Cancer remains one of the leading causes of mortality and morbidity throughout the world. To a significant extent, current conventional cancer therapies are symptomatic and passive in nature. The major obstacle to the development of effective cancer therapy is believed to be the absence of sufficient specificity. Since the discovery of the tumor-oriented homing capacity of mesenchymal stem cells (MSCs), the application of specific anticancer gene-engineered MSCs has held great potential for cancer therapies. The dual-targeted strategy is based on MSCs’ capacity of tumor-directed migration and incorporation and in situ expression of tumor-specific anticancer genes. With the aim of translating bench work into meaningful clinical applications, we describe the tumor tropism of MSCs and their use as therapeutic vehicles, the dual-targeted anticancer potential of engineered MSCs and a putative personalized strategy with anticancer gene-engineered MSCs.

Pancreatic β-cell is the only cell type that produces insulin to regulate glucose metabolism in the body. The insufficiency of insulin-producing β-cells results in dysregulation of blood glucose control, termed as type 1 diabetes (T1D). Both genetic predisposition and environmental factors play a significant role in the development of T1D, and the direct cause of T1D is islet-specific autoimmune-induced insulitis. There are three conventional therapeutic options currently available for the treatment of T1D: insulin therapy, cell-based therapy and immunotherapy. Insulin therapy is passive in Nature., and does not directly address the cause of the disease; cell-based therapy can reverse the consequence of the disease by replacing destroyed β-cells in the diabetic pancreas. The applicable insulin-producing β-cells can be directly obtained from islet transplantation or generated from other cell sources, such as autologous adult stem cells, embryonic stem cells, induced pluripotent stem cells, and even somatic cells through transdifferentiation. However, the replaced β-cells may still encounter the immune attack if the autoimmunity remains active in the body. β-cell replacement alone is unable to cure the disease effectively. Immune intervention addresses the cause of T1D, mainly through up-regulating regulatory T cells (Tregs). Tregs are able to block autoimmune attack to the islets and protect the remaining and/or regenerated β-cells in the pancreas. Apparently, the combination of β-cell replacement and immunotherapy could play a synergistic role in the treatment of T1D. This chapter is intended to summarize the recent progress and analyze the potential synergistic effect of these two options on T1D treatment. Exploring the optimal combination of β-cell replacement and immunotherapy will pave the way to the most effective cure for this devastating disease.

Mesenchymal stem cells (MSCs) have attracted great interest in cancer therapy since the discovery of their tumor tropism. This study was performed to investigate the effects of TNF-related apoptosis-inducing ligand (TRAIL)-engineered MSCs on hepatocellular carcinoma (HCC) ceels (HepG2) under different culture conditions.

Mesenchymal stem cells (MSCs) are the first type of stem cells to be utilized in clinical regenerative medicine, mainly owing to their capacity for multipotent differentiation and the feasibility of autologous transplantation. More recently, the specific tumor-oriented migration and incorporation of MSCs have been demonstrated in various pre-clinical models, highlighting the potential for MSCs to be used as an ideal carrier for anticancer gene delivery. Engineered with specific anticancer genes, MSCs possess the ability of dual-targeting tumor cells. This contrasts with non-engineered native MSCs which have intrinsic pro- and antitumorigenic properties. Engineered MSCs are capable of producing specific anticancer agents locally and constantly. Astute investigation on engineered MSCs may lead to a new avenue toward an efficient therapy for patients with cancer.

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.

Hepatic cirrhosis is the end-stage of chronic liver diseases. 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 diseases. In view of the pathogenic fundamentals of hepatic cirrhosis, cell-based treatment should be aimed to complement or replace damaged liver cells and to correct the imbalanced extracellular matrix regeneration/degradation. Understanding the transition of hepatocyte regeneration to hepatic fibrogenesis during chronic liver injury could guide the appropriate utilization of cell-based therapy. This chapter is intended to describe the characteristics and therapeutic potential of various 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 obstacles for clinical application, their potential interventions on cirrhosis are especially illustrated in terms of the cellular and molecular mechanisms of hepatic fibrogenesis.

Mouse islets are commonly used in diabetes-related studies. Adequate amounts of good quality islets are prerequisites for a reliable investigation. We describe a protocol for islet isolation from mouse pancreas. Three major manipulations are employed in the islet isolation procedure: in situ pancreas perfusion with collagenase, pancreas digestion and islet purification. The whole procedure takes 30–45 min for each individual mouse. By using this protocol, a reasonable number of islets can be obtained in a relatively short period of time. This protocol has been proven to be practicable and reproducible. It can be easily followed by individuals who do not have previous experience in the related research field.

