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Endocrinology Vol. 139, No. 2 713-722
Copyright © 1998 by The Endocrine Society

ARTICLES

KAT45, a Noradrenergic Human Pheochromocytoma Cell Line Producing Corticotropin-Releasing Hormone1

M. Venihaki2, K. Ain, E. Dermitzaki, A. Gravanis and A. N. Margioris

Departments of Clinical Chemistry (M.V., E.D., A.N.M.) and Pharmacology (A.G.), University of Crete School of Medicine, Crete, Greece; and the Division of Endocrinology, Diabetes, and Metabolism, Department of Internal Medicine, University of Kentucky College of Medicine (K.A.), Lexington, Kentucky 40536-0084

Address all correspondence and requests for reprints to: Dr. Andrew N. Margioris, Department of Clinical Chemistry, University of Crete School of Medicine, Heraklion GR-711 10, Crete, Greece. E-mail: andym{at}med.uch.g


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
KAT45 cells were derived from a human pheochromocytoma, which also caused ectopic Cushing’s syndrome, and developed into a cell line spontaneously after the continuous primary culture of the tumor cells. These human pheochromocytoma cells were compared with the extensively characterized PC12 rat pheochromocytoma cell line. KAT45 cells resembled PC12 cells in morphology, proliferation rate, response to cholinergic stimuli, and the development of dendrite-like projections after exposure to nerve growth factor. They produced norepinephrine and epinephrine in a ratio of 50:1, as opposed to production of dopamine by PC12 cells, in amounts 1 order of magnitude higher compared with PC12. Because of the ectopic Cushing’s syndrome in our patient, her normal ACTH level, and the knowledge that PC12 cells and even normal rat chromaffin cells appear to produce CRH, we examined whether KAT45 cells also produced this neuropeptide. Indeed, KAT45 cells released authentic CRH and contained an apparently intact CRH transcript. Nicotine and KCl depolarization stimulated the secretion of CRH, whereas interleukin-1ß, glucocorticoids, and nerve growth factor stimulated its synthesis. In addition to the potential systemic effects of CRH, which in our patient produced ectopic Cushing’s syndrome, CRH can exert paracrine effects within normal or tumoral adrenals. We used KAT45 cells as a model for the study of the local role of CRH. CRH affected several parameters of KAT45 cell metabolism, including their proliferation rate, synthesis of catecholamines, and production of POMC-derived peptides. KAT45 cells, in addition to the data they provided regarding the in vitro profile of a human CRH-producing pheochromocytoma, may prove to be a valuable auxiliary to the PC12 cell line.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
KAT45 CELLS were derived from a human pheochromocytoma, which also caused ectopic Cushing’s syndrome. They developed into cell line spontaneously after the continuous primary culture of the tumor cells. These human pheochromocytoma cells were compared with the extensively characterized PC12 rat pheochromocytoma cell line (1). In the first part of this paper we report our findings regarding KAT45 cell morphology, rate of proliferation, production of catecholamines, and response to cholinergic stimuli. Catecholamines were measured by electrochemical detection after extraction and separation by gradient reverse phase HPLC (RP-HPLC) (2). The proliferation rates were measured by cell counting, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and [3H]thymidine incorporation (3).

Normal rat adrenal chromaffin and PC12 cells produce CRH (4). Our patient had ectopic Cushing’s syndrome with normal ACTH levels. Because of these data, we examined whether KAT45 cells produce CRH. In the second part of this report we describe our findings regarding the presence of the CRH transcript and immunoreactive (IR) CRH in KAT45 cells and their media. The transcript was characterized by Northern blot analysis, while the IR-material by sieve chromatography and RP-HPLC. The release rate of CRH from KAT45 cells was also assessed after exposure to nicotine and depolarizing concentrations of K+. The synthesis rate of CRH was examined after exposure to 7S NGF for 1 week, as it is now known that NGF, in addition to its effect on cell phenotype, also induces the expression of the CRH gene in primary pheochromocytoma cell cultures and in the PC12 cell line (4, 5). The effect of dexamethasone on CRH production was also tested, as glucocorticoids suppress the expression of the CRH gene in the hypothalamus but induce it in placenta (6). Finally, the effect of interleukin-1 (IL-1) was tested because this cytokine now ranks as a major stimulator of CRH production in the hypothalamus (7, 8) and the periphery. Furthermore, it should be noted that the IL-1 cytokines have been shown to be produced locally within the adrenal medulla and have been found to affect both normal adrenal chromaffin and pheochromocytoma cells (9, 10, 11, 12, 13).

