Na-Ca Exchange Mechanism Research Articles

Involvement of a Na-Ca exchange mechanism in contraction induced by low-Na solution in isolated guinea-pig aorta.

The possibility of involvement of a Na-Ca exchange mechanism in the contractile responses induced by a reduction of external Na concentration ([Na]0) has been studied in isolated guinea-pig aorta. Low-Na (11.9 mM) solution (Li-substituted) produced a contraction in ouabain-treated muscles in the presence of phentolamine (10(-6) M). The magnitude of the contraction was dependent on the duration of the pretreatment with ouabain (2 x 10(-5) M). Ca-free solution, but not verapamil (10(-6) M), abolished the contraction induced by low-Na solution. The muscles were loaded with various amounts of Na by incubating the tissue with ouabain and varying [Na]0 (11.9--148.7 mM) in the absence of Ca. The magnitude of the contractions induced in these muscles by low-Na solution containing Ca (2.25 mM) was dependent on the cellular Na content. Loss of cellular Na into low-Na solution followed a single exponential time course and the rate coefficient of Na-loss in the presence of external Ca was about twice as great as in the absence of Ca. Cellular 45Ca uptake in low-Na solution was significantly greater in Na-loaded tissues (pretreated with ouabain for 3 h) than in normal tissues. The 45Ca uptake in low-Na solution was not inhibited by verapamil. These results suggest that the contraction induced by low-Na solution is caused by a Ca influx which is dependent on internal Na (a Na-Ca exchange mechanism).

Potential and tension changes induced by sodium removal in dog Purkinje fibres: role of an electrogenic sodium-calcium exchange.

1. Isolated dog Purkinje fibres were bathed in K-free media or in the presence of ouabain 10(-4) M in order to depress the electrogenic sodium pump activity. Membrane potential and mechanical tension were recorded in the presence of normal external sodium concentration and during lowering or removal of external Na. 2. Lowering or removal of external Na (Na being replaced by choline, Tris, sucrose or Li) induced a hyperpolarization and a contracture which reached a maximum after 1 or 2 min and then decreased progressively. Using Tris, Em increased from -40 +/- 3 to -72 +/- 10 mV (n = 39). The Na-free contracture and hyperpolarization did not occur in the absence of Na pump depression. 3. Tetrodotoxin (1.2 x 10(-5)M), Mn (4 mM), verapamil (1-4 x 10(-5) M) tetraethylammonium (5 mM), 4-aminopyridine (5 mM) and Cs (20 mM, in the presence of ouabain) did not alter the Na-free contracture and hyperpolarization. On the other hand Mn (20 mM), acid media (external pH less than 6.0) and low temperatures depressed or suppressed both the hyperpolarization and contracture. Lanthanum (0.4 mM) did not suppress the hyperpolarization and the contracture. On the contrary the Na-free contracture was generally increased in the presence of La. 4. Caffeine (10 mM) induced strong contractures with no changes in Em, thus demonstrating the possibility for the Purkinje fibers of developing contractures without concomitant hyperpolarizations. 5. It can be concluded that the Na-free contracture and hyperpolarization are not due to changes in passive conductances but are related to the functioning of an electrogenic Na-Ca exchange mechanism which carries inwardly 1 Ca and outwardly 3 or more Na.

Parathyroid hormone stimulates bone resorption via a Na-Ca exchange mechanism.

Parathyroid hormone (PTH) stimulates the release of calcium from bone and is thought to initiate its action by a direct effect on the plasma membrane of bone cells. Although specific membrane receptors for PTH have not yet been identified in bone, they have been characterized in kidney, and PTH does stimulate production of cyclic AMP in bone. However, the mechanism by which PTH causes the release of Ca from bone is not understood. We have determined that several agents that alter ion transport across biological membranes inhibit PTH-stimulated bone resorption in vitro. These agents include ouabain, veratridine and certain monovalent cation ionophores, which transport monovalent cations selectively. The inhibition of bone resorption is dose dependent and reversible within the first 24 h of treatment. All these compounds would be expected to increase intracellular sodium concentration in bone cells. Also, we find that decreasing the extracellular Na+ concentration prevents PTH-stimulated resorption. Therefore, we propose that PTH acts on bone to stimulate Ca release by means of a Na-Ca exchange mechanism. Decreasing the Na gradient across the bone cell plasma membrane would prevent the Na-Ca exchange and thus inhibit the physiological response to PTH.

