Alpha1-adrenergic stimulation and cytoplasmic free calcium concentration in cultured renal proximal tubular cells: Evidence for compartmentalization of quin-2 and fura-2

JOURNAL OF CELLULAR PHYSIOLOGY 1 2 8 4 6 6 4 7 4 (1986)

Alpha,-Adrenergic Stimulation and Cytoplasmic Free Calcium Concentration in Cultured Renal Proximal Tubular Cells: Evidence for Compartmentalization of Quin-2 and Fura-2 MICHAEL S. GOLIGORSKY,* KEITH A. HRUSKA, DAVID J.LOFTUS,

AND

ELLIOT 1. ELSON

Renal Division, Jewish Hospital of St. Louis (M.S.G., K.A.H.), and Department of Biochemistry, Washington University School of Medicine (D.J. L., E. L. E.), St. Louis, Missouri 63110 This study was designed to examine the role of changes in cytoplasmic free calcium concentration ([Ca2+Ii)during the response to a,-adrenergic agonists in cultured renal proximal tubular cells. Experiments were carried out on primary cultures of canine proximal tubular cells grown in defined culture medium on a solid support, on collagen-coated polycarbonate membranes, or on collagen-coated glass coverslips. Quin-2 and fura-2 were used to monitor [Ca2+Ii.The basal level of [Ca2+liwas 101 nM, as measured with quin-2, and 122 nM, as determined using fura-2. Fluorescence flow cytometry revealed that about 85% of the population of proximal tubular cells responded to phenylephrine with an increase in [Ca2+Ii.Phenylephrine (lo-' M) caused an immediate actual increase in [Ca2+Iiby 18 and 24%, as determined with quin-2 and fura-2, respectively, with the peak increase in [Ca2+Iiaveraging 22% and 44% over the basal level (180-300 sec). This effect did not require extracellular calcium. The effect of phenylephrine was abolished by prazosin and verapamil. Fluorescence microscopy of quin-2 or fura-2 loaded cells revealed punctate areas of fluorescence within the cytoplasm suggesting vesicular uptake of the dyes. Pinocytotic entrapment of the dyes was demonstrated by the transfer of cell-impermeant fura-2 across tubular cell monolayers mounted in Ussing chambers. The transfer of the dye was similar to that of a marker of fluid-phase pinocytosis, Lucifer Yellow (LY).This pinocytotic entrapment of Ca2+-indicatorswould lead to underestimation of the actual calcium transients. Microfluorometric study of single proximal tubular cells "scrape-loaded'' with fura-2 revealed a four-fold increase in [Ca2+Iiconcentration following stimulation with phenylephrine. The possibility of a direct effect of the sympathetic nervous system on tubular fluid transport was a controversial issue, until electron microscopic and histochemical studies identified adrenergic nerve terminals surrounding proximal and distal tubules (Muller and Barajas, 1972). More recently, al-and az-adrenergic receptors have been identified at the basolateral membrane of renal tubular cells and have been characterized by means of radioligand binding studies (McPherson and Summers, 1982; Schmitz et al., 1981; Snavely and Insel, 1982). On the basis of this information, studies were performed to correlate al-adrenergic stimulation with changes of fluid transport (Bello-Reuss et al., 1975, 1976; DiBona, 1977; Gottschalk, 1979; Prosnitz and DiBona, 1978; Osswald and Greven, 1981; Osborne et al., 1983; Tomlinson and Wood, 1976; Wood and Tomlinson, 1974). These results demonstrated that al-adrenergic agonists decrease sodium excretion, increase proximal tubular fluid reabsorption, and increase bicarbonate 0 1986 ALAN R. LISS, INC.

flux (Hesse and Johns, 1983; Prosnitz and DiBona, 1978; Chan, 1980).Together with changes in ion transport, qadrenergic agonists stimulate gluconeogenesis in the proximal tubule (Klahr et al., 1973; Kurokawa and Massry, 1973; MacDonald and Saggerson, 1977; Kessar and Saggerson, 1980). The mechanism of tubular cell stimulation by which al-adrenergic agents affect transport functions and gluconeogenesis, remains unknown. Progress in clarifying the mode of action of al-adrenergic agents has been achieved in other tissues such as the hepatocyte and the salivary gland (Exton, 1985; Williamson et al., 1985;

Received February 3,1986; accepted May 13,1986. The results of this study have been presented at the 70th Annual FASEB Meetings, St. Louis, April 12-15,1986. *Towhom reprint requeststcorrespondenceshould be addressed.

