**2. Regulation of pituitary GH secretion**

In 1985, Plotsky and Vale demonstrated in rats that the episodic secretion of GH was under a dual hypothalamic control exerted by growth hormone-releasing hormone *Pituitary Growth Hormone Secretion and Cell Growth Hormone Production: Regulation of Their… DOI: http://dx.doi.org/10.5772/intechopen.108382*

(GHRH) and somatostatin (SS) [7]. According to this model, the determinant of pulsatile GH secretion would be the rhythmic alternate release of hypothalamic GHRH and SS. The existence of a similar intrinsic hypothalamic-somatotroph rhythm (HSR) that governs the pituitary GH secretion was also demonstrated by our group in normal humans [8]. Intrinsic HSR most likely remains functionally active throughout life. However, plasma GH levels are highly age-dependent and show a different pattern of secretion in men and women. Both age and gender physiologically modify the standard pattern of activity, as plasma GH changes reflect. In addition, a number of factors, such as neurotransmitters, other hormones, and metabolic intermediates (amino acids, glucose, and free fatty acids), mainly acting at on the hypothalamic or pituitary level, or both, but also on the pituitary, can modify, both acutely and chronically, the pattern of GH release. Therefore, GH control has traditionally been classically considered as extraordinarily complex, although its complexity has increased over time due to the discovery of new factors that contribute significantly to modify the pituitary secretion of GH, as we will see more ahead. This also reflects the complexity of the multiple actions that GH exerts in the organism, far beyond its role in growth.

### **2.1 GHRH**

Reichlin [9] was the first to propose the existence of a hypothalamic GHreleasing factor. However, it took several years to isolate and characterize this putative factor [10, 11]. The initial isolation of GHRH was facilitated by the existence of human tumors ectopically producing growth hormone-releasing activity [12, 13].

GHRH is a peptide that belongs to the brain-intestinal peptides family, the glucagon secretin family. Two main GHRH forms of GHRH, composed of 40 and 44 amino acids, have been described in the human hypothalamus, occurring as a result of the alternative processing of RNA during the transcription of a GHRH gene located on chromosome 20. These are the result of alternative processing of RNA from the transcript of a single GHRH gene located on chromosome 20. Once it was demonstrated that the GH-releasing activity of GHRH was located between amino acids 1–29, it was possible to produce shorter forms of the peptide (e.g., GHRH-29, known as GRF-29) to be used in clinical applications, such as a provocative test. Since the removal of amino acids 30–44 does not affect the GH-releasing activity of the peptide, shorter forms (e.g., GRF-29) have been synthesized for clinical purposes. On the contrary, modifications at the N-terminal end of the molecule markedly decrease its biologic activity. The half-life of GRF-29 in plasma is very short, about 2 minutes, as it is very rapidly inactivated by circulating proteases. This has been led to the development of potent GHRH agonists and antagonists., as it will be shown later.

GHRH-producing neurons involved in GH control are located primarily in the arcuate nucleus of the hypothalamus from where they reach the median eminence and release the peptide into the primary plexus of the hypothalamic-pituitary portal vascular system. In the pituitary, GHRH binds to its receptor on somatotrophs and induces a rapid, dose-dependent release of GH, but also the transcription of the GH gene as well as the differentiation, growth, and proliferation of these cells.

In addition to this, GHRH-producing cells have also been found in other hypothalamic and extra-hypothalamic structures. These cells, however, do not project to the median eminence but to several other hypothalamic and extrahypothalamic regions. This suggests that apart from its GH-releasing activity, the peptide also plays a neuromodulator role in the central nervous system. In addition, there is GHRH production in numerous tissues and organs, such as the gonads.

GHRH is encoded by a gene located in chromosome 20 (20q11.2), which consists of five exons separated by four introns. Exon I is not coding; exon II codes for the signal peptide and a small N-terminal connecting peptide; exon III encodes the majority of the mature GHRH molecule (including the biologically active peptide); exon IV encodes most of the C-terminal peptide; and exon V encodes the rest of the C-terminal peptide and also contains 3′ untranslated sequences [14] (**Figure 2**).

A variant of the GHRH precursor has been described. It is formed by RNA processing that excludes three nucleotides at the beginning of exon V, leading to the absence of serine-103 in the precursor molecule. This precursor will undergo identical processing to that of the 108 amino acids precursor, with the only difference that in this case the C-terminal peptide will be made up of only 30 amino acids [14].

