Molecular and Cellular Endocrinology, 76 (1991) 45-53 0 1991 Elsevier Scieutific Publishers Ireland, Ltd. 0303-7207/91/$03.50

45

MOLCEL 02454

Cell specific distribution of guanine nucleotlde-binding in rat pituitary tumour cell lines

regulatory proteins

Eyvind J. Paulssen, Ruth H. Paulssen, Trine B. Haugen, Kaare M. Gautvik and Jan 0. Gordeladze Institute of Medical Biochtimistry, University of Oslo, Oslo. Norway (Received 26 July 1990; accepted 3 December 1990)

Key woruk G protein; Adenylyl cyclase; PhosphoIipase C; GH cell; Pituitary; (Rat)

To investigate the effects of guanine nucleotide-binding regulatory proteins (G proteins) on hormonal regulation of prolactin (PRL) synthesis and secretion, the qualitative distribution of G protein a-subunits and their mRNAs was studied in three functionally different pituitary tumour cell lines (GH cells) and normal rat pituitary tissue. Levels of basal and modulated adenylyl cyclase (AC) and phospholipase C (PLC) activities are also included. GH cells and pituitary tissue contained various amounts of mRNAs and protein for G,cr, Gi_zcu,Gi_3’Y and Gory, while mRNA for Gi_,Q was only detected in normal pitlritary tissue. G,a/G,a mRNA was expressed in all pituitary cell lines as well as in pituitary tissue. GJX mRNA and G&G,a mRNA displayed size heterogeneity. These findings may have importance in the understanding of hormone regulation of second messenger systems.

Introduction GH cells are rat pituitary tumour cells that spontaneously produce and secrete prolactin and growth hormone (Tashjian, 1979). GH& and GH3 cells contain receptors for the neuroendocrine peptide hormones thyroliberin (TRH), vasoactive intestinal peptide (VIP) and somatostatin (SRIF) (Hinkle and Tashjian, 1973; Gautvik and Lystad, 1981; Bjprro et al., 1984, 1987), whereas the GH,2C, cells may have lost their functional

Address for correspondence: Eyvind J. Paulssen, Institute of Medical Biochemistry, University of Oslo, P.O. Box 1112, BIindem, N-0317 Oslo 3, Norway.

receptors for TRH and SRIF (Gautvik et al., unpublished observations). Regulation of prolactin synthesis and secretion in pituitary cells by peptide hormones involves receptor interaction and coupling to intermediate guanine nucleotide-binding regulatory proteins (G proteins) that activate or inhibit second me,,enger systems (Gershengom, 1986; Gautvik et al., 1988). Eukaryotic effector systems known to be regulated through G proteins are adenylyl cyclase (AC), phospholipase C (PLC), phospholipase A 2 and receptor operated potassium and calcium ion channels. (For reviews, see Bimbaumer et al., 1987; Spiegel, 1987; Casey and Gilman, 1988; Neer and Clapham, 1988.) The G, protein is believed to be the sole stimulator of A< activity. G, a-subunit (Gsa) is now

