Paradoxical effects of pyrazinoate and nicotinate on urate transport in dog renal microvillus membranes.

The effects of pyrazinoate and nicotinate on urate transport in microvillus membrane vesicles isolated from canine renal cortex were evaluated. An outwardly directed gradient of pyrazinoate stimulated uphill urate accumulation, suggesting urate-pyrazinoate exchange. An inside-alkaline pH gradient stimulated uphill pyrazinoate accumulation, which suggested pyrazinoate-OH- exchange. Pyrazinoate-OH- exchange and urate-OH- exchange were similarly sensitive to inhibitors, implying that both processes occur via the same transport system. In addition, an inward Na+ gradient stimulated uphill pyrazinoate accumulation, suggesting Na+-pyrazinoate cotransport. Inhibitor studies demonstrated that Na+-pyrazinoate cotransport takes place via the same pathway that mediates Na+-lactate cotransport in these membrane vesicles. Previously we found that urate does not share this Na+-dependent cotransport pathway. Nicotinate inhibited transport of pyrazinoate by the anion exchange pathway and the Na+ cotransport pathway, suggesting that it is a substrate for both transport systems. Finally, in the presence of an inward Na+ gradient, low doses of pyrazinoate or nicotinate stimulated urate uptake, and higher doses of pyrazinoate or nicotinate inhibited urate accumulation, thereby mimicking in vitro the paradoxical effects of drugs on renal urate excretion that have been observed in vivo. These findings indicate that the paradoxical effect of uricosuric drugs at low doses to cause urate retention may result at least in part from stimulation of urate reabsorption across the luminal membrane of the proximal tubular cell.

Atrial natriuretic peptide modulates cystic fibrosis transmembrane conductance regulator chloride channel expression in rat proximal colon and human intestinal epithelial cells.

The cystic fibrosis transmembrane conductance regulator (CFTR) is one of the most intensively investigated Cl- channels. Different mutations in the CFTR gene cause the disease cystic fibrosis (CF). CFTR is expressed in the apical membrane of various epithelial cells including the intestine. The major organ affected in CF patients is the lung, but it also causes an important dysfunction of intestinal ion transport. The modulation of CFTR mRNA expression by atrial natriuretic peptide (ANP) was investigated in rat proximal colon and in human intestinal CaCo-2 cells by RNase protection assay and semi-quantitative reverse transcriptase PCR techniques. Groups of rats subjected to volume expansion or intravenous infusion of synthetic ANP showed respective increases of 60 and 50% of CFTR mRNA expression in proximal colon. CFTR mRNA was also increased in cells treated with ANP, reaching a maximum effect at 10(-9) M ANP, probably via cGMP. ANP at 10(-9) M was also able to stimulate both the CFTR promoter region (by luciferase assay) and protein expression in CaCo-2 cells (by Western blot and immunoprecipitation/phosphorylation). These results suggested the involvement of ANP, a hormone involved with extracellular volume, in the expression of CFTR in rat proximal colon and CaCo-2 intestinal cells.

Gates of Janus: cystic fibrosis and diarrhea.

Heat-stable enterotoxin, produced by Escherichia coli, binds to particulate guanylate cyclase to increase cyclic GMP in intestinal cells. This in turn stimulates the cyclic-GMP- or cyclic-AMP-dependent protein kinase, activating the same chloride channel that is defective in cystic fibrosis. It is possible that the relatively high prevalence of cystic fibrosis in humans results from its protective effect against diarrhea.

Thiazide diuretics affect osteocalcin production in human osteoblasts at the transcription level without affecting vitamin D3 receptors.