An interesting aspect, little clarified, is how the transcription of the GHRH gene is regulated. KO mice for the homeobox transcriptional factor Gsh-1 exhibit a dwarf phenotype and abolished GHRH expression [15], and Gsh-1 binds on multiple binding sites of GHRH gene promoter. Co-expression of the transcription factor conditional cAMP response element binding (CREB) protein significantly enhances Gsh-1-induced expression of the GHRH gene, suggesting a cooperative role for the coactivator protein. The Gsh-1 homeobox protein is key for the expression of the GHRH gene.

And what is the role of CREB in the transcription of GHRH gene? In mice in which CREB is lost in the brain, but not in the pituitary, the amount of GHRH peptide is reduced, indicating that CREB is required for efficient production of GHRH in the hypothalamus [16]. This would explain the relationships between CREB and Gsh-1 for hypothalamic GHRH transcription.

#### **Figure 2.**

*Structure of the GHRH gene and transcription of GHRH. As the figure shows, the gene-encoding GHRH is composed by five exons (I–V) and four introns (A–D). GHRH is synthetized as a precursor (pre-pro-GHRH) formed by 108 amino acids (including the signal peptide), which is proteolytically cleaved and gives rise to the mature GHRH (blue rectangle) together with a C-terminal peptide formed by 31 amino acids. As the figure shows, the precursor of GHRH is produced by exon III and a small portion of exon IV (black arrows 3 and 4).*

*Pituitary Growth Hormone Secretion and Cell Growth Hormone Production: Regulation of Their… DOI: http://dx.doi.org/10.5772/intechopen.108382*

Another GHRH gene transcription factor is the nuclear factor of activated T cells (NFAT). Site-directed mutagenesis experiments demonstrated the direct binding of NFAT at five sites of the GHRH promoter, suggesting that NFAT is involved in the depolarization-induced (with high potassium) transcriptional activation of GHRH gene in the hypothalamic neurons producing it [17].

Apart of its hypothalamic production, GHRH is a peptide produced in many human tissues and organs, where it plays a very important physiological and etiopathological role in addition to its main role in the synthesis and release of GH.

#### **2.2 Somatostatin (SS)**

At the end of the 1960s, McCann's group observed that bovine hypothalamic extracts were able to inhibit the release of GH from pituitary cultures. Five years later, Guillemin' group [18–20] reported the identification of the factor responsible for that effect: GH-release-inhibiting factor or SS. Several studies soon demonstrated that SS was not only a GHrelease-inhibiting factor but also a strong inhibitor of a number of secretory events, endo- or exocrine, whose analysis is beyond the objective of this chapter.

Although SS is a tetradecapeptide (SS-14), a form SS-28 has been found in pituitary portal blood, also capable of inhibiting pituitary GH release. Structurally, SS is a tetradecapeptide, although a 28-amino-acid form is also present in pituitary portal blood. Both molecular forms apparently have similar GH-release-inhibiting activity.

SS-producing neurons involved in GH control are found mainly are mainly located in the anterior periventricular and paraventricular nuclei of the hypothalamus. Their axons project to the median eminence, where SS is released into the hypothalamic-pituitary portal circulation to antagonize in a dose-dependent manner the GH-releasing effect of GHRH, and also, although in less degree, the proliferative activities of this GH-releasing peptide.

As with GHRH, SS is synthesized in the form of a precursor, pre-pro-somatostatin, a 116 amino acid peptide (including the 24 amino acid signal peptide) whose biologically active portion is at the C-terminal end. The gene that codes for SS is located on chromosome 3 and consists of two exons separated by an intron [21, 22]. In it, a cAMP response sequence is found between nucleotides −48 and −41 [23]. The transcription of the gene is stimulated by CREB, phosphorylated after an increase in cAMP levels, and the subsequent activation of protein kinase A (PKA). The coding sequence has a high degree of conservation in exons.

The proteolytic processing of pro-somatostatin gives rise to two physiologically important variants, SS-14 and SS-28. SS-14 is a tetradecapeptide that is formed after cleavage by endoproteases of the precursor between amino acids 101 and 102, while SS-28 corresponds to the sequence of the SS-14 form with an N-terminal extension of 14 amino acids [24] (**Figure 3**). The proteolytic cleavage of pro-somatostatin in the basic domain (Arg/Lys) or the monobasic domain (Arg) of the C-terminal end gives rise to SS-14 and SS-28, respectively (**Figure 3**). Both molecular variants are encoded by exon 2.

