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Innovation — the discovery of ways to get more value from limited resources — is critically important for both society’s health and material standard of living. A widely held view is that there would be too little innovation without government support.[1][1] This view is common in the drug

The activity of the cardiac Na+/Ca2+ exchanger (NCX1.1) undergoes continuous modulation during the contraction-relaxation cycle because of the accompanying changes in the electrochemical gradients for Na+ and Ca2+. In addition, NCX1.1 activity is also modulated via secondary, ionic regulatory mechanisms mediated by Na+ and Ca2+. In an effort to evaluate how ionic regulation influences exchange activity under pulsatile conditions, we studied the behavior of the cloned NCX1.1 during frequency-controlled changes in intracellular Na+ and Ca+ (Na[Formula: see text] and Ca[Formula: see text]). Na+/Ca2+ exchange activity was measured by the giant excised patch-clamp technique with conditions chosen to maximize the extent of Na+- and Ca2+-dependent ionic regulation so that the effects of variables such as pulse frequency and duration could be optimally discerned. We demonstrate that increasing the frequency or duration of solution pulses leads to a progressive decline in pure outward, but not pure inward, Na+/Ca2+ exchange current. However, when the exchanger is permitted to alternate between inward and outward transport modes, both current modes exhibit substantial levels of inactivation. Changes in regulatory Ca2+, or exposure of patches to limited proteolysis by α-chymotrypsin, reveal that this “coupling” is due to Na+-dependent inactivation originating from the outward current mode. Under physiological ionic conditions, however, evidence for modulation of exchange currents by Na[Formula: see text]-dependent inactivation was not apparent. The current approach provides a novel means for assessment of Na+/Ca2+ exchange ionic regulation that may ultimately prove useful in understanding its role under physiological and pathophysiological conditions.

The mechanisms underlying the inotropic effect of reductions in [K+]o were studied using recordings of membrane potential, membrane current, cell shortening and [Ca2+]i in single, isolated cardiac myocytes. Three types of mammalian myocytes were chosen, based on differences in the current density and intrinsic voltage dependence of the inwardly rectifying background K+ current IK1 in each cell type. Rabbit ventricular myocytes had a relatively large IK1 with a prominent negative slope conductance whereas rabbit atrial cells expressed much smaller IK1, with little or no negative slope conductance. IK1 in rat ventricle was intermediate in both current density and slope conductance. Action potential duration is relatively short in both rabbit atrial and rat ventricular myocytes, and consequently both cell types spend much of the duty cycle at or near the resting membrane potential. Rapid increases or decreases of [K+]o elicited significantly different inotropic effects in rat and rabbit atrial and ventricular myocytes. Voltage-clamp and current-clamp experiments showed that the effects on cell shortening and [Ca2+]i following changes in [K+]o were primarily the result of the effects of alterations in IK1, which changed resting membrane potential and action potential waveform. This in turn differentially altered the balance of Ca2+ efflux via the sarcolemmal Na+–Ca2+ exchanger, Ca2+ influx via voltage-dependant Ca2+ channels and sarcoplasmic reticulum (SR) Ca2+ release in each cell type. These results support the hypothesis that the inotropic effect of alterations of [K+]o in the heart is due to significant non-linear changes in the current–voltage relation for IK1 and the resulting modulation of the resting membrane potential and action potential waveform.

SEA0400 (SEA) blocks cardiac and neuronal Na+-Ca2+ exchange with the highest affinity of any known inhibitor, yet very little is known about its molecular mechanism of action. Previous data from our lab suggested that SEA stabilizes or modulates the transition of NCX1.1 exchangers into a Na+i-dependent (I1) inactive state. To test this hypothesis, we examined the effects of SEA on mutant exchangers with altered ionic regulatory properties. With mutants where Na+i-dependent inactivation is absent, the effects of SEA were greatly reduced. Conversely, with mutants displaying accelerated Na+i-dependent inactivation, block of NCX1.1 by SEA was either enhanced or unchanged, depending upon the phenotype of the particular mutation. With mutant exchangers where Ca2+i-dependent (I2) inactivation was suppressed, block of exchange currents by SEA was similar to that observed for wild-type NCX1.1. These data strongly support the involvement of I1 inactivation in the inhibitory mechanism of NCX1.1 by SEA, whereas I2 inactivation does not seem to serve an important role. The involvement of processes regulated by intracellular Na+ in the inhibitory mechanism of SEA may prove to be particularly important when considering the potential cardioprotective effects of this agent.

