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Role of inducible nitric oxide synthase in the acute activation of murine vascular smooth muscle BK channels… Yakubovich, Natalia 2002

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R O L E O F I N D U C I B L E NITRIC O X I D E S Y N T H A S E IN T H E A C U T E A C T I V A T I O N O F M U R I N E V A S C U L A R S M O O T H M U S C L E B K C H A N N E L S B Y I N T E R N A L L Y A P P L I E D L I P O P O L Y S A C C H A R I D E by N A T A L I A Y A K U B O V I C H B.Sc, Physiology, The University of British Columbia, 1997 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQTJTJ3EMENTS FOR THE DEGREE OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Physiology) We accept this thesis as conforming to the reqmregl_sJ2B49*d THE UNIVERSITY OF BRITISH C O L U M B I A December 2001 © Natalia Yakubovich, 2001 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my deparment or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Physiology The University of British Columbia Vancouver, Canada Date beimtir&MH ABSTRACT The role of inducible nitric oxide synthase (iNOS) in the acute activation of large conductance, Ca2+-activated K + channels (BK channels) by the internally applied Escherichia coli lipopolysaccharide (LPS) was studied in murine vascular smooth muscle cells. Primary cell culture, immunocytochemistry, and patch clamp recording techniques were utilized. Immunocytochemical studies showed that rat cerebrovascular arteries fixed promptly upon donor rat sacrifice stained negative for iNOS. However, within 1.5 h of brain removal, iNOS-like immunoreactivity could be detected in cerebrovascular smooth muscle cells (CVSMCs) enzymatically dispersed from rat cerebral arteries, suggesting rapid induction of this protein during cell isolation. Confocal microscopy demonstrated localization of iNOS-like immunoreactivity in the cytoplasm and under the sarcolemma of rat CVSMCs. LPS was then applied to the cytoplasmic face of inside-out membrane patches excised from rat CVSMCs within 2.5-8 h of donor rat sacrifice. It was found that 50 ug/ml LPS rapidly and reversibly increased the open probability of B K channels in these patches, leaving the single channel conductance unaltered. Kinetic analysis showed that LPS activated B K channels by shortening long-duration channel closures, while having little effect on the average duration of channel openings. Importantly, the acute activation of B K channels by LPS was not altered in the presence of the non-isoform specific NOS inhibitor N^-nitro-L-arginine (L-NNA, 100 uM). Inside-out patch clamp recordings obtained from wild-type mouse aortic smooth muscle cells (ASMCs) revealed B K channels which had large conductance, were activated by elevation in intracellular calcium and upon membrane depolarization, and were blocked by intracellular tetraethylammonium (25 mM). The effects of LPS on these channels were next compared in wild-type and iNOS knockout (iNOS-KO) mice. Cytoplasmic.application of 50 pg/ml LPS acutely activated B K channels in inside-out patches of A S M C membrane derived from iNOS-K O mice, with a degree of potentiation not significantly different from that observed in wild-type mice. These studies establish that internally applied LPS can activate murine vascular smooth muscle B K channels independently of iNOS expression or activity. The mechanism which underlies this novel LPS response remains to be elucidated. iv T A B L E OF C O N T E N T S A B S T R A C T i i T A B L E OF CONTENTS iv LIST OF FIGURES vii LIST OF TABLES viii LIST OF ABBREVIATIONS ix A C K N O W L E D G E M E N T S xi 1. INTRODUCTION 1 1.1 Lipopolysaccharide plays a major role in the pathogenesis of bacterial meningitis and septic shock 1 1.2 The known effects of LPS on mammalian cell function are mediated by endotoxin binding to membrane-bound or soluble receptors, followed by signal transduction. 5 1.3 Tissue responses to LPS involve induction of iNOS, one of the three isoforms of nitric oxide synthases 9 1.4 iNOS induction leads to activation of large conductance, calcium-activated potassium channels 11 1.5 B K channels are important regulators of myogenic tone in vascular smooth muscle 12 1.6 Molecular structure and properties of B K channels 12 1.7 Rationale and hypothesis 16 2. METHODS = 19 2.1 Isolation and culture of murine vascular smooth muscle cells 19 2.1.1 Experimental animals 19 2.1.2 Coverslip preparation 20 2.1.3 Dispersal of rat cerebrovascular smooth muscle cells 21 2.1.4 Preparation of mouse aortic smooth muscle cells (ASMCs) 21 2.1.5 Identification of isolated VSMCs 24 2.2 Immunocytochemical visualization of iNOS 25 2.2.1 Rat brain tissue preparation 25 2.2.2 Immunofluorescence microscopy on isolated rat CVSMCs and rat cerebral arteries 25 2.2.3 Controls 26 2.2.4 Confocal imaging 27 2.3 Electrophysiological studies on B K channels in freshly prepared murine VSMCs 28 2.3.1 Inside-out patch clamp recordings 28 2.3.2 Preparation and application of test agents 32 2.3.3 Data acquisition and analysis 33 3. RESULTS : 35 3.1 The occurrence of iNOS-like immunoreactivity in rat cerebral arteries and in isolated rat cerebrovascular smooth muscle cells 35 3.1.1 Double-labeling of rat brain sections 35 3.1.2 iNOS-like immunoreactivity in isolated rat CVSMCs 35 3.2 Effects of NOS inhibition on the acute activation of rat cerebrovascular smooth muscle B K channels by internally applied LPS 41 3.2.1 Quantitative analysis of B K channel response to LPS in freshly dispersed rat CVSMCs 41 3.2.2 Effects of NOS inhibitor L - N N A on the activation of B K channels by LPS 50 3.3 The effects of LPS on aortic smooth muscle B K channels in wild-type and iNOS knockout mice 53 3.3.1 Biophysical properties of mouse vascular smooth muscle B K channels. 54 3.3.2 B K channel response to LPS in wild-type and iNOS knockout mice 59 4. DISCUSSION 73 vi 4.1 The occurrence of iNOS-like immunoreactivity in rat CVSMCs 73 4.2 Subcellular localization of iNOS in rat cerebrovascular smooth muscle cells 76 4.3 Potentiation of B K channel response to internally applied LPS in freshly dispersed rat CVSMCs 78 4.4 Effects of NOS inhibition on the acute activation of B K channels by LPS in freshly dispersed rat CVSMCs 80 4.5 The B K channel response to LPS in wild-type and iNOS knockout mice 82 4.6 Possible mechanisms of acute activation of B K channels by LPS 84 4.7 Pathophysiological significance of B K channel interactions with endotoxin 85 4.8 Conclusions 86 5. REFERENCES 88 V l l LIST OF FIGURES FIGURE 1. Schematic representation of the structure of bacterial lipopolysaccharide (LPS) 3 2 Schematic illustration of inducible nitric oxide synthase-mediated activation of B K channels following exposure of vascular smooth muscle cells (VSMCs) to LPS 6 3 A diagram of B K channel a- and p-subunits 14 4 Diagram of cerebral arteries on the ventral surface of the rat brain 22 5 Dual-well patch clamp recording set-up 29 6 A transverse section through the rat posterior communicating artery fixed immediately on donor rat sacrifice and double-labeled for smooth muscle a-actin and iNOS 36 7 A rat C V S M C fixed 1.5 h after donor rat sacrifice and double-immunostained for smooth muscle a-actin and iNOS 38 8 Effect of LPS on the activity of rat vascular smooth muscle B K channels studied in the absence and presence of the NOS inhibitor L - N N A 42 9 Effect of 50 u.g/ml LPS on the open probability of B K channels studied in inside-out membrane patches of rat CVSMCs in the absence and presence of L - N N A 44 10 Effects of 50 pg/ml LPS on the kinetics of a single B K channel in an inside-outpatch of rat C V S M C membrane 47 11 Effect of 50 pg/ml LPS on the gating of B K channels studied in inside-out membrane patches of rat CVSMCs in the absence and presence of L - N N A 51 12 Biophysical characterization of single B K channel currents studied in inside-out membrane patches excised from ASMCs of wild-type mice 55 13 Effect of membrane depolarization on the open probability and gating kinetics of single B K channels studied in inside-out membrane patches excised from ASMCs of wild-type mice 57 14 Effect of T E A + on single B K channel currents in an inside-out patch of A S M C membrane derived from a wild-type mouse 60 15 Effects of LPS on the activity of B K channels present in inside-out membrane patches of A S M C membrane derived from wild-type and iNOS-KO mice 62 16 Effect of 50 ug/ml LPS on the open probability of B K channels studied in inside-out patches of A S M C membrane derived from wild-type and iNOS-KO mice 64 17 Effects,of 50 u,g/ml LPS on the kinetics of a single B K channel in an inside-out patch of A S M C membrane derived from a wild-type mouse 67 18 Effect of 50 pg/ml LPS on the gating of B K channels studied in inside-out patches of A S M C membrane derived from wild-type and iNOS-KO mice 69 viii LIST OF T A B L E S T A B L E 1 Effects of 50 ug/ml LPS on the gating kinetics of B K channels studied in inside-out membrane patches of rat CVSMCs in the absence and presence of L - N N A 49 2 Effects of 50 pg/ml LPS on the gating kinetics of B K channels studied in inside-out patches of A S M C membrane derived from wild-type and iNOS-KO mice 71 LIST OF A B B R E V I A T I O N S ASMC(s) aortic smooth muscle cell(s) B H 4 6(R)-5,6,7,8-tetrahydrobiopterin B K channel large conductance, calcium-activated potassium channel B S A bovine serum albumin Ca 2 + i intracellular free calcium [Ca 2 +]i intracellular free calcium concentration CaM calmodulin CD14 CD 14 receptor cGMP cyclic guanosine monophosphate C L S M confocal laser scanning microscopy CVSMC(s) cerebrovascular smooth muscle cell(s) E. coli Escherichia coli eNOS, NOS3 endothelial nitric oxide synthase ES cells/line embryonic stem cells/line F A D flavin adenine dinucleotide F M N flavin mononucleotide GPI glycosyl-phosphatidylinositol gsc single channel conductance GTP guanosine triphosphate H E K cells human epithelial kidney cells I K B inhibitors of kappa B iNOS, NOS2 inducible nitric oxide synthase iNOS-KO mice inducible nitric oxide synthase gene knockout mice KATP channel ATP-sensitive potassium channel LBP lipopolysaccharide-binding protein L - N A M E N^-nitro-L-arginine methyl ester L - N N A Nm-nitro-L-arginine LPS lipopolysaccharide, endotoxin M A P kinase mitogen-activated protein kinase mCD14 membrane-bound CD 14 receptor N A D P H nicotinamide-adenine-dinucleotide phosphate N F - K B nuclear factor kappa B NHS normal horse serum nNOS,NOSl neuronal nitric oxide synthase NO nitric oxide NOS(s) nitric oxide synthase(s) Po channel open probability P A R protease-activated receptor PBS phosphate buffer saline PFA paraformaldehyde P K G cyclic guanosine monophosphate-dependent protein kinase RT-PCR reverse-transcription polymerase chain reaction sCD 14 soluble CD 14 receptor S.E.M. standard error of the mean s-guanylate cyclase soluble guanylate cyclase T E A + tetraethylammonium TX-100 Triton X-100 xCf fast closed time constant x c m medium closed time constant x c s slow closed time constant ^mean open mean channel open time T m e a n c i o s e d mean channel closed time T 0 f fast open time constant x o s slow open time constant VDCC(s) voltage-dependent calcium charrnel(s) V m membrane potential VSMC(s) vascular smooth muscle cell(s) xi A C K N O W L E D G E M E N T S It has taken me a while to complete this study, but looking at my thesis now, it seems like quite an achievement, and it was so worth it!! The unexpected results and decision to use knockouts, the troublesome primary cell culture and finicky patch clamping - these were just some of the challenges I faced. Yet how thrilling it was to have overcome them, to arrive at clear findings, and to finally be here, ready to defend. This moment would not have been possible, however, without the help of my supervisor Dr. David Mathers. His support and guidance throughout my Master's were immeasurable. Thank you, David Mathers, for teaching me the secrets of patch clamping, and for all your help with cell culture and data analysis. Thank you for teaching me how to think and problem solve, and how to write academic papers. Thank you for being available whenever I needed help - 1 don't know of any supervisor who would come out for his students on weekends! Your support and belief in me throughout my academic journey were invaluable. Dr. Alison Buchan was also very involved in the progress and success of my immunostainings. Thank you, Alison, for your tremendous help in solving problems with staining and in interpreting the data. I am very grateful to Valerie Smith and Sue Curtis for taking the time to teach me the techniques of immunostaining, and to Jodene Eldstrom for all her help with the confocal imaging. Many thanks also go to Dr. Steve Kehl for his excellent lectures in electrophysiology, and for his guidance through difficult times. M y Advisory Committee members Dr. Ray Pederson, Dr. Ed Moore and Dr. Ismail Laher deserve special thanks for their useful suggestions and stimulating discussions. I am also grateful to John Sanker, Joe Tay and Jack Lewis for their technical assistance. I would like to thank Dr. Tony Pearson, Dr. Carol-Ann Courneya and all of my friends for checking on me throughout my grad studies. Your care was always felt and always appreciated. Finally, I extend my thanks to my parents and sister Svetlana for their unconditional support and help throughout this endeavour. Your love and continued faith in me allowed me to keep going despite all the obstacles. I move on now to focus on my studies in Medicine, and yet it is kind of sad to say 'good-bye' to Physiology. I will miss all those grad retreats with Ray, C A . and Margaret, all those hikes and murder mysteries, the outings with the Physiology gang, and just hanging out in the lab... Hard to believe, but there will be times when I wish I were back in Physiology, doing another Master's or a Ph.D. (just kidding). Oh well, it is time to move on. 1 1. INTRODUCTION 1.1 Lipopolysaccharide plays a major role in the pathogenesis of bacterial meningitis and septic shock. Despite intensive research and major advances in diagnosis and treatment, bacterial infections remain an important source of morbidity and mortality throughout the world. Two major complications of bacterial infections include meningitis and septic shock, which are associated with a mortality rate of 10-20% and 40-60%, respectively. In United States alone, meningitis causes approximately 100,000 deaths/year, whereas septic