Both SS-14 and SS-28 are widely distributed in the body, being particularly abundant throughout the brain, but also in the gastrointestinal tract (particularly in the pancreas and stomach), which may be related to its inhibitory functions of endocrine and exocrine secretions. SS also acts as a neurotransmitter and regulator of the immune system. Most likely, these are the reasons for the existence of five receptors (SST1 to SST5) with different localizations and biological activities. Since the fact that SS has anti-proliferative and anti-angiogenic effects and given that its half-life is very

#### **Figure 3.**

*Proteolytic processing of pre-pro-somatostatin. Proteolytic processing of pre-pro-somatostatin releases the signal peptide (formed by 24 amino acids) and then the connection peptide (green rectangle, formed by 64 amino acids). As indicated SS-28 is formed by the 14 amino acids extension (yellow rectangle) to the N-terminal end of SS-14. A: monobasic domain. B: basic domain.*

short (1–3 min), as well as the wide spectrum of its biological responses, led to the need to develop selective stable receptor agonists that are bind strongly to selective SS receptor subtypes, therefore, in addition to blocking GH secretion (e.g., treatment of Acromegaly), these SS agonists are being used for the therapy of neuroendocrine tumors, diabetic complications (nephropathy, retinopathy), anti-neoplastic therapies. More recently, it has been shown that these agonists have anti-inflammatory and anti-nociceptive effects [25].

Before reviewing how GHRH and SS interact in the control of pituitary synthesis and release, we will describe how the pituitary receptors for these hormones are stimulated. Most likely this is different than how they are stimulated in other tissues in which they are expressed.

#### *2.2.1 Pituitary GHRH receptor*

The GHRH receptor (GHRHR) is a 423 amino acid protein (including the signal peptide) that belongs to a subfamily of G-protein-coupled receptors [26, 27]. Its producing gene is located to the short arm of chromosome 7 (7p13–p21) near the epidermal growth factor receptor (EGFR) gene [28].

The gene has a very complex structure [26]. Unlike other G-protein-coupled receptors, which do not have introns, in this gene there is a coding sequence interrupted by several introns. An alternative ribonucleic acid (RNA) processing has been described that results in the insertion of 41 amino acids just before the beginning of the sixth transmembrane domain. This type of processing does not appear to be exclusive to GHRHR [29], although its physiological significance is unknown.

Like the rest of the receptors that use G proteins for intracellular signaling, the GHRHR has seven transmembrane hydrophobic domains, linked to each other by six loops, three intra-cytoplasmatic and three extracellular.

The N-terminal extracellular domain of GHRHR contains a site for N-glycosylation as well as six cysteine residues and an aspartate residue that are conserved in this receptor family, while the third intracellular loop and C-intracellular domain contain several potential phosphorylation sites, which may regulate signaling and receptor internalization.

*Pituitary Growth Hormone Secretion and Cell Growth Hormone Production: Regulation of Their… DOI: http://dx.doi.org/10.5772/intechopen.108382*

GHRHR mRNA has been detected in the pituitary, gonads, placenta, kidney, hypothalamus, and many brain regions, although it is expressed predominantly in the anterior pituitary due to its dependence of Pit-1 (also known as GHF-1).

GHRH interaction with its GHRHR on the somatotroph leads to the activation of the associated Gs protein, allowing the αs subunit to separate from the dimer ßγ. The activation of Gs protein will then have a double effect: (1) It induces the release of GH stored in secretory granules by allowing the entry of Ca++ into the cell, and (2) the αs subunit activates the adenylyl cyclase (AC), which leads to increased cellular levels of cAMP [26, 30, 31]. This, in turn, activates the protein kinase A (PKA), which induces a cascade of phosphorylation and activation of the transcription factor CREB [32]. The activation of CREB induces the pituitary-specific transcription factor Pit-1 [33–35]. Therefore, Pit-1 represents a pathway by which GHRH can increase GH synthesis in the pituitary. Interestingly, Pit-1 also regulates GHRHR synthesis, as if there were some kind of feedback positive between them, and also induces its own expression. In fact, the GHRHR is not expressed in the pituitary of dw/dw mice that lack functional Pit-1 [30]. In turn, the non-activation of CREB leads to somatotroph hypoplasia and dwarfism in mice.

