Journal of the American Society of Nephrology 1995

Isoproterenol Induces Mitogenesis in MCI and LLC-PKTubular Cells1

Gunter Wolf2 and Eric G. Neilson

G. Wolf, Department of Medicine, Division of Nephrol-

ogy and Osteology, University of Hamburg, Ham-

burg, Germany

E.G. Neilson, Penn Center for Molecular Studies ofKidney Diseases, Renal-Electrolyte Division of the De-

parfment of Medicine, University of Pennsylvania, Phil-adelphia, PA

(J. Am. Soc. Nephrol. 1994; 4:1995-2002)

ABSTRACTThis study assessed the effects of exogenous isopro-

terenol on the proliferation of the proximal tubularcell lines MCI and LLC-PK1. Both cell lines express /3-adrenergic receptors as demonstrated by Scatchardanalysis of binding data, receptor-cross linking stud-

es, and mRNA expression for /32-adrenergic recep-tors. Isoproterenol (1O� M) for 15 mm stimulated the

formation of intracellular cAMP in MCT cells (controls,

8.0 ± 0.7; isoproterenol, 12.6 ± 0.89 fmol of cAMP/pg

of protein; P < 0.01). This effect was blocked by the/3-receptor antagonist propranolol (106 M). Isopro-

terenol, in a dose-dependent manner, also inducedproliferation in MCI and LLC-PK1 cells, as measured

by (3H)thymidine incorporation and direct cellcounts. Time-course experiments demonstrated

maximal mitogenesis 48 h after a single dose of I 0�M isoproterenol. This mitogenic effect was mimickedby a stable cAMP analog or cholera toxin, but notby a cGMP analog, indicating that the isoproterenol-mediated growth effects are likely caused by cAMP.These results provide evidence that isoproterenol isa mitogenic growth factor for cultured proximal tu-

bular cells. These findings may be important in the

growth mechanisms involved in the proliferative re-modeling of injured tubules after acute renal failure.

Key Words: Tubular cells. cAMP, f3-recepfors, mitogenesis.

propranolol

I Received March 30. 1993; accepted December 14, 1993.

2 correspondence to Dr. G. wolf, Department of Medicine, Division of Nephrol-

ogy and Osteology, University of Hamburg, University Hospital Eppendorf. Pavil-ion 62. MartinistraMe 52, D-20246 Hamburg, Germany.

1046-6673/0412-1995$03.OO/OJournal of the American Society of Nephrologycopy�ght a) 1994 by the American Society of Nephrology

A lthough proximal tubular cells have a very lowturnover rate under normal conditions, they

have an intrinsic ability to proliferate and regeneratefunctional tubular tissue after ATN ( 1 ,2). This abilityof tubular epithelium plays a central role in the self-healing and survival of the kidney subsequent toacute damage (2). Although several factors includingepidermal growth factor (3), transforming growthfactor-a (4), hepatocyte growth factor (5), and insulin-

like growth factor- 1 (6) have been identified as pos-sible mitogens for cultured proximal tubular cells,their definitive role in tubular proliferation in vivo isnot totally clear. Moreover, it remains controversialwhether the actual concentration of these factor isenhanced in the tubulointerstitial microenvironmentduring regenerative tubular growth (1,2,7).

We have been interested in the possible growth-modulatory actions of vasoactive factors (8). In this

regard, we have previously demonstrated in a seriesof experiments that angiotensin II is not mitogenic,but rather induces cellular hypertrophy of the murineproximal tubular cell line MCT (9, 1 0). To further

extend the hypothesis that vasoactive substancesmay act as tubular growth factors, this study wasperformed to test possible growth effects of the ad-renergic substance isoproterenol in the two tubularcell lines MCT and LLC-PK1 in culture. Our datademonstrate that these cell lines express adrenergicreceptors at the mRNA and protein levels and that

isoproterenol induces mitogenesis through a recep-

tor-mediated mechanism. It is likely that an increasein intracellular cAMP is responsible for this prolif-eration.