46 known as two different protein products of molecular mass 52,000 (a52) and 45,000 (a45) (Northup et al., 1980) and four corresponding mRNAs that originate from the same gene have been described (Bray et al., 1986; Robishaw et al., 1986). Gs has later been shown to also activate ligand regulated calcium channels (Yatani et al., 1988). Gi was named for its inhibitory effect on adenylyl cyclase. G i a-subunit (Gia) is by now known as three different proteins (Gi_la, Gi.2ot and Gi.3a ) from separate genes (Jones and Reed, 1987; Goldsmith et al., 1988; Itoh et al., 1988). G o has two known a-subunits (Goa) distinct from those of Gs and Gi (Van Meurs et al., 1987; Hsu et al., 1990). Both Gt.3a and Goa are believed to activate potassium channels (VanDongen et al., 1988; Mattera et al., 1989), while Goa is involved in the modulation of voltage-dependent Ca2+-channels (Rosenthal et al., 1988). The recently cloned pertussis toxin-insensitive a-subunit of G i G x (Fong et al., 1988; Matsuoka et al., 1988) has yet no known function. The model for activation of GTP binding proteins has been reviewed by several groups (Bimbaumer et al., 1987; Spiegel, 1987; Allende, 1988; Casey and Gilman, 1988; Neer and Clapham, 1988; Weiss et al., 1988). Of the known modulators of PRL synthesis and release, VIP appears to bind to receptors that couple only to the G proteins that activate the AC system (Bjoro et al., 1984). In contrast, TRH and SRIF receptors are probably multifunctional, involving several entities or families of G proteins (Gautvik et al., 1983; Martin, 1983; Gershengom, 1985; Bjoro et al., 1988; Rosenthal et al., 1988). In light of the recently described mode by which G proteins are involved in the action of these hormones in GH cells (Gordeladze et al., 1989) and the occurrence of 'cross talk' between the phospholipase C and the adenylyl cyclase system (Gordeladze e t a l . , 1988; Lo, 1988), it is important to describe G protein occurrence and distribution on the mRNA and protein level in comparison to effector system modulation. The existence of Gsa, Gia and Goa proteins in GH 3 cells has been reported earlier (Rosenthal et al., 1988; Offermanns et al., 1990). In this study we therefore examined the distribution of G protein a-subunits and their corresponding mRNAs (Gsa , Goa , Gi.la, Gi.2a, Gi.3a

and Gza/Gxa) in three GH cell lines (GH4C1, GH3 and GH12C1) and also in normal female rat pituitary gland with Western and Northern blot techniques. The results of basal and hormone- and 5'-guanylyl imidophosphate- (Gpp(NH)p) modulated activity of adenylyl cyclase and phospholipaso C have been included. For comparison, the corresponding parameters have also been examined in liver tissue. Materials and methods

Cell culture GH cell lines (GH4C1, GH3 and GH12C1) (Tashjian, 1979) were grown in plastic tissue culture flasks (Costar) containing Ham's F-10 medium (Ham, 1963) (Gibco) supplemented with 6.5% horse serum and 3% (for GH3) or 5% (for GH12C1 or GH4C1) fetal calf serum (Gibco) at 37°C in a humidified atmosphere of 95% air and 5% CO 2 (Gautvik et al., 1983; Bjoro et al., 1984). Penicillin (50 IU/ml) and streptomycin (50 #g/ml) (G-ibco), and amphotericin B (2.5 ~g/ml) (Flow Laboratories) were added to the culture medium. Culture medium was changed every 2-3 days and always 24 h before harvest. The cells were used in the exponential phase of growth, RNA extraction Total cell RNA was extracted from female Sprague-Dawley rat pituitary and liver and GH cell lines by homogenization and centrifugation through CsC1 as described by Chirgwin et al. (1979). Cytoplasmic RNA was extracted from GH cell lines by lysis with Nonidet P-40 as described by Maniatis etal. (1982). Poly(A) selection of RNA was done using oligo(dT) cellulose type III (Collaborative Research), according to the manufacturer. Northern blot analysis RNA was separated on 1.4% agarose gels in 18% formaldehyde and 3-[N-morpholino]propanesulphonic acid (MOPS) buffer (Maniatis et al., 1982) and capillary blotted onto Hybond-N nylon filters (Amersham) or BioTrans nylon filters (ICN) in Tris-acetate-EDTA buffer (Maniatis et al., 1982).

