T. M. Nosek

Inositol trisphosphate enhances Ca2+ oscillations but not Ca2+-induced Ca2+ release from cardiac sarcoplasmic reticulum

The role of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in excitation-contraction coupling in cardiac muscle is still unclear, although many laboratories are beginning to assume a critical role for this putative second messenger. Earlier studies from this laboratory [Nosek et al. (1986) Am J Physiol 250:C807] found that Ins(1,4,5)P3 enhanced spontaneous Ca2+ release and the caffeine sensitivity of Ca2+ release from myocardial sarcoplasmic reticulum (SR) and proposed an increase in the Ca2+ sensitivity of the release as a possible mechanism. In order to clarify the phyisological relevance of these actions of Ins(1,4,5)P3 and specifically to test the effect of Ins(1,4,5)P3 on the Ca2+ sensitivity of Ca2+ release, we compared the effects of Ins(1,4,5)P3 on Ca2+ oscillations and on Ca2+-induced Ca2+ release (CICR) from the SR in saponin-skinned rat papillary muscle. We found that: (a) 30 μM Ins(1,4,5)P3 enhanced the Ca2+ oscillations (measured by tension oscillations) from the rat cardiac SR, consistent with the previous report on guinea pig tissue; (b) both GTP and GTP[S] enhanced Ca2+ oscillations. The effect was not additive to that of Ins(1,4,5)P3 indicating that two different Ca2+-release pools do not exist in cardiac SR; (c) 30 μM Ins(1,4,5)P3 had no effect on the Ca2+ sensitivity of CICR; (d) Ins(1,4,5)P3 (up to 30 μM) had no effect on SR Ca2+ loading. The studies were performed in the presence of Cd2+ or 2,3-bisphosphoglycerate, agents that inhibit Ins(1,4,5)P3 hydrolysis. These results suggest that: (a) two different mechanisms underlie Ca2+ oscillations and CICR, Ins(1,4,5)P3 influencing Ca2+ oscillations but not CICR; (b) Ins(1,4,5)P3 does not increase the Ca2+ sensitivity of Ca2+ release from the SR; (c) cardiac muscle is different from smooth muscle where Ca2+ release from the SR is dependent upon GTP; (d) the physiological role of Ins(1,4,5)P3 in excitation-contraction coupling in cardiac muscle is minimal. In contrast, Ins(1,4,5)P3 may play a pathological role in cardiac arrhythmogenesis by enhancing spontaneous Ca2+ ocsillations.

The effect of 2,3-butanedione 2-monoxime (BDM) on ventricular trabeculae from the avian heart.

Summary2,3-butanedione 2-monoxime (BDM, 3–30 mm) decreased twitch force of intact ventricular trabeculae isolated from 19-day embryonic chick hearts in a dose-dependent manner. The responses to BDM were rapid and reversible. In an attempt to determine the cellular basis for the inhibitory effect of BDM, experiments were carried out on skinned muscle fibres and isolated myocytes. In trabeculae skinned with Triton X-100, BDM depressed maximum calcium activated force (Fmax) with an IC50 of 14 mm. At 3 mm BDM, the proportional decrease in twitch force in intact tissue was similar to that of Fmax in skinned tissue. At higher BDM concentrations (10 and 30 mm), however, the proportional decrease in twitch force was greater than that of Fmax. BDM (up to 10 mm) had no effect on the normalized force-pCa relationship. In saponin-skinned preparations, BDM (3 and 30 mm) released calcium from the fully loaded sarcoplasmic reticulum to a slightly greater extent in the absence of calcium (pCa 8.5) than in the presence of a fixed level of free calcium (pCa 5.5). Whole cell patch clamping of freshly isolated chick myocytes demonstrated that BDM caused a dose-dependent decrease in the T-and L-type calcium current. Therefore, at low BDM concentrations (3 mm), the decrease in twitch force can be ascribed predominantly to depression of the contractile apparatus while, at higher concentrations of BDM, there is an additional inhibitory effect of BDM on excitation-contraction coupling.

How Valproate and Phenytoin Affect the Ionic Conductances and Active Transport Characteristics of the Crayfish Giant Axon

