Endocrinology: Chemical Transmission

Endocrinology: Chemical Transmission

Autocrine:
Acting on the cells that synthesized them
Example: IGF – 1 which stimulates cell division in the cell which produced it.

Paracrine
Acting on neighbouring cells.
Example: Insulin, secreted by pancreatic b-cells and affecting secretion of glucagon by pancreatic cells.

Endocrine
Acting on cells or organs to which they are carried in the bloodstream or through another aqueous ducting system, such as lymph.
Examples: Insulin, Oestradiol and Cortisol.

Neuroendocrine
This is really paracrine or endocrine, except that the hormones are synthesized in a nerve cell (neurone) which releases the hormone adjacent to the target cell (paracrine), or releases it into the bloodstream, which carries it to the target cell, for example from the hypothalamus to the anterior pituitary gland through the portal system.

Neural
This is neurotransmission, when a chemical is released by one neurone and acts on an adjacent neurone. These chemicals are termed neurotransmitters. Neurotransmitters produce virtually instantaneous effects, for example acetylcholine, whereas some chemicals have a slower onset but longer lasting effect on the target organ, and are termed neuromodulators, for example certain opioids.

Pheromonal transmission
This is the release of volatile hormones, called pheromones, into the atmosphere, where they are transmitted to another individual and are recognized as an olfactory signal.

Basic Principles of Neurotransmission

When the nerve impulse arrives at the terminal, it triggers a calcium – dependent fusion of neurotransmitter packets or vesicles with the nerve terminal plasma membrane (Fig. 2b), followed by release of the neurotransmitter into the gap, or synapse, between the nerve cells. The neurotransmitters and neuromodulators bind to specific plasma membrane receptors, which transmit the information that the neurotransmitter has brought to the receiving cell by means of other membrane proteins and intracellular ‘ second messengers ’ . The neurotransmitters are inactivated by enzymes or taken up into the nerve that released them and metabolized. The release of the neurotransmitter may be modulated and limited by: (i) autoreceptors on the nerve terminal from which it was released, so that further release of the neurotransmitter is inhibited; and (ii) by presynaptic inhibition, when another neurone synapses with the nerve terminal.

An autoreceptor is a receptor located on the neuron (terminals, soma, and/or dendrites), and the function is to bind a specific ligand (such as neurotransmitters or hormones) released by that same neuron. The autorecptor is mainly used as a feedback mechanism to monitor neurotransmitter synthesis and/or release.

Chemical Transport

The movement of chemicals between cells and organs is usually tightly controlled.

Diffusion is the movement of molecules in a fluid phase, in random thermal (Brownian) motion (Fig. 2c). If two solutions containing the same chemical, one concentrated and the other relatively dilute, are separated by a membrane which is completely permeable and passive, the concentrations of the chemical on either side of the membrane will eventually end up being the same through simple diffusion of solutes. This is because there are many molecules of the chemical on the concentrated side, and therefore a statistically greater probability of movement from the more concentrated side to the more dilute side of the membrane. Eventually, when the concentrations are equal on both sides, the net change on either side becomes zero.  Lipophilic molecules such as ethyl alcohol and the steroids, for example estradiol, appear to diffuse freely across all biological membranes.

Facilitated transport is the transport of chemicals across membranes by carrier proteins. The process does not require energy and cannot, therefore, transport chemicals against a concentration gradient. The numbers of transporter proteins may be under hormonal control. Glucose is carried into the cell by transporter proteins (see Chapter 39 ) whose numbers are increased by insulin.

Active transport uses energy in the form of adenosine triphosphate (ATP) or other metabolic fuels. Therefore chemicals can be transported across the membrane against a concentration gradient, and the transport process can be interrupted by metabolic poisons.

Ion channels mediate active transport, and consist of proteins containing charged amino acids that may form activation and inactivation ‘ gates ’ . Ion channels may be activated by receptors, or by voltage changes through the cell membrane.  Channels of the ion Ca 2 + can be activated by these two methods.

Osmosis is the passive movement of water through a semipermeable membrane, from a compartment of low solute concentration to one which has a greater concentration of the solute. ( ‘ Solute ’ refers to the chemical which is dissolved in the ‘ solvent ’ , usually water in biological tissues.) Cells will shrink or swell depending on the concentrations of the solutes on either side of the membrane.

Phagocytosis and pinocytosis are both examples of endocytosis. Substances can enter the cell without having to pass through the cell membrane. Phagocytosis is the ingestion or ‘ swallowing ’ of a solid particle by a cell, while pinocytosis is the ingestion of fluid. Receptor – mediated endocytosis is the ingestion of specifically recognized substances by coated pits. These are parts of the membrane which are coated with specific membrane proteins, for example clathrin.

