Mechanisms of inactivation of the action of aldosterone on collecting duct by TGF-beta.

The purpose of these experiments was to investigate the mechanisms whereby transforming growth factor-beta (TGF-beta) antagonizes the action of adrenocorticoid hormones on Na(+) transport by the rat inner medullary collecting duct in primary culture. Steroid hormones 1) increased Na(+) transport by three- to fourfold, 2) increased the maximum capacity of the Na(+)-K(+) pump by 30-50%, 3) increased the steady-state levels of the alpha(1)-subunit of the Na(+)-K(+)-ATPase by approximately 30%, and 4) increased the steady-state levels of the alpha-subunit of the rat epithelial Na(+) channel (alpha-rENaC) by nearly fourfold. TGF-beta blocked the effects of steroids on the increase in Na(+) transport and the stimulation of the Na(+)-K(+)-ATPase and pump capacity. However, there was no effect of TGF-beta on the steroid-induced increase in mRNA levels of alpha-rENaC. The effects of TGF-beta were not secondary to the decrease in Na(+) transport per se, inasmuch as benzamil inhibited the increase in Na(+) transport but did not block the increase in pump capacity or Na(+)-K(+)-ATPase mRNA. The results indicate that TGF-beta does not inactivate the steroid receptor or its translocation to the nucleus. Rather, they indicate complex pathways involving interruption of the enhancement of pump activity and activation/inactivation of pathways distal to the steroid-induced increase in the transcription of alpha-rENaC.

Amiloride-sensitive Na+ channels in pelvic uroepithelium involved in renal sensory receptor activation.

Stretching the renal pelvic wall increases ipsilateral afferent renal nerve activity (ARNA). This response is enhanced by inhibiting Na+-K+-ATPase with ouabain, suggesting a modulatory role for intracellular Na+ in the activation of mechanosensitive neurons. The messenger RNA for alpha-, beta-, and gamma-subunits of epithelial Na+ channels (ENaC) is found in collecting duct cells. Because ENaC subunits show homology with genes involved in mechanosensation, we examined whether ENaC mRNA could be found in the pelvic wall and whether the ARNA response to increased renal pelvic pressure was modulated by blockers of the Na+ channel. alpha-, beta-, and gamma-subunits are present in the pelvis. The messenger RNA for the beta- and gamma-subunits is readily detected by in situ hybridization throughout the uroepithelium. The ARNA response to increased renal pelvic pressure was reduced by 53 +/- 10% and 40 +/- 10% (P < 0.01) by renal pelvic perfusion with the inhibitors amiloride and benzamil, respectively. Amiloride inhibited the ouabain-induced enhancement of the ARNA response to increased renal pelvic pressure. The magnitude of this inhibition was inversely correlated with the magnitude of the amiloride-mediated blockade of the ARNA response to increased renal pelvic pressure (P < 0.001). Amiloride also reduced the ARNA response to renal pelvic administration of substance P, a mediator of the ARNA response to increased renal pelvic pressure. We conclude that the ENaC complex in the pelvic uroepithelium participates in the activation of renal pelvic mechanosensitive neurons.

Regulation of rENaC mRNA by dietary NaCl and steroids: organ, tissue, and steroid heterogeneity.

Rats on a low-NaCl diet have a high Na+ channel activity in colon and kidney. To address the mechanism of this increased activity, we measured mRNA levels of three Na+ channel subunits in epithelial tissue (rENaC) from rats having been fed either a low (0.13%)- or high (8%)-NaCl diet for 2-3 wk. The size of the mRNA for each of the rENaC subunits as determined by Northern blot was unaffected by diet. RNase protection assay showed heterogeneity of response by organs and subunit. In lung, there was no effect of diet on any of the three subunits. In descending colon, the low-NaCl diet increased beta- and gamma-rENaC mRNA, with no effect on alpha-rENaC mRNA. In the kidney, the response to dietary NaCl was dependent on the region. In cortex and outer medulla, diet had no effect on any of the subunits. Rats fed the low-NaCl diet had greater alpha-rENaC in inner medulla but not beta- or gamma-rENaC mRNA. We next asked whether acute administration of pure glucocorticoid (GC) or mineralocorticoid (MC) hormones to adrenalectomized rats reproduced the effects of a low-NaCl diet. Six hours after administration of GC or MC, a somewhat different heterogeneity occurred. In lung, alpha-rENaC mRNA was increased but only in response to GC. In colon, either GC or MC increased beta- or gamma-rENaC, and there was no effect on alpha-rENaC. In kidney, either GC or MC increased alpha-rENaC, without an effect on beta- or gamma-rENaC. In contrast to the response to a low-NaCl diet, all three regions were similarly affected by acute steroids. These results demonstrate a striking heterogeneity in response to physiological stimuli that regulate ENaC function. The mRNA levels of each of the rENaC subunits can be determined by the type of steroid and by factors unique to the organ and even to the specific region of the kidney.

