Studies on oxygen and volume restrictions in cultured cardiac cells I. A model for ischemia and anoxia with a new approach

Summary A novel incubation unit is described that is highly suitable for thorough studies of oxygen deprivation states. Its application with cultured heart cells is experimentally demonstrated. The release of enzymes, taken as a marker for cell damage, has clearly shown that restriction of the volume of extracellular medium combined with oxygen plus glucose deprivation caused greatest cellular damage. It may be considered as an experimental ischemia-like state. Furthermore, the onset of cellular damage followed a time table very much like that occurring in vivo under similar conditions, more so than any other previously described studies. A time lag between the release of cytoplasmic enzymes and lysosomal enzymes and other observations made in the present study suggests a sequential order of events in which the release of cytoplasmic enzymes occurs at a stage of reversible damage due to oxygen deprivation, whereas the release of lysosomal enzymes may point at irrepairable damage. Supported by grants from The Chief Scientist, Ministry of Health, State of Israel; The Ministry of Education and Sciences, State of Niedersachsen (FRG); and The Foundation for Heart Research from Mr. and Mrs. D. Vidal-Madjar, Paris, France. This study was done as partial fulfilment of Vemuri's Ph.D. thesis in biochemistry.     

Kinetic changes of ethanolamine base exchange activity and increase of viscosity in sarcolemmal membranes of hamster heart during development of cardiomyopathy

The activity of the phospholipid base exchange enzyme specific for ethanolamine has been measured in cardiac sarcolemmal membrane preparations from Syrian golden and UM-X7.1 cardiomyopathic hamsters. In Syrian golden hamsters, the Km of the enzyme for ethanolamine does not change with age, whereas it almost doubles in membranes from cardiomyopathic animals, from the 30th to the 150th day of age. During the same period, the membrane cholesterol content increases by 68% in cardiomyopathic hamsters, whereas it does not change significantly in the Syrian golden hamster strain. As a consequence, in the adult animal, the cholesterol to phospholipid ratio and the viscosity of sarcolemmal membranes are higher in UM-X7.1 strain than in Syrian golden hamsters. A cause consequence relationship between the enzymatic changes and the compositional modifications in the sarcolemma occurring in UM-X7.1 hamsters during the development of cardiomyopahhy is proposed. (Mol Cell Biochem 116: 89–93,1992)     

Salutary effect of tedisamil on post-ischemic recovery rat heart: Involvement of sarcolemmal (Na,K)-ATPase

The in vitro effect of tedisamil on the specific activity and kinetic parameters of the sarcolemmal (Na,K)-ATPase as well as its ex vivo effect on the (Na,K)-ATPase in the isolated, perfused rat hearts was determined. Five mol/l of tedisamil was added 5 min before the onset of 30 min global normothermic ischemia followed by 10 min reperfusion. At the conditions of its maximal cardioprotective effect (heart rate reduction, improved postischemic recovery of left ventricular developed pressure), the hearts were immediately used for isolation of sarcolemmal vesicles. In vitro, 1–100 mol/l of tedisamil produced a concentration-dependent stimulatory effect on (Na,K)-ATPase activity, with a peak seen at 20 mol/l (p < 0.01), while Mg-dependent ATPase was almost unchanged. Kinetic analysis revealed a significant increase in the affinity of the Na-binding sites on ATPase molecule at 20 mol/l of tedisamil. These biochemical findings were confirmed by cytochemistry. Moreover, ex vivo experiments revealed that tedisamil rendered the sarcolemmal (Na,K)-ATPase activity to be a more resistant to detrimental effects of ischemia. In conclusion, the cardioprotective action of tedisamil was accompanied with a better preservation of the specific activity of (Na,K)-ATPase.     

