Intraluminal volume homeostasis

Abstract

Volume homeostasis is a common physiological phenomenon for fluid secreting organs, such as exocrine and endocrine glands. It is a manifestation of a finite intraluminal space and an ever changing demand for secretory fluids. Volume homeostasis addresses issues of fluid secretion, storage and clearance for efficient functioning. Here we discuss the evidence gathered over the past 2-3 decades on serotonin’s role as a feedback inhibitor of secretion in the mammary gland, salivary gland, liver, pancreas, lung, thyroid gland and prostate gland. We propose that serotonin action is a common mechanism of regulating intraductal volume homeostasis. 

In any organism, a large fraction of the body is composed of fluid. There are various specialized fluids that perform specific functions e.g., milk provides nourishment to the infant, saliva, pancreatic juice and bile aid in digestion, prostate fluid contributes to semen etc. With the presence of these different specialized fluids arises the issues of their compartmentalization and volume regulation in relation to their demand and physical space available in the organ (volume homeostasis).

The majority of these secretory organs (including exocrine and endocrine glands) have the basic tissue architecture of an arborized ductal network that terminates into numerous spherical lobules, alveoli or acini.1 These ductal and lobuloalveolar structures are lined by epithelial cells that are responsible for production and secretion of the specialized fluids. In addition, the junctions between these epithelial cells (adherence and tight junctions) compartmentalize the ductal and lobuloalveolar structures from the surrounding stroma and thus define the physical space available for fluid secretion. Because of these reasons the ductal and alveolar epithelial cells are paramount for volume homeostasis.

The repertoire of serotonin's (5-Hydroxytryptamine, 5-HT) actions affect virtually all major organ systems including, cardiovascular, pulmonary, gastrointestinal, genitourinary and the central nervous system.2 The serotonin system is highly complex; consisting of rate limiting biosynthetic enzymes tryptophan hydroxylase (TPH), 5-HT reuptake transporter (SERT) which internalizes 5-HT, monoamine oxidases (MaO) which metabolise 5-HT and an extensive network of >20 different receptors that are divided into seven classes (5-HT₁–5-HT₇) based on pharmacological properties.3

Given the extensive presence and function of 5-HT, here we conduct a highly focused discussion of involvement of 5-HT in volume homeostasis and make the case that this is a common mechanism present across various fluid secreting organs.

Mammary Gland

5-HT action on volume homeostasis has been most extensively studied in the mammary gland. The mammary gland is an exocrine gland that is the most recent acquisition on an evolutionary timescale. It is found in all mammals and its main function is nourishing the infant through milk secretion. Unlike most organs, mammary gland development occurs postnatally in association with pregnancy. The mammary gland secretes milk in a cyclical manner in relation to suckling of the infant and stores milk in between bouts of nursing (milk stasis).4 This necessitates the presence of a reversible feedback inhibitory mechanism to rein in milk secretion in accordance with the volume-space availability of the gland.

A comparative genomic analysis of non-secretory and hyper-secretory mouse mammary glands showed high induction of the 5-HT biosynthetic enzyme, TPH1.5 5-HT biosynthesis was detected during lactation and 5-HT was found in the mammary epithelium and in milk. Interestingly, 5-HT biosynthesis was induced by milk stasis (accumulation) during lactation. 5-HT inhibits milk protein secretion in vivo and in explant cultures.5 Alternatively, 5-HT biosynthesis disruption and 5-HT receptor antagonists significantly enhance secretory features and caused alveolar dilation.5 This led to the conclusion that 5-HT, in an autocrine-paracrine manner, regulates volume homeostasis within the mouse mammary gland. Similar actions of 5-HT in affecting lactation have been observed in humans and bovine.6,7

Human mammary epithelium expresses multiple 5-HT receptors (5-HT_1D, 5-HT_2B, 5-HT_3A and 5-HT₇).8,9 Similar observations have been made in rodents and bovine.8–10 Among these, 5-HT₇ expression is conserved across species and hence has been studied most extensively.8,9 5-HT₇ receptor is localized to the basolateral membrane of mouse and human mammary epithelial cells8,9 as depicted in the model in Figure 1. SERT has also been found in human and mouse mammary gland and is localized to the apical membrane of the mammary epithelial cells6,8,9 (Fig. 1).

