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M X Zhu

Publications and source records attributed to M X Zhu.

At least 19 recordsLinked to original sources
PubMed2001

Expression of Trp3 determines sensitivity of capacitative Ca2+ entry to nitric oxide and mitochondrial Ca2+ handling: evidence for a role of Trp3 as a subunit of capacitative Ca2+ entry channels.

The role of Trp3 in cellular regulation of Ca(2+) entry by NO was studied in human embryonic kidney (HEK) 293 cells. In vector-transfected HEK293 cells (controls), thapsigargin (TG)-induced (capacitative Ca(2+) entry (CCE)-mediated) intracellular Ca(2+) signals and Mn(2+) entry were markedly suppressed by the NO donor 2-(N,N-diethylamino)diazenolate-2-oxide sodium salt (3 microm) or by authentic NO (100 microm). In cells overexpressing Trp3 (T3-9), TG-induced intracellular Ca(2+) signals exhibited an amplitude similar to that of controls but lacked sensitivity to inhibition by NO. Consistently, NO inhibited TG-induced Mn(2+) entry in controls but not in T3-9 cells. Moreover, CCE-mediated Mn(2+) entry into T3-9 cells exhibited a striking sensitivity to inhibition by extracellular Ca(2+), which was not detectable in controls. Suppression of mitochondrial Ca(2+) handling with the uncouplers carbonyl cyanide m-chlorophenyl hydrazone (300 nm) or antimycin A(1) (-AA(1)) mimicked the inhibitory effect of NO on CCE in controls but barely affected CCE in T3-9 cells. T3-9 cells exhibited enhanced carbachol-stimulated Ca(2+) entry and clearly detectable cation currents through Trp3 cation channels. NO as well as carbonyl cyanide m-chlorophenyl hydrazone slightly promoted carbachol-induced Ca(2+) entry into T3-9 cells. Simultaneous measurement of cytoplasmic Ca(2+) and membrane currents revealed that Trp3 cation currents are inhibited during Ca(2+) entry-induced elevation of cytoplasmic Ca(2+), and that this negative feedback regulation is blunted by NO. Our results demonstrate that overexpression of Trp3 generates phospholipase C-regulated cation channels, which exhibit regulatory properties different from those of endogenous CCE channels. Moreover, we show for the first time that Trp3 expression determines biophysical properties as well as regulation of CCE channels by NO and mitochondrial Ca(2+) handling. Thus, we propose Trp3 as a subunit of CCE channels.

Anthranilate Synthase↗
PubMed2001

Identification of common binding sites for calmodulin and inositol 1,4,5-trisphosphate receptors on the carboxyl termini of trp channels.

Homologues of Drosophila Trp (transient receptor potential) form plasma membrane channels that mediate Ca(2+) entry following the activation of phospholipase C by cell surface receptors. Among the seven Trp homologous found in mammals, Trp3 has been shown to interact with and respond to IP(3) receptors (IP(3)Rs) for activation. Here we show that Trp4 and other Trp proteins also interact with IP(3)Rs. The IP(3)R-binding domain also interacts with calmodulin (CaM) in a Ca(2+)-dependent manner with affinities ranging from 10 nm for Trp2 to 290 nm for Trp6. In addition, other binding sites for CaM and IP(3)Rs are present in the alpha but not the beta isoform of Trp4. In the presence of Ca(2+), the Trp-IP(3)R interaction is inhibited by CaM. However, a synthetic peptide representing a Trp-binding domain of IP(3)Rs inhibited the binding of CaM to Trp3, -6, and -7 more effectively than that to Trp1, -2, -4, and -5. In inside-out membrane patches, Trp4 is activated strongly by calmidazolium, an antagonist of CaM, and a high (50 microm) but not a low (5 microm) concentration of the Trp-binding peptide of the IP(3)R. Our data support the view that both CaM and IP(3)Rs play important roles in controlling the gating of Trp-based channels. However, the sensitivity and responses to CaM and IP(3)Rs differ for each Trp.

Amino Acid Sequence↗
PubMed2001

Increased inwardly rectifying potassium currents in HEK-293 cells expressing murine transient receptor potential 4.

