Isoproterenol and 8-bromo-cyclic adenosine monophosphate stimulate the expression of the angiotensinogen gene in opossum kidney cells

Kidney International, Vol. 46 (1994), pp. 703—710

Isoproterenol and 8-bromo-cyclic adenosine monophosphate stimulate the expression of the angiotensinogen gene in opossum kidney cells TIAN-TIAN WANG, MING CHEN, SILVANA LACHANCE, ALINE DELALANDRE,

SERGE CRIERE, and JOHN S.D. CHAN University of Montreal, Maisonneuve-Rosemont Hospital Research Center, Montreal, Quebec, Canada

Isoproterenol and 8-bromo-cyclic adenosine monophosphate stimulate the expression of the angiotensinogen gene in opossum kidney cells. To investigate whether the expression of the renal angiotensinogen (ANG) gene is regulated by f3-adrenoceptors and the cAMP-dependent protein kinase A pathway, we introduced stably the fusion gene containing the 5'-flanking regulatory sequence of the ANG gene with a human growth

hormone (hGH) gene as a reporter, pOGH (ANGN-1498/+18), into opossum kidney (OK) cells. We successfully obtained several stable

gen (ANG) synthesized in renal proximal tubules [10—12]. For example, immunocytochemical studies by Richoux et al [10] using

polyclonal antibodies to pure ANG demonstrated that ANG is localized predominantly in the renal proximal tubule but not in the granular cells of the juxtaglomerular apparatus. Physiological studies in the rat by Seikaly et a! [11] have shown that the level of

luminal Ang II in the renal proximal tubule is as high as iO M whereas the level of plasma Ang II is less than 10 12 M. Most convincingly, the studies of Ingelfinger et al [12] using in situ hybridization techniques showed that the ANG mRNA is present primarily in the renal proximal tubule while renin mRNA is hGH (IR-hGH) secreted into the culture medium. The addition of isoproterenol (10—" M to iO M) stimulated the expression of pOGH present primarily in the juxtaglomerular apparatus and glomeru(ANG N-1498/+ 18) and increased the accumulation of intracellular lar tuft. Most recently, we [13], as well as Ingelfinger et al [14], cAMP. Higher concentrations of isoproterenol (that is, greater than i0 have demonstrated that the ANG mRNA is expressed in opossum transformants with a high expression of the pOGH (ANG N-1498/+ 18) fusion gene. One stable transformant (OK 27) that is able to maintain the expression of pOGH (ANG N-1498/+18) in culture for more than a year was used in the present study. The level of expression of the pOGH (ANG N-1498/+ 18) in OK 27 was evaluated by the amount of immunoreactive-

M) had low or minimal effect. In contrast, the addition of 8-bromo-cAMP

(8-Br-cAMP) and forskolin stimulated the expression of pOGH (ANG N-1498/+18) in a dose-dependent manner. The stimulatory effect of isoproterenol was blocked by the presence of propranolol, atenolol and ICI 118,551. The addition of ICI 118,551, however, was less effective than

atenolol. Furthermore, the stimulatory effect of isoproterenol and 8-BrcAMP on the expression of the pOGH (ANG N-1498/+ 18) was inhibited by the presence of Rp-cAMP (an inhibitor of cAMP-dependent protein kinase A I and II). These studies demonstrated that the expression of the pOGH (ANG N-14981+18) in OK cells is stimulated by j3-adrenoceptors and the cAMP-dependent protein kinase A pathway. Our data indicate

that OK 27 cells provide a useful model to study the regulation of expression of the ANG gene in vitro.

kidney (OK) cells. Thus, the above studies demonstrated unequivocally that the proximal tubule is a major site of intrarenal ANG formation. The regulation of expression of the renal ANG gene in the proximal tubule, however, is unknown. In the present study, we investigated the ANG gene expression in OK cells which have been stably integrated into a fusion gene, pOGH (ANG N-14981+ 18) containing the endogenous 5'-flanking regulatory DNA sequence of the rat ANG gene fused with a

human growth hormone (hGH) gene as a reporter, and studied the effect of isoproterenol, 8-bromo-cAMP (8-Br-cAMP) and forskolin on the expression of pOGH (ANG N-14981+ 18) in OK

cells. Furthermore, we investigated whether the addition of Renal proximal tubular cells are richly innervated by adrenergic nerves [1]. Denervation and activation of renal nerves have been shown to diminish and increase sodium reabsorption by proximal tubules, respectively [2—7]. Thus, it has been suggested that renal nerve activity may participate directly in the development of renal hypertension.