MATERIALS AND METHODS

Cell Culture

Studies were performed in the murine proximaltubular cell line MCT and in pig LLC-PK1 cells. MCTcells are a differentiated proximal tubular cell line,harvested originally from the renal cortex of naiveSJL (H�2S) mice (1 1). They have been extensivelycharacterized in our laboratory and express manyfeatures of proximal tubular cells (6,9- 1 2). LLC-PK1cells are a permanent, well-characterized proximaltubular cell line from pig ( 1 3- 1 5) with some charac-teristics of cells from the distal nephron (see refer-ence 36). LLC-PK1 cells were obtained as passage 200from Dr. Falk Fahrenholz (Max Plack Institute ofBiophysics, Frankfurt, Germany). Both cell lines

Isoproterenol and Tubular Cells

1996 Volume 4 - Number 12 - 1994

were grown in Dulbeccos’s modified Eagle’s medium(DMEM; Gibco, Eggenstein, Germany) supplementedwith 100 u/mL penicillin, 100 �g/mL streptomycin,and 1 0% heat-inactivated fetal calf serum (Gibco) at37#{176}Cin 5% CO2. MCT cells were passaged every 4 to5 days, and LLC-PK1 cells were passaged every 7 to

1 0 days by light trypsination.

Receptor Binding Studies

To test for the expression of /3-adrenergic receptors,binding studies were performed with [3H]propranolol.Subconfluent monolayers of MCT and LLC-PK1 cells

In 24-well plates (original plating density, 5 x 1 0�cells per well) were incubated in DMEM with 1 % BSA,

1 mM phenylmethylsulfonyl fluoride (Sigma, Deisen-hofen, Germany), and 10 U of aprotinin (Sigma) withvarying concentrations of cold propranolol (Sigma:

redissolved in medium) and 2 �L of L-[4-3Hjpropran-olol (23.4 Cl/mmol; New England Nuclear, Dreieich,Germany) at room temperature (22#{176}C)on a shaker for1 h. Nonspecific binding was determined in the pres-

ence of a 1 00-fold excess of cold propranolol and wasless than 1 5% of total bound radioactivity. At the end

of the experiments, cells were washed three timeswith ice-cold phosphate-buffered saline (PBS) con-taming 1 mg/mL BSA, and cell-associated radioactiv-Ity was measured by scintillation spectroscopy aftercells were solubilized in 1 N NaOH with 0.5% Tritonx- 1 00. Nonspecific binding was subtracted from to-tal counts, and data for bound versus free propran-olol were analyzed as previously described with thecomputer program Enzfltter (9: Elsevier-Biosoft,Cambridge, United Kingdom). Results are presentedas Scatchard blots. Each point is the mean of threeto four independent experiments, each performed in

triplicate.To further evaluate specific binding in MCT cells,

receptor cross-linking studies were performed. MCT

cells (5 x 1 0�) were incubated in serum-free DMEM

containing 1 % BSA with 1 00 �L of [3Hjpropranolol (4x 1 o-� M) in the presence and absence of 1 O� M coldpropranolol for 2 h at room temperature. At the endof the incubation period, cells were washed twicewith cross-linking buffer (0. 1 M HEPES, 0. 1 2 M NaC1,5 mM KC1, 1 .2 mM MgSO4, 8 mM D-glucose, and 10mg/mL BSA). The bound [3H]propranolol was cova-

lently cross-linked to its receptor by a 1 5-mm incu-bation with 1 0_6 M freshly prepared disuccinimidylsuberate (Pierce, Rockford, IL) in cross-linkingbuffer. The reaction was terminated by the addition

of 0. 1 M TrIs-HC1 (pH 8.8). The cells were washedwith PBS and solubilized in sodium dodecyl sulfate(SDS)-loading buffer (60 mM Tris-HC1, pH 6.8: 2%SDS, and 100 mM dithiothreitol). Samples wereboiled for 1 0 mm and centrifuged, and the proteinconcentrations were measured in the supernatantsby a modification of the Lowry method, which is

insensitive to the concentrations of SDS and dithio-

threitol used (16). Equal amounts of protein (5 �tg/lane) were loaded on an SDS- 1 0% polyacrylamide

separation gel with a 4% stacking gel. High-molecularRainbow@ markers (Amersham, Braunschweig, Ger-many), which comprise 14,300 to 20,000 d, wereused as standards. At the end of the electrophoresis,the gel was soaked in Amplifye (Amersham) and

fluorographed at -70#{176}Cfor 7 days.