Adrenomedullary CRH, in addition to its potential systemic effects, which in our patient caused ectopic Cushing’s syndrome, may also exert local paracrine-autocrine effects (4, 5). In the last part of this report we present our findings regarding the paracrine effects of CRH on KAT45 cells. Synthetic CRH and/or its antagonist {alpha}-helical ({alpha}h) CRH were tested on the following parameters: 1) proliferation rate, as CRH has been shown to affect the proliferation of normal anterior pituitary corticotrophs (14) as well as that of the mouse corticotropic cell line AtT20 (15); 2) production of catecholamines, as CRH has been shown to directly stimulate the secretion of norepinephrine from neurons in the prefrontal cortex (16, 17, 18, 19), hippocampus (20), and hypothalamus (16, 17) and adrenaline from normal bovine adrenal chromaffin cells (21); and 3) production of ACTH and ß-endorphin (4, 22), as CRH is a major regulator of POMC in pituitary, central and peripheral nervous system, and several nonneuronal tissues that produce it.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
KAT45 cells
The KAT45 cell line grew spontaneously from continuous primary culture of a human pheochromocytoma for more than 9 months. The tumor was excised from a 49-yr-old woman presented with hypertension and clinical stigmata of Cushing’s syndrome. The 24-h urinary vanillylmandelic acid level was 24.7 (normal, <7.6 mg/24 h), the normetanephrine was 5,522 (normal, <500 µg/24-h urine), total metanephrine was 5,635 (normal, <700 µg/24-h urine), norepinephrine was 1,133 (normal, <80 µg/24-h urine), epinephrine was 5.1 (normal, 0.5–20.0 µg/24-h urine), dopamine was 227 (normal, 64–400 µg/24-h urine), and free cortisol was 254 (normal, <100 µg/24-h urine). Her basal plasma ACTH level was 56 (normal, 0–100 pg/ml), epinephrine was 50 (normal, 0–114), norepinephrine was 12,599 (normal, 174–624 pg/ml), and chromogranin A was 1,298 ng/ml (normal, 10–50 ng/ml). On Liddle’s test she did not suppress on 2 mg/day dexamethasone but suppressed after the 8 mg/day dose. Magnetic resonance imaging of the sella was normal. A 5 x 3 x 2-cm pheochromocytoma was resected from the right adrenal gland. Immunohistochemical staining of the tumor was strongly positive for chromogranin, but negative for ACTH. Postoperatively, the patient’s Cushing’s syndrome was resolved with normalization of her appearance and urinary free cortisol. However, the patient remained hypertensive.

Sterile, fresh tissue, from within the tumor capsule was minced and cultured in flasks containing antibiotic-free RPMI 1640 medium with 10% (vol/vol) FBS (all from Life Technologies, Gaithersburg, MD) in humidified 5% CO2 at 37 C.

PC12 cells
The PC12 cells were obtained from two sources: 1) Dr. G. Guroff, Section on Growth Factors, NICHHD, NIH (Bethesda, MD); and 2) the American Type Culture Collection (Rockville, MD). The conditions of their handling in our laboratory have been previously described (2, 3, 4). The PC12 cells were cultured on either flat bottom wells in 12-well plates of 4.5-cm2 surface area/well (Costar Europe, Badhoevedorp, The Netherlands) or on flasks of 75-cm2 surface area, at an initial concentration of 1 x 105 cells/cm2. They were left to grow in Eagle’s MEM (Life Technologies) containing 10 mM L-glutamine, 1.5 mM HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 7.5% horse serum, and 7.5% FCS in an incubator (Forma Scientific, Marietta, OH) at 5% CO2 and 37 C. Differentiation of PC12 to neuron-like cells followed exposure to 7S NGF (25 ng/ml x 1 week). The 7S NGF was provided by Dr. G. Guroff, Section on Growth Factors, NICHHD, NIH.

Proliferation
The proliferation rate was determined under basal conditions and after exposure to CRH. It was measured by 1) cell counting, 2) the MTT (Sigma Chemical Co., St. Louis, MO) method, and 3) [3H]thymidine incorporation. For cell counting, cells were trypsinized, centrifuged, resuspended in RPMI and 10% FCS, and counted visually on a hematocytometer using trypan blue. In addition, the cells were counted on a desktop Coulter counter (Coulter Electronics, Hialeah, FL). For the MTT method, the cells were plated in flat bottom 96-well plates at an initial concentration of 6,000-10,000 cells/well on RPMI-10% FBS. MTT was dissolved in PBS at 5 mg/ml (stock solution). KAT45 cells were plated in flat bottom wells (96 wells/plate) at an initial concentration of 6,000–10.000 cells/well, on RPMI medium containing 10% FBS. The next day (second day of the experiment), this medium was replaced by test medium free of serum containing CRH, {alpha}hCRH, or their vehicles (controls). At the end of each experiment, the media were replaced by fresh test medium. The cells were left to rest for 1 h, and MTT was added at a final concentration of 0.5 mg/ml and incubated for an additional 4 h at 37 C. Living mitochondria metabolize MTT to blue crystals. The MTT media were then replaced by a solution of acid-isopropanol, and the optical density was read on a Dynatech MicroElisa reader (Chantilly, VA) at a wavelength of 600 nm. For the incorporation experiments, the cells were aspirated from 6-well plates and transferred to flat bottom 96-well microtiter plates (Costar, Cambridge, MA). Each well was then pulsed with 1 µCi [methyl-3H]thymidine (SA, 25 Ci/mmol; Amersham Life Science, Aylesbury, UK) for 24 h (a 24-h incubation was found to give the best incorporation) at 37 C in 5% CO2-95% air (3). At the end of the final incubation, the cells were aspirated automatically by a cellular harvester (Minimash 2000, Dynatech, France), transferred to prewet filters, vacuum-washed three times, dried for 90 min at 65 C, cut, and counted in a liquid scintillation ß-counter (4000 series, United Technologies, Packard, Downers Grove, IL).