47

Plasmids containing cDNA clones for five asubunits of G proteins in pGEM-2 were kindly supplied by Dr. Randall R. Reed (Jones and Reed, 1987). The eDNA clone for Gzot was kindly supplied by Dr. Henry K.W. Fong (Fong et al., 1988)' and eDNA for Gxa by Dr. Tohru Kozasa (Matsuoka et al., 1988). 32p labelled RNA probes were made from linearised plasmids using SP6, T7 or T3 polymerase (Pharmaeia Fine Chemicals and Stratagene) as described by Melton et al. (1984). 32p labelled eDNA probes were made from gel purified eDNA fragments containing unique sequences, from the above mentioned plasmids by the method of random priming (Maniatis et al., 1982). mRNA detection was carried out according to Maniatis et al. (1982), with slight modifications as suggested by ICN: prehybridization was done in 5 × Denhardt's solution, 5 × standard saline citrate solution (SSC), 50 mM sodium phosphate pH 6.5, 0.1% sodium dodecyl sulphate (SDS), 250 #g/nil herring sperm DNA and 50% (v/v) for. mamide at 42°C for 2 h. Hybridization took place in the same buffer including approx. 2 x 106 dpm labeUed DNA per ml, also at 42 °C overnight. When RNA probes were used, hybridization solution ~ontained 60% formamide and 1% SDS and hybridization was at 60°C overnight. Filters were washed 4 times in 2 x SSC, 0.1% SDS at room temperature and 2 times 15 rain each at 500C in 0.1 x SSC, 0.1% SDS and then exposed for various times to Hyperfilm MP (Amersham) or XAR-5 film (Kodak.) at - 7 0 ° C with the use of Cronex Lightening Plus intensifying screens (DuPont) before development. Prior to reprobing, probe was removed from the filter at 70°C for 1 h in 70% formamide, 10 mM sodium phosphate pH 6.5.

Preparation of subcellular particulate fractions The medium was removed and the cells scraped in ice-cold 150 mM NaCI, 10 mM Tris-HCl, pH 7.5 and pelleted (700 x g, 10 min, 4°C) (Gautvik et al., 1983). The call pellet was washed once with the same buffer, resuspended in 20 volumes of 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, and eventually homogenized on ice using an Ultra-Turrax (Janke Kunkel, F.R.G.) for 10 s. The homogenate

was subsequently filtered through nylon mesh and centrifuged at 27,000 × g for 30 min at 4°C. Finally, the pellet was resuspended in 10 volumes of Tris-EDTA buffer containing 0.1% bovine serum albumin, using the rotating knife for 5 s. This constituted the crude membrane fraction and contained about 5 mg membrane protein per ml. When membranes were prepared from whole tissue, specimens were minced, washed with ice-cold saline and otherwise processed as described above.

Western blotting and immunostaining SDS-polyacrylamide gel electrophoresis (PAGE) was performed as described (Laemmli, 1970). Cell membrane samples were prepared for electrophoresis by suspending in sample buffer (Laemmli, 1970) and heating to 100°C for 5 min. Proteins were electrophoretically transferred (Towbin et al., 1979) from gel to PVDF lmmobilon-P filters (Millipore) with constant current (100 mA) for ~,bout 12 h in a Bio-Rad Transblot apparatus. After transfer, the filter was incubated 2 h with phosphate-buffered saline (PBS) including 5% fat-free dry milk powder, and subsequently incubated for 6-24 h at room temperature in PBS containing 1% dry milk powder and anti-G protein antisera. After being washed with PBS containing 0.1% Tween 20, the filter was incubated with 12sl-protein A (150,000 cpm/ml, Amersham) in PBS containing 1% dry milk powder for 1 h at room temperature. The filter was then washed extensively with PBS containing 0.1% Tween 20 and dried prior to autoradiography.

G protein a-subunit antibodies Rabbit antisera against synthetic peptides corresponding ~o the predicted C-terminal amino acid sequence of different G protein a-subunits were a generous gift from Dr. Allen M. Spiegel (Simonds et al., 1989). Antisera were used at a final dilution of 1/200-1/400.

