Calcitonin Stimulates Lysosomal Enzyme Release and Uptake in LLC-PK1 Cells

J Am Soc Nephrol 10: 1640–1648, 1999 Calcitonin Stimulates Lysosomal Enzyme Release and Uptake in LLC-PK1 Cells

MASAKO GOTO* and KAZUTOSHI MIZUNASHI† *Institute of Rehabilitation Medicine, Tohoku University School of Medicine, and †Second Department of Internal Medicine, Tohoku University School of Medicine, Sendai, Japan.

Abstract. Renal tubular targeted hormones increase urinary inhibitor), and 10 mM mannose-6-phosphate. Furthermore, excretion of a lysosomal enzyme, N-acetyl-␤-D-glucosamini- calcitonin promoted progression of intracellular membranes dase (NAG). To elucidate the mechanism of this event, the stained by a fluorescence membrane marker, styryl pyridinium calcitonin effect on NAG handling by LLC-PK1 cells was dye, from cell periphery to perinuclear regions (commonly examined. Calcitonin (1 nM to 1 ␮M), phorbol myristate (10 referred to as recycling vesicles) and increased dye release nM to 1 ␮M), and ionomycin (1 to 10 ␮M) promoted NAG from preloaded cells. Fluorescence release from the cells pre- release without any increase in lactate dehydrogenase release loaded with FITC-labeled NAG or albumin was also stimulated or any reduction of mitochondrial dehydrogenase activity. by calcitonin. These calcitonin effects on endocytotic and Treatment with 100 nM calphostin C or 50 ␮M KN-93 par- re-exocytotic pathways were inhibited by 100 nM cytochalasin tially reversed the calcitonin effect on NAG release. Calcitonin D, 100 nM nocodazole, 0.1 to 1 ␮M bafilomycin A1, or 0.1 promoted secretion of fluorescence ceramide, a reporter of mM monodansyl cadaverine. Increased urinary NAG excretion protein transport from Golgi apparatus to cell surface. Calci- has been considered to reflect renal tubular damage. However, tonin-stimulated NAG release was partially inhibited by 10 it was demonstrated here that stimulation of secretory and ␮g/ml brefeldin A, a blocker of protein transport through the recycling pathways may be an alternative mechanism for cal- Golgi apparatus. Calcitonin accelerated cellular uptake of ex- citonin-induced enzymuria, which will become a new indicator ogenous NAG, which was inhibited by low temperature, 0.1 of renal tubular response to this hormone. mM monodansyl cadaverine (receptor-mediated endocytosis