The electrophysiological effects of the benzothiazepine 7-chloro-3,5-dihydro-5-phenyl-1H-4,1-benzothiazepine-2-one (CGP-37157) (CGP) were investigated on the canine (NCX1.1) and Drosophila (CALX1.1) plasmalemmal Na+-Ca2+ exchangers. These exchangers were selected for study because they show opposite responses to cytoplasmic regulatory Ca2+, thereby allowing us to examine the role of this regulatory mechanism in the inhibitory effects of CGP. CGP blocked Na+-Ca2+ exchange current mediated by both transporters with moderate potency (IC50 values = approximately 3-17 microM) compared with other recently reported blockers of Na+-Ca2+ exchange [e.g., 2-[4-[2,5-difluorophenyl) methoxy]phenoxy]phenoxy]-5-ethoxyaniline (KB-R7943) and 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea (SEA0400)]. Experiments using alpha-chymotrypsin to remove autoregulation of Na+-Ca2+ exchange showed that block by CGP was reduced, suggesting that part of the effects of this drug may require intact ionic regulatory mechanisms. For NCX1.1, the inhibition produced by CGP was greater for outward Na+-Ca2+ exchange currents compared with inward currents. When CALX1.1 was examined, the extent of inhibition was similar for both inward and outward exchange currents. Although the extent and potency of CGP-mediated inhibition of Na+-Ca2+ exchange are less than those observed with SEA0400 and KB-R7943, our data demonstrate that CGP constitutes a novel class of plasmalemmal Na+-Ca2+ exchange inhibitors. Moreover, the widespread use of CGP as a selective mitochondrial Na+-Ca2+ exchange inhibitor should be reconsidered in light of these additional inhibitory effects.

Ca(2+)-induced Ca(2+) release (CICR) mediated by sarcoplasmic reticulum resident ryanodine receptors (RyRs) has been well described in cardiac, skeletal and smooth muscle. In brain, RyRs are localised primarily to endoplasmic reticulum (ER) and have been demonstrated in postsynaptic entities, astrocytes and oligodendrocytes where they regulate intracellular Ca(2+) concentration ([Ca(2+)](i)), membrane potential and the activity of a variety of second messenger systems. Recently, the contribution of presynaptic RyRs and CICR to functions of central and peripheral presynaptic terminals, including neurotransmitter release, has received increased attention. However, there is no general agreement that RyRs are localised to presynaptic terminals, nor is it clear that RyRs regulate a large enough pool of intracellular Ca(2+) to be physiologically significant. Here, we review direct and indirect evidence that on balance favours the notion that ER and RyRs are found in presynaptic terminals and are physiologically significant. In so doing, it became obvious that some of the controversy originates from issues related to (i) the ability to demonstrate conclusively the physical presence of ER and RyRs, (ii) whether the biophysical properties of RyRs are such that they can contribute physiologically to regulation of presynaptic [Ca(2+)](i), (iii) how ER Ca(2+) load and feedback gain of CICR contributes to the ability to detect functionally relevant RyRs, (iv) the distance that Ca(2+) diffuses from plasma membranes to RyRs to trigger CICR and from RyRs to the Active Zone to enhance vesicle release, and (v) the experimental conditions used. The recognition that ER Ca(2+) stores are able to modulate local Ca(2+) levels and neurotransmitter release in presynaptic terminals will aid in the understanding of the cellular mechanisms controlling neuronal function.

Linkage regulations tie generic drug approval and, thus, access to essential medications to existing drug patents through potentially long and costly litigation. The linkage regime is in the process of rapidly spreading worldwide through international free trade agreements. Even so, very little is known about how the regulations work in developed nations let alone how they impact public health systems across international borders. The authors constitute a network of eleven health policy experts and practicing lawyers in nine countries including those with mature linkage regulations, those with new regulations, those without regulations but with practices that parallel linkage, and those where regulations are currently subject to intense public debate and litigation.

