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J. Physiol. (1979), 295, pp. 353-363 With 4 text-ftgure Printed in Great Britain

NEPHRON ELECTROLYTE TRANSPORT AND SODIUM-POTASSIUM ADENOSINE TRIPHOSPHATASE ACTIVITY: INFLUENCE OF NICOTINE IN RAT AND RABBIT

BY MICHAEL HORSTER, TRAUDL KONIG, HEIDE SCHMID* AND UDO SCHMIDT* From the Physiologisches Institut der Universitit Mfinchen, Pettenkoferstrasse 12, D-8000 Mitnchen 2, Germany (Received 15 December 1978) SUMMARY

1. The influence upon mammalian renal epithelial transport of L-nicotine was studied in different types of experiments. 2. The kidney in situ (rat), when infused with nicotine (1 mg hr-' kg -1i'.), lowered its absolute and fractional K+-absorption significantly and reversibly, but Na+-absorption did not change. Effects on glomerular function and an irreversible effect upon epithelial Na+ and K+ absorption were prevailing at higher infused amounts (10 or 50 mg hr-1 kg-1). 3. Single dissected nephron segments (collecting tubule, rabbit) were perfused in vitro, and the Na+ and K+ transtubular net flux was measured while L-nicotine (50 ng/ml.) had been added to the contraluminal side of the epithelium. Both, Na+-absorption and K+-secretion were decreased reversibly. 4. The activity of the Na+-K+-activated ATPase was significantly decreased in the cortical collecting tubule and in the proximal convoluted tubule of the rabbit after incubation of single in vitro dissected nephron segments with L-nicotine (50 or 100 ng/ml.). In contrast, nicotine added to a homogenate of renal cortical tissue had no effect on the Na+-transport enzyme or on key enzymes of glycolysis and gluconeogenesis. 5. These observations on the kidney in situ and on defined, perfused and nonperfused nephrons in vitro suggest that L-nicotine has dose-dependent, direct epithelial and mediated systemic effects on mammalian renal ion transport. INTRODUCTION

The abundant clinical, pharmacological, and toxicological work on the effects of the widely used drug tobacco smoke upon mammalian tissues (Larson & Silvette, 1968, 1975; Schievelbein, 1968) offers surprisingly little, if any, information concerning the influence of its constituents on ion transport of epithelial cells; mechanisms of cellular action of nicotine have been studied particularly in exitable tissue. However, transport of H+ and of Cl- by the isolated frog mucosa is reversibly * Present address: Pathologisches Institut der Universitat Tubingen, Liebermeisterstrasse 8, D-7400 Tubingen, Germany. 0022-3751/79/5280-0905 $01.50 © 1979 The Physiological Society 12

PHY 295

M. HORSTER, T. K6NIG, H. SCHMID AND U. SCHMIDT inhibited by nicotine (5 mM) in the serosal bath solution (Dinno, Ando, Dinno, Huang & Rehm, 1977). Nicotine, therefore, appeared to be suitable for a study of correlates between epithelial ion transport and cell enzyme activities in the mammalian kidney. The infusion of L-nicotine was used to study direct and mediated effects upon electrolyte absorption by the entire organ in 8itU. The concentration at the epithelial cell in this type of preparation is unknown. The application of nicotine (L-) to the in vitro perfused and to the non-perfused nephron segment served to evaluate the direct cellular action at concentrations similar to those in human blood during inhalation of tobacco smoke. 354

