Intracellular pH regulation in heart

Intracellular pH (pH_i) is an important modulator of cardiac excitation and contraction, and a potent trigger of electrical arrhythmia. This review outlines the intracellular and membrane mechanisms that control pH_i in the cardiac myocyte. We consider the kinetic regulation of sarcolemmal H⁺, OH⁻ and HCO₃⁻ transporters by pH, and by receptor-coupled intracellular signalling systems. We also consider how activity of these pH_i effector proteins is coordinated spatially in the myocardium by intracellular mobile buffer shuttles, gap junctional channels and carbonic anhydrase enzymes. Finally, we review the impact of pH_i regulatory proteins on intracellular Ca²⁺ signalling, and their participation in clinical disorders such as myocardial ischaemia, maladaptive hypertrophy and heart failure. Such multiple effects emphasise the fundamental role that pH_i regulation plays in the heart.     

The responses of muscle spindles to small, slow movements in passive muscle and during fusimotor activity

We have previously shown that movement detection thresholds at the human elbow joint were less than a degree of joint rotation in the passive limb but were higher if they were measured while subjects co-contracted elbow muscles [A.K. Wise, J.E. Gregory, U. Proske, J. Physiol., 508 (1998) 325-330]. Here we report observations on the responses of muscle spindles of the soleus muscle of the anaesthetised cat to determine their ability to signal small length changes in the passive muscle and during a contraction, under conditions resembling those of the human experiments. After appropriate conditioning of the muscle to control for history effects, primary endings of muscle spindles showed thresholds to ramp stretch at 20 micrometers s-1 of between less than 5 micrometers and 15 micrometers, which translates to 0.05 degrees -0.15 degrees of human elbow joint rotation. Thresholds were much higher following conditioning to introduce slack in the muscle. Since during a voluntary contraction there is likely to be alpha:gamma co-activation, responses of spindles were also recorded during slow stretches (100 micrometers at 20 micrometers s-1) during static fusimotor stimulation, dynamic fusimotor stimulation, combined fusimotor stimulation and fusimotor plus skeletomotor stimulation. Invariably, responses to passive stretch were larger than during motor stimulation. It is concluded that spindles are sensitive enough to signal fractions of a degree of elbow joint rotation and that the rise in threshold observed during a voluntary contraction may be accounted for by the actions of fusimotor and skeletomotor axons on spindle stretch responses.     

Relaxant and contractile responses of porcine pulmonary arteries to a thrombin receptor activating peptide (TRAP)

Recent studies on cloning of the thrombin receptor, which belongs to the family of G-protein-coupled receptors, suggest that thrombin cleaves a peptide from the extracellular N-terminus. A synthetic peptide of 14 amino acids corresponding to the sequence of the newly generated N-terminus was found to possess thrombin-like activity in several cells endowed with thrombin receptors. The relaxant and contractile effects of this thrombin receptor activating peptide (TRAP, Ser-Phe-Leu-Leu-ArgAsn-Pro-Asn-Asp-Lys Tyr-Glu-Pro-Phe) were investigated in porcine pulmonary arteries and compared with the action of thrombin.In PGF₂-precontracted vessels with intact endothelium, TRAP (0.3 – 10 mol/l) caused reversible transient and concentration-dependent relaxation which was absend after mechanical removal of the endothelium. Preincubation of the vessels with N^G-nitro-l-rarginine (200 mol/l) markedly reduced the relaxation. The TRAP-induced relaxation was associated with an increase in cGMP in the arteries. In comparison to thrombin, TRAP (EC₅₀: 0.8 mol/l) was less potent by more than three orders of magnitude.In endothelium-denuded pulmonary arteries TRAP (1–20 mol/l) caused a concentration-dependent contraction which was reversible after washout. The TRAP-induced contractile response was preceded by an increase in generation of inositol 1,4,5-triphosphate (IP₃); the peak of IP₃ accumulation was reached after 30 s. Compared with the contractile effect of thrombin, that of TRAP was weaker by three of magnitude.The vascular effect of TRAP was not inhibited by the thrombin inhibitors hirudin or heparin while the protein kinase C inhibitor staurosporine (0.1 mol/l) preferentially inhibited the tonic phase of contraction.The present findings suggest that TRAP mimics the dualistic vascular effects of thrombin caused by direct activation of thrombin receptors at endothelial and smooth muscle cells. Correspondence to: E. Glusa at the above address     

Relationship between the uptake of 3H-(±) metaraminol and the density of adrenergic innervation in isolated rat tissues

Summary In this study simultaneous measurements of action potentials and force of contraction of isolated ventricular muscle from cat and calf hearts were performed under stimulated steady-state and rested-state conditions. It is shown that time-to-peak force of the rested-state contraction correlates well with electrically induced changes in the duration of the platcau phase of the corresponding action potential. Moreover, adrenaline (0.2 and 0.6 M) and theophylline (2 and 5 mM) increase the amplitude of the rested-state and stimulated steady-state contractions. The concentrations of these drugs were about equi-effective in increasing stimulated steady-state force of contraction. However, the effect of adrenaline on the rested-state contraction is small in comparison to that of theophylline. The effect of both drugs on the rested-state contraction is accompanied by an increase in the plateau amplitude of the corresponding action potential. Since the plateau phase of the cardiac action potential is maintained by the slow inward current mainly carried by Ca ions and since adrenaline and methylxanthines increase this current, it is concluded from the present study that the rested-state contraction may be regulated primarily by Ca ions flowing into the cell during the action potential, while the stimulated steady-state contraction seems to be determined by the amount of Ca ions released from the sarcoplasmic reticulum.     

