Clinical Endocrinology (1991) 35,455-466
Review
Dopamine, the dopamine D2 receptor and pituitary tumours D. F. Wood, J. M. Johnston and D. G. Johnston Unit of Metabolic Medicine, St Mary’s Hospital Medical School, London, UK (Received 2 July 1991; returned for revision 12 August 1991; finally revised 23 August 1991; accepted 10 September 1991)
In the hypothalamic pituitary axis dopamine acts both as a neurotransmitter and as a hormone, a dual role which is, in part, responsible for the complex nature of its action. Whilst dopamine directly inhibits anterior pituitary function, its secretory pattern and biological effects are modified by interactions with other hypothalamic factors and by feedback from the pituitary itself. Within the anterior pituitary the inhibitory effects of dopamine are mediated by membrane bound receptors of the D2 subtype. Recent identification of the gene encoding the D2 receptor, prediction of its protein structure and increasing recognition of the diversity of intracellular events triggered by D2 receptor activation now allow greater insight into intracellular dopamine action. Abnormalities of dopamine activity have been described in association with a number of pituitary tumours. These observations have led to the hypothesis that chronic dopamine deficiency may be involved in the development of such tumours. In this review we aim to re-examine this hypothesis in the light of recent investigations into the aetiology of pituitary tumours and the increasing knowledge of dopamine D2 receptor activity. The aetiology of pituitary tumours
Despite the well recognized clinical syndromes associated with pituitary adenomas, their aetiology has remained largely obscure. However, there is now increasing evidence that these tumours arise from intrinsic pituitary cell defects caused by somatic mutation. Whilst rarely malignant, pituitary tumours exhibit features of neoplasia and morphologically they represent discrete adenomas (Kovacs et al., 1978; Melmed el al., 1983). This is in contrast to pituitary hyperplasia associated with ectopic releasing hormone production (Melmed & Rushakoff, 1987). In general, neoplasia involves expansion of a clonal cell line (Fialkow, 1986). Two recent studies have used the technique of X-chromosome inactivation analysis to study the clonal nature of pituitary Correspondence: D. F. Wood, Unit of Metabolic Medicine, St Mary’s Hospital Medical School, London W2 lPG, UK.
tumours (Alexander et al., 1990; Herman et al., 1990). This technique, developed by Vogelstein et al. (1987), exploits restriction fragment length polymorphisms (RFLP) and differential methylation patterns in two X-linked genes, HPRT and PGK, in female cells. In any female cell only one X-chromosome, either maternally or paternally derived, is active. Therefore if tumour cells are monoclonal in origin, the same X-chromosome will be active in all of them and this can be detected by RFLP and methylation analysis. In the studies cited, the majority of pituitary tumours were found to be monoclonal in nature. Limitations in the experimental method, allowing inclusion of some normal surrounding pituitary tissue, may have been responsible for inappropriate assignment of some of the tumours as polyclonal (Herman et al., 1990). A mechanism whereby somatic mutation may lead to adenoma formation has been identified in some growth hormone (GH) secreting pituitary adenomas. Vallar et al. (1987) identified a subset of these tumours characterized by high GH release and intracellular cAMP accumulation in vitro, events associated with constitutive activation of adenylyl cyclase. These tumours expressed somatic mutations in the a-subunit of the stimulatory G-protein,Gs (see below), which is involved in signal transduction at the growth hormone-releasing hormone receptor and which therefore plays a role in regulating both G H release and somatotroph cell division. The postulated oncogene Gsp which encodes the mutant protein may thus allow unrestrained hormone secretion and cell division (Landis et al., 1989; McCormick, 1989). Expression of Gsp has been identified in 3040% of human GH-secreting pituitary adenomas (Landis et al., 1990; Spada et al., 1990). In two recent studies the role of cAMP as a regulator of somatotroph cell division was demonstrated in transgenic mice. Animals expressing a transgene encoding non-functional cAMP binding protein (CREB, required to mediate the transcriptional effects of CAMP) were found to have somatic hypoplasia and dwarfism (Struthers et al., 1991). Conversely, use of the rat GH gene promoter to express an intracellular form of cholera toxin, which irreversibly activates Gs, produced transgenic mice with somatotroph hyperplasia and gigantism (Burton et al., 1991). However, these animals showed no evidence of pituitary tumour formation. A longer period of hyperplasia or other oncogenic events in addition to the constitutive activation of adenylyl cyclase may be required to instigate neoplastic change. Another piece of evidence to support the theory of an intrinsic pituitary defect in the aetiology of pituitary tumours 455
456
D. F. Wood eta/.
was supplied by in-situ hybridization studies of resected tumours from acromegalic patients (Lloyd et al., 1989). Eighty-seven per cent of these tumours were found to contain prolactin messenger RNA, despite the lack of prolactin gene expression in normal somatotrophs. This may indicate neoplastic transformation of primitive somatomammotrophs or dedifferentiation of existing somatotrophs. Dopamine and pltultary tumorigenesls
Dopamine inhibits both hormone secretion and cell division in the anterior pituitary (see below) (Jacobi & Lloyd, 1981; Cronin et al., 1982). The abnormalities of dopamine activity associated with some pituitary tumours suggest that chronic dopamine deficiency may be involved in their development. This may be produced in a number of ways. Hypothalamic dysfunction has been postulated in the case of prolactinomas (Fine & Frohman, 1978; Saeger & Ludecke, 1983; Landolt & Minder, 1984). However, in the majority of cases prolactin and TSH responses to secretagogues or inhibitors are restored to or towards normal following tumour resection, suggesting that hypothalamic dysfunction is not a primary defect (Tucker et al., 1980; Schlecte & Sherman, 1981; Barbarino et al., 1982; Molitch et al., 1982; Rodman et al., 1984). Abnormal pituitary vasculature with the development of a systemic arterial blood supply has been observed in some pituitary tumours (Weiner et al., 1987; Schecter et al., 1988). This may allow localized ‘escape’ of parts of the pituitary from dopamine input via the hypophyseal portal circulation. However, the acquisition of an arterial blood supply may be a secondary phenomenon as a newly developed ‘tumour circulation’ is observed in many human tumours. Finally, intrinsic pituitary abnormalities may arise whereby somatic mutations occur in the D2 receptor or its intracellular mediators. Such abnormalities, producing an effective intrapituitary dopamine deficiency, are under investigation in several laboratories. Dopamlne actlon at the anterlor pituitary: the doparnine D2 receptor
Classically, dopamine receptors have been divided into two major groups: those which stimulate adenylyl cyclase activity (D1 receptors) and those which inhibit this enzyme (D2 receptors) (Kebabian & Calne, 1979; Enjalbert & Bockaert, 1983).In the past decade investigation into the mechanism of dopamine action in the central nervous system has suggested that further dopamine receptor subtypes exist, based on the diversity of biochemical, pharmacological and physiological responses observed. The evidence for multiple receptor subtypes has been reviewed recently (Andersen et al., 1990)
Clinical Endocrinology (1991) 35
and three further discrete subtypes have been described (D3, D4, D5; Sokoloff et al., 1990; Van To1 et al., 1991; Sunahara et al., 1991). Anterior pituitary dopamine receptors are of the D2 subtype, mediating the inhibitory effects of dopamine on anterior pituitary function. Activation of the D2 receptor produces a variety of intracellular responses in addition to decreasing CAMP concentrations (De Camilli et al., 1979). These include changes in potassium channel activity (Israel et al., 1985; Catelletti et al., 1989) and calcium channel flux (Taraskevich & Douglas, 1978), inhibition of phosphatidyl inositol turnover (Vallar & Meldolesi, 1989) and alteration of intracellular calcium concentrations (Baudry et al., 1986; Enjalbert et al., 1986). Cloning and expression of a cDNA encoding the rat D2 receptor showed it to have typical features of a guanine nucleotide binding protein (G-protein)-coupling receptor (Bunzow et al., 1988). The diverse intracellular effects produced by D2 receptor activation suggest that signal transduction is mediated by several different G-proteins (Senogles et al., 1987; Ohara et al., 1988; Elazar et al., 1989). These events are shown schematically in Fig. 1. The human D2 receptor gene has been localized to chromosome 11 (Grandy et al., 1989a) and shows high homology with the rat and bovine genes (Grandy et al., 1989b;Monsma et al., 1989; Chi0 et al., 1990). In all cases the deduced amino acid sequences contain the seven transmembrane domains and the long third intracytoplasmic loop typial of G-protein coupled receptors (Dohlman et al., 1987; Johnson & Dhanasekaran, 1989). The D2 receptor gene has a number of characteristic features. Whilst most G-proteincoupled receptor genes lack introns within their coding sequences (O’Dowd et al., 1989), the dopamine D2 receptor gene has a more complex organization being at least 12 kb long with a minimum of six introns (Giros et al., 1989). The D2 receptor cDNA encodes a single polypeptide of 415 amino acids with a molecular mass of approximately 47 kDa. This corresponds to the deglycosylated receptor previously described (Jarvie & Niznik, 1989). The overall molecular mass of the D2 receptor is approximately 110-140 kDa although two subunits of 94 kDa and 35 kDa are observed on electrophoresis (Lew et al., 1990). A second receptor isoform of 444 amino acids has been described, containing a 29-amino-acid insertion in the third intracytoplasmic loop (Fig. 2). The two isoforms are generated by alternative RNA splicing (Dal Toso et a11989; Eidne et al., 1989; Giros et al., 1989; Monsma et al., 1989; Chi0 et al., 1990). Both isoforms of the D2 receptor can be identified within the anterior pituitary gland, with the longer form being in greater abundance (Dal Toso et al., 1989). In receptors of this family the third intracytoplasmic loop appears to be important for
Dopamine and pituitary tumours 457
Clinical Endocrinology (1991) 35
Cell
A1
Fig. 1 Signal transduction at the dopamine D2 receptor. Dopamine-D2 receptor binding activates a number of intracellular pathways (see text for references). Dopamine binding inhibits adenylyl cyclase activity via the G-protein Gi , lowering intracellular CAMPconcentration and subsequently causing a reduction in calcium mobilization. Activation of Gk by the bound receptor causes potassium efflux. Inhibition of phosphoinositol bisphosphate hydrolysis occurs, probably mediated by the action of Go G-proteins, which may also have direct effects on intracellular calcium concentrations. Membrane calcium channel activity is altered. The overall effects of D2 receptor activation are to lower intracellular Ca2+ concentrations and this decrease causes the alterations in hormone release and gene transcription. The effects of D2 receptor activation on phospholipase C activity remain to be elucidated. D2R, dopamine D2 receptor; DA, dopamine; Gi, Gk, Go, G-proteins involved in signal transduction; AC, adenylyl cyclase; PIP2, phosphoinositol bisphosphate; IP3, inositol trisphosphate; DAG, diacyl glycerol; PKC, protein kinase C, PLC, phospholipase C.