Drosophila transient receptor potential (Trp) and its mammalian homologues are postulated to form capacitative Ca2+ entry or store-operated channels. Here we show that expression of murine Trp4 in HEK 293 cells also leads to an increase in inwardly rectifying K+ currents. No similar increase was found in cell lines expressing Trp1, Trp3 or Trp6. Consistent with typical characteristics of inward rectifiers, the K+ currents in Trp4-expressing cells were blocked by low millimolar concentrations of Cs+ and Ba2+, but not by 1.2 mM Ca2+, and were only slightly inhibited by 5 mM tetraethylammonium. Single channel recordings of excised inside-out patches revealed the presence of two conducting states of 51 pS and 94 pS in Trp4-expressing cells. The outward current in the excised patches was blocked by 1 mM spermine, but not by 1 mM Mg2+. How Trp4 expression causes the increase in the K+ currents is not known. We propose that Trp4 either participates in the formation of a novel K+ channel or up-regulates the expression or activity of endogenous inwardly rectifying K+ channels.

Animals↗
PubMed2001

Activation of Trp3 by inositol 1,4,5-trisphosphate receptors through displacement of inhibitory calmodulin from a common binding domain.

Mammalian homologues of Drosophila Trp form plasma membrane channels that mediate Ca(2+) influx in response to activation of phospholipase C and internal Ca(2+) store depletion. Previous studies showed that human Trp3 is activated by inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) and identified interacting domains, one on Trp and two on IP(3)R. We now find that Trp3 binds Ca(2+)-calmodulin (Ca(2+)/CaM) at a site that overlaps with the IP(3)R binding domain. Using patch-clamp recordings from inside-out patches, we further show that Trp3 has a high intrinsic activity that is suppressed by Ca(2+)/CaM under resting conditions, and that Trp3 is activated by the following: a Trp-binding peptide from IP(3)R that displaces CaM from Trp3, a myosin light chain kinase Ca(2+)/CaM binding peptide that prevents CaM from binding to Trp3, and calmidazolium, an inactivator of Ca(2+)/CaM. We conclude that inhibition of the inhibitory action of CaM is a key step of Trp3 channel activation by IP(3)Rs.

Amino Acid Sequence↗
PubMed2000

Association of mammalian trp4 and phospholipase C isozymes with a PDZ domain-containing protein, NHERF.

Mammalian homologues of Drosophila Trp have been implicated to form channels that are activated following the depletion of Ca(2+) from internal stores. Recent studies indicate that actin redistribution is required for the activation of these channels. Here we show that murine Trp4 and Trp5, as well as phospholipase C beta1 and beta2 interact with the first PDZ domain of NHERF, regulatory factor of the Na(+)/H(+) exchanger. We demonstrated the association of Trp4 and phospholipase C-beta1 with NHERF in vivo in an HEK293 cell line expressing Trp4 and in adult mouse brain by immuno-coprecipitation. NHERF is a two PDZ domain-containing protein that associates with the actin cytoskeleton via interactions with members of ezrin/radixin/moesin family. Thus, store-operated channels involving Trp4 and Trp5 can form signaling complexes with phospholipase C isozymes via interactions with NHERF and thereby linking the lipase and the channels to the actin cytoskeleton. The interaction with the PDZ protein may constitute an important mechanism for distribution and regulation of store-operated channels.

Amino Acid Sequence↗
PubMed2000

Coassembly of Trp1 and Trp3 proteins generates diacylglycerol- and Ca2+-sensitive cation channels.

To analyze the functional consequences of coassembly of transient receptor potential 1 (Trp1) and Trp3 channel proteins, we characterized membrane conductances and divalent cation entry derived by separate overexpression and by coexpression of both Trp isoforms. Trp1 expression generated a 1-oleoyl-2-acetyl-sn-glycerol (OAG)-activated conductance that was detectable only in Ca(2+)-free extracellular solution. Trp3 expression gave rise to an OAG-activated conductance that was suppressed but clearly detectable at physiological Ca(2+) concentrations. Coexpression of both species resulted in a constitutively active, OAG-sensitive conductance, which exhibited distinctive cation selectivity and high sensitivity to inhibition by intracellular Ca(2+). Trp1-expressing cells displayed only modest carbachol-induced Ca(2+) entry and lacked OAG-induced Sr(2+) entry, whereas Trp3-expressing cells responded to both agents with a substantial divalent cation entry. Coexpression of Trp1 plus Trp3 suppressed carbachol-induced Ca(2+) entry compared with Trp3 expression and abolished OAG-induced Sr(2+) entry signals. We concluded that coassembly of Trp1 and Trp3 resulted in the formation of oligomeric Trp channels that are subject to regulation by phospholipase C and Ca(2+). The distinguished Ca(2+) sensitivity of these Trp1/Trp3 hetero-oligomers appeared to limit Trp-mediated Ca(2+) signals and may be of importance for negative feedback control of Trp function in mammalian cells.