Effect of platelet-activating factor (PAF) on sodium calcium exchange in cardiac sarcolemmal vesicles

Summary The effects of platelet-activating factor (PAF) on Na⁺-dependent calcium uptake in myocardial sarcolemmal vesicles were examined in order to clarify its mechanism of inotropic action on the heart. PAF (40 and 20 µM) significantly inhibited Na⁺-Ca²⁺ exchange by 61% and 37%, respectively. Both initial rate of exchange and maximal exchange were inhibited. The Km for the reaction was not altered but Vmax was lowered 55% by PAF. Lyso-PAF inhibited Na⁺-Ca²⁺ exchange to a similar degree as PAF. CV-3988, a specific PAF receptor antagonist, failed to diminish the inhibitory effect of PAF on Na⁺-Ca²⁺ exchange, suggesting that the effect of PAF on Na⁺-Ca ²⁺ exchange is not via a receptor mechanism. The passive permeability of sarcolemmal vesicles to Ca²⁺ was markedly elevated after PAF treatment. However, this effect could not account for the decrease in Na⁺-Ca²⁺ exchange. Interestingly, passive Ca²⁺ binding to cardiac sarcolemma was increased by 40 µM PAF. This study indicates that a depression of Na⁺-Ca²⁺ exchange probably does not play a role in the negative inotropic effect of PAF on the myocardium under physiological conditions. Its mechanism of action on Na⁺-Ca²⁺ exchange is discussed.     

CHO cell responses to low oxygen: regulation of oxygen consumption and sensitization to oxidative stress

We have investigated the regulation of oxygen consumption and modulation of glutathione levels in CHO-K1 cells under oxygen-limiting conditions. We report here suppression of oxygen consumption and alteration of the supply-dependent relationship as a consequence of prolonged hypoxic or anoxic exposure. The suppression is characterized by an increase in the value of P(o(2)/50) (the oxygen tension at which oxygen consumption is half maximal). Under prolonged anoxia there is also a decrease in the cells' potential to use oxygen. Elevated glucose consumption under low oxygen conditions may contribute to the suppression in respiration. The glutathione concentration remains constant throughout hypoxic exposure but may decrease by as much as 40% under anoxia. The glutathione level in hypoxic and anoxic cells increases by two- and four-fold, respectively, over that of the control cells when exposed to a cytotoxic level of oxygen (93%). This suggests that anoxic and hypoxic exposure sensitizes CHO cells to oxidative stress. (c) 1992 John Wiley & Sons, Inc.     

Ischemic myocardial injury in cultured heart cells: Leakage of cytoplasmic enzymes from injured cells

Summary An in vitro model of myocardial ischemia has been established with primary monolayer cultures of postnatal rat myocardial cells. Ischemic conditions were simulated in vitro by subjecting the myocardial cell cultures to various levels of oxygen and glucose deprivation. The experimental protocol consisted of treatment with 20% or 0% O₂ and 1000, 500 or 0 mg glucose per 1 of medium for 4 or 24 hr. Control cultures were treated with 20% O₂ and 1000 mg glucose. After the ischemic treatments, cultures of beating muscle (M) cells were evaluated for signs of injury, i.e. leakage of cytoplasmic enzymes into the culture medium. Differences were found in leakage of lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) from the cultures that were exposed to partial ischemia of glucose deprivation and from those cultures that were exposed to total ischemia of oxygen and glucose deprivation. Glucose deprivation alone resulted in a slight-to-moderate loss of LDH and CPK from the cells, whereas total ischemia resulted in a significant release of the two cytoplasmic enzymes. When the cultures were allowed to recover after ischemic treatment in complete medium (1000 mg glucose) and a normal atmosphere of 20% O₂, they had levels of LDH leakage comparable to those of control cultures. Cell viability and total protein content of the ischemic cultures did not differ significantly from controls. This study was supported by Research Grant HL 18647 from the National Heart, Lung, and Blood Institute.     

Effect of liposomes on lipid peroxidation and total phospholipids in rabbit ischemic spinal cord model

The effect of spinal cord ischemia (induced by abdominal aorta ligation for 20 minutes) on lipid peroxidation and TPL composition was investigated and discussed in our previous articles. It is known, that partially reduced species of oxygen can be formed under aerobic conditions. For that reason, the effect of ligation release for 60 minutes was observed in experimental animals treated with the selected liposomes. Administration of CP, (CP+SA) and (CP+Chol) liposomes applied 30 minutes before 20 minutes ischemia revealed an ameliorating effect on in vivo and in vitro Fe-dependent peroxidation manifested by TBA-RS accumulation. Combined use of (CP+SA) liposomes with lipophylic form of stobadine (DP 1031) was not more effective. Application of CP liposomes directly before the ligation release slightly increased the antiradical capacity in spinal cord homogenates comparing with not-treated animals. Accumulation of TBA-RS was accompanied by TPL degradation during recirculation period but values of TPL after liposomal treatment were unaffected.     

