The effects of thyrotropin-releasing hormone and potassium depolarization on phosphoinositide metabolism and cytoplasmic calcium in bovine pituitary cells

Biochimica et Biophysica Acta, 1013 (1989) 97-106

97

Elsevier BBAMCR 12366

The effects of thyrotropin-releasing hormone and potassium depolarization on phosphoinosifide metabolism and cytoplasmic calcium in bovine pituitary cells C.A. Wood

* and J.G. Schofield

Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol ( U.K.)

(Received 5 May 1988) (Revised manuscriptreceived 11 August 1988)

Key words: Thyrotropin; Calcium; Potassium depolarization; Phosphoinositide; Cytoplasmiccalcium; Pituitarycell; (Bovine) Addition of thyrotropin-releasing hormone (TRH) (10 nM to 10 # M ) to bovine anterior pituitary cells labelled with [aHlinositol decreased the radioactivity in inositol-containing lipids and increased it in inositol phosphates. TRH aL~o increased the cytop|asmic calcium concentration bipha~ict,,ily. At T R H concentrations below 10 nM, the increase was sustained and sensitive to ir~hibitors of calcium influx through voltage-gated channels, whereas c,mcentrations over 10 nM elicited in addition a rapid transient increase in calcium, which was relatively insensitive to such inhibition. Incubation of the cells in medium containing 25 mM KC| increased the cytoplasmic calc~um concentration by stimulating influx through voltage-gated channels, and markedly enhanced the initial transient i~crease of calcium seen at TRH concentrations above 10 r~M. It did not affect the generation of lnsP3 and it also enhanced the calcium response to ionomycin. It is suggested that stimulation of calcium entry through voltage-gated channels can increase the amount of calcium available for mobilisation by TRH.

Introduction Thyrotropin-releasing hormone (TRH) stimulates the secretion of prolactin from normal anterior pituitary cells and from clonal pituitary cell lines such as G H 3 and GH4C1 cells. In normal bovine anterior pituitary cells [1] and in clonal anterior pituitary tumour cell lines [2,3] TRH causes a rapid elevation of the cytoplasmic free calcium concentration. In the latter cells it has been established that this involves both intracellular calcium mobilisation and calcium influx across the plasma membrane [2,3]. There is considerable evidence that the

* Present address: Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, U.K. Abbreviations: TRH, thyrotropin-releasinghormone; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulphonicacid; InsP, InsP2 and InsP3, inositoJ mono-, bis and trisphosphate; Ptdlns, phosphatidylinositol; Ptdlns4P, phosphatidylinositol 4-phosphate; PtdIns(4,5)P2, phosphatidylinositol 4-bisphosphate; Quin-2 AM, Quin-2 tetraa~etoxymethyl ester; FCS, fetal calf serum; PBS, phosphate-buffered saline; bBSS, buffered bicarbonate saline solution; BSS, buffered saline solution; DMSO, dimethylsulphoxide. Correspondence: C.A. Wood, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, U.K.

stimulation of calcium mobilisation in GH~ and GH4CI cells occurs as a result of the increased hydrolysis of inositol iipids [4-6] and production of Ins(1,4,5)Pa [7]. A similar system operates in normal bovine anterior pituitary cells, since T R H affects the labelling of inositol lipids in them [8] and since InsP 3 mobilises calcium from a bovine pituitary microsome preparation [9]. The stimulation of calcium influx into GH3 and normal rat pituitary cells occurs through dihydropyridine-sensitive calcium channels [3,10,11]. The mechanism by which T R H activates these voltage-gated calcium channels is unclear, evidence that it is indirect, occurring via the mediation of second messengers, being conflicting [12,13]. One aim of this study was to investigate the importance of inositol lipid hydrolysis in the elevation of intracellular calcium concentrations by TRH in a population of normal bovine anterior pituitary cells enriched with lactotrophs. A second objective of the study was to investigate the relative importance of calcium mobiljsation and of calcium entry through voltage-gated channels in the calcium response. In bovine anterior pituitary cells, potassium depolarisation increases 45Ca efflux and the verapamil sensitive release of growth hormone [14] and prolactin [15] by activating voltagegated calcium channels. The effects of potassium de-

0167-4889/89/$03.50 ~ 1989 ElsevierScience Publishers B.V. (Biomedical Division)

98 polarisation on the cytoplasmic calcium response to TRH were therefore determined. Materials and Methods

Materials DNAase I (type II), soya bean trypsin inhibitor (type 1 S), thyrotropin-releasing hormone, Percoll and bovine serun: albumin were obtained from Sigma Chemical Company, Poole, U.K. Collagenase (Clostridium histolyticum) was from BCL, Lewes, U.K. Quin-2 tetraacetoxymethyl ester (Quin-2 AM) was purchased from Lancaster Synthesis, Morcambe, U.K. Other chemicals were obtained from BDH, Poole, U.K. Nitrendipine was a kind gift from Dr. Schramm. Bayer, F.R.G.

Cell dispersion and culture Dispersion of the anterior pituitary cells was carried out as previously described [16]. The cells were maintained in medium M199 with Hanks' salts (Flow Laboratories, Irvine, U.K.), supplemented with 1% Ultroser G (LKB Instruments), 5% fetal calf serum (FCS), 1% MEM non-essential amino acids, fungizone (0.25 ~tg/ml, Gibco, Paisley, U.K.), glutamine (2.5 raM), Penicillin (68 #g/ml), Streptomycin (100 #g/ml) and buffered using 10 mM NaHCO3 and 20 mM Hepes to pH 7.4. M199 was chosen because of its low myo-inositol content (0.05 mg/ml). The dispersed anterior pituitary cells (5.106) were plated onto 90-mm non tissue culture-treated petri dishes containing 7 ml of media and were maintained at 37 °C in a culture cabinet equilibrated with COz/air (3 : 97%) for 72 to 96 h.

