The effect of copper on frog skin: the role of sulphydryl groups.

1. Cu2+ at a concentration of 10-4 M, when applied to the external side of the frog skin produces an increase in the short-circuit current (Isc). 2. This effect was studied in skins of Rana temporaria adapted to cold,(5 degrees C) and room temperature (20 degrees C), skins of Rana pipiens adapted to cold, and the results compared with those obtained previously with Rana ribibunda. 3. The observed effect is less dependent upon the adaptation to cold than upon the functional state of the skin: skins with low short circuit currents have a bigger response to Cu2+ than skins with high Isc. 4. A species difference cannot be ruled out since skins of Rana ribibunda exhibiting high Isc give good responses to Cu2+. 5. 5,5' -dithiobis (2-nitrobenzoic acid), a sulphydryl-oxidizing reagent, produces an effect similar to that of Cu2+, and dithiothreitol an SH-reducing agent, reverses the effect of this ion. 6. Cu2+ also induces an increase in the unidirectional K+ fluxes and unmasks a net outward potassium flux. 7. The outward K+ flux induced by Cu2+ is sensitive to ouabain. 8. It is concluded that Cu2+ increases the permeability of the external barrier of the frog skin to Na+ and K+, probably by reacting with SH groups.     

Invertebrate epithelial Na+ channels: amiloride-induced current-noise in crab gill.

Epithelial sheets (including cuticle) from posterior gills of the freshwater-adapted euryhaline crab Eriocheir sinensis were obtained according to the method of Schwarz and Graszynski ((1989) Comp. Biochem. Physiol. 92A, 601-604; (1989) Verh. Dtsch. Zool. Ges. 82, 211 and (1989) Arch. Int. Physiol. Biochim. 97, C45). With external NaCl-saline, the outward-directed short-circuit current (Isc) could hardly be influenced by external amiloride up to 100 mumol/l but was, on the contrary, strictly dependent on apical Cl- (Onken, Graszynski and Zeiske (1991) J. Comp. Physiol. B 161, 293-301). In absence of external chloride an inward-directed, amiloride-inhibitable Isc was observed which depended on external Na+ (thus, Isc approximately INa) in a two-step, saturating mode. The Isc-block by amiloride obeyed saturation kinetics (half-maximal at less than or equal to 1 mumol/l, suggesting apical Na(+)-channels). Only for Na+ concentrations below 100 mmol/l we found an indication for a competitive interaction between Na+ and amiloride at the channel. Current fluctuation analysis revealed the presence of an amiloride-induced relaxation (Lorentzian) component in the Isc-noise (so-called 'blocker-noise'). The Lorentzian parameter-shifts with increasing amiloride concentration indicate first-order kinetics of the blocker with its apical receptor. Using a 'two-state' blocking model we calculated, for amiloride concentrations between 2 and 5 mumol/l, a mean single-channel current of 0.46 pA and a mean channel density of 250.10(6) cm-2.     

Cyclic AMP stimulation of Cl- secretion by the opercular epithelium: the apical membrane chloride conductance

Chloride secretion (Isc) by the opercular epithelium of the teleost, Fundulus heteroclitus, is stimulated by elevations in intracellular cyclic AMP (cAMP) elicited by beta-adrenergic agonists, such as isoproterenol, and is accompanied by a small but significant increase in the transepithelial conductance (Gt). Cupric ions (Cu2+) have been shown to block the apical membrane Cl- channels in this epithelium, leading to a reduction in both the Isc and Gt (Degnan, '85). In the present studies, the effects of Cu2+ on cAMP-elevated and cAMP-depleted epithelia were observed to define the actions of cAMP in this stimulatory process. At a concentration of 5 X 10(-4) M in the mucosal solution, Cu2+ inhibited the Isc 79.8% and reduced the Gt 39.2%. Isoproterenol produced an attenuated stimulation of the Isc in these tissues compared to untreated controls, but had no effect on the Gt. In tissues bathed bilaterally with Cl- -free Ringer, the Isc was virtually abolished and the Gt was reduced 37.0%; neither Cu2+ nor isoproterenol had any effects on the Isc or Gt under this condition. Simultaneous 2 2Na and 3 6 Cl unidirectional flux determinations indicated that the only effects of both isoproterenol and Cu2+ were on the active Cl- secretory flux. An inhibitor of adenylate cyclase, 2',5' dideoxyadenosine (DDA), reduced the Isc and Gt 39.8% and 20.8% respectively. This inhibitor had no additional effects in Cu2+ -treated tissues and the action of Cu2+ on the Gt was reduced in DDA-treated tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of the Putative Ca2+-Receptor Agonist Gd3+ on the Active Transepithelial Na+ Transport in Frog Skin

