G. L. Smith

Myogenic contraction by modulation of voltage‐dependent calcium currents in isolated rat cerebral arteries.

1. Tissue blood flow and blood pressure are regulated by the spontaneous, myogenic, contraction developed by resistance arteries. However, the cellular mechanisms underlying myogenic contraction are not understood. In this study, the mechanisms of myogenic contraction in cerebral resistance arteries were investigated. 2. The vasoconstriction observed in response to increased pressure in cerebral resistance arteries (myogenic reactivity) was dependent on Ca2+ entry through voltage‐dependent Ca2+ channels, since it was abolished by Ca2+ removal and by dihydropyridine antagonists of voltage‐dependent Ca2+ channels. 3. Myogenic reactivity persisted in a high‐K+ saline, with reduced Ca2+, where membrane potential is presumed to be clamped. Therefore, membrane depolarization alone does not fully account for the increased voltage‐dependent Ca2+ channel opening. 4. Voltage‐dependent Ca2+ currents in single smooth muscle cells isolated from the resistance artery were substantially increased by applying positive pressure to the patch electrode evoking membrane stretch. 5. Myogenic reactivity remained unaffected by ryanodine and therefore was independent of internal ryanodine‐sensitive Ca2+ stores. 6. The myofilament Ca2+ sensitivity was not increased by elevated pressure in alpha‐toxin‐permeabilized arteries. However, pharmacological activation of protein kinase C or G proteins did increase the myofilament Ca2+ sensitivity. 7. Myogenic contraction over the pressure range 30‐70 mmHg could be accounted for by an increase in [Ca2+]i from 100 to 200 nM. 8. It is concluded that modest increases in [Ca2+]i within the range 100‐200 nM can account for that myogenic contraction, and that stretch‐evoked modulation of Ca2+ currents may contribute to the myogenic response.

The effects of taurine on Ca2+ uptake by the sarcoplasmic reticulum and Ca2+ sensitivity of chemically skinned rat heart

1. Caffeine (10 mM) induced a transient contracture in saponin‐treated cardiac trabeculae as a result of Ca2+ release from the sarcoplasmic reticulum (SR). Regular cycles of uptake and release were repeated to stabilize responses. The SR accumulated Ca2+ during the period prior to the addition of caffeine and this was reflected in the size of the caffeine contracture. Increasing the time for Ca2+ loading between successive caffeine exposures resulted in an increase in the amplitude of the contracture. Similarly, the size of the contracture was a function of the calcium ion concentration [( Ca2+]) in the preceding loading period. 2. Taurine (microM‐40‐mM), when included in both loading and caffeine solutions, markedly potentiated the caffeine‐induced contracture. The effect occurs even if taurine was included only in the loading solutions. The potentiating effect was ascribed to a direct action of taurine on the SR, since taurine did not significantly change the [Ca2+] in the loading solutions. 3. The maximal effect of taurine was produced at approximately 5 mM; higher taurine concentrations caused a lesser potentiation of the caffeine contracture. If the solutions were balanced with respect to osmolarity the effect of taurine did not decline at high concentrations. 4. If the [Ca2+] in the loading solutions was increased to produce a caffeine‐induced contracture that peaked close to maximal Ca2(+)‐activated force, taurine caused a fall in the size of contracture and a more variable response. This result could be explained by an increase in the spontaneous release of Ca2+ from the SR in the presence of taurine. 5. In Triton‐skinned trabeculae, taurine (1 mM‐40 mM) increased the Ca2+ sensitivity of the contractile proteins in a dose‐dependent manner but had little effect on maximum Ca2(+)‐activated force. The increase in Ca2+ sensitivity was small: in a typical experiment 30 mM‐taurine reduced the [Ca2+] necessary for half‐maximal activation from 3.02 to 2.56 microM, with no significant change in the shape of the relationship.

Measurement of the dissociation constant of Fluo-3 for Ca2+ in isolated rabbit cardiomyocytes using Ca2+ wave characteristics.

