A Feasibility Study to Evaluate Changes in Urinary Metabolites after OnabotulinumtoxinA Injection for Refractory Overactive Bladder.

Metabolomics analysis of urine before and after overactive bladder (OAB) treatment may demonstrate a unique molecular profile, allowing predictions of responses to treatment. This feasibility study aimed to correlate changes in urinary metabolome with changes in OAB symptoms after intravesical onabotulinumtoxinA (BTX-A) injections for refractory OAB. Women 18 years or older with non-neurogenic refractory OAB were recruited to complete OAB-V8 questionnaires and submit urine samples before and after 100 units intravesical BTX-A injection. Samples were submitted to CE-TOFMS metabolomics profiling. Data were expressed as percent of change from pre-treatment and were correlated with OAB-V8 score improvement. Urinary metabolite changes in the OAB-V8 groups were compared using the Kruskal-Wallis test, and associations between metabolites and OAB-V8 scores were examined using quantile regression analysis. Of 61 urinary metabolites commonly detected before and after BTX-A, there was a statistically significant decrease in adenosine and an increase in N8-acetylspermidine and guanidinoacetic acid levels associated with OAB score improvement, suggesting that intravesical BTX-A injection modifies the urinary metabolome. These urinary metabolites could provide insight into OAB pathophysiology and help identify patients who would benefit most from chemodenervation.

Nanotechnology as a tool to advance research and treatment of non-oncologic urogenital diseases.

Nanotechnology represents an expanding area of research and innovation in almost every field of science, including Medicine, where nanomaterial-based products have been developed for diagnostic and therapeutic applications. Because of their small, nanoscale size, these materials exhibit unique physical and chemical properties that differ from those of each component when considered in bulk. In Nanomedicine, there is an increasing interest in harnessing these unique properties to engineer nanocarriers for the delivery of therapeutic agents. Nano-based drug delivery platforms have many advantages over conventional drug administration routes as this technology allows for local and transdermal applications of therapeutics that can bypass the first-pass metabolism, improves drug efficacy through encapsulation of hydrophobic drugs, and allows for a sustained and controlled release of encapsulated agents. In Urology, nano-based drug delivery platforms have been extensively investigated and implemented for cancer treatment. However, there is also great potential for use of nanotechnology to treat non-oncologic urogenital diseases. We provide an update on research that is paving the way for clinical translation of nanotechnology in the areas of erectile dysfunction (ED), overactive bladder (OAB), interstitial cystitis/bladder pain syndrome (IC/BPS), and catheter-associated urinary tract infections (CAUTIs). Overall, preclinical and clinical studies have proven the utility of nanomaterials both as vehicles for transdermal and intravesical delivery of therapeutic agents and for urinary catheter formulation with antimicrobial agents to treat non-oncologic urogenital diseases. Although clinical translation will be dependent on overcoming regulatory challenges, it is inevitable before there is universal adoption of this technology to treat non-oncologic urogenital diseases.

Flavonoid Nobiletin Attenuates Cyclophosphamide-Induced Cystitis in Mice through Mechanisms That Involve Inhibition of IL-1ß Induced Connexin 43 Upregulation and Gap Junction Communication in Urothelial Cells.

Bladder inflammatory diseases cause various urinary symptoms, such as urinary frequency and painful urination, that impair quality of life. In this study, we used a mouse model of cyclophosphamide (CYP)-induced bladder inflammation and immortalized human urothelial (TRT-HU1) cells to explore the preventive potential of nobiletin (NOB), a polymethoxylated flavone enriched in citrus fruit peel, and investigate its mechanism of action in the bladder. Prophylaxis with PMF90 (60% NOB) attenuated the development of bladder inflammation and urinary symptoms in CYP-treated mice. PMF90 also reduced the upregulation of connexin 43 (Cx43), a major component of gap junction channels, in the bladder mucosa of CYP-treated mice. Stimulation of TRT-HU1 cells with the pro-inflammatory cytokine IL-1ß increased Cx43 mRNA and protein expression and enhanced gap junction coupling-responses that were prevented by pre-treatment with NOB. In urothelium-specific Cx43 knockout (uCx43KO) mice, macroscopic signs of bladder inflammation and changes in voiding behavior induced by CYP treatment were significantly attenuated when compared to controls. These findings indicate the participation of urothelial Cx43 in the development of bladder inflammation and urinary symptoms in CYP-treated mice and provide pre-clinical evidence for the preventive potential of NOB through its anti-inflammatory effects on IL-1ß signaling and urothelial Cx43 expression.

