Stimulus—Response Coupling in Metabolic Sensor Cells

Chapter 34

StimuluseResponse Coupling in Metabolic Sensor Cells Stan Misler

Chapter Outline I. Introduction II. StimuluseSecretion Coupling in the Pancreatic Islet Cells IIA. Role of ATP-Sensitive Kþ Channels in Coupling Metabolism to b-Cell Depolarization IIB. Metabolic Regulation of DepolarizationeSecretion Coupling? IIB1. The “Ca Trigger” IIB2. The “readily releasable pool” (RRP) IIC. The Paradox of StimuluseSecretion Coupling in the Glucagon-Secreting a Cell IID. Implications for Pathophysiology and Therapeutics III. Metabolic Sensing as Protection from Hypometabolic Injury IV. StimuluseSecretion Coupling in Carotid Chemoreceptor Cells

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I. INTRODUCTION The term sensory receptor is usually applied to a limited group of cells, located near the surface of the organism, designed to transduce stimuli from the external environment (e.g. photons of light or air or water-borne molecules or pressure disturbances) into the release of a chemical transmitter, which ultimately signals neurons that project into the central nervous system. However, to maintain a “steady state,” organisms must also detect changes in their internal environment. To do this, specialized internal receptors must sense changes (1) in the levels of circulating metabolic fuels (e.g. nutrient metabolites and O2); (2) in the levels of metabolic wastes (e.g. CO2); (3) in the ionic composition of the extracellular lymph-like fluid which bathes the cells; and (4) in the internal or lumenal tension in hollow tubular organs (e.g. blood vessels and gut). Currently, among the internal receptors, those sensitive to metabolic changes are better understood than those

Cell Physiology Sourcebook. DOI: 10.1016/B978-0-12-387738-3.00034-2 Copyright Ó 2001 Elsevier Inc. All rights reserved.

IVA. Role of O2-Sensitive Kþ Channels in Transduction of the Hypoxic Stimulus IVB. Transduction of Increased pCO2 and Decreased Plasma pH V. StimuluseContraction Coupling in Vascular Smooth Muscle Cells VA. Hypoxic Vasodilation of Coronary and Mesenteric Vessels VB. Hypoxia-Induced Vasoconstriction of Pulmonary Vessels VI. Coupling of Oxygen Sensing to Red Cell Production by Erythropoietin-Secreting Cells Acknowledgments Bibliography

responding to changes in ionic composition or lumenal tension. In this chapter, we shall examine, in some detail, sensory transduction by five types of metabolic sensor cells. 1. Pancreatic islet cells: these cells sense changes in the levels of several circulating metabolites, especially glucose and amino acids, and respond with secretion of the hormone insulin and glucagon. Insulin promotes the uptake of glucose and its storage as glycogen in muscle and liver and maintains fat stores in adipocytes, while glucagon mobilizes fat and glycogen stores. 2. Cardiac and skeletal myocytes and some neurons: these cells curtail their excitability during hypoxia and substrate deprivation, thereby saving themselves from severe hypometabolic injury. 3. Chemoreceptor cells of carotid body: these cells sense changes in the O2 and CO2 content of blood and synapse onto sensory nerve fibers which project into the central nervous system (CNS) and help shape respiratory drive. 601

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4. Vascular smooth muscle cells: these cells sense local changes in O2 tension and respond by regulating their tone, thereby locally controlling blood flow. 5. Hypoxia-secreting renal interstitial cells and liver Ito cells: in response to hypoxia, these cells secrete erythropoetin, the hormone critical for maturation and release from bone marrow of oxygen-carrying erythrocytes. The first four types of metabolic sensor cells share a common property; each makes use of Kþ channels specially attuned to metabolic changes to transduce stimuli. Some also use the metabolic intermediates to modulate more distal processes in their scheme of stimuluseresponse coupling. The fifth cell makes use of what might be called stimulusesynthesis coupling. Familiarity with different patch-clamp recording configurations and basic concepts of excitationesecretion coupling in nerve cells is assumed (see Chapters 20 and 33).

II. STIMULUSeSECRETION COUPLING IN THE PANCREATIC ISLET CELLS The pancreas consists of islands of endocrine tissue, islets of Langerhans, that regulate metabolite uptake, storage and release by liver, adipocytes and skeletal muscle, surrounded by a sea of exocrine tissue, acini and their ducts, that secrete alkaline digestive juices into the duodenum. Each islet contains about 1000 cells. Roughly 65% are insulin-producing b cells, triggered by an increase in serum glucose. These are the largest of the islet cells and have distinct crystals in their secretory granules. Roughly 20% are glucagon-producing a cells, largely triggered by a decrease in serum glucose. The remaining l5% are d or PP cells secreting somatostatin or pancreatic polypeptide. After dispersal of the islet into single cells, a cells can be separated from b cells on the basis of their smaller size and decreased NAD autofluorescence. Though isolated islet cells secrete in response to appropriate stimuli, they secrete best when they are in clusters containing representatives of each cell type. Beta cells are electrically coupled. Alpha, beta and delta cells all “talk” to each other via paracrine interactions; glucagon enhances stimulusinduced insulin secretion, whereas somatostatin inhibits it. Insulin, glucagon and somatostatin all inhibit glucagon secretion. The study of how a rise in plasma glucose leads to rapid secretion of insulin by b cells has provided fertile ground for the interaction of a wide range of disciplines in cell physiology. Since the mid-1970s, it has been widely appreciated that the release of insulin, which is stored in secretory granules, is dependent on extracellular Ca2þ concentration [Ca2þ]o (the calcium hypothesis), requires oxidative metabolism of glucose or other metabolite fuels

Synaptic Transmission and Sensory Transduction

(the fuel hypothesis) and follows the onset of complex electrical activity in the b cells (the depolarizationesecretion coupling hypothesis). These features were first established using techniques of islet perifusion and radioimmunoassay of secreted insulin (Fig. 34.1A). In these experiments, islets are mounted on a filter and exposed to a continuous flow of solution able to trap secreted insulin. Timed aliquots of solution are collected and the insulin content is measured by radioimmunoassay, i.e. the ability of the secreted insulin to displace radioactively labeled insulin bound to an anti-insulin antibody. Note that insulin secretion induced by raising glucose from preprandial levels of 2e3 mM to levels above 5 mM occurs in two phases, a transient “first phase” and a sustained “second phase.” In vivo, the “first phase” may saturate hepatic insulin receptors, as it is often not readily detected in assays of peripheral insulin levels. Note that the “second phase” of release is reversibly reduced by (1) transient reduction of the Ca2þ concentration of the perifusing solution or (2) addition of an inhibitor of glucose metabolism, in this case, sodium azide (NaN3), an inhibitor of the mitochondrial respiration (see Fig. 34.1A). In parallel experiments, it was found that islets bathed in preprandial levels of glucose rapidly released insulin in response to exposure to high [Kþ], provided adequate extracellular Ca2þ was present. When the fuel hypothesis was extensively tested, insulin release was found to be (1) stimulated by amino acids, glycolytic intermediates and ketone bodies shown to be metabolized by b cells but (2) inhibited by substances that specifically block uptake and metabolism of these substrates or by general inhibitors of mitochondrial function. The calcium hypothesis and the depolarizationesecretion coupling hypothesis were tested with intracellular recording from islet cells in intact and perifused islets. Beta cells were found to maintain resting potentials of about 60 mV at fasting glucose concentrations. The resting potential varies with [Kþ]o according to the Nernst equation, hence suggesting that it is largely due to Kþ permeability (PK). Within several minutes of exposure to “secretagogue” concentrations of fuel metabolites, these cells depolarize, coincident with a decline in resting membrane conductance and shortly thereafter exhibit bursts of action potentials whose overshoots are a function of [Ca2þ]o. In cells loaded with a Ca2þ indicator, “spikes” of cytosolic Ca2þ coincide with bursts of action potentials (see Fig. 34.1B). From these observations emerged an early critical unifying hypothesis: fuel metabolism / reduced PK / electrical activity / Ca2þ entry/ insulin release. It is worth mentioning that this scheme also accommodates the actions of a class of hypoglycemic agents, called sulfonylureas. Though not related to fuel metabolites, and themselves not metabolized, sulfonylureas potentiate the

Chapter | 34

StimuluseResponse Coupling in Metabolic Sensor Cells

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FIGURE 34.1 Overview of basic evidence supporting “fuel hypothesis,” “calcium hypothesis” and “depolarizationesecretion coupling hypothesis” of glucose-induced insulin release from pancreatic b cells. (A) Insulin secretion, measured by radioimmunoassay, from isolated intact human pancreatic islets of Langerhans. Note the rapid onset of biphasic insulin secretion on increasing glucose from 1.67 to 16.7 mM, the interruption of the plateau phase by reduction in extracellular [Ca2þ] from 0.2 to 2.0 mM and the termination of secretion by the addition of sodium azide (NaN3), an inhibitor of electron transport by mitochondrial cytochromes. Leucine, glyceraldehyde and acetoacetate produce similar patterns of secretion in the presence of Ca2þ. (B) Simultaneous recording of electrical activity and cytosolic Ca2þ transients from single cryopreserved/thawed human b cells loaded with Ca2þ indicator FURA-2. Note that in the presence of 5 mM extracellular glucose, the cell fires individual or short trains of action potentials and that even single action potentials are sufficient to induce a Ca2þ transient. Subsequent addition of NaN3 abruptly hyperpolarizes the cell to z70 mV and blocks the appearance of Ca2þ transients. (Unpublished experiments by D. Barnett and S. Misler.) (C) Single cell exocytosis detected by amperometry and capacitance. Simultaneous cell-attached patch recording and amperometry from small clamp of islet cells preloaded with serotonin. The amperometric electrode was positioned at the confluence of cells. In 2 mM glucose, the Ion-cell trace shows single-channel openings, which will later be identified as those of ATPsensitive Kþ channels (Kþ(ATP)). After addition of tolbutamide, single channels close and “action currents” develop. After several impulses have fired, amperometric events are noted on the Iamp trace. Those events, slower than those observed when recording for single isolated cells, probably reflect simultaneous exocytotic discharge at several sites, each distant from the electrode. (D) Simultaneous-monitoring membrane capacitance and cytosolic Ca2þ from a voltage-clamped b cell. Note that a 200-ms depolarization from 70 to þ10 mV increases global cytosolic [Ca2þ] by an estimated 100 mm and membrane capacitance by 75 fF. This corresponds to the fusion of 30e60 insulin granules, given that the Cm increase per granule is estimated at 1.25e2.5 fF.