Extracellular
241-270 \
*
*
Fig. 2 Structure of the dopamine D2 receptor. The seven transmembrane domains are shown. Two isoforms, D2[415] and D2[444] are generated by alternative splicing of the primary RNA transcript. The position of the 29 amino acid insert in the long third intracytoplasmic loop is thought to correspond to G-protein binding sites, suggesting different biological activities for the two receptor isoforms.
G-protein coupling (reviewed by Ross, 1989). Receptor heterogeneity produced by alternative RNA splicing may be important in determining the binding to different G-proteins (De Keyser ef al., 1989) and in the production of the diverse intracellular effects characteristic of D2 receptor activation.
Signal transduction at the D2 receptor:G-protein action
Signal transduction at the D2 receptor is effected by a series of G-proteins. G-proteins form part of a newly recognized (and rapidly expanding) superfamily of intracellular proteins
458 D. f . Wood et a/.
Clinical Endocrinology (1991) 35
I
iI
GTF
a-GTP
Effector protein
a-GTP-E
(Ei PI
protein
(E)
1
lntracel lular response
Fig. 3 G-protein action: the cycle of GTP binding and hydrolysis. Activated receptors promote dissociation of GDP from the G-protein a-subunit. GTJ? binding to a in turn promotes dissociation of the 1-y complex and allows activation of intracellular effects. The intrinsic GTP-ase activity of the a-subunit terminates its interaction with an effector and the trimeric G-protein is reconstituted.
characterized by their ability to bind and hydrolyse GTP in a unidirectional cyclical fashion. These proteins act as molecular ‘switches’ to trigger a variety of intracellular responses. The properties of the GTP-ase family are discussed in two recent reviews (Bourne et a[., 1990; 1991). Membrane associated G-proteins are heterotrimeric structures comprising a, /3 and y-subunits. Their role in transmembrane signallinghas been the subject of a number of extensive reviews (Tomlinson et al., 1985; Gilman, 1987; Neer & Clapham, 1988; Ross, 1989; Taylor, 1990). At least eight different G-proteins have been characterized. The a-subunits appear to confer structural and functional specificity whereas the P-y complexes are functionally similar (Bourne et al., 1990). @-Subunitscontain recognition sites for receptors and effector proteins and the guanine nucleotide binding site. The p and y-subunits form a tight complex whose function is not fully elucidated, but which may be involved in anchoring the protein to the cytoplasmic face of the cell membrane and in ‘presenting’ the a-subunit to the receptor (Sternweis, 1986). The cycle of events which follow receptor-G-protein binding is shown in Fig. 3. Activated (ligand-bound) receptors promote dissociation of GDP from the a-subunit and may increase affinity of the a-subunit for GTP-binding (Florio & Sternweis, 1989). GTP-binding in turn promotes dissociation of B-y from the a-GTP complex, allowing the latter to activate intracellular effectors (adenylyl cyclase, phospholipases, etc.). A potential role for the p-y complex in control-
ling other effector proteins remains controversial (Bourne, 1989; Taylor, 1990). Interaction of a-GTP with an effector is terminated by the intrinsic GTP-ase activity of the a-subunit. Subsequently, a-GDP binds to p-y again and reconstitutes the trimeric proteins ready for further activation. In this way, G-proteins are able to transduce and, by means of complex timing mechanisms, to amplify a wide range of transmembrane signals through a smaller set of intracellular effector molecules. The ras proteins form another subset of mammalian GTPases which have been widely investigated because mutations in the ras genes cause neoplastic transformation. The three ras genes, H-ras, K-ras and N-ras, encode 21 kDa proteins (p2Iras)which are involved in the regulation of cell division and differentiation (Barbacid, 1987), partly by transducing growth-promoting signals. Whilst the exact nature of the biochemical processes involved in cell proliferation remains unclear, oncogenic mutations of p2lraScause delayed GTPhydrolysis and prolongation of the active (GTP-bound) state of the protein. This effect is mediated in part by oncoprotein resistance to the action of interacting GTP-ase activating proteins (GAP proteins) (Bourne et af., 1990; Lancet Editorial, 1990). By this means, oncogenic ras mutations result in prolonged and unregulated growth stimulation. A number of similarities between p2lraSand G-protein a-subunits have been observed. Thus Holbrook and Kim (1989) proposed a molecular model for G-protein a-subunits based on the structure of H-ras proteins. Oncogenic mutations of the Gs a-chain have been observed in pituitary (Landis et al., 1989) and thyroid tumours, and of the Gi a-chain in tumours of the adrenal cortex and ovary (Lyons et al., 1990). In all these cases a highly conserved arginine residue (Arg 201 in the Gs a-chain) is replaced. Current data suggest that this amino acid is essential for promoting GTP- hydrolysis and that this process is inhibited in the mutant species (Bourne et al., 1991). Much evidence has thus accumulated from recent work to suggest that events at membrane bound receptors may be involved in human tumorigenesis. As dopamine binding to the D 2 receptor initiates a series of inhibitory responses, then defective D2 receptor activity may be important in the development of adenomatous change. Furthermore, the observed effects of dopamine and dopamine agonists on pituitary tumours reflect the complex nature of dopamine receptor activity. Prolactinomas
Prolactin secretion is under tonic inhibitory control by dopamine (MacLeod & Lehmeyer, 1974; Leblanc et al., 1976). In uitro, dopamine and its agonists inhibit prolactin
Clinical Endocrinology (1991) 35
secretion from both normal and tumorous rat and human pituitary cells (Mashiter et al., 1977; Adams et al., 1979; Maurer, 1980). These effects are associated with a reduction in prolactin gene transcription and mRNA accumulation (Maurer, 1980, 1981, 1982). The regulation of gene transcription involves interactions between regulatory nuclear proteins (trans-factors) with specific DNA sequences (cis-factors). These cis-factors may be within the gene promoter region or form part of enhancer elements lying within or downstream from the structural gene. Such cis-trans interactions allow both tissue specific and hormonal regulation of gene transcription. Much is known about the structure of the prolactin gene and a number of cis and trans-factors have been identified (reviewed by Davis et a[., 1988). However, the mechanisms involved in dopaminergic control of prolactin gene transcription remain unclear. Most of the available data for the prolactin gene relate to the stimulatory effects of CAMP, mediated by CAMP-dependent protein kinase and nuclear protein phosphorylation (Murdoch et al., 1982; Day et al., 1989; Maurer, 1989). Recently a negative cis-acting factor has been located within the rat prolactin gene promoter, possibly involved in tissue-specificgene expression (Zhang et al., 1990). The identification of a negative element raises the possibility of specific inhibition of transcription by dopamine-stimulated intracellular events, over and above its effects on CAMP activity. Hyperprolactinaemia is the most common pituitary disorder seen in clinical practice. In the absence of secondary causes of hyperprolactinaemia (e.g. drugs or hypothyroidism), the majority of patients are found to have a prolactinoma, although visualization of the tumour depends on the degree of sophistication of the imaging technique used (Lancet Editorial, 1980; Burrow et al., 1982). Prolactinomas are commonly small intrasellar microadenomas but, especially in men, they may be large and present with signs of local tumour expansion (Prescott et al., 1982; Grossman & Besser, 1985). The inhibitory effects of dopamine on prolactin secretion led to the introduction of bromocriptine, an ergot-derived long-acting dopamine D2 receptor agonist, as first line treatment for hyperprolactinaemia (Besser et al., 1972; Franks et a[., 1977). Bromocriptine suppresses prolactin secretion and in the majority of cases causes shrinkage of large prolactin secreting tumour (McGregor et al., 1979; Thorner et al., 1980; Prescott et al., 1982; Molitch et al., 1985). However, the inhibitory effects of dopamine are not universally seen. Whilst serum prolactin concentrations generally fall in response to bromocriptine therapy, in up to one-third of reported cases there is either only a small reduction or no response in tumour size (Johnston et al., 1983; Molitch et al., 1985; von Werder et al., 1985; Davis et