Besides their natriuretic and calciuretic effect, thiazide diuretics have been shown to decrease bone loss rate and improve bone mineral density. Clinical evidence suggests a specific role of thiazides on osteoblasts, because it reduces serum osteocalcin (OC), an osteoblast-specific protein, yet the mechanisms implicated are unknown. We therefore investigated the role of hydrochlorothiazide (HCTZ) on OC production by the human osteoblast-like cell line MG-63. HCTZ dose-dependently (1-100 microM) inhibited 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-induced OC release by these cells (maximal effect, -40-50% and p < 0.005 by analysis of variance [ANOVA]) as measured by ELISA. This effect of HCTZ on OC release was caused by a direct effect on OC gene expression because Northern blot analysis revealed that OC messenger RNA (mRNA) levels were reduced in the presence of increasing doses of the diuretic (-47.2+/-4.0%; p < 0.0001 by paired ANOVA with 100 microM 13.6+/-0.49 pmol/mg protein/15 minutes; p < 0.05) in MG-63 cells. Reducing extracellular Ca2+ concentration with 0.5 mM EDTA or 0.5 mM ethylene glycol-bis(beta-amino ethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) only partly prevented the inhibitory effect of the diuretic on OC secretion (maximal effect, -22.5+/-6.9%), suggesting that thiazide-dependent Ca2+ influx is not sufficient to elicit the inhibition of OC secretion. Because OC production is strictly dependent on the presence of 1,25(OH)2D3 in human osteoblasts, we next evaluated the possible role of HCTZ on vitamin D3 receptors (VDR) at the mRNA and protein levels. Both Northern and Western blot analyses showed no effect of HCTZ (1-100 microM) on VDR levels. The presence of EGTA in the culture media reduced slightly the VDR mRNA levels under basal condition but this was not modified in the presence of increasing levels of HCTZ. The OC gene promoter also is under the control of transcription factors such as Yin Yang 1 (YY1) and cFOS. Western blot analysis revealed no changes in YY1 levels in response to HCTZ either in the presence or in the absence of 0.5 mM EGTA in the culture media. In contrast, HCTZ induced a dose-dependent increase in cFOS levels (p < 0.002 by ANOVA), a situation prevented by incubation with EGTA. These studies indicate that HCTZ inhibits OC mRNA expression independently of an effect on VDR, YY1, or extracellular Ca2+ levels but involves changes in cFOS levels. As OC retards bone formation/mineralization, the inhibition of OC production by HCTZ could explain its preventive role in bone loss rate.

Influence of aluminum on the regulation of PTH- and 1,25(OH)2D3-dependent pathways in the rat osteosarcoma cell line ROS 17/2.8.