A denylyl cyclase assay Adenylyl cyclase activity was measured in 20 ~tl aliquots of crude subcellular fractions which were diluted in homogenization buffer to obtain 40-55 btg of protein per assay tube (Gautvik et al., 1983). The total incubation volume was 50/tl and contained 1 mM ATP (including 1.6 × 10 6 cpm of

48

[a-32p]ATP (Amersham), 10 ~M GTP, 2.8 mM MgCI2, 1.4 mM EDTA, 1 mM cAMP (containing approx. 7 × 103 cpm of [8-3H]cAMP (Amersham)), 20 mM creatine phosphate, 0.2 mg/ml creatine kinase, 0.02 mg/ml myokinase and 25 mM TrisHC1 pH 7.4 in the absence or presence of TRI-I (1 /tM), VIP (1/~M), SRIF (1/tM) or Gpp(NH)p (20 /tM). Incubations were carried out at 35 °C for 20 rain. Reactions were stopped with 0.1 ml of a solution comprising 10 mM cAMP, 40 mM ATP and 1% SDS. The [32p]cAMPformed and the [3H]cAMP added to monitor recovery (65-80%) were isolated as described previously using combined Dowex and aluminium oxide chromatography (Salomon et al., 1974). The enzyme activity was linear with time up to 60 rain and prote=~ concentration up to 150 /tg protein (data not shown).

2% sodium cholate were added to each tube, and incubation carried out at 35°C for 5 rain. The reaction was stopped by adding in succession 150 /~1 CHCI3/CH3OH / HCI (1 : 2 : 0.02), 50 /~1 CHC13 and 50/~1 2 M KCI. After vortexing and phase separation at 5000 x g in a microfuge, 100 ~1 aliquots of the aqueous layers were counted in a liquid scintillation counter. In the controls, approx. 2% of the radioactivity was retained in the aqueous phase. The enzyme activity was linear with time up to 20 min and protein concentration up to 100/~g protein (data not shown). Results

Distribution of G protein mRNAs in different pituitary cell lines Poly(A) selected RNA was made from the different G H cell lines and normal rat pituitary and liver tissues. 1 /tg of this RNA (0.5 /tg of liver RNA) was separated on denaturing agarose gels and transferred to nylon filters. The filters were hybridized with 3ep-labelled cDNA and RNA probes for G protein a-subunits. The results are shown in Fig. 1. G,a mRNA is apparently the most abundant G protein a-subunit mRNA present in pituitary tissues as judged by the shorter exposure times (data not shown). It is present i~t all the cell types as a

Phospholipase C assay This method was adapted from the one previously published by Jackowski et al. (1986). Aliquots (20/~1) of dilute3 :,,ucle membrane suspensions (40-5~/~g protein) in 10 mM Tris-HC1 pH 7.4, 1 w,'d EDTA, were mixed with 10/~1 incubatioe mixture (100 mM Tris-HCl pH 6.5, 400/~M GTP, 2.2 mM CaCI2, 1 mM MgCI2 and 10/~1 of TRH (10 ~tM) or Opp(NH)p (400/~M) in 2.4 ml microfuge tubes on ice. 3 /~1 (42,000 cpm) of a [3H]PIP2 (New England Nuclear) stoc',: solution in

2345

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i 2345

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t

i !i

N

•

I' I ~

f.8S

i Ill

N

.

Gs~

Gi_ i~

o

,

Gi_2~

Gi_3a

Goa

Gx~

Fig. 1. Cellulardistribution of G-protein wsubunit mRNAs. 1 ~g each of poly(A)selected RNA from femalerat pituitary(lane 1), GH4C1 cells (lane 2), GH3 cells (lane 3) and GH!2C1 ceils (lane 4), and approximately0.5/tg liver poly(A)+ RNA (lane 5) was separated on denaturingagarose gels and transferred to nylon filter and hybridizedto labelledprobes for G a-subunitsas described in Materialsand Methods.The probes used are indicated under their respectivepanels. The migrationof 28S RNA (4.5 kb) and 18S RNA (1.8 kb) is indicated.The shownmRNAdistributionwas confhmedby repeated experiments.

