Electrical perception of "death message" in chara: analysis of rapid component and ionic process

Electrical response upon wounding was analyzed in Chara corallina. A specimen comprising two adjoining internodal cells was prepared. One cell (victim cell) was killed by cutting and any changes in the membrane potential of the neighboring cell (the receptor cell) were measured. Upon cutting the victim cell, the receptor cell generated four kinds of depolarizations: (1) rapid component, (2) slow and long-lasting component, (3) action potential and (4) small spike. Rapid and slow components were observed in most cells. On the other hand, the action potential and small spike were not always ubiquitous among specimens. When an action potential was generated just after cutting the victim cell, the rapid component could not be observed due to masking by the action potential. It was suggested that both rapid and slow components were generated at the nodal end. On the other hand, action potentials were thought to be generated at the flank of the receptor cell. High turgor pressure of the cell was necessary for generating both rapid and slow components. Experiments under K(+)-induced depolarization unequivocally showed that the Cl(-) channel at the nodal end of the receptor cell was activated upon cutting the victim cell.     

Electrical perception of the 'death message' in Chara: analysis of K+-sensitive depolarization

Wounding electrical responses were studied in Chara corallina. Specimens comprising two adjoining internodal cells were prepared. When one cell (victim cell) was cut, the other cell (receptor cell) generated four kinds of depolarization: (i) rapid depolarization; (ii) long-lasting depolarization; (iii) action potentials; and (iv) small spikes. In the present study, attention was focused on the long-lasting depolarization. A decrease in the electrical resistance suggested activation of ion channel(s). The duration of the depolarization was sensitive to the external ions. K(+) significantly prolonged the depolarization. On the other hand, Ca(2+), Mg(2+) and Na(+) had a tendency to shorten the duration prolonged by K(+). When a nodal end was continuously flushed with a medium lacking K(+), the depolarization was significantly shortened. Treatment of the nodal end with artificial cell sap for 2 min induced a long-lasting depolarization similar to that induced by cutting the victim cell. These findings suggested the involvement of K(+) released from the victim cell in generating the long-lasting depolarization by the receptor cell.     

Blue Light Pulse-Induced Transient Changes of Electric Potential and Turgor Pressure in the Motor Cells of Phaseolus vulgaris L.

Blue light induces both depolarization of membrane potentialin the motor cell and turgor movement in the laminar pulvinusof bean plant. This paper describes the changes of electricpotential and turgor pressure induced in Phaseolus vulgarisL. by blue light pulses. A transient depolarization of membranepotential as large as 40 mV was induced by a short pulse of15 s blue light in motor cells of the laminar pulvinus. Thischange was not an action potential because of the absence ofa refractory period and threshold. The magnitudes of the responsewere dependent on the fluence of light. The response was long-lived,indicating that continuous input of light energy is not requiredfor a sustained response. The potential change was always followedby a transient turgor movement of the pulvinus. A molecular mechanism similar to a model postulated for theblue light response of stomata may operate in the motor cell.However, the direction of the electrical response to blue light(depolarization) in the motor cell was the opposite of thatin the guard cell (hyperpolarization). Turgor change of themotor cell by blue light was also opposite in direction (decrease). (Received February 19, 1988; Accepted June 28, 1988)     

Mechanosensory ion channels in charophyte cells: the response to touch and salinity stress

Mechanosensitive (MS) ion channels are activated by mechanical stress and then transduce this information into electrical signals. These channels are involved in the growth, development and response to environmental stress in higher plants. Detailed analyses of the electrophysiology in higher plants are difficult because such plants are composed of complex tissues. The large cells of the charophytes facilitate electrophysiological measurements and allow us to study MS ion channels at the level of single cells. We draw parallels between the process of touch-perception in freshwater Chara, and the turgor-regulating response to osmotic shock in salt-tolerant Lamprothamnium. In terms of electrophysiology, these responses can be considered in three stages: (1) stimulus perception, (2) signal transmission and (3) induction of response. In Chara the first stage is due to the receptor potential (RPD), a transient depolarization with a critical threshold that triggers action potentials, which are responsible for stages (2) and (3). Receptor potentials are generated by MS ion channels. Action potentials involve a transient influx of Ca(2+) to the cytoplasm, effluxes of K(+) and Cl(-) and a temporary decrease of turgor pressure. Reducing cell turgor increases sensitivity to mechanical stimulation. In Lamprothamnium, a hypotonic shock produces an extended depolarization that resembles an extended RPD and is responsive to osmotic rather than ionic changes. Like the action potential, a critical threshold depolarization triggers Ca(2+) influx, opening of Ca(2+)-sensitive Cl(-) channels and K(+) channels; effluxes that last over an hour and result in turgor regulation. These processes show us, in primal form and at the level of single cells, how mechanoperception occurs in higher plants. Recent progress in research into the role of MS ion channels in the freshwater and salt-tolerant Characeae is reviewed and the relevance of these findings to plants in general is considered.     