Type 1 diabetes (T1D) is the result of the autoimmune response against pancreatic insulin-producing ß-cells. Its ultimate consequence is β-cell insufficiency-mediated dysregulation of blood glucose control. In terms of T1D treatment, immunotherapy addresses the cause of T1D, mainly through re-setting the balance between autoimmunity and regulatory mechanisms. Regulatory T cells play an important role in this immune intervention. An alternative T1D treatment is β-cell replacement, which can reverse the consequence of the disease by replacing destroyed β-cells in the diabetic pancreas. The applicable insulin-producing cells can be directly obtained from islet transplantation or generated from other cell sources such as autologous adult stem cells, embryonic stem cells, and induced pluripotent stem cells. In this review, we summarize the recent research progress and analyze the possible advantages and disadvantages of these two therapeutic options especially focusing on the potential synergistic effect on T1D treatment. Exploring the optimal combination of immunotherapy and β-cell replacement will pave the way to the most effective cure for this devastating disease.

Hepatic cirrhosis is the end-stage of chronic liver diseases. The majority of patients with hepatic cirrhosis die from life-threatening complications occurring at their earlier ages. Liver transplantation has been the most effective treatment for these patients. 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 transplantation of autologous bone marrow-derived mesenchymal stem cells holds great potential for treating hepatic cirrhosis. Mesenchymal stem cells can differentiate to hepatocytes, stimulate the regeneration of endogenous parenchymal cells, and enhance fibrous matrix degradation. Experimental and clinical studies have shown promising beneficial effects. This review is intended to translate the bench study results to the patients' bedside. The potential interventions of mesenchymal stem cells on cirrhosis are illustrated in terms of the cellular and molecular mechanisms of hepatic fibrogenesis.

Pancreatic islet transplantation has the potential to be an effective treatment for type 1 diabetes mellitus. While recent improvements have improved 1-year outcomes, follow-up studies show a persistent loss of graft function/survival over 5 years. One possible cause of islet transplant failure is the immunosuppressant regimen required to prevent alloimmune graft rejection. Although there is evidence from separate studies, mostly in rodents and cell lines, that FK506 (tacrolimus), rapamycin (sirolimus), and mycophenolate mofetil (MMF; CellCept) can damage pancreatic β-cells, there have been few side-by-side, multiparameter comparisons of the effects of these drugs on human islets. In the present study, we show that 24-h exposure to FK506 or MMF impairs glucose-stimulated insulin secretion in human islets. FK506 had acute and direct effects on insulin exocytosis, whereas MMF did not. FK506, but not MMF, impaired human islet graft function in diabetic NOD.scid mice. All of the immunosuppressants tested in vitro increased caspase-3 cleavage and caspase-3 activity, whereas MMF induced ER-stress to the greatest degree. Treating human islets with the GLP-1 agonist exenatide ameliorated the immunosuppressant-induced defects in glucosestimulated insulin release. Together, our results demonstrate that immunosuppressants impair human β-cell function and survival, and that these defects can be circumvented to a certain extent with exenatide treatment.

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.

Purified bone marrow-derived mesenchymal stem cells were autotransplanted to a patient with acute myocardial infarction. The employment of the sub-population of bone marrow-derived mononuclear cells was intended to clarify some disputable outcomes in heterogeneous mononuclear cell therapy. The improvement of myocardial perfusion and cardiac function was observed after delivery of mesenchymal stem cells through a combined procedure of primary intracoronary infusion and secondary intravenous infusion. This procedure is expected to enhance the engraftment efficacy of transplanted cells at infarcted myocardium.

This study concerns the cytogenetic stability of in vitro human bone marrow-derived mesenchymal stem cells (MSCs) in primary culture and after passaging. Bone marrow samples were collected from seven brain malfunction patients involved in autologous MSC transplantation trials. Chromosome preparations from primary MSC cultures and after 3 passages were analyzed by conventional staining and G-banding techniques. All MSCs showed normal diploid karyotypes, 46 XY or 46 XX, without aneuploidy or polyploidy; chromosome structural abnormalities were not detected. The results indicate that the in vitro cultured MSCs retained normal cytogenetics before being transplanted back into the patients.

A local multiorgan donor pancreas procurement program can provide a source for optimized isolation of purified viable islets for transplantation into patients with type 1 diabetes mellitus receiving best medical therapy. Local pancreas donor retrieval with islet isolation and culture conditioning enabled an offer of islets for transplantation for 90% of consecutively processed pancreata. Isolated islets secreted insulin during prolonged follow-up after implantation into patients, yielding metabolic control comparable with that achieved by best medical therapy.