Rp-cAMP (an inhibitor of cAMP-dependent protein kinase A I and H) could block the stimulatory effect of isoproterenol and 8-Br-cAMP. Our results indicate that the expression of pOGH (ANG N-1498/+ 18) fusion gene in OK cells was stimulated by the addition of isoproterenol, 8-Br-cAMP and forskolin. The addition of propranolol (/3, and /32 adrenoceptor blocker), atenolol ((31 adrenoceptor blocker) and ICI 118,551 (j32-adrenoceptor blocker)

The existence of an intrarenal renin-angiotensin system has inhibited the stimulatory effect of isoproterenol. Finally, the

now been generally accepted [8, 9]. Evidence indicated that renal angiotensin (Ang II) is probably derived from the angiotensino-

stimulatory effect of isoproterenol and 8-Br-cAMP was blocked by

the presence of the Rp-cAMP. Methods

Received for publication November 3, 1993 and in revised form March 31, 1994 Accepted for publication April 4, 1994

© 1994 by the International Society of Nephrology

Materials

The fusion gene, pOGH (ANG N-14981+18), containing the 5'-flanking sequence [1498 base-pair (bp)] upstream of the transcriptional site plus 18 bp of Exon I of the rat ANG gene fused

703

704

Wang et a!: OK 27 cells and ANG gene expression

with a hGH gene, has been described previously [15]. The ml of DMEM containing 10% FBS. Then the medium was replaced with fresh medium containing 10% FBS. The selection of stable transformants with high expression of pOOH (ANG N-1498/+ 18) or pTKOH was based upon the effect gift from Dr. Teresa Wang (Dept. of Pathology, Stanford Univer- of the aminoglycoside 0 418 (Geneticin, Oibco Inc.) at 500 p.g/ml sity, Stanford, California, USA). The plasmid, pTKGH, contain- in the media. The stable transformants which were able to grow in ing the thymidine kinase (TK) enhancer/promoter sequence fused the presence of 0 418 and which secreted high levels of IR-hGH to the 5'-end of the hUH gene was purchased from the Nichols into the medium, were further subcloned using the method of limiting dilution. Those cells that had passed through at least Institute of Diagnostics (La Jolla, California, USA). The radioimmunoassay kit for hUH (RIA-hGH) was a gift from three repetitions of limiting dilution and continued to secrete high NIADDK (NIH, Bethesda, Maryland, USA). The double anti- levels of IR-hGH after three months in the presence of 0 418 body RIA procedure was similar to that previously used for the were considered to be stable clones. We have obtained three and radioimmunoassay of ovine placental lactogen (RIA-oPL) [16]. six clones with pOOH (ANG N-1498/+ 18) and pTKGH inteHuman [125I]iodo GH was prepared by a slight modification of grated into their genomes, respectively. In the present study, we the lactoperoxidase method of Thorell and Johannson [17]. characterized two of these clones, clone OK 27 and OK 13 which NIAMDD-hGH-I-1 (AFP-4793 B) was used for both iodination have pOOH (ANO N-1498/+ 18) and pTKOH integrated into and as a hormone standard. The limit of sensitivity of the assay their genomes, respectively. was 0.1 ng/ml. The inter- and intra-assay coefficients of variation were 10% (N = 10) and 12% (N = 10), respectively. Basal expression of pOGH (ANG N-1498/+18) in OK 27 cells The [3H]cAMP assay kit was purchased from Diagnostic ProdOK 27 cells were plated at a density of 1 x io cells/well in plasmid, pRSV-Neo, containing the coding sequence for Neomy-

cm (Neo) with the Rous Sarcoma Virus (RSV) enhancer/promoter sequence fused in the 5'-end of the Neomycin gene was a

ucts Corporation (Los Angeles, California, USA). The cAMP 6-well plates and incubated overnight in DMEM containing 10% assays were performed according to the protocol provided by the FBS. The growth of the cells was arrested by the incubation in supplier. serum-free DMEM for 24 hours. Subsequently, the cells were R(— )-Isoproterenol(+)-bitartrate salt, S(— )-propranolol hy- incubated for 48 hours in the medium containing 10% FBS or up drochloride, S( — )-atenolol, ICI 118,551 and Rp-cAMP (an inhib- to 4 additional days in the medium containing 1% depleted fetal itor of the cAMP-dependent protein kinase A I and II [18]) were bovine serum (dFBS) and five factors (selenium 5 x 10 M), and all purchased from Research Biochemicals Inc. (RBI, Natrick, transferrin (5 j.tg/ml), insulin (1 X 10 M), hydrocortisone (5 X Massachusetts, USA). i0 M) and thyroid hormone, L-T3 (5 x 10_12 M). During Na-'25I was purchased from Dupont, New England Nuclear different time periods, media and cells were harvested and kept at (NEN; Boston, Masschusetts, USA). Calcium chloride was pur- —20°C until the assay for IR-hGH. The cells were extracted with chased from Mallinckrodt, Inc. (Montreal, Quebec, Canada), Geneticin (U 418) was purchased from Bethesda Research Laboratories

