Br. J. Pharmacol. (1990), 101, 615-620
IV"I'l Macmillan Press Ltd, 1990
Pharmacological and biochemical comparison of thyrotropin releasing hormone (TRH) and di-methyl proline-TRH on pituitary GH3 cells Alison M. McDermott, Graham P. Wilkin & *lStephen L. Dickinson Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AZ and *Reckitt and Colman Psychopharmacology Unit, The School of Medical Sciences, Bristol, BS8 1TD 1 The binding of [3H]-thyrotropin releasing hormone ([3H]-TRH) and [3H]-RX77368 (di-methyl proline TRH) and the ability of these peptides to stimulate phosphoinositide hydrolysis were investigated in the GH3 pituitary cell line. 2 For both peptides binding was found to be saturable with a single component (Hill slopes were, for TRH, 0.98 and for RX77368, 1.13). TRH bound with greater affinity than RX77368 Kd values were 16nM and 144 nm respectively. B.. values were 227 fmol mg-' protein for TRH and 123 fmol mg1 protein for RX77368. 3 The rank order of potency of a series of TRH analogues to inhibit binding was the same versus each peptide. However, unlike with saturation analysis, Hill slopes of all displacing ligands were less than 1.0 against both TRH and RX77368 suggeting either multiple binding sites, alteration of affinity state, negative co-operativity or some allosteric interaction. 4 Both peptides stimulated phosphoinositide hydrolysis in a dose-dependent fashion. TRH was more potent than RX77368, ECjo values were 7.9 + 1 nm and 96.3 + 3 nM respectively. 5 These in vitro data suggest that the greater in vivo potency of RX77368 is not the result of enhanced receptor affinity but is more probably due to its greater metabolic stability.
Introduction The tripeptide thyrotropin releasing hormone, TRH, (Figure 1) is widely distributed throughout the central nervous system. It exhibits a broad spectrum of stimulatory effects (Griffiths, 1987) which are probably mediated via receptors very similar to those moderating the endocrine function of the peptide in the pituitary gland (Burt & Taylor, 1980; Dettmar et al., 1983a,b; Sharif et al., 1983). TRH has been found capable of preventing neuronal damage (Freedman et al., 1986; 1989), promoting recovery following spinal trauma (Faden et al., 1981) and relieving weakness and spasticity in motor neurone disease (MND) (Engel et al., 1983). However, its clinical potential in these areas has not been realised, possibly a consequence of the peptide's short biological half life due to its rapid metabolism by pyroglutamyl aminopeptidases and deamidase enzymes in body tissues and fluids (Bassiri & Utiger, 1981; Coggins et al., 1987). RX77368 is one of several analogues synthesized in an attempt to overcome this lability. Here the substitution of two methyl groups at position 3 on the proline ring (Figure 1) renders the molecule much more resistant to enzymatic degradation (Brewster et al., 1981; Griffiths et al., 1982; Metcalf, 1983). RX77368, which has also been found beneficial for the treatment of MND (Guiloff et al., 1987a,b), is endocrinologically equipotent to TRH but shows greater biological activity in several neuropharmacological tests (Dettmar et al., 1980; 1981; 1983a; Metcalf et al., 1981; Sharp et al., 1984ab). The latter has been attributed to the greater metabolic stability hence increased bioavailability of the analogue; however, other factors, for example active metabolites or enhanced potency at the receptor, may play a role. While there are no known active metabolites of RX77368, indeed much of a dose is excreted unchanged (Brewster et al., 1981), there has to date been no data published concerning the direct binding characteristics of the drug, although Hawkins et al. (1986) and more recently Sharif et al. (1989) have shown indirectly that RX77368 has a lower Author for correspondence.
affinity than TRH for mammalian spinal cord and brain receptors. Here we have had the unique opportunity of investigating the binding characteristics of tritiated RX77368 and comparing them to those of the parent compound in whole GH3 pituitary cells, a clonal cell line having receptors for TRH that are coupled to phosphoinositide (PI) hydrolysis and prolactin release (Tashjian et al., 1971; Drummond & Macphee, 1981; Sutton & Martin, 1982). We have also studied the ability of the peptides to stimulate PI hydrolysis in this cell line. A preliminary account of this work has already been published (McDermott et al., 1989). R2
R1 TRH
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as TRH
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I
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NH
MK771
CONH2 Figure 1 The structure of thyrotropin some of its analogues.
releasing hormone (TRH) and
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Methods Cell culture GH3 cells were routinely grown in monolayer culture in 10cm dishes at 370C, under 95% air/5% CO2 in Ham's F10 culture medium supplemented with 15% donor horse serum, 2.5% foetal calf serum and 40 pgmlP' gentamicin. Cells were cultured to about 80% confluency and passaged using 0.05% trypsin in Tris/EDTA. For binding and PI hydrolysis studies, cells were seeded at a concentration of 0I ml on to 13 mm diameter glass coverslips coated with 0.5 pg ml poly-L-lysine in multiwell plates. The culture medium was changed every 3 days and the cells allowed to grow for 9-11 days before use.
from Sigma. [1,2-3H(N)]myo-inositol, [3H]-inositol, (458OCi mmol- 1) and [L-proline-2,3,4,5,3H(N)]-pGlu-His-ProNH2, [3H]-TRH, (119.2 Ci mmol 1) were purchased from New England Nuclear. GH3 cells were a gift from Dr A. Drummond. pGlu-[2,5-3H2] His-3,3'dimethyl Pro-NH2, [3H]-RX77368, (32.7CimmolP , purified and characterized by P.M. Taylor), RX77368 and RX74355 were provided by Reckitt and Colman. MK771 was provided by Merck Sharp and Dohme. CG3509 and CG3703 were from Chemie Grunenthal. All other reagents and solvents were of analytical grade.
Results
Receptor binding studies
Receptor binding studies
The growth medium was aspirated and the cells washed twice with 0.5 ml of Ham's F10 medium, supplemented with 25mM HEPES, 5 mM magnesium sulphate, 14 mm sodium bicarbonate and 400ug mlP- bovine serum albumin, pH 7.4. The cells were then incubated for up to 1 h at 370C in a rotary shaker, in 0.5 ml of medium also containing either [3H]-TRH or [3H]-RX77368. Non-specific binding was determined by the addition of 100paM TRH to the medium. The incubation was terminated by aspirating the medium and the cells were then washed 3 times with 1 ml of ice cold medium. The coverslips were then transferred to vials for scintillation counting.