Doparnine and pituitary turnours 459
al., 1990). Resistance to tumour shrinkage may cause difficulties in the clinical management of some patients with prolactinomas. The mechanism of dopamine resistance is unclear but the above data suggest dissociation of its effects on prolactin secretion from those on cell division. This may reflect the diversity of intracellular pathways triggered by the dopamine receptor. Further work is required to elucidate these pathways involved in different aspects of dopaminergic activity. The incidence of bromocriptine resistance and of intolerable side-effects have led to the investigation of a number of other ergot-based dopamine agonists such as pergolide in the treatment of prolactinomas (Franks et al., 1981; KendallTaylor el a[., 1982). None of these has significant advantages over bromocriptine. The new non-ergot-derived D2 receptor agonist CV205-502 successfully reduces serum prolactin levels and tumour size (Rasmussen et al., 1987; Barnett et al., 1990), effects which are mediated in part by inhibition of prolactin gene transcription (Davis et al., 1989). The results from one double-blind study comparing CV205-502 with bromocriptine in women with hyperprolactinaemia suggested that the non-ergot preparation may produce fewer side-effects (Homburg et af., 1990). However, to date there are no comparative data for patients who have received no previous dopamine agonist therapy, or for those with extrasellar turnours. Further experience of CV205-502 is necessary to define its therapeutic role in the management of prolactinomas. Growth hormone secreting tumours
Dopamine and its agonists reduce G H secretion from cultured rat and human pituitary cells (Adams et al., 1979, 1981; Hanew & Rennels, 1982; Markovitz et al., 1982; Cronin et al., 1984). These effects appear to be mediated by changes in secretion rather than by altered gene transcription (Wood et al., 1987). Conversely, dopamine agonists stimulate basal GH secretion in vivo when given to normal human subjects, through actions at the hypothalamus or above (Besseretal., 1972;Camannietal., 1975; Bansal etal., 1981a, b). Paradoxically in some acromegalics GH secretion is inhibited by dopamine agonist therapy. The reasons for this discrepancy remain unclear. In some patients the response to bromocriptine appears to be correlated with a rise in serum GH in response to TRH administration (Liuzzi et at., 1974b; Lamberts et al., 1982, 1983). A G H response to bromocriptine was found to be more common in those acromegalic patients with a mixed somatomammotrophic tumour and in particular in those with hyperprolactinaemia (Lamberts et al., 1982, 1983, 1985). However, these findings are not universally seen (Bassetti et al., 1988)and, to date, there is no
460
D. F. Wood et al.
reliable clinical indicator to suggest which acromegalic patients will respond to dopamine agonist therapy. Furthermore, although early studies suggested a significant reduction in circulating GH concentrations in up to 75% of patients (Thorner et at., 1975) leading to the widespread use of bromocriptine for the treatment of acromegaly, in other studies the response rate was less encouraging. Taken together, the data show that whilst dopamine agonist therapy is associated with a fall in circulating GH levels in about 50% of cases, normalization of G H and IGF-I concentrations is rarely achieved (Liuzzi et al., 1974a; Camanni et al., 1975; Schwinn et al., 1977; Wass et al., 1977; Halse et al., 1990). Bromocriptine does not cause a significant reduction in tumour size in most acromegalic patients (Oppizzi et al., 1984; Halse et al., 1990), indicating dissociated effects of dopamine on GH secretion and somatotroph cell division.
Clinical Endocrinology (1991) 35
Thyrotrophln secreting tumours
Dopamine inhibits TSH secretion by the normal pituitary both in vivo (Burrow et al., 1977; Scanlon et al., 1979) and in vitro (Foord et al., 1984) and may be involved in controlling the circadian rhythm of TSH secretion (Sowers et al., 1982). Pituitary tumours secreting TSH are rare (Smallridge, 1987). Patients have occasionally been reported to respond to dopamine agonist therapy and in one case dopamine receptors were identified in the resected tumour tissue (Chanson et al., 1984; Spada et al., 1985). However, in general there is no response of TSH secretion to dopamine or dopaminergic drugs (Smith et al., 1982; Shupnik et al., 1986; Samuels et al., 1989; Dunne et al., 1990). The available data suggest that TSH-secreting tumours may be associated with impaired dopamine receptor function, although this must remain speculative until further information becomes available.
The dopamine D2 receptor and pituitary tumorigenesls Gonadotrophln and a-subunit secreting tumours
Tumours secreting gonadotrophins or their subunits have been recognized increasingly and it is clear that many tumours previously classified as ‘non-functioning’ come within this category (Snyder, 1985; Klibanski et al., 1987; Lamberts etal., 1987; Anyaoku et a[., 1989; Kwekkeboom et d.,1989; Wood et al., 1990). The tumours possess dopamine binding sites (Bevan & Burke, 1986; Koga et al., 1987)and in the majority bromocriptine lowers serum gonadotrophin and free a-subunit concentrations (Vance et a!., 1985; Lamberts et al., 1987; Klibanski et al., 1988).Dopamine also reduces u-subunit mRNA accumulation in tumour tissue removed from patients with pure a-subunit secreting tumours, suggesting that these effects are mediated by reduced gene transcription (Klibanski et al., 1988). The reported effects of bromocriptine on tumour size have been variable. In general, no reduction in tumour size has been observed (Barrow et al., 1984; Grossman et al., 1985) but occasional patients clearly respond to dopaminergic therapy, especially when given for prolonged periods (Johnston et al., 1981; Wass et al., 1982; Vance et al., 1985; Klibanski et al., 1987, 1988). In-vitro studies have also suggested a timedependent increase in dopaminergic inhibition of gonadotrophin secretion from tumours maintained in long-term culture (Kwekkeboom et al., 1990). As with other pituitary tumours, therefore, the response of gonadotrophinomas to dopaminergic therapy is variable, particularly with respect to tumour shrinkage. The demonstration of specific dopamine receptors on tumour cell membranes in the absence of a response to bromocriptine therapy suggests that receptor function in these tumours may be defective.