action of glucose even when glucose metabolism is modestly inhibited. They reduce resting PK, depolarize the cell, set off action potentials and stimulate insulin secretion even at preprandial levels of glucose. As we shall discuss in short order, these substances have been found to block directly the Kþ conductance that is physiologically gated by cell metabolism. Critical clues to the validity and the molecular details of this hypothesis have been provided by (1) patch-clamp recording plus molecular biology of channels (cloning and site-directed mutagenesis) and (2) single-cell, real-time monitoring of exocytosis (or fusion of the granule

membrane with the plasma membrane). In patch-clamped b cells, the workings of ATP-inhibited Kþ channels, whose closure underlies cell depolarization, have been analyzed in great molecular detail, in large part due to the identification of this molecule as the b cell’s major sulfonylurea receptor. In patch-clamped b cells, depolarization-induced exocytosis can be measured electrically as an increase in membrane capacitance (Cm), while agonist- (e.g. sulfonylurea-) induced release can be monitored electrochemically as release of packets of native insulin or the false transmitter serotonin previously loaded into insulin granules (see Fig. 34.1C). Using either measure, exocytosis

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is found to be graded with the amount of Ca2þ entry induced by depolarization. (Here it is worth recalling that exocytosis involves simultaneous fusion of the membrane of the secretory granule with the plasma membrane as well as the release of the granule content into the extracellular space. The membrane fusion event, an increase in plasma membrane surface area, is usually detectable as an increase in plasma membrane capacitance. Membrane capacitance is estimated from the ability of the membrane circuit to “phase shift,” or displace in time, a sine wave of voltage excitation. The release of a packet of transmitter [or hormone] can be detected “biologically,” by a receptive patch of membrane, or “electrochemically,” at the surface of an electrode held at a potential sufficient to oxidize or reduce a component of the granule content. In either case, a brief spike of current is produced. When dealing with cells with large secretory granules, one can be more adequately convinced that exocytosis has occurred when it is possible to demonstrate the following points: (1) the jumps in Cm thought to represent single granule fusion events have amplitudes predictable from the surface area of the granule; (2) the single electrochemical spike [or “amperometric event”] coincides in time with jumps in Cm and represents the

FIGURE 34.2 Overview of stimulusesecretion coupling in the pancreatic b cell. The right-hand side shows (1e5) a “consensus scheme” of stimulusesecretion coupling: glucose transport and phosphorylation (1),/ to intermediate metabolism, ATP production/ADP consumption and Kþ(ATP) channel closure (2), / membrane depolarization (3), / opening of voltage-dependent Ca2þ channels (4) and, finally, Ca2þ binding to granule docking complex and granule fusion (5). Non-selective cation channels that form the counterweight to Kþ(ATP) channels at rest are shown to the left of the Glut-2 transporter. Further to the left are the G-protein-linked receptor cascades for muscarinic, cholinergic agonists and the incretin GLP-1. The latter two “priming” compounds produce second messengers capable of releasing Ca2þ from intracellular stores and/or activating protein kinases to modulate channel activity and granule mobilization. Lastly, the contents of the secretory granule, namely insulin and ATP, may “feed back” on the b cell via cell surface receptors and complex signaling cascades.

Synaptic Transmission and Sensory Transduction

release of at least many tens to hundreds of thousands of molecules that are either native to the granule or else preloaded as a marker. In the case of b cells, the preloaded marker is serotonin. It is worth remembering that capacitance and amperometry are complementary techniques; capacitance monitors net increase in total membrane surface area [exocytosis minus endocytosis], whereas amperometry often permits direct counting of quantal release, albeit from a fraction of the cell surface.) On the basis of these and other experiments, over the past decade a more detailed “consensus hypothesis” has emerged for glucose-induced insulin secretion from b cells (Fig. 34.2, right-hand side). Uptake of glucose (predominantly via a Glut-2 type transporter) provides glucose entry into glycolysis (via glucokinase, a low affinity hexokinase) and the mitochondrial Krebs cycle results in the generation of ATP and consumption of ADP (Step 1). This leads to the closure of ATP-inhibited Kþ channels, here abbreviated as Kþ(ATP) (Step 2). Against a background of tonic activity of non-selective cation channels that pass Naþ inward, Kþ(ATP) channel closure results in the depolarization of the b-cell membrane (Step 3). When Vm reaches the threshold for activating voltage-dependent Ca2þ and Naþ channels, electrical activity and voltage-dependent Ca2þ

Chapter | 34

StimuluseResponse Coupling in Metabolic Sensor Cells

entry begins (Step 4). The resultant rise in cytosolic Ca2þ triggers Ca2þ-dependent fusion of insulin granules with the plasma membrane (Step 5). Steps 4 and 5 are reminiscent of the process of depolarizationesecretion coupling, well studied at nerve terminals and other electrically excitable endocrine cells (adrenal chromaffin cells, pituitary lactotropes and melanotropes and terminals of the supraoptic nucleus cells), with subtle differences that shall be discussed later. However, this scheme does not answer some rather old questions. (1) How do enhancers of cAMP serve to restore stimulusesecretion coupling in metabolically and electrically active but non-secreting b cells? (2) How, aside from possibly depolarizing the b cell, do anticipatory or modulatory factors such as gut hormones and acetylcholine work? The anticipatoryemodulatory pathways of receptormediated stimulusesecretion coupling, as well as possible autocrine effects of insulin and ATP released by exocytosis, are outlined on the left of Fig. 34.2 and also will be discussed later. In fact, it may be possible that, under very extreme conditions, modulatory pathways may result in some secretion independent of an inciting Ca2þ current or a detectable rise in global cytosolic Ca2þ. This might occur through the combination of a very localized rise in cytosolic Ca2þ with a vastly increased readiness of the secretory apparatus. However, it should never be forgotten that the “sui generis” of the b cell remains metabolic regulation of cell electrical activity via Kþ(ATP) channels.

IIA. Role of ATP-Sensitive KD Channels in Coupling Metabolism to b-Cell Depolarization How do fuel metabolites produce b-cell depolarization? Early patch-clamp experiments, using the cell-attached mode of recording, revealed the presence of a very interesting Kþ channel that was open at fasting levels of extracellular glucose (2e3 mM), but rapidly closed on raising ambient glucose concentrations to those encountered after a meal (5e7 mM). Usually, channel closure is followed closely by the onset of electrical activity. This channel is open at the cell’s resting potential and its activity is not dependent on membrane voltage. However, the channel is inward rectifying (i.e. single channels pass inward flow of potassium better than outward). These channels have many features predictable from the fuel metabolism hypothesis: addition to the bath of any metabolized fuel closes the channel, yet the channel is reopened subsequently by the addition to the bath of inhibitors of metabolite transport or oxidation. The time course and relative change in channel activity seen in a cellattached patch during a variety of metabolic maneuvers closely parallels the changes in whole-cell resting conductance (Gm), monitored as the change in membrane

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potential in response to a given test pulse of current applied (Fig. 34.3A, B). The latter strongly suggested that the activity of this channel, which tends to move Vm to a level near the potassium equilibrium potential, EK, critically controls the cell’s resting potential. Since this channel population is the predominant type open at rest, its closure

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FIGURE 34.3 ATP-sensitive Kþ channels and electrical activity in canine pancreatic islet b cells. (A) A cell-attached patch was formed with the pore-forming antibiotic nystatin in the pipette. Shortly thereafter, ionchannel activity was recorded before, during and after application of sodium azide (NaN3, 3 mM) to the low-glucose bath. (B) Electrical activity recorded 20 minutes later. By that time, the patch of membrane encompassed by the pipette had been permeabilized by insertion of nystatin channels, thereby permitting recording of membrane current or voltage from the whole cells. Reapplication of NaN3 results in membrane hyperpolarization, abolition of electrical activity and increased membrane conductance (signified by the reductions in transient hyperpolarizations accompanying 5 pA currents applied at arrows). Vm denotes cell membrane potential. (C) Nucleotide gating of Kþ(ATP) channel activity in the excised patch. Addition of 100 micromolar ATP (100 mM) reduces channel activity by threefold (C, upper right panel); further addition of ADP restores channel activity (C, lower left panel), whereas washout of ATP and ADP results in overshoot of channel activity (C, lower right panel) (D.M. Pressel and S. Misler, unpublished data).

