Transmembrane Potentials and Steroidogenesis in Normal and Neoplastic Human Adrenocortical Tissue
JOHN LYMANGROVER,’ A. FRANCES PEARLMUTTER, ROBERTO FRANCO- SAENZ,2 AND MURRAY SAFFRAN3
Departments of Biochemistry and Medicine, Medical College of Ohio, Toledo, Ohio 43614
ABSTRACT. Trans-membrane potentials and ster- oidogenesis were measured in superfused slices of non-tumor and neoplastic human adrenocortical tissue. Non-tumor tissue was obtained at the time of renal transplant or from tissue removed along with tumors.
Non-tumor human adrenocortical tissue had electrophysiological and steroidogenic properties similar to those of the rat and rabbit. In normal me- dium ACTH stimulated steroidogenesis but had no effect on the membrane potential. In K+-free medium, the cells hyperpolarized, and subsequent addition of ACTH caused depolarization.
Trans-membrane potentials of adrenocortical tumors were lower than those of non-tumor cells.
Ommission of K+ from the medium caused hyperpolari- zation of the tumor cells, but the trans-membrane po- tentials did not reach the values of hyperpolarized non- tumor cells. ACTH, added to the K+-free medium, caused little or no change in membrane potential of tumor cells except in one case of a virilizing adenoma, which responded very much like non-tumor tissue.
Except for the virilizing adenoma, tumor tissue slices produced little or no detectable fluorogenic steroid, even in the presence of large amounts of ACTH or cyclic AMP. The virilizing adenoma re- sponded with increased steroidogensis to ACTH and cyclic AMP. (J Clin Endocrinol Metab 41: 697, 1975)
P EARLMUTTER et al. (a) have shown that human adrenocortical tissue be- haves remarkably like rat tissue in its ster- oidogenic response to ACTH and cyclic AMP. Does the human tissue also resemble other species in its electrophysiological properties?
The voltage difference between the in- terior and exterior of a cell depends upon the existence of an ionic gradient across the cell membrane. Nerve and muscle cells main- tain a gradient that results in a voltage of 70 or more mV, negative inside. Most visceral cells have membrane potentials of -20 to -40 mV. Activation of a nerve or muscle cell is accompanied by a characteristic se- quence of changes in membrane potential, the action potential, which appears as a train of rapid depolarizations of the membrane.
Received November 8, 1974.
Supported by NIH grant AM 14132 and GRS grant 94357.
’ Present address: Department of Physiology, Uni- versity of Cincinnati College of Medicine, Cincinnati, Ohio 45219.
2 Department of Medicine.
3 Address correspondence to: M. Saffran, Depart- ment of Biochemistry, Medical College of Ohio, Toledo, Ohio 43614.
The electrical properties of human adre- nocortical cells have not been studied. We took advantage of the availability of samples of human adrenocortical tissue either at the time of renal transplant or removal of adrenal tumors to compare the electrophysiological and steroidogenic properties of non-tumor and tumor tissue. Our results show that the non-tumor human adrenal cortex is similar to that of other species. However, adrenal tumors had lower and more variable mem- brane potentials, depending on the type of tumor.
Materials and Methods
Non-tumor adrenal tissue. Non-tumor adrenal tissue was obtained from 2 kidney transplant donors, 3 non-tumor areas removed along with adrenal tumors, and the cortical areas removed with a pheochromocytoma and an adrenal medul- lary hematoma.
Neoplastic adrenal tissue. Tumor tissue was ob- tained at the time of adrenalectomy or biopsy from 5 patients: 2 carcinomas, 1 probable car- cinoma, and 2 adenomas (Table 1).