shock is responsible for 200,000 deaths annually (Pruitt, 1998; Ingalls et al, 1999; Karima et al, 1999). The absence of effective clinical treatments for meningitis and septic shock reflects the fact that the pathogenesis of these conditions is only partially understood. An important role, however, has been ascribed to excessive stimulation of host immune cells by microbial constituents, leading to the overproduction of inflammatory mediators. These in turn initiate profound vascular changes and tissue injury (Cybulsky et al, 1988; Frei et al, 1993; O'Reilly et al, 1999; Mertineit et al, 2000). In the case of gram-negative bacterial infections, bacterial lipopolysaccharide (LPS, endotoxin) is believed to be of considerable importance in the pathogenesis of both meningitis and septic shock (Cybulsky et al, 1988; Friedland et al, 1993; Tunkel and Scheld, 1993; Karima et al, 1999). LPS is the major glycolipid of the outer membrane of gram-negative bacteria, such as Escherichia coli (E. coli), Salmonella typhimurium and Neisseria meningitidis (Griffiss et al, 1988; Friedland et al, 1993; Garcia-del Portillo et al, 1997). The LPS molecule consists of three major regions: an O-specific chain, a core oligosaccharide, and Lipid A , which anchors the molecule in the outer membrane (Figure 1). The O-specific side chain is composed of repeating oligosaccharide units, and is the most variable and immunogenic region of endotoxin. The core region is a branching oligosaccharide, which is usually phosphorylated and is structurally less variable. Lipid A consists of a phosphorylated disaccharide backbone with bound long-chain fatty acids and is highly conserved among bacterial species. The Lipid A component displays most of the biological activity of the intact LPS molecule (McCabe et al., 1972; Brade et al, 1988; Griffiss et al, 1988; Bayston and Cohen, 1990; Tobias et al, 1999). Following bacterial infection, LPS is released upon lysis of bacterial cells and activates a number of host cell types, including macrophages, endothelial and smooth muscle cells. This leads to the synthesis and release of proinflammatory cytokines, arachidonic acid metabolites, the vasodilatory free radical nitric oxide (NO) and various other mediators (Beutler et al, 1985; Schmidt et al, 1993; Townsend and Scheld, 1993). In bacterial meningitis, these mediators are released in the subarachnoid space and are believed to contribute to the impaired autoregulation and abnormal vasodilation observed in infected patients. The resulting disturbances in cerebral blood flow are believed to account for much of the mortality and morbidity associated with meningitis (Igarashi et al, 1984; Yamashima et al, 1985; Friedland et al, 1993; Beasley and Eldridge, 1994; Mertineit et al, 2000). In sepsis, inflammatory mediators are released systemically in response to an infectious insult. The initial (early phase) cardiovascular response is generally characterized by high cardiac output, low systemic vascular resistance and impaired tissue oxygen utilization, the latter leading to metabolic acidemia. The late phase of septic shock involves impaired myocardial contractility, progressive metabolic acidemia and marked sympathetic adrenergic activation. Low cardiac output, inadequate blood flow distribution and leukocyte-mediated Figure 1 Schematic representation of the structure of bacterial lipopolysaccharide (LPS). The three major regions of the LPS molecule include (i) Lipid A, normally embedded in the outer membrane of the Gram-negative bacteria, (ii) core oligosaccharide, and (iii) a serotype-specific polysaccharide, also known as O-antigen. Different shadings of monosaccharides denote different types of sugars (adapted from Tobias et al., 1999). 4 5 tissue injury are common manifestations of late shock and contribute to the development of multiple organ failure and high mortality (Karima et al., 1999; Mackenzie, 2001). Animal models have demonstrated that similar cardiovascular and inflammatory responses occur in response to both live bacteria and to LPS administration. This suggests that endotoxin plays a major role in the pathogenesis of gram-negative bacterial infections (Cybulsky et al., 1988; Tunkel and Scheld, 1993; O'Reilly et al, 1999). 1.2 The known effects of LPS on mammalian cell function are mediated by endotoxin binding to membrane-bound or soluble receptors, followed by signal transduction. The main effects of LPS on mammalian cells are believed to be mediated via interaction with membrane-bound and soluble LPS receptors. The major cell surface protein which binds LPS is the membrane-bound CD 14 receptor (mCD14), expressed on the surface of monocytes, granulocytes and some B cells (Raetz et al., 1991). mCD14 lacks a transmembrane domain and is anchored via glycosyl-phosphatidylinositol (GPI) membrane protein, although mCD14-mediated responses are not dependent on GPI (Lee et al, 1993). LPS is also known to stimulate CD14-negative cells, such as fibroblasts, endothelial and smooth muscle cells by utilizing soluble CD 14 (sCD14) receptors (see Figure 2), which are released into serum by monocytes (Frey et al, 1992; Durieux et al, 1994; Bufler et ah, 1995; Loppnow et al, 1995). The binding of LPS to CD 14 receptors is markedly enhanced by LPS-binding protein (LBP), found in normal serum (Hailman et al., 1994). The LPS-sCD14 complex subsequently binds to a low affinity surface membrane receptor and induces a complex transduction cascade in target cells (Schletter et al., 1995; Wong et al, 2000). In addition to activating surface membrane 6 Figure 2 Schematic illustration of inducible nitric oxide synthase-mediated activation of B K channels following exposure of vascular smooth muscle cells (VSMCs) to LPS. Extracellular (o) and intracellular (i) sites of the V S M C membrane are shown. LPS binds to soluble CD 14 receptors, a process facilitated by LBP. The LPS-sCD14 complex subsequently binds to V S M C surface receptors and induces expression of inducible nitric oxide synthase (iNOS). iNOS catalyzes NO synthesis, which leads to activation of cGMP-dependent protein kinase (PKG). P K G in turn phosphorylates B K channels, which results in activation of these channels, rapid efflux of K + and relaxation of VSMCs. See text for explanation of abbreviations used (modified from Kirkeboen and Strand, 1999). w < z C3 < X U fa M 8 receptors, LPS has been shown to enter the intracellular compartment of mammalian cells, possibly by means of receptor-mediated endocytosis or via clathrin-coated pits (Kriegsmann et al, 1993; Kharlanova et al, 1994; Detmers et al, 1996; Ghermay et al, 1996; Kitchens and Munford, 1998; Cowan et al, 2001). There is some evidence, however, for the existence of CD14-independent mechanisms of cell activation by LPS. Thus, Haziot et al. (1996) have shown that CD14-deficient mice, although highly resistant to endotoxin shock, still exhibit vasodilatory responses to high concentrations of LPS. Furthermore, these same investigators recently demonstrated that the LPS-induced upregulation of acute-phase proteins, including LBP, fibrinogen and ceruloplasmin, was retained in CD14-deficient mice (Haziot et al, 1998). This suggests the existence of other molecules that recognize LPS, with CD11/CD18 and Toll-like receptors being possible candidates (Ingalls and Golenbock, 1995; Yang et al, 1998; Chow et al, 1999). However, the precise mechanisms of these CD14-independent cellular responses to endotoxin remain to be elucidated. Binding of LPS to CD 14 receptors triggers cell activation via extensive signal transduction, the details of which still need to be fully clarified. Substantial evidence suggests a role for a presently unidentified signal-transducing molecule that interacts with CD 14 at the cell membrane (Blondin et al, 1997; Haziot et al, 1997; Fenton and Golenbock, 1998). Subsequent activation of protein kinase C, tyrosine kinases and mitogen-activated protein (MAP) kinases has been suggested to play an important role in LPS-CD14-mediated signaling (Weinstein et al, 1992; Schumann et al, 1998; Arditi et al, 1995). Activation of this signaling pathway leads to nuclear translocation of the nuclear factor kappa B ( N F - K B ) . N F - K B is normally sequestered in the cytoplasm by association with inhibitory subunits called inhibitors of kappaB (IKB) . Phosphorylation of I K B by I K B kinases leads to ubiquitination and degradation of I K B , unmasking the nuclear localization signal sequence of N F - K B . N F - K B relocates to the nucleus and stimulates transcription of genes important in the inflammatory and immune responses (May and Ghosh, 1998; Sha, 1998; Karima et al, 1999). Mediators such as cytokines, adhesion molecules, acute-phase proteins and an inducible form of nitric oxide synthase (iNOS) are expressed in response to the activation of N F - K B . The resulting host response may include fever, vasodilation, hypotension, leukopenia and even death (Ziegler-Heitbrock, 1995; Liu et al, 1997a). 1.3 Tissue responses to LPS involve induction of iNOS, one of the three isoforms of nitric oxide synthases. Inducible nitric oxide synthase is an important enzyme induced by activated N F - K B in macrophages, neurons, endothelial and vascular smooth muscle cells. iNOS or NOS2 is one of three nitric oxide synthases (NOSs) known to synthetize NO in mammalian cells. The other two isoforms are constitutive neuronal NOS (nNOS or NOS1) and endothelial NOS (eNOS or NOS3). A l l three NOSs are homodimers in their active form, with a molecular weight of 130-160 kDa per monomer, depending on the isoform. Although products of different genes, all NOS isoforms contain binding motifs for the following cofactors: nicotinamide-adenine-dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and 6(R)-5,6,7,8-tetrahydrobiopterin (BH 4) (Nathan, 1992). In addition, all NOSs bind calmodulin (CaM) which is needed for enzymatic activity. Constitutive NOSs (nNOS and eNOS) bind CaM in a Ca2+-dependent manner and are minimally active at resting levels of free 10 C a 2 + in cells ([Ca 2 +]i = 70-100 nM). Inducible NOS binds CaM independently of C a 2 + and displays full activity at resting levels of Ca 2 +j (Cho et al, 1992; Busse and Fleming, 1995). nNOS exists in several splice variants constitutively expressed in various cell types, including neurons, skeletal muscle and pancreatic islet cells. In the brain, nNOS activity has been associated with synaptic plasticity, while in peripheral nerves its activity leads to production of NO, which acts as a neurotransmitter and, for example, relaxes gastrointestinal smooth muscle cells (Bult et al., 1990; Bohme et al., 1991; Bredt et al., 1991; Hope et al., 1991). Unlike nNOS, eNOS is a particulate enzyme found mainly in vascular endothelial cells. eNOS activity is enhanced by membrane shear stress, hypoxia and bradykinin, the latter via an increase in [Ca2+]j following endothelial cell stimulation. NO synthesized by eNOS diffuses into vascular smooth muscle cells and causes vasodilation (Rubanyi et al., 1986; Pohl and Busse, 1989; Fleming et al., 1994; Busse and Fleming, 1995). Inducible NOS is typically expressed in cells under pathophysiological conditions. LPS as well as cytokines, anoxia and ischemia can induce iNOS expression in macrophages, vascular smooth muscle and endothelial cells (Wileman et al., 1995; Iadecola et al., 1995; Clark et al., 1996; Le Cras et al., 1996; Sinz et al, 1999). Regulation of iNOS activity occurs primarily at the transcriptional level (Hecker et al, 1999). However, protein-protein interactions and alternative mRNA splicing have also been reported as iNOS regulation mechanisms (Eissa et al, 1996; Kone and Kuncewicz, 1998; Ratovitski et al, 1999). 11 1.4 iNOS induction leads to activation of large conductance, calcium-activated potassium channels. Once induced, iNOS catalyzes synthesis of NO from the amino acid L-arginine, with the formation of L-citrulline as a by-product (Figure 2). The presence of NO in vascular smooth muscle cells (VSMCs) activates soluble guanylate cyclase (sGC), which converts guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP). cGMP-dependent protein kinase (PKG) is subsequently activated, which in turn phosphorylates a number of target proteins, including large conductance, Ca2+-activated K + channels, also known as B K or maxi-K channels. This results in rapid efflux of potassium ions from VSMCs and membrane hyperpolarization, followed by relaxation of vascular smooth muscle and vasodilation (Robertson et al, 1993; Miyoshi and Nakaya, 1994; Moro et al, 1996; Taguchi et al, 1996). NO is also known to directly activate B K channels in VSMCs (Bolotina et al, 1994). Following exposure to bacterial LPS, NO production is also stimulated in other types of cells, including macrophages, fibroblasts, and endothelial cells (Pang and Hoult, 1997; Noda and Amano, 1997; Radomski et al, 1990). NO-mediated formation of oxygen radicals and stimulated production of eicosanoids account for many of the cytotoxic actions of NO (Salvemini et al, 1993; Beckman et al, 1996). The excessive NO production and B K channel activation partially account for abnormal dilation of systemic and cerebral vessels seen in septic shock and meningitis (Miyoshi and Nakaya, 1994; Greenberg et al, 1995; Hall et al, 1996; Ruetten et al, 1996; Kim et al, 1997; Chen et al, 1999; Mertineit et al, 2000). The important role of iNOS in mediating vascular changes evoked by LPS has been verified in mice with a targeted disruption of the iNOS gene. Carotid artery preparations from iNOS-knockout (iNOS-KO) mice do not develop the LPS-induced hyporesponsiveness to constrictor agents typically 12 seen in vessels from wild-type mice (Gunnett et al, 1998). In addition, LPS produces less hypotension in iNOS-KO mice than is seen in wild-type animals (MacMicking et al., 1995; Nicholson et al, 1999). 