(Gibco-BRL, Burlington, Ontario, Canada). Other reagents were molecular biology grade and obtained either from Sigma Chemicals (St. Louis, Missouri, USA), Bethesda Research Laboratories (GibcoBRL), Boehringer-Mannhcim (Dorval, Quebec, Canada), Pharma-

cia Inc. (Bale d'Urfe, Quebec, Canada) or Promega-Fisher, Inc. (Montreal, Quebec, Canada). Cell culture

The opossum kidney (OK) proximal tubular cell line was obtained from the American Type Tissue Culture Collection (ATCC) (Rockville, Maryland, USA). This cell line is derived from the kidney of a female American opossum and retains several properties of proximal tubular epithelial cells in culture [19—26] and expresses a low level of ANG mRNA [13, 14]. The cells were initially grown in 100 X 20 mm plastic Petri dishes (Uibco) in Dulbecco's Modified Eagle's Medium (DMEM), pH 7.45, supplemented with 10% fetal bovine serum (FBS), 50 U/ml penicillin

and 50 j.tglml streptomycin. The cells were grown in a humidified atmosphere of 95% 02, 5% CO2 at 37°C. For subculturing, the cells were trypsinized (0.05% trypsin and EDTA) and plated at 2.5 >< i04 cells/cm2 in 6-well plates.

DNA transfection

0.1 M NH4 HCO3 after sonication. After gentle agitation overnight at 4°C, cell extracts were centrifuged at 15,000 >< g for 10 minutes

at 4°C to remove the precipitate. The protein content was measured according to the protocol of the Bio-Rad protein kit (Bio-Rad, Richmond, California, USA). The number of cells were counted with a hemocytometer. The total amount of IR-hGH was corrected by the volume of medium and cellular extract and expressed as total nanograms of IR-hGH. The depleted FBS was prepared by incubation with 1% activated charcoal and 1% AG 1 x 8 ion exchange resin (Bio-Rad

Laboratories,) for 16 hours or more at room temperature as described by Samuels et al [29]. This procedure removed endog-

enous steroid and thyroid hormones from the FBS as demonstrated by Samuels, Stanley and Shapiro [29]. Effect of isoproterenol on the expression of pOOH (ANG N-1498/+18) in OK 27 cells in the absence or presence of /3-adrenoceptor antagonists or Rp-cAMP OK 27 cells were plated at a density of ito 2)< i05 cells/well in six-well plates and incubated overnight in DMEM containing 1% dFBS. Then cell growth was arrested by incubation in serum-free

medium for 24 hours. Subsequently, various concentrations of

isoproterenol (i0fl to iO_6 M) were added to the culture

medium containing 1% dFBS. To compare the inhibitory effect of various j3-adrenoceptor Plasmids pOOH (ANO N-1498/+ 18) or pTKGH and pRSVNeo fusion genes were co-transfected (20 itg each) into OK cells antagonists (propanolol, atenolol and ICI 118,551) and Rp-cAMP (1 x 106 cells) utilizing calcium phosphate-mediated endocytosis on the expression of pOOH (ANO N-1498/+ 18) in OK 27 cells, [27, 28]. After transfection, the cells were cultured overnight in 5.0 various concentrations (10_li to 10) of propranolol (/3k and 132

705

Wang et al: OK 27 cells and ANG gene expression

Table 1. Comparison of levels of immunoreactive human growth hormone (IR-hGH) in the media and cellular extracts of OK 27 cells

5

Medium°

4 1 2 3 4

3

1

2

Cell numberc

ng

Days 6.42

0.34

13.17 0.45 19.74 0.30 23.70

1.95

Ratio of IR-hGH

Cellular extractb

0.21 0.02 0.30 0.5 0.8

0.1

0.07 0.20

0.43 106 0.9 X 106

3.3 X 10 1.3 X 106

medium/cellular extract 30:1 44:1 40:1 30:1

OK 27 cells were cultured in monolayer in the DME/HAM F-12

1

0 12

24

36

48

containing 1% depleted fetal bovine serum and five factors. C The total amount of IR-hGH measured in three wells (means SEM of three determinations). b The total amount of IR-hGH measured in the cellular extract of three wells (means SEM of three determinations) C The total numbers of cells (pool of three wells)