Exocytosis is the movement of substances, such as hormones, out of the cell. Chemicals which are stored in the small vesicles or packets are secreted or released from the cell in which they are stored by exocytosis, when the vesicle fuses with the membrane.

Hormone transport in blood. When hormones are secreted into the blood, many are immediately bound to plasma proteins (Fig. 2d). The proteins may recognize the hormone specifically and bind it with high affinity and specificity, for example the binding of sex hormones by sex hormone – binding globulin (SHBG). Other proteins, such as albumin, also bind many hormones, including thyroid hormone and the sex hormones, with much lower affinity. Equilibrium is set up between the free and bound hormone, so that a fixed proportion of the hormone travels free and unbound, while most is carried bound. It is currently believed that only the free fraction of the hormone is physiologically active and available to the tissues and for metabolism. When a hormone is bound to plasma proteins it is physiologically inactive and is also protected from metabolic enzymes in organs such as the liver. Some drugs, such as aspirin, can displace other substances such as anticoagulants from their binding sites, which in the case of anticoagulants may cause haemorrhage.

Lipophilic vs Hydrophilic Transport

Mechanism of Hormone Action I – Membrane Receptors (Receptor Families)

Clinical background
Acromegaly is usually caused by anterior pituitary gland tumours which secrete growth hormone. In 30 to 40% of cases, the tumour is thought to arise due to a somatic mutation affecting transmembrane signalling mechanisms. The stimulatory G – protein Gs is involved in signal transduction at the growth hormone releasing hormone receptor. Mutation of the α – subunit of Gs into the gsp oncogene prolongs the activation phase of the G – protein system, allowing unrestrained hormone synthesis and cell division. The distinctive clinical features of acromegaly and development of the pituitary tumour follow.

Introduction
Hormones interact with target cells through a primary interaction with receptors which recognize the hormones selectively.  There are several different receptor systems, which vary in mechanism and timing (Fig. 3a). Charged ions such as peptides and neurotransmitters bind to receptors on the cell membrane.  This causes a conformational change in other membrane proteins, which activate enzymes inside the cell, resulting in, for example, the synthesis of ‘ second messengers ’ , which activate phosphorylating enzymes.

Uncharged molecules, such as the steroid hormones diffuse into the cell and bind to intracellular receptors (see Chapter 4 ). The hormone – receptor complex binds to specifi c hormone response elements (HRE) on the DNA; the result is that RNA and protein synthesis are altered. The cell will react faster to peptide hormones and neurotransmitters than it will to steroid hormones, which work through relatively slow changes in protein synthesis. Nevertheless, membrane receptors have been discovered for steroid hormones, although the signifi cance of these is not yet clear.

Membrane receptors
Three regions can be distinguished in membrane receptors: the extracellular; the membrane – spanning; and the intracellular domains. The extracellular N – terminal domain has the hormone – binding domain, and also has glycosylation sites. The extracellular domain that binds the receptor is often rich in cysteine residues, which form rigid pockets in which the hormone is bound. The transmembrane region consists of one or more segments, made up of hydrophobic (uncharged) amino acids, arranged helically, whose role may include the anchoring of the receptor in the membrane. Different subunits within the membrane may be held together by means of disulphide linkages (e.g. the insulin receptor, Chapter 39 ). The intracellular domain is the effector region of the receptor, which may be linked with another membrane protein system, a set of enzymes which are guanosine triphosphatases (GTPases). The β – adrenergic receptor is an example of a G – protein – linked receptor. Another class, which includes the insulin receptor, has the intracellular domain as a tyrosine protein kinase. The intracellular region may also have a regulatory tyrosine or serine/ threonine phosphorylation site.

Second Messengers

G protein linked receptors.
These are protein receptors in the cell membrane, with an extracellular domain and an intracellular domain. The peptide chain that forms the protein always spans the membrane. When the hormone binds to the extracellular domain, this causes a change in shape of the receptor. This causes the intracellular domain to activate G proteins. G proteins have three main parts: an a subunit, a b subunit and a g subunit. When activated, firstly the a subunit substitutes a GDP molecule for a GTP molecule. This results in the activation of the G proteins.

They can be either stimulatory or inhibitory, that is they can cause an increased level of enzyme activity or a decreased level of activity in the second messenger systems. Mutations of G proteins can occur and may result in disease (see Clinical scenario above).