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en masse would be needed to make the Kþ conductance of the cell comparable to that of other conductance pathways operative at rest. The latter are non-selective cation conductance channels and anion conductance channels with estimated equilibrium potentials of 0 or z30 mV respectively. This would permit Vm to depolarize to the threshold for activating cell electrical activity (z45 mV). Another hint of the importance of this type of channel was that it is closed by oral hypoglycemic agents. How is this channel gated? In the cell-attached patchrecording configuration, in which the exterior of the channel was isolated from bath glucose by a very tight pipette-to-membrane seal, it is safe to assume that the channel is gated by one or more metabolic intermediates. A priori, cell metabolism might affect a host of metabolic intermediates, ranging from high-energy phosphate compounds (e.g. ATP and ADP), to redox equivalents, to cytosolic Hþ or Ca2þ. Early experiments (see Fig. 34.3C) showed that after excision of the patch, in an inside-out excised patch configuration, the metabolite-regulated Kþ channel was avidly gated by ATP at its inner surface, hence the name Kþ(ATP) channel. Free concentrations of ATP, or its non-hydrolyzable analogs (e.g. AMP-PNP) as low as 10 mM reduce channel activity by half even in the absence of Mg. This suggests that ATP closes this channel by directly “gating” it rather than “phosphorylating” it. In parallel with this, in whole-cell patch experiments where ATP dialyzes out of the cell (due to the absence of ATP in the pipette), resting Gm increases and the cell hyperpolarizes. Interestingly, ATP inhibition of channel activity in the excised (inside-out) patch can be partially reversed by addition of MgADP to the bath. Hence, changes in the relative concentrations of ATP and ADP, which usually change in a reciprocal fashion as metabolism is altered, should be an important factor in physiological channel gating. In most cells, ATP concentrations change little with metabolism, but percentage-wise, ADP levels change more dramatically. Hence the channel may have been misnamed. To be sure, the activity of the Kþ(ATP) channel is also altered by other potential intermediates whose cytosolic concentrations change with metabolic stimulation (e.g. NADH/NAD ratio, Hþ and Ca2þ), but these effects are much smaller and require changes beyond the physiological range. A puzzling question regarding the function of this channel is how its low Kd value (mM) for ATP-induced channel closure in the excised membrane patch can be reconciled with the millimolar-range concentration of ATP in the cytosol. Several factors mitigate this discrepancy: 1. Complex effects of ADP on channel activity. ADP competition for an ATP binding site, combined with allosteric action of ADP at an independent binding site, adjusts the half-maximal effective concentration (Kd) of

Synaptic Transmission and Sensory Transduction

ATP, measured in the presence of ADP, closer to the mM range. 2. Compartmentation of the ATP and steep ATP gradients from source to sink. It is likely that the overwhelming majority of ATP is of mitochondrial origin: mitochondrial inhibition totally prevents glucose-induced channel closure, while ketone bodies, amino acids and their deamination products (e.g. leucine and ketoisovalerate), which directly enter mitochondria, stimulate channel closure even in the face of inhibition of glycolysis. In addition, given the random localization of these channels over the surface of cultured cells, it is likely that the channels are interspersed among ATPases. Hence, steep cytoplasmic gradients of ATP and ADP, due to en passant consumption of ATP and concomitant local production of ADP by neighboring plasma membrane ATPases, should make the ATP/ADP available to the channel different from the average cytosolic concentrations actually measured. 3. Modulation of gating by other metabolic intermediates such as long chain fatty acids. 4. Artifactual enhancement of channel sensitivity to ATP in the excised patch by progressive washout of intrinsic channel inhibitors such as anionic lipids (phosphoinositol phosphates), from the exposed membrane. Polyanionic molecules might specifically compete with ATP for binding to the channel or else contribute to a negative surface charge at the inner surface of the plasma membrane, thereby electrostatically repelling soluble anions. Two features of the metabolite-regulated Kþ(ATP) channel proved critical to its cloning and structureefunction analysis. (1) The channel functions as an inward rectifier Kþ channel and hence could be cloned as a member of that family. (2) The channel is the major locus of action of hypoglycemic (glucose-lowering) sulfonylureas and could be cloned as a sulfonylurea receptor (SUR). The sulfonylureas (e.g. tolbutamide and glyburide [glibenclamide]) enhance glucose-induced insulin release by b cells and are useful in the treatment of non-insulin-dependent diabetes mellitus, in which b cells produce significant quantities of insulin, but release it out of synchrony with the appearance of the glucose load. At concentrations equal to free plasma levels that enhance glucose-induced electrical activity and insulin secretion in intact animals, the sulfonylureas close Kþ(ATP) channels in cell-attached or excised patches alike, while leaving voltage and calcium gated Kþ channels unaffected. Curiously, though Kþ(ATP) channels in myocytes show similar conductance, kinetics and ATP sensitivity, they have greatly reduced sulfonylurea sensitivity. To reconstitute channel activity in a naive cell (e.g. Xenopus oocyte or clonal cell), it was necessary to transfect or inject the cell with cDNA or mRNA coding for

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StimuluseResponse Coupling in Metabolic Sensor Cells

(1) a standard inward rectifier type subunit (Kir6.2), comprised of two membrane-spanning domains separated by an H loop dipping into the bilayer, and (2) the SUR from b cells (SUR1) (Fig. 34.4A, B). SUR 1 is a complex protein with multiple transmembrane domains (hydropathy plot analysis suggests 17!). Structurally, it contains two motifs resembling those of the ATP-binding cassettes of members of the ABC transporter superfamily, the most famous of which are the cystic fibrosis gene product, CFTR, and the multidrug resistance p-glycoprotein. SUR1 has two distinct cytoplasmic loops (or facettes) that bind nucleotide di- and triphosphates in the presence of Mg (NBFs). The channel itself is likely to be an octamer consisting of an inner ring of four Kir6.2 subunits (the confluence of the four H loops forming the ion selective pore) and an outer ring of four SUR1 subunits. The two types of subunits are needed to chaperone each other into the membrane as well as to provide full channel gating; without SUR1, Kir6.2 is not expressed. Clinically, mutations in SUR1 have been identified in persistent hyperinsulinemic, hypoglycemia of infancy, a disease where Kþ(ATP) channel activity is not detected even after metabolic inhibition and b cells are persistently depolarized and secreting. Point mutations to NBF result in channels that are closed by ATP in excised patches, but are not reopened by MgADP, while more extensive mutations result in the absence of channel

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activity under any test circumstance. In contrast, a C-terminal truncated mutant of Kir6.2 (Kir6.2DC26) forms Kþ channels gated by ATP in the absence of Mg. These channels have lower affinity for ATP (Kd z10 mM in the absence of Mg) than the native or Kir6.2/SUR1 cloned channel (Kd z100 mM) but show similar need for ATP cooperativity in channel gating (n ¼ 4). However, these channels lack both inhibition of activity by sulfonylureas and enhancement of activity by MgADP. Hence, while Kir6.2 might be considered the ATP gating subunit, SUR1 with its NBFs is the ATP-hypersensitizing subunit as well as the subunit endowing the channel with special sensitivity to drugs and MgADP. A schematic model of Kþ(ATP) channel gating is presented in Fig. 34.4C. SUR might have a natural ligand, a cytoplasmic protein called endosulfine, recently demonstrated to displace sulfonylureas from SUR binding sites. Analysis of mutant channels has also elucidated the origin of a number of previously puzzling effects. One example is the “refreshment effect” of MgATP. That is, rapid wash-in and wash-out of MgATP, even in concentrations of 10e100 mM, can restore channel activity that otherwise would run down in time in its absence (see Fig. 34.3C). This effect can now be partially explained by the finding that small concentrations of MgATP as well as MgADP activate the channel by binding to a “Walker motif” Kir 6.2

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SUR I FIGURE 34.4 Illustrations depicting Kþ(ATP) channel structure and function. (A) Transmembrane orientations of SUR1 and Kir6.2 subunits based on structural homology, hydropathy and site-directed mutagenesis. (B) Proposed octamer array of SUR1 and Kir6.2 subunits. (C) Simplified reaction scheme in channel gating.

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of the NBF in native forms of SUR1 but not in those SURs whose NBFs have been mutated. Likewise, channel modulation by Mg-guanidine nucleotides also occurs at these sites, suggesting that there is no need to invoke G proteins in GTP or GDP modulation of channel gating (though G-protein-coupled receptors may independently modulate channel activity). Both effects require hydrolyzable nucleotides and Mg, suggesting that some phosphorylation reaction may be involved. A second example is the ability of certain vasodilator drugs, such as diazoxide, to open Kþ(ATP) channels, hyperpolarize the b cell and inhibit glucose-induced insulin secretion. These drugs also bind to the “Walker motif”. A third example is the differential sulfonylurea sensitivity of Kþ(ATP) channels in b cells as compared with those in cardiac myocytes. While both channels have similar Kir6.2 subunits, their SURs differ. Replacing transmembrane domains 13e16 of cardiac SUR (SUR2) with the corresponding region of SUR by engineering of a chimeric SUR restores sulfonylurea sensitivity. Is this finely tuned coupling of cell metabolism to Kþ(ATP) channel activity used as a sensory mechanism by the two other major glucose receptor cells of the body, namely the satiety center of the hypothalamus and the peripheral sweet taste receptors? Recently, Kþ(ATP) channels have been sought in glucose-receptive (GR) neurons of the ventromedial nucleus (VMN) whose activity is associated with suppression of food intake. In these neurons, reduction of bath glucose or addition of the glycolytic inhibitor mannoheptulose results in hyperpolarization and cessation of electrical activity accompanied by an increase in resting Gm; oppositely, tolbutamide produces excitation. In cell-attached patch recordings, cessation of electrical activity is associated with increased activity of a Kþ-selective channel, which is voltage-insensitive. After excision of the membrane patch, the latter channel is inhibited by cytoplasmic ATP. However, this Kþ channel is quite distinct from the Kþ(ATP) channel in b cells: under identical excised patch recording conditions, the Ki for ATP inhibition is 2.3 mM (rather than 20 mM in b cells) and the singlechannel conductance is 135 pS (rather than 55e65 pS in b cells). One very interesting feature of this channel is that it is opened and the cell is hyperpolarized by leptin, a protein released by adipocytes, or fat-storing cells, in proportion to their triglyceride storage. Leptin also opens Kþ(ATP) channels in b cells and appears to do this in a manner dependent on activation of PI-3 kinase. Does leptin serve as a negative feedback hormone, that is signaling adequate energy stores by downregulating both appetite-induced caloric intake and insulin-dependent depot storage? One indication of this comes from obese, hyperphagic, hyperinsulinemic and hyperglycemic db/db rats, where leptin has little influence on the firing rate of VMN neurons. In peripheral taste receptors, glucose chemoreception may occur through G proteins and a second messenger

Synaptic Transmission and Sensory Transduction

cascade. In these cells, application of sucrose or saccharin reduces the amplitude of a voltage-dependent outward Kþ current. This effect can be mimicked by application of membrane-permeable analogs of cAMP. In excised patches, Kþ channels with similar features are closed by exposure of the patch to ATP and cAMP-dependent protein kinase, but not by exposure to ATP alone, suggesting that this Kþ channel is “gated” by phosphorylation.