The distal tubule reabsorbs ~10% of the filtered Mg21, but this is 70–80% of that delivered from the loop of Henle. Because there is little Mg21 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 Mg21 reabsorption is transcellular and active. This review discusses recent evidence for selective and sensitive control of Mg21 transport in the DCT and emphasizes the importance of this control in normal and abnormal renal Mg21 conservation. Normally, Mg21 absorption is load dependent in the distal tubule, whether delivery is altered by increasing luminal Mg21 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 Mg21 uptake was concentration and voltage dependent. Peptide hormones such as parathyroid hormone, calcitonin, glucagon, and arginine vasopressin enhance Mg21 absorption in the distal tubule and stimulate Mg21 uptake into MDCT cells. Prostaglandin E2 and isoproterenol increase Mg21 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 Mg21 transport. Aldosterone does not alter basal Mg21 uptake but potentiates hormone-stimulated Mg21 entry in MDCT cells by increasing hormone-mediated cAMP formation. 1,25-Dihydroxyvitamin D3, on the other hand, stimulates basal Mg21 uptake. Elevation of plasma Mg21 or Ca21 inhibits hormone-stimulated cAMP accumulation and Mg21 uptake in MDCT cells through activation of extracellular Ca21/Mg21-sensing mechanisms. Mg21 restriction selectively increases Mg21 uptake with no effect on Ca21 absorption. This intrinsic cellular adaptation provides the sensitive and selective control of distal Mg21 transport. The distally acting diuretics amiloride and chlorothiazide stimulate Mg21 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 Mg21 balance. Although it is clear that many influences affect Mg21 transport within the DCT, the transport processes have not been identified.

Adenosine 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 Mg21 balance, we determined the effects of adenosine on cellular Mg21 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 Mg21 uptake was measured with fluorescence techniques using mag-fura 2. To assess Mg21 uptake, MDCT cells were first Mg21 depleted to 0.22 6 0.01 mM by being cultured in Mg21-free media for 16 h and then placed in 1.5 mM MgCl2; next, changes in intracellular Mg21 concentration ([Mg21]i) were determined. [Mg21]i returned to basal levels, 0.53 6 0.02 mM, with a mean refill rate, d([Mg21]i)/dt, of 137 6 16 nM/s. Adenosine stimulates basal Mg21 uptake by 41 6 10%. The selective A1 purinoceptor agonist N6-cyclopentyladenosine (CPA) increased intracellular Ca21 and decreased parathyroid hormone (PTH)-stimulated cAMP formation and PTH-mediated Mg21 uptake. On the other hand, the selective A2 receptor agonist 2-[p-(2-carbonyl-ethyl)-phenylethylamino]-59-N-ethylcarboxamidoadenosine (CGS) stimulated Mg21 entry in a concentration- dependent fashion. CGS increased cAMP formation and the protein kinase A inhibitor RpcAMPS inhibited CGS-stimulated Mg21 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 A2 agonist-mediated Mg21 entry. Aldosterone potentiated CGS-mediated Mg21 entry, and elevation of extracellular Ca21 diminished CGS-responsive cAMP formation and Mg21 uptake. Accordingly, MDCT cells possess both A1 and A2 purinoceptor subtypes with intracellular signaling typical of these respective receptors. We conclude that adenosine has dual effects on Mg21 uptake in MDCT cells through separate A1 and A2 purinoceptor pathways.

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 Mg21 balance, we determined the effects of ATP on cellular Mg21 uptake in this segment. The effects of ATP on immortalized mouse distal convoluted tubule (MDCT) cells were studied by measuring Mg21 uptake with fluorescence techniques. The mean basal Mg21 uptake rate was 165 6 6 nM/s. ATP inhibited basal Mg21 uptake and hormone-stimulated Mg21 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 Ca21 concentration, P2X purinoceptors by ionotropicgated channels, and P2Y receptors via G protein-mediated intracellular Ca21 release. The more relatively selective P2X agonists [b,g-methylene ATP (b,g-Me-ATP) and 29- and -39- O-(4-benzoyl-benzoyl)-ATP] inhibited arginine vasopressin (AVP)- and parathyroid hormone (PTH)-mediated Mg21 uptake whereas agonists more selective for P2Y purinoceptors (UTP, ADP, and 2-methylthio-ATP) were without effect. Removal of extracellular Ca21 diminished b,g-Me-ATP-mediated increase in intracellular Ca21 and inhibition of AVPstimulated Mg21 entry. We conclude from this information that ATP inhibited Mg21 uptake in MDCT cells through P2X purinoceptors expressed in this distal convoluted tubule cell line.

