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Leydig cells (LCs) in the testes produce the male sex hormone testosterone (T). Several xenobiotics, including clinical drugs, supplements, and environmental chemicals, are known to disrupt T homeostasis. Notably, some of these xenobiotics are known to activate the pregnane X receptor (PXR), a ligand-dependent nuclear receptor. However, it is currently unknown whether PXR is expressed in LCs and whether PXR activation alters T synthesis in rodent LCs. Therefore, in this study, we sought to determine whether PXR is expressed in rodent LCs and whether pregnenolone 16-alpha carbonitrile (PCN), the prototype agonist of rodent PXR, regulates T biosynthesis in rodent LCs. Hormonal as well as protein and gene expression analyses were conducted in rat primary LCs and MA-10 mouse Leydig cells. Results showed that PXR was expressed at the mRNA and protein level in both rat primary LCs and MA-10 cells. Incubation of rat primary LCs with PCN resulted in a significant decrease in T secretion. This PCN-induced decrease in T secretion was associated with decreased protein expression of key steroidogenic enzymes such as 3β-HSD and CYP17A1. RNA-seq results from MA-10 cells showed that PCN down-regulated the transcripts of steroidogenic enzymes and proteins involved in the T synthesis pathway. Together, these results suggest that PCN, an agonist of rodent PXR, can regulate T biosynthesis in rodent LCs by down-regulating the expression of the steroidogenic enzymes involved in T biosynthesis. Our results are significant as they provide a potential novel mechanism for disruption of testosterone homeostasis by a variety of xenobiotics. [ABSTRACT FROM AUTHOR]

In our previous study, we demonstrated the impact of overexpression of CB1 and CB2 cannabinoid receptors and the inhibitory effect of endocannabinoids (2-arachidonoylglycerol (2-AG) and Anandamide (AEA)) on canine (Canis lupus familiaris) and human (Homo sapiens) non-Hodgkin lymphoma (NHL) cell lines' viability compared to cells treated with a vehicle. The purpose of this study was to demonstrate the anti-cancer effects of the phytocannabinoids, cannabidiol (CBD) and ∆9-tetrahydrocannabinol (THC), and the synthetic cannabinoid WIN 55-212-22 (WIN) in canine and human lymphoma cell lines and to compare their inhibitory effect to that of endocannabinoids. We used malignant canine B-cell lymphoma (BCL) (1771 and CLB-L1) and T-cell lymphoma (TCL) (CL-1) cell lines, and human BCL cell line (RAMOS). Our cell viability assay results demonstrated, compared to the controls, a biphasic effect (concentration range from 0.5 μM to 50 μM) with a significant reduction in cancer viability for both phytocannabinoids and the synthetic cannabinoid. However, the decrease in cell viability in the TCL CL-1 line was limited to CBD. The results of the biochemical analysis using the 1771 BCL cell line revealed a significant increase in markers of oxidative stress, inflammation, and apoptosis, and a decrease in markers of mitochondrial function in cells treated with the exogenous cannabinoids compared to the control. Based on the IC50 values, CBD was the most potent phytocannabinoid in reducing lymphoma cell viability in 1771, Ramos, and CL-1. Previously, we demonstrated the endocannabinoid AEA to be more potent than 2-AG. Our study suggests that future studies should use CBD and AEA for further cannabinoid testing as they might reduce tumor burden in malignant NHL of canines and humans. [ABSTRACT FROM AUTHOR]

Introduction: Osteosarcoma (OSA) is an aggressive form of bone cancer in both dogs and humans. The treatment options for metastatic (stage III) OSA are currently limited and the prognosis is poor. Zoledronate, a second generation aminobisphosphonate, is commonly used for palliation of cancer induced bone pain. Zoledronate has also demonstrated anti-cancer properties and possibly enhances the cytotoxicity of doxorubicin in a canine histiocytosis cell line and human prostatic cancer cell line. The goal of this study was to evaluate the combination effect of zoledronate and various chemotherapeutic drugs in canine OSA cells. Method: Canine OSA cell line (D17), cells from two canine primary OSAs, and MDCK, a canine kidney cell line, were used to evaluate the therapeutic potential of these drugs. Carboplatin, doxorubicin, vinorelbine, toceranib, and isophosphoramide mustard (active metabolite of ifosfamide) were used as chemotherapeutic agents. First, cells were treated with either zoledronate or chemotherapy drug alone for 72 hours. Cell viability was assessed using CellTiter Glo and IC5, IC10, IC20, and IC50 were calculated. Second, cells were treated with a combination of zoledronate and each chemotherapeutic agent at their IC5, IC10, IC20, and IC50 concentrations. After 72 hours, cell viability was assessed by CellTiter Glo. Results and Discussion: Zoledronate, carboplatin, doxorubicin, vinorelbine, and isophosphoramide mustard showed concentration dependent decrease in cell viability. Toceranib showed decreased cell viability only at higher concentrations. When zoledronate was used in combination with chemotherapy drugs, while it showed potential synergistic effects with toceranib, potential antagonistic effects with vinorelbine and isophosphoramide mustard were observed. However, the results differed by cell line and thus, further evaluation is warranted to understand the exact mechanism of action. [ABSTRACT FROM AUTHOR]

