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G Ritchie

Publications and source records attributed to G Ritchie.

At least 19 recordsLinked to original sources
PubMed2002

Clinical efficacy of teflubenzuron (Calicide) for the treatment of Lepeophtheirus salmonis infestations of farmed Atlantic salmon Salmo salar at low water temperatures.

The efficacy of teflubenzuron (Calicide) for the treatment of farmed Atlantic salmon Salmo salar L. infested with sea lice Lepeophtheirus salmonis (Krøyer, 1838), was investigated at low water temperatures in 2 commercial salmon farms. Calicide, coated on commercial feed pellets, was administered orally at 10 mg kg(-1) d(-1) for 7 consecutive days. Fish were randomly sampled and lice numbers recorded from both treated and control groups on 3 or 4 sampling occasions post-medication. Statistically significant reductions in the number of L. salmonis per fish were recorded. Maximum efficacy was observed toward chalimus and preadult stages of L. salmonis, and was achieved approximately 26 d post-medication. No adverse drug reactions or palatability problems were associated with the treatments.

Animals↗
PubMed2001

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.

Adenosine Triphosphate↗
PubMed2001

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.

Animals↗
PubMed2001

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.

Adenosine↗
PubMed2001

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.

Acid-Base Equilibrium↗
PubMed2000

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.

Adrenergic beta-Agonists↗
PubMed2000

Aminoglycosides inhibit hormone-stimulated Mg2+ uptake in mouse distal convoluted tubule cells.

The clinical use of aminoglycosides often leads to renal magnesium wasting and hypomagnesemia. Of the nephron segments, both the thick ascending limb of Henle's loop and the distal tubule play significant roles in renal magnesium conservation but the distal convoluted tubule exerts the final control of urinary excretion. 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. Peptide hormones, such as parathyroid hormone (PTH), glucagon, calcitonin, and arginine vasopressin (AVP) stimulate Mg2+ uptake in MDCT cells that is modulated by extracellular polyvalent cations, Ca2+ and Mg2+. The present studies determined the effect of aminoglycosides on parathyroid hormone (PTH)-mediated cAMP formation and Mg2+ uptake in MDCT cells. Gentamicin, a prototypic aminoglycoside, elicited transient increases in intracellular Ca2+ from basal levels of 102 +/- 13 nM to 713 +/- 125 nM, suggesting a receptor-mediated response. In order to determine Mg2+ transport, MDCT cells were Mg(2+)-depleted by culturing in Mg(2+)-free media for 16 h and Mg2+ uptake was measured by microfluorescence after placing the depleted cells in 1.0 mM MgCl2. The mean rate of Mg2+ uptake, d([Mg2+]i)/dt, was 138 +/- 24 nM/s in control MDCT cells. Gentamicin (50 microM) did not affect basal Mg2+ uptake (105 +/- 29 nM/s), but inhibited PTH stimulated Mg2+ entry, decreasing it from 257 +/- 36 nM/s to 108 +/- 42 nM/s. This was associated with diminished PTH-stimulated cAMP formation, from 80 +/- 2.5 to 23 +/- 1 pmol/mg protein x 5 min. Other aminoglycosides such as tobramycin, streptomycin, and neomycin also inhibited PTH-stimulated Mg2+ entry and cAMP formation. As these antibiotics are positively charged, the data suggest that aminoglycosides act through an extracellular polyvalent cation-sensing receptor present in distal convoluted tubule cells. We infer from these studies that aminoglycosides inhibit hormone-stimulated Mg2+ absorption in the distal convoluted tubule that may contribute to the renal magnesium wasting frequently observed with the clinical use of these antibiotics.

Amiloride↗
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↗
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↗
PubMed1991

Inhibition of sodium-phosphate cotransport in renal brush-border membranes with the stilbenedisulfonate, H2-DIDS.

Membrane proteins involved with sodium/phosphate cotransport across the renal brush border provide the sensitive control for phosphate homeostasis. The present study describes the inhibition of sodium/phosphate cotransport with the stilbenedisulfonate derivatives, DIDS and H2-DIDS. Preincubation of the rat brush-border membrane vesicles with H2-DIDS led to the inhibition of sodium-dependent phosphate uptake with a half maximal concentration, IC50, of about 10 microM. The inhibition was irreversible supporting the notion that H2-DIDS forms covalent bonds with the cotransporter. The cotransporter could be protected by excess sodium phosphate but not sodium chloride, sodium sulfate, sodium succinate, sodium bicarbonate, nor sodium phosphonoformate. These observations suggest that the stilbenedisulfonates may be useful in labeling the sodium/phosphate cotransporter within renal brush-border membranes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

Some characteristics of sodium-independent phosphate transport across renal basolateral membranes.

Sodium-independent phosphate transport was evaluated in porcine renal basolateral membrane vesicles. Phosphate uptake was saturable with an apparent Km 10.1 +/- 1.2 mM and Vmax 13.6 +/- 2.0 nmol (mg protein)-1 min-1, n = 5. Phosphate uptake was trans-stimulated with intravesicle phosphate and was enhanced with a positive transmembrane electrical potential. Arsenate and bicarbonate inhibited phosphate transport but other anions including sulfate and phosphonoformate were without effect. These studies indicate that phosphate uptake across basolateral membranes is present in the absence of sodium, is facilitated, and is specific for phosphate. The apparent affinity and rate of phosphate transport across the basolateral membrane is significantly higher than the respective parameters observed for the brush-border membrane.

Animals↗