Electro- and Pharmacomechanical Coupling in Vascular Smooth Muscle

The resting membrane potential of vascular smooth muscle cells depends largely on the building up of ion gradients and also to some extent on the function of an electrogenic ion pump. However, the role of this membrane potential in determining the mechanical activity of the tissue is rather limited and substances such as noradrenaline and histamine can induce contraction without or with only a limited depolarization of the membrane. This pharmacomechanical coupling is therefore considered the predominant regulatory system in vascular smooth muscle contraction. The absence of action potentials in most of these tissues seems to depend on the rectifying properties of the cell membrane for outward current. The contractile proteins are activated by an increase of the cytoplasmic calcium (Ca), which is supplied from the external membrane and/or from the intracellular Ca store. It is an attractive hypothesis to assume that the outer membrane presents voltage dependent Ca channels which can be activated by depolarization and receptor operated channels which are activated by alpha agonists. Such agonists can also release at the same time Ca from the intracellular stores. The relaxation of the tissue depends on the reuptake of Ca by the intracellular store and on a Ca extrusion across the cell membrane. Some evidence is brought forward that in vascular smooth muscle an ATP-dependent Ca pump is more likely then a Na-Ca exchange mechanism.

Electro- and Pharmacomechanical Coupling in Vascular Smooth Muscle

The resting membrane potential of vascular smooth muscle cells depends largely on the building up of ion gradients and also to some extent on the function of an electrogenic ion pump. However, the role of this membrane potential in determining the mechanical activity of the tissue is rather limited and substances such as noradrenaline and histamine can induce contraction without or with only a limited depolarization of the membrane. This pharmacomechanical coupling is therefore considered the predominant regulatory system in vascular smooth muscle contraction. The absence of action potentials in most of these tissues seems to depend on the rectifying properties of the cell membrane for outward current. The contractile proteins are activated by an increase of the cytoplasmic calcium (Ca), which is supplied from the external membrane and/or from the intracellular Ca store. It is an attractive hypothesis to assume that the outer membrane presents voltage dependent Ca channels which can be activated by depolarization and receptor operated channels which are activated by alpha agonists. Such agonists can also release at the same time Ca from the intracellular stores. The relaxation of the tissue depends on the reuptake of Ca by the intracellular store and on a Ca extrusion across the cell membrane. Some evidence is brought forward that in vascular smooth muscle an ATP-dependent Ca pump is more likely then a Na-Ca exchange mechanism.

Possible mechanism of the inhibitory effect of ouabain on renin secretion from rat renal cortical slices.

1. The effects of ouabain on renin secretion by rat renal cortical slices were studied. 2. Renin secretion was inhibited by 10(-3) M-ouabain in the presence of free Ca (10(-4) to 2.6 x 10(-3) M). Inhibition was blocked at Ca less than 10(-8) M. 3. The effect of free Ca on ouabain-inhibition was shown to be independent of the presence of EGTA, completely reversible, and unrelated to passive leakage of renin from non-viable cells, as assessed by simultaneous release of lactate dehydrogenase activity (LDH). 4. It is proposed that, as a result of inhibition of Na, K-ATPase by ouabain, (a) intracellular Na increases in the renin-secreting juxtaglomerular cells, (b) intracellular Ca increases, via an Na-Ca exchange mechanism, and (c) that Ca accumulation, in some unknown manner, inhibits renin secretion.

Sodium-calcium exchange in regulation of cardiac contractility. Evidence for an electrogenic, voltage-dependent mechanism.