The International Olympic Committee, the World Anti-Doping Agency, and International Sport Federations have banned and restricted the use of many stimulants including prescription and over-the-counter medications and dietary supplements. In addition to elite athletes, people of all ages use stimulants in attempts to improve athletic performance, alter body composition, and increase levels of energy. Here we introduce a seven-stage model designed to facilitate informed decision-making by individuals taking or thinking of taking stimulants for sport, health, and/or appearance reasons. We review for amphetamines, over-the counter sympathomimetics, and caffeine their performance-enhancing and performance-degrading effects, health benefits and mechanisms of action, medical side effects, and legal, ethical, safety, and financial implications.

People with diabetes (DM) have an excess burden of cardiovascular disease (CVD). Impairments in vascular contractility predict poor CVD outcomes, and mitochondria are essential for arterial contraction and relaxation. Mitochondrial dysfunction is observed with DM and insulin resistance. Our laboratory has reported failed vascular mitochondrial adaptation to exercise in DM rats. We hypothesize that repairing vascular mitochondrial function could restore contractility in DM. We employed primary aortic smooth muscle cells (SMC) from a lean, insulin resistant DM rat, Goto-Kakizaki (GK), and the Wistar (W) control to test mitochondrial adaptation to metabolic stress. SMC were exposed to low glucose (LG, 5 mM glucose) or high glucose (HG, 25mM) treatments for 1, 4 and 24 hours. Mitochondrial respiration, superoxide and dynamics (fission and fusion) were measured. Respiration in W SMC exposed to HG was unchanged compared to the LG control, but was decreased after 4 hours of HG treatment in the GK SMC (p≤0.05). GK SMC have significantly increased whole cell production of H 2 O 2 as compared to W SMC, but not mitochondrial superoxide after 4 hours of HG treatment. In contrast, W SMC exposed to HG significantly had increased superoxide relative to LG control (p

The aim of this work was to determine the relationship between peak twitch amplitude and sarcoplasmic reticulum (SR) Ca2+ content during changes of stimulation frequency in isolated canine ventricle, and to estimate the extent to which these changes were dependent upon sarcolemmal Na(+)-Ca2+ exchange. In physiological [Na+]o, increased stimulation frequency in the 0.2-2-Hz range resulted in a positive inotropic effect characterized by an increase in peak twitch amplitude and a decrease in the duration of contraction, measured as changes in isometric force development or unloaded cell shortening in intact muscle and isolated single cells, respectively. Action potentials recorded from single cells indicated that the inotropic effect was associated with a progressive decrease of action potential duration and a marked reduction in average time spent by the cell near the resting potential during the stimulus train. The frequency-dependent increase of peak twitch force was correlated with an increase of Ca2+ uptake into and release from the SR. This was estimated indirectly using the phasic contractile response to rapid (less than 1 s) lowering of perfusate temperature from 37 degrees C to 0-2 degrees C and changes of twitch amplitude resulting from perturbations in the pattern of electrical stimulation. Lowering [Na+]o from 140 to 70 mM resulted in an increase of contractile strength, which was accompanied by a similar increase of apparent SR Ca2+ content, both of which could be abolished by exposure to ryanodine (1 x 10(-8) M), caffeine (3 x 10(-3) M), or nifedipine (2 x 10(-6) M). Increased stimulation frequency in 70 mM [Na+]o resulted in a negative contractile staircase, characterized by a graded decrease of peak isometric force development or unloaded cell shortening. SR Ca2+ content estimated under identical conditions remained unaltered. Rate constants derived from mechanical restitution studies implied that the depressant effect of increased stimulation frequency in 70 mM [Na+]o was not a consequence of a decreased rate of refilling of a releasable pool of Ca2+ within the cell. These results demonstrate that frequency-dependent changes of contractile strength and intracellular Ca2+ loading in 140 mM [Na+]o require the presence of a functional sarcolemmal Na(+)-Ca2+ exchange process. The possibility that the negative staircase in 70 mM [Na+]o is related to inhibition of Ca(2+)-induced release of Ca2+ from the SR by various cellular mechanisms is discussed.

