scholarly journals Insulin stimulates Mg2+ uptake in mouse distal convoluted tubule cells

1999 ◽  
Vol 277 (6) ◽  
pp. F907-F913 ◽  
Author(s):  
Long-Jun Dai ◽  
Gordon Ritchie ◽  
Brian W. Bapty ◽  
Dirk Kerstan ◽  
Gary A. Quamme
Keyword(s):  
Kinase Inhibitor ◽  
Insulin Receptors ◽  
Distal Tubule ◽  
Fold Increase ◽  
Distal Convoluted Tubule ◽  
Maximal Response ◽  
Thick Ascending Limb ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Camp Formation

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 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 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)/d t, 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.

β-Adrenergic agonists stimulate Mg2+ uptake in mouse distal convoluted tubule cells

2000 ◽  
Vol 279 (6) ◽  
pp. F1116-F1123 ◽  
Author(s):  
Hyung Sub Kang ◽  
Dirk Kerstan ◽  
Long-Jun Dai ◽  
Gordon Ritchie ◽  
Gary A. Quamme
Keyword(s):  
Protein Kinase ◽  
Intracellular Signaling ◽  
Distal Tubule ◽  
Distal Convoluted Tubule ◽  
Maximal Response ◽  
Intracellular Camp ◽  
Thick Ascending Limb ◽  
Dependent Manner ◽  
Adrenergic Agonists ◽  
Concentration Dependent Manner

β-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, β-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 Mg2+ uptake with fluorescence techniques. Intracellular free Mg2+ concentration ([Mg2+]i) was measured in single MDCT cells by 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)/d t, of 168 ± 11 nM/s. Isoproterenol stimulated Mg2+ 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 Mg2+ 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 Mg2+ entry rates involve multiple intracellular signaling pathways. Aldosterone potentiated isoproterenol-stimulated Mg2+ uptake (326 ± 31 nM/s), whereas elevation of extracellular Ca2+ inhibited isoproterenol-mediated cAMP accumulation and Mg2+ uptake, 117 ± 37 nM/s. These studies demonstrate that isoproterenol stimulates Mg2+ uptake in a cell line of mouse distal convoluted tubules that is modulated by hormonal and extracellular influences.

PGE2 stimulates Mg2+ uptake in mouse distal convoluted tubule cells

1998 ◽  
Vol 275 (5) ◽  
pp. F833-F839 ◽  
Author(s):  
Long-Jun Dai ◽  
Brian Bapty ◽  
Gordon Ritchie ◽  
Gary A. Quamme
Keyword(s):  
Protein Kinase ◽  
Intracellular Signaling ◽  
Distal Convoluted Tubule ◽  
Maximal Response ◽  
Prostaglandin E ◽  
Intracellular Camp ◽  
Thick Ascending Limb ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Camp Generation

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+]iwere determined. [Mg2+]ireturned to basal levels, 0.53 ± 0.02 mM, with a mean refill rate, d([Mg2+]i)/d t, of 173 ± 8 nM/s. Indomethacin, 5 μM, 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.

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

2000 ◽  
Vol 78 (8) ◽  
pp. 595-602 ◽  
Author(s):  
Hyung Sub Kang ◽  
Dirk Kerstan ◽  
Long-jun Dai ◽  
Gordon Ritchie ◽  
Gary A Quamme
Keyword(s):  
Parathyroid Hormone ◽  
Distal Tubule ◽  
Distal Convoluted Tubule ◽  
Thick Ascending Limb ◽  
Clinical Use ◽  
Cellular Mechanisms ◽  
Nephron Segment ◽  
Tubule Cells ◽  
Renal Magnesium Wasting ◽  
Camp Formation

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, illicited 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 Mg2+-depleted by culturing in Mg2+-free media for 16 h and Mg2+ uptake was measured by microfluorescence after placing the depleted cells in 1.0 mM MgCl2. The mean rate of Mg2+ uptake, d([Mg2+]i)/dt, was 138 ± 24 nM/s in control MDCT cells. Gentamicin (50 µM) 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·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.Key words: intracellular Mg2+, Mg2+ uptake, aminoglycosides, gentamicin, tobramycin, streptomycin, neomycin, parathyroid hormone, microfluorescence, cAMP measurements.