1.5 BK channels are important regulators of myogenic tone in vascular smooth muscle. Myogenic tone is an important determinant of blood pressure and plays a major role in the regulation of regional blood flow in both health and disease. Myogenic tone reflects an intrinsic ability of VSMCs to contract when exposed to increases in intravascular pressure and to dilate under converse conditions (Asano et al, 1987; Meininger, 1991). Myogenic tone is highly dependent on Ca influx through voltage-dependent calcium channels (VDCCs), activated upon increases in intravascular pressure. Calcium-induced calcium release from the sarcoplasmic reticulum generates increases in [Ca 2 +]j and leads to activation of B K channels in the V S M C membrane. Efflux of potassium leads to membrane hyperpolarization and deactivation of VDCCs, followed by a decrease in [Ca 2 +]j and inhibition of further vessel contraction (Nelson et al, 1995; Anwer et al, 1993). B K channels, therefore, play a key role in the negative feedback control of vascular smooth muscle tone. 1.6 Molecular structure and properties of BK channels B K channels belong to a large family of calcium-activated potassium channels, all sharing the common feature of being gated by intracellular Ca 2 + . B K channels exhibit a very high single-channel conductance, in the order of 200-300 pS in symmetrical 140 m M K + solutions. They are activated both by membrane depolarization and elevation of cytosolic Ca , both of which increase the probability of finding these channels in the open state (Nelson et al, 1990; McManus, 1991; Kitazono et al, 1995). B K channels have been shown to be highly 13 selective for K + and are specifically blocked by extracellularly applied iberiotoxin (Galvez et al., 1990). B K channels are also blocked by the K + channel blocker tetraethylammonium (TEA + ) , applied at either the intracellular or extracellular membrane face (Akbarali et al., 1990; Kolb, 1990; Wang and Mathers, 1993; Kehl and Wong, 1996). B K channels exist as a complex of two different kinds of subunits, the pore-forming ot-subunits and regulatory P-subunits (Knaus et al., 1994). Alternative splicing of the oc-subunit and the differential expression of several p-subunit subtypes contribute to the functional diversity of B K channels, which are expressed in most mammalian cell types, with the notable exception of cardiac tissues (Butler et al., 1993; Tseng-Crank et al., 1994; Tseng-Crank et al., 1996; Chang et al., 1997). The a-subunit possesses seven transmembrane domains (S0-S6) as well as four cytoplasmic domains (S7-S10) (Figure 3). The pore forming region of the cc-subunit is localized between S5 and S6 domains, and charged residues of S4 are thought to act as a voltage sensor for channel opening in response to voltage (Jan and Jan, 1992; Diaz et al., 1998). The S9-S10 cytoplasmic domains contribute to the calcium sensitivity of B K channels, while the membrane spanning domain SO of the a-subunit seems to be involved in functional coupling to the P-subunit (Wei et al, 1994; Wallner et al, 1996). Although not an obligatory component of all existing B K channels, P-subunits consist of two membrane-spanning regions connected by a large glycosylated extracellular loop (Figure 3). At least four functionally distinct and tissue-specific subtypes of P-subunit are known to exist in mammalian tissues (Chang et al, 1997). The Pi-subunit is highly expressed in vascular smooth muscle and plays a key role in the functional coupling of intracellular calcium increases to B K channel activation. Thus, the Pi-subunit enhances the apparent calcium sensitivity of B K 14 Figure 3 A diagram of B K channel a- and P-subunits. The pore-forming ct-subunit (SO - SIO functional domains) is regulated by a p-subunit which has two transmembrane regions TI and T2, separated by an extracellular loop. N H 2 denotes the amino-terminus of the oc-subunit located extracellularly (o), and COOH denotes the carboxyl-terminus which is intracellular (i). The amino- and carboxyl-termini of the P-subunit are both intracellular (modified from Wallner et al, 1996). 15 16 channels by eliciting a shift in the half-activation voltage of the channel to more hyperpolarized potentials. The Pi-subunit has also been shown to slow B K channel activation and deactivation, reduce channel inactivation, and increase channel sensitivity to block by iberiotoxin (McCobb et al, 1995; McManus et al, 1995; Dworetsky et al, 1996; Wallner et al, 1996; Behrens et al, 2000; Brenner et al, 2000; Nimigean and Magleby, 2000). In contrast, the P2-subunit predominates in endocrine tissues, while P3 and p 4 subunits are highly expressed in the brain tissue. The P3-subunit is known to confer rapid inactivation upon neuronal B K channels (Behrens et al, 2000; Ha et al, 2000). 1.7 Rationale and hypothesis Previous studies performed in our laboratory revealed a novel action of E. coli LPS on B K channels in rat cerebrovascular smooth muscle cells (CVSMCs) (Hoang et al, 1997; Hoang and Mathers, 1998a). These studies employed CVSMCs which were enzymatically dissociated from rat cerebral arteries using 0.1% trypsin and 0.3% collagenase, and maintained at 4°C for 2-4 days prior to patch clamp recordings of B K channel activity. It was found that acute application of 100 pg/ml LPS to the cytoplasmic face of inside-out patches of C V S M C membrane rapidly and reversibly increased the open probability, P 0 of B K channels by some 3-fold (Hoang et al, 1997). This LPS response was potentiated in the presence of the NOS substrate L-arginine (1 uM) and was suppressed by Nw-nitro-L-arginine (L-NNA, 50 uM), an L-arginine analogue which non-selectively inhibits all NOS isoforms (Hoang and Mathers, 1998b). These findings displayed two novel features. Firstly, they showed that LPS can acutely activate B K channels when applied to the cytoplasmic face of the rat C V S M C membrane. 17 Secondly, this novel effect of LPS on B K channels appeared to be dependent on the activity of a NOS-like enzyme. The acute induction of iNOS in isolated membrane patches used in these studies was clearly impossible. However, the novel LPS response could conceivably be mediated by iNOS pre-induced during isolation of rat CVSMCs. Alternatively, the observed LPS response might be mediated by a novel, constitutive NOS-like enzyme which modulates B K channel activity in cerebrovascular smooth muscle cells. To date, however, studies performed in an attempt to detect constitutive NOS in vascular smooth muscle have resulted in conflicting findings, and iNOS is believed to be the major NOS isoform expressed in VSMCs (Fleming et al, 1991; Gabbott and Bacon, 1993; Charpie and Webb, 1993; Cernadas et al, 1998). One further possibility was that the observed sensitivity of the LPS response to NOS inhibitors and substrates reflected actions of these agents at sites other than nitric oxide synthases (Kontos and Wei, 1996; Das et al, 1999). In view of these considerations, the following general hypothesis was proposed. HYPO THESIS: iNOS mediates the acute activation of murine vascular smooth muscle BK channels by internally applied LPS. In the present study, this hypothesis was tested by employing a combination of primary cell culture, immunocytochemistry and electrophysiological methods. The possibility of rapid iNOS induction during isolation of rat CVSMCs was tested by double-immunostaining rat cerebral arteries and freshly dispersed rat CVSMCs for iNOS and smooth muscle-specific ct-actin. As a functional test for possible iNOS involvement in the acute activation of B K channels by LPS, the LPS response in isolated membrane patches of rat CVSMCs was compared in the absence and presence of a competitive NOS inhibitor L-NNA. In view of the limitations posed by the use of NOS inhibitors in studying the functional importance of NOSs, 18 namely limited specificity and uncertainty around the extent of inhibition, iNOS knockout mice were also employed to study the involvement of iNOS in mediating the BK channel response to LPS. Gene knockout provides a complete and selective elimination of a protein of interest, in our case iNOS, and therefore constitutes an excellent model for studying this protein involvement in a particular cellular process. If iNOS activity is indeed essential for the acute activation of BK channels by LPS, this response should be absent in VSMCs derived from iNOS-KO mice. 19 2. METHODS 2.1 Isolation and culture of murine vascular smooth muscle cells 2.1.1 Experimental animals Adult male Wistar rats and wild-type CD-I white male mice were obtained from U B C Animal Care Centre (Vancouver, B.C.). C57BL/6-NOS2 t m l L a u male mice with a homozygous disruption of the NOS2 gene (iNOS knockout, iNOS-KO mice) were purchased from the Jackson Laboratory (Bar Harbor, ME). Wistar rats and control CD-I mice were housed under regular conditions, while iNOS knockout mice were maintained in specific pathogen-free environment as they are known to be more vulnerable to infection (Wei et al., 1995; Mashimo and Goyal, 1999). iNOS knockout mice used in this study were generated in the Jackson Laboratory according to a protocol described in Laubach et al. (1995). Briefly, a recombinant embryonic stem (ES) line was generated by introducing a targeted mutation into the NOS2 gene in stem cells from the 129 mouse strain. Specifically, the region coding for the calmodulin-binding domain of iNOS was replaced with the neomycin resistance gene, with the intent of disrupting the reading frame of the modified gene. Genetically altered ES cells were then injected into blastocysts from the C57BL/6 (B6) mouse strain, and embryos implanted into pseudopregnant B6 females. Chimeric B6/129 mice carrying the altered gene in their germ cells were used to establish a line with homozygous disruption of the NOS2 gene. Subsequently, backcross breeding to the B6 strain was performed over many generations to achieve >95% uniform B6 genetic background in the genetically altered mice. 20 Importantly, Laubach et al. (1995) demonstrated that upon treatment with a lethal dose of LPS, mice with the above described homozygous disruption of iNOS gene displayed no increases in serum nitrite plus nitrate levels (measured with a Griess assay). In addition, peritoneal macrophages from these mutant mice did not produce NO, measured as nitrite in the culture medium, following stimulation with LPS and interferon-y. Although lysates of these cells contained low levels of abnormal iNOS transcripts which were missing the calmodulin-binding domain (Northern analysis), they contained no iNOS protein (immunoblot analysis) or iNOS enzyme activity (radiolabeled L-arginine to L-citrulline conversion in the absence of Ca 2 + , L-citrulline assay) (Laubach et al., 1995). In view of these findings and the routine monitoring of the genetic integrity of mutant mice by the Jackson Laboratory, experiments to demonstrate that vascular smooth muscle cells from C 5 7 B L / 6 - N O S 2 t m l L a u mice used for our experiments were lacking iNOS expression/activity were not performed. 2.1.2 Cover slip preparation 18 mm circular glass coverslips (Fisher Scientific, Nepean, ON) were mounted onto a holder and cleaned in concentrated HNO3 for 30 min at 60°C, then rinsed thoroughly in distilled H 2 O and dry autoclaved for 30 min. The coverslips were subsequently coated overnight at room temperature (21-23°C) with 10 p.g/ml Poly-D-lysine (Sigma, St. Louis, MO) dissolved in 0.15 M borate sterile buffer solution (Na2B 4 O7-10H 2 O; pH 7.4) for better cell attachment. The coverslips were rinsed again and stored in sterile distilled H2O at 21-23°C until use. 21 2.1.3 Dispersal of rat cerebrovascular smooth muscle cells Donor rats (175-250 g) were sacrificed by an overdose of CO2, and the brain carefully removed using aseptic technique. Cerebral arteries, including the anterior cerebral, middle cerebral, posterior cerebral, posterior communicating and basilar arteries and their first and second order branches (Figure 4) were collected from the ventral surface of the brain with the aid of a dissecting microscope. After removal, the arteries were carefully cleaned of connective tissue and blood clots using fine forceps while in an ice-cold sterile solution containing (in mM): 138 NaCl, 4.5 KC1, 0.5 M g C l 2 , 0.33 Na 2 HP0 4 , 10 HEPES, 5.5 dextrose, pH 7.4. The vessels were then incubated for 30 min at 37°C in 0.06% protease with activity of 9.9 units/mg (Type X X I V ; Sigma), 0.05% collagenase with activity of 414 units/mg (Type 1 A ; Sigma) and 0.04%) trypsin inhibitor (Type US; Sigma). This was followed by washing the arteries in an ice-cold maintenance solution containing (in mM): 70 KC1, 70 K O H , 0.5 M g C l 2 , 50 L-glutamic acid, 20 taurine, 5 creatine, 5 pyruvic acid, 1 K 2 H P 0 4 , 0.5 EGTA,10 HEPES, 5 Na 2 ATP, pH 7.4. Single smooth muscle cells were subsequently isolated by gentle trituration of the digested arteries for 7 min with a sterilized Pasteur pipette flamed to give a tip inner diameter of 1.5 mm. Dispersed cells were plated onto poly-D-lysine coated coverslips and left at 25-27°C for 20 min to allow firm attachment to the substrate. CVSMCs were used for immunocytochemical studies or patch clamp recordings immediately, or kept in the above mentioned maintenance solution at 4°C until use. 2.1.4 Preparation of mouse aortic smooth muscle cells (ASMCs) Donor wild-type and iNOS-KO mice (4-12 weeks old) were sacrificed with a C 0 2 overdose, and thoracic aortae were removed using aseptic technique. Aortic smooth muscle 22 Figure 4 Diagram of cerebral vessels on the ventral surface of the rat brain. Arteries (red), veins (green), and cranial nerves II, IV, V are shown. Int car: internal carotid artery (adapted from Greene, 1959). Z 3 b r a n c h of a n l e T i o T c e T e b T a l after L| AdbeTioi c e r e b r a l vein , - o I ad O T LJ 1TQC1 antenoT cerebral artery • middle ceiebial artery •infundibulum cut chorioid a t t e T L j -basal vein •bosWioT cominunicatincj QTICTLJ (posterior ceTebral cnteTLj inferior cerebral vein via superior betrosal sinus to transverse sinus bos i lQT aTteTq buben 'oT cerebellar artery bataflocculai lobe 24 cells were isolated and maintained as described above for rat cerebrovascular smooth muscle cells, except that 0.06% elastase with activity of 1.4 units/mg (Type HA; Sigma) was added during the enzymatic digestion step and digestion was prolonged to 40 min. 2.1.5 Identification of isolated VSMCs Individual rat cerebrovascular or mouse aortic smooth muscle cells used for patch clamp recordings were routinely confirmed as being VSMCs using immunocytochemistry. Following recordings, cells on coverslips were fixed in freshly dissolved ice-cold 4% paraformaldehyde (PFA) for 6 min and washed in phosphate buffer saline (PBS) containing (in mM): 137 NaCl, 3 KC1, 8 Na 2 HP0 4 , 1.5 K H 2 P 0 4 , 2 NaN 3 , pH 7.4. The coverslips were then treated for 30 min at room temperature with a blocking buffer containing 2% bovine serum albumin (BSA) and 0.3% Triton X-100 (TX-100). Incubation in immunoglobulin-free B S A was required to minimize non-specific binding of antisera, and Triton X-100 was used to increase membrane permeability to antibodies. Cells were then incubated for 24 h at 4°C with mouse monoclonal antibodies specific for smooth muscle a-actin (Sigma), diluted to 1:7000 in PBS containing 0.3% TX-100 and 5% heat-inactivated normal horse serum (NHS; Gibco BRL, Burlington, ON). Following incubation, preparations were washed in PBS (3x5 min) to remove excess antibodies. Cells were subsequently incubated for 1.5 h at room temperature with goat anti-mouse, highly cross-adsorbed secondary antibodies (Molecular Probes, Eugene, OR), labeled with Alexa-594 fluorophore and diluted to 1:7000 in PBS with 0.3% TX-100 and 5% NHS. The latter was added to prevent non-specific binding of secondary antibodies. Coverslips were then washed in PBS, inverted onto glass slides using a mounting medium of PBS and glycerine (1:9), and viewed under a Zeiss Axiophot fluorescence microscope. Cells labeled with Alexa-594 red fluorophore were identified as VSMCs. 