Incubation of [3H]-TRH or [3H]-RX77368 with whole GH3 cells resulted in a time-dependent increase in specific binding at 37°C (Figure 2). [3H]-TRH bound more rapidly than [3H]RX77368. Maximal binding of both peptides was achieved by 60min. The data obtained from experiments in which cells were incubated with various concentrations of [3H]-TRH or [3H]-RX77368 for 1 h, revealed that under these conditions binding of both peptides was saturable (Figure 3a,b). Nonspecific binding was less than 35% for [3H]-TRH and 50% for [3H]-RX77368. Hanes analysis of the data indicated a single binding component for each peptide (Figure 4a,b). Kd and BX,, values were, for TRH 16 nM and 227 fmol mg-1 protein, and for RX77368 144nm and 123fmolmg-t protein. Hill coefficients were 0.98 and 1.13 for TRH and RX77368
Tritiated peptide stability studies On the manufacturer's recommendation, paper chromatography followed by liquid scintillation counting was used to assess the stability of the tritiated ligands. Aliquots of medium containing radioligand that had been incubating with cells (with or without the presence of 40gnml- 1 bacitracin) for 1 h at either 37°C, ambient temperature or 4°C were compared to radioligand incubated without cells.
Phosphoinositide hydrolysis studies Cells were pre-labelled for 2-3 days with 1 uCi mlP-I [3H]-inositol. Under these conditions labelling of the inositol lipids has reached isotopic equilibrium (Drummond et al., 1984; confirmed in our laboratory, data not shown). The growth medium was aspirated and the cells washed twice with 0.5 ml of medium, as described for binding studies but with the addition of 10 mm lithium chloride and a mixture of peptidase inhibitors routinely used in our laboratory (leupeptin 2g ml- 1, chymostatin 1 pg ml-' and bacitracin 40g ml- 1), then pre-incubated in 0.5 ml of this medium for 30 min at 37°C on a rotary shaker. Peptides were added in a volume of 10#p to give the required final concentration, and the incubation continued for a further 15min. The reaction was terminated by transferrence of the coverslips to vials containing 1.5ml chloroform/methanol 2:1 (v/v) then adding 0.5ml water. The mixture was vortexed then left to separate; 0.5 ml of the upper aqueous phase was removed for the determination of total inositol phosphates as described by Cholewinski et al. (1988) following the method of Berridge et al. (1982). An aliquot (200,ul) of the lower phase was removed, dried and counted for the estimation of [3H]-inositol incorporation into phospholipids.
Protein estimation Cellular protein content was determined by the method of Lowry et al. (1951).
Materials Culture media and sera were from Imperial Laboratories. Antibiotics and tissue culture plasticware were from Flow Laboratories. All other tissue culture reagents and TRH were
respectively. The ability of a number of TRH analogues (Figure 1) to inhibit binding was determined by incubating GH3 cells with either [3H]-TRH or [3H]-RX77368 in the presence of various concentrations of analogue (Figure 5). Table 1 shows the concentrations of ligand required to displace 50% of the specific binding. The orders of potency of the analogues to inhibit binding were as follows: versus [3H]-TRH: RX74355 > TRH > = MK771 > = CG3703 > RX77368 > CG3509; versus [3H]-RX77368: RX74355 > = TRH > MK771 > = CG3703 > RX77368 > CG3509. Slopes calculated from Hill plots of the competition curves yielded values of less than 1 for all competing ligands and for both peptides (Table 1). These were confirmed by non-linear regression analysis (GraphPAD INPLOT). 25000 r 20000 c
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Figure 2 Time course of specific binding of [3H]-thyrotropin releasing hormone ([3H]-TRH, [J) and [3H]-dimethyl proline TRH ([3H]RX77368, 0) to GH3 cells. The cells were incubated at 37°C with either [3H]-TRH (5 nM) or [3H]-RX77368 (5OnM) for various times. Maximal binding of both peptides was achieved by 60min. Each value represents the mean of quadruplicate samples from a single experiment; vertical bars show s.e.mean. The discrepancy between actual d.p.m. values reflects the different specific activities of the two radioligands.
TRH AND RX77368 ON GH3 CELLS a
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Concentration (nM) Figure 3 Saturation isotherms for (a) [3H]-thyrotropin releasing hormone ([3H]-TRH) and (b) [3H]-dimethyl proline TRH ([3H]RX77368). Cells were incubated at 370C for 1 h with various concentrations of radioligand alone (total binding 0) or in the presence of 100luM cold TRH (non-specific binding A). Specific binding (El) was determined by subtracting non-specific from total binding. Each value represents the mean of quadruplicate samples from a single experiment; vertical bars show s.e.mean.
Free (nM)
Figure 4 Hanes analysis of (a) [3H]-thyrotropin releasing hormone and (b) ([3H]-dimethyl proline TRH ([3H]-RX77368) binding data. Binding was saturable with a single component (correlation coefficients for TRH and RX77368 were 0.99 and 0.88 respectively). Each value represents the mean of quadruplicate samples from a single experiment. Kd and Bn.. values for TRH were 16nM and 227ffmolmg-' protein (one experiment) and for RX77368 144 nm and 123 fmol mg-1 protein (mean of two experiments).
([3H]-TRH)
Peptide stability studies No significant differences in the proportion of counts occurring in a position equivalent to that of the cold ligand, were observed between radioligand that had been incubated with or without cells indicating that the peptides remained stable throughout the incubation conditions. Changes of temperature or presence of the peptidase inhibitor bacitracin had no effect.
