Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 13 de 13
Filter
Add more filters










Publication year range
1.
Am J Physiol Renal Physiol ; 281(6): F1141-7, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11704566

ABSTRACT

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.


Subject(s)
Adenosine/analogs & derivatives , Adenosine/pharmacology , Kidney Tubules, Distal/metabolism , Magnesium/metabolism , Receptors, Purinergic P1/physiology , Theobromine/analogs & derivatives , Adenosine Triphosphate/pharmacology , Aldosterone/pharmacology , Animals , Calcium/pharmacology , Cell Line, Transformed , Cyclic AMP/biosynthesis , Enzyme Inhibitors/pharmacology , Ion Transport/drug effects , Mice , Parathyroid Hormone/pharmacology , Phenethylamines/pharmacology , Protein Kinase Inhibitors , Purinergic P1 Receptor Agonists , Purinergic P1 Receptor Antagonists , Theobromine/pharmacology , Xanthines/pharmacology
2.
Am J Physiol Renal Physiol ; 281(5): F833-40, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11592941

ABSTRACT

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.


Subject(s)
Adenosine Triphosphate/pharmacology , Kidney Tubules, Distal/metabolism , Magnesium/metabolism , Receptors, Purinergic P2/physiology , Animals , Arginine Vasopressin/pharmacology , Calcium/metabolism , Cell Line, Transformed , Cyclooxygenase Inhibitors/pharmacology , Dinoprostone/pharmacology , Indomethacin/pharmacology , Kinetics , Mice , Nucleotides/pharmacology , Parathyroid Hormone/pharmacology , RNA, Messenger/analysis , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2X , Receptors, Purinergic P2Y1 , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment
3.
Am J Physiol Renal Physiol ; 280(5): F868-78, 2001 May.
Article in English | MEDLINE | ID: mdl-11292630

ABSTRACT

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.


Subject(s)
Calcitriol/pharmacology , Calcium/physiology , Kidney Tubules, Distal/metabolism , Magnesium/metabolism , Animals , Antibodies, Blocking/pharmacology , Blotting, Western , Cattle , Cell Line , Cyclic AMP/metabolism , Cytoplasm/metabolism , Kidney Tubules, Distal/drug effects , Oligodeoxyribonucleotides, Antisense/pharmacology , Sarcoplasmic Reticulum/drug effects , Sarcoplasmic Reticulum/metabolism , Stimulation, Chemical
4.
Physiol Rev ; 81(1): 51-84, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11152754

ABSTRACT

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.


Subject(s)
Kidney Tubules, Distal/metabolism , Magnesium/metabolism , Acid-Base Equilibrium/physiology , Aminoglycosides/toxicity , Animals , Antibiotics, Antineoplastic/toxicity , Cisplatin/toxicity , Diuretics/pharmacology , Hormones/metabolism , Hormones/pharmacology , Humans , Immunosuppressive Agents/toxicity , Ion Transport/drug effects , Ion Transport/physiology , Kidney Tubules, Distal/cytology , Kidney Tubules, Distal/drug effects , Phosphates/deficiency , Phosphates/metabolism , Potassium Deficiency/metabolism , Receptors, Calcium-Sensing , Receptors, Cell Surface/metabolism , Renal Tubular Transport, Inborn Errors/metabolism
5.
Am J Physiol Renal Physiol ; 279(6): F1116-23, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11097631

ABSTRACT

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.


Subject(s)
Adrenergic beta-Agonists/pharmacology , Kidney Tubules, Distal/metabolism , Magnesium/metabolism , Aldosterone/pharmacology , Animals , Cell Line , Cyclic AMP/biosynthesis , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Cytoplasm/drug effects , Cytoplasm/metabolism , Isoproterenol/pharmacology , Kidney Tubules, Distal/drug effects , Mice , Type C Phospholipases/metabolism
6.
Can J Physiol Pharmacol ; 78(8): 595-602, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10958159

ABSTRACT

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.