Pituitary tumours are thus monoclonal in origin, and a proportion respond to the inhibitory effects of dopamine and dopamine agonists in terms of hormone secretion and tumour growth. From our knowledge of the biological events activated by the dopamine receptor, abnormalities of the number, structure or function of dopamine receptors may be implicated in the aetiology of certain tumours. For example, in a study of human prolactinomas which had proved resistant to bromocriptine therapy, Pellegrini et al. (1989) demonstrated a marked reduction in dopamine binding in vitro. Loss of binding sites may be important as a cause of the tumours or may be a secondary phenomenon. The lack of a dopamine sensitive tumour cell line for use in experiments on the dopamine receptor has led our own and other groups to study dopamine receptor activity in rat tumour cells which either fail to bind dopamine or for some other reason fail to regulate prolactin secretion in response to dopaminergic stimulation. We have studied dopamine D2 receptor expression in the GH3 rat pituitary cell line. This cell line lacks high affinity dopamine binding sites (Cronin et al., 1980). Using radiolabelled oligonucleotides complementary to sequences in the D2 receptor, we have identified D2 receptor mRNA in GH3 cells by northern blot analysis (Fig. 4). Furthermore, the putative D2 receptor protein was identified in GH3 cell membrane preparations subjected to SDS-PAGE analysis using a minigel system (Pharmacia Phast System; Fig. 5) (Johnston et al., 1991). Thus in this pituitary tumour model the lack of high affinity dopamine binding does not result from the absence of transcription of the D2 receptor gene, nor does it reflect the fact that the receptor protein cannot be synthesized. Other workers have
Clinical Endocrinology (1991)35
I
Dopamine and pituitary tumours
461
studied the 7315a prolactinoma line, cells which bind dopamine but in which prolactin release is not suppressed (Cronin et al., 1981). D2 receptor messenger RNA and protein have, as expected, been found in these tumour cells (Lew et al., 1990). The defect here may thus be at the postreceptor level and abnormalities of the G-proteins are possible candidates. It is uncertain at present whether or not similar defects occur in human prolactinomas which fail to respond to dopamine agonist therapy.
2
8 1.4
8 5.3
285
B 2.8 kb Summary
Dopamine plays an important role in the hypothalamicpituitary axis where its major effects are to inhibit pituitary hormone secretion and cell division. Chronic dopamine deficiency has been postulated as a cause of pituitary tumour formation and several lines of evidence exist to suggest that a functional deficiency may develop as a result of defective dopamine receptor action. The available data suggest that a number of sites in the dopamine-D2 receptor-second messenger pathways may be implicated. These abnormalities are reflected in the variety of responses to dopamine and its agonists which have been observed in pituitary tumours both in the clinical situation and in cultured cells in uitro. Whilst it seems likely that the primary defect in pituitary tumour formation lies within the pituitary itself, the role of hypothalamic factors in facilitating tumour growth remains to be explored. Further studies of the dopamine receptor and its function will be of value not only in pathophysiological
2.5
8 1.9 18s
8 1-6
Fig. 4 Northern blot analysis of D2 receptor mRNA from normal rat pituitary cells and GH3 rat pituitary tumour cells. Oligonucleotide cDNA sequences complementary to bases I 1 11- 1 140 of the D2 receptor gene (encoding a specific sequence within the third cytoplasmic loop) were synthesized and tail-end labelled with 32P. Bands observed at 2.5 kb correspond to D2 receptor mRNA. Equivalent amounts were detected in northern blots of RNA extracted from normal and tumorous cells. The positions of ribosomal and molecular weight markers are indicated. Lane 1, total RNA from GH3 cells (2 pg); lane 2. total RNA from normal rat anterior pituitary cells (2 p g ) . (From Johnston et d.,1991, with permission.)
I
2
3
4
kb
1m.o
8 97.4
Fig. 5 SDS-PAGE analysis of normal rat (lanes 3 and 4, 3 p g ) and GH3 (lanes 1 and
2, 3 pg) pituitary cell membranes showing silver staining of minigel obtained using the Pharmacia Phast System (Pharmacia Biotechnology Ltd). Partially purified membrane preparations from normal and GH3 cells contained bands visible at 93-96 kDa and 32-36 kDa corresponding to bands observed in rat striatal preparations and attributed to the D2 receptor (Lew et al., 1990). For details of method see Johnston et at. (1991).
8 5S.4
8 36-5
20.1
482 D. F. Wood et e l .
studies of human pituitary adenomas, but also in the development of new pharmacological agents to treat patients Wiih these turnours.
References Adams, E.F., Brajkovich, I.E. & Mashiter. K. (1979) Hormone m e t i o n by dispersed cell cultures of human pituitary adenomas: effects of theophylline, thyrotropin-releasing hormone, somatostatin and 2-bromo-a-ergocryptine. Journal of Clinical Endocrinology and Metabolism, 49, 120- 126. Adams. E.F.. Brajkovich. I.E. & Mashiter, K. (1981) Growth hormone and prolactin secretion by dispersed cell cultures of a normal human pituitary: effects of thyrotrophin-releasing hormone, theophylline, somatostatin and 2-bromo-a-ergocryptine. Acta Endocrinologica,98. 345-351. Alexander, J.M., Biller, B.M.K., Bikkal, H., Zervas. N.T.,Arnold, A. & Klibanski. A. (1990) Clinically non-functioning pituitary tumoun are monoclonal in origin. Journal of Clinical Inoestigation, 86,336-340. Andersen, P.H.. Gingrich, J.A., Bates, M.D., Dearry, A., Falardeau, P., Senogles, S.E. & Caron, M.G. (1990) Dopamine receptor subtypes: beyond the D1/D2 classification. Tips, 11,231 -236. Anyaoku, V., Wood, D.F.. Williams, P., Tan, K. &Johnston, D.G. (1989) Measurement ofserum a-subunit: a rapid sensitive ELlSA technique. Journal of Endocrinology, 123 (Suppl.), 168. Bansal, S., Lee, L. & Woolf, P.D. (1981a) Dopaminergic regulation of growth hormone secretion in normal man: correlation of Ldopa and dopamine levels with the GH response. Journal of Clinical Encocrinology and Metabolism, 53, 301-306. Bansal, S.A., Lee, L.A. & Woolf, P.D. (1981b) Dopaminergic stimulation and inhibition of growth hormone secretion in normal man: studies of the pharmacologic specificity. Journal of Clinical Endocrinology and Metaholism, 53, 1273-1277. Barbacid, M.A. (1987) ras genes. Annual Reuiew of Biochemistry, 56, 779-827. Barbarino, A., Demarinis, L., Anile, C., Menini. E., Merlini, G. & Maira, G. (1982) Dopaminergic mechanisms regulating prolactin secretion in patients with prolactin secreting pituitary adenoma. Long-term studies after selective transphenoidal surgery. Metabolism, 31, 1100-1104. Barnett, P.S., Dawson, J.M., Butler, J., Coskeran, P.B., Maccabe, J.J. & McGregor, A.M. (1990) CV205-502, a new non-ergot dopamine agonist reduces prolactinoma size in man. Clinical Endocrinology. 33,307-3 16. Barrow, D.L., Tindall, G.T., Kovacs. K., Thorner, M.O., Horvath, E. & Hoffman Jnr, J.C. (1984) Clinical and pathological effects of bromocriptine on prolactin-secreting and other pituitary tumoun. Journal of Neurosurzery, 60, 1-7. Bassetti, M., Arosio, M.. Spada, A., Brina, M., Bauoni, N.,Faglia. G. & Giannattasio, G.(1988) Growth hormone and prolactin secretion in acromegaly: correlations between hormonal dynamics and immunocytochemical findings. Journal of Clinical Endocrinology and Metabolism. 67, I 195- 1204. Baudry, M., Evans, J. & Lynch, G. (1986) Excitatory amino acids inhibit stimulation of phospatidyl inositol metabolism by aminergic agonists in hippocampus. Nature, 319,329-33 I. Besser, G.M., Parke, L.. Edwards, C.R., Forsyth, LA. & McNeilly, AS. (1972) Galactorrhoea: successful treatment with reduction of
Clinical Endocrinology (1991) 35
plasma prolactin levels by brom-ergocryptine. British Medical Journal, 3,669-672. Bevan, J.S. & Burke, C.W. (1986) Non-functioning pituitary adenomas d o not regress during bromocriptine therapy but possess membrane-bound dopamine receptors which bind bromocriptine. Clinical Endocrinology, 25, 561-572. Bourne, H.R. (1989) G-protein subunits: who carries what message. Naiure, 337, 504-505. Bourne, H.R., Sanders, D.A. & McCormick, F. (1990) The GTPase superfamily: a conserved switch for diverse cell functions. Nature, 348, 125-132. Bourne, H.R., Sanders, D.A. & McCormick, F. (1991)The GTPasc superfamily: conserved structure and molecular mc
Review
Dopamine, the dopamine D2 receptor and pituitary tumours D. F. Wood, J. M. Johnston and D. G. Johnston Unit of Metabolic Medicine, St Mary’s Hospital Medical School, London, UK (Received 2 July 1991; returned for revision 12 August 1991; finally revised 23 August 1991; accepted 10 September 1991)
In the hypothalamic pituitary axis dopamine acts both as a neurotransmitter and as a hormone, a dual role which is, in part, responsible for the complex nature of its action. Whilst dopamine directly inhibits anterior pituitary function, its secretory pattern and biological effects are modified by interactions with other hypothalamic factors and by feedback from the pituitary itself. Within the anterior pituitary the inhibitory effects of dopamine are mediated by membrane bound receptors of the D2 subtype. Recent identification of the gene encoding the D2 receptor, prediction of its protein structure and increasing recognition of the diversity of intracellular events triggered by D2 receptor activation now allow greater insight into intracellular dopamine action. Abnormalities of dopamine activity have been described in association with a number of pituitary tumours. These observations have led to the hypothesis that chronic dopamine deficiency may be involved in the development of such tumours. In this review we aim to re-examine this hypothesis in the light of recent investigations into the aetiology of pituitary tumours and the increasing knowledge of dopamine D2 receptor activity. The aetiology of pituitary tumours
Despite the well recognized clinical syndromes associated with pituitary adenomas, their aetiology has remained largely obscure. However, there is now increasing evidence that these tumours arise from intrinsic pituitary cell defects caused by somatic mutation. Whilst rarely malignant, pituitary tumours exhibit features of neoplasia and morphologically they represent discrete adenomas (Kovacs et al., 1978; Melmed el al., 1983). This is in contrast to pituitary hyperplasia associated with ectopic releasing hormone production (Melmed & Rushakoff, 1987). In general, neoplasia involves expansion of a clonal cell line (Fialkow, 1986). Two recent studies have used the technique of X-chromosome inactivation analysis to study the clonal nature of pituitary Correspondence: D. F. Wood, Unit of Metabolic Medicine, St Mary’s Hospital Medical School, London W2 lPG, UK.
