The dysbindin-containing complex (BLOC-1) in brain: developmental regulation, interaction with SNARE proteins and role in neurite outgrowth.

Previous studies have implicated DTNBP1 as a schizophrenia susceptibility gene and its encoded protein, dysbindin, as a potential regulator of synaptic vesicle physiology. In this study, we found that endogenous levels of the dysbindin protein in the mouse brain are developmentally regulated, with higher levels observed during embryonic and early postnatal ages than in young adulthood. We obtained biochemical evidence indicating that the bulk of dysbindin from brain exists as a stable component of biogenesis of lysosome-related organelles complex-1 (BLOC-1), a multi-subunit protein complex involved in intracellular membrane trafficking and organelle biogenesis. Selective biochemical interaction between brain BLOC-1 and a few members of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) superfamily of proteins that control membrane fusion, including SNAP-25 and syntaxin 13, was demonstrated. Furthermore, primary hippocampal neurons deficient in BLOC-1 displayed neurite outgrowth defects. Taken together, these observations suggest a novel role for the dysbindin-containing complex, BLOC-1, in neurodevelopment, and provide a framework for considering potential effects of allelic variants in DTNBP1--or in other genes encoding BLOC-1 subunits--in the context of the developmental model of schizophrenia pathogenesis.

Magnesium as NMDA receptor blocker in the traditional Chinese medicine Danshen.

Aqueous extracts of the traditional Chinese medicine Danshen, the dried roots of Salvia miltiorrhiza Bunge (Labiatae), blocked N-methyl-D-aspartate (NMDA) evoked currents in cerebrocortical neurons in vitro. The block of the NMDA-evoked currents was voltage dependent and showed the negative slope conductance reminiscent of the effect of Mg2+ ions. Atomic absorption spectrophotometry (AAS) revealed that aqueous Danshen extracts contained approximately 9mM magnesium. Fractionation of the extracts by high performance liquid chromatography followed by patch clamp recording and AAS indicated that magnesium ions were present in two distinct fractions. One fraction contained approximately 5 mM magnesium and blocked NMDA-induced currents indicating that it contained mostly free Mg2+ ions, while a second fraction did not possess NMDA antagonist activity despite the presence of approximately 4 mM magnesium suggesting that Mg2+ in this fraction was mostly chelated. Following removal of the free Mg2+ by ion exchange chromatography, the previously observed block of the NMDA-induced currents was abolished. These data demonstrate that Danshen contains both free and chelated Mg2+. Free Mg2+ ions account for the NMDA antagonist activity of Danshen in vitro.

Cellular signaling mechanisms underlying pharmacological action of Bak Foong Pills on gastrointestinal secretion.

Bak Foong Pills (BFP, also known as Bai Feng Wan) is an over-the-counter traditional Chinese medicine that has long been used for treating gynecological disorders and improving overall body functions, including gastrointestinal (GI) function. However, the cellular signaling mechanism underlying BFP action, especially on the GI tract, has not been elucidated. In the present study, the human colonic epithelia cell line T(84) was used as a model to investigate the effect of BFP ethanol extract on ion transport in conjunction with the short-circuit current (I(SC)) technique. The results showed that the apical addition of BFP extract produced a concentration-dependent (10-1,000 microg/ml, EC(50) = 120 microg/ml) increase in I(SC). The maximal response was observed at 500 microg/ml with an increase in I(SC) of 24.4 +/- 2.3 microA/cm(2) and apical conductance. The BFP-induced I(SC) was not observed when extracellular Cl(-) was replaced or when treated with Bumetanide (100 microM), an inhibitor of the Na(+)-K(+)-2Cl(-) cotransporter. The BFP-induced I(SC) was insensitive to the Na(+) channel blocker, amiloride, but partially inhibited by the Cl(-) channel blocker, DIDS (100 microM), and completely blocked by DPC (2 mM) or glibenclamide (1 mM) with a significant reduction in the apical conductance. The BFP-induced I(SC) could be mimicked by forskolin (10 microM), but inhibited by a pretreatment of the cells with adenylate cyclase inhibitor, MDL-12330A (10 microM). Pretreatment with EGTA (5 mM) and thapsigargin (10 microM) decreased the BFP-induced I(SC) by 10%. These results demonstrated that BFP ethanol extract exerted a stimulatory effect on gastrointestinal Cl(-) secretion by predominantly activating adenylate cyclase and apical cAMP-dependent Cl(-) channels, with minor contributions from calcium-dependent Cl(-) channels. The effect of BFP may be explored to treat GI disorders such as constipation.