Medical Treatment of Overactive Bladder.

Overactive bladder is a disruptive urinary condition composed of urgency, frequency, and nocturia, which affects a large proportion of men and women. Symptoms are often associated with a decreased quality of life. After optimizing behavioral strategies, pharmacologic intervention is the next consideration for treatment. Therapeutic agents consist primarily of antimuscarinic and ß-agonist medications, as well as off-label use of antidepressants in some cases. These medications, although effective, can be associated with considerable adverse side effects. Combination therapies along with novel therapeutics and drug targets are under investigation.

Generation and Characterization of Immortalized Mouse Cortical Astrocytes From Wildtype and Connexin43 Knockout Mice.

We transduced mouse cortical astrocytes cultured from four litters of embryonic wildtype (WT) and connexin43 (Cx43) null mouse pups with lentiviral vector encoding hTERT and measured expression of astrocyte-specific markers up to passage 10 (p10). The immortalized cell lines thus generated (designated IWCA and IKOCA, respectively) expressed biomarkers consistent with those of neonatal astrocytes, including Cx43 from wildtype but not from Cx43-null mice, lack of Cx30, and presence of Cx26. AQP4, the water channel that is found in high abundance in astrocyte end-feet, was expressed at moderately high levels in early passages, and its mRNA and protein declined to low but still detectable levels by p10. The mRNA levels of the astrocyte biomarkers aldehyde dehydrogenase 1L1 (ALDH1L1), glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP) remained relatively constant during successive passages. GS protein expression was maintained while GFAP declined with cell passaging but was still detectable at p10. Both mRNA and protein levels of glutamate transporter 1 (GLT-1) declined with passage number. Immunostaining at corresponding times was consistent with the data from Western blots and provided evidence that these proteins were expressed at appropriate intracellular locations. Consistent with our goal of generating immortalized cell lines in which Cx43 was either functionally expressed or absent, IWCA cells were found to be well coupled with respect to intercellular dye transfer and similar to primary astrocyte cultures in terms of time course of junction formation, electrical coupling strength and voltage sensitivity. Moreover, barrier function was enhanced in co-culture of the IWCA cell line with bEnd.3 microvascular endothelial cells. In addition, immunostaining revealed oblate endogenous Cx43 gap junction plaques in IWCA that were similar in appearance to those plaques obtained following transfection of IKOCA cells with fluorescent protein tagged Cx43. Re-expression of Cx43 in IKOCA cells allows experimental manipulation of connexins and live imaging of interactions between connexins and other proteins. We conclude that properties of these cell lines resemble those of primary cultured astrocytes, and they may provide useful tools in functional studies by facilitating genetic and pharmacological manipulations in the context of an astrocyte-appropriate cellular environment.

Urothelium-Specific Deletion of Connexin43 in the Mouse Urinary Bladder Alters Distension-Induced ATP Release and Voiding Behavior.

Connexin43 (Cx43), the main gap junction and hemichannel forming protein in the urinary bladder, participates in the regulation of bladder motor and sensory functions and has been reported as an important modulator of day-night variations in functional bladder capacity. However, because Cx43 is expressed throughout the bladder, the actual role played by the detrusor and the urothelial Cx43 is still unknown. For this purpose, we generated urothelium-specific Cx43 knockout (uCx43KO) mice using Cre-LoxP system. We evaluated the day-night micturition pattern and the urothelial Cx43 hemichannel function of the uCx43KO mice by measuring luminal ATP release after bladder distention. In wild-type (WT) mice, distention-induced ATP release was elevated, and functional bladder capacity was decreased in the animals' active phase (nighttime) when Cx43 expression was also high compared to levels measured in the sleep phase (daytime). These day-night differences in urothelial ATP release and functional bladder capacity were attenuated in uCx43KO mice that, in the active phase, displayed lower ATP release and higher functional bladder capacity than WT mice. These findings indicate that urothelial Cx43 mediated ATP signaling and coordination of urothelial activity are essential for proper perception and regulation of responses to bladder distension in the animals' awake, active phase.