Incubation time, hours Fig. 1. Basal expression of the pOGH (ANG N-1498/+18) in OK 27 cells. Cells were incubated for 24 hours in DME/HAM F-12 medium containing 10% fetal bovine serum (FBS). Aliquots (0.5 ml) were taken at different intervals and fresh medium was added to replenish the volume after each aliquot was withdrawn. The amount of IR-hGH expressed at 30 minutes of at least three was 1.8 0.07 ng/ml. Each point represents the mean 0.01 and *** 0.005). determinations (°P 0.05,

three times with phosphate-buffer and harvested with a cellscraper. The cells were weighed and snap-frozen in liquid nitrogen

according to the protocol of the supplier (Diagnostic Products Corp., Los Angeles, California, USA). Then, 0.5 ml of 6% perchioric acid was added to an arbitrary fixed amount of cells

(wet weight), mixed and kept on ice for 30 minutes before

centrifugation at 3,000 X g for 10 minutes. Finally, aliquots of supernatant were taken for the measurement of total cAMP adrenoceptor blockers), atenolol (/31-adrenoceptor blocker), ICI content. 118,551 (132-adrenoceptor blocker) and Rp-cAMP (10h1 to i0 Statistical analysis M) were co-cultured with the isoproterenol (10—v M) for 24 hours.

The experiments were performed at least three times in tripliAt the end of the incubation period, media were collected and cate. The data were analyzed with the Student's t-test or ANOVA kept at — 20°C until assay for IR-hGH. analysis. A probability level of P s 0.05 was regarded as significant. Effect of 8-Br-cAMP and forskolin on the expression of pOGH (ANG N-1498/+18) in OK 27 cells in the absence or presence of Rp-cAMP OK 27 cells were plated at a density of ito 2 X i05 cells/well in six-well plates and incubated overnight in DMEM containing 1% dFBS. Then cell growth was arrested by incubation in serum-free

Results Basal expression of pOGH (ANG N-1498/+18) in OK 27 cells

Figure 1 shows that when OK 27 cells are plated in six-well plates and grown in the medium containing 10% FBS up to 48 medium for 24 hours. Subsequently, various concentrations of hours, the amounts of IR-hGH detected in the medium were 8-Br-cAMP (10 to 10—2 M) or forskolin (10—v to iO M) were time-dependent. The IR-hGH was detectable as early as 30 added to the culture medium containing 1% dFBS and incubated minutes after incubation and increased with time to reach greater than 2.5-fold in 48 hours as compared to that observed after 30 for 16 to 24 hours. To investigate whether Rp-cAMP could block the effect of minutes. Similar results were obtained with the OK 13 cells (not 8-Br-cAMP on the expression of pOGH (ANG N-1498/+ 18) in shown). In non-transfected OK cells, the level of IR-hGH in the OK 27 cells, various concentrations of Rp-cAMP (10 to i0 M) medium was less than 0.1 ng/ml (not shown). Table 1 compares the levels of the IR-hGH in the medium and were co-cultured with 8-Br-cAMP (10 M) for 16 to 24 hours. At the end of the incubation period, medium were collected and kept cellular extracts of OK 27 cells which were incubated for up to four days in the medium containing 1% dFBS and five factors. It at —20°C until assay. appears that the concentration of IR-hGH in the cell extract Effect of isoproterenol on the accumulation of intracellular cAMP and on the expression of pOGH (ANG N-1498/+ 18) in OK 27 cells OK 27 cells were plated at a density of ito 2 x iO cells/well in six-well plates and incubated overnight in DMEM containing 10%

FBS. The cell growth was arrested by incubation in serum-free medium for 24 hours. Subsequently, various concentrations of

isoproterenol (i0_1 to iO M) were added to the culture

remained relatively constant and was less than 5% (range 2 to 3%)

of that detected in the medium. These studies indicate that the IR-hGH expressed by pOGH (ANG N-1498/+ 18) is not stored within the OK cells but is secreted by these cells. Effect of isoproterenol on the expression of pOGH (ANG N-1498/+18) in OK 27 cells in the absence or presence of /3-adrenoceptor antagonists and Rp-cAMP

Figure 2 shows the expression of the pOGH (ANG N-1498/ medium containing 1% dFBS and incubated for various time periods. After incubation, cultured media were taken and kept at + 18) in OK 27 cells in the presence of various concentrations of —20°C for assays of IR-hGH. Then, cell monolayers were washed isoproterenol (10_li to 10 M). A dose-dependent relationship

706

Wang et al: OK 27 cells and ANG gene expression

5

A 9

** 7

*

**

**

5

**

2

0

10h1 1010 10 10

10 10 3

Concentration of isoproterenol, mo/a rity

0

Fig. 2. Effect of isoproterenol on the expression of pOGH (ANG N-1498/

+18) in OK 27 cells. Cells were incubated for up to 24 hours in the presence of various concentrations of isoproterenol. Media were har-

10

10

10-s

B Concentration of propranolol or Ap-cAMP, molarity

vested after 24 hours of incubation and assayed for IR-hGH. Each point represents the mean SD of at least three dishes (*P 0.05, 0.01

and ***p

10

8

0.005). Similar results were obtained from two other

experiments.