Distribution and regulation of ENaC subunit and CFTR mRNA expression in murine female reproductive tract.

The present study investigated the regional distribution and cyclic changes in the mRNA expression of epithelial Na+ channel (ENaC) subunit and cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl- channel, in adult female mouse reproductive tract. In situ hybridization revealed that in contrast to the abundant expression of CFTR, ENaC (alpha, beta, gamma) mRNA signal was not detected throughout the estrus cycle in the ovary and oviduct. Messenger RNA for all ENaC subunits was abundantly detected in the cervical and vaginal epithelia throughout the estrus cycle but for CFTR, mRNA was found only at proestrus. In the uterine epithelium, alphaENaC mRNA was detected at diestrus but not found at any other stage, while CFTR mRNA was only detected at early estrus but not other stages. Semi-quantitative RT-PCR detected mRNA for all ENaC subunits in the uterus throughout the cycle with maximal expression at diestrus and CFTR mRNA was only found in the early stages of the cycle. The involvement of ENaC and CFTR in Na+ absorption and Cl- secretion was demonstrated in cultured endometrial epithelia using the short-circuit current technique and found to be influenced by ovarian hormones. Taken together, these data indicate a main secretory role of the ovary and oviduct and a predominantly absorptive role of the cervix and vagina. The present results also suggest an ability of the uterus to secrete and absorb at different stages of the estrus cycle. Variations in the fluid profiles may be dictated by the regional and cyclic variations in expression of ENaC and CFTR and are likely to contribute to various reproductive events in different regions of the female reproductive tract.

Effect of phenol red and steroid hormones on cystic fibrosis transmembrane conductance regulator in mouse endometrial epithelial cells.

Previous studies have demonstrated that cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-mediated Cl(-)channel found in most epithelia including reproductive tract, could be regulated by various culture conditions. The present study further investigated the effect of phenol red, a pH indicator widely used in growth medium, and steroid hormones, present in the supplement fetal bovine serum (FBS), on primary cultured endometrial epithelial cells by monitoring ion channel activities using the short-circuit current technique. When compared to the results obtained with normal medium supplemented with regular FBS, the forskolin-stimulated I(SC), presumably mediated by CFTR, obtained in phenol red-free medium was significantly reduced, from 16.95+/-1.53 microA/cm(2)(control) to 9.72+/-0.89 microA/cm(2)(medium without phenol red, P< 0.05). The forskolin-activated I(SC)was further attenuated to 5.29+/-0.46 microA/cm(2)in the phenol red-free medium when supplemented with charcoal/ dextran-treated FBS where steroid hormones were removed. Our data suggest that phenol red and steroid hormones present in culture medium and FBS supplement, respectively, may somehow upregulate CFTR expression in vitro. Our study demonstrates the need for carefully choosing the culture media and supplements due to the effect of steroid hormones.

Upregulation of cystic fibrosis transmembrane conductance regulator expression by oestrogen and Bak Foong Pill in mouse uteri.