Phosphoinositide hydrolysis studies Both peptides stimulated inositol phosphate metabolism in GH3 cells. The accumulation of [3H]-phosphates rose in a linear fashion for at least 15 min (Figure 6) by which point a maximum of 16% of the [3H]-inositol-containing phospholipids had been depleted. The response to stimulation by TRH Table 1 Inhibition of [3H]-thyrotropin releasing hormone
([3H]-TRH) and [3H]-dimethyl proline TRH ([3H]RX77368) binding to GH3 cells by various TRH analogues
[3H]-RX77368
[3H]-TRH RX74355 TRH MK771 CG3703 RX77368 CG3509
IC50
Hill slope
ICS0
Hill slope
2.0 ± 0.3 23 + 4 30+4 44 ± 5 295 ± 42 1061 + 218
0.62 0.55 0.71 0.60 0.49 0.42
58 ± 21 80± 16 241 ± 41 316 ±0 2009 ± 230 2990 + 172
0.55 0.40 0.42 0.47 0.37 0.31
Mean + s.e.mean from 1 to 3 experiments in quadruplicates. Concentrations are expressed as nM.
and RX77368 was dose-dependent (Figure 7). TRH was more potent than RX77368, ECjo values were 7.9 + 1 nm (mean + s.e.mean) and 96.3 + 3 nm respectively.
Discussion Saturation analysis revealed that TRH and its dimethyl analogue RX77368 each bind to a single site on GH3 pituitary cells. The dissociation constant obtained for TRH was very similar to that previously reported by Hinkle & Tashjian (1973), although our B,. value was lower. The B,,. value obtained for RX77368, although of a similar magnitude to that of TRH, was somewhat lower indicating the possibilities either that RX77368 binds only to a proportion of the total TRH-binding sites or that TRH binds to some site which is inaccessible to RX77368. The likelihood of two sites is not supported by the similarities of the Hill coefficients for the two ligands. The affinity of RX77368 was nine times lower than that of the parent compound, the latter agreeing well with results obtained by Hawkins et al. (1986) and Sharif et al. (1989) for the inhibition of [3H]-methyl-TRH binding to spinal cord and brain membrane preparations. There was an excellent correlation between the rank order of potency of various analogues to inhibit [3H]-TRH or [3H]-RX77368 binding, strongly suggesting that RX77368 binds to the GH3 cell TRH receptor. Remarkably however, despite saturation analysis indicating a single binding site, under these incubation conditions, the Hill slopes of all the competing ligands against TRH and against RX77368 were lower than 1. This is often taken as an indication that (a) multiple binding sites
618
A.M. McDERMOTT et al. a
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Concentration (>LM) 7 Figure Dose-dependent stimulation of phosphoinositide hydrolysis in GH3 cells by thyrotropin releasing hormone (TRH, 0) and dimethyl proline TRH (RX77368, El). Cells pre-labelled with [3H]inositol were incubated with various concentrations of TRH or RX77368 for 15 min at 370C. Total inositol phosphates were extracted and measured as described under Methods. Values plotted are the mean results from quadruplicate samples from a representative experiment. Two further experiments gave comparable results.
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thought to be minimised by the chosen incubation conditions, indicated by their apparent lack of influence on the saturation curves. Nevertheless, the shape of the inhibition curves, as shown in Figure 5, in particular those using [3H]-RX77368 as the radioligand, appear to be biphasic. Indeed, subsequent non-linear regression analysis indicated that the inhibition data could be better described by a two site rather than one site model. Similar anomalies between association (monophasic) and dissociation kinetics (biphasic) have been reported for rat amygdala membranes (Sharif & Burt, 1983) and GH3 cells (Hinkle et al., 1980). Also Hinkle & Kinsella (1982) have reported a temperature-dependent transformation as
Concentration (nM) Figure 5 Inhibition of binding of (a) [3H]-thyrotropin releasing hormone ([3H]-TRH) and (b) [3H]-dimethyl proline TRH ([3H]RX77368) by various TRH analogues. GH3 cells were incubated at 370C for 1 h with either [3H]-TRH (5 nM) or [3H]-RX77368 (50nM) in the presence of various concentrations of TRH analogue. TRH (0), RX77368 (0), RX74355 (0), MK771 (M), CG3703 (A), CG3509 (A). Values plotted for the inhibition of binding are the mean results of 3 determinations for [3H]-TRH and of 2 determinations for [3H]RX77368.
exist, (b) there is negative co-operativity between binding sites or (c) some allosteric interaction has occurred. The latter two options are the least likely. Indeed, Hinkle et al. (1980) have previously shown that TRH binding is not co-operative. However, while it has been proposed that GH3 cells possess both high affinity membrane bound receptors and possible low affinity uptake or intracellular sites (Gourdji et al., 1973) the contribution of these sites in the present experiments was 3000 r
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2000
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20
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Figure 6 Time course of the accumulation of [3H]-inositol phosphates in GH3 cells stimulated by thyrotropin releasing hormone (TRH, 0) and dimethyl proline TRH (RX77368, D). Cells prelabelled with [3H]-inositol were incubated at 370C with either 1Mm TRH or RX77368 for the times indicated. Total inositol phosphates were extracted and measured as described under Methods. Values plotted are the mean results from quadruplicate samples of a representative experiment. Two further experiments gave comparable results.