Northern Blots

To test for the expression of /3-adrenergic receptortranscripts, northern blots were performed. PolyA�mRNA was isolated from 106 MCT and LLC-PK1 cellswith the FastTracke kit (Invitrogen, San Diego, CA).PolyA� mRNA (1 0 �g) was denatured in formamide/formaldehyde at 65#{176}Cfor 20 mm and separated on a1 .2% agarose gel containing 2.2 M formaldehyde. TheRNA was subsequently vacuum blotted to a nylonmembrane (Zetabind, Cuno, Meriden), UV cross-linked, and prehybridized overnight at 42#{176}Cin 5xSSPE (20X SSPE: 3.6 M sodium chloride, 0.2 M so-dium phosphate, 0.02 M EDTA: pH 7.4), 5x Den-

hardt’s (1 OOx Denhardt’s: 2% Ficoll 400, 2% polyvi-nylpyrrolidone, 2% BSA), 1 50 �g/mL sonicated and

denatured salmon sperm DNA (Sigma), 0. 1 % SDS,and 50% formamide. A 2-kilobase (kb) EcoRI frag-ment of the clone pTF3, which encodes the human

/32-receptor originally cloned from placenta, was sep-arated from its plasmid in low-melt agarose ( 1 7). ThiscDNA cross-hybridizes with murine /3-adrenergicreceptors ( 1 7). The fragment was radioactive labeledwith hexamer primers (1 8) with 5 �tCi of [32P]dATP(3,000 Ci/mmol; New England Nuclear). The labeledprobe was separated from unincorporated nucleo-

tides by Sephadex G-50 spin columns. Northern blotswere hybridized with 1 06 cpm/probe per milliliter ofhybridization buffer (5x SSPE, 2x Denhardt’s, 150

�tg/mL sonicated and denatured salmon sperm DNA,

0. 1 % SDS, 5% dextran sulfate, and 50% formalde-hyde) for 24 h at 42#{176}C.After hybridization, mem-branes were washed twice in 2x SSC (20x SSC: 3 MNaCl, 0.3 M sodium citrate: pH 7.0)-0.5% SDS for 30mm at 22#{176}Cand subsequently in 0.4x SSC-0.5%SDS at 65#{176}Cfor 45 mm. Membranes were then au-toradiographed with intensifying screens at -70#{176}Cfor 3 days.

Measurement of Cellular Proliferation

The incorporation of [3H]thymidine into DNA wasused as one measurement of cellular proliferation.

Cells (1 04/well) were transferred into 96-well cellculture plates (Nunc) in DMEM medium with 10%fetal calf serum. After adherence of cells, the mediumwas switched to DMEM without serum for 24 h.Subsequently, cells were stimulated for up to 72 h

0.1

0.08

a)a)

U. 0.06

�0

� 0.04

0

� 0.02

A. MCT B. LLC-PK1

0.06

0.05

0.04 �#{149}

0.03

0.02

0.01

0 0.5 1 1.5 2

Bound (nM)

43 kD

Wolf and Neilson

Journal of the American Society of Nephrology 1997

with single doses of 10’#{176}to 10� M D-isoproterenol(Sigma) redissolved in medium. Some wells were alsoincubated with isoproterenol and 1 06 M (3-adrenergicreceptor antagonist propranolol (Sigma). In addition,

to test the effect of agents interfering with G� proteins(1 9), MCT cells were incubated with 5 to 1 00 ng/mL

cholera toxin (CT: Sigma) for 24 h in the presence orabsence of 1 O� M isoproterenol. Furthermore, quies-cent MCT cells were also treated with a single doseof 1 O� to 1 O� mol/L of the analogs 8-bromo-cAMP(8-bro-cAMP: Sigma) or 8-bromo-cGMP (8-bro-cGMP:Sigma). All cells were pulsed for the last 6 h with 1�Ci of [3Hjthymidine per well (5 Ci/mmol: Amersham).At the end of the experiment, cells were washed with