Although cystic fibrosis transmembrane conductance regulator (CFTR) has been shown to be expressed in the female reproductive tract, its functional role in the uterus is not fully understood. The present study investigated a possible physiological role of CFTR by comparing the effects of 17beta-oestradiol and Bak Foong Pill (BFP), an over-the-counter Chinese medicine used for centuries for the treatment of various gynaecological disorders, on uterus size and the expression of CFTR in the uterus of ovariectomised mice using RT-PCR. Treatment of ovariectomised mice with 17beta-oestradiol (0.2 mg/kg, p.o.) for 12 days caused a significant increase in uterine wet weight compared to vehicle. However, treatment with BFP (3 g/kg, p.o.) for the same period failed to increase uterine wet weight, indicating a lack of direct oestrogen-like activity of BFP. Analysis of CFTR mRNA expression in the harvested uteri using RT-PCR showed that both 17beta-oestradiol and BFP induced an increase in CFTR mRNA expression in mouse uteri compared to levels observed in vehicle-treated animals. These results suggest that CFTR can be upregulated by oestrogen and BFP, however, the effect exerted by BFP does not seem to be mediated by direct oestrogen-like activity. Regulation of CFTR expression by both oestrogen and gynaecological medication BFP indicates an important role of CFTR in reproductive functions.

Enhanced epithelial Na(+) channel (ENaC) activity in mouse endometrial epithelium by upregulation of gammaENaC subunit.

The amiloride-sensitive epithelial Na(+) channel (ENaC), which is made of three different but homologous subunits, controls the rate of transepithelial Na(+) absorption in a variety of epithelia. The present study investigated the functional role of its subunits in regulating ENaC activity, measured as amiloride sensitive short-circuit current (I(SC)), in the mouse endometrial epithelium under different culture conditions. The treatment of the cultured epithelia with aldosterone (1 microM) or culturing cells on filters coated with concentrated Matrigel resulted in an increase in the amiloride-sensitive I(SC). Semiquantitative RT-PCR demonstrated that the expression of alpha and beta subunits was not significantly altered by these treatments, but an increase in the gamma subunit expression was observed. An 11-fold increase, induced by aldosterone, in the expression of the gamma subunit, but not in the alpha and beta subunits, was confirmed by capillary electrophoresis with laser-induced fluorescence (CE-LIF). The treatment of endometrial cells with antisense against the gammaENaC subunit abolished the aldosterone-enhanced amiloride-sensitive I(SC). The results indicated an important role of gammaENaC subunit in determining ENaC activity, and a possible role of the gammaENaC subunit in interacting with CFTR was also discussed.

Drug-nutrient interactions in transplant recipients.

Drug-nutrient interaction refers to an alteration of kinetics or dynamics of a drug or a nutritional element, or a compromise in nutritional status as a result of the addition of a drug. The potentials for drug-nutrient interaction increase with the number of drugs taken by the patient. Organ transplant recipients are therefore at high risk for drug-nutrient interactions because multiple medications are used to manage graft rejection, opportunistic infections, and other associated complications. Unrecognized or unmanaged drug-nutrient interactions in this patient population can have an adverse impact on their outcomes. This paper reviews the importance of recognizing drug-nutrient interaction when using cyclosporine-based regimens.

Inhibition of amiloride-sensitive Na(+) absorption by activation of CFTR in mouse endometrial epithelium.

Previous studies have demonstrated amiloride-sensitive Na(+) absorption under basal conditions and cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl(-) secretion following neurohormonal stimulation in the mouse endometrial epithelium. The present study investigated the inhibition of amiloride-sensitive Na(+) absorption accompanying activation of CFTR in the mouse endometrium using the short-circuit current ( I(sc)) technique. RT-PCR demonstrated the co-expression of CFTR and epithelial Na(+) channels (ENaC) in primary cultured mouse endometrial epithelia and cultured endometrial monolayers exhibited a basal amiloride-sensitive I(sc) of 5.4 +/- 0.6 microA/cm(2). The amiloride-sensitive current fell to 3.1 +/- 0.5 microA/cm(2) after stimulation with forskolin. When the possible contribution of Na(+) absorption to the I(sc) was eliminated by amiloride (1 microM) or Na(+) replacement, the forskolin-induced I(sc) was not reduced, but rather increased significantly compared with that in the absence of amiloride or in Na(+)-containing solutions ( P < 0.02), indicating that the forskolin-induced I(sc) was mediated by Cl(-) secretion, portion of which may be masked by concurrent inhibition of basal Na(+) absorption if the contribution of Na(+) is not eliminated. When the contribution of Cl(-) to the I(sc) was eliminated by diphenylamine 2,2'-dicarboxylic acid (DPC, 2 mM) or Cl(-) replacement, forskolin now decreased, rather than increased the I(sc), demonstrating the inhibition of Na(+) absorption upon stimulation. Our data suggest an interaction between CFTR and ENaC, which may be the underlying mechanism for balancing Na(+) absorption and Cl(-) secretion across the mouse endometrial epithelium.

