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Pharmacology of the isolated mouse middle cerebral artery Ni, Bai 2004

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P H A R M A C O L O G Y O F T H E I S O L A T E D M O U S E M I D D L E C E R E B R A L A R T E R Y By Ni Bai B . M . , Shenyang Medical College, China, 1996 A THESIS S U B M I T T E D IN P A R T I A L F U F U L L M E N T O F T H E R E Q U I R E M E N T F O R T H E D E G R E E O F M A S T E R O F S C I E N C E IN T H E F A C U L T Y O F G R A D U A T E STUDIES (Department of Pharmacology&Therapeutics) T H E UNIVERSITY O F BRITISH C O L U M B I A O C T O B E R 2004 © Ni Bai, 2004 A B S T R A C T Cerebral arteries play an important role in the regulation of cerebral blood flow through autoregulation to supply oxygen and nutrients for the brain and neurons. Although investigation o f the activity of cerebral blood vessels has a long and distinguished history, there exists a deficit in the pharmacology of the isolated mouse cerebrovascular system. In this thesis, we designed protocols to determine whether the mouse middle cerebral artery ( M C A ) possessed similar properties with respect to myogenic control and responsiveness to vasoconstrictors and vasodilators. We found that the intrinsic tone of the mouse M C A was evoked at low pressures with no significant additional constriction occurring at higher pressures (>50mmHg). Inhibition of nitric oxide (NO) and endothelin-1 (ET-1) altered the extent o f pressure-induced myogenic tone. Unlike the general insensitivity of cerebral arteries to adrenergic receptor stimulation in most other species, the mouse M C A constricted to oc-adrenergic receptor activation. Interestingly, 5-HT and histamine, which are potent vasomotor factors, did not elicit any effect on the mouse M C A . In summary, the mouse M C A has a pharmacological profile that is distinct from other species, including humans; however, similar to findings in other cerebral arteries, the mouse M C A shows intracellular sensitization to C a 2 + following receptor activation. Mouse is the most commonly used species for constructing a genetically modified model to explore the intricacies of cerebrovasculature system; therefore, our study provides pertinent input for the further characterization of the pathophysiology and dysfunction in cerebral vasculature. u T A B L E O F C O N T E N T S A B S T R A C T ii T A B L E O F C O N T E N T S i i i LIST O F T A B L E S v i LIST O F FIGURES v i LIST O F ABBREVIATIONS AND UNITS v i i A C K N O W L E D G E M E N T S ix C H A P T E R 1 C E R E B R O V A S C U L A T U R E AND A U T O R E G U L A T I O N 1 1.1 INTRODUCTION 1 1.2 N E U R O G E N I C C O N T R O L O F C E R E B R A L B L O O D F L O W .....1 1.3 M E T A B O L I C C O N T R O L O F C E R E B R A L B L O O D F L O W 2 1.4 M Y O G E N I C RESPONSE AND C E R E B R A L A U T O R E G U L A T I O N 2 1.4.1 I N T R O D U C T I O N . . . 2 1.4.2 P H Y S I O L O G I C A L S I G N I F I C A N C E 3 1.4.3 M E C H A N I S M S U N D E R L Y I N G T H E M Y O G E N I C R E S P O N S E 3 1.5 E N D O T H E L I U M 4 1.5.1 I N T R O D U C T I O N 4 1.5.2 B L O O D - B R A I N B A R R I E R 4 1.5.3 E N D O T H E L I U M A N D M Y O G E N I C T O N E 4 1.6 M E M B R A N E P O T E N T I A L 5 1.6.1 I N T R O D U C T I O N 5 1.6.2 P H Y S I O L O G I C A L S I G N I F I C A N C E :....6 Membrane potential and vasomotor response 6 Membrane potential and transmural pressure .. . . . 6 Membrane potential and hypoxia 7 Membrane potential and P C O 2 / p H 7 1.7 V A S O A C T I V E M E D I A T O R S 7 1.7.1 N I T R I C O X I D E 7 Synthesis, storage and release 7 Nitric oxide in cerebrovasculature 8 1.7.2 A C E T Y L C H O L I N E 8 Synthesis, storage and release 8 Acetylcholine in cerebrovasculature 8 1.7.3 E N D O T H E L I N 9 Synthesis, storage and release 9 Endothelin in cerebrovasculature 10 1.7.4 5 - H Y D R O X Y T R Y P T A M I N E 10 i i i Synthesis, storage and release 10 5-hydroxytryptamine in cerebrovasculature 10 1.7.5 HIST A M I N E 11 Synthesis, storage and release 11 Histamine in cerebrovasculature 11 1.8 F A C T O R S I N V O L V E D IN T H E R E G U L A T I O N O F V A S O M O T O R R E S P O N S E 1.8.1 C a 2 + 12 1.8.2 M Y O S I N L I G H T C H A I N K I N A S E A N D M Y O S I N L I G H T C H A I N P H O P H A T A S E 13 1.8.3 Rho K I N A S E 13 1.9 T H E TECHNIQUES USED F O R INVESTIGATION O F E R E B R O V A S C U L A R FUNCTIONS 14 C H A R P T E R 2 P H A R M Q C O L O G Y O F I S O L A T E D M O U S E M I D D L E C E R E B R A L A R T E R Y . . . 15 2.1 INTRODUCTION 15 2.2 H Y P O T H E S E S AND O B J E C T I V E 16 2.3 M A T E R I A L S AND M E T H O D S 16 1 2.3.1 T I S S U E P R E P A R A T I O N 16 2.3.2 E X P E R I M E N T A L P R O T O C O L 17 Myogenic tone and endothelial factors 18 Vasoconstrictor effects 18 Vasodilator effects 18 Measurement of intracellular C a 2 + concentration 19 Measurement of membrane potential 19 Solutions and chemicals 20 Expression of results and statistical analysis 20 2.4 R E S U L T S 21 2.4.1 M Y O G E N I C T O N E O F M O U S E M I D D L E C E R E B R A L A R T E R Y 21 2.4.2 V A S O C O N S T R I C T O R R E S P O N S E S 21 2.4.3 V A S O D I L A T O R R E S P O N S E S , 22 2.4.4 [Ca 2 + ]i A N D A R T E R I A L D I A M E T E R 23 2.4.5 M E M B R A N E P O T E N T I A L A N D T H E E F F E C T S O F T R A N S M U R A L R E S S U R E S 23 2.4.6 KC1 A N D A G O N I S T - I N D U C E D D E P O L A R I Z A T I O N O F S M O O T H M U S C L E C E L L S 23 2.5 DISCUSSION 24 2.5.1 INTRINSIC T O N E 24 2.5.2 A G O N I S T - I N D U C E D V A S O C O N S T R I C T I O N 25 iv 2.5.3 A N T A G O N I S T - I N D U C E D . . V A S O D I L A T I O N 26 2.5.4 A R T E R I A L W A L L C A L C I U M A N D S M O O T H M U S C L E C E L L M E M B R A N E P O T E N T I A L 27 2.6 C O N C L U S I O N . . 27 R E F E R E N C E S 44 v LIST O F T A B L E S Table 1.1. The evidence of putative spasmogen 29 Table 2.1. Comparison of vasoactive factors in different species 33 LIST O F FIGURES Fig. 2.1. Myogenic tone of isolated mouse cerebral arteries 34 Fig. 2.2. Agonist-induced constriction of the isolated mouse middle cerebral artery 35 Fig . 2.3. Endothelium-dependent vasodilation in mouse middle cerebral artery 36 Fig. 2.4. Lack of responses to 5-HT and histamine in the mouse middle cerebral artery •. 37 Fig . 2.5. Simultaneous changes in arterial diameter and changes in the fluorescence ratio in mouse middle cerebral artery 38 Fig . 2.6. Recordings of smooth muscle membrane potential in mouse middle cerebral artery 39 Fig. 2.7. Depolarizing effect of increasing concentrations of KC1 on smooth muscle membrane potential of mouse middle cerebral artery 41 Fig. 2.8. A comparison of the depolarizing effects of P E and KC1 in mouse middle cerebral artery 42 vi LIST O F ABBREVIATIONS AND UNITS , The following abbreviations, definitions and units have been used throughout this thesis. A C h acetylcholine A N O V A analysis of variance A T P adenosine triphosphate B B B blood brain barrier B K bradykinin B P blood pressure °C degrees Celsius C a 2 + calcium ion [ C a 2 + ] i intracellular C a 2 + concentration [ C a 2 + ] e extracellular C a 2 + concentration C a C b calcium chloride C a M Ca 2 + -calmodulin C B F cerebral blood flow CI" chloride ion c A M P cyclic adenosine monophosphate C B F cerebral blood flow c G M P cyclic guanidine monophosphate C O 2 carbon dioxide C S F cerebral spinal fluid D A G diacylglycerol EC50 effective dose eliciting 50 % response E C E endothelin-converting enzymes E D R F endothelium-dependent relaxing factor E D T A ethyl en ediaminetetraacetic acid et al et alii (Latin, 'and others') ET-1 endothelin-1 E T A endothelin type A receptor (on vascular smooth muscle) E T B endothelin type B receptor (on endothelium) g gram(s) h hour(s) Hb hemoglobin H 2 O water 5-HT 5-Hydroxytryptamine i. e id est (Latin, 'that is') IP3 inositol-1,4,5-trisphosphate K + potassium ion Kca Ca 2 +-sensitive K + channel KC1 potassium chloride kDa kilodalton L litre(s) L - N A M E Mj-nitro-L-arginine-methyl ester m meter(s) M mole-1"1 M C A middle cerebral artery v i i MetHb methemoglobin M g C h magnesium chloride M i n minute(s) M L C myosin light chain M L C K myosin light chain kinase M L C P myosin light chain phosphatase n number of animals N a C l sodium chloride N E norepinephrine N O S nitric oxide synthase O 2 molecular oxygen OxyHb oxyhaemoglobin p value probability (of incorrectly rejecting the null hypoth P E phenylephrine P H logarithmic unit measuring acidity P K C protein kinase C R B C red blood cell P C O 2 . carbon dioxide tension P G I 2 prostagcyclin P O 2 oxygen tension P R P platelet-rich plasma PSS physiological salt solution R O C receptor-operated channel R y R ryanodine S A H subarachnoid hemorrhage SD standard deviation of the mean S E M standard error of the mean S O D superoxide dismutases S R sarcoplasmic reticulum V S M C vascular smooth muscle cell V S P vasospasm Mathematical prefixes k kilo (10 3) c centi (10"!) m mi l l i (10"3) p. micro (10 6 ) n nano (10"9) vni A C K N O W L E D G E M E N T S I would like to thank my thesis advisor, Dr. Ismail Laher, for the experience I have gained working with him. His knowledge, guidance and profound insight have sharpened my critical thinking during the course of my research. I would also like to thank the members of m y committee: Dr. Catherine C. Y . Pang and Dr. Xiaodong Wang for reading my thesis and offering advice. I had the incredible good fortune to have known Farzad Moien-Afshari , a wonderful colleague and friend in the