Newly synthesized molecules from biosynthetic pathways and resistance to PTH (pseudohypoparathyroidism) (6). Calcitonin exogenous materials carried through endocytic pathways are also increases urinary NAG excretion (6). Therefore, lysoso- transferred to lysosomes. Therefore, lysosomes have been con- mal enzyme excretion may be modified by renal tubular tar- sidered to be the final destination of unidirectionally trans- geted hormones and may be useful as a marker of renal tubular ported soluble macromolecules. However, retrograde traffick- responsiveness to these hormones. ing from lysosomes can occur (1) and lysosomes may be LLC-PK1 cells, which are derived from porcine kidney, maintained by a repeated series of transient fusion and fission express calcitonin receptors, which couple to both adenylyl processes with late endosomes/prelysosome compartments (2). cyclase and phospholipase C (7), and have retained several Moreover, a model of secretory lysosomes fusing directly with features of proximal tubular cells, including high NAG activity the plasma membrane was proposed for mediating lysosomal (8,9). In this study, we investigated the regulatory effects of enzyme release (3). calcitonin on NAG release, cellular uptake of externally sup- Renal proximal tubular cells contain high amounts of plied NAG, protein transport from Golgi apparatus to cell N-acetyl-␤-D-glucosaminidase (NAG), one of the lysosomal surface, intracellular transport of vesicular membranes, and glycolytic enzymes (4). Urinary excretion of this enzyme has re-exocytotic pathway in LLC-PK1 cells. The results will help been used as a marker of renal tubular damage. However, it us further understand hormonal regulation of lysosomal en- shows diurnal variation in healthy subjects similar to the timing zyme handling by renal tubular cells. of circadian rhythm in urinary free cortisol (5). Parathyroid hormone (PTH) promotes urinary NAG excretion in healthy subjects but to a lesser extent in the patients with renal tubular Materials and Methods Materials NAG extracted from bovine kidney was purchased from Boehr- Received November 4, 1998. Accepted March 13, 1999. Correspondence to Dr. Kazutoshi Mizunashi, Second Department of Internal inger Mannheim Biochemicals (Mannheim, German); N-(4,4-dif Medicine, Tohoku University School of Medicine, Seiryo-cho 1-1, Aoba-ku, luoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl) Sendai 980-8574, Japan. Phone: 81 22 717 7165; Fax: 81 22 223 2077; E-mail: sphingosine (BODIPY FL C5-ceramide) and N-(3-triethylammonium- [email protected] propyl)-4-(4-(dibutylamino) styryl) pyridinium dibromide (FM 1-43) 1046-6673/1008-1640 were from Molecular Probes (Eugene, OR); KN-93 was from Seika- Journal of the American Society of Nephrology gaku Co. (Tokyo, Japan); Dulbecco’s modified Eagle’s medium Copyright © 1999 by the American Society of Nephrology (DMEM) was from Life Technologies BRL (Gaithersburg, MD); fetal J Am Soc Nephrol 10: 1640–1648, 1999 Calcitonin-Regulated NAG Handling 1641

calf serum was from Hyclone Laboratories (Logan, UT); all other membrane was determined at 4°C using the same procedure as for materials were obtained from Sigma Chemical Co. (St. Louis, MO). endocytotic experiments.

Cell Culture FITC-Albumin and FITC-NAG Release Assay LLC-PK1 (ATCC CRL 1392) cells were obtained from American The effects of calcitonin on re-exocytotic pathway were also stud- Type Culture Collection (Rockville, MD) and maintained in DMEM ied. Cells were pulsed for 2 h with FITC-albumin (500 ␮g/ml) or supplemented with 10% fetal calf serum. Confluent monolayers were FITC-NAG (200 ␮g/ml). This medium was removed and cells were split once a week and the medium was changed twice a week. washed 6 times with fresh medium. Thereafter, cells were incubated with the indicated agents for 30 min. At 30-min intervals, conditioned NAG Release Experiments medium was collected and replaced with fresh media. The fluores- We examined the effect of salmon calcitonin (sCT) on NAG cence of conditioned medium was measured by a spectrofluorometer

release from LLC-PK1 cells and studied the contribution of protein at excitation 495 nm/emission 520 nm. Fluorescence release was kinase C (PKC), protein kinase A (PKA), and Ca2ϩ/calmodulin- expressed as ␮g equivalent FITC-albumin or FITC-NAG/mg cell dependent protein kinase II (CaMK II) to this action. Cells were protein per 30 min. grown in 10% serum-supplemented medium on 24-well plates for 4 d followed by an additional 18 h of preincubation in serum-free medium containing 0.1% bovine serum albumin (BSA). The stock solution of FITC-Labeling of NAG 10% BSA in phosphate-buffered saline (PBS) was heated at 50°C for NAG (3 mg) extracted from bovine kidney (Boehringer Mannheim, 2 h to inactivate bovine NAG. On the experimental day, cells were Mannheim) was incubated with FITC isomer I (1 mg) in 0.1 M incubated for 30 min with indicated agents and NAG release into the sodium carbonate-bicarbonate buffer, pH 9.0, for2hat20°C. FITC- medium was measured at 30-min intervals. conjugated NAG was purified by eluting Sephadex G-25 column with PBS. Fluorescein/NAG molar ratio was 6.