Separation and measurement of catecholamines
The separation and measurement of catecholamines in culture medium were performed as previously described (2). Briefly, samples of 600 µl were aspirated from each well and transferred to tubes containing 100 µl 0.1 N HCl to ensure the stability of the catecholamines (stable in acidic pH). Subsequently, the catecholamines were extracted by acetone precipitation. The extracts were then evaporated and reconstituted in 200 µl of the mobile phase of the HPLC. Samples of 25 µl (i.e. one eighth of the initially extracted material) were injected into the RP-HPLC (HP-1090, Hewlett-Packard, Waldbronn, Germany) equipped with an electrochemical S100A detector (ESA, Coulochem, Bedford, MA). The detector was set at +0.45 mV (with respect to an H2/H+ couple reference electrode), and the guard cell was set at +0.5 mV. The guard and main columns were RP-18, 5-µm particle size (Hewlett-Packard), whereas the size of the main column was 200 x 4.6 mm. The mobile phase was composed of 25 mM NaH2PO4·H2O, 3.2 mM 1-heptane sulfonic acid sodium salt, 0.5 mM EDTA, and 7.5% methanol. The signals were recorded in an analog digital converter (HP-35900 C, Hewlett-Packard) connected to an HP-1040 computer.

CRH, ACTH, and ß-endorphin assays
Peptides in culture medium were concentrated by C18 reverse phase columns (Sep-Pak, Waters Associates, Milford, MA) after acidification in 2 vol 0.1 N HCl and centrifugation at 1.5 x 103 rpm for 10 min. The supernatants were extracted by activated Sep-Pak cartridges, washed with 20 ml 0.1 N HCl, eluted with 3-ml acetonitrile 0.01–80% HCl, then dried under vacuum (Speed-Vac, Savant Instruments, Farmingdale, NY). The CRH content of the Sep-Pak extracts was assayed by RIA using a rabbit antiserum purchased from Neosystem Laboratoire (Strasbourg, France). The antiserum was raised against human CRH and exhibits 100% cross-reactivity to rat CRH and no cross-reactivity to ovine CRH, human ACTH, human ß-endorphin, LHRH, or arginine vasopressin. The sensitivity of the assay was 1 pg/tube. The intraassay coefficient of variation was 4.4%, and the interassay coefficient of variation was 6.6%. Results are expressed as picograms of IR-CRH per mg total cellular protein determined on whole cellular homogenates. ACTH in the culture medium was measured by chemiluminescence (Nichols Institute Diagnostics, San Juan Capistrano, CA). An acridinium ester-labeled mouse monoclonal ACTH-(1–17) antibody and a biotin-coupled goat polyclonal ACTH antibody exhibiting 100% cross-reactivity to ACTH-(1–39) and no cross-reactivity to ACTH-(1–24), ACTH-(1–10), {alpha}MSH, and ß-endorphin were used. The sensitivity of the assay was 0.5 pg/ml medium. The intraassay coefficient of variation was 3.4%, and the interassay coefficient of variation was 4.6%. Results are expressed as picograms of ACTH per mg total cellular protein. For the measurement of IR-ß-endorphin, a previously described protocol was used (23). Briefly, culture media were first acidified in 5 vol 0.1 N HCl and centrifuged at 10,000 rpm for 20 min, and the supernatant was passed through Sep-Pak cartridges (Waters Associates, Milford, MA), eluted by 0.01 N HCl-80% acetonitrile in tubes containing 1 mg human serum albumin, and then dried by Speed-Vac and reconstituted in RIA buffer. The assay was a solid phase two-site immunoradiometric assay (Nichols Laboratories, Irvine, CA). The antiserum was raised again human ß-endorphin and exhibits a 16% cross-reactivity to ß-lipotropin and no cross-reactivity to ACTH, {alpha}MSH, {alpha}- or ß-neo-endorphin, or the enkephalins and dynorphins. Under our conditions, half-maximal tracer displacement occurred between 20–25 pg, and the sensitivity was 1–2 pg/tube. Serial dilution of a pool of the extracted culture media displaced the radioactive tracer from antiserum in parallel with the synthetic ß-endorphin.

