Assignment 4

The selected paper on vasopressin is "Brain levels of arginine-vasotocin and istocin in dominant subordinate males of a cichlid fish". There were 5 main aspects that the study was looking at, they were as follows:

1. To characterize the quantitative distribution of arginine vasotocin(AVT) and isotocin(IT)
2. To compare the quantitative levels of AVT and IT in the different brain regions and pituitary in males of different social behavior phenotypes
3. To investigate if the hypophysial and extrahypothalamic levels of AVT and IT are coupled or not
4. To asses if stress levels, as measured by circulating cortisol, are correlated with hypophysial levels of AVT
5. To investigate the association between peripheral levels of AVT,  as measured by the concentration of AVT in the pituitary, and urine storage in males of different social status   

The results section is organised poorly as it doesn't have all of the data that was collected . What can be distinguished is that AVT is highest in the pitutary gland followed by the olfactory bulb. The rest of the brain regions that were observed had similar low levels of AVT. The only significant difference observed between dominant and subordinante males was that the subordinante males had higher level of AVT. Similar trends were seen when IT was observed except that the only significant difference that was seen was that the hindbrain of dominent males was the only region of the brain to show higher IT levels that the subordinate male to any significant degree.

As far as a correlation went between the different levels of AVT and IT between the different brain regions are they are mostly absent  The authors use this to conclude that this indicates that the secretion of AVT and IT are mostly independent of each other.

There was not difference between the levels of cortisol in the subordinate and dominant males. There was no graph depicting this yet there was a graph that depicted a similar amount of information. The amount urine that is stored in dominant and subordinate fish was shown. Unlike the amount of cortisol levels that were insignificant there was significantly more urine stored in dominant males than there were in subordinate males.

 The authors claim based on the data that because there is large amount of AVT in the pituitaty it is responsible for the physiological changes that are seen between the dominent and subordinate males. This is all very nice but the levels of peripheral AVT was never measured in the periphery. It looks like they made the assumption that because AVT is in high concentrations in the pituitary, that it is secreted in to the periphery. Even if there is a high level of AVT in the perphiery of the fish it would not indicate that they are responsible for the different phenotypes observed. To determine this, a better study would be to inject the fish with AVT and observe their change in behaviour as they are capable of changing phenotypes when the opportunity arises. If a dominant phenotype were to be observed after the injection then it would indicate more conclusively that AVT is related to the different phenotype as opposed to some other reason that could be associated with a dominant phenotype.  

The authors then talk about the significance of AVP in the olfactory bulb. While this did have a high level of AVP when compared to the rest of the brain regions(except the pituitary) there was no significant difference between the male phenotypes. I don't see why it is that they included this section in the paper as it doesn't contribute to the 5 point that they were trying to prove.

Authors did have a good interpretation of the difference in urine content between the two male phenotypes. Urine is used as a method of communication in species and the different amount of urine and rate or release. This has been known to communicate who is more dominant. A dominant male would need more urine as they release urine more frequently. The amount of urine produced by the different male phenotypes however was not adjusted for size, only the total amount of urine was measured. The dominant males were larger than the subordinate males and would have a larger bladder based on size. A size adjusted amount of urine in the two phenotypes would give a better indication whether or not the difference observed was significant or not.

The discussion about the significant amount of IT in the hindbrain of dominant males was discussed relative to that of its effect on other species. They found in literature that IT has a positive effect on approach-withdrawl tendancies. No behavior was noted during the experiment, this is only speculation in the current experiment.

When the authors looked at whether or not the amount of AVT in the pitutary was related to stress as  determined by blood cortisol levels, none was found. The speculate that this may be because the social groups were stable 5-8 weeks before the data was collected and that the fish became habituated. A mechanism like this had been described in other species. The same experiment could be performed at different time periods during the development to the social structure to see if there is a relationship between AVT and stress.

The difference in AVT and IT levels in the brain region compared to the pituitary apparently indicates that the  secretion of AVT in the brain and the periphery is independent. Peripheral levles of AVT and IT would have to be measure to ensure that the pituitary isn't storing a large amount of these hormones in anticipation of some physiological response. The level of secretion may not be relative to the amount stores in the organ. Further study could be performed. It is unclear as to why this wasn't done as a blood sample was taken to measure the amount of cortisol in the blood.