Subject(s)
Anti-Bacterial Agents/toxicity , Kidney Tubules, Distal/drug effects , Magnesium/metabolism , Parathyroid Hormone/pharmacology , Amiloride/pharmacology , Animals , Calcium/metabolism , Cells, Cultured , Cyclic AMP/metabolism , Gentamicins/toxicity , Kidney Tubules, Distal/metabolism , Mice
7.
Am J Physiol ; 277(6): F907-13, 1999 12.
Article in English | MEDLINE | ID: mdl-10600938

ABSTRACT

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.


Subject(s)
Cyclic AMP/metabolism , Insulin/pharmacology , Kidney Tubules, Distal/physiology , Magnesium/metabolism , Aldosterone/pharmacology , Animals , Biological Transport/drug effects , Cells, Cultured , Cyclic AMP/analogs & derivatives , Cyclic AMP/pharmacology , Cytoplasm/metabolism , Genistein/pharmacology , Kidney Tubules, Distal/cytology , Kidney Tubules, Distal/drug effects , Kinetics , Mice , Microscopy, Fluorescence , Neomycin/pharmacology , Parathyroid Hormone/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Thionucleotides/pharmacology
8.
Cell Calcium ; 26(6): 253-60, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10668563

ABSTRACT

Bronchial epithelial cells respond to extracellular nucleotides from the luminal and basolateral side activating Cl- secretion via [Ca2+]i increase. In this study we investigated the differences of apically (ap) and basolaterally (bl) stimulated [Ca2+]i signals in polarized human bronchial epithelial cells (16HBE14o-). Specifically we investigated the localization of 'capacitative Ca2+ entry' (CCE). 16HBE14o- cells grown on permeable filters were mounted into an Ussing chamber built for the simultaneous measurement of Fura-2 fluorescence and electrical properties. Application of ATP from both sides induced a rapid [Ca2+]i increase and subsequent sustained [Ca2+]i plateau due to transmembraneous Ca(2+)-influx. The use of different nucleotides revealed the following rank order or potency which was very similar for addition from the apical or basolateral side: UTP (EC50 ap: 4 microM, bl: 5 microM) > ATP (EC50 ap: 4 microM, bl: 10 microM) > ADP (n = 4-7 from both sides). 2-MeS-ATP, AMP, adenosine and beta gamma-methylene ATP were ineffective (n = 3 from both sides). The ATP- (ap and bl) induced Ca2+ influx was only abolished by removal of basolateral Ca2+. This was also true for receptor-independent activation of Ca(2+)-influx by intracellular Ca(2+)-store depletion with 2,5 Di-(tert-butyl)-1,4-benzohydroquinone (BHQ) (10 microM). Also in polarized T84 cells the basolateral carbachol and BHQ activated Ca2+ plateau was exclusively sensitive to removal of basolateral Ca2+. We propose that in all polarized epithelial cells the CCE entry pathway is located in the basolateral membrane. We furthermore suggest that Ca2+[i elevating agonists acting from the apical side of the epithelium lead to the opening of a basolateral CCE pathway.


Subject(s)
Bronchi/cytology , Bronchi/metabolism , Calcium/metabolism , Colonic Neoplasms/metabolism , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/pharmacology , Bronchi/drug effects , Calcium Signaling , Cell Membrane/metabolism , Cell Polarity , Colonic Neoplasms/drug therapy , Colonic Neoplasms/pathology , Enzyme Inhibitors/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Humans , Hydroquinones/pharmacology , Tumor Cells, Cultured
9.
Pflugers Arch ; 436(5): 712-6, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9716704