Suppression of CFTR-mediated Cl(-) secretion by enhanced expression of epithelial Na(+) channels in mouse endometrial epithelium.

The present study investigated the effect of enhanced expression of epithelial Na(+) channels (ENaC) on the cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl(-) secretion in the mouse endometrium using the short-circuit current technique. The amiloride sensitivity of the basal current of the cultured endometrial epithelia was found to vary with the magnitude of the basal current, the higher the basal current the greater its sensitivity to amiloride, indicating possible elevation of ENaC expression. However, the magnitude of the forskolin-induced Isc, previously demonstrated to be mediated by CFTR, decreased as the amiloride sensitivity of the basal current increased, suggesting a possible inhibitory effect of elevated expression of ENaC on CFTR-mediated Cl(-) secretion. The Matrigel concentration for culturing the endometrial epithelia was found to affect the amiloride sensitivity of the basal current as well as the forskolin-induced Isc in opposite directions. However, competitive RT-PCR demonstrated that the expression of both ENaC and CFTR was enhanced in Matrigel-treated culture, suggesting that the reduced forskolin-induced Isc with enhanced amiloride sensitivity was not due to a reduction in CFTR expression, but rather suppression of CFTR function by enhanced ENaC expression. In addition to the previously demonstrated inhibition of ENaC by activation of CFTR, the present results reveal possible regulation of CFTR by ENaC. The interaction between the two may be one of the underlying mechanisms for balancing Na(+) absorption and Cl(-) secretion across epithelia.

Pyrimidinoceptors-mediated activation of Ca(2+)-dependent Cl(-) conductance in mouse endometrial epithelial cells.

Previous studies have demonstrated the activation of endometrial Cl(-) secretion through P(2Y2) (P(2U)) purinoceptors by extracellular ATP. The present study further explored the presence of pyrimidine-sensitive receptors in the primary cultured mouse endometrial epithelial cells using the short-circuit current (I(SC)) and whole-cell patch-clamp techniques. UDP induced a transient increase in I(SC) in a concentration-dependent manner (EC(50) approximately 8.84 microM). The UDP-induced I(SC) was abolished after pretreating the epithelia with a calcium chelator, 1, 2-bis-(2-aminophenoxy)-ethane-N,N,N'N'tetraacetic acid-acetomethyl ester (BAPTA-AM), suggesting the dependence of the I(SC) on cytosolic free Ca(2+). The type of receptor involved was studied by cross-desensitization between ATP and UDP. ATP or UDP desensitized its subsequent I(SC) response. However, when ATP was added after UDP, or vice versa, a second I(SC) response was observed, indicating the activation of distinct receptors, possibly pyrimidine-sensitive receptors in addition to P(2Y2) (P(2U)) receptors. Similar results were observed in the patch-clamp experiments where UDP and ATP were shown to sequentially activate whole-cell current in the same cell. The UDP-activated whole-cell current exhibited outward rectification with delay activation and inactivation at depolarizing and hyperpolarizing voltages, respectively. In addition, the UDP-evoked whole-cell current reversed near the equilibrium potential of Cl(-) in the presence of a Cl(-) gradient across the membrane, and was sensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), indicating the activation of Ca(2+)-activated Cl(-) conductance. These characteristics were very similar to that of the ATP-activated whole-cell current. Taken together, our findings indicate the presence of distinct receptors, pyrimidinoceptors and P(2Y2) (P(2U)) receptors in mouse endometrial epithelial cells. These distinct receptors appear to converge on the same Ca(2+)-dependent Cl(-) channels.

Warfarin-acetaminophen drug interaction revisited.