Animals↗
PubMed2000

Trp1, a candidate protein for the store-operated Ca(2+) influx mechanism in salivary gland cells.

The trp gene family has been proposed to encode the store-operated Ca(2+) influx (SOC) channel(s). This study examines the role of Trp1 in the SOC mechanism of salivary gland cells. htrp1, htrp3, and Trp1 were detected in the human submandibular gland cell line (HSG). HSG cells stably transfected with htrp1alpha cDNA displayed (i) a higher level of Trp1, (ii) a 3-5-fold increase in SOC (thapsigargin-stimulated Ca(2+) influx), determined by [Ca(2+)](i) and Ca(2+)-activated K(+) channel current measurements, and (iii) similar basal Ca(2+) permeability, and inhibition of SOC by Gd(3+) but not by Zn(2+), as compared with control cells. Importantly, (i) transfection of HSG cells with antisense trp1alpha cDNA decreased endogenous Trp1 level and significantly attenuated SOC, and (ii) transfection of HSG cells with htrp3 cDNA did not increase SOC. These data demonstrate an association between Trp1 and SOC and strongly suggest that Trp1 is involved in this mechanism in HSG cells. Consistent with this suggestion, Trp1 was detected in the plasma membrane region, the proposed site of SOC, of acinar and ductal cells in intact rat submandibular glands. Based on these aggregate data, we propose Trp1 as a candidate protein for the SOC mechanism in salivary gland cells.

Animals↗
PubMed1999

Modulation of Ca(2+) entry by polypeptides of the inositol 1,4, 5-trisphosphate receptor (IP3R) that bind transient receptor potential (TRP): evidence for roles of TRP and IP3R in store depletion-activated Ca(2+) entry.

Homologues of Drosophilia transient receptor potential (TRP) have been proposed to be unitary subunits of plasma membrane ion channels that are activated as a consequence of active or passive depletion of Ca(2+) stores. In agreement with this hypothesis, cells expressing TRPs display novel Ca(2+)-permeable cation channels that can be activated by the inositol 1,4,5-trisphosphate receptor (IP3R) protein. Expression of TRPs alters cells in many ways, including up-regulation of IP3Rs not coded for by TRP genes, and proof that TRP forms channels of these and other cells is still missing. Here, we document physical interaction of TRP and IP3R by coimmunoprecipitation and glutathione S-transferase-pulldown experiments and identify two regions of IP3R, F2q and F2g, that interact with one region of TRP, C7. These interacting regions were expressed in cells with an unmodified complement of TRPs and IP3Rs to study their effect on agonist- as well as store depletion-induced Ca(2+) entry and to test for a role of their respective binding partners in Ca(2+) entry. C7 and an F2q-containing fragment of IP3R decreased both forms of Ca(2+) entry. In contrast, F2g enhanced the two forms of Ca(2+) entry. We conclude that store depletion-activated Ca(2+) entry occurs through channels that have TRPs as one of their normal structural components, and that these channels are directly activated by IP3Rs. IP3Rs, therefore, have the dual role of releasing Ca(2+) from stores and activating Ca(2+) influx in response to either increasing IP3 or decreasing luminal Ca(2+).

Amino Acid Sequence↗
PubMed1999

The N-terminal domain of the IP3 receptor gates store-operated hTrp3 channels.

In the present work, we studied the interaction and effect of several IP3 receptor (IP3R) constructs on the gating of the store-operated (SOC) hTrp3 channel. Full-length IP3R coupled to silent hTrp3 channels in intact cells but did not activate them until stores were depleted of Ca2+. By contrast, constructs containing the IP3-binding domain activated silent hTrp3 channels in unstimulated cells and restored gating of hTrp3 by IP3 in excised plasma membrane patches. We conclude that the N-terminal domain of the IP3R functions as a gate and is sufficient for activation of SOCs. The sensing and transduction domains of the IP3R are required to maintain SOCs in an inactive state.

Calcium↗
PubMed1999

Control of pollen tube tip growth by a Rop GTPase-dependent pathway that leads to tip-localized calcium influx.

