Evidence For Non Genomic Actions Of Steroid Hormones Biology Essay

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Steroid hormones are a "class of lipids derived from cholesterol." [1] They are essentially steroids that function as hormones; released by endocrine glands, particularly adrenal and gonadal, into the blood stream where they travel to the target cells. Being lipids, they easily pass through cell membranes and bind to the specific receptors to initiate a response. The actions of these hormones has been widely researched and these are sub-classified into two main categories; genomic and non-genomic actions.

The effects of steroids that are mediated by transcription and expression of genes are known as genomic actions. They are distinguished from non-genomic actions primarily by the length of duration of their action which can range from several hours to days. This was first noted by Hans Selye who, in 1942, found that some steroid hormones i.e. anaesthesia, induced their effects remarkably quickly on mice when compared to others.[2] These "non-genomic" actions are characterised by this rapid onset of effects; usually within minutes. For either action to occur the hormone first enters the cell by one of two main methods; passive diffusion where the hormone enters the cell due to a concentration gradient, or by carrier-assisted transport in which specific transporters located on the cell surface bind the hormone, undergoing a conformational change to allow the hormone to be released intracellularly.

To induce a genomic action, the hormone binds to nuclear receptors in the nucleus. The receptor-hormone complex can, in some cases, act as a transcription factor, thereby increasing the affinity for DNA. The complex binds to the DNA at specific sites known as "Hormone-Responsive Elements" which enables gene transcription. This leads to the production of an mRNA molecule which is then translated into its protein that it encodes and it is this protein that ultimately exerts the biological effects. This is a genomic response as the steroid hormone manipulates transcription; a genomic process. A non-genomic action begins in much the same way. It is thought to be mediated by the binding of a hormone to membranous receptors. This binding activates secondary messengers inside the cell that lead to the rapid physiological changes observed. It also leads to the production of secondary messengers such as calcium ion fluxes and cyclic-AMP modulation. However, it is thought to be free of receptors that act in the nucleus. The time limit for this is considerably shorter. Furthermore, another differentiation between the two is the effects of transcription and translation inhibitors such as actinomycin D and cycloheximide respectively. These work by binding to DNA at specific sites thus preventing the formation of mRNA as they do not allow RNA polymerase to function. When these inhibitors are added to a genomic action, we see the action stopped but they have no effect on non-genomic actions.

Figure 1: Genomic versus non-genomic. The diagram highlights the main difference between the two; the hormone binding to the classic nuclear receptor and the complex binding to DNA in the genomic action and the activation of a signal cascade upon binding of hormone to membrane bound receptor in non genomic actions. Diagram adapted from [4]

Hormone

DNA

Hormone receptor

mRNA

Genomic Action

Secondary messenger

Activation

Non-genomic Action

Evidence of such receptors has been accumulated through experiments which show receptors involved that are different to classical receptors. Membrane-bound receptors are found on the cell surface membrane and transmit signals from outside to the inside of the cell. When the steroid binds, the receptor undergoes a conformational change and this sets of a signal cascade inside the cell.

One such example is the effects of progesterone on oocytes. When progesterone is added to these cells, they undergo maturation. As explained above, one characteristic of non genomic actions is the insensitivity to transcriptional and translational inhibitors. The actions of progesterone are not arrested when actinomycin D is added and to strengthen the argument, the effects also occur in unnucleated cells. When progesterone is added, there is a decrease in the level of cAMP. Progesterone was shown to be more effective when it was applied outside the cell rather than being injected inside the cell which supports the idea of membrane receptors. Another form of the classical progesterone receptor, termed XPR, was found on the membrane of these cells. It did not respond to other steroids such as hydrocortisone indicating ligand specificity and also that there are different non-classical receptors for different steroid hormones.

One way to prove the existence of membrane bound receptors is to prove the non-existence of the classical receptors. The way this works is by using immunohistochemistry methods to test for the presence of the classical receptors. This is done by first creating antibodies for the receptors and using RT-PCR to see if the antibodies bind to the receptors. However, there are drawbacks to this method as it may just be that there is a very little concentration of the classical receptor or that it has low sensitivity and hence is not bound to the antibodies.