Other signaling pathways activated after the GHRH-GHRHR interaction involve the Phospholipase C/Inositol phosphatide/Protein kinase C (PLC/IP/PKC) pathway, which is perhaps also responsible for the increase of Ca++ for GH secretion in somatotrophs. In this case, the Ca++ would come from the endoplasmic reticulum. This effect would occur through the stimulation of phospholipase C (PLC) through the complex Gβγ of heterotrimeric proteins G. Activated PLC produces diacylglycerol (DAG) and inositol triphosphate (IP3) that leads to the release of Ca++ from the endoplasmic reticulum. However, this effect has not been seen in some inferior species [36];

#### **Figure 4.**

*Interaction GHRH-GHRHR at the pituitary level and synthesis and secretion of GH. After interacting with its receptor, GHRH activates the associated Gs protein, and the αs subunit is separated from the ßγ dimer (1). This allows: (1) the entry of Ca++ into the cell (1*′*), and the activation of adenylyl-cyclase (AC) (1) leading to increased formation of cAMP from ATP (2). cAMP activates protein kinase A [PKA] that phosphorylates CREB (3). Phosphorylated CREB translocates to nucleus (4) and induces Pit-1, which, in turn, leads to the transcription of the GH gene (5) and also regulates the synthesis of GHRHR (5*′*). On the other hand, the Gβγ complex of heterotrimeric proteins G activates PLC (6) producing DAG and IP3 (7) that leads to the release of Ca++ from the endoplasmic reticulum (7*′*) for the secretion of GH. Another mediator of the effects of GHRH is the mitogenactivated protein kinase (MAPK) pathway, which may mediate the effects of GHRH on the proliferation of somatotrophic cells (8).*

another signaling pathway observed in a wide variety of cell types as a mediator of GHRH effects is the mitogen-activated protein kinase (MAPK) pathway, which may mediate the effects of GHRH on somatotroph cell proliferation [37], although it cannot be ruled out that the proliferative effect is mediated by *c-fos* activated by CREB. These pathways activated by the interaction GHRH-GHRHR are shown in **Figure 4**.

#### *2.2.2 Pituitary SS receptor*

The wide variety of actions of SS and the different organs and tissues in which this hormone acts explain the existence of different types of receptors for this exocrine and endocrine inhibitory hormone. The five types of receptors for SS (SSTR1 to SSTR5) belong to the DRY family of G-protein-coupled receptors, whose common characteristic is the presence of an Asp-Arg-Tyr sequence, located near the boundary between the third transmembrane domain and the second intracellular loop, which is very important for transmission of the SS signal [38, 39]. Each of the receptors is encoded by a different gene, located on different chromosomes, and their coding region lacks introns (as is the case with most G-protein-coupled receptors).

SS receptors show a homology between them that varies between 45% and 61%, and 100 residues are the same in all of them. The highest degree of homology occurs in the sequences corresponding to the seven transmembrane helices, while the NH2 and COOH ends are the most divergent both in sequence and in length [39].

The SSTR2 receptor is the prototype of the SS receptors and is the one that mediates the inhibition that SS exerts on GH secretion in the pituitary gland [40]. This receptor has the same affinity for SS-14 as for SS-28 [39].

The binding of SS with its receptor induces the activation of the related G protein (**Figure 5**), which in the case of the SSTR2 receptor is a Gi protein. The activation of the Gi protein will produce an inhibition of the activity of AC, a reduction in the entry of Ca++ through voltage-gated channels, and the appearance of rectifying potassium currents [39, 41], which leads to hyperpolarization of the somatotropic membrane. The net product of these actions would be the inhibition of the transcription of CREB-dependent genes [42], which counteracts the stimulatory effect of GHRH. However, in the control of GH, SS acts basically by inhibiting the release of the hormone, while its effect on the synthesis of GH would be of minor importance. SS also exerts anti-proliferative effects on mediated somatotrophs, through the activation of a tyrosine phosphatase dependent on the SSTR2 receptor [43, 44].

SS receptors undergo desensitization phenomena after prolonged exposure to agonists [39], which seems to depend on the phosphorylation of serine and threonine residues, located in the third intracytoplasmic loop, by the action of the enzyme BARK (ß**-**adrenergic receptor kinase) [39].