Localization of epithelial sodium channel subunit mRNAs in adult rat lung by in situ hybridization.

The transport of Na+ through amiloride-sensitive sodium channels (ENaC) plays a major role in the absorption of fluid across the pulmonary epithelium. The proteins forming the ENaC channel are encoded by three genes in the rat (alpha-, beta-, and gamma-rENaC). According to Northern blot, all three subunit mRNAs were expressed in adult rat lung. Each subunit was expressed as a single transcript of approximately 3.7, 2.2, and 3.2 kb for alpha-, beta-, and gamma-rENaC, respectively. To localize the alpha-, beta-, and gamma-rENaC subunit mRNAs, we used in situ hybridization. Frozen and paraffin-embedded tissues were hybridized with sense and antisense 35S-labeled riboprobes. The alpha-rENaC mRNA was most abundant and was expressed diffusely in epithelia of the trachea, bronchi, bronchioles, and alveoli. At the alveolar level, alpha-rENaC was expressed in type II cells. The beta- and gamma-rENaC mRNAs were most abundant in the bronchial and bronchiolar epithelia. All three subunits were expressed in the renal cortical collecting duct in a pattern similar to that previously reported by other investigators. Thus the rENaC subunit mRNAs are expressed in regions of the lung where functional Na+ absorption is found. These results are consistent with an important role for ENaC in the absorption of Na+ and fluid across the pulmonary epithelium in all regions of the lung.

Genetic characterization of the "new" Harlan Sprague Dawley Dahl salt-sensitive rats.

In 1994, it was reported that Dahl salt-sensitive SS/Jr rats supplied by Harlan Sprague Dawley were genetically contaminated and resistant to the pressor effects of a high salt diet. Harlan Sprague Dawley subsequently developed new pedigree expansion and production colonies from their foundation colony to supply new, purportedly inbred, Harlan Sprague Dawley SS/Jr (S(HSD)). To evaluate the genetic integrity and salt sensitivity of thse new S(HSD), we performed genotyping (microsatellite DNA markers) and phenotyping (radiotelemetric arterial pressure) of 12 S(HSD), 16 "authentic" SS/Jr from the inbred colony of John Rapp (S(Rapp)), 9 Harlan Sprague Dawley salt-resistant SR/Jr (R(HSD)), and (genotyping only) 6 known "contaminated" Harlan Sprague Dawley Dahl SS/Jr (S*). In the genotyping studies, 20 of 22 markers revealed polymorphisms between S(Rapp) and S* and 18 were polymorphic between S(Rapp) and R(Rapp), but none of the 22 markers revealed polymorphisms between S(Rapp) and the new S(HSD). The phenotyping studies showed that during an ultra-low salt diet, mean arterial pressure was higher (P < .05) in both authentic S(Rapp) (129 +/- 2 mm Hg; mean +/- SE) and new S(HSD) (120 +/- 2 mm Hg) than in R(HSD) (93 +/- 1 mm Hg). A high salt diet increased mean arterial pressure in every S(HSD) and S(Rapp). Increases in mean arterial pressure after 4 weeks of a high salt diet were significantly (P < 0.05) greater in authentic S(Rapp) (+51 +/- 3 mm Hg) than in new S(HSD) (+39 +/- 3 mm Hg). In addition, salt-induced mortality was significantly greater in S(Rapp) (62.5%) than S(HSD) (8.3%) after 8 weeks (P < 0.01). S(HSD) were genotypically indistinguishable from S(Rapp), had an elevated arterial pressure on a low salt diet, and had a pressor response to salt. Thus, the new S(HSD) supplied to us had several characteristics of inbred Dahl SS/Jr and did not have evidence of the previously detected genetic contamination. However, phenotypic characteristics such as body weight, salt-induced hypertension, and mortality were significantly different in S(HSD) compared with S(Rapp). This may reflect genetic differences between these two strains or differences in environmental factors and suggests that the S(HSD) and S(Rapp) may now constitute distinct substrains of Dahl SS/Jr.

rENaC is the predominant Na+ channel in the apical membrane of the rat renal inner medullary collecting duct.