of the TRH receptor complex in GH4C1 cells, another member of the GH family. In their studies, TRH was found to bind initially to a form of the receptor from which it dissociated rapidly, then over the course of 5 min the complex was found to be converted to a form with slow dissociation kinetics. Whilst it may be tempting to speculate about the existence of multiple TRH-receptor subtypes (as has been suggested in brain tissue, Funatsu et al., 1985), given that the available ligands for this receptor are all agonists, the biphasic nature of the competition curves could equally well result from agonist-induced affinity changes and receptor kinetics. Indeed, other workers (Hawkins et al., 1987) have questioned the existence of more than one TRH-receptor subtype. It is interesting to note, from a structure-activity point of view, that the order of potency of the displacing ligands seems not to be related to simple structural modifications of the parent compound. This is demonstrated by the relative Ki values for RX77368 and RX74355, the monomethyl proline derivative, which are of very different magnitudes. Similarly, those of CG3703 and CG3509, which are modifications at the opposite N-terminal end of the tripeptide are very different. The data suggest that both ends of the tripeptide are functionally important and yet small structural changes at either end can have large effects on potency. Both peptides were found to stimulate PI hydrolysis in a dose-dependent fashion, with TRH being twelve times more potent than RX77368. The EC50 value for TRH is similar to that obtained by Hinkle & Tashjian (1973) for prolactin release, the functional correlate for TRH receptor stimulation in this cell line. It is worth noting that the EC50 s for both TRH and RX77368 are very similar to their dissociation constants. This may imply little if any TRH receptor reserve in these cells. Also, it is of interest that, in at least 6 separate experiments in which TRH-stimulated PI turnover had plateaued, RX77368 was capable of achieving a still greater response. Although these intra-experimental findings are con-
TRH AND RX77368 ON GH3 CELLS
sistent within cell-matched experiments carried out at the same time, a statistical difference in efficacy is difficult to show due to inter-experimental variation. Possible differences in efficacy may indicate that TRH is only a partial agonist at its own receptor in GH3 cells, that TRH may regulate, or an active metabolite inhibit, the PI system or, less likely, that TRH is inactivated by a selective uptake process. Differences in metabolism can be ruled out as the experiments were carried out in the presence of peptidase inhibitors. The reduced potency of RX77368 compared with TRH on GH3 cells, may be a direct result of the presence of two methyl groups on the proline ring, since RX74355, the monomethyl derivative, has greater affinity than the parent compound, as indicated by the inhibition studies, and was found to be four
619
times more potent than TRH at stimulating PI turnover (unpublished observation). In conclusion, the greater in vivo potency of RX77368 as established in earlier studies (Dettmar et al., 1980; 1981; Metcalf et al., 1981) cannot be explained, in vitro, by greater affinity or potency at the TRH receptor but is more likely to be due to its enhanced bioavailability, as a result of its reported increased resistance to enzymatic degradation (Brewster et al., 1981; Griffiths et al., 1982; Metcalf, 1983). A.M.McD. is an SERC-CASE student in association with Reckitt & Colman. We thank Dr G. Schofield for the use of his tissue culture facilities, Dr G.W. Bennett for the supply of some materials and Dr D.J. Nutt for helpful discussions.
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SHARP, T., TULLOCH, I.F., BENNETT, G.W., MARSDEN, CA., METCALF, G. & DETTMAR, P.W. (1984b). Analeptic effects of centrally injected TRH and analogues of TRH in the pentobarbitoneanaesthetised rat. Neuropharmacology 23, 339-348. SUTTON, CA. & MARTIN, T.FJ. (1982). Thyrotropin releasing hormone selectively and rapidly stimulates phosphatidyl inositol
turnover in GH pituitary cells: A possible second step of TRH action. Endocrinology, 110, 1273-1280. TASHJIAN, A.H., BAROWSKY, N.J. & JENSEN, D.K. (1971). Thyrotropin releasing hormone: direct evidence for stimulation of prolactin production by pituitary cells in culture. Biochem. Biophys. Res. Commun., 43, 516-523. (Received May 2, 1990 Revised June 28, 1990 Accepted July 11, 1990)
IV"I'l Macmillan Press Ltd, 1990
Pharmacological and biochemical comparison of thyrotropin releasing hormone (TRH) and di-methyl proline-TRH on pituitary GH3 cells Alison M. McDermott, Graham P. Wilkin & *lStephen L. Dickinson Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AZ and *Reckitt and Colman Psychopharmacology Unit, The School of Medical Sciences, Bristol, BS8 1TD 1 The binding of [3H]-thyrotropin releasing hormone ([3H]-TRH) and [3H]-RX77368 (di-methyl proline TRH) and the ability of these peptides to stimulate phosphoinositide hydrolysis were investigated in the GH3 pituitary cell line. 2 For both peptides binding was found to be saturable with a single component (Hill slopes were, for TRH, 0.98 and for RX77368, 1.13). TRH bound with greater affinity than RX77368 Kd values were 16nM and 144 nm respectively. B.. values were 227 fmol mg-' protein for TRH and 123 fmol mg1 protein for RX77368. 3 The rank order of potency of a series of TRH analogues to inhibit binding was the same versus each peptide. However, unlike with saturation analysis, Hill slopes of all displacing ligands were less than 1.0 against both TRH and RX77368 suggeting either multiple binding sites, alteration of affinity state, negative co-operativity or some allosteric interaction. 4 Both peptides stimulated phosphoinositide hydrolysis in a dose-dependent fashion. TRH was more potent than RX77368, ECjo values were 7.9 + 1 nm and 96.3 + 3 nM respectively. 5 These in vitro data suggest that the greater in vivo potency of RX77368 is not the result of enhanced receptor affinity but is more probably due to its greater metabolic stability.
Introduction The tripeptide thyrotropin releasing hormone, TRH, (Figure 1) is widely distributed throughout the central nervous system. It exhibits a broad spectrum of stimulatory effects (Griffiths, 1987) which are probably mediated via receptors very similar to those moderating the endocrine function of the peptide in the pituitary gland (Burt & Taylor, 1980; Dettmar et al., 1983a,b; Sharif et al., 1983). TRH has been found capable of preventing neuronal damage (Freedman et al., 1986; 1989), promoting recovery following spinal trauma (Faden et al., 1981) and relieving weakness and spasticity in motor neurone disease (MND) (Engel et al., 1983). However, its clinical potential in these areas has not been realised, possibly a consequence of the peptide's short biological half life due to its rapid metabolism by pyroglutamyl aminopeptidases and deamidase enzymes in body tissues and fluids (Bassiri & Utiger, 1981; Coggins et al., 1987). RX77368 is one of several analogues synthesized in an attempt to overcome this lability. Here the substitution of two methyl groups at position 3 on the proline ring (Figure 1) renders the molecule much more resistant to enzymatic degradation (Brewster et al., 1981; Griffiths et al., 1982; Metcalf, 1983). RX77368, which has also been found beneficial for the treatment of MND (Guiloff et al., 1987a,b), is endocrinologically equipotent to TRH but shows greater biological activity in several neuropharmacological tests (Dettmar et al., 1980; 1981; 1983a; Metcalf et al., 1981; Sharp et al., 1984ab). The latter has been attributed to the greater metabolic stability hence increased bioavailability of the analogue; however, other factors, for example active metabolites or enhanced potency at the receptor, may play a role. While there are no known active metabolites of RX77368, indeed much of a dose is excreted unchanged (Brewster et al., 1981), there has to date been no data published concerning the direct binding characteristics of the drug, although Hawkins et al. (1986) and more recently Sharif et al. (1989) have shown indirectly that RX77368 has a lower Author for correspondence.