Mechanism of Hormone Action I – Membrane Receptors (Adenylate Cyclase)

Adenylate cyclase system. The hormone binds to the receptor, which activates a membrane G protein, which moves to the receptor (Fig. 3b). In the inactive state, the G protein binds GDP, which is exchanged for GTP, and a subunit of the G protein activates adenylate cyclase to convert ATP to the second messenger cyclic AMP. Adenylate cyclase is situated on the plasma membrane, but does not itself bind the hormone. Once formed in the cytoplasm, cAMP activates the catalytic subunit of a specific protein kinase (PKA), which forms part of a cascade of intracellular phosphorylations resulting in the cellular response.  Since just one molecule of hormone can result in the production of many molecules of cAMP, this is a very effi cient means of amplifying the receptor – hormone interaction. Once formed, cAMP is rapidly broken down by the enzyme phosphodiesterase.

An example of a hormone operating through adenylate cyclase is epinephrine, through the adrenergic β – receptor.

Hormones can produce inhibitory effects on a cell, and this may be achieved through the fact that some G proteins, such as GI , may inhibit adenylate cyclase, thus inhibiting the formation of cAMP. An example of this mechanism in action is the inhibition of adenylate cyclase through the binding of norepinephrine to the α – 2 – receptor on the presynaptic nerve terminal.

Mechanism of Hormone Action I – Membrane Receptors (Inositol Triphosphate System)

Inositol triphosphate system. In this system, the hormone – receptor – G – protein complex interaction triggers the membrane enzyme phospholipase C (PLC), which catalyses the hydrolysis of phosphoinositol (PIP2) to two important metabolites, inositol triphosphate (IP3) and diacylglycerol (DAG; Fig. 3c). IP3 generates, from the endoplasmic endothelium, increased free Ca 2 + , which together with DAG promotes the activation and migration to the membrane of the enzyme protein kinase C (PKC).

PKC may also be mobilized through the entry of Ca 2+ into the cell. Examples of hormones and neurotransmitters which activate the system are epinephrine acting on α – 1 receptors and acetylcholine on muscarinic cholinergic receptors. These systems are important clinically since they provide substantial numbers of possible targets for drugs.

Receptor antagonists
Receptor antagonism is an important aspect of endocrinology and drug use generally, not only in terms of the study of the hormone – receptor interaction, but also in therapeutic terms, since antagonists play a large part in the treatment of endocrine disease. The molecule which binds to the receptor and elicits the normal cellular response is termed the agonist . The ligand which binds, but elicits no response, is the antagonist . Antagonists act at the membrane in different ways. For example the β – receptor blocker propranolol competes with epinephrine at its binding site. The anticonvulsant phenytoin blocks ion channels.

Mechanism of Hormone Action II – Intracellular Receptors

Intracellular receptors

Lipophilic hormones, such as steroids and the thyroid hormones, pass easily through the plasma membrane into the cell, where they combine with specifi c receptor proteins (Fig. 4a).

In the inactive state, for the subfamily of glucocorticoid, progesterone, estrogen and androgen receptors, the receptor is bound to a heat shock protein (HSP 90; Fig. 4b).

When the hormone binds to the receptor, the HSP dissociates from it, the receptors form homodimers and the hormone – receptor complex binds to DNA at specific sites, termed hormone response elements (HREs), which lie upstream from transcription initiation sites. Transcription and subsequent protein synthesis are altered. The thyroid hormone and retinoic acid receptors are not associated with HSPs in their inactive state, and are able to associate with their response elements on the DNA in the absence of the hormones, and act as transcription inhibitors (see also below). Activation of receptors expressing the actions of the hormones appears to be achieved through phosphorylation, although at present this process is poorly understood.

Mechanism of Hormone Action II – Nature of the Steroid Receptor

Nature of the steroid receptor

The steroid receptors form part of a larger ‘ superfamily ’ of nuclear DNA – binding receptors, including androgen, estrogen, glucocorticoid, thyroid and vitamin D receptors (Fig. 4c). They all have two main regions, a hydrophobic hormone – binding region and a DNA – binding region, which consists of two ‘ zinc fingers ’, rich in cysteine and basic amino acids. The structures of the receptors are known. Region 1 is the DNA – binding region, and is the most conserved among the members of the receptor family, in that it has a high sequence homology from receptor to receptor, as shown in Fig. 4c. It is thought that the first zinc finger determines the specificity of the binding of the receptor to DNA, while the second finger stabilizes the receptor to its response element of the DNA. Regions 2 and 3 of the receptors determine the hormone specificity of binding, and are not well conserved among the different receptors.

