Biomedical subjects

L C Garg

Publications and source records attributed to L C Garg.

At least 55 records · Page 3Linked to original sources

PubMed1990

Adenosine and its analogs stimulate phosphoinositide hydrolysis in the kidney.

Renal blood flow, glomerular filtration rate and sodium excretion are known to be affected by adenosine. The present studies were undertaken to investigate the actions of adenosine and its analogs (both agonists and antagonists) on phosphoinositide (PI) hydrolysis in the outer medullary slices. Adenosine was found to cause a dose-dependent stimulation of PI hydrolysis (ED50, 2.8 microM) in renal slices from outer medulla. The adenosine analogs 5'-(N-cyclopropyl)-carboxamidoadenosine (NCCA) and 5'-N-ethylcarboxamidoadenosine (NECA) also stimulated PI hydrolysis in renal medulla. Stimulation of PI hydrolysis was blocked by the adenosine antagonists: aminophylline, 1,3-dipropyl-7-methylxanthine (DMX) and 8-(p-sulfophenyl)-theophylline (8-SPT). Caffeine not only antagonized adenosine-stimulated PI hydrolysis but also increased PI hydrolysis independently. These results indicate that adenosine stimulates PI hydrolysis in renal medulla through a receptor-mediated mechanism.

Adenosine↗

PubMed1990

Stimulation of phosphoinositide hydrolysis in renal medulla by vasopressin.

Arginine vasopressin (AVP) interacts with V1 and V2 receptors to stimulate hydrolysis of phosphoinositides (PI) and formation of cAMP, respectively. The effects of AVP on V2 receptors in the kidney are well characterized. In order to determine whether V1 receptors, coupled to phospholipase C for hydrolysis of PI, are also present in the kidney, we investigated the effects of AVP on PI hydrolysis in tissue slices from the cortex, outer medulla, and inner medulla of the rabbit kidney. We found that 10(-6) M AVP produced a significant increase in PI hydrolysis in the inner and outer medulla but not in the cortex. In the inner medulla, AVP (10(-10) M) produced a greater than 50% increase in PI hydrolysis; the effect was much greater at higher concentrations. AVP-stimulated PI hydrolysis was blocked by a V1 antagonist but not by a V2 antagonist. Increasing the osmolality of the incubation to 600 mosmol/kg water also abolished the effect of AVP on PI hydrolysis in the inner medulla. Furthermore, AVP did not stimulate PI hydrolysis (even in isoosmotic media) in isolated inner medullary collecting duct cells which make a major portion of the inner medulla. Our results indicate: 1) V1 receptors linked to PI system are not present in the inner medullary collecting duct cells but are probably present in blood vessels and/or interstitial cells of the renal medulla; and 2) AVP-stimulated PI hydrolysis in the inner medulla is modulated by the osmolality of the extracellular fluid.

Animals↗

PubMed1990

Stimulation of phosphoinositide hydrolysis by oxytocin in renal epithelial cells.

Recently, it has been reported that oxytocin (OT) produces diuresis by its interaction with OT receptors in the kidney. LLC-PK1 cells have been used as a model system for renal epithelial cells. To determine if OT stimulates receptor-mediated phosphoinositide (PI) hydrolysis in LLC-PK1 cells as it does in nonrenal cell systems, we measured the release of PI hydrolysis products in LLC-PK1 cells by OT and a selective OT agonist (AK-2-60) in the absence and presence of a selective OT antagonist (KB-5-21). In addition, we determined the effect of an increase in osmolality of the incubation medium on OT-stimulated PI hydrolysis in LLC-PK1 cells. The methods involved the incubation of LLC-PK1 cells with [3H]inositol for its incorporation into membrane PI and the measurement of the release of [3H]inositol phosphates in the presence of LiCl which prevents dephosphorylation. The osmolality of the incubation media was increased from 300 mOsmol/kg of H2O to 600, 900 and 1200 mOsmol/kg of H2O by addition of NaCl and urea. In an iso-osmotic incubation medium OT (10(-11) M) produced a greater than 100% increase in PI hydrolysis in LLC-PK1 cells. The OT agonist, AK-2-60, produced a significant increase in PI hydrolysis in LLC-PK1 cells at 10(-8) M concentration. The effects of both OT and its agonist were concentration-dependent and were blocked by the OT antagonist, KB-5-21. An increase in osmolality of the incubation media decreased OT-stimulated PI hydrolysis in LLC-PK1 and abolished completely the effect of OT at 1200 mOsmol/kg of H2O.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Interaction of a positive regulatory factor(s) with a 106-base pair upstream region controls transcription of metallothionein-I gene in the liver.