A critical transition from a non-secretory mammary epithelium (during pregnancy) to a secretory epithelium (post-partumlactation) is the closure of epithelial tight junctions.11 Tight junction closure compartmentalizes milk secretion and accumulation chamber from the rest of the gland. This is important as persistent leakage of milk into the interstitium is a strong signal for the gland to undergo involution and subsequent tissue remodeling that occurs after weaning.12 Using in vitro models of differentiated human and mouse mammary epithelium,13 5-HT has been shown to breach the tight junction barrier8,14 (Fig. 1). Similar disruptive action of 5-HT on tight junctions has been observed in the bovine mammary glands.15 This action of 5-HT is mediated by its 5-HT₇ receptor. Interestingly, 5-HT action on tight junctions was found to be biphasic where 5-HT initially strengthens tight junctions but upon sustained exposure causes disruption of tight junctions.14 This biphasic response is due to an elegant switch in the signaling downstream of the 5-HT₇ receptor. The biphasic response to 5-HT is postulated to play an important role during milk stasis which is accompanied by increased intraluminal pressure. Strengthening of tight junctions prevents the leakage of milk into the interstitium in between bouts of nursing (increased pressure). However, upon weaning continued milk stasis breaches the tight junction and induces mammary gland involution.16,17

The impact of 5-HT on milk secretion has been validated in vivo in both bovine and humans via administration of SERT inhibitors (SSRIs) which increase the bioavailability of 5-HT. SSRIs delay the onset of copious milk secretion in primiparous women.6 In bovine, SSRIs suppress milk secretion and accelerate involution (via breach of epithelial tight junctions).15 Hence 5-HT acts as a critical regulator of milk secretion and milk volume in the mammary glands. Other actions of 5-HT in the mammary gland include regulating mammary epithelial turnover via regulating processes such as cell shedding and apoptosis.18 These effects are likely mediated by other receptors present in the mammary epithelium and will not be discussed in detail in this article.

Salivary Glands

The salivary glands are exocrine glands with classic ductal and acinar structure and produce saliva whose main function is to keep food moist while eating. In mammals like humans they also secrete digestive enzymes such as amylases.19 Immunohistochemical analysis of rat submaxillary salivary glands showed the presence of 5-HT in the epithelial cells of excretory ducts and acini.20 The same cells that were positive for 5-HT were also found to contain the G_i coupled (inhibitory) 5-HT_1A receptor suggesting an autocrine regulation by 5-HT. In addition, the rat submaxillary glands were also found to contain 5-HT_4b and 5-HT_7a (G_s coupled stimulatory) receptors indicating a separate autocrine-paracrine role of 5-HT.21 Given the specific (and physiologically opposite) 5-HT receptors, there appears to be more than one autocrine-paracrine action of 5-HT in the rat submaxillary glands.

Analogously, rat parotid salivary glands show opposing effect of 5-HT on secretion of saliva and enzyme amylase.19,22,23 5-HT treatment decreases salivary flow rate but increased amylase secretion. This might be due to action via specific 5-HT receptor subsets which need to be experimentally tested in the parotid gland.

In perfused rat submaxillary glands, 5-HT decreased the saliva flow rate initiated by acetylecholine (Ach).19,22,23 Analysis in the dispersed cell aggregates from the gland showed cAMP accumulation in response to 5-HT suggesting a receptor-mediated action of 5-HT in regulating saliva volume and protein content.19,22,23 Similar cAMP accumulation in response to 5-HT was observed in mouse and opossum submaxillary glands and rat sublingual and parotid glands.

