Biomedical subjects

J Teulon

Publications and source records attributed to J Teulon.

At least 19 recordsLinked to original sources

PubMed2002

An inward rectifier K(+) channel at the basolateral membrane of the mouse distal convoluted tubule: similarities with Kir4-Kir5.1 heteromeric channels.

In this study, K(+) channels present in the basolateral membrane of the distal convoluted tubule (DCT) were investigated using patch-clamp methods. In addition, Kir4.1, Kir4.2 and Kir5.1 inward rectifier channels were investigated using RT-PCR and immunohistochemistry (Kir4.1). DCTs were microdissected from collagenase-treated mouse kidneys. One type of K(+) channel was detected in about 50 % of cell-attached patches from the DCT basolateral membrane; this channel was inwardly rectifying and had an inward conductance (g(in)) of approximately 40 pS at an external [K(+)] of 145 mM. The current-voltage relationship was linear when inside-out patches were exposed to a Mg(2+)-free medium. Mg(2+) at a concentration of 1.2 mM considerably reduced the outward conductance (g(out)), yielding a g(in)/g(out) ratio of approximately 4.7. The polycation spermine (5 x 10(-7) M) reduced the open probability (P(o)) by 50 %. Channel activity was dependent upon the intracellular pH, with acid pH decreasing, and basic pH increasing, P(o). Internal ATP (2 mM) and Ca(2+) (up to 10(-3) M) had no effect. Channel activity declined irreversibly when the inner side of the patch was exposed to Mg(2+). Kir4.1, Kir4.2 and Kir5.1 mRNAs were all detected in the DCT. The Kir4.1 protein co-localised with the Na(+)-Cl(-) cotransporter, which is specific to the DCT, and was located on basolateral membranes. The DCT K(+) channel differs from other functionally identified renal K(+) channels with regard to its inhibition by spermine and insensitivity to internal ATP and Ca(2+). At the current state of knowledge, the channel is similar to Kir4.1-Kir5.1 and Kir4.2-Kir5.1 heteromeric channels, but not to Kir4.1 or Kir4.2 homomeric channels.

Adenosine Triphosphate↗

PubMed2001

Vasopressin-stimulated chloride transport in transimmortalized mouse cell lines derived from the distal convoluted tubule and cortical and inner medullary collecting ducts.

BACKGROUND: The fine control of NaCl absorption takes place in the distal parts of the renal tubule, but the regulation of Cl(-) transport in this region has not been fully elucidated. We have analysed the effects of dD-arginine vasopressin (dDAVP) on Cl(-) fluxes in cultured mouse distal convoluted tubule (mpkDCT), cortical collecting duct (mpkCCD) and inner medullary collecting duct (mpkIMCD) cell lines. METHODS: RT-PCR and Western blotting were used to detect the amiloride-sensitive sodium channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) mRNAs and protein in cultured mpkDCT, mpkCCD and mpkIMCD cells. Cl(-) fluxes were analysed by measuring the short-circuit current (I(sc)) and bidirectional (36)Cl(-) fluxes on confluent cells grown on filters. RESULTS: All three cell lines expressed ENaC and CFTR and had I(sc) stimulated by dDAVP. The rise in I(sc) caused by dDAVP (10(-8) M) was inhibited by amiloride, and to a lesser extent by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) in all three cell lines. The dDAVP-dependent I(sc) measured under apical Na(+)-free condition was reduced by Cl(-) channel blockers with a profile (NPPB>glibenclamide>DIDS), similar to that for rat CFTR. dDAVP stimulated the apical-to-basal (36)Cl(-) flux and to a lesser extent the basal-to-apical (36)Cl(-) flux under open-circuit condition in all three cultured cell lines. Adding NPPB to the apical side reduced the basal-to-apical (36)Cl(-) flux but not the opposite (36)Cl(-) flux from dDAVP-treated cells. CONCLUSION: These results indicate that dDAVP stimulates the bi-directional flux of Cl(-), resulting in net Cl(-)absorption, in these cultured mouse distal and collecting duct cells. I(sc) experiments also suggest the presence of a minor component of electrogenic Cl(-) secretion, possibly mediated by CFTR.