tumours (Alexander et al., 1990; Herman et al., 1990). This technique, developed by Vogelstein et al. (1987), exploits restriction fragment length polymorphisms (RFLP) and differential methylation patterns in two X-linked genes, HPRT and PGK, in female cells. In any female cell only one X-chromosome, either maternally or paternally derived, is active. Therefore if tumour cells are monoclonal in origin, the same X-chromosome will be active in all of them and this can be detected by RFLP and methylation analysis. In the studies cited, the majority of pituitary tumours were found to be monoclonal in nature. Limitations in the experimental method, allowing inclusion of some normal surrounding pituitary tissue, may have been responsible for inappropriate assignment of some of the tumours as polyclonal (Herman et al., 1990). A mechanism whereby somatic mutation may lead to adenoma formation has been identified in some growth hormone (GH) secreting pituitary adenomas. Vallar et al. (1987) identified a subset of these tumours characterized by high GH release and intracellular cAMP accumulation in vitro, events associated with constitutive activation of adenylyl cyclase. These tumours expressed somatic mutations in the a-subunit of the stimulatory G-protein,Gs (see below), which is involved in signal transduction at the growth hormone-releasing hormone receptor and which therefore plays a role in regulating both G H release and somatotroph cell division. The postulated oncogene Gsp which encodes the mutant protein may thus allow unrestrained hormone secretion and cell division (Landis et al., 1989; McCormick, 1989). Expression of Gsp has been identified in 3040% of human GH-secreting pituitary adenomas (Landis et al., 1990; Spada et al., 1990). In two recent studies the role of cAMP as a regulator of somatotroph cell division was demonstrated in transgenic mice. Animals expressing a transgene encoding non-functional cAMP binding protein (CREB, required to mediate the transcriptional effects of CAMP) were found to have somatic hypoplasia and dwarfism (Struthers et al., 1991). Conversely, use of the rat GH gene promoter to express an intracellular form of cholera toxin, which irreversibly activates Gs, produced transgenic mice with somatotroph hyperplasia and gigantism (Burton et al., 1991). However, these animals showed no evidence of pituitary tumour formation. A longer period of hyperplasia or other oncogenic events in addition to the constitutive activation of adenylyl cyclase may be required to instigate neoplastic change. Another piece of evidence to support the theory of an intrinsic pituitary defect in the aetiology of pituitary tumours 455
456
D. F. Wood eta/.
was supplied by in-situ hybridization studies of resected tumours from acromegalic patients (Lloyd et al., 1989). Eighty-seven per cent of these tumours were found to contain prolactin messenger RNA, despite the lack of prolactin gene expression in normal somatotrophs. This may indicate neoplastic transformation of primitive somatomammotrophs or dedifferentiation of existing somatotrophs. Dopamine and pltultary tumorigenesls
Dopamine inhibits both hormone secretion and cell division in the anterior pituitary (see below) (Jacobi & Lloyd, 1981; Cronin et al., 1982). The abnormalities of dopamine activity associated with some pituitary tumours suggest that chronic dopamine deficiency may be involved in their development. This may be produced in a number of ways. Hypothalamic dysfunction has been postulated in the case of prolactinomas (Fine & Frohman, 1978; Saeger & Ludecke, 1983; Landolt & Minder, 1984). However, in the majority of cases prolactin and TSH responses to secretagogues or inhibitors are restored to or towards normal following tumour resection, suggesting that hypothalamic dysfunction is not a primary defect (Tucker et al., 1980; Schlecte & Sherman, 1981; Barbarino et al., 1982; Molitch et al., 1982; Rodman et al., 1984). Abnormal pituitary vasculature with the development of a systemic arterial blood supply has been observed in some pituitary tumours (Weiner et al., 1987; Schecter et al., 1988). This may allow localized ‘escape’ of parts of the pituitary from dopamine input via the hypophyseal portal circulation. However, the acquisition of an arterial blood supply may be a secondary phenomenon as a newly developed ‘tumour circulation’ is observed in many human tumours. Finally, intrinsic pituitary abnormalities may arise whereby somatic mutations occur in the D2 receptor or its intracellular mediators. Such abnormalities, producing an effective intrapituitary dopamine deficiency, are under investigation in several laboratories. Dopamlne actlon at the anterlor pituitary: the doparnine D2 receptor
Classically, dopamine receptors have been divided into two major groups: those which stimulate adenylyl cyclase activity (D1 receptors) and those which inhibit this enzyme (D2 receptors) (Kebabian & Calne, 1979; Enjalbert & Bockaert, 1983).In the past decade investigation into the mechanism of dopamine action in the central nervous system has suggested that further dopamine receptor subtypes exist, based on the diversity of biochemical, pharmacological and physiological responses observed. The evidence for multiple receptor subtypes has been reviewed recently (Andersen et al., 1990)
Clinical Endocrinology (1991) 35
and three further discrete subtypes have been described (D3, D4, D5; Sokoloff et al., 1990; Van To1 et al., 1991; Sunahara et al., 1991). Anterior pituitary dopamine receptors are of the D2 subtype, mediating the inhibitory effects of dopamine on anterior pituitary function. Activation of the D2 receptor produces a variety of intracellular responses in addition to decreasing CAMP concentrations (De Camilli et al., 1979). These include changes in potassium channel activity (Israel et al., 1985; Catelletti et al., 1989) and calcium channel flux (Taraskevich & Douglas, 1978), inhibition of phosphatidyl inositol turnover (Vallar & Meldolesi, 1989) and alteration of intracellular calcium concentrations (Baudry et al., 1986; Enjalbert et al., 1986). Cloning and expression of a cDNA encoding the rat D2 receptor showed it to have typical features of a guanine nucleotide binding protein (G-protein)-coupling receptor (Bunzow et al., 1988). The diverse intracellular effects produced by D2 receptor activation suggest that signal transduction is mediated by several different G-proteins (Senogles et al., 1987; Ohara et al., 1988; Elazar et al., 1989). These events are shown schematically in Fig. 1. The human D2 receptor gene has been localized to chromosome 11 (Grandy et al., 1989a) and shows high homology with the rat and bovine genes (Grandy et al., 1989b;Monsma et al., 1989; Chi0 et al., 1990). In all cases the deduced amino acid sequences contain the seven transmembrane domains and the long third intracytoplasmic loop typial of G-protein coupled receptors (Dohlman et al., 1987; Johnson & Dhanasekaran, 1989). The D2 receptor gene has a number of characteristic features. Whilst most G-proteincoupled receptor genes lack introns within their coding sequences (O’Dowd et al., 1989), the dopamine D2 receptor gene has a more complex organization being at least 12 kb long with a minimum of six introns (Giros et al., 1989). The D2 receptor cDNA encodes a single polypeptide of 415 amino acids with a molecular mass of approximately 47 kDa. This corresponds to the deglycosylated receptor previously described (Jarvie & Niznik, 1989). The overall molecular mass of the D2 receptor is approximately 110-140 kDa although two subunits of 94 kDa and 35 kDa are observed on electrophoresis (Lew et al., 1990). A second receptor isoform of 444 amino acids has been described, containing a 29-amino-acid insertion in the third intracytoplasmic loop (Fig. 2). The two isoforms are generated by alternative RNA splicing (Dal Toso et a11989; Eidne et al., 1989; Giros et al., 1989; Monsma et al., 1989; Chi0 et al., 1990). Both isoforms of the D2 receptor can be identified within the anterior pituitary gland, with the longer form being in greater abundance (Dal Toso et al., 1989). In receptors of this family the third intracytoplasmic loop appears to be important for
Dopamine and pituitary tumours 457
Clinical Endocrinology (1991) 35
Cell
A1
Fig. 1 Signal transduction at the dopamine D2 receptor. Dopamine-D2 receptor binding activates a number of intracellular pathways (see text for references). Dopamine binding inhibits adenylyl cyclase activity via the G-protein Gi , lowering intracellular CAMPconcentration and subsequently causing a reduction in calcium mobilization. Activation of Gk by the bound receptor causes potassium efflux. Inhibition of phosphoinositol bisphosphate hydrolysis occurs, probably mediated by the action of Go G-proteins, which may also have direct effects on intracellular calcium concentrations. Membrane calcium channel activity is altered. The overall effects of D2 receptor activation are to lower intracellular Ca2+ concentrations and this decrease causes the alterations in hormone release and gene transcription. The effects of D2 receptor activation on phospholipase C activity remain to be elucidated. D2R, dopamine D2 receptor; DA, dopamine; Gi, Gk, Go, G-proteins involved in signal transduction; AC, adenylyl cyclase; PIP2, phosphoinositol bisphosphate; IP3, inositol trisphosphate; DAG, diacyl glycerol; PKC, protein kinase C, PLC, phospholipase C.