In this communication we show that Gd3+ acts as an activator of the apical sodium channel (ENaC) in frog skin epithelia. Application of Gd3+ to the apical solution of frog skin epithelia increased the Na+ absorption measured as the amiloride-inhibitable short-circuit current (Isc). The stimulation was dose dependent with a concentration for half-maximal stimulation (EC50) of 0.023 mM. The change in Isc was found to correlate with the net Na+ flux, confirming that Gd3+ enhances Na+ absorption. By monitoring the cellular potential (Vsc) with microelectrodes during addition of Gd3+, it was found that Vsc depolarized as Isc rose, indicating that Gd3+ affects apical Na+ permeability (PNa). This was confirmed by measuring the I/V relations of the apical membrane. In the presence of benzimidazolylguanidin (BIG), a drug known to abolish the Na+ self-inhibition, Gd3+ had no effect on Isc. The Na+ self-inhibition was investigated using fast changes of the apical Na+ concentration on K+-depolarized epithelia. BIG was found to abolish the Na+ self-inhibition and to activate the basal Na+ transport, whereas Gd3+ only activated the basal Na+ transport but had no effect on the self-inhibition. These results indicate the existence of an alternative nonhormonal mechanism to Na+ self-inhibition, via which both Gd3+ and BIG act, possibly components of the Na+ feedback inhibition system.     

Autoregulation of apical membrane Na+ permeability of tight epithelia. Noise analysis with amiloride and CGS 4270

Noise analysis of the Na+ channels of the apical membranes of frog skin bathed symmetrically in a Cl-HCO3 Ringer solution was done with amiloride and CGS 4270. Tissues were studied in their control states and after inhibition of transepithelial Na+ transport (Isc) by addition of quinine or quinidine to the apical solution. A critical examination of the amiloride-induced noise indicated that the single channel Na+ currents (iNa) were decreased by quinine and quinidine, probably because of depolarization of apical membrane voltage. Despite considerable statistical uncertainty in the methods of estimation of the Na+ channel density with amiloride-induced noise (NA, see text), the striking observation was a large increase of NA with amiloride inhibition of the rate of Na+ entry into the cells. NA was increased to 406% of control, whereas Isc was inhibited to 8.6% of control by 6 microM amiloride. Studies were done also with the Na+ channel blocker CGS 4270. Noise analysis with this compound was advantageous, permitting iCGSNa and NCGS to be measured in individual tissues with a relatively small inhibition of Isc. As with amiloride, inhibition of Isc with CGS 4270 caused large increases of the Na+ channel density (approximately 200% at approximately 35% inhibition of the Isc). Quinine and quinidine caused an approximately 50% increase of Na+ channel density while inhibiting iNa by approximately 60-70%. As inhibition of Na+ entry leads to an increase of Na+ channel density, a mechanism of autoregulation appears to be a major factor in adjusting the apical membrane Na+ permeability of the cells.     

Effects of aldosterone on (Na+ + K+)-ATPase of amphibian sodium-transporting epithelial cells (A6) in culture

Stimulation by aldosterone of sodium reabsorption can be reproduced on a cell line, A6, derived from the renal tissue of Xenopus laevis. These cells organize themselves as a polarized epithelium carrying out unidirectional sodium transport, reflected by the short-circuit current (Isc). Isc response to aldosterone starts to be apparent after a latency period of 2-3 h; the full hormonal effect takes much longer. On the other hand, (Na+ + K+)-ATPase activity and density in ouabain binding sites did not increase before several hours of treatment. At that stage, while Isc more than trebled, Na+ pump activity and density went up by less than 50%. A significant influence of aldosterone on the way the Na+ pump operates is considered unlikely, since cell interaction with ouabain remained unchanged (Kd approximately 18 nM). Furthermore, the close correspondence of hormonal effect, in relative terms, on (Na+ + K+)-ATPase activity vs density, argues against a significant degree of recruitment of spare pump units. Thus aldosterone effect on Na+ pump probably results from increased biosynthesis of the enzyme. The aldosterone dependent Na+ pump stimulation is apparently unrelated to sodium available for transport. The hormone seems to act on Na+ pump directly.     