Kidney stone formation and the gut microbiome are altered by antibiotics in genetic hypercalciuric stone-forming rats.

Antibiotics can alter the gut microbiome (GMB), which may be associated with stone disease. We sought to determine the effect that antibiotics have on the GMB, urine ion excretion and stone formation in genetic hypercalciuric stone-forming (GHS) rats. 116th generation GHS rats were fed a fixed amount of a normal calcium (1.2%) and phosphate (0.65%) diet, and divided into three groups (n = 10): control (CTL) diet, or supplemented with ciprofloxacin (Cipro, 5 mg/day) or Bactrim (250 mg/day). Urine and fecal pellets were collected over 6, 12 and 18 weeks. Fecal DNA was amplified across the 16S rRNA V4 region. At 18 weeks, kidney stone formation was visualized by Faxitron and blindly assessed by three investigators. After 18 weeks, urine calcium and oxalate decreased with Bactrim compared to CTL and Cipro. Urine pH increased with Bactrim compared to CTL and Cipro. Urine citrate increased with Cipro compared to CTL and decreased by half with Bactrim. Calcification increased with Bactrim compared to CTL and Cipro. Increased microbial diversity correlated with decreased urinary oxalate in all animals (R = - 0.46, p = 0.006). A potential microbial network emerged as significantly associated with shifts in urinary pH. Bactrim and Cipro differentially altered the GMB of GHS rats. The Bactrim group experienced a decrease in urine calcium, increased CaP supersaturation and increased calcification. The GMB is likely a contributing factor to changes in urine chemistry, supersaturation and stone risk. Further investigation is required to fully understand the association between antibiotics, the GMB and kidney stone formation.

Mechanosensitive Vaginal Epithelial Adenosine Triphosphate Release and Pannexin 1 Channels in Healthy, in Type 1 Diabetic, and in Surgically Castrated Female Mice.

BACKGROUND: Distension of hollow organs is known to release adenosine triphosphate (ATP) from the lining epithelium, which triggers local responses and activates sensory nerves to convey information to the central nervous system. However, little is known regarding participation of ATP and mediators of ATP release, such as Pannexin 1 (Panx1) channels, in mechanisms of vaginal mechanosensory transduction and of changes imposed by diabetes and menopause, conditions associated with vaginal dysfunction and risk for impaired genital arousal. AIM: To investigate if intravaginal mechanical stimulation triggers vaginal ATP release and if (a) this response involves Panx1 channels and (b) this response is altered in animal models of diabetes and menopause. METHODS: Diabetic Akita female mice were used as a type 1 diabetes (T1D) model and surgical castration (ovariectomy [OVX]) as a menopause model. Panx1-null mice were used to evaluate Panx1 participation in mechanosensitive vaginal ATP release. Vaginal washes were collected from anesthetized mice at baseline (non-stimulated) and at 5 minutes after intravaginal stimulation. For the OVX and Sham groups, samples were collected before surgery and at 4, 12, 22, 24, and 28 weeks after surgery. ATP levels in vaginal washes were measured using the luciferin-luciferase assay. Panx1 mRNA levels in vaginal epithelium were quantified by quantitative polymerase chain reaction. OUTCOMES: The main outcome measures are quantification of mechanosensitive vaginal ATP release and evaluation of impact of Panx1 deletion, OVX, and T1D on this response. RESULTS: Intravaginal mechanical stimulation-induced vaginal ATP release was 84% lower in Panx1-null (P < .001) and 76% lower in diabetic (P < .0001) mice compared with controls and was reduced in a progressive and significant manner in OVX mice when compared with Sham. Panx1 mRNA expression in vaginal epithelium was 44% lower in diabetics than that in controls (P < .05) and 40% lower in OVX than that in the Sham (P < .05) group. CLINICAL TRANSLATION: Panx1 downregulation and consequent attenuation of mechanosensitive vaginal responses may be implicated in mechanisms of female genital arousal disorder, thereby providing potential targets for novel therapies to manage this condition. STRENGTHS & LIMITATIONS: Using animal models, we demonstrated Panx1 involvement in mechanosensitive vaginal ATP release and effects of T1D and menopause on this response and on Panx1 expression. A limitation is that sex steroid hormone levels were not measured, precluding correlations and insights into mechanisms that may regulate Panx1 expression in the vaginal epithelium. CONCLUSIONS: Panx1 channel is a component of the vaginal epithelial mechanosensory transduction system that is essential for proper vaginal response to mechanical stimulation and is targeted in T1D and menopause. Harroche J, Urban-Maldonado M, Thi MM, et al. Mechanosensitive Vaginal Epithelial Adenosine Triphosphate Release and Pannexin 1 Channels in Healthy, in Type 1 Diabetic, and in Surgically Castrated Female Mice. J Sex Med 2020;17:870-880.