N-Acetyl-␤-D-Glucosaminidase Activity Assay NAG activities were determined using 4-methylumbelliferyl-N- Monitoring Transport from Golgi Apparatus to Cell acetyl-␤-D-glucosaminide as substrate, from which NAG liberates Surface 4-methylumbelliferone at pH 4.5 (10). One unit of NAG activity was As a reporter for secretory activity, we used BODIPY FL C - ␮ 5 defined as the amount of NAG catalyzing the liberation of 1 mol of ceramide, which enters cells via spontaneous transfer and specifically 4-methylumbelliferone per minute at 37°C. NAG release was ex- labels the Golgi complex before its metabolites are transported to the ␮ pressed as nU/ g cell protein per min. Protein was determined by a cell surface (11). For staining of the Golgi apparatus, cells were modified method of Bradford using Bio-Rad Protein Assay (Bio-Rad, ␮ incubated for 2 h with 5 M BODIPY FL C5-ceramide. After label- Richmond, CA). ing, cells were washed 3 times with medium and treated with 1 ␮M sCT for 30 min. Cellular fluorescence was observed with a confocal LDH Activity Assay and Mitochondrial Dehydrogenase laser microscope (Leica, Heidelberg, Germany) excited with a Activity Assay 488-nm laser and detected with 530-nm band-pass filter. Cellular To determine whether cell integrity was affected by the exposure to fluorescence intensity was measured with the cells grown on 96-well sCT, phorbol myristate (PMA), and ionomycin, LDH activity was plates using fluorometer (Fluoroskan Ascent; Libsystems, Helsinki, measured in the incubation medium and cell lysates. Cells were Finland) at excitation 485 nm/emission 538 nm. treated with 1 ␮M sCT, 100 nM PMA, or 10 ␮M ionomycin for 30 min. LDH released into medium during the next 3 h was measured Monitoring Endocytosis and Exocytosis using using LDH-cytotoxic test Wako (Wako Junyaku, Osaka Japan). The Membrane Markers reduction of mitochondrial dehydrogenase activity is an early sign of functional impairment (sublethal injury) of cells. The enzyme activity, Using the fluorescent dye FM 1-43, which is virtually nonfluores- the ability to reduce tetrazolium salt WST-1 (succinate-tetrazolium cent in aqueous medium and is inserted into the outer leaflet of surface reductase activity), during 3 h incubation after 30 min preincubation membrane where it becomes intensely fluorescent (12), we investi- with sCT, PMA, or ionomycin was measured using a Cell Counting gated the effects of calcitonin on endocytosis and exocytosis. In ␮ kit (Dojindo, Kumamoto, Japan). endocytosis experiments, cells were exposed to 5 M FM 1-43 with or without 1 ␮M sCT for 30 min and then incubated with FM 1-43 for the indicated additional time. For monitoring exocytosis, cells were Endocytosis Experiments stained with 5 ␮M FM 1-43 for 4 h followed by rapid twice washing The endocytotic pathway may also be involved in the modulatory with fresh medium and then treated with 1 ␮M sCT for 30 min. effects of sCT, PMA, and ionomycin on lysosomal enzyme targeting. Membrane-incorporated FM 1-43 was observed with confocal micro- After 30 min preincubation with 1 ␮M sCT, 100 nM PMA, or 10 ␮M scope excited with 488 nm laser and detected with 605-nm-long pass ionomycin, cells were incubated for 30 min with FITC-labeled NAG filter. The changes in cellular fluorescence intensity were monitored (200 ␮g/ml) or FITC-labeled albumin (200 ␮g/ml) after the indicated with the cells grown on 96-well plates using fluorometer at excitation time intervals. Incubation was stopped by rinsing the cells 6 times 485 nm/emission 590 nm. with Hanks’ balanced salt solution containing 10 mM Hepes, pH 7.4, and cells were solubilized by 0.1% Triton X-100 in PBS. The intracellular fluorescence was determined using a spectrofluorometer at Statistical Analyses excitation 495 nm/emission 520 nm. Cellular uptake of FITC-albumin All values are presented as means Ϯ SD. The significance of and FITC-NAG were expressed as ␮g fluorescence/mg cell protein differences was tested by one-way ANOVA. Differences were con- per 30 min. Binding of FITC-NAG or FITC-albumin to plasma sidered significant if P Ͻ 0.05. 1642 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 1640–1648, 1999

