Rat Cortical Collecting Duct Research Articles

Nifedipine-activated Ca(2+) permeability in newborn rat cortical collecting duct cells in primary culture.

To characterize Ca(2+) transport in newborn rat cortical collecting duct (CCD) cells, we used nifedipine, which in adult rat distal tubules inhibits the intracellular Ca(2+) concentration ([Ca(2+)](i)) increase in response to hormonal activation. We found that the dihydropyridine (DHP) nifedipine (20 microM) produced an increase in [Ca(2+)](i) from 87.6 +/- 3.3 nM to 389.9 +/- 29.0 nM in 65% of the cells. Similar effects of other DHP (BAY K 8644, isradipine) were also observed. Conversely, DHPs did not induce any increase in [Ca(2+)](i) in cells obtained from proximal convoluted tubule. In CCD cells, neither verapamil nor diltiazem induced any rise in [Ca(2+)](i). Experiments in the presence of EGTA showed that external Ca(2+) was required for the nifedipine effect, while lanthanum (20 microM), gadolinium (100 microM), and diltiazem (20 microM) inhibited the effect. Experiments done in the presence of valinomycin resulted in the same nifedipine effect, showing that K(+) channels were not involved in the nifedipine-induced [Ca(2+)](i) rise. H(2)O(2) also triggered [Ca(2+)](i) rise. However, nifedipine-induced [Ca(2+)](i) increase was not affected by protamine. In conclusion, the present results indicate that 1) primary cultures of cells from terminal nephron of newborn rats are a useful tool for investigating Ca(2+) transport mechanisms during growth, and 2) newborn rat CCD cells in primary culture exhibit a new apical nifedipine-activated Ca(2+) channel of capacitive type (either transient receptor potential or leak channel).

Ca2+ mediates the effect of inhibition of Na+-K+-ATPase on the basolateral K+ channels in the rat CCD.

We investigated the effect of inhibiting Na+-K+-ATPase on the basolateral 18-pS K+ channel in the cortical collecting duct (CCD) of the rat kidney. Inhibiting Na+-K+-ATPase with strophanthidin decreased the activity of the 18-pS K+ channel and increased the intracellular Ca2+ to 420 nM. Removal of extracellular Ca2+ abolished the effect of strophanthidin. When intracellular Ca2+ was raised with 5 microM ionomycin or A-23187 to 300, 400, and 500 nM, the activity of the 18-pS K+ channel in cell-attached patches fell by 40, 85, and 96%, respectively. To explore the mechanism of Ca2+-induced inhibition, the effect of 400 nM Ca2+ on channel activity was studied in the presence of calphostin C, an inhibitor of protein kinase C, or KN-93 and KN-62, inhibitors of calmodulin-dependent kinase II. Addition of calphostin C or KN-93 or KN-62 failed to block the inhibitory effect of high concentrations of Ca2+ . This suggested that the inhibitory effect of high concentrations of Ca2+ was not mediated by protein kinase C or calmodulin-dependent kinase II pathways. To examine the possibility that the inhibitory effect of high concentrations of Ca2+ was mediated by the interaction of nitric oxide with superoxide, we investigated the effect of 400 nM Ca2+ on channel activity in the presence of 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron) or N(omega)-nitro-L-arginine methyl ester. Pretreatment of the tubules with 4,5-dihydroxy-1,3-benzenedisulfonic acid or N(omega)-nitro-L-arginine methyl ester completely abolished the inhibitory effect of 400 nM Ca2+ on channel activity. Moreover, application of 4,5-dihydroxy-1,3-benzenedisulfonic acid reversed the inhibitory effect of strophanthidin. We conclude that the effect of inhibiting Na+-K+-ATPase is mediated by intracellular Ca2+ and the inhibitory effect of high concentrations of Ca2+ is the result of interaction of nitric oxide with superoxide.

Cyclic AMP increases cell surface expression of functional Na,K-ATPase units in mammalian cortical collecting duct principal cells.