The origin and regulatory mechanisms of tonic tension (Ca current-independent component of contractility) were investigated in frog atrial muscle under voltage-clamp conditions. Tonic tension was elicited by depolarizing pulses of 160 mV (Em = +90 mV, i.e., close to E ca) and 400--600 ms long. An application of Na-free (LiCl) or Ca-free Ringer's solutions resulted in a fast (less than 120 s), almost complete abolition of tonic tension. When [Na]o was reduced (with LiCl or sucrose as the substitutes), the peak tonic tension increased transiently and then decreased below the control level. The transient changes in tonic tension were prevented by using low-Na, low-Ca solutions where the ratios [Ca]0/[Na]40 to [Ca]o/[Na]4o were kept constant (1.1 X 10(-8) mM-3 to 8.7 X 10(-13) mM-5). Na-free (LiCl) solution elicited contractures accompanied by a membrane hyperpolarization or by an outward current even when the Na-K pump was inhibited. 15 mM MnCl2 (or 3 mM LaCl3) inhibited the development of the Na-free contracture and the related part of hyperpolarization or the outward current. In conclusion, our results indicate that tonic tension is regulated by a Na-Ca exchange mechanism. Furthermore, they suggest that this exchange could be electrogenic (exchanging three or more Na ions for one Ca ion) and thus voltage dependent. The possible contribution of an electrogenic Na-Ca exchange in the maintenance of cardiac membrane potential is discussed.

Na-Ca exchange and tension development in guinea-pig aorta.

1. In guinea-pig aorta, removal of external K produced contraction which reached maximum (about 1 g) after about 3 h. Excess K (65.4 mM) produced contraction (about 1 g) at a faster rate of increase than was observed in K-free solution. 2. Verapamil, nifedipine and diltiazem (10−7–10−5 M) and the raising of [Mg]0 (8–32 mM) had no effect on the contraction induced by K-free solution, although they relaxed the contraction induced by high-K solution. Verapamil (10−6 M) also relaxed the contractions induced by other depolarizing agents, TEA (40 mM) and Ba (15 mM). 3. Following incubation in Ca- and K-free solution for 3 h, the readdition of Ca produced contraction at a faster rate of increase than in a K-free normal Ca containing solution. Verapamil (10−6 M) and Mg (16 mM) had exhibited little effect on these Ca-induced contractions. The Ca-induced contractions in high-K solution, on the other hand, were significantly suppressed in the presence of verapamil (10−6 M) and Mg (16 mM). 4. After incubation of the muscle in K-free solution for 1 h, Na depletion caused a more rapid and greater contraction than was seen in untreated muscle. This tension development was also unaffected by verapamil (10−6 M) and Mg (16 mM). 5. Substitution of Sr for Ca produced contraction in K-free or high-K solution. Sr-induced contraction in K-free solution was scarcely affected by verapamil (10−6 M), while Sr-induced contraction in high-K solution was inhibited. 6. 45Ca entry into smooth muscle during the contractions was measured by the modified “La method”. The 45Ca uptake was markedly increased both in K-free and high-K solutions. Verapamil (10−6 M) and Mg (16 mM) had no significant effect on the 45Ca uptake increase clicited by K-free solution, whereas they significantly inhibited the 45Ca uptake increased by high-K solution. 7. It is suggested that the contraction induced by K-free solution is initiated by a mechanism different from that underlying the depolarization-induced contraction, and that it is produced by an influx of Ca through Na-Ca exchange mechanism. The data also suggest that the Ca binding site on the Na-Ca exchange carrier is occupied by Sr ions but not by Mg ions. It is likely that such a carrier system is not influenced by organic “Ca antagonists”.

The Time-dependent and Dose-dependent Effects of the Sulfhydryl Blocker N-Ethylmaleimide on the Tonic Tension in Bullfrog Atrium

The kinetics of the inhibitory action by N-ethylmaleimide (NEM) on ICa-independent tonic tension in bullfrog atrial muscle was studied under voltage-clamped conditions, using the double sucrose gap method. The higher the concentration of NEM became, the larger was the rate of decrease of the tonic tension. In addition, the tension decreased with time of exposure to NEM. When the rate of the decrease of the tonic tension was plotted as a function of NEM concentration, the curve showed saturation kinetics of the Michaelis-Menten type with respect to NEM concentration, following the characteristics of a carrier-mediated process. Moreover, in the presence of excess Ca or half quantity of Na in Ringer solution, the curve showed sigmoidal kinetics. This probably indicates that the condition of either an excess of Ca or a reduction of Na in Ringer solution is apparently a cooperative effector of the inhibitory action of NEM, suggesting that the carrier protein responsible for the generation of tonic tension may be an allosteric protein. Finally, the relationship between ICa-independent tonic tension and the Na-Ca exchange mechanism which is supposed to be a powerful candidate for generating tension was discussed.