Once the basic components that generate a secretory rhythm of GH have been established, the task should be to understand how this rhythm may begin, how it can be modulated by factors external to said system, and what could be the relationships between its basic components: GH, GHRH, and SS. That is, is there a signal associated with GHRH that causes the interruption of SS release or is SS release linked to the termination of GHRH secretion, as initially thought? Is the end of GHRH secretion directly dependent on the increase in GH produced by the peptide, or is there a GH-associated signal that stimulates SS release and blocks GHRH secretion?

Before answering these questions, there is the need to analyze how the pituitary transcription of GH is regulated by transcription factors, as well as the structural organization of Pit-1, the main one of these factors. After that, the mechanisms that *Pituitary Growth Hormone Secretion and Cell Growth Hormone Production: Regulation of Their… DOI: http://dx.doi.org/10.5772/intechopen.108382*

#### **Figure 5.**

*Interaction of SS-SSTR2 at the pituitary level and its effects on GH secretion. In the pituitary gland, binding of SS to its pituitary receptor SSTR2 (1) activates (2) the Gi-related inhibitory protein. This leads to inhibition (3,* **−***) of AC. Consequently, ATP cannot give rise to cAMP (4,* **=***). The lack of the necessary cAMP prevents (5,*  **=***) the activation of PKA, which then cannot initiate the phosphorylation cascade (6,* **=***) that would activate CREB, and non-phosphorylated CREB cannot translocate to the cell nucleus (7,* **=***) to initiate the transcription of specific genes, including GH (8,* **=***). Then, the secretion of GH would be blocked (***=***). Furthermore, activated Gi blocks (9, −) the channels involved in the entry of Ca++ into the cell (X). This Ca++ is necessary for the release of GH. Moreover, activated Gi leads to the appearance (10,* **+***) of rectifying potassium currents, which leads to hyperpolarization of the somatotropic membrane. Overall, SS blocks GH secretion and partially affects GH synthesis.* **+***: stimulates;* **−***: inhibits***; =***: interrupts or cuts;* **X***: disrupts; blue arrows: stimulates. Red arrows: inhibits.*

regulate the hypothalamic secretion of GHRH and SS will be reviewed. Given that they are neurosecretory products, it seems logical that neurotransmitters play the main role in such regulation.

### **2.3 Regulation of the transcription of the gene GH-N**

Pit-1 is the most important of the transcription factor involved in controlling the expression of the GH-N gene. It is a highly conserved protein, belonging to the family of transcription factors with POU domains that are a subclass of the homeobox genes involved in cell development and differentiation processes [42, 45]. In man, the gene encoding Pit-1 is located on chromosome 20 and is made up of six exons and five introns.

Pit-1 is not only expressed in the pituitary but also in many other territories where it may be involved in the control of cell proliferation. In the pituitary, Pit-1 not only controls the expression of GH and PRL, but also that of the ß chain of TSH, that of the GHRH gene, and, curiously, that of its own gene. Pit-1 is also, as we have already described, a key factor for the development, differentiation, and survival of somatotrophs. The absence of Pit-1, due to mutations, produces alterations in cell development and hormonal synthesis [46, 47]. On the contrary, the overexpression of this factor has been related to the appearance of pituitary adenomas [48, 49].

#### **Figure 6.**

*Schematic structure of the promoter of Pit-1. The human Pit-1 promoter is Pit-1-dependent and autoregulated***.** *Activating sequence of basal Pit-1 self-transcription (blue figure, +) is located at position −55 bp in the promoter, while a second sequence of Pit-1, in this case inhibitor of self-transcription (red figure, −), is located at position +15 bp. The transcriptional activity is negatively regulated by Oct-1 and mediated by the octamer-binding site OTF (red figure). Intracellular levels of cAMP positively affect Pit-1 transcription acting through two cAMPresponsive elements (CRE9 localized in the proximal promoter region). PTF: pituitary transcription factor, a cell-type-specific TATA element that seems not to be conserved in the human promoter. TRE: TPA-responsive element, located at position −490 in the proximal promoter region, whose possible function could be to mediate a complete transcriptional shut-off of the human Pit-1 gene when the DNA-binding activity of the Pit-1 protein is inhibited by mitotic phosphorylation. (TPA: 12-O-tetradecanoylphorbol-13-acetate responsive element).*

Basal transcription of the Pit-1 gene is self-regulated by two binding sequences for Pit-1 itself (**Figure 6**): an activating sequence located at position −55 and an inhibitory sequence, located at position +15. In addition, the transcription of this gene would be regulated by factors capable of increasing the levels of cAMP and activating cAMP response-element-binding (CREB). Likewise, the activation of the protein kinase C-dependent signaling pathway seems to play an important role, having described the existence of a response element to AP-1 (*activating protein-1*), which exerts an inhibitory effect on transcription [50].

