The Cardiovascular System is made up of the heart, the arterial system, the venous system and the microcirculatory systems, coupled with the neuro - humoral influence, form the entire circulation. The CVS main function is the rapid transport of O2, glucose, fatty acids, vitamins, amino acids, drugs and water to all the tissues in the body. It also removes CO2, urea and creatine which are metabolic waste products.
Cardiovascular disease claims more lives each year than cancer, chronic lower respiratory diseases, accidents, and diabetes mellitus combined. In patients with all types of CVD, including coronary heart disease, peripheral arterial disease, chronic heart failure, and stroke, the vascular endothelium is the primary site of dysfunction. [1]
Hypertension
Hypertension is also known as high blood pressure. Blood pressure can be defined as the force of blood pushing against the walls of arteries as it flows through them. As blood passes through the artery it pushes against the inside of the artery walls. The more pressure the blood exerts on the artery walls, the higher the blood pressure will be. Blood pressure is highest when the heart beats to push blood out into the arteries (systolic pressure). When the heart relaxes to fill with blood again, the pressure is at its lowest point (diastolic pressure). Normal blood pressure is considered to be approximately 120/80 mmHg, where 120 is the systolic pressure and 80 being the diastolic pressure. If the systolic and diastolic blood pressures are both high than this can results in hypertension.
In a report issued on May 14, 2003 in the Journal of the American Medical Association, Dr Katharina Wolf-Maier and colleagues proved that Europeans have a 60% higher prevalence of hypertension compared with North America. Average BP was 136/83 mm Hg in the European countries and 127/77 mm Hg in Canada and the United States among men and women combined who were 35 to 74 years of age [2].
1.3 The Smooth Muscle Cells of the Aorta
The aorta commences immediately after the aortic valve. One of its main functions is to supply the tissues of the body with oxygenated blood for their nutrition. The tunica media of the aorta sits between the tunica intima and tunica adventitia. It consists of elastic tissue and smooth muscle cells. It is an involuntary non striated muscle. Smooth muscle cells contains myosin, a thick filament and actin, a thin filament. These filaments slide against each other to produce a contraction and vice versa to cause a relaxation. Motor neurons are not required to stimulate smooth muscle. However the motor neurons of the autonomic nervous system can cause the smooth muscle to contract or relax. This is dependent on the neurotransmitter release. For example, Noradrenaline (NA) is a neurotransmitter released from the adrenal medulla. The adrenal medulla functions as an endocrine gland which means the neurotransmitter is carried in the blood to the target tissue rather than being released from the neuron at a synapse. The hormones bind to the adrenergic receptor. Noradrenaline causes the smooth muscle of the aorta to contract.
1.4 Nitric Oxide Pathway
In comparison to NA, nitric oxide (NO) another neurotransmitter, is a main neurotransmitter which relaxes the smooth muscle cells of the aorta. NO is a gaseous signalling molecule, it is also known as Endothelium Derived Relaxing Factor. It is synthesized from a group of enzymes called nitric oxide synthases (NOS). One of the main activators of NOS enzyme activity are changes in cellular calcium levels. The constitutive isoforms of NOS, eNOS and nNOS, show increased activity following increases in calcium, and therefore calmodulin. During systole, NO is released from endothelial cells that line the aorta and other blood vessels. It can also be produced due to sheer stress. The NO diffuses into the underlying smooth muscle cells causing them to relax and thus allowing blood to pass through the lumen of the vessel easily. Nitric oxide is used by the endothelium to signal the surrounding smooth muscle to relax. This causes vasodilation and therefore an increase in blood flow. It acts through cyclic GMP, this activates protein kinase G which causes inhibition of the myosin light chain and leads to smooth muscle relaxation.
1.6 Endothelial Function
It has also been shown that endothelial cells play a role in the relaxation of arterial smooth muscle. Rubbing of the intimal surface of the aorta removed the capacity of the aortic preparations to relax in response to acetylcholine (ACh). This strongly suggested that the endothelial cells were necessary for relaxation. It has also been shown ACh, acting on the muscarinic receptors of the endothelial cells, stimulates the release of a substance(s) that causes relaxation of the vascular smooth muscle [3].
However another study has shown that relaxation occurs in rat aortic rings with and without functional endothelium, pre contracted to the same extent by noradrenaline. Red wine polyphenol compounds (RWPC) produced complete relaxation of vessels with and without endothelium. However, 1000 fold higher concentrations were needed to relax endothelium denuded rings compared to those with functional endothelium [4].