IIB. Metabolic Regulation of DepolarizationeSecretion Coupling? Recall that closure of Kþ(ATP) channels only depolarizes the cell. Calcium must enter the cells and insulin granules must be ready to be released. Does metabolism alter the “calcium trigger” or maintain the “readily releasable pool” of insulin granules?

IIB1. The “Ca Trigger” When b cells, exposed to sufficient glucose, a cAMP enhancer and near physiological temperature, are depolarized for many tens to hundreds of milliseconds, they exocytose in a Ca2þ-entry-dependent manner. Under these conditions, several studies have shown an approximately 3rd power dependence of exocytosis, as measured by amperometry or capacitance, on the total Ca2þ entering the cell (i.e. the integral of the Ca2þ current). Though most of the Ca2þ current is carried by L-type Ca2þ channels, in a subset of human b cells, where they are present in sufficient density, lower threshold T-type Ca2þ channels also support exocytosis. We might expect changes in cell metabolism to affect the gating of Ca2þ channels via phosphorylation or lipidation. To date, data supporting the hypothesis that enhanced glucose enhances Ca2þ channel activation or slows inactivation have been inconsistent. Another paradigm, however, which illustrates other aspects of depolarizationesecretion coupling may provide some other clues. Beta cells in intact islets secrete pulses of insulin that are time-linked to “bursts” of electrical activity. Longer “bursts” evoke larger pulses of insulin. Contrary to the situation at most nerve terminals and despite quite sizeable Ca2þ currents, it is very rare to see an isolated action potential, or a brief depolarization (10e50 ms in duration), provoke quantal release (here measured by amperometry). However, when the depolarization is extended out to many tens to several hundreds of milliseconds, release routinely commences in the course of the depolarization and often continues for several seconds after Ca2þ entry ceases, while cytosolic Ca2þ is falling (Fig. 34.5). These data suggest that Ca2þ might have to “jump through a few hoops” before triggering secretion and that it might set up a “chain reaction” that outlasts the peak Ca2þ concentration (Fig. 34.6).

Chapter | 34

StimuluseResponse Coupling in Metabolic Sensor Cells

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FIGURE 34.5 Aspects of Ca dependence of exocytosis in b cells (A) Relationship of capacitance increase to total Ca2þ entry (integral of Ca2þ current QCa) during b-cell depolarization. Ca2þ entry was altered by changing the membrane potential to which the cell was voltage-clamped for the 200-ms test stimulus (unpublished data by D. Barnett and S. Misler). (B) Time course of amperometric events during trains of action potentials stimulated at 1 Hz (upper panel) and 4 Hz (lower panel) Note that none of the 11 action potentials occurring at 1 Hz stimulation results in quantal release, while at 4 Hz stimulation quantal release begins after several action potentials. (C) Time course of amperometric events recorded during continuous depolarization to þ10 mV, where Ca2þ entry is maximal. Note that the first release event is seen z120 ms after start of voltage-clamp pulse, while release events continue, albeit at a low frequency, for nearly 3 s after depolarization is completed (unpublished data by Z. Zhou and S. Misler).

FIGURE 34.6 Calcium metabolism in b cells: potential sites for modulation of depolarizationesecretion coupling. After calcium enters the cytoplasm, it is likely that much of it rapidly binds to either fixed or mobile buffers, the latter perhaps resembling the tetra-clawed EGTA. Some of the bound Ca2þ unbinds to diffuse further. Diffusing Ca2þ may then bind to one of the following: (1) a Ca2þ-binding protein (e.g. synaptotagmin) that is part of the docking complex that anchors a granule to the membrane and thereby directly triggers exocytosis, (2) a protein kinase, whose activity enhances the movement of granules along “motorized” tracks, (3) a binding site on Ca2þ-gated channels in Ca2þ storage organelles, including the secretory granule itself, which thereby triggers Ca2þ-induced Ca2þ release. Calcium uptake into organelles (granules, mitochondria and ER) is probably slower (on the time scale of hundreds of milliseconds to seconds). After fusion with the plasma membrane, granule membrane may be retrieved piecemeal by a local “pinch off” mechanism, by using dynamin, or as part of a large endocytic “slurp”.

Several possibilities for this “slow-to-start, slowto-stop” pattern of secretion are currently under investigation. First, with large-sized granules that are not clustered in any regular array, Ca2þ channels and granule

release sites may not be all that closely co-localized. To be sure, the interspersed cytosol has many Ca2þ-buffering molecules. Hence, if the site where Ca2þ triggers exocytosis were, on average, a distance of 1e2 granule radii

SECTION | V

(say 200e300 nm) from the inner mouth of the Ca2þ channel site, local cytoplasmic buffers would need to be saturated before Ca2þ could appear at the release site. This would cause a slight delay in the onset of exocytosis. At this distance, during continuous Ca2þ entry there might be good spatial equilibration of Ca2þ so that the Ca2þ concentration at the release site might closely follow the average Ca2þ concentration of the cell. This is in no way counterintuitive because it is well appreciated that Ca2þ and cytosolic Ca2þ concentrations as low as 1.5 mM evoke respectable rates of release (Fig. 34.7B). Conversely, once Ca2þ no longer enters the cell, the buffer might serve as a continuing source for Ca2þ and support a “secretagogue” level of free cytosolic Ca2þ for some time. This feature has been examined and modeled extensively in adrenal chromaffin cells but has only been explored in a cursory manner in b cells. Second, Ca2þ entry might be causing regenerative release of Ca2þ from some intracellular stores, thus providing a sort of propagating Ca2þ wave, while the increase in cytosolic Ca2þ might be stimulating Ca2þ uptake by other stores for later use. To date, while it is well appreciated that Ca2þ can be released from ER stores by the generation of IP3, blockade of Ca2þ release from ER stores has yielded only small changes in depolarization-induced rises in cytosolic Ca2þ or exocytosis. However, the picture may be more complex in other situations. For example, when GLP-1 is applied to voltage-clamped b cells in the presence of 20 mM Ca2þ, “spontaneous” spikes of cytosolic Ca2þ occur between depolarizations. To be sure, there are multiple stores of intracellular Ca2þ, aside from the IP3releasable ones in ER. For example, mitochondria- and

hormone-containing granules might be expected to release as well as “mop-up” Ca2þ, but little is known about their release and uptake rates during various patterns of electrical stimulation or exposure to hormones and transmitters. Some recent evidence suggests that depletion of the Ca2þ content of the acidic compartment of cytoplasmic organelles, which includes secretory granules, reduces depolarization-induced Ca2þ release. In a metabolically stimulated cell where the Krebs cycle intermediates, NAD-derived redox equivalents and local ATP concentrations may be changing, it would not be surprising that changes in cell Ca2þ metabolism in response to Ca2þ influx might be a very dynamic feature. Third, entering Ca2þ might rapidly recruit granules into a small, “readily releasable pool” waiting to be triggered.

IIB2. The “readily releasable pool” (RRP) In cells designed for rapid secretion, it is convenient to assume that vesicles are lined up against the plasma membrane and ready to fuse as soon as the appropriate level of second messenger is achieved in the cytoplasm. Morphologically, in b cells, this does not appear to be the case (see Fig. 34.6). When viewed under the electron microscope, very few of the z10 000 granules in the b cell are within one granule diameter of the membrane. Physiologically, experiments with single cells reveal that secretion is actually quite “wimpy” unless “revved up” by other maneuvers; with repeated depolarizations, exocytosis “poops out” and may take many tens of seconds to recover. This suggests that the RRP of available granules is actually

+1mMIBMX 10 s Cm

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Synaptic Transmission and Sensory Transduction

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FIGURE 34.7 Enhancement of cytosolic cAMP appears to increase the pool of readily releasable granules. Test by repetitive stimulation. (A) Note that in the “control,” repetitive depolarization produces rapid rundown of capacitance response so that by the fourth depolarization there is no evidence of further net exocytosis. In contrast, after addition of membrane permeable cAMP or a phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX), not only is the response to initial depolarization enhanced, but response to 4th depolarization is nearly as large as the response to the 1st depolarization in the “control”. In this cell, total Ca2þ entry over the entire series of depolarizations differed by less than 15% across the two runs. Perforated patch recording from rat b cell. Test by cell dialysis. (B) Cells with similar baseline capacitance (4e6 pF at break-in to the cell at time 0) were dialyzed against pipette solutions containing 1.5 mM Ca2þ versus 1.5 mM Ca2þ þ 150 mM cAMP. Note that, in the presence of cAMP, not only was the maximum rate of rise of capacitance, abbreviated (dCm/dt) max, on average nearly 2.5-fold greater, but the total capacitance increase over the entire 6 min of recording was on average nearly twofold greater.