At the end of the article the authors state that it is important to collect information on peptide levels relative to social behavior. More data should be collected to confirm the observations made in the paper as they make some assumptions without measuring important factors. A lot is also speculated based on what has been observed in other species without relating the data back to the current experiment, they demonstrate good aspects that should be studied in the future.

Levels of the hormones studies in females would have been interesting to see and how they compare to the levels observed in the subordinate males seeing as these males mimic the appearance and behavior of the females. 

Assignment 3

     Vasopressin has many functions in mammals other than the regulation of water retention and homeostasis. it has been seen to influence sexual behavior. This however is mostly attributed to the abundance and distribution of V1 receptors.

     The precursor sequence to vasopressin has be found and sequenced in many different mammals and members of Eutheria. Even though there is differences in vasoressin between different species there is ahighly conserved single precursor gene for vasopressin in every specie. This lack of phenotypic variability in species indicates that the structure of the hormone is important to its function and that it is quite specific. This could be in part due to its small size, only 9 amino acids. The vaiability that does exist is found in the other regions of the pro-hormone.

     Problems concerning vasopressin can arise not only when the vasopressin core protein is mutated but when the precursor protein is mutated as well. This can be a cause for diabetes insipidus. Mutations in the vasopressin pro-hormone result in large increases in urine production. Even though the vasopressin portion of the pro-hormone may not be mutated the portion of the gene encoding the neurophisin or signal peptide. Mutations in the signal peptide can result in inproper sorting in the magnocellular neurons of the supraoptic and paraventricular nuclei of the peptide. Mutations in the neurophysin can result in improper binding to vasopressin.

     The complete loss of vasopressin rats resulted in diabetes insipidus, this could however be controlled with vasopressin being taken in throught the diet. It also demonstrated that there was a decrease in the stress response of the rats as well. Even single nucleotide polymorphisms can have drastic effects on the stress response and anxitey. At the genomic level a change from an adenine to a guanine changes how vasopressin is represed by CArG binding factor. This is leads to vasopressin over expression.

     Vasopressin primarily acts on two classes of receptor, the V1 and V2 receprots. V1 receptors have been found in smooth muscle, liver and kidneys, this could be attributed to its ability to act on water retention and homeostasis. These receptors are also found in the brain and have been linked to the regulation of social behavior. The V2 receptor is found in periferal tissues. Vasopressin works to bind to the V2 receptor by binding with the alpha subunit of the of the G-protein. There is a lot more variatioon in these receptors than in vasopressin itself. In  the V2 receptor the Pro322 residue is conserved allowing for movement in the recepor. There are 2 mutations linked to this residue that have been linked to nephrogenic diabetes insipidus.

Assignment 2

     Vasopressin is a small peptide hormone that is 9 amino acids long. It is encoded for by a gene that is on the 20th chromosome and is at the AAA64291 locust. The precursor protein is 164 amino acids long. Vasopressin itself starts at the 20th amino acid and ends at the 28th amino acid and has the sequence cyfqncprg.

Figure 1: Amino acid sequence of Vasopressin and Oxytosin

Some of the domains that have been identified on the human version of the vasopressin precursor are a signaling peptide (aa 1-19), neurophysin (aa 32-122), a neurohypophysial hormone region (aa 39-166) and a glycopeptide (aa 124-164). The signal domain is not a typical promoter that is found regulating other genes. It is thought that this is an atypical promoter to promote cell specific expression of the protein. The role of the neurophysin is not thought to be biologicaly active, but instead is thought to help in the protection of vasopressin from enzymatic cleavage. The tyrosine residue of vasopressin is essential for association with neurophysin and transport within the cell. The glycopeptide is used to help vasopressin fold when there is low amounts of glycopeptides in the cell. Once vasopressin has been folded properly it can be secreted.

The receptor for vasopressin has been determined to be the vasopressin V2 receptor. It is 164 amino acids long spans the membrane several times. The gene encoding for this receptor is located on the gene region Xq28. It consists of three exons and two introns. An extra cellular component of the receptor binds with vasopressin that leads to a cascade the results in the cAMP being formed and eventually having more aquaporins inserted in to the membrane of the collecting ducts. The V2 receptor is a G protein. The 8th residue of vasopressin interacts with the first extracellular loop to initiate the cascade that eventually results in aquaporins being added to the collection tubule.