Sodium ions, calcium ions, blood pressure regulation, and hypertension: a reassessment and a hypothesis

An attempt is made to elucidate the cellular mechanisms which may account for the well-documented correlation between sodium metabolism and peripheral vascular resistance. As a starting point, the evidence that the Na electrochemical gradient across the vascular smooth muscle cell plasma membrane (sarcolemma) plays an important role in cell calcium regulation is reviewed. Because there is significant resting tension ("tone") in most resistance vessels, the ionized Ca2+ level ([Ca2+]1) in the smooth muscle fibers in these vessels must be maintained above the contraction threshold. Consequently, the Ca transport system in the sarcolemma, presumably an Na-Ca exchange mechanism, must be set so as to hold [Ca2+]1 at this suprathreshold level. Any change in the Na gradient will then be reflected as a change in [Ca2+]1 and, therefore, in steady vessel wall tension and peripheral resistance. The correlation between Na metabolism and hypertension could then be accounted for if a circulating agent, perhaps the "natriuretic hormone," affects the Na gradient (across the sarcolemma) and, therefore, [Ca2+]1 and tension.

The influence of sodium on calcium fluxes in pinched‐off nerve terminals in vitro.

1. The influence of internal and external Na concentrations on Ca movements have been measured in pinch-off presynaptic nerve terminals (synaptosomes). Ca uptake is enhanced when external Na (Nao) is replaced by Li, choline or dextrose, in Na-loaded synaptosomes. Depletion of internal Na (Nai) abolishes the stimulatory effect of external Na removal. 2. Ca uptake from Na-depleted media is proportional to [Na]i -2, and averages about 1-5 mumole Ca/g synaptosome protein per minute when [Na]i is approximately 137 mM. This may correspond to a Ca influx of about 0-1 p-mole/cm-2 sec. 3. External Na is a competitive inhibitor of the Nai-dependent Ca uptake. The interrelationship between [Na]o, [Ca]o and Ca uptake indicate that two external Na ions may compete with one Ca at each uptake site. 4. The distribution of particles with Nai-dependent Ca uptake activity parallels the distribution of synaptosomes in the preparative sucrose gradient. Thus, this Ca uptake activity is probably a property of the pinched-off nerve terminals per se, and not of the mitochondria which may contaminate the synaptosome fraction. 5. The Nai-dependent Ca uptake mechanism requires an intact surface membrane, since synaptosomes subjected to osmotic lysis lose the ability to accumulate Ca by this route. 6. Ca efflux into Ca-free media is largely dependent upon the presence of external Na. The curve relating Ca efflux to [Na]o is sigmoid, and suggests that more than one external Na ion (perhaps 2 or 3) is needed to activate the efflux of each Ca ion. 7. The net Ca gain exhibited by Na-loaded synaptosomes incubated in Na-depleted media can be accounted for by the increased Ca uptake and decreased Ca loss observed under these conditions. 8. Treatment of synaptosomes with cyanide or 2,4-dinitrophenol decreases Ca uptake and enhances Ca efflux into Na-containing media. This results in a net loss of Ca from the terminals, even in the presence of external Ca. 9. In contrast to the Ca efflux from synaptosomes, the Ca efflux from brain mitochondria is not dependent upon external Na, and is reduced by succinate, a substrate which is known to fuel mitochondrial respiration. 10. The temperature coefficient (Q10) of the Nai-dependent Ca uptake is about 3. 11. The Nai-dependent Ca uptake is reduced at low pH. The relationship between this Ca uptake and pH approximates a titration curve with a pKa of about 5-6. 12. The data indicate that Ca transport in rat brain presynaptic terminals may involve a carrier-mediated Na-Ca exchange mechanism, and that some of the energy required for Ca extrusion may come from the Na electrochemical gradient across the surface membranes.