affinity than TRH for mammalian spinal cord and brain receptors. Here we have had the unique opportunity of investigating the binding characteristics of tritiated RX77368 and comparing them to those of the parent compound in whole GH3 pituitary cells, a clonal cell line having receptors for TRH that are coupled to phosphoinositide (PI) hydrolysis and prolactin release (Tashjian et al., 1971; Drummond & Macphee, 1981; Sutton & Martin, 1982). We have also studied the ability of the peptides to stimulate PI hydrolysis in this cell line. A preliminary account of this work has already been published (McDermott et al., 1989). R2
R1 TRH
0J
N7 CONH2 -
as TRH
RX74355
N
0
Rl "
NH
CONH2
J
r R2
RX77368
asTRH O
0CH2
L
%NH
NH
I
CG3509
N
iCH3 CH3
CONH2 as TRH
O'QNH
CH3 S
as TRH
CG3703 M
NH
MK771
CONH2 Figure 1 The structure of thyrotropin some of its analogues.
releasing hormone (TRH) and
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Methods Cell culture GH3 cells were routinely grown in monolayer culture in 10cm dishes at 370C, under 95% air/5% CO2 in Ham's F10 culture medium supplemented with 15% donor horse serum, 2.5% foetal calf serum and 40 pgmlP' gentamicin. Cells were cultured to about 80% confluency and passaged using 0.05% trypsin in Tris/EDTA. For binding and PI hydrolysis studies, cells were seeded at a concentration of 0I ml on to 13 mm diameter glass coverslips coated with 0.5 pg ml poly-L-lysine in multiwell plates. The culture medium was changed every 3 days and the cells allowed to grow for 9-11 days before use.
from Sigma. [1,2-3H(N)]myo-inositol, [3H]-inositol, (458OCi mmol- 1) and [L-proline-2,3,4,5,3H(N)]-pGlu-His-ProNH2, [3H]-TRH, (119.2 Ci mmol 1) were purchased from New England Nuclear. GH3 cells were a gift from Dr A. Drummond. pGlu-[2,5-3H2] His-3,3'dimethyl Pro-NH2, [3H]-RX77368, (32.7CimmolP , purified and characterized by P.M. Taylor), RX77368 and RX74355 were provided by Reckitt and Colman. MK771 was provided by Merck Sharp and Dohme. CG3509 and CG3703 were from Chemie Grunenthal. All other reagents and solvents were of analytical grade.
Results
Receptor binding studies
Receptor binding studies
The growth medium was aspirated and the cells washed twice with 0.5 ml of Ham's F10 medium, supplemented with 25mM HEPES, 5 mM magnesium sulphate, 14 mm sodium bicarbonate and 400ug mlP- bovine serum albumin, pH 7.4. The cells were then incubated for up to 1 h at 370C in a rotary shaker, in 0.5 ml of medium also containing either [3H]-TRH or [3H]-RX77368. Non-specific binding was determined by the addition of 100paM TRH to the medium. The incubation was terminated by aspirating the medium and the cells were then washed 3 times with 1 ml of ice cold medium. The coverslips were then transferred to vials for scintillation counting.
Incubation of [3H]-TRH or [3H]-RX77368 with whole GH3 cells resulted in a time-dependent increase in specific binding at 37°C (Figure 2). [3H]-TRH bound more rapidly than [3H]RX77368. Maximal binding of both peptides was achieved by 60min. The data obtained from experiments in which cells were incubated with various concentrations of [3H]-TRH or [3H]-RX77368 for 1 h, revealed that under these conditions binding of both peptides was saturable (Figure 3a,b). Nonspecific binding was less than 35% for [3H]-TRH and 50% for [3H]-RX77368. Hanes analysis of the data indicated a single binding component for each peptide (Figure 4a,b). Kd and BX,, values were, for TRH 16 nM and 227 fmol mg-1 protein, and for RX77368 144nm and 123fmolmg-t protein. Hill coefficients were 0.98 and 1.13 for TRH and RX77368
Tritiated peptide stability studies On the manufacturer's recommendation, paper chromatography followed by liquid scintillation counting was used to assess the stability of the tritiated ligands. Aliquots of medium containing radioligand that had been incubating with cells (with or without the presence of 40gnml- 1 bacitracin) for 1 h at either 37°C, ambient temperature or 4°C were compared to radioligand incubated without cells.
Phosphoinositide hydrolysis studies Cells were pre-labelled for 2-3 days with 1 uCi mlP-I [3H]-inositol. Under these conditions labelling of the inositol lipids has reached isotopic equilibrium (Drummond et al., 1984; confirmed in our laboratory, data not shown). The growth medium was aspirated and the cells washed twice with 0.5 ml of medium, as described for binding studies but with the addition of 10 mm lithium chloride and a mixture of peptidase inhibitors routinely used in our laboratory (leupeptin 2g ml- 1, chymostatin 1 pg ml-' and bacitracin 40g ml- 1), then pre-incubated in 0.5 ml of this medium for 30 min at 37°C on a rotary shaker. Peptides were added in a volume of 10#p to give the required final concentration, and the incubation continued for a further 15min. The reaction was terminated by transferrence of the coverslips to vials containing 1.5ml chloroform/methanol 2:1 (v/v) then adding 0.5ml water. The mixture was vortexed then left to separate; 0.5 ml of the upper aqueous phase was removed for the determination of total inositol phosphates as described by Cholewinski et al. (1988) following the method of Berridge et al. (1982). An aliquot (200,ul) of the lower phase was removed, dried and counted for the estimation of [3H]-inositol incorporation into phospholipids.
Protein estimation Cellular protein content was determined by the method of Lowry et al. (1951).
