Fakhra Amin* and Bilqees Bano
Department of Biochemistry, Faculty of life sciences Aligarh Muslim, University Aligarh, UP, 202002, India
Received date: February 04, 2014; Accepted date: March 06, 2014; Published date:March 12, 2014
Citation: Amin F, Bano B (2014) Effect of Amitriptyline an Antidepressant Drug on Structural and Functional Properties of Brain Cystatin. J Mol Genet Med 08:103. doi: 10.4172/1747-0862.1000103
Copyright: © 2014 Amin F, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Purification of buffalo brain cystatin; Thiol proteinases inhibitors; Amytriptyline; Depression
BC: Brain Cystatin
Cysteine proteinases such as cathepsins B, H, L and S , have myriad of biologic activities attributed to them, like proliferation, differentiation, ageing and cancer. Cystatins, the ubiquitous non-covalent competitive inhibitors are involved in the regulation and protection of cells from unwanted proteolysis . Cystatins also play a vital role in the homeostasis of intracellular and extra cellular matrix whose recycling is important otherwise it may cause a number of neurodegerative diseases like Alzheimer and amlyoid angiopathy .
An antidepressant is a psychiatric medication used to alleviate mood disorders, such as major depression insomnia, drossiness muscle stiffness, nervousness and loss of appetite. The Drugs including the monoamine oxidase inhibitors (MAOIs), tricyclic antidepressants (TCAs), and serotonin-norepinephrine reuptake inhibitors (SNRIs) are most commonly used for psychiatric problems drugs. Amytriptyline Hydrochloride is a tricyclic antidepressant drug. It is a white, odorless, crystalline compound which is freely soluble in water. In terms of its mechanism of action, amytriptyline inhibits serotonin degradation and noradrenaline reuptake almost equally. Amytriptyline (Figure1) is also approved for pain associated with the nerves (neuropathic pain), and to prevent migraine headaches. The recommended therapeutic concentration of amitriptyline is 75 mg/day  and the concentration of amitriptyline in brain after crossing the blood –brain barrier is 1500 ng/g – 2000 ng/g wet brain tissue . Typically lower dosages 10 to 50 mg daily are required for pain control .
The marginal stability of the native conformation of proteins is also a delicate balance of various interactions in proteins (van der Waals, electrostatic, hydrogen bonds and disulphide bridges)  which is affected by pH, temperature or addition of small molecules such as substrates, coenzymes, inhibitors and activators that bind especially to the native state and thus alter this fragile equilibrium leading to a number of slightly different conformational states of proteins which differ drastically in their function. In this regard, most drugs exert their activity on interaction with proteins. Moreover drug accumulation at off-target sites in the body leads to unintended adverse reactions [8,9], however drug/ligand induced protein structure conformational alterations  are prime problems complicating drug medical therapy by affecting the cellular proteins. Keeping in view the importance of cystatins in maintenance of proper brain function, the effect of Amytriptyline on the integrity of a newly purified cysteine proteinase inhibitor from buffalo brain has been explored. Most of the antidepressants drugs target neurotransmitters serotonin, although norepinephrine is affected to some degree as well . In fact antidepressant medications are the first-line treatment for people meeting current diagnostic criteria for major depressive disorder. Depression is associated with reduced levels of the monoamines in the brain, such as serotonin. Most antidepressants are designed to enhance the level of serotonin. The selective serotonin and noradrenaline re-uptake inhibitors (SNRIs) are thought to restore the levels of these neurotransmitters in the synaptic cleft by binding at their re-uptake transporters preventing the re-uptake and subsequent degradation of serotonin and noradrenaline. This re-uptake blockade leads to the accumulation of monoamines in the synaptic cleft and their concentration then returns to within the normal range. This action of SNRIs is thought to contribute to the alleviation of the symptoms of depression. Since antidepressants are spread widely throughout the body may also influence serotonergic processes in the periphery. The serotonin transporter also plays a crucial role in the homeostatic regulation of serotonin in the gut and plasma (Figure 1and 2).
Since amitriptyline is believed to be a potent inhibitor of noradrenaline re-uptake at the adrenergic nerve endings  The mechanism in figure 2 and 3 shows that it increases both serotonin and norepinephrine level and this helps in reducing depression.
