~olecu~r and Cellular E~docr~nulo~, 82 (1991) 293-301 0 1991 Elsevier Scientific Publishers Ireland, Ltd. 0303.7207/91/$03.50

293

MOLCEL 02660

Ele~trop~ysiologica~ properties of rat calcitonin-secreting

cells

Hans Scheriibl, Giinter Schultz and Jiirgen Hescheler Pharmakologisches Institut der Freien Universitiit Berlin, D-1000 Berlin 33, F.R.G.

(Received 24 May 1991; accepted 3 September 1991)

Key words: Action potential; Calcium channel; Sodium channel; Potassium current: M~dulla~ thyroid carcinoma; Caicitoninsecreting cell; (Rat cell line rMTC 44-2)

Summdry

The spontaneous electrical activity of calcitonin-secreting cells (C-cells) appears to play an important role in the coupling of fluctuations in the extracellular Ca2+ to changes in the intracellular Ca2+ concentration and thus for calcitonin secretion. Using the patch clamp technique, we have investigated the spontaneous electrical activity and the underlying ionic currents in C-cells of the rMTC 44-2 cell line. With 1.2 mM external Ca2+, the membrane potential was -46.1 + 1.7 mV (n = 58) and about 30% of the cells spontaneously fired action potentials. Rising the external Ca” to 1.8 mM caused the cells to depolarize to -42.1 rf 2.1 mV (n = 56) and spontaneous electrical activity was seen in about 70% of cells. Under voltage clamp conditions, tetrodotoxin-sensitive voltage-dependent Naf currents, outwardrectifying Kf currents and isradipine-, w-conotoxin-sensitive as well as isradipine- and o-conotoxin-insensitive Ca2+ currents were observed. These voltage-dependent currents appear to be the major ionic currents contributing to action potentials in C-ceils and to participate in caicitonin secretion.

Introduction

Cazf entry via voltage-dependent Ca2+ channels during action potentials is known to elicit hormone secretion from pituita~ cells (Schlegel et al., 1987). Similarly to pituitary cells, calcitonin-secreting cells (C-cells) are able to generate Nat- and Ca*+-dependent action potentials (Tischler et al., 1976; Sand et al., 1981, 1986; Kawa, 1988). Moreover, rises of the external Ca2+ stimulate calcitonin secretion and induce either a

Address for correspondence: Hans Scheriibl, Pharmakologisches Institut der Freien UniversitHt Berlin, Thielallee 6973, D-1000 Berlin 33, F.R.G. Tel. 0308386689; Fax 0308315954.

steady increase or oscillations of the cytosolic Ca2+ concentration (Heynen and Franchimont, 1974; Fried and Tashjian, 1986; Haller-Brem et al., 1987; Eckert et al., 1989). Therefore, it appears reasonable to suppose an interrelationship between rises of the extracellular Ca*+, oscillations of the cytosolic Ca2’, calcitonin secretion and the occurrence of action potentials in C-cells. Applying the nystatin modification and the conventional patch clamp technique (Hamill et al., 1981; Horn and Marty, 1988) to C-cells of the rat cell line rMTC 44-2 (Gage1 et al., 1980; Zeytinoglu et al., 1983; Scheriibl et al., 19891, we have studied spontaneously occurring action potentials and we addressed the issue of whether particular voltage-dependent ion channels underlie the exquisite Ca2+ sensitivity of C-cells. Here

we report on the stimulatory effect on action potentials of small rises of the external Ca2’ and on the contributing voltage-dependent Na”, K+ and Ca*+ currents. ~aterjaIs and methods Cell culture

C-cells of the rat medullary thyroid carcinoma cell line rMTC 44-2 were kindly provided by Dr. F. Raue, Med. Klinik, University of Heidelberg, F.R.G. The cells were grown in monolayer culture, using Dulbecco’s modified Eagle’s medium (DMEM} (Biochrom, Berlin, F.R.G.) supplemented with 15% (v/v) horse serum and 2.5% (v/v) fetal calf serum (Gibco, Paisley, U.K.). The medium was changed every other day and cells were subcultured every week. After continuous cultivation for up to ten passages, the cells were replaced by fresh material from the 47th passage frozen stock. Electrophysiology

