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The purpose of this study was to determine the basal levels of dopamine (DA) and to examine the enzymes involved in DA metabolism in different microdissected nephron segments from rat kidneys. Segments were incubated with DA (50 nM) or DA plus monoamine oxidase (MAO) or catechol-O-methyl transferase (COMT) inhibitors. Basal DA levels were higher in the proximal convoluted tubule (PCT, 10.8+/-3.7 pg/mm) and in the medullary collecting duct (MCD, 10.9+/-4.0 pg/mm) than in the medullary thick ascending limb of Henle's loop (MTAL, 4.9+/-0.9 pg/mm) (P<0.05). The percentage of exogenously added DA that was not metabolised was similar in both PCT (67+/-13%) and MCD (65+/-5%) and lower in MTAL (35+/-7%), suggesting that MTAL is a major site of DA metabolism. Inhibition of MAO (pargyline 1 mM) significantly increased the basal content of DA and the percentage of the added non-metabolised DA (to 95+/-10%) in PCT but had no effect on MTAL or MCD. Conversely, inhibition of COMT (nitecapone or Ro-41-0960, both 1 mM) slightly increased the basal levels of DA only in MTAL, whereas the percentage of added DA not metabolised rose to 97+/-10% in MTAL and to 91+/-15% in MCD. COMT inhibition had no effect in PCT. In conscious rats pargyline (50 mg/kg) increased urinary DA from 680+/-34 to 1,128+/-158 ng/d/100 g BW (P<0.01) while nitecapone (40 mg/kg) produced a slight non-significant increment. Our results show that DA is present all along the rat nephron and that renal DA is metabolised continuously and predominantly by MAO in proximal segments, and by COMT in the more distal ones.

The increasing number of available genetically manipulated mice makes it necessary to develop tools and techniques for examining the phenotypes of these animals. We have developed a straightforward and rapid method for the isolation of large quantities of single tubule fragments from the mouse kidney. Immunohistochemistry, electron microscopy, and fluorescence microscopy were used to evaluate the viability, functional characteristics, and morphology of proximal tubules (PT), and collecting ducts from cortex (CCD) and inner stripe of the outer medulla (ISOMCD). Tubules were isolated using a modified collagenase digestion technique, and selected under light microscopy for experimentation. Electron microscopy and trypan blue exclusion showed that a large portion of unselected proximal tubules were damaged by the digestion procedure. The selected tubules, however, all excluded trypan blue, indicating that the plasma membrane had remained intact. Immunocytochemistry on isolated CCD showed normal distribution of H(+)-ATPase, pendrin, and anion exchanger-1 (AE-1) staining. The pH-sensitive dye 2',7'-bis(2-carboxylethyl)-5(6)-carboxyfluorescein (BCECF) was used to measure Na(+)-dependent and -independent intracellular pH (pH(i)) recovery rates in PT, and in single intercalated cells of CCD and ISOMCD fragments. Na(+)-dependent pH(i)-recovery was 0.144+/-0.008 (PT), 0.182+/-0.013 (CCD), and 0.112+/-0.010 pH units/min. (ISOMCD). Na(+)-independent pH(i) recovery was found in all three segments (PT: 0.021+/-0.002, CCD: 0.037+/-0.002, ISOMCD: 0.033+/-0.002 pH units/min) and was sensitive to concanamycin. In summary, we have developed a new technique for rapid and straightforward preparation of large quantities of defined tubule fragments from mouse kidney. Using this technique, the first measurements of plasma membrane vacuolar H(+)-ATPase activities in mouse PT and collecting duct were made. This technique will facilitate further characterization of kidney function in normal and genetically manipulated animals.

Targeted oncogenesis in transgenic mice, where an oncogene is placed under the control of the regulatory sequences of a cell-specific gene, has been used to derive lines of differentiated kidney epithelial cells derived from proximal or distal tubules or from the collecting duct. These renal cell lines were obtained from kidneys of transgenic mice harboring the large-T and little-t antigens placed under the control of regulatory sequences of the L-type pyruvate kinase gene. This review summarizes the main properties of these differentiated cell lines, which are useful ex vivo cell systems for pharmacological and toxicological studies.

