Biomedical subjects

G A Quamme

Publications and source records attributed to G A Quamme.

At least 19 recordsLinked to original sources

PubMed1999

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.

Aldosterone↗

PubMed1998

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.

Amino Acid Sequence↗

PubMed1998

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.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1998

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.

Aldosterone↗

PubMed1998

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.

Amiloride↗

PubMed1998

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.

Animals↗

PubMed1997

Chlorpromazine activates chloride currents in Xenopus oocytes.

Xenopus oocytes are frequently used for in vivo expression of DNA and RNA, especially those encoding ion channel proteins. Accordingly, it is important to understand the expression and control of endogenous conductances. Ionic currents were studied in native Xenopus oocytes with two-microelectrode voltage-clamp technique to characterize the actions of chlorpromazine (CPZ) and trifluroperazine (TFP), two widely used antipsychotic drugs. External application of CPZ or TFP markedly stimulated endogenous conductances in a dose-dependent and reversible fashion. The current-voltage (I-V) relationship was non linear and dependent on the presence of external chloride. The CPZ-activated currents were inhibited by Cl- channel blockers. Although the removal of external Ca2+ had no effect on CPZ-induced conductances, the injection of BAPTA, a Ca2+ chelator, abolished endogenous activity. Thapsigargin also inhibited channel activity suggesting that CPZ acts through intraoocyte Ca2+ release. The calmodulin inhibitors, calmidazolium and W-7, failed to mimic the action of CPZ. These data provide evidence for external or internal phenothiazine receptors which when activated by CPZ induces Ca(2+)-dependent Cl- channel activity in endogenous native oocytes.

Animals↗

PubMed1997

Caffeine decreases intracellular free Mg2+ in isolated adult rat ventricular myocytes.

Caffeine has been extensively used to study intracellular Ca2+ control and contraction-relaxation in cardiomyocytes. The effects of caffeine on intracellular free Mg2+ concentration, [Mg2+]i, were studied in isolated adult rat ventricular myocytes by fluorescent techniques using mag-fura-2. Basal [Mg2+]i was 0.62 +/- 0.02 mM, n = 54, in quiescent cells and 0.73 +/- 0.02 mM, n = 23, in electrically-stimulated adult rat ventricular myocytes. Caffeine, 20 mM, significantly decreased [Mg2+] in both quiescent (-0.17 +/- 0.01 mM) and electrically-stimulated (-0.16 +/- 0.01 mM) adult ventricular myocytes. Ryanodine, a blocker for Ca(2+)-release channels of the sarcoplasmic reticulum, did not have any effect on basal [Mg2+]i, 0.67 +/- 0.02 mM nor on caffeine-induced reduction in [Mg2+]i, -0.16 +/- 0.01 mM in quiescent cardiomyocytes or electrically-stimulated cells; 0.74 +/- 0.03 mM and -0.11 +/- 0.03 mM, respectively. Ruthenium red, an inhibitor of mitochondrial Ca2+ uptake, also failed to alter basal [Mg2+]i, or caffeine-induced reduction in [Mg2+], in either quiescent or electrically-stimulated cells. The effects of caffeine on [Mg2+]i, may be important in considering the use of this drug to study contraction/function studies in heart cells.

Animals↗

PubMed1997

Cellular mechanisms of chlorothiazide and cellular potassium depletion on Mg2+ uptake in mouse distal convoluted tubule cells.

The use of the distally-acting diuretic, chlorothiazide, has been reported to have important effects on renal magnesium handling. The cellular mechanisms of chlorothiazide action on Mg2+ uptake was investigated in immortalized mouse distal convoluted tubule (MDCT) cells. Intracellular free Mg2+ concentration was determined by microfluorescence. Mg2+ transport was measured as a function of change in intracellular Mg2+ concentration with time following placement of Mg2+-depleted cells into a buffer containing 1.5 mM magnesium. The uptake rate of Mg2+ into Mg2+-depleted cells was 179 +/- 28 nM/second. Mg2+ uptake was dependent on the membrane voltage as membrane hyperpolarization enhanced uptake whereas depolarization diminished transport. Chlorothiazide increased Mg2+ uptake by 58%, from 179 +/- 28 to 283 +/- 23 nM/second. The ability of chlorothiazide to stimulate Mg2+ uptake in MDCT cells was concentration-dependent and related to the diuretic-induced hyperpolarization of the plasma membrane. These studies support the notion that acute chlorothiazide administration enhances renal magnesium conservation through its effects on Mg2+ transport within the distal convoluted tubule. Since chronic chlorothiazide administration may result in hypokalemia as well as hypomagnesemia, Mg2+ uptake was determined in potassium-depleted MDCT cells. Mg2+ uptake was diminished, 80 +/- 24 nM/second, in potassium depleted cells. Hyperpolarization of the plasma membrane with the cell permanent anion, SCN-, corrected Mg2+ uptake in potassium depleted cells suggesting that the basis for diminished uptake may, in part, be due to depolarization of the membrane voltage. In summary, acute chlorothiazide stimulates Mg2+ transport in MDCT cells. We postulate that chronic chlorothiazide use may lead to hypokalemia that in turn diminishes Mg2+ transport in the distal tubule resulting in urinary magnesium-wasting.