Abnormal endothelial function is an early marker of CVD and appears to be an ideal target for preventive therapy. Evidence suggests the important role dietary factors play in modulating endothelial function in patients at risk and those with existing CVD [5]. It has been proven in USDA research that wild blueberries rank number 1 in antioxidant activity compared with 40 other fruits and vegetables [6]. These antioxidants are highly concentrated in the blue pigments of wild blueberries. Antioxidants and flavonoids are dietary components that are associated with improving chances of preventing CVD.
1.7 Potassium Channels
Potassium channels play an important role in smooth muscle contraction. K+ channels are a specific class of membrane proteins and have been found in all organisms and various types of cells including vascular smooth muscle cells (VSMC). They play an essential role in the physiological regulation of vascular tone and blood flow, stabilization of membrane potential, release of hormones or transmitters, and control of cell volume, etc. K+ channel opening or closing can be regulated by different stimuli such as a change in cell membrane potential and small active molecules called ligands. Thus, the opening of K+ channels by vasodilators in VSMC increases K+ efflux, which causes membrane hyperpolarization. This closes voltage-dependent Ca2+ channels, decreasing Ca2+ entry, which leads to vasodilation. In contrast, inhibition of K+ channels may contribute to vasoconstriction as well as decreasing the ability of an artery to dilate. The ability of smooth muscle in the blood vessel to constrict is affected by the change in [Ca2+]i, which is largely controlled by the membrane potential. As a major regulator of membrane potential in VSMC, K+ channel activity is therefore an important determinant of vascular tone, arterial diameter, peripheral resistance, and blood pressure.
Potassium channels can be grouped into different classes:
1) Voltage dependent K+ channels are activated by the depolarisation of the membrane. They play a major role in returning a depolarized cell back to its resting state during an action potential. Voltage-gated potassium (Kv) channels play a key role in setting the resting membrane potential and shaping action potential repolarization.3,4-diaminopyridine has been found to act very potently in selectively blocking the potassium channels. This has been examined and proven in squid axon membranes. The potency being about 50 times higher than that of 4-aminopyridine which is another inhibitor. Recent studies have also shown that contraction of smooth and skeletal muscles is enhanced by both 4-AP and 3,4-diaminopyridine (3,4-DAP). It is reported that in squid giant axons 3,4-DAP blocks potassium channels at micromolar concentrations in a highly specific manner[7].
2) Ca2+ - activated K+ channels which are dependent on the intracellular calcium to open its channel. There are 3 types of Kca channels- high conductance channels, intermediate conducting channels and small conductance channels. High conductance channels are also known as maxi channels. These are the most studied channel of the three. Their gating is voltage dependent and their channels are opened by membrane depolarisation and by micromolar concentrations of calcium.
Two types of Ca+ activated K+ channels have been identified on the lumenal surface of the endothelium in rat aortas. One which is sensitive to apamin (KAp) and one sensitive to charybdotoxin. (KCh). KAp are inhibited by nanometer concentrations of apamin but are insensitive to charybdotoxin. KCh channels were, in contrast, insensitive to apamin but were inhibited by charybdotoxin.
Large conductance Ca+2 activated K+ channels are inhibited by nanomolar concentrations of charybdotoxin (BK channels). Small conductance Ca2+ activated K+ channels are inhibited by nanomolar concentrations of apamin (SK channels). Intermediate conductance which is also inhibited by nanomolar concentrations of charybdotoxin (IK channels)
KCh channels are responsible for Ach evoked hyperpolarization. Both resting and stimulated changes in [Ca2+]i in endothelium are influenced by membrane potential changes which influence the driving force for Ca2+ entry. The function of the KAp channels are unclear. They may be transiently activated by large spikes in [Ca2+]i, but most of the time [Ca2+] would appear to be below the concentration needed for activation of KAp channels.[8]
Tetraethylammonium ion (TEA+) is also another drug that blocks calcium-activated potassium channels in smooth muscle cells. [9] It is a small ion that is thought to block open K+ channels by binding either to an internal or to an external site.
3) Inward Rectifying K+ channels - these channels favour the influx rather then the efflux of K+ ions. They are found in excitable and non- excitable cells including the smooth muscle cells of the aorta. They are activated by hyperpolarisation and the K+ ions move from the extracellular space to the intracellular space of the cell. These channels are blocked or inhibited by micromolar concentrations of barium.
5) ATP sensitive K+ channels which are opened in response to a decrease in intracellular ATP. These channels have been found in smooth muscle cells of the pulmonary, coronary and mesenteric arteries. These channels are blocked by glibenclamide.
