Changes in the Ca2+-Transport Processes of Red Cells during Cold Storage in ACD

In order to characterize Ca²⁺-transport in red cells stored in ACD Ca²⁺-loading and Mg²⁺-depletion by the ionophore A23187, CaATPase activity determination in intact cells and an accurate Ca²⁺-influx technique were adapted to preserved blood. Active Ca²⁺-efflux (pump) was measured in rejuvenated cells loaded by Ca²⁺ with A23187. The rate of Ca²⁺-pump declined only slightly during 3 weeks of storage (from 80 ± 15 to 66 ± 17 μmoles Ca²⁺/l. cells/min) and a marginal trend of decrease in the Ca:ATP ratio was observed (from 1.96 ± 0.15 to 1.88±0.11). Passive Ca²⁺-influx (leak) was studied in regenerated cells in which the Ca²⁺-pump was blocked with 0.2 mm lanthanum. Ca²⁺-influx showed a slow increase during the first 3 weeks of storage (from 0.4±0.16 to 1.25±0.4 μmoles Ca²⁺/l. cells/min), later it increased rapidly. Passive Ca²⁺ leak and exchange transport were studied in unregenerated, phosphate ester-depleted cells. In this case passive Ca²⁺-influx increased 2–3-fold as early as 1–2 d after storage in ACD. This initial increase was followed by a continuous slow enhancement that reached a flux of 3.5±0.7 μmoles Ca²⁺/l. cells/min after 3 weeks of storage. The passive Ca²⁺-permeability increase that occurred during storage could be readily compensated by the Ca²⁺-pump without causing metabolic imbalance. The Ca²⁺-transport, of unregenerated stored cells, however, showed impairment under certain conditions (A23187+EDTA and lanthanum treatments, ghost preparation). The Ca²⁺-induced shape changes were reversible and ran parallel with the cell Ca²⁺ level during Ca²⁺-pumping up to 5 weeks of storage. This finding indicates a direct relationship between cell Ca²⁺ and shape.     

Influence of metabolic inhibition by NaCN on electrical and mechanical activities of frog atrial fibers: Studies using current and voltage clamp

Voltage clamp experiments have documented that after 10 min NaCN treatment: (1) there is a reduction in both action potential duration and plateau amplitude associated with a reduction in the contractile strength; (2) in Ringer tetrodotoxin there is a marked reduction in the magnitude of the slow inward current and the corresponding tension; (3) there is a negative shift in the reversal potential of the slow inward current.In previous work it was suggested that during metabolic inhibition the lack of high energy compounds leads to an increase in the intracellular free Ca²⁺ concentration, thereby decreasing the Ca²⁺ driving force. However, the present study has shown that excess Ca²⁺ in the bathing solution was unable to increase the slow inward current amplitude during NaCN treatment, although the $\text{I}\text{V}$ relationship showed a restoration of the Ca²⁺ driving force. On the other hand the application of 10^?7m adrenaline during poisoning induced a rapid increase in both the slow inward current and the phasic contraction amplitude. It is concluded that NaCN reduces the slow inward current and the corresponding tension, decreasing both Ca²⁺ driving force and slow channel conductance. The marked increase in both slow inward current and phasic component of contraction with adrenaline suggests that under these conditions cyclic AMP is made available in spite of metabolic inhibition, thereby preventing the increase of internal free Ca²⁺ concentration, and also increasing the slow channel conductance.     

Influence of mild hypothermia on myocardial contractility and circulatory function