Animals↗

PubMed1997

Phosphate depletion diminishes Mg2+ uptake in mouse distal convoluted tubule cells.

Hypophosphatemia caused by phosphate depletion is associated with renal magnesium wasting. The cellular mechanisms of phosphate depletion were investigated in an immortalized mouse distal convoluted tubule (MDCT) cell line. Intracellular free Mg2+ concentration. [Mg2+]i was determined by microfluorescence. Mg2+ transport was assessed as a function of change in [Mg2+]i with time following placement of Mg(2+)-depleted cells into a buffer containing 1.5 mM magnesium. The uptake rate of Mg2+ into Mg(2+)-depleted cells cultured in normal phosphate, 1.0 mM, was 175 +/- 21 nM/second. Depletion of phosphate in the culture media was associated with a significant decrease in Mg2+ uptake, which was dependent on the degree of phosphate depletion and on the time cultured in phosphate-deficient media. Cells cultured for 16 hours in 0.3 mM and 0 mM phosphate possessed Mg2+ uptake rates of 105 +/- 18 nM/second and 15 +/- 12 nM/second, respectively. Diminished Mg2+ uptake was rapidly induced following placement in low phosphate and was fully reversed following readdition of phosphate to the culture media. The effects of phosphate depletion on Mg2+ uptake was post-translational in nature as fully up-regulated MDCT cells with maximal Mg2+ uptake was associated with a rapid decrease (within 30 min) in Mg2+ transport when placed in phosphate-deficient media. Although Mg2+ uptake is altered by the transmembrane voltage, diminished Mg2+ uptake associated with phosphate depletion was not dependent on changes in membrane voltage. Further, it was not associated with a sustained increase in intracellular Ca2+ concentration. Chlorothiazide, probably through hyperpolarization of the plasma membrane, stimulates Mg2+ uptake in normal. 283 +/- 23 nM/second, and phosphate-depleted cells, 203 +/- 29 nM/second, but failed to entirely correct the defective transport. These studies demonstrate that magnesium wasting associated with hypophosphatemia and phosphate depletion is due, in part, to diminished Mg2+ transport in the distal convoluted tubule. The evidence is that the actions of phosphate deficiency are through alterations of Mg2+ transport across the luminal membrane of the distal convoluted tubule cell.

Animals↗

PubMed1997

Renal magnesium handling: new insights in understanding old problems.