Reissner's membrane epithelium forms much of the barrier that produces and sustains the large ionic differences between cochlear endolymph and perilymph. We have reported that Reissner's membrane contributes to normal cochlear function by absorbing [Na.sup.+] from endolymph via amiloride-sensitive channels in gerbil inner ear. We used mouse Reissner's membrane to 1) identify candidate genes involved in the [Na.sup.+] transport pathway, 2) determine whether their level of expression was regulated by the synthetic glucocorticoid dexamethasone, and 3) obtain functional evidence for the physiological importance of these genes. Transcripts were present for [alpha]-, [beta]-, and [gamma]-subunits of epithelial [Na.sup.+] channel (ENaC); corticosteroid receptors GR (glucocorticoid receptor) and MR (mineralocorticoid receptor); GR agonist regulator 11[beta]hydroxysteroid debydrogenase (HSD) type 1 (11[beta]-HSD1); [Na.sup.+] transport control components SGK1, Nedd4-2, and WNKs; and [K.sup.+] channels and [Na.sup.+]-[K.sup.+]-ATPase. Expression of the MR agonist regulator 11[beta]-HSD2 was not detected. Dexamethasone upregulated transcripts for [alpha]- and [beta]-subunits of ENaC (~6- and ~3-fold), KCNK1 (~3-fold), 1l[beta]-HSD1 (~2-fold), SGK1 (~2-fold), and WNK4 (~3-fold). Transepithelial currents from the apical to the basolateral side of Reissner's membrane were sensitive to amiloride ([IC.sub.50] ~0.7 [micro]M) and benzamil ([IC.sub.50] ~0.1 [micro]M), but not EIPA ([IC.sub.50] ~34 [micro]M); amiloride-blocked transepithelial current was not immediately changed by forskolin/IBMX. Currents were reduced by ouabain, lowered bath [Na.sup.+] concentration (from 150 to 120 mM), and [K.sup.+] channel blockers (XE-991, [Ba.sup.2+], and acidification from pH 7.4 to 6.5). Dexamethasone-stimulated current and gene expression were reduced by mifepristone, but not spironolactone. These molecular, pharmacological, and functional observations are consistent with [Na.sup.+] absorption by mouse Reissner's membrane, which is mediated by apical ENaC and/or other amiloride-sensitive channels, basolateral [Na.sup.+]-[K.sup.+]-ATPase, and [K.sup.+]-permeable channels and is under the control of glucocorticoids. These results provide an understanding and a molecular definition of an important transport function of Reissner's membrane epithelium in the homeostasis of cochlear endolymph. inner ear; corticosteroids; endolymph homeostasis; gene expression; vibrating probe

Glucocorticoids stimulate cation absorption by semicircular canal duct epithelium via epithelial sodium channel. Am J Physiol Renal Physiol 286: F1127-F1135, 2004. First published March 2, 2004; 10.1152/ajprenal. 00387.2003.--The semicircular canal duct (SCCD) epithelium is a vestibular epithelial domain that was recently shown to actively contribute to endolymph homeostasis by [Cl.sup.-] secretion under control of [[Beta].sub.2]-adrenergic stimulation. By analogy to other [Cl.sup.-] secretory epithelia, we hypothesized that SCCD also provides an active absorptive pathway for [Na.sup.+] under corticosteroid control. Measurements of short-circuit current ([I.sub.sc]) demonstrated stimulation (7-24 h) by the glucocorticoids hydrocortisone (E[C.sub.50] 13 nM), corticosterone (33 nM), prednisolone (70 nM), and dexamethasone (13 nM) over physiologically and therapeutically relevant concentrations and its block by amiloride (I[C.sub.50] 470 nM) and benzamil (57 nM), inhibitors of the epithelial sodium channel (ENaC). [I.sub.sc] was also partially inhibited by basolateral ouabain and [Ba.sup.2+], indicating the participation of [Na.sup.+]K+-ATPase and a [K.sup.+] channel in Na+ transport. By contrast, aldosterone stimulated [I.sub.sc] only at unphysiologically high concentrations (E[C.sub.50] 102 nM). The action of all steroids was blocked by mifepristone (RU-486; [K.sub.d] ~0.3 nM) but not by spironolactone ([K.sub.d] ~0.7 [micro]M). Expression of mRNA for the [alpha]-, [Beta]-, and [gamma]-subunits of ENaC was demonstrated in the presence and absence of glucocorticoids. These findings are the first to identify SCCD in the vestibular labyrinth as a site of physiologically significant ENaC-mediated [Na.sup.+] absorption and osmotically coupled water flux. They further demonstrate regulation of [Na.sup.+] transport by natural and therapeutic glucocorticoids. The results provide for the first time an understanding of the therapeutic benefit of glucocorticoids in the treatment of Meniere's disease, a condition that is associated with increased luminal fluid volume. rat; inner ear; vestibular labyrinth; dexamethasone; prednisolone; cortisol (hydrocortisone)