Figure 1. Effect of salmon calcitonin (sCT), phorbol 12-myristate 13-acetate (PMA), and ionomycin on N-acetyl-␤-D-glucosaminidase (NAG) ␮ Œ F ␮ ࡗ release from LLC-PK1 cells. Treatment with 1 M sCT ( ), 100 nM PMA ( ), and 10 M ionomycin ( ) increased NAG release (A). Dose-dependent effects of sCT (1 nM to 1 ␮M), PMA (0.1 nM to 1 ␮M), or ionomycin (0.1 to 10 ␮M) on NAG release are presented in Panel B. Number of experiments (n) ϭ 16. *P Ͻ 0.05 versus control (E).

Results effect on the calcitonin action on NAG release (data not pre- Effect of sCT, PMA, and ionomycin on NAG Release sented). The effects of sCT and ionomycin on NAG release and Ceramide Transport from Golgi Apparatus to the were significantly reduced in the presence of 50 ␮M KN-93 Cell Surface (CaMK II inhibitor) (Table 1). Calcitonin promoted release of

Treatment with sCT stimulated NAG release from LLC-PK1 BODIPY FL C5-ceramide fluorescence from the preloaded cells in a dose-dependent manner (Figure 1). PMA and iono- cells (Figure 2). Brefeldin A is a potent drug that blocks coated mycin mimicked the calcitonin action. Ionomycin reproduced vesicle formation from Golgi apparatus and induces a revers- the initial increase in NAG activity, whereas PMA reproduced ible block of protein secretion. The effects of sCT and PMA on the calcitonin action in a more sustained manner. PKA activa- NAG release were partially reversed by this agent (Table 1). tors (100 ␮M forskolin, 100 ␮M dibutyryl cAMP, 100 ␮M SpcAMP, 100 ␮M 8-bromo cAMP) had no significant effect Effect of sCT, PMA, and Ionomycin on Cell Integrity on NAG release (data not shown). Calcitonin-induced NAG and Viability release was suppressed by the pretreatment with 100 ␮M H-7 Treatment with sCT, PMA, or ionomycin had no effect on (nonspecific serine/threonine kinase inhibitor) (data not LDH release. Ionomycin had no effect on mitochondrial dehy- shown) and 100 nM calphostin C (selective PKC inhibitor) drogenase activity. PMA and sCT did not reduce but rather (Table 1), whereas 100 ␮M RpcAMPS (PKA inhibitor) has no increased the enzyme activity (135% of control cells). Collec-

Table 1. Inhibitory effects of calphostin C, KN-93, and brefeldin A on the action of salmon calcitonin (sCT), phorbol ␤ a myristate (PMA), and ionomycin on N-acetyl- -D-glucosaminidase (NAG) release from LLC-PK1 cells NAG Release (nU/␮g cell protein per min) Condition Control sCT (1 ␮M) PMA (100 nM) Ionomycin (10 ␮M)