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

1998 ◽  
Vol 275 (3) ◽  
pp. F353-F360 ◽  
Author(s):  
Brian W. Bapty ◽  
Long-Jun Dai ◽  
Gordon Ritchie ◽  
Lucie Canaff ◽  
Geoffrey N. Hendy ◽  
...  
Keyword(s):  
Distal Convoluted Tubule ◽  
Dependent Manner ◽  
Cellular Mechanisms ◽  
Polyvalent Cation ◽  
Concentration Dependent Manner ◽  
Nephron Segment ◽  
The Mean ◽  
Free Media ◽  
Cation Sensing ◽  
Camp Formation

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 ∼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.

Sevoflurane Inhibits Guanosine 5′-[γ-thio]triphosphate–stimulated, Rho/Rho-kinase–mediated Contraction of Isolated Rat Aortic Smooth Muscle

2003 ◽  
Vol 99 (3) ◽  
pp. 646-651 ◽  
Author(s):  
Jingui Yu ◽  
Koji Ogawa ◽  
Yasuyuki Tokinaga ◽  
Yoshio Hatano
Keyword(s):  
Smooth Muscle ◽  
Kinase Inhibitor ◽  
Isometric Force ◽  
Rho Kinase ◽  
Force Transducer ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Aortic Smooth Muscle ◽  
Gamma S ◽  
Kinase Pathway

Background The Rho/Rho-kinase signaling pathway plays an important role in mediating Ca2+ sensitization of vascular smooth muscle. The effect of anesthetics on Rho/Rho-kinase-mediated vasoconstriction has not been determined to date. This study is designed to examine the possible inhibitory effects of sevoflurane on the Rho/Rho-kinase pathway by measuring guanosine 5'-[gamma-thio]triphosphate (GTP gamma S)-stimulated contraction and translocation of RhoA (one of the three Rho subtypes) and Rock-2 (one of the two Rho-kinase subtypes) from the cytosol to the membrane in rat aortic smooth muscle. Methods GTP gamma S-induced contraction of rat aortic endothelium-denuded rings was measured using an isometric force transducer, and GTP gamma S-stimulated membrane translocation of RhoA and Rock-2 in smooth muscle cells was detected with Western blotting in the presence and absence of sevoflurane. Results GTP gamma S (10(-4) m) induced a sustained contraction, which was significantly inhibited by the Rho-kinase inhibitor, Y27632 (3 x 10(-6) m). Before treatment with GTP gamma S, RhoA and Rock-2 were detected primarily in the cytosolic fraction. GTP gamma S (10(-4) m) stimulated the translocation of RhoA and Rock-2 from the cytosol to the membrane, which was sustained for more than 60 min. Sevoflurane (1.7, 3.4, and 5.1%) concentration dependently inhibited the GTP gamma S-induced constriction of rat aortic smooth muscle with a reduction of constriction of 52-75% (P < 0.01, n = 8), and attenuated the translocation of RhoA and Rock-2 by 31-66% and 34-78%, respectively (P < 0.05-0.01, respectively; n = 4). Conclusion The current findings show that sevoflurane depresses the GTP gamma S-stimulated contraction and translocation of both Rho and Rho-kinase from the cytosol in a concentration-dependent manner, indicating that sevoflurane is able to inhibit vasoconstriction mediated by the Rho/Rho-kinase pathway in rat aortic smooth muscle.

Effects of potassium channel modulators on myotropic responses of aortic rings of pregnant rats

2000 ◽  
Vol 278 (2) ◽  
pp. H567-H576 ◽  
Author(s):  
C. Cadorette ◽  
B. Sicotte ◽  
M. Brochu ◽  
J. St-Louis
Keyword(s):  
Membrane Potential ◽  
Potassium Channels ◽  
Basal Membrane ◽  
Maximal Response ◽  
Dependent Manner ◽  
Response Curves ◽  
Concentration Dependent Manner ◽  
Pregnant Rats ◽  
Potassium Channel Modulators ◽  
High Conductance