The ductal epithelium of the semicircular canal forms much of the boundary between the [K.sup.+]-rich luminal fluid and the [Na.sup.+]-rich abluminal fluid. We sought to determine whether the net ion flux producing the apical-to-basal short-circuit current ([I.sub.sc]) in primary cultures was due to anion secretion and/or cation absorption and under control of receptor agonists. Net fluxes of [sup.22]Na, [sup.86]Rb, and [sup.36]Cl demonstrated a basal-to-apical C1- secretion that was stimulated by isoproterenol. Isoproterenol and norepinephrine increased [I.sub.sc] with an E[C.sub.50 ]of 3 and 15 nM, respectively, and isoproterenol increased tissue cAMP of native canals with an E[C.sub.50 ]of 5 nM. Agonists for adenosine, histamine, and vasopressin receptors had no effect on [I.sub.sc]. Isoproterenol stimulation of [I.sub.sc] and cAMP was inhibited by ICI-118551 (I[C.sub.50] = 6 [micro]M for [I.sub.sc]) but not by CGP-20712A (1 [micro]M) in primary cultures, and similar results were found in native epithelium. [I.sub.sc] was partially inhibited by basolateral [Ba.sup.2+] (I[C.sub.50] = 0.27 mM) and ouabain, whereas responses to genistein, glibenclamide, and DIDS did not fully fit the profile for CFTR. Our findings show that the canal epithelium contributes to endolymph homeostasis by secretion of [Cl.sup.-] under [beta]-adrenergic control with cAMP as second messenger, a process that parallels the adrenergic control of [K.sup.+] secretion by vestibular dark cells. The current work points to one possible etiology of endolymphatic hydrops in Meniere's disease and may provide a basis for intervention. anion secretion; vestibular labyrinth; receptors; endolymph

Abstract Background Sodium absorption by semicircular canal duct (SCCD) epithelial cells is thought to contribute to the homeostasis of the volume of vestibular endolymph. It was previously shown that the epithelial cells could absorb Na+ under control of a glucocorticoid hormone (dexamethasone) and the absorptive transepithelial current was blocked by amiloride. The most commonly-observed target of amiloride is the epithelial sodium channel (ENaC), comprised of the three subunits α-, β- and γ-ENaC. However, other cation channels have also been observed to be sensitive in a similar concentration range. The aim of this study was to determine whether SCCD epithelial cells absorb only Na+ or also K+ through an amiloride-sensitive pathway. Parasensory K+ absorption could contribute to regulation of the transduction current through hair cells, as found to occur via vestibular transitional cells [S. H. Kim and D. C. Marcus. Regulation of sodium transport in the inner ear. Hear.Res. doi:10.1016/j.heares.2011.05.003, 2011]. Results We determined the molecular and functional expression of candidate cation channels with gene array (GEO GSE6197), whole-cell patch clamp and transepithelial recordings in primary cultures of rat SCCD. α-, β- and γ-ENaC were all previously reported as present. The selectivity of the amiloride-sensitive transepithelial and cell membrane currents was observed in Ussing chamber and whole-cell patch clamp recordings. The cell membrane currents were carried by Na+ but not K+, but the Na+ selectivity disappeared when the cells were cultured on impermeable supports. Transepithelial currents across SCCD were also carried exclusively by Na+. Conclusions These results are consistent with the amiloride-sensitive absorptive flux of SCCD mediated by a highly Na+-selective channel, likely αβγ-ENaC. These epithelial cells therefore absorb only Na+ via the amiloride-sensitive pathway and do not provide a parasensory K+ efflux from the canals via this pathway. The results further provide caution to the culture of epithelial cells on impermeable surfaces.