We have already reported the purification and characterization of buffalo brain cystatin (BC) identified to be a variant of cystatin type-I  BC is a double sub-unit structure having a molecular weight 44.2 kDa protein, Fluorescence spectroscopy was employed to assess any structural changes in brain cystatin on interaction with Amytriptyline. Functional changes were analyzed by assessment of the inhibitory potential of the drug-bound protein. Literature shows that amitriptyline binds to amino acids tyrosine, tryptophan, valine, leucine, isoleucine and phenylalanine our purified protein contains these amino acids thus amitriptyline after binding with purified protein leads to inactivation of cystatin thus it is a physiologic event .
Papain (99% purity) and amytriptyline was obtained from Sigma Chemical Company (St. Louis, USA). The solutions were prepared in 50 mM phosphate buffer of pH 7.4. Salts were purchased from Merck (India). The protein concentration was determined spectro-photometrically. All other reagent was of analytical grade and double distilled water was used throughout.
Purification of Brain Cystatin
Fresh brain tissue (150 grams) was homogenized in 50 mM sodium phosphate buffer of pH 7.5 (30 ml) containing 1% NaCl, 3mM EDTA and 2% n-butanol. After centrifugation at 11000 rpm for 15 minutes at 4°C residue was discarded and the supernatant was further processed. The procedure involved a combination of alkaline treatment (pH 11.0), ammonium sulphate fractionation and gel filtration chromatography. Buffalo brain was homogenized and fractionated with ammonium sulfate between 40-60%, it was then dialyzed against 50 mM sodium phosphate buffer pH 7.4 containing 0.1 M NaCl. Elution profile showed two protein peak one major and one minor named as peak-I and peak-II. Peak-I corresponding to high molecular weight. Cystatin had significant inhibitory activity and protein content; however peak-II with insignificant proteins concentration and low inhibitory activity was not taken into consideration for further studies. Peak-I renamed as BC was then purified with fold purification of 384.72 and yield of 64.13%. Papain inhibitory fractions of peak -I were pooled, concentrated and checked for purity. Five milliliter fractions were collected and assayed for protein by the method of Lowry et al. (1951) and inhibitory activity against papain by the method of Kunitz . Homogeneity of the preparation was investigated by 7.5% PAGE .
Fluorescence spectra of brain cystatin with amytriptyline
Brain cystatin (BC) (1µM) was incubated for 30 min with increasing concentrations of amytriptylinein 0.05 M sodium phosphate buffer pH 7.5 in a final reaction volume of 1ml at room temperature. Drug solutions were prepared in the same buffer. Fluorescence measurements were carried out on a Shimadzu Spectrofluorimeter model RF-540 equipped with a data recorded DR-3 at 298K. The fluorescence was recorded in wavelength range of 300-400 nm after exciting the protein at 280 nm. The slits were set at 10 nm for excitation and emission. The path length of the sample was 1 cm. The data was analyzed by stern-Volmer equation
Stern- volmer constant
The fluorescence quenching was analyzed by the Stern-Volmer equation
Where F0 and F are the fluorescence intensities in the absence and presence of quencher respectively, Ksv the stern-volmer quenching constant and [Q] is the concentration of the quencher.
Determination of binding constant [K] and number of binding sites (n)
Where K and n are the binding constant and number of binding sites respectively thus a plot of Log [(Fo-F)/F versus [Q] can be used to determine K as well as n.
UV spectra of cystatin in the presence of Amytriptyline
The UV measurement of brain cystatin in the presence and absence of antidepressant was made in the range of 200-300 nm and the inhibitor (Cystatin) concentration was fixed at 1µM while the drug concentration was varied to different extent. Absorption spectra were recorded on a double beam Shimadzu UV-vis spectrophotometer UV-1700 using a cuvette of 1 cm path length.
Interaction of Amytriptyline with Cystatin
For macromolecules, the fluorescence measurements can give some information about the binding of small molecules with proteins, such as the binding constants, binding sites and binding affinity. Fluorescence intensity of a compound may get affected by interactions of protein with drugs.
Fluorescence Spectra of cystatin with Amytriptyline
The fluorescence spectra of cystatin (1µM) in the presence of different concentrations of amytriptyline were recorded in the range of 300-400 nm upon excitation at 280 nm. Amytriptyline caused quenching of the intrinsic fluorescence of cystatin (Figure 4) with no shift in wavelength; however with the increase in Amytriptyline concentration, fluorescence intensity was further decreased (Table 1).
The maximum decrease in fluorescence intensity (48%) occurred at 8 µM concentration. These results indicated that there were interactions between Amytriptyline and brain cystatin and the binding reactions resulted in non-fluorescent complex. Fluorescence quenching data was analyzed by the Stern-Volmer equation as given in the methods section data is presented in table 2.
