ imaging with Fura‐2 revealed that alcohol causes a rapid (1–2 min), dose‐dependent increase in [Ca2+]i, which persisted for the duration of the experiment (5 or 7 min). The alcohol‐induced increase in [Ca2+]i was observed in Ca2+‐free media, suggesting intracellular Ca2+ release. Pre‐treatment of CGN cultures with an inhibitor (2‐APB) of the inositol‐triphosphate receptor (IP3R), which regulates Ca2+ release from the endoplasmic reticulum (ER), blocked both the alcohol‐induced rise in [Ca2+]i and the neuronal death caused by alcohol. Similarly, pre‐treatment with BAPTA/AM, a Ca2+‐chelator, also inhibited the alcohol‐induced surge in [Ca2+]i and prevented neuronal death. In conclusion, alcohol disrupts [Ca2+]i homeostasis in CGN by releasing Ca2+ from intracellular stores, resulting in a sustained increase in [Ca2+]i. This sustained increase in [Ca2+]i may be a key determinant in the mechanism underlying alcohol‐induced neuronal death.

Protease-Activated Receptor 4 Induces Bladder Pain through High Mobility Group Box-1

Pain is the significant presenting symptom in Interstitial Cystitis/Painful Bladder Syndrome (IC/PBS). Activation of urothelial protease activated receptor 4 (PAR4) causes pain through release of urothelial macrophage migration inhibitory factor (MIF). High Mobility Group Box-1 (HMGB1), a chromatin-binding protein, mediates bladder pain (but not inflammation) in an experimental model (cyclophosphamide) of cystitis. To determine if PAR4-induced bladder hypersensitivity depends on HMGB1 downstream, we tested whether: 1) bladder PAR4 stimulation affected urothelial HMGB1 release; 2) blocking MIF inhibited urothelial HMGB1 release; and 3) blocking HMGB1 prevented PAR4-induced bladder hypersensitivity. HMGB1 release was examined in immortalized human urothelial cultures (UROtsa) exposed to PAR4-activating peptide (PAR4-AP; 100 μM; 2 hours) or scrambled control peptide. Female C57BL/6 mice, pretreated with a HMGB1 inhibitor (glycyrrhizin: 50 mg/kg; ip) or vehicle, received intravesical PAR4-AP or a control peptide (100 μM; 1 hour) to determine 1) HMGB1 levels at 1 hour in the intravesical fluid (released HMGB1) and urothelium, and 2) abdominal hypersensitivity to von Frey filament stimulation 24 hours later. We also tested mice pretreated with a MIF blocker (ISO-1: 20 mg/kg; ip) to determine whether MIF mediated PAR4-induced urothelial HMGB1 release. PAR4-AP triggered HMGB1 release from human (in vitro) and mice (in vivo) urothelial cells. Intravesical PAR4 activation elicited abdominal hypersensitivity in mice that was prevented by blocking HMGB1. MIF inhibition prevented PAR4-mediated HMGB1 release from mouse urothelium. Urothelial MIF and HGMB1 represent novel targets for therapeutic intervention in bladder pain conditions.

Macrophage Migration Inhibitory Factor Mediates PAR-Induced Bladder Pain

Introduction Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, is constitutively expressed in urothelial cells that also express protease-activated receptors (PAR). Urothelial PAR1 receptors were shown to mediate bladder inflammation. We showed that PAR1 and PAR4 activator, thrombin, also mediates urothelial MIF release. We hypothesized that stimulation of urothelial PAR1 or PAR4 receptors elicits release of urothelial MIF that acts on MIF receptors in the urothelium to mediate bladder inflammation and pain. Thus, we examined the effect of activation of specific bladder PAR receptors on MIF release, bladder pain, micturition and histological changes. Methods MIF release was measured in vitro after exposing immortalized human urothelial cells (UROtsa) to PAR1 or PAR4 activating peptides (AP). Female C57BL/6 mice received intravesical PAR1- or PAR4-AP for one hour to determine: 1) bladder MIF release in vivo within one hour; 2) abdominal hypersensitivity (allodynia) to von Frey filament stimulation 24 hours after treatment; 3) micturition parameters 24 hours after treatment; 4) histological changes in the bladder as a result of treatment; 5) changes in expression of bladder MIF and MIF receptors using real-time RT-PCR; 6) changes in urothelial MIF and MIF receptor, CXCR4, protein levels using quantitative immunofluorescence; 7) effect of MIF or CXCR4 antagonism. Results PAR1- or PAR4-AP triggered MIF release from both human urothelial cells in vitro and mouse urothelium in vivo. Twenty-four hours after intravesical PAR1- or PAR4-AP, we observed abdominal hypersensitivity in mice without changes in micturition or bladder histology. PAR4-AP was more effective and also increased expression of bladder MIF and urothelium MIF receptor, CXCR4. Bladder CXCR4 localized to the urothelium. Antagonizing MIF with ISO-1 eliminated PAR4- and reduced PAR1-induced hypersensitivity, while antagonizing CXCR4 with AMD3100 only partially prevented PAR4-induced hypersensitivity. Conclusions Bladder PAR activation elicits urothelial MIF release and urothelial MIF receptor signaling at least partly through CXCR4 to result in abdominal hypersensitivity without overt bladder inflammation. PAR-induced bladder pain may represent an interesting pre-clinical model of Interstitial Cystitis/Painful Bladder Syndrome (IC/PBS) where pain occurs without apparent bladder injury or pathology. MIF is potentially a novel therapeutic target for bladder pain in IC/PBS patients.