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 1a,25-dihydroxyvitamin D [1,25(OH)2D3] on magnesium transport. In this study, we examined the effect of 1,25(OH)2D3 on distal cellular magnesium uptake and the modulation of this response by extracellular Ca21 and Mg21 in an immortalized mouse distal convoluted tubule (MDCT) cell line. MDCTcells possess the divalent cation-sensing receptor (CaSR) that responds to elevation of extracellular Ca21 and Mg21 concentrations to diminish peptide hormone-stimulated Mg21 uptake. Mg21 uptake rates were determined by microfluorescence in Mg21-depleted MDCT cells. Treatment of MDCT cells with 1,25(OH)2D3 for 16–24 h stimulated basal Mg21 uptake in a concentration-dependent manner from basal levels of 164 6 5 to 210611 nM/s, representing a 2863% change. Pretreatment with actinomycin D or cycloheximide abolished 1,25(OH)2D3- stimulated.Mg21 uptake (154 6 18 nM/s), suggesting that 1,25(OH)2D3 stimulates Mg21 uptake through gene activation and protein synthesis. Elevation of extracellular Ca21 inhibited 1,25(OH)2D3-stimulated Mg21 uptake (143 6 5 nM/s). Preincubation of the cells with an antibody to the CaSR prevented the inhibition by elevated extracellular Ca21 of 1,25(OH)2D3-stimulated Mg21 uptake (202 6 8 nM/s). Treatment with an antisense CaSR mRNA oligodeoxynucleotide also abolished the effects of extracellular Ca21 on 1,25(OH)2D3-responsive Mg21 entry. This showed that elevated extracellular calcium modulates 1,25(OH)2D-mediated responses through the CaSR. In summary, 1,25(OH)2D3 stimulated Mg21 uptake in MDCT cells, and this is dependent on de novo protein synthesis. Elevation of extracellular Ca21, acting via the CaSR, inhibited 1,25(OH)2D3-stimulated Mg21 entry. These data indicate that 1,25(OH)2D3 has important effects on the control of magnesium entry in MDCT cells and these responses can be modulated by extracellular divalent cations.

b-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, b-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 Mg21 uptake with fluorescence techniques. Intracellular free Mg21 concentration ([Mg21]i) was measured in single MDCT cells by using microfluorescence with mag-fura-2. To assess Mg21 uptake, MDCT cells were first Mg21 depleted to 0.22 6 0.01 mM by culturing in Mg21-free media for 16 h and then placed in 1.5 mM MgCl2, and the changes in [Mg21]i were determined. [Mg21]i returned to basal levels, 0.53 6 0.02 mM, with a mean refill rate, d([Mg21]i)/dt, of 168 6 11 nM/s. Isoproterenol stimulated Mg21 entry in a concentration-dependent manner, with a maximal response of 252 6 11 nM/s, at a concentration of 1027 M, that represented a 50 6 7% increase in uptake rate above control values. This was associated with a sixfold increase in intracellular cAMP generation. Isoproterenol-stimulated Mg21 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, isoproterenolmediated Mg21 entry rates involve multiple intracellular signaling pathways. Aldosterone potentiated isoproterenolstimulated Mg21 uptake (326 6 31 nM/s), whereas elevation of extracellular Ca21 inhibited isoproterenol-mediated cAMP accumulation and Mg21 uptake, 117 6 37 nM/s. These studies demonstrate that isoproterenol stimulates Mg21 uptake in a cell line of mouse distal convoluted tubules that is modulated by hormonal and extracellular influences.

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 Mg21 uptake with fluorescence techniques using mag-fura 2. To assess Mg21 uptake, MDCT cells were first Mg21 depleted to 0.22 6 0.01 mM by culturing in Mg21-free media for 16 h and then placed in 1.5 mM MgCl2, and the changes in intracellular Mg21 concentration ([Mg21]i) were measured with microfluorescence. [Mg21]i returned to basal levels, 0.53 6 0.02 mM, with a mean refill rate, d([Mg21]i)/dt, of 164 6 5 nM/s. Insulin stimulated Mg21 entry in a concentration-dependent manner with maximal response of 214 6 12 nM/s, which represented a 30 6 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 6 3 pmol·mg protein21 ·5 min21). Genistein, a tyrosine kinase inhibitor, diminished insulin-stimulated Mg21 uptake (169 6 11 nM/s), but did not change insulin-mediated cAMP formation (47 6 5 pmol·mg protein21 ·5 min21). PTH stimulates Mg21 entry, in part, through increases in cAMP formation. Insulin and PTH increase Mg21 uptake in an additive fashion. In conclusion, insulin mediates Mg21 entry, in part, by a genistein-sensitive mechanism and by modifying hormone-responsive transport. These studies demonstrate that insulin stimulates Mg21 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.