Fecal transplant modifies urine chemistry risk factors for urinary stone disease.

Urinary stone disease (USD) is a major health concern. There is a need for new treatment modalities. Recently, our group provided evidence for an association between the GMB composition and USD. The accessibility of the Gut Microbiome (GMB) makes it an attractive target for investigation and therefore, in these studies we have evaluated the extent to which the whole gut microbial community in fecal transplants can affect urinary stone risk parameters in an animal model. Fresh fecal pellets were collected from Zucker lean rats, homogenized in PBS (100 mg/mL), filtered through a 70 µm strainer and then orally gavaged into C57BL/6NTac germ-free mice. Twenty-four hours urine collections and GMB analysis were performed over time for 1 month. Kidney and gut tissue were harvested from transplanted mice for western blot analysis of expression levels of the Slc26a6 transporter involved in oxalate balance. Urinary calcium decreased after fecal transplant by 55% (P < 0.001). Urinary oxalate levels were on average 24% lower than baseline levels (P < 0.001). Clostridiaceae family was negatively correlated with urinary oxalate at 4 weeks after transplant (r = -0.83, P < 0.01). There was a 0.6 unit average increase in urinary pH from a baseline of 5.85 (SE ± 0.028) to 6.49 (SE ± 0.04) (P < 0.001) after transplant. There was a concomitant 29% increase in gastrointestinal alkali absorption (P < 0.001) 4-weeks after fecal transplant. Slc26a6 expression increased by 90% in the cecum after transplant. Our results suggest that the gut microbiome may impact metabolism, alters urinary chemistry, and thereby may influence USD; the accessibility of the GMB can potentially be leveraged for therapeutic interventions.

Gap junction mediated signaling between satellite glia and neurons in trigeminal ganglia.

Peripheral sensory ganglia contain the somata of neurons mediating mechanical, thermal, and painful sensations from somatic, visceral, and oro-facial organs. Each neuronal cell body is closely surrounded by satellite glial cells (SGCs) that have properties and functions similar to those of central astrocytes, including expression of gap junction proteins and functional dye coupling. As shown in other pain models, after systemic pain induction by intra-peritoneal injection of lipopolysaccharide, dye coupling among SGCs in intact trigeminal ganglion was enhanced. Moreover, neuron-neuron and neuron-SGC coupling was also detected. To verify the presence of gap junction-mediated coupling between SGCs and sensory neurons, we performed dual whole cell patch clamp recordings from both freshly isolated and short term cultured cell pairs dissociated from mouse trigeminal ganglia. Bidirectional gap junction mediated electrical responses were frequently recorded between SGCs, between neurons and between neurons and SGCs. Polarization of SGC altered neuronal excitability, providing evidence that gap junction-mediated interactions between neurons and glia within sensory ganglia may contribute to integration of peripheral sensory responses, and to the modulation and coordinaton of neuronal activity.