49

single dominant band with molecular size of 1.9 kilobases (kb). Gi.~a mRNA is detected only in normal pituitary as a single band of approximately 3.5 kb, and not in any pituitary tumour cells or fiver. Cross-hybridization of this probe with Gi.~a mRNA could explain the signals of lower molecular size that occur in GH~ and GH~2C~ mRNA. Gi.ia mRNA (2.4 kb) is found in all pituitary derived cells and is also detected in liver RNA. Gi.~a mRNA (3.5 kb) is present in all the studied cell types in approximately equal amounts. Goa mRNA shows size heterogeneity, revealing five distinct mRNAs with approximate molecular sizes of 6.2, 3.8, 3.3, 3.0 and 1.6 kb with an uneven relative distribution between pituitary derived cells. GH~2C~ cells and normal pituitary gland contain all these mRNA species, whereas in GH 3 and GH~C~ cells the smaller molecular weight mRNA species appear less abundant. Goa mRNA is not detected in rat fiver. G~a mRNA appeared in GH cells and pituitary tissue as three major bands representing mRNAs of approximate molecular sizes 4.0, 3.3 and 2.4 kb. There were major differences in the relative distribution of the 4.0 and 3.3 kb mRNAs. Levels of the 2.4 kb species, that was the most abundant in pituitary tissue, GH3 and GH~2C~ cells, were more constant.

Western blot analysis of G protein a-subunits in membrane fractions The relative distribution of G proteins in crude cell membranes from GH cells and rat pituitary and fiver tissue was measured with specific antisera against synthetic peptides (Simonds et al., 1989) as described in Materials and Methods. Fig. 2 shows the result of such experiments. Gsa protein (approximate MW 46 (upper arrow) and 42 kDa (lower arrow)) was found with various quantities in GH cell types; there appeared to be more Gsa protein in GH 3 and GH4C~ calls than in GH12C1 cells. Somewhat less was found in pituitary tissue. Little or none was detected in rat fiver membranes. Gi.~a/Gi.2 a protein (approx. MW 40 kDa) levels were high in rat pituitary tissue and generally lower in the other cell types. Gi_3a protein (approx. MW 42 kDa) was detected in GH cells

GHI GH3 GH4 Pit Liv

Gse

BID Q

Goa

q ~ ! ~ ~ • ~"~,.' . Gi-I/i-ea

i

Fig. 2. ImmunoLlot analysis of G protein a-subunit distributiori. Crude membrane fractions (20 ~tg per lane) of GH!2C~, GH 3 and GH4C 1 cells, pituitary (Pit) and liver (Liv) were separated on 10~ SDS-PAGE and transferred to PVDF membranes as described in Materials and Methods. The occurrence of G proteins was detected with antibodies against C-terminal sequences of Gsa (approx. MW 46 kDa (upper arrow) and 42 kDa (lower arrow)), Gi.la/Gi.2a (40 kDa), Gi.3a (41 kDa) and Goa (40 kDa) prior to incubation with 125I-protein A and autoradiography. The distribution pattern was confirmed in repeated experiments.

and liver, but tittle or none was found in pituitary cell membranes. Goa protein (approx. MW 40 kDa) was most abundant in pituitary and GH12C1 cells and less in other GH cells.

Measurements of basal and modulated activity of adenylyl cyclase and phospholipase C Activities of adenylyl cyclase (AC) and phospholipase C (PLC) were measured in crude membrane fractions as described in Materials and

50 TABLE 1 BASAL, HORMONE- AND Gpp(NH)p-MODULATED ADENYLYL CYCLASE (AC) ACTIVITY IN CRUDE MEMBRANE FRACTIONS FROM GHi2Cl, GH3 AND GH4C 1 CELLS, FEMALE RAT PITUITARY AND RAT LIVER AC activity was measured in the presence of 1 mM ATP, 20 pM GTP, 0.4 mM free Mg 2+ and 100 ~tM Ca 2+. Enzyme activities are expressed as pmoles cAMP per mg protein per rain and given as means :t: SD of triplicate determinations. Modulations of the AC activity were performed (for 20 rain at 35°C) in the presence of either 1 /~M thyroliberin (TRH), 1 pM vasoactive intestinal polypeptide (VIP), 10/tM somatostatin (SRIF) or 20 pM of the non-hydrolyzable GTP analogue Gpp(NH)p. The assay was repeated twice with similar results. Tissues/cells