Extracellular
241-270 \
*
*
Fig. 2 Structure of the dopamine D2 receptor. The seven transmembrane domains are shown. Two isoforms, D2[415] and D2[444] are generated by alternative splicing of the primary RNA transcript. The position of the 29 amino acid insert in the long third intracytoplasmic loop is thought to correspond to G-protein binding sites, suggesting different biological activities for the two receptor isoforms.
G-protein coupling (reviewed by Ross, 1989). Receptor heterogeneity produced by alternative RNA splicing may be important in determining the binding to different G-proteins (De Keyser ef al., 1989) and in the production of the diverse intracellular effects characteristic of D2 receptor activation.
Signal transduction at the D2 receptor:G-protein action
Signal transduction at the D2 receptor is effected by a series of G-proteins. G-proteins form part of a newly recognized (and rapidly expanding) superfamily of intracellular proteins
458 D. f . Wood et a/.
Clinical Endocrinology (1991) 35
I
iI
GTF
a-GTP
Effector protein
a-GTP-E
(Ei PI
protein
(E)
1
lntracel lular response
Fig. 3 G-protein action: the cycle of GTP binding and hydrolysis. Activated receptors promote dissociation of GDP from the G-protein a-subunit. GTJ? binding to a in turn promotes dissociation of the 1-y complex and allows activation of intracellular effects. The intrinsic GTP-ase activity of the a-subunit terminates its interaction with an effector and the trimeric G-protein is reconstituted.
characterized by their ability to bind and hydrolyse GTP in a unidirectional cyclical fashion. These proteins act as molecular ‘switches’ to trigger a variety of intracellular responses. The properties of the GTP-ase family are discussed in two recent reviews (Bourne et a[., 1990; 1991). Membrane associated G-proteins are heterotrimeric structures comprising a, /3 and y-subunits. Their role in transmembrane signallinghas been the subject of a number of extensive reviews (Tomlinson et al., 1985; Gilman, 1987; Neer & Clapham, 1988; Ross, 1989; Taylor, 1990). At least eight different G-proteins have been characterized. The a-subunits appear to confer structural and functional specificity whereas the P-y complexes are functionally similar (Bourne et al., 1990). @-Subunitscontain recognition sites for receptors and effector proteins and the guanine nucleotide binding site. The p and y-subunits form a tight complex whose function is not fully elucidated, but which may be involved in anchoring the protein to the cytoplasmic face of the cell membrane and in ‘presenting’ the a-subunit to the receptor (Sternweis, 1986). The cycle of events which follow receptor-G-protein binding is shown in Fig. 3. Activated (ligand-bound) receptors promote dissociation of GDP from the a-subunit and may increase affinity of the a-subunit for GTP-binding (Florio & Sternweis, 1989). GTP-binding in turn promotes dissociation of B-y from the a-GTP complex, allowing the latter to activate intracellular effectors (adenylyl cyclase, phospholipases, etc.). A potential role for the p-y complex in control-
ling other effector proteins remains controversial (Bourne, 1989; Taylor, 1990). Interaction of a-GTP with an effector is terminated by the intrinsic GTP-ase activity of the a-subunit. Subsequently, a-GDP binds to p-y again and reconstitutes the trimeric proteins ready for further activation. In this way, G-proteins are able to transduce and, by means of complex timing mechanisms, to amplify a wide range of transmembrane signals through a smaller set of intracellular effector molecules. The ras proteins form another subset of mammalian GTPases which have been widely investigated because mutations in the ras genes cause neoplastic transformation. The three ras genes, H-ras, K-ras and N-ras, encode 21 kDa proteins (p2Iras)which are involved in the regulation of cell division and differentiation (Barbacid, 1987), partly by transducing growth-promoting signals. Whilst the exact nature of the biochemical processes involved in cell proliferation remains unclear, oncogenic mutations of p2lraScause delayed GTPhydrolysis and prolongation of the active (GTP-bound) state of the protein. This effect is mediated in part by oncoprotein resistance to the action of interacting GTP-ase activating proteins (GAP proteins) (Bourne et af., 1990; Lancet Editorial, 1990). By this means, oncogenic ras mutations result in prolonged and unregulated growth stimulation. A number of similarities between p2lraSand G-protein a-subunits have been observed. Thus Holbrook and Kim (1989) proposed a molecular model for G-protein a-subunits based on the structure of H-ras proteins. Oncogenic mutations of the Gs a-chain have been observed in pituitary (Landis et al., 1989) and thyroid tumours, and of the Gi a-chain in tumours of the adrenal cortex and ovary (Lyons et al., 1990). In all these cases a highly conserved arginine residue (Arg 201 in the Gs a-chain) is replaced. Current data suggest that this amino acid is essential for promoting GTP- hydrolysis and that this process is inhibited in the mutant species (Bourne et al., 1991). Much evidence has thus accumulated from recent work to suggest that events at membrane bound receptors may be involved in human tumorigenesis. As dopamine binding to the D 2 receptor initiates a series of inhibitory responses, then defective D2 receptor activity may be important in the development of adenomatous change. Furthermore, the observed effects of dopamine and dopamine agonists on pituitary tumours reflect the complex nature of dopamine receptor activity. Prolactinomas
Prolactin secretion is under tonic inhibitory control by dopamine (MacLeod & Lehmeyer, 1974; Leblanc et al., 1976). In uitro, dopamine and its agonists inhibit prolactin
Clinical Endocrinology (1991) 35
secretion from both normal and tumorous rat and human pituitary cells (Mashiter et al., 1977; Adams et al., 1979; Maurer, 1980). These effects are associated with a reduction in prolactin gene transcription and mRNA accumulation (Maurer, 1980, 1981, 1982). The regulation of gene transcription involves interactions between regulatory nuclear proteins (trans-factors) with specific DNA sequences (cis-factors). These cis-factors may be within the gene promoter region or form part of enhancer elements lying within or downstream from the structural gene. Such cis-trans interactions allow both tissue specific and hormonal regulation of gene transcription. Much is known about the structure of the prolactin gene and a number of cis and trans-factors have been identified (reviewed by Davis et a[., 1988). However, the mechanisms involved in dopaminergic control of prolactin gene transcription remain unclear. Most of the available data for the prolactin gene relate to the stimulatory effects of CAMP, mediated by CAMP-dependent protein kinase and nuclear protein phosphorylation (Murdoch et al., 1982; Day et al., 1989; Maurer, 1989). Recently a negative cis-acting factor has been located within the rat prolactin gene promoter, possibly involved in tissue-specificgene expression (Zhang et al., 1990). The identification of a negative element raises the possibility of specific inhibition of transcription by dopamine-stimulated intracellular events, over and above its effects on CAMP activity. Hyperprolactinaemia is the most common pituitary disorder seen in clinical practice. In the absence of secondary causes of hyperprolactinaemia (e.g. drugs or hypothyroidism), the majority of patients are found to have a prolactinoma, although visualization of the tumour depends on the degree of sophistication of the imaging technique used (Lancet Editorial, 1980; Burrow et al., 1982). Prolactinomas are commonly small intrasellar microadenomas but, especially in men, they may be large and present with signs of local tumour expansion (Prescott et al., 1982; Grossman & Besser, 1985). The inhibitory effects of dopamine on prolactin secretion led to the introduction of bromocriptine, an ergot-derived long-acting dopamine D2 receptor agonist, as first line treatment for hyperprolactinaemia (Besser et al., 1972; Franks et a[., 1977). Bromocriptine suppresses prolactin secretion and in the majority of cases causes shrinkage of large prolactin secreting tumour (McGregor et al., 1979; Thorner et al., 1980; Prescott et al., 1982; Molitch et al., 1985). However, the inhibitory effects of dopamine are not universally seen. Whilst serum prolactin concentrations generally fall in response to bromocriptine therapy, in up to one-third of reported cases there is either only a small reduction or no response in tumour size (Johnston et al., 1983; Molitch et al., 1985; von Werder et al., 1985; Davis et
