Lysosomotropic agents increase vinblastine efflux from mouse MDR proximal kidney cells exhibiting vectorial drug transport.

Vinblastine (VBL) transport and efflux were studied in mouse proximal tubule PKSV-PR cells and in their multidrug-resistant derivatives PKSV-PRcol50 cells. The PKSV-PRcol50 cells produced more mdr1b transcripts and had higher resistance to various drugs. PKSV-PRcol50 cells had a predominantly basal-to-apical flux of [3H]VBL, 2.7 times larger than that in PKSV-PR cells. This flux was partially inhibited by verapamil (VRP) (10 microM) and cyclosporin A (CsA) (200 nM). [3H]VBL efflux was also greater in PKSV-PRcol50 than in PKSV-PR cells. Treatment with NH4Cl (30 mM), a lysosomotropic weak base, and concanamycin A (CCM A) (20 nM), an inhibitor of the vacuolar H+/ATPase, further increased [3H]VBL efflux from PKSV-PRcol50 cells. The cytoplasmic pH (pHcyt) of these drug-resistant cells transiently increased in the presence of NH4Cl deltapHcyt: +0.4). CCM A caused a moderate, delayed increase in pHcyt (deltapHcyt: +0.1) and made the acidic intralysosomal compartment more alkaline (deltapHlys: +1.3). VRP and CsA prevented the NH4Cl- and CCM A-induced [3H]VBL efflux from PKSV-PRcol50 cells. However, VRP (10 microM) did not significantly affect pHcyt of PKSV-PRcol50 cells, the NH4Cl-and CCM A-induced pHcyt responses, and the effect of CCMA on pHlys. Thus, lysosomotropic agents may affect the kinetics of [3H]VBL efflux. Our results also suggest that the inhibitory action of VRP on VBL efflux was not directly mediated by a pH-dependent process in these drug-resistant renal proximal tubule cells.

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.

Extracellular ATP raises cytosolic calcium and activates basolateral chloride conductance in Necturus proximal tubule.

1. Extracellular nucleotides modulate ionic transport mechanisms in various epithelia. In the present study, we investigated the effects of extracellular ATP on the intracellular free Ca+2 concentration ([Ca2+]i) and electrophysiological properties of Necturus maculosus proximal convoluted tubule (PCT). 2. ATP raised [Ca2+]i in microdissected fura-2-loaded PCTs (half-maximal effect, approximately mumol 1(-1) ATP). The initial ATP-induced changes in [Ca2+]i were not blunted by the removal of external Ca2+ nor by the presence of Ca2+ channel blockers, but were abolished by thapsigargin and suramin. The sequence for the potency of various agonists on [Ca2+]i was 2-methylthioATP (2MeSATP) = ADP = ATP >> UTP, 2',3',-O-(4-benzoilbenzoil) ATP (BzATP), alpha, beta-methylene ATP (AMPCPP), adenosine. 3. In vivo electrophysiological measurements showed that 100 mumol 1(-1) peritubular ATP added to a Ringer solution reduced the basolateral cell membrane potential (Vm) and increased the cell membrane input conductance. In a low Cl- solution, this ATP-induced depolarization was enhanced. These effects were inhibited by 1 mmol l-1 SITS, consistent with the activation of a basolateral Cl- conductance. 4. The ATP-induced change in Vm was reproduced by ADP but not by UTP or adenosine, and was prevented by suramin. 5. The ATP-induced membrane depolarization was not influenced by thapsigargin, BAPTA AM, or staurosporine and was not reproduced by manoeuvres increasing [Ca2+]i or intracellular cAMP content. 6. We conclude that, in Necturus PCT, a P2y receptor mobilizes Ca2+ mainly from intracellular pools and increases a basolateral Cl- conductance, GCl. The activation of GCl occurs by a mechanism which is not related either to an increase in [Ca2+]i or cAMP content, or to PKC activation.

Disturbed myeloperoxidase-dependent activity of neutrophils in cystic fibrosis homozygotes and heterozygotes, and its correction by amiloride.

The present study addresses the question of a possible linkage between the cystic fibrosis (CF) genetic autosomal recessive disorder and disturbance in neutrophil function. Neutrophil-dominated chronic airway inflammation is present at an early age in children with CF, even in the absence of detectable infection. As evidenced by extracellular superoxide anion release (measured by lucigenin luminescence) or intracellular hydrogen peroxide production (measured by 2',7'-dichlorofluorescein (DCF) fluorescence), no significant difference in the nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase activity of isolated neutrophils was observed in noninfected CF children (homozygotes), their mothers or fathers (CF heterozygotes), and controls. In contrast, both myeloperoxidase (MPO)-dependent oxygenation activity (measured by luminol luminescence) and chloramine release were increased significantly in both CF homozygotes and heterozygotes as compared with controls. In the presence of either amiloride (a sodium channel inhibitor and sodium/proton antiport blocker) or EIPA (5-ethyl-N-isopropyl-amiloride, a specific inhibitor of the antiport), or choline buffer, intracellular MPO activity was decreased significantly in controls and in the CF homozygotes and heterozygotes, thus bringing intracellular MPO-dependent activity in CF subjects back to the level of controls. Extracellular release of MPO, measured by an ELISA to provide an activity-independent assessment of the enzyme, was increased only in CF homozygotes, and was decreased by amiloride and choline buffer, but not by EIPA. We conclude that a modification of intracellular pH and/or ionic concentrations may be related to the altered MPO enzymatic activity observed in CF neutrophils.

