Correlation analysis of genetic diversity and population structure of Houttuynia cordata Thunb with regard to environment.

To study the levels of genetic diversity, and population structure, of Houttuynia cordata Thunb, the genetic background and relationships of populations were analyzed in terms of environmental factors. The genetic diversity and population structure of H. cordata were investigated using sequence-related amplified polymorphisms and correlation with environmental factors was analyzed using the SPSS software. Two thousand one hundred sixty-three sites were amplified from 41 pairs of primers, 1825 of which were polymorphic, and the percentage of polymorphic loci was 84.37%; the percentage of polymorphic sites was 72.14 and 67.77% at the species and population level, respectively. The observed number of alleles was 1.52 and 1.30 at species and population level, respectively. The effective number of alleles was 1.38 and 1.24 at species and population level, respectively. The Nei's diversity was 0.26 and 0.15 at species and population level, respectively. The Shannon's information index was 0.87 and 0.63 at species and population level, respectively. The genetic differentiation coefficient of populations was 0.51, and 12 populations were divided into three classes based on D = 0.20; the genetic diversities of different populations are correlated at different significance levels (P < 0.05) with environmental factors. Genetic differentiation existed among populations and the populations exhibited heteroplasmy.

The challenge of pancreatic cancer therapy and novel treatment strategy using engineered mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have attracted significant attention in cancer research as a result of their accessibility, tumor-oriented homing capacity, and the feasibility of auto-transplantation. This review provides a comprehensive overview of current challenges in pancreatic cancer therapy, and we propose a novel strategy for using MSCs as means of delivering anticancer genes to the site of pancreas. We aim to provide a practical platform for the development of MSC-based therapy for pancreatic cancer.

TRAIL-engineered pancreas-derived mesenchymal stem cells: characterization and cytotoxic effects on pancreatic cancer cells.

Mesenchymal stem cells (MSCs) have attracted great interest in cancer therapy owing to their tumor-oriented homing capacity and the feasibility of autologous transplantation. Currently, pancreatic cancer patients face a very poor prognosis, primarily due to the lack of therapeutic strategies with an effective degree of specificity. Anticancer gene-engineered MSCs specifically target tumor sites and can produce anticancer agents locally and constantly. This study was performed to characterize pancreas-derived MSCs and investigate the effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-engineered MSCs on pancreatic cancer cells under different culture conditions. Pancreas-derived MSCs exhibited positive expression on CD44, CD73, CD95, CD105, negative on CD34 and differentiated into adipogenic and osteogenic cells. TRAIL expression was assessed by both enzyme-linked immunosorbent assay and western blot analysis. Different patterns of TRAIL receptor expression were observed on the pancreatic cancer cell lines, including PANC1, HP62, ASPC1, TRM6 and BXPC3. Cell viability was assessed using a real-time monitoring system. Pancreatic cancer cell death was proportionally related to conditioned media from MSC(nsTRAIL) and MSC(stTRAIL). The results suggest that MSCs exhibit intrinsic inhibition of pancreatic cancer cells and that this effect can be potentiated by TRAIL-transfection on death receptor-bearing cell types.

Stability and nonlinear optical properties of ZnO nanoparticles.

Photoluminescence (PL) of ZnO nanoparticles of different surface states and sizes grown by several methods has been measured. The origin of luminescence and dependence of the luminescence spectrum shape and intensity on 325 nm excitation laser power are studied. Strong ultraviolet emission at 3.26 eV, weak violet emission around 3.12 eV and weak green emission at 2.40 eV have been observed in 16 nm nanoparticles capped by octylamine grown by non-hydrolytic method. The nanoparticles are stable under high power laser radiation and their PL intensity increases nonlinearly with an increasing laser power. As the nanoparticle size decreases to 12 nm, high power laser produces nonradiative centers which may quench the luminescence in a degree. Nanoparticles of 8 nm capped by PVP and uncapped nanoparticles of 14 nm are unstable and their luminescence depends on the excitation laser power. High power laser can quench O vacancy emission and enhance ultraviolet emission in PVP capped nanoparticles while vacancy emission can not be quenched in uncapped nanoparticles.