CQ-ADRENERGIC AGONISTS AND CYTOPLASMICCALCIUM

Putney, 1979;Reinhart et al., 1984). In these tissues, a1adrenergic stimulation results in an immediate increase of plasma membrane phosphatidylinositol 4’5’-biphosphate hydrolysis and production of inositol triphosphate which is responsible for mobilization of calcium from a nonmitochondrial stores (Charest et al., 1983; Blackmore et al., 1982; Williamson et al., 1985; Exton, 1985). The present studies were designed to examine the mode of action of the al-adrenergic agonist phenylephrine (PE) in proximal tubular cells (PTC), in particular, its effect on [Ca2+]iconcentrations. Recent studies have shown that ax-adrenergic stimulation of the proximal tubule cells results in increased production of inosito1 triphosphate (Freiberg et al., 1986). However, stimulation of a calcium transient in the cytosol of these cells remains to be demonstrated.

MATERIALS AND METHODS Preparation of proximal tubular segments and cell culture techniques Mongrel dogs of either sex were fed standard Purina dog chow and had free access to water. Under pentobarbital anesthesia nephrectomy was performed, and the renal artery was perfused with ice-cold saline. Isolated proximal tubular segments were prepared as described by Vinay et al. (19811,and Hruska et al. (1986). Isolated tubular segments from the band IV (proximal tubules) of the Percoll gradient were seeded onto cell culture flasks or coverslips at a density of 2.5 x lo-* segments/ml. The growth medium was initially 45% Dulbecco’s modified Eagle’s medium (MEM), 45% Hamm’s F-12, 5% fetal calf serum, and 5% donor horse serum. After 3 days, the growth medium was changed to serum free 50% Dulbecco’s MEM, 50% Hamm’s F-12 with the following additions: transferrin, 5 pg/ml; insulin, 3 pg/mk PGEI, 25 ng/ml; hydrocortisone,5 x lO-’M, and triiodothyronine, 5 x 10-=M. Cells were used from confluent monolayers at 10-14 days. At confluence, the cell layer exhibited numerous hemicysts. Characterizations of the cells and their identification with proximal tubular epithelium has been described by Hruska et al. (1986). Alternatively, cells were seeded at hyperconfluent densities upon collagen-coated Nucleopore polycarbonate membranes (0.8 pm, 13 mm-Nucleopore Corp., Pleasanton, CA) glued to rubber rings (Millipore, Bedford, MA). Growth conditions were similar to those described above, except that the cells were used in studies 14-21 days after initiation of cultures. At this time cells responded to phenylephrine with a 30% increase in glucose production (manuscript in preparation), suggesting an intracellular coupling between a1-adrenoreceptorand gluconeogenesis. Spectrofluorometry For the study of quin-2 fluorescence, cells were preincubated in Dulbecco’s MEM for 24 h and then scraped from the culture flask with a rubber policeman. Quin-2 loading was performed essentially according to Tsien et al. (1982). Quin-2 acetoxymethyl ester at a concentration of 50 pM was added to cell suspensions of lo7 celldm1 for 20 min and then diluted ten-fold with fresh medium. The incubation was continued for 1h, after which the cells were centrifuged at 500g for 3 min and washed three times with Krebs-Henseleit buffer. In a separate

467

series of experiments cells were loaded with 5 pM quin2 (instead of 50 pM) to reduce the effect of Ca2+ buffering (Harvey et al., 1985). The estimated intracellular concentration of quin-2 under these conditions decreased to 0.1 mM (regular loading procedure resulted in 0.50.6 mM quin-2). Both rotocols resulted in similar basal [Ca2+]ilevels and Gag+ transients. The effect of heavy metal chelators, diethylenetriaminepentaacetic acid (DTPA) and tetrakis[2-pyridylmethyl]ethylenediamine (TPEN),which prevent interference of fluorescence measurements (Arslan et al., 1985) of trace amounts of zinc was examined in preliminary studies. A small increase in quin-2 fluorescence was observed only when cells were loaded with 5 pM quin-2, with no effect on Ca2+ transients produced by hormone addition. Changes in intracellular quin-2 fluorescence were measured in 2 ml samples of continuously stirred cell suspensions in 1cm cuvettes at 37°C using a SLM 4880 spectrofluorometer(SLM Instruments, Urbana, IL).The slit widths were 4 nm for excitation and 8 nm for emitted light. Transformation of the fluorescent signal to [Ca2+]iwas performed according to Tsien et al. (1982). Alternatively, cells were loaded with fura-2/AM (20 pM) as described by Tsien et al. (1985). By alternating excitation wavelengths from 340-390 nm and monitoring fluorescence intensity at 505 nm, the ratio 340/390 was derived (Grynkiewicz et al., 1985).Calculations were performed using KD of fura-2 for calcium of 365 nM (Tsien et al., 1985). For microfluorometric studies fura-2 loading was performed by means of the “scrape-loading” technique as described by McNeil et al. (1984) and studied 8-12 h later as cells attached to collagen-coatedcoverslips.