*

between isoproterenol concentrations and the stimulation of expression of pOGH (ANG N-1498!+18) was observed for iso-

I

*

proterenol at lO M to i0 M. It appears that maximal stimulation of expression of the pOGH (ANG N-1498/+ 18) was found with i09 NI isoproterenol. At concentrations greater than

i0 M, it appears that the stimulatory effect of isoproterenol is minimal or inhibited. Similar results were observed from two other experiments. Figure 3 shows that the addition of either propranolol (b_fl to i0 M), or Rp-cAMP (10_li to i0 M) (Fig. 3A) or ICI 118,551 (10_li to 10 M) or atenolol (10 to 10 M) (Fig. 3B) inhibits the stimulatory effect of isoproterenol (10 M) on the expression of the pOGH (ANG N-14981+ 18) in OK 27 cells in a dosedependent manner. The effective dose for the inhibition of the stimulated expression (by isoproterenol) of the pOGH (ANG

N-1498/+18) was found with i0 M propranolol (P 0.05), 10_il M Rp-cAMP (P 0.05) and lO M atenolol (P 0.01).

ICI 118,511 was effective in inhibiting the effect of isoproterenol

** 5

** 4 0

10

10

10

10

Concentration of atenolol or ICI 118,551, mo/a rity Fig. 3. The inhibitoiy effect of 13-adrenoceptor antagonists or Rp-CAMP on the expression of pOGH (ANG N-1498/+18) in OK 27 cells stimulated by isoproterenol. A. Cells were incubated for up to 24 hours in the presence of

isoproterenol (10 M) and various concentrations of propranolol (b_fl to iO M), or Rp-cAMP (10" to iO M). B. Cells were incubated in the presence of isoproterenol (10—p M) and various concentrations of atenolol

(10_il to i0 M) or ICI 118,551 (lo_fi to i0 M), Media were harvested and assayed for the level of IR-hGH. Each point represents the mean SD of at least of three dishes (*P

0.01 and ***

0.05, 0.005) only at a concentration equal to or greater than iO M (P 0.05). (propranolol, A—L; Rp-cAMP, O—O; atenolol, • •; ICI These studies suggest that the stimulatory effect of isoproterenol 118,551, A----A. is mediated via the f3-adrenoceptors and cAMP-dependent protein kinase A I and II in the expression of the fusion gene.

Effect of 8-Br-cAMP or forskolin on the expression of pOGH (ANG N-1498/+18) in OK 27 cells and OK 13 cells

stimulatory effect of 8-Br-cAMP on the expression of the pOOH Figure 4 shows the expression of the pOGH (ANG N-1498/ (ANG N-1498/+ 18) in OK 27 cells in a dose-dependent manner. + 18) in OK 27 cells in the presence of various concentrations for Maximal and half-maximal inhibition of the stimulated expression

8-Br-cAMP (10 to 10 NI) (Fig. 4A) or forskolin (b0 to iO of the pOGH (ANG N-14981+ 18) was found with 10 and iO M) (Fig. 4B). A dose-dependent relationship between hormone concentrations and the stimulation of expression of pOGH (ANG N-1498/+ 18) was observed for both 8-Br-cAMP and forskolin. Maximal stimulation of expression of the pOGH (ANG N-14981

+ 18) was found with 10 M 8-Br-cAMP (Fig. 4A) or 10 M forskolin (Fig. 4B). The half-maximal effects observed for 8-BrcAMP and forskolin were i0 M and b0_6 M, respectively.

Figure 5 shows that the addition of Rp-cAMP inhibits the

M Rp-cAMP (P 0.05). These studies suggest that the cAMPdependent protein kinase A I and II is probably involved in the

expression of the fusion gene. The addition of Rp-cAMP also inhibits the stimulatory effect of forskolin on the expression of the pOGH (ANG N-1498/+ 18) in OK 27 cells (not shown).

In OK 13 cells, neither 8-Br-cAMP (10 M) nor forskolin (10 M) could stimulate the expression of pTKGH compared to the controls (absence of 8-Br-cAMP or forskolin; Fig. 6).