25 2.2 Immunocytochemical visualization of iNOS 2.2.1 Rat brain tissue preparation Donor rats were sacrificed by C O 2 overdose, and brains were dissected out carefully to minimize damage to cerebral vessels. Brains were fixed within 8 min of animal sacrifice in 4% PFA for 2 h at room temperature. The tissue was washed in PBS (3x10 min) and stored in PBS containing 20% sucrose for 24 h at 4°C to improve tissue preservation. Brains were frozen for 45 s in 2-methyl-butane cooled to -60°C in liquid nitrogen, and then cut into 40 pm thick sections using a cryostat. Brain sections showing transverse views through the posterior communicating artery were placed in wells containing PBS and utilized for immunofluorescence studies. 2.2.2 Immunofluorescence microscopy on isolated rat CVSMCs and rat cerebral arteries Double-immunostaining was used to co-localize iNOS and smooth muscle-specific cc-actin in isolated rat CVSMCs and cross-sections of rat cerebral arteries. Dispersed rat CVSMCs prepared as outlined in Section 2.1.3. were fixed in 4% PFA within 1.5-2 h of donor animal sacrifice and subsequently blocked in 2% B S A and 0.3%> TX-100 in PBS. A mixture of the following primary antibodies was then applied to cells for 24 h at 4°C: monoclonal mouse anti-smooth-muscle-a-actin IgG at a dilution of 1:7000 (Sigma) and polyclonal rabbit anti-iNOS antibodies at a dilution of 1:100 (Transduction Laboratories, Lexington, K Y ) . The anti-smooth muscle cc-actin antibodies were generated against a synthetic decapeptide encoding the N H 2 terminus of smooth muscle-specific a-actin, while anti-iNOS antibodies were raised against a 21 kDa peptide encoding the carboxyl terminus of mouse macrophage iNOS. Preparations were then washed in PBS and incubated for 1.5 h at room temperature with a 26 mixture of the following highly cross-adsorbed secondary antibodies: Alexa-594 labeled goat anti-mouse IgG at a dilution of 1:7000 (Molecular Probes) and Alexa-488 labeled goat anti-rabbit IgG at a dilution of 1:2000 (Molecular Probes). Alexa-594 red fluorophore (with absorption/fluorescence emission maxima of 590 nm/617 nm) was used to visualize smooth muscle cc-actin-like immunoreactivity, while Alexa-488 green fluorophore (with absorption/fluorescence emission maxima of 495 nm/519 nm) detected iNOS-like immunoreactivity. These fluorophores were selected on the basis of a minimum overlap of their emission spectra to minimize bleed-through. Rat brain sections prepared as noted in Section 2.2.1. were blocked in 2% B S A and 0.3% TX-100 for 1 h at room temperature, and double-labeled in a similar fashion. Following immunostaining, floating tissue sections were deposited onto glass Poly-D-lysine coated coverslips and subsequently mounted onto glass slides using a mixture of PBS and glycerine (1:9). Double-labeled rat brain tissue and rat CVSMCs were subsequently viewed using a Zeiss Axiophot fluorescence microscope. Specimens with a high intensity of fluorescence and a low level of background staining were later scanned under a Biorad M R C 600 confocal laser scanning microscope. 2.2.3 Controls Control immunofluorescence experiments on VSMCs and brain sections included single-labeled specimens with assessment for bleed-through as well as incubations with omission of primary antibodies to assess the specificity of secondary antibodies. Highly cross-adsorbed secondary antibodies were selected for the experiments to reduce the possibility of cross-reactivity. Cells and tissue sections were also incubated with mouse anti-smooth muscle a-actin primary antibodies, followed by anti-rabbit secondary antibodies or rabbit anti-iNOS 27 primary followed by anti-mouse secondary antibodies to rule out cross-reactivity between these species. A l l controls were carried out in parallel with the experimental tissues for consistency of the timing of incubations and antibody concentrations. Specificity of mouse anti-smooth-muscle-a-actin antibodies (Sigma) was confirmed by immunostaining Human Epithelial Kidney (HEK) cells, with expected absence of labeling. Specificity of rabbit anti-iNOS antibodies (Transduction Laboratories) was demonstrated by negative labeling of rat cerebral vessels fixed immediately upon removal from donor animals (see Section 3.1.1.). Since the immunogenic peptide used to raise rabbit anti-iNOS antibodies was not available from Transduction Laboratories, antigen-preabsorption studies to confirm antibody specificity were not carried out. The specificity of the anti-iNOS primary antibodies used in this study has been previously documented (Coers et al., 1998). 2.2.4 Confocal imaging Confocal laser scanning microscopy (CLSM) allows collection of a series of X - Y plane images at different Z positions through the thickness of the sample, producing a three dimensional representation of the object. Each X - Y image (an optical section) is produced by scanning a point of laser light across the sample in the X - Y plane and recording the brightness intensity of each point (named a pixel). Then the plane of focus is shifted by a sub-micron increment along Z axis (a Z step, which is set to match pixel size) and the sample is scanned again. The major advantage of confocal microscopy over conventional light microscopy is that it makes it possible to optically section specimens while eliminating most of the out-of-focus light, thereby allowing for examination of thick specimens with high resolution of final images (Laurent etal, 1994). 28 In the present study, double-labeled rat brain sections and rat CVSMCs were scanned using a Bio-Rad M R C 600 microscope equipped with a double dichroic filter set, chosen to select for the appropriate excitation and emission wavelengths of the two fluorophores. Two stacks of X - Y plane images (one stack for each fluorophore) were collected simultaneously for each specimen and then processed using the NTH Image program to produce maximum intensity projections. To generate a single maximum intensity projection from a Z-series of consecutive images, the intensity of a pixel in one X - Y image was compared to the corresponding pixel in the next image, and the pixel with the greatest intensity was retained and used for comparison with the next image. The final maximum intensity projection was a two-dimensional image representing the maximum intensity for each pixel from all the images in the stack. The maximum intensity projections were thus obtained for both Alexa-594 (red) and Alexa-488 (green) fluorophores, and then imported into Adobe Photoshop program for further processing. Red and green images were also merged together to assess for co-localization of labeled molecules within the specimen. 2.3 Electrophysiological studies on BK channels in freshly prepared murine VSMCs 2.3.1 Inside-out patch clamp recordings B K channel activity was recorded using patch clamp recording techniques standard in our laboratory (Wang and Mathers, 1993; Hoang et al, 1997). The recording set-up included a dual-well recording chamber (Figure 5) mounted onto the stage of an inverted, phase-contrast microscope (Olympus CK, Tokyo, Japan, x300 magnification). The microscope was in turn mounted onto an air-suspended table (Kinetic Systems Inc., Boston, M A ) , and along with the 29 Figure 5 Dual-well patch clamp recording set-up. Following formation of a gigaohm seal onto one of dispersed VSMCs in the right-hand compartment, the patch electrode was withdrawn from the cell surface and rapidly transferred through air to the cell-free compartment on the left, filled with solution. The inside-out membrane patch so obtained was then voltage-clamped and single B K channel activity was monitored. A reference electrode (not shown) was placed in the right-hand chamber and the two compartments were connected via an agar-filled salt bridge. The patch in the recording chamber was perfused with test solutions via the inlet and outlet ports, as indicated (adapted from Hoang, 1997). 30 31 perfusion system and the head stage of a List EPC-7 patch clamp amplifier (Medical Systems Corp., Greenvale, NY) was shielded from electromagnetic radiation by a Faraday cage. Patch electrodes were pulled from borosilicate glass (1.5 mm outer diameter x 0.75 mm inner diameter, Frederik Haer Corp, Brunswick, ME) using a two-stage vertical puller (David Kopf 700C, Tujunga, CA) to give tip outer diameter of approximately 1 um. The electrodes were fire-polished just before use to produce a clean and smooth tip, and had a final resistance of 10-15 M f l when filled with experimental solution. Rat CVSMCs and mouse ASMCs prepared as outlined in Sections 2.1.3. and 2.1.4. were normally used for single channel recordings within 2.5-8 h of donor animal sacrifice. For each experiment, a coverslip with dispersed cells was transferred into the larger compartment of the dual-well recording chamber filled with a holding solution containing (in mM): 135 NaCl, 4 KC1, 1.8 CaCl 2 , 1 M g C l 2 , 10 HEPES, 5 dextrose, pH 7.4. A patch electrode was lowered onto a V S M C and a gigaohm seal was formed using standard techniques (Hamill et al., 1981). To form an inside-out patch, the electrode was withdrawn from the cell surface, creating a membrane vesicle at the electrode tip. The electrode was then rapidly transferred through air to the cell-free compartment of the recording chamber and lowered into the experimental solution, resulting in a rupture of the outer component of the membrane vesicle. The extracellular face of the excised membrane patch was exposed to solution A within the patch electrode containing (in mM): 140 KC1, 1.48 CaCl 2 , 10 HEPES, 3 EGTA, pH 7.4 (free calcium concentration 50 nM). The cytoplasmic face of inside-out membrane patches was typically bathed in solution B of composition (in mM): 140 KC1, 2.78 CaCl 2 , 10 HEPES, 3 EGTA, pH 7.4 (free calcium concentration 0.65 uM). Membrane patches were then voltage-clamped to the desired 32 membrane potential V m , which in most cases was +30 mV, and single B K channel currents were recorded at 21-23°C. Free calcium concentration in experimental solutions was calculated using the Max Chelator program (Standford University, CA). A free calcium concentration in solution B on the intracellular side of the membrane, [Ca 2 +]j = 0.65 p M was chosen as it resulted in appropriate open probability of B K channels. The very low free C a 2 + concentration in the electrode solution (50 nM) was selected to prevent activation of B K channels by inward C a 2 + currents during depolarization of membrane patches (Rubart et al., 1996; Marion and Tavalin, 1998). The identity of B K channels in inside-out patches was confirmed by their large conductance (>200 pS in symmetrical 140 m M K + ) , activation on membrane depolarisation at low levels of Ca 2 +j (0.65 uM), and block by 25 mM tetraethylammonium (TEA + , Sigma), a K + channel blocker, applied to the intracellular aspect of the excised patch membrane. The calcium dependency of B K channels was assessed by bathing the patches in solution B containing 5nM Ca 2 + j , with expected suppression of B K channel openings (Brayden and Nelson, 1992; Wang and Mathers, 1993). 2.3.2 Preparation and application of test agents Solutions bathing the cytoplasmic face of isolated patches could be rapidly exchanged for test solutions using a gravity-fed perfusion system. This system consisted of several 60 ml cylindrical reservoirs mounted above the stage of the microscope and connected to the cell-free compartment of the recording chamber via a short length of plastic tubing. The recording compartment contained 1-1.5 ml of solution at any time and the perfusion rate was maintained at about 5 ml/min. Under these conditions, solutions bathing the inside-out membrane patch could be effectively replaced within 30 s. Following perfusion with 10 ml of test solution, 33 perfusion was turned off and B K channel activity was recorded for later analysis. Test agents were washed out by perfusion with 10-15 ml control solution B. For studies on rat and mouse VSMCs, 50 ug/ml LPS, E. coli serotype 0127:B8 (Sigma) was freshly dissolved in solution B and applied to the cytoplasmic face of isolated membrane patches. The patches were also bathed in 5nM free C a 2 + solution B of composition (in mM): 140 KC1, 10 HEPES, 3 EGTA, 0.27 CaCl 2 , pH 7.4 and subsequently, in 100 uM free C a 2 + solution B containing (in mM): 140 KC1, 10 HEPES, 2.68 EGTA, 2.78 CaCl 2 , pH 7.4 to confirm that the currents observed were flowing through Ca2+-dependent K + channels. Finally, 25 m M tetraethylammonium chloride salt (TEA + ) was freshly dissolved in solution B and applied to inside-out membrane patches for the same purpose. In patch clamp experiments on rat CVSMCs involving N^-nitro-L-arginine (L-NNA; Sigma), this agent was first solubilized in 0.1 N HC1 and stored at 4°C as a stock solution. 