Fluorescence flow cytometry of quin-2 loaded PTC Individual cell fluorescence of quin-2 was studied by means of flow cytometry (Coulter Electronics, Hialeah, FL). The flow cytometer was interfaced with a multiparameter data acquisition and display system. Excitation was 347 & 10 nm from an argon laser, and fluorescence was monitored at 480-515 nm. For each measurement fluorescence of 10,000-25,000 cells was collected, and fluorescence intensity was plotted against the number of counted cells. After the basal fluorescence of the cell suspension was determined, M phenylephrine was added to the cuvette, and recording was immediately repeated. The time required for completion of the fluorescence measurements was 40-120 sec. Under the experimental conditions phenylephrine did not affect the autofluorescence of the cells. Fluorescence microscopy and microfluorometry Proximal tubular cells (3°C) grown on coverslips, as described above, were incubated for 5-15 min in KrebsHenseleit buffer with an addition of either Lucifer Yellow CH (1 mg/ml), quin-2K (50 pM), or fura-2K (20 pM). The distribution of dyes was examined with a Zeiss IM35 microscope equipped for epifluorescence. Photographs were taken with Kodak Tri-X pan 400 ASA film. Microfluorometric measurements of a single cell calcium concentration was performed according to Tsien et al. (1985).Fura-2 loaded cells attached to collagen-coated coverslips were mounted into Sykes-Mooreflow-through chambers and placed on the stage of a Nikon Optiphot microscope equipped for epifluorescence (Episcopic-fluo-

468

GOLIGORSKY, HRUSKA, LOFTUS, AND ELSON

rescence attachment EF-D). Focusing on a single cell was performed using UV-40 oil objective and a pinhole diaphragm and corrected via a CF-eyepiece lens. On the excitation pathway, two BV dichroic mirrows (Nikon) were placed one with an interference filter at 340 nm (Oriel Corp., Stratford, C n , and the other with an interference filter at 381 nm (Oriel Gorp.). During experiments filters were alternated manually. Emitted light was collected at 480-515 nm. A calibration curve was built using 20 p M fura-2 in EGTA-bufferedintracellularlike solution with different final concentrations of [Ca2+]i. During experiments fluorescence intensity at 340 nm and 380 nm excitation wavelength was monitored, background and autofluorescencewere subtracted from these values, and the ratio 340/380 was obtained. The conversion of 340/380 ratios to the respective concentrations of [Ca2’]i was performed using the calibration curve. Transfer of Lucifer Yellow CH and fura-2 across cell monolayers To study pinocytosis (or transcytosis) PTC grown on collagen-coated polycarbonate membranes were mounted in a modified Ussing chamber. Both sides were bathed in Krebs-Henseleit buffer, pH 7.4, thermostated at 37°C. Solutions were gently mixed by bubbling 95% o&% C02. After 30 min equilibration fluorescent dye was added to one side of the membrane; solution on the contralateral side (without added dye)was changed every h. After 3 h of fluorescence measurements, each side was washed and fluorescent dye was added to the opposite side. Fluorescence measurements were then continued for an additional 3 h. A marker of fluid phase pinocytosis, Lucifer Yellow CH (Swanson et al., 1985) was used at a concentration of 0.25 mg/ml; quin-2, 20 pM, and fura-2, 2 pM. Net transfer was calculated by subtraction of the amount of the dye transfered in the direction basolateral-luminal side from the luminal-basolateral. Fluorescence of the samples was measured with a spectrofluorometer. Analysis of the data The data are presented as mean f SEM. The statistical difference was calculated using paired Student’s t test. The tracings presented below are representative of at least three experiments.

I

+Ii

Quin-2 I

K

I Min

[ca‘j nM

K t -

I

I

0 1 2 3 4 5 6 M I N Fig. 1. The effect of phenylephrine on [Ca2+]i concentration in suspensions of PTC as assessedwith quin-2(upper panel) and fwa-2 (lower panel).These results are typical of nine and five separate experiments with quin-2 and fwa-2,respectively.

M there was an incremental response of quin-2 fluorescence. Quin-2 fluorescence changes in response to lo-* M PE were similar to those of M (data not shown). The basal level of [Ca2+]iin suspensions of PTC measured with fura-2 averaged 122 rt 18 nM (n = 5), in fair agreement with the results obtained using quin-2. Exposure of the cells to M PE resulted in an increase [Ca2+]ias measured by changes in fura-2 fluorescence (Fig. 1B; Table 1). Are all PTC responsive to PE? To assess uniformity of the cellular response to “1adrenergic agonists, suspensions of PTC cells were studied by means of flow cytometry. Cells were loaded with quin-2 as previously described, and the fluorescence of individual cells was determined. Figure 2 demonstrates that exposure to PE resulted in a shift of quin-2 fluorescence intensity which was contributed by only 24.5 f 0.9% (n = 5) of the total number of cells counted, the value obtained by a simple subtraction of the curves. However, when Kolmogorov-Smirnov test of normality was applied to approximate the curves in Figure 2, the data were best fitted to a hypothetical bimodal distribution. Thus, comparison of the curves in Figure 2 by mere subtraction is invalid. Figure 3 demonstrates that 85% of the population of PTC responded to the administration of PE with an increase of fluorescence. Appropriate corrections for the actual increase in [Ca2+]iin the PEresponsive population of PTC were made (Table 1).