Chapter | 34

StimuluseResponse Coupling in Metabolic Sensor Cells

quite small and that maintaining it under high output demand is a non-trivial matter. However, in b cells, two ideal sources of “physiological revving” of secretion are (1) activation of protein kinases (A and C) and (2) the provision of ATP by enhanced substrate metabolism. Protein kinases are well known to facilitate the attachment of a variety of granules to tracks (microtubules, actin filaments) that bring them in close proximity to the membrane. In b cells, during feeding, insulin secretion is “primed” even before serum glucose rises, by release of two substance that activate protein kinases, acetylcholine from vagal fibers and incretins (e.g. glucagon-like peptide [GLP-1]) from enterochromaffin cells. Activation of muscarinic ACh receptors results in activation of phospholipase C and production of IP3 and diacylglycerol. Activation of incretin receptors results in activation of protein kinase A pathway and enhanced cytosolic cAMP levels. These ACh and GLP-1 pathways might enhance distal processes in excitationesecretion coupling, along with increasing the rate of b-cell depolarization in response to glucose, by affecting ATP-sensitive Kþ channels and/or the nonselective cation channels. Hence both background electrical activity and insulin release- and metabolite-induced electrical activity and insulin release could be enhanced. Changes in cytosolic MgATP would be expected to alter the fueling of vesicle transport processes as well as the molecular motors (e.g. NSF) that untwine paired attachment proteins (SNAREs) on granules and the target membrane. The latter permits SNAREs subsequently to intertwine with their cohorts on the complementary membrane, thereby accomplishing vesicle docking. In b cells, glucose is known to enhance insulin secretion evoked by elevated [Kþ]o even when Kþ(ATP) channels are already closed, while metabolic inhibition can block insulin secretion, even as cytosolic [Ca2þ] rises into the micromolar range. Experiments monitoring exocytosis from single b cells support the motion that the RRP is small, easily depletable and requires constant replenishment by processes that are cAMP, MgATP and temperature dependent. Three approaches have been used. In the first approach, the cell is rapidly depolarized, in the presence versus absence of a modulator, until there is no further increase in capacitance. An estimate of the total number of granules exocytosed provides a measure of RRP, while the time to recovery of full response provides a measure of the refilling rate. Then, a rough estimate of size and dynamics of vesicle pools, in the presence versus absence of a modulatory factor, is obtained by examining the time course of release after introduction of a given concentration of Ca2þ into cell. A final approach is to measure the capacitance increase, in the presence versus absence of the modulator, in response to instantaneous “uncaging,” by flash photolysis, of Ca2þ bound to a chelator introduced into the cytosol, or “uncaging” a fraction of the modulator, now

611

bound to a chelator, in the presence of a fixed level of cytosolic Ca2þ. Sample experiments demonstrating that stimulators of cytosolic cAMP enhance initial Ca2þ-entrydependent, depolarization-evoked release, as well as maintain release with repeated depolarization, all with little attendant change in Ca2þ entry are shown in Fig. 34.7A. Sample experiments demonstrating that cAMP enhances both the initial maximum rate of exocytosis and the total exocytosis in cells dialyzed against a pipette with fixed concentrations of Ca2þ are shown in Fig. 34.7B. More recently, similar experiments using all three approaches have been performed at varying cytosolic levels of MgATP. ATP appears to modulate RRP as well. Lastly, to be sure, anticipation of future high rates of release requires activity-stimulated, energy-dependent insulin synthesis followed by processing in specialized membrane compartments. With prolonged continuous stimulation, b cells undergo several phases of insulin release; after an initial spurt of release lasting several minutes, insulin secretion wanes and then, beginning 10e15 min later, rises to a plateau that is sustained for hours. Also, islets rechallenged with glucose several hours after an initial brief bout of secretion display greater “peak” insulin release on the second round of stimulation and do this with newly synthesized insulin. Thus islets display a crude but effective form of “memory” for previous stimulation. As activity-stimulated protein synthesis can be triggered by changes in cell Ca2þ, cAMP and ATP levels, their relative contributions to synthesis of competent new insulin granules is receiving intense scrutiny.

IIC. The Paradox of StimuluseSecretion Coupling in the Glucagon-Secreting a Cell Alpha cells secrete glucagon at preprandial levels of glucose (2e3 mM); secretion is further enhanced by amino acids such as arginine and depressed by higher levels of glucose. Curiously, under some conditions, the glucagon secretion is stimulated by tolbutamide and inhibited by diazoxide, both at concentrations that alter insulin secretion from b cells. So, do a cells have Kþ(ATP) channels and, if so, do these channels contribute to a-cell function? In the presence of 0e3 mM glucose, a cells generate spontaneous Naþo- and Ca2þ o -dependent action potentials from their resting potential of z60 mV. They hyperpolarize on their exposure to increased glucose (5e20 mM); they depolarize and show enhanced spike frequency on exposure to 10 mM arginine. Under voltage-clamp control, depolarization of the a cell results in Ca2þ-entry-dependent increases in cytosolic Ca2þ and increases in membrane capacitance, the latter enhanced by agents that increase cytosolic cAMP (e.g. b2 adrenergic agonists). Single-channel and whole-cell patch-clamp recording from a cells reveals inward rectifier Kþ currents that have

612

SECTION | V

similar single-channel conductance and kinetics to those in b cells as well as similar Kd for inhibition by ATP and activation by MgADP and PIP2. In situ hybridization identifies Kir6.2 and SUR1 subunits of characteristic of b-cell Kþ(ATP) channels on glucagon-bearing cells; in fact, the densities of these subunits are even higher than those in b cells. With apparently functional Kþ(ATP) channels in place, the a cell’s inability to respond to glucose or metabolic inhibition is probably related to their extraordinarily high cytosolic [ATP] and [ATP]/[ADP] ratio that remains virtually unchanged after increases in extracellular glucose. (a Cells are much less efficient transporters and metabolizers of glucose than are b cells.) In contrast, the a cell’s response to arginine may be related to the depolarizing effect of the electrogenic transport of this cationic amino acid coupled to the low threshold for excitability of this cell type (i.e. low resting membrane conductance and abundance of low-voltage-activated [T-type] Ca2þ channels). (Arginine also depolarizes b cells without closing Kþ(ATP) channels, although it usually does not enhance electrical activity unless suprathreshold concentrations of glucose are also present.) However, the crucial element of physiological regulation of the a cell, the inhibition of its electrical activity and secretion by glucose, remain unexplained on the basis of Kþ(ATP) channel activity. It is possible that the inhibitory effect of glucose results largely from a paracrine interaction within the islet. a Cells display a robust g-aminobutyric acid (GABA) activated Cl current which, when stimulated, can abolish arginine-enhanced electrical activity. b Cells are known to secrete GABA along with insulin. Hence secretagogue-induced b-cell exocytosis might be the “suppressor” of a-cell activity.

IID. Implications for Pathophysiology and Therapeutics Diabetes mellitus, a disease characterized by hyperglycemia and dysregulation of metabolite use and culminating in multiorgan system damage, comes in two general varieties. The insulin-dependent, ketosis-prone variety (IDDM) is usually an autoimmune disease of rapid onset, characterized by destruction of b cells as a result of massive assault by antibodies and cytokines. In contrast, the noninsulin-dependent, non-ketosis-prone variety (NIDDM) is a spectrum of diseases characterized by increasing insulin resistance coupled with poorly timed, inadequate insulin secretion. One of its earliest indicators of this condition is the loss of the transient “first phase” of glucose-induced insulin secretion, thought to be “primed” by acetylcholine released by the vagus nerve and GLP-1 released by enterochrommaffin cells. Accompanying this is a “compensatory” increase in insulin release during the prolonged “second phase.” Unfortunately, this contributes to

Synaptic Transmission and Sensory Transduction

downregulation or “tonic tune out” of insulin receptors. Possible defects in NIDDM have been proposed for every link along the b-cell’s stimulusesecretion coupling cascade (i.e. from reduced affinity of glucose transporters to reduced efficiency of intracellular handling of Ca2þ). A recently explored model for NIDDM features insulin resistance of the b cell with chronically decreased release of Ca2þ from intracellular (ER) stores. No doubt, the single cell approaches outlined above will be increasingly brought to bear to dissect out cellular mechanisms underlying NIDDM. Those approaches may be expected to spur the development of more “physiologically targeted” pharmacotherapy for NIDDM, including carefully timed, pre-prandial administration of (1) GLP-1 and other gutsecreted incretins and (2) highly selective sulfonylureatype agents of shorter onset and duration of action than those currently marketed. To be sure, two realizations, (1) that a cells contain sulfonylurea-inhibited Kþ(ATP) channels possibly of identical structure, and (2) that constant activation of a cells coupled with waning of b-cell function, may contribute to the progressive sulfonylurea insensitivity, are now promoting synthesis of hypoglycemic drugs more selectively targeted at b-cell function. It is worth noting that sulfonylureas also modify the gating of some Cl conductance channels in b cells as well as the activity of Naþ-Kþ ATPase and the efficacy of depolarizationesecretion coupling in a manner similar to the effects of protein kinase C enhancement. As mentioned above, the NBFs of SUR resemble those of CFTR. Spare CFTR subunits appear to interact with a variety of Cl channels and even with the epithelial Naþ channel (EnaC) in epithelial cells. Do spare SUR subunits likewise interact with integral membrane or membrane associated proteins in b cells to modulate a variety of events in stimulussecretion coupling?