The differential transcription of the cloned mouse metallothionein-I (MT-I) gene in tissues was studied in unfractionated and fractionated nuclear extracts from rat liver and brain. MT-I gene transcription was 10-fold greater in liver nuclear extract than in brain nuclear extract, whereas the level of transcription of the histone H4 gene was almost identical in both tissue extracts. 5' Deletion analysis of upstream sequences revealed that a 106-base pair (bp) region located between the -148- and -42-bp positions with respect to the transcription start site was responsible for the higher level of expression of the MT-I gene in the liver. Preincubation of the liver extract with the 106-bp fragment resulted in a significant decrease in MT-I gene transcription in the liver extract. In contrast, MT-I gene transcription in the brain nuclear extract was not altered by preincubation with the 106-bp upstream sequence. Mixing liver and brain extract did not diminish MT-I gene transcription normally occurring in liver nuclear extract. Preincubation of brain nuclear extract with the MT-I gene had no inhibitory effect on transcription of MT-I gene in liver nuclear extract. These studies suggest that neither an inhibitor nor a negative trans-acting factor in the brain is responsible for the differential transcription of MT-I gene; rather a positive regulatory factor(s) in the liver which interacts with the 106-bp upstream region contributes to the higher level of MT-I gene expression in this tissue.

Animals↗

PubMed1989

A 37-base pair element in the far upstream spacer region can enhance transcription of rat rDNA in vitro and can bind to the core promoter-binding factor(s).

Previous studies in this laboratory have demonstrated that a 174-base pair (bp) rat rDNA spacer region located more than 2 kilobase pairs upstream of the initiation site, can enhance rat rDNA transcription in vitro independent of its orientation or distance or when inserted downstream of the initiation site. Further dissection of this region showed that transcription of a rDNA fragment containing just 37 bp of the spacer sequence, located between -2.183 and -2.219 kilobase pairs upstream of the initiation site, is 8-fold greater than that of the rDNA fragment devoid of the spacer element. Electrophoretic mobility shift assay demonstrated specific interaction of the 37-bp DNA fragment with a cellular protein(s). The spacer DNA competed for essential transcription factors as demonstrated by the absence of transcription following preincubation of the extract with the 37-bp fragment. Similar competition was also observed when a 58-bp PolI promoter was substituted for the enhancer fragment. The binding of the factor(s) to the enhancer element was not altered when coding and noncoding strands of the 37-bp oligodeoxynucleotide were used separately in the competition assay. Since the 37-bp enhancer region and the core promoter do not exhibit any significant sequence homology, the factor(s) appears to interact with these cis-acting elements in a sequence-independent manner.

Adenocarcinoma↗

PubMed1989

A cis-acting sequence within the rat ribosomal DNA enhancer region can modulate RNA polymerase II-directed transcription of the metallothionein I gene in vitro.

Plasmids were constructed by inserting a 557-bp or 174-bp spacer fragment of rat ribosomal (r)DNA containing an enhancer element(s) at -148 bp upstream from a cloned mouse metallothionein gene (pMT-I). Transcription of these plasmids in a fractionated nuclear extract from a rat hepatoma resulted in 5 to 20-fold stimulation of MT-I gene transcription. This enhancement occurred independent of orientation of the enhancer or its distance from the metallothionein gene promoter or in the presence of the MT-I gene enhancer, and was sensitive to low levels of alpha-amanitin. Stimulation of MT-I gene transcription under the direction of the rDNA spacer element also occurred in HeLa nuclear extract, albeit to a smaller extent. Prior incubation of the nuclear extract with the 557-bp or 174-bp fragment resulted in as much as 5- to 10-fold stimulation of MT-I gene transcription. No significant effect on MT-I gene transcription was observed following preincubation with other DNAs. Preincubation of the extract with three subfragments of the 174-bp spacer inhibited MT-I gene transcription, which suggests that the majority of the 174-bp domain is required for binding to the negative regulatory factor(s) for MT-I gene transcription and that the subfragments can only interact with the positive core promoter-binding factor. The 37-bp subfragment, which has been shown to interact with a positive rDNA trans-acting factor, could also interact with a positive polymerase II (pol II) trans-acting factor. These studies have demonstrated that the 174-bp rat rDNA spacer element containing the pol I enhancer can also modulate pol II-directed transcription.

Animals↗

PubMed1989

Cholinergic receptors in renal medullary collecting duct cells.