1.5 Hydrogen Sulfide Gasotransmitter
Hydrogen sulphide (H2S) is a gasotransmitter which acts on KATP- channels on smooth muscle cells. H2S also possesses vasorelaxing properties [10]. H2S is produced endogenously in rat arteries from cysteine by cystathionine - y - lyase enzyme (CSE). Endogenously produced H2S dilates rat resistance arteries (11). H2S also relaxes the human artery (11). H2S production is inhibited by D,L-propargylglycine, an inhibitor of CSE. A recent study by D. George, (2007), indicate that CSE protein is expressed in human arteries, that human arteries synthesize H2S, and that higher concentrations of H2S relax human arteries, in part by opening KATP channels [11]. Another study has proven that in isolated vascular smooth muscle cells (SMCs), H2S directly increased KATP channel currents and hyperpolarized membrane. Intravenous bolus injection of H2S transiently decreased blood pressure of rats by 12- 30 mmHg, which was antagonized by prior blockade of KATP channels. H2S relaxed rat aortic tissues in vitro in a KATP channel-dependent manner [12].
1.8 Polyphenols
It has been suggested that and increase in polyphenol intake provides protection against coronary heart disease and stroke. Polyphenols are found in great supply in blueberries. They are considered one of the best sources of antioxidants among fruit and vegetables. Blueberries contain polyphenols, anthocyanins, and also flavonoid.
Polyphenols belong to the broad family of naturally-occurring physiologically-active nutrients. They can be divided into subgroups: bioflavonoids, anthocyanins and proanthocyanidin which are found primarily in the berry nectars.
Compared to other fruits and vegetables, a high antioxidant capacity has been reported for lowbush blueberries. The antioxidant capacity is most significantly correlated with the contents of total phenolics and anthocyanins [13].
1.9 Blueberry Diets
Blueberry components such as polyphenols have been shown to prevent lipid oxidation in liposomes and LDL oxidation in vitro and in vivo. Berry extracts have been shown to decrease the vulnerability of the vessel to oxidate stress and inflammatory insults [14]. Numerous studies have demonstrated marked improvement in endothelial function in spontaneously hypertensive rats (SRH) treated with antioxidant rich dietary ingredients. A recent study shows that dietary wild blueberry enrichment affects the vasomotor tone in developmental phase of essential hypertension in the aorta of SHRs. Therefore they investigated the possible influence wild blueberries may have on the Ach induced endothelium - dependent vasoralaxation and phenoylephrine (Phe) induced vasoconstriction in the aorta of young SHRs, as well as the possible mechanism by which blueberries may exert their action on endothelial function in an animal model with dysfunctional endothelium [15].
1.10 Other Juices
Cranberry juice (CBJ) is also rich in polyphenols. This juice has similar vasorelaxing properties similar to those of red wine. Vasodilation using cranberry juice is dependent on the endothelium being intact. CBJ vasodilation is dependent on endothelial cell nitric oxide formation. The effects of the CBJ on the rat aorta are similar to those observed effects of Concord grape juices and various wines at a 1:100 dilution. It can also be noted that CBJ has vasodilatory effects down to a 1:5,000 dilution in aorta from spontaneously hypertensive rats [16]
1.11 Conclusion
The present study was designed to look at the effect of blueberry juice in causing vasorelaxation. Blueberry juice was chosen as it is one of the richest sources of polyphenols known. We also investigated the role of potassium channels in mediating relaxation of the smooth muscle in response to blueberry juice. As well as directly examining the role of voltage gated potassium channels, we also investigated the role of the H2S gasotransmitter pathway and the inherent role of Katp channels.
Chapter Two:
Aims & Materials
2. Aims & Objectives
The aim of this project was to prove that blueberry juice can affect the functional properties of the rat aorta by decreasing vascular constriction by suppressing the α1 adrenergic receptor agonist mediated contraction.
The overall aim of this project is to find new a new means of reducing hypertension and possibly a new treatment.
Chapter Three:
Materials & Methods
3. Materials and Methods
3.1 Materials
3.1.1 Krebs solution
Krebs solution is a vital experimental component which ensures that the aortic rings are able to survive during experimental procedures. Krebs solution contains the following components in 1 litre of distilled H20:
Na Cl 5.8g/L NaH2PO4 0.207g/L NaCOOH3 1.6g/L
Glucose 1.8g/L MgSO4 0.1805g/L CaCL2 1.8g/L
NaHCO 3 2.1g/L KCL 0.298g/L
All components were heated in a water bath at 37oáµ’C and bubbled with 95% oxygen and 5% CO2 for 30minutes. CaCl2 was then added and the solution was continuously bubbled and maintained at 37áµ’C for the rest of the day.