Tissue slices. The adrenal was sliced (0.5 mm) with a Stadie-Riggs tissue slicer and 30-100 mg portions were placed in the tissue holder. No
| ACTH | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Duration of Symp- toms | Plasma cortisol | Urinary | Stimu- | Dexameth supp. 17 17 OH KS | Urine DHA5 mg/24 hr. | Plasma testos- terone ng/100 ml | ||||||
| lation3 | ||||||||||||
| 17 | 17 KS2 hr. | 17 17 OH KS mg/24 hr. | ||||||||||
| Pa- tient Age Sex | AM µg/100 | ml PM | mg/24 OH' | |||||||||
| mg/24 hr. | ||||||||||||
| 300- | 4-8 | |||||||||||
| (7- | (3- | (3- | (5- | 500% | mg | (4- | (0.2- | (F < 90 | ||||
| Normal range | 15) | 6) | 10) | 18 | incr. | incr. | <3 | 8) | 1.13) | ng) | Clinical and pathological data | |
| W.T. 29 F | 6-8 yrs. | 7.5 | 20 | 7 | 14 | 31 | 29 | 8 | 22 | 0.14 | 326 | Virilizing adrenal tumor; well encapsulated right adrenocortical |
| 11 | 10 | 0.08 | 339 | adenoma; 9 g; 3.5 cm diam. | ||||||||
| J.D. 39 F | 10 years | 26.4 | 24.5 | 17 | 21 | 14 | 27 | 11 | 43 | 0.7 | 124 | Cushing's syndrome with mixed hypercortisolism and severe virilization; |
| 11 | well-encapsulated left adrenocortical adenoma; 10 g; 2.5 cm diam. | |||||||||||
| C.O. 23 F | 1 year | 16 | 18 | 11 | 201 | 31 | 219 | 12 | 195 | 21 | 104 | Cushing's syndrome with mild hypercortisolism and virilization; well- |
| 35 | encapsulated right adrenocortical adenoma (carcinoma?); 90 g; | |||||||||||
| 7.5 x 4.0 x 5.2 cm. | ||||||||||||
| M.S. 63 F | 1 year | 13 | 22 | 16 | 90 | not | done | not | done | 3.5 | 171 | Cushing's syndrome with hypercortisolism and virilization; poorly |
| differentiated adrenocortical carcinoma of right adrenal; widespread metastases; 1,520 g. | ||||||||||||
| H.W. 54 M | 2 months | 28 | 28 | 38 | 16 | not | done | not | done | not done | not done | Cushing's syndrome; poorly differentiated adenocarcinoma of right adrenal; widespread metastases; inoperable. Biopsy specimen obtained. |
’ 17-hydroxycorticosteroids.
2 17-ketosteroids.
3 40 Units infused during 8 hours.
’ Dexamethasone suppression test, 2 mg every 6 h for 2 days.
5 Dehydroepiandrosterone.
” Episodic hypersecretion occurred in W.T.
attempt was made to separate the adrenal zones. Superfusion with medium began within 30 min of obtaining the tissue from the operating room.
Superfusion system & steroid measurement. The superfusion system for the adrenal tissue has been described in detail (2,3). Slices were placed in a tissue holder and superfused at a rate of 4 ml/min with a solution containing Na+, 144 mM; K+, 4.7 mM; Ca++, 2.7 mM; Cl-, HCO37, 25 mM; Mg++, 1.2 mM; H2PO4-, 1.2 mM; glucose, 11 mM; bubbled with 95% O2/ 5% CO2. Potassium-free medium was pre- pared by omitting 4.7 mM KCI from the solution. The effluent from the tissue holder was pumped into the analytical system, which is designed to record continuously the fluorescence formed from corticosteroids released into a stream of medium by adrenal slices. The analytical system extracts the steroids from the medium with a stream of methylene chloride, and the methyl- ene chloride is in turn extracted with the ethanol- H2SO4 reagent. Fluorescence formed by heating the ethanol-H2SO4 extract is detected in the flow cell of a Turner Model 111 fluorometer and is recorded on a strip chart. In this procedure corti- sol has 44% of the fluorescence of corticosterone and aldosterone does not fluoresce. Testosterone and several other C19 steroids did not fluoresce very much (Table 2). Because the normal human adrenal secretes approximately 90% cortisol (4), steroidogenesis is expressed as cortisol.
The apparatus is adjusted to zero fluores- cence with the medium alone, and is calibrated with known amounts of cortisol. The fluorescence due to the steroid appears as a peak on the record. The area under the peak is directly proportional to the amount of steroid.
After calibration of the system, the tissue holder containing adrenal slices is introduced into the apparatus. An increase in fluorescence appears immediately due to the steroids released by the tissue. This release becomes stabilized in about 1 h, and the base-line production of corticoids is established, approximately 0.16 ng/mg/min, expressed as cortisol.