PBS, incubated for 5 mm with 200 �zL/well of trypsin/EDTA, and harvested on glass-filter paper with an

automated cell harvester. Radioactivity was meas-ured by liquid scintillation spectroscopy. Data fromfive to six experiments with four replicates werepooled. For the measurement of cell counts, 5 x 10�

cells were transferred into each well of a six-wellplate, made quiescent in serum-free medium, andstimulated for 48 h as appropriate. After the stimu-

latlon period, cells were trypsinized, washed in PBS,and counted in a Neubauer hemacytometer.

Intracellular cAMP Concentrations

To test whether isoproterenol actually stimulatesintracellular cAMP in MCT cells, 2 x 1 O� quiescentcells were treated for 1 5 mm with 1 0� M isoproter-

enol. Additional wells were preincubated for 5 mmwith 1 0_6 M propranolol before the isoproterenol wasadded. After harvesting, cells were washed in ice-cold PBS with 4 mM EDTA and were subsequentlylysed in 50 mM Tris-HC1/4 mM EDTA (pH 7.5). Intra-cellular cAMP was measured in duplicate from each

experiment (N = 6) in the supernatants by a commer-cial competitive radioimmunoassay (20: Amersham).

Protein was determined in aliquots by a modificationof the Lowry method ( 1 6). Values are expressed asfemtomoles of intracellular cAMP per micrograms ofprotein.

Statistical Analysis

Results are expressed as means ± SE. Statistical

significance between different groups was firsttested with the nonparametric Kruskal-Wallis testfor multiple comparIsons. Individual groups weresubsequently tested with the Wilcoxon-Mann-Whit-

ney test, considering P < 0.05 as significant.

RESULTS

Expression of /3-Receptors

We used two well-characterized permanent celllines in these studies that express properties of prox-

imal tubules In culture. MCT cells have demonstrated

features of proximal tubules (9- 1 2), and LLC-PK1cells have some characteristics remniscent of cells

derived from the distal part of the nephron (13-15).Because there has been some uncertainty as towhether proximal tubules express /3-adrenoreceptorsIn vivo (21-23). we first tested MCT and LLC-PK1cells for the presence of these receptors in ligand-

binding studies with I3Hlpropranolol. Figure 1 dem-onstrates that both cell lines express /3-adrenocep-tors, although with different affinities (Kd of 23 nMfor MCT cells; Kd of 633 nM for LLC-PK1 cells). Al-though we cannot exclude the possibility that the celllines may express subtypes of /3-adrenoceptors withdifferent affinities for [3H]propranolol, computeranalysis of binding data with the program Enzfittersuggests that the best fit assumes a single class of

receptors. Both cell lines also express mature tran-scripts for f32-adrenergic receptors, as detected witha cDNA probe encoding the human /32-adrenergic

Bound [nM]

Figure 1 . Demonstration of specific /3-adrenoceptors in MCTand LLC-PK1 cells by Scatchard plots of ligand-binding data.Kd, 23 nM for MCT cells; Kd, 633 nM for LLC-PK1 cells. Eachpoint is the mean ofthree to four independent experiments,each performed in triplicate.

A. B.MCT LLC-PK1 1 2

// //68 kD

2.2kb 1�.. �

Figure 2. (A) Expression of mRNA encoding the �i2-adrener-gic receptor in MCT and LLC-PK1 cells. PolyA� mRNA (10 �g)was loaded onto the gel. Autoradiography was performedfor 72 h. (B) Cross-linking of (3H)propranolol to its receptorin MCT cells in the absence of competitor (Lane 1). Inclusionof iO� M isoproterenol in the incubation medium almostcompletely abolished the specific cross-linking to thereceptor (Lane 2). After separation on a SDS-1O% poly-acrylamide gel (4% stacking gel), a single band of 60 to65 kd is visible. Fluorography of the gel was performed at-70#{176}Cfor I wk.

No Prop D iO.6 M Prop

16.

14.

� 12.

,.-. 10.Eo_ 8.C.)