Cyclic AMP (cAMP) stimulates the transport of Na(+) and Na,K-ATPase activity in the renal cortical collecting duct (CCD). The aim of this study was to investigate the mechanism whereby cAMP stimulates the Na,K-ATPase activity in microdissected rat CCDs and cultured mouse mpkCCD(c14) collecting duct cells. db-cAMP (10(-3) M) stimulated by 2-fold the activity of Na,K-ATPase from rat CCDs as well as the ouabain-sensitive component of (86)Rb(+) uptake by rat CCDs (1.7-fold) and cultured mouse CCD cells (1.5-fold). Pretreatment of rat CCDs with saponin increased the total Na,K-ATPase activity without further stimulation by db-cAMP. Western blotting performed after a biotinylation procedure revealed that db-cAMP increased the amount of Na,K-ATPase at the cell surface in both intact rat CCDs (1.7-fold) and cultured cells (1.3-fold), and that this increase was not related to changes in Na,K-ATPase internalization. Brefeldin A and low temperature (20 degrees C) prevented both the db-cAMP-dependent increase in cell surface expression and activity of Na,K-ATPase in both intact rat CCDs and cultured cells. Pretreatment with the intracellular Ca(2+) chelator bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid also blunted the increment in cell surface expression and activity of Na,K-ATPase caused by db-cAMP. In conclusion, these results strongly suggest that the cAMP-dependent stimulation of Na,K-ATPase activity in CCD results from the translocation of active pump units from an intracellular compartment to the plasma membrane.

Identification of receptor ligands by screening phage-display peptide libraries ex vivo on microdissected kidney tubules.

A novel method to identify receptor ligands for defined renal tubular segments has been developed. Ex vivo screening of phage-display peptide libraries on isolated intact rat proximal convoluted tubules (PCT) and cortical collecting ducts (CCD) allowed the direct access of phage to the basolateral surface of tubular epithelial cells. Two distinct peptide motifs were selected for CCD and PCT, indicating differential expression of some membrane receptors on the basolateral surface of defined kidney tubule segments. Using the linear peptide motif ELRGD(R/M)AX(W/L), recovered from freshly isolated rat CCD, mediated 16-fold selectivity of phage binding to CCD compared with PCT. Binding to CCD was 39-fold higher than that of a random control phage. Binding and subsequent internalization of phage, most likely by an integrin-mediated endocytosis pathway, was abolished by the addition of the corresponding synthetic peptide. Furthermore, the results demonstrate that presentation and flanking amino acids determine the specific binding properties of RGD ligands to their putative integrin receptors. The results emphasize the need of a native cell system for the identification of renal epithelial cell surface ligands. Such ligands are of potential relevance for the analysis of interactions between extracellular matrix and kidney tubules or for the development of improved vectors for kidney-specific drug delivery or gene transfer.

Vasopressin Regulates Water Flow in a Rat Cortical Collecting Duct Cell Line Not Containing Known Aquaporins

Transepithelial water movements and arginine-vasopressin (AVP)-associated ones were studied in a renal cell line established from a rat cortical collecting duct (RCCD(1)). Transepithelial net water fluxes (J(w)) were recorded every minute in RCCD(1) monolayers cultured on permeable supports. Spontaneous net water secretion was observed, which was inhibited by serosal bumetanide (10(-5) m), apical glibenclamide (10(-4) m) and apical BaCl(2) (5 x 10(-3) m). RT-PCR, RNAse protection and/or immunoblotting experiments demonstrated that known renal aquaporins (AQP1, AQP2, AQP3, AQP4, AQP6 and AQP7) were not expressed in RCCD(1) cells. AVP stimulates cAMP production and sodium reabsorption in RCCD(1) cells. We have now observed that AVP significantly reduces the spontaneous water secretory flux. The amiloride-sensitive AVP-induced increase in short-circuit current (I(sc)) was paralleled by a simultaneous modification of the observed J(w): both responses had similar time courses and half-times (about 4 min). On the other hand, AVP did not modify the osmotically driven J(w) induced by serosal hypertonicity. We can conclude that: (i) transepithelial J(w) occurs in RCCD(1) cells in the absence of known renal aquaporins; (ii) the "water secretory component" observed could be linked to Cl- and K = secretion; (iii) the natriferic response to AVP, preserved in RCCD(1) cells, was associated with a change in net water flux, which was even observed in absence of AQP2, AQP3 or AQP4 and (iv) the hydro-osmotic response to AVP was completely lost.