Sieve chromatography and RP-HPLC
The mol wt of IR-CRH was assessed by gel filtration chromatography. Briefly, pooled culture media were acidified in 2 vol 0.1 N HCl and centrifuged at 1 x 105 rpm for 10 min, and the supernatant was extracted by activated Sep-Pak cartridges, washed with 20 ml 0.1 N HCl, eluted with 3 ml acetonitrile 0.01–80% HCl, and dried under vacuum (Speed-Vac). The samples were reconstituted in 0.5 ml 10% formic acid containing 0.5% defatted BSA and 6 M urea and chromatographed on Sephadex G-50 (0.9 x 60 cm; bed volume, 40 ml; flow rate, 1.5 ml/h; 1-ml fractions collected). Fractions were dried under vacuum and reconstituted for RIA. After chromatographies of the samples, the G-50 column was calibrated with blue dextran and synthetic human CRH.

For the characterization of IR-CRH by RP-HPLC, KAT-45 cells in culture were scrapped, pooled, homogenized in 5 vol 0.1% trifluoroacetic acid (TFA), and centrifuged at 10,000 rpm for 20 min; the supernatant was dried under vacuum, were reconstituted in 30 µl 25% acetonitrile-0.1% TFA, and centrifuged again for 10 min; and one third of the supernatant was injected into the HPLC connected to a 0.2 x 30-cm C18 µBondapack column (Waters Associates, Milford, MA). A linear gradient program was used employing the TFA-acetonitrile solvent system shown in Fig. 3Go. Solvent A was composed of 20% acetonitrile-0.1% TFA, and solvent B was composed of 80% acetonitrile-0.1% TFA. The flow rate was 1 ml/min. Samples of 1 ml were collected. The retention time (Rt) of the IR-CRH extracted from the KAT45 cells was determined by RIA after evaporation of the HPLC fractions and reconstitution in RIA medium. After the sample runs, 20 ng diluted synthetic CRH and CRH oxidized by 5% H2O2 were run, and their Rt values were determined by RIA.



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  		Figure 3. Basal and nicotine-induced norepinephrine secretion. KAT45 cells produce norepinephrine and epinephrine in a ratio of 50:1. Their total production of catecholamines is 1 order of magnitude higher than that of PC12 cells. Nicotine stimulated the secretion of norepinephrine from KAT45 cells in a dose-dependent manner. Thus, a 15-min exposure of KAT45 cells to nicotine at 10-6 M increased the concentration of norepinephrine in the medium approximately 3 times. This effect lasted for about 60 min. The vertical lines at the top of each barsignify ±SEM (n = 6). The same letter identifies statistically compared pairs. *, P < 0.05; **, P < 0.01.

 
Northern blot
Isolation of total RNA from human placenta (positive control) and KAT45 cells was performed using guanidinium thiocyanate-acid phenol-chloroform. Total RNA was quantified by UV absorption at 260 nM. Each sample was diluted in denaturing buffer composed of 50% formamide, 18% formaldehyde, 1 x FRB [20 mM 3-N-morpholinol propanesulfonic acid (MOPS), 5 mM sodium acetate, and 1 mM EDTA, pH 7.0], heated at 65 C for 5 min, and centrifuged at 6000 rpm for 5 min. Thirty microliters (containing 20 µg total RNA) per sample were size-fractionated by electrophoresis on a formaldehyde-agarose (1.5%) gel containing 1 µg/ml ethidium bromide, run at room temperature in 1 x FRB, and transferred to GeneScreen nylon membrane as previously described (24). Blots were heated at 80 C for 120 min. Prehybridization was performed in a solution containing 5 x SSC (standard saline citrate), 50 mM phosphate buffer (pH 6.5), 5 x Denhardt’s solution [Ficoll 1%, polyvinylpyrrolidone 1%, BSA (Pentax Fraction V) 1%], 0.1% SDS, 250 mg/ml sheared single strand salmon sperm DNA, and 250 mg/ml transfer RNA (Boehringer Mannheim, Indianapolis, IN) for 12 h at 42 C. A synthetic 42-mer oligonucleotide corresponding to amino acids 22–27 of the human sequence was used as a probe (24). The probe was labeled at the 3'-end with [{alpha}-32P]deoxy-ATP (1200 Ci/mmol; Amersham, Arlington Heights, IL) and terminal deoxynucleotidyl transferase (Boehringer Mannheim) to a specific activity of 0.8 x 10-9 dpm/mg. Hybridization was performed in the prehybridization buffer containing 1 x 10-6 dpm/ml labeled CRH probe at 42 C. Blots were washed in 0.2 x SSC-0.1% SDS for 1 h at 42 C and exposed at -70 C to Kodak XR film (Eastman Kodak, Rochester, NY) in the presence of intensifying screens.