Materials Cell culture medium was obtained from KC Biological (Lenexa, KS); TMB-8 from Aldrich Chemical (Milwaukee, W; prazosin hydrochloride from Pfizer (New York, NY); L-phenylephrine hydrochloride, verapamil hydrochloride, Quin-2 AM, quin-2iK, and Lucifer Yellow were from Sigma Chemical (St. Louis, MO). Fura-2 AM and Fura-2iK, DTPA, and TPEN were from Molecular Probes, Inc. (Junction City, OR). RESULTS Effects of PE on [Ca2+li In this series of experiments, the basal level of [Ca2+]i in suspensions of PTC averaged 100.9 rt 11.3 nM (n = 9). Primary cultures of proximal tubular cells exhibited Specificity of the effect of P E and the source of an immediate (10-20 sec) increase of the quin-2 fluoresCa2+ mobilization cence signal when exposed to phenylephrine (Fig. 1A; Table 1).L-phenylephrine hydrochloride (PE) was inefDifferent “1 -adrenerpic antagonists were employed to fective at a dose of low7M; however, with doses of 10-% ascertain t h o specificiiy of th; PE-induced changes in

469

CYI-ADRENERGICAGONISTS AND CYTOPLASMICCALCIUM

TABLE 1.Phenylephrine-inducedchanges in cytoplasmic calcium concentration % Increase over basal level fsec)

Quin-2 Fura-2

Actual increase in [Ca2+]i in the PE-responsive subpopulation of EYTC 180-300 sec 20-60 S ~ C

Basal level

20-60

100.9 f 11.3 nM (n = 9) 122.5 f 18.0 nM

15.2 f 1.8*

19.2 f 2.0*

18.0 f 2.12*

22.0 f 2.4*

20.0 f 2.9*

36.9 f 4.8*

24.0 f 3.4*

44.0 f 5.7*

S ~ C

180-300 sec

*P c .05 as compared to basal level.

quin-2 fluorescence. Pretreatment of the cells with 40 nM prazosin for 3 min completely abolished the characteristic effect of M PE (Fig. 4A). The calcium channel blocker verapamil, which has been reported to antagonize the crl-adrenoreceptor-mediatedeffects of catecholamines on hepatocytes and in myocardium (Blackmore et al., 1979; Nayler et al., 19821, was also tested. As shown in Figure 4B, verapamil(25 pM)pretreatment for 3 min blocked an increase in [Ca2+]iinduced by PE. This effect was similar to that of prazosin. To further evaluate the specificity of the PE effect, the possibility of inducing refractoriness by repeated stimulation with PE was examined. As shown in Figure 4C, repeated treatment of PTC with M PE resulted in no additional effect on quin-2 fluorescence. Hence, it is

A

10000 Cells

B

2247 Cells

suggested that the changes in [Ca2+]iinduced by PE are related to a specific interaction with cY1-adrenoreceptors. In order to evaluate the requirement for extracellular calcium, cells loaded with quin-2 were resuspended in nominally calcium-free Krebs-Henseleit buffer (prepared with Ca-free water, 18kohm, with an addition of 200 pM EGTA). Figure 4D demonstrates that basal [Ca2+]i under these conditions, was decreased to the range of 50 nM. However, exposure to PE resulted in an immediate increase in [Ca2+]i, indicating that at least in a short-term experiment extracellular calcium is not essential for the PE-induced increase in [Ca2+Ii. FFC Data.

Bimodal Model

cn

J J

w V

n

U

w

IZ 3

0 V

0

0

10

30

20

l o g fluorescence

Log of Fluorescence Intensity

20

SO

SO

40

(arbitrary scale)

-W A 6

- A-

A Bimodal f i t

Fig. 2. Phenylephrine-inducedshift in fluorescence intensity of quin2-loaded EYTC in suspension. Panel A represents distribution of fluorescence intensity in a resting population of PTC (10,000cells were counted in this experiment) and immediately following administration of phenylephrine into the cuvette of the flow cytometer. Hatched area demonstrates an increase in fluorescence upon exposure to phenylephrine. Panel B-computer-derived substraction of the curves from panel A, reflecting phenylephrine-inducedshift in fluorescence intensity of quin2. This shift was contributed by 22.5% of the total counted cells. In five separate experiments this subpopulation of the cells averaged 24.5 f 0.9% of the total number of cells counted.

B Bimodal f i t

-----_component -__-__- component component

a A

b component B

Fig. 3. The data on Figure 2 were plotted and fitted to hypothetical bimodal distributions. The resultant curves demonstrate that the bulk of the cells (85%) responded to phenylephrine with an increase in fluorescenceintensity.

