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Chapter 2


Prolactin (PRL) was the first of the pituitary hormones to be biochemically identified and purified,1,2 and hyperprolactinemia caused by hormone-secreting tumors is the most common human pituitary disease. Only recently, however, have the physiology and biochemistry of prolactin actions yielded to contemporary analytic methods to reveal a biology that is at once elegantly simple and sublimely complex. PRL has been identified in the pituitary glands of members of all vertebrate classes, and it has diverse effects on osmoregulation, metabolism, reproduction, metamorphosis, migratory behavior, parental behavior, and lactation.36 In most species, especially mammals, PRL has a specialized role in the postmating phase of reproduction. The predominant mammalian actions of PRL are stimulation of lactation and maternal behavior, and inhibition of reproductive function. Associated with the specialization of PRL in mammals, novel genes that encode placentally derived lactogens have evolved. PRL does not perform any indispensable function for the survival of the individual, but gestation and lactation lie at the core of the mammalian life cycle, and they place extreme demands on physiology. Adaptations in the control of PRL secretion and its physiologic actions have therefore been integral to the biology of all mammals, and abnormalities of PRL secretion are a relatively common cause of endocrine disease. The deepening understanding of PRL actions on both physiologic and molecular levels has facilitated improved therapeutic approaches to diseases of PRL secretion and has opened opportunities to use the physiology of PRL and lactation in new ways.

The Evolutionary Biology of Prolactin

The Prolactin Family

PRL and growth hormone (GH) are related at the primary amino acid sequence level.7 PRL has been identified in all of the vertebrate classes, and it has been inferred that the PRL and GH genes arose from a duplication of an ancestral gene at least 400 million years ago, at about the time of the origin of vertebrates.8 Deeper relationships with other hormones are less certain, but erythropoietin shares substantial primary sequence similarity, as well as three-dimensional structural features, with PRL and GH, suggesting that all three of these hormones share an ancient common ancestry. In addition to PRL and GH, which have been conserved in all vertebrate lineages, a wide variety of derivative genes have appeared in specific vertebrate groups by duplication of the PRL or GH gene. The most familiar of these are the various mammalian placental lactogens.9,10

Placental Lactogens

Placental lactogens (PLs) are synthesized during pregnancy in most, but not all, eutherian mammals. Species that apparently do not produce any placental lactogens are distributed among many mammalian families and include familiar species such as pigs, horses, and dogs.11 Primates (including humans) synthesize a PL that is encoded by a gene duplicated within the GH locus.8 The GH locus in humans encompasses five genes spanning a region of about 50 kb on the long arm (q22-24) of chromosome 17. This locus includes two PL or, preferably, chorionic somatomammotropin (CS) genes (ICSH-1 and ICSH-2). The ICSH-1 and ICSH-2 genes, although slightly divergent at the nucleotide sequence level, encode identical proteins, and the genes are coexpressed in the placenta during gestation. In nonprimates (e.g., rodents, ruminants), the PLs have descended evolutionarily from duplications of the PRL gene.8 Multiple PL genes and nonlactogenic PRL-like genes have evolved from PRL. In mice, placental lactogen-I (PL-I) is synthesized early during gestation, appearing immediately after implantation. PL-I expression is extinguished at about midgestation and is replaced by PL-II. Both of the mouse PL genes are synthesized in trophoblast giant cells. In species that synthesize PLs, including humans, the major stimulus to mammary gland development during pregnancy is presumably PLs, rather than pituitary PRL. PL levels generally rise in correlation with placental growth, and their secretion is controlled by both positive and negative regulators.12 Loss of PLs and placental steroids at parturition is accompanied by elevation of pituitary PRL secretion, and a corresponding shift to pituitary-dominated regulation of mammary gland function during lactation. The importance of this shift is that pituitary PRL is strongly regulated by a suckling-induced neuroendocrine reflex, which allows nursing activity to determine directly the lactational stimulus to the mammary glands.