For electrophysiological investigations, cells were cultured on small glass slides (density about lO,OOO/cm*) for 4-7 days. Then the glass slide was transferred into a chamber (0.2 ml) mounted on an inverted microscope. The attached cells were-continuously superfused at a constant rate of about 5 ml/mm. We used the whole-cell configuration of the patch clamp technique as described by Hamill et al. (1981). The patch electrodes were prepared from Jencons (Leighton Buzzard, U.K.) glass and had an average resistance between 5 and 7 Mfl when filled with standard saline. GR seals were established by suction with a negative pressure of about - 15 cm H,O, and after disruption of the membrane patch, a whole-cell configuration was obtained. In order to record action potentials for periods of up to 1 h, we alternatively accessed the cytoplasma by using the nystatin method (Horn and Marty, 1988; Falke et al., 1989). With freshly prepared nystatin (stock soIution 30 mg/ml, dissolved in dimethyl sulfoxide (DMSO)), acceptable values for access resistance below 50 Ma were achieved after 3-25 min. All experiments were performed at 36-37°C except for the determina-

tions of Naf currents, which were recorded at room temperature (24°C). In 23 experiments, the background conductance was determined after blockage of Na”, CaZf and Ki currents by tetrodotoxin, Cd2+, Ba’+ and Cs+, respectively; it was 0.56 i 0.09 nS (mean I SEMI. This Ohmic conductance may represent leakage conductance or the presence of voltage-independent ion channels. The membrane capacitance, measured as current response to a ramp pulse, amounted to 13.0 f 0.4 pF (n = 67). For the calculation of the Na+, Ca*+ or K” current densities as depicted in the current-voltage relations (IV curves), experiments with seal resistances below 20 Go were excluded because they gave significantly smaller currents. Solutions

External solution El contained (in mM): 135 NaCl, 1.2 CaCl,, 1 MgCl,, 5.4 KCl, 10 glucose and 10 Hepes (pH 7.4 with NaOH, 37°C). Solutions E2-E4 contained (in mM): 135 or 125 tetraethylammoniumC1 (TEA-Cl), 1 MgCl,, 10 glucose, 10 Hepes (pH 7.4 with tetraethylammonium-OH, 37°C) and 1.2 CaCl, (solution E2) or 10.8 CaCl, (solution E3) or 10.8 BaCl, (solution E4). Pipette solution 11 contained (in mM): 90 I(+-aspartate, 50 KCI, 4 MgCl,, 10 Hepes (pH 7.4 with KOH, 37’0, 3 Na,-ATP and was supplemented with freshly prepared nystatin (100-200 pg/ml). 12 contained (in mM): 100 CsCl, 40 CsOH, 4 MgCl,, 3 Na,-ATP, 10 Hepes (pH 7.4 with CsOH, 37”C), 10 EGTA, 6.0 CaCl,; the calculated free concentration of Ca2+ in this solution was 0.1 PM. 13 contained (in mM): 100 K+-aspartate, 50 KOH, 4 MgCI,, 3 Na,-ATP, 10 Hepes (pH 7.4 with KOH, 37”C), 10 EGTA, 6.0 CaCl 2. Statistics

Data are presented as the mean + SEM. Statistical significance was assessed by the Wilcoxon rank sum test. Results Spontaneous action potentials

Applying the nystatin method of the patch clamp technique (Horn and Marty, 1988; Falke et