Microelectrode studies of amphotericin B on Na+ and K+ conductance in bullfrog cornea

Addition of 10(-5) M amphotericin B to the tear solution of an in vitro preparation of the frog cornea increased the transepithelial conductance, gt, and decreased the apical membrane fractional resistance, f(R0), in the presence or absence of tear Na+ and Cl-. In the presence of tear Na+ and Cl-, amphotericin B increased the short-circuit current, Isc, from 3.9 to 8.8 microA.cm-2 and changed the intracellular potential, V0, from -48.5 to -17.9 mV probably due to a higher increase in the Na+ than in the K+ conductance. In the absence of tear Na+ and Cl-, amphotericin B decreased Isc from 5.5 to about 0 microA.cm-2 due to K+ (and possibly Na+) flux from cell to tear and changed V0 from -35.4 to -63.6 mV due to the increase in conductance of both ions. Increase in the tear K+ from 4 to 79 mM (in exchange for choline), in the presence of amphotericin B and absence of tear Na+ and Cl-, decreased f(R0) from 0.09 to 0.06, increased gt from 0.23 to 0.31 mS, increased Isc from 0.63 to 7.3 microA.cm-2, and changed V0 from -65.5 to -17.3 mV due to the change in EK in the presence of a high conductance in the tear membrane. Similar effects were observed with an increase of tear Na+. Results support the concept that the Na+ conductance opened by amphotericin B in the apical membrane is greater than the K+ conductance. Previously observed transepithelial effects of the ionophore may be explained mostly on the basis of its effect on the apical membrane.     

Divalent cations reduce the electrogenic transport of monovalent cations across rumen epithelium

The rumen epithelium of sheep and goats showed an increase in short circuit current ( Isc) and transepithelial conductance (gt) upon mucosal removal of divalent cations. A divalent-sensitive Isc and gt were present in Na+, K+ or Rb+ buffer, but nearly abolished in mucosal NMDG+ (N-methyl-D-glucamine) buffer. High K buffer, addition of BaCl2 or of ouabain on the serosal side also reduced or abolished the divalent-sensitive Isc. Mucosal Ca2+ was more potent in blocking Isc, but had the same potency as Mg2+ in blocking gt. A prolonged mucosal deprivation of Mg2+ ions increased gt, potential difference and basal as well as the Ca2+-sensitive Isc. Mucosal addition of Mg2+ had a smaller effect on gt after serosal preincubation with Ba. The data suggest that rumen epithelial cells exhibit an apical non-selective cation conductance, which permits the passage of monovalents in the mucosal absence of divalents. The development of a divalent-sensitive Isc in Na buffer requires Na+/K+ pumps and K+ recycling through Ba2+-sensitive K+ conductances on the basolateral side. This Isc is blocked by extracellular Ca2+ and both extracellular and intracellular Mg2+ ions. A prolonged deprivation of mucosal Mg2+ alone seems to affect intracellular Mg2+ in this Mg2+-absorbing tissue.     

External Copper Inhibits the Activity of the Large-Conductance Calcium- and Voltage-sensitive Potassium Channel from Skeletal Muscle

We have characterized the effect of external copper on the gating properties of the large-conductance calcium- and voltage-sensitive potassium channel from skeletal muscle, incorporated into artificial bilayers. The effect of Cu2+ was evaluated as changes in the gating kinetic properties of the channel after the addition of this ion. We found that, from concentrations of 20 microM and up, copper induced a concentration- and time-dependent decrease in channel open probability. The inhibition of channel activity by Cu2+ could not be reversed by washing or by addition of the copper chelator, bathocuproinedisulfonic acid. However, channel activity was appreciably restored by the sulfhydryl reducing agent dithiothreitol. The effect of copper was specific since other transition metal divalent cations such as Ni2+, Zn2+ or Cd2+ did not affect BK(Ca) channel activity in the same concentration range. These results suggest that external Cu2+-induced inhibition of channel activity was due to direct or indirect oxidation of key amino-acid sulfhydryl groups that might have a role in channel gating.     

Tetracyclines reduce Na+/K+ pump capacity in Calu-3 human airway cells.