C’)

0I-

x

2.

a)� 20

E0.15C.)

C�10

5

24 h

*

io.7 MIsoproterenol

Controls

48 h 72 h

- 20

�- 15

� 10

)C

o�

Isoproterenol and Tubular Cells

1998 Volume 4 . Number 12 . 1994

receptor (Figure 2A). The size of the transcripts inour murine and porcine tubular cell lines (2.2 kb)was identical to that in human placenta (1 7). Tofurther test for the protein expression of /3-adre-

noceptors in MCT cells, cross-linking experimentswere performed with [3H)propranolol. Figure 2B dem-onstrates that covalent cross-linking of [3Hjpropran-olol to cell membranes of MCT cells and separationunder denaturing conditions resulted in a band ofapproximately 60 to 65 kd. The inclusion of 1 0� Mcold propranolol almost completely abolished thebinding of the radioactive ligand, indicating the spec-ificity of this interaction (Figure 2B, Lane 2).

Proliferation Studies

We used the incorporation of [3H]thymidine intoDNA to test the possible mitogenic effects of isopro-terenol on the cultured tubular cell lines. Figure 3

revealed that the incubation of quiescent MCT cellswith different concentrations of isoproterenol (10_�0

to 10�) for 24 h induces, in a dose-dependent man-ner, significant increases in [3Hjthymidine incorpo-

ration. Higher concentrations of isoproterenol (� 1 O�M) were toxic for both cell lines because the platedcells rounded, lifted off of the plate. and incorporatedless [3Hjthymidine than unstimulated controls. Theproliferation induced with 1 0_8 to 1 O� M isoprotere-nol was blocked by coincubation with 1 0_6 M pro-pranolol (Figure 4). Time-kinetic experiments dem-onstrate that stimulation of MCT cells with J#{216}_7Misoproterenol produces maximal incorporation of [3H]thymidine after 48 h of incubation (Figure 5). Asshown in Table 1 , 1 0 M Isoproterenol also induces

a significant Increase in [3H]thymidine incorporationin LLC-PK1 cells. This mitogenic effect of isoprotere-nol on LLC-PK3 cells was also abolished in the pres-

ence of propranolol (10� M Isoproterenol for 24 h,15.4 ± 0.7: iO’� M Isoproterenol + 106 M propranololfor 24 h, 1 2.0 ± 0.9 x 1 O� cpm/well: P < 0.05: N

25

**

control 10-SM 104M 1O-7M 1O-8M 1O-�M 1O-IOM

Figure 3. Dose-dependent stimulation of (3H)thymidine in-corporation. Quiescent MCT cells were incubated for 24 hwith single doses of 1O�#{176}to 1O� M isoproterenol. Highconcentrations of Isoproterenol (� I O� M) were apparentlytoxic because cells had the tendency to lift off ofthe plate.. P< 0.05, #{149}-P< 0.01 versus unstimulated controls; N= 12.

Controls iC

Figure 4. Incubation of quiescent MCT cells for 24 h withdifferent doses of isoproterenol in the presence or absenceof 106 M propranolol (Prop). Isoproterenol (1O_8 to 1O� M)significantly stimulated (3H)thymidine incorporation in MCTcells. This effect was blocked by coincubation with the �-

adrenergic receptor antagonist propranolol. P< 0.05 ver-sus controls; t P< 0.05 versus cells treated with isoprotere-nol only; N= 12.

30

25

Figure 5. Time course of proliferation induced by a singledose of 1O’� M isoproterenol in MCT cells. A maximumproliferation was observed 48 h after incubation with asingle dose of 1O-� M isoproterenol. P< 0.05 versus unstim-ulated controls; ‘ P< 0.01 versus unstimulated controls; N= 12.

1 2). Doses of 1 0_8 M isoproterenol for 48 h alsoresulted in a significant increase in total cell numberin both cell lines (Table 2).