lab. He was always wil l ing to set aside time to help out with things I was working on. It would have been difficult to accomplish what I did without his cheerfulness and willingness help. I also want to acknowledge my previous colleagues Anie M i n and Adrian Hui who taught me the techniques and were wil l ing to help me anytime. I would like to thank Shaila Jamaluddin Merchant who encouraged me all the time and provided invaluable help for my thesis. The short but enjoyable time working with her in the lab is unforgettable. Most importantly, I would like to send my deepest thanks to my family: my parents, my brother and my husband who always stood by me and without whom none of this would have been possible. Their unconditional love and support are the most valuable thing I have had in my life. Finally, I would like to acknowledge the financial support from Natural Sciences and Engineering Research Council of Canada. IX C H A P T E R 1 C E R E B R A L V A S C U L A T U R E AND A U T O R E G U L A T I O N 1.1 INTRODUCTION Although the brain is only about 2% of the total body weight in humans, it consumes 15-20% of the total cardiac output due to the high-energy demand o f brain cells and neurons, and the inability of brain to store essential nutrients (oxygen and glucose). Nerve cells begin to die within 3-5 minutes thereby causing irreversible brain damage i f the brain is completely deprived of blood. To provide a sufficient and uninterrupted blood supply, cerebral circulation possesses a characteristic feature to maintain relatively constant blood flow in the brain over a wide range of arterial blood pressures (Busija and Heistad 1984a). The independence o f cerebral blood flow (CBF) from arterial blood pressure is called cerebral autoregulation, which is a relatively robust response and occurs even following the development of atherosclerosis (Heistad et al. 1980). Autoregulation is critical to satisfy the metabolic needs of the brain despite the fluctuations in arterial blood pressure. Autoregulation also protects downstream vessels from damage resulting from arterial hypertension (Faraci and Heistad 1990). It has been reported that a variety of modulators, including neurogenic, metabolic and myogenic factors, are involved in the autoregulatory response. Among these regulators, myogenic factors may be predominant during arterial hypertension, while other factors are likely more important during the decline of blood pressure. Autoregulation of C B F is easily abolished by trauma, hypoxia or other noxious stimuli in the brain. 1.2 N E U R O G E N I C R E G U L A T I O N IN C E R E B R A L V A S C U L A T U R E Cerebral blood vessels are extensively innervated with inputs from sympathetic parasympathetic and sensory fibers (Busija and Heistad 1984a). It has also been reported that central pathways, existing exclusively within the brain itself, innervate cerebral resistance. vessels; however, evidence has shown that cerebral autoregulation occurs despite removal of 1 various neural influences, suggesting that the nerve supply of the cerebral arteries is not necessary for this response (Rapela et al. 1967,Waltz et al. 1971).. Although the relationship between neurogenic modulation and cerebral autoregulation is controversial, it is clear that nerve stimulation modifies the autoregulation curve, shifting it toward higher pressure levels. Other features such as the speed of vascular response, may also be modulated by the neurogenic factors (Salanga and Waltz 1973). 1.3 M E T A B O L I C R E G U L A T I O N IN C E R E B R A L V A S C U L A T U R E The brain has a high and rather stable global metabolic rate of oxygen consumption in sleep, resting wakefulness, and while performing motor and/or sensory work. Only in pain and in anxiety are increases seen in total cerebral oxygen uptake (by 20% to 30%) (Ingvar et al.1976, Kety 1975). Cerebral blood flow, a main determinant of the oxygen and glucose supply, is also relatively high (approximately 50ml/100g/min) and stable with increases in pain and anxiety. Since oxygen tension ( P O 2 ) variations around the normal level (approximately 100 mmHg) do not influence C B F , or even tend to increase during enhanced brain activity, it is unlikely that the suppressed local oxygen is the principal messenger to adjust flow to match metabolism. However variations in arterial C 0 2 tension ( P C O 2 ) exert a profound influence on C B F . Hypercapnia causes dilatation, while hypocapnia induces constriction. ( G O T O H et al. 1961). On the other hand, marked hypoxia causes clear-cut cerebral vasodilatation (see detailed discussion below). The additional factors that couple flow to metabolism are H + , K7, C a z -and adenosine concentration. 1.4 M Y O G E N I C RESPONSE IN C E R E B R A L A U T O R E G U L A T I O N 1.4.1 I N T R O D U C T I O N Myogenic response is defined as a phenomenon in which blood vessel's respond to transmural pressure elevation with constriction, and to pressure reduction with dilation. This behavior is inherent to smooth muscle and is independent of neural, metabolic and hormonal influences 2 (Johnson 1978). The myogenic response is a unique property of resistance vessels (with lumen diameter of 70-200um) by which blood flow and blood pressure are regulated. Although the myogenic response is mainly observed in arterioles, it can be occasionally demonstrated i n arteries, venules, veins and lymphatics. 1.4.2 P H Y S I O L O G I C A L S I G N I F I C A N C E The most important functions of the vascular myogenic response are the establishment of basal vascular tone and autoregulation of blood flow and capillary hydrostatic pressure. Basal vascular tone establishes an underlying arteriolar constriction upon which other control mechanisms produce vasodilation or vasoconstriction. The local regulation of blood flow protects capillary beds from large increases in hydrostatic pressure during variation in systemic arterial pressure (Johnson 1978). 1.4.3 M E C H A N I S M S U N D E R L Y I N G T H E M Y O G E N I C R E S P O N S E Uchida and colleagues have been credited with the initial suggestion that the myogenic response may reflect an improved excitation-contraction coupling resulting from membrane depolarization and increased C a 2 + permeability (Uchida and Bohr 1969). Although the exact signal transduction mechanisms underlying myogenic tone remain to be elucidated, it is clear that the phenomenon resides within the vascular smooth muscle cell, and can be modulated by a variety of mechanisms, such as vascular smooth muscle depolarization, mechanosensitive channels, nonselective cation channels, K + , CI", and voltage gated C a 2 + channels (Davis and H i l l , 1999). Moreover, other possibilities have also been suggested, including the control o f myosin light chain-phosphorylation, the smooth muscle binding proteins caldesmon and calponin, protein kinase C (PKC) , G-proteins, the adenylate cyclase, arachidonic acid pathways, and the cytoskeleton and extracellular matrix (Davis and H i l l , 1999). 3 1. 5 E N D O T H E L I U M 1.5.1 I N T R O D U C T I O N Endothelium is located at the interface between the blood and the vessel wall . The endothelial cells are in close contact and form a layer that prevents blood cell interaction with the vessel wall . Moreover, the dynamic tissue performs many other active functions, such as the secretion o f vasoactive substances (endothelin-1, nitric oxide) to regulate vascular smooth muscle tone, and hence C B F , the regulation of coagulation, leukocyte adhesion, and vascular smooth muscle cell proliferation and migration. 1.5.2 B L O O D - B R A I N B A R R I E R In most of vessels, endothelial tissue has small spaces between each individual cell, thereby substances can move readily between the inside and the outside of the vessel. However, in the brain, the endothelial cells are joined by tight junctions making up the so-called blood-brain barrier ( B B B ) , which allows some materials (glucose, iron, amino acids, peptides, small organic acids) to access the brain, but prevents others from passing. The B B B has several important functions: 1) prevents the brain from- "foreign substances" in the blood that may injure the brain tissue; 2) protects the brain from hormones and neurotransmitters in the rest o f the body; 3) maintains homeostasis in the brain. 1.5.3 E N D O T H E L I U M A N D M Y O G E N I C T O N E The hypothesis that vascular endothelium was prerequisite to myogenic responsiveness was suggested by Harder (Harder, 1987) and was confirmed by Katusic who showed that endothelium removal from canine basilar arterial rings prevented the active force development by stretch. However, subsequent studies demonstrated that the mechanical denudation of endothelium did not retard the generation of the myogenic response on various types of vessels. • 4 Currently, it is widely accepted that the myogenic response is the consequence of the mechanical stimuli that directly act on the vascular smooth muscle to evoke vasoconstriction independent from the intact endothelium. On the other hand, the stretch or distension o f vascular wall augments the production of endothelium-derived vasoconstrictors or decreased the release of an endothelium-derived relaxing factor which may modify the myogenic response (Meininger et al., 1992). 