Stem cells and hepatic cirrhosis.

Hepatic cirrhosis is defined as the histological development of regenerative nodules surrounded by fibrous bands in response to chronic liver injury, which leads to portal hypertension and end-stage liver disease. The majority of patients with hepatic cirrhosis die from life-threatening complications at early age. Liver transplantation has been the most effective treatment for patients with hepatic cirrhosis. Since liver transplantation is critically limited by the shortage of available donor livers, searching for an effective alternative therapy has attracted great interest in preclinical studies. The encouraging advances in stem cell research have paved the way towards the treatment of the end-stage of chronic liver disease. In view of the pathogenic fundamentals of hepatic cirrhosis, stem cell-based treatment should be aimed to complement or replace damaged liver cells and to correct the imbalanced extracellular matrix regeneration/degradation. This review is intended to describe the characteristics and therapeutic potential of various liver repair-related stem cells, including hepatocytes, liver progenitor cells, hematopoietic stem cells, mesenchymal stem cells, embryonic stem cells and induced pluripotent stem cells. Since autologous adult stem cells have the least number of obstacles for clinical application, their potential interventions on cirrhosis are especially illustrated in terms of the cellular and molecular mechanisms of hepatic fibrogenesis.

A combined procedure to deliver autologous mesenchymal stromal cells to patients with traumatic brain injury.

BACKGROUND: There is increasing evidence of therapeutic benefits from bone marrow (BM)-derived mesenchymal stromal cells (MSC) in various animal models with neurologic disorders. It is of great interest to apply the approach to clinical patients, i.e. to take the investigations from laboratory bench to the patient's bedside. This clinical trial was performed to assess the safety and feasibility of a combined procedure to deliver autologous MSC to patients with traumatic brain injury. METHODS: MSC were isolated by BM aspiration and expanded in culture. Seven patients received autologous cell transplantation. A primary administration of 10(7)-10(9) cells was applied directly to the injured area during the cranial operation; a second dose of 10(8)-10(10) cells was infused intravenously. All patients were followed up regularly for 6 months. RESULTS: There was no immediate or delayed toxicity related to the cell administration within the 6-month follow-up period. Neurologic function was significantly improved at 6 months after cell therapy. DISCUSSION: The procedure used is safe and feasible at ordinary medical facilities without additional invasive procedures for the patient. The combined cell delivery procedure is expected to enhance the engraftment efficacy of transplanted cells at injured brain tissue, thereby promoting neurologic recover.

Adenosine modulates Mg(2+) uptake in distal convoluted tubule cells via A(1) and A(2) purinoceptors.

tk;1Adenosine plays a role in the control of water and electrolyte reabsorption in the distal tubule. As the distal convoluted tubule is important in the regulation of renal Mg(2+) balance, we determined the effects of adenosine on cellular Mg(2+) uptake in this segment. The effect of adenosine was studied on immortalized mouse distal convoluted tubule (MDCT) cells, a model of the intact distal convoluted tubule. The rate of Mg(2+) uptake was measured with fluorescence techniques using mag-fura 2. To assess Mg(2+) uptake, MDCT cells were first Mg(2+) depleted to 0.22 +/- 0.01 mM by being cultured in Mg(2+)-free media for 16 h and then placed in 1.5 mM MgCl(2); next, changes in intracellular Mg(2+) concentration ([Mg(2+)](i)) were determined. [Mg(2+)](i) returned to basal levels, 0.53 +/- 0.02 mM, with a mean refill rate, d([Mg(2+)](i))/dt, of 137 +/- 16 nM/s. Adenosine stimulates basal Mg(2+) uptake by 41 +/- 10%. The selective A(1) purinoceptor agonist N(6)-cyclopentyladenosine (CPA) increased intracellular Ca(2+) and decreased parathyroid hormone (PTH)-stimulated cAMP formation and PTH-mediated Mg(2+) uptake. On the other hand, the selective A(2) receptor agonist 2-[p-(2-carbonyl-ethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS) stimulated Mg(2+) entry in a concentration-dependent fashion. CGS increased cAMP formation and the protein kinase A inhibitor RpcAMPS inhibited CGS-stimulated Mg(2+) uptake. Selective inhibition of phospholipase C, protein kinase C, or mitogen-activated protein kinase enzyme cascades with U-73122, Ro-31-8220, and PD-98059, respectively, diminished A(2) agonist-mediated Mg(2+) entry. Aldosterone potentiated CGS-mediated Mg(2+) entry, and elevation of extracellular Ca(2+) diminished CGS-responsive cAMP formation and Mg(2+) uptake. Accordingly, MDCT cells possess both A(1) and A(2) purinoceptor subtypes with intracellular signaling typical of these respective receptors. We conclude that adenosine has dual effects on Mg(2+) uptake in MDCT cells through separate A(1) and A(2) purinoceptor pathways.