Control 4.7 Ϯ 0.5 22.7 Ϯ 1.4b 23.4 Ϯ 1.0b 14.6 Ϯ 2.0b Calphostin C (100 nM) 3.6 Ϯ 0.5b 14.0 Ϯ 1.3c 13.3 Ϯ 2.9d KN-93 (50 ␮M) 3.9 Ϯ 0.2b 17.3 Ϯ 1.1c 6.0 Ϯ 0.4e Brefeldin A (10 ␮g/ml) 3.2 Ϯ 0.4b 15.9 Ϯ 2.2c 14.2 Ϯ 1.5d 14.0 Ϯ 1.6

a LLC-PK1 cells were treated with sCT, PMA, or ionomycin for 30 min after a 30-min preincubation with calphostin C or KN-93. In the brefeldin A case, experiments were conducted in the presence of brefeldin A after a 30-min preincubation with brefeldin A. n ϭ 8. b P Ͻ 0.05 versus control. c P Ͻ 0.05 versus sCT treatment. d P Ͻ 0.05 versus PMA treatment. e P Ͻ 0.05 versus ionomycin treatment. J Am Soc Nephrol 10: 1640–1648, 1999 Calcitonin-Regulated NAG Handling 1643

Figure 2. Effects of calcitonin on the release of BODIPY FL C5-ceramide fluorescence from the preloaded cells. (A) Calcitonin promoted the ␮ release of BODIPY FL C5-ceramide fluorescence from the preloaded cells. Top panel, control; Bottom panel, sCT-treated cells (1 M). (B) Cellular fluorescence intensity measured using fluorometer are expressed by the ratio with the 0 min value as 1. F, calcitonin; E, control. n ϭ 8. *P Ͻ 0.05 versus control.

tively, these observations indicated that cell viability was not baseline and calcitonin-induced uptake of NAG and albumin significantly affected in these experimental conditions. (Table 2). NAG contains mannose-6-phosphate (M6P), which is a recognition marker for mannose-6-phosphate receptor

Effects of sCT on Albumin and NAG Uptake (MPR). LLC-PK1 cells may reuptake NAG through receptor- Treatment with sCT, PMA, or ionomycin promoted cellular mediated endocytosis via MPR on the plasma membrane. To uptake of externally supplied NAG and albumin with the test this possibility, endocytosis experiments were conducted maximum effect at 60 to 90 min (Figure 3). Binding of FITC- in the presence of M6P. Treatment with M6P partially blocked NAG or FITC-albumin to plasma membrane determined at 4°C both baseline and calcitonin-stimulated NAG uptake but had was not affected by these agents (Figure 3). Monodansyl no effect on albumin internalization (Table 2). We also inves- cadaverine (MDC) inhibits clustering and internalization of the tigated the role of proton pump, actin, and microtubule in the ligand-receptor complexes into clathrin-coated vesicles. We stimulatory effect of calcitonin using Bafilomycin A1, cy- used this agent to investigate the possible role of receptor- tochalasin D, or nocodazole. Treatment with these antagonists mediated endocytosis in NAG uptake. MDC strongly inhibited inhibited calcitonin-stimulated uptake of albumin (Table 2). 1644 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 1640–1648, 1999

Figure 3. Effect of sCT, PMA, and ionomycin on cellular uptake of FITC-labeled albumin or FITC-labeled NAG. Treatment with 1 ␮M sCT (Œ), 100 nM PMA (F), and 10 ␮M ionomycin (ࡗ) increased uptake of FITC-labeled albumin (A) and FITC-labeled NAG (B). Binding of FITC-albumin or FITC-NAG on the plasma membrane determined at 60 to 90 min (arrows) was not affected with these agents. Treatment with 0.1 mM monodansyl cadaverine (MDC; ) suppressed cellular uptake of FITC-labeled albumin and FITC-labeled NAG. n ϭ 8. *P Ͻ 0.05 versus control (E).