We have shown that Rop1At, a pollen-specific Rop GTPase that is a member of the Rho family of small GTP binding proteins, acts as a key molecular switch controlling tip growth in Arabidopsis pollen tubes. Pollen-specific expression of constitutively active rop1at mutants induced isotropic growth of pollen tubes. Overexpression of wild-type Arabidopsis Rop1At led to ectopic accumulation of Rop1At in the plasma membrane at the tip and caused depolarization of pollen tube growth, which was less severe than that induced by the constitutively active rop1at. These results indicate that both Rop1At signaling and polar localization are critical for controlling the site of tip growth. Dominant negative rop1at mutants or antisense rop1at RNA inhibited tube growth at 0.5 mM extracellular Ca(2+), but growth inhibition was reversed by higher extracellular Ca(2+). Injection of anti-Rop antibodies disrupted the tip-focused intracellular Ca(2+) gradient known to be crucial for tip growth. These studies provide strong evidence for a Rop GTPase-dependent tip growth pathway that couples the control of growth sites with the rate of tip growth through the regulation of tip-localized extracellular Ca(2+) influxes and formation of the tip-high intracellular Ca(2+) gradient in pollen tubes.

Arabidopsis↗
PubMed1998

Biochemical characterization of benomyl inhibition on endometrial growth during decidualization in rats.

The antimitotic action of the systemic benzimidazole carbamate compound, benomyl, the basis for its fungitoxicity, was assessed in a mammalian system by selected biochemical endpoints of endometrial proliferation during decidualization in rats. The deciduoma, artificially induced on Day 4 of pseudopregnancy (PG), represents the maternal portion of the placenta that attains maximal growth between Days 9-11 PG. Deciduoma induction by surgical uterine trauma normally prolongs PG into the decidualization process. Measured endometrial parameters were the wet weight, protein for hypertrophy, DNA indicative of hyperplasia; enzymatic biomarkers- isocitrate dehydrogenase (ICDH) and the matrix metalloproteinases (MMPs); and serum progesterone which hormonally maintains decidual growth. Benomyl was administered by oral gavage in daily doses (500 mg/kg/rat in corn oil for 5 days, PG Days 5-9) and animals were sacrificed on PG Day 10. Benomyl caused significant reduction (P < 0.001) in endometrial wet weight, protein and DNA concentrations. ICDH activity was also significantly reduced (P < 0.01) following benomyl treatment. Of the two MMP species (72 and 92 kDa), whereas the 72 kDa was only slightly affected, the 92 kDa MMP was suppressed 2-3 fold by benomyl. Benomyl was without effect on the progesterone concentration. The findings suggest that during decidualization in rats, the anti-deciduogenic, antimitotic action of post-traumal benomyl treatment which occurred via the biochemical molecules (protein, DNA, ICDH and the MMPs) apparently was not mediated by progesterone.

Animals↗
PubMed1998

Time-dependent relationship between the estrogen receptors and the matrix metalloproteinases following deciduoma induction in rats.

The purpose of this study was to investigate time-related interactions between the estrogen receptors, mediators of steroidal regulation of uterine growth, and an extracellular regulatory enzyme, the matrix metalloproteinases (MMPs) engaged in connective tissue degradation and remodeling that are fundamental to implantation and placentation. Pseudopregnant rats, in which the decidual response, the basis for decidualization, was surgically induced on day 4 of pseudopregnancy (PG), were sacrificed on PG days 3, 6, 9, and 15 for retrieval of uterine tissues for assays: the radioligand binding assay for the estrogen receptors and substrate zymography for the MMPs. Following increases on PG day 3, there were time-dependent decreases in the cytosolic low and high capacity estrogen receptors during deciduoma development (PG days 6-9) and regression (PG day 15) in both the endometrium and myometrium. Moreover, whereas the low capacity estrogen receptor levels were only slightly decreased (PG days 6-15), the high capacity receptors were reduced on day 6 (P < 0.001) and were completely diminished during PG days 9 and 15. In contrast, the MMPs (92 and 72 kDa) activities were increased from PG days 6-15 (P < 0.05) over the pre-decidual induction values on PG day 3 in both uterine compartments. The results suggest that deciduoma induction can modulate the concentration of cytosolic estrogen receptor subtypes and MMP activities in rats. The inverse time-dependent interrelationship between these cellular and extracellular components during deciduoma development and regression imply that the remodeling role of the MMPs may be enhanced by the reduced cytosolic estrogen receptor/estrogen action.

Animals↗
PubMed1998

Antiproliferative effects of inducible nitric oxide synthase inhibition on decidualization in pseudopregnant rats.