ATP inhibits Mg(2+) uptake in MDCT cells via P2X purinoceptors.

Nucleotides have diverse effects on water and electrolyte reabsorption within the distal tubule of the nephron. As the distal tubule is important in control of renal Mg(2+) balance, we determined the effects of ATP on cellular Mg(2+) uptake in this segment. The effects of ATP on immortalized mouse distal convoluted tubule (MDCT) cells were studied by measuring Mg(2+) uptake with fluorescence techniques. The mean basal Mg(2+) uptake rate was 165 +/- 6 nM/s. ATP inhibited basal Mg(2+) uptake and hormone-stimulated Mg(2+) entry by 40%. Both P2X (P2X1-P2X5 subtypes) and P2Y2 receptor subtypes were identified in MDCT cells using differential RT-PCR. Activation of both receptor subtypes with selective agonists increased intracellular Ca(2+) concentration, P2X purinoceptors by ionotropic-gated channels, and P2Y receptors via G protein-mediated intracellular Ca(2+) release. The more relatively selective P2X agonists [beta,gamma-methylene ATP (beta,gamma-Me-ATP) and 2'- and -3'-O-(4-benzoyl-benzoyl)-ATP] inhibited arginine vasopressin (AVP)- and parathyroid hormone (PTH)-mediated Mg(2+) uptake whereas agonists more selective for P2Y purinoceptors (UTP, ADP, and 2-methylthio-ATP) were without effect. Removal of extracellular Ca(2+) diminished beta,gamma-Me-ATP-mediated increase in intracellular Ca(2+) and inhibition of AVP-stimulated Mg(2+) entry. We conclude from this information that ATP inhibited Mg(2+) uptake in MDCT cells through P2X purinoceptors expressed in this distal convoluted tubule cell line.

1,25(OH)(2)D(3) stimulates Mg2+ uptake into MDCT cells: modulation by extracellular Ca2+ and Mg2+.

The distal convoluted tubule plays a significant role in renal magnesium conservation. Although the cells of the distal convoluted tubule possess the vitamin D receptor, little is known about the effects of 1alpha,25-dihydroxyvitamin D [1,25(OH)(2)D(3)] on magnesium transport. In this study, we examined the effect of 1,25(OH)(2)D(3) on distal cellular magnesium uptake and the modulation of this response by extracellular Ca2+ and Mg2+ in an immortalized mouse distal convoluted tubule (MDCT) cell line. MDCT cells possess the divalent cation-sensing receptor (CaSR) that responds to elevation of extracellular Ca2+ and Mg2+ concentrations to diminish peptide hormone-stimulated Mg2+ uptake. Mg2+ uptake rates were determined by microfluorescence in Mg2+ -depleted MDCT cells. Treatment of MDCT cells with 1,25(OH)(2)D(3) for 16-24 h stimulated basal Mg2+ uptake in a concentration-dependent manner from basal levels of 164 +/- 5 to 210 +/- 11 nM/s, representing a 28 +/- 3% change. Pretreatment with actinomycin D or cycloheximide abolished 1,25(OH)(2)D(3)-stimulated(.)Mg2+ uptake (154 +/- 18 nM/s), suggesting that 1,25(OH)(2)D(3) stimulates Mg2+ uptake through gene activation and protein synthesis. Elevation of extracellular Ca2+ inhibited 1,25(OH)(2)D(3)-stimulated Mg2+ uptake (143 +/- 5 nM/s). Preincubation of the cells with an antibody to the CaSR prevented the inhibition by elevated extracellular Ca2+ of 1,25(OH)(2)D(3)-stimulated Mg2+ uptake (202 +/- 8 nM/s). Treatment with an antisense CaSR mRNA oligodeoxynucleotide also abolished the effects of extracellular Ca2+ on 1,25(OH)(2)D(3)-responsive Mg2+ entry. This showed that elevated extracellular calcium modulates 1,25(OH)(2)D-mediated responses through the CaSR. In summary, 1,25(OH)(2)D(3) stimulated Mg2+ uptake in MDCT cells, and this is dependent on de novo protein synthesis. Elevation of extracellular Ca2+, acting via the CaSR, inhibited 1,25(OH)(2)D(3)-stimulated Mg2+ entry. These data indicate that 1,25(OH)(2)D(3) has important effects on the control of magnesium entry in MDCT cells and these responses can be modulated by extracellular divalent cations.