The disulfinde bond that gives vasopressin a ring can be seen in Figure 1. Also seen in figure 1 is oxytosin. It can be noted how similar they are in sequence only varying at the 8th residue. There is evidently not a lot of variation that exist in these molecules and have them still have the same structure. This becomes even more evident when the protein sequence for vasopressin of multiple animals are lined up. The protein itself is highly conserved especially throughout the four mamals.

Figure 2: Multiple alingment of prepropeptide of Vasopressin.
vasopressin is highlted in yellow

The neurophysin domain that come after the vasopressin domain is also well conserved between species. It is probably due to the fact that neurophysin is necessairy for proper folding of vasopressin. The rest of the domains are not well conserved between all of the species. This is not surprising as the organisms selected are very evolutionary divers. A closer look however will indicate the mammals share a large amount of sequence similarity in the glycopeptide sequence. There is a lot more to be learned about the structure of vasopressin and its receptor.

Assignment 1

     Hormones are biological molecules that are synthesized by an organism, they are secreted in the body to regulate metabolism and other processes in the body. One of these hormones is vasopressin(VP) also known as anti-diuretic hormone(ADH). This is a peptide hormone that is stored in the anterior pituitary where it is secreted in to the blood stream. The hormone was first identified in 1895 by Oliver and Schafer they demonstrated its existence when they removed the pituitary and saw a change in blood pressure. There is more than the hormone vasopressin that is associated with the pituitary gland. It wasn't until 1952 that vasopressin was first isolated from the pituitary by Vigneau and colleagues. He was also able to isolate oxytosin and was rewarded a nobel prize in 1955 for his work on these two hormones. Much work has been done on these hormones since 1952 and a vast amount of knowledge on it functions and structure has been learned.

     Vasopressin in humans is encoded from a region on the 20th chromosome. While there are equivalent proteins in other organisms, they are found on different chromosomes, the 2nd chromosome in mice and the 3rd chromosome in rats, it is believed that vasopressin originated from the duplication of a vasotensin gene. This mutation must have taken place many millenia ago as vasopressin like molecules have been observed in invertebrates such as hydra. In bony fish the vasopressin like molecule is called vasotecin.

     In humans the gene encoding for vasopressin is located on the 20th chromosome, more specifically at the AAA61291 locust. The gene encoding for the vasopressin is 164 amino acids long. There is much modification that vasopressin undergoes before it is the 9 amino acid protein that is secreted in to the blood stream. Some of the domains that are attached before the final product is obtained is a signaling peptide (aa 1-19). There is a vasopressin/ neurophysin at the 32-122 aa region and a glycopeptide at region 124-164 of the peptide sequence. The end product for vasopressin is  Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly. The cystine residues form a disulfide bond that give a ring structure. The structure of vasopressin is very similar to the structure of oxytosin. They differ only at the 3rd and 8th position.

There are multiple levels of control that function to regulate the secretion of vasopressin. There are two receptors in the body for this purpose, one is located in the heart while the other is in the hypothalamus. The sensor in the hypothalamus measures osmatic pressure. The second sensor in the heart measures changes in blood volume. These two systems work together, as water is lost from the body blood volume goes down and the osmatic pressure in the blood stream goes up. This triggers a release of vasopressin to restore the water balance in the body and maintain homeostasis.

     One of the functions of vasopressin to maintain proper water balance in the body. This is achieved by impacting the permeability of the nephrons in the kidneys to be more permeable to water and urea. The permeability of the terminal inner mendullary collecting duct is the region of the nephron that has its permeability affected. To affect the permeability of the nephron to water vasopressin is secreted from the anterior pituitary through cyclase coupled vasoreceptor. Vasopressin then binds to V2 receptors. This results in an increase in the production of cAMP. This causes PKA to phosporylate aquaporines at select serine residues. This causes the vesicles harboring the aquaporines to insert them in to the membrane of the nephron increasing its permeability to water. This aids in water re-uptake making the urine more concentrated. The method that vasopressin works to increase the nephrons permeability is less well understood than the system for the re-uptake of water. As with water vasopressin binds to the V2 receptor which results in the increased production of cAMP. Which activates PKA and results in the phosphorylation of UT-A1 at select serine residues. Epac is then phosphorylated which results in the accumulation of UT-A1 in the apical membrane.