The aim of this study was to assess the involvement in decidual proliferation of nitric oxide (NO), a regulator of many cellular processes, that is synthesized from L-arginine by NO synthase. The investigation was conducted on pseudopregnant (PG) rats in which the decidual cell reaction, the basis for the decidualization process, was surgically induced by uterine trauma on PG Day 4. Groups of animals (n = 5) were pretreated with either 2 doses/day of N(G)-nitro-L-arginine methyl ester (L-NAME) that inhibits NO synthase, or twice daily doses of L-NAME plus L-arginine combined. Drug application times coincided with 3 hr after lights on or 3 hr before lights off. The two treatment regimens (PG Days 1-4 or 5-8) respectively preceded or followed decidual induction. Animals were sacrificed at mid-light on PG Day 9, the day of maximal growth response to the deciduogenic stimulus. Parallel, time-dependent increases in both NO synthase activity and decidual growth occurred mainly in the endometrium. L-NAME produced reductions in endometrial and myometrial growth that were reversed by the combined L-NAME plus L-arginine treatments. These inhibitory effects by L-NAME were caused by only the pretraumal (PG Days 1-4) administration. Hormonally, circulating progesterone levels were similarly affected by this early treatment and may also contribute to the reduced decidual sensitivity. In contrast, serum estradiol, along with the zinc metalloenzymes, alkaline phosphatase and the matrix metalloproteinases--prominent decidualization biomarkers--were all unaffected by either the pre- or post-decidual induction dosings. The study demonstrates that inducible NO synthase/endogenous NO may physiologically participate in uterine metabolism during the decidual cell reaction. Moreover, by virtue of L-NAME inhibition of the decidual response, it appears that NO synthase/NO may influence decidual growth either by directly increasing uterine sensitivity to the deciduogenic stimulus or by indirectly affecting endometrial vascularity and subsequent availability of decidual metabolites.

Alkaline Phosphatase↗
PubMed1997

Temporal glucocorticoid treatment: modulation of periodic endometrial responses during decidualization and pregnancy in rats.

The synthetic glucocorticoid, dexamethasone (Dex) was administered subcutaneously (1.5 mg/day/rat) in 3-days pretreatment regimens (Days 2-4, 4-6, 6-8, 8-10 and 10-12) to pseudopregnant rats in which decidualization was surgically induced and to pregnant rats. Variability in endometrial growth during decidualization and in the fetoplacental homeostasis of pregnancy was assessed at the end of each treatment period (Days 4, 6, 8, 10 and 12). During decidualization, endometrial growth (wet weight, protein and DNA) displayed significant (p < 0.05) time-dependent inhibitory profiles which rose steeply from Day 4 to Day 6 and declined thereafter to Day 10 in fairly well defined linear patterns. For the endometrial enzymes (isocitrate dehydrogenase, alkaline phosphatase and the matrix metalloproteinases--72 and 92 kDa), although the inhibitory patterns were inconsistent, a Days 6-8 treatment regimen seemed to be critical. By contrast Dex treatment induced progressive inhibition in serum progesterone concentrations from Day 2, to peak levels by Day 12. This indicates that time-related Dex inhibition of endometrial growth appeared not to be progesterone-mediated since the endometrial and progesterone inhibitory profiles were not in synchrony. The inhibitory effect of Dex under the pregnancy status demonstrated that birth potentials, fetal and placental weights, all had similar response patterns which rose from Day 4 to Day 8 and then underwent reductions to Day 12. Collectively, the results indicate that there was time dependency in growth inhibition by Dex at the endometrial and fetoplacental levels. Maximal sensitivity to drug exposure essentially coincided with the immediate post-traumal (decidualization) and postimplantation (pregnancy) periods.

Animals↗
PubMed1990

[Effect of diphenylhydantoin sodium in causing deformity and that of folic acid preparations for its prevention].

Pregnant rats were divided into four groups. The groups A, B and C were intraperitoneally injected with DPH during the 9 to 11 days of gestation in doses of 75 Mg/kg/day. The groups A and B were supplemented with a mixture of folic acid (FA) or with FA alone in the food fed during pregnancy. The results showed that DPH induced a significant decrease in the body weights and lengths of fetal rats (P less than 0.01), but induced an increase in the incidences of subcutaneous bleeding (P less than 0.05) as well as skeletal and internal malformation (P less than 0.05). The supplement of FA or a mixture of FA in the food fed during pregnancy exhibited partial preventive effects on DPH to induce teratogenicity. The effect of a mixture of FA was better than that with FA alone in reducing the incidences of internal abnormalities and agenesis of the bones of the distant phalanges and subcutaneous bleeding.

Abnormalities, Drug-Induced↗