Labelling of the cells with [ SH]inositol In experiments where cells were to be labelled with [3H]inositol, they were plated as described above, except that 45 mm non tissue culture-treated petri dishes containing 3 ml of media were used. To minimise the concentration of unlabelled inositol in the medium, its FCS content was reduced to 1%. The medium contained 2 /~Ci/ml of myo-[2-3H]inositol (Amersham, U.K.; 10-20 Ci/mmol). The cells were maintained in culture for 3-4 days.

immunocytochemistry Immunocytological staining was carried out to determine the proportion of lactotrophs in the cell preparation. Cells which had been maintained in culture on tissae culture-treated plates were fixed by incubation for 30 minutes at room temperature in a freshly prepared solution containing paraformaldehyde (4 g), glutaraldehyde (25%; 0.2 ml) and sodium phosphate (0.13 M, pH 7.4) 100 ml. The petri dishes were then washed with phosphate-buffered saline (PBS). (containing NaHzPO4 (10 rnM, pH 7.5) in 0.9% (v/v) NaCI) and air-dried. The cells were incubated for 90 min at

room temperature in 2.2 ml PBS containing 45 #1 goat serum (Vector Laboratories, Peterborough, U.K.) and 0.3% Triton X-100. They were then washed twice in PBS and incubated for 18 h at 0 °C in PBS containing a 1:1000 dilution of rabbit anti-bovine prolactin antiserum (a kind gift from Dr. M. Wallis, Department of Biological Sciences, University of Sussex). After this incubation the cells were washed twice in PBS and then incubated for 30 rain at room temperature with biotinylated rabbit anti-lgG antiserum (Vector Laboratories, Peterborough, U.K.). They were then washed twice in PBS and incubated for 60 rain at room temperature in PBS containing 9 # l / m l avidin DH solution and 9 # l / m l biotinylated horseradish peroxidase H (both from Vector Laboratories, Peterborough, U.K.). This solution had been mixed and incubated for 30 rain prior to use. The cells were washed twice in PBS and then incubated for 7 rain at room temperature in a solution containing 50 p,l diaminobenzidine tetrahydrochloride (50 mg/ml in 0.1 M Tris), 200 #1 Tris (0.1 M) and 250 #l hydrogen peroxide (0.02%). The cells were finally washed with tap water. The proportion of lactotrophs (which stained with anti° prolactin) was 48 + 14% (three cell preparations).

Measurement of intracellular calcium concentrations Cells and medium were decanted from the dishes and the cells were recovered by centrifugation (250 g, 10 min). They were washed in a buffered bicarbonate saline solution (bBSS; containing Nacl 129 mM; KCI 4.7 raM; NaH2PO 4 2.7 mM; NaHCO 3 4.6 mM; CaCI 2 1 mM; MgCI 2 1.2 mM; Hepes 11.3 raM; glucose 5.2 raM; albumin 0.05% (w/v); and adjusted to pH 7.4 at 370C). They were then resuspended at a density of 4.106 cells per ml in bBSS containing Quin-2 AM at a final concentration of 20 /~M, gassed with O2/CO= (95/5%) and incubated at 370C for 30 rain. To minimise precipitation, the Quin-2 AM solution was diluted by adding bBSS with rapid mixing to the appropriate volume of a 40 mM stock solution of Quin-2 AM in DMSO. The loaded cells were washed and resuspended (8.106 cells per ml) in a bicarbonate-free, Hepesbuffered saline solution (BSS; containing NaCI 140 mM; KCI 4.4 mM;KH2PO 4 1.2 mM; MgSO4 1.2 raM; CaCI a 1 mM; Hepes 10 mM; glucose 5 raM; albumin 0.05% (w/v); adjusted to pH 7.4 at 37°C and stored at room temperature prior to use. Measurements of the intracellular calcium concentrations was carried out in BSS as described previously [16].

Extraction of metabolites labelled with [3H]inositol The cells were harvested by centrifugation, washed once in oxygenated BSS and then resuspended at 5- 10 7 per ml in BSS containing LiCI (5 raM). They were incubated in a shaking water-bath at 37 °C for 5 min prior to use. In some experiments cell suspensions were

99

preincubated with effectors for the appropriate length of time. Aliquots of 95 #1 were then removed from the treated or untreated cell suspensions and added to glass test tubes containing 5 /d of either BSS or agonist. Experiments were terminated by the addition of 1.5 ml of ice-cold chloroform/methanol/12 M HCI (20:40: 1, v/v). The samples were left on ice for a further 30 rain. The phases were broken by addition of 0.8 ml chloroform and 0.75 ml EDTA (5 mM). The samples were mixed for 60 s using a vortex mixer and then centrifuged at 500 × g for 5 rain. The aqueous phase was separated from the organic phase and the aqueous and organic phases were backwashed with synthetic organic and aqueous phases, respectively. After centrifugation at 500 x g for 5 rain, the aqueous phases were removed and combined. The organic phases were combined, dried down and stored at - 2 0 " C for 2-3 days.