Statistical analysis
The concentration of IR-CRH in the culture medium was normalized according to the total cellular protein content after resuspension and homogenization at the end of each experiment (4). The secretion of epinephrine and dopamine in the culture medium is expressed as either the mean (nanograms) ± SEM of five or six experiments (per well per mg total cell protein) or as percent changes compared with control wells. Total cellular protein in each well was measured by a modification of the Bradford Coomassie Brilliant Blue G250 method (Serva, Heidelberg, Germany) using BSA as standard (25). For the statistical evaluation of our data, we used ANOVA followed by multiple comparison Fisher’s least significance difference test and the Newman-Keuls test. For data expressed as the percent change over controls, we used the nonparametric test of Kruskal-Wallis for several independent samples.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Morphology and proliferation
Figure 1AGo depicts KAT45 cells 24 h after plating at low power. KAT45 cells exhibit a size variation that tends to disappear as cells grow. Figure 1BGo depicts a high power view of KAT45 cells, the phenotype of which remained unchanged after freezing and thawing. Figure 1CGo depicts a high power view of PC12 cells; the morphological features and size of these cells resemble those of KAT45 cells. Figure 1DGo depicts the effect of 7S NGF at 25 ng/ml for 1 week on KAT45 cells, whereas Fig. 1EGo shows its effect on PC12 cells. Figure 2Go, depicts the proliferation rate of KAT45 cells under basal conditions measured by cell counting of trypan-excluding cells (Fig. 2AGo). The doubling time, under our culture conditions, was approximately 24–48 h, which is similar to that of PC12 cells (data not shown). MTT assays of the steepest part of the proliferation curve confirmed these results (Fig. 2BGo, upper curve).



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  		Figure 1. Morphology. A, KAT45 cells 24 h after plating. B, KAT45 cells at high power. C, PC12 cells at high power. D, KAT45 cells after exposure to 7S NGF (25 ng/ml) for 1 week. E, Effect of NGF on PC12 cells.

 


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  		Figure 2. Proliferation rates. A, The number of trypan blue-excluding cells at 24-h intervals after plating. B, Upper curve, MTT assessment of KAT45 cell proliferation at the steepest part of the curve. B, Lower curve, Suppressive effect of CRH at 10-6 M on the proliferation of KAT45 cells. The effect of CRH was not dose dependent. Exposure of KAT45 cells to the specific CRH antagonist {alpha}hCRH at 10-5 M in addition to CRH blocked its inhibitory effect. {alpha}hCRH did not have any effect of its own.

 
Production of catecholamines
KAT45 cells produced norepinephrine and epinephrine in a ratio of 50:1 and small amounts of dopamine, whereas PC12 cells produced mainly dopamine. The total amount of catecholamines produced by KAT45 cells was approximately 100 times higher than that of PC12. Short exposure of KAT45 cells to nicotine stimulated their release of catecholamines in a manner similar to PC12 cells. Figure 3Go depicts the effect of nicotine at 10-6 M; it caused a highly significant increase in norepinephrine release at 15 min that lasted up to 60 min.

Production of CRH
Figure 4Go depicts the concentration pattern of IR-CRH in the medium of KAT45 cells over a period of 3 days under basal conditions. It also shows the characterization of the released IR-peptide and its transcript. Specifically, Fig. 4AGo depicts the concentration of IR-CRH in the culture medium 3, 6, 9, 12, 12, 48, and 72 h after the removal of FBS-enriched culture medium and its replacement by plain medium containing aprotinin and BSA. The concentration of IR-CRH in the medium exhibited a wave-like pattern, a phenomenon also observed in other chromaffin cells (4). The mean concentration of IR-CRH ranged from 20–30 pg/mg total cellular protein or approximately 0.5–1 x 10-8 M. The inset of Fig. 4AGo depicts the elution profile of KAT45-derived CRH on sieve chromatography, which is similar or identical to that of synthetic human CRH. Figure 4BGo depicts the RP-HPLC chromatographic profile of KAT45-derived IR-CRH. A gradient of acetonitrile (CH3CN) was used (indicated by the broken line) in the presence of 0.1 N TNF. The IR-CRH from the KAT45 cells had two peaks. The first had the Rt of oxidized Met(O) CRH (formed during the extraction procedures), whereas the second peak had the Rt of nonoxidized human/rat CRH standard. The inset of Fig. 4BGo (at the top) depicts the Northern blot signal of KAT45 total RNA after hybridization with a 42-mer CRH oligo (right) compared with the signal of total RNA from human placenta (left). The calculated size of both hybridization signals was the same as that of the authentic transcript.



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  		Figure 4. Characterization of CRH. KAT45 cells produced CRH. A, Basal secretion of IR-CRH from KAT45 cells over a period of 72 h. The vertical lines signify ±SEM (n = 6). The inset of A depicts the elution profile of KAT45-derived CRH on sieve chromatography, which is similar to that of synthetic human/rat CRH. B, The gradient RP-HPLC profile of IR-CRH extracted from KAT45 culture medium. The broken line indicates the acetonitrile gradient. The Rt of the first peak is that of oxidized Met(O) CRH, and that of the second peak is that of human/rat CRH. The inset of B (top) depicts the Northern blot signal of KAT45-derived total RNA hybridized with a 42-mer CRH oligonucleotide (right) compared with the hybridization signal of total RNA from human placenta (left).