An example of this method of this is seen by the action of testosterone on the concentration of intracellular calcium in IC-21 macrophages[3]. Primers were produced that were complementary to classic androgen receptors. Despite multiple presence-detecting techniques such as RT-PCR and blotting, these receptors were not found. A conjugate of testosterone and bovine serum-albumin (BSA) was mixed with a fluorescent dye - fluorescein isothiocyanate (FITC) - and when this was applied to the cells, there was an intracellular calcium response. This demonstrated the presence of membrane bound receptors.

Another example of membrane-bound receptors can be seen by the effects of the steroid hormone 1α,25(OH)2D3. This is shown to have the characteristic rapid effect of non-genomic steroids which stimulate calcium transport in intestines. When 1α,25(OH)2D3 is applied to the intestinal cells, there is a rapid increase in the amount of Ca2+ seen leaving the intestine. Furthermore, even when actinomycin D is added, there is no change. Actinomycin D is an inhibitor of transcription and it works by binding to DNA at specific sites, thereby inhibiting the formation of mRNA by preventing RNA polymerase to function. If this was a genomic effect, we would see this process stopped as actinomycin D would prevent transcription.[5],[8]

Furthermore, the effects of oestradiol also aid in the evidence of the presence of non-classical receptors. In endometrial cells, there was rapid elevation in the concentration of calcium ions when induced by oestradiol and this effect was not inhibited by tamoxifen; an antagonist for the classical oestrogenic receptor. It was also shown that oestrogen bound to certain sites on the membrane, suggesting the presence of non-classical receptors. There was evidence for the existence of membrane bound receptors which was seen by three main methods; an influx in calcium, activation of mitogen-activated protein kinase (MAPK) and the levels of cAMP. The latter two are secondary messenger systems that, when activated, regulate cellular activities. These actions are thought to occur via non-classical receptors on the membrane but the evidence for this is unreliable. This finding was proved by producing a conjugate of oestradiol and BSA (E2-BSA). The BSA molecule acted as a large molecule preventing the oestradiol entering the cell and thereby acting via genomic mechanisms. As rapid effects were seen, it was suggestive of non-genomic actions acting via membrane receptors. However, the binding efficiency of these two is very low. Hence, it is not reliable to use E2-BSA as a replacement for oestrogen as sometimes there is unconjugated E2 present in the mix which could be the reason for the changes observed. Furthermore, there is also a possibility of the oestradiol dissociating from the BSA molecule and entering the cell which would lead to genomic actions.[9],[10]

A problem that arises with the effects of oestradiol is that the structure of the membrane bound receptor was found to be identical to the nuclear receptors. This caused further controversy as the oestrogen receptors, ERα and ERβ, do not have transmembrane domains. A proposed mechanism for this is that these receptors are translocated to the plasma membrane where they are involved in protein-protein interactions to set off an MAPK cascade.

The difference between the two actions has long since brought about the question of their mechanism of action. Research has been, and is continuing, on whether or not these two mechanisms work by different receptors. It has been accepted that the genomic action is mediated by "classical" receptors. However, this started a controversy as to whether or not the classical receptors also work for non-genomic actions as this means that little about the function of the receptors is known.

There are many different mechanisms by which non-genomic actions occur and these are highlighted via a classification method. This was created in 1998 in Germany and is known as the "Mannheim classification". This system attempts to place different mechanisms into a system. [6]

The response is first split according to whether it is direct i.e. steroid alone is sufficient or indirect i.e. the steroid require co-agonists to induce an action. This is further split into specific and non-specific responses equating to a receptor that is specific to the ligand and no receptor at all, respectively. The specific group is again sub-classified into two branches; the classical steroid receptor or a non-classical steroid receptor. This classification helps to differentiate between the three main mechanisms of rapid steroid actions:

Side-effects: ligand specificity is more or less the same for both the genomic and non-genomic actions of the steroid hormone

Non specific, direct effects. In this mechanism, there is no receptor present. The rapid non genomic actions occur due to a high concentration of the steroid hormone that effects the fluidity of the membrane

The involvement of non-classical receptors.

In conclusion, steroids are known to act in one of two methods; genomically by binding on nuclear receptors and acting on the DNA and non-genomically by binding to membrane receptors and setting off a secondary messenger cascade to carry out the effects. There is a whole host of evidence present for non-genomic actions. While this is not doubted, the mechanism of these "membrane receptors" are at the centre of controversy regarding the character of these receptors is under scrutiny.

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