Mechanism of Hormone Action II – Oestrogen & Thyroid Hormone Receptors

Clinical background

Estrogen stimulates the proliferation of breast cancer tissue and exposure to estrogens may be important in the pathogenesis of this disease. During the treatment of women with breast cancer it is routine practice to establish the presence (ER + ve) or absence (ER – ve) of estrogen receptors in cancer cells. Women who have ER + ve tumours are more likely to respond to endocrine manipulation following surgery and/or chemotherapy (50 – 60% response rate in ER + ve cancers, 5 – 10% in ER − ve tumours). The most commonly used endocrine therapy is the drug tamoxifen which has estrogen – antagonist effects in the breast, probably mediated by the recruitment of corepressors for estrogen receptor action. It produces a signifi cant fall in tumour recurrence and death rates for women with ER +ve disease, irrespective of age. The possible use of tamoxifen and the newer, selective estrogen receptor modulator drugs (SERMs, e.g. raloxifene, toremifine) for the prevention of breast cancer are under investigation. Trastuzumab, a humanized IgG1 against human epidermal growth factor receptor – 2 (HER – 2 + ), is now used to treat early breast cancer that overexpresses HER – 2.

Estrogen receptors

Two distinct, main receptor forms have been discovered, called ER – α and ER – β respectively. They have different affinities for estradiol and different anatomical distribution. For example only ER – α has been found in the liver, and ER – β is the predominant form in prostate. These differences may account, in part, for the wide diversity of estrogen action in different tissues and under different physiological and pathological states. It has been found, for example, that in healthy ovarian tissue the β form predominates, but in ovarian cancer the a form predominates.

It is possible that the β form somehow regulates the activity of the α form. The α and β forms have several nuclear coactivators and repressors, and their activity depends also on their rates of turnover.

Estrogen receptor antagonists have found a powerful use in the prevention and treatment of breast cancer (see Clinical scenario above). These compounds interfere with the processing of the normal intracellular hormone – receptor interaction.

This can occur at one or more of several sites (Fig. 4d). The receptor itself may be blocked or post – receptor – binding events, for example receptor dimerization, receptor turnover or mRNA or protein synthesis, may be inhibited. Examples of estrogen receptor blockers are the SERMS (selective estrogen receptor modulators) such as tamoxifen, raloxifene and toremifene.

These are interesting because they appear to act as agonists in some tissues such as bone and liver cells, and may therefore be important preventive measures for reducing the rate of development of osteoporosis and for lowering blood cholesterol.

SERMS may act by activating as yet unidentified coactivators or corepressors and may modulate estrogen receptor turnover. Their action may also be dictated by whether they combine with ER – a or ER – b receptors.

Thyroid hormone receptors

Like other members of the nuclear receptor family, thyroid hormone receptors function as hormone – activated transcription factors. In contrast to steroid hormone receptors, however, thyroid hormone receptors bind to DNA in the absence of hormone, leading usually to transcriptional repression. When thyroid hormone binds to the receptor, however, it causes a conformational change in the receptor that changes it to function as a transcriptional activator. As with many other receptors, several isoforms have been discovered. Currently, four different isoforms are recognized, namely: α – 1, α – 2, β – 1 and β – 2. These different forms appear to be very important in development; different isoforms are expressed at different stages of development and in different organs and tissues. For example α – 1, α – 2 and β b – 1 are expressed in virtually all tissues in which thyroid hormones act, but β – 2 is synthesized mainly in the developing ear, and in the anterior pituitary gland and hypothalamus. Receptor α – 1 is the first isoform detected in the conceptus, and the β form appears to be essential for normal brain development shortly after birth.

Types of Hormones

Steroid Hormones

  • Steroid hormones are lipophilic (fat-loving) – meaning they can freely diffuse across the plasma membrane of a cell
  • They bind to receptors in either the cytoplasm or nucleus of the target cell, to form an active receptor-hormone complex
  • This activated complex will move into the nucleus and bind directly to DNA, acting as a transcription factor for gene expression
  • Examples of steroid hormones include those produced by the gonads (i.e. estrogen, progesterone and testosterone)
    • Vitamin D
    • Estrogen
    • Glucocorticoid
    • Thyroid
    • Retinoic acid
    • Androgen
    • Mineralocorticoid
    • Progesterone
    • Peroxisome profilerator-activated receptor (PPAR)

Peptide Hormones

  • Peptide hormones are hydrophylic and lipophobic (fat-hating) – meaning they cannot freely cross the plasma membrane
  • They bind to receptors on the surface of the cell, which are typically coupled to internally anchored proteins (e.g. G proteins)
  • The receptor complex activates a series of intracellular molecules called second messengers, which initiate cell activity
  • This process is called signal transduction, because the external signal (hormone) is transduced via internal intermediaries
  • Examples of second messengers include cyclic AMP (cAMP), calcium ions (Ca2+), nitric oxide (NO) and protein kinases
  • The use of second messengers enables the amplification of the initial signal (as more molecules are activated)
  • Peptide hormones include insulin, glucagon, leptin, ADH and oxytocin

Amine Hormones

  • Amine hormones are derived from the amino acid tyrosine and include adrenaline, thyroxin and triiodothyronine
  • Amine hormones do not all share identical properties and have properties common to both peptide and steroid hormones