Effect of scavengers of active oxygen species and pretreatment with acetyl-salicylic acid on the injury to cultured endothelial cells by thrombin-stimulated platelets

Summary Thrombin-stimulated human platelets adhere to and injure cultured human endothelial cells. We hypothesize that generation of active oxygen species by the stimulated platelets are involved in the injury. To confirm this, catalase [final concentration (8.25 μg/ml)], superoxide dismutase (SOD) (10 μg/ml), ofd-mannitol (9 mg/ml) were added to the cell culture medium before the experiments. Platelet suspension (200.000/μl) and thrombin (4 U/ml) were added and the culture dishes shaken for 15 min at room temperature. In separate experiments the endothelial cells were pretreated with acetylsalicylic acid (0.05, 0.1, or 0.5 mM) to test whether the arachidonic acid metabolism of the endothelial cells is involved in the injury process. In preliminary experiments we were able to confirm that platelets, when stimulated by thrombin, produce chemiluminescence which was suppressed by mannitol but not by catalase or SOD. The degree of injury to cultured endotheial cells by thrombin-stimulated platelets, as measured by release of⁵¹Cr from prelabeled endothelial cells, was reduced significantly with the presence of mannitol, but only moderately when catalase or SOD had been added. Morphometric quantification based on scanning electron micrographs of the endothelial cells after exposure to thrombin-stimulated platelets in the presence of catalase or mannitol showed a reduced number of injured cells. Pretreatment of the endothelial cells with acetylsalicylic acid did not cause any significant change in the degree of endothelial cell injury as measured by the⁵¹Cr release. It is concluded that active oxygen species, in particular hydroxyl radicals, may be generated during thrombin stimulation of platelets and cause injury to the endothelial cells. This work was supported by the Norwegian Research Council for Science and the Humanities and the Norwegian Council for Cardiovascular Diseases. We express our gratitude for their grants.     

Studies on tamoxifen encapsulated in lipid vesicles: Effect on the growth of human breast cancer MCF-7 cells

Tamoxifen is a nonsteroidal estrogen-receptor modulator widely used in the treatment of breast cancer. Apoptosis has been reported to be a major mechanism for its antitumor effect. In the current studies, an endeavor was made to investigate the efficacy of vesicularly encapsulated tamoxifen on human breast cancer MCF-7 cells. Phospholipid-based vesicular systems viz. conventional liposomes and elastic-membrane liposomes were employed to encapsulate the drug. The MTT colorimetric assay was used to determine the efficacy of the tested formulations. The results demonstrated composition-dependent strong inhibition in the viability of MCF-7 cells with encapsulated tamoxifen vis-à-vis free drug. The encouraging findings from the current work construe immense potential of the lipid-based vesicular systems in the treatment of breast cancer.     

Development and gene expression of nuclear transplants generated by transplantation of cultured cell nuclei into non-enucleated eggs in the medaka Oryzias latipes

To develop nuclear transplantation techniques for the medaka Oryzias latipes, nuclei of cultured cells from transgenic fish were transplanted into unfertilized eggs of the orange-red variety of O. latipes, without enucleation, in two experimental series. In the first experimental series, fibroblast cells cultured from the adult caudal fin were used as donors, which carried the green fluorescent protein (GFP) gene driven by the promoter of the medaka elongation factor 1alpha-A gene. Wild-type body color was another donor genetic marker used in this experimental series. In the second experimental series, cells cultured from 6-day-old embryos were used as donors, which carried the GFP genetic marker driven by the promoter of the medaka beta-actin gene. From more than 1000 eggs transplanted in each experiment, a considerable number of nuclear transplants developed to various embryonic stages showing stage- and tissue-specific expression of the donor genetic markers, although the expression was mosaic in many cases. Three and six of the transplanted eggs in the first and second experimental series (0.3 and 0.5%, respectively) hatched, and the hatchlings expressing the genetic markers survived for up to 3 weeks. The chromosome number varied among cells in a single transplant embryo. The results obtained in these experiments may help future cloning efforts in fish.     