We studied the effect of tetracyclines on the Na+/K+ pump activity in Calu-3, a human airway cell line. To estimate Na+/K+ pump capacity on the basolateral membrane, an ouabain-sensitive component of the short-circuit current (Isc) was measured in the presence of nystatin, an ionophore of Na+. The application of ouabain (1 mM) to the basolateral solution completely inhibited the Isc generated by adding nystatin (50 microM) to the apical solution. Tetracycline (TC), minocycline (MC), or demethylchlortetracycline (DC) at 0.5 mM applied to the apical but not to the basolateral solution also decreased the nystatin-induced Isc. Neither phlorizin- nor diphenylamine-2-carboxylic acid-sensitive Isc was affected by TC, MC, or DC. These results indicate that tetracyclines may permeate only through the apical membrane with the result that the Na+/K+ pump's capacity for Na+ extrusion should be suppressed without a decrease in Cl- transport.     

Paths of ion transport across canine fetal tracheal epithelium

Fluid secretion by the fetal sheep lung is thought to be driven by secretion of Cl- by the pulmonary epithelium. We previously demonstrated Cl- secretion by tracheal epithelium excised from fetal dogs and sheep. In this study we characterized the ion transport pathways across fetal canine tracheal epithelium. The transport of Na+ and Cl- across trachea excised from fetal dogs was evaluated from transepithelial electrical properties and isotope fluxes. Under basal conditions the tissues were characterized by a lumen-negative potential difference (PD) of 11 mV and conductance of 5.2 mS/cm2. The short-circuit current (Isc) was 43 microA/cm2 (1.6 mueq.cm-2.h-1). Basal Na+ flows were symmetrical, but net Na+ absorption (1.1 mueq.cm-2.h-1) could be induced by exposure of the luminal surface to amphotericin B (10(-6) M). Bilateral replacement of Na+ reduced Isc by 85%. Replacement of submucosal Na+ or exposure to submucosal furosemide (10(-4) M) reduced net Cl- secretion by 60-70%. Luminal exposure to indomethacin (10(-6) M) induced a 50% decrease in Isc, whereas isoproterenol (10(-6) M) increased Isc by 120%. The properties of the Cl- secretory pathway across fetal dog trachea are consistent with the model proposed for Cl- secretion across adult dog trachea and other Cl- -secreting tissues (e.g., bullfrog cornea and shark rectal gland). The absence of basal Na+ absorption by fetal dog trachea probably reflects limited apical membrane Na+ permeability.     

Endothelin: a potent stimulator of intestinal ion secretion in vitro.

Effects of endothelin (ET) on electrical properties and Na+ and Cl- fluxes in stripped rabbit ileal mucosa were investigated in vitro in Ussing chambers. Results demonstrate that serosal addition of ET-1, ET-2, ET-3 or the precursor 38 amino acid 'big endothelin' produce dose-dependent increases in short-circuit current (Isc) with maximal effects at approx. 100 nM, 100 nM, 10 nM and 100 nM, respectively and half-maximal effects at 1.4 nM, 5 nM, 1.4 nM and 20 nM, respectively. Mucosal addition of ET-3 failed to elicit a response. Changes in Isc elicited by ET-3 are accompanied by decreases in net fluxes of both Na+ and Cl-. The cyclooxygenase inhibitors, indomethacin and piroxicam, inhibited the increase in Isc produced by ET-3 and indomethacin also abolished the changes in Na+ and Cl- fluxes produced by ET-3. However, no changes in the release of PGE2, thromboxane B2 or 6-keto-prostaglandin F1 alpha could be detected up to 20 min after the addition of ET-3. Preincubation of tissues with neuronal agonists or antagonists, antihistamines or an LTD4/LTE4 receptor antagonist, SKF 104353, failed to alter the response to ET-3. Furthermore, removal of serosal Ca2+ also failed to inhibit the change in Isc produced by ET-3. These results indicate that endothelin is a potent intestinal secretagogue which does not appear to elicit its response through stimulation of PGE2, thromboxane A2 or prostacyclin.     

The neurosecretory effect of ouabain on isolated rabbit ileal mucosa

Ouabain, when added to fluid bathing rabbit ileal mucosa mounted in a flux chamber, transiently increases short circuit current, implying a paradoxical secretory response. To determine the cause of this change, we studied unidirectional fluxes of 36Cl and 23Na and the effects of ion substitution, of reduced Ca concentration, verapamil, tetrodotoxin and atropine. Ouabain 0.1 mM, transiently increased the serosal to mucosal flux of Cl and Na, increased Isc and PD and reduced ion conductance. The Isc response to ouabain was diminished by reducing the bath fluid concentration of Cl, of Ca, and by adding verapamil. Tetrodotoxin both delayed and reduced the maximal Isc response; atropine had no effect. We conclude that ouabain acts by releasing a neurotransmitter of unknown identity and by increasing the serosal to mucosal flux of Cl.     