Interestingly, extensive study of insect (blowfly) salivary glands has shown a prominent role of 5-HT (via cAMP pathway) in regulating salivary secretions via modulation electrochemical gradients driving ion transport.24,25 In this system, the 5-HT receptors are localized on the basolateral membrane similar to that seen in the mammary gland as depicted in the model in Figure 1.26

Liver

The liver is the largest internal organ and the largest gland in the human body. The highly specialized tissues of liver perform various vital functions like detoxification, glycogen storage, protein synthesis, hormone production, decomposition of red blood cells and bile secretion for digestion and emulsification of lipids.27

The biliary tree consists of arborized bile ducts lined by cholangiocytes which are epithelial cells that contribute to bile secretion.28 Cholangiocytes are normally mitotically dormant, but proliferate in response to blockage of bile duct (and subsequent bile accumulation and increased pressure).29

In rats, cholangiocytes synthesize and secrete 5-HT which is increased upon blockage of bile duct (Bile duct ligation-BDL).30 The increased 5-HT has two distinct effects on the biliary tree; it inhibits the proliferation of cholangiocytes and it inhibits the cholangiocyte secretion that contributes towards bile.30 Analogously, Gcoupled (inhibitory) 5-HT_1A and 5-HT_1B receptors have been found in the basolateral membrane of cholagiocytes and mediate both actions of 5-HT.30 5-HT via 5-HT_1A and 5-HT_1B inhibits bile and bicarbonate secretion even in presence of the stimulant secretin.

In addition to the autocrine signaling to inhibit bile secretion, 5-HT also stimulates the stromal cells in a paracrine manner to produce TGFβ(1).31 Increased TGFβ(1) counters the autocrine inhibition of bile secretion and proliferation in cholangiocytes by suppressing 5-HT synthesis.

Interestingly, similar to mammary gland, cholestasis induces changes in the tight junction protein distribution.32 This affects the barrier function in an effort to contain bile intraluminally. 5-HT has been shown to affect mammary epithelial tight junction permeability8,14 (Fig. 1), however such action of 5-HT in liver has not been explored.

Pancreas

The pancreas consists of two secretory systems, endocrine (insulin secreting β cells) and exocrine (digestive enzyme secreting epithelial cells) system. The 5-HT physiology in the endocrine system will not be discussed here as it is not a part of the volume-homeostatic system.8,14 The exocrine system consists of an extensive ductal network that needs two components operating oppositely and cyclically (digestive and post-digestive periods); a stimulatory input that initiates secretion and a negative feedback input that inhibits secretions when not required. Pancreatic exocrine stimulators are well studied and have been previously reviewed in reference 33. In terms of negative feedback, a direct autocrine-paracrine inhibition of exocrine secretion has been documented with 5-HT identified as one of the highly potent local inhibitors.34 Other local factors include arginine, vasopressin, substance P, etc.33

Pancreatic duct cells secrete HCO₃^- ions to control the basal fluid secretion of pancreatic juice.35 Solitary serotonin producing cells are dispersed across the acinar and ductal epithelium of the exocrine pancreas.35 Isolated guinea pig pancreatic exocrine duct explants have been found to respond to 5-HT.33 Exogenous 5-HT decreased the HCO₃^- dependent basal fluid secretion of the explants. This inhibitory action of 5-HT was observed even in presence of stimulatory signals like secretin or Acetylcholine (ACh). Interestingly, 5-HT acted specifically through the basolateral side as apical application of 5-HT had no effect. This effect of 5-HT was mediated by 5-HT₃ receptor. 5-HT₃is a ligand-gated cation channel which by allowing flow of Na⁺ and Ca²⁺ ions along the basolateral membrane, dissipates the gradient necessary for intracellular HCO₃^- accumulation. This intracellular HCO₃^- accumulation is essential for HCO₃^- secretion into the lumen. Additionally, increasing intraductal pressure in vivo, decreased fluid secretion and this decrease could be attenuated by 5-HT₃ antagonist.33 Hence, the combined evidence documenting 5-HT action on pancreatic exocrine system suggests that the solitary 5-HT cells act as pressure sensors to regulate fluid secretion.