The role of hormonal status in the development of aluminum (Al)-dependent renal osteodystrophy, which is characterized by reduced bone matrix deposition, still remains largely unknown. To address this question, we used the osteoblast-like osteosarcoma cell line ROS 17/2.8 to evaluate the role of Al on parathyroid hormone (PTH)- and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-dependent activities in these cells. Al (1 microM) caused an inhibition of basal and 1,25(OH)2D3-induced alkaline phosphatase, but only at low doses (< 1 nM) of the steroid. Al partly inhibited basal osteocalcin (OC) secretion in ROS cells (p < 0.001), and the dose-dependent increase in 1,25(OH)2D3-induced OC release by these cells was also reduced by 1 microM Al at low concentrations of the steroid (< or = 1 nM), whereas high doses of 1,25(OH)2D3 (> or = 5 nM) totally prevented the inhibiting effects of Al. Al also had strong inhibitory actions on PTH-dependent cAMP production by ROS cells over the concentration range tested (0.5-50 nM). This inhibitory action of Al was also observed for PTH-related peptide- (PTHrp, 50 nM) but not for Isoproterenol-dependent (100 nM) cAMP formation. To evaluate more fully the mechanism of this inhibition of cAMP formation, we investigated the effect of Al on toxin-modulated, G protein-dependent regulation of cAMP formation and on the activation of adenylate cyclase by Forskolin. Cholera toxin (CT, 10 micrograms/ml), applied to cells for 4 h prior to PTH challenge, enhanced cAMP production about 2-fold above PTH alone (p < 0.001), a process that was further stimulated by Al. Pertussis toxin (PT, 1 microgram/ml, 4 h) did not modify basal PTH-dependent cAMP formation by ROS cells. However, PT treatment prevented the inhibitory effect of Al on cAMP formation by these cells (p < 0.025). The stimulation of adenylate cyclase by Forskolin (0.1 and 1 microM), which bypasses G protein regulation, was not modified by Al, indicating that Al does not affect adenylate cyclase directly. Northern blot analysis of PTH receptor mRNA levels showed that Al did not modify PTH receptor message in ROS cells. Likewise, Western blot analyses of G protein subunits showed that Al did not significantly alter Gs alpha subunit levels, in accordance with the results obtained for cAMP-dependent formation in response to CT. In contrast, Gi alpha-1 and Gi alpha-2 subunits were decreased by Al treatment, consistent with PT-restricted increases in cAMP formation in Al-treated ROS cells. Taken together, these results suggest that Al has multiple actions in osteoblast-like ROS cells. The effects of Al are modulated by hormonal control of the pathways investigated. Al affects 1,25(OH)2D3-regulated functions only when this steroid is low. Al has large inhibitory effects on PTH- and PTHrp-dependent cAMP formation. This last feature is related to the ability of Al to alter the G protein transducing pathway for PTH/PTHrp-dependent formation of cAMP since it does not affect adenylate cyclase activity directly and does not affect the PTH receptor message level. Thus, Al has stronger deleterious effects in osteoblast-like cells with an already compromised 1,25(OH)2D3 status and can modulate specifically PTH/PTHrp-mediated cAMP formation at the postreceptor level.

Cardiac L-type calcium channel alpha 1-subunit is increased by cyclic adenosine monophosphate: messenger RNA and protein expression in intact bone.

L-type calcium channels have been identified previously in both osteoblast-like osteosarcoma cell lines and primary cultures of osteoblasts using numerous techniques such as patch clamp recording, drug inhibited 45Ca2+ uptake, and Fura-2 measurements, but intact bone has not been investigated. Using reverse-transcription polymerase chain reaction (RT-PCR) we found that the three major isoforms of the alpha 1-subunit of L-type calcium channels, (alpha 1C, alpha 1D, and alpha 1S) are present in RNA extracted from ROS 17/2.8 osteosarcoma cells, rat femur, and rat skull. Sequencing of most of the alpha 1C-subunit from rat femur and ROS cells revealed that the splice variants in osteosarcoma cells and intact bone differ, but there are no unique sequence variations compared with those found in other tissues. Northern blot analysis of ROS cell RNA indicated that cyclic adenosine monophosphate (cAMP), but not 1 alpha, 25-dihydroxyvitamin D3, increased the messenger RNA (mRNA) of the alpha 1C-subunit. Western blot of ROS cell lysates revealed a band of more then 220 kDa, the amount of which increased in cells treated with cAMP. Using confocal microscopy combined with immunohistochemistry in ROS cells, intact bone, and cartilage, we found that the alpha 1C-subunit of this channel is expressed in osteoblasts and chondrocytes suggesting this channel may be a pathway for signal transduction in intact tissue, because it is in osteosarcoma cell lines and primary osteoblasts grown in tissue culture.

1 alpha,25-dihydroxyvitamin D3 analogs featuring aromatic and heteroaromatic rings: design, synthesis, and preliminary biological testing.