Doparnine and pituitary turnours 459
al., 1990). Resistance to tumour shrinkage may cause difficulties in the clinical management of some patients with prolactinomas. The mechanism of dopamine resistance is unclear but the above data suggest dissociation of its effects on prolactin secretion from those on cell division. This may reflect the diversity of intracellular pathways triggered by the dopamine receptor. Further work is required to elucidate these pathways involved in different aspects of dopaminergic activity. The incidence of bromocriptine resistance and of intolerable side-effects have led to the investigation of a number of other ergot-based dopamine agonists such as pergolide in the treatment of prolactinomas (Franks et al., 1981; KendallTaylor el a[., 1982). None of these has significant advantages over bromocriptine. The new non-ergot-derived D2 receptor agonist CV205-502 successfully reduces serum prolactin levels and tumour size (Rasmussen et al., 1987; Barnett et al., 1990), effects which are mediated in part by inhibition of prolactin gene transcription (Davis et al., 1989). The results from one double-blind study comparing CV205-502 with bromocriptine in women with hyperprolactinaemia suggested that the non-ergot preparation may produce fewer side-effects (Homburg et af., 1990). However, to date there are no comparative data for patients who have received no previous dopamine agonist therapy, or for those with extrasellar turnours. Further experience of CV205-502 is necessary to define its therapeutic role in the management of prolactinomas. Growth hormone secreting tumours
Dopamine and its agonists reduce G H secretion from cultured rat and human pituitary cells (Adams et al., 1979, 1981; Hanew & Rennels, 1982; Markovitz et al., 1982; Cronin et al., 1984). These effects appear to be mediated by changes in secretion rather than by altered gene transcription (Wood et al., 1987). Conversely, dopamine agonists stimulate basal GH secretion in vivo when given to normal human subjects, through actions at the hypothalamus or above (Besseretal., 1972;Camannietal., 1975; Bansal etal., 1981a, b). Paradoxically in some acromegalics GH secretion is inhibited by dopamine agonist therapy. The reasons for this discrepancy remain unclear. In some patients the response to bromocriptine appears to be correlated with a rise in serum GH in response to TRH administration (Liuzzi et at., 1974b; Lamberts et al., 1982, 1983). A G H response to bromocriptine was found to be more common in those acromegalic patients with a mixed somatomammotrophic tumour and in particular in those with hyperprolactinaemia (Lamberts et al., 1982, 1983, 1985). However, these findings are not universally seen (Bassetti et al., 1988)and, to date, there is no
460
D. F. Wood et al.
reliable clinical indicator to suggest which acromegalic patients will respond to dopamine agonist therapy. Furthermore, although early studies suggested a significant reduction in circulating GH concentrations in up to 75% of patients (Thorner et at., 1975) leading to the widespread use of bromocriptine for the treatment of acromegaly, in other studies the response rate was less encouraging. Taken together, the data show that whilst dopamine agonist therapy is associated with a fall in circulating GH levels in about 50% of cases, normalization of G H and IGF-I concentrations is rarely achieved (Liuzzi et al., 1974a; Camanni et al., 1975; Schwinn et al., 1977; Wass et al., 1977; Halse et al., 1990). Bromocriptine does not cause a significant reduction in tumour size in most acromegalic patients (Oppizzi et al., 1984; Halse et al., 1990), indicating dissociated effects of dopamine on GH secretion and somatotroph cell division.
Clinical Endocrinology (1991) 35
Thyrotrophln secreting tumours
Dopamine inhibits TSH secretion by the normal pituitary both in vivo (Burrow et al., 1977; Scanlon et al., 1979) and in vitro (Foord et al., 1984) and may be involved in controlling the circadian rhythm of TSH secretion (Sowers et al., 1982). Pituitary tumours secreting TSH are rare (Smallridge, 1987). Patients have occasionally been reported to respond to dopamine agonist therapy and in one case dopamine receptors were identified in the resected tumour tissue (Chanson et al., 1984; Spada et al., 1985). However, in general there is no response of TSH secretion to dopamine or dopaminergic drugs (Smith et al., 1982; Shupnik et al., 1986; Samuels et al., 1989; Dunne et al., 1990). The available data suggest that TSH-secreting tumours may be associated with impaired dopamine receptor function, although this must remain speculative until further information becomes available.
The dopamine D2 receptor and pituitary tumorigenesls Gonadotrophln and a-subunit secreting tumours
Tumours secreting gonadotrophins or their subunits have been recognized increasingly and it is clear that many tumours previously classified as ‘non-functioning’ come within this category (Snyder, 1985; Klibanski et al., 1987; Lamberts etal., 1987; Anyaoku et a[., 1989; Kwekkeboom et d.,1989; Wood et al., 1990). The tumours possess dopamine binding sites (Bevan & Burke, 1986; Koga et al., 1987)and in the majority bromocriptine lowers serum gonadotrophin and free a-subunit concentrations (Vance et a!., 1985; Lamberts et al., 1987; Klibanski et al., 1988).Dopamine also reduces u-subunit mRNA accumulation in tumour tissue removed from patients with pure a-subunit secreting tumours, suggesting that these effects are mediated by reduced gene transcription (Klibanski et al., 1988). The reported effects of bromocriptine on tumour size have been variable. In general, no reduction in tumour size has been observed (Barrow et al., 1984; Grossman et al., 1985) but occasional patients clearly respond to dopaminergic therapy, especially when given for prolonged periods (Johnston et al., 1981; Wass et al., 1982; Vance et al., 1985; Klibanski et al., 1987, 1988). In-vitro studies have also suggested a timedependent increase in dopaminergic inhibition of gonadotrophin secretion from tumours maintained in long-term culture (Kwekkeboom et al., 1990). As with other pituitary tumours, therefore, the response of gonadotrophinomas to dopaminergic therapy is variable, particularly with respect to tumour shrinkage. The demonstration of specific dopamine receptors on tumour cell membranes in the absence of a response to bromocriptine therapy suggests that receptor function in these tumours may be defective.