470

GOLIGORSKY, HRUSKA, LOFTUS, AND ELSON

[ca2+li

150

too nM

I. P y .

B

A 25pM

pF

1 0 5 ~

Ve r

10-5M

I min

I

n

10-5

PE

c

4

Ca-free medium

nM

150 I min

n

50

'1

t

10-SM PE

9E

lmin

D

himin

n

Fig. 4. Specificity of phenylephrine-induced increase in [Ca2+]icon- D)the effect of phenylephrine on [Ca2+liconcentration during incubacentration and the source of calcium mobilization. A) prazosin pretreat- tion in Ca-freemedium. For details of experimental protocol see text. ment; B) pretreatment with verapamil; C) homologous desensitization;

Fluorescence microscopy of quin-2 loaded cells: evidence for compartmentalization of quin-2 Quin-2 has been shown to be distributed homogeneously within the cytoplasm of lymphocytes (Tsien et al., 1982) and Ehrlich carcinoma cells (Arslan et al., 1985). However, Rink and Pozzan (1985) suggested that this does not necessarily imply to other cells. Fluorescence microscopy of PTC loaded with quin-2 demonstrated the expected cytoplasmic distribution of the fluorophore (Fig. 5A). However, multiple fluorescent spots were visible throughout the cytoplasm. Furthermore, when cell impermeant quin-2 or fura-2 were added to the medium, a bright punctate fluorescence within the unstained cytoplasm of PTC was observed (Fig. 5B). This suggested that quin-2 and fura-2 were not distributed exclusively within the cytoplasm of PTC. The only type of transcellular traffic that is spatially separated from the cytoplasm in these cells is represented by pinocytosis Wan Deurs and Christensen, 1984). In order to examine the possibility of pinocytotic entrapment of quin-2 or fura-2, PTC were grown on collagencoated polycarbonate membranes and mounted in the Ussing-type chamber. Cell-impermeant fura-2/Kt together with a marker of fluid-phase pinocytosis, LY, were added alternatively to the luminal or the basolateral compartment of the chamber. The transfer of dyes to the contralateral side was studied. Figure 6A shows the time course of transfer of LY from the luminal to the basolateral bath and vice versa. The luminal-basolateral transfer rate was three times higher than that occurring in the opposite direction. Assuming that the basolateral-luminal vector represents predominantly

paracellular traffic of the dye and subtracting these values from that obtained for luminal-basolateral transfer, the resulting net transfer characterizes fluid-phase transcytosis of LY in PTC. This process is linear during the time-course of the experiment, is inhibitable by 4"C, and exhibits a linear relationship with the concentration of the dye (data not shown). All of these features are characteristic of fluid-phase pinocytosis (Swanson et al., 1985). Luminal-basolateral transfer of a cell-impermeant fura-2/Kf also predominated over that occurring in the opposite direction (Fig. 6B) and net transfer of the dye was similar to that of LY. This data on the membrane-impermeant form of fura-2 indicates that the dye can be trapped and translocated to the basolateral side of PTC by means of fluid-phase transcytosis. In order to quantitate the relative amount of the dye in the vesicular compartment, PTC were loaded with quin-2/AM and quin-2/K+. Ca-independent fluorescence of quin-2 was then measured in both sets of samples. Fluorescence of the cells loaded with quin-2/Kf averaged 16%of that obtained in the cells loaded with quin2lAM. This indicated, that about 16% of the total cellular content of the dye was entrapped in the vesicular compartment. Single cell microfluorometric measurements of [Ca2+]i concentration In order to overcome the compartmentalization of fura2, PTC were scrape-loaded NcNeil et al., 1984)with the dye and studied 8-12 h later as described in Materials and Methods. No punctate fluorescence was visible in the cytoplasm. Sykes-Moore chamber was constantly

al-ADRENERGIC AGONISTS AND CYTOPLASMICCALCIUM

471

A

B Fig. 5A. Digitalized image of 340 nm fluorescence of the proximal tubular cell loaded with 50 pM quin-2 acetoxymethyl ester for 45 min. Fig. 5B. Fluorescence microscopy of proximal tubular cells incubated with 20 pM fura-2/Kf for 10 min. Punctate fluorescence of fura-2 is

perfused at a rate of 4 mVmin with oxygenated 95% 0 2 + 5% C02 Krebs-Henseleit-bicarbonate buffer, at room temperature, pH 7.4, to which the desired additions were made. A typical experiment is shown in Figure 7. The single cell was alternatively excited with light at 340 nm and 380 nm wavelengths. After background and resting state fluorescence measurements were made, PE was added to the perfusate for 3-5 min and then washed out. The lower panel represents the calculated ratio 3401 380 changes during the experiment, and the respective [Ca2+]iconcentrations. Table 2 summarizes the results obtained. Basal levels of [Ca2+]iconcentration obtained with this technique are lower than that reported by

distributed throughout the cytoplasm. Cells were excited at 340 nm, and fluorescence was collected at 490-510 nm. The similar fluorescent pattern was observed when preparations were excited at 360 and 390

-.

quin-2 or fura-2 in cell suspensions. PE resulted in a rapid four-fold increase in [Ca2+]i,with a subsequent decline of [Ca2+]itowards the basal level. All the cycle was completed within 2-5 min. These results confirm the data obtained in cell suspensions with quin-2 and fura-2. They also emphasize the importance of the mode of cell loading with a fluorescent indicator.