1.6 M E M B R A N E P O T E N T I A L 1.6.1 I N T R O D U C T I O N Electrophysiological events occurring at the plasma membrane regulate many cellular functions within excitable cells. With respect to vascular smooth muscle cells, one of the principal events primarily regulated by changes in resting membrane potential (E m ) is activation of contractile elements and cell shortening. The precise degree of E m involved in activation o f smooth muscle cells varies depending upon the vascular types as well as the species. The resting E m of cerebrovascular smooth muscle cell studied in an organ bath is between - 6 0 to - 7 0 m V (Harder 1980; Lombard et al. 1986). This is maintained largely by the cel l permeability to K + (Hirst, 1989). Three physiologically relevant types of channels which may play a role in the regulation of E m have been identified; they are delayed rectifier K + channel (Okabe et al. 1987), Ca 2 +-sensitive K + channel (Kc a ) (Benham et al. 1986; Wilde and Lee 1989), and ATP-sensitive K + channel (Standen et.al. 1989). The K c a channel is activated as intracellular C a 2 + increases, but may be sensitive to extracellular C a 2 + as well . The A T P -sensitive K + channel is turned off as intracellular A T P increases beyond some critical value and is not only sensitive to A T P but also regulated by a variety of intracellular mediators including p H and P 0 2 (Ashcroft 1988; Davies 1990). 5 1.6.2 P H Y S I O L O G I C A L S I G N I F I C A N C E Membrane potential and vasomotor response Membrane potential is an important determinant o f intracellular C a 2 + concentration ( [Ca ]i), hence regulating the vasomotor response. The hyperpolarization mediated b y the extrusion of K + reduces the open probability o f C a 2 + channels, leading to vasorelaxation. On the other hand, when K + channels close there is resultant depolarization and vasoconstriction. The classical model of electromechanical coupling considers that membrane potential determines [Ca 2 + ] i . In turn, [Ca 2 + ] i also can regulate membrane potential by modulating the open probabilities of ion channels, suggesting that there is a network of positive and negative feedback loops regulating [Ca 2 + ]i as well as membrane potential (Carl et al. 1996). Membrane potential and transmural pressure Increasing transmural pressure in an isolated cerebral arterial segment depolarizes muscle cells, induces spontaneous action potential generation, and reduces internal diameter (Harder 1984). Either pressure or the subsequent mechanical deformation of the vascular muscle wall activates arterial muscle by depolarizing the membrane and increasing C a 2 + permeability. The "sensor " translating the mechanical stimulus into the observed biophysical process in arterial muscle is not yet well defined (Smeda and Daniel 1988; Osol and Halpern 1985; Halpern and Osol 1985). One of putative "pressure-sensors" is a "stretch-activated" ion channel either in the vascular muscle, endothelial cell, or both. The majority o f mechanosensitive ion channels are nonspecific and exhibit conductance for multiple ion species with the greatest level of selectivity being either for cations or anions (Morris 1990). These mechanosensitive channels are considered as plausible mediators for the muscle cell depolarization and action potential generation observed in cerebral arteries upon elevations in transmural pressure. 6 Membrane potential and hypoxia In the cerebrovascular system, hypoxia primarily increases the open probability and mean open time of Kca channels which hyperpolarize vascular smooth muscle cell to mediate vasorelaxation, thereby increasing C B F . Hypoxia may also reduce tone by stimulating the activity o f an A T P sensitive K + channel (Daut et al. 1990). When the level of intracellular A T P drops due to hypoxia, there is an augmentation of signal K + channel activity, which increases the outward current, hyperpolarizes and then relaxes arterial muscle. Membrane potential and PCO2/pH Reduction of extracellular p H either by increases in P C O 2 or in H + concentration dilates cerebral arteries. This dilation is mediated by membrane hyperpolarization in isolated cerebral arteries (Harder 1982). Reduction of P C O 2 or elevation of p H depolarizes and contracts cerebral arteries. Both of these effects are apparently mediated at least in part by changes in the conductance of K + . In patch-clamped isolated cerebral arterial muscle cells, reduction of p H from 7.4 to 7.20 greatly increases the peak outward current by 35% (Harder and Madden 1985). 1.7 V A S O A C T I V E M E D I A T O R S 1.7.1 N I T R I C O X I D E .Synthesis, storage and release In 1980, Furchgott and Zawadzki discovered that nitric oxide (NO) is the endothelium-dependent relaxing factor (EDRF)(Furchgott and Zawadzki, 1980). Endogenous nitric oxide (NO) is converted from L-arginine by nitric oxide synthase (NOS); in addition, N O may also be formed from endogenous nitrate ion. Being a gaseous mediator, N O is not stored in cells but produced in response to a number of agonists, such as acetylcholine, bradykinin and substance P. After being produced, N O rapidly diffuses from its synthesis site to surrounding tissues. The 7 family of N O S consists of three isoforms known as: neuronal N O S (nNOS), found in epithelial and neuronal cells; endothelial N O S (eNOS), formed in endothelial cells, and inducible ( iNOS) , produced in macrophages and smooth muscle cells. NO in the cerebrovasculature The relaxation of cerebral smooth muscle by N O released from endothelial cells is due to the stimulation of soluble guanylate cyclase in smooth muscle cell, which results in the formation of cyclic guanidine monophosphate (cGMP) (Rapoport and Murad 1983). The latter activates cGMP-dependent protein kinase, which leads to an upregulated extrusion of C a and vasodilation (Lincoln, 1994). Moreover, cGMP-independent pathways such as activation of K c a channels,, inhibition of vasoconstrictor (20-hydroxyeicosatetraenoic acid) production, and direct inhibitory effects on C a 2 + influx and C a 2 + release are also responsible for the NO-induced relaxation (Alonso-Galicia et al. 1997; Blatter and Wier 1994; Bolotina et al. 1994; Kannan et al. 1997). 1.7.2 A C E T Y L C H O L I N E .Synthesis, storage and release Acetylcholine (ACh) is synthesized from acetyl-CoA and choline by the enzyme choline acetyltransferase. The availability of choline may serve as the rate-limiting step for the synthesis of A C h . After synthesis, A C h is transported into and stored in synaptic vesicles of the nerve ending. The release of A C h from vesicles requires the entry of C a 2 + and the stimulation of an interaction between proteins associated with the vesicles and the membrane. The action o f A C h is terminated by metabolism to acetate and choline by the enzyme acetylcholinesterase. ACh in cerebrovasculature 8 ACh-induced vasorelaxation is due to the presence of muscarinic receptors located on endothelial cells. In cerebral arteries, the stimulation of these receptors, primarily M 3 subtype, increases the influx of C a 2 + that activates the synthesis of N O from L-arginine and elicits vasodilation (Moncada and Higgs 1995). In the absence of endothelium, A C h induces vasoconstriction by its direct action on smooth muscle cells mediated by the reduction of cycl ic adenosine monophosphate ( cAMP) , the stimulation of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) release, and the activation of G-protein. Cholinergergic vasodilation may be impaired by multiple mechanisms in the ischemic brain and may play a role in the pathogenesis of cerebrovasculature disease. Moreover, a deficit o f cerebral A C h is a prominent finding in Alzheimer's disease (Geaney et al. 1990). 1.7.3 E N D O T H E L I N Synthesis, storage and release Endothelins (ETs) are peptide vasoconstrictors produced from the cleavage of big ET-1 by endothelin-converting enzyme (ECE) which is primarily located in endothelial cells of blood vessels. Although ET-1 is mainly produced in endothelial cells, the synthesis also occurs in other cell types such as vascular smooth muscle cells. Under normal condition, small amount o f ET-1 is produced and released from cellular vesicles to mediate vascular tone in vivo. In pathological conditions, such as hypoxia and vasospasm, ET-1 production is greatly upregulated (Weir, 1999). Three different E T peptides (ET-1, ET-2, ET-3) with minor variations in amino acid sequence have been identified in humans. ETs act by at least three different receptor subtypes including E T A , E T b - 1 , and E T B - 2 . The E T A and E T B - 2 receptor are localized in vascular smooth muscle cells and mediate the vasoconstrictor effect (Arai, 1990; Sakurai, 1990). The E T B - 1 subtype 9 also occurs on vascular endothelial cells and induces the endothelium-dependent vasodilator effect. ET-1 in the cerebrovasculature ET-1 is a potent and long-lasting constrictor which functions from the adventitial but not the luminal side of cerebral arteries (Yanagisawa, 1988; Masaki , 1991). ET-1 acts by a C a 2 + -dependent mechanism partly mediated by the activation of P K C and phospholipase A 2 (Murray, 1992). Also , E T causes C a 2 + influx via a voltage-operated C a 2 + channel (Luscher, 1992). Moreover, ET-1 activates smooth muscle proliferation and is involved in some forms o f hypertension and a number of cerebrovascular disorders, including vasospasm. 1.7.4 5 - H Y D R O X Y T R Y P T A M I N E Synthesis, storage and release 5-Hydroxytryptamine (5-HT) is synthesized through a 2-step process involving a tetrahydrobiopterin-dependent hydroxylation reaction catalyzed by tryptophan-5-monooxygenase and a decarboxylation catalyzed by aromatic L-amino acid decarboxylase. 5-H T is located in several tissues including circulating platelets, central neurons, and cerebral mast cells. After release from serotonergic neurons, most of the released 5-HT is recaptured by an active reuptake mechanism. 