Role of pannexin 1 channels in load-induced skeletal response.

The pannexin 1 (Panx1) channel is a mechanosensitive channel that interacts with P2X7 receptors (P2X7R) to form a functional complex that has been shown in vitro to play an essential role in osteocyte mechanosignaling. While the participation of P2X7R in skeletal responses to mechanical loading has been demonstrated, the role of Panx1 and its interplay with P2X7R still remain to be determined. In this study, we use a global Panx1-/- mouse model and in vivo mechanical loading to demonstrate that Panx1 channels play an essential role in load-induced skeletal responses. We found that absence of Panx1 not only disrupts the P2X7R-Panx1 signaling complex, but also alters load-induced regulation of P2X7R expression. Moreover, lack of Panx1 completely abolished load-induced periosteal bone formation. Load-induced regulation of ß-catenin and sclerostin expression was dysregulated in Panx1-/- , compared to wild-type, bone. This finding suggests that Panx1 deficiency disrupts Wnt/ß-catenin signaling by lowering ß-catenin while favoring inhibition of bone formation by increasing load-induced sclerostin expression. This study demonstrates the existence of a Panx1-dependent mechanosensitive mechanism that not only modulates ATP signaling but also coordinates Wnt/ß-catenin signaling that is essential for proper skeletal response to mechanical loading.

A new perspective on mechanisms governing skeletal complications in type 1 diabetes.

This review focuses on bone mechanobiology in type 1 diabetes (T1D), an area of research on diabetes-associated skeletal complications that is still in its infancy. We first provide a brief overview of the deleterious effects of diabetes on the skeleton and of the knowledge gained from studies with rodent models of T1D. Second, we discuss two specific hallmarks of T1D, low insulin and high glucose, and address the extent to which they affect skeletal health. Third, we highlight the mechanosensitive nature of bone tissue and the importance of mechanical loading for bone health. We also summarize recent advances in bone mechanobiology that implicate osteocytes as the mechanosensors and major regulatory cells in the bone. Finally, we discuss recent evidence indicating that the diabetic bone is "deaf" to mechanical loading and that osteocytes are central players in mechanisms that lead to bone loss in T1D.

P2X7R-Panx1 Complex Impairs Bone Mechanosignaling under High Glucose Levels Associated with Type-1 Diabetes.

Type 1 diabetes (T1D) causes a range of skeletal problems, including reduced bone density and increased risk for bone fractures. However, mechanisms underlying skeletal complications in diabetes are still not well understood. We hypothesize that high glucose levels in T1D alters expression and function of purinergic receptors (P2Rs) and pannexin 1 (Panx1) channels, and thereby impairs ATP signaling that is essential for proper bone response to mechanical loading and maintenance of skeletal integrity. We first established a key role for P2X7 receptor-Panx1 in osteocyte mechanosignaling by showing that these proteins are co-expressed to provide a major pathway for flow-induced ATP release. To simulate in vitro the glucose levels to which bone cells are exposed in healthy vs. diabetic bones, we cultured osteoblast and osteocyte cell lines for 10 days in medium containing 5.5 or 25 mM glucose. High glucose effects on expression and function of P2Rs and Panx1 channels were determined by Western Blot analysis, quantification of Ca2+ responses to P2R agonists and oscillatory fluid shear stress (± 10 dyne/cm(2)), and measurement of flow-induced ATP release. Diabetic C57BL/6J-Ins2Akita mice were used to evaluate in vivo effects of high glucose on P2R and Panx1. Western blotting indicated altered P2X7R, P2Y2R and P2Y4R expression in high glucose exposed bone cells, and in diabetic bone tissue. Moreover, high glucose blunted normal P2R- and flow-induced Ca2+ signaling and ATP release from osteocytes. These findings indicate that T1D impairs load-induced ATP signaling in osteocytes and affects osteoblast function, which are essential for maintaining bone health.

Pannexin-1 and P2X7-Receptor Are Required for Apoptotic Osteocytes in Fatigued Bone to Trigger RANKL Production in Neighboring Bystander Osteocytes.