Intrarenal administration of cholinergic agents produces diuresis. However, neither cholinergic innervation or specific cholinergic receptors have been shown to be present in the kidney. Recently, we have demonstrated that carbachol, a cholinergic agent, stimulates phosphoinositide hydrolysis in the inner medullary collecting duct (IMCD) cells. The effect was blocked by atropine (a cholinergic antagonist), suggesting that phosphoinositide hydrolysis occurs through the interaction of carbachol with specific cholinergic receptors in these cells. Therefore, we examined the cholinergic receptors in IMCD cells by measurement of radioligand binding of a cholinergic receptor antagonist, I-quinuclidinyl (phenyl-4-3H)benzilate([3H]QNB). The IMCD cells were prepared from rabbit kidneys by incubating the inner medullary slices with collagenase and treating the isolated cells with hypotonic solution to lyse cells other than IMCD cells. Binding of [3H]QNB to IMCD cells was measured at 37 degrees C for 60 min in the absence (total binding) and the presence (nonspecific binding) of 100 microM atropine (a muscarinic receptor antagonist). The specific binding (the difference between total and nonspecific binding) of [3H]QNB to IMCD cells was saturable with a Bmax (maximum binding sites) of 27.5 fmol/mg of protein and Kd (dissociation constant) of 0.27 nM. Atropine, but not hexamethonium (a nicotinic antagonist), was able to displace [3H]QNB from IMCD cells with a Ki of 0.1 microM. It is, therefore, concluded that specific high affinity muscarinic receptors are present in IMCD cells. These receptors may play a role in producing the pharmacologic actions of cholinergic agents on the kidney.

Animals↗

PubMed1989

Cholinergic stimulation of phosphoinositide hydrolysis in renal medullary collecting duct cells.

Recently, we have demonstrated that carbachol, a cholinergic agonist, stimulates the hydrolysis of phosphoinositides (PI) in the inner medullary (IM) slices from the rabbit kidney. In order to localize the effects of carbachol in the IM, we measured PI hydrolysis in IM collecting duct (CD) cells which form approximately 50% of the IM and play an important role in determining the final composition of the urine. The IMCD cells were prepared from IM slices of the rabbit kidney by treatment with collagenase followed by addition of water to lyse the cells other than IMCD cells. To measure PI hydrolysis, the IMCD cells were incubated with [3H]inositol for its incorporation into PI before measurement of inositol phosphates (IP) released and accumulated in the presence of LiCl which prevents the dephosphorylation of IP. Carbachol (1 mM) produced greater than 16-fold increase in the release of IP (from 1.53 +/- 1.34% in control to 26.26 +/- 4.59% in drug-treated) in the isolated IMCD cells. The effect was concentration-dependent with an EC50 (50% maximum effective concentration) of 4 microM carbachol. Carbachol-stimulated PI hydrolysis was blocked completely by 1 microM atropine, a muscarinic antagonist, and not by 1 microM hexamethonium, a nicotinic antagonist. The nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium iodide (1 mM), had no significant effect on PI hydrolysis in the IMCD cells. We conclude that the stimulation of PI hydrolysis by cholinergic agents in the IMCD cells occurs through their interaction with muscarinic receptors and this process may play a role in the diuretic and natriuretic effects of these agents.

Animals↗

PubMed1988

Effects of aldosterone on NEM-sensitive ATPase in rabbit nephron segments.

Aldosterone (aldo) treatment of animals stimulates the rate of H+ secretion in the collecting duct, a process which may involve an H+-ATPase sensitive to inhibition by NEM (N-ethylmaleimide). Therefore, we determined NEM-sensitive ATPase activity in distal nephron segments from three groups of adrenalectomized (adx) rabbits maintained on different doses of aldo (in an osmotic minipump) for seven days. Group 1 was given 1.5 micrograms aldo/100 g body wt/day, whereas groups 2 and 3 were maintained on 5 micrograms and 50 micrograms of aldo/100 g body wt/day, respectively. Aldo concentrations in the plasma of groups 1, 2 and 3 were 10.4 +/- 0.8, 70 +/- 7 and 408 +/- 133 ng/dl, respectively. There was a significant increase in NEM-sensitive ATPase activity in connecting tubule (CNT) and cortical, outer and inner medullary duct segments (CCD, OMCD and IMCD) but not in cortical thick ascending limb (CTAL) and distal convoluted tubule (DCT) in group 2 as compared to group 1. A further increase in plasma concentration of aldo (group 3) did not produce any more increase in NEM-sensitive ATPase activity in the CNT, CCD, OMCD and IMCD, but did increase the enzyme activity in the DCT. These results are consistent with the hypothesis that aldo increases H+ secretion in the connecting tubule and collecting duct segments by increasing the activity of NEM-sensitive H+-ATPase activity in these segments.