Conflicting theories on the existence of several renal arginase isoenzymes remain in debate. Because the activity of arginase is high in two embryologically different nephron segments of the Meriones shawi kidney, namely the cortical (CPST) and medullary (OSPST) proximal straight tubule and the outer medullary collecting duct (OMCD), we postulate that these nephron segments may contain different isoforms. Isolated nephron segments were dissected from collagenase-treated kidneys. Tubules were permeabilized with Triton X-100 (0.25%) and incubated with increasing Arg concentrations to characterize the arginase activity. The results were as follows: (1) in OMCD, one arginase isoform (E1), characterized by a high Arg affinity (1.160 mM), was present; (2) in CPST, two arginase isoforms were discovered - one, E1, had a similar Km (1.407 mM) to that found in OMCD whereas the other (E2) had a low affinity for Arg (Km =18.8 mM); and (3) in OSPST, two isoenzymes were present - E1 which had a Km of 1.478 mM and the second isoform that we named E2 which had a Km of 9.07 mM. In addition, arginase located in CPST and OMCD was strongly inhibited by Orn and Lys. The Ki value for Lys varied between 1.635 and 2.288 mM. Therefore, this work demonstrates that two arginase isoforms are present in the kidney of Meriones shawi. Isoform E1 is present in the proximal tubule and the collecting duct whereas isoform E2 is restricted to the proximal tubule.

Changes in activity and abundance of renal Na,K-ATPase were evaluated during cold exposure and hibernation of the jerboa Jaculus orientalis by measuring the hydrolytic activity, the number of units and the transport activity of Na,K-ATPase in isolated nephron segments. As compared to controls, jerboas exposed to cold (6 degrees C) for 4-5 weeks displayed mild diuresis, decreased urinary osmolality and increased kaliuresis. In cold-exposed jerboas, Na,K-ATPase hydrolytic activity was reduced in the medullary thick ascending limb and enhanced in the cortical and outer medullary collecting duct, whereas it was not altered in other nephron segments. The number of Na,K-ATPase units and the activity of Na,K-pump, determined by [3H]-ouabain binding and by ouabain-sensitive rubidium uptake respectively, changed in parallel with the hydrolytic activity in the medullary thick ascending limb and cortical collecting duct. The maximal rate of activity (Vmax) of Na,K-ATPase was not modified further during hibernation. Thus, cold exposure, but not the onset of hibernation, induces segment-specific changes in the abundance and activity of Na,K-ATPase units which are likely to be related to the entry into hibernation, but not to the maintenance of some renal functions during deep hibernation.

Previous studies have demonstrated the presence of two populations of Na,K-ATPase with distinct kinetic, pharmacological and immunological characteristics along the rabbit nephron, indicating that the proximal segments of the nephron express exclusively the alpha 1 isoform of the catalytic subunit, whereas the collecting duct expresses an alpha 3-like isoform. Because pharmacological studies have shown the existence of two populations of Na,K-ATPase with different sensitivities to ouabain in the rat cortical collecting duct, which may result from the presence in the same nephron segment of the two isoforms demonstrated in the different segments of the rabbit nephron, the present study was undertaken to characterize the properties of Na,K-ATPase along the rat nephron. Results indicate that each segment of the rat nephron contains two subpopulations of Na,K-ATPase: a component highly sensitive to ouabain (IC50 approximately 5.10(-6) M) which is recognized by an anti-alpha 3 antibody and another moiety of lower affinity for ouabain (IC50 approximately 5.10(-4) M) which is recognized by an anti-alpha 1 antibody. Whether these two subpopulations correspond to different isoforms of the alpha subunit of Na,K-ATPase (alpha 1 and alpha 3-like) remains to be determined.

Dopamine decreases tubular sodium reabsorption, attributed in part to Na-K-ATPase inhibition in the proximal convoluted tubule (PCT). Because the final regulation of sodium excretion occurs in the collecting duct, where specific dopamine DA1 binding sites have been demonstrated, we examined the effects of dopamine, as well as of DA1 and DA2 receptor agonists on Na-K-ATPase activity and on the number of units in Madin-Darby canine kidney (MDCK) cells, which retain differentiated properties of the renal cortical collecting tubule epithelium. Dopamine (10(-5) M) inhibited pump activity (by 50%) and reduced the number of units. This effect was reproduced by the DA1 agonist SKF 38393, which inhibited pump activity in a dose- and time-dependent manner (maximum, 10(-5) M). The DA2 agonist quinpirole hydrochloride was without effect, either alone or in combination with SKF 38393. Inhibition of pump activity by dopamine was totally abolished by H7 (100 microM), an inhibitor of protein kinase (PK), but partially by 2',5'-dideoxy-adenosine (DDA) and H4, respective inhibitors of cAMP production and PKA, which suggests that the dopamine effect on Na-K-ATPase activity may be linked to activation of both PKC and PKA. In these cells, amiloride addition during preincubation did not alter the effect of dopamine on Na-K-ATPase activity; in contrast, furosemide increased further the inhibitory effect of dopamine on the enzyme activity. Monensin addition (10(-3) M) reversed the inhibitory effect of dopamine after a 30-min preincubation.(ABSTRACT TRUNCATED AT 250 WORDS)