Osteocyte apoptosis is required to induce intracortical bone remodeling after microdamage in animal models, but how apoptotic osteocytes signal neighboring "bystander" cells to initiate the remodeling process is unknown. Apoptosis has been shown to open pannexin-1 (Panx1) channels to release adenosine diphosphate (ATP) as a "find-me" signal for phagocytic cells. To address whether apoptotic osteocytes use this signaling mechanism, we adapted the rat ulnar fatigue-loading model to reproducibly introduce microdamage into mouse cortical bone and measured subsequent changes in osteocyte apoptosis, receptor activator of NF-κB ligand (RANKL) expression and osteoclastic bone resorption in wild-type (WT; C57Bl/6) mice and in mice genetically deficient in Panx1 (Panx1KO). Mouse ulnar loading produced linear microcracks comparable in number and location to the rat model. WT mice showed increased osteocyte apoptosis and RANKL expression at microdamage sites at 3 days after loading and increased intracortical remodeling and endocortical tunneling at day 14. With fatigue, Panx1KO mice exhibited levels of microdamage and osteocyte apoptosis identical to WT mice. However, they did not upregulate RANKL in bystander osteocytes or initiate resorption. Panx1 interacts with P2X7 R in ATP release; thus, we examined P2X7 R-deficient mice and WT mice treated with P2X7 R antagonist Brilliant Blue G (BBG) to test the possible role of ATP as a find-me signal. P2X7 RKO mice failed to upregulate RANKL in osteocytes or induce resorption despite normally elevated osteocyte apoptosis after fatigue loading. Similarly, treatment of fatigued C57Bl/6 mice with BBG mimicked behavior of both Panx1KO and P2X7 RKO mice; BBG had no effect on osteocyte apoptosis in fatigued bone but completely prevented increases in bystander osteocyte RANKL expression and attenuated activation of resorption by more than 50%. These results indicate that activation of Panx1 and P2X7 R are required for apoptotic osteocytes in fatigued bone to trigger RANKL production in neighboring bystander osteocytes and implicate ATP as an essential signal mediating this process.

Regulation of prostaglandin EP1 and EP4 receptor signaling by carrier-mediated ligand reuptake.

After synthesis and release from cells, prostaglandin E2 (PGE2) undergoes reuptake by the prostaglandin transporter (PGT), followed by cytoplasmic oxidation. Although genetic inactivation of PGT in mice and humans results in distinctive phenotypes, and although experiments in localized environments show that manipulating PGT alters downstream cellular events, a direct mechanistic link between PGT activity and PGE2 (EP) receptor activation has not been made. Toward this end, we created two reconstituted systems to examine the effect of PGT expression on PGE2 signaling via two of its receptors (EP1 and EP4). In human embryonic kidney cells engineered to express the EP1 receptor, exogenous PGE2 induced a dose-dependent increase in cytoplasmic Ca(2+). When PGT was expressed at the plasma membrane, the PGE2 dose-response curve was right-shifted, consistent with reduction in cell surface PGE2 availability; a potent PGT inhibitor acutely reversed this shift. When bradykinin was used to induce endogenous PGE2 release, PGT expression similarly induced a reduction in Ca(2+) responses. In separate experiments using Madin-Darby Canine Kidney cells engineered to express the PGE2 receptor EP4, bradykinin again induced autocrine PGE2 signaling, as judged by an abrupt increase in intracellular cAMP. As in the EP1 experiments, expression of PGT at the plasma membrane caused a reduction in bradykinin-induced cAMP accumulation. Pharmacological concentrations of exogenous PGE2 induced EP4 receptor desensitization, an effect that was mitigated by PGT. Thus, at an autocrine/paracrine level, plasma membrane PGT regulates PGE2 signaling by decreasing ligand availability at cell surface receptors.

Pannexin 1 channels play essential roles in urothelial mechanotransduction and intercellular signaling.