Physicians and pharmacists routinely advise patients receiving warfarin to take acetaminophen for pain or fever because of its relative safety; however, a recent study questioned the safety of such practice. A comprehensive search of MEDLINE and IPA for human studies and case reports from 1966-1999 revealed evidence that acetaminophen may potentiate the effect of warfarin by a mechanism that has yet to be elucidated. Due to lack of a safer alternative, acetaminophen still should be the analgesic and antipyretic of choice in patients taking warfarin, as long as excessive amounts and prolonged administration (> 1.3 g acetaminophen/day for > 2 wks) are avoided. With the high degree of interpatient variability and the unpredictability of various drug-drug interactions with warfarin, close and frequent monitoring of international normalized ratios is the key for safe oral anticoagulation therapy.

Activation of an adenosine 3',5'-cyclic monophosphate-dependent Cl- conductance in response to neurohormonal stimuli in mouse endometrial epithelial cells: the role of cystic fibrosis transmembrane conductance regulator.

Previous studies have demonstrated that Cl- secretion by the mouse endometrial epithelium is under neurohormonal influence. The present study characterized the Cl- conductance activated by a number of agonists in the mouse endometrial epithelial cells using the whole-cell voltage-clamp technique. Adrenaline (1 microM), prostaglandin (PG) E2 (5-10 microM), and PGF2alpha (100 microM) activated a whole-cell current that exhibited a linear I-V relationship as well as time- and voltage-independent characteristics. However, the current magnitude varied with different agonists. The agonist-activated current could be mimicked by an adenylate cyclase activator, forskolin (10 microM), and suppressed by an adenylate cyclase inhibitor, MDL12330A, suggesting the involvement of cAMP. Current characteristics remained the same after cation replacement, leaving Cl- as the major permeant ion species in the solutions. The reversal potential of the agonist-induced current was close to the equilibrium potential of Cl- in the presence of a Cl- gradient, indicating the activation of Cl- conductance. The agonist-induced current was inhibited by the Cl- channel blocker diphenylamine 2,2'-dicarboxylic acid (DPC), but not by the Cl- channel blocker 4,4'-diisothiocyanatostibene-2, 2'-disulfonic acid (DIDS). The anion selectivity sequence of the current was NO3->Br->Cl->I-. The observed electrophysiological properties of the agonist-induced Cl- conductance were consistent with those reported for the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl- channel expressed in many epithelia. The expression of CFTR in the mouse endometrial cells was also demonstrated by Western blot analysis. It appears that neurohormonal regulation of the uterine fluid in the mouse endometrium converges on the cAMP-activated Cl- channel, presumably CFTR.

Prolongation of the QT interval related to cisapride-diltiazem interaction.

Cisapride, a cytochrome P450 3A4 (CYP3A4) substrate, is widely prescribed for the treatment of gastrointestinal motility disorders. Prolongation of QT interval, torsades de pointes, and sudden cardiac death have been reported after concomitant administration with erythromycin or azole antifungal agents, but not with other CYP3A4 inhibitors. A possible drug interaction occurred in a 45-year-old woman who was taking cisapride for gastroesophageal reflux disorder and diltiazem, an agent that has inhibitory effect on CYP3A4, for hypertension. The patient was in near syncope and had QT-interval prolongation. After discontinuing cisapride, the QT interval returned to normal and symptoms did not recur. We suggest that caution be taken when cisapride is prescribed with any potent inhibitor of CYP3A4, including diltiazem.

N-(4-hydroxyphenyl)retinamide induces apoptosis in T lymphoma and T lymphoblastoid leukemia cells.