Table 2. Effects of monodansyl cadaverine (MDC) and mannose-6-phosphate (M6P) on endocytosis and role of proton pump, actin, and microtubule in cellular uptake of albumina

FITC-Albumin Uptake (␮g/mg cell FITC-NAG Uptake (␮g/mg cell protein protein per 30 min) per 30 min) Condition Control sCT (1 ␮M) Control sCT (1 ␮M)

Control 1.93 Ϯ 0.17 3.22 Ϯ 0.25b 1.24 Ϯ 0.06 1.89 Ϯ 0.11b MDC (0.10 mM) 1.34 Ϯ 0.18b 2.17 Ϯ 0.15c 0.89 Ϯ 0.05b 1.31 Ϯ 0.05c M6P (10 mM) 2.14 Ϯ 0.24 3.57 Ϯ 0.24 1.13 Ϯ 0.11b 1.52 Ϯ 0.14c Bafilomycin A1 0.05 ␮M 1.36 Ϯ 0.14b 1.87 Ϯ 0.27c 0.1 ␮M 1.22 Ϯ 0.16b 1.27 Ϯ 0.20c 1 ␮M 1.13 Ϯ 0.13b 1.31 Ϯ 0.33c Cytochalasin D (100 nM) 1.79 Ϯ 0.18 2.56 Ϯ 0.12c Nocodazole (100 nM) 2.05 Ϯ 0.18 2.54 Ϯ 0.24c Brefeldin A (10 ␮g/ml) 1.69 Ϯ 0.22b 2.43 Ϯ 0.29c

a Cells were treated with sCT for 30 min in combination with MDC, after a 30-min preincubation with Bafilomycin A1, cytochalasin D, and nocodazole, or in the presence of brefeldin A after a 30-min preincubation with brefeldin A. Cellular uptake of FITC-labeled albumin or FITC-labeled NAG was measured at 60 to 90 min. Endocytosis experiments were also conducted in the presence of M6P. n ϭ 8. b P Ͻ 0.05 versus control. c P Ͻ 0.05 versus sCT treatment.

Baseline and calcitonin-stimulated albumin uptake was also loss in fluorescence intensity. Treatment with sCT promoted partially inhibited by brefeldin A. this progression of the fluorescence membranes to perinuclear regions. The fluorescence intensity of calcitonin-treated cells Effects of Calcitonin on the Intracellular Progress of was higher at 60 to 90 min than control cells (Figure 4). Endocytosed Membranes Intracellular membranes stained by FM 1-43 were found in Effect of sCT on the Release of the Fluorescence from a cluster of vesicles in both peripheral (probably early endo- the Cells Preloaded with FM 1-43, FITC-Albumin, or somes) and perinuclear regions (commonly referred to as re- FITC-NAG cycling vesicles) of the cell. During endocytosis, FM 1-43 In the fluorescence release experiments with the cells pre- progressed from cell periphery to perinuclear region with little loaded with FM 1-43, sCT promoted decrease of fluorescence J Am Soc Nephrol 10: 1640–1648, 1999 Calcitonin-Regulated NAG Handling 1645

Figure 4. Effects of calcitonin on the intracellular progress of endocytosed membranes. (A) Treatment with sCT (1 ␮M) for 30 min promoted the progression of membrane-incorporated N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide (FM 1-43) from cell periphery to perinuclear region. Top panel, control; Bottom panel, sCT-treated cells (1␮M). (B) The fluorescence intensity of the sCT-treated cells (F) was higher at 60 to 90 min than control cells (E) (cellular fluorescence intensity is expressed as ␮M equivalent FM 1-43 in methanol). n ϭ 8. *P Ͻ 0.05 versus control.