Cystatin (1 µM) was incubated with various concentration of Amytriptyline varying from 2 µM to 8 µM for 30 min. The fluorescence was recorded in wavelength region 300-400 nm, exciting wavelength was 280 nm. The slits were set at 10 nm for excitation and emission. The path length of the sample was 1 cm.
Determination of ΔG0 of interaction between Amytriptyline and Cystatin
The forces of interaction between drugs and biomolecules include hydrophobic force, electrostatic interactions, vander Waals interactions and hydrogen bonds. In order to identify the interacting forces between amytriptyline with cystatin, the thermodynamic parameters i.e. free change (ΔG0) of the interactions were calculated from the following equations (Table2).
The value obtained for cystatin – amytriptyline interaction was -36.97 kJ/mol indicating the reaction to be spontaneous.
UV-vis absorption spectra of Amytriptyline cystatin complex
The interaction between Amytriptyline-cystatin was studied from UV-VIS absorption spectral data as given in method section (Figure 5). Cystatin concentrations were fixed at 1µM while the Amtriptyline concentration was varied from 2 µM-8 µM.
The UV-vis absorption spectra were computed at all the antidepressant concentrations. However, no profound changes were noted at 2-8 µM Amytriptyline. The spectra obtained for BC at 2 µM Amytriptyline shows two distinct positive peaks at 210 nm and 240 nm, the gross conformation of Cystatin at all concentrations of Amytriptyline was found to be unaffected. The UV absorption intensity of Cystatin increased with the variation of Amytriptyline concentration. The Addition of drug did not result in the shift of λmaxtowards longer wavelength. However there was quenching in fluorescence. This evidence clearly indicated the interaction and some complex formation between Amytriptyline and Brain Cystatin. [19,20].
Brain Cystatin concentrations was fixed at 1µM while the Amytriptyline concentration was varied from 2 µM-10 µM. Absorption spectra of native BC and in presence of Amytriptyline were recorded in the range of 200-300 nm ,a cuvette of 1 cm path length for 30minin the final reaction volume of 1 ml in 0.05 M sodium phosphate buffer pH 7.5.
Inhibitory activity of Cystatin in the presence of Amytriptyline
Effect of Amytriptyline on Cystatin function was assessed by monitoring changes in its anti-proteolytic activity by caseinolytic assay of papain  1µM of Cystatin was incubated with increasing concentration of Amytriptyline (2-8µM). The results obtained are summarized in (Table 3) When Cystatin incubated with increasing concentration of Amytriptyline, it resulted in rapid decline of anti-proteolytic activity showing 58% loss at 6 µM concentration of Amytriptyline with half of its inactivation taking place at a concentration of 8 µM. The data obtained also indicated that inactivation of Cystatin by Amytriptyline is concentration dependent.
Changes in the inhibitory activity of cystatin (1 µM) after incubation with increasing concentration of Amytriptyline (2 -8 µM) for 30 min in the final reaction volume of 1 ml in 0.05 M sodium phosphate buffer pH 7.5. Cystatin inhibitory activity without amytriptyline was taken as 100%.
All data are expressed as mean ± S.E for four different sets of experiments statical significance was conducted employing ONE WAY ANOVA a probability level of 0.05 was selected showing results are significant.
Antipsychotic and antidepressant drugs are imperative in the treatment of schizophrenia and other brain disorders. These drugs exert their therapeutic effects at least in part through perturbation of the dopamine, noradrenalin and serotonin neurotransmitter systems in the brain, but additional molecular mechanisms of action are likely to contribute to their clinical effects.
The MAOIs monoamine oxidase (Antidepressants) prevent the breakdown of monoamine neurotransmitters and therefore increase the concentration of the neurotransmitters (serotonin and dopamine) leading to depression in the brain.
Amytriptyline is a medication used to treat various forms of depression, pain associated with the nerves (neuropathic pain), and to prevent migraine headaches. It is often used to manage nerve pain resulting from cancer treatment. Amytriptyline acts to block reabsorption of chemicals that transmit nerve messages to the brain.
It produces its effects on the central nervous system via its actions on synaptic transmission. In general, the antidepressants effect the transmitters, like catecholamine’s and acetylcholine. They also increase the transmission in the brain and may affect the secretion of anterior pituitary hormones by regulating the hypothalamic secretions . The aim of the present study was to investigate the effect of antidepressants Amytriptyline on structural and functional modification of cystatin. To the best of our knowledge interaction of amitriptyline with cystatin has been done for the first time. However amitriptyline alone [when not bind with cystatin] increases serotonin level and the side effect of amitriptyline is that it binding with cystatin creates proteinase-antiproteinase imbalance. Investigations reveal that amitriptyline binds with cystatin reducing the effective concentration of amitriptyline therefore effectiveness of amitriptyline in depression is undercut by cystatin.