We demonstrate that N-(4-hydroxyphenyl)-all-trans-retinamide (4-HPR), a synthetic retinoic acid (RA) derivative, is a potent and selective inducer of apoptosis in malignant T lymphoid cells, but has little effect on normal lymphoid cells of the thymus or spleen. 4-HPR and its stereoisomer, 9-cis-4-HPR, are 50 to > 150 times more potent than 7 other retinoids in killing CEM-C7 human T lymphoblastoid leukemia cells and P1798-C7 murine T lymphoma cells. 4-HPR's apoptotic action requires the intact molecule bearing both the retinoid moiety and the hydroxyphenol ring; 4-HPR remains unmetabolized after uptake into CEM-C7 and P1798-C7 cells for up to 24 hours. We also show that glucocorticoid (GC)-resistant variants are equally susceptible to 4-HPR as are GC-sensitive cells. Thus, 4-HPR may be potentially important as a new chemotherapeutic drug for use as alternative to, or in combination with, conventional drugs for treating lymphoid malignancies.

Glucocorticoid/oxysterol-induced DNA lysis in human leukemic cells.

Both glucocorticoids and oxysterols, steroids with quite different known transduction pathways, cause the death of lymphoid cells. Dual TUNEL/propidium iodide assays on sensitive human leukemic CEM-C7 clones treated with either steroid were clearly positive by 48 h, consistent with apoptosis. Both steroids evoked two distinctive types of DNA lysis: cleavage into large fragments of several different sizes and the classic "ladders", multiples of approximately 200 base pairs. Conventional gel electrophoresis showed that a small proportion of total DNA had undergone laddering 36-48 h after treatment with glucocorticoid or 24 h after oxysterol exposure. On field inversion gel electrophoresis of cellular DNA both steroids caused an increase in an array of large DNA fragments <50 kb in size. A 50 kb fragment appeared 36 h after treatment with either steroid, but only oxysterol treatment caused a significant increase in a 300 kb fragment. Oxysterol treatment did not result in DNA fragmentation in the resistant M10R5 subclone, which retained sensitivity to glucocorticoids. We conclude that glucocorticoids and oxysterols kill these cells with similar, but not identical, patterns of DNA lysis which occur just before or concomitant with the onset of cell death.

N-(4-hydroxyphenyl)retinamide prevents development of T-lymphomas in AKR/J mice.

N-(4-Hydroxyphenyl)retinamide (4-HPR), a synthetic retinoic acid derivative, has chemopreventive effects on several types of cancer. We recently showed that 4-HPR is a potent inducer of apoptosis in malignant, but not normal, T-lymphoid cells in vitro. To test 4-HPR's effect in vivo, we used the virus-induced T-lymphoma in AKR/J mice as a model system. The AKR/J mice were fed 4-HPR at 0, 1 or 2 mmole/kg diet, and the animals were monitored as to tumor development, plasma level of 4-HPR, body weight, appetite, and general health. Our results show that in a 19-week period, 4-HPR prevented T-lymphoma development by 40% and 50% of animals fed 1 and 2 mmole 4-HPR/ kg diet, respectively. In the plasma, 4-HPR reached micromolar levels without causing any observable deleterious side-effects. Thus, 4-HPR is potentially useful in chemoprevention of lymphoid cancers.

Retinoic acid inhibition of serum-induced c-fos transcription in a fibrosarcoma cell line.

We investigated the mechanism by which retinoic acid causes growth arrest and flat reversion of SSV-NRK, simian sarcoma virus-transformed normal rat kidney cells. Northern analysis revealed that both chronic (7 days) and acute (6 h) retinoic acid treatment of serum-stimulated SSV-NRK cells caused a 6-fold decrease in c-fos mRNA levels. In addition, nuclear run-on experiments showed that retinoic acid regulated c-fos expression in SSV-NRK cells at the transcriptional initiation level. Attenuation of c-fos transcription was equal in both retinoic acid-treated and control cells, and no increased c-fos mRNA turnover was detected in retinoic acid-treated cells. Furthermore, there was no observed change in the c-fos mRNA levels after only 30 min of retinoic acid treatment, suggesting that a mechanism involving the interruption of the signal transduction mechanism at the membrane level is unlikely. Because it has been shown that c-fos expression plays a pivotal role in mitogenesis of quiescent fibroblasts, we conclude that the retinoic acid-mediated down-regulation of c-fos expression is a mechanism for growth inhibition in SSV-NRK cells.