Abstract Background The ionic composition of the luminal fluid in the vestibular labyrinth is maintained within tight limits by the many types of epithelial cells bounding the lumen. Regulatory mechanisms include systemic, paracrine and autocrine hormones along with their associated intracellular signal pathways. The epithelium lining the semicircular canal duct (SCCD) is a tissue that is known to absorb sodium and calcium and to secrete chloride. Findings Transport function was assessed by measurements of short circuit current (Isc) and gene transcript expression was evaluated by microarray. Neither ATP nor UTP (100 microM) on the apical side of the epithelium had any effect on Isc. By contrast, basolateral ATP transiently increased Isc and transepithelial resistance dropped significantly after basolateral ATP and UTP. P2Y2 was the sole UTP-sensitive purinergic receptor expressed. Isc was reduced by 42%, 50% and 63% after knockdown of α-ENaC, stimulation of PKC and inhibition of PI3-K, while the latter two increased the transepithelial resistance. PKCdelta, PKCgamma and PI3-K were found to be expressed. Conclusions These observations demonstrate that ion transport by the SCCD is regulated by P2Y2 purinergic receptors on the basolateral membrane that may respond to systemic or local agonists, such as ATP and/or UTP. The sodium absorption from endolymph mediated by ENaC in SCCD is regulated by signal pathways that include the kinases PKC and PI3-K. These three newly-identified regulatory components may prove to be valuable drug targets in the control of pathologic vestibular conditions involving dysfunction of transport homeostasis in the ear, such as Meniere's disease.

Background and Objective: In many patients with hepatocellular carcinoma (HCC), cytochrome P450 3A4 (CYP3A4) expression has been reported to be significantly reduced in the tumor liver tissue. Moreover, this CYP3A4 repression is associated with decreased CYP3A4-mediated drug metabolism in the tumor liver tissue. However, the underlying mechanisms of CYP3A4 repression are not fully understood. We have previously shown that Mg2+/Mn2+-dependent phosphatase 1A (PPM1A) positively regulates human pregnane X receptor (hPXR)-mediated CYP3A4 expression in a PPM1A expression-dependent manner. We sought to determine whether PPM1A expression is downregulated and whether PPM1A downregulation is correlated with CYP3A4 repression in the tumor liver tissue of HCC patients. Methods: Quantitative RT-PCR and western blot analyses were performed to study mRNA and protein expression, respectively. Cell-based reporter gene assays were conducted to examine the hPXR transactivation of CYP3A4 promoter activity. Results: Arginase-1 and glypican-3 gene expression studies confirmed that the tumor samples used in our study originate from HCC livers but not non-hepatocellular tumors. mRNA and protein expression of PPM1A and CYP3A4 was found to be significantly repressed in the tumor liver tissues compared to the matched non-tumor liver tissues. In the reporter gene assays, elevated PPM1A levels counteracted the inhibition of hPXR-mediated CYP3A4 promoter activity by signaling pathways that are upregulated in HCC, suggesting that decreased PPM1A levels in HCC could not fully counteract the hPXR-inhibiting signaling pathways. Conclusions: Together, these results are consistent with the conclusion that PPM1A downregulation in the tumor liver tissue of HCC patients correlates with CYP3A4 repression. Downregulation of PPM1A levels in the tumor liver tissue may contribute to reduced hPXR-mediated CYP3A4 expression, and provide a novel mechanism of CYP3A4 repression in the tumor liver tissue of HCC patients. [ABSTRACT FROM AUTHOR]

Adverse pharmacokinetic interactions between illicit substances and clinical drugs are of a significant health concern. Illicit substances are taken by healthy individuals as well as by patients with medical conditions such as mental illnesses, acquired immunodeficiency syndrome, diabetes mellitus and cancer. Many individuals that use illicit substances simultaneously take clinical drugs meant for targeted treatment. This concomitant usage can lead to life-threatening pharmacokinetic interactions between illicit substances and clinical drugs. Optimal levels and activity of drug-metabolizing enzymes and drug-transporters are crucial for metabolism and disposition of illicit substances as well as clinical drugs. However, both illicit substances and clinical drugs can induce changes in the expression and/or activity of drug-metabolizing enzymes and drug-transporters. Consequently, with concomitant usage, illicit substances can adversely influence the therapeutic outcome of coadministered clinical drugs. Likewise, clinical drugs can adversely affect the response of coadministered illicit substances. While the interactions between illicit substances and clinical drugs pose a tremendous health and financial burden, they lack a similar level of attention as drug-drug, food-drug, supplement-drug, herb-drug, disease-drug, or other substance-drug interactions such as alcohol-drug and tobacco-drug interactions. This review highlights the clinical pharmacokinetic interactions between clinical drugs and commonly used illicit substances such as cannabis, cocaine and 3, 4-Methylenedioxymethamphetamine (MDMA). Rigorous efforts are warranted to further understand the underlying mechanisms responsible for these clinical pharmacokinetic interactions. It is also critical to extend the awareness of the life-threatening adverse interactions to both health care professionals and patients. [ABSTRACT FROM AUTHOR]