METHODS

In vitro perfumed and non-perfu8ed nephron segments The general methods applied in this study have previously been reported from this laboratory (Horster & Larsson, 1976; Horster & Schmidt, 1978; Horster, 1978a, b). The description will stress recent improvements of the microdissection and in vitro microperfusion techniques. Microdi88ection of nephron segments. Renal slices were cut from rabbit kidneys and plunged from the knife into a special Krebs-Ringer solution (see below) with 5 vol. % rabbit serum added, kept at 4 00 and constant pH (7.4). Slices were transferred from this solution into a thermostatically controlled dissection chamber, containing the bath medium at identical conditions but without protein. All solutions were kept at pH 7-4 by adjusting the flow rate of 95 % 0,/5 % C00. During dissections of individual nephron segments, proteolytic enzymes such as collagenase or trypsin have not been used. These enzymes are commonly applied in mashed 'isolated tubule' fragment preparations or in renal cell preparations; besides not being necessary for microdissection in the rabbit they are, above all, harmful in various tissues since not only the basement membrane but moreover cell membrane receptor properties may be altered. The dissection chamber is illuminated by darkfield transmission light (Wild-Heerbrugg). Tweezers are used for dissection at x 20 to x 70 magnification under a zoom-stereomicroscope (Leitz; Wild). The techniques used or developed in this laboratory for dissecting nephron segments from fresh renal slices are specific for each segment in cortex and medulla. In vitro perfusion of the collecting tubule. The dissected nephron segment was slowly aspirated into a fluid-filled special pipette and transferred into the perfusion chamber. The chamber had been constructed for in vitro nephron perfusion and built into a recently developed inverted microscope (Zeiss IM 35) to allow optical control of the nephron segment during perfusionanalysis at x 20 to x 630 magnification. The bath medium in this chamber was identical to that used for dissection, cooled to 15 00 until the collecting tubule had settled on the cover glass, and warmed up to 37 00 thereafter. Glass pipettes used for perfusion of individual nephron segments in vitro and for collection of the perfused fluid were prepared on a microforge (Stoelting) to fit coaxially. The perfusate could be exchanged within 10 sec to apply a solution containing L-nicotine (50 ng/ml. of perfusate) in some experiments while the nephron segment remained in place. Each collecting tubule in all experiments at different bath concentration of L-nicotine, and in the few tubules in which the substance was applied luminally, served as its own control. The pipettes holding the tubule and the tubule ends were sealed off from the extratubular medium by larger pipettes containing a highly viscous silicone preparation (Sylgard). The collecting tubule was perfused either by pump (Sage) or by gravity from a fluid reservoir at rates of 8-12 nl./min. Perfusate composition (mM) was: NaCl 150-0; K2HPO4 2-5; MgSO4 1-2; CaCl2 1-0; pH 7-4. Bath composition was: NaCl 115-0; NaHCO, 25-0; KC1 5-0; CaCl2 1 0; MgS04 1-2; NaH2PO4 1 0; Na acetate 10 0; glucose 5X5; all bath solutions were constantly gassed with 02/CO2 (95 %/5 %) to maintain pH (7-4) which had been set by HCl-titration, and to provide adequate stirring. Dialysed t14C]inulin (5-15 mc/m-mole; Amersham) had been added to the perfusate. The protocol for dialysis, however, had not been sufficient to eliminate all of the inulin-free 14C activity. Therefore, another isotope, [3H]inulin (NEN), was dialysed completely (polysaccharide membrane; Carbide Union)