Myocardial contractility depends on temperature. We investigated the influence of mild hypothermia (37–31°) on isometric twitch force, sarcoplasmic reticulum (SR) Ca²⁺-content and intracellular Ca²⁺-transients in ventricular muscle strips from human and porcine myocardium, and on in vivo hemodynamic parameters in pigs. In vitro experiments: muscle strips from 5 nonfailing human and 8 pig hearts. Electrical stimulation (1 Hz), simultaneous recording of isometric force and rapid cooling contractures (RCCs) as an indicator of SR Ca²⁺-content, or intracellular Ca²⁺-transients (aequorin method). In vivo experiments: 8 pigs were monitored with Millar-Tip (left ventricular) and Swan-Ganz catheter (pulmonary artery). Hemodynamics parameters were assessed at baseline conditions (37°), and after stepwise cooling on cardiopulmonary bypass to 35, 33, and 31°C. Hypothermia increase isometric twitch force significantly by 91 ± 16% in human and by 50 ± 9% in pig myocardium (31 vs. 37°C; p < 0.05, respectively). RCCs or aequorin light emission did not change significantly. In anesthetized pigs, mild hypothermia resulted in an increase in hemodynamic paramters of myocardial contractility. While heart rate decreased from 111 ± 3 to 73 ± 1 min⁻¹, cardiac output increased from 2.4 ± 0.1 to 3.1 ± 0.3 l/min, and stroke volume increased from 21 ± 1 to 41 ± 3 ml. +dP/dt_max increased by 25 ± 8% (37 vs. 31°C; p < 0.05 for all values). Systemic and pulmonary vascular resistance did not change significantly during cooling. Mild hypothermia exerts significant positive inotropic effects in human and porcine myocardium without increasing intracellular Ca²⁺-transients or SR Ca²⁺-content. These effects translate into improved hemodynamics parameters of left ventricular function. Received: 26 June 2000, Returned for revision: 20 July 2000, Revision received: 11 October 2000, Accepted: 17 October 2000     

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.     

A 4-h hyperglycaemic excursion induces rapid and slow changes in major electrolytes in blood in healthy human subjects

Hyperglycaemia is well known to cause reductions in plasma Na⁺ levels or even hyponatraemia due to an osmotically induced dilution of the interstitium and blood. It is, however, unclear whether this dilution is significantly counteracted by ion regulatory homeostatic mechanism(s) or not. Furthermore, the effects of moderate hyperglycaemia on other major ions are less well known. To further clarify these questions, we measured the changes in blood osmolarity and concentrations of Na⁺, K⁺, Cl^?, Mg²⁺ and Ca²⁺ during a 4-h-long experimental hyperglycaemia in healthy subjects rendered temporarily insulin deficient using the hyperglycaemic clamp. Hyperglycaemia, 16.8?mM, was rapidly imposed from a baseline of 4.4?mM by intravenous somatostatin and glucose infusions in 19 healthy subjects (10?m, 9 f; age 36?±?5?years (mean?±?SD); BMI 22.7?±?2.9?kg/m2). Subsequently, glycaemia was returned to basal and measurements continued until all dynamic changes had stopped (at ~8?h). Osmolarity increased from 281.8?±?0.7 to 287.9?±?0.7, while Na⁺ decreased from 143.9?±?0.3 to 138.7?±?0.2, Cl^? from 101.7?±?0.2 to 99.5?±?0.1, Ca²⁺ from 1.98?±?0.04 to 1.89?±?0.02 and Mg²⁺ from 0.84?±?0.01 to 0.80?±?0.00?mM. All these changes were rapidly reaching stable levels. K⁺ increased from 4.02?±?0.02 to 4.59?±?0.02?mM (P?<?0.0001) also reaching stable levels but with some delay. Na⁺, Cl^?, Mg²⁺ and Ca²⁺ are essentially determined by blood dilution, and their values will remain diminished as long as the hyperglycaemia lasts. Partial suppression of insulin-stimulated Na⁺/K⁺ pumping lead to increased K⁺ levels. The combination of elevated K⁺ and decreased Mg²⁺ and Ca²⁺ levels may lead to an altered excitability, which is particularly relevant for diabetic patients with heart disease.     

Differences in Ca-Handling and Sarcoplasmic Reticulum Ca-Content in Isolated Rat and Rabbit Myocardium

We made novel measurements of the influence of rest intervals and stimulation frequency on twitch contractions and on sarcoplasmic reticulum (SR) Ca²⁺-content (using rapid cooling contractures, RCCs) in isolated ventricular muscle strips from rat and rabbit hearts at a physiological temperature of 37 °C. In addition, the frequency-dependent relative contribution of SR Ca²⁺-uptake and Na⁺/Ca²⁺-exchange for cytosolic Ca²⁺-removal was assessed by paired RCCs. With increasing rest intervals (1–240 s) post-rest twitch force and RCC amplitude decreased monotonically in rabbit myocardium (after 240 s by 45±10% and 61±11%, respectively P<0.05, n=14). In contrast, rat myocardium (n=11) exhibited a parallel increase in post-rest twitch force (by 67±16% at 240 sP <0.05) and RCC amplitude (by 20±14%P<0.05). In rabbit myocardium (n=11), increasing stimulation frequency from 0.25 to 3 Hz increased twitch force by 295±50% (P<0.05) and RCC amplitude by 305±80% (P<0.05). In contrast, in rat myocardium (n=6), twitch force declined by 43±7% (P<0.05), while RCC amplitude decreased only insignificantly (by 16±7%). The SR Ca²⁺-uptake relative to Na⁺/Ca²⁺-exchange (based on paired RCCs) increased progressively with frequency in rabbit, but not in rat myocardium (66±2% at all frequencies). We conclude that increased SR Ca²⁺-load contributes to the positive force–frequency relationship in rabbits and post-rest potentiation of twitch force in rats. Decreased SR Ca²⁺-load contributes to post-rest decay of twitch force in rabbits, but may play only a minor role in the negative force–frequency relationship in rats. SR Ca²⁺-release channel refractoriness may contribute importantly to the negative force-frequency relationship in rat and recovery from refractoriness may contribute to post-rest potentiation.     