Aromatic compounds 2a-c, analogs of 1 alpha, 25-dihydroxyvitamin (calcitriol, 1), and heteroaromatic compounds 4a-c and 5a-c, analogs of 19-nor-1 alpha, 25-dihydroxyvitamin D3 (3), were designed to simulate the topology of their biologically potent parent compounds while avoiding previtamin D equilibrium. Convergent and facile total syntheses of the analogs (+)-2b, (+)-2c, (-)-4b, and (-)-5b were achieved via carbonyl addition of regiospecifically formed organolithium nucleophiles to the enantiomerically pure C,D-ring ketone (+)-17, characteristic of natural calcitriol (1). Likewise, hybrid analogs 20a-c were prepared to determine whether incorporation of a known potentiating side chain would lead to increased biological activity. Preliminary in vitro biological testing showed that aromatic analogs (+)-2b, (+)-2c, and 20a-c as well as heteroaromatic analogs (-)-4b and (-)-5b have very low affinities for the calf thymus vitamin D receptor but considerable antiproliferative activities in murine keratinocytes at micromolar concentration. No biological advantage was observed in this keratinocyte assay for the doubly modified hybrid analogs 20a-c over the singly modified parent (+)-2b. Analog (+)-2b, but surprisingly not the corresponding analog 20b differing from (+)-2b only in the side chain, showed considerable activity in nongenomic opening of calcium channels in rat osteosarcoma cells.

Contribution of amiloride-insensitive pathways to alveolar fluid clearance in adult rats.

The contributions of amiloride-sensitive and -insensitive fractions of alveolar fluid clearance in adult ventilated rats were studied under control conditions and after beta-adrenergic stimulation. Rats were instilled with a 5% albumin solution containing terbutaline (10(-4) M) or dibutyryl-cGMP (DBcGMP; 10(-4) M) with or without the cyclic nucleotide-gated cation channel inhibitor l-cis-diltiazem (10(-3) M) and/or amiloride (10(-3) M). Alveolar fluid clearance over 1 h was 18 +/- 2% in controls. In controls, amiloride inhibited 46 +/- 15% of alveolar fluid clearance, whereas l-cis-diltiazem had no inhibitory effect. Terbutaline and DBcGMP stimulated alveolar fluid clearance by 85 +/- 3 and 36 +/- 5%, respectively. Amiloride and l-cis-diltiazem inhibited nearly equal fractions of terbutaline-stimulated alveolar fluid clearance when given alone. Amiloride and l-cis-diltiazem given together inhibited a significantly larger fraction of alveolar fluid clearance in terbutaline-stimulated rats and in DBcGMP-stimulated rats. Based on these data, terbutaline stimulation recruited both amiloride-sensitive and l-cis-diltiazem-sensitive pathways. In contrast, DBcGMP mainly recruited l-cis-diltiazem-sensitive pathways. Therefore, the amiloride-insensitive fraction of Na+-driven alveolar fluid clearance may be partly mediated through cyclic nucleotide-gated cation channels and activated by an increase in intracellular cGMP.

Metabolites and analogs of 1alpha,25-dihydroxyvitamin D(3): evaluation of actions in bone.

Analogs of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] activate both genomic mechanisms via the nuclear vitamin D(3) receptor (nVDR) and nongenomic pathways via the plasma membrane vitamin D(3) receptor (pmVDR). Both of these pathways are normally activated by 1alpha,25(OH)(2)D(3), but as a result of synthesis of numerous analogs of 1alpha,25(OH)(2)D(3) these pathways can be distinguished. We used increasing doses of vitamin D(3) analogs to determine their potencies of action on these two distinct pathways, measuring calcium channel potentiation as an indicator of the nongenomic action and measuring increases in osteocalcin mRNA and protein release and bone resorption as indicators of genomic action. We found that both 25(OH)-16,23E-diene-D(3) (R) and 1alpha,25(OH)(2)-16,23E-diene-D(3) (A) are 10-fold more potent than 1alpha,25(OH)(2)D(3) for activation of the nongenomic pathway because double bonds in the side chain and the D ring increase the affinity for calcium channel potentiation. While the C-1alpha-hydroxyl group is not necessary for potentiation of calcium channels, methyl groups at this position can alter the affinity for calcium channel potentiation. On the other hand, 1000 fold higher concentrations of nongenomic analogs were needed compared to 1alpha,25(OH)(2)D(3) to increase osteocalcin mRNA or protein release. 1alpha,25-Dihydroxy-16-ene-23-yne-26,27-hexafluorovitamin D(3), (E) is an agent that is 10 fold more potent than 1alpha,25(OH)(2)D(3) at increasing osteocalcin mRNA and protein release, whereas 1alpha,25(OH)(2)-3-epi-D(3) increases osteocalcin mRNA and protein with a potency over 10 fold lower than 1alpha,25(OH)(2)D(3). These results suggest that double bonds in the side chain and the D ring stabilize action on the nongenomic pathway whereas F(6) on the terminal portion of the side chain increases potency for nVDR. On the other hand, while the C-1alpha-hydroxyl group is necessary for activation of genomic events via nVDR, the activation of nongenomic events occurs in the absence of this group.