Nonlactogenic Prolactin Relatives

Nonlactogenic members of the PRL gene family are synthesized by the placenta of nonprimates. Although the physiologic activities of these PRL-related proteins have not yet been established, the expression patterns for some of these proteins are tightly regulated during gestation,10 and this has been interpreted to indicate that the proteins are functionally important. In mice and rats, at least six nonlactogenic PRL-like proteins (PLP-A through PLP-F) are present. In addition, mice synthesize two proteins, namely, proliferin and proliferin-related protein, which have been proposed to act as regulators of angiogenesis.13 Nonlactogenic PRL-like protein genes have also been extensively characterized in cattle.14 One curious feature of the PRL-like gene family is the apparently rapid evolutionary divergence of members of this family. These proteins generally share less than 25% sequence identity with PRL, but all share two pairs of cysteine residues that are conserved throughout the PRL and GH superfamily.10 Information regarding receptors for the nonlactogenic PRL-related proteins is scant. Proliferin binds to the mannose-6-phosphate insulin-like growth factor-2 (IGF-2) receptor.15 In mice, some of the non-lactogenic PRL relatives are essential for preventing fetal death during physiologic stresses, such as hypoxia.16,17 Therefore, the species specificity of PRL relatives may reflect the reproductive stresses that were relevant during the evolutionary divergence of different mammalian groups.

Extrapituitary Prolactin

Many mammalian tissues, including the human mammary gland and uterine decidua, express the PRL gene. In addition, various tissues metabolize PRL to alternative forms that may be biologically active. PRL is synthesized by both the decidua and the uterine myometrium in humans.18 High concentrations of PRL are present in the amniotic fluid; these can be traced to both decidually synthesized hormone and plasma PRL that is transported across the placenta into the amniotic fluid. The synthesis of human PRL in extrapituitary sites is controlled by a promoter that is distinct from the pituitary PRL promoter. Human extrapituitary PRL messenger RNA (mRNA) has a distinct 5′ untranslated sequence corresponding to an additional exon (exon 1A)19 (Fig. 2-1). Exon 1A and the promoter elements associated with it are located about 8000 base pairs (bp) distal to the initiation site for pituitary PRL transcription. In rodents, the evidence for a distinct extrapituitary PRL promoter is less certain than in the human. It is conceivable that rodents use other mechanisms, such as growth factors that control the conventional pituitary PRL promoter, to provide for regulation of PRL synthesis in extrapituitary tissues. The mammary gland is an important site of PRL synthesis and secretion. PRL is present in significant concentrations in milk, and milk PRL is absorbed by the neonatal gut and causes changes in the maturation of the hypothalamic neuroendocrine system.20 Pituitary PRL is transported out of the circulation, across the mammary epithelium, and into the alveolar lumen, and locally synthesized PRL is secreted into milk.18 To date, no disease states have been connected to dysregulation of extrapituitary PRL secretion. The lack of any clear proof that symptoms are present that are caused by either hypersecretion of extrapituitary PRL—say, from a PRL-secreting ectopic tumor—or loss of extrapituitary PRL gene expression makes it difficult to surmise the normal functional roles of extrapituitary PRL in humans. It has been suggested that locally synthesized PRL in the mammary gland might act as a growth factor for both normal breast epithelium and breast cancer cells.21 In mice, decidual PRL prevents fetal loss late in pregnancy by inhibiting the expression of multiple genes that are detrimental to pregnancy, including inflammatory cytokines.22 A recent pilot study reports a lack of endometrial PRL expression in some women with unexplained infertility and patients with repeated miscarriages.23

The Biochemistry of Prolactin

Human PRL is synthesized as a prehormone that is encoded by an mRNA with an open reading frame of 684 bases. The native gene for PRL is divided into five exons, and the initiation site for translation is in exon 124 (see Fig. 2-1). Preprolactin (pre-PRL) is 227 amino acids in length, with a deduced molecular weight of nearly 26,000. Cleaving the signal peptide from the N-terminus of pre-PRL results in a mature polypeptide that is 199 residues in length and has a molecular weight of nearly 23,000 (23k PRL). On the basis of the fact that the bacterially synthesized recombinant 23k PRL monomer binds to the PRL receptor (PRL-R) and transduces functional signals, it is clear that no additional modifications are essential for the core functions of PRL. PRL folds itself into a tertiary structure that includes three intrachain disulfide bridges, two of which are conserved in all members of the PRL-GH family, and one that links residues 4 and 11 in the N-terminus, which is unique to PRL and its closest relatives.7 Four α-helical domains in PRL are arranged so that helices 1 and 2 run antiparallel to helices 3 and 4. This general molecular architecture of PRL has been conserved with GH and other homologous proteins and also has evolved independently in several families of cytokines.25 The convergent evolution of hormone and cytokine ligand architecture apparently has been driven by the properties of receptors that bind these hormones and transduce signals to the intracellular space.