Materials Culture media and sera were from Imperial Laboratories. Antibiotics and tissue culture plasticware were from Flow Laboratories. All other tissue culture reagents and TRH were
respectively. The ability of a number of TRH analogues (Figure 1) to inhibit binding was determined by incubating GH3 cells with either [3H]-TRH or [3H]-RX77368 in the presence of various concentrations of analogue (Figure 5). Table 1 shows the concentrations of ligand required to displace 50% of the specific binding. The orders of potency of the analogues to inhibit binding were as follows: versus [3H]-TRH: RX74355 > TRH > = MK771 > = CG3703 > RX77368 > CG3509; versus [3H]-RX77368: RX74355 > = TRH > MK771 > = CG3703 > RX77368 > CG3509. Slopes calculated from Hill plots of the competition curves yielded values of less than 1 for all competing ligands and for both peptides (Table 1). These were confirmed by non-linear regression analysis (GraphPAD INPLOT). 25000 r 20000 c
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8. 15000
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-
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30
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120
Time (min)
Figure 2 Time course of specific binding of [3H]-thyrotropin releasing hormone ([3H]-TRH, [J) and [3H]-dimethyl proline TRH ([3H]RX77368, 0) to GH3 cells. The cells were incubated at 37°C with either [3H]-TRH (5 nM) or [3H]-RX77368 (5OnM) for various times. Maximal binding of both peptides was achieved by 60min. Each value represents the mean of quadruplicate samples from a single experiment; vertical bars show s.e.mean. The discrepancy between actual d.p.m. values reflects the different specific activities of the two radioligands.
TRH AND RX77368 ON GH3 CELLS a
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Concentration (nM) Figure 3 Saturation isotherms for (a) [3H]-thyrotropin releasing hormone ([3H]-TRH) and (b) [3H]-dimethyl proline TRH ([3H]RX77368). Cells were incubated at 370C for 1 h with various concentrations of radioligand alone (total binding 0) or in the presence of 100luM cold TRH (non-specific binding A). Specific binding (El) was determined by subtracting non-specific from total binding. Each value represents the mean of quadruplicate samples from a single experiment; vertical bars show s.e.mean.
Free (nM)
Figure 4 Hanes analysis of (a) [3H]-thyrotropin releasing hormone and (b) ([3H]-dimethyl proline TRH ([3H]-RX77368) binding data. Binding was saturable with a single component (correlation coefficients for TRH and RX77368 were 0.99 and 0.88 respectively). Each value represents the mean of quadruplicate samples from a single experiment. Kd and Bn.. values for TRH were 16nM and 227ffmolmg-' protein (one experiment) and for RX77368 144 nm and 123 fmol mg-1 protein (mean of two experiments).
([3H]-TRH)
Peptide stability studies No significant differences in the proportion of counts occurring in a position equivalent to that of the cold ligand, were observed between radioligand that had been incubated with or without cells indicating that the peptides remained stable throughout the incubation conditions. Changes of temperature or presence of the peptidase inhibitor bacitracin had no effect.
Phosphoinositide hydrolysis studies Both peptides stimulated inositol phosphate metabolism in GH3 cells. The accumulation of [3H]-phosphates rose in a linear fashion for at least 15 min (Figure 6) by which point a maximum of 16% of the [3H]-inositol-containing phospholipids had been depleted. The response to stimulation by TRH Table 1 Inhibition of [3H]-thyrotropin releasing hormone
([3H]-TRH) and [3H]-dimethyl proline TRH ([3H]RX77368) binding to GH3 cells by various TRH analogues
[3H]-RX77368
[3H]-TRH RX74355 TRH MK771 CG3703 RX77368 CG3509
IC50
Hill slope
ICS0
Hill slope
2.0 ± 0.3 23 + 4 30+4 44 ± 5 295 ± 42 1061 + 218
0.62 0.55 0.71 0.60 0.49 0.42
58 ± 21 80± 16 241 ± 41 316 ±0 2009 ± 230 2990 + 172
0.55 0.40 0.42 0.47 0.37 0.31
Mean + s.e.mean from 1 to 3 experiments in quadruplicates. Concentrations are expressed as nM.
and RX77368 was dose-dependent (Figure 7). TRH was more potent than RX77368, ECjo values were 7.9 + 1 nm (mean + s.e.mean) and 96.3 + 3 nm respectively.
Discussion Saturation analysis revealed that TRH and its dimethyl analogue RX77368 each bind to a single site on GH3 pituitary cells. The dissociation constant obtained for TRH was very similar to that previously reported by Hinkle & Tashjian (1973), although our B,. value was lower. The B,,. value obtained for RX77368, although of a similar magnitude to that of TRH, was somewhat lower indicating the possibilities either that RX77368 binds only to a proportion of the total TRH-binding sites or that TRH binds to some site which is inaccessible to RX77368. The likelihood of two sites is not supported by the similarities of the Hill coefficients for the two ligands. The affinity of RX77368 was nine times lower than that of the parent compound, the latter agreeing well with results obtained by Hawkins et al. (1986) and Sharif et al. (1989) for the inhibition of [3H]-methyl-TRH binding to spinal cord and brain membrane preparations. There was an excellent correlation between the rank order of potency of various analogues to inhibit [3H]-TRH or [3H]-RX77368 binding, strongly suggesting that RX77368 binds to the GH3 cell TRH receptor. Remarkably however, despite saturation analysis indicating a single binding site, under these incubation conditions, the Hill slopes of all the competing ligands against TRH and against RX77368 were lower than 1. This is often taken as an indication that (a) multiple binding sites
618
A.M. McDERMOTT et al. a
.) c
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C
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.0
._
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0 a) U)
0-
-o~
100 000 0.0001 0.001 0.01
0.1
1
10
100
1000
Concentration (>LM) 7 Figure Dose-dependent stimulation of phosphoinositide hydrolysis in GH3 cells by thyrotropin releasing hormone (TRH, 0) and dimethyl proline TRH (RX77368, El). Cells pre-labelled with [3H]inositol were incubated with various concentrations of TRH or RX77368 for 15 min at 370C. Total inositol phosphates were extracted and measured as described under Methods. Values plotted are the mean results from quadruplicate samples from a representative experiment. Two further experiments gave comparable results.