Does the use of recombinant AAV5 in pulmonary gene therapy lead to lung damage?

This study investigated whether repeated administration of recombinant adeno-associated virus type 5 (rAAV5) to the airways induces inflammatory processes in the lungs of BALB/c-mice, with mechanical and histologic changes. Saline was instilled intratracheally in the control group, and rAAV5-green fluorescence protein (GFP) (4x10(11)particles) in the virus group (VR). These groups were subdivided into four subgroups: one dose analyzed 3 weeks later (VR1d3w) and two doses analyzed 1 (VR2d1w), 2 (VR2d2w) and 3 weeks (VR2d3w) after the second dose. Lung morphometry, mechanical parameters, airway responsiveness, rAAV5-GFP transduction and the expression of inflammatory cytokines were investigated. No significant differences in lung mechanics, airway responsiveness, and morphometry were observed. Re-administration of rAAV5 vector resulted in a decrease in GFP mRNA expression in the VR2d3w group. There was no evidence of inflammatory response or apoptosis in any group. rAAV5 did not induce an inflammatory process, mechanical or morphometric changes in the lungs. AAV5 may be an appropriate vector for lung gene therapy.

WNK4 kinase regulates surface expression of the human sodium chloride cotransporter in mammalian cells.

Pseudohypoaldosteronism type II (PHA II) is caused by mutations of two members of WNK ((with no lysine (k)) kinase family. WNK4 wild type (WT) has been shown to inhibit the activity and surface expression of sodium chloride cotransporter (NCC) when expressed in Xenopus oocytes. Here, we have studied NCC protein processing in mammalian cells in the presence or absence of WNK4 WT and its mutants, E562K and R1185C, by surface biotinylation, Western blot, co-immunoprecipitation (Co-IP) and immunostaining. WNK4 WT significantly reduced NCC surface expression in Cos-7 cells (58.9+/-6.8% vs 100% in control, P<0.001, n=6), whereas its mutant E562K has no significant effect on NCC surface expression (92.9+/-5.3% vs 100%, P=NS, n=6). Another mutant R1185C still partially reduces surface expression of NCC (76.2+/-11.8% vs 100%, P<0.05, n=6). The reduction of NCC surface expression by WNK4 WT (62.9+/-3.3% of control group) is not altered by WT dynamin ((61.8+/-3.7% (P=NS)) or its mutant K44A ((65.4+/-14.1% (P=NS)). A Co-IP study showed that both WNK4 WT and WNK4 E562K interact with NCC. Furthermore, a proton pump inhibitor, bafilomycin A1, partially reverses the inhibitory effect of WNK4 WT on NCC expression. Our data suggest that WNK4 WT significantly inhibits NCC surface expression, which is not owing to an increase in clathrin-mediated endocytosis of NCC, but likely results from enhanced degradation of NCC through a lysosomal pathway.

24R,25-(OH)2 vitamin D3 inhibits 1alpha,25-(OH)2 vitamin D3 and testosterone potentiation of calcium channels in osteosarcoma cells.