295

al., 19891, a major disturbance of the cytoplasmic milieu was avoided, and membrane potentials could be recorded for periods of up to 1 h. The mean values of membrane potential (V,) obtained shortly after establishing electrical access were -46.1 k 1.7 mV (n = 58) in standard saline with 1.2 mM Ca2+ and - 42.1 _t 2.1 (n = 56) when external Ca*+ was 1.8 mM. The membrane potential appeared to be largely determined by a K+ conductance, since 135 mM KC1 and 10 mM TEA-Cl depolarized the cells to -5.1 + 2.0 mV (n = 3) and - 16.0 f 2.9 mV (n = 3), respectively (not shown). Whereas only 18 out of 58 (about 30%‘) cells exhibited spontaneous action potentials in normal saline with 1.2 mM Ca’+, electrical activity was seen in up to 70% of the examined cells (39/56) with 1.8 mM external Ca*+. The different patterns of electrical activity observed in single C-cells at 1.8 mM external Ca*+ are summarized in Fig. 1. Fig. 1C demonstrates the spiking activity seen in the majority of cells. The mean frequency of the action potentials was 3.4 k 0.4 Hz (n = 22), and in most cases trains of action potentials were observed. Similarly to pituitary cells (Ingram et al., 19861, the spontaneous action potentials in C-cells were initiated from minor depolarizing fluctuations of V, which built up to a characteristic slow depolarizing prepotential. These small depolarizing fluctuations were responsible for eliciting spikes, when they reached the threshold of -45 to -40 mV. Even in the 30% of cells (17/56) that failed to fire action potentials, such fluctuations were seen (Fig. 1A). The maximal amplitude of action potentials was 65 mV, and the reversal mostly occurred between -20 and 0 mV. The mean duration of action potentials was 123.8 f 9.2 ms at half height from baseline and 10.7 k 1.9 ms at half height from threshold. About 10% of C-cells exhibited plateau potentials with or without interspersed single spikes (Fig. 1B); their amplitude was 21.4 + 2.5 mV, and the reversal of the spikes occurred around -20 mV. The plateau potentials lasted between 300 ms and 700 ms. In the experiments where the electrode was placed into a cluster of cells, plateau potentials were seen more frequently; in those instances the plateau potentials lasted for periods of up to several seconds (not shown). Bursts of spikes as reported for pancre-

A 0 mV -60 1 s 6

C 0 mV -60

D 0 mV -60 Fig. 1. Different patterns of spontaneous electrical activity in C-cells. A: Minor potential fluctuations. B: Plateau potentials with interspersed single spikes. C: Trains of action potentials. D: ‘Bursts’ of spikes. The cytoplasma was accessed using the nystatin method. The bath solution contained 1.8 mM Ca*+.

atic p-cells (Rorsman and Trube, 1986) were seen only in a few cells (Fig. 10). They reached to peak potentials at about -20 mV, and their maximal frequency amounted to 16 Hz. Suppression of spontaneous tetrodotoxin and isradipine

action potentials by

Sand et al. (1986) have clearly shown that both the Naf channel blocker tetrodotoxin (TTX) and the Ca2+ channel blocker D-600 block depolarization-induced action potentials in C-cells from human medullary thyroid carcinoma. We reinvestigated the effects of TTX and the Ca*+ channel blocker isradipine (PN 200 110) on the spontaneous electrical activity in rMTC 44-2 cells. Both TTX (0.1-l PM) and isradipine (0.1-l PM) reversibly suppressed the spontaneous action potentials (Fig. 2). In contrast to TTX, isradipine

296

1 pM

Tetrodotoxin

B

1 @A lsradipine

0

mV

f

-60 [

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2s Fig. 2. Effects of tetrodotoxin (TTX) and isradipine on spontaneous action potentials. The extracellular solution El (1.2 mivi CaLf ) was superfused. ‘RX (1 PM, A) and isradipine (1 FM, B) reversibly suppressed action potentials. The cytoplasma was accessed by the nystatin method.

even eliminated the minor potential fluctuations preceding action potentials. As previously reported for melanotrophs (Taraskevich and Doug-

las, 1989), adding 100 nM Bay K 8644, a Ca2+ channel agonist, induced trains of spikes (not shown) or at higher concentrations (above 1 PM) 6

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normal saline (El) supplemented with 50 /.LM Cd*+. K+ was Fig. 3. Whole-cell recordings of Na+ currents. The bath contained replaced by Cs+ in the pipette solution (12). A: Superimposed original traces are shown for depolarizations starting from a holding potential of -80 mV to test potentials of -50, -40, -30, -20, - 10, 0, 10, 20 and 30 mV. B: Naf inward current-voltage relation. Peak current densities are plotted versus test potentials before (full squares) and after adding 1 PM TTX (open squares). Means + SEM from seven experiments are shown.