Recent research has provided new concepts in our understanding of renal magnesium handling. Although the majority of the filtered magnesium is reabsorbed within the loop of Henle, it is now recognized that the distal tubule also plays an important role in magnesium conservation. Magnesium absorption within the cTAL segment of the loop is passive and dependent on the transepithelial voltage. Magnesium transport in the DCT is active and transcellular in nature. Many of the hormonal (PTH, calcitonin, glucagon, AVP) and nonhormonal (magnesium-restriction, acid-base changes, potassium-depletion) influences that affect magnesium transport within the cTAL similarly alter magnesium absorption within the DCT. However, the cellular mechanisms are different. Actions within the loop affect either the transepithelial voltage or the paracellular permeability. Influences acting in the DCT involve changes in active transcellular transport either Mg2+ entry across the apical membrane or Mg2+ exit from the basolateral side. These transport processes are fruitful areas for future research. An additional regulatory control has recently been recognized that involves an extracellular Ca2+/Mg(2+)-sensing receptor. This receptor is present in the basolateral membrane of the TAL and DCT and modulates magnesium and calcium conservation with elevation in plasma divalent cation concentration. Further studies are warranted to determine the physiological role of the Ca2+/Mg(2+)-sensing receptor, but activating and inactivating mutations have been described that result in renal magnesium-wasting and hypermagnesemia, respectively. All of these receptor-mediated controls change calcium absorption in addition to magnesium transport. Selective magnesium control is through intrinsic control of Mg2+ entry into distal tubule cells. The cellular mechanisms that intrinsically regulate magnesium transport have yet to be described. Familial diseases associated with renal magnesium-wasting provide a unique opportunity to study these intrinsic controls. Loop diuretics such as furosemide increase magnesium excretion by virtue of its effects on the transepithelial voltage thereby inhibiting passive magnesium absorption. Distally acting diuretics, like amiloride and chlorothiazide, enhance Mg2+ entry into DCT cells. Amiloride may be used as a magnesium-conserving diuretic whereas chlorothiazide may lead to potassium-depletion that compromises renal magnesium absorption. Patients with Bartter's and Gitelman's syndromes, diseases of salt transport in the loop and distal tubule, respectively, are associated with disturbances in renal magnesium handling. These may provide useful lessons in understanding segmental control of magnesium reabsorption. Metabolic acidosis diminishes magnesium absorption in MDCT cells by protonation of the Mg2+ entry pathway. Metabolic alkalosis increases magnesium permeability across the cTAL paracellular pathway and stimulates Mg2+ entry into DCT cells. Again, these changes are likely due to protonation of charges along the paracellular pathway of the cTAL and the putative Mg2+ channel of the DCT. Cellular potassium-depletion diminishes the voltage-dependent magnesium absorption in the TAL and Mg2+ entry into MDCT cells. However, the relationship between potassium and magnesium balance is far from clear. For instance, magnesium-wasting is more commonly found in patients with Gitelman's disease than Bartter's but both have hypokalemia. Further studies are needed to sort out these discrepancies. Phosphate deficiency also decreases Mg2+ uptake in distal cells but it apparently does so by mechanisms other than those observed in potassium depletion. Accordingly, potassium depletion, phosphate deficiency, and metabolic acidosis may be additive. The means by which cellular potassium and phosphate alter magnesium handling are unclear. Research in the nineties has increased our understanding of renal magnesium transport and regulation, but there are many in

Absorption↗

PubMed1997

Mechanisms of amiloride stimulation of Mg2+ uptake in immortalized mouse distal convoluted tubule cells.

The distal convoluted tubule reabsorbs approximately 10% of the filtered magnesium, which is approximately 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 ofamiloride. Intracellular free Mg2+ concentration ([Mg2+]i) was determined on single MDCT cells using microfluorescence with mag-fura 2. Basal [Mg2+]i was 0.53 +/- 0.01 mM, which is approximately 2% of the total cellular magnesium. To assess Mg2+ uptake, MDCT cells were first Mg2+ depleted (0.22 +/- 0.01 mM) by culturing in Mg2+-free media for 8-16 h and then placed in 5 mM MgCl2, and the [Mg2+]i was determined. [Mg2+]i returned to basal levels (0.50 +/- 0.04 mM) with refill rate, d([Mg2+]i)/dt, of 181 +/- 33 nM/s. Mg2+ entry rate was concentration dependent; a concentration of approximately 0.1 mM resulted in half-maximal uptake rates. Mg2+ uptake was inhibited by La3+ (36 +/- 17 nM/s), Mn2+ (56 +/- 25 nM/s), and nitrendipine (52 +/- 18 nM/s), but not Ca2+ (225 +/- 70 nM/s). 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 +/- 54 nM/s, respectively). Depolarization with external KCl diminished Mg2+ uptake (57 +/- 25 nM/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 microM) increased Mg2+ uptake (235 +/- 79 nM/s) in a concentration-dependent manner (half-maximal concentration of 35 microM 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.

Amiloride↗

PubMed1997

Acid-base changes alter Mg2+ uptake in mouse distal convoluted tubule cells.