The Ca(2+) dissociation constant (K(d)) of Fluo-3 was determined using confocal fluorescence microscopy in two different situations: (i) within the cytosol of a permeabilised cardiomyocyte; and (ii) in an intact cardiomyocyte after incubation with the acetoxymethyl ester form of Fluo-3 (AM). Measurements were made on isolated rabbit ventricular cardiomyocytes after permeabilisation by a brief treatment with beta-escin (0.1mg/ml) and equilibration with 10 microM Fluo-3. The K(d) of Fluo-3 within the cytosol was not significantly different from that in free solution (558 +/- 15 nM, n=6). Over a range of cytoplasmic [Ca(2+)], the minimum [Ca(2+)] values between Ca(2+) waves was relatively constant despite changes in wave frequency. After loading intact cardiomyocytes with Fluo-3 by incubation with the -AM, spontaneous Ca(2+) waves were produced by incubation with strophanthidin (10 microM). By assuming a common minimum [Ca(2+)] in permeabilised and intact cells, the intracellular K(d) of Fluo-3 in intact myocytes was estimated to be 898 +/-64 nM (n=6). Application of this K(d) to fluorescence records shows that Ca(2+) waves in intact cells have similar amplitudes to those in permeabilised cells. Stimulation of cardiac myocytes at 0.5 Hz in the absence of strophanthidin (room temperature) resulted in a Ca(2+) transient with a maximum and minimum [Ca(2+)] of 1190 +/- 200 and 158 +/- 30 nM (n=11), respectively.

The Relationship between Intracellular [Ca2+] and Ca2+ Wave Characteristics in Permeabilised Cardiomyocytes from the Rabbit

Spontaneous sarcoplasmic reticulum (SR) Ca2+ release and propagated intracellular Ca2+ waves are a consequence of cellular Ca2+ overload in cardiomyocytes. We examined the relationship between average intracellular [Ca2+] and Ca2+ wave characteristics. The amplitude, time course and propagation velocity of Ca2+ waves were measured using line‐scan confocal imaging of β‐escin‐permeabilised cardiomyocytes perfused with 10 μM Fluo‐3 or Fluo‐5F. Spontaneous Ca2+ waves were evident at cellular [Ca2+] > 200 nM. Peak [Ca2+] during a wave was 2.0–2.2 μM; the minimum [Ca2+] between waves was 120–160 nM; wave frequency was ≈0.1 Hz. Raising mean cellular [Ca2+] caused increases in all three parameters, particularly Ca2+ wave frequency. Increases in the rate of SR Ca2+ release and Ca2+ uptake were observed at higher cellular [Ca2+], indicating calcium‐sensitive regulation of these processes. At extracellular [Ca2+] > 2 μM, the mean [Ca2+] inside the permeabilised cell did not increase above 2 μM. This extracellular‐intracellular Ca2+ gradient could be maintained for periods of up to 5 min before the cardiomyocyte developed a sustained and irreversible hypercontraction. Inclusion of mitochondrial inhibitors (2 μM carbonyl cyanide m‐chlorophenylhydrazone and 2 μM oligomycin) while perfusing with > 2 μM Ca2+ abolished the extracellular‐intracellular Ca2+ gradient through the generation of Ca2+ waves with a higher peak [Ca2+] compared to control conditions. Under these conditions, cardiomyocytes rapidly (< 2 min) developed a sustained and irreversible contraction. These results suggest that mitochondrial Ca2+ uptake acts to delay an increase in [Ca2+] by blunting the peak of the Ca2+ wave.