The terminal nephron segment, the inner medullary collecting duct (IMCD), absorbs Na+ by an electrogenic process that involves the entry through an apical (luminal) membrane Na+ channel. To understand the nature of this Na+ channel, we employed the patch clamp technique on the apical membrane of primary cultures of rat IMCD cells grown on permeable supports. We found that all ion channels detected in the cell-attached configuration were highly selective for Na+ (Li+) over K+. The open/closed transitions showed slow kinetics, had a slope conductance of 6-11 pS, and were sensitive to amiloride and benzamil. Nonselective cation channels with a higher conductance (25-30 pS), known to be present in IMCD cells, were not detected in the cell-attached configuration, but were readily detected in excised patches. The highly selective channels had properties similar to the recently described rat epithelial Na+ channel complex, rENaC. We therefore asked whether rENaC mRNA was present in the IMCD. We detected mRNA for all three rENaC subunits in rat renal papilla and also in primary cultures of the IMCD. Either glucocorticoid hormone or mineralocorticoid hormone increased the amount of alpha-rENaC subunit mRNA but had no effect on the mRNA level of the beta-rENaC or gamma-rENaC subunits. From these data, taken in the context of other studies on the characteristics of Na+ selective channels and the distribution of rENaC mRNA, we conclude that steroid stimulated Na+ absorption by the IMCD is mediated primarily by Na+ channels having properties of the rENaC subunit complex.

Anion secretion by the inner medullary collecting duct. Evidence for involvement of the cystic fibrosis transmembrane conductance regulator.

It is well established that the terminal renal collecting duct is capable of electrogenic Na+ absorption. The present experiments examined other active ion transport processes in primary cultures of the rat inner medullary collecting duct. When the amiloride analogue benzamil inhibited electrogenic Na+ absorption, cAMP agonists stimulated a transmonolayer short circuit current that was not dependent on the presence of Na+ in the apical solution, but was dependent on the presence of Cl- and HCO3-. This current was not inhibited by the loop diuretic bumetanide, but was inhibited by ouabain, an inhibitor of the Na+/K+ pump. The current was reduced by anion transport inhibitors, with a profile similar to that seen for inhibitors of the cystic fibrosis transmembrane conductance regulator (CFATR) Cl- channel. Using several PCR strategies, we demonstrated fragments of the predicted lengths and sequence identity with the rat CFTR. Using whole-cell patch-clamp analysis, we demonstrated a cAMP-stimulated Cl- current with characteristics of the CFTR. We conclude that the rat inner medullary collecting duct has the capacity to secrete anions. It is highly likely that the CFTR Cl- channel is involved in this process.

Netropsin and bis-netropsin analogs as inhibitors of the catalytic activity of mammalian DNA topoisomerase II and topoisomerase cleavable complexes.

This study examined the ability of netropsin and related minor groove binders to interfere with the actions of DNA topoisomerases II and I. We evaluated a series of netropsin dimers linked with flexible aliphatic chains of different lengths. These agents are potentially able to occupy longer stretches of DNA than the parental drug as a result of bidentate binding. Both netropsin and its dimers were found: (i) to inhibit the catalytic activity of isolated topoisomerase II and (ii) to interfere with the stabilization of the cleavable complexes of topoisomerase II and I in nuclei. Dimers with linkers consisting of 0-4 and 6-9 methylene groups (n) were far more inhibitory than netropsin against isolated enzyme and in the nuclear system. The compound with n = 5 was less active than netropsin in both assays while the dimer with n = 10 inhibited only the isolated enzyme. The comparison of dimers with fixed linker length (n = 2) but varying number of N-methylpyrrole residues (from 1 to 3) revealed that the inhibitory properties were enhanced with increasing number of N-methylpyrrole units. For dimers with varying linker length, drug ability to inhibit catalytic activity of isolated topoisomerase II was positively correlated with calf thymus DNA association constants. In contrast, no such correlation existed in nuclei. However, the inhibitory effects in the nuclear system were correlated with the association constants for poly(dAdT). The results indicate that bidentate binding can significantly enhance anti-topoisomerase activity of netropsin related dimeric minor groove binders. However, other factors such as the length of the linker, the number of pyrrole moieties and the nature of the target (isolated enzyme/DNA versus chromatin in nuclei) also contribute to these activities.

Effects of minor groove binding drugs on camptothecin-induced DNA lesions in L1210 nuclei.