The contribution of potassium channels [ATP-sensitive potassium (KATP) and high-conductance calcium-activated potassium (BKCa) channels] in the resistance of aortic rings of term pregnant rats to phenylephrine (Phe), arginine vasopressin (AVP), and KCl was investigated. Concentration-response curves to tetraethylammonium (TEA), a nonselective K+ channel inhibitor, were obtained in the absence or presence of KCl. TEA induced by itself concentration-dependent responses only in aortic rings of nonpregnant rats. These responses to TEA could be modulated in both groups of rings by preincubation with different concentrations of KCl. Concentration-response curves to Phe, AVP, and KCl were obtained in the absence or presence of cromakalim or NS-1619 (KATP and BKCa openers, respectively) and glibenclamide or iberiotoxin (KATPand BKCa inhibitors, respectively). Cromakalim significantly inhibited the responses to the three agonists in a concentration-dependent manner in both groups of rats. Alternatively, in the pregnant group of rats, glibenclamide increased the sensitivity to all three agonists. NS-1619 also inhibited the response to all agonists. With AVP and KCl, its effect was greater in aortic rings of pregnant than nonpregnant rats. Finally, iberiotoxin increased the sensitivity to all three agents. This effect was more important in aortic rings of nonpregnant rats and was accompanied by an increase of the maximal response to Phe and AVP. These results suggest that potassium channels are implicated in the control of basal membrane potential and in the blunted responses to these agents during pregnancy.

scholarly journals Quercetin Is a Flavonoid Breast Cancer Resistance Protein Inhibitor with an Impact on the Oral Pharmacokinetics of Sulfasalazine in Rats

Pharmaceutics ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 397
Author(s):  
Yoo-Kyung Song ◽  
Jin-Ha Yoon ◽  
Jong Kyu Woo ◽  
Ju-Hee Kang ◽  
Kyeong-Ryoon Lee ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Breast Cancer Resistance Protein ◽  
Fold Increase ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Cancer Resistance ◽  
Concentration Dependent Manner ◽  
Resistance Protein

The potential inhibitory effect of quercetin, a major plant flavonol, on breast cancer resistance protein (BCRP) activity was investigated in this study. The presence of quercetin significantly increased the cellular accumulation and associated cytotoxicity of the BCRP substrate mitoxantrone in human cervical cancer cells (HeLa cells) in a concentration-dependent manner. The transcellular efflux of prazosin, a stereotypical BCRP substrate, was also significantly reduced in the presence of quercetin in a bidirectional transport assay using human BCRP-overexpressing cells; further kinetic analysis revealed IC50 and Ki values of 4.22 and 3.91 μM, respectively. Moreover, pretreatment with 10 mg/kg quercetin in rats led to a 1.8-fold and 1.5-fold increase in the AUC8h (i.e., 44.5 ± 11.8 min∙μg/mL vs. 25.7 ± 9.98 min∙μg/mL, p < 0.05) and Cmax (i.e., 179 ± 23.0 ng/mL vs. 122 ± 23.2 ng/mL, p < 0.05) of orally administered sulfasalazine, respectively. Collectively, these results provide evidence that quercetin acts as an in vivo as well as in vitro inhibitor of BCRP. Considering the high dietary intake of quercetin as well as its consumption as a dietary supplement, issuing a caution regarding its food–drug interactions should be considered.

scholarly journals Reversal of Cancer Multidrug Resistance (MDR) Mediated by ATP-Binding Cassette Transporter G2 (ABCG2) by AZ-628, a RAF Kinase Inhibitor

2020 ◽  
Vol 8 ◽  
Author(s):  
Jing-Quan Wang ◽  
Qiu-Xu Teng ◽  
Zi-Ning Lei ◽  
Ning Ji ◽  
Qingbin Cui ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Kinase Inhibitor ◽  
Efflux Pump ◽  
Simulation Analysis ◽  
Chemotherapeutic Agents ◽  
Dynamics Simulation ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Raf Kinase

Overexpression of ABCG2 remains a major impediment to successful cancer treatment, because ABCG2 functions as an efflux pump of chemotherapeutic agents and causes clinical multidrug resistance (MDR). Therefore, it is important to uncover effective modulators to circumvent ABCG2-mediated MDR in cancers. In this study, we reported that AZ-628, a RAF kinase inhibitor, effectively antagonizes ABCG2-mediated MDR in vitro. Our results showed that AZ-628 completely reversed ABCG2-mediated MDR at a non-toxic concentration (3 μM) without affecting ABCB1-, ABCC1-, or ABCC10 mediated MDR. Further studies revealed that the reversal mechanism was by attenuating ABCG2-mediated efflux and increasing intracellular accumulation of ABCG2 substrate drugs. Moreover, AZ-628 stimulated ABCG2-associated ATPase activity in a concentration-dependent manner. Docking and molecular dynamics simulation analysis showed that AZ-628 binds to the same site as ABCG2 substrate drugs with higher score. Taken together, our studies indicate that AZ-628 could be used in combination chemotherapy against ABCG2-mediated MDR in cancers.