The effect of oligomycin on the electrical activity of frog atrial muscle and the effect of variation of [Ca2+]0 concentration

The effect of oligomycin on electrical activity in frog atrial fibers has been studied using the current and voltage clamp double sucrose gap technique. Also, the accompanying contraction has been measured. Current clamp studies showed that oligomycin reduced both peak amplitude and duration of the action potential, and also the contractile force; the effect increasing with concentration. Recovery occurred following washout, but this also was dependent on concentration on inhibitor, with recovery not being achieved if concentration of inhibitor exceeded a given value. Voltage clamp demonstrated a marked reduction in the slow inward current amplitude and a negative shift of the reversal potentials. In addition, oligomycin induced a slight increase in the delayed outward current, this increase not occurring in the early repolarization phase of the action potential. In the presence of oligomycin with excess [Ca²⁺]₀ the action potential shortened, but its amplitude increased. Excess [Ca²⁺]₀ during inhibition also increased the slow inward current amplitude and shifted the reversal potential toward a more positive value. These results suggest that when the coupling of respiration with oxidative phosphorylation is inhibited the level of ATP in the sarcoplasm is decreased, leading to an increase in intracellular free Ca²⁺ concentration, thereby reducing the transmembrane Ca²⁺ concentration gradient. This decreases the Ca²⁺ driving force which is Ca-dependent and alters the action potential amplitude, together with the slow channel conductance (which is Ca²⁺ dependent) leading to a reduction of excitation-contraction coupling.     

High extracellular sodium and digoxin-induced arrhythmias in guinea-pig ventricular myocardium

Oscillatory fluctuations in diastolic potential [4, 10] and triggered rhythmical activity [4] are well known manifestations of digitalis intoxication and are attributed to intracellular Ca²⁺ overload consecutive to an elevated intracellular Na⁺ [7]. In the present paper we report the electrophysiological effects of elevated extracellular Na⁺ on the spontaneous rhythmical activity and oscillatory fluctuations in digoxin-intoxicated ventricular cells. Our results show that by increasing extracellular Na⁺ from 135 mm to 175 mm, we were able to abolish spontaneous rhythmical activity but were not able to suppress the damped sinusoidal oscillations in the resting membrane potential. We suggest that Na⁺Ca²⁺ exchange would be stimulated by a greater Na⁺ gradient, resulting in an enhanced Ca²⁺ extrusion.     

Nongenomic Actions of Thyroid Hormone and Intracellular Calcium Metabolism

Nongenomic actions of thyroid hormone include several that involve or require calcium. Actions of thyroid hormone at the plasma or intracellular membranes include stimulation of membrane glucose transport and of the Na⁺/H⁺ antiporter (exchanger) by mechanisms that require liberation of intracellular calcium and stimulation of the cell membrane and sarcoplasmic reticulum calcium pumps (Ca²⁺-ATPases). These pumps not only transport Ca²⁺, but also are regulated by the intracellular calmodulin-Ca²⁺ complex (plasma membrane/sarcolemma) or calmodulin-dependent protein kinase II phosphorylation of phospholamban (sarcoplasmic reticulum). Intracellular calcium ion concentration may also be subject to regulation by other nongenomic effects of iodothyronines, such as those on the Na⁺/H⁺ antiporter or sodium current, that secondarily affect the Na⁺/Ca²⁺ exchanger. Certain of these nongenomic actions of thyroid hormone, e.g., Na⁺/H⁺ exchanger, Ca²⁺-ATPase, are now recognized to begin at a recently described hormone receptor on a heterodimeric structural membrane protein, integrin αvβ3. The thyroid hormone signal at this receptor is further transduced by the mitogen-activated protein kinase (MAPK; extracellular regulated kinase1/2, ERK1/2) pathway.     