Three isoforms of Pit-1 have been described, of which two of them with molecular weights of 31 kDa and 33 kDa are jointly called Pit-1 or Pit-1a and represent the majority of Pit in the pituitary. The third isoform, Pit-2 or Pit-1b, appears by alternative RNA processing; it retains its ability to bind DNA and may be involved in the differentiation process of somatotrophs [51].

Structurally, three regions are distinguished in Pit-1: (1) a DNA-binding zone located at the C-terminal end, in which two domains can be differentiated, a homeodomain (POUHD) of 60 amino acids found throughout the family of transcription factors with POU domains, and a specific POU domain (POUs) of 75 amino acids located at the N-terminus of the POUHD domain. The POUHD domain is the one that comes into contact with the DNA and is the one that contains the Pit-1 nuclear transfer signal. For its part, the POUs domain does not directly contact the DNA, but surely increases the binding of POUHD to it, stabilizing the DNA-protein complex [42]. (2) A region responsible for the activation of transcription, which comprises the 72 amino acids from the N-terminal end of the molecule and binds to site 1 of the response element. (3) A "bridge" region located between the two above.

In addition to Pit-1, GH transcription in man is increased by GHRH and glucocorticoids and inhibited by SS and activin [46]. GHRH increases intracellular cAMP that produces an increase in the synthesis of CREB proteins that bind to the two CREs (proximal and distal) of the GH promoter (**Figure 7**) increasing the transcription of the gene. On the contrary, both SS and activin counteract these actions by inhibiting the synthesis of cAMP. In the case of glucocorticoids, their effect is produced by the direct action of their receptor on the response elements located at the promoter and first exon level (**Figure 7**), facilitating the access of other transcription factors (especially Pit-1 and CREB) to their specific binding sequences [42].

*Pituitary Growth Hormone Secretion and Cell Growth Hormone Production: Regulation of Their… DOI: http://dx.doi.org/10.5772/intechopen.108382*

#### **Figure 7.**

*Structural organization of the GH promoter. The figure shows the location of the DNA response elements for the different transcription factors of the GH-N gene. GRE: glucocorticoid response element. CRE: cAMP response elements (dCRE: distal and pCRE: proximal). 1 and 2 represent, respectively, the binding sites for the DNAbinding domain and for the transactivation domain of the specific transcription factor Pit-1. NF-1 (nuclear factor-1) represents the binding sequence of the silencers of said family (found in rat). Sp-1 (specificity protein 1) provides GHRH responsiveness in the GH gene through activation of adenylate cyclase. The lower part of the figure shows the location of the second GRE at intron A of the gene. Ex: Exon. In: Intron. bp: base pairs.*

#### **2.4 Neurotransmitter regulation of GHRH and SS secretion**

Our group demonstrated that GH responses to an exogenous GHRH challenge and other stimuli for GH secretion, in humans, were strongly dependent on the functional status of the intrinsic HSR at the time of testing [8]. From this it is concluded that: (a) GHRH was not able to interrupt HSR the hypothalamic rhythm that governs GH secretion, and (b) stimuli capable of altering this rhythm must act mainly by inhibiting the release of SS, rather than by stimulating endogenous GHRH. In fact, a continuous infusion of GHRH does not alter the pulsatile GH secretion. The maintenance of pulsatile GH secretion when GHRH is continuously infused supports these conclusions.

On this basis, the role of the main neurotransmitters in the control of GH secretion was reevaluated.

#### *2.4.1 Adrenergic pathways and GHRH and SS secretion*

Central adrenergic pathways play an important role in the control of GH in mammals [52]. Pharmacological blockade of catecholamine synthesis or release leads to the abolition of pulsatile GH secretion, an effect reversed by the administration of α2 adrenergic receptor agonists. Conversely, pharmacological blockade of α1-adrenergic receptors does not modify the latter phenomenon [53].