3.1.2 Noradrenaline
Noradrenaline (NA) was purchased in 2ml vials at a concentration of 1mg/ml, MW = 169. A stock solution of 1mM was made in Krebs solution. 2.5ïl of this stock solution was added to the tissue bath containing 25ml to give a final concentration of 0.1ïm.
3.1.3 Achetylcholine
Achetylcholine was purchased as Acetylcholine bromide (MW = 226). A 10mM stock was made up using Krebs solution. 1/10 dilutions of the stock were made, and then 25ul of the ACH solutions were added to the tissue bath of 25mls to give final concentrations of 10ïM, 1ïM, and 0.1ïM.
3.1.4 Nw-Nitro-L-arginine methyl ester hydrochloride
A working stock of L-NAME (100µM) was prepared in Krebs Solution. 25ul of L-NAME was added to each 25ml tissue chamber giving a final concentration of 100µM.
3.1.5 3,4- Diaminopyridine
3,4-diaminopyridine (MW = 109) was dissolved in distilled water to a concentration of 0.11M. pH adjusted. 228ul of this stock was added to the 25ml tissue baths giving a final concentration of 1mM.
3.1.6 4- Aminopyridine
4-aminopyridine (MW = 94.13) was dissolved in distilled water to a concentration of 100mM. pH adjusted. 250ul of this stock was added to the 25ml tissue baths giving a final concentration of 1mM.
3.1.7 D,L-proparygylglycine
Proparygylglycine (MW = 113.11) was prepared by diluting PPG in H2O to give a stock of 0.09M. From this stock, 568µl was added to each 25ml tissue bath giving a final concentration of 2mM.
3.1.8 Blueberry Juice
Pure blueberry juice was investigated - manufactured by Biona. The juice was added to the tissue bath to yield concentrations of 1:1000, 1:500, 1:250, 1:100: 1:50, 1:10. pH adjusted
3.2 Methods
3.2.1Calibration of equipment
All equipment was calibrated every morning before commencing the experimental procedures. The contraction and relaxation of the mounted aortic rings in the organ bath was measured by the use of inductive force transducer and stored on the MacLab software chart. The programme chart was opened on the computer, the speed set at 4ms and the voltage to 2mV. The range was set and then zeroed. The transducer was calibrated using a small weight with a weight of 3.64g. The weight was tied onto a piece of string and hung from the transducer. After a reading was attained, the units were changed to grams and applied. The calibration allowed the transducer to produce a wave on the monitor reflective of the contraction that occurred.
The chambers within the organ bath were first cleaned out with a small brush to remove any dirt that may have gathered with it. The chambers were than washed out three times with Krebs solution. The chambers were then filled to the 25ml mark with Krebs solution. It was than left to reach 37áµ’C and was continuously bubbled with 95% oxygen and 5% CO2.
3.2.2 Tissue preparation
Adult Sprague - Dawley rats of both sexes were used in this experiment. However the majority of rats used were female rats. These weighed about 250g approximately. The resided in a temperature controlled envoirnment of 21 - 23áµ’C. The rats had access to fresh water and were fed once a day with adlibtum food. They were killed by carbon dioxide asphyxiation.
The thoracic aorta was removed and placed in warm Krebs solution of approximately 37áµ’C. Once removed, the lumen was flushed with Krebs solution which removed the blood clots. The surrounding connective tissue was cleaned away and the aorta was cut into 2.5mm segments approximately. Whilst scrapping away the excess connective tissue and fat, tissue was kept moist with warm Krebs by using a syringe. Care was taken to avoid rubbing of the endothelial cell layer with instruments used.
The thoracic aorta was than mounted onto two triangular shaped hooks and clipped up over the two chambers of the organ bath. One hook was connected to the force - displacement transducer while the other was hooked to the bottom of the chamber. The aortic rings were left for 30 minutes to equilibrate in the Krebs solution.
The tension on the aortic rings was than increased from 0g of tension to 2g of tension.
3.3 Experimental Protocol
3.3.1 Noradrenaline stimulated contraction of aortic rings
The contractile response to Noradrenaline (NA) was examined on a daily basis. The two segments of the aorta from the animal tissue were exposed to NA (0.1µM). It was added to each 25ml chamber in the tissue bath. Tissue that did not respond was considered not to be viable and was not used in the experiment. It roughly took 10 minutes for the contraction to plateau.