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 Mg21, Ca21, and neomycin. The present studies determined the effect of Mg21/ Ca21 sensing on hormone-mediated cAMP formation and Mg21 uptake in MDCT cells. MDCT cells were Mg21 depleted by culturing in Mg21-free media for 16 h, and Mg21 uptake was measured by microfluorescence after placing the depleted cells in 1.5 mM MgCl2. The mean rate of Mg21 uptake was 164 6 5 nM/s in control MDCT cells. Activation of Mg21/Ca21 sensing with neomycin did not affect basal Mg21 uptake (155 6 5 nM/s). We have previously reported that treatment of MDCT cells with either glucagon or arginine vasopressin (AVP) stimulated Mg21 entry. In the present studies, the addition of extracellular Mg21 or Ca21 inhibited glucagon- and AVP-stimulated cAMP formation and Mg21 uptake in concentration-dependent manner with half-maximal concentrations of ,1.5 and 3.0 mM, respectively. Exogenous cAMP or forskolin stimulated Mg21 uptake in the presence of Mg21/Ca21 sensing activation.We infer from these studies that Mg21/Ca21-sensing mechanisms located in the distal convoluted tubule may play a role in control of distal magnesium absorption.

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 Ca21 signaling, it was shown that the polyvalent cation-sensing mechanism is sensitive to extracellular magnesium concentration ([Mg21]o) and extracellular calcium concentration ([Ca21]o) in concentration ranges normally observed in the plasma. Moreover, both [Mg21]o and [Ca21]o were effective in generating intracellular Ca21 transients in the presence of large concentrations of [Ca21]o and [Mg21]o, respectively. These responses are unlike those observed for the Casr in the parathyroid gland. Finally, activation of the polycationsensitive mechanism with either [Mg21]o or [Ca21]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 [Mg21]o as well as in [Ca21]o.

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 Mg21 uptake in an immortalized mouse distal convoluted tubule (MDCT) cell line. Intracellular free Mg21 concentration ([Mg21]i) was determined on single MDCT cells using microfluorescence with mag-fura 2. To determine Mg21 entry rate into MDCT cells, they were first Mg21 depleted ([Mg21]i, 0.22 6 0.01 mM) by culturing in Mg21-free media for 16 h and then placed in 1.5mMMgCl2. The rate of change in [Mg21]i as measured as a function of time, d([Mg21]i)/dt, was 164 6 5 nM/s in control cells. We have shown that glucagon or arginine vasopressin (AVP) stimulates Mg21 entry by 63% and 15%, respectively. Incubation of MDCT cells with aldosterone for 16 h did not change the rate of Mg21 uptake (172 6 8 nM/s). However, aldosterone potentiated glucagon- and AVP-stimulated Mg21 uptake rate up to 330 6 39 and 224 6 6 nM/s, respectively. Aldosterone also potentiated glucagon- and AVP-induced intracellular cAMP accumulation in a concentration-independent manner. As cAMP stimulates Mg21 entry in MDCT cells, it is inferred that aldosterone may stimulate Mg21 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 Mg21 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.

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 Mg21 transport in this segment of the distal tubule. Glucagon and AVP increased cellular cAMP concentrations by about fivefold above basal levels in normal and Mg21-depleted cells. Intracellular free Mg21 concentration ([Mg21]i) was determined on single MDCT cells using microfluorescence with mag-fura 2. To assess Mg21 uptake, MDCT cells were first Mg21 depleted (0.22 6 0.01 mM) by culturing in Mg21-free media for 16 h and then placed in 1.5 mM MgCl2, and the [Mg21]i was determined. [Mg21]i returned to basal levels, 0.53 6 0.02 mM, with a mean refill rate, d([Mg21]i)/dt, of 164 6 5 nM/s. Both glucagon and AVP stimulated Mg21 uptake into MDCT cells, 196 6 11 and 189 6 6 nM/s, respectively, at concentrations of 3 3 1027 M and 1027 M, respectively. Enhanced Mg21 uptake for each of the hormones was concentration dependent and inhibited by the channel blocker, nifedipine. Hormone stimulation of Mg21 entry was not dependent on protein synthesis. 8-Bromo-cAMP, 1024 M, enhanced Mg21 uptake (225 6 13 nM/s), whereas phorbol esters were without effect. Finally, protein kinaseAinhibition prevented glucagon and AVP stimulation of Mg21 uptake, supporting the notion that the cAMP pathway is important as expected in the hormone action. These studies demonstrate that glucagon and AVP stimulate Mg21 uptake in MDCT cells and suggest that these hormones act to control magnesium conservation in the convoluted segment of the distal tubule.