ABSTRACT

We have previously investigated, in studies of rat distal colonic mucosa, the effect of ATP added to the basolateral side on ion transport and [Ca2+]i. It was demonstrated that ATP acts via a P2Y1 receptor to increase [Ca2+]i and NaCl secretion. In the present study we investigated the effect of luminally added nucleotides (ATP, UTP) on transepithelial voltage (Vte) and resistance (Rte) in Ussing chamber experiments on rat distal colonic mucosa. Both nucleotides induced a rapid and transient (within 30 s) change of Vte to lumen-positive values (resting Vte: -2+/-1 mV; peak Vte after 100 micromol/l ATP: +2.4+/-1.1 mV) and a decrease of Rte from 89. 9+/-10.3 to 83.8+/-9.1 Omegacm2 (n=10). Similar values were obtained with luminal UTP (n=15). The estimated EC50 values for both nucleotides were approximately 6 micromol/l. The ATP-induced Vte effect was nearly completely sensitive to Ba2+. Addition of the K+ channel blocker Ba2+ (1 mmol/l) to the luminal solution reversibly inhibited 77+/-4% (n=5) of the ATP-induced Vte effect. Experiments to identify the respective P2 receptor subtype revealed the following rank order of potency at 500 micromol/l agonist: UTP>/=ATP>>2-methylthio-ATP=ADP>>adenosine> AMP>beta, gamma-methylene-ATP (n=5). This closely resembles the published rank order for the P2Y2 receptor. Using the reverse-transcriptase polymerase chain reaction (RT-PCR) technique P2Y2 receptor-specific mRNA was detected in total RNA extracted from isolated crypts. In summary these data indicate that luminal ATP and UTP act via a P2Y2 receptor in the luminal membrane of colonic mucosa to elicit a transient K+ secretion.


Subject(s)
Adenosine Triphosphate/pharmacology , Colon , Intestinal Mucosa/drug effects , Intestinal Mucosa/metabolism , Potassium/metabolism , Receptors, Purinergic P2/drug effects , Receptors, Purinergic P2/metabolism , Adenosine/pharmacology , Adenosine Diphosphate/pharmacology , Adenosine Monophosphate/pharmacology , Adenosine Triphosphate/analogs & derivatives , Animals , Barium/pharmacology , Biological Transport/drug effects , Female , In Vitro Techniques , Ion Channels/drug effects , Male , Membrane Potentials/drug effects , Polymerase Chain Reaction , Rats , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2Y2 , Thionucleotides/pharmacology , Uridine Triphosphate/pharmacology
10.
Pflugers Arch ; 436(2): 227-32, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9594022

ABSTRACT

It has previously been shown in studies of a renal epithelial cell line that nonselective cation (NSC) channels are activated by exposure to hypertonic solution. We have also found such channels in excised patches of colonic crypt cells. They require high Ca2+ activities on the cytosolic side and a low ATP concentration for their activation and have not been recorded from cell-attached patches of colonic crypts. We examine here whether this type of channel is activated by hypertonic cell shrinkage. Bath osmolality was increased by addition of 25, 50 or 100 mmol/l mannitol. Cell-attached and whole-cell patch recordings were obtained from rat base and mid-crypt cells. In whole-cell recordings we found that addition of 50 or 100 mmol/l mannitol depolarized these cells significantly from -78+/-2.0 to -66+/-3.8 mV (n=22) and from -78+/-1. 3 to -56+/-2.6 mV (n=61), respectively, and reduced the whole-cell conductance from 20+/-8.0 to 14+/-6.6 nS (n=7) and from 20+/-3.0 to 9.8+/-1.6 nS (n=19), respectively. In cell-attached patches K+ channels with a single-channel conductance of approximately 16 pS were found in most recordings. The activity of these channels (NxPo, N=number, Po=open channel probability) was reduced from 2.08+/-0.37 to 0.98+/-0.23 (n=15) by the addition of 50 mmol/l mannitol and from 1.75+/-0.26 to 0.77+/-0.20 (n=12) by 100 mmol/l mannitol. No NSC channel activity was apparent in any of these recordings. Previously we have shown that the 16-pS K+ channel is controlled by cytosolic Ca2+ ([Ca2+]i). Therefore we measured [Ca2+]i by the fura-2 method and found that hypertonic solution reduced [Ca2+]i significantly (n=16). These data indicate that exposure of rat colonic crypts to hypertonic solutions does not activate NSC channels; [Ca2+]i falls in hypertonic solution leading to a reduction in the value of K+ channel NxPo, a reduced whole-cell conductance and depolarization of mid-crypt cells. These processes probably assist volume regulation inasmuch as they reduce KCl losses from the cell.