Pituitary GH12C1 GH 3 GH4C! Liver

Adenylyl cyclase activity (pmoles cAMP/mg membrane protein/rain) Basal

TRH

VIP

SRIF

Gpp(NH)p

78.5 + 6.3 * 4.8+0.2 5.2+0.3 5.1+0.2 18.8+0.6 *

166 + 12 ** 4.9+ 0.1 8.0+ 1.1 ** 9.8+ 0.3 ** 126 + 7 2.**

187 ± 13 ** 19.3+ 0.6 ** 18.6± 1.1 ** 28.4± 0.9 ** 115 ± 8 **

56 ± 3.3 ** 5.1±0.3 4.0+0.1 ** 3.4+0.1 ** 18.6±0.6 **

183 ± 15 * * 9.6± 0.8 ** 18.3± 0.5 ** 30.6+ 1.2 ** 41.5± 2.7 **

= Glucagon (1/tM). * p = a < 0.05, Wilcoxon rank test (basal tissue/cells vs. basal GH 3 cell AC). * * p = a < 0.05, Wilcoxon rank test (modulated vs. basal AC).

Methods. Both basal and hormone- or Gpp(NH)p-modulated enzyme activities were registered. Tables 1 and 2 show the results of these experiments. Basal AC activity was significantly higher in the pituitary than in liver (4-fold) and than in GH cells (15-fold) (Table 1). TRH gave a 2-fold increase in AC activity in GH4C! cells, an enhancement comparable to that in the normal pituitary gland. TRH had less effect on AC in GH3 cells and no effect in GH12C~ cells. VIP showed a stronger stimulation of AC activity in the GH cell lines ~,'. to 6-fold) than in the normal pituitary (about 2.5-fold). SRIF inhibited AC activity in GH 3, GH4C 1 and the pituitary gland by about 20-30~, but did not affect the GH12C~ AC activity. A~enylyl cyclase was highly susceptible to glucagon and VIP in liver tissue, the maxima being about that in pituitary tissue. The effect of Gpp(NH)p on adenylyl cyclase was less than the effect exerted by VIP in GH12C~ cells and fiver. In the other tissues examined, the effect of this non-hydrolyzable GTP analogue approximated that of VIP. Basal phosphofipase C (PLC) activity varied within the studied cell types and tissues (Table 2) but exhibited a different pattern from that of the basal AC activity. The pituitary contained a relatively high level of basal PLC activity, GH~2C~,

GH 3 and GH4C 1 cells showed an intermediate level and there was tittle or no basal PLC activity in fiver cells. Gpp(NH)p activates PLC more than does TRH in pituitary cells. The relative increase

TABLE 2 BASAL, TRH- AND Gpp(NH)p-STIMULATED PHOSPHOLIPASE C (PLC) ACTIVITY IN CRUDE MEMBRANE FRACTIONS FROM RAT PITUITARY, PITUITARY CELL LINES OR LIVER PI.C activity was estimated in the presence of 42,000 cpm of [3H]PiP2 (phosphatidyl inositol bisphosphate), 20 /tM GTP and 100/tM Ca 2+. Enzyme activities are expressed as 103 cpm of IP3 (inositol trisphosphate) formed per mg protein per rain and tabulated as m e a n ± S D of tripficate determinations. Stimulations of PLC activity were achieved (for 5 min at 35°C) in the presence of either 1/tM TRH or 20 ttM Gpp(NH)p. The assay was repeated twice with similar results Tissues/ cells