Intracardiac injection of matrigel induces stem cell recruitment and improves cardiac functions in a rat myocardial infarction model

Matrigel promotes angiogenesis in the myocardium from ischemic injury and prevents remodelling of the left ventricle. We assessed the therapeutic efficacy of intracardiac matrigel injection and matrigel‐mediated stem cell homing in a rat myocardial infarction (MI) model. Following MI, matrigel (250 μl) or phosphate‐buffered solution (PBS) was delivered by intracardiac injection. Compared to the MI control group (MI‐PBS), matrigel significantly improved left ventricular function (n= 11, P < 0.05) assessed by pressure–volume loops after 4 weeks. There is no significant difference in infarct size between MI‐matrigel (MI‐M; 21.48 ± 1.49%, n= 10) and MI‐PBS hearts (20.98 ± 1.25%, n= 10). The infarct wall thickness of left ventricle is significantly higher (P < 0.01) in MI‐M (0.72 ± 0.02 mm, n= 10) compared with MI‐PBS (0.62 ± 0.02 mm, n= 10). MI‐M hearts exhibited higher capillary density (border 130.8 ± 4.7 versus 115.4 ± 6.0, P < 0.05; vessels per high‐power field [HPF; 400×], n= 6) than MI‐PBS hearts. c‐Kit⁺ stem cells (38.3 ± 5.3 versus 25.7 ± 1.5 c‐Kit⁺ cells per HPF [630×], n= 5, P < 0.05) and CD34⁺ cells (13.0 ± 1.51 versus 5.6 ± 0.68 CD34⁺ cells per HPF [630×], n= 5, P < 0.01) were significantly more numerous in MI‐M than in MI‐PBS in the infarcted hearts (n= 5, P < 0.05). Intracardiac matrigel injection restores myocardial functions following MI, which may attribute to the improved recruitment of CD34⁺ and c‐Kit⁺ stem cells.     

The impact of cell fixation on coherent anti‐stokes Raman scattering signal intensity in neuronal and glial cell lines

A number of studies require sample fixation, aimed to preserve cells in a physiological state with minimal changes of morphology and intracellular molecular content. Sample fixation may significantly distort experimental data, which makes the data interpretation process more challenging. It is particularly important for study of lipid‐related diseases, where the biochemical and morphological characteristics of the cells need to be well preserved for an accurate data analysis. This study investigates the effects of formaldehyde and ethanol (EtOH) fixatives on coherent anti‐stokes Raman scattering (CARS) signal of proteins and lipids in major cellular compartments of neuronal and glial cells. We found that both fixatives induce alteration of proteins and lipids signal in studied cell lines. Furthermore, the impact of sample preservation methods on CARS signal varies between cell lines. For instance, our data reveals that EtOH fixation induces ~45% increase of CARS signal of proteins in the nucleolus of neuronal cells and ~35% decrease of CARS signal in glial cells. The results indicate that aldehyde fixation is a preferable method for preservation of neuronal and glial cells prior to CARS imaging, as it less affects both CARS signal and intracellular distribution of proteins and lipids.      

Sulfation and glucuronidation of acetaminophen by cultured hepatocytes reproducing in vivo sex-differences in conjugation on matrigel and type 1 collagen

Summary The sulfate and glucuronide conjugation of acetaminophen (APAP) by hepatocytes cultured on Matrigel or type 1 collagen was compared to APAP metabolism in vivo. The metabolic fate of low (15 mg/kg), medium (125 mg/kg), and high (300 mg/kg) doses of APAP injected intraperitoneally were determined in male and female rats. Males excreted more APAP as the sulfate conjugate than females, which correlated with the twofold greater APAP sulfotransferase activity in the male vs. females (301±24 vs. 156±18 pmol · mg⁻¹ protein · min⁻¹). Also, as sulfate conjugation became saturated, there was a dose-related shift in APAP metabolism from sulfate to glucuronide conjugation in both sexes. After death, the livers of the same animals were perfused with collagenase and the hepatocytes cultured in modified Waymouth’s medium on either Matrigel or rat-tail collagen, with various doses of APAP (0, 0.125, 0.25, 0.5, and 1.0 mM). Sex differences in APAP sulfation and glucuronidation persisted in culture for up to 4 days, with sulfation predominating in the male similar to in vivo. With increasing APAP concentration (dose), there was a saturation of sulfate conjugation and a shift to glucuronidation as observed in vivo. Sex differences in APAP sulfation and glucuronidation were no longer significant by Day 4 in culture. Sulfation, and to a lesser extent, glucuronidation, were more stable on Matrigel than collagen. We concluded that APAP metabolism of freshly isolated hepatocytes could replicate in vivo sex differences in conjugation, and that Matrigel was superior to collagen as substrate.     