Summary: The purpose of this study was to determine the effect of valproic acid (VPA) and phenytoin (DPH) on chloride conductance and electrogenic Na+‐K+ active transport in the crayfish giant axon. Exposure of the axon to a medium containing no chloride (isethionate substitution) produced no change in the resting potential but significantly decreased the resting conductance and +dV/dtmax and increased the action potential duration. The response of axons bathed in a Cl‐‐free medium to VPA was not significantly different from that of axons bathed in Ch‐containing medium; the resting potential, total membrane conductance (gM), +dV/dtmax, and ‐dV/dtmax decreased, while the action potential duration increased. These results indicate that VPA does not produce its effects on membrane properties through an influence on chloride conductance. Similarly, DPH does not produce its effects through a change in chloride conductance; DPH decreases +dV/dtmax, ‐dV/dtmax, and gM, increases the duration of the action potential, and is without effect on the resting and action potentials with or without chloride in the bathing medium. Ouabain depolarized control axons and axons pretreated with VPA or DPH to an equal extent. From these data and other electrophysiologic characteristics of the axons, the resting Na+ and K+ conductances and the net pump current and coupling ratio were calculated. VPA significantly decreased gK but was without effect on gNa. That VPA was without effect on any property of the electrogenic Na+‐K+ active transport process indicates that the depolarization and decrease in total membrane conductance in response to VPA are due to the decrease in gK> produced by this compound. Even though VPA did not affect active transport at rest, it did suppress the response of active transport to drive, suggesting that VPA inhibits posttetanic potentiation. In contrast, DPH decreased g K and gNa so as to maintain the gnJgK ratio constant. This effect, coupled with a lack of an influence on the electrogenic active transport process, explains the absence of an effect of DPH on the resting membrane potential. The decrease in g M by DPH accounts for the increase in magnitude of the response to drive noted in the presence of DPH. These membrane effects suggest cellular mechanisms by which VPA and DPH can interfere with the spread of high‐frequency electrical activity away from an epileptic focus.

Effects of chloride on the electrical and mechanical properties of guinea pig ventricle

The purpose of this study was to investigate the influence of chloride on the electrical and mechanical properties of the guinea pig ventricular myocardium. Bathing media were made chloride free by substituting the relatively impermeant anion gluconate, isethionate, or sulfate. Removal of chloride increased contractility and decreased the duration of the action potential. Additional experiments explored the influence of chloride free media on electrogenic calcium influx estimated from the magnitude of the action potential in cells partially depolarized by potassium (the slow response). In the absence of chloride, transient increases occurred in the magnitude of the slow response while the positive inotropic effect was maintained. These experiments suggest that the effects of chloride free media are mediated secondarily by an enhanced calcium influx.

Inositol trisphosphate has no direct effect on the contractile apparatus of skinned cardiac muscles

Alpha-adrenoceptor stimulation produces a positive inotropic effect in heart muscle via mechanisms that are not well understood. The purpose of our study was to test the hypothesis that the increase in inositol 1,4,5 trisphosphate [Ins(1,4,5)P3] concentration that accompanies alpha stimulation contributes to the inotropic effect by increasing the calcium sensitivity of the contractile proteins, an effect which Ins(1,4,5)P3 has been shown to have in skeletal muscle. We determined the calcium sensitivity of the contractile apparatus of small, chemically skinned bundles from papillary muscles of rabbit, rat and dog hearts. These preparations were chosen because they exhibit a range of sensitivity to alpha agonists. In addition, we measured the calcium sensitivity of chemically skinned, single fibers from rabbit psoas muscle. All preparations were skinned with Triton X-100, a non-ionic detergent that disrupts the sarcolemmal, sarcoplasmic reticular, and mitochondrial membranes. In all cardiac preparations, we found that 38 μM Ins(1,4,5)P3 had no effect on either the calcium sensitivity or maximum calcium-activated force. Thus, there was no correlation between inotropic response to alpha stimulation and myocardial response to Ins(1,4,5)P3. On the other hand, the maximum calcium-activated force of skinned skeletal muscle was slightly increased by Ins(1,4,5)P3. Moreover, Ins(1,4,5)P3 significantly increased the sensitivity of these fibers to calcium.

Influence of Ionic Strength on Contractile Force and Energy Consumption of Skinned Fibers From Mammalian and Crustacean Striated Muscle

Increased ionic strength decreases maximal calcium-activated force (Fmax) of skinned muscle fibers via mechanisms that are incompletely understood. In detergent-skinned fibers from either rabbit (psoas) or lobster (leg or abdomen), Fmax in KCl-containing solutions was less than in potassium methanesulfonate (KMeSO3), which we showed previously was the least deleterious salt for adjusting ionic strength. In either salt, lobster fibers were considerably less sensitive to elevated ionic strength than rabbit fibers. Trimethylamine N-oxide (TMAO, a zwitterionic osmolyte found in high concentration in cells of salt-tolerant animals) increased Fmax, especially in high KCl solutions. In this regard, TMAO was more effective than a variety of other natural or synthetic zwitterions. In rabbit fibers, increasing ionic strength decreases Fmax but has little effect on contractile ATPase rate measured simultaneously using a linked-enzyme assay. Thus high salt increases the tension-cost of contraction (i.e. ratio ATPase/Fmax). At both high and low salt, TMAO decreases tension-cost. Given a simple two-state model of the cross-bridge cycle, these data indicate that ionic strength and TMAO affect the apparent detachment rate constant. High ionic strength KCl solutions extract myosin heavy- and light-chains, and troponin C from rabbit fibers. This extraction is virtually abolished by TMAO. Natural zwitterions, such as TMAO, have been shown to protect proteins against destabilization by high salt or other denaturatants. Our data indicate that, even in the best of salts, destabilization of the actomyosin complex may play a role in the effect of high ionic strength on the contractile process.