5′-Guanylimidodiphosphate stimulation of slow Ca2+ current in myocardial cells

Positive inotropic agents which elevate cyclic AMP, such as catecholamines, histamine, and methylxanthines, increase the number of membrane slow cationic channels available for voltage activation. 5′-Guanylimidodiphosphate (GPP(NH)P), an agent known to directly activate adenylate cyclase in a variety of broken cell preparations, was tested for its ability to induce slow Ca²⁺ channels in embryonic chick ventricular myocardial cells. The preparations used were reaggregated cell culture and intact noncultured ventricular myocardium. When the myocardial cells were rendered inexcitable by tetrodotoxin (which blocks the fast Na⁺ channels), addition of any of the positive inotropic agents stated above gives rise to slowly-rising overshooting electrical responses (the “slow response”) accompanied by contractions within 1 to 3 min. The addition of GPP(NH)P (10^?5 to 10^?3m) also induced the slow response within 5 to 30 min. The slow responses induced by GPP(NH)P were blocked by agents known to block slow channels and Ca²⁺ current, such as Mn²⁺ (1 mm) and verapamil (10^?6m). GTP did not induce the slow response. These results support the hypothesis that the intracellular level of cyclic AMP controls the availability of the slow Ca²⁺ channels in the myocardial sarcolemma.     

Excitation-contraction coupling in gastric muscles

Several mechanisms contribute to the regulation of force generated by gastric muscles. Phasic contractions in the stomach are triggered by the propagation of electrical slow waves. These events are associated with an influx of Ca²⁺ and an increase in intracellular Ca²⁺ sufficient to elicit contraction. Entry of Ca²⁺ may be supplemented by the release of Ca²⁺ from intracellular stores. Excitatory agonists enhance the amplitude of slow waves, increase the amplitude of Ca²⁺ transients, and increase the force of phasic contractions. Inhibitory agonists have opposite effects. Excitatory agonists may also enhance release of Ca²⁺ from stores via the production of IP₃. Excitatory and inhibitory agonists may also regulate the sensitivity of the contractile apparatus for Ca²⁺ and therefore alter the contractile response to a given change in intracellular Ca²⁺.     

Activation of the SK potassium channel-calmodulin complex by nanomolar concentrations of terbium

Small conductance Ca²⁺-activated K⁺ (SK) channels sense intracellular Ca²⁺ concentrations via the associated Ca²⁺-binding protein calmodulin. Structural and functional studies have revealed essential properties of the interaction between calmodulin and SK channels. However, it is not fully understood how the binding of Ca²⁺ to calmodulin leads to channel opening. Drawing on previous biochemical studies of free calmodulin using lanthanide ions as Ca²⁺ substitutes, we have used the lanthanide ion, Tb³⁺, as an alternative ligand to study the activation properties of SK channels. We found that SK channels can be fully activated by nanomolar concentrations of Tb³⁺, indicating an apparent affinity >100-fold higher than Ca²⁺. Competition experiments show that Tb³⁺ binds to the same sites as Ca²⁺ to activate the channels. Additionally, SK channels activated by Tb³⁺ demonstrate a remarkably slow deactivation process. Comparison of our results with previous biochemical studies suggests that in the intact SK channel complex, the N-lobe of calmodulin provides ligand-binding sites for channel gating, and that its ligand-binding properties are comparable to those of the N-lobe in isolated calmodulin.     

Electrical and contractile consequences of Na+ or Ca2+ gradient reduction in cultured heart cells