NEPHRON FUNCTIONS AND NICOTINE

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and used simultaneously for volume flux measurements in two cortical collecting tubules. The bath leak activity of 14C could now be quantified and the luminal 14C activity was corrected, using the recovery of [3H]inulin which was virtually complete, to calculate the transmural net volume flux. Tubule fluid analy~is. The fluid collected from the single perfused collecting tubule was taken up directly into a calibrated constriction pipette. The fluid samples were delivered individually into a special Petri-dish filled with mineral oil. The concentrations of Na+ and of K+ were measured in 4-5 nl. fluid samples using a helium-glow photometer (Aminco). The accuracy of the analysis was 2*3% (S.D.) for Na+ and 6-4% for K+ in multiple determinations of standard solutions within the pertinent concentration range. Na+ and K+ concentrationsoflthe perfusate and bath bulk solutions were measured by flame photometry (Zeiss), chloride concentrations were measured by titration (Eppendorf). The total osmotic pressure, as determined in a laboratory osmometer (Roebling), was equal on both sides of the epithelium. The accuracy of the in vitro perfusion methods was evaluated by perfusing ["4C]inulin (5-15 mc/m-mole) directly from the perfusion pipette into the collection pipette. During this procedure, carried out before and following the experiment, the pipette tips were covered with a drop of Sylgard (Dow Coming) to prevent fluid evaporation. This perfusate was collected quantitatively and analysed for ["4C]inulin concentration in a Liquid Scintillation Counter (Packard 3320). The bath fluid (about 2 ml.) was changed routinely every 8-10 min and analysed for apparent [I4C]inulin activity throughout the experiment. The leak flux of 14C, but not inulin, into the bath during successful perfusions was in the range of 1-2 % of the perfused amount. The data from the tubule fluid analysis are expressed as mean values (+ S.D.) of the pre-experimental control periods, post-experimental control periods, and the mean values during tubule perfusion while the bath contained L-nicotine (50 mg/ml.) Each of these periods lasted for about 60 mi. Analy1s8 of nephron Na-K-ATPase activity. Single micro-dissected nephron segments, the proximal convoluted tubule and the cortical collecting tubule, were transferred individually from the dissection chamber into special Petri-dishes containing the bath medium at 37 TC and pH 7-4. Three different concentrations of L-nicotine were used for incubation and prepared for each experiment: 0, 50 and 100 ng/ml. of bath medium. At least three individual nephron segments of the proximal and cortical tubule from each kidney \were incubated Ifor 90 min in each of these solutions. At the end of the incubation period, the tubule was transferred by forceps directly into a drop of 1 pl. volume of bidistilled water on a specially prepared glass slide. All transfer procedures were carried out under direct observation through a stereomicroscope ( x 20 to x 140 magnification). Each glass slide, containing two nephron segments in separate drops of water, was set on an aluminum block in dry ice. The nephron segment froze immediately upon contact. Several glass slides were processed this way, stored into a special aluminum rack carrier, and placed into a Pyrex glass vacuum tube. The subsequent steps, including lyophilization, weighing of the tubule, as well as the enzymatic cycling system have previously been described (Schmidt & Horster. 1977). The enzyme activity was expressed in moles of inorganic phosphate liberated per kilogram dry weight of tissue per hour at 37 'C (MKH units). Na+-EK-ATPase was calculated by subtracting the ouabain-insensitive Mg2+ATPase from the total Na-K-ATPase. Renal Na+- and K+-ab8orption. These studies were carried out on Wistar rats (n = 19), body weight 150-260 g. The animals (male, n = 11; female, n = 8) had been kept on a normal laboratory diet (Altromin standard; tap water). Anesthesia was initiated with intraperitoneal Na pentobarbitone (2.5 mg/t00 g body wt.) and was maintained with i.v. Na pentobarbitone (Ringer solution 1-25 mg/ml.). The animal was prepared for analysis of renal functions as described previously (Zink & Horster, 1977). Total volume administration (Ringer solution) was 06-0-8 ml./hr per 100 g of body wt. The inulin (Inutest, Laevosan) concentration was adjusted to maintain a plasma inulin level of 100-120 mg/100 ml. plasma. L-nicotine was given i.v. at 1 mg hr-1 kg-', as well as at 10 or 50 mg hr-' kg-'. The standard protocol was as follows: after inulin equilibration within the extracellular space (about 45 min), three to five pre-experimental control collections of final urine were made. Each collection period was 15-20 min. L-Nicotine infusion was begun and after an equilibration period of 20 min, four to six experimental collections were made, followed by three to five post-experimental urine collection periods. Clearance values of 12-2

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inulin and absolute absorption of electrolytes were measured and calculated as described previously (Horster & Valtin, 1971; Zink & Horster, 1977). Results have been expressed as mean value of pre- and post-experimental control periods which were not different, and as mean value of all experimental periods (± S.D.).