5-HT in the cerebrovasculature The serotonergic innervation of cerebral blood vessels was first postulated in 1976 (Chan-Palay 1976; G O T O H et al. 1961). However, the existence of a serotonergic innervation of the cerebrovasculature remains to be elucidated as studies also showed consistent results suggesting that the serotonergic innervation was in fact the noradrenergic sympathetic innervation (Saito and Lee 1987). 10 A s 5 - H T does not cross the B B B , its influence on the cerebral circulation cannot be directly assessed. Depending on the sites of investigation and the methods employed, 5 - H T elicits vascular relaxation and constriction; Most studies on the vascular effects of 5-HT showed that this amine was a powerful constrictor agent, while 5-HT was also reported to induce vasodilation in small pial arteries (Harper and MacKenzie 1977). The function of 5-HT is exerted upon its interaction with specific receptors identified as 5 H T 1 . 7 . The majority o f cerebral arteries express a 5-HTi-mediated contractile response, although mixed 5 - H T i / 5 - H T 2 characteristic is also observed. 5-HT is involved in the pathogenesis of cerebral vasospasm following subarachnoid hemorrhage and also in some cerebrovascular diseases such as stroke, trauma, and migraine (Pappius 1991). 1.7.5 H I S T A M I N E Synthesis, storage and release Histamine is formed from histidine by histidine decarboxylase and is mainly stored in lungs, skin and intestinal mucosa. Histamine is released from mast cells by a secretory process during inflammatory or allergic reactions. Histamine in the cerebrovasculature Intravascularly applied histamine may not affect C B F as long as blood pressure is kept within the autoregulatory range since circulating histamine does not readily cross the B B B (Oldendorf 1971). Extravascularly applied histamine induced pial arterial dilation in most species, while contractile action of histamine was also found in cerebral arteries (Miranda et al. 1992; Ottosson et al. 1988b; Toda 1990; Van Riper and Bevan 1991). Histamine produces its action via specific receptors, which are H i , H 2 , and H 3 . Histamine dilates blood vessels by acting on H i receptor in humans arid a combined action on H i and H 2 receptors in some experimental animals. Histamine-induced vasodilation is partly endothelium-11 dependent, whereas at high concentrations, it produces vasoconstriction in cerebral arteries which does not involve specific histamine receptors. Histamine can stimulate inositol phospholipid hydrolysis which results in synthesis of IP3 and then release of C a 2 + from the intracellular store to activate the contractile apparatus (Berridge and Irvine 1984). Additionally, activation of an unselective cation channel which consequently upregulates the influx of C a 2 + may also be involved in the contractile action of histamine (Karashima and Kuriyama 1981). Due to its potent enhancement of permeability and. dilatory effects on cerebral vessels, an increased concentration of histamine may induce brain edema. Whether histamine acts as a mediator of vasospasm is still under debate (Table 1.1). 1.8 F A C T O R S I N V O L V E D I N T H E R E G U L A T I O N O F V A S O M O T O R R E S P O N S E 1.8.1 C A L C I U M The importance of C a 2 + i n smooth muscle constriction and arteriolar tone was directly established three decades ago (Uchida 1969; Nakayama, 1982). The advent of C a 2 + sensitive fluorescent dyes together with video-based imaging and photometer techniques allows researchers to investigate Ca dynamics in resistance vessels. Smooth muscle contraction or dilation is mainly controlled by the changes in intracellular [ C a 2 + ]j. Intracellular C a 2 + increases during contraction as a consequence of C a 2 + influx from the extracellular space or its release from intracellular stores. Binding molecules for C a 2 + in the myoplasm and in the sarcoplasmic reticulum (SR) act as cell buffers which endow a relatively large capacity for C a 2 + storage with the low free concentrations in the cells (Horowitz et al. 1996). The sarcolemma is a barrier across which a 10,000-fold concentration gradient exists between extracellular C a 2 + ([Ca 2 + ] e ) and [Ca 2 +]j. C a 2 + enters the sarcoplasm via receptor-operated channels (ROCs), voltage-dependent channels(L type), and the N a + / C a 2 + exchanger. 12 C a 2 + is removed from the sarcoplasm by a plasma membrane A T P a s e as w e l l as the N a + / C a 2 + exchanger The second Ca 2 +-integrating system is in the SR which is an intracellular C a 2 + pool with more than 10 times the C a 2 + needed to trigger a single contraction. At least two types of C a 2 + channels are present in the SR. One is sensitive IP3; the other type is sensitive to ryanodine (RyR) and caffeine. The efflux of C a 2 + from the C a 2 + store is linked to the binding of the second messenger IP 3 or R y R to their receptors on the SR. The reduction in stored C a 2 + stimulates the release of a C a 2 + influx factor, which facilitates the entry o f C a 2 + through the plasmalemma via depletion-operated C a 2 + channels. On the other hand, the drop in lumenal free C a 2 + makes it easier for sarcolemmal C a 2 + ATPase to pump C a 2 + into the SR against a lesser concentration gradient. 1.8.2 M Y O S I N L I G H T C H A I N K I N A S E A N D M Y O S I N L I G H T C H A I N P H O P H A T A S E A major factor in the regulation of vasomotor action is the phosphorylation of myosin. The degree of myosin phosphorylation depends on the relative activity o f myosin light chain kinase ( M L C K ) and myosin light chain phosphatase ( M L C P ) (Stull et al. 1998). M L C K (105 kDa) is a Ca2 +-dependent protein kinase and its primary regulator is C a 2 + / Ca 2 + -calmodulin (CaM). When C a 2 + binds to calmodulin, there is a conformational change rendering it capable o f activating M L C K , which subsequently phosphorylates serine 19 of the myosin light chain ( M L C ) . The phosphorylation M L C greatly increases actin-activated myosin Mg-ATPase activity, which ultimately initiates smooth muscle contraction. 1.8.3 Rho K I N A S E The Ca 2 +-mediated pathway has long been regarded as the principle mechanism by which M L C phosphorylation is regulated; however, a second pathway regulating the phosphorylation state o f M L C was found by analyzing muscle cells. Studies show that various physiological stimuli 13 can induce smooth muscle contraction also in the absence of an increase in the free cytosolic C a 2 + concentration (Bradley and Morgan 1987; Himpens et al. 1990; Rembold 1990). Subsequent studies revealed that this "Ca2+-independent" regulation occurs through the inhibition of myosin phosphatase and involves the monomelic GTP-binding protein RhoA (Bradley and Morgan 1987; Hirata et al. 1992; Gong et al. 1996; Somlyo and Somlyo 1994). Activation of RhoA leads to the activation of Rho-kinase which in turn phosphorylates the regulatory myosin-binding subunit of myosin phosphatase and results in the inhibition of the enzyme (Amano et al. 1996; Hirata et al. 1992; Kimura et al. 1996; Kureishi et al. 1997). Thus increased actomyosin interaction can be achieved through Ca2+-mediated M L C K activation and through Rho-dependent inhibition of RLC dephosphorylation, both leading to increased R L C phosphorylation. 1.9 T H E T E C H N I Q U E S F O R T H E INVESTIGATION O F C E R E B R O V A S C U L A R FUNCTIONS During 1930s, Forbes and Fog performed the first systematic attempts to analyze cerebrovascular smooth muscle reactivity using a cranial window technique, comprised of a craniotomy and the superfusion of artificial cerebrospinal fluid on the exposure area. This preparation could visualize pial arteriole diameter hence examine the alterations of cerebral circulation following changes in blood pressure and resulting from the administration of various agents (Purves, 1972). It was not until the beginning of 1970s that isolated vessel techniques were developed (Nielsen and Owman 1971). Nowadays, the techniques (isometric and isotonic preparation) enable more careful and precise quantitation in resistance arteries. Particularly, the isotonic preparation more closely mimics the physiological condition and serves as an useful approach to distinguish the effect of a desired objective from flow, metabolic, neural, and endothelial influences in the cerebrovasculature. 