DISCUSSION Accumulating evidence [recently reviewed (Exton, 1985; Williamson et al., 19891 suggests that [Ca2+]i mediates the response to q-adrenergic stimulation in diverse systems such as the hepatocyte, lacrimal and

472

GOLIGORSKY, HRUSKA, LOFTUS, AND ELSON

B

A

Fura-2/ K+

2.4350.23%

Bosoloterol -+

0.982 0.06%

0.93A. 0.15%

8

HOURS

Y

Fig. 6. Comparison between Lucifer Yellow CH and fura-21K+ trans- monolayers; solution on the contralateral side was changed every hour fer across the PTC monolayers. Cells were grown on polycarbonate and fluorescence of samples were counted. After 3 h of incubation, membranes coated with collagen and mounted in Ussing chambers. monolayers were washed three times with Krebs-Henseleit buffer, and Monolayerswere incubated in Krebs-Henseleitbuffer, pH 7.4, at 37”C, fluorescent dye was added to the opposite side. Fluorescence measurewith continuous gentle bubblin of 95% o2/5%COz. Lucifer Yellow ments were continued for an additional 3 h. CH (0.25 mg/ml) and/or fura-2K% (2 llM, were added to one side of the

--

TABLE 2. Phenylephrine-induced changes in cytoplasmic calcium concentration of single proximal tubular cells

SINGLE CELL FURA-2 FLUORESCENCE

ul

Basal level (n = 14) Perfusion with phenylephrine (n = 7 ) Wash-out of phenylephrine (n = 5)

57.1 f 6.5 nM 242.9 f 34.9 nM* 137.0 f 7.4 nM**

*P i.05, as compared to basal level. **P < .05,as compared to peak level.

.IE 0.2 V

Fig. 7. Microfluorometricmeasurement of [Ca2+]iconcentration in a single cell “scraploaded” with fura-2. The upper panel represents an actual recording obtained at two wavelengths 340 nm and 380 nm. The lower panel represents calculated ratios 340380, and the respective levels of [Ca2+liconcentration.

parotid gland, heart, smooth muscle, and adipocyte. In renal tubular cells, the mechanism of action is unknown. PE ultimately produces an increase in fluid transport and gluconeogenesis (Exton, 1985;Williamson et al., 1985;McPherson and Summers, 1982). The present studies demonstrate that PE produces a prompt increase in [Ca2+]i in PTC. Fluorescence flow

cytometry indicates that a high proportion (85%)of the cells are responsive; this result corroborates the findings from morphological and functional studies (Hruska et al., 1986) that the cell culture system used provides a pure population of proximal tubule cells. The data are limited, however, by the fact that the cells accumulated dye into pinocytotic vesicles, as demonstrated by punctate fluorescence within the cytoplasm of cells. There is good morphologic evidence of abundant clathrin-coated pits and the formation of pinocytotic vesicles in PTC (Rodman et al., 1984; Van Dews and Christensen, 1984). This process resulted in compartmentalization of fluorescent probes, as expressed by a punctate fluorescence distribution within the cytoplasm, as studied by fluorescence microscopy. Vectorial apical-basolateral translocation of the cell-impermeant fura-2 favors the suggestion of pinocytotic (transcytotic) vesicle entrapment of the dye. Similar compartmentalization of fura-2 has been recently demonstrated in mast cells (Almers and Neher, 1985). Under conditions used to measure fluorescence changes of quin-2 or fura-2 in PTC, the dye is distributed in two partially separated compartments within the cell: in the cytoplasm and within pinocytotic vesicles. The indicator which is trapped in the pinocytotic compart-

al-ADRENERGIC AGONISTS AND CYTOPLASMICCALCIUM

ment is not immediately exposed to changes in cytosolic concentration of calcium (about 16%of the total cellular dye content is trapped in this compartment). However, this fraction of indicator dye will be picked up during the calibration procedure, and will increase artificially Fm, and Fmin range. Consequently, the scale for calibration of fluorescence changes will be broadened, thus underestimating the calculated values of actual changes in [ca2+1i. The data on the basal levels of [Ca2+]iare in a good agreement with those reported previously (Hruska et al., 1986), and with the results obtained by other methods (Lorenzen et al., 1984; Murphy and Mandel, 1982; Snowdown and Borle, 1984). The fluorescence response to cq-adrenergic stimulation was prompt (10-20 sec). The specificity of this change in [Ca2+]i was demonstrated by pretreatment with prazosin and verapamil, which completely abolished response to phenylephrine. The ability to induce homologous desensitization of [Ca2+]iresponse further supported the specificity of the phenylephrine effect. We conclude that PE induces an increase in [Ca2+]iin cultured renal PTC.PE-induced increase in [Ca2+]ican be blocked at the level of the receptor (prazosin and verapamil) and does not require extracellular calcium. In addition, quantitation of the fluorescence signals obtained with quin-2 and fura-2 in PTC requires caution, since these Ca2 indicators are partially compartmentalized in pinocytotic vesicles, thus underestimating the calculated values of Ca2+ transients. When the entrapment of the dye was prevented by using scrape-loading protocol, PE resulted in a four-fold increase in [Ca2+]i concentration. This mode of cell loading with the dye can, perhaps, be used in other cells like macrophages, endothelium, mast cells etc., which are characterized by a significant rate of endocytosis. +