III. METABOLIC SENSING AS PROTECTION FROM HYPOMETABOLIC INJURY The hyperpolarization and increased Kþ(ATP) channel activity displayed by b cells exposed to metabolic inhibitors that reduce cytosolic [ATP]/[ADP] suggests that a safeguard against hypoxic damage of an excitable cell might be hypoxia-induced opening of Kþ(ATP) channels. This mechanism appears to apply to skeletal and cardiac myocytes and to some neurons. As in b cells, in these cells metabolically sensitive channels are very often Kþ(ATP) channels consisting of Kir and SUR subunits, though often the SUR subunit (SUR2) has much lower affinity for some sulphonylureas than does SUR1. In addition, in skeletal myocytes, which produce lactate under hypoxic conditions, the Kþ(ATP) channel displays pHi sensitivity that is the reverse of Kþ(ATP) channels in b cells; in these myocytes,

Chapter | 34

StimuluseResponse Coupling in Metabolic Sensor Cells

intracellular acidosis vigorously opens Kþ(ATP) channels even at fixed [ATP]/[ADP]. Recent evidence suggests that, in highly active regions of the brain, those cell types with higher density of Kþ(ATP) channels show most rapid excitability block in response to hypoxia and survive it best. However, what may be an adaptive feature to individual cells may have disastrous consequences for the entire organism. In skeletal muscle, a drop in tension development by some motor units results, via spinal reflex, in recruitment of other less active motor units. More extended fatigue due to inexcitability may preserve enough ATP to maintain sarcolemmal integrity and avert leak of myoplasmic contents such as myoglobin. (The latter condition, known as rhabdomyolysis, is the chief cause of extended incapacity and systemic illnesses, such as with acute renal failure, seen after intense bouts of exercise.) However, in contrast, in cardiac ventricle, block of excitability or excitation contraction coupling in one part of the electromechanical syncitium may predispose to an arrhythmia or severely dyskinetic contraction, while in neurons, excitability block can result in a depressed level of central consciousness and even coma. As an example of hypoxia-induced excitation block, let us examine block of excitationecontraction coupling in the cardiac ventricular myocyte. Historically, this phenomenon was key to the original discovery of the Kþ(ATP) current. In these cells, deprivation of substrate or oxygen, or inhibition of substrate metabolism, was found to result in shortening of Ca2þ o -dependent plateau phase of the action potential with attendant abbreviation or block of generation of contractile force. This was found to have occurred prior to detectable changes in membrane potential or voltagedependent Naþ and Ca2þ currents and it was accompanied by major augmentation of resting Kþ conductance. Critically, plateau phase shortening and reduced contraction were reversed by intracellular injection of MgATP. As shown in Fig. 34.8, the key to this phenomenon is very basic. The plateau phase of the ventricular action potential represents a delicate moment-to-moment balance between two opposing tendencies. These are (1) the “depolarizing tendency” of the voltage-dependent, but slowly inactivating Ca2þ conductance and (2) the “repolarizing tendency” of the sum of a variety of Kþ currents that are either slowly activating or slowly emerging from “inward rectification” (i.e. polyamine or Mg block). Increases in background Kþ current provided by opening of Kþ(ATP) channel tip the balance in favor of repolarization, as soon as the huge inward Naþ current, underlying the rapid upstroke of the action potential, wanes. However, shortening of the broadly propagating action potential may reduce the refractory period and promote local impulse re-entry or rebound excitation. In addition, the period of reoxygenation may enhance arrhythmogenicity by promoting large transient inward currents (Iti) which result in spontaneous

613

0 Vm (mV) –85 GK+(IR) GCa2+ GK+(DR) GK+(ATP)

Tension 250 ms

FIGURE 34.8 Kþ(ATP) channels and cardiac excitability. A steady-state enhancement of Kþ(ATP) conductance (GKþ(ATP)), resulting from metabolic inhibition, shortens the time courses of the ventricular action potential and twitch tension and alters the time courses and magnitudes of the underlying ionic conductances. Solid lines indicate control; dashed lines indicate effects of metabolic inhibition.

depolarizations. Hence, it would appear to be better for the ventricle to anticipate rather than react to metabolic deprivation. Physiologically, this is exactly what happens: ventricular hypoxia results in dilation of coronary arterioles leading to greater supply of oxygen and metabolites (see Section VA). However, as often happens in critical physiological processes, there is yet another factor at work. Short bouts of hypoxia, with opening of Kþ(ATP) channels, may actually “precondition” myocytes and ameliorate some of the effects of subsequent, more prolonged bouts. In that case, does prevention of hypoxia-induced opening of Kþ(ATP) channels, as might occur in the presence of a sulfonylurea, predispose to hypoxia-induced cell injury? Controversy still surrounds this issue. It is worth mentioning other mechanisms that might contribute to ischemia-induced contractile failure. These include (1) altered [Ca2þ]i “homeostasis,” (2) changes in the binding affinity of the contractile apparatus for Ca2þ induced by accumulation of acid equivalents and (3) activation of other Kþ channels, such as muscarinic and G-protein gated Kþ channels, by metabolic intermediates generated during hypoxia.

IV. STIMULUSeSECRETION COUPLING IN CAROTID CHEMORECEPTOR CELLS Neural control of respiratory drive is critical in maintaining physiologic levels of plasma O2, CO2 and Hþ. In the rhythmic, neuromuscular process of breathing, trains of action potentials, generated by pacemaker cells in the CNS,

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SECTION | V

trigger motor neurons and activate periodic contractions of the inspiratory muscles (diaphragm and intercostal muscles). This action expands the chest, thereby sucking air of high-O2, low-CO2 content into the lungs. Gas exchange consists of net CO2 diffusion from capillary to adjacent lung air spaces (alveoli) and net O2 diffusion from alveoli to the capillary. Relaxation of inspiratory muscles, sometimes combined with the contraction of expiratory muscles, expels the air of high CO2, low O2 content from the lungs. A major stimulus for altering the pattern of respiratory drive is a drop in the partial pressure of O2 dissolved in plasma (i.e. plasma pO2); a secondary stimulus is a rise in plasma pCO2 or a fall in pH. The carotid body is the organ that senses changes in plasma pO2, pCO2 and pH and mediates changes in CNS respiratory drive. Located at the bifurcation of the carotid artery, a branch of the aorta, the carotid body consists of a central core of chemoreceptor (or glomus) cells; these originate from the neural crest. Glomus cells synapse on dendritic endings of sensory nerve fibers that comprise the carotid sinus nerve travelling into the CNS. The glomus cell core is surrounded by more superficial glial, or sustentacular, cells, as well as by a dense network of highly porous capillaries. The glomus cell is the site of transduction of changes in plasma pO2, pCO2 and pH into changes in electrical activity of the afferent carotid sinus nerve. Decreases in pO2, as well increases in pCO2 or decreases in pH, lead to an increase in release of dopamine and probably to an increase in as-yet-unidentified transmitters from glomus cells, as well as to an increased action

Synaptic Transmission and Sensory Transduction

potential frequency in the dopamine-sensitive carotid sinus nerve. This contributes to increased respiratory drive.

IVA. Role of O2-Sensitive KD Channels in Transduction of the Hypoxic Stimulus Two divergent views have emerged concerning chemotransduction of hypoxia by glomus cells (Fig. 34.9A). The first theory proposed that hypoxia induces cell depolarization, followed by opening of voltage-gated Ca2þ channels, Ca2þ influx and synchronized exocytotic release of dopamine. This might give rise to sufficiently large excitatory postsynaptic potentials in the dendritic regions of single sinus nerve fibers to trigger propagating action potential. An alternative view proposed that hypoxia induces quantal release of transmitter in a Ca2þ-dependent manner that is independent of voltage-gated Ca2þ entry but dependent on the discharge of Ca2þ from intracellular stores. The latter should increase asynchronous quantal release of transmitter, generating a rise in the frequency of miniature excitatory postsynaptic potentials and should enhance ongoing electrical activity in the low-threshold carotid sinus nerve fiber. These contrasting viewpoints have arisen from data generated with different preparations from different species. There are several lines of evidence supporting the classical scheme for “depolarizationesecretion coupling” in glomus cells. First, glomi are electrically excitable sensory cells and contain HVA Ca2þ currents, delayed rectifier Kþ currents and, in some cases, voltage-dependent

FIGURE 34.9 Stimulusesecretion coupling in carotid chemoreceptor cells. (A) O2-induced transmitter release. Key steps in classical scheme of depolarizationesecretion coupling are outlined. Decreased pO2 results in decreased O2 binding to the intracellular receptor coupling to a specific voltagedependent Kþ channel (1). This results in the closure of the channel (2), cell depolarization (3), increased probability of the opening of HVA Ca2þ channels (4), increased calcium entry (5) and the enhanced rate of fusion of dopamine-containing vesicles with the plasma membrane (6). Alternatively (see path beginning with 1a), hypoxia-induced rundown of the mitochondrial proton gradient might result in reduced ATP generation, slow release of Ca2þ from mitochondria or other stores and, ultimately, a slow rise in spontaneous quantal release. (B) CO2 and H-induced transmitter release. Increased pCO2 or H entry (1b) results in decreased cytosolic pH and stimulation of the Naþ-H exchanger (2). The resultant influx in Naþ stimulates the Ca2þ-Naþ exchanger (3). This, in turn, increases Ca2þ influx (4) and Ca2þ-dependent exocytosis (5).