A variety of biochemical variants of 23k PRL, which appear to have altered functions, have been identified. PRL has a tendency to aggregate and form intermolecular disulfide bridges spontaneously when in solution at high concentrations. High-molecular-weight variants (sometimes referred to as “big” PRLs) may arise by virtue of multimerization, glycosylation, or cross-linking with other proteins. Only a small fraction of human PRL is glycosylated, whereas in some other species, such as swine, glycosylated PRL represents a large portion of both pituitary and plasma hormone.26 Glycosylation may alter the relative potency of PRL by changing its receptor-binding characteristics or by modifying its pharmacokinetic properties in the animal (plasma half-life, partitioning between plasma and interstitial compartments, etc.). PRL is metabolized by tissue uptake and by proteolysis in the circulation or in cells. Proteolysis also produces a 16kDa PRL fragment that has been proposed to have antiangiogenic bioactivity.27

Recent studies implicate the cathepsin-cleaved 16kDa PRL as causative of postpartum (or peripartum) cardiomyopathy (PPCM). PPCM is the acute onset of heart failure in women during late-stage pregnancy or the first several months post partum. Hilfiker-Kleiner and colleagues discovered that PPCM developed in mice with selective deletion of the STAT3 gene in cardiac myocytes.28 STAT3 has been shown to be a critical factor modulating cardiac angiogenesis, an important part of the normal cardiac hypertrophy that occurs during pregnancy.29 In a series of elegant studies, they demonstrated that the absence of STAT3 led to increased production of reactive oxygen species, upregulation of cathepsin-D, and elevation of the 16kDa fragment of PRL. Treatment of these mice with bromocriptine, which reduces secretion of 23kDa PRL from the pituitary, resulted in enhanced cardiac function and prevention of postpartum mortality. Preliminary studies have shown that circulating levels of 16kDa are barely detectable in healthy nursing women but are elevated in some women with PPCM.28 A few clinical case reports have found that women with PPCM may respond favorably to treatment with bromocriptine.28,30,31

Phosphorylated PRL has reduced potency in standard bioassays, and it antagonizes the actions of the predominant unphosphorylated form.32 Actions of kinases or phosphatases in either the pituitary or individual target tissues may have an important effect on the bioactivity of PRL in vivo.

The Ontogeny and Physiology of Prolactin Secretion

Development of Lactotrophs

PRL is synthesized by lactotrophs, which are acidophilic cells that represent 20% to 50% of the anterior pituitary cell population. The lactotrophs are the last of the pituitary cell types to fully differentiate and, coincidentally, the most likely to give rise to pituitary adenomas. Pituitary PRL mRNA synthesis begins at 12 weeks in human gestation and is preceded by GH synthesis by at least 4 weeks.8,33 In rodents, the pattern is similar, with the GH gene being expressed several days before PRL, and with dual-functioning somatolactotrophs being observed before fully differentiated lactotrophs.34 Control of pituitary development and lactotroph differentiation depends on the orchestrated expression of a series of intrinsic, tissue-specific regulatory molecules that act as “molecular switches” to induce the sequence of developmental changes that lead up to full pituitary differentiation. Many of the intrinsic factors that have been implicated in pituitary development are evolutionarily related to “homeotic mutation” genes, which were first identified by their dramatic effects on development in fruit flies.33 Some genetic diseases of the pituitary, pituitary tumors, and physiologic states of hormone deficiency or excess can be attributed to dysfunction of these regulatory molecules.