Adrenalectomy↗

PubMed1988

Vasopressin stimulates phosphoinositide hydrolysis in LLC-PK1 cells.

LLC-PK1 cells have been shown to possess vasopressin (VP) receptors (V2 type) that are coupled to adenyl cyclase to generate adenosine 3,5'-cyclic monophosphate (cAMP). To determine whether VP also stimulates phosphoinositide (PI) hydrolysis to generate inositol phosphate (IP) and diacylglycerol (DAG) messenger system in LLC-PK1 cells, we measured the release of IP in LLC-PK1 cells in the absence and presence of various concentrations of VP. In addition, we also determined the effect of an increase in osmolality of the incubation medium on VP-stimulated PI hydrolysis in LLC-PK1 cells. The methods involved the incubation of LLC-PK1 cells with [3H]inositol for its incorporation into membrane PI and the measurement of the release of [3H]IP in the presence of LiCl which prevents dephosphorylation. The osmolality of the incubation media was increased from 300 to 600, 900, and 1,200 mosmol/kgH2O by the addition of NaCl and urea. In an isosmotic incubation medium, VP (10(-8) M) produced a 100% increase in PI hydrolysis in LLC-PK1 cells. The effect was much greater at higher concentrations of the hormone. There was no effect of osmolality in VP-stimulated PI hydrolysis in LLC-PK1 cells up to 600 mosmol/kgH2O, but PI hydrolysis decreased significantly when the osmolality of the incubation medium was increased to 900 or 1,200 mosmol/kgH2O. Our results suggest that in LLC-PK1 cells, VP stimulates PI hydrolysis probably through VP receptors that are coupled to phospholipase C. Furthermore, VP-stimulated PI messenger system in LLC-PK1 cells is influenced by osmolality of the extracellular fluid.

Animals↗

PubMed1988

Ouabain-insensitive K-adenosine triphosphatase in distal nephron segments of the rabbit.

An electrogenic H-ATpase sensitive to inhibition by N-ethyl-maleimide has been reported to be present in renal distal tubules. In contrast to another H-ATPase (gastric H-K-ATPase), the renal enzyme is not stimulated by K+ and is not inhibited by vanadate. However, our preliminary observations indicated that a K-stimulated ATPase (K-ATPase) sensitive to inhibition by vanadate is present in renal medullary collecting duct (MCD). To localize and further characterize this renal tubular K-ATPase, we measured K-ATPase activity in eight specific segments of the rabbit nephron. K-ATPase activity was the difference in ATPase activity in the presence and absence of KCl but in the presence of ouabain (to inhibit Na-K-ATPase). ATPase activity was determined by a fluorometric microassay in which ATP hydrolysis is coupled to the oxidation of NADH. There was a significant K-ATPase activity (expressed as pmol.min-1.mm-1) in the connecting tubule (CNT, 17.0 +/- 3.3), cortical collecting duct (CCD, 6.6 +/- 0.7), and MCD (8.8 +/- 1.7), but not in the proximal segments and the thick ascending limbs. The renal tubular K-ATPase was not only inhibited by vanadate but also by omeprazole and SCH 28080 (relatively specific inhibitors of gastric H-K-ATPase). It is concluded that K-ATPase present in the CNT, CCD, and MCD has some properties in common with gastric H-K-ATPase. However, the physiological role of K-ATPase in the distal nephron segments remains to be elucidated.

Adenosine Triphosphatases↗

PubMed1988

Cholinergic stimulation of phosphoinositide hydrolysis in rabbit kidney.