Metabolic alkalosis leads to renal magnesium conservation, whereas metabolic acidosis is associated with urinary magnesium wasting. Micropuncture studies suggest that these actions affect magnesium transport in the distal tubule. The cellular mechanisms of acid-base changes were investigated in an immortalized mouse distal convoluted tubule (MDCT) cell line. Intracellular free Mg2+ concentration ([Mg2+]i) was determined by microfluorescence using the Mg(2+)-responsive dye, mag-fura 2. Mg2+ transport was assessed as a function of change in [Mg2+]i with time following placement of Mg(2+)-depleted cells into a buffer containing 1.5 mM magnesium. The uptake rate of Mg2+, d([Mg2+]i)/dt, into Mg(2+)-depleted cells determined with a buffer solution of pH 7.4 was 178 +/- 21 nM/s. Mg2+ uptake at pH 8.0 was markedly increased 278 +/- 35 nM/s, whereas transport at pH 6.0 was significantly reduced to 121 +/- 15 nM/s. Mg2+ uptake at pH 7.4 was not stimulated with 20 or 40 mM bicarbonate, nor were the differences in Mg2+ uptake with pH associated with changes in membrane voltage. Mg2+ uptake was stimulated with membrane hyperpolarization at pH 6.0 but not at pH 8.0. Chlorothiazide (10(-4) M), which stimulates Mg2+ uptake by hyperpolarizing the membrane voltage, increased uptake at pH 6.0, 59 +/- 14%, but decreased it at alkaline pH of 8.0, -55 +/- 3%. Accordingly, MDCT cells become refractory to the stimulating effects of hyperpolarization at alkaline pH values. These studies show that protons may directly affect Mg2+ transport in MDCT cells.

Acid-Base Equilibrium↗

PubMed1996

Na+/Ca2+ exchanger in epithelial cells of the porcine cortical thick ascending limb.

Intracellular Ca2+ concentration ([Ca2+]i) plays an important role in the signal transduction processes within cortical thick ascending limb (CTAL) cells. Control of [Ca2+]i was investigated in isolated CTAL cells with microfluorescent techniques. CTAL cells pretreated with ouabain to elevate intracellular Na+ concentration ([Na+]i) had basal [Ca2+]i of 86 +/- 2 nM. Removal of extracellular Na (Nao+) or voltage depolarization with KCl (in the presence of Nao+) resulted in a rapid and reversible maximal elevation of [Ca2+]i (1,023 +/- 72 nM, n = 28), which was dependent on the presence of external Ca2+ (Cao2+). The rise in [Ca2+]i was inhibited with La3+, Mg2+, amiloride, and bepridil. The increments of [Ca2+]i with either removal of Nao+ 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.

Animals↗

PubMed1996

Modulation of Na+/Ca2+ exchange in epithelial cells of porcine thick ascending limb.

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+]i) 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+]i (79 +/- 3 nM). Substitution of extracellular NaCl (50 mM) with KCl resulted in the rapid elevation of [Ca2+]i to maximal levels of 795 +/- 60 nM (n = 17). The increments of [Ca2+]i were associated with [Na+]i. We next determined the modulation of Na+/Ca2+ exchange activity with phosphorylative inhibitors. Pretreatment of cells with calmidazolium, a Ca(2+)-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 half-maximal [Ca2+]i 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+]i. 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.

Animals↗

PubMed1995

Altered [Ca2+]i mobilization in diabetic cardiomyocytes: responses to caffeine, KCl, ouabain, and ATP.

To study the mechanisms mediating intracellular calcium transients involved in diabetic cardiac dysfunction, changes in intracellular calcium concentration ([Ca2+]i) in response to stimulation by caffeine, ouabain, KCl and ATP were studied in single cardiomyocytes (quiescent or electrically-stimulated) isolated from streptozotocin (STZ) diabetic rats. [Ca2+]i was measured by fluorescence microscopy using fura-2. Peak [Ca2+]i response to caffeine (20 mM) and decline of [Ca2+]i (-peak d[Ca2+]i/dt) were decreased in diabetic myocytes. Insulin treatment corrected these depressed [Ca2+]i responses. The data suggest a reduced sarcoplasmic reticulum (SR) calcium content and a depressed Na-Ca exchange activity in diabetic myocytes. Insulin deficiency may play a causal role in these changes. The maximum [Ca2+]i increase in response to ouabain was reduced in diabetic cells while the sensitivity of diabetic myocytes to ouabain was increased. This may be a result of depressed Na-K ATPase and elevated [Na+]i as previously reported. The KCl (12.5-50 mM)-induced [Ca2+]i increase was enhanced in diabetic cells. Caffeine (20 mM) and dichlorobenzamil (DCB, 10 microM) blocked this [Ca2+]i transient to a smaller degree in diabetic cells, but nitrendipine effects were similar in diabetic and control cells. These effects may be due to the increased L-channel activity and altered features, such as different responses to Ca-channel blockers, in diabetes which has previously been reported. The maximum response of [Ca2+]i to exogenous ATP was increased in diabetic cells while the sensitivity remained unchanged. The mechanisms underlying this enhanced response may be similar to the KCl-induced [Ca2+]i changes in diabetes.