Separation of the inositol phosphates The aqueous phases were applied directly to 1 ml AG1-X8 (formate form) anion-exchange resin columns (Bio-Rad) and the inositol phosphates were eluted in a similar way to that described by Berridge et al. [17]. [3H]Inositol was eluted by 10 ml H20, inositol 1,2cyclicmonophosphate and glycerophosphoinositol by 20 ml of 5 mM sodium tetraborate plus 60 mM ammonium formate, and inositol monophosphates, bisphosphates and trisphosphates by stepwise addition of 20, 8 and 8 ml of 0.1 M formic acid containing 0.1, 0.4 and 0.8 M ammonium formate, respectively.

Separation of the inositol lipids The lipids were deacylated and then separated by anion-exchange chromatography as described by Creba et al. [18] and Downes and Wusteman [19]. Briefly, the material dried from the organic layer was redissolved in 1 ml of chloroform, and 0.2 ml methanol and 0.2 ml of 1 M sodium hydroxide in methanol/water (19:1, v/v) were added. The samples were mixed using a vortex mixer and incubated at room temperature for 20 min. Chloroform (1 ml), methano~ (0.6 ml) and H20 (0.6 ml) were then added. The samples were mixed using a vortex mixer for 30 s and centrifuged at 500 x g for 5 min. 1 ml of the aqueous layer was transferred to AG1-X8 columns prepared as for the inositol phosphate separations. Unbound radioactivity was eluted from the column by 15 ml H20, glycerophosphoinositol by 20 ml 5 mM sodium tetraborate containing 180 mM ammonium formate, glycerophosphoinositol 4-phosphate by 16 ml 0.1 M formic acid containing 0.3 M ammonium formate, and glycerophosphoinositol 4,5-bisphosphate by 10 ml 0.1 M formic acid containing 0.75 M ammonium formate. The radioactivity associated with these fractions was determined by scintillation counting.

Results

The effect of TRH on [3H]inositol radioactivity in phosphatidyi inositides and inositol phosphates TRH (1 #M) caused a time-dependent decrease in the radioactivity in the inositol lipids (Fig. 1). Radioactivity in Ptdlns (4,5)P2 had decreased to a minimum of 85 + 3% (n = 7) of the control with;n 5 s (Fig. lc); in PtdIns4P and Ptdlns radioactivity had started to decrease within 5 s and by 60 s had fallen to 84 + 5% (n = 4) and 86 + 4% of the control, respectively (Figs. l b and la). Radioactivity in all three lipids had returned to unstimulated levels by 300 s. TRH increased the radioactivity in inositoi phosphates (Fig. 2). Radioactivity in InsP 3 was maximally increased ~,~ 171 + 10% (n = 7) of the control 5 s after the addition of TRH (1 ~M) and by 60 s had fallen to 125 + 11% of the control, remaining at this level for at least 300 s (Fig. 2a). In InsP 2 radioactivity was maxi-

T line (sec)

100 o

80

7C 0

~o

~2,o ~,8o 2~,o 31o

l

I

100~

N

~

9o

~

Bo

8

70

°r

,oo/C

~~"~

Ptdlns4-P

JI Ptdlns45-P2

Fig. 1. Time-dependent alterations in inositol lipids in response to TRH. Bovine anterior pituitary cells were labelled for 66 h with 2 # C i / m l myo-[2-'~ltlinositol, when the radioactivity in the phosphoinositides was: Ptdlns4 10553+ 1370 cpm; Ptdlns4P 5675 5:594 cpm; and Ptdlns(4,5)P2 4048 5:320 cpm (mean + S.E.M. for 5.10 (' cells). TRH (1 #M; O) or buffer ( o ) were added at time zero and the data show the percent change in radioactivity in (a) Ptdlns; (b) Ptdlns4P; and (c) Ptdlns(4,5)P2. Data are mean :t: S.E.M. of at least three separate experiments. * P < 0.05.

100

really elevated to 152 4. 1% (n 4) of control at 10 s and had returned to unstimulated levels (Fig. 2b) by 60 s. An increase in radioactivity in InsP was not observed until 60 s after the addition of TRH but thereafter continued to increase in a linear fashion for at least 300 s (Fig. 2c). Even at 10/~M TRH the increase in InsP3 had not reached a plateau (Fig. 3), which indicates that the ECs0 is probably greater than 0.8 ~M. =

100

65O

Q

T

I

O v

Q w

OU

ge Ee_

~x _o~

5(:

o @

=,E

~..3

ou_.

The effect of TRH on intracellular calcium concentrations The resting calcmm concentration in the dispersed cells averaged at 180 + 10 nM (mean + S.E.M. for 20 preparations: range 115-256 nM). As shown in Fig. 4, TRH caused a dose-dependent increase in the intraceUular calcium concentration, At concentrations above 10 nM, TRH caused a transient spike of calcium followed by the attainment of a new steady-state level. At lower TRH concentrations the transient spike was less evident and the elevation of the steady-state calcium concentration predominated. After TRH (1 /~M) the transient

g~

~g

C~

lnM

'0 1OhM O.IjJM 1JJM IOJ~M TRH

Fig. 3. The dose dependences of effects of TRH on lnsP3 levels and intracellular calcium concentrations. InsP3 measurements (0) were made at 5 s after the addition of TRH at the concentrations shown. The calcium elevation (e) was the maximum rise of calcium above unstimulated levels. The data represent the mean+ $.E.M. of at least four separate experiments (e); and the range of means obtained in two separate experiments(0).