Trans-membrane potentials. The procedure for trans-membrane potential recordings was that de- scribed by Matthews (5). Finely drawn glass electrodes, filled with 1.5 M K-citrate solution and with initial resistances of 50-100 megohms, were aged in 0.9% NaCl solution for several days
| Steroid | % Fluorescence |
|---|---|
| Corticosterone | 100.00 |
| Testosterone | 0.41 |
| Dehydroepiandrosterone | 0.37 |
| Androsterone | 0.01 |
| Androstan-3,17-dione | 0.05 |
| Androst-5-ene-3,7-dione | 0.47 |
| Androst-5-ene-33, 178-diol | 0.43 |
| Androst-4-ene-3,17-dione | 0.01 |
prior to use. The electrodes were positioned with the aid of a dissecting microscope. Trans-mem- brane potentials were visualized and recorded with a Bioelectric P-1A amplifier system and a Tektronix 5031 storage oscilloscope and Westron- ics D 11A pen recorder. When the tip of the elec- trode penetrates the membrane, a sharp deflec- tion of the trace on the oscilloscope screen or the recording paper occurs. If the seal around the electrode is imperfect, the tip is quickly ejected from the cell and the trace returns to base line. A good seal is signalled by a potential that rises quickly to a nearly final level, settles within a few seconds to a slightly higher value, and maintains that value for 10 s or more. The quality of the recording is dependent upon the quality of the electrodes. Often a good electrode will seal into a cell and remain there for an hour or more. The emergence of an electrode from a cell is signalled by the rapid return of the trace to base line.
Under the dissecting microscope it is relatively easy to differentiate between medullary, cap- sular, and fasciculata-reticularis zones in the human adrenal gland and to direct the tip of the electrode into a population of cells in the desired zone. No attempt was made to distinguish be- tween zona fasciculata and zona reticularis. Samples of tumor slices were taken from the center of a cross-section of the tumor to avoid, as much as possible, contamination with extra- tumor tissue. In some experiments tumor and nontumor tissue from the same patient were studied simultaneously.
The tissue was superfused in Krebs medium at 37 C for 1-4 h until consistent potentials were observed before the experiments were begun. The tissue was usually studied for 24 h and in some instances for up to 48 h. Steroidogenesis and membrane potentials were measured simul-
taneously on different slices of the same tissue sample.
Membrane potentials are expressed as mean and standard errors of a population of cells in the tissue slices. Statistical analysis was per- formed using Student’s t test for unpaired data; P values <0.05 were considered to indicate statistically significant differences between groups. In the figs. 95% confidence limits are shown when the number of observations was 3 or more.
Materials. Cyclic AMP and dibutyryl cAMP were obtained from Schwarz/Mann. Porcine ACTH was obtained from Sigma Chemical Company and as the Third International Standard, from Dr. D. Bangham, National Institute for Medical Re- search, Mill Hill, London.
Clinical laboratory methods. Plasma cortisol was estimated by the fluorometric method of Rudd et al. (6). Urinary 17-ketosteroids were measured by the procedure of Drekter et al. (7). Urinary 17-hydroxycorticoids were determined by a modified Porter-Silber method (8). Gas-liquid chromatography was used to separate and meas- ure the urinary 17-ketosteroids, from which the excretion of dehydroepiandrosterone could be estimated (9). Testosterone was measured in plasma with the radioimmunoassay kit furnished by ICN Pharmaceuticals, Inc., Portland, Ore- gon 97208.
-mV
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Results
Resting membrane potential (Fig. 1). The mean resting membrane potential of 44 cells in slices of nontumor adrenal cortex was -56.7 + 1.9 SE mV. The resting membrane potentials of 182 cells in tumor slices were lower, ranging from a near-normal value of -48.7 ± 1.3 mV in the adenoma of patient J.D. to - 13 ± 1.2 mV in cells of an adrenal carcinoma (patient H.W.).
Potential after ACTH in normal medium (Fig. 2). Superfusion of non-tumor adrenal
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slices with 20 and 100 mU of ACTH per ml in normal medium did not alter significantly the membrane potential 80-90 min after introduction of ACTH, although the higher dose resulted in a higher mean potential of -64.6 mV.