Animals↗

PubMed2001

CFTR disruption impairs cAMP-dependent Cl(-) secretion in primary cultures of mouse cortical collecting ducts.

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in the renal cortical collecting duct (CCD) has not yet been fully elucidated. Here, we investigated the effects of deamino-8-D-arginine vasopressin (dDAVP) and isoproterenol (ISO) on NaCl transport in primary cultured CCDs microdissected from normal [CFTR(+/+)] and CFTR-knockout [CFTR(-/-)] mice. dDAVP stimulated the benzamyl amiloride (BAm)-sensitive transport of Na(+) assessed by the short-circuit current (I(sc)) method in both CFTR(+/+) and CFTR(-/-) CCDs to a very similar degree. Apical addition of 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) or glibenclamide partially inhibited the rise in I(sc) induced by dDAVP and ISO in BAm-treated CFTR(+/+) CCDs, whereas dDAVP, ISO, and NPPB did not alter I(sc) in BAm-treated CFTR(-/-) CCDs. dDAVP stimulated the apical-to-basal flux and, to a lesser extent, the basal-to-apical flux of (36)Cl(-) in CFTR(+/+) CCDs. dDAVP also increased the apical-to-basal (36)Cl(-) flux in CFTR(-/-) CCDs but not the basal-to-apical (36)Cl(-) flux. These results demonstrate that CFTR mediates the cAMP-stimulated component of secreted Cl(-) in mouse CCD.

Amiloride↗

PubMed1999

Two types of voltage-dependent potassium channels in outer hair cells from the guinea pig cochlea.

Cell-attached and cell-free configurations of the patch-clamp technique were used to investigate the conductive properties and regulation of the major K(+) channels in the basolateral membrane of outer hair cells freshly isolated from the guinea pig cochlea. There were two major voltage-dependent K(+) channels. A Ca(2+)-activated K(+) channel with a high conductance (220 pS, P(K)/P(Na) = 8) was found in almost 20% of the patches. The inside-out activity of the channel was increased by depolarizations above 0 mV and increasing the intracellular Ca(2+) concentration. External ATP or adenosine did not alter the cell-attached activity of the channel. The open probability of the excised channel remained stable for several minutes without rundown and was not altered by the catalytic subunit of protein kinase A (PKA) applied internally. The most frequent K(+) channel had a low conductance and a small outward rectification in symmetrical K(+) conditions (10 pS for inward currents and 20 pS for outward currents, P(K)/P(Na) = 28). It was found significantly more frequently in cell-attached and inside-out patches when the pipette contained 100 microM acetylcholine. It was not sensitive to internal Ca(2+), was inhibited by 4-aminopyridine, was activated by depolarization above -30 mV, and exhibited a rundown after excision. It also had a slow inactivation on ensemble-averaged sweeps in response to depolarizing pulses. The cell-attached activity of the channel was increased when adenosine was superfused outside the pipette. This effect also occurred with permeant analogs of cAMP and internally applied catalytic subunit of PKA. Both channels could control the cell membrane voltage of outer hair cells.

4-Aminopyridine↗

PubMed1998

Cl- absorption across the thick ascending limb is not altered in cystic fibrosis mice. A role for a pseudo-CFTR Cl- channel.