Protein-tyrosine Phosphatase Reduces the Number of Apical Small Conductance K+ Channels in the Rat Cortical Collecting Duct

Previous studies have demonstrated that an increase in the activity of protein-tyrosine kinase (PTK) is involved in the down-regulation of the activity of apical small conductance K(+) (SK) channels in the cortical collecting duct (CCD) from rats on a K(+)-deficient diet (). We used the patch clamp technique to investigate the role of protein-tyrosine phosphatase (PTP) in the regulation of the activity of SK channels in the CCD from rats on a high K(+) diet. Western blot analysis indicated that PTP-1D is expressed in the renal cortex. Application of 1 microm phenylarsine oxide (PAO) or 1 mm benzylphosphonic acid, agents that inhibit PTP, reversibly reduced channel activity by 95%. Pretreatment of CCDs with PAO for 30 min decreased the mean NP(o) reversibly from control value 3.20 to 0.40. Addition of 1 microm herbimycin A, an inhibitor of PTK, had no significant effect on channel activity in the CCDs from rats on a high K(+) diet. However, herbimycin A abolished the inhibitory effect of PAO, indicating that the effect of PAO is the result of interaction between PTK and PTP. Addition of brefeldin A, an agent that blocks protein trafficking from Golgi complex to the membrane, had no effect on channel activity. Moreover, application of colchicine, a microtubule inhibitor, or paclitaxel, a microtubule stabilizer, had no effect on channel activity. In contrast, PAO still reduced channel activity in the presence of brefeldin A, colchicine, or paclitaxel. Furthermore, the effect of PAO on channel activity was absent when the tubules were bathed in 16% sucrose-containing bath solution or treated with concanavalin A. We conclude that PTP is involved in the regulation of the activity of SK channels and that inhibition of PTP may facilitate the internalization of the SK channels.

Dopamine D4 receptor isoform mRNA and protein are expressed in the rat cortical collecting duct.

We reported previously [Am. J. Physiol. 271 (Renal Fluid Electrolyte Physiol. 40): F391-F400, 1996] that dopamine inhibits vasopressin (AVP)-dependent water permeability and Na+ transport in the rat cortical collecting duct (CCD) apparently through a D4 dopamine receptor. The present experiments used RT-PCR of total RNA extracted from microdissected rat CCD to determine whether the D4 and D1A dopamine receptor isoforms are expressed. Specific primers were used to amplify three regions of the D4 cDNA. All three gave products with 98-100% nucleotide identity to the known rat D4 sequence; however, there was an extra 6-bp insert at the 3' end of the second transmembrane region that was identical to the human and mouse sequences but which had not been documented in the rat sequence. D4 receptor protein was also localized exclusively to the CCD and medullary collecting ducts by immunohistochemistry. Two regions of the D1A dopamine receptor message were also amplified by RT-PCR of RNA from rat CCD and were verified by sequencing and immunohistochemistry. We conclude that both D4 and D1A dopamine receptors are expressed in the rat CCD, but the physiological effects are attributable to a D4 receptor.

Neuronal nitric oxide synthase is expressed in principal cell of collecting duct.

We used the RT-PCR technique and immunocytochemical methods to determine the expression of endothelial nitric oxide synthase (eNOS) or neuronal nitric oxide synthase (nNOS) in the cortical collecting duct (CCD) in rats on high-K+ diet. The microdissected CCDs of the rat kidney were lysed, and RT-PCR was carried out using rat nNOS and eNOS gene-specific primers. Southern analysis showed the presence of mRNA of nNOS but not eNOS in the CCD. The presence of nNOS in the CCD was further confirmed by light microscopy. We used the polyclonal nNOS antibody in immunocytochemical studies of the isolated CCD. We found that immunoreactivity to nNOS was present in the CCD and heterogeneous with positive and negative immunostaining. We performed the immunocytochemical studies in the split-open CCD and found that the immunoreactivity to nNOS was detected only in principal cells but not in intercalated cells. We conclude that nNOS is expressed in the rat CCD in rats on high-K+ diet. The presence of nNOS in the CCD is heterogeneous and mainly located in principal cells.

Reaction of nitric oxide with superoxide inhibits basolateral K+ channels in the rat CCD.

