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If the standard error associated with the Km value is large (>25%) and/or if the Km value falls outside the range of substrate concentrations studied discount 50mg acarbose overnight delivery diabetic diet livestrong, it is prudent to repeat this exper- iment with a new range of substrate concentrations that bracket the estimated Km value purchase 25 mg acarbose otc diabetes symptoms for dogs. When the Eadie-Hofstee plot suggests the involvement of two kinetically distinct enzymes in the formation of a particular metabolite discount acarbose 25 mg online diabetes pregnancy signs symptoms, the data should be fitted to a dual-enzyme model according to the following equation: Vmax1 Á ½ŠS Vmax2 Á ½ŠS vtotal ¼ v1 þ v2 ¼ þ ð8Þ Km1 þ ½ŠS Km2 þ ½ŠS where vtotal is the overall rate of metabolite formation at substrate [S] cheap acarbose 25 mg with mastercard dr oz diabetes diet video, Vmax1 and Vmax2 are the maximal velocities of the reaction, and Km1 and Km2 are the Michaelis-Menten constants for enzyme 1 and enzyme 2, respectively. For simplicity, the following discussion assumes that enzyme 1 is the high-affinity (low-Km) enzyme and that enzyme 2 is the low-affinity (high-Km) enzyme. It further assumes that Km1 and Km2 differ by at least an order of magnitude and that the range of substrate concentrations extended well below Km1 and up to or above Km2. Under such conditions, enzyme 2, the high-Km enzyme, contributes negligibly to vtotal at low substrate concentrations, and the range of substrate concentrations where this is largely true can be identified by visual inspection of the Eadie-Hofstee plot; (Fig. These “enzyme 1” data are plotted on an Eadie-Hofstee plot to obtain Km1 and Vmax1. Subsequently, v2 (which equals vtotal À v1) is calculated, and the data are plotted on an Eadie-Hofstee plot to obtain Km2 and Vmax2. When Km1 and Km2 differ by less than an order of magnitude, or when the range of substrate concentrations does not bracket both Km1 and Km2, it may not be possible to determine the kinetic constants of the individual enzymes. Two enzymes with similar Km values toward the same substrate have frequently been observed, and these will result in an Eadie-Hofstee plot consistent with single-enzyme kinetics. Applying the dual-enzyme model for such situations will not help; instead, reaction-phenotyping data must be used to tease out the role of the two enzymes. These result in an S-shaped curve on a (substrate) versus rate graph and a “hook”- shaped line graph on an Eadie-Hofstee plot. When allosteric interactions are In Vitro Study of Drug-Metabolizing Enzymes 323 Figure 23 Depictions of a reaction catalyzed by two kinetically distinct enzymes. The- oretical data illustrate the method used to determine the kinetic constants when two enzymes are involved in the same reaction. Note that the direct plot (left) does not effectively indicate that two enzymes might be involved in a given reaction. However, this is readily achieved by a concave-appearing Eadie-Hofstee plot (middle graph). The kinetic constants (Km and Vmax) of the high-affinity (low-Km) enzyme are determined using the initial rates observed at low substrate concentrations (solid line in the middle graph). Then, the contribution of the low-Km enzyme is subtracted and the kinetic constants for the high- Km enzyme are determined (dotted line in the middle graph). It is evident that the relative contribution of the high-Km enzyme increases (and that of the low-Km enzymes decreases) as the substrate concentration is increased. The Hill equation is: n Vmax Á ½ŠS v ¼ n ð9Þ S50 þ ½ŠS where S50 is analogous to (but not identical to) Km (i. When n is greater than 1, it indicates positive cooperativity (substrate activation); when n is less than 1, it indicates negative cooperativity (substrate inhibition) (109). A Hill coefficient of 2 implies the presence of two discrete (nonoverlapping) substrate-binding sites on the enzyme, whereas a Hill coefficient of, say, 1. Correlation Analysis: Sample-to-Sample Variation in the Metabolism of the Drug Candidate Correlation analysis is one of the four basic approaches to reaction phenotyping. The experimental conditions for examining the in vitro metabolism of the drug candidate by a bank of human liver microsomes are based on the results of experiments described in Steps 2 and 3 (i. In order to obtain clinically relevant results, the metabolism of the drug candidate by human liver microsomes must be examined at pharmacologically relevant concentrations of the drug candidate, as illustrated for lansoprazole 5-hydroxylation in Figure 20. In our laboratory, reaction phenotyping is carried out with a bank of human liver microsomal samples (e. Banks of human liver microsomes intended for correlation analysis are commercially available as kits (e. The latter determination also provides a measure of the statistical significance of any correlations. Correlation analysis provides valuable information on the extent to which the metabolism of a drug candidate will potentially vary from one subject to the next (i. However, when two or more enzymes contribute to metabolite