Renal cortical enzyme analysis (1) Na+-K+-ATPase. Lyophilized rat kidney slices were homogenized in a cooled acqua bidest.solution (DAB 7) containing serum albumin (0-05 %). The final tissue concentration was 100 jag/ 100 ul. of solution. The reagents and analytical procedures have been described in detail (Schmidt & Horster, 1978) and are summarized below. Reagent for total ATPase (A): Tris-HOl-buffer, 0-1 M, adjusted to pH 7-4; NaCl 54-6 mm; KCl 4-9 mx; MgCl2 2 mM; EDTA 0-1 mm; bovine albumin 0-05 mm. The reagent for the ouabain-insensitive ATPase (D) was similar except that the KCl was omitted and ouabain (0-01 M) was added. Analytical procedures: homogenate (5 u1.) was incubated with medium A or medium D, with or without nicotine (0-4-5-0 mm) for 15 min at 36 'C. The reaction was stopped by boiling, cooled in an ice bath, and the Pi-reagent (200 jl.) was added. Standard solutions of Pi (5 to 20 x 10-i M) were carried throughout the analysis. In an additional analysis, homogenate (2 id.) was added to 18 j1. nicotine and incubated for 30 min at 37 0C. Then, incubation medium (5 jel.) was added to medium A or medium D (245 ul.). (2) Fructoee-1,6-biephosphlate (FBPase). The tissue homogenate was prepared as above. The reagent was: Tris-HCl-buffer 0-1 mm, pH 8-0; MgCl2 5 mM; NADP 0-5 nm; mercaptoethanol 24 mm; phosphoglucoisomerase 20 #sg/ml. (3-5 #u./ml.); glucose-6-p-dehydrogenase 10 ,zg/ml. (0.7 .s./ml.); fructose- 1,6-bisphosphatase 0-6 mM; bovine serum albumin 0-05 %. Also, NADPH standard solutions (0-7 to 3-0 x 10-M ) were used throughout the procedure. Homogenate (5 ,ul.) was added to 200 jl. of the reagent, with or without nicotine (as above), and incubated for 30 min at 37 0C. The reaction was stopped and aliquots of NaOH (0-1 N; 150 ,ul.) were added. The resulting flourescence was read at 460 nm after excitation at 340 nm, as previously reported in detail (Schmidt, Schmid & Guder, 1978). (3) Pho8phofructokinase. The tissue homogenate was prepared as above. The reagent was: Tris-HUl-buffer, 0-1 M, pH 8-0; fructose-6-phosphatase 2 mM; ATP 1-8 mm; NADH 0-2 mM; MgCl2 5mM; 5'-AMP 1mM; amytal 2mM; NaHPO4 20mM; bovine serum albumin 0-05% aldolase 2-5 jg/ml.; glycero-p-dehydrogenase/triose-p-isomerase 2-5 jug/ml. The fructose-1,6bisphosphatase standards (0-3 to 1-2 x 104 M) were carried throughout the procedure. Homogenate (1O jl.) was added to reagent (190 j1.) with or without nicotine as above, and incubated for 30 min at 37 0C. After the reaction-stop, HCI (1 N; 2 ,l.) was added to the incubation medium to destroy the excess NADH; the indicator reagent (NaOH 6-6 N, 200 ,1. with 10 mm-imidazole) was added to 8 #1. incubation medium and incubated for 10 min at 60 0C. Fluorescence was read at 360 nm after excitation at 340 nm (Farrand). RESULTS

Na+- and K+-transport across the epithelium of the collecting tubule Individual experiments (n = 9) and Na+ net transport within each period are depicted in Fig. 1 and summarized in Fig. 4C. The mean value of pre-experimental control sodium absorption (n = 32) was 7-76 + 2-97 (S.D.) equiv cm-' sec-1 10-12; post-experimental control was 6-98 + 2-34 (n = 27). In contrast, Na+ absorption was significantly (P < 0-002) lower, compared with the pre-experimental control, in all but one collecting tubule during the experimental period (n = 28) when L-nicotine (50 ng/ml.) had been added to the medium in contact with the tubule basement membrane. Na transtubular net flux was 2-61 + 1-15 equiv cm-1 sec-1 10-12. The fractional change (%) ofNa+ net transport induced by L-nicotine was apparently not related to the level of control net flux. Restoration of Na+ flux during the post-

NEPHRON FUNCTIONS AND NICOTINE

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Fig. 1. Influence of L-nicotine (50 ng/ml.) in the bath medium upon Na+ net transport across the in vitro dissected and perfused cortical collecting tubule (rabbit). Mean values of control and experimental periods (n = 87) in the same tubule (n = 9) are linked by continuous and dashed lines.