The electrical potential difference, short circuit current and Na+ and Cl- transport across different segments of sheep colon

1. The electrical potential difference (pd) and short circuit current (Isc) across the sheep colon descendens was significantly higher than across the sheep colon ascendens. 2. The ion equivalent of the Isc and the net Na+ transport from the mucosal (m) to the serosal (s) side of the short-circuited sheep colon descendens were identical, while the net Na+ transport across the colon ascendens exceeded the ion equivalent of the Isc. 3. There was a net m-s Cl- transport across both short-circuited colon segments, indicating that Cl-, like Na+, is absorbed by active transport. 4. The results suggest that active Na+ transport across the sheep colon descendens occurs entirely by an electrogenic mechanism, whereas active Na+ transport across the sheep colon descendens occurs entirely by an electrogenic mechanism, whereas active Na+ transport across sheep colon ascendens probably occurs by both an electrogenic and an electrically silent mechanism.     

The identity of the current carriers in canine lingual epithelium in vitro

Ion transport across the lingual epithelium has been implicated as an early event in gustatory transduction. The fluxes of isotopically labelled Na+ and Cl- were measured across isolated canine dorsal lingual epithelium under short-circuit conditions. The epithelium actively absorbs Na+ and to a lesser extent actively secretes Cl-. Under symmetrical conditions with Krebs-Henseleit buffer on both sides, (1) Na+ absorption accounts for 46% of the short-circuit current (Isc); (2) there are two transcellular Na+ pathways, one amiloride-sensitive and one amiloride-insensitive; (3) ouabain, added to the serosal solution, inhibits both Isc and active Na+ absorption. When hyperosmotic (0.25 M) NaCl is placed in the mucosal bath, both Isc and Na+ absorption increase; net Na+ absorption is at least as much as Isc. Ion substitution studies indicate that the tissue may transport a variety of larger ions, though not as effectively as Na+ and Cl-. Thus we have shown that the lingual epithelium, like other epithelia of the gastrointestinal tract, actively transports ions. However, it is unusual both in its response to hyperosmotic solutions and in the variety of ions that support a transepithelial short-circuit current. Since sodium ion transport under hyperosmotic conditions has been shown to correlate well with the gustatory neural response, the variety of ions transported may likewise indicate a wider role for transport in taste transduction.     

Ion transport in an immortalized rat submandibular cell line SMG-C6

The immortalized rat submandibular epithelial cell line, SMG-C6, cultured on porous tissue culture supports, forms polarized, tight-junction epithelia facilitating bioelectric characterization in Ussing chambers. The SMG-C6 epithelia generated transepithelial resistances of 956+/-84Omega.cm2 and potential differences (PD) of -16.9 +/- 1.5mV (apical surface negative) with a basal short-circuit current (Isc) of 23.9 +/- 1.7 microA/cm2 (n = 69). P2 nucleotide receptor agonists, ATP or UTP, applied apically or basolaterally induced a transient increase in Isc, followed by a sustained decreased below baseline value. The peak DeltaIsc increase was partly sensitive to Cl- and K+ channel inhibitors, DPC, glibenclamide, and tetraethylammonium (TEA) and was completely abolished following Ca2+ chelation with BAPTA or bilateral substitution of gluconate for Cl-. The major component of basal Isc was sensitive to apical Na+ replacement or amiloride (half-maximal inhibitory concentration 392 nM). Following pretreatment with amiloride, ATP induced a significantly greater Isc; however, the poststimulatory decline was abolished, suggesting an ATP-induced inhibition of amiloride-sensitive Na+ transport. Consistent with the ion transport properties found in Ussing chambers, SMG-C6 cells express the rat epithelial Na+ channel alpha-subunit (alpha-rENaC). Thus, cultured SMG-C6 cells produce tight polarized epithelia on permeable support with stimulated Cl- secretory conductance and an inward Isc accounted for by amiloride-sensitive Na+ absorption.     