There is general agreement that the action of iso-proterenol in many systems is mediated by an in-crease in intracellular cAMP (22,23). Consequently,we were interested in whether a stable, exogenousanalog of cAMP (8-bro-cAMP) exerts any effect on theproliferation of MCT and LLC-PK1 cells. Concentra-tions of 1 O� to 1 0� M of the cAMP analog for 24 hsignificantly increased [3Hjthymidine incorporationinto DNA in both cell lines. In contrast, 1 0� M 8-bro-cGMP had no significant effect on mitogenesis inMCT and LLC-PK1 cells (Table 3).

The signal for the generation of intracellular cAMPafter /3-adrenergic stimulation is mediated by stimu-

Wolf and Neilson

Journal of the American Society of Nephrology 1999

TABLE I . Effect of different concentrations ofisoproterenol for 24 h on (3H)thymidineincorporation into quiescent LLC-PK1 cells#{176}

Concentration(M)

(3H)ThymidineIncorporation

x iO� cpm/well

Control Media I 1.1 ± 0.410-If 11.9±0.7iO-� 12.0±0.8108 13.4± 1.010-i 15.4±0.7�10_6 13.5±0.T

0 Values are means ± SE; N= 12; cpm, counts per minute.bp<0 001

C p< 0.05.

TABLE 2. Cell number of MCI and LLC-PK1 cellsafter 48 h of stimulation with differentconcentrations of isoproterenol#{176}

Concentrations(M)

X I 0� cells

MCI cells LLC-PK1 cells

Control medium 0.71 ± 0.04 0.91 ± 0.8710-10 0.83 ± 0.04b 1.69 ± 049b

10-6 0.87 ± 0.05b 1.13 ± 0.22b10-6 1.14±0.17b 0.92±0.27

0 Values are means ± SE: N = 6.

bp<005

TABLE 3. Effect of different concentrations of acAMP analog for 24 h on (3H)thymidineincorporation into quiescent MCI and LLC-PK1 cells#{176}

Concentration (M)

(3H)Thumidine IncorporationX 10� cpm/well

MCT cells LLC-PK1 cells

Control Media 8.6 ± 0.5 10.4 ± 0.2iO-� 8-bro-cAMP I 1.2 ± 0.8b 13.0 ± 0.5c10-i 8-bro-cAMP I 1.0 ± 0.5b 13.6 ± 0.4b10� 8-bro-cAMP 9.6 ± 0.4 ND10-i 8-bro-cGMP 8.4 ± 1.0 1 1.4 ± 0.6

0 Values are means ± SE; N = 10-12: cpm, counts per minute; ND,

not done.bp<001

C p< 0.05.

latory Ge-proteins ( 1 9,22), which can be activated by

CT. Various concentrations of CT (5 to 100 ng/ml)for 24 h significantly stimulated proliferation in MCTcells, indicating that the activation of Ge-protein,even in the absence of isoproterenol, leads to mito-

genesis (control media, 8.6 ± 0.2; 5 ng/mL CT, 15.2± 0.4; 50 ng/mL CT, 1 1 .2 ± 0.2; 100 ng/mL CT, 14.6± 0.2 x i0� cpm/well: P < 0.05 for all CT valuesversus control media: N = 10).

Intracellular cAMP Concentrations

To test whether the isoproterenol concentrationthat induces mitosis actually increases cAMP for-mation, quiescent MCT cells were treated for 1 5 mm

with 1 #{216}_7M isoproterenol in the presence or absenceof 10_6 M propranolol. We have previously demon-strated that MCT cells have an adenylate cyclase that

is positively and negatively regulated by differentstimulants (20). Table 4 shows that 1 o7 M isoproter-enol significantly induces the formation of intracel-

lular cAMP. This stimulatory effect is abolished bypreincubation and coincubatlon with 106 M pro-

pranolol. The same concentration of propranololalone had no significant effect on intracellular cAMPlevels in MCT cells (Table 4).