TABiE 1. K+ secretion of the isolated, in vitro perfused collecting tubule (rabbit) and the effect of L-nicotine (50 ng/ml.) in the bath solution K+ net flux (bath to lumen) (l1wl12equiv cm-' sec-1) Pre-control 2-35 ± 0-47 (5) Experimental 0-71 ± 0-62 (5) Post-control 1-83 ± 0-60 (5) No. of experiments in parentheses.

experimental period was incomplete in most tubules (- 12 %), although the difference between experimental and post-experimental phase was as significant (P < 0-002), as between the experimental and pre-experimental phase. This observation is unlikely to be related to the natural course of an in vitro preparation; rather, the substance per se may not have been removed completely from the basolateral cell surface despite several exchanges of the bath medium, or the effects of the substance on transport last longer than the presence of the agent. When L-nicotine (50 ng/ml.) was applied with the perfusate (n = 4) to the luminal cell surface, it had no effect upon Na+ net flux across the collecting tubule. K+ net transport in the collecting tubule (n = 5) is listed in Table 1 as the mean flux rate (n = 27) for each of the three periods. The effect of nicotine (50 ng/ml.) was reversible and significant (P < 0-002). However, the post-experimental control values (post-control) -were smaller than the pre-experimental periods (P < 0-05). The secretion of K appeared to be affected more than the absorption of Na. Net fluid flux across the cortical collecting tubule (n = 14) was 0-027 + 0-020 nl. min-'.mm1.

358

M. HORSTER, T. KONIG, H. SCHMID AND U. SCHMIDT

Na+-K+-ATPase activity in single nephron segments The enzyme activities of the proximal convoluted tubule and the cortical collecting tubule are given individually in Fig. 2. and have been summarized in Fig. 4B. Control enzyme activities of this series were 6*94 MKH + 0X89 (n = 5) for the proximal tubule, and 5*67 + 1 26 (n = 6) for the cortical. These values are in close agreement with those reported in previous studies (Schmidt, Schmid, Funk & Dubach, 1974; Schmidt & Horster, 1977, 1978). The enzyme

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from the same kidney, when incubated for 90 min in the medium to which L-nicotine (50 ng/ml.) had been added, was drastically lowered (Fig. 2). The mean value (MKH) was 1-91 + 0-77 (n = 8) for the cortical tubule, and 0*39 + 0-28 for the proximal. A further and sugnificant (P = 0.02) reduction of enzyme activity in the cortical tubule (n = 7) to 1 10 + 0*38 MKH was observed after incubation of single dissected tubules in a higher concentration of L-nicotine (100 ng/ml.). Enzyme activities of the proximal tubule, in contrast, were not different at these two concentrations; also, mean values at 50 and 100 ng/ml. (0.32 + 0.20 MKH, n = 5) were not different from zero. In two experiments, as indicated in Fig. 1 and 2 by filled circles, collecting tubules were analysed for Na+-K+-ATPase activity following the in vitro perfusion of

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the segment (90 min) while the bath medium contained L-nicotine (50 ng/ml.). As might have been inferred from the non-perfused cortical collecting tubule, both the sodium flux and the Na+-K+-ATPase activity within the same tubule were decreased.

L-nicotine on renal Na+ and K+ transport These experiments on the rat kidney in situ are not directly comparable with those on single dissected in vitro perfused tubules of rabbit, because the concentration of the substances under study at the site of action is unknown. The concentration of L-nicotine in a modified Ringer infusate at pH 7 40 was varied in an inital series until

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Fig. 3. Influence of I.v. application of L-nicotine (1 mg hr-4 kg-l) upon in 8ictu renal Na (Fig. 3A) and K+ (Fig. 3B) absorption (rat). The regression lines (control) of Na+ orKe absorption have been calculated from the pre. and post-experimental control periods. Values from experimental periods of Na+ absorption (Fig. 3A) fall upon the control regression line, whereas those for K+ absorption (Fig. 3B) are significantly lower. The range of tubular load and of absorption is due to differences in renal weight.