3.3.2 The vasorelaxant response of the aortic rings to acetylcholine
Acetylcholine (ACh) is a NO-dependent vasorelaxing hormone and has the ability to induce more than 50% relaxation in aortic rings that were pre-contracted with NA (1µM). Ach was added to the tissue bath to ensure the tissue sample had retained its ability to relax after it had been contracted by NA (0.1µM). Ach was added to both tissue chambers giving the following final concentrations of 0.1µM, 1µM, 10µM.
3.3.3 The effect of L-NAME on vasorelaxant response of rat aortic rings to Blueberry Juice
L-NAME is a NO synthase inhibitor. Aortic rings were exposed to L-NAME (100µM) 30 minutes before the addition of NA (1µM). Blueberry juice were then examined by the addition of the juice to the tissue chambers to give a final L-NAME was not reported by itself.
3.3.4 The effect of 3,4-Diaminopyridine on vasorelaxant response of the rat aortic rings to Blueberry Juice
Is a voltage gated potassium channel blocker. Aortic rings were exposed to 3,4-Diaminopyridine (1mM) for 15 minutes prior to the addition of NA(1µM). Blueberry Juice was than added to the tissue chambers giving a final concentration of 1:100, 1:500, 1:250, 1:100, 1:50, 1:10.
3.3.5 The effect of 4-Aminopyridine on vasorelaxant response of the rat aortic rings to Blueberry Juice
Another voltage gated potassium channel blocker. Aortic rings were exposed to 4-aaminopyridine (1mM) for 15 minutes prior to the addition of NA (1µM). Blueberry Juice was than added to the tissue chambers giving a final concentration of 1:100, 1:500, 1:250, 1:100, 1:50, 1:10.
3.3.6 The effect of D,L-proparygylglycine on vasorelaxant response of the rat aortic rings to Blueberry Juice
A H2S pathway inhibitor. Aortic rings were exposed to PPG (2mM) for 15 minutes prior to the addition of NA(1µM). Blueberry Juice was added to the tissue chambers giving a final concentration of 1:100, 1:500, 1:250, 1:100, 1:50, 1:10.
3.4 Statistics
All data was entered into the SuperAnova and Dunnet's one tailed t test was used for statistical analysis. All results are expressed as a mean ± s.e.mean for n experiments. A P value of P<0.01 or P<0.05 were considered statistically significant.
Chapter Four
Results
4. Results
4.1 Lab Chart Image - % contraction with Blueberry Juice
Fig 1.The ability of blueberry juice to induce relaxation of a pre-contracted aorta with NA (1µM). X-Axis Blueberry Juice concentrations inserted at 10 minute intervals. Y-Axis: % contraction that occurred in the presence of blueberry juices.
4.2 The effect of blueberry juice on rat aortic rings in a pre-contracted aorta with NA
The ability of blueberry juice to induce relaxtion of a pre-contracted aorta with NA (0.1µM). Blueberry juice was evaluated at the concentrations of 1:1000, 1:500, 1:250, 1:100, 1:50, 1:10.
Fig. 2 The effect of blueberry juice on rat aortic rings pre-contracted with NA (1uM). Values shown for percentage relaxation are ± s.e.m for n= 8. 100% contraction = 1.62g ± 0.740552 N = 8 F (6,49) = 7.565 P<0.05
Blueberry juice induced a significant P(<0.05) relaxation effect in rat aortic rings pre-contracted by NA (0.1uM), at the concentractions of 1:250, 1:100, 1:50, 1:10.
4.3 The effect of Ach on rat aortic rings pre-contracted with NA
Ach induced relaxation of the rat aorta pre contracted with NA (1µM). Ach was added at 10 minute intervals at concentrations of 0.1µM, 1µM, 10µM.
Fig. 2 The effect of Ach on the rat aortic rings pre-conntracted with NA (0.1µM). Values shown for % contraction are ± s.e.m for n=7
F (3,16) = 6.114
Acetylcholine induced a significant (P<0.05) relaxation effect in the rat aortic rings pre-contracted by NA, at the concentrations of 1µM, 10 µM.
4.4 The effect of D,L-proparygylglycine on the vasorelaxant response of the rat aortic rings to Blueberry Juice
The ability of blueberry juice to induce relaxation in the presence of PPG (2mM). Aortic rings were exposed to PPG for 15 minutes prior to the addition of NA (0.1µM). Blueberry juice was evaluated at the three highest concentrations of 1:100, 1:50, 1:10 respectively.
Fig. 4 The effect of blueberry juice on rat aortic rings pre contracted with NA (1µM), with and without PPG. Values shown for percentage relaxation are ± s.e.m for n=7
F (3,8) =6.082
In the presence of PPG blueberry juice induced a significant (P<0.05) relaxation effect in rat aortic rings pre contracted by NA, at concentrations of 1:100, 1:50.