Urothelial cells respond to bladder distension with ATP release, and ATP signaling within the bladder and from the bladder to the CNS is essential for proper bladder function. In other cell types, pannexin 1 (Panx1) channels provide a pathway for mechanically-induced ATP efflux and for ATP-induced ATP release through interaction with P2X7 receptors (P2X7Rs). We report that Panx1 and P2X7R are functionally expressed in the bladder mucosa and in immortalized human urothelial cells (TRT-HU1), and participate in urothelial ATP release and signaling. ATP release from isolated rat bladders induced by distention was reduced by the Panx1 channel blocker mefloquine (MFQ) and was blunted in mice lacking Panx1 or P2X7R expression. Hypoosmotic shock induced YoPro dye uptake was inhibited by MFQ and the P2X7R blocker A438079 in TRT-HU1 cells, and was also blunted in primary urothelial cells derived from mice lacking Panx1 or P2X7R expression. Rinsing-induced mechanical stimulation of TRT-HU1 cells triggered ATP release, which was reduced by MFQ and potentiated in low divalent cation solution (LDPBS), a condition known to enhance P2X7R activation. ATP signaling evaluated as intercellular Ca2+ wave radius was significantly larger in LDPBS, reduced by MFQ and by apyrase (ATP scavenger). These findings indicate that Panx1 participates in urothelial mechanotransduction and signaling by providing a direct pathway for mechanically-induced ATP release and by functionally interacting with P2X7Rs.

Human liver cell trafficking mutants: characterization and whole exome sequencing.

The HuH7 liver cell mutant Trf1 is defective in membrane trafficking and is complemented by the casein kinase 2α subunit CK2α''. Here we identify characteristic morphologies, trafficking and mutational changes in six additional HuH7 mutants Trf2-Trf7. Trf1 cells were previously shown to be severely defective in gap junction functions. Using a Lucifer yellow transfer assay, remarkable attenuation of gap junction communication was revealed in each of the mutants Trf2-Trf7. Electron microscopy and light microscopy of thiamine pyrophosphatase showed that several mutants exhibited fragmented Golgi apparatus cisternae compared to parental HuH7 cells. Intracellular trafficking was investigated using assays of transferrin endocytosis and recycling and VSV G secretion. Surface binding of transferrin was reduced in all six Trf2-Trf7 mutants, which generally correlated with the degree of reduced expression of the transferrin receptor at the cell surface. The mutants displayed the same transferrin influx rates as HuH7, and for efflux rate, only Trf6 differed, having a slower transferrin efflux rate than HuH7. The kinetics of VSV G transport along the exocytic pathway were altered in Trf2 and Trf5 mutants. Genetic changes unique to particular Trf mutants were identified by exome sequencing, and one was investigated in depth. The novel mutation Ile34Phe in the GTPase RAB22A was identified in Trf4. RNA interference knockdown of RAB22A or overexpression of RAB22AI34F in HuH7 cells caused phenotypic changes characteristic of the Trf4 mutant. In addition, the Ile34Phe mutation reduced both guanine nucleotide binding and hydrolysis activities of RAB22A. Thus, the RAB22A Ile34Phe mutation appears to contribute to the Trf4 mutant phenotype.

Mechanosensory responses of osteocytes to physiological forces occur along processes and not cell body and require αVß3 integrin.

Osteocytes in the lacunar-canalicular system of the bone are thought to be the cells that sense mechanical loading and transduce mechanical strain into biomechanical responses. The goal of this study was to evaluate the extent to which focal mechanical stimulation of osteocyte cell body and process led to activation of the cells, and determine whether integrin attachments play a role in osteocyte activation. We use a novel Stokesian fluid stimulus probe to hydrodynamically load osteocyte processes vs. cell bodies in murine long bone osteocyte Y4 (MLO-Y4) cells with physiological-level forces <10 pN without probe contact, and measured intracellular Ca(2+) responses. Our results indicate that osteocyte processes are extremely responsive to piconewton-level mechanical loading, whereas the osteocyte cell body and processes with no local attachment sites are not. Ca(2+) signals generated at stimulated sites spread within the processes with average velocity of 5.6 µm/s. Using the near-infrared fluorescence probe IntegriSense 750, we demonstrated that inhibition of αVß3 integrin attachment sites compromises the response to probe stimulation. Moreover, using apyrase, an extracellular ATP scavenger, we showed that Ca(2+) signaling from the osteocyte process to the cell body was greatly diminished, and thus dependent on ATP-mediated autocrine signaling. These findings are consistent with the hypothesis that osteocytes in situ are highly polarized cells, where mechanotransduction occurs at substrate attachment sites along the processes at force levels predicted to occur at integrin attachment sites in vivo. We also demonstrate the essential role of αVß3 integrin in osteocyte-polarized mechanosensing and mechanotransduction.