The basolateral membrane of the rat cortical collecting duct (CCD) principal cell is K+ conductive. Recently, two different K+ channels have been described, namely a small- and an intermediate-conductance K+ channel (s-K+ and i-K+) which most likely are responsible for the macroscopic K+ conductance. K+ channel activity was investigated at the single-channel level using the patch-clamp technique. Patch-clamp recordings were obtained from enzymatically isolated CCD segments and freshly isolated CCD cells using conventional cell-free, cell-attached, cell-attached-nystatin and slow-whole-cell methods. Both K+ channels showed rundown behaviour after excision. In an excised inside-out oriented membrane, K+ channels could be activated by simultaneous addition of 0.1 mmol/l (cyclic guanosine monophosphate (cGMP) and 0.1 mmol/l MgATP to the bath. The i-K+ was activated in 13 out of 45, the s-K+ in 15 out of 45, cases. No activation of either channel was observed with cGMP alone (0.1 mmol/l), MgATP alone (0.1 mmol/l), cGMP and guanosine triphosphate (GTP) (0.1 mmol/l each) or cyclic adenosine monophosphate (cAMP) and MgATP (0.1 mmol/l each) n = 15, 11, 7, 8, respectively). The activated s-K+ could be blocked by KT 5823 (n = 8), a specific inhibitor of a cGMP-dependent protein kinase (PKG). An inhibition of the activated i-K+ was seen in seven cases. The membrane potential hyperpolarized significantly after application of dibutyryl-cGMP (0.1 mmol/l, n = 6) or nitroprusside (10 mumol/l, n = 5), which is known to liberate NO and thus increase the intracellular cGMP level.(ABSTRACT TRUNCATED AT 250 WORDS)

There is ample evidence of calcium being an intracellular second messenger during volume regulatory processes in various cells including inner medullary collecting duct (IMCD) cells. Therefore, we measured intracellular calcium concentrations (Cai) under anisotonic conditions in primary cultures of IMCD cells using the Fura-2 technique. Basal steady-state calcium at 600 mosmol/l was found to be 110 +/- 4 nmol/l; n = 119. Exposure to hypotonic medium (300 mosmol/l, reduction of sucrose) resulted, within 1 min, in a strong increase in calcium to 563 +/- 87 nmol/l (n = 7; P < 0.01), followed by a decrease over 4-6 min to twice the initial values. The calcium increase was smaller (260 +/- 14 nmol/l; n = 5; P < 0.05) when the osmotic pressure was decreased by reducing NaCl instead of sucrose. Stepwise reduction of osmolarity to either 500 or 400 mosmol/l increased calcium by a significantly smaller extent, suggesting a threshold for calcium influx between 400 and 300 mosmol/l. In hypotonic calcium-free solutions no significant increase in calcium was observed. Verapamil (40 mumol/l), D-600 (40 mumol/l), diltiazem (40 mumol/l), and nifedipine (40 mumol/l) inhibited the hypotonically induced calcium influx in decreasing order of potency. Lanthanum (La3+) and gadolinium (Gd3+) had no effect. Membrane depolarization by incubation in potassium-rich solution diminished calcium influx. Preincubation with cytochalasin B (50 mumol/l for 30 min) resulted in a lower basal calcium level and attenuated the calcium increase during hypotonic shock. These results demonstrate an increased calcium influx during hypotonic shock in IMCD cells in culture mediated by channels whose nature (stretch activated and/or voltage dependent) remains to be determined. The transient increase in Cai in turn may trigger inorganic and organic osmolyte fluxes observed previously.