A comparison of the effects of ATP and tetracaine on spontaneous Ca2+ release from rat permeabilised cardiac myocytes

1 Fluo‐3 fluorescence measurements were made in isolated β‐escin permeablised rat cardiac myocytes using confocal microscopy. Perfusion of a mock intracellular solution containing 0.22‐0.23 μm Ca2+ and 5 mm ATP elicited regular waves of Ca2+ (approximately every 5 s) due to spontaneous release of Ca2+ from the sarcoplasmic reticulum (SR). 2 An approximately linear relationship was noted between Ca2+ wave velocity (v) and amplitude (σ). Under the control conditions the ratio of velocity to amplitude (v/σ) varied little and was 99.8 ± 2.5 m s−1μm−1(n = 78). 3 Reduction of [ATP] in the bathing solution to 0.5 and 0.2 mm ATP progressively decreased Ca2+ wave frequency and propagation velocity while increasing the amplitude. The changes in Ca2+ wave characteristics in 0.5 mm ATP were similar to those observed during perfusion with 50 μm tetracaine. In 0.2 mm ATP the decline of [Ca2+] during a Ca2+ wave was slowed suggesting a lowered rate of Ca2+ re‐uptake by the SR Ca2+ pump. 4 Reduction of [ATP] to 0.1 mm abolished Ca2+ waves after 15‐20 s. Returning the [ATP] to 5 mm caused a burst of high frequency and large amplitude waves. Mean velocity of the first wave on returning to 5 mm ATP was larger than normal but the v/σ value was 32 ± 6 % of control (n = 6). In the similar burst on removal of 100 μm tetracaine v/σ was higher than control (166 ± 9 %, n = 6). 5 Rapid application of caffeine (10 mm) was used to assess the SR Ca2+ content. This showed that SR Ca2+ increased as [ATP] was reduced or [tetracaine] was increased. The highest SR Ca2+ content was observed after perfusion with 0.1 mm ATP, which was 245 ± 15 % of control values. 6 Returning [ATP] from 0.1 mm to 5 mm caused a burst of high frequency, large amplitude Ca2+ waves. But recovery after incubation with 300 μm tetracaine resulted in SR Ca2+ release with no coherent wave pattern. The reason for this discrepancy is discussed.

Transient outward K+ current reduction prolongs action potentials and promotes afterdepolarisations: a dynamic‐clamp study in human and rabbit cardiac atrial myocytes

The shape of the cardiac atrial action potential is influenced by the flow of a transient outward K+ current (ITO) across atrial muscle cell membranes. Whether changes in ITO could alter atrial cell action potentials in ways that could affect mechanisms of abnormal heart rhythms (arrhythmias) is unclear, because currently available ITO blocking drugs are non‐selective. We used the ‘dynamic‐clamp’ technique, for the first time in atrial cells isolated from patients, and from rabbits, to electrically simulate selective changes in ITO during action potential recording. We found that ITO decrease prolonged atrial cell action potential duration and, under β‐adrenergic‐stimulation, provoked abnormal membrane potential oscillations (afterdepolarisations) that were preventable by ITO increase or a β‐blocker. These results help us better understand the contribution of ITO to atrial cell action potential shape and mechanisms of arrhythmia, with potential implications for both the development and treatment of atrial fibrillation.

Spontaneous frequency of rabbit atrioventricular node myocytes depends on SR function

Spontaneous Ca(2+) release from the sarcoplasmic reticulum (SR) appears to play an important role in cardiac sinoatrial node pacemaking. However, comparatively little is known about the role of intracellular Ca(2+) in the atrioventricular node (AVN). Intracellular Ca(2+) was therefore monitored in cells isolated from the rabbit AVN, using fluo-3 in conjunction with confocal microscopy. These cells displayed spontaneous Ca(2+) transients and action potentials. Ca(2+) transients were normally preceded by a small, slow increase (ramp) of intracellular Ca(2+) which was sometimes, but not always, accompanied by Ca(2+) sparks. During the Ca(2+) transient, intracellular [Ca(2+)] increased initially at the cell periphery and propagated inhomogeneously to the cell centre. The rate of spontaneous activity was decreased by ryanodine (1muM) and increased by isoprenaline (500nM); these changes were accompanied by a decrease and increase, respectively, in the slope of the preceding Ca(2+) ramp, with no significant change in Ca(2+) spark characteristics. Rapidly reducing bathing [Na(+)] inhibited spontaneous activity. These findings provide the first information on Ca(2+) handling at the sub-cellular level and link cellular Ca(2+) cycling to the genesis of spontaneous activity in the AVN.