a significant and reversible effect upon renal ion transport was evident. The amount of L-nicotine infused in all experimental periods was 1 0 mg hr-' kg-1 body wt, corresponding to 3.5 jutg min' in the animals of this series. Ren absorption ofNad was99360/n al 0 + 32) ofthefiltered load(equivin-i (1 m 10) mn pre- and post-experimental controls (n = 81). During the experimental periods (n = 32), renal Na+ absorption was almost identical (99.30A%;±l0.32). In Fig. 3, the range of tubular Na+ and K+ load (abscissa) and corresponding renal Na+ and K+ absorption ordinatee) is due to differences in renal weight. In contrast to renal Na+ handling, the absorbed amount of K+ was importantly lowered by L-nicotine when compared with control absorption (Fig. 3B). Specifically, 98w57 3 (± 2.94) of the filtered K+ were absorbed during control periods ('control' line in Fig. 3B), whereas a significantly (P = 0001) and consistently smaller amount of 81r 23 % (± 7.35) was absorbed during the experimental phase (n = 39). However, the recovery from the nicotine effect was not always complete; mean post-experimental control was 94.79 % ( + 2-78). Thus, 17 34 % of the filtered amount of potassium were excreted under the influence of L-nicotine (1 mg hr-1 kg-'). To test the total effective range of concentration, nicotine was infused at 10 or

M. HORSTER, T. KONIG, H. SCHMID AND U. SCHMIDT 360 50 mg hr-4 kg-1 in four experiments. As well as the absorption of potassium and sodium being reduced, the glomerular filtration rate was decreased by 34.7 % (± 18-2) when compared with the pre-experimental control. This effect was not reversible within the post-experimental periods of at least 90 min duration. Enzyme activities in rat renal cortex The substance L-nicotine was also studied in homogenate preparations of rat renal cortex. The range of concentrations used (0-10 mM) was similar to that in other TABSx 2. Effect of L-nicotine on enzyme activities in rat renal cortex L-Nicotine (mM) 0

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protocols of this series. Standard deviations of enzyme activity values were consistently below 20 % of the mean values listed in Table 2. Neither the activities of the transport enzyme Na-K-ATPase nor those of key enzymes of gluconeogenesis (fructose-bisphosphatase) and glycolysis (phosphofructokinase) were significantly altered by L-nicotine in this type of tissue preparation. DISCUSSION

The effects of nicotine on renal epithelial function observed in this study might offer some insight into its cellular mode of action. Ion transport. The net fluxes of the Na+ and K+ ion across the in vitro perfused renal collecting tubule decreased within 45 min when L-nicotine (50 ng/ml.) was in contact with the basolateral cell membranes. After removal of the substances from the bath, ion transport rates returned to near control level within 45-60 min (Fig. 1 and Table 1). The values for Na+ absorption and K+ secretion during control flux confirm those of a previous study under similar experimental conditions (Stoner, Burg & Orloff, 1974). A qualitatively similar response to nicotine was found in situ when the substance was applied (1 mg hr-1 kg-') intravenously; renal net transport of K+ decreased reversibly (Figs. 3B, 4A). At higher infusion rates (10 mg hr-1 kg-') of nicotine, the in situ transport of both potassium and Na+ decreased, as did the glomerular filtration rate. Glomerular and tubular effects were irreversible at an infusion rate of 50 mg hr-1 kg-'. These two sets of experiments suggest that L-nicotine may affect an epithelial transport mechanism which Na+ and K+ have in common. The isolated, in vitro perfused renal tubule provides complete control of the compartments bordering the