Neuropsychological correlates of normal variation in emotional response to visual stimuli.

Although the neural substrates of induced emotion have been the focus of numerous investigations, the factors related to individual variation in emotional experience have rarely been investigated in older adults. Twenty-six older normal subjects (mean age, 54) were shown color slides to elicit emotions of sadness, fear, or happiness and asked to rate the intensity of their emotional responses. Subjects who experienced negative emotion most intensely showed relative impairment on every aspect of the Wisconsin Card Sorting Test. Intense positive emotion was associated with relatively impaired performance on the Rey Complex Figure Test. The volume of frontal brain structures, however, was not associated with emotion responses. Hemisphere-specific executive dysfunction was associated with greater intensity of emotional experience in normal older subjects. The role of these differences in intensity of induced emotion and impairment in executive function in daily social and vocational activity should be investigated.

Preclinical study of a Kv11.1 potassium channel activator as antineoplastic approach for breast cancer

Potassium ion (K+) channels have been recently found to play a critical role in cancer biology. Despite that pharmacologic manipulation of ion channels is recognized as an important therapeutic approach, very little is known about the effects of targeting of K+ channels in cancer. In this study, we demonstrate that use of the Kv11.1 K+ channel activator NS1643 inhibits tumor growth in an in vivo model of breast cancer. Tumors exposed to NS1643 had reduced levels of proliferation markers, high expression levels of senescence markers, increased production of ROS and DNA damage compared to tumors of untreated mice. Importantly, mice treated with NS1643 did not exhibit significant cardiac dysfunction. In conclusion, pharmacological stimulation of Kv11.1 activity produced arrested TNBC-derived tumor growth by generating DNA damage and senescence without significant side effects. We propose that use of Kv11.1 channels activators could be considered as a possible pharmacological strategy against breast tumors.

Elevated Urine Levels of Macrophage Migration Inhibitory Factor in Inflammatory Bladder Conditions: A Potential Biomarker for a Subgroup of Interstitial Cystitis/Bladder Pain Syndrome Patients

OBJECTIVE To investigate whether urinary levels of macrophage migration inhibitory factor (MIF) are elevated in interstitial cystitis/bladder pain syndrome (IC/BPS) patients with Hunner lesions and also whether urine MIF is elevated in other forms of inflammatory cystitis. METHODS Urine samples were assayed for MIF by enzyme-linked immunosorbent assay. Urine samples from 3 female groups were examined: IC/BPS patients without (N = 55) and with Hunner lesions (N = 43), and non-IC/BPS patients (N = 100; control group; no history of IC/BPS; cancer or recent bacterial cystitis). Urine samples from 3 male groups were examined: patients with bacterial cystitis (N = 50), radiation cystitis (N = 18) and noncystitis patients (N = 119; control group; negative for bacterial cystitis). RESULTS Urine MIF (mean MIF pg/mL ±  standard error of the mean) was increased in female IC/BPS patients with Hunner lesions (2159 ± 435.3) compared with IC/BPS patients without Hunner lesions (460 ± 114.5) or non-IC/BPS patients (414 ± 47.6). Receiver operating curve analyses showed that urine MIF levels discriminated between the 2 IC groups (area under the curve = 72%; confidence interval 61%-82%). Male patients with bacterial and radiation cystitis had elevated urine MIF levels (2839 ± 757.1 and 4404 ± 1548.1, respectively) compared with noncystitis patients (681 ± 75.2). CONCLUSION Urine MIF is elevated in IC/BPS patients with Hunner lesions and also in patients with other bladder inflammatory and painful conditions. MIF may also serve as a noninvasive biomarker to select IC/BPS patients more accurately for endoscopic evaluation and possible anti-inflammatory treatment.

