Introduction: Mechanisms of positive inotropic effects

Summary This review deals with the principal mechanisms which are known to play a role in positive inotropism: 1) The myoplasmic Ca²⁺ concentration may be increased by increases in cyclic AMP. Beside receptor-mediated stimulation (isoprenaline) or direct stimulation (forskolin) of the adenylate cyclase, the cyclic AMP may be increased by phosphodiesterase inhibition; 2) Cyclic AMP-independent activation of Ca²⁺ channels can be brought about by alpha-adrenergic agents (phenylephrine) or so-called calcium agonists; 3) Only a small increase in myoplasmic Na⁺ concentration can greatly enhance the force of contraction by an increase in the intracellular Ca²⁺ concentration. This is possible by inhibition of the Na⁺/K⁺-ATPase (glycosides) or by prolongation of the open state of Na⁺ channels (DPI 201-106); 4) A direct inhibition of the Na⁺/Ca²⁺ exchange has been discussed for amiloride; 5) A prolongation of the action potential induced by K⁺ channel-inhibiting agents such as 4-amino-pyridine may increase the myoplasmic Ca²⁺ concentration by a prolongation of the slow Ca²⁺ inward current; 6) An increased Ca²⁺ sensitivity of the contractile proteins has been demonstrated for a number of compounds in vitro; the contribution of such an effect to the overall positive inotropism is unknown because a calcium sensitizer without any effects on calcium or sodium movements is not yet available.     

Role of calcium influx in the regulation of sugar transport in resting left atrial muscle

The role of external Ca²⁺ in the regulation of sugar transport in isolated resting atria of rats and guinea pigs was studied by measuring the tissue/medium distribution of ¹⁴C-labelled 3-methylglucose and of ⁴⁵Ca. Omission of Ca²⁺ from the medium strongly antagonized the stimulation of sugar transport by insulin, hyperosmolarity (100 mM mannitol) or Na⁺-pump inhibition (K⁺ -free medium). Basal sugar transport was not affected by Ca²⁺ omission but was increased when 0.5 mM EGTA was also added. The Ca²⁺ antagonist drug D-600 decreased ⁴⁵Ca influx and also inhibited sugar-transport stimulation by the above 3 treatments in the rat, while in the guinea pig it antagonized only insulin-stimulated transport. The stimulation of sugar transport by a high adrenaline concentration and the inhibition by a low concentration were both antagonized by Ca²⁺ omission or D-600. The results illustrate the important role of Ca²⁺ influx in the control of sugar transport by hormonal and other modulators and are consistent with the hypothesis that cytoplasmic Ca²⁺ regulates glucose transport in muscle.     

Na+/K+ pump inhibition induces cell shrinkage in cultured chick cardiac myocytes

Summary Myocardial cell swelling occurs in ischemia and in reperfusion injury before the onset of irreversible injury. Swelling has been attributed to failure of the Na⁺/K⁺ pump and the accumulation of intracellular Na⁺. To evaluate the role of the pump-leak model of cell volume maintenance, short term changes in cell volume in response to Na⁺/K⁺ pump inhibition were studied in aggregates of cultured embryonic chick cardiac myocytes using optical and biochemical methods. Exposure to 100 M ouabain over 20 min induced cell shrinkage of approximately 10%. Cell water was also decreased by Na⁺/K⁺ pump inhibition; incubation for 1 hr either in the presence of 100 M ouaain or in K⁺-free solution reduced cell water by 18.4% and 28.4% respectively. When exposed to ouabain in the absence of extracellular Ca²⁺, the aggregates swelled by approximately 15%, indicating that extracellular Ca²⁺ was required for the ouabain-induced shrinkage to occur. Ouabain still caused shrinkage, however, in the presence of the Ca²⁺ channel blockers verapamil (10 M) and nifedipine (10 M), suggesting that Na⁺/Ca²⁺ exchange, rather than Ca²⁺ channels, is the route for Ca²⁺ influx during Na⁺/K⁺ pump inhibition. Efflux of amino acids (taurine, aspartate, glutamate, glycine and alanine) from confluent monolayers of chick heart cells exposed to ouabain for 20 min was nearly double that observed in control solution. These results suggest that, during Na⁺/K⁺ pump inhibition, chick heart cells can limit accumulation of intracellular sodium by means of Na⁺/Ca²⁺ exchange, and that a rise in intracellular [Ca²⁺], also mediated by Na⁺/Ca²⁺ exchange, promotes the loss of amino acids and ions to cause cell shrinkage. Therefore, swelling during ischemic injury may not result from Na⁺/K⁺ pump failure alone, but may reflect the exhaustion of alternative volume regulatory transport mechanisms.     