Dopamine decreases tubular sodium reabsorption, attributed in part to Na/K-ATPase inhibition in the proximal convoluted tubule (PCT). Because the final regulation of sodium excretion occurs in the collecting duct, where we have demonstrated specific dopamine DA1 binding sites, we examined the effects of dopamine, and of DA1 and DA2 receptor agonists on the Na/K pump in the microdissected rat cortical collecting duct (CCD) and in Madin-Darby canine kidney (MDCK) cells, a line derived from the dog distal nephron. Dopamine inhibited pump activity in CCD by approximately 40%-50%, an effect proportionally larger than in the PCT. Unlike in the latter, the effect of dopamine was reproduced by the DA1 agonist fenoldopam, which inhibited the CCD pump in dose-dependent manner (maximum, 10 microM). The DA2 agonist quinpirole was without effect, either alone or in combination with fenoldopam. These actions on Na/K-ATPase paralleled in reciprocal fashion effects on adenylate cyclase: dopamine or fenoldopam, but not quinpirole, produced a significant increase in cAMP content, and the stimulation by dopamine was blocked by SCH 23390. Inhibitors of cAMP phosphodiesterase (3-isobutyl-1-methyl-xanthine and theophylline), as well as forskolin and dibutyryl-cAMP, mimicked the effect of dopamine on the pump, underscoring the role of increased cAMP in this phenomenon. Both dopamine and fenoldopam inhibited Na/K-ATPase activity in MDCK cells. The results indicate that besides the PCT dopamine inhibits Na/K-ATPase activity in cells of the distal nephron, where its effect on the pump appears to be more pronounced and is mediated by activation of the DA1 receptor. The natriuretic effect of dopamine is probably exerted at both proximal and distal nephron sites.

Both aldosterone and dexamethasone are known to stimulate renal Na-K-ATPase activity although their action is restricted to specific nephron segments: the collecting tubule, the target site for mineralocorticoids, and the thick ascending limb and distal convoluted tubule, the target sites for glucocorticoids. As this stimulation by corticosteroids is very fast, we attempted to establish whether it occurs through de novo synthesis of new Na-K-ATPase units or by increasing the specific activity of the Na-K-ATPase units already present. For this purpose we studied the effects of aldosterone and dexamethasone on Na-K-ATPase specific activity in microdissected nephron segments from adrenalectomized rabbits. This specific activity was determined by the ratio of ATPase activity over the apparent number of catalytic units, as measured by specific 3H ouabain binding. In the proximal tubule, neither adrenalectomy nor steroid replacement altered Na-K-ATPase activity or the apparent number of catalytic sites. In other nephron segments, adrenalectomy reduced Na-K-ATPase activity and specific 3H ouabain binding concomitantly, and therefore left this enzyme's specific activity unaltered. In the cortical and outer medullary collecting tubules, 10 micrograms/kg aldosterone simultaneously restored both the activity and apparent number of catalytic units of Na-K-ATPase to their control levels, and therefore did not modify the specific activity of the pump. Conversely, 100 micrograms/kg dexamethasone increased Na-K-ATPase activity in the thick ascending limb and distal convoluted tubule without changing the apparent number of catalytic units.(ABSTRACT TRUNCATED AT 250 WORDS)

Mineralo- and glucocorticoids stimulate renal Na-K-ATPase activity if given over a few days, but their immediate effect on the enzyme (i.e. within the time period required to alter electrolyte transport) remains controversial. We evaluated the short-term (3 h) in vivo effect of physiologic and pharmacologic doses of the natural glucocorticoid (corticosterone) and mineralocorticoid (aldosterone), and of a semisynthetic glucocorticoid (dexamethasone) on Na-K-ATPase activity in cortical collecting tubules microdissected from adrenalectomized rats. This nephron segment was chosen because it is a major target site for both classes of corticoids. Neither corticosterone (0.006, 0.6, and 5 mg/100 g body wt, IM), nor dexamethasone (5 mg/100 g body wt, IM) or aldosterone (10 micrograms and 50 micrograms, IV) altered significantly Na-K-ATPase activity in the cortical collecting tubule of these animals 3 h after administration. These results confirm our previous observations in the mouse, and suggest that the enhancing effect of corticosteroids on the renal enzyme seen after longer intervals represents a secondary phenomenon, possibly related to augmentation of the sodium load presented to the pump.

This study was initiated to define the dose- and time-dependence of triiodothyronine (T3) action on Na-K-ATPase in single microdissected nephron segments. For this purpose, the activity and the number of catalytic sites of Na-K-ATPase, as determined by the specific binding of 3H-ouabain, were measured following a single injection of T3 to rabbits thyroidectomized since 8-12 days. Triiodothyronine restored both the activity and the number of catalytic sites of Na-K-ATPase in a dose-dependent manner in all nephron segments where the enzyme was decreased following thyroidectomy, i.e., the proximal and the collecting tubule. At a dose of 50 micrograms/kg bw, T3 restored Na-K-ATPase activity and 3H-ouabain binding with the same kinetics. However, the kinetics depended on the nephron segments: in the proximal tubule, Na-K-ATPase stimulation occurred after a 12 h period of latency and was completed within 24 h whereas in the collecting tubule, the stimulation was biphasic with a first increase within the first 3 h and a second increase concomitantly to that observed in the proximal tubule. These results indicate that thyroid hormones regulate Na-K-ATPase activity by altering the number of catalytic sites of the enzyme. This control depends on two different mechanisms which differ by their time-dependence.