intensity of the cells (Figure 5). Treatment with sCT stimulated sis. To determine the relationship between endocytosis and release of the fluorescence from the cells preloaded with FITC- re-exocytosis, MDC were used. Pretreatment with MDC re- labeled albumin or NAG (Figure 6). Ionomycin or PMA reduced both baseline and calcitonin-stimulated release of fluo- produced calcitonin-stimulated release of the fluorescence, but rescence from the cells preloaded with FITC-albumin or FITC- with a certain delay in PMA experiments. Pretreatment with NAG (Figure 6). MDC (0.02 to 0.1 mM) also reduced sCT KN-93 reduced both baseline and calcitonin-stimulated release effects on endogenous NAG release by 12 to 21%. of fluorescence (Figure 6). The stimulatory effects of calcitonin on the release of fluorescence were also inhibited by the Discussion pretreatment with Bafilomycin A1, cytochalasin D, or nocoda- The major findings of the present study are that calcitonin zole (Table 3). increases NAG release and uptake, probably by promoting protein transport from the Golgi apparatus to the cell surface, The Relationship between Exocytosis and Endocytosis receptor-mediated endocytosis, and re-exocytotic pathway. Endocytosis results in removal of membrane components Calcitonin-induced NAG release was not associated with any from cell surface. Exocytosis may be required to complement increase of LDH release or reduction of mitochondrial dehy- the membrane deficit produced by endocytosis. Therefore, drogenase activity. Fluorescence release from the cells pre- triggered exocytosis may be related to the triggered endocyto- loaded with FITC-labeled NAG or albumin was dependent on 1646 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 1640–1648, 1999

Figure 5. Effects of calcitonin on the release of fluorescence from the endocytosed membranes. (A) Calcitonin promoted decrease of fluorescence intensity of the cells preloaded with FM 1-43. Top panel, control; Bottom panel, sCT-treated cells (1 ␮M). (B) Data are expressed by the ratio with the 0 min value as 1. F, calcitonin; E, control. n ϭ 8. *P Ͻ 0.05 versus control. microtubular function and blocked by receptor-mediated endo- Protein transport from endoplasmic reticulum and distal Golgi cytosis inhibitor. compartments to plasma membrane is known to be PKC- Lysosomal enzyme release is known to be modified by dependent (13) and is reversibly blocked by brefeldin A, which secretagogues, Ca2ϩ ions, PKC, or hormones in several cells. disassembles Golgi and prevents coated vesicle formation from Recently, Rodrı´guez et al. demonstrated Ca2ϩ-dependent fu- the Golgi complex and trans-Golgi network (14,15). Inhibitory sion of lysosomes with plasma membrane and, as a result, effects of brefeldin A on calcitonin- or PMA-regulated NAG lysosomal content release from fibroblasts and epithelial cells release also indicate that protein traffic through Golgi complex (3). The results in the present study indicate that PKC and is involved in the stimulatory effects of calcitonin and PMA on

CaMK II constitute important mediators also in calcitonin- intracellular NAG traffic in LLC-PK1 cells. induced NAG release from LLC-PK1 cells. PKC and intracellular Ca are known to be related to endo- Newly synthesized lysosomal enzymes reach lysosomes di- cytotic pathways in other cells (16,17). After lysosomes fuse rectly from the trans-Golgi network or indirectly via endocy- with plasma membrane and lysosomal enzymes are secreted tosis from the cell surface. The experiment with BODIPY FL into extracellular space, cation-independent (CI-) MPR appear

C5-ceramide indicated that sCT promotes protein transport on the plasma membrane and the released lysosomal enzymes from Golgi complexes to plasma membrane in LLC-PK1 cells. can be internalized (18). Calcitonin, PMA, and Ca ionophore J Am Soc Nephrol 10: 1640–1648, 1999 Calcitonin-Regulated NAG Handling 1647

Figure 6. Effect of sCT, PMA, and ionomycin on release of the fluorescence from the cells preloaded with FITC-labeled albumin or NAG. sCT (1 ␮M Œ), PMA (100 nM F), and ionomycin (10 ␮M ࡗ) increased the release of the fluorescence from the cells preloaded with FITC-labeled albumin. Thirty minutes of pretreatment with MDC or KN-93 inhibited the sCT action (, 0.1 mM MDC; ‚, MDC ϩ sCT; Ⅺ,50␮M KN-93; Ⅺ, KN-93 ϩ sCT) (A). Similar results were observed in the cells preloaded with FITC-NAG (B). n ϭ 8. *P Ͻ 0.05 versus control; ‡P Ͻ 0.05 versus sCT treatment.