291

ward currents (not shown). Moreover, 1 PM TTX reversibly suppressed the voltage-dependent Na+ currents (see Fig. 3B). Na+ current inactivation was measured by 500 ms conditioning pulses preceding a test pulse to 0 mV. Inactivation was seen from prepotentials of - 100 mV onwards and was complete at about - 30 mV. The mean potential of half-inactivation was -49 mV in four experiments (not shown). With Na+ and K+ currents Calcium currents. eliminated by substituting TEA-Cl for NaCl and Cs+ for K+, Ca*+ inward currents were elicited by 300 ms pulses to various test potentials from a holding potential of -80 mV (Fig. 4). The dependency of these inward currents on the concentration and the type of the divalent charge carrier was studied with 1.2 mM Ca*+ (Fig. 4A), 10.8 mM Ca2+ (Fig. 4B) and 10.8 mM Ba2+ (Fig. 4C) in the external solution. Peak current densities were plotted versus test pulses for 1.2 mM and 10.8 mM Ca2+ in Fig. 5A. A maximal inward current density of 9.3 + 0.7 pA/pF (n = 12) and of 23.1 f 1.3 pA/pF (n = 7) was observed for 1.2 mM Ca2+ at - 10 mV and for 10.8 mM Ca*+ at 0 mV, respectively. The different threshold potentials of -50 mV for 1.2 mM Ca*+ and of - 40 mV for 10.8 mM Ca*+ are most likely due to the screening effect of surface charges by divalent cations (Ohmori and Yoshii, 1977; Hagiwara and Ohmori, 1982; Wilson et al., 1983).

caused a continuous depolarization (Scheriibl et al., 1990). Therefore, it seems reasonable to suggest that a dihydropyidine-sensitive Ca*+ current participates in the slow depolarizing phase at the beginning of the action potentials. Voltage-clamp studies on C-cells

As the analysis of the action potentials had suggested the existence of voltage-dependent Naf, Ca*+ and Kt channels, we proceeded to study the C-cells by voltage-clamp experiments. To characterize the voltageSodium currents. dependent Na+ currents we blocked Ca2+ currents by Cd*+ and K+ currents by substituting Cs’ for K’ in the internal solution. Fig. 3A shows a series of superimposed current records taken from a holding potential of -80 mV and employing depolarizing test pulses. The Na*+ currents reached a peak value within 0.3-3 ms after the onset of the clamp-pulse and rapidly inactivated to a steady-state level less than 5% of this peak within 7 ms. The current-voltage relation (IV curve> is depicted in Fig. 3B. A maximal Na+ current density of 87.7 f 7.6 pA/pF (n = 7) was recorded at - 10 mV. The threshold potential of - 60 mV was close to the threshold of - 50 mV observed for depolarizatioh-induced action potentials. The apparent reversal potential was around 45 mV. Replacing external NaCl by choline-Cl or TEA-Cl totally eliminated the inA

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Fig. 4. Whole-cell recordings of inward currents through Ca2+ channels. A: 300 ms current traces for depolarizations to -40, - 30, - 20 and - 10 mV (top panel) and 0, 10 and 20 mV (bottom panel). The holding potential was - 80 mV; solutions were 12 and E2 (1.2 mM Ca*+ 1. Panels B and C show 300 ms current traces for depolarizations to -30, - 20, - 10 and 0 mV (top panels) and 10, 20, 30 mV (bottom panels). The holding potential was - 80 mV. Solutions in B were 12 and E3 (10.8 mM Ca*+ ). Solutions in C were I2 and E4 (10.8 mM Ba*+ 1. Representative experiments out of 12 are shown.

298

Applying 3 s long depolarization pulses from - 80 to - 10 mV, we determined two inactivation time constants, T, and TV; with 1.2 mM external Ca*+ they were 168 ms and 4.9 s, respectively (n = 5). Inactivation time constants as long as 4.9 s at 37°C have not been described for other cells (Eckert and Chad, 1984). Since calcium-dependent calcitonin secretion is known to be inhibited by Ca2+ channel blockers (Hishikawa et al., 19851, the TTX-resistant inward currents through Ca2+ channels were further characterized by these pharmacological tools. Applying test pulses to 0 mV from a holding potential of -80 mV we studied the (maximal) effects of isradipine, w-conotoxin and Cd*+ (Fig. 5B). Isradipine (1 PM) suppressed 32.8 k 3.7% (n = 5) of the Ba2+ inward current. The isradipine-sensitive Ba2+ current could as well (entirely) be blocked by 10 PM methoxyverapamil or 10 /IM diltiazem (not shown). Combining w-cono-