Lungs

In the lungs there are two set of cells that secrete serotonin. Both are collectively called Pulmonary NeuroEndocrine Cells (PNEC). One set of cells is organized into clusters called NeuroEpithelial Bodies (NEB) and are widely studied.33 The second set of cells is arranged in a solitary manner diffused across the airway epithelium and are called NeuroEpithelial Cells (NEC). Physiologically, NECs are quite different than NEBs and only recently began to be investigated.36 5-HT physiology in the NEBs is part of the oxygen sensing apparatus in lung36,37 and hence will not be discussed here. The NECs are widely distributed within the lung epithelium and postulated to be critical in early lung development. These cells extend long dendritic cytoplasmic process along the basement membrane making direct contact with several neighboring epithelial cells as well as epithelial-mesenchymal interaction with subbasement membrane mesenchyme.38,39 Mechanical stretch induces TPH1 and 5-HT release by NECs. Blocking NEC mechanoreceptors attenuates the release of 5-HT.36,37 Mechanical stretch in turn stimulates proliferation of lung epithelial cells and induction of extracellular matrix (ECM) and differentiation of alveolar cells.

Complementing the above study, cultured guinea pig lungs from late gestation fetuses show a 5-HT dose-dependent decrease in the rate of fluid secretion, and activated reabsorption of fluid in the lung.36,37 This phenomenon is critical for the transition of the lungs during childbirth. Furthermore, 5-HT receptor antagonism blocks these effects. Taken altogether, serotonin is induced by stretch, and causes fluid clearance in the late gestation developing lung thus, functioning as a regulator of volume homeostasis in the developing lung.

Thyroid

The thyroid gland is one of the largest endocrine gland and controls energy metabolism, growth and function of many other organ systems of the body. It performs these functions by release of two hormones, triiodothyronin (T3) and thyroxine (T4). Unlike the exocrine glands discussed earlier in this article, the thyroid mainly consists of spherically structures called thyroid follicles made of thyroid epithelial cells. These follicular cells synthesize the thyroid hormones. The centers of these follicles are filled with colloid, which is a proteinacious depot of thyroid hormone precursors. In addition to follicular cells, specific cells nestled between the follicles called parafollicular cells are also present. 5-HT has been localized to both follicular and parafollicular cells.40 Interestingly, 5-HT is also present in the colloid in the follicular lumen and its concentration in the colloid is greater than the follicular cells.41,42 This suggests secretion of 5-HT by the follicular cells. However, localization of SERT only in the follicular cells41,42 has led to the hypothesis that 5-HT is synthesized by parafollicular cells and acts in a paracrine manner on the follicular cells. Hence, the source of 5-HT synthesis still remains unclear.

Chronic co-treatment of rats with L-tryptophan (a 5-HT precursor that increases 5-HT production) and a SERT inhibitor (increases 5-HT availability) reduced serum levels of thyroid hormone.41–43 Alternatively, chemical inhibition of 5-HT synthesis (DL-p-chlorophenylalanine) or administration of broad spectrum 5-HT receptor antagonist increases serum levels of thyroid hormone.44 These observations indicate that 5-HT regulates thyroid hormone secretion likely in an autocrine-paracrine manner. However, in this case an indirect effect of 5-HT via influence on neural—pituitary axis cannot be ruled out.

Prostate

The prostate gland is an exocrine organ in males that secretes and stores a slightly acidic fluid that contributes towards the final semen. Prostate structure is typical of an exocrine gland with a ductal architecture ending into acini that secrete the fluid. In the prostate, distinct 5-HT producing cells have been found and are referred to as the neuroendocrine (NE) cells.44 The scientific community studying the prostate gland has delved into intensive study of NE cell's involvement in prostate cancer, however there are only a few studies addressing the structure and function of NE cells in normal prostate physiology.