Effect of Ca2+-independent myosin light chain kinase on different skinned smooth muscle fibers

In various skinned smooth muscle fiber preparations, (porcine carotid artery, rat tail artery, chicken gizzard and Taenia coli from guinea pig) a Ca²⁺-independent myosin light chain kinase (MLCK) initiated a contraction in absence of Ca²⁺. While the Ca²⁺ insensitive MLCK was effective on the vertebrate smooth muscles it did not act on the invertebrate skinned skeletal muscle preparation from Limulus and anterior byssus retractor muscle from Mytilus edulis. The results indicate that in vertebrate smooth muscles phosphorylation is sufficient for activation and that there is no obligatory role for an additional mechanism in initiation of contraction.     

The action of Ca2+, Mg2+ and H+ on the contraction threshold of frog skeletal muscle

Summary The dependence of the threshold potential for contraction on pH and the concentration of Ca²⁺ and Mg²⁺ in the bathing solution was measured in frog skeletal muscle.Decreasing the pH from 10.3 to 4.65 resulted in a threshold shift to more positive potentials. Between pH 6.5 and 8.5 the contraction threshold was almost pH-independent.Increasing [Ca²⁺]_o (in the concentration range 0.5–50 mM) shifted the curves relating contraction threshold to pH to less negative potentials and diminished the overall pH-dependence.The contraction threshold exhibited a similar dependence on [Ca²⁺]_o and [Mg²⁺]_o, the two curves running parallel in the concentration range of 5–50 mM, but Mg^2– was onlyc. 0.6 as effective as Ca²⁺.To explain these results a surface charge model is proposed which assumes that two acidic groups, ₁ and ₂, and one basic group, ₃, reside at the outer surface of the membrane of the T-system. Alterations in the extracellular medium exert their influence on the electro-mechanical coupling process by changing the surface potential. The groups will be titrated by protons and their charges screened off by the divalent cations. In addition, Ca²⁺ was supposed to bind with a weak dissociation constant (23 M) to the two acidic groups.The chosen charge densities are: ₁ = –0.0085/Ų[= –1e/(10.8Å)²], ₂ = –0.0037/Ų[= –1e/(16.4Å)²], ₃ = 0.0028/Ų[= +1e/(18.9Å)²] with intrinsic dissociation constantsK _H1=10^–2.0 M,K _H2=10^–4.1 M, andK _H3=10^–8.5 M.The measured threshold values are satisfactorily described by this model except at extreme alkaline and acid pH values.Supported by Deutsche Forschungsgemeinschaft, Bad Godes-berg-SFB 114 Bionach.     

Contractile properties of chemically skinned fibers from pregnant rat myometrium: existence of an internal Ca-store

Smooth muscle fibers isolated from pregnant rat myometrium were skinned by saponin treatment. Properties of the contractile system and the involvement of an intracellular source of activator calcium were studied. (1) In the presence of 4 mM total EGTA, skinned fibers contracted in a concentration-dependent manner to micromolar applications of calcium ions. Rat myometrium exhibited a high calcium sensitivity (80% of the maximum contraction was achieved in the presence of 10⁻⁶ M Ca²⁺). (2) Several divalent cations induced concentration-dependent contractile responses in skinned uterine muscle. The rank order of potency was: Ca²⁺>Mn²⁺>Sr²⁺>Ba²⁺. All these divalent cation-activated contractions were antagonized by trifluoperazine in a concentration-dependent manner. (3) pretreatment of skinned fibers with cAMP (5×10⁻⁵–5×10⁻⁴ M) depressed cation-activated contractions. This effect was dependent on the free cation concentration. (4) In the presence of a low EGTA concentration (0.1 mM) the pCa-tension curve was shifted to the right with a 2.5-fold increase in the Ca-concentration required to induce half-maximum contraction. (5) After Ca-loading (10⁻⁶ M Ca²⁺ for 3 min in low EGTA-containing solution), total replacement of K⁺ ions by choline induced a small and tonic contraction. In these conditions, the ionophore A23287 (5×10⁻⁸ M –5×10⁻⁵ M) and inositol 1,4,5-triphosphate (InsP₃; 2×10⁻⁶ M – 2×10⁻⁵ M) also produced contractions of skinned uterine fibers even in the presence of NaN₃ (5 mM) and of NaCN (5 mM). Repeated K⁺-choline substitution or InsP₃ addition induced repeated contractile responses. (6) Ryanodine (5×10⁻⁵ M), procaine (15 mM) and vanadate (2.5×10⁻⁴ M) strongly depressed the Insp₃-induced contractile response. It is concluded that in physiological conditions, a Ca-calmodulin-myosin light chain kinase complex is implicated in the contractile process of rat uterine smooth muscle, and that a part of the activator calcium can be supplied by an intracellular store (presumably sarcoplasmic reticulum).     