Aldosterone, when added in vitro to defined nephron segments, dissected from adrenalectomized rabbits, restores the depressed Na+-K+-ATPase activity in the thick ascending limb of the loop of Henle and in the cortical collecting tubule within 1 h of incubation. The direct effect is specific and, as demonstrated for the cortical collecting tubule, dose-dependent. The time course of aldosterone-mediated Na+-K+-ATPase activation is compatible with the conjecture of changes in the enzyme lipid environment.

The effects of long-term thyroidectomy and of subsequent triiodothyronine administration on kidney Na-K-ATPase were studied at the level of single nephron segments and were compared to the short-term effects previously reported. After 8-11 weeks, thyroidectomy resulted in a marked decrease in Na-K-ATPase activity in all the segments of the rabbit nephron, the proximal tubule, the thick ascending limb of Henle's loop, the distal convoluted tubule and the collecting tubule. Within this delay, thyroidectomy also decreased the ouabain-insensitive Mg-ATPase activity, the basal and hormone-stimulated adenylate-cyclase activity, and the volume of tubular epithelium in all the segments where these parameters were measured. Administration of 50 micrograms/kg body weight triiodothyronine to 8-11 weeks thyroidectomized rabbits did not restore Na-K-ATPase activity in any nephron segment within 48 h. These observations are different from those reported in animals thyroidectomized only 1 week before study since, within this latter delay, thyroidectomy altered specifically Na-K-ATPase activity, this action was observed on the proximal and collecting tubules exclusively and, triiodothyronine administration corrected Na-K-ATPase alterations after 48 h. Results of the present study indicate that in the long term, thyroidectomy has a wide spectrum of renal effects which involves the whole nephron and most cellular functions. The tubular involution induced by long-term thyroidectomy is probably responsible for the inability of kidney cells to quickly increase their Na-K-ATPase activity in response to hormonal stimulation.

Forskolin is a unique diterpene that may directly activate the catalytic subunit of adenylate cyclase. We therefore examined the effect of 50 microM forskolin on osmotic water permeability in rabbit cortical collecting tubules perfused in vitro. Forskolin increased net volume flux (Jv, from 0.30 to 1.22 nl/mm/min, P less than 0.02) in all tubules. The hydro-osmotic effect of forskolin was similar with respect to magnitude and time course to that produced by a maximal dose (250 microU/ml) of arginine vasopressin. An additive effect on Jv and Lp was not observed when maximal concentrations of forskolin and arginine vasopressin were given simultaneously. The compound d(CH2)5Tyr(Et) VAVP, which noncompetitively inhibits the vasopressin receptor, significantly reduced collecting tubular hydro-osmotic response to arginine vasopressin. In contrast, the hydro-osmotic response to forskolin was maintained in the presence of d(CH2)5 Tyr(Et)VAVP. However, the hydro-osmotic response to forskolin could be inhibited by 1.0 microM guanine 5'-(beta,gamma-imido) triphosphate (GppNHp) and by the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). These results demonstrate that forskolin exerts an hydro-osmotic effect in the mammalian nephron which occurs independent of the vasopressin receptor. Guanine nucleotide regulatory proteins may modulate the osmotic water permeability effect of forskolin. Finally, calmodulin is required for full expression of the effect of forskolin to increase osmotic water flux.

In order to investigate the presence of renal kallikrein, the localization of kallikrein-like proteolytic activity along a single nephron was determined in rabbits. Single nephrons were dissected into 8 segments under a microscope. Activity was fluorometrically measured with two different substrates (benzoyl-L-arginine ethyl ester: BAEE and prolyl-phenylalanyl-arginine-methylcoumarin amide: MCA). Proteolytic activity could be detected in the early (S1), the middle (S2), and the terminal (S3) portions of the proximal tubule and in the granular portion of the distal tubule (DCTg). With MCA, the specific activity in S1, S2, S3 and DCTg was 0.77 +/- 0.08, 0.28 +/- 0.10, 0.13 +/- 0.05, and 0.27 +/- 0.05 pmoles/microgram/min, respectively. The activity in DCTg was inhibited by aprotinin but that in the proximal tubules was not inhibited. No activity was found in the glomerulus, the thick ascending limb of Henle's loop, the bright portion of the distal tubule, and the light portion of the cortical collecting tubule. The inhibition of the activity by aprotinin in DCTg suggests that intrarenal kallikrein could be localized only in DCTg.