The cortical thick ascending limb (CTAL) absorbs Cl- via a Na+-K+-Cl- cotransport at the apical membrane and several Cl- channels at the basolateral membrane, including a 9-pS channel having several properties of the cystic fibrosis transmembrane conductance regulator (CFTR). Having checked that CFTR mRNA is present in the mouse CTAL, we investigated whether this channel is a CFTR molecule by applying the patch-clamp technique to CTALs microdissected from CFTR knockout mice (cftrm1Unc). The 9-pS channel was active in cell-attached patches from tubules of mice homozygous for the disrupted cftr gene [CFTR (-/-)] at the same frequency and with the same activity (NPo) as in normal [CFTR (+/+)] or heterozygous [CFTR (+/-)] mice. The conductive properties of the channel, studied on inside-out patches, were identical in CFTR (-/-), CFTR (+/+), and CFTR (+/-) tubules, as were the sensitivities to internal pH and internal ATP, two typical features of this channel. In addition, the Cl- absorption in isolated, microperfused CTALs and the Na+-K+-Cl- cotransport activity were identical in CFTR (-/-), CFTR (+/+), and CFTR (+/-) mice. These results show that the 9-pS Cl- channel is distinct from CFTR, and that the CFTR protein has no influence on the Cl- absorption in this part of the renal tubule.

Adenosine Triphosphate↗

PubMed1996

Modulation by purines of calcium-activated non-selective cation channels in the outer hair cells of the guinea-pig cochlea.

1. The cell-attached and cell-free configurations of the patch-clamp technique were used to investigate whether external ATP and its derivatives modulate channel activity in outer hair cells freshly isolated from the guinea-pig cochlea. 2. Submicromolar concentrations of ATP stimulated a non-selective cation channel with a conductance of about 25 pS. The ATP-elicited stimulation was partly blocked by the membrane-permeant blocker 3',5-dichlorodiphenylamine-2-carboxylic acid (DCDPC), and mimicked by the calcium ionophore, ionomycin, suggesting that the channel activated by ATP is identical to a previously reported calcium-activated non-selective (CAN) cation channel. 3. The P2x agonist beta, gamma-methylene-ATP (beta, gamma-MeATP, 10 microM) and the P2Y agonist 2-methyl-thio-ATP (2-MeSATP, 1 microM) both activated CAN channels. The effect of ATP was inhibited by the P2 antagonist suramin but not by the P2Y antagonist Reactive Blue 2. These results suggest that both purinergic receptors are involved in the ATP-evoked response and that internal calcium acts as a second messenger for opening CAN channels. 4. In contrast, adenosine inhibited CAN channels. This effect was reproduced by the A2 agonist 5'-N-ethylcarboxyamidoadenosine (NECA) and the permeant cAMP analogue 8-bromo-adenosine 3',5'-cyclic monophosphate (8-Br-cAMP), but not by the A1 agonist N6-cyclo-hexyladenosine (CHA). CAN channels were also inhibited when the catalytic subunit of protein kinase A was applied internally on inside-out patches, suggesting that adenosine A2 receptor downregulates CAN channels via a cAMP-dependent phosphorylation.

Adenosine Triphosphate↗

PubMed1996

Inhibition of a small-conductance cAMP-dependent Cl- channel in the mouse thick ascending limb at low internal pH.

1. A small-conductance Cl- channel that is stimulated by ATP and protein kinase A has been identified in the basolateral membranes of cortical thick ascending limbs (CTALs) of the mouse nephron. The present study uses the cell-attached and inside-out variants of the patch-clamp technique to investigate the pH sensitivity of this channel. 2. The open-state probability (Po) was dependent upon the internal pH in inside-out patches. Expressed as a percentage of the Po value at pH 7.2, Po increased to about 180% at pH 7.6, and decreased to 25% at pH 6.8. Po was close to zero at pH 6.4. The internal pH had no effect on the channel unit conductance. 3. The effect of pH on the CTAL Cl- channel was assessed in intact cells using NH4Cl to acidify the intracellular compartment. Experiments with the pH-sensitive fluorescent dye 2',7'-(carboxyethyl)-5'(6')-carboxy fluorescein penta-acetoxymethyl ester (BCECF) indicated that 1 mmol l-1 NH4Cl acidified the cytoplasm by 0.15 pH units and 5 mmol l-1 NH4Cl by 0.34 pH units. These concentrations of NH4Cl reduced the activity of the CTAL Cl- channel by 24 and 82% in cell-attached patches, showing that moderate changes in internal pH substantially altered the activity of this channel. NH4+ had no direct effect on channel activity. 4. Inhibition at low pH is a newly discovered property of small-conductance Cl- channels in epithelia, which might help discriminate between types of Cl- channel.