For years, experimental evidence led to the postulate that GH secretion induced by central α2-adrenergic stimulation was mediated by increased GHRH release.

However, consistent with our observations described above [8], GH responses to any challenge must depend on the following: (a) potentiation of endogenous GHRH release, while SS tone is physiologically low, or (b) a primary inhibitory effect on

SS release, accompanied by a secondary stimulation of GHRH secretion. In the first case, the responses would be sporadic, depending on the functional status of HSR at the time of the test. In the second case, a significant GH release should always be observed. The rationale for this is also based on the fact that, although there is no GH secretion in the absence of GHRH, the GH-releasing effect of this peptide cannot be observed in the presence of a physiologically or pharmacologically elevated hypothalamic SS production tone. On this basis, we demonstrated in humans [54], dogs [55, 56], and rats [57] that α2-adrenergic pathways primarily act in GH control by inhibiting SS release, rather than inducing GHRH release, although hypothalamic release of this peptide is also stimulated by α2-adrenergic pathways. Blockade of α2-adrenergic receptors has been shown to stimulate SS release in rabbits [58], so there appears to be no interspecies difference in the role of α2-adrenergic pathways in GH control. Both the α1- and β-adrenergic systems antagonize the α2-effect, although the former appears to have no physiological relevance in man.

β-Adrenergic antagonists enhance GH responses to GHRH [59, 60] and other stimuli, such as insulin-induced hypoglycemia [59], which logically is due to inhibition of hypothalamic release of SS.

In summary, the adrenergic system performs two antagonistic functions in the regulation of human GH: facilitator, mediated by α2-adrenergic receptors that act mainly by inhibiting the release of SS and secondarily by inducing the release of GHRH; and inhibitory, dependent on β-adrenergic activity, which stimulates SS secretion and inhibits GHRH release [61, 62].

#### *2.4.2 Dopaminergic pathways and GHRH and SS secretion*

Studies on the role of dopaminergic pathways in GH secretion have yielded contradictory results; stimulant and inhibitory effects have been described. Vance et al. [63] reported that dopamine (DA) increased GH secretion caused by GHRH in humans, suggesting an inhibitory effect of DA on the hypothalamic release of SS, exerted at the level of the median eminence. However, administration of centrally acting DA agonists, such as bromocriptine [63] and CV 205–502 [64], has also been shown to enhance both spontaneous GH secretion and GHRH-induced release. Therefore, DA would presumably inhibit SS release at least at two levels (the hypothalamus proper and the median eminence).

*In vitro* studies have indicated that DA stimulates SS secretion from the median eminence; furthermore, an increase in SS release in the pituitary portal blood has been observed after intraventricular injection of DA in rats [54]. On the other hand, our group has reported that central DA receptor blockade with metoclopramide was able to enhance the GH response to GHRH during periods of physiologically increased delivery of SS to the pituitary, but not when the SS tone appeared to be low [65]. This suggested a stimulatory role for DA in hypothalamic SS release, but also that this could be mediated by an indirect mechanism: negative DA modulation of NA release. This hypothesis is not inconsistent with the aforementioned studies [63, 64] showing that DA agonists centrally facilitate GH release. In fact, centrally acting DA agonists, such as bromocriptine, induce a biphasic pattern of GH secretion: inhibition followed by rebound stimulation.

In summary, everything suggests that the central role of DA in the control of GH in humans depends mainly on its effects on adrenergic transmission to SS neurons. Therefore, DA would act as a modulator rather than a direct regulator of GH secretion.

*Pituitary Growth Hormone Secretion and Cell Growth Hormone Production: Regulation of Their… DOI: http://dx.doi.org/10.5772/intechopen.108382*

#### *2.4.3 Cholinergic pathways and GHRH and SS secretion*

Evidence indicates that cholinergic pathways play a key role in the control of GH secretion in both humans and laboratory animals [54, 66, 67]. Inhibition of central cholinergic pathways with muscarinic receptor-blocking drugs (atropine, pirenzepine) strikingly decreases GH release induced by a number of physiologic and pharmacologic stimuli [68, 69]; however, these have no effect on the GH secretion elicited by insulin-induced hypoglycemia. Conversely, enhancement of cholinergic tone with muscarinic cholinergic agonists potentiates GHRH-stimulated GH secretion and stimulates basal GH release as well.