Transduction of pressure signal to electrical signal upon sudden increase in turgor pressure in Chara corallina

By taking advantage of large cell size of Chara corallina, we analyzed the membrane depolarization induced by decreased turgor pressure (Shimmen in J Plant Res 124:639–644, 2011). In the present study, the response to increased turgor pressure was analyzed. When internodes were incubated in media containing 200 mM dimethyl sulfoxide, their intracellular osmolality gradually increased and reached a steady level after about 3 h. Upon removal of dimethyl sulfoxide, turgor pressure quickly increased. In response to the increase in turgor pressure, the internodes generated a transient membrane depolarization at its nodal end. The refractory period was very long and it took about 2 h for full recovery after the depolarizing response. Involvement of protein synthesis in recovery from refractoriness was suggested, based on experiments using inhibitors.     

Inhibition of Programmed Cell Death in Tobacco Plants during a Pathogen-Induced Hypersensitive Response at Low Oxygen Pressure

The hypersensitive response (HR) of plants to invading pathogens is thought to involve a coordinated activation of plant defense mechanisms and programmed cell death (pcd). To date, little is known about the mechanism underlying death of plant cells during this response. In addition, it is not known whether suppression of pcd affects the induction of other defense mechanisms during the HR. Here, we report that death of tobacco cells (genotype NN) infected with tobacco mosaic virus (TMV) is inhibited at low oxygen pressure. In contrast, virus replication and activation of defense mechanisms, as measured by synthesis of the pathogenesis-related protein PR-1a, were not inhibited at low oxygen pressure. Bacterium-induced pcd was also inhibited at low oxygen pressure. However, pcd induced by TMV or bacteria was not inhibited in transgenic tobacco plants expressing the mammalian anti-pcd protein Bcl-XL. Our results suggest that ambient oxygen levels are required for efficient pcd induction during the HR of plants and that activation of defense responses can be uncoupled from cell death. Furthermore, pcd that occurs during the interaction of tobacco with TMV or bacteria may be distinct from some cases of pcd or apoptosis in animals that are insensitive to low oxygen or inhibited by the Bcl-XL protein.     

Transport Systems of Ventricaria ventricosa: Asymmetry of the Hyper- and Hypotonic Regulation Mechanisms

Hyper- and hypotonic stresses elicit apparently symmetrical responses in the alga Ventricaria. With hypertonic stress, membrane potential difference (PD) between the vacuole and the external medium becomes more positive, conductance at positive PDs (Gmpos) increases and KCl is actively taken up to increase turgor. With hypotonic stress, the membrane PD becomes more negative, conductance at negative PDs (Gmneg) increases and KCl is lost to decrease turgor. We used inhibitors that affect active transport to determine whether agents that inhibit the K(+) pump and hypertonic regulation also inhibit hypotonic regulatory responses. Cells whose turgor pressure was held low by the pressure probe (turgor-clamped) exhibited the same response as cells challenged by hyperosmotic medium, although the response was maintained longer than in osmotically challenged cells, which regulate turgor. The role of active K(+) transport was confirmed by the effects of decreased light, dichlorophenyldimethyl urea and diethylstilbestrol, which induced a uniformly low conductance (quiet state). Cells clamped to high turgor exhibited the same response as cells challenged by hypo-osmotic medium, but the response was similarly transient, making effects of inhibitors hard to determine. Unlike clamped cells, cells challenged by hypo-osmotic medium responded to inhibitors with rapid, transient, negative-going PDs, with decreased Gmneg and increased Gmpos (linearized I-V), achieving the quiet state as PD recovered. These changes are different from those exerted on the pump state, indicating that different transport systems are responsible for turgor regulation in the two cases.     