DISCUSSION

The regenerative growth process of proximal tu-bules after acute necrosis due to ischemia or othercause is principally characterized by extensive mito-sis (1 ,2). Although many growth factors and cyto-kines have been demonstrated to be mitogens forcultured tubular cells (24), a role for these factors intubular proliferation in vivo is unclear (7,25). An

increase in vascular resistance with an activation ofsympathetic nerves has been proposed by some inacute renal failure (26). Studies performed in micemore than 20 yr ago clearly demonstrated that the

TABLE 4. Intracellular concentrations of cAMP inMCI cellsa

Concentration (M)fmol of cAMP/

� of Protein

Control media 8.0 ± 0.7iO-� isoproterenol 12.6 ± 0.89blO6propranolol 7.0 ± 2.11iO-� isoproterenol + 10_6 6.2 ± 0.84c

propranolol

0 Values are means ± SE; N = 6. Incubation with stimulants for 15mm.

b p< � versus control media.

C p< � versus cells treated with isoproterenol only.

Isoproterenol and Tubular Cells

2000 Volume 4 . Number 12 ‘ 1994

Injection of isoproterenol produces cell proliferationin the kidney (27,28). However, the cell type in thekidney that proliferates after isoproterenol injectionis not known, nor have those interesting studies beenfollowed up more recently. Because we have beeninterested in the function of vasoactive peptides as

possible renal growth factors (8), we examined thebiologic response of isoproterenol on the growth oftwo different, but well-characterized, tubular cell

lines.We first determined whether MCT and LLC-PK1

cells express /3-adrenergic receptors in culture. Thispart of the study was not performed to extensively

characterize all of the subtypes of /3-adrenergic recep-tors. but merely served to demonstrate that growtheffects may be mediated by the binding of ligands totheir putative receptors. Northern blot analysis re-vealed that both cell lines clearly express mRNA for

132-adrenergic receptors, although the transcripts arenot very abundant. Ligand-binding studies with [3Hjpropranolol, which binds to /31.2-adrenergic receptors,

demonstrate the presence of high-affinity receptors.We have further tested the protein expression of /31,2-

receptors by cross-linking the ligand to its putativereceptor. One major band of 60 to 65 kd was detected

after SDS-polyacrylamide electrophoresis. The bind-ing and subsequent cross-linking of [3Hjpropranololwere specific, because it was almost completely dis-placed by cold isoproterenol. This is in good agree-ment with studies estimating the size of membraneglycoproteins representing /3-adrenergic receptors as64 kd (17,29). Ligand-binding studies with [3HIpro-pranolol demonstrate the presence of receptors in

both cell lines, although MCT cells express receptorsthat exhibit an approximately 27-fold higher affinitythan the receptors present on LLC-PK1 cells (MCT

cells, Kd of 23 nM; LLC-PK1 cells, Kd of 633 nM).Whether proximal tubular cells in vivo express /3-

adrenergic receptors is arguable, because species dif-ferences may exist (2 1 ,22). Several groups have eval-uated the effects of /3-receptor antagonists on thetransport of various ions in the proximal tubule (30-33). For example, in studies in isolated proximalconvoluted tubules, the addition of catecholaminesenhances sodium reabsorption via /3-adrenergic

receptors (30,32). In contrast, others found no effectof /31.2 blockade on the antinatriuretic response tolow-level renal stimulation (33). Functional studiesapplying electrophysiologic methods suggest thepresence of /32-receptors in isolated proximal tubules

microdissected from rabbit and canine kidneys (31).In addition, recent data indicate that 1 O� M isopro-terenol stimulates renin production in primary cul-

tures of rabbit proximal tubules by the activation ofadenylate cyclase (34). In contrast, autoradiographicstudies on rat kidney slices reveal that distal tubulespreferentially express /32-receptors (34). These differ-

ences in functional studies and autoradiographic re-sults need clarification by further experiments.

We selected MCT and LLC-PK1 cells for these stud-ies. Part of the reason for chosing these cells was tocompare the effects of isoproterenol with previous

observations demonstrating that the vasoactive fac-tor angiotensmn II is hypertrophogenic, but not prolif-erative (9,10). A major advantage of clonal cell linesis the relative constancy of features between experi-ments, a fact that does not necessarily apply to pri-mary isolates. Although LLC-PK1 cells have manyproperties of proximal tubular cells, they may ex-press some characteristics reminiscent of distal tu-

bules, such as vasopressin-stimulated adenylate cy-clase activity (36). In contrast, MCT cells have beenshown by several investigators to express in vitroonly features of proximal tubules (6,9, 1 1,20,37,38),suggesting that the demonstration of /3-adrenergicreceptors in our studies cannot be attributed to con-

tamination with cells of distal tubular origin or genederepression after viral transformation. More studiesare currently underway to define subtypes of cate-

cholamine receptors in LLC-PK1 and MCT cells. How-ever, as with all cell culture studies, the immediaterelevance of our experiments to the in vivo situationof regenerative tubular growth, although suggestedby the early experiments in whole kidneys (27,28),remains to be established.