Pharmacological properties of endothelin receptor subtypes mediating positive inotropic effects in rabbit heart

1994 ◽  
Vol 266 (6) ◽  
pp. H2220-H2228 ◽  
Author(s):  
H. Kasai ◽  
M. Takanashi ◽  
C. Takasaki ◽  
M. Endoh
Keyword(s):  
Partial Agonist ◽  
Ventricular Myocardium ◽  
Rabbit Heart ◽  
Maximal Response ◽  
Second Phase ◽  
Receptor Subtypes ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Inotropic Effects ◽  
Rabbit Ventricular Myocardium

The positive inotropic effect (PIE) of endothelin (ET) isoforms, ET-1 and ET-3, was similar in that 1) the PIE was associated with prolongation of isometric contractions, 2) the maximal response was approximately 60% of that to isoproterenol (Isomax), 3) the PIE was associated with acceleration of PI hydrolysis, and 4) it was selectively antagonized by phorbol 12,13-dibutyrate. Because the concentration-response curve for ET-1 was biphasic (whereas that for ET-3 was monophasic), ET-1 had a PIE greater than ET-3 up to 10(-8) M. ET-1 induced a PIE at 3 x 10(-14) M and higher, which reached a plateau of 10-20% of Isomax at 10(-12) M (first phase); the curve became steeper at 10(-9) M and higher (second phase), achieving the maximal response at 10(-7) M to 3 x 10(-7) M. An ETA-selective antagonist, BQ-123, did not affect the PIE of ET-1 up to 10(-7) M; it abolished the first phase at 10(-6) M but did not affect the second phase. BQ-123 at 10(-8) to 10(-6) M antagonized the PIE of ET-3, [Thr2]sarafotoxin S6b, and [Glu9]sarafotoxin S6b in a concentration-dependent manner. The PIE of ET-3 was abolished by 10(-6) M BQ-123. An ETB-selective partial agonist IRL-1620 neither elicited a PIE nor affected the PIE of ET-3. These findings indicate that the PIE of ET receptor agonists on rabbit ventricular myocardium cannot be totally explained by occupancy of the ETA or ETB receptor.

scholarly journals Extracellular ATP inhibits transport in medullary thick ascending limbs: role of P2X receptors

2009 ◽  
Vol 297 (5) ◽  
pp. F1168-F1173 ◽  
Author(s):  
Guillermo B. Silva ◽  
Jeffrey L. Garvin
Keyword(s):  
Nitric Oxide ◽  
Oxygen Consumption ◽  
Oxidative Phosphorylation ◽  
Receptor Antagonist ◽  
P2x Receptors ◽  
Extracellular Atp ◽  
Thick Ascending Limb ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Nitric Oxide Synthase Inhibitor

Absorption of NaCl by the thick ascending limb (TAL) involves active transport and therefore depends on oxidative phosphorylation. Extracellular ATP has pleiotropic effects, including both stimulation and inhibition of transport and inhibition of oxidative phosphorylation. However, it is unclear whether ATP alters TAL transport and how this occurs. We hypothesized that ATP inhibits TAL Na absorption by reducing Na entry. We measured oxygen consumption in TAL suspensions. ATP reduced oxygen consumption in a concentration-dependent manner. The purinergic (P2) receptor antagonist suramin (300 μM) blocked the effect of ATP on TAL oxygen consumption (147 ± 15 vs. 146 ± 16 nmol O2·min−1·mg protein−1). In contrast, the adenosine receptor antagonist theophylline did not block the effect of ATP on oxygen consumption. When Na-K-2Cl cotransport and Na/H exchange were blocked with furosemide (100 μM) plus dimethyl amiloride (100 μM), ATP did not inhibit TAL oxygen consumption (from 78 ± 13 to 98 ± 5 nmol O2·min−1·mg protein−1). The Na ionophore nystatin (200 U/ml) increased TAL oxygen consumption to a similar extent in both ATP- and vehicle-treated samples (368 ± 41 vs. 397 ± 47 nmol O2·min−1·mg protein−1). The nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester (3 mM) blocked the ATP effects on TAL oxygen consumption (157 ± 10 vs. 165 ± 15 nmol O2·min−1·mg protein−1). The P2X-selective receptor antagonist NF023 blocked the effect of ATP on oxygen consumption, whereas the P2X-selective agonist β-γ-Me-ATP reduced oxygen consumption in a concentration-dependent manner. We conclude that ATP inhibits Na transport-related oxygen consumption in TALs by reducing Na entry and P2X receptors and nitric oxide mediate this effect.

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