We previously demonstrated that nitric oxide (NO) stimulates the basolateral small-conductance K+ channel (SK) via a cGMP-dependent pathway [M. Lu and W. H. Wang. Am. J. Physiol. 270 (Cell Physiol. 39): C1336-C1342, 1996]. Because NO at high concentration has been shown to react with superoxide (O-2) to form peroxynitrite (OONO-) [W. A. Pryor and G. L. Squadrito. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L699-L722, 1995 and M. S. Wolin. Microcirculation 3: 1-17, 1996], we extended our study to examine, using patch-clamp technique, the effect of high concentrations of NO on SK in cortical collecting duct (CCD) of rat kidney. Addition of NO donors [100-200 microM S-nitroso-N-acetyl-penicillamine (SNAP) or sodium nitroprusside (SNP)] reduced channel activity, defined as the product of channel number and open probability, to 15 and 25% of the control value, respectively. The inhibitory effect of NO was completely abolished in the presence of 10 mM Tiron, an intracellular scavenger of O-2. NO donors, 10 microM SNAP or SNP, which stimulate channel activity under control conditions, can also inhibit SK in the presence of an O-2 donor, pyrogallol, or in the presence of an inhibitor of superoxide dismutase, diethyldithiocarbamic acid. The inhibitory effect of NO is still observed in the presence of exogenous cGMP, suggesting that the NO-induced inhibition is not the result of decreased cGMP production. We conclude that the inhibitory effect of NO on channel activity results from an interaction between NO and O-2.

Nitric oxide links the apical Na+ transport to the basolateral K+ conductance in the rat cortical collecting duct.

We have used the patch clamp technique to study the effects of inhibiting the apical Na+ transport on the basolateral small-conductance K+ channel (SK) in cell-attached patches in cortical collecting duct (CCD) of the rat kidney. Application of 50 microM amiloride decreased the activity of SK, defined as nPo (a product of channel open probability and channel number), to 61% of the control value. Application of 1 microM benzamil, a specific Na+ channel blocker, mimicked the effects of amiloride and decreased the activity of the SK to 62% of the control value. In addition, benzamil reduced intracellular Na+ concentration from 15 to 11 mM. The effect of amiloride was not the result of a decrease in intracellular pH, since addition 50 microM 5-(n-ethyl-n-isopropyl) amiloride (EIPA), an agent that specifically blocks the Na/H exchanger, did not alter the channel activity. The inhibitory effect of amiloride depends on extracellular Ca2+ because removal of Ca2+ from the bath abolished the effect. Using Fura-2 AM to measure the intracellular Ca2+, we observed that amiloride and benzamil significantly decreased intracellular Ca2+ in the Ca2+-containing solution but had no effect in a Ca2+-free bath. Furthermore, raising intracellular Ca2+ from 10 to 50 and 100 nM with ionomycin increased the activity of the SK in cell-attached patches but not in excised patches, suggesting that changes in intracellular Ca2+ are responsible for the effects on SK activity of inhibition of the Na+ transport. Since the neuronal form of nitric oxide synthase (nNOS) is expressed in the CCD and the function of the nNOS is Ca2+ dependent, we examined whether the effects of amiloride or benzamil were mediated by the NO-cGMP-dependent pathways. Addition of 10 microM S-nitroso-n-acetyl-penicillamine (SNAP) or 100 microM 8-bromoguanosine 3':5'-cyclic monophosphate (8Br-cGMP) completely restored channel activity when it had been decreased by either amiloride or benzamil. Finally, addition of SNAP caused a significant increase in channel activity in the Ca2+-free bath solution. We conclude that Ca2+-dependent NO generation mediates the effect of inhibiting the apical Na+ transport on the basolateral SK in the rat CCD.

Receptors for bradykinin and prostaglandin E coupled to Ca 2+ signalling in rat cortical collecting duct