Topoisomerase I inhibition detected in mammalian cells can be correlated with reduced tumor growth. Camptothecin specifically inhibits topoisomerase I by stabilization of a covalently linked DNA-enzyme complex and associated DNA single-strand breaks. Whether perturbations in nuclear DNA structure can alter camptothecin-induced DNA damage was examined using the non-intercalative DNA minor groove binders distamycin, Hoechst 33258 and DAPI (4',6-diamidino-2-phenylindole). L1210 nuclei were treated with camptothecin alone or in the presence of single minor groove binders. DNA-protein crosslinks and single-strand breaks were determined using potassium-sodium dodecyl sulfate precipitation and alkaline elution respectively. Distamycin produced a dose-dependent decrease in DNA-protein crosslinks and strand breaks. This effect was reduced if nuclei were treated with camptothecin prior to distamycin addition. Distamycin was unable to reverse lesions once induced or to prevent repair of damage upon camptothecin removal. Hoechst 33258 and DAPI also decreased camptothecin-induced DNA damage. The order of inhibitory potency was: distamycin greater than Hoechst greater than DAPI. This order corresponded to the molecular weights as well as to the size of the nucleotide binding sites of the drugs. Identifying agents which alter such DNA lesions should provide better understanding of the chemotherapeutic activity of camptothecin as well as help elucidate new leads for drug combinations of improved therapeutic benefit.

Effect of minor groove binding drugs on mammalian topoisomerase I activity.

Three minor groove binding drugs, distamycin A, bisbenzimide (Hoechst 33258) and 4',6-diamidino-2-phenylindole (DAPI), were examined for their abilities to modulate the activity of topoisomerase I purified from L1210 cells. At 0.5 and 1.0 microM, distamycin stimulated topoisomerase I relaxation of supercoiled DNA by 38 and 13%, respectively, while increasing the drug concentration above 2.0 microM resulted in inhibition. Inhibition was reversible. Complete relaxation could be achieved even in the presence of inhibitory concentrations of distamycin if the incubation time with topoisomerase I was increased from 7.5 to 120 min. The velocity of topoisomerase I mediated relaxation was reduced by 2 microM distamycin at DNA levels ranging from 350 to 2000 ng/reaction. Hoechst 33258 and DAPI inhibited topoisomerase I relaxation in a concentration-dependent manner. Hoechst 33258 and distamycin were equivalent in their abilities to inhibit topoisomerase I, whereas DAPI had a lesser effect (e.g. relaxation was reduced by 50% with 2.7 microM distamycin and 2.8 microM Hoechst 33258 compared to 5 microM DAPI). This study suggests that ligand binding in the minor groove can be a factor in the regulation of topoisomerase I activity.

DNA minor groove binding agents interfere with topoisomerase II mediated lesions induced by epipodophyllotoxin derivative VM-26 and acridine derivative m-AMSA in nuclei from L1210 cells.

This study demonstrated that agents capable of interacting with the minor groove in nuclear DNA interfere with topoisomerase II mediated effects of antitumor drugs such as VM-26 and m-AMSA. Distamycin, Hoechst 33258, and DAPI were used as agents capable of AT-specific binding in the minor groove of DNA while producing no profound long-range distortion of DNA structure. In intact nuclei from L1210 cells, these minor groove binders inhibited the induction of topoisomerase II mediated DNA damage (DNA-protein cross-links and DNA double-strand breaks) by VM-26 and m-AMSA. The inhibitory effects of distamycin reflected prevention of formation of new lesions but not reversal of preexisting damage. The minor groove binders did not differentiate between lesions induced by an intercalator, m-AMSA, or by a DNA-nonbinding drug, VM-26. All three groove binders inhibited DNA breaks more strongly than DNA-protein cross-links. The inhibitory potency correlated with the size of minor groove binders and the size of their DNA-binding sites: distamycin (5 bp) greater than Hoechst 33258 (4 bp) greater than DAPI (3 bp). The results showed that DNA minor groove binders are a new type of modulators of the action of topoisomerase II targeted drugs.

Modulation of topoisomerase II catalytic activity by DNA minor groove binding agents distamycin, Hoechst 33258, and 4',6-diamidine-2-phenylindole.