An injection of acetylcholine (ACh) into renal artery is known to cause diuresis. In brain and other organs, cholinergic agents have been shown to produce their actions through the phosphoinositide (PI) second messenger system. To determine if cholinergic agents also produce activation of the PI messenger system in the kidney, we investigated the effects of carbachol (a stable analog of acetylcholine) on PI hydrolysis in the cortex, outer medulla and inner medulla of the rabbit kidney. PI hydrolysis was determined by measuring the formation of inositol phosphates in response to stimulation by carbachol in the presence of 8 mM lithium. Carbachol, 1 mM, was able to stimulate PI hydrolysis in the inner medulla and outer medulla (622 and 388% over control values, respectively), but not the cortex. The response to carbachol in the inner medulla was concentration-dependent (EC50 = 10(-5) M). The response was blocked by 1 microM atropine and not by 1 microM hexamethonium. The nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium iodide did not stimulate PI hydrolysis. The effect of carbachol was dependent upon the presence of calcium ions. Substitution of alpha-ketoglutarate for glucose inhibited the response to carbachol in the inner medulla, suggesting a specific substrate requirement in PI metabolism. It is concluded that cholinergic agents produce stimulation of PI hydrolysis through muscarinic receptors in the inner medulla. Whether PI second messenger system in the kidney is involved in the diuretic effect of cholinergic agents remains to be determined.

Animals↗

PubMed1988

Effects of osmolality on phosphoinositide hydrolysis in renal medulla.

We have reported previously that carbachol stimulates hydrolysis of phosphoinositides (PIs) in the renomedullary slices when incubated in a buffer of 300 mOsm/kg of H2O. However, the mammalian renal medulla has a hypertonic environment that changes with the state of hydration of the animal. In order to determine if the change in renal osmolality produces a change in the response of the inner medulla to hormones and neurotransmitters, we determined the effects of osmolality on carbachol-stimulated hydrolysis of PIs in the inner medullary slices of the rabbit kidney. The hydrolysis was determined by incorporation of [3H]inositol into PIs and the release of [3H]inositol phosphates in the presence and absence of 1 mM carbachol. The osmolality of the incubation media was increased from 300 to 1200 mOsm/kg of H2O in increments of 300 mOsm/kg of H2O by addition of either urea, NaCl, mannitol or an equiosmolar mixture of urea and NaCl. Increasing the osmolality of the incubation media by any one of these solutes decreased carbachol-stimulated release of inositol phosphates in the inner medullary slices of the rabbit kidney. Our results suggest that the effect of carbachol on PI messenger system in the renal medulla in vivo will depend on the tissue osmolality that itself depends on the state of hydration of the animal.

Animals↗

PubMed1987

An enhancer element in the far upstream spacer region of rat ribosomal RNA gene.

To determine whether far upstream 5'-flanking sequences control rat rDNA transcription, we constructed plasmids containing several 5' and 3' deletions within the nontranscribed spacer region. In vitro transcription of these plasmids identified three enhancer regions, designated A, B, and C, which can dramatically stimulate transcription from the core promoter. Further analysis of region B showed that the enhancer element lies between -2.183 and -2.357 kilobase pairs upstream of the initiation site. The plasmid containing the 174-base pair enhancer element could stimulate rRNA gene transcription as much as 10-20-fold relative to transcription of the plasmid containing only the core promoter. This enhancer was not another promoter domain and could function irrespective of its orientation or distance from the promoter or when inserted downstream of the initiation site. Computer analysis of known sequences of enhancer regions A and C did not reveal any significant homology between these DNA segments and the 174-base pair enhancer element. Competition assay demonstrated that the enhancer element B forms a stable complex with the transcription factor(s) and that interaction between the enhancer and the factor(s) was essential for the stimulation of rDNA transcription. This is the first report of a mammalian ribosomal rDNA enhancer element that exhibits the characteristics of an RNA polymerase II enhancer.

Animals↗

PubMed1987

Accurate transcription of mouse metallothionein-I gene in a fractionated nuclear extract from a rat hepatoma.

Nuclear extract from Morris hepatoma 3924A was fractionated by DEAE-Sephadex chromatography. The fraction eluting with 300 mM (NH4)2SO4 (DE-C) was used for transcribing cloned mouse metallothionein-I (MT-I) gene in a run-off assay. This fraction contained the majority of RNA polymerase II as well as the transcription factor(s). Accuracy of MT-I DNA transcription was confirmed by S1 nuclease mapping. Low concentrations (1 microgram/ml) of alpha-amanitin inhibited the reaction, indicating that RNA polymerase II directed the transcription. Unfractionated nuclear extracts from the hepatoma or a rat mammary adenocarcinoma as well as whole cell extract obtained from the mammary tumor also transcribed MT-I gene. The extent of transcriptional activity was in the following order: hepatoma nuclear fraction DE-C greater than whole cell extract derived from rat mammary adenocarcinoma cells greater than nuclear extract derived from rat hepatoma or rat mammary adenocarcinoma cells. These studies have demonstrated that a fractionated nuclear extract obtained from a tissue supports efficient and accurate RNA polymerase II-mediated transcription of MT-I DNA.