TRPC3 mediates pyrimidine receptor-induced depolarization of cerebral arteries

We tested the hypothesis that TRPC3, a member of the canonical transient receptor potential (TRP) family of channels, mediates agonist-induced depolarization of arterial smooth muscle cells (SMCs). In support of this hypothesis, we observed that suppression of arterial SMC TRPC3 expression with antisense oligodeoxynucleotides significantly decreased the depolarization and constriction of intact cerebral arteries in response to UTP. In contrast, depolarization and contraction of SMCs induced by increased intravascular pressure, i.e., myogenic responses, were not altered by TRPC3 suppression. Interestingly, UTP-evoked responses were not affected by suppression of a related TRP channel, TRPC6, which was previously found to be involved in myogenic depolarization and vasoconstriction. In patch-clamp experiments, UTP activated a whole cell current that was greatly reduced or absent in TRPC3 antisense-treated SMCs. These results indicate that TRPC3 mediates UTP-induced depolarization of arterial SMCs and that TRPC3 and TRPC6 may be differentially regulated by receptor activation and mechanical stimulation, respectively.     

Gradients of turgor, osmotic pressure, and water potential in the cortex of the hypocotyl of growing ricinus seedlings : effects of the supply of water from the xylem and of solutes from the Phloem

To evaluate the possible role of solute transport during extension growth, water and solute relations of cortex cells of the growing hypocotyl of 5-day-old castor bean seedlings (Ricinus communis L.) were determined using the cell pressure probe. Because the osmotic pressure of individual cells (πⁱ) was also determined, the water potential (ψ) could be evaluated as well at the cell level. In the rapidly growing part of the hypocotyl of well-watered plants, turgor increased from 0.37 megapascal in the outer to 1.04 megapascal in the inner cortex. Thus, there were steep gradients of turgor of up to 0.7 megapascal (7 bar) over a distance of only 470 micrometer. In the more basal and rather mature region, gradients were less pronounced. Because cell turgor ≈ πⁱ and ψ ≈ 0 across the cortex, there were also no gradients of ψ across the tissue. Gradients of cell turgor and πⁱ increased when the endosperm was removed from the cotyledons, allowing for a better water supply. They were reduced by increasing the osmotic pressure of the root medium or by cutting off the cotyledons or the entire hook. If the root was excised to interrupt the main source for water, effects became more pronounced. Gradients completely disappeared and turgor fell to 0.3 megapascal in all layers within 1.5 hours. When excised hypocotyls were infiltrated with 0.5 millimolar CaCl₂ solution under pressure via the cut surface, gradients in turgor could be restored or even increased. When turgor was measured in individual cortical cells while pressurizing the xylem, rapid responses were recorded and changes of turgor exceeded that of applied pressure. Gradients could also be reestablished in excised hypocotyls by abrading the cuticle, allowing for a water supply from the wet environment. The steep gradients of turgor and osmotic pressure suggest a considerable supply of osmotic solutes from the phloem to the growing tissue. On the basis of a new theoretical approach, the data are discussed in terms of a coupling between water and solute flows and of a compartmentation of water and solutes, both of which affect water status and extension growth.     

Temporal relationship between cytosolic free Ca(2+) and membrane potential during hypotonic turgor regulation in a brackish water Charophyte Lamprothamnium succinctum

Internodal cells of a brackish water charophyte, Lamprothamnium succinctum, regulate turgor pressure in response to changes in external osmotic pressure by modifying vacuolar concentrations of KCl. An increase in cytosolic concentration of free Ca(2+) ([Ca(2+)](c)) is necessary for the progress of turgor regulation induced by hypotonic treatment. Initial changes in membrane potential and [Ca(2+)](c) upon hypotonic treatment were measured to examine the temporal relationship between the two parameters. Fura-dextran (potassium salt, M(r) 10,000, anionic) that had been injected into the cytosol was used to measure [Ca(2+)](c). Membrane potential and membrane conductance under a current-clamp condition were also measured. Decrease in external osmotic pressure by 0.16 Osm induced a simultaneous increase in [Ca(2+)](c) with both depolarization of the membrane and increase in the membrane conductance. Decrease in external osmotic pressure by 0.05 Osm induced a simultaneous increase in [Ca(2+)](c) with membrane depolarization but the increase in membrane conductance started later than the other two processes. There was a close temporal relationship between the increase in [Ca(2+)](c) and membrane depolarization on the initial response of turgor regulation induced by hypotonic treatment.     