 
Effectors of CRH secretion
Exposure of KAT45 cells to nicotine or to a depolarizing concentration of KCl resulted in an immediate increase in IR-CRH in the culture medium. Figure 5AGo depicts the effect of nicotine (10-6 M) on CRH compared with levels in parallel controls. The stimulatory effect of nicotine on CRH peaked at 40 min, whereas its effect on norepinephrine peaked earlier, at 15 min. Although the costorage and corelease of classical neurotransmitters with neuropeptides is a characteristic of neural crest-derived cells, the concentration of neurotransmitters in the culture medium depends not only on the rate of their release but also on the rate of their reuptake (26, 27). Thus, the asynchronous peaks we find may be due to reuptake of norepinephrine by KAT45 cells. Figure 5BGo depicts the effect of K+-induced depolarization, which peaked at 20 min.



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  		Figure 5. Regulation of CRH secretion. Nicotine and K+ exerted an acute stimulatory effect on CRH production from KAT45 cells, suggesting the involvement of secretion mechanisms. A, Time-dependent effect of nicotine at 10-6 M on CRH secretion over a period of 40 min. B, Effect of K+-induced depolarization. The effects of both stimuli were highly reproducible. The vertical lines at the top of each bar signify ±SEM (n = 6).

 
Effectors of CRH synthesis
Glucocorticoids exert a strong suppressive effect on hypothalamic CRH, whereas the IL-1 cytokines have a strong stimulatory effect (7, 8, 9, 10, 11). Both factors exerted a delayed effect on KAT45-derived CRH, suggesting the involvement of synthesis rather than secretion. Thus, dexamethasone at 10-6 M did not have any effect up to the fifth day of culture, when it stimulated CRH in a highly significant manner (Fig. 6AGo). Similarly, IL-1ß at 10 ng/ml exerted a highly significant stimulatory effect only after 24 h or more (Fig. 6BGo). Nerve growth factor (NGF) also appeared to involve CRH synthesis. Figure 6CGo depicts the effect of 7S NGF (25 ng/ml x 1 week) on CRH production compared with that in parallel controls (KAT45 cells cultured in parallel for the same number of days and not exposed to NGF). PC12 rat and primary cultures of human pheochromocytoma cells respond to NGF in a similar manner (4, 5).



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  		Figure 6. Regulation of CRH synthesis. Glucocorticoids, IL-1 cytokines, and NGF exerted a stimulatory effect on CRH production from KAT45 cells. A, Effect of dexamethasone (10-6 M), which had a statistically significant stimulatory effect on IR-CRH only on the fifth day of culture. B, Effect of IL-1ß. Short exposure (<24 h) of KAT45 cells to IL-1ß (10 ng/ml) did not affect the concentration of IR-CRH in the culture medium. However, the effect of IL-1ß was significant after 24, 36, and 48 h of incubation, suggesting the involvement of CRH synthesis. C, Effect of NGF compared with parallel control cells not exposed to NGF. The rate of IR-CRH accumulation in the medium of KAT45 cells exposed to 7S NGF for 1 week was statistically higher than that in the control cells. The vertical lines signify ±SEM (n = 6). The same letter identifies statistically compared groups. *, P < 0.05; **, P < 0.01.

 
Effects of CRH on KAT45 cells
CRH affects the proliferation of several types of cells, including normal anterior pituitary corticotrophs (14) and the corticotropic cell line ATt20 (15). CRH had a similar effect on KAT45 cells. Figure 2BGo, lower curve, depicts the suppressive effect of CRH at 10-6 M on the proliferation of KAT45 cells. The trend of declining proliferation became statistically significant by the fourth day of culture. This effect of CRH appears to be pharmacological rather than physiological, as lower concentrations of CRH were ineffective (data not shown). Furthermore, the specific antagonist {alpha}hCRH did not have any effect of its own (data not shown), suggesting that endogenous CRH does not play a role in the proliferation process of KAT45 cells.

CRH stimulates the synthesis of catecholamines in the locus coeruleus (19), hippocampus (20), prefrontal cortex (16, 18), hypothalamus (16, 17), adrenal chromaffin (4, 21), and PC12 rat pheochromocytoma cells (4). CRH had a similar stimulatory effect on KAT45 cells. A short exposure of KAT45 cells to CRH (<12 h) did not cause any significant effect, suggesting that CRH does not influence the release of catecholamines, a finding in agreement with previously published data (4, 21). Longer exposure (36 h or more) resulted in a highly significant stimulation, suggesting the involvement of biosynthetic mechanisms. Figure 7AGo depicts the dose-dependent stimulatory effect of CRH on norepinephrine secretion from KAT45 cells exposed to it for 36 h (upper curve). The simultaneous addition of the CRH antagonist {alpha}hCRH in excess (10-5 M) abolished the stimulatory effect of CRH to a significant degree (lower curve), suggesting the involvement of specific CRH receptors. However, {alpha}hCRH did not have any significant effect of its own (data not shown).