200

15(

10( 20C "b

InsP2

150

100' 28( •c

InsP

~t

EO(

I0C

60

120 180 Time (sec)

240

300

Fig. 2. Time-dependentalterations in inositol phosphates in response to TRH. Cells were labelled for 66 h as for Fig. 1, when radioactivity in inositol phosphates was: [nsP 3166+459; [nsP2 355=1=49; and InsP3 513±71 (mean+S.E.M. for 5.106 cells). TRH (5/LI, final concentration 1/zM; e ~ e ) or buffer (5 pl, o) was added at time zero and the data show the percent change in radioactivity in (a) lnsP3; (b) InsP2, and (c) InsP. All experiments were carded out in the presence of 5 mM LiCI. Data are mean_+S.E.M. of at least three separate experiments. * P < 0.05, * * P < 0.01.

peak cytoplasmic calcium concentration averaged at 460 + 46 nM (mean + S.E.M. for 20 preparations: range 284-1209 nM), and the steady-state attained subsequently averaged at 252 4- 13 nM (mean 4- S.E.M. for 20 preparations: range 138-383 nM). To investigate how the transient and sustained phases of the calcium response depended on T R H concentration, cells were exposed to a low dose of T R H and the effect of increasing the concentration was examined. Adding TRH to increase the concentration from 1 nM to 0.1/~M caused a large transient calcium response, but only increased the sustained calcium component slightly over the rise caused by 1 nM T R H (Fig. 5). In four such experiments the transient calcium rise caused by T R H (0.1/~M) was decreased from 136 4- 12 nM to 113 4- 20 nM by TRH (1 nM) whereas the sustained calcium rise decreased from 91 4- 12 nM to 10 4- 8 nM. This would be expected if 1 nM T R H is able to elicit a nearly maximal sustained response without causing an appreciable transient rise. The overall dependence of the peak calcium response on T R H concentration is shown in Fig. 3: an ECs0 of approx. 20 nM was observed. By comparing the dose-response curve for the elevation of calcium with that for InsP 3 (Fig. 3) it is apparent that TRH evoked a calcium rise at concentrations which caused no observable change in InsP 3 accumulation.

The effects of EGTA and calcium-channel antagonists on the TRH-induced calcium rise The importance of calcium influx to the two phases was investigated using EGTA (2.5 raM), which reduces the extraceUular calcium concentration below 100 nM and so prevents calcium influx• In the presence of

101 1000

400

0

0

300

800

200 600 100

L

40C 201

TRH

0.1jJM

0 60 E -i _u o

500

sec

400

u

300

60 sec u o

8O0 r-

- -

200 10(3

r

o

60(:

0

0 L

Fig. 5. The effect of a low concentration of TRH on the elevation of intracellular calcium concentrations caused by a higher concentration of TRH. Traces show the effect on cytoplasmic calcium concentration of: (a) TRH (0.1/~M) alone, (b) TRH (0.1 #M) added after addition of TRH (1 riM). Times of additions are indicated by the arrows. These traces are typical of results averaged in text.

r" u sO u

40C

E 200 :3 _H o u

g

"OOc o

L.

2000~"~ 400fd " 2

0

0

~

ot I Fig. 4. The dose-dependent effect of TRH on intracellular calcium concentrations. The four panels show four cuvettes from a typical experiment in which bovine anterior pituitary cells from a single dispersion were loaded with Quin-2 and their intracellular calcium concentrations measured as described in the text. TRH was added at the times indicated by the arrows at: (a) 1 FM; (b) 0.1 ~M, (c) 10 nM and (d) 1 nM.

nitrendepine confirmed that the sustained phase of the response to 0.1 #M TRH was more sensitive than the transient phase to the calcium channel antagonist (Fig. 8). A difference in inhibition of the two phases was also seen following 120 s exposure to verapamil (20 #M). This antagonist did not significantly alter the initial spike of calcium release (TRH alone 198 + 28 nM, with verapamil 163 ± 27 nM: n = 4) but markedly reduced the later steady-state calcium rise (TRH alone 110 + 22 nM, with verapamil 14 ± 4 nM (n = 4; P < 0.05). The effect of potassium depolarisation on the intracellular calcium concentration in the presence and absence of TRH Depolarisation of pituitary cell suspensions by exposing them to an external potassium concentration of 25

E~EGTA the sustained phase was absent and only the transient spike of calcium concentration was seen in response to TRH (1/~M; Fig. 6). The contribution of voltage-gated channels to the calcium responses caused by high and low TRH concentrations was assessed using two antagonists of these channels, nitrendepine and verapamil [20]. Nitrendepine (1/~M), which inhibited the sustained calcium rise caused by a low TRH concentration (1 nM) by 70%, inhibited the sustained phase of the rise caused at a higher TRH concentration (0.1 #M) more than the initial transient phase (Table I and Fig. 7). Varying the concentration of

4OO TRH

o u

~U

EGTA

Fig. 6. The effect of calcium removal on tile TRH-induced elevation of intracellular calcium concentrations: The two traces were obtained using two cuvettes. TRH (1/~M) was added to the upper trace at ~he first arrow. EGI-A (2.5 mM) and TRH (1 #M) were added to the lower trace at the two arrows. These traces are typical of the results averaged in the text.