formation, corre- lation analysis may lack the statistical power to establish the identity of each enzyme. Statistically significant correlations should always be confirmed with a visual inspection of the graph because there are two situations that can produce a misleadingly high correlation coefficient: (1) the regression line does not pass through or near the origin and (2) there is an outlying data point that skews the correlation analysis, as illustrated in Figure 25. Correlation analysis works particularly well when a single enzyme dominates the formation of a particular metabolite. This approach success- fully identifies the enzymes involved when each enzyme contributes 25% or more to metabolite formation, but it will likely not identify an enzyme that contributes only approximately 10%. A graphical representation of the application of multivariate analysis to the results of a reaction phenotyping experiment is shown in Figure 26, on the basis of an examination of the sample-to-sample variation in the 1-hydroxylation of bufuralol (12 mM) by a panel of human liver microsomes. The sample-to-sample variation in bufuralol 1-hydroxylation correlates reasonably well with In Vitro Study of Drug-Metabolizing Enzymes 327 Figure 25 Common pitfalls in correlation analysis. Correlation analysis is suspected when the regression line is unduly affected by a single outlying data point, or when the regression line does not pass near the origin. When two enzymes contribute significantly to metabolite formation, their identity and relative con- tribution can be established by performing correlation analysis in the presence and absence of an inhibitor of one of the participating enzymes (preferably the major contributor). This approach works even when one of the enzymes contributes substantially less than 25% to metabolite formation, as was demonstrated by 328 Ogilvie et al. Chemical and Antibody Inhibition Chemical and antibody inhibition represent the second and third approaches to reaction phenotyping. As in the case of correlation analysis, chemical and antibody inhibition experiments must be conducted with pharmacologically relevant concentrations of the drug candidate in order to obtain clinically relevant results. Therefore, appropriate solvent and preincubation controls should be included in all chemical inhibition experiments. The lack of specificity can complicate the interpretation of chemical inhibition experiments. If a drug candidate is metabolized by a high-affinity enzyme, the con- centration of a competitive chemical inhibitor must be increased with increasing concentration of the drug candidate in order to achieve a high degree of inhi- bition. A good rule of thumb is to use multiples (generally up to 10-fold) of the lowest inhibitor concentration, which is calculated from the following equation: ½DrugŠÁKiðinhibitorÞ Lowest½InhibitorŠ¼ ð10Þ KmðDrugÞ where [Drug] is the intended final concentration of the drug candidate added to the microsomal incubation, Ki is the inhibition constant of the inhibitor for a given enzyme, and Km is the Michaelis constant of the drug candidate (as determined in Step 3). This method of calculating of the lowest concentration of inhibitor is applicable to competitive inhibitors but not to noncompetitive or metabolism-dependent inhibitors. A range of inhibitor concentrations is rec- ommended to demonstrate concentration dependence. For example, if the lowest concentration of the chemical inhibitor were calculated to be 1 mM (from the above equation), then the range of inhibitor concentration should span at least 10-fold (e. If both enzymes contribute to metabolite formation, the inhibitory effect of the chemical on one enzyme may be offset by its activating effect on the other enzyme. When chemical inhibition experiments are conducted with a relatively metabolically stable drug candidate (one that must be incubated with relatively high concentrations of human liver microsomes for a relatively long time in order to generate quantifiable levels of metabolite), it is important to take into account the metabolic stability of the inhibitors themselves. Lack of metabolic stability makes some compounds poor choices as chemical inhibitors despite their selectivity. Finally, appropriate controls should be included in each chemical inhibi- tion experiment to evaluate whether any of the chemical inhibitors interfere with the chromatographic analysis of the metabolites of interest and whether metabolite formation is inhibited by any of the organic solvents used to dissolve the chemical inhibitors. Unfortu- nately, the utility of this method is limited by the availability of specific inhibitory antibodies and by nonspecific effects associated with the addition of antiserum and ascites fluid to the microsomal incubation. The use of antiserum (for polyclonal antibodies) and ascites fluid (for monoclonal antibodies) rather than purified antibodies often necessitates adding a large amount of albumin and other proteins to the micro- somal incubation. For this reason, control (preimmune) serum and ascites fluid should be included as negative controls in antibody inhibition experiments.