Interaction of (Na + K + 2 Cl) cotransport and the Na/K pump in cultured chick cardiac myocytes

We have recently reported the presence of an electroneutral (Na + K + 2 Cl) cotransport mechanism that is bumetanide-sensitive and maintains Cl_i above its electrochemical equilibrium in cultured chick heart cells. In steady state, (Na + K + 2 Cl) cotransport is inwardly directed and so contributes to the Na influx that must be counterbalanced by the activity of the Na/K pump to maintain Na_i homeostasis. We now show that manipulating (Na + K + 2 Cl) cotransport by restoring Cl_o to a Cl-free solution indirectly influences Na/K pump activity because the bumetanide-sensitive recovery of a _infNa ^supi to its control level and the accompanying hyperpolarization could be blocked by 10^–4M ouabain. In another protocol, when the Na/K pump was reactivated by restoring K_o (from 0.5 mM to 5.4 mM) and removing ouabain, the recovery of a_Na was attenuated by 10^–4M bumetanide. The relatively slow rate of ouabain dissociation coupled with the activation of Na influx by (Na + K + 2 Cl) cotransport clearly establishes the interaction of these transport mechanisms in regulating Na_i. Although (Na + K + 2 Cl) cotransport is electroneutral, secondary consequences of its activity can indirectly affect the electrophysiological properties of cardiac cells.     

Effects of 16,16-dimethyl prostaglandin E2 on glycoprotein and lipid synthesis of gastric epithelial cells grown in a primary culture

Summary We investigated the biosynthesis of phospholipid, neutral lipids, glycoproteins, and DNA in primary cultures of rat oxyntic mucosal cells. In addition, responses of these biosynthetic pathways to the gastric protective agent 16,16-dimethyl prostaglandin E₂ (dmPGE₂) were studied. Cultured gastric cells under control conditions synthesized glycoprotein in a linear manner over time. The cells responded to dmPGE₂ with an increase in glycoprotein synthesis without an effect on DNA synthesis. Investigations of lipid synthesis showed that phospholipid was produced in a linear fashion by these cells, however, no effect of exogenously administered dmPGE₂ on its rate of formation was discernible. In contrast, the incorporation of labeled palmitate into neutral lipids revealed that triglyceride biosynthesis was significantly increased by the addition of dmPGE₂ to the culture medium, which could be further enhanced by the administration of the phosphodiesterase inhibitor, isobutyl methyl xanthine. Cyclic nucleotide involvement was further suggested by our finding that triglyceride synthesis in cultured gastric mucous cells could be increased a comparable amount by the addition of both dbcAMP and dbcGMP to the medium. The possible relationship between these biochemical alterations and the gastric protective action of dmPGE₂ is discussed. This work was supported by grant DK33239 from the National Institutes of Health, Bethesda, MD. The dmPGE₂ was a generous gift of the Upjohn Company, Kalamazoo, MI.     

Effect of cumulus cell coculture and oxygen tension on the in vitro developmental competence of bovine zygotes cultured singly