Inorganic phosphate decreases the Ca2+ content of the sarcoplasmic reticulum in saponin-treated rat cardiac trabeculae.

1. Measurements of [Ca2+] were made in saponin‐permeabilized rat ventricular trabeculae using the fluorescent indicator Indo‐1. Application of caffeine (20 mM) caused a transient rise in [Ca2+] within the preparation as a result of Ca2+ release from the sarcoplasmic reticulum (SR). The size of the caffeine‐induced Ca2+ transient was related to the amount of Ca2+ accumulated by the SR prior to addition of caffeine. Caffeine‐induced Ca2+ release was abolished by ryanodine (10 microM), an inhibitor of SR Ca2+ release. 2. At a bathing [Ca2+] of 0.2 microM, the amount of Ca2+ released from the SR on addition of caffeine was sufficient to generate a tension transient. Ca2+ and tension responses were stabilized by application of caffeine at regular intervals (2 min). Addition of 10 mM inorganic phosphate (Pi) induced a transient increase in [Ca2+] within the preparation due to a net release of Ca2+ from the SR. The amplitude of subsequent caffeine‐induced Ca2+ transients were reduced to 65 +/‐ 7.5% (mean +/‐ S.D., n = 13) of control. In addition, the accompanying tension transient fell to 45 +/‐ 6.9% of control. Removal of Pi caused a transient decrease in the [Ca2+] within the preparation consistent with a net increase in Ca2+ uptake by the SR. Subsequent caffeine‐induced Ca2+ and tension transients returned to control levels. 3. Inclusion of Pi (2‐30 mM) in the perfusing solution decreased the size of caffeine‐induced Ca2+ and tension transients in a dose‐dependent manner. 4. Addition of 10 mM ADP caused a transient increase in [Ca2+] and depressed subsequent caffeine‐induced Ca2+ transients to a greater extent than 10 mM Pi. Despite the reduction in Ca2+ release from the SR, tension responses were larger in the presence of 10 mM ADP than under control conditions. This is a consequence of an increase in Ca(2+)‐activated force by ADP. 5. A decrease in the amplitude of caffeine‐induced Ca2+ transients also occurred on changing from a solution containing 1 mM ADP and 10 mM Pi to a solution with 10 mM ADP and 1 mM Pi. This confirms the previous observation that ADP is more effective than Pi at reducing caffeine‐induced Ca2+ released from the SR. 6. Spontaneous oscillations of [Ca2+] and tension occurred in the presence of 0.5 microM Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of creatine phosphate and inorganic phosphate on the sarcoplasmic reticulum of saponin‐treated rat heart.