707

Wang Ct a!: OK 27 cells and ANG gene expression

A

**

4

5

*

I03

3

2

1

0

10

10

1 lY-2

8-Br-cAMP 1 0-3

10-s

10-s

1O-

Rp-cAMP 0

1

1 0-

10

Concentration of 8-Br-cAMP, molarity

Concentration, molarity

B 8

*** 6

Fig. 5. The inhibitoiy effect of Rp-cAMP on the expression of pOGH (ANG N-14981+18) in OK 27 cells stimulated by 8-Br-cAMP. Cells were incubated

for up to 24 hours in the presence of 8-Br-cAMP (10—s M) and various concentrations of Rp-cAMP (10—u to iO M). Media were harvested and assayed for the level of IR-hGH. Each point represents the mean SD of at least of three dishes (*P 0.05, P 0.01 and *** 0.005).

** 30

4

20

2 0

10

10

10-s

Concentration of forskolin, molarity Fig. 4. Effect of 8-bromo-cAMP (A) and forskolin (B) on the expression of pOGH (ANG N-14981+18) in OK 27 cells. Cells were incubated for up to 24 hours in the presence of various concentrations of 8-bromo-cAMP or forskolin. Media were harvested after 24 hours of incubation and assayed

for IR-hGH. Each point represents the mean SD of at least three dishes 0.01 and *** 0,005). (*P 0.05, **D

10 Control 8-Br-cAMP 1O Forskolin 10

Concentration, molarity Fig. 6. Effect of 8-Br-cAMP and forskolin on the expression of pTKGH in OK 13 cells. Cells were incubated for 24 hours in the absence or presence of 8-Br-cAMP (10—i M) or forskolin (10- M). Media were harvested 24 hours after the incubation and assayed for IR-hGH. Each point represents

the mean SD of a minimum of three dishes. The empty bar on the extreme left is without added hormones. The stippled bar is from Effect of isoproterenol on the accumulation of cAMP and on the expression of pOGH (ANG N-14981+18) in OK 27 cells

8-Br-cAMP (10—s M) treated cells and the solid bar on the extreme right 0.01 and *** is from forskolin (10—s M) treated cells (*P 0.05, 0.005).

Table 2 shows that the incubation with 10_11 M and io M isoproterenol for two hours stimulates the accumulation of cAMP by 1.3- and 1.9-fold as compared to control (without the addition of isoproterenol), respectively. Isoproterenol at concentration of iO M had no stimulatory effect on intracellular cAMP. Similarly,

10_li M and iO M isoproterenol stimulated the expression of pOGH (ANG N-1498/+18) by 154% and 162%, respectively, whereas isoproterenol at i0 M had no stimulatosy effect. After

24 hours of incubation, 10_li M and iO M isoproterenol stimulated the accumulation of cAMP by 1.4- and 1.6-fold, as compared to control, respectively; iO M isoproterenol had no

effect. On the other hand, the expression of pOGH (ANG N-14981+ 18) was stimulated only at a concentration of io M isoproterenol (P 0.05), whereas 10_li M and 1O M had no significant stimulatory effect.

Discussion Our previous studies [14, 30] have shown that OK cells can express the fusion genes containing the 5'-flanking sequence of the ANG gene fused with either the hGH gene or the chioramphenicol acetyl-transferase (CAT) coding sequence when these fusion genes were transiently transfected into OK cells. There are two shortcomings with transient gene transfection experiments. The first is that the fusion gene remains within the cell for only a short time period, that is, less than two weeks. Thus, transient gene transfections must be done repeatedly which sometimes led to results that were not reproducible due to the variation of DNA transfection efficiency. The second problem is that the physical

708

Wang et al: OK 27 cells and ANG gene expression

Table 2. Effect of isoproterenol on the accumulation of cAMP and on the expression of pOGH (ANG N-1498/+ 18) in OK 27 cells

concentrations had minimal or no stimulatory effect (Fig. 2).

Incubation period Isoproterenol

lished observations). At present, we have no explanations for

inolarity

Control

iO 10

10_lI ap

24 Hours

2 Hours cAMP pmol/300 pg

IR-hGH nglml

cAMP pmol/300 pg

IR-hGH ag/nil

0.110 0.002 1.796 0.06 0.115 0.001 3.724 0.100 0.001 2.051 0.10 0.110 0.008 3.869 0.210 0008b 2.916 o.lla 0.180 O.OO4 4.951 0.140

0.002a 2.770

0.07' 0.160

0.003a 4.038

0.11 0.15

0.07' 0.14

0.05

1P s 0.001 environment of the naked plasmid is not similar to the endogenous gene which should be bound or surrounded by nucleosomes. Thus, the molecular mechanism(s) of regulation of the endogeneous ANG gene could be different from the transiently transfected fusion gene. In this regard, a cell line with the fusion gene stably integrated into its genome that is able to express the fusion gene under the stimulation of various factors would be an ideal in vitro system. Hence, the main objective of our studies was to develop such a cell line. Clones OK 27 and OK 13 are stable transformants with pOOH (ANG N-1498/+ 18) and pTKGH integrated into OK cellular genomes, respectively. These clones have been grown in the