Experimental evidence for negative turgor pressure in small leaf cells of Robinia pseudoacacia L versus large cells of Metasequoia glyptostroboides Hu et W.C.Cheng. 1. Evidence from pressure‐volume curve analysis of dead tissue.

This paper provides a mini‐review of evidence for negative turgor pressure in leaf cells starting with experimental evidence in the late 1950s and ending with biomechanical models published in 2014. In the present study, biomechanical models were used to predict how negative turgor pressure might be manifested in dead tissue, and experiments were conducted to test the predictions. The main findings were as follows: (i) Tissues killed by heating to 60 or 80 °C or by freezing in liquid nitrogen all became equally leaky to cell sap solutes and all seemed to pass freely through the cell walls. (ii) Once cell sap solutes could freely pass the cell walls, the shape of pressure‐volume curves was dramatically altered between living and dead cells. (iii) Pressure‐volume curves of dead tissue seem to measure negative turgor defined as negative when inside minus outside pressure is negative. (iv) Robinia pseudoacacia leaves with small palisade cells had more negative turgor than Metasequoia glyptostroboides with large cells. (v) The absolute difference in negative turgor between R. pseudoacacia and M. glyptostroboides approached as much as 1.0 MPa in some cases. The differences in the manifestation of negative turgor in living versus dead tissue are discussed.     

Studies on mechano-perception in the Characeae: transduction of pressure signals into electrical signals

Mechano-perception by Chara cells was studied with an emphasis on the role of the nodal complex in transducing pressure signals into electrical signals. Three types of experimental material were used: (1) tandem internodal cells connected by a single layer of nodal cells; (2) single internodal cells, from which either apical or basal nodes were removed by ligation and cutting; (3) single internodes from which both nodes had been removed. Exposure to a hypertonic solution (sorbitol or sucrose) induced a depolarization at the node in 1 and 2. Depolarization did not occur at the ligated end of the cell in 2, or at all in 3. Addition of K+ increased the magnitude of the response, whilst it was significantly decreased by the divalent cations, Ca2+ and Mg2+. Electrical resistance decreased at the node during the depolarization, showing that a passive diffusion potential was responsible. I suggest that the change in the trans-nodal hydraulic pressure difference mechanically stretches the plasma membrane, and this induces the electrical depolarization.     

A New Turgor/Membrane Potential Probe Simultaneously Measures Turgor and Electrical Membrane Potential

Abstract: A new combined turgor/membrane potential probe (T-EP probe) monitored cell turgor and membrane potential simultaneously in single giant cells. The new probe consisted of a silicone oil-filled micropipette (oil-microelectrode), which conducted electric current. Measurements of turgor and hydraulic conductivity were performed as with the conventional cell pressure probe besides the membrane potential. In internodal cells of Chara corallina, steady state turgor (0.5-0.7 MPa) and resting potentials (-200 to ?220 mV) in APW, and hydraulic conductivity (0.07 to 0.21 × 10～⁵ m s^?1 MPa^?1) were measured with the new probe, and cells exhibited healthy cytoplasmic streaming for at least 24 h during measurements. When internodal cells of Chara corallina were treated with 30, 20, 10, and 5 mM KCI, turgor responded immediately to all concentrations, and the osmotic changes in the medium were measured. Action potentials, which brought the membrane potential to a steady depolarization that measured the concentration difference of K⁺ in the medium, were induced in a concentration — dependent delay and occurred only 30, 20, and 10 mM of KCl. When the solution was changed back to APW, the repolarization of membrane potential consisted of a quick and a following slow phase. During the quick phase, which took place immediately and lasted 1 to 3 min, the plasma membrane remained activated. The membrane was gradually deactivated in the slow phase, and entirely deactivated when the membrane potential recovered to the resting potential in APW. Although the activated plasma membrane was permeable to K⁺, no major ion channels were activated on the tonoplast, and therefore, internodal cells of Chara corallina did not regulate turgor when osmotic potential changed in the surrounding medium.     