Chapter | 34

StimuluseResponse Coupling in Metabolic Sensor Cells

Naþ currents. Second, in some isolated cell preparations, glomus cells respond to hypoxia by depolarizing and generating action potentials. (However, in other preparations, glomus cells fire action potentials only in response to current injection or release of the cell from sustained hyperpolarization. These cells show no change in passive electrical activity in response to hypoxia.) Third, in glomus cells that fire in response to hypoxia, depolarization increases both cytosolic Ca2þ, measured with intracellular dyes, and dopamine release, measured by amperometry. In these cells, depolarizationesecretion coupling is reduced by exposure to blockers of the HVA-type Ca2þ channels or by reduction in extracellular Ca2þ. A very exciting development in chemoreceptor physiology consistent with the depolarizationesecretion coupling scheme outlined above is the discovery that, in whole-cell recordings, lowering ambient pO2 selectively and reversibly reduces the outward Kþ current flowing through delayed rectifier Kþ(DR) channels of the glomus cells. Reducing pO2 from 160 mmHg to 90 mmHg reduces peak Kþ(DR) current by z30%. This effect is not dependent on the concentrations of ATP (0e3 mM) or Ca2þ (<1 nMe0.5 mM) in the pipette. In outside-out patches of membrane, reduced ambient pO2 reversibly decreases the probability of opening (Po) of a 20-pS Kþ(DR) type channel (Fig. 34.10). The first reports suggested that in vitro this channel was most responsive to changes in O2 over the range of pO2 values between 110 and 150 mmHg, in contrast to the intact carotid sinus nerve that actually fires optimally at pO2s <70 mmHg.

(A)

615

However, the O2 sensitivity of the channel can be shifted into a more physiological range by the addition of a membrane-permeant analog of cAMP and recent data have shown Kþ(DR)-type channel activity pO2s ranging from 20 to 150 mmHg. In whole-cell, current-clamp recordings, the effect of a reduction in pO2 is (1) an increase in the rate of cell depolarization on release from maintained hyperpolarization, (2) an increase in the frequency of spike activity in the resultant short train impulses and (3) an increase in AP overshoot. Hence, in glomus cells with some intrinsic spontaneous electrical pacemaker activity, O2-dependent changes in Kþ current could alter the frequency of action potentials and large Ca2þ transients, thereby increasing [Ca2þ]-dependent transmitter release onto the afferent nerve. In glomus cells with little automaticity that maintain resting potentials of between 50 and 40 mV, closure of Kþ(DR)type channels could still cause steady-state depolarization “generator potential” and trigger electrical activity de novo. More extensive perforated-patch recordings will be needed to determine the range of electrical activity patterns exhibited by these cells. These data suggest that O2 maintains the activity of a delayed rectifier type Kþ channel through a novel gating mechanism. Several possibilities for the molecular mechanism of O2 transduction and its relationship to channel gating have been suggested, but there is no definitive evidence for any of them. The first is that a heme protein, analogous to a subunit of the O2-carrying protein hemoglobin, is attached to the channel, or to a functional subunit

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Vc(mV)

–80

FIGURE 34.10 Origin of low-pO2-induced enhancement of electrical activity in carotid glomus chemoreceptors. (A) Reduction in ambient pO2 results in increased frequency of spontaneous AP activity, partly due to reduced rate of repolarization of the AP. Hence, any background depolarizing current might be more effective in raising the membrane potential toward threshold for firing a spike. (Reproduced from The Journal of General Physiology, 1989, Vol. 93, p. 979, Fig. 14. By permission of the Rockefeller University Press.) (B) Reduction in ambient pO2 reduces the probability (Po) that a Kþ(DR) channel will open on depolarizing the cell from 80 to þ20 mV. Lowest traces in each column represent the average of many individual channel current traces. (Idealized traces adapted from Ganfornina and Lopez Barneo, 1991.)

616

SECTION | V

of the channel. In this way, a change in configuration of the protein, on losing O2, would alter channel gating. The second is the presence of an oxidase which, on reduction of cytosolic pO2, produces less hydrogen peroxide (H2O2), consequently altering the concentration of redox intermediates and thereby of channel conformation. The alternative view of chemotransduction of hypoxia in glomus cells is that the rise in intracellular Ca2þ necessary for dopamine secretion is due to Ca2þ release from intracellular stores and that voltage-activated currents play only a secondary role, perhaps allowing replenishment of intracellular Ca2þ stores. This hypothesis has arisen from data generated from both isolated glomus cells and in situ carotid cell bodies. Mitochondrial poisons, such as cyanide, produce a condition known as histotoxic hypoxia, which mimics true hypoxia in stimulating carotid body nerve activity and respiratory drive. These poisons produce increases in intracellular Ca2þ that are unaffected by pharmacologic maneuvers designed to abolish or enhance the action potential, hence suggesting that increases in cytosolic Ca2þ arise from intracellular stores. Hypoxia and mitochondrial poisons produce a rise of cell NAD(P)H. Additionally, studies from in situ carotid bodies have shown (1) that action potentials induced in carotid sinus nerve by hypoxia are not blocked by drugs that block outward Kþ currents and (2) that whole cell glomus membrane resistance is not changed in response to hypoxia. A proposed mechanism whereby hypoxia induces intracellular Ca2þ release is that low O2 decreases mitochondrial efficiency, perhaps slowing electron transfer in the respiratory chain, and reducing the proton gradient across the mitochondrial inner membrane. A consequence of this could be the release of Ca2þ from mitochondria or decreased ATP production, resulting in slow Ca2þ release from other intracellular stores (e.g. the ER). For this mitochondrial-based hypothesis to work, it might be necessary that O2-trapping properties of mitochondrial cytochrome oxidase and Ca2þ-storage properties of mitochondria in glomus cells differ significantly from their counterparts in other tissues.

IVB. Transduction of Increased pCO2 and Decreased Plasma pH Glomus cells rapidly equilibrate pHi and pHo. Hence, it is probable that with acidic stimuli the cell is actually sensing a fall in pHi. But how does an increase in [Hþ]i result in Ca2þ o -dependent transmitter release, especially as Hþi-induced release is insensitive to blockers of HVA Ca2þ channels and HVA Ca2þ channels are often inhibited by reduction in pHi? The proposed link between increased [Hþ]i and Ca2þ entry in promoting dopamine release is that increased [Hþ]i activates an Naþo-Hþi exchanger which, in turn, elevates Naþ and recruits a Naþ-Ca2þ

Synaptic Transmission and Sensory Transduction

exchanger. The activity of the latter exchanger is often augmented by cell depolarization; this may, in fact, occur because a drop in pHi reduces Kþ(DR) type Kþ current much as a drop in pO2 does. An increase in background “spontaneous” quantal release of dopamine, which is not phase-linked to action potential activity, could increase steady-state depolarization of carotid nerve fibers and, perhaps, augment on-going impulse activity (see Fig. 34.9B). In considering this scheme, it should be cautioned that thus far, good evidence exists only for activation of an Naþ-Hþ exchanger. To be sure, respiratory drive varies significantly according to age and physiologic status. Human fetuses do not exhibit regular respiratory movements in utero, whereas newborn infants often display periodic breathing different from breathing patterns seen in older children and adults. Disease states produce abnormal breathing patterns, such as CheyneeStokes breathing, the diamond-shaped changes in respiratory excursions followed by a period of nonbreathing (apnea), seen in stroke, congestive heart failure and chronic hypoxia. In infants, prolonged apnea despite persistent hypoxia or hypercarbia can culminate in sudden infant death syndrome (SIDS). Recent data suggest that carotid body glomus cells from animals raised under hypoxic conditions show blunted electrophysiologic responses to hypoxia compared to cells from healthy animals. Disorders of breathing such as apnea and SIDS are seen in higher frequency in infants at increased risk for chronic hypoxia due to conditions such as prematurity or second-hand cigarette smoke exposure. Studies on the mechanism of changes in carotid body chemoreception in normal development and in various disease states should be a fruitful area for further inquiry.

V. STIMULUSeCONTRACTION COUPLING IN VASCULAR SMOOTH MUSCLE CELLS VA. Hypoxic Vasodilation of Coronary and Mesenteric Vessels Smooth muscle cells of resistance arterioles are very sophisticated metabolic sensors. In fact, they encompass a complete vasodilator reflex system in a single cell. In response to a drop in ambient pO2, these cells reduce their tension generation. This results in vessel relaxation and the local redistribution of O2 supply to O2-consuming tissue. As in the case of the cardiac myocyte, a key to the explanation of this phenomenon is the increase in Kþ conductance and membrane hyperpolarization, which precedes the fall in tension. It is well appreciated that resistance vessels in the coronary and mesenteric (gut) circulation display resting tone. Their myocytes maintain a resting potential of 40 to 50 mV and have an abundance of Kþ(ATP) and

Chapter | 34

StimuluseResponse Coupling in Metabolic Sensor Cells

L-type (HVA) Ca2þ channels. In fact, in smooth muscle of mesenteric artery, the Kd value (mM) for ATP-induced closure of Kþ(ATP) channels is roughly double that in cardiac myocytes, in principle making Kþ(ATP) channels of these myocytes more sensitive to hypoxia than the Kþ(ATP) channels in the heart. Under these circumstances, small changes in resting potential would be expected to affect resting tone. A small depolarization (of 5e10 mV) caused by closure of Kþ(ATP) channels should result in increased opening of HVA Ca2þ channels and subsequent vasoconstriction (Fig. 34.11A). In contrast, a small hyperpolarization of 5e10 mV, affected by addition of a Kþ(ATP) channel opener or a dihydropyridine Ca2þ channel antagonist, should reduce resting tone and result in vasodilation. These predictions have been borne out experimentally. A modest drop in microenvironment pO2, insufficient to affect cardiac excitationecontraction coupling, is sufficient to reduce cytosolic ATP in vascular smooth muscle cells, open Kþ(ATP) channels and cause vasodilation. (In bulk cardiac muscle in situ, preferential opening of smooth muscle Kþ(ATP) channels might be further augmented by the release of adenosine by active cardiac tissue; extracellular adenosine has been shown to activate Kþ(ATP) channels via a G-protein-dependent mechanism.) Hence preferential hypoxia-induced arteriolar dilation, with its attendant increases in local O2 delivery, may spare cardiac myocytes the risk of hypoxia-induced alterations in excitationecontraction coupling. This scheme might

(A)

617

work for the regulation of local blood supply to the brain and gut as well as to the heart.