14 C H A P T E R 2 P H A R M O C O L O G Y O F I S O L A T E D M O U S E M E D D L E C E R E B R A L A R T E R Y 2.1 INTRODUCTION Cerebral arteries supply oxygen and glucose to the brain; remove waste products and regulate the ionic environment of the cerebral circulation (Thorin et al. 1997a). They also play an important role in the regulation of cerebral blood flow through autoregulation. Although much is known about the cerebral circulation, many essential mechanisms of autoregulation and features of pathological changes in the cerebral circulation, such as subarachnoid hemorrhage and stroke, have not been elucidated (Table 1.1). Due to these shortcomings, various animal models have been developed to improve our understanding of the pathophysiology of the cerebral circulation with the goal of establishing strategies for the prevention and treatment o f cerebral circulatory disorders. To better understand cerebral ischemia, mice lacking both alleles for neurotrophin 4 or deficient in a single allele for brain-derived neurotrophic factor were selected (Endres et al. 2003). Likewise, in order to identify mechanisms of oxygen-induced cerebral vasoconstriction, mouse models either overexpressing or lacking extracellular superoxide dismutase (SOD) were constructed (Demchenko et al. 2002). B y overexpressing transforming growth factor-betal, transgenic mice were also used to explore the etiology o f cerebrovascular abnormalities prominent in Alzheimer's disease (Buckwalter et al. 2002). Thus there are a variety of murine models of cerebrovascular disease where a number of molecular and structural elements of cerebral disorders have been gleamed. However, the pharmacological properties of mouse cerebral arteries have not been described, and there exists a deficit in our knowledge of the mouse cerebrovascular system. Thus, we designed protocols that would determine whether the cerebral blood vessels of the mouse possessed similar properties with respect to myogenic control and responsiveness to 15 vasoconstrictors and vasodilators. We undertook this study by using two forms o f vasoconstrictors — increases in transmural pressure because pressure-induced regulation o f artery diameters is a key component of cerebral autoregulation, and receptor agonists known to have vasoconstrictor efficacy in the cerebral circulation of other species. 2.2 H Y P O T H E S E S A N D O B J E C T I V E M y hypothesis was that the mouse cerebral circulation should respond to changes in transmural pressure as well as vasoconstrictors, such as 5-HT and vasodilators, such as A C h , in a comparable manner as for cerebral vessels from other species. Likewise, we also examined the effects of several known endothelium-dependent vasodilators. In addition, I used the pressurized isolated mouse middle cerebral artery ( M C A ) to simultaneously measure changes o f E m and arterial diameter or changes in intracellular C a 2 + and arterial diameter in response to these vasoactive stimuli. 2.3 M A T E R I A L S A N D M E T H O D S 2.3.1 T I S S U E P R E P A R A T I O N The mice used in this study were housed in a temperature- and humidity-controlled environment with a 12-hour light/dark cycle and free access to food and water. Male C D - I mice weighing between 38 — 45g were injected with sodium pentobarbital (30mg/kg) and heparin sulfate (500U/kg) intraperitoneally. After loss of all reflexes, the mouse was decapitated and the brain was removed from the cranium and placed in ice-cold (4°C) physiological salt solution (PSS). The mouse M C A (intraluminal diameter, 80 - 90/xm) was dissected and transferred to the chamber of a pressure myograph that contained two glass micropipettes (tip diameter 35 — 45/im). The distal cannula was occluded and theproximal one was connected to a pressure servo system (Living Systems Instrumentation, Burlington, V T , U S A ) . The vessel 16 was mounted on the proximal cannula with a single strand of braided 4-0 silk suture, and blood was removed by gently perfusing PSS into the vessel. The other end o f the vessel was then mounted on the distal cannula. Each vessel was bathed in PSS, which continually circulated at a flow rate of 20 — 30 ml/min from a reservoir. A gas mixture containing 95%02 and 5% CO2 was used to aerate the PSS . After gassing, the p H was maintained between 7.35 — 7.45. A circulating water bath and a glass heat exchange coil were used to maintain the temperature between 36.5 ~ 37°C. After the vessel was positioned between the tips of the two cannulae, the chamber was placed on the stage of an inverted microscope. A Video Dimension Analyzer measured inner diameter and data were recorded on a personal computer. 2.3.2 E X P E R I M E N T A L P R O T O C O L Myogenic tone and endothelial factors After a M C A was mounted, intravascular pressure was gently increased to 80 m m H g at which point the bath temperature was raised to 37°C. The artery segment was equilibrated for 60 minutes at 80 mmHg during which time the vessel spontaneously developed pressure-induced myogenic tone. The pressure-diameter relationship was obtained by increasing the intraluminal pressure from lOmmHg to H O m m H g in lOmmHg increments, and 5 minutes allowed for the vessel to achieve a new steady-state diameter at each new pressure. To determine the role of nitric oxide (NO) in modulating the intrinsic tone, M}-ni t ro-L-arginine-methyl ester ( L - N A M E ) ( lOpM) , a N O synthase (NOS) inhibitor, was added to the external reservoir for 30 minutes and the pressure-diameter curve was obtained in an identical way. 17 To further examine whether endothelin contributes the generation of pressure-induced tone, bosentan ( l p M ) , an E T A and E T B receptor antagonist, was applied following the same procedure in a separate series of experiments and the pressure-diameter curve was recorded. Vasoconstrictor effects A l l responses to vasoconstrictors were studied at 20mm Hg . The constrictor effects o f endothelin-1 (ET-1, l O p M - 0.1 pM) , phenylephrine (PE, I n M - O. lmM), U46619 ( l O p M -lOuM) , 5-HT ( I n M - lOuM) , and histamine ( I n M - l O p M ) were studied by making cumulative additions to the external reservoir to construct cumulative concentration-response curves. In addition, the responses to various concentrations of KC1 were examined. Vasodilator effects M C A was pretreated with P E (5uM) at 20mm Hg. After a sustained constriction was evoked, tissue was exposed to A C h ( I n M - 50uM) added to the external reservoir, and the final maintained diameters were recorded. To further clarify whether N O was involved in the response to A C h , the vessel was exposed to L - N A M E ( I J U M ) for 15 minutes following washout of A C h . The identical protocol was used to study the vasodilator effects of bradykinin ( B K , O . l n M -10/uM), and substance P ( l p M - O. luM) . Moreover, in other experiments, 5-HT ( I n M -10uM), and histamine ( I n M - lOpM) were also added in P E pre-constricted vessels to determine possible vasodilatory effects in mouse M C A . To study whether N O is involved in the vasodilatation of 5-HT and histamine, L - N A M E (1/iM) was incubated for 15 minutes after the pre-constriction by P E (5pM) at 20mm Hg. At the end of each experiment, PSS was substituted with calcium-free PSS to obtain the maximal passive diameter of each vessel. 18 Measurement of intracellular Ca2+ concentration Mouse M C A s were first cannulated in a pressure-myograph chamber as described above. The arteries were then incubated with the Ca 2 +-sensitive fluorescent dye, fura-2 A M 5/nM (20ul o f anhydrous D M S O was used to dissolve 50|4,g fura-2 A M and was diluted with 10ml o f PSS to yield a final concentration of 5^M) , and pluronic acid (0.01%; wt/vol) for 30 minutes at room o temperature in oxygenated PSS followed by washout with PSS for 30 minutes at 37 C (Coombes et al. 1999; Gollasch et al. 1998; Knot and Nelson 1998b; /Lagaud et al. 2002a; L o h n et al. 2001b). Excitation was performed using a 75w xenon arc. The excitation wavelength was alternatively switched between 340 - 380 nm using a diffraction grating, and the emission was recorded at 510nm. The emitted fluorescence was converted to relative measures of [ C a 2 + ] i using Felix quantitative ratio fluorescence software (Photon Technology International; Monmouth Junction, NJ). The measurements in each vessel were normalized to the maximum recorded in response to 60 m M KC1. Ratio was measured and compared at different bath concentrations of P E ( l O n M - l O u M ) and KC1 (20,40,60,and 80mM). In these experiments, changes in intracellular Ca 2 + and changes in arterial diameter were recorded simultaneously. Measurement of membrane potential Mouse M C A was studied using a pressure myograph as described above. The chamber was then transferred to the stage of an inverted microscope (Ziess, Axiovert 25) where changes i n E m and in internal diameter were recorded simultaneously. E m was measured in vascular smooth muscle cells ( V S M C s ) of arteries by inserting microelectrodes pulled from borosilicate , glass with filament (OD: 1.2mm, ID: 0.69mm) using a Flaming/Brown micropipette puller (model p-97, Sutter Instrument Co.). Microelectrodes were backfilled with 3 M KC1 solution (tip resistance 100 - 150 M Q ) and mounted on an electrode holder (Axon Instrument, Inc.) connected to a micromanipulator (MP-285, S U T T E R Instrument Comp.), which was used to 19 move the electrodes towards the arterial wall for insertion. A reference electrode ( A g - A g C l ) was positioned in the solution inside the chamber. Microelectrodes pierced the vessels from the adventitial side and the electrical signal obtained was amplified using an AxoClamp 2 B amplifier (Axon Instruments, Inc.). The amplified signal was continuously monitored and recorded by a personal computer using Axoscope 8.1 software (Axon Instrument, Inc.). E m was measured at different transmural pressures and also after 1 5 - 2 0 minutes incubation with P E or different concentrations of KC1. The criteria for accepting a record were i) a sharp negative deflection in potential upon entry; ii) stable recording for at least one minute after entry; and i i i ) a sharp positive deflection to OmV upon exiting from the recorded cell. Solutions and chemicals The PSS consisted of the following (in m M ) : N a C l 119, KC1 4.7, K H 2 P 0 4 1.18, N a H C 0 3 24, M g S 0 4 7 H 2 0 1.17, C a C l 2 1.6, glucose 5.5 and E D T A 0.026. Ca 2 +-free solution was PSS containing no C a C l 2 and 2.0 m M E G T A . Equimolar concentrations of K + replace N a + for depolarizing solutions of KC1. U46619 was obtained from Cayman Chemical (Ann Arbor, M I ) . F u r a 2 - A M and Pluronic® F127 were from Molecular Probes. Other drugs were purchased from Sigma (St. Louis, M O ) . Expression of results and statistical analysis 2 + Myogenic tone at each pressure was expressed as a percent constriction=100%x[(Z)ca -free -Z)PSS)/Dca 2 +-free], where D is the diameter in calcium free (Z)ca2+-free) or Ca 2 +containing PSS (£>PSS). Percent dilation was calculated using the equation 100%x[(Z)d -Db)/(£>ca 2 +-free -Z)b)], where D is the diameter upon stabilization after drug addition (d), baseline (b) or C a 2 + free. Constrictor responses of arteries loaded with fura2-AM were not different from those without the incubation with the dye. 20 A l l results were expressed as mean ±SE of n experiments. Data were analyzed with N C S S 2000 and P A S S 2000 software using analysis of variance ( A N O V A ) and/or repeated-measures A N O V A with multiple comparisons performed by Bonferroni's test when appropriate. -LogECso (pDi) was calculated by Graphpad Prism, version 3.02. The results o f statistical tests were considered statistically significant atp<0.05. 