The homeobox transcription factors are a diverse class of developmental regulatory proteins that share sequence similarities in their DNA-binding regions and are sequentially activated during organogenesis. Two pituitary homeobox proteins (Ptx1 and Ptx2) are expressed in multiple anterior (head and face) tissues before the development of Rathke’s pouch, and they continue to be expressed in some differentiated pituitary cells. Rathke’s pouch homeobox protein (Rpx) is expressed first in neural structures associated with the head region and then in Rathke’s pouch. During the formation of Rathke’s pouch, a subgroup of LIM-related homeobox proteins are synthesized (P-LIM, Lhx3, and Lhx4), and these genes continue to be expressed in specific regions of the pituitary throughout life. Properly timed extinction of expression is, for certain genes, as important during development as is their appropriate induction. Rpx must be turned off after Rathke’s pouch has been formed, so that genes that are specific to later stages of pituitary differentiation can be turned on. The transcription factor that downregulates Rpx expression is PROP-1 (Prophet of Pit-1). PROP-1 turns off Rpx and turns on Pit-1, leading to differentiation of some of the hormone-producing cells of the pituitary gland, including lactotrophs.35

In the adult pituitary gland, a population of stem-like cells provides new progenitors for all of the hormone-secreting cells, including lactotrophs.36

Pit-1 is essential for differentiation of both PRL- and GH-secreting cells, hence its alternative name, GH factor-1 (GHF-1).37 An early developing subpopulation of thyrotrophs is also dependent on Pit-1. The Pit-1 protein shares close sequence similarity with two other transcription factors within regions referred to as the POU (Pit, Oct, Unc)-specific domain, and the POU-homeodomain.38 Pit-1 expression in the developing pituitary gland precedes the synthesis of hormones and is necessary for the expression of GH, PRL, and thyroid-stimulating hormone (TSH) in fetal pituitaries. Variant forms of Pit-1 are encoded by alternatively spliced mRNAs and may differentially control expression of individual hormones. Pit-1 binds not only to DNA sequences in the GH, PRL, and TSH genes, but also to autoregulatory sites in the Pit-1 promoter. Autoactivation of Pit-1 transcription is one means of preserving phenotypic stability in differentiated pituitary cells. The factors that act after Pit-1 to drive the differentiation of lactotrophs from somatotroph progenitors are not known. Estrogen receptors synergize with Pit-1 to induce PRL, but not GH, gene expression. Estrogen therefore may be one of the factors that drive the ultimate differentiation of lactotrophs.3942

Several extrinsic factors are involved in lactotroph differentiation. Estrogen is an important positive regulator of lactotroph development. Lactotrophs are greater in number and contain more PRL per cell in females during their reproductive years. Estrogen acts directly on lactotrophs to stimulate PRL synthesis and cell proliferation. Estrogen-induced galanin secretion from lactotrophs is an important mediator of these estrogen actions43,44 and involves signaling through the classic estrogen receptor isoform alpha (ERα).45 Paracrine factors produced by other anterior pituitary cell types include basic fibroblast growth factor (B-FGF or FGF-2), which has a specific positive stimulatory effect on lactotrophs.46 Likewise, epidermal growth factor (EGF) stimulates lactotrophs and may act as a developmental regulatory factor as well as a physiologic stimulator of PRL secretion.47 As will be presented in subsequent sections, the same factors that drive vectorial differentiation of pituitary cells can participate in regulating the tides of hormone secretion on a physiologic time scale and in disorders of hormone secretion.

Regulation of Pituitary Prolactin Synthesis and Secretion

In mammals, PRL secretion is normally restrained by the action of dopamine (DA), which is secreted from the hypothalamus.48 Although the levels of other pituitary hormones are modulated by inhibitory secretagogues such as somatostatin, PRL is the only such hormone that is secreted at unrestrained high levels when completely isolated from the positive trophic influences of the hypothalamus. This unconventional situation is unique to mammals. Control of PRL secretion in birds and other nonmammals is more conventional in the sense that positively acting secretagogues are the predominant regulators of PRL secretion.49,50 Lactotrophs are excitable cells in that they display spontaneous membrane depolarizations associated with calcium ion influx, and their resting membrane potential is influenced by neurotransmitters and peptide neuromodulators.