Animals↗

PubMed1996

Functional evidence for a Ca2+/polyvalent cation sensor in the mouse thick ascending limb.

The effects of extracellular polyvalent cations on the cytosolic free Ca2+ concentration ([Ca2+]i) of isolated segments of the mouse nephron were investigated using fura 2 microfluorometry. Extracellular Ca2+ concentration ([Ca2+]o), gadolinium (Gd3+), and neomycin (Neo) increased the [Ca2+]i in cortical thick ascending limb (CTAL) tubules with effective doses (ED50) of approximately 3.5 mM for Ca2+, 20 microM for Gd3+, and 40 microM for Neo. This effect was reproduced by Ba2+ but not by Mg2+. High [Ca2+]o inhibited the responses to Gd3+, Neo, and Ba2+. The Gd(3+)- and Neo-evoked [Ca2+]i transients persisted in the absence of external Ca2+ and were abolished by the depletion of internal Ca2+ stores with thapsigargin (TG). The responses to rises in [Ca2+]o were similarly inhibited by TG and slightly reduced by 20 microM La3+ but not by 10 microM nifedipine. Mn2+ also mobilized a TG-sensitive internal Ca2+ store and stimulated its own entry. External Ca2+, Gd3+, and Neo induced small but significant increases in [Ca2+]i in distal convoluted tubule, cortical collecting duct, and outer medullary collecting duct segments, transiently increased [Ca2+]i in some medullary TAL (MTAL) tubules, but had no effect on descending thin limb. We conclude that a Ca(2+)-mobilizing Ca2+/polyvalent cation sensor resembling that of the parathyroid gland cells is predominantly located in the mouse CTAL but also in the MTAL and, to a lesser extent, in more distal segments.

Animals↗

PubMed1995

A small-conductance Cl- channel in the mouse thick ascending limb that is activated by ATP and protein kinase A.

1. Chloride channels were identified in the basolateral membrane of isolated cortical thick ascending limbs (CTALs) of the mouse nephron by the patch-clamp technique. A channel with a conductance of 45 pS, previously shown to be Cl- selective, was detected in 21% of cell-attached patches when CTAL fragments were pre-incubated with 10 mumol l-4 forskolin for at least 15 min. The same channel was found in only 8.5% of cell-attached patches formed on unstimulated tubules. 2. Another channel with a smaller conductance (7-9 pS) was found in 42.8% of cell-attached patches and 57% of inside-out patches in unstimulated CTAL tubules, but in 82-87% of patches from forskolin-treated tubules. 3. The small channels was Cl- selective (Cl(-)-to-Na+ permeability ratio, PCl/PNa = 9.8) with the permeability sequence: NO3- > Br- > Cl- > F- > gluconate. Channel activity decreased (Br-) or disappeared (NO3-) at negative voltages. At 140 mmol l-1, I- completely inhibited channel activity at all voltages, but a PI/PCl ratio of 1.6 was estimated using a low I- concentration (10 mmol l-1). 4. Internal adenosine triphosphate (ATP) increased normalized current (nPo) in 48% of inside-out patches containing Cl- channels from unstimulated tubules and in 63% of patches from forskolin-treated CTAL tubules. The non-hydrolysable ATP analogue, adenosine 5'-adenylyl imidodiphosphate (AMP-PNP) did not increase channel activity. 5. Adding the catalytic subunit of protein kinase A to the bath in the presence of ATP increased the activity of the small channel in 58% of inside-out patches from unstimulated tubules, but it had no effect on the 45 pS channel. 6. The Cl- channel blockers 5-nitro-2-(3-phenylpropylamine)-benzoic acid (NPPB), 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) or glibenclamide, all at 0.1 mmol l-1, and diphenylamine-2-carboxylic acid (DPC), at 1 mmol l-1, inhibited the small channel activity by 80-100% in inside-out patches. 7. These results indicate that two Cl- channels with contrasting properties mediate the basolateral step of NaCl absorption in the thick ascending limb of the loop of Henle.