2.4 R E S U L T S 2.4.1 M Y O G E N I C T O N E O F M O U S E M I D D L E C E R E B R A L A R T E R Y Myogenic tone of mouse M C A was initiated at lOmmHg where the arteries were constricted by 3 .4±2 .1% (n=15). As intravascular pressure was increased, myogenic constriction increased to 20.6±2.4% (n=15) at 90mmHg, which was not significantly different from the myogenic constriction that reached a plateau at 50mmHg (Fig. 2.1 A ) . The addition of L - N A M E ( l O u M ) to arteries equilibrated in PSS potentiated the pressure-induced constriction at all pressures between 20 and 110 mmHg. A t 90mmHg, L - N A M E potentiated vascular tone by 12.4±0.4%(n=4). The myogenic response o f vessels equilibrated with bosentan ( l u M ) was attenuated by 7.2±1.8%(n=4) at 90mmHg(Fig. 2. IB) . 2.4.2 V A S O C O N S T R I C T O R R E S P O N S E S Application of a depolarizing solution of raised K + evoked constriction (Fig. 2.2A). Constriction occurred with low concentrations of KC1, such that with 8 m M the constriction was 3.6±2.1%(n=6) in the mouse M C A . With cumulative additions of KC1, the maximum constriction achieved was 63 .3±3 .3% at 66mM; increasing the concentration of KC1 to 114mM did not increase tone further. The EC 5 o for KC1 was 31.0 ± 4 .1mM. The contractile response to ET-1 , a combined E T A and E T B receptor agonist, was also examined. There was a significant contractile effect of ET-1 on the mouse M C A . ET-1 (50nM) 21 generated the greatest constriction (47.8 ± 4.2%, n=6) amongst all agonists studied. A t this concentration, the arteries responded weakly to adrenergic receptor stimulation, such that only 2.9±0.6% constriction was elicited by PE , an ai-adrenoceptor selective agonist. Although P E could constrict vessels to 45.8 ±4.5%(n=8), a higher concentration (50/iM) was required. U46619, a stable analog of thromboxaneA2, produced a 13.9±2.6%(n=8) constriction at a concentration of 50nM. The maximum constriction generated by U46619 (5jtiM) was 34.8±3.5%(n=8). The p D 2 values of p D 2 values of ET-1 , P E and U46619 are 8.8±0.5, 6.0± 0.3, and 7.1±0.3, respectively. 5-HT ( l O n M - l O u M , n=6), a potent vasoconstrictor of cerebral arteries in most other species (e.g. rabbit, dog, monkey, human etc.) did not induce any contractile response in mouse M C A (Fig. 2.4A). The inhibition of N O production did not unveil constriction to 5-HT (10/ iM, n=6) (Fig. 2.4B). 2.4.3 V A S O D I L A T O R R E S P O N S E S ACh-induced dilation of mouse M C A was studied in arteries that possessed an intact endothelium and had been pre-contracted by P E (5uM) or U46619 ( lOnM) at 20mmHg. A C h ( I n M - 50/iM) induced a maximal dilation of 58.7±11.8% at 10/xM (n=6), as shown in F ig . 2.3A. Using another M C A segment, a stronger concentration-related vasodilation was exhibited by the addition of B K (0.1 n M - 10 fiM), which reached a peak value of 82.4±10.4% (n=4) at lOpiM. Another endothelium-dependent vasodilator substance P (50nM) produced maximum dilation 42.9 ±9 .2%, which was lower than the maximal vasodilation induced by B K and A C h . Inhibition of N O synthase with L - N A M E (1/^.M) attenuated the vasodilatory responses induced by B K (Fig. 2.3B), A C h and substance P (data not shown). The p D 2 values of B K , A C h and substance P are 8.6±0.4, 7.4±0.5, and 10.3±1.5, respectively. Their efficacy with a decreasing order is B K >ACh > substance P. 22 Histamine either dilates or constricts the cerebral arteries of most species; however, the cumulative application o f histamine ( I n M - 10/xJVl, n=6) in the mouse M C A failed to produce any vasomotor actions (Fig. 2.4A). The inhibition of N O production did not unmask histamine (10/xM, n=6) to exhibit vessel constriction (Fig. 2.4C). 2.4.4 [Ca 2 + ]i A N D A R T E R I A L D I A M E T E R The fluorescence ratio at 340/380 was measured in fura-2 loaded M C A as an indicator o f [Ca 2 +]i.. We compared the effects of P E (10/iM) and KC1 (40mM) in the same artery segments. These bath concentrations were equally effective in causing vasoconstriction (42.3 ± 2.3% vs. 45.8 ± 4.5%, respectively, n=5, paired t-test, = 0.54) (Fig. 2.5A). The normalized 340/380 fluorescence ratio was higher in the presence of KC1 40 raM compared to P E 10/ iM (0.99 ± 0.003,n=6 vs.0.92 ± 0.02, n=6,p< 0.01) (Fig. 2.5B). 2.4.5. M E M B R A N E P O T E N T I A L A N D T H E E F F E C T S O F T R A N S M U R A L P R E S S U R E S A s the transmural pressure was increased, smooth muscle cell Em became more positive. The Em was significantly more negative at a low pressure of 20mmHg (-52.6 ± 0.90mV) compared to higher pressures: 40mmHg (-42.3 ± 1.99mV), 60mm H g (-35.3 ± 2.12mV) and 80mmHg (-37.3 ± 1.75mV). The differences in Em of smooth muscle cells at 60 and 80mmHg were not statistically significant (Fig. 2.6 A & B) . 2.4.6. KC1 A N D A G O N I S T - I N D U C E D D E P O L A R I Z A T I O N OF S M O O T H M U S C L E C E L L S The resting Em of the mouse M C A at a transmural pressure of 20mmHg was -52.6 ± 0.9mV (n = 12). The addition of KC1 depolarized smooth muscle cell in a concentration dependent manner: KC1 20mM (-38.5 ± 3.3mV, n=12), 4 0 m M (-29.5 ± l . l m V , n =15), 60mM (-30.6 ± 0.9mV, n = 19), and 80mM (-29.0 + 1.4mV, n = 6). A significant difference in Em between 23 2 0 m M KC1 and higher concentrations of K + was observed (Fig. 2.7). Likewise, P E ( l O u M ) depolarized the membrane significantly in mouse M C A s (-43.4 ± 2.7, n = 15) compared to control (-52.6 ± 0.9, n=25, /><0.01)(Fig. 2.6A&2.8). Whereas at the same level of constriction, the Em was significantly more negative in the presence of P E ( lOpM) compared to 4 0 m M K C 1 (-43.4 ± 2.7mV vs. -29.5 ± l . l m V , n =15,/><0.01) (Fig. 2.8). 2.5 D I S C U S S I O N 2.5.1. I N T R I N S I C T O N E Intrinsic tone results from intravascular pressure activating myogenic constriction of small arteries, and may be more important physiologically than the efficacy or potency of any single contractile or relaxant agonist in the regulation of basal vascular tone. In the cerebral circulation, the ability to autoregulate blood flow over a broad range of perfusion pressures is almost completely dependent on the regulation of intrinsic tone (Rosenblum and Wormley 1995a). Our data demonstrated that the intrinsic tone of the mouse M C A was evoked at low pressures with no significant additional constriction occurring at higher pressures (>50mmHg). This implies that myogenic vasoconstriction in the mouse M C A only partially protects the capillary vessels, and that at higher pressures; other protective mechanisms may be activated. Nevertheless, the maximum constriction generated by the raised intraluminal pressure in mouse M C A is similar to that exhibited in isolated cerebral arteries from different species which range between 20-30% (Wallis et al. 1996; Ishiguro et al. 2002)(Table2.2). The application o f bosentan impaired the development of myogenic tone of mouse M C A ; while L - N A M E . potentiated vessel constriction. Our results are compatible with previous studies, suggesting that intrinsic tone includes the influences of both vasoconstrictor (e.g. ET-1) and vasodilator (e.g. N O ) molecules released from the vascular endothelium (Smeda and Payne 2003). 24 2.5.2 A G O N I S T - I N D U C E D V A S O C O N S T R I C T I O N Extracellular K + concentration, which is 3-5mM in central nervous system interstitial tissue, increases to 10-12mM when neurons are activated. In pathological conditions, such as traumatic brain injury and stroke, extracellular K + can increase to 50 — 80mM to depolarize the vascular smooth muscle and cause constriction (Sykova 1983). In rat cerebral arteries, 2 1 m M K + induces dilation, and maximal constriction is produced by 8 1 m M K + (Golding et al. 2000). In the mouse M C A , we found that 2 0 m M KC1 elicited a constriction and concentration-dependent constrictions were maximal with 6 6 m M KC1. ET-1 elicits potent and long-lasting constriction. The vasoconstrictor potency of ET-1 exceeds that of P E and U46619 in the mouse M C A . The sensitivity of mouse M C A to ET-1 is similar to that reported in isolated human and canine cerebral arteries (Kaito et al. 1995), but lower than in cat isolated M C A (Saito et al. 1989) (Table2.2). The cerebral vasculature has an extensive adrenergic innervation arising predominantly from the superior cervical ganglia. Under normal conditions, these nerves may not have an important role since cerebrovascular tone is largely influenced by changes in intraluminal flow and intravascular pressure (Busija and Heistad 1984b; Bevan and Laher 1991 ; Bevan 1997). Cerebral arteries from a number of species, including humans, are relatively insensitive to adrenoceptor vasoconstrictor agonists and frequently require high (mM) concentrations of agonists (Laher and Bevan 1985b). Compared to a-adrenergic vasoconstriction in cerebral arteries from human, monkey, canine, and rabbit (Laher and Bevan 1985c; Sasaki et al. 1985a; Thorin et al. 1997b), the PE-evoked contractile response in mouse M C A is more pronounced. This suggests that adrenoceptor sensitivity of cerebral arteries may be species-related and/or the vasomotor response is mediated by a class of adrenergic receptors distinct from the classical ones (Laher, 1986). 