The distal convoluted tubule reabsorbs - 10% of the filtered magnesium, which is -70% of that delivered to it from the loop of Henle. The cellular mechanisms of magnesium transport in the distal convoluted tubule are not known. Amiloride has been reported to promote magnesium conservation. Studies were performed on immortalized mouse distal convoluted tubule (MDCT) cells to characterize distal magnesium transport and the effects of amiloride. Intracellular free Mg”+ concentration ( [Mg2+]i) was determined on single MDCT cells using microfluorescence with mag-fura 2. Basal [Mg”+]i was 0.53 t 0.01 mM, which is -2% of the total cellular magnesium. To assess Mg2+ uptake, MDCT cells were first Mg2+ depleted (0.22 t 0.01 mM) by culturing in Mg2+-free media for 8-16 h and then placed in 5 mM MgC12, and the [Mg2+]i was determined. [Mg2+]i returned to basal levels (0.50 t 0.04 mM) with refill rate, d([Mg2+]i)ldt, of 181 t 33 r&I/s. Mg2+ entry rate was concentration dependent; a concentration of -0.1 mM resulted in half-maximal uptake rates. Mg”+ uptake was inhibited by La3+ (36 t 17 nM/s), Mn2+ (56 t 25 r&I/s), and nitrendipine (52 t 18 nM/s), but not Ca2+ (225 t 70 nnlvs). Mg2+ uptake was influenced by the transmembrane voltage; hyperpolarization either with the addition of valinomycin or the substitution of bath NaCl with NaSCN stimulated Mg2+ influx (205 ? 3 and 561 t 54 r&I/s, respectively). Depolarization with external KC1 diminished Mg2+ uptake (57 t 25 r&I/s). These data provide evidence for novel Mg2+ entry pathways in MDCT cells that are specific for Mg2+ and activated by an increase in transmembrane voltage. Because amiloride leads to a hyperpolarization of the apical membrane, we postulated that amiloride may enhance Mg2+ transport by influencing the membrane voltage. Amiloride (50 pM) increased Mg2+ uptake (235 t 79 nM/s) in a concentration.-dependent manner (half-maximal concentration of 35 uM amiloride). Accordingly, the distal diuretic, amiloride, inhibits Na+ transport, hyperpolarizes the apical membrane, and results in a stimulation of Mg2+ uptake in MDCT cells. These results provide the cellular basis for the clinical use of amiloride to bring about renal magnesium conservation.

We have provided functional and molecular evidence for the presence of Na+/Ca2+ exchange in isolated porcine cortical thick ascending limb (CTAL) cells. The present studies were designed to show that this exchange activity may be modulated by phosphorylative processes. Control of intracellular Ca2+ concentration ( [Ca2+];) was determined in isolated CTAL cells with microfluorescence. CTAL cells were pretreated with ouabain to elevate intracellular Na+ concentration ([Na+]i) from 10 to 20 mM. These cells had normal basal [Ca2+]; (79 2 3 nM). Substitution of extracellular NaCl (50 mM) with KC1 resulted in the rapid elevation of [Ca2+]; to maximal levels of 795 t 60 nM (n = 17). The increments of [Ca2+]; were associated with [Na+]i. We next determined the modulation of Na+lCa2+ exchange activity with phosphorylative inhibitors. Pretreatment of cells with calmidazolium, a Ca2+- calmodulin inhibitor, resulted in a shift of the [Na+]i dependence curve to the right. Pretreatment with okadaic acid, a phosphatase 1 and 2A inhibitor, increased the Na+/Ca2+ exchanger activity resulting in halfmaximal [Ca2+]; increase near normal [Na+]i of 12 mM. Furthermore, in the presence of okadaic acid in normal CTAL cells, pretreatment with ouabain and the elevation of [Na+]i was not required to elicit increments in [Ca2+];. These data indicate that Na+/Ca2+ exchange is present in CTAL cells and the exchange activity appears to be modulated, directly or indirectly, by phosphorylation events.

Intracellular Ca2+ concentration ( [Ca2+]i) plays an important role in the signal transduction processes within cortical thick ascending limb (CTAL) cells. Control of [Ca2+]; was investigated in isolated CTAL cells with microfluorescent techniques. CTAL cells pretreated with ouabain to elevate intracellular Na+ concentration ([Na+]i) had basal [Ca2+]. 10 f 86 IT 2 nM. Removal of extracellular Na (Na,f ) or voltage depolarization with KC1 (in the presence of Na,f) resulted in a rapid and reversible maximal elevation of [Ca2+]i (1,023 t 72 nM, n = ZS), which was dependent on the presence of external Ca 2+ (Caf’). The rise in [Ca2+]i was inhibited with La3+, Mg2+, amiloride, and bepridil. The increments of [Ca2+]i with either removal of Naz or voltage depolarization were dependent on pretreatment with ouabain and increases in [Na+]i. The presence of a Na+/Ca2+ exchanger was confirmed with hybridization techniques, and the isoform was identified by sequencing the alternative splicing site within the intracellular loop. A gene transcript that encodes a portion of the intracellular loop of the renal Na+/Ca2+ exchanger was amplified from cortical tissue and single CTAL cells by reverse transcription-polymerase chain reaction, using primers flanking the alternative splicing site. Southern hybridization and DNA sequencing demonstrated the isoform contained exons B and D, which is characteristic of one isoform (NACA3) of the renal Na+/Ca2+ exchanger. The results provide both functional and molecular evidence for a Na+/Ca2+ exchanger in thick ascending limb cells of the porcine kidney.