It has been hypothesized that cholinergic synapses may be the final pathway for a variety of stimuli inducing GH secretion. The administration of anti-SS antibodies blocks the inhibitory effect of atropine on GHRH-elicited GH release in normal rats. Moreover, the acetylcholinesterase inhibitor pyridostigmine increases GH responses to a maximal stimulating dose of GHRH even when there is an abnormally high SS tone, as in obesity. Therefore, the facilitating role of cholinergic pathways on GH secretion appears to be mediated by an inhibition of hypothalamic SS release. In fact, while muscarinic cholinergic agonists stimulate basal GH secretion and its response to GHRH, muscarinic antagonists inhibit both responses [68, 69], immunoneutralization of GHRH by a GHRH-specific antibody does not affect the secretion of GH induced by pyridostigmine [70].

Because α2-adrenergic and muscarinic cholinergic pathways appear to play an equally important role in GH neuroregulation in humans, both by inhibiting hypothalamic SS release, it has been investigated the functional relationships between these two neurotransmitter pathways. Our findings obtained showed that while α2-adrenergic activation was able to overcome the inhibitory effect of muscarinic cholinergic blockade on GHRH-induced GH secretion, blockade of α2−adrenergic receptors counteracted the stimulating action of the pyridostigmine on said response [61]. Given the above, we speculated that in the control of GH, the α2-adrenergic neurons are located distally to the cholinergic neurons, so that the latter contribute to GH secretion by modulating the functional activity of the former, as occurring in peripheral tissues. The fact that known stimuli for SS inhibition acting through adrenergic neurons (e.g., galanin and insulin hypoglycemia) are able to overcome the inhibitory effect of atropine on GH secretion is consistent with this theoretical scheme, which, moreover, does not exclude the possibility that the cholinergic input directly reaches SS neurons and inhibits them, as acetylcholine does in hypothalamic cultures *in vitro*.

Thus, the degree of cholinergic activity likely determines the amount of catecholamines released into the synaptic cleft of SS neurons. This, in turn, activates either SS-stimulating β-adrenergic receptors (responsive to low concentrations of catecholamines) or SS-inhibiting α2-adrenergic receptors (responsive only to high catecholamine concentrations). To better understand the actions of these three neurotransmission pathways on GH secretion, see **Figure 8**.

#### *2.4.4 Other neurotransmitters acting on GHRH and SS secretion*

Given the complexity of hypothalamic structures and the relationships between different neurotransmitters in the control of hypothalamic functions, it seems logical that neurotransmitters other than those analyzed may play a role in the control of the hypothalamic discharge of GHRH and SS release into portal blood. This is the case for serotonin, gamma-aminobutyric acid (GABA), nitric oxide (NO), and endogenous opioids.

#### **Figure 8.**

*Main control of GH by neurotransmitters. The main role is played by adrenergic signals (CA) to somatostatinproducing neurons (SS). Depending on the amount of CA released into the synaptic cleft, inhibitory α2-R receptors (high CA input) or stimulatory β2-R adrenoceptors (low CA supply) are activated in SS neurons (see text). This, in turn, would be positively modulated by ACh supply to CA neurons, but negatively by DA. However, a direct inhibitory effect of ACh on SS neurons, or a stimulatory effect of DA on SS release in the median eminence (?), cannot be excluded. On the other hand, the stimulatory effect of CA on GHRH release, mediated by stimulation of α2-adrenergic receptors in the GHRH neuron, appears to be less important than the inhibition of SS. Therefore, SS appears to be the main determinant of the secretion pattern of GH. Blue arrows: stimulation. Red arrows: inhibition.*

### *2.4.4.1 Serotonin*

In rats, serotonin stimulates GH secretion, whereas in humans, stimulation or inhibition or even no effect has been described [53]. These discrepancies could be due to the low specificity of the drugs used, but also to the fact that serotonin acts on multiple types of receptors [71]. However, serotonin fibers have been found in periventricular nucleus and the arcuate nucleus where the SS and GHRH-producing neurons are located, respectively, so this neurotransmitter must play some role in the control of GH secretion. Indeed, administration of an anti-GHRH antibody suppresses serotonininduced GH secretion in rats [72] and hypoglycemia-induced GH release in man is abolished by administration of serotonin antagonists [73]. In turn, the administration of L-tryptophan, a precursor of serotonin, increases the release of GH.