The presence of cyclic AMP-stimulated protein kinase substrates and evidence for endogenoudogenous protein kinase activity in various Na+, K+-ATPase preparations from brain, heart and kidney.

Protein kinase catalyzed the phosphorylation of membrane preparations possessing Na⁺, K⁺-ATPase activity from beef brain, beef heart and dog kidney. Cyclic AMP (10^?6m) stimulated phosphorylation from 1.9 to 2.8-fold for nearly all preparations in the presence of exogenous protein kinase. Cyclic AMP in the absence of exogenous protein kinase stimulated phosphorylation in all preparations examined except highly purified dog outer medulla kidney Na⁺, K⁺-ATPase, suggesting the presence of endogenous protein kinase activity in the former preparations, but a removal of protein kinase after purification of Na⁺, K⁺-ATPase. Cyclic AMP stimulated the phosphorylation of histone in the presence of the beef brain Na⁺, K⁺-ATPase-containing membrane fragments, further substantiating the presence of endogenous protein kinase activity in this preparation. The phosphorylated products were hydroxylamine-insensitive. Phosphorylation of glycerol-precipitated membrane preparations exhibiting various specific Na⁺, K⁺-ATPase activities including zero, in heat-denatured preparations, suggested that the level of phosphorylation is not influenced by Na⁺, K⁺-ATPase activity. SDS-polyacrylamide gel electrophoresis of a phosphorylated, highly purified dog outer medulla kidney Na⁺, K⁺-ATPase preparation suggested that cyclic AMP stimulated phosphorylation of the larger of the two Na⁺, K⁺-ATPase peptide components.     

Influence of adrenalin on sugar transport in soleus,a red skeletal muscle

The effect of adrenalin on the transport of the nonmetabolized sugar, 3-methylglucose, was studied in the isolated rat soleus. Basal sugar transport was inhibited and Na⁺ and K⁺ gradients were increased by 10^?8 to 10^?3 M adrenalin. Isoproterenol and phenylephrine had the same effects which were due to beta-adrenoceptor interaction. Adrenalin was also inhibitory under anoxic conditions and in the presence of palmitate and of low levels of insulin. External Na⁺, K⁺ or Ca²⁺ were not required for its effect and the data suggest that it involves a change in the apparent V_max of transport rather than in K_m. Inhibition of sugar influx was not always paralleled by lower internal Na⁺ levels, nor was it correlated with changes in ATP or G-6-P; this excludes several possible mechanisms for the effect. The failure of high adrenalin concentrations to stimulate sugar transport (as in diaphragm and heart muscle) is attributed to the high oxidative capacity of this red muscle.     

Mechanisms underlying the insulinostatic effect of peptide YY in mouse pancreatic islets