In freshly isolated rat cortical collecting ducts (CCD) we measured intracellular Ca 2+ activity ([Ca 2+] i) with the Fura-2 method. Bradykinin (BK) induced a transient and biphasic increase in ([Ca 2+] i). This increase was concentration dependent and was half maximal at a concentration of 15 nM. The B 2 receptor antagonist HOE 140 (100 nM, n = 6) completely abolished BK (100 nM) induced increase in ([Ca 2+] i). The B, receptor agonist des-Arg 9-bradykinin (100 nM, n = 4) had no effect on ([Ca 2+] i). In the absence of extracellular Ca 2+, the maximal increase in [Ca 2+] i induced by BK was diminished and the secondary plateau phase was completely abolished. Prostaglandin E 2 (PGE 2) elevated ([Ca 2+] i) also concentration-dependently and biphasically. A half maximal effect was reached with 1 nM PGE 2. The secondary plateau phase was absent when extracellular Ca 2+ was removed. Sulprostone (100 nM, n = 6) mimicked the PGE 2 (100 nM) induced increase in ([Ca 2+] i). The effect of BK (100 nM) on [Ca 2+] i was not inhibited by the cyclooxygenase inhibitor indomethacin (10 μM, n = 5). Dopamine (1 μM, n = 4) did not significantly alter ([Ca 2+] i). BK and PGE 2 regulate [Ca 2+] i in the rat CCD via release of Ca 2+ from intracellular Ca 2+ stores as well as via Ca 2+ influx from extracellular space. BK directly modulates [Ca 2+] i through B 2 receptors. EP 1 receptors are most likely to be responsible for the PGE 2 induced increase in [Ca 2+] i.

Moxonidine inhibits Na+/H+ exchange in proximal tubule cells and cortical collecting duct

The imidazoline receptor agonist moxonidine has been recently introduced as an antihypertensive therapy. Imidazoline specific binding sites have also been found in the kidney. Moxonidine induced natriuresis and diuresis in clearance studies in rats. Related substances such as various guanidinium derivatives have been shown to inhibit Na+/H+ exchange in several preparations. We therefore examined whether the renal effects of moxonidine could be mediated by an inhibition of the Na+/H+ exchanger. Intracellular pH (pHi) was measured microfluorimetrically with BCECF in proximal LLC-PK1 cells and in the principal cells of rat cortical collecting ducts (CCD). In LLC-PK1 cells moxonidine (10 mumol/liter) had no effect on the basal pH1; however, it reduced the Na+/H+ activity reversibly by 43 +/- 4% (N = 26) when the exchanger was activated by cellular acidification. In rat CCD cells moxonidine slightly decreased basal pHi by 0.08 +/- 0.03 pH units (N = 12). After acidification the recovery rate of pHi was reduced with moxonidine by 45 +/- 6% (N = 18). The effects of moxonidine could be mimicked in both cell types by inhibitors of the Na+/H+ exchanger (HOE 694, amiloride). In the presence of the imidazoline receptor antagonist idazoxan (10 mumol/liter) the effects of moxonidine were almost completely inhibited. The alpha 2-antagonist yohimbine (10 mumol/liter) did not significantly alter the effects of moxonidine in both cell types. These data suggest that in LLC-PK1 and in rat CCD cells, Na+/H+ is inhibited by moxonidine via an activation of the imidazoline receptor.

Nitric oxide-induced hyperpolarization stimulates low-conductance Na+ channel of rat CCD.

We used the patch-clamp technique in the split-open cortical collecting duct (CCD) to investigate the effect of nitric oxide (NO) on the low-conductance (6-pS) Na+ channel that can be blocked by 1 microM amiloride. We confirmed that the number of Na+ channels increased significantly in CCDs of rats on a low-Na+ diet (17). Application of 100 microM N(G)-nitro-L-arginine methyl ester (L-NAME), an agent that blocks endogenous NO synthase, reduced NPo [the product of channel number (N) and open probability (Po)] to 45% of the control value. The effect of L-NAME was specific, since addition of D-NAME, which does not inhibit NO synthase, did not change the activity of the Na+ channel. That the effect of L-NAME results from inhibition of NO synthase is further confirmed by experiments in which addition of an exogenous NO donor, either 10 microM S-nitroso-N-acetyl penicillamine or sodium nitroprusside (SNP), restored the Na+ channel activity when it had been blocked by L-NAME. The action of NO involves a guanosine 3',5'-cyclic monophosphate (cGMP)-dependent pathway, since 100 microM 8-bromo-cGMP (8-BrcGMP) mimicked the effect of SNAP on K+ channels. However, 100 microM 8-BrcGMP did not alter the activity of Na+ channels in inside-out patches, suggesting an indirect action. Because the Na+ channel is activated by hyperpolarization (19) and NO stimulates basolateral K+ channels (16), we tested whether hyperpolarization mediated the effect of NO. In perforated whole cell recordings, addition of L-NAME depolarized the cell membrane from -73 to 51 mV, and application of 10 microM SNP repolarized the membrane to -68 mV. Furthermore, the L-NAME-induced decrease in NPo was effectively restored by 25 mV hyperpolarization of the patch membranes, and addition of 2 mM Ba2+ also abolished the effect of L-NAME. We concluded that the stimulatory effect of NO on the Na+ channel is an indirect effect mediated by a NO-induced increase of basolateral K+ conductance.