100 uM L - N N A in holding solution was applied to cells for at least 30 min at 21-23°C prior to excision of membrane patches. The same concentration of inhibitor was present in the recording chamber when monitoring B K channel currents in the presence and absence of LPS. 2.3.3 Data acquisition and analysis Patch current signals were subjected to active filtering (DC-2 kHz, -3 dB Bessel), sampled at 8 kHz and stored on VHS videotape for later analysis using commercial computer software (Instrutech Corporation, NY) . A threshold for event detection was set at 50% of the mean open channel current amplitude. Frequency distributions for channel open times, closed times and current amplitudes were obtained by analyzing 500-1200 channel transitions in each data set. 34 Current amplitude distributions for single B K channels were fitted by Gaussian functions with the mean current amplitude obtained at the peak of the fitted curve. Frequency distributions of channel open and closed times were plotted on a logarithmic time scale. This transformed the exponential function y = A m a x •e"t/T into a curve with peak amplitude, A m a x at the time constant, x, and an area proportional to the number of events in that component (Sigworth and Sine, 1987). Simplex maximization of likelihood was used to fit exponential terms to these distributions (Colquhoun and Sigworth, 1983). The probability, P 0 of finding a single B K channel in the open state was calculated from the relation, P 0 = (Ti + 2-T2 + ... + N-T N ) / N-T.O, where N was the total number of B K channels in the patch, estimated under conditions which strongly favored channel opening. T t o t was the total duration of a recording, and T i , T 2 , . . . T N were the total times when at least 1,2, ... N channels are open. A l l data were expressed as mean ± standard error of the mean (S.E.M.). The parametric A N O V A test followed by the Tukey post-hoc test were used to evaluate differences between experimental groups. Data sets for which a P value was less than 0.05 were considered significantly different. 35 3. RESULTS 3.1 The occurrence of iNOS-like immunoreactivity in rat cerebral arteries and in isolated rat cerebrovascular smooth muscle cells 3.1.1 Double-labeling of rat brain sections To examine the possibility of iNOS expression in vascular smooth muscle, rat brains fixed immediately upon removal from donor animals were double-labeled for iNOS and smooth muscle-specific oc-actin using immunocytochemical methods. Figure 6 illustrates the results of double-labeling of one such specimen, showing a transverse section through the left posterior communicating artery. Shown in each panel is a maximum intensity projection obtained from a stack of consecutive X - Y plane images (pixel size 0.56 pm), taken 0.6 pm apart through the 40 pm thick specimen. Extensive a-actin-like immunoreactivity shown in panel A identified smooth muscle cells in the media of the vessel wall. Panel B shows the same vessel cross-section viewed to detect a green fluorophore identifying iNOS. It can be seen that iNOS-like immunoreactivity was essentially absent in the smooth muscle layer of this vessel specimen. This result was confirmed on examining other double-labeled specimens. It was concluded that iNOS-like immunoreactivity was undetectable in vascular smooth muscle cells fixed immediately upon brain removal from donor animals. 3.1.2 iNOS-like immunoreactivity in isolated rat CVSMCs We next investigated the occurrence of iNOS-like immunoreactivity in enzymatically dispersed rat CVSMCs fixed 1.5 h after donor rat sacrifice and double-labeled for smooth muscle a-actin and iNOS. Shown in Panels A and B of Figure 7 are optical sections through 36 Figure 6 A transverse section through the rat posterior communicating artery fixed immediately on donor rat sacrifice and double-labeled for smooth muscle a-actin and iNOS. A. Extensive labeling for smooth muscle a-actin (Alexa-594, red) was seen in the vessel wall. B. iNOS-like immunoreactivity (Alexa-488, green) was essentially absent. 40 um thick tissue section was scanned using C L S M methods, with subsequent image processing to produce maximum intensity projections shown. 38 Figure 7 A rat C V S M C fixed 1.5 h after donor rat sacrifice and double-immunostained for smooth muscle a-actin and iNOS. A . Smooth muscle a-actin-like immunoreactivity (Alexa-594, red) was present mainly under the sarcolemma. B. Extensive iNOS-like immunoreactivity (Alexa-488, green) was seen throughout the cytoplasm, with sparing of the central nuclear region. C. Overlay of the optical sections seen in A and B from the same Z-level in the cell. Yellow/orange staining in the sub-membrane region of the cell indicated co-localization of the two immunoreactivities. Cells were scanned using C L S M methods. CD 40 one such cell demonstrating ct-actin and iNOS immunostainings. Stacks of X - Y plane images (one for each fluorophore) were collected at 0.2 pm increments through this cell, with a pixel size of 0.28 pm. The two individual X - Y plane images presented in Panels A and B came from . the same Z-level. Panel A illustrates the pattern of smooth muscle a-actin-like immunoreactivity observed, which was mainly under the sarcolemma, with some additional cytosolic staining. Panel B of Figure 7 illustrates iNOS-like immunoreactivity in the same focal plane of the cell. As in most images of CVSMCs examined, extensive cytosolic labeling for iNOS was detected, with the central, nuclear region of the cell remaining unstained. These results indicated that within 1.5 h of donor animal sacrifice, rat CVSMCs displayed iNOS-like immunoreactivity, suggesting that iNOS was rapidly induced during enzymatic dispersion of these cells from intact arteries. To further investigate the pattern of iNOS distribution within single vascular smooth muscle cells, images of iNOS and ct-actin immunostainings taken at the same Z-level in the cell were merged to give a sub-cellular co-localization map of the labeled molecules. Panel C of Figure 7 illustrates the results of overlaying individual X - Y plane images of the two immunostainings for the above mentioned cell. Yellow/orange staining in the periphery of the cell indicated that iNOS and ct-actin immunostainings co-localized within 0.28 pm of each other, as determined by the image pixel size. This was a true co-localization of the two immunoreactivities, because X - Y plane images of the two fluorophores came from the same Z-level in the cell. Hence, the yellow/orange points were a consequence of overlaying red and green pixels in the same focal plane, rather than vertically superimposing pixels from different focal planes one upon the other. The same pattern of co-localization was found in the majority of rat CVSMCs examined, suggesting that iNOS is likely to be present in the actin-rich region 41 under the sarcolemma of vascular smooth muscle cells, and therefore might be present in membrane patches isolated from these cells for patch clamp recordings. 3.2 Effects of NOS inhibition on the acute activation of rat cerebrovascular smooth muscle BK channels by internally applied LPS 3.2.1 Quantitative analysis of BK channel response to LPS in freshly dispersed rat CVSMCs The recordings of B K channel activity employed inside-out membrane patches excised from rat CVSMCs within 2.5-8 h of donor animal sacrifice. 50 pg/ml LPS was applied to the cytoplasmic face of isolated membrane patches and single B K channel currents were monitored. The recorded currents were analyzed to determine the effect of LPS on B K channel open probability (P0), single channel conductance (gsc) and gating kinetics. Figure 8A shows currents flowing through a single B K channel in a membrane patch excised from a C V S M C within 3 h of donor rat sacrifice. This inside-out patch was bathed in symmetrical 140 m M K + and voltage-clamped to a membrane potential V m = -30 mV. On application of 50 pg/ml LPS to the cytoplasmic face of this membrane patch, the open probability of the single B K channel it contained markedly increased. This potentiation was observed within 30 s of the start of LPS perfusion, and was rapidly reversed upon wash-out of endotoxin. The results obtained from a number of experiments of this type are summarized in Figure 9. It was found that LPS increased the open probability of single B K channels, from an average P 0 = 0.047 ± 0.018 to P 0 = 0.569 ± 0.089, which is a 12-fold increase (n = 8 patches, P < 0.05, A N O V A ) . 42 Figure 8 Effect of LPS on the activity of rat vascular smooth muscle B K channels studied in the absence and presence of the NOS inhibitor L-NNA. Both inside-out membrane patches were excised and isolated from CVSMCs within 3 h of donor rat sacrifice. LPS (50 ug/ml) was applied to the intracellular aspect of these membrane patches, voltage-clamped to V m = -30 mV in symmetrical 140 m M K + with [Ca 2 +]j = 0.65 uM. A . Rapid, reversible activation of a single B K channel by LPS in a membrane patch studied in the absence of L-NNA. B. Application of LPS to a second membrane patch studied in the presence of 100 u M L - N N A at the cytoplasmic membrane face also activated the single B K channel it contained. Numbers on the right of the presented traces indicate patch current levels when B K channels are closed (0) or open (1). Recordings were made at a bandwidth of DC-2 kHz. 44 Figure 9 Effect of 50 ug/ml LPS on the open probability of B K channels studied in inside-out membrane patches of rat CVSMCs in the absence and presence of L-NNA. n = 8 patches were studied in the absence of L-NNA, data being obtained before (Control) and during LPS application (LPS). A further n = 6 patches were studied in the continuous presence of 100 uM L-NNA, data being obtained before (L-NNA) and during application of LPS (L-NNA/LPS). * Significantly different from the Control value (P < 0.05, A N O V A ) . ** Significantly different from the L-N N A value, but not significantly different from the LPS value obtained in the absence of L-N N A . A l l data were obtained at V m = -30 mV with [Ca2 +]i = 0.65 uM. 45 46 Recordings of single B K channel currents were further analyzed to determine the effect of LPS on the conductance and kinetic properties of B K channels. It was found that LPS (50 p-g/ml) had no significant effect on the single B K channel conductance, with g s c = 235 ± 23 pS in control solution and 239 ± 21 pS on perfusion with LPS (n = 6 patches, P > 0.05, A N O V A ) . The effect of LPS on the distribution of open times of single B K channels was next examined and is shown in Figure 1 0 A for one of the isolated patches. In both the absence and presence of LPS, B K channel open time distributions were well described by the sum of two exponential terms, that is y = A0{-e'M + Aos-e^™. Here, the fast and slow open time constants x 0f and x o s governed the amplitude terms A 0 f and A o s , respectively. Measurement of these parameters allowed calculation of the mean open time of B K channels using the relation T m e a n open = t 0 f • Aof / (A 0 f + AQS) + tos-Aos/(A 0f + A o s ) . Table 1 shows the results obtained on averaging kinetic parameters from 7 inside-out patches and performing A N O V A analysis to evaluate differences between experimental groups. It can be seen that LPS had no significant effect on the values of x0f, x o s and T m e a n open • It was concluded that LPS did not significantly alter the average duration of B K channel openings. The effect of LPS on the distribution of closed times for single B K channel currents in one inside-out patch is shown in Figure 10B. In both the absence and presence of 50 ug/ml LPS, closed time distributions were well described by the sum of three exponential terms, that is y = A c f e_ t /TCf + Acm-e^ 1 0 " 1 + Acs-e^ 1 ", where xCf, x c m and x c s are fast, medium and slow closed time constants, respectively. Measurement of these parameters allowed calculation of the mean closed time of B K channels as x m e a n closed = fcf -Acf/(A c f + A c m + Ac S ) + x c m - A c m / ( A c f + A c m + Ac S ) + x c s - A c S / ( A c f + Acm + A c s ) . When kinetic parameters for 7 such patches were averaged and Figure 10 Effects of 50 pg/ml LPS on the kinetics of a single B K channel in an inside-out patch of rat C V S M C membrane. The patch was voltage-clamped to V m = -30 mV in [Ca 2 +]j = 0.65 uM. A . Open time distributions obtained in Control (954 channel openings) and LPS-containing solutions (724 openings). Each distribution was well-described by the sum of two exponential terms (smooth curves) using the following fit parameters, defined in the text. Control: x 0 f= 0.12 ms; x o s = 2.4 ms. LPS: x 0 f= 0.16 ms; x o s = 6.8 ms. B. Corresponding closed time distributions from the same recordings used in A . These distributions were well-described by the sum of three exponential terms (smooth curves) using the following parameters. Control (961 closings): x Cf= 0.14 ms; x c m = 3.5 ms; x c s = 47.8 ms. LPS (686 closings): xCf = 0.10 ms; x c m = 0.90 ms; x c s = 5.4 ms. 48 49 Table 1 Effects of 50 p.g/ml LPS on the gating kinetics of B K channels studied in inside-out membrane patches of rat CVSMCs in the absence and presence of L-NNA. Patches were voltage-clamped to V m = -30 mV with [Ca 2 +]j = 0.65 pM. See text for explanation of parameter symbols used. Kinetic parameters were obtained from n = 7 patches untreated with L - N N A (Control versus LPS) and n = 6 patches studied in the presence of 100 p M L - N N A at the cytoplasmic membrane face (L-NNA versus L - N N A + LPS). Values given are mean ± S.E.M. * Significantly different from the corresponding Control value (P < 0.05, A N O V A ) . ** Significantly different from the value in L-NNA, but not significantly different from the LPS value obtained in absence of L-NNA. Parameter (ms) Control LPS L - N N A L - N N A + LPS . x 0f 0.7 ±0 .1 1.0 ±0.2 0.9 ±0.1 1.6 ±0 .5 x o s 9 ±2 .3 14 ±4 .0 12 ±2.5 18 ±3 .3 Tmeanopen 7 ±2.1 12 ± 3.4 10 ±2 .6 14 ± 3.5 T c f 0.7 ±0.2 0.3 ±0.1 0.5 ±0 .1 0.5 ± 0.2 x c m 23 ±6 .8 6 ±2 .9 19 ±9 .0 3 ± 0.7 T C S 350 ± 5 4 64 ± 2 7 * 423 ± 72 31 ± 7 ** Xmeanclosed 119±40 7.2 ± 2.9 * 110 ± 21 4 . 1 ± 2 . 3 * * experimental groups compared using A N O V A , it was found that LPS had no significant effect on the values of T c f and x c r n , as shown in Table 1. However, the time constant governing long-duration closures, x c swas significantly reduced in the presence of LPS, as demonstrated in Table 1 and Figure 11. Consequently, the value of Tmean closed was also reduced (see Table 1). These observations indicated that LPS increased B K channel open probability by accelerating the rate of reopening from long-duration channel closures. 