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

PubMed1995

Effects of internal pH on the nonselective cation channel from the mouse collecting tubule.

We investigated the effects of internal pH on Ca-activated, nucleotide-inhibited nonselective cation channels in the basolateral membranes of mouse collecting tubules, using the inside-out variant of the patch clamp technique. pH modulated the channel open probability (Po), giving a bell-shaped curve peaking at pH 6.8/7.0: Po at pH 6.0 was 11 +/- 6% of Po at pH 7.2 and 32 +/- 7% at pH 8.0. The open and closed time distributions, best fitted to the sum of two exponentials, were differently sensitive to acid and alkaline conditions. Low pH reduced the short and long open times to 38 and 24% of their pH 7.2 values, while high pH produced a 4-fold increase in the long closed time. As previously reported, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) induced a quasi-permanent opening of the channel. The inhibition of the channel produced by high pH disappeared in the presence of SITS, while the inhibition produced by low pH was unaffected. These results suggest that the pH dependence of the channel is due to two separate mechanisms. pH was without effect on the ATP-evoked inhibition of the channel, while high pH profoundly reduced the steepness of the AMP inhibition curve, without altering the half-maximal inhibitory AMP concentration.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

PubMed1995

Extracellular ATP and UTP trigger calcium entry in mouse cortical thick ascending limbs.

The effects of extracellular nucleotides on the cytosolic free Ca2+ concentration ([Ca2+]i) of mouse cortical thick ascending limb (CTAL) segments were investigated using the Ca(2+)-sensitive fluorescent probe fura 2. ATP (50% effective dose, ED50, 40 microM) transiently increased [Ca2+]i, while adenosine (a P1 purinoceptor agonist), N6-cyclohexyladenosine (an A1 agonist), AMP, ADP (a P2t agonist), beta, gamma-methyleneadenosine 5'-triphosphate (a P2x agonist), or 2-methylthioadenosine 5'-triphosphate (a P2y agonist) all had little or no effect. CTAL tubules were also sensitive to UTP. The responses to 100 microM ATP and UTP were similar but not additive. Both [Ca2+]i responses were strongly inhibited by 300 microM suramin (a P2 purinoceptor antagonist). Adenosine 5'-O-(3- thiotriphosphate) and ITP were slightly less potent than ATP, while GTP and CTP had no effect. The absence of external Ca2+ or the presence of 50 microM nifedipine similarly and markedly reduced the ATP- and UTP-evoked [Ca2+]i transients. We conclude that mouse CTAL tubules possess nucleotide receptors that are equally sensitive to ATP and UTP and that transiently elevate [Ca2+]i by triggering Ca2+ entry via a nifedipine-sensitive pathway.

Adenine Nucleotides↗

PubMed1995

Hypertonic NaCl enhances adenosine release and hormonal cAMP production in mouse thick ascending limb.