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  		Figure 7. Paracrine effects of CRH. Synthetic human recombinant CRH exerted a dose-dependent stimulatory effect on norepinephrine and ß-endorphin production. A, Stimulatory effect of CRH on norepinephrine production (upper curve) from KAT45 cells exposed to it for 36 h. Shorter exposures were ineffective. The addition of the CRH antagonist {alpha}hCRH in excess (10-5 M) shifted the dose-response curve to the right (lower curve), suggesting that this effect involved specific CRH receptors. B, Dose-dependent effect of CRH on IR-ß-endorphin production. C, Time-dependent effect of CRH.

 
Exposure of KAT45 cells to CRH, in addition to its effect on proliferation and catecholamines, affected the production of POMC-derived peptides from KAT45 cells. The concentration of ß-endorphin in the culture medium of near-confluence KAT45 cells was 49.8 ± 10.5 pg/mg total cellular protein (n = 6) 3 h after the replacement of FCS-rich medium with test medium. At 6 h, its concentration was 53.5 ± 8 pg/mg total cellular protein; at 9 h, it was 37.2 ± 4; at 12 h, it was 52 ± 10; and at 24 h, it was 75.8 ± 12. The concentration of ACTH in the medium was similar to that of ß-endorphin, but had a tendency to decline rapidly, as previously described (4), probably due to the presence of specific pheochromocytoma-derived proteases. Indeed, the concentration of ACTH, which peaked at 22.7 ± 4 pg/mg total cellular protein (n = 6) at 3 h, decreased to 20.6 ± 4 at 6 h, 13.4 ± 2 at 9 h, 6.2 ± 0.5 at 12 h, and 6.5 ± 0.1 at 24 h. The release of both ACTH and ß-endorphin increased by more than 100% after exposure to a depolarizing concentration of KCl (data not shown). Figure 7BGo shows that the stimulatory effect of CRH on IR-ß-endorphin release from KAT45 cells was dose dependent, whereas Fig. 7CGo shows that its time-dependent response peaked at 9 h. Similar responses were obtained for ACTH (data not shown).


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
KAT45 cells derive from a human pheochromocytoma causing Cushing’s syndrome
They developed spontaneously into a cell line after continuous culture of the tumor cells. KAT45 cells exhibit several characteristics of the PC12 cell line, which was established by Greene and Tischler in 1976 from a transplantable rat adrenal pheochromocytoma (1). This cell line is extensively characterized and represents the only pheochromocytoma cell line currently available. The PC12 cell line is used as tool for the in vitro study of pheochromocytomas and the NGF-mediated change in cell phenotype to mature neurons. They synthesize and store dopamine and neuropeptides. Several subclones have recently emerged, differing from the initial cell line in their size, ability to attach to plastic surfaces, and production of neuropeptides. The American Type Culture Collection, which provides an original clone, cannot identify the passage number of their cells, which grow as floating clusters. Indeed, in our laboratory, two of our PC12 subclones grow in floating clusters, and one (presented in our photograph) attaches to culture surfaces. Similar to the latter, our KAT45 cells attach to culture surfaces. As far as catecholamines are concerned, the PC12 cells produce mainly dopamine, whereas the KAT45 cells produce large amounts of catecholamines, mainly norepinephrine and epinephrine in a ratio of 50:1, and some dopamine. Compared with PC12 cells, KAT45 produced much higher quantities of catecholamines. The proliferation rate of KAT45 cells was similar to that of our PC12 subclones, with a doubling time of 1–2 days. KAT45 cells responded to 1-week exposure to 7S NGF by changing their phenotype in a manner similar to that of PC12 cells. The development of dendrite-like projections in only a portion of the cells exposed to NGF is difficult to explain, but appears not to be restricted to KAT45 cells, because we observed the same phenomenon in our PC12 cells.

Our patient had ectopic Cushing’s syndrome with normal ACTH levels, a finding suggesting that its cause was ectopic production of CRH from the tumor and release into the systemic circulation. However, it is now widely suspected that the production of CRH by pheochromocytomas may be a rather common phenomenon. On the other hand, the development of Cushing’s syndrome from the systemic action of tumoral CRH is probably quite rare. Although we know of no study that evaluates the percentage of pheochromocytomas producing pathophsiologically relevant amounts of CRH, it appears that in most cases CRH produced by this type of tumor does not exert any systemic effect. Indeed, CRH has been shown to be produced in primary cultures of human pheochromocytomas, a small minority of which caused ectopic Cushing’s syndrome (28, 29, 30, 31, 32, 33). Furthermore, it now appears that CRH is also produced by normal human adrenal medulla (28, 29, 34, 35) and by the medulla of rodents (11, 36, 37, 38), cows (39, 40, 41), and dogs (42). Based on these data we have hypothesized that the production of CRH may represent the preservation of a physiological characteristic of adrenal chromaffin cells rather than a tumor-induced phenomenon. Indeed, dispersed normal rat adrenal chromaffin cells and the PC12 rat pheochromocytoma cell line produce comparable amounts of CRH (4). In the few cases in which there is development of Cushing’s syndrome from a systemic effect of CRH, we beleive that this reflects a failure of the normal barriers against it rather than a dramatic elevation of its concentration in plasma. The precise mechanism of this failure is not readily apparent, but it may be an enhanced bioavailability of CRH secondary to a decreased concentration of the CRH-binding proteins or a parallel increase in the sensitivity of anterior pituitary corticotrophs to CRH.