Summary Peptide YY is an insulinostatic peptide which is released into the circulation from the intestinal mucosa upon food intake. Peptide YY is also co-stored with glucagon in the secretory granules of the pancreatic alpha cells. We examined the mechanisms underlying the insulinostatic effect of peptide YY in isolated mouse pancreatic islets. We found that peptide YY (0.1 nmol/l-1 mol/l) inhibited glucose (11.1 mmol/l)-stimulated insulin secretion from incubated isolated islets, with a maximal inhibition of approximately 70% observed at a dose of 1 nmol/ 1 (p<0.001). Also in perifused islets the peptide (1 nmol/l) inhibited insulin secretion in response to 11.1 mmol/l glucose (p<0.001). Furthermore, peptide YY inhibited glucose-stimulated cyclic AMP formation (by 67%, p<0.05), and insulin secretion stimulated by dibutyryl cyclic AMP (p<0.01). In contrast, the peptide was without effect both on the cytoplasmic Ca²⁺ concentration in dispersed mouse islet-cell suspensions as measured by the FURA 2-AM technique, and on insulin release in isolated islets, when stimulated by the protein kinase C-activator 12-O-tetradecanoyl phorbol 13-acetate. Finally, in pre-labelled perifused islets, peptide YY caused a small and transient increase in the ⁸⁶Rb⁺ efflux (p<0.001), but only in the absence of extracellular Ca²⁺. We conclude that peptide YY inhibits glucose-stimulated insulin secretion from isolated mouse islets by inhibiting two different steps in the cyclic AMP cascade, that is, both the accumulation and the action of the cyclic nucleotide. In contrast, the data suggest that protein kinase C, K⁺ channels, the cytoplasmic Ca²⁺ concentration or other processes directly regulating the exocytosis are not involved in the signal transduction underlying peptide YY-induced inhibition of insulin secretion.Abbreviations PYY Peptide YY - TPA 12-O-tetradecanoylphorbol 13-acetate     

Inhibitory mechanisms of kinetin,a plant growth-promoting hormone,in platelet aggregation

Kinetin has been shown to have anti-aging effects on several different systems including plants and human cells. The aim of this study was to examine the detailed inhibitory mechanisms of kinetin in platelet aggregation. In this study, kinetin concentration-dependently (50-150 μM) inhibited platelet aggregation in human platelets stimulated by agonists. Kinetin (70 and 150 μM) also concentration-dependently inhibited intracellular Ca²⁺ mobilization and phosphoinositide breakdown in platelets stimulated by collagen (1 μg/ml). Kinetin (70 and 150 μM) significantly inhibited thromboxane A₂ formation stimulated by collagen (1 μg/ml) and arachidonic acid (60 μM) in human platelets. In addition, kinetin (70 and 150 μM) significantly increased the formation of cyclic AMP. Intracellular pH values were measured spectrofluorometrically using the fluorescent probe BCECF-AM in platelets. The thrombin-evoked increase in pHi was markedly inhibited in the presence of kinetin (70 and 150 μM). Rapid phosphorylation of a platelet protein of molecular weight (M_r) 47 000 (P47), a marker of protein kinase C activation, was triggered by collagen (1 μg/ml). This phosphorylation was inhibited by kinetin (70 and 150 μM). In conclusion, these results indicate that the anti-platelet activity of kinetin may be involved in the following pathways: kinetin's effects may initially be due to inhibition of the activation of phospholipase C and the Na⁺/H⁺ exchanger. This leads to lower intracellular Ca²⁺ mobilization, followed by inhibition of TxA₂ formation and then increased cyclic AMP formation, followed by a further inhibition of the Na⁺/H⁺ exchanger, ultimately resulting in markedly decreased intracellular Ca²⁺ mobilization and phosphorylation of P47. These results suggest that kinetin has an effective anti-platelet effect and that it may be a potential therapeutic agent for arterial thrombosis.     

The release of calcium from heart mitochondria by sodium

The release of Ca²⁺ from heart mitochondria could be induced by addition of 20 to 50 mm Na⁺ to the incubation medium. Of the other cations tested, K⁺, Rb⁺, Cs⁺, and Mg²⁺ were without effect, whereas some release was induced by Li⁺. In the presence of ruthenium red, which prevented the re-binding of the released Ca²⁺, 2 to 5 mm Na⁺ were sufficient to produce a measurable release of Ca²⁺. The amount of Ca²⁺ freed from the mitochondria was proportional to the amount of Na⁺ added, and to the concentration of Ca²⁺ present in the mitochondria. The release appeared to be a biphasic process, and in the first 15 s up to 3 nmol Ca²⁺ per mg of mitochondrial protein could be dissociated from the mitochondria.The phenomenon was more evident at pH 6.5 than under slightly alkaline conditions. The significance of this release in the process of heart contraction is discussed.     