Subject(s)
Cell Size , Colon/cytology , Colon/physiology , Hypertonic Solutions , Potassium Channels/physiology , Animals , Calcium/metabolism , Cell Membrane/physiology , Cytosol/metabolism , Electric Conductivity , Mannitol/administration & dosage , Patch-Clamp Techniques , Rats
11.
Pflugers Arch ; 434(1): 77-83, 1997 May.
Article in English | MEDLINE | ID: mdl-9094258

ABSTRACT

Under resting conditions the mammalian distal colon is a NaCl-absorptive epithelium. NaCl absorption occurs at surface cells in colonic crypts. Intracellular Ca2+ or cAMP are important second messengers that activate NaCl secretion, a function that is most pronounced in crypt bases. In the present study we examined the effect of extracellular ATP on isolated crypts of rat distal colon using the fura-2 technique. Intracellular Ca2+ ([Ca2+]i) was measured spectrofluorimetrically either by photon counting or video imaging. ATP reversibly increased [Ca2+]i in crypt base cells with an EC50 of 4.5 micromol/l (n = 11). This [Ca2+]i increase was composed of an initial peak, reflecting intracellular store release, and a secondary plateau phase reflecting transmembrane influx. Digital video imaging revealed that agonist-induced [Ca2+]i elevations were most marked at the crypt base. In the middle part of the crypt ATP induced smaller increases of [Ca2+]i (peak and plateau) as compared to basal cells and in surface cells this [Ca2+]i transient was even further reduced. Attempts to identify the relevant P2-receptor demonstrated the following rank order of potency: 2MeS-ATP > ADP >/= ATP >> AMP > UTP > AMP-PCP > adenosine. In Ussing chamber experiments ATP (1 mmol/l) functioned as a secretagogue, increasing transepithelial voltage (Vte) and equivalent short-circuit current (Isc): Delta Isc = -36.4 +/- 5.4 microA/cm2, n = 17. Adenosine itself (1 mmol/l) induced an increase of Isc of similar magnitude to that induced by ATP: Delta Isc = -55. 1 +/- 8.4 microA/cm2, n = 9. The effect of adenosine, but not that of ATP, was fully inhibited by the A1/A2-receptor antagonist 8-(p-sulphophenyl)theophylline, 0.5 mmol/l, n = 4. Together these data indicate that: (1) basolateral ATP induces [Ca2+]i in isolated rat colonic crypts and acts as a secretagogue in the distal rat colon; (2) a basolateral P2Y-receptor is responsible for this ATP-induced NaCl secretion; (3) the ability of ATP to increase Isc in Ussing chamber experiments is not mediated via adenosine; and (4) the agonist-induced [Ca2+]i signals are mostly located in the crypt base, which is the secretory part of the colonic crypt.


Subject(s)
Adenosine Triphosphate/pharmacology , Calcium/metabolism , Colon/drug effects , Intestinal Mucosa/drug effects , Receptors, Purinergic P2/drug effects , Animals , Colon/metabolism , Dose-Response Relationship, Drug , Female , Intestinal Mucosa/metabolism , Ion Transport/drug effects , Male , Rats
12.
Pflugers Arch ; 432(6): 1011-22, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8781195