-40

-20

20

40

toxin (10 PM) and isradipine (1 PM) reduced the inward current by 69 f 6.7% (n = 5). A third, w-conotoxin- and isradipine-insensitive component of the Ba2+ current (about 30% of the entire Ba*+ current) was completely blocked by 0.1 mM Cd2+ (see Fig. 5B) or 0.1 mM Ni*+ (not shown). Thus, different types of Ca2+ currents appear to underlie the observed whole-cell Ca*’ current (Nowycky et al., 19851. Low thresholdactivated Ca2+ channel currents with fast inactivation kinetics were, however, not observed (in Na+ free solutions) (n = 114). Potassium currents. Fig. 6A shows the outward currents measured during 300 ms pulses to various test potentials from a holding potential of - 80 mV. There was a transient component with a rapid decay during the first 50 ms, which was followed by a sustained component. The current density at the end of the 300 ms pulse was plotted versus the test potential in Fig. 6B. The IV curve

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channel currents. A: Current-voltage relations of the Ca 2f inward current. Peak current densities were Fig. 5. Whole-cell Ca” corrected for leakage and plotted versus test potentials for 1.2 mM Ca2+ (squares, II = 12) and 10.8 mM Ca2+ (circles, n = 7). Means + SEM are shown. Solutions were 12 and E2 (1.2 mM Ca2+ ) or E3 (10.8 mM Ca2+ 1. B: Pharmacological characterization of the Ca2+ inward current. Test pulses (200 ms) to 0 mV were applied from a holding potential of -80 mV. Isradipine (1 PM), (0.1 mM) were consecutively applied. The bath solution with isradipine (I PM) plus w-conotoxin (w-CT, 10 PM) and Cd” w-conotoxin contained 10.8 mM Ba2+ and 1 mM Mg2+; It was superfused for 2 min. Solutions were I2 and E4. CON, control.

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inhibit Ca*+-dependent K+ currents (Burgess et al., 19811, a major component of the outward currents appears to consist of this type of K+ currents. 4-Aminopyridine completely suppressed the transient component of the outward current and reduced the slowly inactivating outward currents of rMTC cells by maximally 40.5 f 4.1% at a concentration of 2 mM (n = 6) (Fig. 7C). Since 4-aminopyridine is known to effectively inhibit transient K+ currents of the A-type (Cook, 1988), it appears reasonable to suggest that A-type currents represent the transient component of the outward currents and may play a role in the frequency of action potentials (Castle et al., 1989). Apamin (lo-500 nM, II = 51, glibenclamide (0.1-3 PM, II = 4) or charybdotoxin (30-100 nM, it = 5) had no effect on the outward currents (for apamin, see Fig. 70). Discussion

Electrophysiological studies of many endocrine cell types have provided evidence to suggest that the electrical properties of their cell membranes play a central role in the regulation of the secretory process (Poisner and Trifaro, 1985). Since hormone secretion from C-cells as well as from parathyroid cells is exquisitely sensitive to changes in the extracellular Ca*+ concentration (Brown, 19911, the electrical properties of C-cells have attracted much interest (Ozawa and Sand, 1986; Brauneis et al., 1990; Scheriibl et al., 1990; Yamashita and Hagiwara, 1990). In particular, voltage-dependent Ca2+ channels have been implicated to play a crucial role in the Ca2+ sensing mechanism in C-cells (Hishikawa et al., 1985; Raue et al., 1989; Scheriibl et al., 1989). Our present electrophysiological studies further strengthen this idea: with physiological extracellular Ca*+ concentrations, voltage-dependent Ca2+ channels became detectable in the range of the normal resting membrane potential ( - 50 to - 40 mV>, and the amplitude of the Ca*+ inward currents depended on the extracellular Ca2+ concentration. Since the Ca2+ inward currents displayed almost no inactivation at potentials of -40 mV to -30 mV, it appears reasonable to suggest that the slow inactivation kinetics of the Ca*+ channels allow for the possibility of steadystate Ca2+ influx in C-cells. The coupling of the