Papaverine blockade of an inward slow Ca2+ current in guinea pig heart.

The membrane potentials of the ventricular fibers at the epicardial surface of isolated perfused guinea pig hearts were recorded with intracellular microelectrodes. Papaverine ($\text{15 mg}\text{100 ml}$) markedly depressed the rate of rise (to about 10V/s) within 10–15 min, prolonged the duration of the action potential, and abolished the contractions, i.e., the hearts became electromechanically uncoupled. The rate of rise or overshoot of this slowly-rising action potential was not affected by tetrodotoxin (TTX). Papaverine also abolished within 3 min the slowly-rising overshooting, plateau-like responses (with accompanying contractions) induced by isoproterenol (10^?7m) in hearts perfused with 27 mm K⁺ (cells depolarized to ?40 mV) to inactivate the fast Na⁺ channels. The effects of papaverine were readily reversible. The cyclic AMP levels of the ventricles were elevated nearly 3 fold by papaverine, and the ATP levels were not significantly depressed. Verapamil and metabolic inhibitors did not increase the cyclic AMP levels. These results suggest that papaverine has three major actions: (1) it blocks fast Na⁺ channels; (2) it directly blocks the inward slow Ca²⁺ current (like verapamil does), but leaves a TTX-insensitive slow cation current (presumably Na⁺) operational in 2.7 mm K⁺; and (3) it increases cyclic AMP levels, presumably by acting as a phosphodiesterase inhibitor.     

SBA-15 with Crystalline Walls Produced via Thermal Treatment with the Alkali and Alkali Earth Metal Ions

Crystalline walled SBA-15 with large pore size were prepared using alkali and alkali earth metal ions (Na⁺, Li⁺, K⁺ and Ca²⁺). For this work, the ratios of alkali metal ions (Si/metal ion) ranged from 2.1 to 80, while the temperatures tested ranged from 500 to 700 °C. The SBA-15 prepared with Si/Na⁺ ratios ranging from 2.1 to 40 at 700 °C exhibited both cristobalite and quartz SiO₂ structures in pore walls. When the Na⁺ amount increased (i.e., Si/Na increased from 80 to 40), the pore size was increased remarkably but the surface area and pore volume of the metal ion-based SBA-15 were decreased. When the SBA-15 prepared with Li⁺, K⁺ and Ca²⁺ ions (Si/metal ion = 40) was thermally treated at 700 °C, the crystalline SiO₂ of quartz structure with large pore diameter (i.e., 802.5 Å) was observed for Ca⁺² ion-based SBA-15, while no crystalline SiO₂ structures were observed in pore walls for both the K⁺ and Li⁺ ions treated SBA-15. The crystalline SiO₂ structures may be formed by the rearrangement of silica matrix when alkali or alkali earth metal ions are inserted into silica matrix at elevated temperature.     

Inhibition of slow action potentials of guinea pig atrial muscle by adenosine: a possible effect on Ca2+ influx.