0) C
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thought to be minimised by the chosen incubation conditions, indicated by their apparent lack of influence on the saturation curves. Nevertheless, the shape of the inhibition curves, as shown in Figure 5, in particular those using [3H]-RX77368 as the radioligand, appear to be biphasic. Indeed, subsequent non-linear regression analysis indicated that the inhibition data could be better described by a two site rather than one site model. Similar anomalies between association (monophasic) and dissociation kinetics (biphasic) have been reported for rat amygdala membranes (Sharif & Burt, 1983) and GH3 cells (Hinkle et al., 1980). Also Hinkle & Kinsella (1982) have reported a temperature-dependent transformation as
Concentration (nM) Figure 5 Inhibition of binding of (a) [3H]-thyrotropin releasing hormone ([3H]-TRH) and (b) [3H]-dimethyl proline TRH ([3H]RX77368) by various TRH analogues. GH3 cells were incubated at 370C for 1 h with either [3H]-TRH (5 nM) or [3H]-RX77368 (50nM) in the presence of various concentrations of TRH analogue. TRH (0), RX77368 (0), RX74355 (0), MK771 (M), CG3703 (A), CG3509 (A). Values plotted for the inhibition of binding are the mean results of 3 determinations for [3H]-TRH and of 2 determinations for [3H]RX77368.
exist, (b) there is negative co-operativity between binding sites or (c) some allosteric interaction has occurred. The latter two options are the least likely. Indeed, Hinkle et al. (1980) have previously shown that TRH binding is not co-operative. However, while it has been proposed that GH3 cells possess both high affinity membrane bound receptors and possible low affinity uptake or intracellular sites (Gourdji et al., 1973) the contribution of these sites in the present experiments was 3000 r
C
0
2000
E ,._10
.-O
1000F o 0
20
40
60
80
100
Time (min)
Figure 6 Time course of the accumulation of [3H]-inositol phosphates in GH3 cells stimulated by thyrotropin releasing hormone (TRH, 0) and dimethyl proline TRH (RX77368, D). Cells prelabelled with [3H]-inositol were incubated at 370C with either 1Mm TRH or RX77368 for the times indicated. Total inositol phosphates were extracted and measured as described under Methods. Values plotted are the mean results from quadruplicate samples of a representative experiment. Two further experiments gave comparable results.
of the TRH receptor complex in GH4C1 cells, another member of the GH family. In their studies, TRH was found to bind initially to a form of the receptor from which it dissociated rapidly, then over the course of 5 min the complex was found to be converted to a form with slow dissociation kinetics. Whilst it may be tempting to speculate about the existence of multiple TRH-receptor subtypes (as has been suggested in brain tissue, Funatsu et al., 1985), given that the available ligands for this receptor are all agonists, the biphasic nature of the competition curves could equally well result from agonist-induced affinity changes and receptor kinetics. Indeed, other workers (Hawkins et al., 1987) have questioned the existence of more than one TRH-receptor subtype. It is interesting to note, from a structure-activity point of view, that the order of potency of the displacing ligands seems not to be related to simple structural modifications of the parent compound. This is demonstrated by the relative Ki values for RX77368 and RX74355, the monomethyl proline derivative, which are of very different magnitudes. Similarly, those of CG3703 and CG3509, which are modifications at the opposite N-terminal end of the tripeptide are very different. The data suggest that both ends of the tripeptide are functionally important and yet small structural changes at either end can have large effects on potency. Both peptides were found to stimulate PI hydrolysis in a dose-dependent fashion, with TRH being twelve times more potent than RX77368. The EC50 value for TRH is similar to that obtained by Hinkle & Tashjian (1973) for prolactin release, the functional correlate for TRH receptor stimulation in this cell line. It is worth noting that the EC50 s for both TRH and RX77368 are very similar to their dissociation constants. This may imply little if any TRH receptor reserve in these cells. Also, it is of interest that, in at least 6 separate experiments in which TRH-stimulated PI turnover had plateaued, RX77368 was capable of achieving a still greater response. Although these intra-experimental findings are con-
TRH AND RX77368 ON GH3 CELLS
sistent within cell-matched experiments carried out at the same time, a statistical difference in efficacy is difficult to show due to inter-experimental variation. Possible differences in efficacy may indicate that TRH is only a partial agonist at its own receptor in GH3 cells, that TRH may regulate, or an active metabolite inhibit, the PI system or, less likely, that TRH is inactivated by a selective uptake process. Differences in metabolism can be ruled out as the experiments were carried out in the presence of peptidase inhibitors. The reduced potency of RX77368 compared with TRH on GH3 cells, may be a direct result of the presence of two methyl groups on the proline ring, since RX74355, the monomethyl derivative, has greater affinity than the parent compound, as indicated by the inhibition studies, and was found to be four
619
times more potent than TRH at stimulating PI turnover (unpublished observation). In conclusion, the greater in vivo potency of RX77368 as established in earlier studies (Dettmar et al., 1980; 1981; Metcalf et al., 1981) cannot be explained, in vitro, by greater affinity or potency at the TRH receptor but is more likely to be due to its enhanced bioavailability, as a result of its reported increased resistance to enzymatic degradation (Brewster et al., 1981; Griffiths et al., 1982; Metcalf, 1983). A.M.McD. is an SERC-CASE student in association with Reckitt & Colman. We thank Dr G. Schofield for the use of his tissue culture facilities, Dr G.W. Bennett for the supply of some materials and Dr D.J. Nutt for helpful discussions.