102 TABLE ! Effect of nitrendipine on the elevation of cytoplasmic calcium concentration caused by low and high concentrations of TRH Cells were loaded with Quin-2 and the calcium concentrations measured as described under Materials and Methods. Where appropriate nitrendipine was added 120 s prior to TRH. Where TRH was used at 0.1 ~tM, data are: (1) the transient spike of calcium release, and: (2) the sustained phase of calcium entry. In these experiments the mean cytoplasmic concentration in untreated cells was 185 :l: 13 nM (n : 8) and in the presence of nitrendipine (1 #M) was 162+16 nM (n ffi 6).

¢o

70~,~i

JO

0¢,j 0 c ~,

_u 0

U

CL

01

I

TRH (1 nM) (n = 4)

TRH (0,1/tM) (n = 6)

81 ± 12

263 ± 36

58 :~ 11

(100~)

(100~)

(100~)

24±4 ** (29 4- 3¢$)

215±34 (73 4- 8~)

24±9 * (52 + 16~)

1

None Nitrendipine (1/~M)

2

P < 0,05.

* * P < 0,01.

mM increased the intracellular calcium concentration from 217 + 13 nM to 524 + 55 nM ( n - 6). This increase was completely prevented by nitrendepine at 1 ~tM, inhibition having an ICs0 of 20 nM (Fig. 9). The calcium rise therefore depends on entry of calcium through nitrendepine-sensitive channels. As shown in Fig. 10, increasing the external potassium-ion concentration to 25 mM greatly enhanced the initial phase of the calcium respowse to TRH (0.1 #M): the average enhancement for seve.~t cell dispersions was from 242 :t: 34 nM to 1003 + 304 nM t P < 0.05). Enhancement was only seen at high concentrations of

!

1

Increment in intracellular calcium concentrations (nM) caused by

Addition

*

350-

10

I

100

I

1000

Nitrendipine (nM)

Fig. 8. The dose-dependent inhibition by nitrendipine of the elevation of intracellular calcium concentrations by TRH. Data show the effects of nitrendipine at the concentrations shown on the early spike of calcium release (e) and on the sustained phase of calcium elevation (o) caused by TRH (0.1 ~tM). Nitrendepine was added 120 s before TRH. Data are the mean + S.E.M. of three separate cell preparations. * P < 0.05, * * P < 0.01.

TRH: at 1 nM, TRH had little effect on the calcium concentration in cells depolarised by potassium (Table II). This suggests that potassium depolarisation enhances the amount of calcium mobilised by TRH. However, the enhancement was progressively inhibited by increasing concemrations of nitrendipine (Fig. 11 and Table III) which indicated that it was dependent upon calcium entry. Further experiments were undertaken to investigate whether the enhancement could be explained by an increase in the amount of calcium available for release or in the ability of TRH to elicit an increase in InsP3. Increasing the external concentration of potassium ions 10ol

SO0[ o

'°°I

E ~-, D v~

r-

LO

~00~~.~ ol

t.~O

"

"

.... u' E o u"6

T~H 60 sec

°'tr'T'°'°e r,

"6==

1

lO

lOO

lOOO

Nitrendipine (nM)

Fig, 7, Inhibition of nitrendipine of the calcium elevation caused by TRH, Effect of TRH (0,1/~M) on the intracellular calcium concentration in the absence (a) and presence (t) of nitrendipine (1 /~M). Additions were made at the times indicated by the arrows. Ethanol, the solvent for nitrendipine, had no effect on intracellular calcium at the final concentration used (0.1~, v/v). The traces are typical of experiments averaged in Table I.

Fig. 9. The dose-dependent inhibition by nitrendipine of the elevation of intracellular calcium concentrations by potassium ions. Data show the calcium rise observed on increasing the external KCI concentration to 20 mM in the presence of the given concentrations of nitrendepine, as a percentage of the calcium rise in the absence of nitrendepine. The nitrendipine was added 120 s prior to the addition of potassium ions (20 mM). Data represent the mean+ S.E.M. for three separate cell preparations. * P < 0.05, * * P < 0.01.

103 BOC 60C 400 200 TRH

0

6 0 sec 160(;

140(3 120(3

be due to enhanced inositol phosphate accumulation. Moreover, the increase in cytoplasmic calcium induced by ion,~,mycin was also greatly increased by potassium depolarisation, from 387 + 18 nM to 2842 + 403 nM (n = 3; P < 0.05, Fig. 12). This suggests that the amount of calcium available for release by TRH and ionomycin is increased following potassium depolarisation. Other agents which increase intracellular calcium concentrations also enhance the increase in cytoplasmic calcium induced by TRH (0.1 #M). Thus Bay K8644, which activates voltage-gated calcium channels, and Momany peptide (Growth Hormone Releasing Peptide, GHRP) which increased the intracellular calcium con-

100C 800

'°°r

[

400 Q

2oo~,-~ O[

TkH

K*

200

Fig. 10. The effect of a depolarising concentration of potassium ions on the elevation of intracellular calcium concentrations caused by TRH. Traces show the effect on cytoplasmic calcium concentration of Ca) TRH (0.1 ~M) alone and (b) TRH (0.1 pM) added 120 s after addition of KC! (final concentration 20 raM). Additions were made at the times indicated by the arrows. The traces are typical of the experiments averaged in Tables I1 and V.