Effects of sodium nitroprusside in the rat cortical collecting duct are independent of the NO pathway

Recently we described K+ channels in the basolateral membrane of principal cells of rat cortical collecting duct (CCD) which are regulated by a cGMP-dependent protein kinase (Pflugers Arch 429:338-344, 1995). We examined the effects of the NO-liberator sodium nitroprusside (SNP) on single channel activity and membrane voltage (Vm) in principal cells of rat CCD, and on transepithelial voltage, lumen-to-bath Na+ fluxes, and osmotic water permeability in isolated perfused rat CCD tubules. While in patch clamp experiments SNP (10 microM) hyperpolarized principal cells from -54 +/- 10 mV to -71 +/- 5 mV (N = 5) and increased the activity of the described K+ channels from 0.05 +/- 0.03 to 0.45 +/- 0.14 (N = 5) in cell-attached and from 0.04 +/- 0.02 to 0.25 +/- 0.05 (N = 4) in excised patch clamp experiments, it had no effect on basal or AVP-dependent transepithelial voltage, Na+ fluxes, or the osmotic water permeability. In addition, neither 50 microM SIN-1, another liberator of NO, nor 1 mM L-NAME, an inhibitor of the NO-synthase, changed Vm significantly. Furthermore, in cGMP-assays SNP failed to increase intracellular cGMP in CCD segments. Thus, we conclude that in the rat CCD transport is not regulated via the NO-pathway and that SNP acts as an cGMP independent activator of K+ channels in the basolateral membrane of these cells.

Endothelial hypoxic stress proteins

Top of pageAbstract Moxonidine inhibits Na+/H+ exchange in proximal tubule cells and cortical collecting duct. The imidazoline receptor agonist moxonidine has been recently introduced as an antihypertensive therapy. Imidazoline specific binding sites have also been found in the kidney. Moxonidine induced natriuresis and diuresis in clearance studies in rats. Related substances such as various guanidinium derivatives have been shown to inhibit Na+/H+ exchange in several preparations. We therefore examined whether the renal effects of moxonidine could be mediated by an inhibition of the Na+/H+ exchanger. Intracellular pH (pHi) was measured microfluorimetrically with BCECF in proximal LLC-PK1 cells and in the principal cells of rat cortical collecting ducts (CCD). In LLC-PK1 cells moxonidine (10 mol/liter) had no effect on the basal pHi; however, it reduced the Na+/H+ activity reversibly by 43 4% (N = 26) when the exchanger was activated by cellular acidification. In rat CCD cells moxonidine slightly decreased basal pHi by 0.08 0.03 pH units (N = 12). After acidification the recovery rate of pHi was reduced with moxonidine by 45 6% (N = 18). The effects of moxonidine could be mimicked in both cell types by inhibitors of the Na+/H+ exchanger (HOE 694, amiloride). In the presence of the imidazoline receptor antagonist ida-zoxan (10 mol/liter) the effects of moxonidine were almost completely inhibited. The 2antagonist yohimbine (10 mol/liter) did not significantly alter the effects of moxonidine in both cell types. These data suggest that in LLC-PK1 and in rat CCD cells, Na+/H+ is inhibited by moxonidine via an activation of the imidazoline receptor. Keywords: moxonidine, hypertension, Na+/H+ exchange, blood pressure control, imidazoline-receptor agonist, antihypertensive drugs

Cell Shrinkage Activates a Cation Conductance in Principal Cells of Rat Cortical Collecting Duct