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They are often wedge-shaped with the wide base towards the periphery of the organ acarbose 50mg without prescription diabetes insipidus name origin. Infarcts are usually pale but may become hemorrhagic if there is either a minor collateral circulation (e cheap acarbose 50 mg mastercard diabetes symptoms checklist. When an infarct extends to a serosal surface generic 50mg acarbose with amex diabetes symptoms dry mouth, it is covered by fibrinous (fibrin- containing) exudate initially proven 25mg acarbose diabetes labs, which may then cause fibrous (collagen- containing) adhesions as it heals. In most infarcts, inflammatory cells are noted in the periphery of large lesions, because that is where the remaining arterial circulation allows delivery of leukocytes. In the first few days neutrophils predominate, and then macrophages and fibroblasts appear until the lesion is replaced by fibrosis (scar). Old infarcts tend to be pale, shrunken and depressed beneath the surface of the organ. Edema of the surrounding tissues can cause severe problems when the tissue compartment is constrained, such as in stroke or compartments of the extremities. Bleeding may occur from mucosal surfaces of infarcted organs, such as hematuria (blood in urine), hemoptysis (blood in sputum), and intestinal bleeding from renal, lung, and bowel infarcts respectively. The cardiovascular system is responsible for transporting nutrients, oxygen, carbon dioxide and non- usable metabolic products between various organs in the body. The demands on the cardiovascular system vary greatly during a "normal day" for most of us. Getting out of bed in the morning requires major changes in the cardiovascular system. The lecture will primarily focus on the autonomic nervous system and its role in regulating cardiac function. In addition, some mechanisms responsible for local regulation of blood flow in peripheral vessels will be discussed. After reviewing the basic circuitry for regulating cardiac function, we will ask members of the class to help demonstrate cardiovascular responses to minor physiologic stress. Cardiac output (the volume of blood pumped by the heart per minute) is varied by altering the heart rate or the volume of blood pumped during contraction. The heart rate is controlled by the autonomic nervous system and will be discussed below. The volume of blood pumped during each beat is determined by several factors: 1- The volume of blood delivered to the heart. The autonomic nervous system can regulate delivery of the blood from Cardiac Reflexes - Brian Kobilka, M. The autonomic nervous system can direct cardiac myocytes to change the strength of contraction. When resistance is high, the ventricle cannot empty completely and therefore delivers less volume per contraction. Carotid body and aorticarch baroreceptors detect The autonomic nervous changes in blood pressure. The sympathetic nervous system regulates The sympathetic nervous vascular resistance and system regulates salt and regional blood flow. Large vessels in the abdomen and lower extremities serve as a reservoir for blood. The central nervous system receives information about the performance of the cardiovascular system from several sources. The information is processed at several levels in the central nervous system, but the final integration is accomplished in the dorsal motor nucleus of the vagus, and the vasomotor center located in the medulla and the lower third of the pons. Adjustments in cardiovascular function are made via sympathetic and parasympathetic modulation of the heart rate, and cardiac contractility, as well as sympathetic modulation of arterial resistance, venous capacitance, and renal function. The autonomic nervous system consists of the sympathetic and parasympathetic nervous systems. The vasomotor center controls the sympathetic output to the heart and blood vessels. Parasympathetic innervation of the heart originates in the dorsal motor nucleus of the vagus. Under conditions of normal cardiovascular function both sympathetic and parasympathetic Cardiac Reflexes - Brian Kobilka, M. Modulation of function is accomplished by either increasing or decreasing the basal level of activity to specific organs. Higher levels of central nervous system control over cardiovascular function arise in the cerebral cortex, limbic system and the hypothalamus. These centers exert control over cardiovascular function by modulating the activity of the medullary centers. Some sympathetic control is preserved in patients with low cervical cord transections. Acetylcholine released from postganglionic vagal fibers is rapidly degraded by acetylcholinesterase. Sympathetic nerves originate in the intermediolateral columns of the lower cervical and upper thoracic spinal cord. The preganglionic fibers synapse in the sympathetic ganglia which lie adjacent to the vertebral column. The adrenal medulla is a specialized sympathetic ganglia that releases epinephrine and norepinephrine into the systemic circulation. The neurotransmitter released from the sympathetic nerve terminal is primarily norepinephrine while both epinephrine and norepinephrine are released from the adrenal medulla. As discussed below, specific adrenergic receptor subtypes are more responsive to epinephrine while others are more responsive to norepinephrine. Most of the sympathetic nerves going to the heart either synapse in, or pass through the stellate ganglia (fusion of the last cervical and first thoracic). The right stellate ganglia has a greater effect on heart rate and the left has a greater effect on contractility. Most of the resistance and capacitance vessels to