Magnesium transport in the renal distal convoluted tubule.

The distal tubule reabsorbs approximately 10% of the filtered Mg(2+), but this is 70-80% of that delivered from the loop of Henle. Because there is little Mg(2+) reabsorption beyond the distal tubule, this segment plays an important role in determining the final urinary excretion. The distal convoluted segment (DCT) is characterized by a negative luminal voltage and high intercellular resistance so that Mg(2+) reabsorption is transcellular and active. This review discusses recent evidence for selective and sensitive control of Mg(2+) transport in the DCT and emphasizes the importance of this control in normal and abnormal renal Mg(2+) conservation. Normally, Mg(2+) absorption is load dependent in the distal tubule, whether delivery is altered by increasing luminal Mg(2+) concentration or increasing the flow rate into the DCT. With the use of microfluorescent studies with an established mouse distal convoluted tubule (MDCT) cell line, it was shown that Mg(2+) uptake was concentration and voltage dependent. Peptide hormones such as parathyroid hormone, calcitonin, glucagon, and arginine vasopressin enhance Mg(2+) absorption in the distal tubule and stimulate Mg(2+) uptake into MDCT cells. Prostaglandin E(2) and isoproterenol increase Mg(2+) entry into MDCT cells. The current evidence indicates that cAMP-dependent protein kinase A, phospholipase C, and protein kinase C signaling pathways are involved in these responses. Steroid hormones have significant effects on distal Mg(2+) transport. Aldosterone does not alter basal Mg(2+) uptake but potentiates hormone-stimulated Mg(2+) entry in MDCT cells by increasing hormone-mediated cAMP formation. 1,25-Dihydroxyvitamin D(3), on the other hand, stimulates basal Mg(2+) uptake. Elevation of plasma Mg(2+) or Ca(2+) inhibits hormone-stimulated cAMP accumulation and Mg(2+) uptake in MDCT cells through activation of extracellular Ca(2+)/Mg(2+)-sensing mechanisms. Mg(2+) restriction selectively increases Mg(2+) uptake with no effect on Ca(2+) absorption. This intrinsic cellular adaptation provides the sensitive and selective control of distal Mg(2+) transport. The distally acting diuretics amiloride and chlorothiazide stimulate Mg(2+) uptake in MDCT cells acting through changes in membrane voltage. A number of familial and acquired disorders have been described that emphasize the diversity of cellular controls affecting renal Mg(2+) balance. Although it is clear that many influences affect Mg(2+) transport within the DCT, the transport processes have not been identified.

beta-Adrenergic agonists stimulate Mg(2+) uptake in mouse distal convoluted tubule cells.