extracellular Ca2 + to the intracellular Ca2+ concentration regulates hormone secretion; however, not only in C-cells but as well, for example, in pituitary cells (Ramsdell and Tashjian, 1985). This implies that the particular Ca*+ sensitivity of C-cells may involve an amplification mechanism of the Ca*+ signal and/or a particular sensitivity of the transmembraneous Ca*+ influx. In contrast to the particular properties of the Ca2+ currents, the voltage-dependent Nat and K+ currents of C-cells displayed properties similar to the ones described in other endocrine cells (Fenwick et al., 1982; Ozawa and Sand, 1986; Cobbett et al., 1987; Chen et al., 1990). C-cells are well known to generate Na+- and Ca2+-dependent action potentials in response to membrane depolarization (Ozawa and Sand, 1986; Kawa, 19881. By applying the nystatin method of the patch clamp technique and with 1.8 mM external Ca*+, we were able to observe spontaneously firing action potentials in up to 70% of rMTC 44-2 cells. In agreement with the previous reports on both Na+- and Ca*+-dependent action potentials, both tetrodotoxin and the Ca2+ channel blocker isradipine reversibly suppressed spontaneous electrical activity in C-cells. Since isradipine but not tetrodotoxin eliminated the slow depolarizations preceding action potentials, it appears reasonable to suggest that a dihydropyridine-sensitive Ca*+ current contributes to the slow depolarizations and thus to the generation of action potentials. Coincident measurements of the cytosolic free Ca*+ and action potentials in excitable neuronal (Gorman and Thomas, 1978) or endocrine cells (Schlegel et al., 1987) have shown that Ca*+ entry during action potentials is responsible for oscillations of cytosolic Ca *+ in these cells. In C-cells, the oscillations as well as the steady increase of cytosolic Ca* + induced by high external Ca2+ (Eckert et al., 1989) may similarly be due to action potentials that are elicited by high external Ca’+. The frequency as well as the duration of action potentials could thus determine the Ca*+ influx and thereby tightly regulate calcitonin secretion from C-cells. As the electrical activity in various endocrine cells, for example in p-cells of the pancreas (Sherman and Rinzel, 1991) is known to be synchronized by gap junctions and as C-cells

301

are known to be grouped as clusters within the thyroid gland (Pearse, 19661, synchronization of the electrical activity may similarly occur in C-cells and its possible contribution to the Ca2+ sensitivity warrants further investigation. Since calcitonin secretion is regulated not only by the intracellular Ca*+ concentration but as well by protein kinase A or C activation (HallerBrem et al., 1988; Raue et al., 1991), it will be interesting to know the effects of protein kinase A or C activation on the electrical activity. Furthermore, the electrical properties observed in C-cells of the rMTC 44-2 cell line should be reexamined in isolated mammaIian C-cells. Acknowledgements We are indebted to Dr. Friedhelm Raue, Medizinische Klinik, Universitat Heidelberg, Heidelberg, F.R.G., for providing a start culture of the rMTC 44-2 cell line. We thank Inge Reinsch, Monika BigaIke and Wolfgang Stamm for excellent technical assistance. This study was supported by grants from the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, and a DFG fellowship to H.S. References Brauneis, U., Fajtova, V.T. and Tillotson, D.L. (1990) 72nd Annual Meeting of the Endocrine Society, Atlanta, Abstr. 1431. Brown, A.M. (19911 Physiol. Rev. 71, 371-411. Burgess, G.M., Claret, M. and Jenkinson, D.H. (1981) J. Physiol. 317, 67-90. Castle, N.A., Haylett, D.G. and Jenkinson, D.H. (1989) Trends Neural. Sci. 12, 59-65. Chen, C., Zhang, J., Vincent, J.D. and Israel. J.M. (1990) J. Physiol. 425, 29-42. Cobbett, P., Ingram, CD. and Mason, W.T. 61987) J. Physiol. 392, 273-299. Cook. N.S. (1988) Trends Physiol. Sci. 9, 21-28. Eckert, R. and Chad, J.E. (1984) Prog. Biophys. Mol. Biol. 44, 215-267. Eckert, R.W., Scheriibl, H., Petzelt, C., Raue, F. and Ziegler, R. (1989) Mol. Cell. Endocrinol. 64, 267-270. Falke, L.C., Gilles. K.D., Pressel, D.M. and Misler, S. (1989) FEBS Lett. 251, 167-172. Fenwick, E.M., Marty, A. and Neher, E. (1982) J. Physioi. 331, 599-635. Fried. R.M. and Tashjian, A.X. (1986) J. Biol. Chem. 261, 7669-7674. Gage], R.F., Zeytinoglu, F.N., Voelkel, E.F. and Tashjian, Jr., A.H. (19801 Endocrinology 107, 516-523.

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