The customary practice in bovine in vitro embryo production (IVP) is to handle oocytes and embryos in groups; although there are several reasons for establishing an IVP system for individual embryos that allows for following a single oocyte from retrieval through development to the blastocyst stage. To date, reports of individual IVP are inconsistent, and in most cases, resulted in unsatisfactory blastocyst rates. The objective of this study was to develop an efficient system for routine in vitro culture of individual bovine embryos. Single culture of zygotes in 2 different culture volumes (20 and 500 μL) yielded less than 3% blastocysts in experiment 1. In an attempt to improve these results, cumulus cells were added to the culture medium in experiment 2, after which blastocyst rates increased from 2.9 to 21.8% (P < 0.05). The third experiment revealed that an atmospheric oxygen tension, which is commonly used with somatic cell coculture, was not beneficial during individual embryo-cumulus cell coculture, because it resulted in lower blastocyst rates (Odds ratio 0.57, P < 0.001) and in lower blastocyst cell numbers (P < 0.05), when compared to culture in 5% oxygen. Grouped vs. single culture and reduced oxygen tension did not have a significant effect on cleavage and hatching rates. In experiment 4, three different cumulus cell coculture conditions during individual culture were tested and compared with the cleavage, blastocyst and hatching rates, and cell number of group culture (73.2%, 36.4%, 66.7% and, 155.1 ± 7.26, respectively). The outcome variables after individual embryo culture on a 5-day-old cumulus cell monolayer (74.1%, 38.2%, 71.9% and 133.4 ± 9.16, respectively), and single culture in the presence of added cumulus cells (69.9%, 31.9%, 66.7% and 137.3 ± 8.01, respectively) were not significantly different from those obtained after group culture (P < 0.05). Though, individual culture in a cumulus cell conditioned medium significantly reduced both the cleavage (59.0%) and blastocyst rates (6.3%). These results demonstrate that single culture of bovine zygotes can be fully sustained by coculture with cumulus cells in a low oxygen environment; implementation of these findings in our IVP system produced blastocysts comparable in quantity and quality to those obtained by group culture. These results were consistently achieved after acquiring experience and expertise in the handling of single zygotes.     

Effect of magnesium on calcium influx activated by glutamate and its agonists in cultured cerebellar granule cells

The effects of Mg²⁺ on the glutamate-, kainate-, N-methyl-d-aspartate- and quisqualate-induced influx of⁴⁵Ca²⁺ were studied in cultured cerebellar granule cells. The N-methyl-d-aspartate- and quisqualate-evoked influx was totally and the kainate- and glutamate-evoked influx partially blocked in 1.3 mM extracellular Mg²⁺. The increase in influx induced by kainate, quisqualate and glutamate was maximal at 0.1 mM Mg²⁺, whereas N-methyl-d-aspartate was most effective in totally Mg²⁺-free media.d-2-Amino-5-phosphonovalerate blocked partially and phencyclidine completely the enhancement of Ca²⁺ influx by 1 mM quisqualate in 0.1-mM Mg²⁺ medium. The effect of 10 M quisqualate was also significantly inhibited by antagonists specific for different glutamate receptor subtypes, including N-methyl-d-aspartate, (RS)-amino-3-hydroxy-5-methyl-4-isozazolepropionate and metabotropic recptors. This evidences a heterogeneous action of quisqualate, mediated by different glutamate receptor subtypes in 0.1 mM Mg²⁺ medium. The efficacy of quisqualate in inducing influx of Ca⁺ and the selectivity of antagonists for different receptors are also modified by extracellular Mg²⁺.     

Control of intracellular glutathione and its effect on ultraviolet radiation-induced K+ efflux in cultured rose cells

Abstract Modification of the ‘intracellular concentration of reduced glutathione’ (IC-GSH) affected the response of cultured rose cells (Rosa damascena) to ultraviolet radiation (UV)-induced leakage of K⁺. High IC-GSH induced by incubation of cells in 10 mol m^?3 GSH (IC-GSH increased linearly with time from 20 to about 600 μmol g^?1 in 61.2 ks) caused cells to become significantly less sensitive to UV. Low IC-GSH induced by treatment with 1 mol m^?3 buthionine sulphoximine (BSO) plus 1 mol m^?3 diethylmaleate (DEM) (IC-GSH decreased from 20 to about 3 μg g^?1 in 61.2 ks) reduced, rather than increased, the UV-sensitivity of the cells. However, treatment with DEM also induced a large transient K⁺ leakage; and treatment with BSO induced a slight leakage. The K⁺ leaked was recovered by 3.24 ks. Following K⁺ recovery, the DEM-treated cells showed almost complete insensitivity to UV, and BSO-treated cells showed a slightly reduced sensitivity to UV. These results are in agreement with our previous findings that other treatments (heat, cycloheximide, UV), which also cause a transient leakage of K⁺, also reduce the induction of K⁺ leakage by a subsequent UV treatment. We conclude that high IC-GSH may play a role in protecting plant cells from UV-induced K⁺ leakage. Increased UV-sensitivity with low ICGSH was not observed, we believe, because of the transient K⁺ leakage, though the mechanism of reduced sensitivity to UV induced by transient leakage of K⁺ is not known at this time. Treatment with UV did not reduce the IC-GSH, showing that this is not the mechanism by which UV induces K⁺ leakage.