The normal electrical and contractile activity of cultured neonatal rat ventricular cells is characterized and compared to activity seen in low [Ca²⁺]₀ and low [Na⁺]₀ solutions. In 0 Ca²⁺/0.5 mm EGTA solutions electrical activity is altered: the maximum diastolic potential (m.d.p.), maximum rate of rise ($\text{+}\text{V}\text{?}{}_{\mathrm{<mtext>max</mtext>}}$), and overshoot (o.s.) are reduced, while duration is increased. Low [Ca²⁺]₀ activity is insensitive to TTX and blocked by La³⁺. In low [Na⁺]₀ solutions electrical activity is generally absent; when present $\text{+}\text{V}\text{?}{}_{\mathrm{<mtext>max</mtext>}}$ and o.s. are decreased while duration is increased. Low [Na⁺]₀ activity is blocked by La³⁺. These data suggest the presence of one La³⁺-sensitive slow inward current channel. The absence of spontaneous electrical activity in low [Na⁺]₀ solutions suggests an inhibition of automaticity. To determine if this inhibition is due to a reduction of the Na⁺ gradient, drugs which raise [Na⁺]_i were examined. Both monensin (a Na⁺ ionophore) and ouabain inhibit the occurrence of spontaneous action potentials (cells respond to stimulation) indicating a dependence of pacemaker activity on a normal Na⁺ gradient. During Na⁺ gradient reduction, asynchronous subcellular contractile activity occurs independent of membrane potential fluctuation. This asynchronous activity is inhibited by La³⁺ or when Ca²⁺₀ is absent, but continues in the presence of verapamil (normal activity is blocked by all three conditions). The Na⁺Ca²⁺ exchange system is unaffected by verapamil but blocked by La³⁺, while both these drugs block the slow inward current. These data indicate that the Na⁺Ca²⁺ exchange system can directly supply Ca²⁺ (independent of electrical activity at the membrane) to intracellular sites that support contractile activity.     

Role of Calcium Loading in Early Afterdepolarizations Generated by Cs+ in Canine and Guinea Pig Purkinje Fibers

Ca²⁺-Loading and EAD. introduction: Our previous observations indicate that the Na²⁺:Ca²⁺ exchange current (I_Na:Ca) plays an important role in early afterdepolarizations occurring at more negative Vm (L-EAD). The purpose of these studies was to examine the role of Ca²⁺-loading, which stimulates I_Na:Ca, in generation of L-EAD. Methods and Results: Purkinje strands and preparations of ventricular myocardium from dogs and guinea pigs were superfused with oxygenated physiologic buffer solutions at 37°C, To induce EADs, [K⁺]₀ was reduced to 2.0 to 3.0 mM and [Cs⁺]₀, (3.6 to 4.0 mM) was added at slow rates of ≤ 0.3 Hz. Isometric contraction in canine Purkinje strands and guinea pig papillary muscles doubled in 1-hour exposure to Cs⁺ and low [K⁺]₀ at slow rates, and the uptake of ⁴⁵Ca²⁺ was approximately doubled after 30 minutes. Forty-three percent of Purkinje fibers developed L-EAD after a latent period of 17 to 123 minutes of exposure. Ouabain (0.2 μM) suppressed LEAD within 10 minutes reversibly. Ca²⁺-loading (low [Na⁺]₀ or high [Ca²⁺]₀ for 5 to 10 minutes before exposure to Cs⁺, low [K⁺]₀, and slow rates resulted in rapid development of L-EAD in all preparations during subsequent exposure. In Ca²⁺-loaded preparations, delayed afterdepolarizations (DADs) as well as L-EADs developed. Conclusion: Reduction of K⁺ currents with Cs⁺, low [K⁺]₀, and slow rates induced L-EAD in a fraction of Purkinje fibers after a latent period during which Ca²⁺-loading of the sarcoplasmic reticulum occurred, while fibers preloaded with Ca²⁺ developed L-EAD rapidly and uniformly. These findings indicate that Ca²⁺-loading is a critical condition for the development of L-EAD. Early suppression of L-EAD by ouabain suggests a dependence of L-EAD on low [Na⁺]₁. These findings implicate I_Na:Ca in the generation of L-EAD.     

Ionic mechanism of morphological changes of cultured myocardial cells on successive incubation in media without and with Ca2+