ABSTRACT

We have previously shown that a new type of K+ channel, present in the basolateral membrane of the colonic crypt base (blm), is necessary for cAMP-activated Cl- secretion. Under basal conditions, and when stimulated by carbachol (CCH) alone, this channel is absent. In the present patch clamp-study we examined the ion channels present in the blm under cell-attached and in cell-excised conditions. In cell-attached recordings with NaCl-type solution in the pipette we measured activity of a K+ channel of 16 +/- 0.3 pS (n = 168). The activity of this channel was sharply increased by CCH (0. 1 mmol/l, n = 26). Reduction of extracellular Ca2+ to 0.1 mmol/l (n = 34) led to a reversible reduction of activity of this small channel (SKCa). It was also inactivated by forskolin (5 micromol/l, n = 38), whilst the K+ channel noise caused by the very small K+ channel increased. Activity of non-selective cation channels (NScat) was rarely observed immediately prior to the loss of attached basolateral patches and routinely in excised patches. The NScat, with a mean conductance of 49 +/- 1.0 pS (n = 96), was Ca2+ activated and required >10 micromol/l Ca2+ (cytosolic side = cs). It was reversibly inhibited by ATP (<1 mmol/l, n = 13) and by 3',5-dichloro-diphenylamine-2-carboxylate (10-100 micromol/l, n = 5). SKCa was also Ca2+ dependent in excised inside-out basolateral patches. Its activity stayed almost unaltered down to 1 micromol/l (cs) and then fell sharply to almost zero at 0.1 micromol/l Ca2+ (cs, n = 12). SKCa was inhibited by Ba2+ (n = 31) and was charybdotoxin sensitive (1 nmol/l) in outside-out basolateral patches (n = 3). Measurements of the Ca2+ activity ([Ca2+]i) in these cells using fura-2 indicated that forskolin and depolarization, induced by an increase in bath K+ concentration to 30 mmol/l, reduced [Ca2+]i markedly (n = 8-10). Hyperpolarization had the opposite effect. The present data indicate that the blm of these cells contains a small-conductance Ca2+-sensitive K+ channel. This channel is activated promptly by very small increments in [Ca2+]i and is inactivated by a fall in [Ca2+]i induced by forskolin.


Subject(s)
Calcium/pharmacology , Colon/physiology , Ion Channels/physiology , Potassium Channels/physiology , Adenosine Triphosphate/pharmacology , Animals , Barium/pharmacology , Carbachol/pharmacology , Cations , Cell Membrane/chemistry , Cell Membrane/physiology , Chloride Channels/physiology , Chlorides/metabolism , Colforsin/pharmacology , Colon/chemistry , Cyclic AMP/pharmacology , Electric Conductivity , Electrophysiology , Rats
13.
Pflugers Arch ; 432(1): 112-20, 1996 May.
Article in English | MEDLINE | ID: mdl-8662275

ABSTRACT

Acetylcholine-controlled exocrine secretion by pancreatic acini has been explained by two hypotheses. One suggests that NaCl secretion occurs by secondary active secretion as has been originally described for the rectal gland of Squalus acanthias. The other is based on a "push-pull" model whereby Cl- is extruded luminally and sequentially taken up basolaterally. In the former model Cl- uptake is coupled to Na+ and basolateral K+ conductances play a crucial role, in the latter model, Na+ uptake supposedly occurs via basolateral non-selective cation channels. The present whole-cell patch-clamp studies were designed to further explore the conductive properties of rat pancreatic acini. Pilot studies in approximately 300 cells revealed that viable cells usually had a membrane voltage (Vm) more hyperpolarized than -30 mV. In all further studies Vm had to meet this criterion. Under control conditions Vm was -49 +/- 1 mV (n = 149). The fractional K+ conductance (fK) was 0.13 +/- 0.1 (n = 49). Carbachol (CCH, 0.5 micromol/l) depolarized to -19 +/- 1.1 mV (n = 63) and increased the membrane conductance (Gm) by a factor of 2-3. In the seeming absence of Na+ [replacement by N-methyl-D-glucamine (NMDG+)] Vm hyperpolarized slowly to -59 +/- 2 mV (n = 90) and CCH still induced depolarizations to -24 +/- 2 mV (n = 34). The hyperpolarization induced by NMDG+ was accompanied by a fall in cytosolic pH by 0.4 units, and a very slow and slight increase in cytosolic Ca2+. fK increased to 0.34. The effect of NMDG+ on Vm was mimicked by the acidifying agents propionate and acetate (10 mmol/l) added to the bath. The present study suggests that fK makes a substantial contribution to Gm under control conditions. The NMDG+ experiments indicate that the non- selective cation conductance contributes little to Vm in the presence of CCH. Hence the present data in rat pancreatic acinar cells do not support the push-pull model.


Subject(s)
Pancreas/physiology , Animals , Carbachol/pharmacology , Chlorides/metabolism , Electric Conductivity , Electrophysiology , Meglumine/pharmacology , Pancreas/cytology , Pancreas/drug effects , Patch-Clamp Techniques , Pilot Projects , Potassium Channels/physiology , Rats
SELECTION OF CITATIONS
SEARCH DETAIL
...