Adenosine 3',5'-cyclic monophosphate (cAMP), accumulated in the presence of adenosine, was measured in medullary portions of mouse thick ascending limbs of Henle's loop, suspended either in classic extracellular buffer or in the presence of added NaCl. Under control conditions (140 mmol/l NaCl), adenosine (< 10(-5) mol/l) and N6-cyclohexyladenosine, an A1 adenosine receptor agonist, inhibit the cAMP accumulation induced by arginine vasopressin (AVP). On the other hand, high concentrations of adenosine and CGS-21680, an A2 adenosine receptor agonist, stimulate cAMP formation. Addition of NaCl (+300 mmol/l) to extracellular buffer stimulates the release of endogenous adenosine. It also enhances A2 receptor-induced cAMP accumulation but suppresses A1 receptor-mediated inhibition of adenylyl cyclase. This hypertonic NaCl medium also potentiates the stimulatory action of AVP on adenylyl cyclase. The modifications of tubular responses to both AVP and A1 and A2 agonists, brought about by hypertonic NaCl, were all inhibited by adenosine deaminase, thereby demonstrating the involvement of endogenous adenosine. Adenosine, the release and the effects of which are modulated by hypertonic NaCl, thus appears to act as an endogenous physiological modulator of kidney medulla function.

Adenosine↗

PubMed1994

A calcium-activated nonselective cationic channel in the basolateral membrane of outer hair cells of the guinea-pig cochlea.

The patch-clamp technique was used to investigate ion channels in the basolateral perilymph-facing membrane of freshly isolated outer hair cells (OHCs) from the guinea-pig cochlea. These sensory cells probably determine, via their motile activity, the fine tuning of sound frequencies and the high sensitivity of the inner ear. A Ca(2+)-activated nonselective cationic channel was found in excised inside-out membrane patches. The current/voltage relationship was linear with a unit conductance of 26.3 +/- 0.3 pS (n = 15) under symmetrical inger conditions. The channel excluded anions (PNa/PCl = 18 where PNa/PCl denotes the relative permeability of Na to Cl); it was equally permeant to the Na+ and K+ ions and exhibited a low permeability to N-methyl-D-glucamine and Ba2+ or Ca2+. Channel opening required a free Ca2+ concentration of about 10(-6) mol/l on the internal side of the membrane and the open probability (Po) was maximal at 10(-3) mol/l (Po = 0.72 +/- 0.06, n = 12). Adenosine 5'mono-, tri- and di-phosphate reduced Po to 29 +/- 14 (n = 5), 42 +/- 10 (n = 8) and 51 +/- 12 (n = 5) % of control Po, respectively, when they were added at a concentration of 10(-3) mol/l to the internal side. The channel was partially blocked by flufenamic acid (10(-4) mol/l) and 3',5'-dichlorodiphenylamine-2-carboxylic acid (DCDPC, 10(-5) mol/l). This type of channel, together with Ca(2+)-activated K+ channels, might participate in the control of membrane potential and modulate the motility of OHCs.

Adenine Nucleotides↗

PubMed1994

A ubiquitous non-selective cation channel in the mouse renal tubule with variable sensitivity to calcium.

Basolateral membranes of microdissected collagenase-treated fragments of renal tubules from the mouse were examined using the cell-attached and the cell-free variants of the patch-clamp technique. With a K(+)-rich solution in the pipette, a highly active, inwardly rectifying K+ channel was observed on intact cells of the cortical collecting tubule (CCT). The mean inward and outward conductances were 38.5 +/- 3.1 pS and 17.3 +/- 1.8 pS, respectively (n = 4). In contrast, cell-attached patches were usually inactive when a Na(+)-rich solution filled the patch pipette. However, another type of channel with a conductance of 20-30 pS exhibited a sparse activity in 4/20 CCT. In excised, inside-out patches, the most frequent channel in CCT had an ohmic unit conductance of 27.1 +/- 1.2 pS (n = 17), excluded anions (PCl/PNa = 0.09), discriminated little between NH4+, K+ and Na+ (PNH4/PNa = 1.5; PK/PNa = 0.9), and was much less permeable to Ca2+ and Ba2+ than to Na+ (PCa/PNa = 0.09; PBa/PNa approximately 0). The cation channel was moderately voltage-dependent, showing a decreased open probability (Po) at negative voltages. It was activated by internal calcium (threshold: 1 mumol/l-0.1 mmol/l calcium), and inhibited by the adenine nucleotides ATP, ADP and AMP with half-maximal inhibition of Po at 1.2 mumol/l AMP. As in other cell models, 3',5'-dichlorodiphenylamine-2-carboxylic acid blocked channel activity when added to the internal surface of the membrane patch. Extending our study to other parts of the renal tubule, we found that the basolateral membranes of the proximal (pars recta), distal convoluted, connecting and outer medullary collecting tubules, the thin descending limb and the medullary thick ascending limb all contained a similar Ca- and ATP-sensitive cation channel. The calcium sensitivity varied from one part to another.