Estimation of myofibrillar responsiveness to Ca2+ in isolated rat ventricular myocytes

 To estimate myofibrillar responsiveness to Ca²⁺, we used the relation between cell length and intracellular [Ca²⁺] ([Ca²⁺]_i) during tetanic contractions of isolated ventricular myocytes. Enzymatically isolated rat ventricular myocytes were loaded with fura-2 AM (4 μM for 10 min) and excited alternately at 340 nm and 380 nm. The ratio (R) of fura-2 fluorescence at these wavelengths [F(340)/F(380), an index of [Ca²⁺]_i] and cell length (L) were measured simultaneously. Following treatment with thapsigargin (0.2 μM), myocytes were stimulated at 10 Hz for 10 s to produce a tetanic contraction every min and an instantaneous plot of R vs L (R-L trajectory) was constructed. The R-L trajectory followed the same path during cell shortening and re-lengthening, suggesting that dynamic equilibrium between R and L was achieved during tetanus. Increasing the extracellular [Ca²⁺] from 1 to 8 mM extended the R-L trajectory without a substantial shift of the relation. The Ca²⁺-sensitizing thiadiazinone derivative, EMD57033 (1 μM), shifted the R-L trajectory to the left (sensitization of the myofibrils to Ca²⁺), whereas the non-selective phosphodiesterase inhibitor, 3-isobutyl-1-methylxantine (200 μM), shifted the R-L trajectory to the right (desensitization of the myofibrils to Ca²⁺), in agreement with previous results obtained using skinned preparations. We conclude that the R-L trajectory is useful for estimating the myofibrillar responsiveness to Ca²⁺ in isolated myocytes and may be beneficial for the evaluation of inotropic agents. Received: 13 March 1998 / Received after revision: 4 May 1998 / Accepted: 2 June 1998     

Propagation of excitation-contraction coupling into ventricular myocytes

This paper examines the [Ca²⁺]_i transient in isolated rat heart cells using a laser scanning confocal microscope and the calcium indicator fluo-3. We find that the depolarization-evoked [Ca²⁺]_i transient is activated synchronously near the surface and in the middle of the heart cell with similar kinetics of activation. The time of rise of the transient did not depend on whether the sarcoplasmic reticulum (SR) Ca-release was abolished (by thapsigargin and ryanodine). The synchrony of activation and the similarity of levels of [Ca²⁺]_i at the peripheral and deeper myoplasm (regardless of the availability of SR Ca-release) shows that sarcolemmal Ca channels and SR Ca-release channels are distributed throughout the rat heart cell and that the propagation of the action potential into the interior of the cell is rapid. In addition, the activation of calcium release from the SR by CICR is rapid (2 ms) when compared to the time-course of calcium influx via the sarcolemmal Ca channel.     

Re-acceleration of the down-regulated contraction kinetics in the rat tracheal smooth muscle

The contraction kinetics of smooth muscle show a down-regulation after the transient rise found during sustained contraction. We tried to find out therefore if the contraction kinetics of rat tracheal smooth muscle can be re-accelerated during sustained activation. A 2 s length vibration (100 Hz sinusoidal; amplitude=6% of the muscle length) produces an immediate fall in the force developed by the activated muscle. A biexponential function was fitted to the force recovery. The reciprocal of the time constant,t ₂, describing the slow component of force recovery, reflects the kinetics of contraction. The contraction kinetics reach their highest levels (t ₂=4.9±0.1 s,n=166) about 30 s after the onset of electrical field stimulation. Three experimental groups were activated by either 10 M serotonin (5-HT), 100 M acetylcholine (ACh), or by 2 M ACh for 50 min. Approximately 10 vibrations were applied to each preparation after an 8 min activation in order to observe stabilized down-regulated contraction kinetics.t ₂ values were calculated from the force recovery after vibration and averaged 11.2±0.2 s (n=141), 11.5±0.2 s (n=137), and 11.1±0.3 s (n=84), respectively. After 50 min of continuous chemical activation, the preparation was stimulated additionally by the neurogenic release of acetylcholine. Thet ₂ of post-vibration force recovery, as measured after 30 s of neural activation, showed no change in the specimens basically activated by 100 M ACh (11.0±0.4 s,n=51). A decline int ₂, indicating accelerated kinetics, was observed in the groups which had been stimulated by 10 M 5-HT (5.9±0.2 s,n=51) and 2 M ACh (5.6±0.2 s,n=47). The re-accelerating effect of the second stimulus could be reproduced recurrently. The down-regulated contraction kinetics can be re-accelerated either by activating another receptor type in addition to the one already maximally stimulated or by increasing the stimulus mediated by one of the receptor types from half maximal to maximal strength. However, this is only possible if the additional activation is strong enough, as indicated by an increase in active force. It could be demonstrated that the slowing of the cross-bridge cycling rate is the result of a regulatory process and not the result of substrate deficiencies or refractoriness in the regulatory of contractile proteins.