3.2.2 Effects of NOS inhibitor L-NNA on the activation of BK channels by LPS In order to investigate a possible mediating role of iNOS or another NOS-like enzyme in the acute activation of rat cerebrovascular smooth muscle B K channels by LPS, patch clamp experiments were repeated in the presence of a competitive NOS inhibitor L-NNA. This non-isoform specific NOS antagonist is known to inhibit iNOS expressed in LPS-stimulated rat vascular smooth muscle cells with an IC50 in the range of 1-10 uM (Palacios et al., 1996). These experiments employed freshly isolated rat CVSMCs which had been pre-incubated in 100 uM L - N N A for at least 30 min prior to membrane patch excision. The same concentration of L - N N A was present in all solutions applied to the cytoplasmic face of the isolated membrane patches. Figure 8B shows B K channel current in one such inside-out patch, voltage-clamped to V m = -30 mV in symmetrical 140 m M K + solutions. Even in the presence of L-NNA, application of 50 u,g/ml LPS to the intracellular aspect of this membrane patch resulted in rapid and reversible activation of the single B K channel present. This was seen in all 6 patches studied, and the A N O V A analysis confirmed that the magnitude of B K channel potentiation by LPS was unaffected by the presence of the NOS inhibitor L - N N A (Figure 9). 51 Figure 11 Effect of 50 pg/ml LPS on the gating of B K channels studied in inside-out membrane patches of rat CVSMCs in the absence and presence of L-NNA. Shown is the time constant of long-duration channel closures, x c s obtained before (Control) and during LPS application (LPS) in n = 7 patches untreated with L-NNA. A further n = 6 patches were studied in the continuous presence of 100 uM L - N N A at the intracellular membrane face, data being obtained before (L-NNA) and during LPS application (L-NNA/LPS). * Significantly different from the Control value (P < 0.05, A N O V A ) . ** Significantly different from the L - N N A value, but not significantly different from the LPS value obtained in absence of L-NNA. A l l data were obtained at V m = -30 mV with [Ca 2 +]j = 0.65 pM. 53 Specifically, an 11.5-fold increase in B K channel open probability was observed, from P 0 = 0.062 ± 0.018 in L - N N A to P 0 = 0.714 ± 0.078 in LPS and L-NNA. Also of note, pre-incubation in L - N N A did not itself alter the open probability of B K channels (see Figure 9). When measured in the presence of 100 p M L-NNA, no change in B K channel conductance was seen on application of 50 pg/ml LPS, with g s c = 251 ± 23 pS in L - N N A alone, and g s c = 249 ± 22 pS in L - N N A + LPS (n = 4, P > 0.05, A N O V A ) . As shown in Table 1, L-N N A itself had no effect on the values of x0f, x o s and x m e a n open, nor were any changes seen in these parameters on application of 50 pg/ml LPS in the presence of L - N N A (Table 1). Similarly, L - N N A (100 pM) had no effects on xcf, x c m , x c s or x m e a n closed, and most importantly did not significantly alter the effects of LPS on the latter two parameters (Table 1). Even in the presence of L-NNA, LPS increased B K channel open probability by reducing x c s which governed long-duration channel closures, thereby reducing the average duration of the closed state (Table 1, Figure 11). In summary, the presence of the NOS inhibitor L - N N A did not affect the magnitude or the mechanism of B K channel potentiation by LPS. These findings greatly reduced the likelihood of iNOS being involved in the acute activation of rat cerebrovascular smooth muscle B K channels by LPS. 3.3 The effects of LPS on aortic smooth muscle BK channels in wild-type and iNOS knockout mice To further study the putative role of iNOS in the acute response of B K channels to LPS, a second series of patch clamp experiments was carried out on freshly dispersed ASMCs from wild-type and iNOS-KO mice. Although cloned mouse B K channels have been studied extensively (Horrigan et al, 1999; Horrigan and Aldrich, 1999), mouse vascular smooth muscle 54 B K channels received little attention in the past (Drab et al., 1997; Brenner et al., 2000). Therefore, the biophysical properties of these channels were characterized first in wild-type CD-I mice. 3.3.1 Biophysical properties of mouse vascular smooth muscle BK channels Experiments employed inside-out membrane patches excised from ASMCs within 2.5-8 h of donor mouse sacrifice. Single B K channel currents detected in one such patch, voltage-clamped to V m = + 30 mV in symmetrical 140 m M K + solutions are shown in Figure 12A. The calcium dependency of these currents was demonstrated by reversible suppression ofthese currents on reducing free C a 2 + concentration at the cytoplasmic face of the channel ([Ca 2 + ]j) from 0.65 p.M to 5 nM (Figure 12A). The single channel current was then plotted against voltage for mouse B K channels studied in 8 such patches, and the results are shown in Figure 12B. The straight line was fitted to this plot by linear regression and yielded an average single channel conductance, g s c = 218 ± 5 pS and a reversal potential (zero current intercept), E K = +2.3 mV, very close to the expected value of 0 mV in symmetrical 140 m M K + . Mouse B K channel gating was strongly voltage-dependent when examined in 0.65 uM Ca 2 + i , with activation upon membrane depolarization. Figure 13 A shows the single channel activation curve obtained on averaging data from 11 patches. These data were well fit by the smooth curve drawn to the Boltzmann relation P 0 = 1/(1 + e" K ( V m" V i /2 ) ) , where K is a slope factor describing the steepness of voltage-dependence and V1/2 is the membrane potential at which P 0 = 0.5. The best fit was obtained using K = 0.15 mV"1 and V]/2 = +44 mV. Next, mean open time and mean closed time were obtained for mouse B K channels in 8 patches and averages were plotted against membrane potential in Figure 13B. It can be seen 55 Figure 12 Biophysical characterization of single B K channel currents studied in inside-out membrane patches excised from ASMCs of wild-type mice. A . Reversible abolition of B K channel currents on reducing the concentration of free C a 2 + at the intracellular aspect of the patch membrane from [Ca 2 +]j = 0.65 p M to [Ca 2 +]j = 5 nM. This patch was voltage-clamped to V m = +30 mV. Channel closed and channel open current levels are indicated by 0 and 1 respectively. B. Current-voltage relation of single B K channels exposed to symmetrical 140 m M K + solutions. Data were obtained from n = 8 patches. The straight line fitted by linear regression had a slope of 218 pS and a reversal potential of +2.3 mV. A 0.65 p M [Ca 2 +]i -1 - 0 5 n M [Ca 2 +]i 0.65 \sM [Ca 2 +]i I U 10 57 Figure 13 Effect of membrane depolarization on the open probability and gating kinetics of single B K channels studied in inside-out membrane patches excised from ASMCs of wild-type mice. A. Activation curve for single B K channels studied in n = 11 patches in the presence of [Ca ]j = 0.65 pM. The smooth curve is the best-fit Boltzmarm relation, drawn to the equation P 0 = 1/(1 + e K ( V m V i / 2 ) ) with K = 0.15 mV' 1 and \ m = +44 mV. B. Effect of membrane potential on the mean open time (open circles) and mean closed time (closed circles) of single B K channels. Data were obtained from n = 8 patches studied with [Ca 2 +]i = 0.65 pM. OPEN PROBABILITY 1.00-, 0.75-0.65 uM [Ca2+]j 0.50-1J -100 -SO -60 -40 -20 0 20 40 60 80 100 MEMBRANE POTENTIAL (mV) 10000.0-, 1000.0 100.0 10.0 1.0 0.1 o Mean Open Time , i • Mean Closed Time 1* i 5 5 o i -100 -80 -60 -40 -20 0 20 40 60 80 100 MEMBRANE POTENTIAL (mV) 59 that on depolarization, the B K channel open probability increased mainly due to a reduction in the mean channel closed time. However, depolarization also lead to a small increase in the average duration of B K channel openings. Figure 14 demonstrates that mouse B K channels were susceptible to block by T E A + applied to the cytoplasmic face of inside-out membrane patches. All-points histograms of single B K channel currents were obtained from one inside-out patch before, during, and after bath application of 25 m M T E A + . Marked reduction in the mean current amplitude associated with the open state was observed upon application of T E A + , as was expected for a fast type channel blocker known to inhibit B K channels with an IC50 of 20-50 m M when applied intracellularly (Furuya et al, 1989; Kehl and Wong, 1996; Wu et al, 1996). For 5 inside-out patches voltage-clamped to V m = +30 mV, mean single channel current was reduced to 37 ± 7 % of the control value. In summary, the above findings confirmed the identity of B K channels in inside-out membrane patches from mouse ASMCs. . 3.3.2 BK channel response to LPS in wild-type and iNOS knockout mice The effects of LPS on aortic smooth muscle B K channels were next compared in wild-type and iNOS-KO mice. Inside-out membrane patches were excised from freshly dispersed ASMCs and voltage-clamped to V m = +30 mV in symmetrical 140 m M K + solutions. 50 pg/ml LPS was applied to the cytoplasmic face of B K channels and the results obtained are illustrated in Figure 15. It can be seen that LPS profoundly activated B K channels in inside-out patches from both mouse strains. A N O V A analysis demonstrated that LPS significantly increased the P 0 of B K channels derived from both wild-type and iNOS-KO mice, as shown in Figure 16. Moreover, the degree of potentiation in open probability did not differ significantly between the Figure 14 Effect of T E A + on single B K channel currents in an inside-out patch of A S M C membrane derived from a wild-type mouse. All-points histograms of current are shown for one such B K channel before (Control), during (TEA + ) , and after (Wash) application of 25 m M T E A + to the cytoplasmic membrane face. Each histogram contains two peaks (arrows), corresponding to mean current amplitude when the channel is closed (~ 0 pA) and when it is open. Note that T E A + application markedly reduced mean current in the open state. Each histogram is accompanied by a representative trace of B K channel current. In these traces, 0 denotes closed channel current level while 1 denotes current in the open state. A l l data were obtained at V m = +30 mV with [Ca 2 +]i = 0.65 uM. 61 CONTROL COUNTS 100 1—t r JL l -0- *w PI IV rf 25 mM T E A + COUNTS Y 0- J ^ f l i ^ J l v J ^ ^ ~i i i i i i i COUNTS T—i—r—~r* T 1-A 0- ' i V ^ v ^ N ^ 5pA AMPLITUDE (pA) 100 ms 62 Figure 15 Effects of LPS on the activity of B K channels present in inside-out membrane patches of A S M C membrane derived from wild-type and iNOS-KO mice. LPS (50 pg/ml) was applied to the cytoplasmic face of both membrane patches, voltage-clamped to V m = +30 mV in symmetrical 140 m M K + and [Ca 2 +]j = 0.65 pM. A. Rapid, reversible activation of a single B K channel by LPS in an A S M C membrane patch derived from a wild-type mouse. B. Application of LPS to the patch derived from an iNOS-KO mouse also activated the single B K channel it contained. Numbers on the right of the presented traces indicate patch current levels when B K channels are closed (0) or open (1). Bandwidth of recordings was DC-2 kHz. 63 64 Figure 16 Effect of 50 pg/ml LPS on the open probability of B K channels studied in inside-out patches of A S M C membrane derived from wild-type and iNOS-KO mice. Shown are data obtained before (Control) and during application of LPS (LPS) in patches derived from wild-type (left-hand panel, n = 6) and iNOS-KO mice (right-hand panel, n = 5). * Significantly different from the corresponding Control value (P < 0.05, A N O V A ) . ** Significantly different from the corresponding Control value, but not significantly different from the LPS value seen in wild-type mice. A l l data were obtained at V m = +30 mV with [Ca2+]j = 0.65 pM. 65 A)!|iqeqojd uedo 66 two mouse strains, with an average 5-fold increase in P 0 in wild-type mice (n = 6) and an 8-fold increase in iNOS-KO mice (n = 5). Of note, control open probability did not differ significantly between the two strains (P > 0.05, A N O V A ) . The effect of LPS on the conductance of mouse B K channels was then examined. As found previously for rat B K channels, LPS had no significant effect on the conductance of B K channels from either wild-type or iNOS-KO mouse strains. In wild-type mice, g s c values of 207 ± 14 pS and 202 ± 14 pS were obtained in control solution and on perfusion with LPS, respectively (n = 6, P > 0.05, A N O V A ) . In iNOS-KO mice, the respective conductance values were 202 ± 19 pS and 197 ± 19 pS (n = 5, P > 0.05, A N O V A ) . Control values of single channel conductance did not differ between the two strains (P > 0.05, A N O V A ) . Kinetic analysis was performed on single B K channel currents obtained from wild-type mice to determine the effects of LPS on gating parameters. As found previously for rat channels, mouse B K channels displayed open time distributions which were well described by the sum of two exponential terms (Figure 17A). Closed time distributions required the sum of three exponentials in both the presence and absence of LPS (Figure 17B). Again as found in the rat, application of LPS (50 p-g/ml) significantly altered only the time constant governing long-duration channel closures, T c s, decreasing the value of this parameter, and hence lowering the value of x m e a n c i 0 S ed (n = 5, P < 0.05, A N O V A ) . This is illustrated in Figure 18 and Table 2. Time constants x0f, x o s , x m e a n open, xCf, x c m were all unaffected by LPS (Table 2). The gating behaviour of B K channels derived from iNOS knockout mice was qualitatively and quantitatively similar to that seen in channels obtained from wild-type mice (Table 2). Application of LPS to these channels again reduced the value of x c s , lowering 67 Figure 17 Effects of 50 pg/ml LPS on the kinetics of a single B K channel in an inside-out patch of A S M C membrane derived from a wild-type mouse. This patch was voltage-clamped to V m = +30 mV in [Ca ]j = 0.65 pM. A. Open time distributions obtained in Control (532 channel openings) and LPS-containing solutions (791 openings). Each distribution was well-described by the sum of two exponential terms (smooth curves) using the following fit parameters, defined in the text. Control: t 0 f = 3.9 ms; x o s = 13.2 ms. LPS: x 0 f = 1.7 ms; x o s = 6.7 ms. B. Corresponding closed time distributions from the same recordings used in A . These distributions were well-described by the sum of three exponential terms (smooth curves) using the following parameters. Control (522 closings): x C f= 1.1 ms; Xcm 31.2 ms; x c s — 223 ms. LPS (660 closings): x c f = 0.9 ms; x c m = 3.4 ms; x c s = 14.5 ms. 68 Vi PM *- Vi S O O O O Q © o r— <o <o •* <o o l »- - I — i 1 1 1 —r S N O U V A H 3 S H O SMOixvAMasao Vi o Pi H z o u ° S 8 5 SNOiivAHasao SMOixvAHasao PP 69 Figure 18 Effect of 50 p.g/m.1 LPS on the gating of B K channels studied in inside-out patches of A S M C membrane derived from wild-type and iNOS-KO mice. Shown is the time constant of long-duration channel closures, x c s obtained before (Control) and during LPS application (LPS) in patches derived from wild-type (left-hand panel, n = 5) and iNOS-KO mice (n = 5). * Significantly different from the corresponding Control value (P < 0.05, A N O V A ) . ** Significantly different from the corresponding Control value, but not significantly different from the LPS value seen in wild-type mice. A l l data were obtained at V m = +30 mV with [Ca 2 +], = 0.65 uM. 70 (sui) S 0 J ^ 71 Table 2 Effects of 50 pg/ml LPS on the gating kinetics of B K channels studied in inside-out patches of A S M C membrane derived from wild-type and iNOS-KO mice. Patches were voltage-clamped to V m = +30 mV with [Ca 2 +]j = 0.65 pM. See text for explanation of parameter symbols used. Kinetic parameters were obtained in wild-type mice (n = 5 patches) and iNOS-KO mice (n = 5 patches) in absence and presence of LPS (Control versus LPS). Values given are mean ± S.E.M. * Significantly different from the corresponding Control value obtained in absence of LPS (P < 0.05, A N O V A ) . ** Significantly different from the iNOS-KO Control value, but not significantly different from the LPS value obtained in wild-type mice. WILD-TYPE MOUSE iNOS-KO MOUSE Parameter (ms) Control LPS Control LPS 1.0 + 0.3 10+1.2 8 ± 1.2 0.5 ± 0.2 7 + 2.2 31 + 11 ** 9 ±2 .8 ** x0f 1.9 ±0 .4 1.1 ±0 .3 1.0 ±0 .3 T o s 15 ±4.2 12 ±4 .8 7 ±1 .4 Xmean open 12 + 4.0 10 ± 4.3 6 ±1 .6 X c f 0.8 ±0 .1 26 ±7 .4 207 ± 59 Xmean closed 70 ± 15 0.6 ±0 .2 0.8 ±0 .1 11 ±2 .4 30± 13 46 ± 9 * 114 ± 2 0 14 ±2.9 * 91 ± 16 Xmean closed but leaving all other kinetic parameters unchanged (Figure 18, Table 2). It was concluded that the effects of LPS on B K channel open probability and kinetics were quite independent of the expression of iNOS in mouse ASMCs. 