Similar results were obtained from two other clones with pOOH (ANG N-1498/+ 18) stably integrated into their genome (unpubthese observations, although one possible explanation may be that high concentrations of isoproterenol (that is, greater than io— M)

might desensitize the /3-adrenoceptors. Indeed, studies by Lefkowitz and co-workers [341 have shown that /3-adrenoceptors are subject to desensitization by its own agonists. Furthermore,

studies by Cheng et al [25] demonstrated that isoproterenol at concentrations of 10—8 M to iO M has no stimulatory effect on cAMP formation in OK cells. Our present studies (Table 2) are in agreement with the studies of Cheng et al [25]. Thus, it is possible that high concentrations of /3-adrenoceptor agonists may desensitize its own receptors in OK cells. More experiments are needed to clarify these observations.

Another possible explanation might be that the number or density of /3-adrenoceptors in OK cells is low compared to the

proximal tubules in vivo. We had performed transient gene transfection experiments by cotransfecting OK cells with pOCAT (ANG N-1498/+ 18) (a plasmid containing the 5'-flanking region of the rat AJ'TG gene fused with the coding seqence for chloramphenicol acetyl transferase as reporter [30]) and plasmid contain-

ing either the /3k- or /32-adrenoceptor eDNA (a gift from Dr. Michel Bouvier, Dept. of Biochemistry, University of Montreal, Quebec, Canada). Our studies showed that the addition of isopro-

medium containing 500 gg/ml of 0418 for more than three terenol (i0 54) stimulates the expression of pOCAT (ANG N-1498/ months and expressed a high amount of IR-hGH into the + 18) by a factor of least two- to four-fold higher as compared to medium. The expression of pOOH (ANG N-1498/+ 18) in OK 27 control (unpublished observations). Thus, our data appear to support cells was time-dependent (Fig. 1). The levels of IR-hGH in the possibility that the number of /3-adrenoceptors in OK cells may cellular extracts were consistently less than 5% of that found in be low. More studies are definitely warranted to clarify the low the media (Table 1). These studies indicate that IR-hGH is not stimulatoty effect of isoproterenol in OK cells. stored in the cell. It is interesting to note that 8-Br-cAMP could stimulate directly Catecholamines are known to increase fluid and solute transport the expression of the ANO-GH fusion gene in OK cells (Fig. 4) by the renal proximal tubule [4—6]. However, the mechanisms by but not in mouse hepatoma HEPA 1-6 cells [35]. In HEPA 1-6 which adrenoceptors mediate the stimulatory action of cat- cells, we had shown that 8-Br-cAMP alone had no effect on the echolamines remain undefined. Functional 13-adrenoceptors along expression of the ANG gene but 8-Br-cAMP could enhance the the proximal tubule of the rat kidney and on the rabbit proximal stimulatory effect of dexamethasone [35]. At present, the rea-

straight tubules have been reported [31, 32]. Recent studies by son(s) for these different responses is not clear. One possible Taniguchi et al [33] also located the f32-adrenergic receptor mRNA explanation may be that there is a tissue-specific response bein the early proximal convoluted tubule. These studies suggest that tween the kidney and the liver. Indeed, this possibility is sup(3-adrenoceptors may contribute a significant role in catecholaminestimulated electrolyte transport. Our present studies showed that the

ported by the recent studies of Schunkert et al [36] that the expression of ANG mRNA in the liver and kidney is tissue-

addition of low concentrations of isoproterenol, or 8-Br-cAMP or specific in rats with chronic experimental heart failure. On the forskolin directly stimulate the expression of pOOH (ANO N-14981 other hand, based on our previous studies of the DNA structure + 18) in OK 27 cells (Figs. 2 and 4), whereas the addition of of the 5'-flanking sequence of the rat ANG gene [16], we observed 8-Br-cAMP or forskolin did not stimulate the expression of pTKGH that the DNA sequence nucleotides [N-795 to N-788 (TGACGin OK 13 cells (Fig. 6). Furthermore, the addition of either /3-adreTAC)] are identical to the CRE (TGACGTCA) of the human noceptor antagonists (propranolol, atenolol and IC! 118,551) (Fig. 3) angiotensinogen gene [37], somatostatin (SMS) gene (38), vasoor Rp-cAMP (Fig. 5) blocked the stimulatoryi effect of isoproterenol and 8-Br-cAMP in a dose-dependent manner. These studies indicate that /3-adrenoceptors and cAMP-dependent protein kinase A path-

active intestinal peptide (VIP) gene [39], a-chorionic gonado-

way are involved in the expression of the ANG gene in OK cells.