We examined whether a nonselective cation (NSC) conductance is activated in the freshly isolated rat cortical collecting duct (CCD) by osmotically induced cell shrinkage. Membrane voltages (Vm), whole cell conductances (Gc) and single-channel currents were measured with the patch-clamp method. Cellular Ca2+ ([Ca2+]i) and Na+ ([Na+]i) activities were estimated fluorimetrically with fura-2 and sodium-binding benzofuran isophthalate. Increasing extracellular osmolality by 50 or 100 mosm/1 depolarized Vm by 6 ± 1 (n = 26) and 11 ± 3 mV (n = 12), respectively. Gc was increased under these conditions by 10 ± 7 (n = 9) and 46 ± 17% (n = 6), respectively. The depolarization was partly inhibited by either amiloride (n = 6) or Gd3+ (n = 4, each 10 µmol/l) and completely inhibited by amiloride plus Gd3+ (n = 13) or removal of Na+ (n = 6). Cell-attached NSC channels could neither be observed under control conditions nor after increasing osmolality. [Ca2+]i did not significantly change upon increasing osmolality by 50 mosm/1 (n = 11) and decreased slightly by 15 ± 3 nmol/l (n = 11) when the osmolality was increased by 100 mosm/1. [Na+]i was significantly increased upon increasing osmolality (+100 mosm/1) by 14 ± 2 mmol/l (n = 23). This increase in [Na+]i was reduced to 8 ± 2 mmol/l (n = 9) in the presence of amiloride (10 µmol/ 1), to 11 ± 3 mmol/l (n = 8) in the presence of Gd3+ (10 µmol/l) and to 6 ± 1 mmoI/1 (n = 7) with both inhibitors. These data indicate that osmotically induced cell shrinkage activates a NSC conductance in rat CCD cells.

Ca<sup>2+</sup> Entry in Isolated Rat Cortical Collecting Duct Is pH- and Voltage-Sensitive

We studied effects of extracellular pH (pH₀), voltage and Ca2+-channel blockers on the basal and swelling-induced increase in intracellular Ca2+ ([Ca2+]i) activity of isolated rat cortical collecting duct (CCD) segments. [Ca2+]i and membrane voltages (Vm) were measured using the fura-2 method and the slow-whole-cell patch-clamp technique, respectively. Fura-2 fluorescence ratio (FR) at a pHo of 7.4 was 1.04 ± 0.02 (n = 121), corresponding to a calculated [Ca2+]i of 97 ± 6 nM. Depolarization of Vm by changes in extracellular K+ reduced FR (n = 6), and hyperpolarization of Vm increased FR (n = 6). Lowering pHo to 6.0 decreased FR by 0.07 ± 0.01 (n = 20), elevation of pHo to 8.0 induced an increase in FR by 0.14 ± 0.01 (n = 67) exclusively via Ca2+ influx. Ca2+ influx showed an increased Mn2+ quench of fura-2 fluorescence with alkaline pHo and a decreased quench with acidic pHo. The alkaline pHo-induced increase in FR was completely abolished by Ni2+ (5 mM, n = 6) and La3+ (10 µM, n = 8) and partially by Gd3+ (10 µM, n = 7). Cell swelling induced by a decrease in extracellular osmolality by 130 mosm led to an increase in FR of 0.46 ± 0.05 (n = 20). The swelling-induced increase was reduced at a pHo of 6.0 to 0.27 ± 0.06 (n = 10) and enhanced at pHo of 8.0 to 0.57 ± 0.10 (n = 8). Ni2+ (n = 6) and Gd3+ (n = 7) had no, and La3+ (n = 8) only a partial inhibitory effect on the swelling-induced increase in FR. Ni2+ quenched fura-2 fluorescence during cell swelling. Verapamil diminished the basal and swelling-activated Ca2+ influx only at a concentration > 1 µM. Neither nifedipine (1 µM, n = 7) nor nimodipine (1 µM, n = 5) had any effect on the swelling-induced increase in FR. In rat CCD [Ca2+]i is sensitive to pHo and Vm. Basal and cell swelling-induced Ca2+ influx is apparently mediated via different pathways. L-type Ca2+ channels are most likely not responsible for this Ca2+ permeability.

CGMP-activating peptides do not regulate electrogenic electrolyte transport in principal cells of rat CCD.