T R H 0 1000

b

60

sec

E

800 o

had no effect on inositol phosphate levels in the presence or absence of TRH (Table IV). Thus the enhancement of the TRH-induced calcium rise was unlikely to

I e

600

8 E u

400

T R H ~

0 u

b

TABLE II

_=

Effect of a depolaris#zg concentration of potassium ions on the elevation of cytoplasmic calcium concentrations caused by T R H Cells were loaded with Quin-2 and the calcium concentration measured as described under Materials and Methods. The potassium concentration of the medium was increased to 25 mM 120 s prior to the addition of TRH. In these experiments the mean cytoplasmic concentration of cells in medium containing 5 mM KCI was 196 5=12 nM (n = 7) and in medium containing 25 mM KCI was 484 + 46 nM (n 6).

---

containing 5 mM KCI

* P < 0.05.

200

K • 0 400

200

Elevation of intracellular calcium concentration above control (nM) caused by TRH in medium

TRH (1 nM) (n=3) TRH (0.1 ~tM) (n = 7)

v U

25 mM KC!

79+ 7

43+ 23

242 + 34

1003 ~-304 *

c~K, T R j~ Nitrendipine

C

Fig. 11. The effect of nitrendipine on the potentiation of the TRH-induced elevation of intracellular calcium concentrations caused by TRH. Traces show the effect on cytoplasmic calcium concentration of: (a) TRH (0.1/~M) alone; (b) TRH (0.1 pM) added after addition of KC! (final concentration 20 mM); and (c) TRH (0.1 #M) added after KC! (final concentration 20 raM) to cells in the presence of nitrendipine (1 pM). Additions were made at the times indicated by the arrows. These traces are typical of results averaged in Table lit.

104 TABLE III

TABLE IV

Inhibition by nitrenedipine of the potentiation of the TRH induced elevation of cytoplasmic calcium concentrations caused by potassium depolarisation

Effect of TRH on radioaclivity in inositol monophosphate in medium containing normal or high concentrations of potassium ions

Cells were loaded witb Qw;n-2 and the calcium concentrations were measured as described under Materials and Methods. Where appropriate nitrendipine was added 120 s before KCI, which was added 120 s before TRH. In these experiments the average increase in calcium concentration caused by 0.1/~M TRH alone was 344-8~ of that caused by TRH in the presence of 25 mM KCI. Increase in cytoplasmic calcium concentration (percent maximum) caused by

Addition

25 mM KC! None (n = 3)

100

0.1/~M TRH after 25 mM KCI

For experimental details see Materials and Methods. Bovine anterior pituitary cells were labeEed for 66 h with 2 laCi/ml myo-[2-3H]inositol. Where appropriate the potassium concentration of the medium was increased to 25 mM 120 s prior to the addition of TRH and the incubation terminated after a further 5 rain. Addition

None (n = 4) TRH (0.1/tM) (n = 4)

Radioactivity in [3H]InsP (cpm) in medium containing 5 mM KC!

25 mM KCI

2 855 + 66

2 829 4- 262

7385 4- 874

6421 4-1038

100

Nitrendipine (10 n M )

(n = 3) Nitrendipine (1/~M) (n=3)

46+ 13 24- 1 " *

62+ 16 424- 8 "

TABLE V

Modification of the TRH-induced elevation of cytoplasmic calcium by agents which increase intracellular calcium concentrations

* /'<0.05. ** P < 0.01.

centration themselves, both slightly enhanced the elevation of intracellular calcium caused by TRH (Table V).

Cells were loaded with Quin-2 and the calcium concentrations were measured as described under Materials and Methods. The agents were added 120 s prior to the addition of TRH. Effector

Discussion In bovine anterior pituitary cells, as in clonal pituitary GH3 cells [3A], TRH decreases the levels of the 200(

BayK8644 (1 ~tM)

150(

GHRP (1/tM) (n=4) KC! (25 mM) (n = 7)

(n=4)

C

1000

~0

~oo

Elevation of intracellular calcium concentration (nM) caused by (a) effector alone

(b) TRH (0.I ~aM) alone

(c) TRH (0.I pM) after the effector

101+24

158+15

246+ 49

74+13

1584-15

269+ 84

294 + 44

242 + 34

1003 + 304 *

* P < 0.05. o

8

60 sec

IJ

E ,_~ 300C "b _u 250C 8000 150Q 100( K*

.50( ion(: n¥cin

Fig. 12. The effect of a depolarising concentration of potassium ions on the amount of calcium available for release by ionomycin. Traces show the effect on cytoplasmic concentration of: (a) ionomycin (0.1 ~aM) and (b) ionomycin (0.1/tM) added after addition of KCI (final concentration 20 mM). Time of additions are shown by the arrows. These traces are typical of results averaged in the text.

three inositol lipids whilst causing the accumulation of inositol mono-, bis- and trisphosphates. The timecourses of the changes suggest that PtdIns(4,5)P2 is rapidly hydrolysed to yield InsP3. No attempt was made in this study to distinguish between the InsP3 isomers, but comparison of the time-courses with those in clonal pituitary GH4C1 cells [6], GH3 cells [21] and other cell types including hepatocytes, HL60 cells, pancreatic acinar cells and the parotid gland [22,23] suggest that the rapidly elevated lnsP 3 is likely to be Ins(1,4,5)P3 rather than Ins(1,3,4)P3. It is widely accepted that the agonist-induced hydrolysis of PtdIns(4,5)P 2 to Ins(1,4,5)P3 mediates calcium mobilisation (see Refs. 24, 25 and 26 for reviews), and Ins(1,4,5)P3 is known to release calcium from a non-mitochondrial intracellular store in a variety of cell types including GH3 cells [27] and bovine anterior pituitary cell microsomes [9]. It is