The normal secretory pattern of PRL is a series of daily pulses, occurring every 2 to 3 hours, which vary in amplitude so that the bulk of the hormone is secreted during rapid eye movement (REM) sleep. REM sleep is the dominant organizer in men and nonparous women and occurs mostly during the latter half of the sleep phase. Thus, the highest levels of PRL generally occur during the night in humans.51 In nocturnal rodents, the relationship to the light cycle is reversed, so higher PRL secretion occurs during the daytime, which is the inactive phase. It is unclear how REM and PRL secretion are linked. Infusion of PRL increases REM activity in the electroencephalogram (EEG),52,53 suggesting that it is PRL that induces REM sleep, and not vice versa.

In lactating women, suckling is a potent stimulator of PRL secretion. This classic neuroendocrine reflex originates with the stimulation of sensory nerve endings in the nipple and is transmitted via the spinal cord and brain stem, ultimately to the hypothalamus. Stress and sexual orgasm are also potent stimulators of PRL secretion. Stress-induced PRL secretion varies with the duration, degree, and modality of the stressor. The relative contributions of PRL-releasing (PRF) and PRL-inhibiting factors to these PRL secretory events remain controversial.


As was mentioned earlier, the major regulatory input to lactotrophs is inhibitory, provided in the form of DA produced within the hypothalamus. The primary PRL-regulating DA neurons are the tuberoinfundibular dopaminergic (TIDA) cells, which have their cell bodies in the arcuate nucleus of the hypothalamus; they release DA in the median eminence and the pituitary stalk (Fig. 2-2). A secondary tuberohypophysial dopaminergic system has cell bodies in the rostral caudate and paraventricular nuclei, and these neurons release DA in the posterior pituitary.48 The type 2 isoforms (D2) of the DA receptor mediate the direct inhibitory actions of DA on PRL secretion, synthesis, and cell proliferation. Targeted disruption of the D2 receptor in mice leads to a phenotype of PRL hypersecretion and lactotroph proliferation.54

Dopamine is synthesized by a two-step reaction in which tyrosine conversion to levodopa is catalyzed by tyrosine hydroxylase, and levodopa is converted to DA by the action of aromatic amine decarboxylase. As is the case for catecholamine synthesis in other cells, the momentary rate of DA synthesis in the TIDA neurons is determined by the activity of tyrosine hydroxylase. The negative feedback mechanism for controlling PRL release is to increase tyrosine hydroxylase activity in the TIDA neurons, thereby increasing the amount of DA available for release from the median eminence. PRL receptors are located in both the arcuate nucleus (site of the TIDA perikarya) and the median eminence.55 Therefore, circulating PRL may feed back on TIDA neurons at their terminals, which lay outside the blood-brain barrier, or systemic PRL may enter the cerebrospinal fluid via the choroid plexus. The choroid plexus expresses high levels of a short isoform of the PRL-R, which may serve to transport PRL across the blood-brain barrier. Levels of PRL in the cerebrospinal fluid reflect changes in PRL in the systemic circulation.56 Isolated PRL deficiency resulting from targeted gene disruption in the mouse causes decreased DA in the median eminence but does not affect DA levels in other regions of the hypothalamus.57

Activation of D2 receptors in lactotrophs has at least two main actions that result in inhibition of PRL. D2 receptors are members of the heptahelical G protein–coupled receptor superfamily, and they activate the αi subunits, which leads to inhibition of cyclic adenosine monophosphate (cAMP) synthesis.48 In addition, D2 receptors activate a G protein–coupled, inwardly rectifying potassium channel, which instantaneously causes hyperpolarization of the lactotroph membrane and closes voltage-gated calcium channels.58 Cytoplasmic calcium levels fall because of decreased influx of extracellular calcium, and the reduction in cytosolic free calcium decreases the exocytosis of secretory vesicles.

Dopamine-induced membrane hyperpolarization opposes the actions of some stimulatory factors such as thyrotropin-releasing hormone (TRH), which acts predominantly to increase influx of extracellular calcium by depolarizing the lactotroph membrane. Inhibition of cAMP by DA also opposes the actions of stimulatory factors such as vasoactive intestinal peptide (VIP), which acts via a positive effect on cAMP. This action decreases PRL release in the short to intermediate term. Second, because cAMP is mitogenic in lactotrophs, as well as in other pituitary cells, activation of Gi signaling by DA is antimitogenic. Lactotroph proliferation is important for physiologic elevation of PRL release during lactation. The proliferative action of cAMP on lactotrophs is understood to be an important promoter of pituitary tumor growth, thereby contributing to pathologic hyperprolactinemia.35

Other hypothalamic factors, as well as DA, and local pituitary peptides can inhibit PRL secretion. Somatostatin inhibits PRL secretion and acts through both cAMP-dependent and cAMP-independent mechanisms.59 Calcitonin has been shown to inhibit PRL secretion and may be secreted from the hypothalamus.60 Endothelin-1 is produced by lactotrophs and inhibits PRL secretion; transforming growth factor-β1 can act as a paracrine inhibitor of PRL.61,62 The biologic significance of these factors in pituitary development and physiology has not yet been established.