25 Cerebral arteries from most species contract in response to 5-HT. The vasoconstriction ranges from 40 to 100% of the maximum constriction elicited by 124mM KC1 (Young et al. 1986). In isolated human and cat cerebral arteries, 5-HT induces endothelium-independent constriction (Conde et al. 1991), while 5-HT causes an unstable contractile response in rabbit M C A (Thorin et al. 1997c). In our study, 5-HT did not provoke changes in arterial tone in the mouse M C A , and the inhibition of N O did not unmask any constrictions to 5-HT. This brings into question the usefulness of the mouse as a model for migraine and stroke research, diseases in which 5-H T is thought to play important roles (Nilsson et al. 1999; Wester et al. 1992). 2.5.3 A N T A G O N I S T - I N D U C E D V A S O D I L A T I O N A C h induces vasodilation of isolated rabbit, feline and guinea pig M C A s (Brayden and Wellman 1989; Brayden 1990; Dong et al. 2000). A C h also relaxes the mouse M C A , and the sensitivity of mouse M C A to A C h is higher than that in human cerebral arteries (Tsukahara et al. 1989). Inhibition of N O S with L - N A M E suppressed the Ach- , B K - and substance P -induced dilations in mouse M C A , indicating the involvement of the endothelial-dependent relaxing factors. B K has greater efficacy than A C h in isolated mouse M C A , a finding that is consistent with experiments conducted in vivo (Rosenblum and Wormley 1995b). Histamine dilates isolated human (Ottosson et al. 1988a) monkey (Ayajiki et al. 1992) rat (Benedito et al. 1991b) cerebral arteries. Also histamine elicited vasoconstriction in rabbit cerebral arteries (Laher and Bevan 1985a, Gokina and Bevan 2000). However, our study showed that the histamine was unable to evoke either dilation or constriction in the mouse M C A , either in the absence or presence of L - N A M E (Fig. 2.4C). This finding also raises the question of choosing the isolated mouse M C A to investigate the etiology of brain edema and vasospasm in which histamine is one of putative mediators. 26 2.5.4 A R T E R I A L W A L L C a 2 + A N D S M O O T H M U S C L E C E L L M E M B R A N E P O T E N T I A L Depolarization induced by KC1 causes vascular constriction by an increase in [Ca' +]j due to opening of voltage-gated C a 2 + channels (Nelson et al. 1990). Agonists such as P E induce arterial constriction through a combination of increased C a 2 + entry and augmented intracellular Ca 2 +sensitization (Somlyo and Somlyo 1994). In our study both P E and KC1 induced significant membrane depolarization; however, at the same level of constriction, the KCl- induced depolarization was significantly higher than P E . These findings combined with direct measurements of intracellular calcium indicating that a significant role for agonist-induced C a 2 + sensitization in cerebrovasculature. Increases in transmural pressure also caused membrane depolarization of mouse M C A . This membrane depolarization may contribute to pressure-induced myogenic tone in mouse cerebral vessels. The mechanism of this effect could be attributable to depolarization-induced opening o f L-type calcium channels in V S M C as demonstrated in rat cerebral artery. In rat M C A , both myogenic tone and membrane depolarization reach a plateau at a transmural pressure o f lOOmmHg (Knot and Nelson 1998a). However, our data indicate that both myogenic tone and membrane depolarization reach a plateau at 50-60mmHg. 2.6 C O N C L U S I O N In summary, this study focuses on the response of the mouse M C A to a number o f pharmacological agents that are modulators in the cerebrovascular circulation under normal and pathophysiological states. We demonstrated that the mouse M C A i) constricts to a-adrenergic receptor activation. This is unlike the general insensitivity o f cerebral arteries to adrenergic receptor stimulation in most other species, including humans; ii) is insensitive to 5-HT, which is a potent constrictor in the cerebral circulation in other species; iii) is insensitive to histamine, which causes vasodilation in various species, and iv) increases in tone produced by P E is 27 accompanied by smaller changes in membrane potential and intracellular Ca compared to tone produced by membrane depolarization with raised K + . 28 Table 1.1 Evidence of putative spasmogen Putative spasmogen For Against Neurogenic factors Adrenergic, Cholinergic or Peptidergic nerves 1) Innervation of adventitia of cerebral blood vessels 2) Lesion of the A 2 nucleus, an ascending pathway for N E release, prevents V S P 3) N E uptake altered by Hb 4) Neurogenic vasodilation is suppressed by oxyHb 5) Phentolamine inhibits V S P 6) Continuous electrical stimulation of the trigeminal ganglion causes vasodilation and increases C B F ; a trigeminal lesion causes vasoconstriction. 1) Loss of staining of nerves following exposure to blood does not correlate with V S P 2) Sympathectomy and bilateral superior cervical' ganglionectomy does not reverse V S P 3) No obvious changes in sympathetic / parasympathetic perivascular neural networks found in rat V S P 4) Cerebral arteries dilate to electrical stimulation in the presence of tetrodotoxin Biogenic amines Histamine, N E 1) Histamine and N E produce vasoconstriction 2) N E metabolites in C S F detected in V S P 3) A N E periarterial nerve plexus is depleted of fluorescence by V S P 4) N E uptake decreases to about 60% after S A H 5) Selective lesions of the medullary catecholamine nuclei prevent V S P 6) Reduced tyrosine hydroxylase like immunoreactivity occurs in S A H 1) Cerebrovascular smooth muscle is relatively insensitive to a-adrenergic vasoconstrictors 2) Multi-receptor has little effect on V S P 3) Contractility to N E and histamine of vasospatic vessels does not differ from control 4) Phenoxybenzamine does not reserve V S P 5-HT 1) Injection o f 5-HT into the subarachnoid space causes V S P 2) 5-HT metabolism is activated in V S P 3) Phenoxybenzamine prevents the 5HT constriction 4) Pronounced network of 5-HT immunoreactive nerve fibers after S A H 1) Injection of blood and 5-HT into the subarachnoid space evokes only a transient constriction 2) Chronic V S P is insensitive to the 5-HT antagonist (cyproheptadine) 3) No elevation of 5-HT in C S F after S A H 4) Decline of 5-HT levels does not change the severity of V S P 5) Contractility of vasospatic vessels to 5-HT is 29 unchanged 6) Augmented 5-HT constriction in V S P is due to the suppressed release of N O Eicosanoids Prostaglandins 1) Prostaglandins (F 2 a , E 2 , A l , B l , B2) cause constriction 2) Elevation o f different prostaglandins occurs in S A H 3 ) P G I 2 is reduced in V S P 4) Sudoxicam and meclofenamate have a marked inhibitory effect on the development of V S P 1) Prostaglandin synthesis inhibitors are not effective in reversing V S P Thromboxanes 1) Thromboxanes induce vasoconstriction 2) Thromboxane synthetase inhibitors ameliorate V S P 1) Thromboxane A 2 is not increased in spastic vessels Leukotrienes 1) Leukotriene D4 evokes vasoconstriction 2) Intraventricular injection of intermediates of leukotriene induces V S P 1) No detectable changes of leukotrienes in C S F after S A H Endothelin 1) ET-1 , big ET-1 , E C E increased in S A H 2) E T A and E T B receptor m R N A doubled in vasospastic cerebral arteries 3 ) E T receptor binding increased after S A H 4) Inhibitors of E T receptors and E C E retard V S P and inhibit the ischemic damage 1) No elevation of ET-1 in C S F after S A H 2) E T levels (CSF, plasma) does not correlate with development of V S P 3 ) In a double-hemorrhage canine model, the inhibition of ET receptors and E C E fails to significantly affect V S P 4) ET-1 antibody does not reverse V S P Blood and C S F 1) Incubation o f blood-CSF mixture causes cerebral vasoconstriction 2) Chronic V S P occurs after the application of R B C s and the degree of V S P is proportional to volume of R B C mass 3 ) Hemolysate induces potent contraction 4) P R P constriction due to 5-HT release 5) Intracisternal injection of blood lacking R B C s does not produce V S P or narrowing 1) PRP contraction is transient 2) Fresh autologous R B C s resuspended in P R P produces no V S P 3 ) Intracisternal injections of washed R B C s induces no arterial narrowing 6hr after injection 4) Normal clear C S F has no contractile activity 5) Chronic V S P not produced by white blood cells plus PRP Hb 1) OxyHb causes severe chronic V S P 1) Pure human oxyHb dose not produce severe 3 0 -2) OxyHb inhibits production of N O 3) OxyHb stimulates release of endothelin and prostaglandin 4) OxyHb produces other potential vasoconstrictors such as hemin, iron, and bilirubin 5) OxyHb can auto-oxidize to release 0{ and produce OH" 6) OxyHb-induced contraction has similar pharmacology as xanthochromic C S F 7) Hb damages perivascular nerves 8) Hb is synergistic with K + , A T P , 5-HT, fibrin degradation products, and hypoxia 9) Hb increases intracellular C a 2 + 10) Hb present in spastic