The medullary thick ascending limb (MAL), but not the medullary collecting tubule (MCT), has been shown to have an impaired adenylate cyclase (AC) responsiveness to ADH and a selective hypoplasia in Brattleboro diabetes insipidus (DI) rats. Since chronic ADH administration has been found to increase epithelium volume and basolateral membrane surface area in MAL but not in MCT, we investigated whether chronic ADH infusion would affect the hormone-sensitive AC and the Na-K-ATPase activity--two markers of the basolateral membrane--in single micro-dissected portions of thick ascending limb and collecting tubule in DI rats. Results indicate that 1. in MAL of ADH-treated rats, AC responses to in vitro AVP and glucagon and Na-K-ATPase activity increased to the same extent as did epithelium volume (60-80%); 2. changes in the other segments were independent of any morphological alteration. In the cortical thick ascending limb, AVP and glucagon-sensitive AC decreased by 30-40% whereas Na-K-ATPase activity did not change. In the collecting tubule, AC response to in vitro AVP was not altered by ADH-treatment but glucagon-sensitive AC dropped by 50% and Na-K-ATPase activity doubled, independently of any variation in plasma aldosterone and glucagon levels. These results show that, in the MAL, the ADH-induced variations in enzyme activity are a reflection of the enlargement of the basolateral membrane surface area. Further studies are needed to clarify the origin of enzymatic alterations in the other segments.

The effect of vasoactive intestinal peptide (VIP) upon adenylate cyclase (AC) activity has been determined in defined microdissected renal tubules isolated from collagenase-treated rabbit kidneys. In the presence of 10 microM GTP, 1 microM VIP gave marked stimulations of AC over basal values in the bright portion of the distal convoluted tubule (DCTb) (10.1-fold), and in the collecting tubule isolated from the inner stripe of the outer medulla (OMCTi, 7.8-fold). Less pronounced effects of VIP were found in the medullary collecting tubule isolated from the outer stripe (2.5-fold) and in the granular portion of the distal convoluted tubule (2.0-fold). VIP stimulation of AC activity in these segments amounted to 25 to 40% of the effect elicited by other agonists (arginine vasopressin, calcitonin or parathyroid hormone) in their respective target segments. A low response to VIP was observed in the cortical thick ascending limb (1.8-fold) which represented less than 5% of the calcitonin-stimulated AC activity. In the thin descending limb VIP produced a slight and variable stimulation of AC. VIP was without effect upon AC in the convoluted and straight portions of the proximal tubule, the medullary thick ascending limb and the cortical collecting tubule. Half-maximal stimulation of AC by VIP was observed at 26 +/- 10 nM (n = 3) in OMCTi and at 19 nM (n = 2) in DCTb. Related peptides glucagon, secretin and PHI gave lower stimulations of AC compared to VIP in OMCTi. Conversely for rat OMCTi, under identical conditions, glucagon was much more effective than VIP.

Na-K-ATPase activity was determined in 10 segments of the rat nephron using a fluorometric microassay method [4]. The enzyme activity showed three peaks (greater than 200 pmol ADP min-1 mm-1) along the nephron of normal rats. These peaks were in the S1 portion of the proximal tubule, the medullary thick ascending limb from the inner stripe and the distal convoluted tubule. Feeding the rats a low potassium diet for 8 weeks produced a significant decrease in Na-K-ATPase activity in the cortical collecting duct, but no significant change in this enzyme in any other segment. The low potassium diet did not produce a significant change in Mg-ATPase in any nephron segments. We conclude that Na-K-ATPase activity along the rat nephron shows a pattern that is qualitatively similar to that seen in the rabbit nephron [4]. However, quantitatively the Na-K-ATPase activity in the rat nephron is greater than in the corresponding segments of the rabbit nephron. The results are consistent with the greater rate of glomerular filtration and Na+ reabsorption per rat nephron. Furthermore, our results suggest that the decrease in potassium excretion during potassium deficiency is modulated, at least in part, by the level of Na-K-ATPase activity in the cortical collecting duct.