VB. Hypoxia-Induced Vasoconstriction of Pulmonary Vessels In contrast to coronary, mesenteric and cerebral vessels, pulmonary artery and its smooth muscle constrict in response to a drop in pO2. This hypoxic pulmonary vasoconstriction (HPV) constitutes an adaptive response in the lung bed because it ensures that areas of the lung that are poorly oxygenated will receive less blood flow; the extra blood flow is “redirected” towards better oxygenated areas to optimize gas exchange. HPV is critical in fetal pulmonary development when the maturing air spaces are filled with secreted fluid rather than inspired air. Under these conditions, HPV maintains the relatively high pulmonary vascular resistance that shunts venous return around the low flow pulmonary bed, through the ductus arteriosus of the cardiac septum and into the left heart. With inflation of the newborn’s lungs to air containing substantially higher pO2, O2-induced pulmonary vasodilation occurs, thereby promoting blood flow through the pulmonary circulation. In the adult, HPV is useful in maintaining moment-tomoment matching of local ventilation to perfusion; this reduces the risk of hypoxia that can occur when a portion of the lung is poorly inflated. However, when the areas of local poor ventilation are widespread, or with chronic hypoxia (such as at high altitudes), chronic HPV is accompanied by

(B)

Ca2+

K+

ATP

Low Ca2+

–50 mV –35 mV

High Ca2+ Ca2+ ↓pO2

–50 mV

↓pO2

K+

Low Ca2+ –30 mV

High Ca2+

FIGURE 34.11 Hypoxia alters stimulusecontraction coupling in vascular myocytes. (A) Model of hypoxia-induced vasodilation in cardiac, cerebral and mesenteric vessels. In normoxic conditions, Kþ(ATP) channels are largely closed, but voltage-dependent Ca2þ channels are open, Vm z35 mV, and cytosolic Ca2þ is low. With a decrease in pO2, cytosolic ATP levels drop, resulting in the opening of Kþ(ATP) channels, repolarization to z50 mV, closure of Ca2þ channels, a drop in cytosolic Ca2þ and myocyte relaxation. (B) Model of hypoxia-induced vasoconstriction in pulmonary arteries. In normoxic conditions, Vm z50 mV, O2-sensitive Kþ channels are largely open and voltage-dependent Ca2þ channels are largely closed. Hence, cytosolic Ca2þ levels are low and there is little resting tension. With a decrease in pO2, O2-sensitive Kþ channels close, resulting in depolarization, opening of Ca2þ channels, Ca2þ entry and myocyte contraction.

618

SECTION | V

smooth muscle proliferation (i.e. “work hypertrophy of muscle”). The end result is the development of increased resistance and pressure in the total pulmonary vascular bed. This “pulmonary artery hypertension” imposes an increased “afterload” on the right ventricle, thus becoming a stimulus for its hypertrophy. What cellular mechanisms support HPV? Pulmonary artery myocytes maintain a low resting tension, a Vm of z40 mV and a resting cytosolic [Ca2þ] of <100 nM. They respond to progressive hypoxia (e.g. a slow fall in pO2 from 150 to 15 mmHg) with a 15-mV depolarization, which is not affected by changes in [Ca2þ]o, followed by Ca2þ o dependent increases in both cytosolic Ca2þ and tension. Given that Vm in these cells shows a Nernstian relationship to [Kþ]o, these results suggest that membrane depolarization is due to a large decrease in Ca2þ o -independent resting membrane conductance (e.g. GK). However, depolarization ultimately results in a small increase in GCa. This is sufficient to provide entry to trigger contraction. Tension is maintained so long as Ca2þ entry through voltage-gated Ca2þ channels exceeds Ca2þ efflux via the Naþ-Ca2þ exchanger. Whole-cell voltage-clamp experiments have provided good evidence that a voltage-activated, delayed rectifier type Kþ current seen at Vm values positive to 50 mV and, hence, open at rest, is significantly inhibited by hypoxia. Depolarization of a few mV would be sufficient to open HVA Ca2þ channels and result in vasoconstriction (see Fig. 34.11B). Hence, as in the carotid chemoreceptor, hypoxia-induced reduction in the activity of Kþ(DR) type channels in a cell with a very low background Gm, appears to be responsible for depolarization and stimuluseresponse coupling. An important paradox, which remains to be explained, is how pulmonary artery myocytes manage to “hide” Kþ(ATP) channels, known to be present in the sarcolemma, during hypoxia. In these cells, Kþ(ATP) channel openers abort high [Kþ]o-induced tension increase, while sulfonylureas enhance tension generation, as they do in resistance vessels, yet hypoxia does not open these channels. Are mechanisms similar to those in pancreatic a cells at play here?

VI. COUPLING OF OXYGEN SENSING TO RED CELL PRODUCTION BY ERYTHROPOIETIN-SECRETING CELLS The ultimate metabolic sensors are the erythropoietin-(epo-) secreting cells of the renal interstitium and liver. These modified fibroblasts respond slowly to hypoxia by increasing their synthesis and constitutive secretion of epo which, in turn, serves as a maturation factor for erythrocyte precursors in bone marrow. Epo binds to a membrane receptor, which serves as a scaffold for the activation of numerous growth and division signaling pathways

Synaptic Transmission and Sensory Transduction

including JAK/STAT and ras/MAP kinases; epo also increases the expression of Bcl-Xl, an anti-apoptotic peptide. Mature erythrocytes are virtually cytoplasmic bags of hemoglobin (Hb) which bind O2. In high pO2 and pH environs (arterioles), Hb binds O2, while at lower pO2 and pH environs, Hb gives off its O2. In this way Hb ferries O2. The net effect of this receptoreeffector cell pair is that hypoxia can ensure an increase in blood O2 carriage for up to 120 days, the life span of an erythrocyte. A clue to the mechanism of O2-reception epo-producing cells is that their exposure to cobalt, nickel and manganese mimics the effects of hypoxia, whereas exposure to metabolic (mitochondrial) inhibitors such as cyanide cannot. This suggests that the O2 sensor is an Hb-type molecule, which can be locked into a deoxygenated form, rather than a molecule involved in red-ox transfer, which uses O2 as the final acceptor. The epo gene has been cloned and its cis-regulatory element has been found to bind a hypoxically inducible factor (HIF) that can transfer from the cytoplasm into the nucleus (Fig. 34.12A). Epo regulation is

(A)

fibroblast HIF O2

mature erythrocytes epo

(B) REG REP

bone marrow stem cells

myoblast epo tet

REG

FIGURE 34.12 Native and bioengineered stimulusesynthesis coupling in erythropoietin production. (A) Stimulusesynthesis coupling in renal interstitial fibroblasts and hepatic Ito cells likely consists of triggering of epo gene transcription by a hypoxically induced factor (HIF), which is freed to enter the nucleus after O2 dissociates from a heme pigment receptor. Newly synthesized epo is packaged and constitutively released. The kidneys are an excellent primary site for an O2 sensor because under control conditions they are perfused with z20% of the cardiac output. Circulating epo binds to erythroblasts (erythrocyte stem cells) in bone marrow and triggers their proliferation and maturation to enucleated erythrocytes that circulate as hemoglobin-packed, O2 carrying sacs for an average of 120 days. (B) Genetically engineered stimulusesynthesis coupling for epo production in skeletal myocytes. Myoblasts are co-transfected with (i) a regulator gene (REG) under the control of mature muscle cells and (ii) a reporter gene (REP) coding for epo and under control of a tetracycline-sensitive product of the regulator gene. These cotransfected myoblasts are injected into muscle where they fuse with multinucleated myocytes. Under the control of exogenously added tetracycline (tet), myocytes are induced to synthesize and secrete epo.

Chapter | 34

StimuluseResponse Coupling in Metabolic Sensor Cells

an example of a widespread system for gene induction by O2; this system includes pathways for induction vascular growth factors and changing isoforms of glycolytic enzymes in metabolically active cells. Erythropoietin production wanes in chronic renal failure, as scar tissue replaces metabolically active cells. While epo can be replaced by weekly injection, the recombinant molecule is costly. To circumvent this, significant effort has recently been made to engineer cells that might tonically secrete epo on exposure to a simple, gene-activating stimulus. Primary myoblasts are cotransfected with two retroviruses and then injected into muscle where they fuse with mature, multinucleated myocytes. One of the transfecting retroviruses contains a regulator, or reverse transactivation, gene (REG) designed to be under the control of a promoter only activated in mature myocytes. The second retrovirus contains the reporter gene (REP), coding for epo, but under control of both the protein coded by REG and tetracycline (tet). Once in the mature muscle, the REG protein and tet control can tonically activate the epo gene. Significantly, when mice are injected with these engineered myoblasts, epo secretion can be switched on and off over months, depending on the availability of tet in the drinking water.

ACKNOWLEDGMENTS Original data traces shown here were obtained in our laboratory through the support of NIH grant DK37380. I thank Todd Owyoung for preparing Figures 34.2 and 34.6, David Bryant for preparing Figures 34.9e34.11 and Drs David Pressel and David Barnett for sharing insights and delicate turns of phrase during the preparation of a prior version of this chapter. This chapter is dedicated to the memory of Golda Hazak, wit and sage of Kiryat Yam.

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