ACKNOWLEDGMENTS Expert assistance of Ms. Helen Odle in the preparation of this manuscript is gratefully acknowledged. Dr. Goligorsky is a Sabbatical-Fellow at Jewish Hospital, Washington University Medical School and a recipient of the award from the National Kidney Foundation of Eastern Missouri and Metro East, Inc. This study was supported in part by the National Institutes of Health Grants AM09976 and AM32087. LITERATURE CITED Almers, W., and Neher, E. (1985) The Ca signal from fura-2 loaded mast cells depends strongly on the method of dye-loading. FEBS Lett., 192:13-18. Arslan, P., Di Virgilio, F., Beltrame, M., Tsien, R.V., and Pozzan, T. (1985) Cytosolic Ca2+ homeostasis in Ehrlich and Yoshida carcinomas. J. Biol. Chem., 260:2719-2727. Bello-Reuss,E., Colindres, R., Pastoriza-Munoz,E., Mueller, R.A., and Gottschalk, C.W. (1975)Effects of acute unilateral renal denervation in the rat. J. Clin. Invest., 56208-217. Bello-Reuss, E., Trevino, D.L., and Gottschalk, C.W. (1976) Effect of renal sympathetic nerve stimulation on proximal water and sodium reabsorption. J. Clin. Invest., 571104-1107. Blackmore, P.F., El-Refai,M., and Exton, J. (1979) a-Adrenergic blockade and inhibition of A23187 mediated Ca2+ uptake by the calcium antagonist verapamil in rat liver cells. Molec. Pharmacol., 15498606. Blackmore, P.R., Hughes, B.P., Shuman, E.A., and Exton, J.H. (1982) m -Adrenereic activation of DhosDhorvlase in liver cells involves mobilization 2 intracellular Ealcik without influx of extracellular calcium. J. Biol. Chem., 257190-197.