Phospholipase C activity (103 cpm IP3/mg membrane protein/min) Basal

Pituitary GH12C1 GH 3 GH4C 1 Liver

35.6±1.7 12.6±0.4 9.3 ± 0.5 6.1±0.2 1.3±0.1

* * * *

TRH

Gpp(NH)p

78.3±4.2 ** 13.9±0.2 35.3 ± 1.8 * * 17.7+1.1 ** 1.3±0.0

295 ±13 ** 79.4± 3.1 ** 72.5 ± 2.7 * * 40.9± 1.8 ** 1.6± 0.1

a < 0.05, Wilcoxon rank test (basal tissue/cells vs. basal GH 3 cell AC). ** p ffi a < 0.05, Wilcoxon rank test (modulated vs. basal AC). * p-

51 in PLC activity upon stimulation was close to equal for these cell types. Discussion

GH cells have for years been used as a model system to study aspects of hormone-induced alterations in the synthesis and secretion of prolactin (PRL) and growth hormone (GH). Many of the known hormone receptors that interact with G proteins have been found in these cells. The GH12C1, G H 3 and GH4C1 cells differ in respect to production and re!ease of PRL and GH. Thus a comparison of functional properties of these three cell fines could contribute to the understanding of interactions between hormone receptors, G proteins and cellular effector systems. In this study, we have shown that both GH cells and normal rat pituitary contain mRNAs for G protein subunits Gsa, Gi.2 a, Gi.3a, Goa and Gxa and also that they contain various quantities of the corresponding a-subunit proteins. Gsa is predominantly present in its 46 kDa form. It depicts the most dominant G protein a-subunit mRNA in the cells studied, but not the dominating a-subunit protein. The differences in the quantities of G~a protein are more pronounced (Fig. 2) than those of Gsa mRNA (Fig. 1), GH 3 and GH4C1 cells disF!aying more Gsa protein than GH12C1 cells and pituitary. There is a low abundance of Gsa protein in fiver in accordance with the mRNA data. Pituitary tissue cells, but not GH cells, express Giqa mRNA. As the pituitary gland contain more cell types than just somatotrophs and mammotrophs, it is premature to conclude whether the difference in expression of tiffs a-subunit is due to cell type differences or represents a major difference between stable and growing pituitary cells. Ongoing in situ hybridization experiments may enlighten the reasons for this difference in mRNA expression. Gi.la/Gi.2ot antiserum revealed protein levels that reflect the sum of the distribution of the corresponding mRNAs in all studied cell types and tissues. Finally, the amount of G i.3a protein mirrors the distribution of the corresponding mRNA with the major exception that pituitary membranes contain little or no Gi.30l protein.

Goa mRNAs display size heterogeneity. This study confirms previous observations (Casey et al., 1988; Hsu et al., 1990) but adds to the number of different Goa mRNAs. Cross-hybridization with other known a-subunit mRNAs is unlikely, as the pattern of distribution (for Gi.2a and Gi.3a) or the molecular size (for Gsa ) differ. Goa proteins migrate as one band on SDS-PAGE, which is according to previous reports (Jones and Reed, 1987) and also does not conflict with a recent report describing two Goa proteins of identical size (Hsu et al., 1990). The distribution of the different Goa mRNA species differs between GH cells and is in accordance with the Go a-subunit protein distribution pattern. The Gs a-subunit is suggested to belong to a family of gone products from a single gone (Bray et al., 1986; Robishaw et ~., 1986). Our results may indicate that this is indeed the case for Goa as well. Goa mRNA is not observed in liver tissue in this study. Gxa mRNA is detected in normal pituitary, GH cells and liver as multiple mRNAs, in discordance with earlier studies that only show the presence of these mRNAs in brain, spleen and adrenal tissues (Fong et al., 1988; Matsuoka et al., 1988). Furthermore, we report size heterogeneity of G~a mRNA. Gxa antibodies were net available to us during the cause of this study. Fig. 2 also shows an inverse proportion of Gs to Gi/Go: cells rich in Gs (GH3 and GH42C1) are generally low in their content of G o and Gi.l/Gi. 2. Basal adenylyl cyclase (AC) activity measured in pituitary and liver cells differs significantly from that in GH cells (Table 1). This difference may reside in the tumour nature and increased growth rate of the GH cells. There is, however, a 4-fold higher basal AC activity in pituitary tissue compared with that in fiver. The levels of basal AC activity do not correlate to the relative levels of any single a-subunit protein in the cell fines and tissues studied (Fig. 2). This could implicate that the level of G s, which is believed to be the sole stimulator of adenylyl cyclase, is not alone important for basal AC activity. This would also be in accordance with previous findings (Cerione et al., 1985). The mentioned inverse relationship between G~ and G i / G o is also not reflected in basal AC activity, indicating that this ratio is not of major