The serotonin producing cells are widely distributed in the prostate epithelium, but are more abundant in the major ducts and irregularly distributed in the acini.45 Analogous to the lung, there are at least two different subpopulations of 5-HT producing cells. One subpopulation (mainly in the peripheral zone) responds to the factors present at birth and at puberty and are suggested to play a role in prostatic growth and differentiation.46,47 These will not be discussed in detail in this article. The second subpopulation mainly resides in prostatic duct system, and are not influenced by the same factors but are postulated to be involved in homeostatic regulation of the secretory process of the gland.45 The morphology of these cells is similar to those found in lung epithelium, where these cells extend dendrite-like processes underneath and between adjacent epithelial cells.45 Based purely on the morphological similarly and physiological comparison with other organs it is postulated that these 5-HT producing cells directly regulate the secretion of adjacent epithelial cells in a paracrine manner.46,47 Along similar lines as that of lung, the prostate 5-HT secreting cells may also be induced to secrete 5-HT by a stretch-activated mechanism. These hypotheses remain to be experimentally validated.

Conclusions

Based on the literature discussed here, it is evident that 5-HT plays a central role in volume homeostasis of various fluid-secreting organs (including endocrine and endocrine). There appears to be a common pattern of 5-HT system in these secretory organs where 5-HT is secreted upon fluid accumulation and/or increased intraluminal pressure (Fig. 1). However, it is important to note that in organs like salivary glands, the wide spread localization of 5-HT may be a manifestation of 5-HT uptake via SERT and only a few subset of cells may actually be synthesizing 5-HT. 5-HT mainly acts as a feedback inhibitor of fluid secretion. Although 5-HT receptor expression in the organs varies widely (Table 1), they are mainly found to be located on the basolateral side of the epithelial cells (Fig. 1). Additionally, 5-HT regulation of volume homeostasis in these secretory organs is conserved across multiple species and classes.

All this evidence points to the hypothesis that 5-HT regulation of secretion and volume homeostasis is a very basic and evolutionarily ancient phenomenon that has been adapted and used to suit the needs of various organ systems as they evolved. The abundant repertoire of 5-HT receptors may be a manifestation of this divergent evolution. This hypothesis is supported by the findings that on an evolutionary timeline 5-HT is a very old molecule with function in both plant and animal kingdom.45,48 Also, 5-HT is linked to ion transport and fluid secretion right from establishment of body axis and first epithelial differentiation49 to intestinal fluid and ion transport.50–52

Figures and Tables

Figure 1 Mammary epithelial serotonin system and its mechanism of action. Diagramatic representation of mammary epithelial serotonin system. Mammary epithelial cells synthesize serotonin and secrete it into their surroundings. This serotonin acts in an autocrine-paracrine manner through its receptors. Among these, 5-HT₇ receptor has been localized to the baso-lateral side of the mammary epithelial cells. 5-HT through the 5-HT₇ receptor generates two signals; a cAMP-PKA signal and a cAMP-p38^MAPK signal. Serotonin reuptake transporter (SERT) is present on the apical membrane of the mammary epithelial cells and is involved in recycling and metabolism of mammary serotonin.

Table 1 Serotonin receptor expression in ductal organs

Organ/Tissue	Receptor	Function	References
Mammary Gland
Epithelial cells (immortal and primary)	5HT7	Tight junction regulation	53
Isolated epithelial cells	5HT1D, 5HT2B, 5HT3A	Unknown	8, 14
Bovine epithelium	5HT1B, 5HT2A, 5HT2B, 5HT4, 5HT7	Suppression milk gene expression	9
Salivary Glands
Submandibular gland homogenate	5-HT1A, 5HT4B, 5HT7A	Inhibit fluid secretion	10
Liver
Cholangiocytes	5HT1A, 5-HT1B	Inhibit Proliferation and inhibit bile secretion	21
Pancreas
Isolated interlobular ducts	5HT3	Inhibit HCO3^- secretion	30
Lungs
Neuroepithelial bodies	5HT3	Oxygen sensing	33
Neuroepithelial cells	?	Inhibit secretion and induce fluid resorbtion	38, 39
Thyroid
Follicular cells	5-HT1 and 5-HT2	Biphasic action on growth and inhibition of hormone secretion	40
Prostate
Human prostate homogenate	5HT1A, 5HT1B, 5HT1D	Growth promoting in cancers	42