Adenocarcinoma↗

PubMed1987

Effects of potassium bicarbonate on distal nephron Na-K-ATPase in adrenalectomized rabbits.

Na-K-ATPase activity in the connecting tubule (CNT) and cortical collecting duct (CCD) has been shown to be influenced by KCl both in the presence and in the absence of aldosterone. To investigate if the aldosterone-independent effect of K+ on Na-K-ATPase can be produced by other K+ salts, we studied the effects of dietary KHCO3 on Na-K-ATPase and ouabain-insensitive Mg-ATPase activities in four nephron segments of adrenalectomized (ADX) rabbits. The segments examined were: the distal convoluted tubule (DCT), CNT, CCD and medullary collecting duct (MCD). All diets were similar in composition except their KHCO3 contents which were 100, 300, 500 and 700 meq/kg in groups 1 to 4 respectively. Increasing KHCO3 in the diet increased K+ excretion (7 X) and urine pH (6.6 to 8.3). Na-K-ATPase activity in the CCD increased greater than 200% as dietary KHCO3 was increased to 700 meq/kg. There was a linear relation between Na-K-ATPase activity in this segment and steady state plasma K+ as well as K+ excretion in the urine. However, Na-K-ATPase activity in the CCD was lower in KHCO3-fed ADX rabbits than the KCl-fed animals studied previously under similar conditions. There were no significant differences in Na-K-ATPase activities in DCT, CNT and MCD among the four groups given different KHCO3-diets. It is concluded that dietary intake of KHCO3 can also influence Na-K-ATPase activity in the CCD independent of aldosterone.

Adrenalectomy↗

PubMed1987

Effects of hydrochlorothiazide on Na-K-ATPase activity along the rat nephron.

Na-K-ATPase activity was determined in seven nephron segments of five-week-old, spontaneously hypertensive rats (SHR) with or without continuous hydrochlorothiazide (HCTZ) treatment for seven days. For comparison, the effects of HCTZ treatment on Na-K-ATPase activity in the nephron segments of age-matched normotensive Wistar-Kyoto rats (WKY) were also determined. Na-K-ATPase activity in proximal convoluted tubule (PCT), medullary thick ascending limb (MTAL), cortical thick ascending limb (CTAL), distal convoluted tubule (DCT) and cortical collecting duct (CCD) was significantly lower in HCTZ-treated SHR compared to control (untreated) SHR. However, there was no significant difference in Na-K-ATPase activity in proximal straight tubule (PST) and medullary collecting duct (MCD) between HCTZ-treated and control SHR. HCTZ treatment also produced a significant decrease in blood pressure (BP) and creatinine clearance (CCr) in SHR. On the other hand, HCTZ treatment did not produce a significant change in Na-K-ATPase activity in PCT, PST, MTAL, CTAL and MCD, in BP or in CCr in WKY. However, HCTZ treatment produced a decrease in the enzyme activity in the DCT and an increase in the enzyme activity in the CCD in WKY. The decrease in Na-K-ATPase activity in almost all nephron segments from SHR may be due to a significant decrease in CCr produced by HCTZ. On the other hand, a decrease in Na-K-ATPase activity in the DCT with an increase in the enzyme activity in the CCD from WKY suggest that renal compensation to the natriuretic effect of HCTZ occurs by an increase in Na+ reabsorption in the CCD.

Animals↗

PubMed1987

Role of DNA topoisomerase I in the transcription of supercoiled rRNA gene.

The fraction DE-B obtained by fractionating an extract from rat mammary adenocarcinoma cells on a DEAE-Sephadex column was used for transcribing linear and supercoiled rRNA gene (rDNA). This fraction, which is known to contain RNA polymerase I and essential transcription factors, also contains DNA topoisomerase I activity. Inhibition of this topoisomerase activity by the selective inhibitor camptothecin markedly diminished transcription of supercoiled rDNA, and at a concentration of 150 microM, camptothecin almost completely inhibited DNA topoisomerase I activity and supercoiled rDNA transcription. Addition of exogenous calf thymus DNA topoisomerase I to the sample containing the drug restored the ability of the extract to transcribe supercoiled rDNA. Camptothecin, even at a concentration of 500 microM, had no significant effect on the transcription of linear rDNA. These studies show that relaxation of supercoiled rDNA by DNA topoisomerase I is essential for its transcription. The preferential inhibition of rRNA synthesis in vivo following treatment with camptothecin is probably due to selective camptothecin inhibition of DNA topoisomerase I activity.

Adenocarcinoma↗