73 4. DISCUSSION 4.1 The occurrence of iNOS-like immunoreactivity in rat CVSMCs The present study was designed to investigate the possible mediating role of iNOS in the acute activation of B K channels by LPS in isolated patches of murine vascular smooth muscle cell membrane. We first investigated the occurrence of iNOS in dispersed rat cerebrovascular smooth muscle cells. iNOS-like immunoreactivity was not observed in the smooth muscle cells of cerebral arteries fixed within a few minutes of donor rat sacrifice (see Figure 6). This is in agreement with other studies on cerebral arteries from healthy young rats fixed in situ by perfusion with paraformaldehyde and immunostained for iNOS (Clark et al., 1996; Iadecola et al., 1996). Immunostaining for iNOS is also reported to be absent in uninjured rat aortas, rat mesenteric and pancreatic arteries, as well as pig coronary arteries and rabbit aortas, all tissues being fixed by perfusion in situ or immediately on isolation from donor animals (Al-Mufti et al, 1998; Banning et al, 1999; Behr et al, 1999; Cernadas et al, 1998; Bardell and MacLeod 2001). Furthermore, previous studies have demonstrated the absence of iNOS activity (as measured by L-citrulline assay) and iNOS protein (determined by Western blot) in aortas and mesenteric arteries freshly isolated from young healthy rats (Cernadas et al, 1998; Bardel and MacLeod, 2001). Zheng et al. (1999) showed that iNOS mRNA (assessed by reverse-transcription polymerase chain reaction, RT-PCR), iNOS activity (determined by relaxation of precontracted endothelium-denuded vessels on addition of L-arginine) and iNOS immunostaining were all undetectable in freshly isolated rat aortas. However, within 2-3 h of maintenance of endothelium-denuded aortas in an organ bath, an L-arginine-induced relaxant 74 response became apparent, which reached a maximum at 5 h. This response was abolished in the presence of the iNOS inhibitor aminoguanidine. The appearance of the L-arginine-induced relaxation response coincided with the appearance of iNOS mRNA, as detected by RT-PCR. After 5 h incubation in an organ bath, the smooth muscle layer of these rat aortas also stained positive for iNOS (Zheng et al, 1999). This and other studies indicated that, even though iNOS is undetectable in freshly isolated arteries, iNOS expression and activity can be detected in endothelium-denuded vascular preparations within several hours of incubation in standard physiological solutions to which no exogenous LPS or cytokines have been added (Zehetgruber et al, 1993; K i m et al, 1997; Zheng et al, 1999). In our study, iNOS-like immunoreactivity was detected in dispersed rat cerebrovascular smooth muscle cells within 1.5 h of donor rat sacrifice (see Figure 7), yet it was absent in cerebral arteries fixed minutes after animal sacrifice (Figure 6). This suggests that iNOS was rapidly induced during isolation of CVSMCs from intact arteries. The nature of the factors responsible for this rapid induction of iNOS in rat CVSMCs remains unclear. It is possible that contamination of the bathing media with LPS may play a role in iNOS induction, as previously suggested (Rees et al, 1990). Alternatively, the rapid induction of iNOS in rat CVSMCs may be initiated by the stress and hypoxia which accompany death of animals and removal of blood vessels from the donor rats. Stress activates immediate early genes (Senba and Ueyama, 1997), products of which can bind to the promoter region of the iNOS gene and enhance iNOS expression (Hecker et al, 1999). In addition, hypoxia inducible factor-1 is known to be expressed in cells exposed to hypoxia, which is at least partially responsible for iNOS induction via the hypoxia-responsive element in the iNOS promoter region (Melillo et al, 1995; Jung et al, 2000). These regulatory 75 features may contribute to the protective effect of iNOS expression in minimizing long-term neurological deficits following traumatic brain injury, as it was shown that iNOS-KO mice exhibit greater deficits in cognitive performance following controlled cortical impact than do control mice (Sinz et al, 1999). However, other studies suggest that nNOS activation and sustained overproduction of NO via iNOS expression during cerebral ischemia or traumatic brain injury are neurotoxic, whereas eNOS activation in endothelial cells is neuroprotective, as it leads to vasodilation and increased blood flow to ischemic brain regions (Hara et al, 1996; Lo et al, 1996; Iadecola et al, 1997; Wada et al, 1998). Although trace amounts of LPS, stress and hypoxia might be responsible for iNOS induction during isolation of rat CVSMCs, the appearance of iNOS mRNA and iNOS protein in vascular tissues typically proceeds with a slower time course than that observed in the present study. Thus, a 2-4 h lag exists between the stimulation of rat ASMCs or endothelium-denuded rat aortic rings by LPS and/or cytokines and the appearance of iNOS mRNA, and a 5-6 h lag is seen before the appearance of iNOS protein and enzyme activity (Fleming et al, 1991; Hattori and Gross, 1995; Durante et al, 1996; Kim et al, 1997). Similarly, a 12 h delay has been reported for detection of iNOS mRNA and iNOS enzymatic activity in the brain and iNOS immunoreactivity in cerebral vessels following transient cerebral ischemia in the rat (Iadecola et al, 1996). After traumatic brain injury in rats, brain iNOS mRNA expression was detected within 2-6 h, and iNOS labeling was evident in cerebral vessels at 24 h (Clark et al, 1996; Sinz etal, 1999). However, iNOS mRNA was detected in rat heart tissue as early as 40 min after LPS administration in vivo, peaking at 4-8 h (Liu et al, 1997b). In situ hibridization performed on this heart tissue at 4 h revealed iNOS mRNA localization mainly in the VSMCs of arterioles, 76 but also in myocytes and infiltrated inflammatory cells. Similarly, in rat glomerular mesangial and possibly endothelial cells, iNOS mRNA levels significantly increased 60 min following either in vivo or in vitro administration of LPS, iNOS immunostaining was seen within 2 h of the administration of LPS to rats, and NO generation in plasma and in glomerular preparations significantly increased 2-4 h after the in vivo LPS treatment (Sade et al., 1999). These findings are more consistent with our observations of rapid induction of iNOS in cerebrovascular smooth muscle cells and may also explain the rapid hemodynamic changes observed in septic shock. The rapid iNOS induction in rat CVSMCs could be mediated via rapid initiation of NF-K B activation via degradation of I K B , which has been observed in cytokine-stimulated ASMCs from old rats (Yan et al., 1999). 4.2 Subcellular localization of iNOS in rat cerebrovascular smooth muscle cells iNOS is known to exist both in a free cytoplasmic form and in a post-translationally modified form which is associated with organellar membranes (Vodovotz et al., 1995). Given that freshly dispersed rat CVSMCs stained positive for iNOS immunoreactivity, we next investigated the subcellular localization of iNOS in these cells. Confocal microscopy indicated that iNOS-like immunoreactivity in CVSMCs was predominantly cytoplasmic in nature (see Figure 7B). However, iNOS immunoreactivity was also detected in an actin-rich, sub-sarcolemmal region of the cells, with iNOS and a-actin immunoreactivities being co-localized within 0.28 urn of each other (see Figure 7C). Ultrastructural studies indicate that in VSMCs of LPS-stimulated rats, iNOS is localized primarily in cytoplasmic vesicles and in the sarcoplasmic reticulum (Ishiwata et al., 1997). Peripheral sarcoplasmic reticulum is known to come as close as 10 nm to the sarcolemma of 77 VSMCs (Laporte and Laher, 1997). Therefore, iNOS immunoreactivity observed under the sarcolemma of rat CVSMCs probably reflects iNOS associated with the sarcoplasmic reticulum rather than with the sarcolemma itself. Our findings are in accord with other studies which reported both cytoplasmic and plasma membrane bound pools of iNOS in LPS-stimulated microglia, as well as iNOS localization along plasma membrane, T-tubules, contractile fibers, and membranous organelles in non-stimulated rat cardiac myocytes (Wood et al., 1994; Buchwalow et al., 2001). The enzyme was also shown to localize under the postsynaptic folds of human and rat neuromuscular junctions, that is under the sarcolemma of skeletal muscle fibers (Yangetal., 1997). The presence of iNOS immunoreactivity in the sub-sarcolemmal region of rat CVSMCs employed in our study makes it conceivable that iNOS was present in membrane patches isolated from CVSMCs for patch clamp recordings, even i f iNOS were to be associated with sarcoplasmic reticulum. Fragments of the sarcoplasmic reticulum are often present in inside-out membrane patches excised from VSMCs (Xiong et al, 1992). However, based on our findings it is difficult to say whether sub-sarcolemmal iNOS in CVSMCs is actually in close proximity to the B K channel structural domains. Although iNOS might be present under the sarcolemma of the rat CVSMCs used in the present study, the enzyme is not necessarily functionally active at all times in this cell region. It has been reported that iNOS associates with caveolins in mouse skeletal muscle (Gath et ah, 1999). Caveolins comprise a group of structural scaffolding proteins found in plasmalemmal signal-transducing domains termed caveolae. Since association with caveolins represses eNOS enzyme activity in endothelial cells, similar regulation may hold true for iNOS present in the membrane-bound region (Garcia-Cardena, 1997; Feron et al., 1998; Rizzo et al, 1998). 78 Nevertheless, the presence of iNOS under the sarcolemma of rat CVSMCs opens up the possibility of this enzyme being functionally active in this cell region, and exerting a modulatory effect on membrane-bound proteins and ion channels. 4.3 Potentiation of BK channel response to internally applied LPS in freshly dispersed rat CVSMCs Since iNOS may indeed be present at the cytoplasmic aspect of isolated membrane patches, we next investigated its potential role in the acute activation of B K channels by internally applied LPS. When rat CVSMCs were dispersed using 0.1% trypsin and 0.3%> collagenase and maintained at 4°C for 2-4 days prior to patch clamp recordings (Protocol 1), application of 100 pg/ml LPS to the cytoplasmic aspect of excised membrane patches resulted in a 3-fold increase in B K channel open probability (Hoang et al., 1997; Hoang and Mathers, 1998a). In the present study, using rat CVSMCs freshly isolated from cerebral arteries by means of 0.05%> collagenase, 0.06%> protease and 0.04%> trypsin inhibitor (Protocol 2), the open probability of B K channels increased 12-fold on application of 50 pg/ml LPS (see Figure 9). These results demonstrated that the response of B K channels to internally applied LPS was not dependent on a protracted period of cell culture. Given the apparent potentiation in the LPS response with the change in protocol used for cell isolation, we next explored whether the mechanism of modulation of B K channel activity by LPS had been altered as well. Analysis of B K channel gating kinetics showed that in membrane patches isolated from freshly dispersed rat CVSMCs, LPS facilitated channel opening by shortening long-duration channel closures, while having no significant effect on mean channel open time (see Table 1 and Figure 11). This was similar to findings in rat CVSMCs prepared according to Protocol 1 79 (Hoang and Mathers, 1998a). Similarly, LPS did not change the conductance of single B K channels in cells isolated using either of the protocols. It was concluded that, although the B K channel response to LPS was potentiated in CVSMCs isolated using Protocol 2, this response remained kinetically similar to that seen in cells prepared using Protocol 1. Preliminary studies on rat CVSMCs isolated using-Protocol 2 but maintained at 4°C for 3 days prior to recordings revealed an LPS response similar in magnitude to that seen in freshly dispersed cells. This indicated that the loss of sensitivity of B K channels to LPS seen with Protocol 1 was not simply due to the prolonged period of cell maintenance in vitro prior to patch clamp recordings. A major difference between the two methods of cell isolation was the use of 0.1% trypsin for cell dispersion in Protocol 1 (Hoang et al., 1997). Trypsin is known to activate protease-activated receptor-2 (PAR-2), which belongs to a family of G-protein-coupled receptors. These P A R receptors require proteolytic cleavage to be self-activated by a newly exposed, tethered ligand sequence within the amino-terminal of the receptor itself (Glusa et al., 1997). A number of studies have demonstrated an NO-mediated relaxation of precontracted arteries in response to trypsin, this effect being dependent on the presence of endothelium (Glusa et ai, 1997; Hamilton et al, 1998; Sobey and Cocks, 1998). In VSMCs, trypsin has 2"i* also been shown to induce mitogenesis, activate N F - K B , and increase intracellular free Ca concentration, the latter leading to contraction of endothelium-denuded arteries (Bono et al, 1997; Komuro et al, 1997; Moffatt and Cocks, 1998; Bretschneider et al, 1999). It is possible, therefore, that trypsin used for dispersion of rat CVSMCs in Protocol 1 activated PAR-2 receptors on these cells and somehow changed cell function, leading to a diminished B K channel response to LPS. 80 Alternatively, trypsin could have digested some residues on the channel protein itself and altered its function and sensitivity to LPS. Incubation with intracellular trypsin, for example, abolishes B K channel inactivation in rat adrenal chromaffin cells, and reduces intracellular calcium sensitivity of B K channels derived from rabbit colonocytes (Salomao et al., 1992; Solaro and Lingle, 1992). Trypsin is also known to contaminate many commercial enzyme preparations including collagenase and protease. Therefore, we cannot entirely discount possible effects of trypsin on PAR-2 receptors or B K channels in the present study. However, trypsin inhibitor was routinely added during the enzymatic digestion of rat CVSMCs to minimize this possibility. 