vessel walls vasospasm in monkeys 2) Most studies use impure Hb in vitro 3) Hb contains endotoxin, stromal proteins, and phospholipids that cause vasoconstriction and inflammation 4) Hb contractile potency is increased after combination with low-molecular-weight components of erythrocytes N O 1) Excessive production of N O causes cellular injury 2) Nitrotyrosin and peroxynitrite contribute to chronic V S P 3) Endothelial cells provide sufficient N O to damage their own cellular function 4) Innervation by NOS-containing nerve fibers 5) Hb vasocontriction occurs via the reduction in N O delivery 6) Inducible N O S is upregulated in V S P 1) N O is a vasodilator Free Radicals Superoxide, Hydroxyl radical 1) L ip id peroxides are elevated in C S F post-SAH 2) S A H causes a marked elevation in CSF uric acid (xanthine oxidase product) 3) Injection xanthine, xanthine oxidase, ferric choride, metHb, iron and E D T A mixture into the cisterna magna produces vasocontriction 4) S A H generates lipid peroxides 5) C S F Glutathione peroxidase increases in V S P 6) Gene transfer of extracellular SOD attenuates 1) SOD and catalase fail to protect against oxyHb-induced V S P 2) Lipid peroxidation may be a result of V S P rather than its cause 3) Inhibitors of lipid peroxidation do not prevent V S P 4) Allopurinal prevents the elevation in uric acid but does not inhibit V S P or vascular damage 31 V S P 7) Some free radical scavengers ameliorate V S P through affecting oxyHb constriction 8) Vitamin E prevents S A H cerebral hypoperfusion Abbreviation: A T P : Adenosine triphosphate; C B F : Cerebral blood flow; C S F : Cerebral spinal fluid; E T : Endothelin; E C E : Endothelin converting enzyme; E D T A : Ethylenediaminetetraacetic acid, H b : Hemoglobin; 5-HT: 5-hydroxytryptamine; metHb: methemoglobin; N O : Nitric oxide; N O S : Nitric oxide synthase; N E : Norepinephrine; O x y H b : Oxyhaemoglobin; P G I 2 : Prostagcyclin; P R P : Platelet-rich plasma; R B C ; Red blood cell; S A H : Subarachnoid hemorrhage; S O D : Superoxide dismutases; V S P : Vasospasm 32 Table 2.1 Comparison of vasoactive factors in different species Mouse Human Monkey Rat Rabbit Canine Feline Tone 20.6±2.4 % 23.4±4.2% (Wallis et al., 1996) 38.0±2.0% (Jarajapu and Knot, 2002) 19.7 ± 2 . 2 % (Ishiguro et al 2002) 13.2% (Harder et al., 1989) ET-1 (+) 8.8±0.07 (+) 8.9 (Pierre, 1999) (+) 8.5 (Hansen-Schwartz et al. 2003a) (+) 8.2 (Petersson et al., 1996) (+) 8.08 (Kaito et a l , 1995) (+) 9.81-10.86 (Saitoetal 1989) P E (+) 6.0± 0.1 Insensitive (Laher and Bevan, 1985) (+) 6.0 (Sasaki,et al, 1985) Small, Unstable contraction (Thorin et al., 1997) (+) 4.8 (Sasaki et al., 1985) U-46619 (+) 7.1±0.1 7.5 (Wallis et al., 1996) (+) 9.4 (Sasaki,et al, 1985) (+) 14.5 (Momoi et al., 2003) (+) 8.9 (Sasaki, et al., 1985) 5-HT (N/A) (+) 7.8 (Conde et al., 1991) (+) 6.8 (McCulloch, 1984) (-) 8.2 (Hansen-Schwartz et al. 2003b) Unstable contraction (Thorin et al., 1997) (+) 8.5 (McCulloch and Edvinsson, 1984) (+) 7.7 (Conde et al., 1991) A C h (-) 7.4±0.2 (-) 5.7 (Tsukahara etal., 1989) (N/A) (Ayajiki et al., 2002) (-) 5.8 (Momoi et al., 2003) (-) 6.3 (Gilbert et al., 2001) (-) 5.3 (Nakagomi et al., 1988) (-) 10.4 (Edvinsson, 1977) B K (-) 8.6±0.2 (-) 7.89 (Wahl et al. (-) (Waiters, 1971) (-) (Dacey et al., 1988) (-) (Whalley, 1983) (+) (Whalley et al., 1987) (-) 6.6 (Wahl etal. 1983) 33 1983) Substance P (-) 10.3±0.7 (-) (Wallis et. al., 1996) (N/A) (Dacey et al., 1988) (-) (Stubbs et al., 1992) (-) 8.9 (Edvinsson et al., 1981) Histamin e (N/A) (-) 7.3 (Ottosson et al., 1988) (-) (Ayajiki et al., 1992) (-) 5.3 (Benedito et al. 1991a) (+) (Gokina and Bevan, 2000) (-) (Kitamura et al., 1995) (-) 5.3 (Edvinsson, 1975) (+): constriction; (-): dilation; (N/A) : no response; Values are -LogEDso 34 A Fig . 2.1. Myogenic tone, of isolated mouse cerebral arteries. (A) A s the intraluminal pressure was increased at 5 minutes intervals, mouse M C A ' s (n=15) contracted spontaneously and achieved a new steady-state diameter. The maximum active constriction was 20.6 + 2.4% at 90mmHg,which was not significantly different from the myogenic constriction that reached a plateau at 50mmHg. (B) Representative trace o f effect of L - N A M E , a N O S inhibitor, and bosentan, an E T A / E T B receptor antagonist, on the myogenical tone mouse M C A . The addition o f L - N A M E ( IOJUM) potentiated pressure-induced contraction (n=4), while the myogenic response of vessels equilibrated with bosentan ( 1 / J M ) was attenuated (n=4). 35 A Fig. 2.2. Agonist-induced constriction of the isolated mouse M C A . (A) Vasoconstriction was induced at concentrations as low as 8 m M and the EC50 o f KC1 was 31.0+ lO.OmM. Maximum constriction (63.3 + 3.3%, n=6) occurred with 66 raM KC1. (B) Concentration response curves for the constrictor effect of three distinct receptor activators studied at 20mmHg. Cumulative concentrations of agonists were applied vessels allowed to reach stabilized constriction. Maximum constriction (47.8 + 4.2%) was evoked by ET-1 (50nM, n=6). P E was able to generate its greatest constriction (45.8 ± 4 . 5 % ) at 50/xM (n=8). U46619 elicited its highest constriction (34.8 ± 3 . 5 % ) at 5 / i M (n=8). The p D 2 values of ET-1 , P E and U46619, were 8.8±0.2M, 6.0± 0 .1M and 7.1±0.1M, respectively. The rank order of efficacy is ET-1 >PE >U46619 (repeated measures A N O V A , p O . O l ) . 36 A lOO-i Log Concentration (M) B Fig. 2.3. Endothelium-dependent vasodilation in mouse M C A . (A) Cumulative concentrations were applied and vessel diameters allowed to reach a maintained value. The greatest vasodilator response (82.4 + 5.1%) occurred with 5pM BK (n=4), while maximal vasodilation with ACh was 58.7 + 6.0% (n=6) and with substance P was 42.9 + 5.3% (n=5). (B) A representative recording shows the inhibitory action of L - N A M E (lpM) to the dilation evoked by BK (O.lnM - lOpM) after pre-constriction with U46619 (lOnM). In the presence of L-NAME, BK-induced vasodilation was greatly attenuated. Maximal vasodilation occurred with SNP (lpM), a directly acting vasodilator that is a NO donor. 37 5 min. w: Wash v: Vasopressin Fig. 2.4. Lack of responses to 5-HT and histamine in the mouse M C A . (A) Trace showing that 5-HT (lOuM) and histamine (lOuM) induced neither constriction, nor dilation in PE (luM) pre-constricted mouse M C A (n=6). (B) L - N A M E (luM) was applied for 15 minutes before addition of PE (luM). 5-HT (lOuM) was not able to constrict the vessel further, whereas vasopressin (0.5uM) generated additional tone (n=6). (C) After washout, L - N A M E (luM) was again incubated for 15 minutes and histamine (lOuM) did not evoked additional tone either, whereas vasopressin (0.5uM) elicited additional vasoconstriction, and SNP (luM) caused maximal dilation (n=6). 38 A Intraluminal Diameter (pin) 50 PSS OCa PSS K C I 20 - 80 m M P E l O n M - lOnM B [K*] 40mM PE 10mVI • B % Constriction vzzzzz Normalized Ratio Fig. 2.5. Simultaneous changes in arterial diameter and changes in the 340/380 fluorescence ratio in mouse M C A loaded with fura-2. (A) After being incubated with the Ca2+-sensitive fluorescent dye, fura-2 A M and pluronic acid, mouse MCAs was challenged with increasing concentrations of KCI (20mM - 80mM, n=5) and PE (lOnM -•- lOuM, n=5). A representative trace shows that a more pronounced rise in the fluorescence ratio occurred with KCl-induced constriction compared to PE. (B) Although PE (lOuM) and KCI (40mM) caused similar constriction (42.3 ± 2.3% vs. 45.8 ± 4.5%, n=5, paired t-test,/?= 0.54), the normalized 340/380 fluorescence ratio was higher in the presence of KCI 40 mM compared to PE 10/iM (0.99 ± 0.003 vs.0.92 ± 0.02,n=5,/K 0.01). 39 A Fig. 2.6. Recordings of smooth muscle membrane potential in mouse M C A . (A) E m was measured in vascular smooth muscle cells ( V S M C s ) of the mouse M C A by inserting 40 microelectrodes. The measurement was done either at rest (PSS) or in the presence o f P E ( I O L I M ) at a transmural pressure of 20mmHg. A sharp negative deflection signaled the entry o f microelectrode into a V S M C , and a sharp positive deflection to OmV indicated microelectrode exit from the recorded cell. (B) Depolarizing effect of stepwise-increases in transmural pressure on smooth muscle Em of mouse M C A . A t low pressure (20mmHg), the Em was significantly more negative (-52.6 ± l .OmV) compared to higher pressures: 40mmHg (-42.3 ± 2.OmV), 60mm Hg (-35.3 ± 2.1mV) and 80mmHg (-37.3 ± 1.8mV). The differences in Em of smooth muscle cells at 60 and 80mmHg were not statistically significant. The numbers in parentheses represent the sample size. (Repeated measures A N O V A with multiple comparisons performed by Bonferroni's test */?<0.01 compared to other pressures, ** /?<0.01 compared to other pressures). 41 [KCI] (mM) 20 40 60 80 5-E, TO C & 2. c 2 st E <o 2 •10--20H -30--40 -50H - 6 0 J (15) (19) (6) (12) * (17) Fig. 2.7. Depolarizing effect of increasing concentrations of K C I on smooth muscle Em o f mouse M C A . The application of different concentrations of K C I shows concentration-dependent depolarization. Mouse M C A smooth muscle cells were significantly more depolarized at K C I 20mM (-38.5 ± 3.3mV, n=12), 4 0 m M (-29.5 ± l . l m V , n =15), 6 0 m M (-30.6 ± 0.9mV, n = 19), and 80mM (-29.0 ± 1.4mV, n = 6) compared to control -52.6 ± 0.9mV(n=25), which was the resting Em o f 5 m M K C I (PSS) at 20mmHg (p<0.01). Moreover, the Em of 2 0 m M K C I had a significant difference compared to that o f higher concentrations o f K C I . The numbers in parentheses represent the sample size. 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