beta-Adrenergic agonists influence electrolyte reabsorption in the proximal tubule, loop of Henle, and distal tubule. Although isoproterenol enhances magnesium absorption in the thick ascending limb, it is unclear what effect, if any, beta-adrenergic agonists have on tubular magnesium handling. The effects of isoproterenol were studied in immortalized mouse distal convoluted tubule (MDCT) cells by measuring cellular cAMP formation with radioimmunoassays and Mg(2+) uptake with fluorescence techniques. Intracellular free Mg(2+) concentration ([Mg(2+)](i)) was measured in single MDCT cells by using microfluorescence with mag-fura-2. To assess Mg(2+) uptake, MDCT cells were first Mg(2+) depleted to 0.22 +/- 0.01 mM by culturing in Mg(2+)-free media for 16 h and then placed in 1.5 mM MgCl(2), and the changes in [Mg(2+)](i) were determined. [Mg(2+)](i) returned to basal levels, 0.53 +/- 0.02 mM, with a mean refill rate, d([Mg(2+)](i))/dt, of 168 +/- 11 nM/s. Isoproterenol stimulated Mg(2+) entry in a concentration-dependent manner, with a maximal response of 252 +/- 11 nM/s, at a concentration of 10(-7) M, that represented a 50 +/- 7% increase in uptake rate above control values. This was associated with a sixfold increase in intracellular cAMP generation. Isoproterenol-stimulated Mg(2+) uptake was completely inhibited with RpcAMPS, a protein kinase A inhibitor, and U-73122, a phospholipase C inhibitor, and partially blocked by RO 31-822, a protein kinase C inhibitor. Accordingly, isoproterenol-mediated Mg(2+) entry rates involve multiple intracellular signaling pathways. Aldosterone potentiated isoproterenol-stimulated Mg(2+) uptake (326 +/- 31 nM/s), whereas elevation of extracellular Ca(2+) inhibited isoproterenol-mediated cAMP accumulation and Mg(2+) uptake, 117 +/- 37 nM/s. These studies demonstrate that isoproterenol stimulates Mg(2+) uptake in a cell line of mouse distal convoluted tubules that is modulated by hormonal and extracellular influences.

B lymphoblastoid cell lines as efficient APC to elicit CD8+ T cell responses against a cytomegalovirus antigen.

Potent and readily accessible APC are critical for development of immunotherapy protocols to treat viral disease and cancer. We have shown that B lymphoblastoid cell lines (BLCL) that stably express CMV phosphoprotein 65 (BLCLpp65), as a result of retroviral transduction, can be used to generate ex vivo CTL cultures that possess cytotoxicity against CMV and EBV. In this report, we demonstrate that the EBV-specific cytotoxicity in the BLCLpp65-primed culture had a spectrum of EBV-Ag recognition similar to that of the BLCL-primed counterpart, suggesting that retroviral transduction and expression of the CMV Ag would not compromise the Ag-presenting capacity of BLCL. In addition, BLCLpp65 appeared to present multiple natural pp65 epitopes, because pp65-specific CTL, which recognized different CMV clinical isolates, were generated in BLCLpp65-primed cultures from individuals with various HLA backgrounds. Consistent with a polyclonal expansion of virus-specific CTL, T cell lines established from the BLCLpp65-primed CTL cultures expressed different TCR-Vbeta Although most of the virus-specific T cell isolates were CD8+, EBV-specific CD4+ lines were also established from BLCLpp65-primed cultures. Western blot analysis revealed that the CD8+ lines, but not the CD4+ line, expressed granzyme B, consistent with features of classic CTL. Thus, our results suggested that BLCL stably expressing a foreign Ag might be used as a practical APC to elicit CD8+ T cell responses.

Insulin stimulates Mg2+ uptake in mouse distal convoluted tubule cells.