Depression of Axonal Excitability by Valproate Is Antagonized by Phenytoin

Summary: Standard electrophysiologic techniques were employed to determine the effects of the two anticonvulsants, valproic acid (VPA) and phenytoin (DPH), on the membrane excitability properties of the crayfish giant axon. VPA, 4 mM, produces a depolarization of the membrane that is associated with a decrease in the resting membrane conductance (gM)‐ VPA also attenuates the increases in gNa and gK that are responsible for the depolarization and repolarization of the action potential; it decreases the magnitude, rate of depolarization and repolarization, and conduction velocity of the propogated action potential while increasing its duration. DPH has some effects on membrane properties that are qualitatively similar to those of VPA; 0.11 mM DPH also decreases gM, gNa; and gK. Unlike VPA, DPH does not have a significant effect on the magnitude of either the resting or action potential. Pretreatment of axons with DPH reduces the effect of VPA on the magnitude, rate of depolarization and repolarization, and duration of the action potential while completely preventing the effects of VPA on resting potential, conduction velocity, and membrane conductance. These experiments and others on the effects of K+ depolarization on membrane properties demonstrate that part, but not all, of the influence of VPA on the membrane is secondary to its depolarizing effect. The results reported here on a membrane model suggest at least part of the cellular basis for the anticonvulsant properties of VPA and DPH, alone and in combination.

Alterations of Myocardial Contraction Associated with a Structural Heart Defect in Embryonic Chicks

Ablation of cardiac neural crest at stages 8-10 produces a structural heart defect (persistent truncus arteriosus, PTA) in embryonic chicks. PTA is associated with decreased myocardial contractility, as indicated by decreased left ventricular ejection fraction. We compared the force of small ventricular strips from normal and defective chick hearts. In intact muscle, ablation of the neural crest leads to a 30-50% decrease in twitch force at any level of extracellular Ca2+ (0.45-20 mM) at embryonic days (ED) 7 and 15, relative to sham-operated controls. These differences could reflect defects at the level of the contractile apparatus and/or in the excitation-contraction coupling process. To distinguish changes of the contractile apparatus, we used detergent skinned preparations. The maximal Ca(2+)-activated force (Fmax) at ED15 was not significantly different between control and experimental embryos. At ED 7, however, Fmax was reduced by 36% in experimental preparations. Electron-micrographs showed that the organization and orientation of the myofibrils was similar in experimental and control ventricles. At ED 14, however, the average myofibrillar diameter was significantly increased in experimental ventricles. The content of the major myofibrillar proteins (myosin heavy chain, actin, and tropomyosin), determined from polyacrylamide gel electrophoresis and Coomassie Blue staining, normalized to total protein, was not statistically different in experimental and control ventricles at ED7. At ED15, however, content of these proteins was doubled in experimental ventricles. These data suggest a possible defect of the contractile apparatus at both ED 7 and 15, since the ratio of Fmax/myosin is reduced in the experimental hearts.

Ruthenium red affects the contractile apparatus but not sarcoplasmic reticulum Ca2+ release of skinned papillary muscle

Ruthenium red has been shown to have a positive inotropic effect on isolated perfused hearts. The cellular mechanism of this action is not clear. Ruthenium red is able to block the Ca2+ release channel in isolated sarcoplasmic reticulum (SR) vesicle and reconstituted channel preparations. However, the effect of ruthenium red on SR Ca2+ release has not been studied in skinned cardiac muscle preparations. In the present study we investigated the actions of ruthenium red on both the characteristics of force generation by the contractile apparatus and Ca2+ release from the SR in chemically skinned rat papillary muscle. Ruthenium red (2 and 10 μM) significantly increased the Ca2+ sensitivity of the contractile apparatus (decreasing Ca2+ required for the half-maximal response from 1.56±0.04 μM to 1.46±0.05 μM) but had no effect on the maximal Ca2+-activated force in triton X-100 treated fibers. This result may suggest one explanation for the positive inotropic effect of ruthenium red on the heart. On the other hand, ruthenium red had no significant effect on either caffeine-induced Ca2+ release or Ca2+-induced Ca2+ release from the SR in saponin-skinned muscle fibers. Lack of a blocking effect on SR Ca2+ release by ruthenium red in skinned fibers suggests that the SR Ca2+ channels in intact preparations have characteristics that are different from those of either vesicular or reconstituted channel preparations.

Inotropic influence of macrocyclic polyethers on tracheal smooth muscle

Incubated guinea pig tracheal smooth muscle exhibited both positive and negative inotropic responses to a variety of crown ether analogs that ranged in size from 12-crown-4 to 30-crown-10 and included molecules whose lipophilicity was modified by the addition of benzo- and cyclohexo-substituents on the basic molecular framework. The inotropic influence of crown ethers may not only be due to their ionophoretic capabilities but may result from their ability to affect alterations in membrane physiology.