References BASSIRI, R.M. & UTIGER, R.D. (1981). Metabolism and excretion of endogenous thyrotropin releasing hormone in humans. J. Clin. Invest., 52, 1616-1619. BERRIDGE, M.J., DOWNES, C.P. & HANLEY, M.R. (1982). Lithium amplifies agonist dependent phosphatidylinositol responses in brain and salivary glands. Biochem. J., 20, 587-595. BREWSTER, D., HUMPHREY, MJ. & WEARING, M.V. (1981). Metabolism and pharmacokinetics of TRH and an analogue with enhanced neuropharmacological potency. Neuropeptides, 1, 153165. BURT, D.R. & TAYLOR, R.L. (1980). Binding studies for thyrotropin releasing hormone in sheep nucleus accumbens resemble pituitary receptors. Endocrinol., 106, 1416-1423. CHOLEWINSKI, AJ., HANLEY, M.R. & WILKIN, G.P. (1988). A phosphoinositide linked peptide response in astrocytes: evidence for regional heterogeneity. Neurochem. Res., 13, 389-394. COGGINS, P.J., McDERMOTT, J.R., SNELL, C.R. & GIBSON, A.M. (1987). Thyrotropin releasing hormone degradation by rat synaptosomal peptidases: production of the metabolite His-Pro. Neuropeptides, 10, 147-156. DETTMAR, P.W., FORTUNE, D., LYNN, A.G., METCALF, G. &
MORGAN, B.A. (1980). Biological evaluation of a TRH analogue with a modified proline residue. Neuropharmacol., 19, 1247-1248. DETTMAR, P.W., FORTUNE, D., LYNN, A.G., METCALF, G., MORGAN,
B.A. & TULLOCH, I.F. (1981). Neuropharmacological evaluation of TRH analogues with modified proline residues. Br. J. Pharmacol., 73, 263P. DETTMAR, P.W., LYNN, A.G., METCALF, G. & MORGAN, B.A. (1983a). Brain TRH receptors are the same as pituitary TRH receptors. J. Pharm. Pharmacol., 35, 399-400. DETTMAR, P.W., LYNN, A.G. & METCALF, G. (1983b). A comparison between TRH and RX77368 to provoke the release of TSH and prolactin in the rat. In Thyrotropin Releasing Hormone. ed. Griffiths, E.C. & Bennett, G.W. p. 362. New York: Raven Press. DRUMMOND, A.H. & MACPHEE, C.H. (1981). Phosphatidyl inositol metabolism in GH3 pituitary tumour cells stimulated by TRH. Br. J. Pharmacol., 74, 967P. DRUMMOND, A.H., BUSHFIELD, M. & MACPHEE, C.H. (1984). Thyrotropin releasing hormone stimulated [3H]-inositol metabolism in GH3 pituitary tumour cells. Mol. Pharmacol., 25, 201-208. ENGEL, W.K., SIDDIQUE, T. & NICOLOFF, J.T. (1983). Effect on weakness and spasticity in amyotrophic lateral sclerosis of thyrotropin releasing hormone. Lancet, ii, 73-75. FADEN, A.I., JACOBS, T.P. & HOLADAY, J.W. (1981). thyrotropin releasing hormone improves neurologic recovery after spinal trauma in cats. New Engl. J. Med., 305,1063-1067. FREEDMAN, J., HOKFELT, T., JONSSON, G. & POST, C. (1986). TRH counteracts neuronal damage induced by substance P antagonist. Exp. Brain Res., 62, 175-178. FREEDMAN, J., HOKFELT, T., POST, C., BRODIN, E., SUNDSTROM, E., JONSSON, C., TERENIUS, L., LEANDER, S., FISCHER, J.A. & VERHOFSTAD, A. (1989). Immunohistochemical and behavioural analysis of spinal lesions induced by a substance P antagonist and protection by thyrotropin releasing hormone. Exp. Brain Res., 74, 279-292. FUNATSU, K., TESHIMA, S. & INANAGA, K. (1985). Various types of Thyrotropin-Releasing Hormone receptors in discrete brain regions and the pituitary of the rat. J. Neurochem., 45, 390-397. GOURDJI, D., TIXIER-VIDAL, A., MORIN, A., PRADELLES, P., MORGAT, J., FROMAGOT, P. & KERDELHUE, B. (1973). Binding of
a tritiated thyrotropin-releasing factor to a prolactin secreting clonal cell line (GH3). Exp. Cell. Res., 82, 39-46. GRIFFITHS, E.C., McDERMOTT, J.R. & SMITH, A.J. (1982). Mechanisms of brain inactivation of centrally acting thytrotropin releasing hormone (TRH) analogues: a high performance liquid chromatography study. Reg. Peptides, 5, 1-11. GRIFFITHS, E.C. (1987). Clinical applications of thyrotropin releasing hormone. Clin. Sci., 73, 449-57. GUILOFF, R.J., ECKLAND, D.J.A., DEMAINE, C., HOARE, R.C.,
MACRAE, K.D. & LIGHTMAN, S.L. (1987a). Controlled acute trial of a thyrotropin releasing hormone analogue (RX77368) in motor neuron disease. J. Neurol. Neurosurg. Psychiat., 50, 1359-1370. GUILOFF, RJ., STALBERG, E., ECKLAND, D.J.A. & LIGHTMAN, S.L.
(1987b). Electrophysiological observations in patients with motor neuron disease receiving a thyrotropin releasing hormone analogue (RX77368). J. Neurol. Neurosurg. Psychiat., 50, 1633-1640. HAWKINS, E.F., BEYDOUN, S.R., HAWN, C.K. & ENGEL, W.K. (1986). Analogs of thyrotropin releasing hormone: hypotheses relating receptor binding to net excitation of spinal lower motor neurons. Biochem. Biophys. Res. Commun., 138, 1184-1190. HAWKINS, E.F., WADE, R. & ENGEL, W.K. (1987). Lack of usefulness of DN-1417 for characterisation of a CNS receptor for thyrotropin releasing hormone. J. Neurochem., 49, 239-245. HINKLE, P.M. & TASHJIAN, A.H. (1973). Receptors for thyrotropin releasing hormone in prolactin producing rat pituitary cells in culture. J. Biol. Chem., 248, 6180-6186. HINKLE, P.M., LEWIS, D.G. & GREER, T.L. (1980). Thyrotropin releasing hormone-receptor interaction in GH3 cells. Endocrinology, 106, 1000-1005. HINKLE, P.M. & KINSELLA, P.A. (1982). Rapid temperature-dependent transformation of the thyrotropin-releasing hormone-receptor complex in rat pituitary tumour cells. J. Biol. Chem., 257, 54625470. LOWRY, O.H., ROSEBROUGH, N.J., FARR, A.L. & RANDALL, R.J.
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