The following studies identified and characterized atria1 natriuretic peptide (ANP) receptor-mediated Ca2+ transients in cortical thick ascending limb (CTAL) cells. Primary cell cultures were prepared from porcine kidneys by immunodissection, and intracellular Ca2+ concentration ([Ca”+]J was determined in single cells with microfluorometry. ANP (10m7M ) and its analogue,C -type natriuretic peptide (CNP, 10m7M ), elicited Ca2+t ransients [ 104 t 6 (basal levels) to 653 & 112 nM (stimulated) and from 84 t 4 to 209 & 18 nM, respectively]. Receptor-mediated [ Ca2+]i increase was dose-dependenwt ith a 50% effective concentration (EC,,) of N lo-lo M. The increment in [Ca”‘]i was due to internal release and influx across the plasma membrane. Prior treatment of ANP or CNP (10q7 M) did not markedly affect a post application of either ANP or CNP. The truncated analogue of ANP, C-ANP-(4-23), which preferentially binds to clearance receptors, elicited an increase in [Ca”+]i (82 & 1 to 427 t 41 nM). 8-Bromoguanosine 3’,5’-cyclic monophosphate (8-BrcGMP) did not alter [Ca”+]i, but pretreatment of CTAL cells with 8-BrcGMP for 30 min before agonist treatment prevented ANP-induced Ca2+ signals [83 t 5 (basal) to 88 t 5 nM (stimulated)]. These results are evidence for the existence of clearance ANP receptors in CTAL cells that may have biological functions and clearance. The functional responseso f these signal interactions may have important consequenceosn hormone actions with the CTAL.

Intracellular free Mg2+ concentration ( [Mg2+]i) has beeni mplicated in the pathogenesiso f hypertension. It has been postulated that Mg2+ through its antagonistic effects on intracellular Ca2+ concentration may affect tension and contractility of vascular smooth muscle cells. An established cell line of rat thoracic aorta cells (AlO) was cultured on glass cover slips, and [Mg2+], was determined by fluorescent techniques on single cells with the use of mag-fura-2. Basal [Mg2+]i was 0.52 t 0.02 mM (n = 15). Vascular smooth muscle cells were challenged with A23187 plus 5 mM MgC12 to rapidly elevate [ Mg2+ ]i. [Mg2+]i increased to a peak of 1.03 ,t 0.09 mM within l-2 s and then quickly declined to below basal levels, 0.30 & 0.03 mM, within 45-60 s despite the continued presence of A23187 and external Mg2+. The rapid removal of the Mg2+ challenget o belowb asall evelss uggeststh e presenceo f intracellular transport mechanisms,li kely in intracellular compartments or organelles. Spatial imaging studies indicated that Mg2+ is heterogeneously distributed within the cell with the greatest variations in the perinuclear region, the area of most cytosolic organelles. Vanadate, an inhibitor of P-type adenosinetriphosphatases, inhibited the removal rate from 10.2 t 0.9 to 6.8 t 1.0 FM/S. Inhibitors of intracellular Ca2+ mobilization, thapsigargin, dantrolene, and 3,4,5-trimethoxybenzoic acid 8- (diethylamino)octyl ester, inhibited Mg2+ sequestration. Ryanodine and caffeine had no effect on Mg2+ removal. Ruthenium red did not inhibit Mg2+ sequestration, but oligomycin B slowed its removal. These studies demonstrated that [Mg2+]i in vascular smooth muscle cells is carefully controlled by active mechanisms involving intracellular and plasma membrane transporters. Alteration of this control may play a role in aberrant vasoconstriction.