Table 3. Role of proton pump, actin, and microtubule in the of the lysosomal enzymes in urine containing the highest stimulatory action of calcitonin on release of the proportion of mannose-6-phosphorylated form (23). Calcitonin fluorescence from the cells preloaded with FITC- receptors distribute along the nephron from proximal straight labeled albumina tubule to cortical collecting duct (24). Lysosomal enzymes released from some portion of renal tubules can be recycled by FITC-Albumin Release (␮g/ml cell protein per 30 min) the tubular cells at the more distal portion of nephron. Others Condition have suggested that lysosomal enzymes bound to MPR at cell ␮ Control sCT (1 M) surface of fibroblasts mediate degradation of matrix proteogly- Control 1.12 Ϯ 0.10 1.91 Ϯ 0.12b cans (25). In the distal tubule, urine pH is less than 5 under Bafilomycin A1 appropriate conditions (26). Lysosomal acid hydrolase can 0.1 ␮M 0.85 Ϯ 0.21b 1.40 Ϯ 0.12c actively degrade intraluminal materials at this site. Therefore, 1 ␮M 0.75 Ϯ 0.17b 1.37 Ϯ 0.12c lysosomal enzyme release from proximal renal tubular cells Cytochalasin D (100 nM) 1.16 Ϯ 0.07 1.67 Ϯ 0.12c may have two implications: enzyme reuse by more distal Nocodazole (100 nM) 1.02 Ϯ 0.10 1.57 Ϯ 0.11c tubular cells and degradation of intraluminal substrates at the distal portion of the nephron. a After 2-h loading of 500 ␮g/ml FITC-albumin, LLC-PK cells 1 Most receptor-ligand complexes accumulate at clathrin- were treated with sCT after a 30-min preincubation with Bafilomycin A1, cytochalasin D, or nocodazole and then coated pits of plasma membrane, which bud off to yield clath- fluorescence release from the preloaded cells was measured. n ϭ 8. rin-coated vesicles. The vesicles distribute throughout the pe- b P Ͻ 0.05 versus control. ripheral and perinuclear cytoplasm and transport membrane c Ͻ P 0.05 versus sCT treatment. components, directly or indirectly, back to plasma membrane. The experiments using membrane marker FM 1-43 and release stimulated endocytotic pathway as well as exocytotic traffic in of the fluorescence from the cells preloaded with FITC-albumin or FITC-NAG indicated that calcitonin promotes the re- LLC-PK1 cells. Inhibitory effects of MDC and M6P on baseline and calcitonin-induced NAG uptake indicated that NAG cycling pathway. Delivery of endocytosed proteins to the cell uptake takes place by receptor-mediated endocytosis via MPR surface is known to be regulated by intracellular Ca (17,27). Inhibitory effects of KN-93 on baseline and calcitonin-stimu- in LLC-PK1 cells. Several putative physiologic functions have been attributed lated release of fluorescence from the cells preloaded with to M6P-dependent endocytosis. One of the potential functions FITC-albumin indicated that the return pathway from endo- may consist in transfer of lysosomal enzymes from one cell somes to cell surface is regulated by CaMK II also in LLC-PK1 type to another (19,20). A proportion of lysosomal enzymes in cells. Cytochalasin D and nocodazole experiments indicated human urine are efficiently internalized by cultured hepato- that the re-exocytotic pathway is also dependent on the func- cytes via the CI-MPR (21,22), and NAG consists of about 25% tion of microfilaments and microtubules in these cells. Brefel- 1648 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 1640–1648, 1999 din A is known to suppress not only protein transport through 11. Ktistakis NT, Kao CY, Wang RH, Roth MG: A fluorescent lipid the trans-Golgi network but also early endocytic pathway analogue can be used to monitor secretory activity and for (28,29). 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