Adenine Nucleotides↗

PubMed1994

Renal cells transformed with SV40 contain a high-conductance calcium-insensitive potassium channel.

The inside-out variant of the patch-clamp technique was used to investigate the properties of a K+ channel occurring in 20% of patches from two renal cell lines transformed with the wild-type simian virus 40 (SV40) (Vandewalle et al. J. Cell. Physiol. 141: 203-221, 1989). This channel was practically absent from the primary cultures of renal cortical cells from which the cell lines were originally derived. With identical K(+)-rich solutions on both sides of the membrane patch, the channel showed an inwardly rectifying current-voltage relationship with unit conductances of 151.8 +/- 4.8 pS at negative and 86.4 +/- 5.9 pS at positive voltages (n = 18). When K+ in the bath was replaced by Na+, a mean reversal potential of 57.0 +/- 5.2 mV (n = 6) was observed from which a K(+)-to-Na+ permeability ratio of 13 was calculated. The channel was insensitive to internal Ca2+ and was blocked by internal Ba2+. No clear dependence on voltage was apparent. This channel bears no resemblance to any epithelial K+ channel and may be a novel type of K+ channel. Its occurrence in two transformed cell lines with quite distinct phenotypes, one of proximal cells (RC.SV1) and the other of thick ascending limb cells (RC.SV2), suggests that transformation by SV40 might be responsible for its appearance.

Animals↗

PubMed1993

K+ fluxes mediated by Na(+)-K(+)-Cl- cotransport and Na(+)-K(+)-ATPase pumps in renal tubule cell lines transformed by wild-type and temperature-sensitive strains of Simian virus 40.

The relative contributions of Na(+)-K(+)-ATPase pumps and Na(+)-K(+)-Cl- cotransport to total rubidium (Rb+) influx into primary cultures of renal tubule cells (PC.RC) and cells transformed either with the wild-type or a temperature-sensitive mutant of the simian virus 40 (SV40), were measured under various growth conditions. The Na(+)-K(+)-ATPase-mediated component represented 74% and 44-48% of total Rb+ influx into PC.RC and SV40-transformed cells, respectively. Proliferating transformed cells showed substantial ouabain-resistant bumetanide-sensitive (Or-Bs) Rb+ influx (41-45% of total) which indicated the presence of a Na(+)-K(+)-Cl- cotransport. The Or-Bs component of Rb+ influx was greatly reduced when temperature-sensitive transformed renal cells (RC.SVtsA58) grown in Petri dishes or on permeable filters were shifted from the permissive (33 degrees C) to the restrictive temperature (39.5 degrees C) to arrest cell growth. The ouabain-sensitive Rb+ influx mediated by the Na(+)-K(+)-ATPase, the total and amiloride-sensitive Na+ uptakes were not modified following inhibition of cell proliferation. A similar fall in the Or-Bs influx was obtained when renal tubule cells transformed by the wild-type SV40 (RC.SV) were incubated with the K+ channel blocker, tetraethylammonium (TEA) ion, which we had previously shown to arrest cell growth without affecting cell viability (Teulon et al.: J. Cell. Physiol., 151:113-125, 1992). Reinitiation of cell growth by removal of TEA or return to 33 degrees C of the temperature-sensitive cells restored the Or-Bs component of Rb influx. Taken together, these results indicate that the Na(+)-K(+)-Cl- cotransport activity is critically dependent on cell growth conditions.