NEPHRON FUNCTIONS AND NICOTINE 361 luminal and basolateral cell surfaces. The bath concentration of nicotine during perfusion analysis was defined (50ng/ml.) and kept constant; the tissue to bath mass relation (2 x 10-1 ng vs. 2 x 106 ng) is likely to have prevented any important metabolic conversion of the substance under study. The changes of Na+ and K+ net transport in the single tubule seem to indicate that a direct and reversible, inhibitory effect of nicotine might occur on some transport component at the basolateral membrane. For the in situ preparation, the concentration of L-nicotine in the kidney appears to be important, as inferred from the wide range of tubular and vascular effects; however, the effective concentration of the substances at the epithelial cell can not be quantified in this type of preparation. Nicotine, given at a rate of 1 mg hr-1 kg-1, decreased renal net K+ transport without affecting net Na+ transport. This selective effect of nicotine upon renal K+ and Na+ transport deserves attention (Fig. 3). It has been observed in the isolated mucosa of the frog stomach that nicotine causes a marked inhibition of net H+ transport (Dinno et al. 1977). The well established relation of renal H+ and K+ net transport, on the other hand, is thought to depend on the intracellular H+ concentration (Malnic, de Mello Aires & Giebisch, 1971) or on the luminal H+ concentration (Boudry, Stoner & Burg, 1976). In the light of these proposals, the reduced net K+ transport in the present in situ and in vitro studies might be interpreted as follows: a decrease in H+ secretion by nicotine might have caused the alteration in K+ transport rate in situ. In contrast, the concomitant changes in the single collecting tubule of Na+ transport, of K+ transport (Table 1), and of Na+-K+-activated ATPase (Figs. 2, 4B) point to an additional or primary effect of nicotine (50 ng/ml.) on the Na+-extruding enzyme system. Enzyme activities. The analysis of the Na+-K+-activated ATPase (Figs. 2, 4B) in the same nephron segment lends support to the notion of a direct and primary effect of nicotine on epithelial Na+ and K+ transport. The enzyme activity decreased significantly (P < 0*002) during the incubation of the dissected segment (nonperfused, n = 6; perfused n = 2) with L-nicotine (50 ng/ml., i.e. 3-1 x 1o-7 M). The further change in enzyme activity after incubation in 100 ng/ml. was not significant. The action of nicotine was not restricted to the collecting tubule; the proximal tubule, an epithelium of different biophysical and biochemical properties, but comparable in Na+-K+ATPase activity (Schmidt & Horster, 1977), responded in a similar way to the substance. The Na+-extruding enzyme and key enzymes of glycolysis and gluconeogenesis were also measured in cortical homogenates of the rat kidney (Table 2). The failure of this preparation to respond to nicotine might at first sight be disturbing in view of the effect of nicotine in the single intact nephron segment. However, this seeming discrepancy emphasizes that the structural integrity of epithelial cell membranes is a prerequisite for receptor-enzyme interaction. The preparation of membrane fraction by homogenization or by proteinases, such as collagenase and trypsin, is known to destroy or inactivate specific receptor functions of transporting cell membranes (Schmidt & Horster, 1978). The influence of tobacco smoke constituents upon ion transporting epithelia in rat and rabbit does not imply identical effects on other tissues or species, such as human. The ability to detoxify nicotine has been shown to differ not only between organs of the same species, but also between species: the

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Fig. 4. Synopsis of the principal effects Of L-ncotine upon renal tubular electrolyte transport and enzyme activity. A, influence of i.v. application Of L-nicotlne upon Na+ and K+ absorption in the rat kidney. B, changes in the activity of Na+-K+-ATPase of single dissected nephron segments (rabbit; proximal tubule, cortical collecting tubule), when L-ncotine (50 or 100 ng/ml.) had been added to the bath medium in vitro. 0r proximal; 0d collecting. C, effect Of L-n cotine (50 ng/ml.) in the bath medium upon Nah net transport rate in the dissected, in tro perused cortical collecting tubule of the rabbit. The effects of L-nicotine on Kt net transport in the cortical collecting tubule are listed in Table 1. It is noteworthy that cotinine, the major oxidation product of nicotine which is biologically active but less toxic than nicotine (Larson & Silvette, 1975), was tested in similar clearance experiments (eight rats) and by homogenate enzyme as -ATPase analysis of cortical tissue: renal Na+ and K+ absorption as wellNaW-K activities were not altered at concentrations of 25 mg hr-' kg-' (i.v.) and of 0*0510.0 nmm-cotinne (medium). rate of renal detoxification is higher by a factor of 5 in the rabbit when compared with the rat (in: Schievelbein, 1968). However, the substance appears to be an interesting tool for further studies on the quantitative relationship between ion transport rates (Na+, K+, H+) and enzyme activities in mammalian cells. Exposure of epithelial cells and tissues in culture to the nicotine stimulus could yield additional information on chronic metabolic changes. Part of this work has been presented in abstract form at the I. Wissensch. Sympos. Forschungsrat Rauchen und Gesundheit, Hamburg, West Germany, 1978. Nicotine (L-) was kindly

prepared (500 mg/ml.) and tested gas-chromatographically by Dr R. Hallermeyer. This work was supported financially by the Forschungsgesellschaft Rauchen und Gesundheit mbH,

Hamburg.

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