Cystatins are very important in the pathogenesis of depression, although it does not bind with serotonin. The published results have revealed that serotonin unfolds cystatin, which renders cystatin unable to regulate the activity of cathepsins leading to a proteinase - anti proteinase imbalance this may result in many chronic diseases. Several studies have shown that cystatins also bind with antidepressant monoamine oxidase inhibitor (MAOI) .
As a result MAOI may not be able to act on monoamine oxidase (MAO) whose function is to degrade serotonin and therefore destruction of serotonin continues, which lead to depression. Thus our study shows the involvement of cystatin in the pathway leading to depression.
Drug–protein interaction studies are used to determine the ability of antidepressants to analyze its interaction to number of binding sites binding of a protein. Drug binding is evaluated at various drug concentrations and the Information derived from binding studies has been used to determine the affinity of amytriptyline for cystatin. However, binding parameters are only the first step in understanding overall pharmacologic effects. .In the present study some binding reaction Parameters, including the binding constants and the numbers of the binding sites has been determined.
Intrinsic fluorescence of protein provides considerable information about protein structure, in terms of quenching, enhancement of intensity and spectral shift which are used for elucidation of related structure-dynamics in proteins . In the present study, fluorescence spectroscopic measurements were undertaken to gain insight into the interaction of cystatin with amytriptyline. It was found that in the presence of drug fluorescence intensity decreased indicating a conformational change in the protein (Figure 4).
When 1 µM of Cystatin was incubated with 2 µM of amytriptyline for 30 min and the samples were analyzed by fluorescence, the fluorescence emission spectrum showed no shift in λmax,with slight decline in fluorescence intensity as compared to native. The negative value of ΔG° reveals that the interaction process is spontaneous. Our finding is similar to the binding of Congo red with human serum albumin .The value for the number of binding sites (n) equal to 0.89 showing that there is one independent binding site of interaction. The Ksv value reported for amytriptyline - cystatin complex was 1.15 x 105/ Indicating the quenching in fluorescence to be static. These data are in accordance with interactions of lung cystatin with other drugs .
Absorption spectral measurements of Cystatin in the presence of amytriptyline provided information related to changes in their structure. The two positive peaks observed at 210 and 240 nm for amytriptyline - cystatin complexes at all concentrations studied, showed contribution from phenylalanine and histidine residues (Figure 5) . When cystatin incubated along with amytriptyline for varying concentration it caused decrease in antiproteolytic activity, there was 58% loss in antiproteolytic activity at 6µM of Amytriptyline concentration. This loss in antiproteolytic activity increased to 62% at 8µM of amitriptyline,(Table-2) showing a concentration dependent effect. These results have shown similarity with other cystatins . The phenomena of binding of drug with cystatin cannot be generalized. The present study shows that cystatin binds with amitriptyline however in other cases antidepressants lead to unfolding of cystatin therefore study of cystatin with more drugs is required to see the effect of other antidepressant drugs on cystatin.
However results prove that cystatin binds with amitriptyline reducing its effective concentration and it also shows the disbalance of crucial regulator cystatin for thiol proteinase activities which is a side effect of the drug.
It is well known that effectiveness of amitriptyline after some time reduced this may be the effect of cystatin interaction with amitriptyline however the complete molecular mechanism of action of psychotropic drugs are still not known and the study needs to be extended with other antipsychotic drugs.
According to our hypothesis the reduced action of amitriptyline may be due to its binding with cystatin, which also reduce the activity of cystatin which rendering it unable to regulate the function of cathepsins leading to a proteinase - anti proteinase imbalance a cause of many chronic diseases [22,27]. The study has sheds some light on cystatin function with regard to amitriptyline action. With regard to the mechanism of action of amitriptyline, other changes may also be considered that take place with chronic antidepressant use to maintain homeostasis, including alterations in the density and functioning of serotonin receptors, transporters, and enzymes [28-31].
However further studies are needed to study the in vivo effect of amitriptyline that is to use a depressed rat model which may be given different doses of amitriptyline and then the activity of cystatin in brain tissues as well as the level of serotonin may be measured. The information may be used for in-depth knowledge about cystatin function and to understand the mechanism of action of amitriptyline.
Acknowledgments Facilities provided by Aligarh Muslim University are gratefully acknowledged.