Intracellular Mg”+ plays an important role in cell physiology. Studies were performed on MDCK cells and primary cortical thick ascending limb (CTAL) cells to determine hormonal influences on intracellular Mg2+ control. Free Mg2+ ( [Mg2+]i) was measured by fluorescence with mag-fura-2. Addition of 8bromoguanosine 3’,5’-cyclic monophosphate @-BrcGMP, 10e4 M) to subconfluent MDCK cells resulted in rapid increases in [Mg2+]i from basal levels of 552 it 6 PM to peak concentrations of 682 t 5 PM, whereas 8BrcAMP (10v4 M) led to significant decreases in [Mg2+]i from 538 of 5 to 362 t 17 PM. These effects of cyclic nucleotides were dose dependent with half-maximal concentrations (EC,,) of - 10e5 M for both increments in [Mg2+]i with cGMP and decrements in [Mg”+]i with CAMP. Atria1 natriuretic peptide (ANP) and cGMP increased Mg2+ in porcine primary CTAL cells from 525 t 12 to 592 & 18 PM and from 538 & 8 to 609 t 18 PM, respectively. The increment in [Mg2+]i with ANP was dose responsive with EC& values of - lo-l1 M suggesting that these effects may be of physiological importance. Parathyroid hormone and calcitonin and their second messenger, CAMP, diminished Mg2+ by -80 PM. The EC& value for calcitonin was in the order of 10mg M. The changes in [Mg2+]i, whether increases with ANP or cGMP and decreases with PTH, calcitonin, or CAMP, were rapid in nature and independent of changes in intracellular free Ca2+ concentration. These data indicate that [Mg”+]i is influenced by peptide hormones and their second messengers likely through activation of appropriate protein kinases. The functional role of these changes remains to be determined.

Magnesium reabsorption and regulation within the kidney occur principally within the cortical thick ascending limb (cTAL) cells of the loop of Henle. Fluorometry with the dye, mag-fura- 2, was used to characterize intracellular Mg2" concentration (IMg2j11) in single cTAL cells. Primary cell cultures were prepared from porcine kidneys using a double antibody technique (goat anti-human Tamm-Horsfall and rabbit anti-goat IgG antibodies). Basal [Ig2+"] was 0.52±0.02 mM, which was - 2% of the total cellular Mg. Cells cultured (16 h) in high magnesium media (5 mM) maintained basal [Mg2j1i, 0.48±0.02, in the normal range. However, cells cultured in nominally magnesium- free media possessed [Mg2J1j, 0.27±0.01 mM, which was associated with a significant increase in net Mg transport, (control, 0.19±0.03 and low Mg, 0.35±0.01 nmol. mg-' protein min-') as assessed by 'Mg uptake. Mg2'-depleted cells were subsequently placed in high Mg solution (5 mM) and the Mg2" refill rate was assessed by fluorescence. [Mg2"1 returned to normal basal levels, 0.53±0.03 mM, with a refill rate of 257±37 nM/s. Mg2" entry was not changed by 5.0 mM Ca2" or 2 mM Sr2+, Cd2+, Co2+, nor Ba2+ but was inhibited by Mn2+ = La3+ .,Gd3+ Ni2+ , Zn2+ Be2+ at 2 mM. Intracellular Ca2` and "Ca uptake was not altered by Mg depletion or Mg2+ refill, indicating that the entry is relatively specific to Mg2e. Mg2+ uptake was inhibited by nifedipine (117±20 nM/s), verapamil (165±34 nM/s), and diltiazem (194±19 nM/s) but enhanced by the dihydropyridine analogue, Bay K 8644 (366±71 nM/s). These antagonists and agonists were reversible with removal and IMg2+Jj subsequently returned to normal basal levels. Mg2+ entry rate was concentration and voltage dependent and maximally stimulated after 4 h in magnesium-free media. Cellular magnesium depletion results in increases in a Mg2+ refill rate which is dependent, in part, on de novo protein synthesis. These data provide evidence for novel Mg2+ entry pathways in cTAL cells which are specific for Mg2` and highly regulated. These entry pathways are likely involved with renal Mg2` homeostasis.

Basal free Mg2’ concentration was 0.49 t 0.03 mM in normal single Madin-Darby canine kidney (MDCK) cells as measured by fluorescence with the aid of mag-fura-2. Accordingly, Mg2+ may enter the cell down a transmembrane electrical gradient. The present study describes some aspects of Mg2+ entry into the established MDCK cell line. MDCK cells were Mg2’-depleted (0.26 t 0.01 mM) by culturing in Mg2’-free media for 16-20 h. Cells were subsequently exposed to 5 mM MgCL,, and intracellular M$+ concentration ( [ Mg2+] i) was monitored with fluoresence. [Mg+]i returned to normal basal levels, 0.56 t 0.05 mM, with a refill rate of 272 t 39 nM/s, n = 4. M$+ entry was not changed by 5.0 mM external Ca2+ but was completely inhibited with 5.0 mM La3+. Intracellular Ca2+ concentration was not altered by Mg+ depletion or during M$+ repletion. Mg2+ uptake was inhibited by verapamil (0 t 27 nM/s, n = 3), was inhibited less so by diltiazem (141 t 34 nM/s, n = 3), and was not affected by nifedipine (300 t 53 nM/s, n = 6). These inhibitors were fully reversible on removal, and [Mg”‘]i returned to normal levels. These data indicate the presence of a unique M$+ entry pathway in MDCK cells that may be important in M$+ homeostasisT. he model of Mg2+ refill into M$+- depleted cells may be useful in other cell types.