skin, skeletal muscle and viscera are richly innervated by sympathetic nerves. Release of norepinephrine from sympathetic nerve terminals in these vessels leads to vasoconstriction through alpha 1 adrenergic receptors; however, during exercise, circulating epinephrine released from the adrenal medulla activates beta 2 receptors in skeletal muscle vessels leading to dilatation of these vessels. Cerebral, coronary and pulmonary vessels are poorly innervated and are poorly responsive to sympathetic stimulation. Under maximal sympathetic stimulation, blood flow to the brain, heart and lungs is preserved at the expense of other organs. Catecholamines modulate renal blood flow, fluid and electrolyte balance and renin release. Activation of the sympathetic nervous system under severe stress such as the fight or flight response, or strenuous, prolonged physical exertion leads to a generalized release of catecholamines (predominantly norepinephrine) from all sympathetic nerve terminals throughout the body as well as a release of catecholamines from the adrenal gland into the circulation. For example, blood vessels in skeletal muscle dilate to increase blood flow to muscles, while blood vessels in the abdominal viscera constrict, diverting blood from intestines. This organ and tissue specific response to catecholamine release is accomplished by structural and functional diversity in the family of adrenergic receptors that respond to catecholamines. Similar diversity exists in the family of muscarinic receptors that respond to acetylcholine. Muscarinic and adrenergic receptors are structurally and functionally similar plasma membrane receptors that form the interface between the autonomic nervous system and the cardiovascular system. The activated G protein goes on to modulate one or more cellular enzymes or ion channels. There are 9 subtypes of adrenergic receptors (Alpha 1a, b and c, Alpha 2 a, b and c, Beta 1, Beta 2 and Beta 3) and 5 subtypes of muscarinic receptors (m1-m5).

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They are delivered to the terminals by fast axoplasmic transport and are the only type of vesicle to be found in axons (see Calakos and Scheller 1996) generic acarbose 50mg with visa diabetes medications table. This suggests that they have different functions and regulatory processes which buy acarbose 25 mg overnight delivery medications for diabetes, since they contain peptides discount 25mg acarbose with amex diabetes mellitus type 2 and high blood pressure, agrees with the finding that their release requires higher frequencies of nerve stimulation than does that of the classical neurotransmitters buy acarbose 25mg with mastercard diabetes prevention diet and exercise. Electron microscopy certainly shows that their membranes are recovered after fusion with the axolemma but precisely how this occurs is unresolved. One possibility is that they are retrieved intact from the active zone, immediately after release has taken place. Alternatively, they could become incorporated into, and mix with, the components of the axolemma but are reformed after sorting of the different membrane elements (see Kelly and Grote 1993). Recent studies of exocytosis from retinula cells of the Drosophila fly suggest that both these processes for membrane retrieval can be found within individual cells. These studies have shown that there is rapid recovery of vesicular membrane from the active zone. However, a second slower process exists which takes place at sites remote from the active zone and involves the formation of invaginations in the axolemma. This process is thought to precede endocytosis because the formation of these invaginations is followed by the appearance of tubular cisternae within the nerve terminal from which new vesicles bud-off (Koenig and Ikeda 1996). This finding raises the interesting question of whether these different processes lead to the formation of two different populations of synaptic vesicles with different release characteristics. The reserve pool would then comprise vesicles which are docked, more remotely, on the neuronal cytoskeleton. It is thought that vesicles move from one pool to the other as a result of the actions of protein kinases which effect cycles of phosphorylation/dephosphorylation of proteins, known as synapsins, which are embedded in the vesicle membranes. Although they account for only about 9% of the total vesicular membrane protein they probably cover a large proportion of their surface. Recent evidence suggests that, while synapsins might have a role in synaptogenesis, they also regulate the supply of vesicles to the release pool (Hilfiker et al. Experiments in vitro have shown that dephosphorylated synapsin I causes growth and bundling of actin filaments which are a major component of neuronal microfilaments. Such findings form the basis of the hypothesis that synapsin I forms a ternary complex with transmitter storage vesicles and the neuronal cytoskeleton, thereby confining vesicles to a reserve pool (Fig. Phosphorylated synapsin dissociates from the vesicles and F-actin, reduces the number of vesicle anchoring sites, and so frees the vesicles to the release pool. This process would enable synapsin to act as a regulator of the balance between the releasable and reserve pools of vesicles. By contrast, injection of dephosphorylated synapsin I into either the squid giant axon or goldfish Mauthner neurons inhibits transmitter release. It has also been suggested that synapsin promotes vesicle clustering by a process which is not dependent on phosphorylation. It achieves this by forming cross-bridges between vesicles and by stabilising the membranes of the aggregated