Action Spectrum of Light Pulse-Induced Membrane Depolarization in Pulvinar Motor Cells of Phaseolus

In addition to circadian changes in the membrane potential andleaf movement, light applied to the pulvinus causes changesin both the membrane potential and the pulvinar movement inPhaseolus vulgaris L. Even after a short pulse of light, a transientdepolarization of the membrane occurs and leaf movement is observed.Decreases of turgor pressure of the motor cells are always precededby the depolarization. The direction of the leaf movement canbe explained by the decrease of turgor pressure in the motorcells on the irradiated side of the pulvinus. Using the OkazakiLarge Spectrograph at the National Institute for Basic Biology,we determined the action spectrum of the membrane depolarizationinduced by light pulses (30 s) in motor cells of Phaseolus.The pulvinus was left exposed to air during measurement of themembrane potential with microelectrodes. The action spectrumobtained was in the range of 300 to 730 nm. It had the highestpeak at 460 nm with lower peaks at 380 nm and 420 nm. Almostno sensitivity was observed at wavelengths shorter than 360nm and longer than 520 nm. Red and far-red light had no effecton the depolarization of the motor cell. The features of theaction spectrum are almost the same as those of the Blue-Typeresponse in plants. (Received January 9, 1997; Accepted February 14, 1997)     

Light and turgor affect the water permeability (aquaporins) of parenchyma cells in the midrib of leaves of Zea mays

In response to light, water relation parameters (turgor, half-time of water exchange, T(1/2), and hydraulic conductivity, Lp; T(1/2) proportional 1/Lp) of individual cells of parenchyma sitting in the midrib of leaves of intact corn (Zea mays L.) plants were investigated using a cell pressure probe. Parenchyma cells were used as model cells for the leaf mesophyll, because they are close to photosynthetically active cells at the abaxial surface, and there are stomata at both the adaxial and abaxial sides. Turgor ranged from 0.2 to 1.0 MPa under laboratory light condition (40 micromol m(-2) s(-1) at the tissue level), and individual cells could be measured for up to 6 h avoiding the variability between cells. In accordance with earlier findings, there was a big variability in T(1/2)s measured ranging from 0.5 s to 100 s, but the action of light on T(1/2)s could nevertheless be worked out for cells having T(1/2)s greater than 2 s. Increasing light intensity ranging from 100 micromol m(-2) s(-1) to 650 micromol m(-2) s(-1) decreased T(1/2) by a factor up to five within 10 min and increased Lp (and aquaporin activity) by the same factor. In the presence of light, turgor decreased due to an increase in transpiration, and this tended to compensate or even overcompensated for the effect of light on T(1/2). For example, during prolonged illumination, cell turgor dropped from 0.2 to 1.0 MPa to -0.03 to 0.4 MPa, and this drop caused an increase of T(1/2) and a reduction of cell Lp, i.e. there was an effect of turgor on cell Lp besides that of light. To separate the two effects, cell turgor (water potential) was kept constant while changing light intensity by applying gas pressure to the roots using a pressure chamber. At a light intensity of 160 micromol m(-2) s(-1), there was a reduction of T(1/2) by a factor of 2.5 after 10-30 min, when turgor was constant within +/-0.05 MPa. Overall, the effects of light on T(1/2) (Lp) were overriding those of turgor only when decreases in turgor were less than about 0.2 MPa. Otherwise, turgor became the dominant factor. The results indicate that the hydraulic conductivity increased with increasing light intensity tending to improve the water status of the shoot. However, when transpiration induced by light tends to cause a low turgidity of the tissue, cell Lp was reduced. It is concluded that, when measuring the overall hydraulic conductivity of leaves, both the effects of light and turgor should be considered. Although the mechanism(s) of how light and turgor influence the cell Lp is still missing, it most likely involves the gating of aquaporins by both parameters.     