Late potentiating effect of low-K ringer solution on the contractility of the bullfrog ventricle.

1) The electrical and mechanical activity of bullfrog ventricle was investigated during and after a prolonged exposure to a K-depleted fluid, which was applied for the purpose of increasing the intracellular Na ions in exchange for K ions.2) There was a gradual increase in twitch tension during immersion in the K-depleted fluid in addition to the immediate potentiation by this solution. The action potential was slightly depressed in height during the immersion.3) There was also a relative reduction in the rate of relaxation of twitch with the gradual increase in tension, and the twitch pattern became a type of rapid onset and slow relaxation.4) The muscles treated with cold K-depleted fluid for an extremely long period of time (24 hr) showed a similar augmentation in contractility and a particularly high sensitivity to extracellular Na-depletion for generating a Na-lack contracture.5) Possible mechanisms relating the intracellular ionic environment to the myocardial contractility were discussed, and attempts were made to interprete the results in terms of Na-Ca exchange mechanism across the cell membrane or Na-Ca competition at the subcellular membrane sites.

Tension fall after contraction of bullfrog atrial muscle examined with the voltage clamp technique.

1) Tension fall after termination of a depolarization was studied on the bullfrog atrial muscle under voltage clamp by the double glycerol-gap technique.2) The tension fall was composed of three different phases:(a) initial retardation, (b) principal rapid relaxation, and (c) final slow relaxation.3) The initial and second phases appeared exponential with time after repolarization. At 17°C, the time constant of the former was 0.07-0.08sec and that of the latter, 0.3-0.4sec for 25 to 80mV depolarizations.4) The tension and the rate of tension fall during the second principal phase appeared almost proportional to the maximal tension at the end of depolarization, and strongly depended on the membrane potential levels before and after repolarization.5) The principal tension fall during relaxation at different potential levels was also exponential, and the time constant increased in a hyperbolic manner with depolarization.6) The relationship between the logarithm of late tension around 1 sec after repolarization and the membrane potential level during relaxation was linear for the depolarizing side. For the hyperpolarizing side the tension, becoming independent of the potential, appeared as a mere function of time.7) These results are discussed in connection with possibilities that the initial retardation of tension fall relates to probable Ca influx immediately after repolarization and the second principal phase of relaxation, to the Na-Ca exchange mechanism.

Membrane currents responsibile for contraction and relaxation of the bullfrog ventricle.

1) The membrane potentials, currents, and contractile responses were measured in the bullfrog ventricular trabeculae under voltage clamped conditions by means of the double glycerol-gap technique, and the membrane currents responsible for initiation of contraction and relaxation were studied.2) For the initiation of twitch contraction, the fast Na current was found to play a minor role, producing less than 6% of the maximum tension, whereas the slow current system was most important.3) Under constant presence of tetrodotoxin (10-7g/ml), 50% Na-depletion produced a marked augmentation of the active slow inward current as well as contractile tension and a conspicuous decrease in the threshold voltages, indicating the presence of Ca-Na antagonism in this slow current system. These effects, however, became temporary when external Na concentration was further decreased to 25% or totally eliminated.4) Na depletion experiments also revealed that a steady outward current due to elimination of leaky Na inward current was accompanied by a sustained tonic contracture, while reintroduction of Na ions elicited a steady inward current and a relaxation of contracture.5) Kinetics of tension development due to rectangular depolarization and relaxation due to repolarization appeared quite different, the former showing a complication and the latter a simple exponential curve in Nadeficient condition. Na ions accelerated the relaxation by decreasing the time constant and appeared necessary for rapid relaxation.6) These results suggest a presence of more than two types of excitationcontraction systems in the bullfrog ventricle; the fast and slow inward currents responsible for the initiation of phasic contraction and some Na-Ca exchange mechanism responsible for that of tonic contraction and for the relaxation of phasic and tonic contractions.