52

importance for the non-stimulated activity of this effector. VIP is believed only to act through Gs and thereby stimulating adenylyl cyclase (Bjoro et al., 1988). TRH, which mainly activates phospholipase C (Martin, 1983; Gershengorn, 1985), is believed also to act upon AC through Gs (Gordeladze et al., 1988). AC activity is also under inhibitory control via one or more Gi protein (Boyd et al., 1988). Maximum activation of G proteins by non-hydrolyzable analogues, such as Gpp(NH)p in these experiments, could reflect the net effect of regulatory signals on this enzyme system. We find that VIP activates AC just as well as Gpp(NH)p in pituitary, GH 3 and GH4C1 cells and better than Gpp(NH)p in GH12C1 cells and liver (Table 1). There is no obvious correlation between the stimulatory effects of VIP or Gpp(NH)p on adenylyl cyclase and the Gs a-subunit distribution or the ratio of Gs to Gi. Somatostatin (SRIF) has no detectable inhibitory effect on AC activity in GH12C~ cells, that may lack a functional SRIF receptor (Gautvik et al., unpublished observations), nor in liver. In the other cell types, however, it displays measurable effects. Basal phospholipase C (PLC) activity shows a different pattern from that of AC (Table 2). The pituitary gland also here exhibits the highest enzyme levels, approximately 3-fold higher than in GH12C 1 cells. It is interesting to note that basal PLC levels in all cell types and tissues correlate to relative protein levels of Goa. The absence of the Gt_ ~a species from GH cells is also noteworthy in this context. Activation of phospholipase C by TRH is less than the maximum attainable enhancement measured in the presence of Gpp(NH)p. TRH has been postulated to stimulate IP3 formation through a PTX-insensitive G protein (Aub et al., 1986), implicating the existence of a G~a/Gza-like mediator in GH cells. G~a/Gza mRNA, being previously detected in brain (Fong et al., 1988; Matsuoka et al., 1988), is also present in normal pituitary or the GH cell lines, indicating the presence of the corresponding protein. These observations do not exclude the possibility that G J G ~ mediates TRH stimulation of PLC in GH cells.

GH cells have developed into a useful tool to study receptor regulation of second messenger systems. This study shows the distribution of G prorein subunit mRNAs and polypeptide levels in these cells. Our results indicate that there are differences between three GH cell lines regarding the expression of G proteins. This knowledge is of importance for the understanding of functional systems involved in hormone synthesis and secretion in GH cells and the rat pituitary gland, and should be a relevant basis for further studies that may involve hormone regulation or specific inhibition of G protein expression.

Acknowledgements We thank Dr. Randall R. Reed for supplying the eDNA clones for the G protein Gsa, Goa and three Gi a-subunits, Dr. Henry K.W. Fong for the Gza clone and Dr. Tohru Kozasa for the Gxa clone. The authors are also grateful to Dr. Allen M. Spiegel for supplying the G protein antisera. E.J.P. and R.H.P. are recipients of grants from the Norwegian Society against Cancer, T.B.H. is supported by the Norwegian Research Council for Science and the Humanities. This work received economical support from the Anders Jahre Foundation for the Promotion of Sciences, Oslo, and Insulinfondet, Copenhagen, Denmark. The authors are grateful for the technical assistance from Ms. Aase Karine Fjeldheim, Ms. Hanne Thilesen and Ms. Annette Belset.

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