Disruption of calcium homeostasis in cardiomyocytes underlies cardiac structural and functional changes in severe sepsis.

Sepsis, a major cause of morbidity/mortality in intensive care units worldwide, is commonly associated with cardiac dysfunction, which worsens the prognosis dramatically for patients. Although in recent years the concept of septic cardiomyopathy has evolved, the importance of myocardial structural alterations in sepsis has not been fully explored. This study offers novel and mechanistic data to clarify subcellular events that occur in the pathogenesis of septic cardiomyopathy and myocardial dysfunction in severe sepsis. Cultured neonatal mice cardiomyocytes subjected to serum obtained from mice with severe sepsis presented striking increment of [Ca(2+)]i and calpain-1 levels associated with decreased expression of dystrophin and disruption and derangement of F-actin filaments and cytoplasmic bleb formation. Severe sepsis induced in mice led to an increased expression of calpain-1 in cardiomyocytes. Moreover, decreased myocardial amounts of dystrophin, sarcomeric actin, and myosin heavy chain were observed in septic hearts associated with depressed cardiac contractile dysfunction and a very low survival rate. Actin and myosin from the sarcomere are first disassembled by calpain and then ubiquitinated and degraded by proteasome or sequestered inside specialized vacuoles called autophagosomes, delivered to the lysosome for degradation forming autophagolysosomes. Verapamil and dantrolene prevented the increase of calpain-1 levels and preserved dystrophin, actin, and myosin loss/reduction as well cardiac contractile dysfunction associated with strikingly improved survival rate. These abnormal parameters emerge as therapeutic targets, which modulation may provide beneficial effects on future vascular outcomes and mortality in sepsis. Further studies are needed to shed light on this mechanism, mainly regarding specific calpain inhibitors.

Contribution of pannexin1 to experimental autoimmune encephalomyelitis.

Pannexin1 (Panx1) is a plasma membrane channel permeable to relatively large molecules, such as ATP. In the central nervous system (CNS) Panx1 is found in neurons and glia and in the immune system in macrophages and T-cells. We tested the hypothesis that Panx1-mediated ATP release contributes to expression of Experimental Autoimmune Encephalomyelitis (EAE), an animal model for multiple sclerosis, using wild-type (WT) and Panx1 knockout (KO) mice. Panx1 KO mice displayed a delayed onset of clinical signs of EAE and decreased mortality compared to WT mice, but developed as severe symptoms as the surviving WT mice. Spinal cord inflammatory lesions were also reduced in Panx1 KO EAE mice during acute disease. Additionally, pharmacologic inhibition of Panx1 channels with mefloquine (MFQ) reduced severity of acute and chronic EAE when administered before or after onset of clinical signs. ATP release and YoPro uptake were significantly increased in WT mice with EAE as compared to WT non-EAE and reduced in tissues of EAE Panx1 KO mice. Interestingly, we found that the P2X7 receptor was upregulated in the chronic phase of EAE in both WT and Panx1 KO spinal cords. Such increase in receptor expression is likely to counterbalance the decrease in ATP release recorded from Panx1 KO mice and thus contribute to the development of EAE symptoms in these mice. The present study shows that a Panx1 dependent mechanism (ATP release and/or inflammasome activation) contributes to disease progression, and that inhibition of Panx1 using pharmacology or gene disruption delays and attenuates clinical signs of EAE.