105 likely, therefore, that the InsP3, which accumulates in response to TRH in bovine anterior pituitary cells can release intraceUular calcium to form the mobilisation phase of the TRH response. The data presented here show that the time-course of the change in cytoplasmic free calcium concentrations in bovine pituitary cells was dependent on the concentration of TRH added. Higher concentrations of TRH were required to cause the rapid initial transient calcium spike than the slower sustained calcium rise. Thus, increasing the concentration of TRH to 0.1 #M outside cells exposed to TRH at 1 nM caused a large transient calcium response, because 1 nM TRH is not high enough to cause an appreciable transient rise, but caused only a small further increase in the sustained calcium component over that caused by 1 nM TRH. Moreover, it appeared that the source of cytoplasmic calcium changed as the TRH concentration increased. The transient phase seen at high TRH appeared to depend on mobilisation of internal stored calcium, as in clonal pituitary G H 3 cells [2] and single rat lactotrophs [30], whereas the sustained phase seen at lower TRH appeared to be caused by increased entry of external calcium. Thus the transient phase was affected more than the sustained phase by nitrendipine or verapamil, which block entry of external calcium through the high voltage-activated, persistent, calcium channel [28,29]. Moreover, removal of extracellular calcium concentration by EGTA completely abolished the sustained phase but only partially reduced the initial transient phase of calcium elevation, probably reflecting some depletion of intracellular calcium stores. It is of interest to consider the implications of the difference in concentration dependence of these two modes of calcium elevation. Comparison of the dose-dependences showed that TRH caused the sustained rise in calcium at concentrations too low to produce significant increases in InsP3. This contrasts with GHa cells in which TRH stimulated Ins(1,4,5)P3 and calcium elevations are closely coupled [31]. Electrophysiological studies indicate that TRH activates calcium channels in bovine lactotrophs [12,32]. One conclusion might be that low concentrations of TRH can activate the nitrendipine-sensitive increase in calcium entry without causing Ins/a-dependent calcium mobilisation, which suggests that calcium entry may not be stimulated by changes in the metabolism of the inositol lipids. However, other explanations are possible which would be consistent with a single primary action of TRH on phospholipase C. It is possible that there is a calcium entry mechanism which is more sensitive to Ins(1,4,5)P3 than calcium mobilisation. Alternatively, progressive activation of protein kinase C by diacylglycerol as the concentration of TRH is increased might prevent the stimulatory effect of an inositol phosphate on calcium entry, resulting in a calcium change at high TRH concentrations which was predominantly due

to mobilisation. Recent data indicate that activation of protein kinase C does close voltage-gated calcium channels in anterior pituitary tumour cell lines [33,34]. A more trivial explanation might be that most of the InsP3 measured in these studies was inactive Ins(1,3,4)Pa, and that we could not detect the small change in Ins(1,4,5)P3 or a metabolite of it which stimulates calcium entry. Depolarising the cells by increasing external potassium caused a rapid increase in the cytoplasmic calcium concentration which was inhibited by the dihydropyridine calcium-channel antagonist, nitrendipine, with an ICs0 similar to that observed in GH 3 cells [35]. Thus the rise required calcium entry through voltage-gated channels, which have been demonstrated in identified bovine lactotrophs [15], and it was of interest to see how the activation of these channels affected the ability of TRH to enhance intracellular calcium concentrations. In contrast to GH 3 cells [36], potas.~ium depolarisation greatly potentiated the initial transient calcium response to high TRH concentrations in bovine anterior pituitary cells, which involves calcium mobilisation. It did not enhance the ability of low TRH concentrations to cause a sustained rise in calcium, an effect which relies mainly on entry of extracellular calcium. This suggested that potassium depolarisation increases calcium mobilisation rather than entry in response to TRH. Other agents, such as Bay K8644 and GHRP, which increase intracellular calcium concentrations although to a lesser extent than potassium, also potentiated the TRH-induced calcium elevation. The potentiation of calcium mobilisation was apparently due to an increase in the amount of calcium available for mobilis,mon from intracellular stores rather than increased availability of the mobilising messenger InsP 3. Potassium depolarisation had no effect on TRHinduced InsF s accumulation, which precludes the possibility that potentiation results from increased Ins/'3 production as observed in brain slices [37]. Moreover, the effect of ionomycin, which releases calcium from intracellular stores without generating Ins/'3 [38], was also potentiated by potassium depolarisation. It appears that the stimulation of calcium influx through channels activated by depolarisation increased the cytoplasmic calcium concentration and led to increased calcium uptake into intracellular stores which are used in the calcium mobilisation response to TRH. This is supported by the observation that nitrendipine inhibited the potassium-induced rise in calcium and the potassium-induced potentiation of the TRH response in parallel. Evidence obtained using normal anterior pituitary cells [11,15] and anterior pituitary turnout cell lines [10,11] indicates that the influx of calcium through the plasma membrane and the mobilisation of intracellular calcium are both required for the stimulation of prolactin secretion in response to TRH. In this study, we