1. Ventricular trabeculae from rat heart were permeabilized by treatment with saponin. In the presence of 150 nM Ca2+, application of 20 mM caffeine released Ca2+ from the sarcoplasmic reticulum (SR), resulting in a transient contracture. Ca2+ released from the SR was detected using fura‐2 fluorescence. The amplitudes of the caffeine‐induced Ca2+ transients were used to assess SR Ca2+ content. 2. In the absence of creatine phosphate (CP), introduction of 5‐30 mM inorganic phosphate (Pi) caused a net release of Ca2+ from the SR. Subsequent caffeine‐induced Ca2+ and tension transients were smaller in the presence of Pi. Under these conditions, 30 mM Pi decreased the caffeine‐induced Ca2+ transients by 45 +/‐ 3.1% (mean +/‐ S.D., n = 14). On removal of Pi, the [Ca2+] transiently decreased and the caffeine‐induced Ca2+ transients returned to control levels over 4‐6 min. 3. In the presence of CP (5‐15 mM), the Ca2+ transients were unaffected by the introduction of Pi (5‐30 mM) or slightly increased in amplitude. Pi (30 mM) significantly increased the caffeine‐induced Ca2+ transients by 7 +/‐ 8.8% (mean +/‐ S.D., n = 19, P < 0.05) in the presence of 15 mM CP. The release of Ca2+ on addition of Pi and decrease in [Ca2+] on Pi withdrawal was less pronounced or absent completely in the presence of CP. The inhibitory effects of Pi on caffeine‐induced Ca2+ release became apparent as the [CP] was decreased from 5 to 0 mM. 4. In the presence of the creatine phosphokinase inhibitor dinitro‐fluorobenzene (DNFB) the effects of Pi (in the presence of CP) were qualitatively similar to the results obtained in the absence of CP, although the decrease in caffeine‐induced Ca2+ release was less pronounced. 5. These results suggest that the rise in [Pi]i during ischaemia or anoxia will have little effect on the regulation of Ca2+ by the SR while the [CP]i remains above 5 mM. However, as the [CP] decreases below 5 mM, the accumulation of Pi within the cytosol will progressively reduce the SR Ca2+ content. CP may act in conjunction with endogenous creatine phosphokinase to modify the response of the SR to Pi, and possible mechanisms are considered.

Comparative effects of inorganic phosphate and oxalate on uptake and release of Ca2+ by the sarcoplasmic reticulum in saponin skinned rat cardiac trabeculae.

1. Ventricular trabeculae from the right ventricle of rat heart were suspended in a 6 microliters bath and ‘skinned’ with saponin (50 mg ml‐1). Preparations were perfused with solutions mimicking the intracellular milieu and the [Ca2+] within the bath was monitored continuously using fura‐2. 2. Application of 20 mM caffeine released Ca2+ from the sarcoplasmic reticulum (SR), resulting in a transient increase in the fura‐2 fluorescence ratio. Caffeine‐induced Ca2+ transients were smaller in the presence of 30 or 60 mM inorganic phosphate (Pi). This depressive effect of Pi on SR function was reversed by 10 mM creatine phosphate (CP). Caffeine‐induced Ca2+ transients were also reduced in the presence of 10 mM oxalate, although this effect was not reversed by CP. 3. When perfusion was stopped in the presence of 30 or 60 mM Pi, the [Ca2+] within the bath remained constant. However, when the flow was stopped in the presence of 60 mM Pi and 10 mM CP, a prolonged decrease in [Ca2+] occurred, consistent with precipitation of calcium phosphate within the SR. A similar decrease in [Ca2+] was observed when perfusion was stopped in the presence of 2 or 20 mM oxalate, in the absence or presence of CP. 4. The SR was Ca2+ depleted by withdrawal of ATP and exposure to 20 mM caffeine. Perfusion was then stopped and ATP reapplied, resulting in a maintained decrease in [Ca2+] within the bath, due to SR Ca2+ uptake. Net Ca2+ uptake was markedly reduced in the presence of 30 mM Pi. In contrast, 20 mM oxalate increased Ca2+ uptake and the [Ca2+] within the bath continued to fall over 2‐3 min. 5. Introduction of Pi released Ca2+ from the SR. Ryanodine (100 microM) abolished caffeine‐induced Ca2+ release while Pi‐induced Ca2+ release was unaffected. Pi‐induced Ca2+ release was reduced in the constant presence of 20 mM caffeine or 10 mM CP and was abolished completely by disruption of the SR membrane with Triton X‐100. Pi‐induced Ca2+ release occurred after abolition of SR Ca2+ uptake by ATP withdrawal. 6. These results suggest that the Pi‐induced decrease in releasable Ca2+ does not result from precipitation of calcium phosphate within the SR lumen. Pi inhibits net SR Ca2+ uptake, but this appears to result from activation of a ryanodine‐insensitive Ca2+ efflux pathway rather then inhibition of Ca2+ uptake. Possible mechanisms are considered, including reversal of the SR Ca2+ pump.