Pituitary tumours are thus monoclonal in origin, and a proportion respond to the inhibitory effects of dopamine and dopamine agonists in terms of hormone secretion and tumour growth. From our knowledge of the biological events activated by the dopamine receptor, abnormalities of the number, structure or function of dopamine receptors may be implicated in the aetiology of certain tumours. For example, in a study of human prolactinomas which had proved resistant to bromocriptine therapy, Pellegrini et al. (1989) demonstrated a marked reduction in dopamine binding in vitro. Loss of binding sites may be important as a cause of the tumours or may be a secondary phenomenon. The lack of a dopamine sensitive tumour cell line for use in experiments on the dopamine receptor has led our own and other groups to study dopamine receptor activity in rat tumour cells which either fail to bind dopamine or for some other reason fail to regulate prolactin secretion in response to dopaminergic stimulation. We have studied dopamine D2 receptor expression in the GH3 rat pituitary cell line. This cell line lacks high affinity dopamine binding sites (Cronin et al., 1980). Using radiolabelled oligonucleotides complementary to sequences in the D2 receptor, we have identified D2 receptor mRNA in GH3 cells by northern blot analysis (Fig. 4). Furthermore, the putative D2 receptor protein was identified in GH3 cell membrane preparations subjected to SDS-PAGE analysis using a minigel system (Pharmacia Phast System; Fig. 5) (Johnston et al., 1991). Thus in this pituitary tumour model the lack of high affinity dopamine binding does not result from the absence of transcription of the D2 receptor gene, nor does it reflect the fact that the receptor protein cannot be synthesized. Other workers have
Clinical Endocrinology (1991)35
I
Dopamine and pituitary tumours
461
studied the 7315a prolactinoma line, cells which bind dopamine but in which prolactin release is not suppressed (Cronin et al., 1981). D2 receptor messenger RNA and protein have, as expected, been found in these tumour cells (Lew et al., 1990). The defect here may thus be at the postreceptor level and abnormalities of the G-proteins are possible candidates. It is uncertain at present whether or not similar defects occur in human prolactinomas which fail to respond to dopamine agonist therapy.
2
8 1.4
8 5.3
285
B 2.8 kb Summary
Dopamine plays an important role in the hypothalamicpituitary axis where its major effects are to inhibit pituitary hormone secretion and cell division. Chronic dopamine deficiency has been postulated as a cause of pituitary tumour formation and several lines of evidence exist to suggest that a functional deficiency may develop as a result of defective dopamine receptor action. The available data suggest that a number of sites in the dopamine-D2 receptor-second messenger pathways may be implicated. These abnormalities are reflected in the variety of responses to dopamine and its agonists which have been observed in pituitary tumours both in the clinical situation and in cultured cells in uitro. Whilst it seems likely that the primary defect in pituitary tumour formation lies within the pituitary itself, the role of hypothalamic factors in facilitating tumour growth remains to be explored. Further studies of the dopamine receptor and its function will be of value not only in pathophysiological
2.5
8 1.9 18s
8 1-6
Fig. 4 Northern blot analysis of D2 receptor mRNA from normal rat pituitary cells and GH3 rat pituitary tumour cells. Oligonucleotide cDNA sequences complementary to bases I 1 11- 1 140 of the D2 receptor gene (encoding a specific sequence within the third cytoplasmic loop) were synthesized and tail-end labelled with 32P. Bands observed at 2.5 kb correspond to D2 receptor mRNA. Equivalent amounts were detected in northern blots of RNA extracted from normal and tumorous cells. The positions of ribosomal and molecular weight markers are indicated. Lane 1, total RNA from GH3 cells (2 pg); lane 2. total RNA from normal rat anterior pituitary cells (2 p g ) . (From Johnston et d.,1991, with permission.)
I
2
3
4
kb
1m.o
8 97.4
Fig. 5 SDS-PAGE analysis of normal rat (lanes 3 and 4, 3 p g ) and GH3 (lanes 1 and
2, 3 pg) pituitary cell membranes showing silver staining of minigel obtained using the Pharmacia Phast System (Pharmacia Biotechnology Ltd). Partially purified membrane preparations from normal and GH3 cells contained bands visible at 93-96 kDa and 32-36 kDa corresponding to bands observed in rat striatal preparations and attributed to the D2 receptor (Lew et al., 1990). For details of method see Johnston et at. (1991).
8 5S.4
8 36-5
20.1
482 D. F. Wood et e l .
studies of human pituitary adenomas, but also in the development of new pharmacological agents to treat patients Wiih these turnours.
References Adams, E.F., Brajkovich, I.E. & Mashiter. K. (1979) Hormone m e t i o n by dispersed cell cultures of human pituitary adenomas: effects of theophylline, thyrotropin-releasing hormone, somatostatin and 2-bromo-a-ergocryptine. Journal of Clinical Endocrinology and Metabolism, 49, 120- 126. Adams. E.F.. Brajkovich. I.E. & Mashiter, K. (1981) Growth hormone and prolactin secretion by dispersed cell cultures of a normal human pituitary: effects of thyrotrophin-releasing hormone, theophylline, somatostatin and 2-bromo-a-ergocryptine. Acta Endocrinologica,98. 345-351. Alexander, J.M., Biller, B.M.K., Bikkal, H., Zervas. N.T.,Arnold, A. & Klibanski. A. (1990) Clinically non-functioning pituitary tumoun are monoclonal in origin. Journal of Clinical Inoestigation, 86,336-340. Andersen, P.H.. Gingrich, J.A., Bates, M.D., Dearry, A., Falardeau, P., Senogles, S.E. & Caron, M.G. (1990) Dopamine receptor subtypes: beyond the D1/D2 classification. Tips, 11,231 -236. Anyaoku, V., Wood, D.F.. Williams, P., Tan, K. &Johnston, D.G. (1989) Measurement ofserum a-subunit: a rapid sensitive ELlSA technique. Journal of Endocrinology, 123 (Suppl.), 168. Bansal, S., Lee, L. & Woolf, P.D. (1981a) Dopaminergic regulation of growth hormone secretion in normal man: correlation of Ldopa and dopamine levels with the GH response. Journal of Clinical Encocrinology and Metabolism, 53, 301-306. Bansal, S.A., Lee, L.A. & Woolf, P.D. (1981b) Dopaminergic stimulation and inhibition of growth hormone secretion in normal man: studies of the pharmacologic specificity. Journal of Clinical Endocrinology and Metaholism, 53, 1273-1277. Barbacid, M.A. (1987) ras genes. Annual Reuiew of Biochemistry, 56, 779-827. Barbarino, A., Demarinis, L., Anile, C., Menini. E., Merlini, G. & Maira, G. (1982) Dopaminergic mechanisms regulating prolactin secretion in patients with prolactin secreting pituitary adenoma. Long-term studies after selective transphenoidal surgery. Metabolism, 31, 1100-1104. Barnett, P.S., Dawson, J.M., Butler, J., Coskeran, P.B., Maccabe, J.J. & McGregor, A.M. (1990) CV205-502, a new non-ergot dopamine agonist reduces prolactinoma size in man. Clinical Endocrinology. 33,307-3 16. Barrow, D.L., Tindall, G.T., Kovacs. K., Thorner, M.O., Horvath, E. & Hoffman Jnr, J.C. (1984) Clinical and pathological effects of bromocriptine on prolactin-secreting and other pituitary tumoun. Journal of Neurosurzery, 60, 1-7. Bassetti, M., Arosio, M.. Spada, A., Brina, M., Bauoni, N.,Faglia. G. & Giannattasio, G.(1988) Growth hormone and prolactin secretion in acromegaly: correlations between hormonal dynamics and immunocytochemical findings. Journal of Clinical Endocrinology and Metabolism. 67, I 195- 1204. Baudry, M., Evans, J. & Lynch, G. (1986) Excitatory amino acids inhibit stimulation of phospatidyl inositol metabolism by aminergic agonists in hippocampus. Nature, 319,329-33 I. Besser, G.M., Parke, L.. Edwards, C.R., Forsyth, LA. & McNeilly, AS. (1972) Galactorrhoea: successful treatment with reduction of
Clinical Endocrinology (1991) 35
plasma prolactin levels by brom-ergocryptine. British Medical Journal, 3,669-672. Bevan, J.S. & Burke, C.W. (1986) Non-functioning pituitary adenomas d o not regress during bromocriptine therapy but possess membrane-bound dopamine receptors which bind bromocriptine. Clinical Endocrinology, 25, 561-572. Bourne, H.R. (1989) G-protein subunits: who carries what message. Naiure, 337, 504-505. Bourne, H.R., Sanders, D.A. & McCormick, F. (1990) The GTPase superfamily: a conserved switch for diverse cell functions. Nature, 348, 125-132. Bourne, H.R., Sanders, D.A. & McCormick, F. (1991)The GTPasc superfamily: conserved structure and molecular mc