tropin (a-CG) gene [40] and phosphoenolpyruvate (PEPCK) gene [41] with the exception that the last two nucleotides are in reverse order. Thus, we speculate that ANG N-795 to N-788 might be the

Thus, these studies raise the possibility that activation of renal nerves

authentic CRE of the rat ANG gene. Indeed, we are currently

(that is, via /3-adrenoceptors) may have a regulatory role on the

investigating this possibility. In summary, our studies showed that the ANG-GH fusion gene, pOOH (ANG N-1498/+ 18) stably integrated into the genome of

expression of the local intrarenal renin-angiotensin system. However, more experiments are definitely required to confirm this possibilty in vivo.

OK cells can be expressed in culture. The addition of low

We were surprised that the effect of isoproterenol at io— M was concentrations of isoproterenol (b_U as to i0 M), or 8-Br-

not more than 1.5-fold higher than the control, whereas higher cAMP or forskolin directly stimulated the expression of the

Wang et a!: OK 27 cells and ANG gene expression

709

ANG-GH fusion gene. Furthermore, the stimulatory effect of

13. Ciw JSD, CHAN AHH, Nm Z-R, SIKSTROM R, LACHANCE S,

isoproterenol could be blocked by the presence of 13-adrenoceptor antagonists and Rp-cAMP. These studies demonstrate unequivo-

expression of the angiotensinogen gene in cultured opossum kidney

cally that there are kidney-specific transacting factors which interact with the 5'-fianking sequence of the ANG gene to promote its expression. The 13-adrenoceptors and the cAMP-

HA5HIM0T0 5, CARRIERE S: Thyroid hormone, L-T3, stimulates the (OK) cells. JAm Soc Nephrol 2:1360—1367, 1992

14. INGELFINGER JR, BounouNEs B, JUN FF, TAG S: High glucose

downregulates expression of renin angiotensin system (RAS) in opossum kidney cells. (abstract) Pediatr Nephrol 29:344A, 1991

dependent protein kinase A I and II are probably involved in the 15. CHA.N JSD, Cl-IAN AHH, JIANG Q, NiE Z-R, LACI-IANCE S, CARRIERE, 5: Molecular cloning and expression of the rat angiotensinogen gene. signal transduction pathways in the expression of the renal ANG Pediatr Nephrol 4:429—435, 1990 gene. Our studies raise the possibility that the increase of intra- 16. Cns.r JSD, ROBERTSON HA, FRIESEN HG: Maternal and fetal concellular cAMP which is induced by the activation of renal nerves centration of ovine placental lactogen measured by radioimmunoassay. Endocrinology 102:1606—1613, 1978 (that is, via 13-adrenoceptors) may stimulate the expression of the renal ANG gene. The local formation of renal ANG II might then 17. Thoiu. JI, JOHANNsON BG: Enzymatic iodination of polypeptides with 1251 of high specific activity. Biochim Biophys Acta 251:363—369, 1971 modulate the physiology of the renal proximal tubular cells (that 18. LANDSBERG CR, JASTORFF B: In vitro phosphorylation of microtubuleis, sodium and fluid reabsorption). Thus, local renal RAS might associated protein 2: Differential effects of cyclic AMP analogues. J play a significant role in the modulation of sodium reabsorption. Neurochem 45:1212—1222, 1985 Finally, OK cells with pOGH (ANG N-1498/+ 18) integrated into 19. KOYAMA H, GOODPASTURE C, MILLER MM, TEPLITZ RL, RIGGS AD: Establishment and characterization of a cell line from the American their genome provide an excellent in vitro model system to study opossum (Didelphys virginia). In Vitro (Rockville) 14:239—246, 1978 the regulation of expression of the renal ANG gene. 20. POLLOCK AS, WARNOCK DG, STREWLER GJ: Parathyroid hormone inhibition of Na-H antiporter activity in a cultured renal cell line.

Acknowledgments This work was supported by a grant from the Medical Research Council

of Canada (MRC) (#MT-11568) and in part from the "Fonds de Ia Recherche en Sante du Québec" (FRSQ). We also thank NIDDK and NHPP, University of Maryland, School of Medicine (Drs. S. Raitii and A. Parlow) for the gift of hGH-RIA kit (Award #31730). We thank Mrs. Ilona Schmidt for her secretarial assistance, and Drs. Kenneth D. Roberts and Michèle Gagnan-Brunette for their comments.

Reprint requests to John S.D. Chan, Ph.D., University of Montreal,

Maisonneuve-Rosemont Hospital, Research Center, 5415 L 'Assomption Blvd., Montrea4 Quebec, Canada HiT 2M4.

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