(1) Incubation of cultured mouse myocardial cells in medium containing Ca²⁺ after brief pre-incubation in Ca²⁺-free medium caused morphological changes, such as full contraction of myofibrils and balloon formation of the cell membrane (“Ca²⁺ paradox phenomenon”). (2) When myocardial cells were pre-incubated in Ca²⁺-free medium, and then incubated in medium containing various concentrations of Ca²⁺, both the percentage of cells showing morphological changes and the rate of Ca²⁺ uptake increased with increase in the Ca²⁺ concentration. (3) Pre-incubation in medium containing 10^?7m Ca²⁺ or less was necessary for induction of both morphological changes and excess uptake of Ca²⁺ during incubation in medium containing Ca²⁺. (4) When myocardial cells were pre-incubated in Ca²⁺-free medium containing various concentrations of Na⁺, and then incubated in medium containing Ca²⁺, both the percentage of cells showing morphological changes and the rate of Ca²⁺ uptake increased with increase in the Na⁺ concentration in the pre-incubation medium. (5) Various treatments that inhibited excess uptake of Ca²⁺ by the cells inhibited the genesis of morphological changes. Thus the observed morphological changes of myocardial cells were due to excess uptake of Ca²⁺ by the myocardial cells. (6) When myocardial cells were incubated in medium containing various concentrations of Ca²⁺ and a fixed concentration of Na⁺, the intracellular concentration of Na⁺ increased with decrease in the Ca²⁺ concentration of the medium. Cells that had been preloaded with a higher concentration of Na⁺ took up the Ca²⁺ faster. (7) Conditions that inhibited Na⁺ uptake by the cells during pre-incubation without Ca²⁺ inhibited Ca²⁺ uptake by the cells during subsequent incubation with Ca²⁺. These results suggest that Ca²⁺ uptake by myocardial cells during incubation in medium with Ca²⁺ depends upon the intracellular Na⁺ concentration.     

Serotonin stimulation of cAMP-dependent plasticity in Aplysia sensory neurons is mediated by calmodulin-sensitive adenylyl cyclase

Calmodulin (CaM)-sensitive adenylyl cyclase (AC) in sensory neurons (SNs) in Aplysia has been proposed as a molecular coincidence detector during conditioning. We identified four putative ACs in Aplysia CNS. CaM binds to a sequence in the C1b region of AC-AplA that resembles the CaM-binding sequence in the C1b region of AC1 in mammals. Recombinant AC-AplA was stimulated by Ca²⁺/CaM. AC-AplC is most similar to the Ca²⁺-inhibited AC5 and AC6 in mammals. Recombinant AC-AplC was directly inhibited by Ca²⁺, independent of CaM. AC-AplA and AC-AplC are expressed in SNs, whereas AC-AplB and AC-AplD are not. Knockdown of AC-AplA demonstrated that serotonin stimulation of cAMP-dependent plasticity in SNs is predominantly mediated by this CaM-sensitive AC. We propose that the coexpression of a Ca²⁺-inhibited AC in SNs, together with a Ca²⁺/CaM-stimulated AC, would enhance the associative requirement for coincident Ca²⁺ influx and serotonin for effective stimulation of cAMP levels and initiation of plasticity mediated by AC-AplA.     

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.     

Slow Ca2+ and Na+ responses induced by isoproterenol and methylxanthines in isolated perfused guinea pig hearts exposed to elevated K+.

The induction of Ca²⁺ channels in cardiac muscles by catecholamines (CA) was studied in a physiological preparation, the isolated perfused guinea pig heart. The heart was rendered inexcitable by elevating external [K⁺] to 27 mm, which depolarizes the cells to about ?40 mV. Isoproterenol (10^?7m) infusion induced excitability within 1 min, i.e., slowlyrising electrical potentials with concomitant contraction in response to stimulation. Their shape and duration was similar to that of the plateau component of the normal action potential, but their maximum rate of rise was only 6 to 12 V/s. The slow responses persisted for over 2 h. Methylxanthines (MX) (caffeine and theophylline, 3 mm) mimicked the catecholamines, except that they were not blocked by propranolol. The slow responses were blocked by: (a) Ca²⁺-free solution, (b) 1 mm Mn²⁺, and (c) verapamil (0.05 μg/ml), suggesting that an inward Ca²⁺ current plays a key role in the response. In hearts perfused with normal Ringer, these conditions greatly depressed the contractions at a time when there was little or no effect on the shape and duration of the action potentials. Sr²⁺ and Ba²⁺ could replace Ca²⁺ in maintaining the response. Na⁺ is also required for the responses, because the magnitude, duration, and rate of rise of the response diminished in lowered [Na⁺]_o; the responses disappeared in 0 to 30 mm Na⁺. Ouabain (10^?5m) suppressed the CA- or MX-induced response. Continuous perfusion with dc-AMP or ATP (10^?4m) slowly induced the slow responses even in the presence of propranolol. The Ca²⁺ ionophore, X-537A, also induced the slow responses, but its effect was blocked by propranolol, suggesting that its action was mediated by endogenous CA release. Metabolic poisons, hypoxia, or ischemia blocked the slow responses. The following conclusions were drawn from the results: (a) the induced slow response is dependent on both Ca²⁺ and Na⁺ ions; (b) the underlying mechanism requires both Na⁺ and Ca²⁺ for its operation; (c) c-AMP is implicated in the control of the activities of this mechanism; (d) the production and/or maintenance of the mechanism is energy dependent; and (e) inactivation of the slow response mechanism results in electromechanical uncoupling.     