Adenosine decreases the force of contraction in atrial muscle, the uptake of Ca²⁺, the plateau phase and duration of the action potential. In contrast to acetylcholine which increases K⁺ permeability the effects of adenosine are antagonized by caffeine and are not blocked by atropine. It has been suggested that these effects of adenosine are mediated by a depression of the cell membrane to Ca²⁺ permeability. In order to test this hypothesis we attempted to determine whether adenosine had inhibitory effects on the slow action potential of potassium-depolarized (20 mm) atrial muscle treated with norepinephrine (5 × 10^?5m). With the slow action potentials it appears that Ca²⁺ carries the inward current since (1) the overshoot varies according to the Nernst equation upon changes in extracellular [Ca²⁺], (2) changes in inward ionic flow associated with the action potential are paralleled by changes in developed tension. Adenosine at micromolar concentrations reduced within seconds the rate of rise and amplitude of the action potential. The action potentials lost their all-or-none nature and appeared graded with adenosine. The muscle became completely inexcitable at concentrations as low as 10.2 μm. These effects of adenosine could either be reversed within seconds by enzymatic degradation of adenosine or by raising the extracellular [Ca²⁺]. These findings suggest that adenosine depresses the membrane permeability to Ca²⁺ either directly or through an indirect membrane stabilizing effect mediated by permeability changes in Na⁺ or K⁺ ions. This effect of adenosine possibly occurs on the extracellular side of the membrane since adenosine can only exist extracellularly and large and charged adenosine derivatives (ATP, ADP, AMP, c-AMP, NAD and NADP) that probably do not penetrate the cell cause similar effects.     

Increased Potassium Permeability by Calcium in Hypochromic Red Blood Cells

The red blood cell (RBC) content of Na⁺ and K⁺ were measured both on fresh cells from normal, heterozygous β-thalassaemic and iron-deficiency-anaemic subjects, and on the same cells incubated for 24 h, at 37° C, either in presence or in absence of Calcium (Ca²⁺). Ca²⁺ did not increase membrane permeability to Na⁺, but increased the K⁺ loss, both from normal cells and to a greater degree much more from hypochromic cells. Glucose largely prevented the K⁺ loss from hypochromic cells incubated either in absence or in presence of Ca²⁺, probably maintaining an adequate level of ATP during the incubation. EDTA only partially decreased the permeability to K⁺ in hypochromic cells incubated for 24 h at 37° C, possibly removing Ca²⁺ bound to the cell membrane. The results suggest that Ca²⁺ does not represent the primary cause of K⁺ leak in hypochromic cells, but it is able to enhance a pre-existing peculiar abnormality of the cell membrane when the ATP level slows down.     

Characterization of the Cation‐Binding Capacity of a Potassium‐Adsorption Filter Used in Red Blood Cell Transfusion

A K⁺‐adsorption filter was developed to exchange K⁺ in the supernatant of stored irradiated red blood cells with Na⁺. To date, however, the filter's adsorption capacity for K⁺ has not been fully evaluated. Therefore, we characterized the cation‐binding capacity of this filter. Artificial solutions containing various cations were continuously passed through the filter in 30 mL of sodium polystyrene sulfonate at 10 mL/min using an infusion pump at room temperature. The cation concentrations were measured before and during filtration. When a single solution containing K⁺, Li⁺, H⁺, Mg²⁺, Ca²⁺, or Al³⁺ was continuously passed through the filter, the filter adsorbed K⁺ and the other cations in exchange for Na⁺ in direct proportion to the valence number. The order of affinity for cation adsorption to the filter was Ca²⁺>Mg²⁺>K⁺>H⁺>Li⁺. In K⁺‐saturated conditions, the filter also adsorbed Na⁺. After complete adsorption of these cations on the filter, their concentration in the effluent increased in a sigmoidal manner over time. Cations that were bound to the filter were released if a second cation was passed through the filter, despite the different affinities of the two cations. The ability of the filter to bind cations, especially K⁺, should be helpful when it is used for red blood cell transfusion at the bedside. The filter may also be useful to gain a better understanding of the pharmacological properties of sodium polystyrene sulfonate.     

C-peptide stimulates rat renal tubular Na+, K+-ATPase activity in synergism with neuropeptide Y