Calcium influx via L-type calcium channels in osteoblast cells causes a rapid (in seconds) elevation in intracellular calcium initiated by plasma membrane receptors for 1alpha, 25-dihydroxyvitamin D3 (1alpha,25-D3). 24R,25-Dihydroxyvitamin D3 (24,25-D3) alone, in concentrations up to 200 nM, does not cause potentiation of calcium currents in osteoblasts, but it does inhibit the current potentiation by 1alpha,25-D3. To determine how various steroids interact in their potentiation of calcium channels, the action of vitamin D3 analogues and testosterone with calcium channels in the rat osteoblast-like cell line ROS 17/2.8 was investigated. Bath additions of both 1alpha,25-D3 and testosterone at doses below K1/2 (the dose causing 50% left shift in the current-voltage relationship) are additive in their ability to potentiate calcium channels. When 1alpha,25-D3 and testosterone are added together at concentrations that would cause a maximal shift in the current-voltage relationship by each agent alone (Vmax), the effect of these steroids is not additive. Taken together these data suggest one population of calcium channels is activated by 1alpha, 25-D3 or testosterone. The shift in the current-voltage relationship caused by 1alpha,25-D3 is reduced by 1beta,25-dihydroxyvitamin D3 (1beta,25-D3), an agent which is thought to act specifically on the plasma membrane receptor for 1alpha,25-D3, but the potentiation caused by testosterone is not blocked by 1beta,25-D3. However, 24, 25-D3 inhibits the left shift in the peak current-voltage relationship mediated by either 1alpha,25-D3 and testosterone. This result implies that 1) 1beta,25-D3 directly displaces 1alpha,25-D3 but not testosterone from its plasma membrane receptor, and 2) the rapid (in seconds) stimulatory effects of 1alpha,25-D3 and testosterone on calcium channels are mediated by separate plasma membrane receptors for testosterone and 1alpha,25-D3, which are blocked by another receptor for 24,25-D3. The interaction of these three receptors with L-type calcium channels is pertussis toxin-sensitive.

Hormones increase mRNA of cyclic-nucleotide-gated cation channels in airway epithelia.

Previous studies have shown that the mRNA of cyclic-nucleotide-gated nonselective cation (CNG) channels is expressed in rat airway epithelia and that these channels contribute to sodium-mediated short-circuit currents in cultured rat tracheal epithelia. Patch-clamp studies from human A549 cells indicate that these channels contribute to cGMP-stimulated L-cis-diltiazem- and dichlorobenzamil-inhibited whole-cell sodium currents. This study demonstrates that mRNA for primary and secondary subunits of CNG channels, halphaCNG1 and hbetaCNG1 respectively, are expressed in several human airway cell lines, including normal and cystic fibrosis bronchial airway cells, in normal and cystic fibrosis tracheal airway cell lines and nasal polyp tissue from a cystic fibrosis patient. The mRNA of ralphaCNG1 in rat lung increased in response to increased circulating glucocorticoids and decreased in animals with lowered circulating glucocorticoids after aminoglutethimide treatment. Likewise the mRNA of halphaCNG1 increased in the presence of glucocorticoids in cultured alveolar airway cells. The mRNA of alphaCNG1 in rat lung was also increased in response to a low-salt diet and lowered in animals fed a high-salt diet. Likewise the mRNA of alphaCNG1 was increased in response to increased aldosterone and decreased in animals given spironolactone. These results suggest that mRNA for alphaCNG1 increases in response to elevated glucocorticoids or mineralocorticoids. Because alphaCNG1 is a functional sodium entry channel in both rat and human airway epithelial cells, if channel protein is also elevated this channel could mediate an increase in sodium absorption across lung epithelia in response to circulating hormones.

Modification of Ca2+-activated K+ channels in cultured medullary thick ascending limb cells by N-bromoacetamide.