4.5 The effect of 4-Aminopyridine on the vasorelaxant response of the rat aortic rings to Blueberry Juice
The ability of blueberry juice to induce relaxation in the presence of 4-Aminopyridine (1mM). Aortic rings were exposed to 4-Aminopyridine for 15 minutes prior to the addition of NA (1µM). Blueberry juice was evaluated at the three highest concentrations of 1:100, 1:50, 1:10.
Fig. 5 The effect of blueberry juice on rat aortic rings pre-contracted with NA, with and without 4-Aminopyridine. Values shown for percentage relaxation are ± s.e.m for n=5
F (2,39)= 4.256
In the presence of 4-Aminopyridine, blueberry juice induced a significant (P<0.05) relaxation effect in rat aortic rings pre-contracted by NA, at concentrations of 1:100.
4.6 The effect of 3, 4-Diaminopyridine on the vasorelaxant response of the rat aortic rings to Blueberry Juice.
The ability of blueberry juice to induce relaxation in the presence of 3,4- Diaminopyridine (1mM). Aortic rings were exposed to 3,4-Diaminopyridine for 15 minutes prior to the addition of NA (1µM). Blueberry juice was evaluated at the three highest concentrations of 1:100, 1:50, 1:10 respectively.
Fig. 6 The effect of blueberry juice on rat aortic rings pre-contracted with NA, with and without 3,4-Diaminopyridine. Values shown for percentage relaxation are ± s.e.m for n=7
F (2,39)= 4.256
In the presence of 3.4-Diaminopyridine, blueberry juice induced a significant (P<0.05) relaxation effect in rat aortic rings pre-contracted by NA, at concentrations of 1:100 and 1:50.
4.7 The effect of L-NAME & PPG on vasorelaxant response of the rat aortic rings to Blueberry Juice
The ability of blueberry juice to induce relaxation in the presence of L-NAME & PPG. Aortic rings were exposed to L-NAME (100µM) for 30 minutes prior to the addition of PPG (2mM) and 15 minutes prior to the addition of NA (1µM). Blueberry juice was evaluated at the three highest concentrations of 1:100, 1:50, 1:10 respectively.
Fig. 7 The effect of blueberry juice on rat aortic rings pre-contracted with NA, with L-NAME and PPG, without L-NAME and PPG and with PPG but no L-NAME . Values shown for percentage relaxation are ± s.e.m for n=4 F (2,33) =0.350
In the presence of L-NAME & PPG, blueberry juice induced no significant (P<0.05) relaxation effect in rat aortic rings pre-contracted by NA, at concentrations of 1:100 and 1:50 or 1:10
Chapter Five:
Discussion
5. Discussion
In this project, we document for the first time the role that potassium channels play in mediating relaxation of rat aortic smooth muscle in response to blueberry juice. Our research was carried out using aortic ring preparations from Sprague Dawley rats. Previous studies conducted have used various juices such as cranberry, grapefruit fruit juice as well as red wine to mediate relaxation of the smooth muscle cells via the nitric oxide pathway and H2S pathway. It is the polyphenol content within the blueberries that induces relaxation of the smooth muscle cells. Blueberry juice has been shown to have the highest polyphenol content among tested fruit and vegetables, and therefore an ideal juice to use for our research project. There is growing evidence that oxidative stress contributes to the pathogenesis of hypertension and endothelial dysfunction. Thus, dietary antioxidants may beneficially influence blood pressure (BP) and endothelial function by reducing oxidative stress [17]* The aim of this project was to prove that blueberry juice can affect the functional properties of the rat aorta by decreasing vascular constriction by suppressing the α1 adrenergic receptor agonist mediated contraction.
5.1 Summary of Results:
5.1.1 The effect of blueberry juice on smooth muscle contraction
Fig 1 and 2 both show the effect blueberry juice has on a pre - contracted aorta with noradrenaline NA (0.1µM). Fig. 1 being an example of an individual result taken from the lab chart. It shows the effects that each dose of blueberry juice had on a single pre-contracted aorta. Six concentrations were used: 1:1000, 1:500, 1:250, 1:100, 1:50 and 1:10. The final three concentrations, 1:100, 150 and 1:10 had the most significance, where P<0.05. It proved that blueberry juice had vasorelaxing properties.
We repeated this experiment to get n=8. Fig. 2 represents the data we retrieved. It shows that blueberry juice induced a relaxation of the smooth muscle cells and was most significant in the following concentrations, 1:250, 1:100, 1:50, 1:10, where P<0.05.