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Chan, Y.L. (1980) Adrenergic control of bicarbonate absorption in the proximal convoluted tubule of the rat kidney. Mug. Arch., 388159164. Charest, R., Blackmore, P.F., Berthon, B., and Exton, J.M. (1983) Changes in free cystolic Ca" in hepatocytes following al-adrenergic stimulation. J. Biol. Chem., 2588769-8773. DiBona, G.F. (1977) Neurogenic regulation of renal tubular sodium reabsorption. Am. J. Physiol., 233F73-F81. Exton, J.M. (1985) Mechanisms involved in a-adrenergic phenomena. Am. J. Physiol., 248:E633-E647. Freiberg, M., Harrison, S., and Filburn, C.R. (1986) al-Adrenergic regulation of phosphoinositide metabolism and gluconeogenesis in rat renal proximal tubules. (Abstr.) Kidney Internat., 29:353. Gottschalk, C.W. (1979) Renal nerves and sodium excretion. Ann. Rev. Physiol., 41:229-240. Grynkiewicz, G., Poenie, M., Tsien, R.Y. (1985) A new generation of Ca2' indicators with greatly improved fluorescence properties. J. Biol. Chem., 260 3440-3450. Harvey, D.J., Godber, J.F., Timmerman, M.P., Castell, L.M., and Ashley, S.S. (1985) Measurement of free Ca2+ changes and total Ca2' release in a single striated muscle fibre using the fluorescent indicator quin-2. Biochem. Biophys. Res. Commun., 1281180-1189. Hesse, I.F.A., and Johns, E.J. (1983)Renal tubular al-adrenergic receptors mediate the antinatriuretic response to renal nerve stimulation in the rabbit. J. Physiol. (London), 339:32P. Hruska, K., Goligorsky, M., Scoble, J., Moskowitz, D., Westbrook, S., and Mills, S. (1986) The effeds of parathyroid hormone on cytosolic calcium in renal proximal tubular primary cultures. Am. J. Physiol. (in press). Kessar, P., and Saggerson, E.D. (1980) Evidence that catecholamines stimulate renal gluconeogenesis through an a1-type of adrenoreceptor. Biochem. J., 190119-123. Klahr, S., Nawar, T., and Schoolwerth,A.C. (1973) Effects of catecholamines on ammoniogenesis and gluconeogenesis by renal cortex in vitro. Biochem. Biophys. Acta, 304:161-168. Kurokawa, K., and Massry, S. (1973) Evidence for stimulation of renal gluconeogenesis by catecholamines. J. Clin. Invest., 52961-964. Lorenzen, M., Lee, C., and Windhager, E. (1984) Cytosolic Ca2+ and Na+ activities in perfused proximal tubules of Necturus kidney. Am. J. Physiol., 247F93-Fl02. MacDonald, D.W.R., and Saggerson, E.D. (1977) Hormonal control of gluconeogenesis in tubule fragments from renal cortex of fed rats. Biochem. J., 168:33-42. McNeil, P.L., Murphy, R.F., Lanni, F., and Taylor, D.L. (1984)A method for incorporating macromolecules into adherent cells. J. Cell Biol., 981556-3564. McPherson, G.A., and Summers, R.J. (1982)A study of el-adrenorecep tors in rat renal cortex: comparison of [3Hfprazosin binding with a1adrenoreceptor modulating gluconeogenesis under physiological conditions. Br. J. Pharmacol., 77177-184. Muller, J., and Barajas, L. (1972) Electron microscopic and histochemical evidence for a tubular innervation in the renal cortex of the monkey. J. Ultrastruct. Res., 41:533-549. Murphy, E, and Mandel, L. (1982) Cytosolic free calcium levels in rabbit proximal tubules. Am. J. Physiol., 242:C124-C128. Nayler, W.G., Thompson, J.E., and Jarrott, B. (1982)The interaction of calcium antagonists (slow channel blockers) with myocardial alpha adrenoreceptors. J. Molec. Cell. Cardiol., 14185-188. Osborne, J.L., Holdaas, M., Thames, M.D., and DiBona, G.F. (1983) Renal adrenoreceptor mediation of antinatriuretic and renin secretion responses to low frequency renal nerve stimulation in the dog. Circ. Res., 53:298-305. Osswald, M., and Greven, J. (1981)Effects of adrenergic activators and inhibitors on kidney function. In: Handbook of Experimental Pharmacology. M. Osswald, ed. Springer-Verlag,New York, Vol. 54m, pp. 243-288. Prosnitz, E.M., and DiBona, G.F. (1978) Effect of decreased renal sympathetic nerve activity on renal tubular sodium reabsorption. Am. J. Physiol., 235F557-F563. Putney, J.W. (1979)Stimulus-permeabilitycoupling: Role of calcium in receptor regulation of membrane permeability. Pharmacol. Rev., 30209-245. Reinhart, P.M., Taylor, W.M., and Bygrave, F.L. (1984)The mechanism of cq-adrenergic agonist action in liver. Biol. Rev., 59511-557. Rink, T.J., and Pozzan, T. (1985) Using quin-2 in cell suspensions. Cell Calcium, 6113-144. Rodman, J.S., Kerjashki, D., Merisko, E., and Farquhar, M.G. (1984) Presence of an extensive clathrin coat on the apical plasmalemma of the rat kidney proximal tubular cells. J. Cell Biol., 989630-1636. Schmitz. J.M.. Graham. R.M., Sagalowsky. A.. and Pettinger, W.A. (1981)Renal al- and aradrener& receptors: biochemical and phar~

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GOLIGORSKY, HRUSKA, LOFTUS, AND ELSON

macological correlations. J. Pharmacol. Exp. Ther., 219400406, Snavely, M.D., and Insel, P.A. (1982) Characterization of alpha-adrenergic receptor subtypes in the rat renal cortex. Mol. Pharmacol., 22532-546. Snowdown, K., and Borle, A. (1984) Measurement of cytosolic free calcium in mammalian cells with aequorin. Am. J. Physiol., 247C396-C409. Swanson, J.A, Yirinec, B.D., and Silverstein, S.C. (1985)Phorbol esters and horseradish peroxidase stimulate pinocytosis and redirect the flow of pinocytosed fluid in macrophages. J. Cell Biol., 100:851-859. Tomlinson, R.W., and Wood, A.W. (1976) Catecholamine-induced changes in ion transport in short-circuitedfrog skin and the effect of &blockade. J. Physiol., 257:515-530. Tsien, R., Pozzan, T., and Rink, T. (1982)Calcium homeostasis in intact

lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J. Cell Biol., 94:325-334. Tsien, R.Y., Rink, T.J., and Poenie, M. (1985)Measurement of cytosolic free Ca2+ in individual small cells using fluorescence microscopy with dual excitation wavelengths. Cell Calcium, 6:145-157. Vinay, P., Gougoux, A., and Lemieux, G. (1981) Isolation of a pure suspension of rat proximal tubules. Am. J. Physiol., 241:F403-F411. Williamson, J.R., Cooper, R.H., Joseph, S.K., and Thomas, A.P. (1985) Inositol triphosphate and diacylglycerolas intracellular second messengers in liver. Am. J. Physiol., 248C203-C216. Wood, A.W., and Tomlinson, R.W. (1974) The effect of catecholamines on ion transport in the toad bladder. Biochem. Biophys. Acta, 367375-384.