Ca2+-activated K+ channels were studied in cultured medullary thick ascending limb (MTAL) cells using the patch-clamp technique in the inside-out configuration. The Ca2+ activation site was modified using N-bromoacetamide (NBA). 1 mM NBA in the bath solution, at 2.5 microM Ca2+ reduces the open probability, Po, of the channel to less than 0.01, without an effect on single-channel conductance. NBA-modified channels are still Ca2+-sensitive, requiring 25 mM Ca2+ to raise Po to 0.2. Both before and after NBA modification channel openings display at least two distributions, indicative of more than one open state. High Ca2+ (1 mM) protects the channels from modification. Also presented is a second class of Ca2+-activated K+ channels which are normally present in MTAL cells which open infrequently at 10 microM Ca2+ (Po = 0.01) but have a Po of 0.08 at 1 mM Ca2+. We can conclude (i) that NBA modifies the channel by shifting Ca2+-sensitivity to very high Ca2+, (ii) that NBA acts on a site involved in Ca2+ gating, and (iii) that a low affinity channel is present in the apical cell membrane with characteristics similar to those of normal channels modified with NBA.

cGMP-dependent protein kinase regulation of a chloride channel in T84 cells.

Chloride channels at the apical membrane of intestinal epithelial cells are involved in the excessive fluid secretion in diarrhea and diminished secretion in cystic fibrosis (CF). Diarrhea induced by heat-stable toxin from Escherichia coli is associated with elevated guanosine 3',5'-cyclic monophosphate (cGMP) in intestinal epithelial cells, but it is unknown whether chloride secretion is regulated by cGMP directly or via cGMP-dependent protein kinase (PKG). Single-channel recordings (inside-out excised patches) from the apical membrane of T84 cells reveal a 10-pS chloride channel with a linear current-voltage relationship, which is opened when an endogenous membrane-bound PKG is activated with ATP (1 mM) and cGMP (100 microM). Soluble PKG (200 nM) isolated from bovine lung, added to the intracellular face of patches, also opens this channel. No activation occurs with Ringer solution alone or only ATP or cGMP. Addition of nonhydrolyzable forms of ATP (AMP-PNP, 1 mM) or a combination of ATP, cGMP, plus H-8 (5 microM), an inhibitor of PKG, also does not stimulate the channel. The catalytic subunit of adenosine 3',5'-cyclic mono-phosphate-dependent protein kinase (PKA, 200 nM, with 1 mM ATP) activates a channel with similar characteristics. The 10 pS channel has a PNa/PCl ratio of 0.06, an anion selectivity of Br- (1.2) greater than Cl- (1.0) greater than I- (0.8) greater than F- (0.4), and a low affinity for the chloride channel blockers, 4,4-dinitrostilbene-2,2-disulfonic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

The renal cGMP-gated cation channel: its molecular structure and physiological role.

Cyclic nucleotide-gated cation channels, which are permeable to monovalent and divalent cations, are expressed in a number of tissues. cDNAs encoding cGMP-gated cation channel subunits have been cloned in retinal rods, cones, olfactory neuroepithelium, pineal gland, aorta, testis, heart, and most recently kidney. Patch clamp studies have identified and characterized cGMP-gated cation channels in the cortical collecting duct (CCD) and inner medullary collecting duct (IMCD). cGMP-gated cation channels in kidney share many biophysical and molecular properties with the retinal rod cGMP-gated channel. However, unlike the retinal rod channel, the cGMP-gated cation channel in kidney is inhibited by cGMP and stimulated by increased calcium levels. In the IMCD the cGMP-gated cation channel mediates electrogenic sodium absorption which is inhibited by ANP via cGMP. Recently, cGMP-gated cation channel poly(A+) RNA has been identified in other nephron segments by RT-PCR and in situ PCR hybridization. Furthermore, cGMP-gated cation channel protein has also been identified in all nephron segments by Western blot analysis. These observations suggest that cGMP-gated cation channels, or closely related gene products, may play an important physiological role in all nephron segments. Hormones that increase intracellular cGMP may regulate sodium, and perhaps calcium, uptake in nephron segments proximal to the IMCD. Increases in cell sodium and calcium may regulate other transport and signaling pathways.