Superfusion of tumor slices from patient J.D. with normal medium containing 10 mU ACTH per ml did increase the trans- membrane potential significantly above the resting potential. The membrane potential of cells of the tumor of patient C.O. was in- creased by the addition of 10 mU ACTH per ml to the medium; the increase was of borderline significance.
Potential in K+-free medium (Fig. 3). Non- tumor adrenocortical cells were hyper- polarized significantly to a mean of -72.0 ± 3.1 mV after 30 min in K+-free medium. All of the tumor samples were hyper- polarized significantly in K+-free medium except the tumor of C.O. None of the tumor samples reached the high values of hyper- polarized non-tumor cells, with the possible exception of the tumor of J.D.
Potential after ACTH in K+-free medium (Figs. 4-7). The addition of ACTH to the K+-free medium bathing the hyperpolarized non-tumor cells resulted in depolarization of the cells (Fig. 4). Fig. 5 is a continuous
ACTH (10 mU/ml)
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tracing of the membrane potential of a single cell in K+-free medium to which ACTH was added; depolarization began almost im- mediately after ACTH was introduced, then accelerated after 40 min to reach the very low value of - 11.7 mV in 70 min.
Hyperpolarized cells of the tumor of pa-
ACTH 10mU/ml in K- free medium
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tient W.T. were also depolarized by ACTH. A single cell of a slice of this tumor was im- paled while bathed in K+-free medium. The membrane potential was -72 mV. While the same cell was impaled, ACTH was added to the K+-free medium. The mem- brane potential fell to -63 mV within 5 min of the addition of ACTH, and to -45 and -25 mV at 15 and 25 min, respectively (Fig. 6). Transient action potential-like spikes were noted in 5 cells of this tumor when the membrane potentials were about -35 to -45 mV (Fig. 7). These spikes ranged from 10-20 mV in height and 200- 300 msec in duration. Neither of the other 2 tumors tested in this way was depolarized by ACTH. Cells of the tumors of patients C.O. and M.S. were not tested because the cells did not hyperpolarize above - 40 mV on exposure to K+-free medium.
ng/mg Cortisol
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Steroid formation (Figs. 8-11). Two sam- ples of non-tumor adrenal slices from a kid- ney transplant donor responded to a total dose of 25 mU of ACTH with increases of 22.5 and 23.5 ng of cortisol per mg tissue over the basal production. Doses of 50 mU produced a mean increase of 27.7 ng per mg, but variation from slice to slice was con- siderable. The 95% confidence limits were 12.4→ 43.0 ng/mg for the response to 50 mU (Fig. 8).
Non-tumor tissue from patient W.T. re- sponded to 25 m U of ACTH with the produc- tion of 20.6 ng/mg of steroid, and slices of the tumor produced 29.4 ng/mg with the same dose of ACTH (Fig. 8). Slices of this tumor responded to cyclic AMP also (Fig. 9.)
Non-tumor tissue of patient J.D. was re- sponsive to 5 mU of ACTH, producing 12.6 ng cortisol per mg. However, tumor slices of the same patient did not respond to doses of ACTH up to 2000 mU (Fig. 8). Non-tumor tissue from patient C.O. responded to 50 mU of ACTH with 14.3 ng cortisol per mg, but the tumor failed to respond to doses up to 500 mU (Fig. 8). Non-tumor slices from pa- tients M.S. and H.W: could not be obtained; the tumor slices did not respond to doses of ACTH up to 1000 and 5000 mU, respec- tively (Fig. 8). Cyclic AMP did not stimulate
FLUORESCENCE
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steroidogenesis in slices of the tumors from M.S. and H.W.
The response of adrenal slices from a kid- ney transplant donor to total doses of ACTH from 1-25 mU in normal medium increased linearly with the log dose (Fig. 10).
The basal level of cortisol production by non-tumor tissue in normal medium was 0.16 ng/mg/min (±0.027, S.E.). Continuous superfusion with ACTH (10 mU/ml at 4 ml/min) increased cortisol production to 1.3 ng/mg/min in normal medium.