4.4 Effects of NOS inhibition on the acute activation of BK channels by LPS in freshly dispersed rat CVSMCs In marked contrast to earlier results obtained from cells prepared using Protocol 1 (Hoang and Mathers, 1998b), the LPS response in freshly isolated CVSMCs prepared according to Protocol 2 was not altered in the presence of the non-isoform specific NOS inhibitor, L-N N A , even at a concentration of 100 uM (see Figures 9, 11 and Table 1). This new observation suggested that activation of any NOS isoform, including iNOS, is not required for the acute stimulation of B K channels by endotoxin. If iNOS activity were indeed essential for the acute stimulation of B K channels by internally applied LPS, the LPS response should be always suppressed by NOS inhibitors, regardless of the protocol used for cell isolation. It remains unclear why in rat CVSMCs prepared using Protocol 1, the LPS response developed a marked sensitivity to block by NOS inhibitors. There is growing evidence that some types of VSMCs express constitutive as well as inducible forms of NOS. Thus the 81 neuronal isoform of NOS (nNOS) has been detected by immunocytochemistry and by Western blot methods in the adventitia and media of rat aorta, and in the media of bovine carotid arteries and hamster systemic arteries (Schwarz et al, 1999; Segal et al, 1999; Brophy et al, 2000). Human VSMCs harvested during abdominal surgery also express a constitutive isoform of NOS of the endothelial type, detected by Northern blot hybridization (Trovati et al, 1999). It has been also shown that nNOS and eNOS readily form plasma membrane-associated complexes with cytoskeletal and transmembrane proteins (Brenman et al, 1996; McDonald et al, 1997; Feron et al, 1998). Assemblies of this type, formed in rat CVSMCs following isolation according to Protocol 1, might account for the sensitivity of the LPS response to NOS inhibitors seen in this preparation. It has also been shown that L-arginine analogues, including L-NNA, have cellular effects unrelated to competitive inhibition of nitric oxide synthases. L - N N A (250 pM) blocked dilation of cat pial arterioles in response to hypercapnia or ATP-sensitive K + (K A TP ) channel agonists such as pinacidil, and these effects were reversed by L-arginine (Kontos and Wei, 1996). Since pinacidil and hypercapnia dilated arterioles independently of activation of soluble guanylate cyclase, the authors suggested that arginine analogues modulate activity of K A T P channels independently of increased NO production, possibly via an arginine site on the channels themselves (Kontos and Wei, 1996). Kontos and Wei (1998) further showed that in cat cerebral arterioles, L-arginine binding, possibly to the sulfonylurea receptor, was essential for K A T P channel opening by agonists such as pinacidil. The L-arginine analogue and NOS inhibitor Nffl-nitro-L-arginine methyl ester, L - N A M E reversed M g 2 + - or Ni 2 +-induced relaxation of endothelium-denuded precontracted rat aortic rings (Das et al, 1999). This effect was unrelated to inhibition of iNOS as it persisted 82 following pre-incubation of aortic rings with the iNOS inhibitor aminoguanidine. It was suggested that L - N A M E acts directly on some vascular smooth muscle plasma membrane protein, affecting its reactivity to divalent cations and hence reversing the relaxation of ASMCs (Das et al., 1999). Therefore, in studies performed in our laboratory, L - N N A may have acted via an NOS-independent membrane protein and thereby modulated the B K channel response to LPS under certain conditions of isolation or maintenance of rat CVSMCs. Alternatively, the trypsin used for cell isolation in Protocol 1 might have altered the function of CVSMCs by interacting with PAR-2 receptors or with B K channels themselves, rendering B K channel responses sensitive to NOS inhibitors (Solaro and Lingle, 1992; Bono et al., 1997; Moffatt and Cocks, 1998). 4.5 The BK channel response to LPS in wild-type and iNOS knockout mice To further examine the role of iNOS in the acute activation of B K channels by internally applied LPS, the LPS response was compared in wild-type and iNOS knockout mice. The biophysical properties of native B K channels were characterized firstly in aortic smooth muscle cells obtained from wild-type mice. These channels were shown to have a large conductance (218 pS in symmetrical 140 m M K+solutions), were activated by high [Ca 2 +]j and upon membrane depolarization, and were blocked by intracellular T E A + (25 mM) (see Figures 12, 13, 14). Brenner et al. (2000) also reported the C a 2 + and voltage dependence of B K channels in mouse CVSMCs. Drab et al. (1997) detected B K channels in differentiating mouse VSMCs, which were activated by C a 2 + and blocked by iberiotoxin. Single B K channels in mouse ASMCs described in our study had similar biophysical properties to B K channels in vascular smooth muscle cells of the rat (Wang and Mathers, 1993; Wang and Wu, 1997) and the rabbit 83 (Morales et al., 1996; Snetkov et al., 1996). These mouse aortic smooth muscle B K channels displayed conductance and C a 2 + and voltage sensitivity similar to those of single B K channels in mouse cortical neurons and skeletal muscle fibers (Liu et al., 1999; Mallouk et al., 2000). Next, the B K channel response to LPS in mouse ASMCs was investigated. The response appeared smaller in magnitude but was kinetically similar to that seen in rat CVSMCs. Thus, in wild-type mice, application of 50 pg/ml LPS to the cytoplasmic face of inside-out membrane patches of ASMCs resulted in a 5-fold increase in B K channel open probability (see Figure 16). This response involved the facilitation of channel reopening from long-duration closures (see Table 2 and Figure 18). Moreover, LPS was also found to rapidly activate B K channels in patches of A S M C membrane derived from iNOS-KO mice, with a degree and mechanism of potentiation not significantly different from that seen in wild-type mice (see Figures 16, 18 and Table 2). Taken together, the unaltered LPS response in wild-type and iNOS-KO mice and the absence of inhibition of LPS response in rat CVSMCs by the NOS inhibitor L-NNA, these data provide clear evidence that neither the expression nor the activation of iNOS is a prerequisite for the acute stimulation of B K channels by endotoxin. It should be noted, however, that even though mice lacking in expression of selected genes are excellent models for establishing the functional importance of a particular gene product, there are still limitations in the use of transgenic animals for experimental studies. In our case, there might have been a developmental compensation in the iNOS-KO mice which occurred in response to deletion of the iNOS gene. There could have been another gene product which was upregulated to replace the function of the iNOS gene, and as a result, no change in LPS response was observed between wild-type and iNOS-KO mice (Meng et al., 1998; Godecke and Schrader, 2000). 84 4.6 Possible mechanisms of acute activation of BK channels by LPS The present results do not support the hypothesis that iNOS plays a mediating role in the acute activation of murine vascular smooth muscle B K channels by internally applied LPS. This means that LPS can activate these channels by a mechanism independent of the well-established iNOS-mediated pathway in vascular smooth muscle cells. At present, the nature of this novel mechanism remains unclear. However, the fact that LPS exerts its effects rapidly when applied to the cytoplasmic membrane surface suggests that it acts either on cytoplasmic or membrane spanning domains of the B K channel subunits or on some sub-sarcolemmal proteins modulating the function of these channels. LPS, for example, might enhance B K channel sensitivity to intracellular calcium, either by facilitating the interaction between a and P i channel subunits or by acting on the calcium-sensing S9-S10 cytoplasmic domain of the a-subunit (Wei et al., 1994, Brenner et al., 2000; Nimigean and Magleby, 2000). This seems plausible given that intracellular C a 2 + activates B K channels by shortening long duration channel closures (Wang and Mathers, 1993). Similar changes in B K channel gating were observed in our experiments upon application of LPS to the cytoplasmic membrane face. Since vascular smooth muscle B K channels are also subject to modulation by protein kinases, an alternative mechanism of LPS action may be an altered interaction between a protein kinase and the B K channel protein (Taniguchi et al., 1993; Minami et al., 1993; Esguerra et al., 1994; Wang et al, 1999). LPS, for example, is known to activate microtubule-associated protein kinases in in vitro preparations (Ding et al., 1993). This possibility can be tested using known inhibitors of protein kinases (Xiong et al., 1995). 85 4.7 Pathophysiological significance of BK channel interactions with endotoxin Since iNOS-KO mice remain susceptible to hypotension and death following LPS administration, important iNOS-independent mechanisms of LPS toxicity clearly exist (Laubach et al, 1995; MacMicking et al, 1995; Parratt, 1997; Cobb et al, 1999; Hollenberg et al, 2000). Apart from the induction of expression of various mediators, LPS may have more immediate effects on target cells, for example, via an interaction with ion channels. Extracellular LPS (0.01-1.0 ng/ml) has been shown to enhance whole-cell L-type Ca channel currents in rat VSMCs, within 78 s of application to the bath (Wilkinson et al, 1996). Yet, LPS (1-100 ng/ml) added to the bath solution led to inhibition of the L-type C a 2 + channel currents in cell-attached membrane patches of rat pheochromocytoma cells, an effect which required 3.5 min (Simard et al, 1996). Blunck et al (2001) recently demonstrated that extracellular LPS (2-20 ng/ml) activates single B K channels in outside-out membrane patches of human macrophages. This effect was dependent on the availability of membrane-bound CD 14 receptors for LPS and was strongly sensitive to the conformation of Lipid A . Furthermore, activation of B K channels was essential for LPS-induced cytokine production by these macrophages, since cytokine production was strongly inhibited by the selective B K channel blocker paxilline and by the nonselective K + channel blockers quinine and T E A + . It was concluded that the interaction between mCD14, LPS and the B K channel comprises an early and important step in transmembrane signaling in these cells (Blunck et al, 2001). LPS has been shown to enter intracellular compartments of target cells both in vitro and in vivo (Whiteley et al, 1990; Kharlanova et al, 1994; Detmers et al, 1996; Ghermay et al, 1996; Kitchens and Munford, 1998; Cowan et al, 2001). For example, LPS (0.01-1.0 pg/ml) was rapidly endocytosed in cultured rat cardiomyocytes, followed by association of endotoxin 86 with the Golgi complex, endosomes, lysosomes, sarcomeres and the plasma membrane (Cowan et al, 2000; Cowan et ah, 2001). LPS internalization was independent of CD14 receptor binding, and was shown to be obligatory for activation of endotoxin-dependent signal transduction in cardiomyocytes, since inhibition of endocytosis attenuated N F - K B activation, TNF-ot production and iNOS expression in these cells. The importance of endotoxin internalization in transduction of LPS signals within target cells has been also demonstrated in human neutrophils and rat Kupffer cells (Detmers et al., 1996; Lichtman et al., 1996). LPS internalization has been reported to occur in vascular endothelial cells following intravenous injection of endotoxin (Whiteley et al., 1990; Kang et ah, 1995), and may very well occur in VSMCs during endotoxemia. The mean cytosolic concentration of LPS is unlikely to reach 50 u,g/ml in cells exposed to LPS in vivo. However, internalized endotoxin can form dense intracellular aggregates when taken up by mammalian cells (Kitchens and Munford, 1998; Vasselon et al., 1999; Cowan et al., 2001). Therefore, high local concentrations of endotoxin may arise within target cells, with possible intracellular effects of LPS on the function of ion channels and other proteins. This may constitute another mechanism of LPS toxicity in vivo, and contribute to the hemodynamic disturbances seen in septic shock and meningitis. 4.8 Conclusions In summary, the present study provides strong experimental evidence that iNOS is not involved in the acute activation of murine vascular smooth muscle B K channels by internally applied LPS. Our findings contribute to understanding the newly discovered interactions between LPS and ion channels and the role of these interactions in endotoxemia. This study, 87 therefore, has the potential to facilitate the development of novel agents aimed at alleviating cardiovascular disturbances seen in meningitis and septic shock. 88 5. REFERENCES Akbarali, FL, Nakajima, T., Wyse, D.G. and Giles, W. (1990). Ca2+~activated K + currents in smooth muscle. Canadian Journal of Physiology & Pharmacology, 68(11): 1489-94. Al-Mufti, R.A., Williamson, R.C. and Mathie, R.T. (1998). 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