The effects of distamycin, Hoechst 33258, and 4',6-diamidine-2-phenylindole (DAPI) on the catalytic activity of topoisomerase II from L1210 cells were determined. These compounds were used as model agents capable of AT-specific binding in the minor groove of DNA while producing no profound long-range alterations to the DNA structure. Two types of reactions catalyzed by topoisomerase II were examined, relaxation of supercoiled DNA and decatenation of highly catenated DNA. Distamycin at low concentrations (0.2-2 microM) substantially stimulated relaxation of supercoiled pBR322 DNA. Higher drug levels (25-50 microM) resulted in a potent inhibition of relaxation. At the stimulatory concentrations of distamycin, only completely relaxed reaction products were observed, as in the absence of the drug. The onset of inhibition (caused by 5-10 microM distamycin) was accompanied by the appearance of partially relaxed intermediates. Similar inhibition of relaxation was observed for Hoechst 33258 and DAPI but, unlike distamycin, these agents produced only marginal stimulation of relaxation when added in low noninhibitory concentrations. Another reaction of topoisomerase II, decatenation of catenated kinetoplast DNA, was also inhibited by distamycin, Hoechst 33258, and DAPI at concentrations similar to those inhibiting the relaxation reaction. This study demonstrates that agents binding to the minor groove of DNA represent a new class of drugs interfering with topoisomerase II and provides possibilities for modulation of this important enzyme.

Topoisomerase-II-mediated lesions in nascent DNA: comparison of the effects of epipodophyllotoxin derivatives, VM-26 and VP-16, and 9-anilinoacridine derivatives, m-AMSA and o-AMSA.

This study compares the effects of the epipodophyllotoxin derivatives, VM-26 and VP-16, and the 9-anilinoacridine derivatives, m-AMSA and o-AMSA, on nascent and mature DNA. Two types of lesion which are putatively mediated by topoisomerase II, DNA-protein crosslinks and DNA double-strand breaks, were analyzed in drug-treated nuclei from 3H/14C labelled L1210 cells. Potassium/dodecyl sulfate precipitation assay was used to assess DNA-protein crosslinks in mature and nascent (1 min old) DNA. Both epipodophyllotoxins and m-AMSA showed a strong preference for nascent DNA. DNA double-strand cleavage induced by VM-26 and m-AMSA also showed a preference for nascent DNA as indicated by neutral elution technique. Sedimentation on neutral sucrose gradients revealed that these drugs generated highly degraded fragments (under 30 S) in nascent DNA substantially faster than in mature DNA. Lesions in nascent DNA were diminished substantially by the omission of ATP or the addition of novobiocin. The ability to induce lesions in nascent DNA correlates with cytotoxic potency of the agents studied. The results suggest that replicating DNA may constitute a preferential target for antitopoisomerase II drugs.

Assessment of preferential cleavage of an actively transcribed retroviral hybrid gene in murine cells by deoxyribonuclease I, bleomycin, neocarzinostatin, or ionizing radiation.

Preferential cleavage induced by bleomycin, neocarzinostatin, or ionizing radiation in a transcribed cellular gene was evaluated through comparisons with deoxyribonuclease I. The glucocorticoid-inducible LTL gene (a hybrid viral gene derived from mouse mammary tumor virus DNA) previously described [Zaret, K. S., & Yamamoto, K. R. (1984) Cell (Cambridge, Mass.) 38, 29-38] served as the specific DNA target. A Southern blot analysis was used to specifically assess cleavage of the LTL gene in nuclei isolated from cells either treated or untreated with the synthetic glucocorticoid dexamethasone. Hypersensitivity of the gene to bleomycin or neocarzinostatin, which paralleled deoxyribonuclease I hypersensitivity, was evident only in nuclei isolated from dexamethasone-treated cells. Like deoxyribonuclease I, sites of dexamethasone-inducible drug hypersensitivity were coincident with the binding region for the glucocorticoid receptor found within the regulatory sequences of the LTL gene. In contrast, no hypersensitivity to ionizing radiation was evident. Although bleomycin and neocarzinostatin showed qualitatively similar preferences for the transcribed LTL gene, quantitative evaluations of damage to total cellular DNA by filter elution showed that the relative specificity of bleomycin for the hypersensitive region was much less than that of either deoxyribonuclease I or neocarzinostatin.

Neurospora glucamylase and a mutant affected in its regulation.

Neurospora glucamylase is a glucose-repressible extracellular enzyme. The enzyme was purified to homogeneity and found to have a molecular weight of 82,000 and to release glucose from either maltose or amylose. The rate of glucamylase synthesis increases more than 100-fold when cells are transferred from a glucose-containing medium to a glucose-free medium. Increased from a glucose-containing medium to a glucose-free medium. Increased production of glucamylase begins within 30 min of the transfer. Glucamylase is rapidly secreted into the medium. A mutant affecting the ability of glucose to repress the synthesis of the glucose-repressible extracellular enzymes glucamylase and invertase has been isolated and studied. The mutant constitutively synthesizes and secretes a glucamylase which is indistinguishable from the wild-type enzyme.