A comparison of the response of non- tumor slices of patient W.T. to doses of 25 mU of ACTH in normal and K+-free medium over a prolonged period is shown in Fig. 11. In normal medium the initial sensitivity to ACTH is maintained for 11 h. After 1 h in K+-free medium, the steroidogenic response to ACTH was the same as in normal medium. Only after longer exposure to K+-free me- dium was the sensitivity to ACTH de- creased, reaching 10% after 4 h. When the tissue was then re-exposed to normal me- dium for 1 h, full steroidogenic response to ACTH was restored.
Discussion
The mean resting membrane potential of non-tumor human zona fasciculata-retic- ularis cells in normal medium ( -56.7 + 1.9 mV) was similar to that of other species. Matthews (5) reported mean resting
potentials of -66.2 mV for newborn rabbits, -70.5 mV for newborn rats and -71.5 mV for kittens. Fawcett (10) confirmed the rela- tively high resting membrane potential in adult rats measured in vivo. Joseph et al. (11) found a mean resting membrane potential of -56.9 mV for fetal rabbit adrenocortical cells. Such resting membrane potentials are higher than in most other cells except for nerve and muscle (12).
Matthews (5) has also shown that the transmembrane potential of the newborn rabbit adrenocortical cell was sensitive to changes in external potassium concentra- tion but not to changes in sodium concen- tration. A 10-fold increase in [K+] decreased the membrane potential of the adreno- cortical cells of the rabbit by 44 mV. On exposure to K+-free medium, rabbit adreno- cortical cells were hyperpolarized within 50 min from -67 mV to -86 mV and then declined to -70 mV 70 min later. Addition of ACTH to cells hyperpolarized by K+-free medium resulted in an accelerated depolari- zation (13). Transient action potential-like
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changes appeared after 30-60 min exposure to ACTH in K+-free medium (13,14). The “action potentials” ranged from 10-60 mV in amplitude and lasted from 600-1800 msec.
The electrophysiological properties of the adrenal cortex of non-human species were discussed by Matthews and Saffran (13). The membrane potential depends largely upon the concentration gradient of K+ across the cell membrane (5). The gradient is maintained by the sodium pump that pumps Na+ out of the cell and K+ into the cell. Removal of K+ from the external medium makes the K+-gradient steeper and therefore increases the trans-membrane potential at first. With prolonged exposure to a K+-free environment the sodium pump is impaired (14), resulting in a passive movement of Na into and K out of the cell. The K-gradient is decreased, and the membrane potential falls. An initial hyperpolarization and subse- quent slow depolarization was seen with rabbit adrenal cells exposed to K+-free me- dium for a prolonged period (13). ACTH accelerated the depolarization of adreno- cortical cells hyperpolarized by K-free medium (13), but ACTH had no detectable effect in normal medium (15). ACTH can cause accelerated depolarization through in- creased permeability of the membranes to ions. There is little evidence for changes in the concentrations of K+ in adrenal cells with ACTH (16).
Ouabain, an inhibitor of the sodium pump, not only abolishes the trans-membrane po- tential of adrenocortical cells, but also pre- vents the steroidogenic action of ACTH (13). When Ca++ was completely removed, then ACTH was no longer able to stimulate steroidogenesis and the membrane potential fell to zero.
The significance of the effect of ACTH on the membrane potential remains to be estab- lished. Both in man and in laboratory ani- mals the extraordinarily high trans-mem- brane potential and the depolarization of ACTH-responsive cells support the con-
cept that the membrane is a site of the regulatory effect of ACTH on the adrenal cortex. It is conceivable that loss of respon- siveness to ACTH may occur at the mem- brane level, with retention of the ability of the cell to respond to the intracellular sec- ond messenger, cyclic AMP; however, in the instances that were examined, response to ACTH and to cyclic AMP seemed to go hand in hand.
Adrenocortical tumor cells differed from non-tumor cells in several ways. Cortisol production by tumors failed to respond to ACTH or to cyclic AMP. Tumor cells had lower trans-membrane potentials to begin with and, although they were hyperpo- larized by omission of K+ from the medium, the transmembrane potentials did not reach the extreme values seen with non-tumor cells. ACTH, in contrast to its depolarizing effect on hyperpolarized non-tumor cells, had little effect on tumor cells.