Extracellular urea concentration modulates cAMP production in the mouse MTAL.

Ionic reabsorption along the ascending limb of Henle's loop (TAL) is controlled by hormonal stimulation. Most of the hormones that affect this reabsorption regulate ionic transporter activity via cAMP, and some of these hormonal actions have been shown to be modulated by interstitial osmolarity. We studied the early effects of increasing extracellular urea concentration on the production of cAMP induced by arginine vasopressin (AVP) and forskolin in a suspension of medullary portions of TAL (MTAL) prepared from mouse kidney. The addition of urea, performed fifteen minutes before adenylyl cyclase stimulation, decreased both AVP- and forskolin-induced cAMP production. This effect, observed both in the presence and the absence of phosphodiesterase inhibition, was optimal with 300 mmol/liter urea. Addition of urea to the extracellular medium disturbed several cellular parameters, but the decrease in cAMP production appeared to be mediated by the activation of both the protein kinase A and a phosphatase rather than by the modifications in phospholipid metabolism. Since cAMP is the major cytosolic transductional factor in MTAL cells, urea present in the medullary interstitium may thus be considered as an important modulator of hormonal actions in this segment of the nephron.

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.

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.

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.

Microfluorometric studies of intracellular Ca2+ and Na+ concentrations in normal human labial gland acini.

The responses of human labial salivary acini to muscarinic, adrenergic, and substance P peptidergic stimulation were studied using the fluorescent indicators fura 2 for intracellular Ca2+ concentration ([Ca2+]i) and sodium-binding benzofuran isophthalate for intracellular Na+ concentration ([Na+]i). Of the agents tested (carbachol, epinephrine, isoproterenol, and substance P) only the muscarinic agonist carbachol increased [Ca2+]i substantially above basal levels (three to fourfold; half-maximal effect approximatley 1 microM). Experiments with the Ca(2+)-adenosinetriphosphatase inhibitor thapsigargin indicated the presence of both thapsigargin-sensitive and thapsigargin-insensitive intracellular Ca2+ stores, both of which were mobilized by carbachol. [Na+]i in resting labial acini was approximately 20 mM. On stimulation with carbachol, [Na+]i rose transiently to more than three times this value and then partially recovered. This carbachol-induced rise in [Na+]i was largely blocked by bumetanide, a specific inhibitor of the Na(+)-K(+)-2Cl- cotransporter. These results are consistent with an intact muscarinic fluid secretory response in human labial acini with transepithelial Cl- secretion driven via Na(+)-K(+)-2Cl- cotransport and the secretion of fluid presumably following Cl- loss via an apical Ca(2+)-dependent anion channel, as observed in salivary acini from other species.

Activation of the Na(+)-K(+)-2Cl- cotransporter in rat parotid acinar cells by aluminum fluoride and phosphatase inhibitors.

The bumetanide-sensitive component of pHi recovery from an NH4Cl-induced acute alkaline load was used as a measure of Na(+)-K(+)-2Cl- cotransport activity in rat parotid acini. Acinar treatment with NaF/AlCl3 (15 mM NaF plus 10 microM AlCl3) induced a 5-fold stimulation in the initial rate of bumetanide-sensitive pHi recovery. This effect was dependent on NaF concentration (K1/2 approximately 7 mM) and was blunted in the presence of the Al3+ chelator desferal mesylate suggesting that it might be due to the aluminofluoride ion, AlF-4. NaF/AlCl3 treatment did not increase acinar intracellular cAMP levels but did result in an increase in intracellular calcium concentration (from 87 +/- 5 to 181 +/- 2 nM) and in acinar cell shrinkage (12 +/- 1%). But the stimulation of the Na(+)-K(+)-2Cl- cotransporter by NaF/AlCl3 persisted in acini which had been depleted of their intracellular Ca2+ stores. In these acini no effect of NaF/AlCl3 on intracellular calcium or cell volume was observed, indicating that stimulation of the cotransporter was not secondary to either of these phenomena. The effect of NaF/AlCl3 on the cotransporter was blocked by the protein kinase inhibitor K252a indicating the involvement of a protein phosphorylation event. This result is consistent with either NaF/AlCl3-dependent protein kinase activation or phosphatase inhibition. The stimulation of the cotransporter by NaF/AlCl3 was mimicked by the protein phosphatase inhibitor calyculin A; however, this effect was not blocked by K252a suggesting that a different protein kinase from that associated with NaF/AlCl3 may be involved. The data indicate that the Na(+)-K(+)-2Cl- cotransporter in this tissue is under tight regulatory control, in all likelihood via multiple protein kinase/phosphatase systems. The physiological roles of these regulatory events in modulating acinar fluid secretion driven by the Na(+)-K(+)-2Cl- cotransporter remain to be elucidated.