Effect of chloride on pH microclimate and electrogenic Na+ absorption across the rumen epithelium of goat and sheep

Active Na+ absorption across rumen epithelium comprises Na+/H+ exchange and a nonselective cation conductance (NSCC). Luminal chloride is able to stimulate Na+ absorption, which has been attributed to an interaction between Cl-/HCO3- and Na+/H+ exchangers. However, isolated rumen epithelial cells also express a Cl- conductance. We investigated whether Cl- has an additional effect on electrogenic Na+ absorption via NSCC. NSCC was estimated from short-circuit current (Isc) across epithelia of goat and sheep rumen in Ussing chambers. Epithelial surface pH (pHs) was measured with 5-N-hexadecanoyl-aminofluorescence. Membrane potentials were measured with microelelectrodes. Luminal, but not serosal, Cl- stimulated the Ca2+ and Mg2+ sensitive Isc. This effect was independent of the replacing anion (gluconate or acetate) and of the presence of bicarbonate. The mean pHs of rumen epithelium amounted to 7.47 +/- 0.03 in a low-Cl- solution. It was increased by 0.21 pH units when luminal Cl- was increased from 10 to 68 mM. Increasing mucosal pH from 7.5 to 8.0 also increased the Ca2+ and Mg2+ sensitive Isc and transepithelial conductance and reduced the fractional resistance of the apical membrane. Luminal Cl- depolarized the apical membrane of rumen epithelium. 5-Nitro-2-(3-phenylpropylamino)-benzoate reduced the divalent cation sensitive Isc, but only in low-Cl- solutions. The results show that luminal Cl- can increase the microclimate pH via apical Cl-/HCO3- or Cl-/OH- exchangers. Electrogenic Na+ absorption via NSCC increases with pH, explaining part of the Cl- effects on Na+ absorption. The data further show that the Cl- conductance of rumen epithelium must be located at the basolateral membrane.     

Bioelectric properties and ion flow across excised human bronchi

Bioelectric properties and ion transport of excised human segmental/subsegmental bronchi were measured in specimens from 40 patients. Transepithelial electric potential difference (PD), short-circuit current (Isc), and conductance (G), averaged 5.8 mV (lumen negative), 51 microA X cm-2, and 9 mS X cm-2, respectively. Na+ was absorbed from lumen to interstitium under open- and short-circuit conditions. Cl- flows were symmetrical under short-circuit conditions. Isc was abolished by 10(-4) M ouabain. Amiloride inhibited Isc (the concentration necessary to achieve 50% of the maximal effect = 7 X 10(-7) M) and abolished net Na+ transport. PD and Isc were not reduced to zero by amiloride because a net Cl- secretion was induced that reflected a reduction in Cl- flow in the absorptive direction (Jm----sCl-). Acetylcholine (10(-4) M) induced an electrically silent, matched flow of Na+ (1.7 mueq X cm-1 X h-1) and Cl- (1.9 mueq X cm-12 X h-1) toward the lumen. This response was blocked by atropine. Phenylephrine (10(-5) M) did not affect bioelectric properties or unidirectional ion flows, whereas isoproterenol (10(-5) M) induced a small increase in Isc (10%) without changing net ion flows significantly. We conclude that 1) Na+ absorption is the major active ion transport across excised human bronchi, 2) Na+ absorption is both amiloride and ouabain sensitive, 3) Cl- secretion can be induced by inhibition of the entry of luminal Na+ into the epithelia, and 4) cholinergic more than adrenergic agents modulate basal ion flow, probably by affecting gland output.     

Effect of divalent copper ions on heat denaturation of DNA

Using the thermal denaturation method the effect of bivalent copper of (4-10(-6)-10(-3)) M concentrations on the helix-coil transition of DNA was studied in the solution of Na+ concentrations 10(-3)-10(-1) M. Unlike the previous studies, this paper makes allowance for the effect of impurity ions present in DNA and deionized water. It has been shown that in the region of low Cu2+ and Na+ concentrations, thermal stability increases, the melting range extends and the denaturation curves become asymmetric. At concentrations more than approximately 3-10(-5) M Cu2+, melting temperature starts to fall, and the range reduces to 1-1.5 degrees at [Cu2+] greater than or equal to 2-10(-4) M. As [Cu2+] reaches these values, the denaturation curve asymmetry and melting range increase again, which is due to the inversion of the relative stability of AT- and GC-pairs. Employing experimental and phase-transition-theory data for homopolymers, the constants of Cu2+ binding with phosphates and DNA bases were calculated. The concentration dependence of the DNA denaturation parameters was shown to be governed by the superposition of binding Cu2+ with phosphates and nucleic acid bases.