Effects of nitric oxide donors on vascular smooth muscles depend on a type of vascular smooth-muscle preactivation

The abilities of such therapeutic nitrovasodilators as sodium nitroprusside (SNP) and glyceryl trinitrate (GTN) to dilate vascular smooth muscles (VSM) and affect intracellular calcium concentration level ([Ca²⁺]_i) in a rat tail artery were tested under different types of preactivation. To shed light on mechanisms underlying possible differences in the action of these two nitric oxide (NO) donors, simultaneous measurements of [Ca²⁺]_i and contractile force were done. All vascular rings were precontracted either using a high-K⁺-Krebs solution or phenylephrine (PE). It was shown that the effect of both NO donors strongly depended on a type of VSM preactivation. The EC₅₀ for GTN under K⁺ stimulation of VSM comprised (2.48±1.6)×10⁻⁵ M, whereas the mean EC₅₀ under PE stimulation was (3.05±2.3)×10⁻⁴ M (p<0.05, n=9). The EC₅₀ for SNP under K⁺ stimulation of VSM comprised (1.09±0.47)×10⁻⁷ M, whereas the EC₅₀ under PE stimulation was (8.01±2.4)×10⁻⁶ M (p<0.05, n=9). GTN demonstrated a significant discrepancy in the magnitude of changes in [Ca²⁺]_i and related VSM relaxant responses, indicating the ability of GTN to relax VSM in the absence of a proportional decrease in [Ca²⁺]_i. The main peculiarity of SNP action under K⁺ stimulation as compared to PE stimulation was the transient decrease in [Ca²⁺]_i while relaxation was sustained. Therefore, both NO donors demonstrated their ability to produce vasorelaxation as a result of an alteration in myofilament calcium sensitivity. These data clearly indicate that the sensitivity of VSM to NO donors is higher under K⁺ depolarization than that seen under PE stimulation, indicating that Ca²⁺ entry through voltage-operated calcium channels is more sensitive to NO as compared to calcium mobilization by means of Ca²⁺ entry through receptor-operated calcium channels or intracellular Ca²⁺ release, or both.     

Platelet membrane fluidity and intraplatelet Ca2+ mobilization are affected in uraemia

Abstract: In present investigations, platelet membrane fluidity and intraplatelet Ca²⁺ mobilization were analysed in uraemic platelets by fluorescence techniques. Thirteen non-dialyzed uraemic patients and 16 control subjects were examined. Anisotropy of DPH-probe, measured at 37°C, was significantly higher in control (0.2236 ± 0.0050) than in uraemic platelets (0.1969 ± 0.0082; p < 0.01). There was no difference between control (109.8 ± 6.0 nm ) and uraemic platelets (100.0 ± 7.3 nm ) when the basal [Ca²⁺]_i in resting platelets was determined. Activation of platelets by ADP (12.5 μm ) or by thrombin (0.1 U/ml) resulted in an increase in [Ca²⁺]_i. It was significantly higher (p* < 0.003 for ADP and p* < 0.009 for thrombin, respectively) in control platelets (383.6 ± 56.3 nm and 2031.0 ± 298.8 nm , respectively) than in uraemic ones (191.0 ± 21.3 nm and 838.7 ± 144.1 nm , respectively). The amount of released Ca²⁺ was higher in control platelets activated by both ADP and thrombin (157.6 ± 21.4 nm and 409.3 ± 71.0 nm , respectively) than in uraemic platelets (76.7 ± 15.7 nm and 203.0 ± 29.3 nm , respectively) and the differences were significant (p < 0.01 and p* < 0.01, respectively). These results indicate an abnormal intracellular Ca²⁺ mobilization in uraemic platelets. Both increased membrane fluidity and decreased Ca²⁺ mobilization should be considered as a possible reason of reduced fibrinogen receptor exposure on uraemic platelets.