We examined whether the decrease in cardiac contractility in streptozotocin-induced diabetes in the rat is accompanied by reduced or excessive loading of the sarcoplasmic reticulum (SR) with Ca2+. Pooled SR Ca2+ content and fractional release on stimulation were estimated with rapid cooling contracture (RCC) and twitch height measurements, respectively. Interval-force relation was studied to assess the ability of diabetic tissue to alter the relative contribution of SR Ca2+ for contraction. Two months after injection with streptozotocin, peak isometric contraction and steady-state RCC decreased in parallel to approximately 50% of control values. The time to peak force development and complete relaxation was prolonged to 156 and 161% in diabetes in the presence of 1.25 and 2.5 mM extracellular Ca2+ concentration [Ca2+]o, respectively. A stepwise increase in the rate of stimulation from 0.2 to 0.5 and 1.0 Hz resulted in a negative force staircase, the slope of which was identical in control and diabetic animals in each [Ca2+]o tested. Postrest contractions and RCC, after variable test intervals, were significantly depressed after 0.2 and 0.5 Hz stimulation in diabetic muscles at 1.25 mM [Ca2+]o. This defect of SR Ca2+ availability was reversed by increasing the stimulation frequency to 1.0 Hz or by elevating [Ca2+]o to 2.5 mM. The results suggest that the marked reduction of developed tension in diabetic tissues was a consequence of depleted SR Ca2+ stores, rather than a result of chronic SR Ca2+ overloading. The maintained integrity of the interval-force relation in the presence of diabetes implies that the cellular mechanisms responsible for frequency- and time-dependent alterations in SR Ca2+ availability are not disturbed at this stage of disease.

The mechanisms underlying the inotropic effect of reductions in [K(+)](o) were studied using recordings of membrane potential, membrane current, cell shortening and [Ca(2+)](i) in single, isolated cardiac myocytes. Three types of mammalian myocytes were chosen, based on differences in the current density and intrinsic voltage dependence of the inwardly rectifying background K(+) current I(K1) in each cell type. Rabbit ventricular myocytes had a relatively large I(K1) with a prominent negative slope conductance whereas rabbit atrial cells expressed much smaller I(K1), with little or no negative slope conductance. I(K1) in rat ventricle was intermediate in both current density and slope conductance. Action potential duration is relatively short in both rabbit atrial and rat ventricular myocytes, and consequently both cell types spend much of the duty cycle at or near the resting membrane potential. Rapid increases or decreases of [K(+)](o) elicited significantly different inotropic effects in rat and rabbit atrial and ventricular myocytes. Voltage-clamp and current-clamp experiments showed that the effects on cell shortening and [Ca(2+)](i) following changes in [K(+)](o) were primarily the result of the effects of alterations in I(K1), which changed resting membrane potential and action potential waveform. This in turn differentially altered the balance of Ca(2+) efflux via the sarcolemmal Na(+)-Ca(2+) exchanger, Ca(2+) influx via voltage-dependant Ca(2+) channels and sarcoplasmic reticulum (SR) Ca(2+) release in each cell type. These results support the hypothesis that the inotropic effect of alterations of [K(+)](o) in the heart is due to significant non-linear changes in the current-voltage relation for I(K1) and the resulting modulation of the resting membrane potential and action potential waveform.

Rest potentiation, believed to be due to increased utilization of sarcoplasmic reticular calcium, was converted to rest depression by BAY K 8644 (1 microM). Plateau height and duration of the postrest beat were enhanced by BAY K 8644, suggesting an enhancement of extracellular calcium entry. Caffeine (3 mM) also produced depression at all rest intervals, although to a lesser extent than BAY K 8644. Compared with BAY K 8644, treatment with caffeine resulted in an elevation of plateau amplitude and a shortening of action potential duration. Action potential configuration changes induced by rest were unaltered by caffeine despite reduction in rest potentiation. Caffeine-induced rest depression was associated with an increase in the time to peak tension. This was not observed with BAY K 8644. Treatment with both caffeine (3 mM) and BAY K 8644 (1 microM) greatly prolonged time to peak tension. Action potential duration and plateau height were either maintained or increased. Less rest depression was observed with the combination than with either agent alone. These results suggest that 1) BAY K 8644 and caffeine inhibit rest potentiation by different mechanisms, and 2) caffeine-induced inhibition of calcium uptake by the sarcoplasmic reticulum may enhance the effect of BAY K 8644-induced increase in calcium influx on the contractile apparatus.