Adenosine Triphosphate↗

PubMed1994

Effect of pH on intracellular free Mg2+ in isolated adult rat cardiomyocytes.

Changes in intracellular pH (pHi) alters the cytosolic concentrations of many electrolytes including Ca2+ and Na+. The present studies determined the effect of pHi on intracellular Mg2+ ([Mg2+]i) activity in isolated adult rat ventricular myocytes. Intracellular magnesium, [Mg2+]i, and calcium, [Ca2+]i, concentrations were measured with microfluorometry. Basal intracellular [Mg2+]i was 634 +/- 27 microM, n = 42 cells, and was not changed following electrical stimulation (0.5 Hz/s) which resulted in transient increases in [Ca2+]i and cell contractions. An NH4Cl pulse was used to rapidly alkalize and acidify the cytosol. Intracellular Mg2+ concentration within single cells decreased by 129 +/- 13 microM with rapid alkalinization of pH from basal levels of 7.1 to 7.6 following a NH4+ pulse. Removal of NH4Cl bathing solution resulted in cytosolic acidification, pH 6.9, and an increase in [Mg2+]i, from 467 +/- 47 to 569 +/- 41 microM. Intracellular [Ca2+] rose with acidification 80 +/- 4 to 149 +/- 19 nM, n = 5, and returned to normal levels, 89 +/- 5 nM, following recovery of pHi. Intracellular acidosis following exposure to 5% CO2/20 mM HCO3- solutions resulted in a significant increase in [Mg2+]i, to 778 +/- 63 microM. These results indicate that intracellular pH may have significant effects on [Mg2+]i in adult cardiomyocytes.

Ammonium Chloride↗

PubMed1994

Hormone-mediated Ca2+ transients in isolated renal cortical thick ascending limb cells.

Peptide hormones control salt reabsorption in cortical thick ascending limb (cTAL) cells of the loop of Henle. These agonists act, in part, through alterations on intracellular Ca2+ ([Ca2+]i). Primary cell cultures were prepared from porcine kidneys using a double antibody technique (goat antihuman Tamm-Horsfall and rabbit antigoat IgG antibodies). [Ca2+]i was determined in single cells with fluorescent techniques using fura-2. Parathyroid hormone (PTH) and arginine vasopressin (AVP) transiently increased [Ca2+]i in a dose-dependent manner. [Ca2+]i maximally increased from 85 +/- 5 nmol/l to 608 +/- 99 nmol/l with PTH, 10(-6) M, and to 766 +/- 162 nmol/l with AVP, 10(-7) M. The increment in [Ca2+]i by both hormones was by intracellular Ca2+ release and entry through plasma membrane Ca2+ channels. 8-Bromo-adenosine-3',5'-cyclic monophosphate (8-BrcAMP), 10(-4) M, increased [Ca2+]i (basal 83 +/- 3 to 427 +/- 121 nmol/l) but only from internal sources as nifedipine (10 mumol), ([Ca2+]i changes: 86 +/- 4 to 390 +/- 29 nmol/l) and removal of bath Ca/+o, ([Ca2+]i changes: 84 +/- 6 to 517 +/- 142 nmol/l), were without effect on agonist-induced [Ca2+]i. Thapsigargin, 1.5 mumol, completely abolished the AVP- and cyclic adenosine monophosphate-(cAMP)-induced Ca2+ transients, and partially inhibited PTH-mediated Ca2+ transients by about 50%. Pretreatment with 8-BrcAMP inhibited the PTH and AVP responses likely through depletion of cAMP-sensitive Ca2+ stores. Activation of protein kinase C (PKC) with phorbol esters inhibited PTH and AVP responses and 8-BrcAMP-induced [Ca2+]i transients.(ABSTRACT TRUNCATED AT 250 WORDS)

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