106 have observed that stimulation of calcium influx through voltage-gated channels can enhance the transient phase of the calcium response to TRH. The ability of agents which promote calcium entry to increase the amount of calcium released by agonists which mobilise calcium would prime the cell to give a larger response to a given concentration of the second agonist. It would be interesting to discover whether the effect on the agonist-induced calcium rises is mirrored by an enhancement of the secretory response. Acknowledgements

The authors thank Dr. M.J. Wallis of the School of Biological Sciences, University of Sussex for the gift of the anti-bovine prolactin serum. This work was supported by a grant from the M.R.C. to J.G.S.C.A.W. was supported by an M.R.C. studentship. References 1 Schofield, J.G. (1983) FEBS Lett. 159, 79-82. 2 Gershengorn, M.C. and Thaw, C. (1985) Endocrinology 116, 591-596. 3 Martin, T.F.J. and Kowalchyk, J.A. (1984) Endocrinology 114, 1517-1526. 4 Rebecchi, M.J. and Gershengom, M.C. (1983) Biochem. J. 216, 287-294. 5 MacPhee, C.H. and Drummond, A.H. (1984) Mol. Pharmacol. 25, 193-200. 6 Tashjian, A.H., Heslop, J.P. and Berridge, M.J. (1987) Biochem. J. 243, 305-308. 7 Gershengorn, M.C. (1985) Recent Prog. Hormone Res. 41, 607-646. 8 Hollingshead, M., Thompson, M.G. and Schofield, J.G. (1985) Biochem. Soc. Trans. 13, 189-190. 9 Spat, A,, Lukas, G.L., Eberhardt, I., Kiesei, L. and Runnebaum, B. (1987) Biochem. J. 244, 493-496. 10 Tan, K.-N. and Tashjian, A.-H. (1984) J. Biol. Chem. 259, 427-431. 11 Enyeart, J. Sheu, S.-S. and Hinlde, P.M. (1.85) Am. J. Physiol. 248, C507-516. 12 Dubinsky, J.M. and Oxford, G.S. (1985) Proc. Natl. Acad. Sci. USA 82, 4282-4286. 13 Mason, W.T., Bicknell, K.J., Cobbett, P., Waring, D.W. and

Ingram, C.D. (1986) in Neuroendocrine Molecular Biology. (Fink, G., Harmar, A.J. and McKerns, K.W., eds.), pp. 376-398, Plenum Press, New York. 14 Schofield, J.G. and Bicknell, R.J. (1978) Mol. Cell. Endocrinol. 9, 255-268. 15 Ingram, C.D., Bicknell, R.J. and Mason, W.T. (1986) Endocrinology 119, 2508-2518. 16 Schofield, J.G., Kahn, A. and Wood, A. (1988) J. Endocrinol. 116, 393-401. 17 Berridge, M.J., Dawson, R.M.C., Downes, C.P., Heslop, J.R. and Irvine, R.F. (1983) Biochem. J. 212, 473-482. 18 Creba, J.A., Downes, C.P., Hawkins, P.T., Brewster, G., Michell, R.H, and Kirk, C.J. (1983) Biochem. J. 212, 733-747. 19 Downes, C.P. and Wusteman, M.M. (1983) Biochem. J. 216, 633-640. 20 Triggle, D.J. (1988) in Handbook of Experimental Pharmacology 83 (Baker, B.F., ed.), pp. 115-134, Springer Verlag, Berlin. 21 Dean, N.M. and Moyer, J.B. (1987) Biochem. J. 242, 361-366. 22 Burgess, G.M., McKinney, J.S., Irvine, R.F. and Putney, J.W. (1985) Biochem. J. 232, 237-243. 23 Hawkins, P.T. Stephens, L. and Downes, C.P. (1986) Biochem. J. 238, 507-516. 24 Berridge, M.J. (1984) Biochem. J. 220, 345-360. 25 Berridge, M.J. (1987) Annu. Rev. Biochem. 56, 159-193. 26 Downes, C.P. and Michell, R.H. (1985) In Molecular Mechanisms of Transmembrane Signalling. (Cohen, P. and Housley, M. eds.), pp. 3-56, Elsevier, Amsterdam. 27 Gershengorn, M.C., Geras, E., Purrello, V.S. and Rebecchi, M.J. (1984) J. Biol. Chem. 259, 10675-10681. 28 Titeler, M., DeSouza, E.B. and Kuhar, M.J. (1985) J. Neurochem. 44, 1955-1958. 29 Cohen, CJ. and McCarthy, R.T. (1985) Biophys. J. 47, 513a. 30 Malgaroli, A., Valler, L., Elahi, F.R., Pozzan, T., Spada, A. and Meldolesi, J. (1987) J. Biol. Chem. 262, 13920-13927. 31 Ramsdell, J.S. and Tashjian, A.H. (1986) J. Biol. Chem. 261, 5301-5306. 32 lngram, C.D. and Mason, W.T. (1985) J. Physiol. 364, 52P. 33 Marchetti, R. and Brown, A.M. (1988) Am. J. Physiol. 254, C206-C210. 34 Lewis, D.L. and Weight, F.F. (1988) Neuroendocrinology 47, 169-175. 35 Shangold, G.A., Kongasamut, S. and Miller, RJ. (1985) Life Sci. 36, 2209-2215. 36 Drummond, A.H. (1985) Nature 315, 752-754. 37 Baird, J.G. and Nahorski, S.R. (1986) Biochem. Biophyso Res. Commun. 141, 1130-1137. 38 Albert, P.R. and Tashjian, A.H. (1984) J. Biol. Chem. 269, 15350-15360.