Animals↗

PubMed1993

[Arginine]vasopressin hydrolyses phosphoinositides in the medullary thick ascending limb of mouse nephron.

NaCl reabsorption across the thick ascending limb of Henle's loop (TAL) is stimulated by several hormones, in particular vasopressin acting through V2 receptors and cyclic AMP production. This study used suspensions of medullary TAL (mTAL) tubules from the mouse nephron to investigate the possibility that, besides activating adenylyl cyclase, vasopressin also stimulates phospholipase C via V1 receptor occupancy. Two different methods, phosphoinositide labelling and inositol trisphosphate (InsP3) radioimmunoassay, were used to show that [arginine]vasopressin (AVP) rapidly stimulated the formation of InsP3, which peaked at 200%-250% of control within the first minute of incubation with 10 nmol/l vasopressin at 37 degrees C, and declined to basal level after 5-10 min. Dose/response curves for InsP3, established at 30 degrees C and 37 degrees C using radioimmunoassay, showed a half-maximal stimulation of InsP3 production at about 1 nmol/l AVP and a maximal response at 10 nmol/l. Similar values were obtained for the response to AVP in terms of cAMP accumulation. InsP3 content in the presence of higher concentrations of AVP (1 mumol/l) was significantly lower (P < 0.001) than in the presence of 10 nmol/l AVP, giving a bell-shaped appearance to the dose/response curve at 37 degrees C but not at 30 degrees C. The V2 receptor agonist, 1-deamino-[8-D-Arg]vasopressin (dAVP) did not stimulate the formation of InsP3, and the V1 receptor antagonist d(CH2)5[Tyr(Me)2]AVP inhibited AVP-induced InsP3 formation, which therefore appeared to be mediated by V1 receptor occupancy. Under the same conditions, AVP also induced the formation of diradylglycerol via V1 receptor activation, with an analogous dose/response curve.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Transformation of renal tubule epithelial cells by simian virus-40 is associated with emergence of Ca(2+)-insensitive K+ channels and altered mitogenic sensitivity to K+ channel blockers.

We compared the pattern of K+ channels and the mitogenic sensitivity to K+ channel blocking agents in primary cultures of rabbit proximal tubule cells (PC.RC) (Ronco et al., 1990) and two derived SV-40-transformed cell lines exhibiting specific functions of proximal (RC.SV1) and more distal (RC.SV2) tubule cells (Vandewalle et al., 1989). First, K+ channel equipment surveyed by the patch-clamp technique was modified after SV-40 transformation in both cell lines; although a high conductance Ca(2+)-activated K+ channel [K+200 (Ca2+)] remained the most frequently recorded K+ channel, the transformed state was characterized by emergence of three Ca(2+)-insensitive K+ channels (150, 50, and 30 pS), virtually absent from primary culture, contrasting with reduced frequency of two Ca(2+)-sensitive K+ channels (80 and 40 pS). Second, quinine (Q), tetraethylammonium ion (TEA) and charybdotoxin (CTX), at concentrations not affecting cell viability, all decreased 3H-TdR incorporation and cell growth in PC.RC cultures, but only TEA had similar effects in transformed cells. The latter were further characterized by paradoxical effects of Q that induced a marked increase in thymidine incorporation. Q also exerted contrasting effects on channel activity: it inhibited the [K+200 (Ca2+)] when the channel was highly active, with a Ki (0.2 mM) similar to that measured for 3H-TdR incorporation in PC.RC cells (0.3 mM), but increased the mean current through poorly active channels. TEA blocked all K+ channels with conductance greater than or equal to 50 pS, including the [K+200 (Ca2+)], in a range of concentrations that substantially affected cell proliferation. The unique effect of TEA on SV-40-transformed cells might be related to broad inhibition of K+ channels.

Animals↗