K+ channels in the basolateral membrane of rat cortical collecting duct (CCD) are regulated by a cGMP-dependent protein kinase (J. Hirsch and E. Schlatter. Pfluegers Arch. 429: 338-344, 1995). Conflicting data exist on the effects of cGMP-activating agonists on Na+ transport in these cells. Thus we tested members of the family of peptides that increase intracellular cGMP [cardiodilatin/atrial natriuretic peptide (CDD/ANP), brain natriuretic peptide, C-type natriuretic peptide, urodilatin, guanylin, and uroguanylin], as well as bradykinin +/- CDD/ANP on membrane voltages (Vm) of principal cells of isolated rat CCD using the slow whole cell patch-clamp technique (E. Schlatter, U. Fröbe, and R. Greger. Pfluegers Arch. 421: 381-387, 1992). None of the agonists tested changed Vm significantly. There was also no effect of dibutyryl guanosine 3',5'-cyclic monophosphate (DBcGMP) on AVP-dependent lumen-to-bath Na+ flux, transepithelial voltage, or osmotic water permeability in isolated perfused rat CCD. Finally, CDD/ANP increased intracellular cGMP only in glomeruli but not in CCD. Thus the findings provide no evidence for control of electrogenic electrolyte transport by these natriuretic peptides in principal cells of rat CCD, and the agonist that physiologically regulates the cGMP-dependent K+ channels remains to be identified.

Dopamine inhibits AVP-dependent Na+ transport and water permeability in rat CCD via a D4-like receptor

We studied the receptor responsible for dopamine action in isolated perfused cortical collecting ducts (CCD) from rats treated with deoxy-corticosterone. (Critical experiments were repeated in CCD from untreated rats with the same results.) At doses > or = 1 microM, dopamine inhibited arginine vasopressin (AVP)-dependent Na+ and water transport (measured by the unidirectional lumen-to-bath 22Na+ flux and the transepithelial voltage) and osmotic water permeability (Pf). The effects of dopamine were not reversed by the dopamine-1 (D1) antagonist SCH-23390, and no inhibition was produced by the D1 agonists fenoldopam or SKF-81247. When Na+ transport and Pf were stimulated with 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate plus 3-isobutyl-1-methylxanthine, dopamine did not inhibit, suggesting a "D2-type" receptor. However, the D2 agonist quinpirole had no effect on the AVP-dependent transepithelial voltage (VT), and the D2 and D3 antagonists domperidone and pimozide did not reverse dopamine inhibition of VT. The only agent tested that reversed the effects of dopamine was the D4-specific antagonist clozapine. We conclude that dopamine inhibition of salt and water transport in the CCD is mediated by a D4-like receptor.

Characteristics of a rat cortical collecting duct cell line that maintains high transepithelial resistance

This study describes the establishment of a rat kidney cortical collecting duct (CCD) clonal cell line (RCCD1 cells) that maintains high transepithelial resistance and specific hormonal sensitivities. Immortalized cells were obtained by infection of primary cultured CCD cells with the wild-type simian virus 40. Grown on Petri dishes, RCCD1 cells are organized as monolayers of cuboid cells separated by tight junctions and form domes. Grown on permeable filters, confluent RCCD1 cells exhibit high transepithelial resistance (Rt: 2390 +/- 140 omega. cm2), transepithelial potential difference (PD) of -10.5 +/- 1.2 mV lumen negative, an associated short-circuit current (Isc) of 4.3 +/- 0.5 microA/cm2, and generated significant Na+, K+, H+ and HCO3- gradients, reflecting Na+ and H+ reabsorption and K+ and HCO3- secretion. RCCD1 cells exhibit features of both principal (PC) and intercalated (IC) cells. Consistent with PC phenotype, about 50% of the cells were positively stained by a PC-specific agglutinin. In situ hybridization studies revealed the presence of alpha, beta and gamma subunit mRNAs of the amiloride-sensitive epithelial Na+ channel and alpha 1 and beta 1 subunits of Na(+)-K(+)-ATPase. Moreover, Na(+)-K(+)-ATPase was immunolocalized at the basolateral side of the cells. Arginine vasopressin (AVP) induced a significant increase in both cellular cAMP content and Isc. Amiloride decreased in a dose-dependent manner Isc from untreated and AVP-treated RCCD1 cells. In addition, a barium-sensitive K+ conductance was evidenced in the apical side of the cells. Consistent with IC phenotype, isoproterenol (ISO) provoked a large increase in cellular cAMP and stimulated Isc. The effect of ISO on Isc was blocked by 5 x 10(-3) M DPC, a chloride channel blocker. Finally, AVP plus ISO had additive effect on Isc. Taken together, these results provide evidence that the RCCD1 cell line has maintained many of the original properties of rat CCD from which they were derived.