1 Post-rest inotropy in canine ventricular myocardium has proved a useful indicator of sarcoplasmic reticular calcium release. This phenomenon is converted to rest depression by the calcium channel activator (agonist), Bay K 8644 as well as other chemically diverse agents such as caffeine and ryanodine. 2 Rapid cooling contractures and post-rest contraction amplitude were used as independent measures of sarcoplasmic reticular calcium content and release. Simultaneous recordings of transmembrane action potentials and their accompanying contractions were obtained to determine the association between electrophysiological and mechanical events. The present study was designed to elucidate the mechanism by which Bay K 8644, caffeine and ryanodine alter force production after variable periods of rest. 3 Bay K 8644 (1 μm) increased steady state contraction in response to a constant train of stimulation, caused rest-depression after 2 and 8 min rest, prolonged action potential duration and increased action potential plateau amplitude. Augmented steady state tension was not accompanied by any change in time to peak tension or rapid cooling contracture amplitude. However, the post-rest rapid cooling contracture was greatly diminished compared to that observed prior to Bay K 8644 treatment. 4 Caffeine (3 and 5 mm) caused rest-depression with an increase in steady state contraction amplitude. Along with this there was a slight decrease in action potential duration and plateau amplitude and an increase in time to peak tension. The rapid cooling contractures were virtually abolished at all conditioning intervals. The effect of caffeine on twitch tension and cooling contracture is consistent with the ability of this compound to inhibit calcium sequestration by the sarcoplasmic reticulum. 5 A combination of Bay K 8644 and caffeine caused significantly less rest-depression than that seen with Bay K 8644 alone. The augmented twitch tension was accompanied by a long time to peak tension and action potential duration. However, there was no increase in the amplitude of the rapid cooling contracture, either after a regular train of stimulation or after rest, compared to that seen after Bay K 8644. 6 Ryanodine (10 nm), produced rest-depression, reduced steady state twitch tension and augmented the rest-depression produced by Bay K 8644. The steady state rapid cooling contracture remained unchanged when both agents were present simultaneously, while the post-rest rapid cooling contracture was significantly depressed compared to that observed with Bay K 8644 alone. 7 Bay K 8644 and ryanodine appear to have similar actions with respect to promoting diastolic loss of calcium from the sarcoplasmic reticulum. Although caffeine also decreases post-rest potentiation, it antagonizes rest-depression caused by Bay K 8644. The data from these experiments suggest that this reversal is a result of depressed intracellular calcium buffering and enhanced myofilament sensitivity produced by caffeine in the presence of increased transmembrane calcium influx promoted by Bay K 8644.

The mechanism of the negative force staircase in thin rat ventricular trabeculae was investigated and compared with the positive force staircase in dog ventricular muscles of comparable diameter. Increasing stimulus frequency from 0.2 to 0.5 and 1 Hz resulted in a stepwise reduction of twitch amplitude that was demonstrated in both 1.25 and 2.5 mM external calcium concentration ([Ca]o). The negative staircase was associated with no change in the amplitude of postrest contraction or rapid-cooling contracture at either [Ca]o investigated. Hence, neither decreased loading nor overloading of the rat sarcoplasmic reticulum with calcium during the steady state can be invoked as a likely explanation for the negative staircase. The results are consistent with a frequency-dependent increase in the refractoriness of the sarcoplasmic reticular calcium release process or a decrease in the amount of trigger for calcium release, assuming that the amount of calcium present in the release pool is constant from one frequency to the next. In contrast to the rat, canine ventricular muscle exhibited a positive force staircase, the slope of which depended on [Ca]o, as well as a frequency-dependent increase in postrest contraction and rapid-cooling contracture. Data obtained from this series of experiments suggests that increased filling of the release pool within the sarcoplasmic reticulum with calcium underlies the inotropic effect of high-frequency stimulation in canine ventricular muscle.