The /3-adrenergic receptor is the prototype of a G�-protein-mediated activation of adenylate cyclase, re-

sulting in an increase in intracellular cAMP afterligand binding (23,39). We have tested whether the

stimulation of MCT cells with isoproterenol resultedin an intracellular increase in cAMP. We and othershave previously demonstrated that this cell line ex-presses a hormone-responsive adenylate cyclase thatis stimulated by parathyroid hormone and inhibitedby angiotensin II (20,38). Isoproterenol stimulatescAMP formation in MCT cells, suggesting an adenyl-

ate cyclase coupled to the /3-adrenergic receptor. Ear-her investigations did not find a /3-adrenergic-de-pendent adenylate cyclase in proximal tubular cellsbut localized the enzyme in distal convoluted tubules

and cortical collecting ducts (39). In contrast, morerecent studies provided functional evidence for anisoproterenol-sensitive adenylate cyclase system inthe pars recta of canine proximal tubules (30).

A cAMP analog, but not 8-bromo-cGMP, mimickedthe isoproterenol-mnduced proliferation in MCT andLLC-PK1 cells. Furthermore, the activation of aden-ylate cyclase by the stimulation of Ge-proteins withCT also resulted in an increase in [3Hjthymidine in-corporation into quiescent MCT cells, indicating thatthe mitogenic effects of isoproterenol are most likelymediated by an increase in intracellular cAMP. Aden-osine nucleotides have been previously shown tostimulate mitogenesis in various renal cell types.

Wolf and Neilson

Journal of the American Society of Nephrology 2001

Kartha and Toback convincingly demonstrated that

ADP (200 �M) is a powerful mitogen for monkeykidney epithelial cells of the BSC-1 line, although

cAMP was less effective (40). This mitogenic effectof ADP was associated by the induction of immediate

early gene expression like Egr- 1 (4 1). Schulze-Lohoffand colleagues have extensively studied the effectsof ATP and analogues on the proliferation of cultured

rat mesangial cells (42). Exogenous ATP significantlystimulated the mitogenesis of cultured mesangial

cells via P2-purinerglc receptors.Several Investigators have studied the effect of

renal ischemia associated with tubular proliferationon the expression of immediate early genes, and fun-damental differences in the Initiation of growth com-pared with tubular hypertrophy have been workedout (43,44). Early induction of the genes c-fos andEgr-1 was a common feature in those studies (43,44).Both genes have cAMP-responsive elements in their5’-flanking promoter region, and c-fos induction bycAMP or CT has been demonstrated in different cell

types (45,46). Those observations may suggest amechanism as to how an increase in intracellular

cAMP after isoproterenol stimulation of MCT cells istransduced into cellular mitogenesis.

In conclusion, exogenous isoproterenol inducesproliferation through specific /3-adrenergic receptorsin two different tubular cell lines. It is likely that thismitogenic effect is mediated by an increase in intra-cellular cAMP. However, the role of i3-adrenergic sub-stances in the regeneration of functional tubulartissue after acute damage In vivo remains to be es-tablished.

ACKNOWLEDGMENTS

Parts of the studies were supported by Grant Wo 460/2- 1 from the

Deutsche Forschungsgemeinschaft. a grant to G. Wolf from the Paul

and Cilli Weill Foundation, Frankfurt, and Grants DK-45191. DK-

07006, AR-20553. DK-30280, and DK-41 1 10 from the NIH. We

thank Drs. Rolf AK. Stahl and Fuad N. Ziyadeh for their critical

reading of the manuscript and helpful discussions.

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