vesicles, thereby enabling them to cluster in the active zone without fusing with each other or the axolemma. When synapsin dissociates from the vesicles, as occurs during neuronal excitation, this membrane-stabilising action is lost. This would enable fusion of the membranes of vesicles, clustered near the active zone, with the axolemma. This scheme is supported by evidence that vesicles near the active zone have much lower con- centrations of synapsin than those located more remotely (Pieribone et al. For instance, it has been suggested that they might also regulate the kinetics of release, downstream of the docking process. An increase in intracellular Ca2‡ triggers phosphorylation of synapsin I which dissociates from the vesicular membrane. This frees the vesicles from the fibrin microfilaments and makes them available for transmitter release at the active zone of the nerve terminal and Scheller 1996). The following sections will deal with those factors about which most is known and which are thought to have a prominent role in exocytosis. The extent to which this scheme explains release from large dense-cored vesicles is unclear, not least because these vesicles are not found near the active zone. The processes leading to docking and fusion of the vesicle with the axolemma membrane are thought to involve the formation of a complex between soluble proteins (in the neuronal cytoplasm)and those bound to vesicular or axolemma membranes. Much of this evidence is based on studies of a wide range of secretory systems (including those in yeast cells)but which are thought to be conserved in mammalian neurons. How the interconversion of these complexes occurs and which components trigger these processes is poorly understood. Proteins such as rab 3A, Ca2‡ binding proteins and Ca2‡ channels are likely to be involved. How all these processes are influenced by Ca2‡ is uncertain but another vesicle membrane-bound protein, synaptotagmin, is widely believed to effect this regulatory role (Littleton and Bellen 1995). This tail binds Ca2‡ and could enable synaptotagmin to act as a Ca2‡-sensor but, although it is found in adrenergic and sensory neurons, it appears to be absent from motor neurons. Its transmembrane structure resembles that of connexins which form gap junctions and has provoked the theory that neuronal excitation might cause synaptophysin to act as a fusion pore. For a detailed review of the role of all these factors in the exo- cytotic cycle, see Benfanati, Onofri and Giovedi 1999. Early experiments using stimulated sympathetic nerve/end-organ preparations in situ, or synaptosomes, indicated that release of [3H]noradrenaline was attenuated by exposure to unlabelled, exogenous transmitter. This action was attributed to presynaptic adrenoceptors, designated a2-adrenoceptors, which were functionally distinct from either a1-orb-adrenoceptors. Later experiments have confirmed that a2-adrenoceptors comprise a family of pharmacologically and structurally distinct adrenoceptor subtypes. For instance, autoreceptors can only be synthesised in the cell bodies of neurons and are delivered to the terminals by axoplasmic transport. Yet a2-adrenoceptors have not been found in either the cell bodies or axons of sympathetic nerves. Such findings fuel speculation that feedback inhibition of transmitter release might involve a transsynaptic mechanism. These are found on the cell bodies of noradrenergic neurons in the nucleus locus coeruleus of the brainstem. When activated, they depress the firing rate of noradrenergic neurons in the nucleus. This means that changes in the concentration of noradrenaline in the medium bathing these somatodendritic a2-autoreceptors will modify the firing rate of central noradrenergic neurons. Autoreceptor-mediated feedback control of transmitter release will obviously depend on enough transmitter accumulating in the synapse to activate the receptors. If the trains of stimuli are either too short, or their frequency too low, then transmitter release is not augmented by the administration of autoreceptor antagonists, implying that there is no autoreceptor activation (Palij and Stamford 1993). Conversely, at higher frequencies and long trains of stimulation, it becomes harder to inactivate the autoreceptors with antagonist drugs, presumably because of competition with increased concentrations of transmitter in the synapse. These receptors are thought to be located on the terminals of, and to modulate transmitter release from, one type of neuron, but are activated by transmitter released from a different type of neuron (Laduron 1985). For example, noradrenaline has been proposed to modulate release of a wide range of transmitters (e. It is therefore hard to be certain that heteroceptors are actually located on the terminals of the [3H]labelled neuron and to rule out the possibility that they form part of a polysynaptic loop. To avoid this problem, a few studies have used synaptosomes to test the effects of one transmitter on K‡-evoked release of another. Evidence suggests that co-transmitters in a terminal have their own autoreceptors and, in some cases, activation of their own presynaptic receptor can influence the release of the co-stored, classical transmitter. However, in other cases, feedback modulation of release of classical and their asso- ciated co-transmitters seems to have separate control mechanisms. This would suggest that either the two types of transmitter are concentrated in different nerve terminals or that mechanisms for regulating release target different vesicles located in different zones of the terminal (Burnstock 1990).

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