Turgor regulation in the salt-tolerant alga Chara longifolia

Chara longifolia is a salt‐tolerant Charophyte which regulates its turgor inresponse to osmotic stress. Membrane depolarization, in creased membrane conductance, and cessation of cytoplasmic streaming (due to increase in cytoplasmic Ca^{2 +} ) precede regulation in response to hypotonic stress. Measurements of these three parameters are presented here with simultaneous turgor measurements. Variability in the occurrence, rate and extent of turgor regulation in individual cells was correlated with magnitude of the stress. Hypertonic stress showed the same slow time course as was found previously, requiring several days for complete regulation. Fifty μ M nifedipine, a Ca^{2 +} channel blocker, inhibited turgor regulation. In the presence of 5 μ M nifedipine, turgor regulation was delayed. An increase in conductance preceded regulation, but membrane depolarization was less and no detectable change in cytoplasmic streaming was observed, requiring modifications to a previously presented model for turgor regulation. There was no significant difference in ⁴⁵Ca^{2 +} influx under control and stress conditions. However, the control flux was insensitive to nifedipine, whereas under stress the flux is inhibited 54% by nifedipine. We suggest that osmotic stress results in a rapid increase in a nifedipine‐sensitive Ca^{2 +} entry mechanism, followed very quickly by a decrease in the control entry mechanism.     

In vivo extraction of Arabidopsis cell turgor pressure using nanoindentation in conjunction with finite element modeling

Turgor pressure in plant cells is involved in many important processes. Stable and normal turgor pressure is required for healthy growth of a plant, and changes in turgor pressure are indicative of changes taking place within the plant tissue. The ability to quantify the turgor pressure of plant cells in vivo would provide opportunities to understand better the process of pressure regulation within plants, especially when plant stress is considered, and to understand the role of turgor pressure in cellular signaling. Current experimental methods do not separate the influence of the turgor pressure from the effects associated with deformation of the cell wall when estimates of turgor pressure are made. In this paper, nanoindentation measurements are combined with finite element simulations to determine the turgor pressure of cells in vivo while explicitly separating the cell‐wall properties from the turgor pressure effects. Quasi‐static cyclic tests with variable depth form the basis of the measurements, while relaxation tests at low depth are used to determine the viscoelastic material properties of the cell wall. Turgor pressure is quantified using measurements on Arabidopsis thaliana under three pressure states (control, turgid and plasmolyzed) and at various stages of plant development. These measurements are performed on cells in vivo without causing damage to the cells, such that pressure changes may be studied for a variety of conditions to provide new insights into the biological response to plant stress conditions.     

Interaction of myogenic mechanisms and hypoxic dilation in rat middle cerebral arteries

The goal of this study was to determine how myogenic responses and vascular responses to reduced Po(2) interact to determine vascular smooth muscle (VSM) transmembrane potential and active tone in isolated middle cerebral arteries from Sprague-Dawley rats. Stepwise elevation of transmural pressure led to depolarization of the VSM cells and myogenic constriction, and reduction of the O(2) concentration of the perfusion and superfusion reservoirs from 21% O(2) to 0% O(2) caused vasodilation and VSM hyperpolarization. Myogenic constriction and VSM depolarization in response to transmural pressure elevation still occurred at reduced Po(2). Arterial dilation in response to reduced Po(2) was not impaired by pressure elevation but was significantly reduced at the lowest transmural pressure (60 mmHg). However, the magnitude of VSM hyperpolarization was unaffected by transmural pressure elevation. This study demonstrates that myogenic activation in response to transmural pressure elevation does not override hypoxic relaxation of middle cerebral arteries and that myogenic responses and hypoxic relaxation can independently regulate vessel diameter despite substantial changes in the other variable.     

Water transport parameters and regulatory processes inEremosphaera viridis

Summary The water relations parameters and the osmoregulatory response ofEremosphaera viridis were investigated both by using the pressure probe technique and by analyzing the intracellular pool of osmotically active agents. In the presence of various concentrations of different salts a biphasic osmoregulatory response was recorded, consisting of a rapid decrease in turgor pressure due to water loss followed by an increase in turgor pressure to the original turgor pressure value (depending on the salt). The values of turgor pressure, volumetric elastic modulus and hydraulic conductivity depended on the composition of the media. Nonelectrolytes did not cause a turgor recovery after the initial water efflux. The second phase of turgor regulation in the presence of salts was characterised by the intracellular accumulation of ions and sugars and required at least 24 hr. Analysis of the cell sap showed that the increase in the internal osmotic pressure was mainly achieved by accumulation of sucrose. Additionally, accumulation of glucose was observed in illuminated cells in the presence of Rb and K. Electron micrographs suggested that the sucrose was produced by degradation of starch granules. Turgor pressure recovery after salt stress seemed to be dependent on temperature and is well correlated with the according photosynthetic activity. The data suggest that a temperature-dependent enzyme which is activated by potassium or rubidium is involved in the regulatory response.