The resting membrane potentials of the tumors were significantly lower than normal cells. As a group, the carcinomas exhibited the lowest resting membrane potentials, about - 14 mV. The adenomas were inter- mediate between normal cells and car- cinomas.
The resting membrane potentials of tumor cells of other tissues have been shown to be significantly lower than their normal counterparts (17-19). For example, Balitsky and Shuba (17) observed that the average trans-membrane potential of rhabdomyo- sarcoma cells was -10 mV whereas adja- cent non-malignant muscle cells had a mem- brane potential of -90 mV. Cone and Ton- gier (20) and McDonald et al. (21) have shown that there is a correlation between trans-membrane potential and rapidity of growth of normal cells in culture. The rapidly dividing cells had lower resting membrane potentials.
ACTH (20 mU/ml) given for 2 h to non- tumor human zona fasciculata-reticularis cells in normal medium caused no signifi- cant change in membrane potential. A
larger dose, 100 mU/ml, yielded higher membrane potentials but the difference was not significant. The adrenocortical tumors from patients J.D. and C.O. showed a slight, but statistically significant, hyper- polarization when ACTH was added to the tissue in normal medium. However, such rapid hyperpolarization is not typical of normal adrenal cortical tissue from a number of species we have studied (13), although some other cell types have been shown to hyperpolarize in response to peptide hor- mones or cyclic AMP (22-25). Non-tumor cells hyperpolarized by K+-free medium, were quickly depolarized by exposure to ACTH. Unlike other species, “action- potentials” were not observed in non-tumor human adrenocortical cells. Perhaps action potentials could be evoked in human cells by different doses of ACTH from those used in our experiments.
The tumor tissues were unresponsive to ACTH, except for the tumor from patient W.T., which was responsive to ACTH in vitro, both by increased steroidogenesis and by depolarization in K+-free medium. This tumor was unique in displaying “ac- tion-potentials” similar to those seen in normal cells of other species (13) during depolarization by ACTH.
A paradox in our experiments is the low or undetectable production of fluorogenic steroid by slices of adrenocortical tumors from patients with hyperadrenocorticalism. The superfusion apparatus is limited to 100 mg of tissue, but the tumors ranged in weight from 9-1500 g and that of H.W. was large and inoperable. Even a very low rate of cortisol production by a few tissue slices mean a considerable total production by the tumor. Moreover, the tumor tissue in vivo may rely upon a supply of steroid precur- sors from the blood, which is absent under in vitro conditions.
Completely “normal” human adreno- cortical tissue is impossible to obtain be- cause of the previous in vivo history of the tissue. The non-tumor adrenal tissue from
tumor-bearing patients came from an envir- onment characterized by an unregulated production of corticoids. The tissues from patients with pheochromocytoma and me- dullary hematoma were exposed to the stress of surgery. The adrenals in the accident victims that served as kidney donors were exposed to the stress of the accident and its sequelae. In spite of the different and ab- normal pre-experimental histories of the non-tumor adrenal tissues, the samples gave similar electrical and steroidogenic pictures and responded very much like rat adrenal tissue, which was taken from resting normal animals with a minimum of disturbance (1). All samples of non-tumor human adrenal tissue which we examined responded to ACTH with a prompt and vigorous increase in steroid formation.
This is the first exploration of the electro- physiological properties of non-tumor and neoplastic human adrenocortical tissue. Further work is needed to extend and ex- plain the significance of the results.
Acknowledgments
We thank Eloise Rapino and Michael Levin for assistance. Tissue samples were obtained through the cooperation of Drs. W. Blakemore, J. F. Brunner, C.D. Cobau, R. A. Gandy, Jr., K. A. Kropp, A. A. Mancini, F. I. Regueyra, and R. T. Tidrick.
We acknowledge helpful discussion with Dr. E. K. Matthews in this work.
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International Symposium on Urinary Stone Formation
International Symposium on Urinary Stone Formation
March 29 to April 1, 1976 Davos, Switzerland
Language: English
For further information please write to:
Prof. H. Fleisch, Department of Pathophysiology Murtenstrasse 35, CH-3008 Bern/Switzerland