It has been previously reported that the consumption of blueberry-enriched diets attenuates the arterial contractile response to α1-adrenergic stimuli and affects vasomotor tone via endothelium-related pathways [17]. Other experimental evidence has also suggested that isolated polyphenols and flavonoids elicit endothelium dependent vasorelaxation in experimental animals, as well as human subjects [17]. Our results coincide with these results. We show that the polyphenol content in blueberry juice induces vasorelaxation of the smooth muscle of the aorta via the suppression of the α1-adrenergic receptor agonist.
5.1.2 Viability of the Aorta
Fig. 3 Shows the effect of Ach on rat aortic rings pre-contracted with NA (0.1µM). Acetylcholine (Ach) was used to test the viability of the aorta. It induced relaxation in the rat aorta, pre contracted with NA (0.1µM). Three concentrations were used: 0.1, 1 and 10 µM. The two higher doses, 1 and 10 µM had the most significant effect in causing vasodilation. It has been demonstrated previously that Ach requires the presence of endothelial cells, and the Ach, acting on muscarinic receptors of the cells, stimulates the release of a substance(s) that causes relaxation of the vascular smooth muscle. It is proposed that this may be one of the principal mechanisms for Ach-induced vasodilation in vivo [18]. Our results prove that the endothelium was intact and present when our experiments were being carried out. Other studies carried out, where isolated preparations had failed to relax when Ach was added had their endothelial cells unintentionally rubbed off during the course of their preparation for experiments [19].
4.1.3 Role H2S plays in mediating relaxation in the presence of Blueberry Juice
Fig. 4 Shows the effect of D,L-proparygylglycine (PPG) on the vasorelaxant response of the rat aortic rings to blueberry juice. PPG is an inhibitor of the hydrogen sulphide (H2S) synthesizing enzyme, cystathionine - y - lyase (CSE). Our results show the effect that blueberry juice had on rat aortic rings pre contracted with NA (0.1µM), with and without PPG. It compared the three highest concentrations of blueberry juice: 1:100, 1:50, 1:10. It shows that when PPG was present, it blocked the vasorelaxing effect of blueberry juice a 100% at 1:100 concentration. It blocked the vasorelaxing effect of blueberry juice approximately 85% with the 1:50 concentration. These results were significant in that P<0.05.
The blockage of the H2S gasotransmitter by PPG indicates that H2S is involved in the relaxation of the vascular tissues. This is an effect mediated by the activation of ATP-sensitive K+ (KATP) channels in vascular SMCs. It has been shown that H2S directly alters the activity of KATP channels without the involvement of second messengers [20]. It has also been shown that the endogenous production of H2S in the cardiovascular system is likely regulated by nitric oxide, whereas the vasorelaxant effect of nitric oxide is inhibited by H2S [20].
5.1.4 Role of Potassium Channels in mediating smooth muscle relaxation by blueberry juice
In a study carried out by MT Nelson et al, 1995, he concluded that regulation of arterial smooth muscle membrane potential through activation or inhibition of K+ channel activity provides an important mechanism to dilate or constrict arteries. He also stated that KV, KCa, KIR, and KATP channels serve unique functions in the regulation of arterial smooth muscle membrane potential and K+ channels integrate a variety of vasoactive signals to dilate or constrict arteries through regulation of the membrane potential in arterial smooth muscle [20].
In Fig. 5 We compare the effect of blueberry juice on rat aortic rings pre-contracted with NA, with and without 4AP. 4AP along with 3,4 diAP are drugs that block voltage gated potassium channels. Contraction of smooth and skeletal muscles is enhanced by both 4-AP and 3,4-diaminopyridine [22]. 4AP blocked the potassium channels approximately 80% at the 1:100 concentration. This was significant as P<0.05. These results indicate that potassium channels, and in this case voltage gated potassium channels, are involved mediating relaxation in response to blueberry juice.
3,4-diAminopyridine has been shown to have a potency 50 times higher than that of 4AP [22]. Fig. 6 Shows the effect of blueberry juice on rat aortic rings pre-contracted with NA, with and without 3,4-diaminopyridine. Our results coincides with the previous study mentioned, as 3,4-diaminopyridine had a stronger potency and blocked the potassium channel 100% at the 1:100 concentration of blueberry juice and blocked the channel 95% at the 1:50 concentration of blueberry juice. The effect of blueberry juice on its own, with no 3,4-diAP, showed a much higher relaxation rate. From this result we can now say that voltage gated potassium channels are involved in mediating relaxation in response to blueberry juice.