Insulin has been shown to be a magnesium-conserving hormone acting, in part, through stimulation of magnesium absorption within the thick ascending limb. Although the distal convoluted tubule possesses the most insulin receptors, it is unclear what, if any, actions insulin has in the distal tubule. The effects of insulin were studied on immortalized mouse distal convoluted tubule (MDCT) cells by measuring cellular cAMP formation with radioimmunoassays and Mg2+ uptake with fluorescence techniques using mag-fura 2. To assess Mg2+ uptake, MDCT cells were first Mg(2+) depleted to 0.22 +/- 0.01 mM by culturing in Mg2+-free media for 16 h and then placed in 1.5 mM MgCl2, and the changes in intracellular Mg2+ concentration ([Mg2+]i) were measured with microfluorescence. [Mg2+]i returned to basal levels, 0.53 +/- 0.02 mM, with a mean refill rate, d([Mg2+]i)/dt, of 164 +/- 5 nM/s. Insulin stimulated Mg2+ entry in a concentration-dependent manner with maximal response of 214 +/- 12 nM/s, which represented a 30 +/- 5% increase in the mean uptake rate above control values. This was associated with a 2.5-fold increase in insulin-mediated cAMP generation (52 +/- 3 pmol. mg protein(-1). 5 min(-1)). Genistein, a tyrosine kinase inhibitor, diminished insulin-stimulated Mg2+ uptake (169 +/- 11 nM/s), but did not change insulin-mediated cAMP formation (47 +/- 5 pmol. mg protein(-1). 5 min(-1)). PTH stimulates Mg2+ entry, in part, through increases in cAMP formation. Insulin and PTH increase Mg2+ uptake in an additive fashion. In conclusion, insulin mediates Mg2+ entry, in part, by a genistein-sensitive mechanism and by modifying hormone-responsive transport. These studies demonstrate that insulin stimulates Mg2+ uptake in MDCT cells and suggest that insulin acts in concert with other peptide and steroid hormones to control magnesium conservation in the distal convoluted tubule.

Simultaneous ex vivo expansion of cytomegalovirus and Epstein-Barr virus-specific cytotoxic T lymphocytes using B-lymphoblastoid cell lines expressing cytomegalovirus pp65.

Cytomegalovirus (CMV) infection and Epstein-Barr virus (EBV)-induced lymphoproliferative disease are serious complications associated with allogeneic stem cell transplantation. Immunotherapy using ex vivo expanded, virus-specific cytotoxic T lymphocytes (CTL) has been explored and proven to be effective in therapeutic or prophylactic regimens for CMV and EBV infections. To generate CTL specific for both CMV and EBV, we engineered EBV-transformed B-lymphoblastoid cell lines (BLCL) to express CMV pp65 for use as antigen-presenting cells (APC). BLCL were transduced with a recombinant retrovirus encoding pp65, the immunodominant CMV polypeptide. Western blot analysis and immunocytochemistry confirmed the expression of pp65 in the transduced cells. Peripheral blood mononuclear cells (PBMC) from healthy CMV seropositive donors were stimulated with autologous pp65-expressing BLCL weekly for 3 weeks. Chromium release assays showed that the resulting CTL cultures possessed specific cytotoxicity against EBV and CMV. Recombinant vaccinia viruses encoding individual CMV peptides were used to demonstrate that this CMV-specific cytotoxicity was specific for pp65. Assays on CD4- and CD8-depleted CTL fractions indicated that CD8(+) CTL mediated the pp65-specific cytotoxicity. These CMV/EBV-specific CTL recognized CMV- and EBV-infected targets sharing HLA class I antigens, but not HLA mismatched targets. Our results demonstrate that BLCL can be used as APC to stimulate expansion of EBV- and CMV-specific CTL simultaneously. These findings have potential implications for posttransplant CMV and EBV immunotherapy in recipients of allogeneic stem cell transplants.

PGE2 stimulates Mg2+ uptake in mouse distal convoluted tubule cells.

Prostaglandins have diverse effects on renal electrolyte reabsorption, inhibiting NaCl absorption in the thick ascending limb and modulating sodium and calcium transport in cortical collecting cells. It is unclear what effect, if any, prostaglandins have on tubular magnesium handling. The effects of prostaglandin E2 (PGE2) were studied on immortalized mouse distal convoluted tubule (MDCT) cells by measuring cellular cAMP formation with radioimmunoassays and Mg2+ uptake with fluorescence techniques. Intracellular free Mg2+ concentration ([Mg2+]i) was measured on single MDCT cells using microfluorescence with mag-fura 2. To assess Mg2+ uptake, MDCT cells were first Mg2+ depleted to 0.22 +/- 0.01 mM by culturing in Mg2+-free media for 16 h and then placed in 1.5 mM MgCl2, and the changes in [Mg2+]i were determined. [Mg2+]i returned to basal levels, 0.53 +/- 0.02 mM, with a mean refill rate, d([Mg2+]i)/dt, of 173 +/- 8 nM/s. Indomethacin, 5 microM, diminished basal Mg2+ uptake, suggesting that endogenous prostaglandins may stimulate Mg2+ entry in control cells. PGE2 stimulated Mg2+ entry in a concentration-dependent manner with maximal response of 311 +/- 12 nM/s, at a concentration of 10(-7) M, which represented an 80 +/- 3% increase in uptake rate above control values. This was associated with a sixfold increase in intracellular cAMP generation. PGE2-stimulated Mg2+ uptake was completely inhibited with the Rp diastereoisomer of adenosine 3',5'-cyclic monophosphothionate (Rp-cAMPS), a protein kinase A inhibitor, and U-73122, a phospholipase C inhibitor, and partially by chelerythrine, a protein kinase C inhibitor. Accordingly, PGE2-mediated Mg2+ entry rates involve multiple intracellular signaling pathways. These studies demonstrate that PGE2 stimulates Mg2+ uptake in a cell line of MDCT.