It is now well established that the immune system cross-talk with the two stress axes, the hypothalamic-pituitary-adrenal axis and the sympathetic system (central and peripheral). Indeed, systemic and local macrophage-derived IL-1 cytokines affect the two axes centrally at the level of the hypothalamus (where they stimulate the production of CRH) and at the level of the locus coeruleus (where they stimulate the production of noradrenaline) (7, 8, 43). In the periphery, exposure of normal adrenal medulla to recombinant human IL-1ß increases both the production of CRH and that of catecholamines (4, 12). Our findings are in complete agreement with these data. Our human pheochromocytoma cells respond to recombinant human IL-1ß by increasing the rate of their CRH synthesis, suggesting that the response of normal adrenal chromaffin cells to these cytokines may be preserved in some pheochromocytomas.

It is now known that in a growing number of extracranial sites, glucocorticoids stimulate the expression of the CRH gene instead of suppressing it. For example, it has been found that in primary cultures of human placental cytotrophoblasts (6) and in human pheochromocytomas (5), glucocorticoids stimulate the production of CRH. In agreement with these data, we have found that glucocorticoids stimulate the production of CRH in our cells. The physiological significance of this phenomenon and the mechanism involved are currently speculative.

Several reports have shown that NGF affects the production of CRH from neural crest-derived cells. Indeed, NGF increases the CRH messenger RNA content of primary cultures of human pheochromocytomas by 10-fold (5) and the production of the CRH peptide by PC12 rat pheochromocytoma cells (4). This phenomenon may be associated with the acquisition of neuron-like characteristics after exposure to NGF (4). Our KAT45 data strengthen these observations, suggesting that the NGF-mediated neuronal transformation of pheochromocytoma cells may be functionally linked to the expression of the CRH gene, as the two phenomena appear to develop in parallel. It is possible that CRH may mediate some of the effects of NGF. However, preliminary data on the effect of CRH or its antagonist on the change in our cell phenotype were not promising. Thus, it is possible that the changes in CRH production observed after NGF treatment might be a nonspecific phenomenon, part of a major reorganization of the cellular metabolism initiated by NGF. Indeed, NGF-treated cells look completely different from parallel controls.

It is our hypothesis that the biological role of peripherally produced CRH is local, and any potential systemic effect must first overcome the protective barrier of the circulating CRH-binding protein. A local effect of CRH on adrenals and their tumors is probable because they both contain specific CRH-binding sites (21, 44, 45). In agreement with this hypothesis, we have found that several parameters of PC12 and KAT45 cells appear to be affected by CRH, including their proliferation rate (a rather pharmacological effect), production of catecholamines, and POMC-derived peptides. In our hands, the stimulatory effect of CRH on the production of catecholamines was dose and time dependent and was blocked by the specific antagonist {alpha}hCRH, suggesting the mediation of CRH receptors. In this regard, the response of our cells to CRH was similar to that of normal chromaffin cells (21) and catecholaminergic neurons in the central nervous system (16, 17, 19, 20). In general, CRH is a potent effector of catecholamine metabolism, stimulating the synthesis of tyrosine hydroxylase (46, 47), the rate-limiting enzyme in their synthetic pathway. In the central nervous system, the stimulatory effect of CRH on the production of catecholamines appears to be exerted in a paracrine manner via the close anatomical proximity of the CRH and the noradrenergic neurons (48, 49). Thus, the close association between the hypothalamic-pituitary-adrenal and catecholaminergic stress axes appears to be retained in both the normal adrenal medulla and at least some pheochromocytomas.

CRH affects the proliferation of normal anterior pituitary corticotrophs (14) and the corticotropic cell line AtT20 (15). We found that CRH suppressed the proliferation of KAT45 cells. However, this effect does not appear to be of physiological significance, as it occurred only at the highest dose of CRH, and {alpha}hCRH, the specific CRH antagonist, did not have any effect of its own, suggesting that KAT45 cells were not under tonic inhibition by endogenous CRH as in the case of endogenous opioids in which the addition of opioid antagonists exerts a major effect on their proliferation (3).

In conclusion, our KAT45 cells, in addition to the data they provided regarding the in vitro profile of a human CRH-producing pheochromocytoma, may prove to be a valuable auxiliary to the PC12 cell line in the study of neuronal differentiation and the neuro-immuno-hormonal interactions regulating the production of catecholamines in normal and tumorous adrenal medulla.


   Footnotes
 
1 This work was supported by the Greek Ministry of Industry, Energy, and Technology (Grant PENED 91 ED 10), the Medicon Hellas Co., and the PEPAGNH University Hospital of Heraklion, Crete, Greece. Back

2 Present address: Division of Endocrinology, Department of Pediatrics, Childrens Hospital, Harvard Medical School, 300 Longwood Avenue, Boston, Massachusetts 02115. Back

Received April 11, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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