Summary This study was performed in order to test the hypothesis that the connecting peptide of proinsulin, C-peptide, might in itself possess biological activity. Renal tubular Na⁺, K⁺-ATPase, which is a well-established target for many peptide hormones, was chosen as a model. Rat C-peptide (I) was found to stimulate Na⁺, K⁺-ATPase activity in single, proximal convoluted tubules dissected from rat kidneys. C-peptide increased the Na⁺ affinity of the enzyme and all subsequent studies were performed at non-saturating Na⁺ concentrations. C-peptide stimulation of Na⁺, K⁺-ATPase activity occurred in a concentration-dependent manner in the dose range 10^–8–10^–6 mol/l. The presence of neuropeptide Y, 5×10^–9 mol/l, enhanced this effect and stimulation of Na⁺, K⁺-ATPase activity then occurred in the C-peptide dose range 10^–11–10^–8 mol/l. C-peptide stimulation of Na⁺, K⁺-ATPase activity was abolished in tubules pretreated with pertussis toxin. It was also abolished in the presence of FK 506, a specific inhibitor of the Ca²⁺-calmodulin-dependent protein phosphatase 2B. These results indicate that C-peptide stimulates Na⁺, K⁺-ATPase activity, probably by activating a receptor coupled to a pertussis toxin-sensitive G-protein with subsequent activation of Ca²⁺-dependent intracellular signalling pathways.Abbreviations PTX Pertussis toxin - NPY neuropeptide Y - PCT proximal convoluted tubule - BSA bovine serum albumin - dB cAMP dibutyryl cyclic adenosine monophosphate - PP2B Ca²⁺/calmodulin-dependent protein phosphatase 2B - PKC protein kinase C - [Ca²⁺] intracellular calcium concentration     

Mechanism of Ca2+ resistance in adult heart cells isolated with trypsin plus Ca2+

Ca²⁺-resistant heart cells prepared with trypsin and Ca²⁺ leak Na⁺ and K⁺ more slowly than Ca²⁺-susceptible cells prepared without trypsin and Ca²⁺. The two preparations show similar leak rates for amino acids and nucleotides. Cells prepared with Ca²⁺ alone show low ion leak rates, but the yield of rod-shaped cells is less than half that when trypsin is present. Cells prepared with trypsin alone show high ion leak rates. The Na⁺-K⁺ ATPase activity of Ca²⁺-susceptible cells appears to be approximately three-fold greater than that of Ca²⁺-resistant cells. Imposing a Na⁺-K⁺ leak by the addition of gramicidin D causes no stimulation of Na⁺-K⁺ ATPase in Ca²⁺-susceptible cells, but stimulates the activity of Ca²⁺-resistant cells up to that of the Ca²⁺-susceptible cells. Ca²⁺-resistant cells appear to contain more K⁺ and less Na⁺ than Ca²⁺-susceptible cells. Treatment of Ca²⁺-resistant cells with ouabain (1 mm) for 5 min changes the $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ balance to approximately that of the Ca²⁺-susceptible cells, and induces a similar degree of Ca²⁺ susceptibility. We therefore conclude that treatment with trypsin plus Ca²⁺ confers Ca²⁺ resistance by keeping the permeability of the sarcolemma to Na⁺ and K⁺ sufficiently low to allow the Na⁺-K⁺ ATPase and $\text{Na}{}^{\mathrm{+}}\text{Ca}{}^{\mathrm{+}}$ exchanger to maintain normal gradients of Na⁺, K⁺ and Ca²⁺. The agent responsible for maintaining low ion permeability appears to be Ca²⁺ itself, while trypsin increases the yield and purity of the Ca²⁺-resistant cells.     

Regulation of86Rb outflow from pancreatic islets

Summary Theophylline (1.4 mM), cyclic AMP (1.0 mM) and dibutyryl-cyclic AMP (0.5 mM) decreased⁸⁶Rb fractional outflow rate from pancreatic islets perifused in the absence of glucose. In the presence of glucose (16.7 mM), however, the same drugs provoked a modest increase in⁸⁶Rb fractional outflow rate. The increase in⁸⁶Rb outflow evoked by theophylline in the presence of glucose was suppressed by quinine, suggesting that it may result from an increase in cytosolic Ca²⁺ concentration. It is proposed that changes in the cyclic AMP content of islet cells may participate in the regulation of K⁺ conductance by insulin secretagogues.     