5.1.5 Nitric Oxide Synthase
Inhibitors of NO synthesis such as L-monomethyl arginine (L-NMMA) or N-nitro-L-arginine methyl ester (L-NAME) cause endothelium-dependent contractions in isolated arteries and inhibit endothelium-dependent relaxations to a variety of agonists [23]. Fig. 7 compares three tests: the effect of blueberry juice on rat aortic rings pre-contracted with NA, with L-NAME and PPG, without L-NAME and PPG and finally with PPG but without L-NAME.
When L-NAME was present with PPG, there was no significant result, the smooth muscle relaxed, P>0.05. For this experiment with L-NAME and PPG combined, we were hoping to achieve a sustained contraction without any relaxation. In another study carried out, oral administration of L-NAME has shown a marked and sustained hypertension effect [24]. It has also been shown the effect of oral treatment of L-NAME for 6 weeks on vascular reactivity of the aorta in Wistar-Kyoto rats. Systolic blood pressure increased in the L-NAME group by 80 mm Hg systolic but not in controls [25]. Therefore our results did not match those of other studies.
Reasons why L-NAME and PPG may not have worked in our project include: damage to the endothelium when hanging the aorta, damage to the aorta during the dissection, too low a concentration of L-NAME and/or PPG used, the length of time the drugs were left to equilibrate with the aorta, may have been too long a time frame or too short a time frame.
5.1.6 Limitations encountered and Future Studies
Due to time restrictions we did not get to complete all the experiments we would have liked to. We concentrated on voltage gated potassium channels. If more time was given, we would have liked to have looked into the role that other potassium channels (KCa, KIR, KATP channels) that could have played in mediating relaxation of the smooth muscle in response to blueberry juice. The effect of blueberry juice on calcium-activated-potassium-channels could have been tested by inhibiting their channel with tetraethylammonium ion (TEA+). ATP sensitive channels could be inhibited by glibenclamide, while inward rectifying K+ channels could inhibited by barium, to see the effect blueberry juice has on them.
Another limitation we encountered in the laboratory included a minimum supply of aortic tissues. We received one aorta each day for 7 weeks, which meant that gathering data was a slow process. Wrong concentrations of noradrenaline were used in the first two weeks experiments, therefore these results had to be removed from our overall data. We also had to make sure the Krebs solution within the tissue chambers were kept at a constant temperature, 37áµ’C. We noticed that the force of contraction was reduced significantly when the temperature was lower 37áµ’C.
Future studies could be carried out on the flavonoids and polyphenols within the blueberry juice. This could be accomplished by extracting the flavonoids and polyphenols from the blueberry juice. Another study has shown that a 15-day daily consumption of 100 g flavanol-rich dark chocolate (88mg flavonols/1 OOg of dark chocolate) decreased BP and improved endothelium-dependent relaxation in patients with essential hypertension [26]. If more time and funds were aliquoted to us, we could have continued our study by investigating a blueberry juice enriched diet, in spontaneous hypertensive rats (SHR).
5.1.7 Conclusion
In the last number of years there has been a dramatic increase in health and financial consequences due to CVD. A scientific search for more effective and at the same time a more affordable means of tackling CVD has become a necessity. The endothelium is the primary site of dysfunction in all types of CVD therefore appears to be an ideal target for a preventive approach. In the last decade flavonoids and polyphenols, have been positively associated with cardiovascular health protection. Accumulating evidence has documented a positive link between consumption of foods containing these compounds, and their effect on health beyond nutrition. Wild blueberries rank high among functional foods due to their high antioxidant capacity, as well as their high content of bioactive compounds, which may have multiple effects on health beyond antioxidant protection.
Following this experiment, I found that a significant conclusion could be made regarding the role of potassium channels in mediating relaxation of rat aortic smooth muscle in response to blueberry juice. Significant results were found in almost all our graphs seen in Chapter 4 indicating blueberry juice is very effective at mediating relaxation. The results show that voltage gated potassium channels are involved in the vasodilatory effects mediated by blueberry juice. We also noted that the vasodilatory effect induced by blueberry juice is also partially mediated by the H2S pathway as seen in Fig.4. There was no significant result when L-NAME and PPG were added together; therefore we could not rule out the possible role the nitric oxide pathway might play in the relaxation of the smooth muscle in the presence of blueberry juice.
Our overall results demonstrate that blueberry juice is a very effective vasorelaxing agent, with potential implications for treatment and prevention of hypertension. Our findings also demonstrate that this effect is mediated at least partially by potassium channels.