Mg2+/Ca2+ sensing inhibits hormone-stimulated Mg2+ uptake in mouse distal convoluted tubule cells.

The distal convoluted tubule plays a significant role in renal magnesium conservation. An immortalized mouse distal convoluted tubule (MDCT) cell line has been extensively used to study the cellular mechanisms of magnesium transport in this nephron segment. MDCT cells possess an extracellular polyvalent cation-sensing mechanism responsive to Mg2+, Ca2+, and neomycin. The present studies determined the effect of Mg2+/Ca2+ sensing on hormone-mediated cAMP formation and Mg2+ uptake in MDCT cells. MDCT cells were Mg2+ depleted by culturing in Mg2+-free media for 16 h, and Mg2+ uptake was measured by microfluorescence after placing the depleted cells in 1.5 mM MgCl2. The mean rate of Mg2+ uptake was 164 +/- 5 nM/s in control MDCT cells. Activation of Mg2+/Ca2+ sensing with neomycin did not affect basal Mg2+ uptake (155 +/- 5 nM/s). We have previously reported that treatment of MDCT cells with either glucagon or arginine vasopressin (AVP) stimulated Mg2+ entry. In the present studies, the addition of extracellular Mg2+ or Ca2+ inhibited glucagon- and AVP-stimulated cAMP formation and Mg2+ uptake in concentration-dependent manner with half-maximal concentrations of approximately 1.5 and 3.0 mM, respectively. Exogenous cAMP or forskolin stimulated Mg2+ uptake in the presence of Mg2+/Ca2+ sensing activation. We infer from these studies that Mg2+/Ca2+-sensing mechanisms located in the distal convoluted tubule may play a role in control of distal magnesium absorption.

Extracellular Mg2(+)- and Ca2(+)-sensing in mouse distal convoluted tubule cells.

An immortalized cell line (designated MDCT) has been extensively used to investigate the cellular mechanisms of electrolyte transport within the mouse distal convoluted tubule. Mouse distal convoluted tubule cells possess many of the functional characteristics of the in vivo distal convoluted tubule. In the present study, we show that MDCT cells also possess a polyvalent cation-sensing mechanism that is responsive to extracellular magnesium and calcium. Southern hybridization of reverse transcribed-polymerase chain reaction (RT-PCR) products, sequence determination and Western analysis indicated that the calcium-sensing receptor (Casr) is expressed in MDCT cells. Using microfluorescence of single MDCT cells to determine cytosolic Ca2+ signaling, it was shown that the polyvalent cation-sensing mechanism is sensitive to extracellular magnesium concentration ([Mg2+]o) and extracellular calcium concentration ([Ca2+]o) in concentration ranges normally observed in the plasma. Moreover, both [Mg2+]o and [Ca2+]o were effective in generating intracellular Ca2+ transients in the presence of large concentrations of [Ca2+]o and [Mg2+]o, respectively. These responses are unlike those observed for the Casr in the parathyroid gland. Finally, activation of the polycation-sensitive mechanism with either [Mg2+]o or [Ca2+]o inhibited parathyroid hormone-, calcitonin-, glucagon- and arginine vasopressin-stimulated cAMP release in MDCT cells. These studies indicate that immortalized MDCT cells possess a polyvalent cation-sensing mechanism and emphasize the important role this mechanism plays in modulating intracellular signals in response to changes in [Mg2+]o as well as in [Ca2+]o.