Na+/H+ exchange and its inhibition in cardiac ischemia and reperfusion

Summary The characterization of various ion transport systems has led to a better understanding of the effects, which seem to take part in the impairment of ischemic and reperfused cardiac tissue. This review discusses the role of the Na⁺/H⁺ exchange system in the pathophysiology of ischemia and reperfusion and the beneficial effects of its inhibition.At the onset of ischemia intracellular pH (pH_i) decreases due to anaerobic metabolism and ATP hydrolysis, leading to an activation of Na⁺/H⁺ exchange. This in turn increases intracellular Na⁺ (Na⁺ _i) and activates Na⁺/K⁺ ATPase, with a consecutive increase of energy consumption. Since cellular Na⁺ and Ca⁺⁺ transport are coupled by the Na⁺/Ca⁺⁺ exchange system, which depends on the Na⁺ gradient, the high Na⁺ _i leads to increased intracellular Ca⁺⁺ (Ca⁺⁺ _i). After a certain period, Na⁺/H⁺ exchange is inactivated by a decrease of extracellular pH.In case of reperfusion the acid extracellular fluid is washed out, which reactivates Na⁺/H⁺ exchange, leading to an unfavourably fast restoration of pH_i and a second time to Na⁺ and Ca⁺⁺ _i overflow.High Ca⁺⁺ _i is assumed to be one of the main reasons for ischemic and reperfusion injury, like arrhythmias, myocardial contracture, stunning and necrosis.It seems that the inhibition of Na⁺/H⁺ exchange can interrupt this process at an early phase and prevent or delay the consequences of ischemia and reperfusion as demonstrated by numerous investigators.     

Studies on the role of calcium and cyclic nucleotides in the control of TSH secretion.

The role of putative mediators in the control of thyrotropin (TSH) secretion has been investigated by monitoring hormone release from isolated anterior cells in response to agents and conditions which modify cyclic nucleotide concentrations or calcium fluxes. The secretory response to 5-min pulses of TRH, raised K⁺ concentration and A-23187 was diminished within 5 min when Ca²⁺-free perifusion was begun 80 sec prior to the pulse. In contrast, the response to theophylline and IBMX was unaffected under these conditions. Both IBMX and dibutyryl cyclic AMP potentiated the effects of TRH and raised K⁺concentration but not that of A-23187. Methoxyverapamil inhibited TSH secretion stimulated by TRH, raised K⁺ concentration and IBMX but not that induced by A-23187. Calcium efflux showed a similar temporal profile to hormone secretion in response to TRH, IBMX and raised K⁺ concentration. It is proposed that one interpretation of these findings is that there is an interrelationship between calcium ions and the cyclic nucleotides in the control of TSH secretion and that cyclic nucleotides may act at the level of the Ca²⁺ channel to modulate Ca²⁺ entry.     

NCLX is an essential component of mitochondrial Na+/Ca2+ exchange

Mitochondrial Ca²⁺ efflux is linked to numerous cellular activities and pathophysiological processes. Although it is established that an Na⁺-dependent mechanism mediates mitochondrial Ca²⁺ efflux, the molecular identity of this transporter has remained elusive. Here we show that the Na⁺/Ca²⁺ exchanger NCLX is enriched in mitochondria, where it is localized to the cristae. Employing Ca²⁺ and Na⁺ fluorescent imaging, we demonstrate that mitochondrial Na⁺-dependent Ca²⁺ efflux is enhanced upon overexpression of NCLX, is reduced by silencing of NCLX expression by siRNA, and is fully rescued by the concomitant expression of heterologous NCLX. NCLX-mediated mitochondrial Ca²⁺ transport was inhibited, moreover, by CGP-37157 and exhibited Li⁺ dependence, both hallmarks of mitochondrial Na⁺-dependent Ca²⁺ efflux. Finally, NCLX-mediated mitochondrial Ca²⁺ exchange is blocked in cells expressing a catalytically inactive NCLX mutant. Taken together, our results converge to the conclusion that NCLX is the long-sought mitochondrial Na⁺/Ca²⁺ exchanger.