Activation of cyclic GMP-dependent protein kinase blocks alcohol-mediated cell death and calcium disruption in cerebellar granule neurons

Alcohol during brain development leads to the widespread neuronal death observed in fetal alcohol spectrum disorders (FASD). In comparison, the mature brain is less vulnerable to alcohol. Studies into maturation-acquired alcohol resistance uncovered a protective mechanism that reduces alcohol-induced neuronal death through nitric oxide-cGMP-cyclic GMP-dependent protein kinase (NO-cGMP-cGK) signaling. However, the downstream processes underlying this neuroprotection remain unclear. Alcohol can disrupt levels of intracellular calcium ([Ca2+]i) in vulnerable neuronal populations to trigger cell death in both in vivo and in vitro models of FASD. Since cGK has been demonstrated to regulate and inhibit intracellular Ca2+ release, we examined the hypothesis that cGK confers alcohol resistance by preventing [Ca2+]i disruptions. Alcohol resistance, determined by neuronal survival after 24 h of alcohol exposure, was examined in primary cerebellar granule neuron (CGN) cultures derived from 5 to 7 day-old neonatal mice with an activator, 8-Br-cGMP, and/or an inhibitor, Rp-8-pCPT-cGMPS, of cGK signaling. Intracellular Ca2+ responses to alcohol were measured by ratiometric Ca2+ imaging in Fura-2-loaded CGN cultures after 8-Br-cGMP treatment. Our results indicate that activating cGK with 8-Br-cGMP before alcohol administration provided neuroprotection, which the cGK inhibitor, Rp-8-pCPT-cGMPS, blocked. Alcohol exposure elevated [Ca2+]i, whereas 8-Br-cGMP pretreatment reduced both the level of the alcohol-induced rise in [Ca2+]i as well as the number of cells that responded to alcohol by increasing [Ca2+]i. These findings associate alcohol resistance, mediated by cGK signaling, to reduction of the persistent and toxic increase in [Ca2+]i from alcohol exposure.

Macrophage Migration Inhibitory Factor Mediates Protease-Activated Receptor 4-Induced Bladder Pain Through Urothelial High Mobility Group Box 1

Macrophage migration inhibitory factor (MIF) mediates pain although the mechanisms are not well understood. Urothelial activation of protease activated receptor 4 (PAR4) results in urothelial MIF release, urothelial high mobility group box 1 (HMGB1) release and bladder pain in mice without bladder inflammation. All three effects are prevented by MIF inhibition while intravesical disulfide HMGB1 alone can induce bladder pain. This study utilizes genetic MIF deletion to determine whether MIF mediates PAR4‐induced bladder pain and is upstream of HMGB1‐induced bladder pain. Wild type (C57/BL6) and MIF knockout (KO) mice were treated with intravesical PAR4 activating peptide or disulfide HMGB1 and tested for abdominal mechanical hypersensitivity at baseline (before treatment) and 24 h after injection. Micturition parameters and bladder histology were examined after behavioral test. Real‐time PCR and western blotting measured HMGB1 mRNA and protein levels in the bladders of naïve wild type and MIF KO mice, while immunofluorescence measured HMGB1 protein levels in the urothelium of both strains. Intravesical PAR4 activation resulted in abdominal mechanical hypersensitivity in wild‐type mice but not MIF KO mice. Intravesical disulfide HMGB1 induced abdominal mechanical hypersensitivity in both strains. Neither treatment resulted in significant changes in micturition or bladder histology in either strain. HMGB1 mRNA and protein levels were higher in MIF KO mouse bladders and the urothelium of MIF KO bladder had greater immunostaining than the wild‐type strain. MIF is a pivotal molecule mediating PAR4‐induced bladder pain and regulating urothelial HMGB1 production and release to elicit bladder pain.

Clonidine as a preoperative sedative: CLONIDINE AS A PREOPERATIVE SEDATIVE

The purpose of this study was to -examine the use of oral clonidine as a preoperative sedative prior to parenteral moderate sedation. Initially, four patients were given 0.2 mg oral clonidine but reduced to 0.1 mg clonidine due to -significant drops in blood pressure. Oral clonidine doses of 0.1 mg were then given to 19 patients preoperatively. In all these patients, blood pressure measurements decreased, but there were no significant differences in amounts of sedative agents needed in the clonidine group and the control group (N = 80). The conclusions reached suggest that clonidine has an advantage over other preoperative sedation agents in anxious patients exhibiting hypertension and tachycardia. However, the preop ideal dose required to reduce the amount of sedative drugs used as well as provide anxiolysis remains unknown. In further studies, different doses should be explored to determine what dosage of clonidine may offer hemodynamic protection as well as decrease sedative drugs needed.