Glucagon and arginine vasopressin stimulate Mg2+ uptake in mouse distal convoluted tubule cells.

Glucagon and arginine vasopressin (AVP) enhance renal magnesium conservation through actions within the loop of Henle and the distal tubule. Studies were performed on an immortalized mouse distal convoluted tubule (MDCT) cell line to characterize the cellular actions of these hormones on Mg2+ transport in this segment of the distal tubule. Glucagon and AVP increased cellular cAMP concentrations by about fivefold above basal levels in normal and Mg(2+)-depleted cells. Intracellular free Mg2+ concentration ([Mg2+]i) was determined on single MDCT cells using microfluorescence with mag-fura 2. To assess Mg2+ uptake, MDCT cells were first Mg2+ depleted (0.22 +/- 0.01 mM) by culturing in Mg(2+)-free media for 16 h and then placed in 1.5 mM MgCl2, and the [Mg2+]i was determined. [Mg2+]i returned to basal levels, 0.53 +/- 0.02 mM, with a mean refill rate, d([Mg2+]i/dt, of 164 +/- 5 nM/s. Both glucagon and AVP stimulated Mg2+ uptake into MDCT cells, 196 +/- 11 and 189 +/- 6 nM/s, respectively, at concentrations of 3 x 10(-7) M and 10(-7) M, respectively. Enhanced Mg2+ uptake for each of the hormones was concentration dependent and inhibited by the channel blocker, nifedipine. Hormone stimulation of Mg2+ entry was not dependent on protein synthesis. 8-Bromo-cAMP, 10(-4) M, enhanced Mg2+ uptake (225 +/- 13 nM/s), whereas phorbol esters were without effect. Finally, protein kinase A inhibition prevented glucagon and AVP stimulation of Mg2+ uptake, supporting the notion that the cAMP pathway is important as expected in the hormone action. These studies demonstrate that glucagon and AVP stimulate Mg2+ uptake in MDCT cells and suggest that these hormones act to control magnesium conservation in the convoluted segment of the distal tubule.

Aldosterone potentiates hormone-stimulated Mg2+ uptake in distal convoluted tubule cells.

The distal convoluted tubule reabsorbs significant amounts of filtered magnesium that is under hormonal control. In this study, we describe the effects of aldosterone on Mg2+ uptake in an immortalized mouse distal convoluted tubule (MDCT) cell line. Intracellular free Mg2+ concentration ([Mg2+]i) was determined on single MDCT cells using microfluorescence with mag-fura 2. To determine Mg2+ entry rate into MDCT cells, they were first Mg2+ depleted ([Mg2+]i, 0.22 +/- 0.01 mM) by culturing in Mg(2+)-free media for 16 h and then placed in 1.5 mM MgCl2. The rate of change in [Mg2+]i as measured as a function of time, d([Mg2+]i)/dt, was 164 +/- 5 nM/s in control cells. We have shown that glucagon or arginine vasopressin (AVP) stimulates Mg2+ entry by 63% and 15%, respectively. Incubation of MDCT cells with aldosterone for 16 h did not change the rate of Mg2+ uptake (172 +/- 8 nM/s). However, aldosterone potentiated glucagon- and AVP-stimulated Mg2+ uptake rate up to 330 +/- 39 and 224 +/- 6 nM/s, respectively. Aldosterone also potentiated glucagon- and AVP-induced intracellular cAMP accumulation in a concentration-independent manner. As cAMP stimulates Mg2+ entry in MDCT cells, it is inferred that aldosterone may stimulate Mg2+ uptake through intracellular signaling pathways involving cAMP. The actions of aldosterone were dependent on de novo protein synthesis, as pretreatment of the cells with cycloheximide inhibited aldosterone potentiation of hormone stimulation of Mg2+ uptake and cAMP accumulation. These studies with MDCT cells suggest that aldosterone may modulate the effects of hormones acting within the distal convoluted tubule to control magnesium absorption.