Prolactin-Releasing Factors

A wide variety of stimulatory PRL secretagogues have been identified over the years, and it is likely that additional PRFs will be identified in the future. Known stimulators of PRL secretion include, but are not limited to, steroids (estrogen63), hypothalamic peptides (TRH, oxytocin, VIP,64,65 pituitary adenylate cyclase activating peptide [PACAP],66 and galanin67), and local pituitary factors (growth factors such as EGF68 and FGF-2,69 angiotensin II,70 and, again, PACAP71 and galanin43).

TRH is a potent and rapid stimulator of PRL release in vitro via a set of calcium-mediated pathways activated by a Gq-coupled receptor. However, the relative contribution of TRH to physiologic control of lactotrophs is not clear. VIP acts through cAMP to stimulate PRL synthesis and release on an intermediate to long-term basis. The importance of VIP as a positive lactotrophic factor is supported by two types of evidence. With the use of antibodies against VIP, the secretion of PRL can be inhibited to a very low level.47 In addition, VIP appears to be the primary PRF in birds and other nonmammals,72,73 suggesting that this positive mechanism may have been in place before the evolution of the dopaminergic inhibitory system in mammals. Oxytocin secretion is tightly coupled with PRL secretion during lactation, and both are secreted in response to nipple stimulation. The potential role of oxytocin as a PRF, given that it can reach the anterior pituitary through the short portal system, has remained controversial. Oxytocin antagonism partially suppresses PRL secretion,48 so this peptide is likely to provide some portion of the physiologic stimulus for PRL release. PACAP stimulates PRL synthesis and release. Galanin is synthesized in both the pituitary and the hypothalamus. In the pituitary, it colocalizes with PRL in lactotroph secretory granules and acts by autocrine and paracrine mechanisms to stimulate lactotrophs.43

A putative PRL-releasing peptide (PrRP) from the hypothalamus was identified by searching for ligands that activate an orphan pituitary G protein–coupled receptor. The mature peptide that was identified from bovine hypothalamus is a 20 amino acid molecule that originally was reported to cause rapid secretion of PRL from isolated pituitary cells.74 However, subsequent studies have failed to confirm that PrRP acts on lactotrophs to stimulate PRL release.75 Rather, PrRP may act within the hypothalamus to indirectly elevate PRL by inhibiting DA release. Antagonists of serotonin or opioid receptors inhibit PRL secretion under physiologically meaningful stimuli. Conversely, antidepressants that inhibit serotonin reuptake (fluoxetine [Prozac], etc.) may increase PRL secretion in humans and in laboratory animals. Serotonin and opioids are important indirect regulators of PRL by virtue of their actions on DA and releasing factor secretion in the hypothalamus.

Lactotrophs display a large degree of functional heterogeneity within the anterior pituitary. This heterogeneity is manifested as differences in morphology (i.e., secretory granule size and density), basal hormone release, electrical activity, and response to releasing and inhibiting factors. Assay of hormone release from single cells has revealed not only substantial cell-to-cell variations in function, but also marked temporal variations in a single cell.76

Transcription regulators that control the development of the anterior pituitary lactotrophs also participate in controlling PRL synthesis during adult life. Prominent among these factors is the Pit-1 protein. Pit-1 binds to two regions of the human PRL gene, the proximal promoter (within 250 bp of the transcription start) and a distal enhancer (beyond −1300 bp) (see Fig. 2-1). Multiple Pit-1–binding sites are present in each of these regions. Transcription regulators such as cAMP and estrogen receptors can control PRL gene expression by influencing Pit-1 activity.39,63

Pathophysiology of Prolactin Secretion

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