Specificity and modes of the anion exchanger in dog renal microvillus membranes.

The transport of various organic anions via the pathway that mediates the exchange of urate or p-aminohippurate (PAH) for OH- or Cl- in dog renal microvillus membrane vesicles was investigated. The pH gradient-stimulated uptakes of tracer urate and PAH were significantly inhibited by 5 mM PAH, n-valerate, lactate, beta-hydroxybutyrate, pyruvate, acetoacetate, maleate, succinate, alpha-ketoglutarate, oxaloacetate, and cis-aconitate but not by 5 mM acetate, malate, oxalate, or citrate. the pH dependence of inhibition suggested that it was in their monovalent forms that these acid anions interacted with the urate exchange pathway. Outwardly directed gradients of succinate, lactate, and PAH stimulated uphill urate accumulation. Imposition of an inside-alkaline pH gradient stimulated the uphill accumulation of lactate and succinate. Na+ cotransport pathways for lactate and succinate were also present. In the presence of an inwardly directed Na+ gradient, lactate stimulated the uphill accumulation of urate, indicating that the pathways mediating Na+-lactate cotransport and lactate-urate exchange coexisted in at least some membrane vesicles. We conclude that the anion exchange pathway for urate in dog renal microvillus membrane vesicles has affinity for additional organic anions and can function in multiple exchange modes. Exchange of luminal urate or Cl- for intracellular organic anions or OH- is a possible mechanism for effecting uphill anion reabsorption in the proximal tubule.

Vitamin D3 analogs stimulate calcium currents in rat osteosarcoma cells.

1 alpha, 25-Dihydroxyvitamin D3 (1,25-D3) rapidly (within seconds) shifts the threshold for activation of inward calcium currents to more negative and physiological potentials. To determine whether the opening of calcium channels in bone cells is mediated by the cytosolic 1,25-dihydroxyvitamin D3 receptor (VDR), several natural metabolites 1,25-D3, 25-hydroxyvitamin D3, and 24R,25-dihydroxyvitamin D3 and synthetic analogs 25-hydroxy-16,23E-diene D3 (HO), 25-hydroxy-23-yne D3 (Y), and 1 alpha, 25-dihydroxy-16-ene-23-yne-26,27-F6 D3 (EO) were tested on dihydropyridine-sensitive inward barium currents. In order to probe the structural specificity at the 1 position of the steroid for stimulation of barium currents, we used several synthetic 1-(1'-hydroxyethyl) (NP, ON, NN, OP) and 1-(2'-hydroxyethyl)-25-hydroxyvitamin D3 analogs (14w-1 alpha and 14-w1 beta). Using the perforated patch-clamp technique, we found that the naturally occurring vitamin D3 analogs gave nearly the same rank order potency for stimulation of barium currents as their affinity for VDR with 1,25-D3 being the most potent analog. Using the synthetic analogs which have minimal affinity for VDR, we found that the compounds without 1-OH group but with additional double bonds in positions 16 and 23 or with a triple bond in position 23 retained high affinity for calcium channel activation. Furthermore, 1-hydroxyethyl-25-hydroxyvitamin D3R isomers at the 1' position had greater affinity than the S isomers at this position, and a beta oriented 2'-hydroxyethyl group gave the homolog greater affinity than did the alpha-oriented 2'-hydroxyethyl group. The fact that these synthetic analogs cause rapid effects on calcium channels and show pharmacological specificity different from the binding to the cytosolic vitamin D3 receptor suggests that calcium influx is mediated by a distinct signal transduction pathway. The high and physiological affinity of 1,25-D3 (50 pM) suggests that it is a biological regulator of calcium channels.