Beta-adrenergic upregulation of the Na(+)-K(+)-2Cl- cotransporter in rat parotid acinar cells.

We used the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6')-carboxyfluorescein to monitor the recovery of the intracellular pH (pHi) of rat parotid acini from an NH4(+)-induced alkaline load. This recovery was markedly inhibited by the loop diuretic bumetanide and by Cl- removal, indicating that it is largely due to NH4+ entry via the basolateral Na(+)-K(+)-2Cl- cotransporter. The rate of recovery of pHi was enhanced threefold by pretreatment (37.5 s) with isoproterenol (K1/2 = 21.5 nM) or norepinephrine (in the presence of phentolamine), and blocked by the beta 1-specific antagonist atenolol, indicating an upregulation of cotransport activity by beta 1-adrenergic stimulation. The effect of isoproterenol was prevented by protein kinase inhibitors and mimicked by cAMP analogues, and by maneuvers known to increase cytosolic cAMP levels in these cells, consistent with the involvement of protein kinase A. Physiologically, such an upregulation of the acinar Na(+)-K(+)-2Cl- cotransporter would lead to an increase in acinar chloride uptake across the basolateral membrane, and consequently, an increase in overall chloride and fluid secretion. Prevention of this upregulation by beta-blockers and possibly by other commonly used clinical agents may account for the dry mouth and dry eyes experienced by some patients taking these medications.

cAMP-activated chloride channel in the basolateral membrane of the thick ascending limb of the mouse kidney.

The properties of an anion-selective channel observed in basolateral membranes of microdissected, collagenase-treated, cortical thick ascending limbs of Henle's loop from mouse kidney were investigated using patch-clamp single-channel recording techniques. In basal conditions, single Cl- currents were detected in 8% of cell-attached and excised, inside-out, membrane patches whereas they were observed in 24% of cell-attached and 67% of inside-out membrane patches when tubular fragments were preincubated with Forskolin (10(-5) M) or 8-bromo-cAMP (10(-4) M) and isobutylmethylxanthine (10(-5) M). The channel exhibited a linear current-voltage relationship with conductances of about 40 pS in both cell-attached and cell-free membrane configurations. A PNa+/PCl- ratio of 0.05 was estimated in the presence of a 142/42 mM NaCl concentration gradient applied to inside-out membrane patches. Anionic selectivity of the channel followed the sequence Cl- greater than Br- greater than NO3- much greater than F-; gluconate was not a permeant species. The open-state probability of the channel increased with membrane depolarization in cell-attached, i.e., in situ membrane patches. In excised, inside-out, membrane patches, the channel was predominantly open with the open-state probability close to 0.8 over the whole range of potentials tested (-60 to +60 mV). The channel activity was not a function of internal calcium concentration between 10(-9) and 10(-3) M. We suggest that this Cl- channel, whose properties are distinct from those in other epithelia, could account for the well-documented conductance which mediates Cl- exit in the basolateral step of NaCl absorption in thick ascending limb of Henle's loop.

A cation channel in the thick ascending limb of Henle's loop of the mouse kidney: inhibition by adenine nucleotides.

1. Patch-clamp single-channel current recordings were used to study the inhibition of Ca2+-activated non-selective cation channels by internal nucleotides in patches excised from basolateral membranes of the thick ascending limb of Henle's loop of the mouse kidney. 2. The application of ATP, ADP or AMP to the cytoplasmic face of excised inside-out membrane patches reduced the open-state probability of the channels (Po) in a dose-dependent way without effect upon the unitary current amplitude. Dose-response curves gave half-maximal inhibitory concentrations of 20, 21 and 2.5 microM for ATP, ADP and AMP, respectively, while the Hill coefficient was close to one in all three cases. 3. Cyclic AMP partially inhibited channel activity (Po = 35 +/- 17% of control) only at high, unphysiological concentrations (10(-3) M) while adenosine (10(-3) M) had very little effect (Po = 83 +/- 7% of control). 4. Replacement of adenine with other purines (guanine, hypoxanthine) or pyrimidine (uridine) bases very largely reduced inhibitory activity. Cyclic GMP had no effect. 5. Non-hydrolysable analogues of ATP, AMP-PNP (10(-3) M) and ATP-gamma-S (5 x 10(-4) M), were effective inhibitors of the channel (Po = 24 +/- 7 and 9 +/- 4% of control, respectively.