The role of mitochondria in cytoplasmic Ca2+ cycling.

It has been known for a long time that isolated mitochondria are able to accumulate large amounts of calcium ions. Before the discovery that endoplasmic reticulum (ER) was the main Ca(2+)-storing cellular organelle, mitochondria were thought to play a major role in cytoplasmic Ca(2+) homeostasis (Carafoli, 2002). After IP(3) was discovered and it was shown that IP(3) receptors were localized in ER membrane and that Ca(2+)-binding proteins such as calsequestrin could store large amounts of Ca(2+) in the ER, the role of mitochondria in the regulation of cytoplasmic Ca(2+) was questioned. However, in recent years, mostly due to the development of new methods, there has been increasing evidence that mitochondria could be an important cytoplasmic Ca(2+) sink, especially under conditions of a high cellular Ca(2+) load.

Expression of members of the S100 Ca2+-binding protein family in guinea-pig smooth muscle.

The calcium-binding proteins of the S100 family show tissue-specific expression. In this study, the mRNA and protein expression of five S100 calcium-binding proteins was investigated in different guinea-pig smooth muscle preparations. Transcripts of cDNA of S100A1, S100A4, S100A6 and S100A10 were amplified from smooth muscle RNA of neonatal and adult urinary bladder, ileum, aorta and from freshly isolated and cultured urinary bladder smooth muscle cells. S100B was not detectable in smooth muscle RNA, but was seen in control RNA isolated from brain. The pattern of S100 mRNA expression did not change during postnatal development and cell culture of smooth muscle. The structural homology of guinea-pig S100 proteins reverse transcription-polymerase chain reaction (RT-PCR) sequences compared to other species was 85-93% (human), 83-88% (rat) and 81-87% (mouse). Protein expression of S100A4, S100A6 and S100A10 was investigated in aorta, ileum, bladder and cultured bladder smooth muscle cells by Western blot analysis using polyclonal antibodies against guinea-pig-specific S100 immunogenic peptide sequences raised in rabbits. The results show that the proteins S100A4, S100A6 and S100A10 are expressed in the smooth muscle of ileum, bladder and aorta. S100A4 and S100A6 proteins are also expressed in cultured smooth muscle cells. The results of this study suggest that the calcium-binding proteins S100A1, S100A4, S100A6 and S100A10, but not S100B, are expressed in guinea-pig smooth muscle, and could be potentially involved in the regulation of cytoplasmic Ca(2+)-concentration and/or in signal transduction in smooth muscle.

The amount of acetylcholine mobilisable Ca2+ in single smooth muscle cells measured with the exogenous cytoplasmic Ca2+ buffer, Indo-1.

Single smooth muscle cells from guinea pig urinary bladder were voltage clamped with patch electrodes containing 1 mM Indo-1. As Indo-1 entered the cell, delta[Ca2+]i in response to Ca2+ influx with ICa (1 s steps to -10 mV) was progressively decreased. delta F410 was used as a measure of the Ca2+ amount bound to Indo-1. Within less than 2 min after establishment of the whole-cell configuration, the fraction of Ca2+ entering the cell with ICa which binds to Indo-1 became constant, suggesting that Indo-1 completely overrides the endogenous Ca2+ buffers. Under these conditions, delta F410 was satisfactorily fitted with the time integral of ICa during 1 s long steps. Acetylcholine (ACh, 50 microM) was rapidly applied to Indo-1 loaded cells to induce IP3-induced Ca2+ release (IICR), which peaked within about 1 s. From delta F410 in response to ICa and ACh and from the time integral of ICa the amount of Ca2+ released during IICR was estimated to be 680 attomole (680 x 10(-18) mole), corresponding to 230 microM for 3 pl of accessible cytoplasmic volume.

Effect of membrane potential on the initiation of acetylcholine-induced Ca2+ transients in isolated guinea pig coronary myocytes.

The muscarinic stimulation of single voltage-clamped coronary arterial smooth muscle cells of the guinea pig was used to evaluate the effect of membrane potential on the inositol 1,4,5-tris-phosphate (IP3)-mediated changes of ionized [Ca2+] in the cytoplasm (Ca2+ transient) measured with indo 1. When applied at the membrane potential of -50 mV, 10 micromol/L acetylcholine (ACh) induced a [Ca2+]i increase after the mean latency of 2.6+/-0.9 s. The latency was reduced to 1.1 +/- 0.3 s when the same dose was applied at a holding potential of +50 mV. In paired experiments in the same cells, the latency of response at +50 mV was reduced by a factor of 2.2 +/- 0.3 compared with the response at -50 mV. Supramaximal [ACh] (100 micromol/L) induced Ca2+ transients with a 0.4 +/- 0.1-s latency, which was independent of membrane potential. When applied repetitively at -50 mV, ACh induced Ca2+ transients with a progressively reduced amplitude and slower rate of rise. Depolarization to +50 mV accelerated the rate of rise of the Ca2+ transient by a factor of 3.4 +/- 0.4 without affecting the amplitude. The modulation of the initiation of Ca2+ transient by a 100-mV depolarization can be explained by an approximately threefold increase in the rate of IP3 accumulation.

Dissociation of subsarcolemmal from global cytosolic [Ca2+] in myocytes from guinea-pig coronary artery.

1. Changes in cytosolic Ca2+ concentration (delta[Ca2+]c) were measured by global indo-1 fluorescence and compared with changes in subsarcolemmal Ca2+ concentration (delta[Ca2+]sl) indicated by Ca(2+)-activated K+ currents (IK(Ca)). 2. At -50 mV holding potential, 10mM caffeine increased both IK(Ca) and [Ca2+]c without measurable delay. While IK(Ca) peaked within 0.3 +/- 0.16 s (mean +/- S.D.) and decayed to 50% within 0.4 +/- 0.2 s, delta[Ca2+]c peaked within 1.5 +/- 0.5 s and decayed to 50% within 5.2 +/- 1.0 s. The different time courses support the idea that [Ca2+]sl and [Ca2+]c deviate. 3. When 10 mM caffeine was applied 20 s after an initial 2 s caffeine application, IK(Ca) was suppressed to 22 +/- 5% and delta [Ca2+]c to 40 +/- 4%. During the following 1 min caffeine-free period, IK(Ca) recovered to 61 +/- 7% while delta [Ca2+]c remained at 40 +/- 3%. The differences between IK(Ca) and delta[Ca2+]c suggest that Ca2+ deprivation and Ca2+ refilling is faster in peripheral than in central sarcoplasmic reticulum (SR). 4. During the loading period of indo-1, a spontaneous delta[Ca2+]c of 30-80 nM appeared both at -50 mV and at more positive potentials. The amplitude of spontaneous delta[Ca2+]c increased with the amplitude, the frequency or the fusion of spontaneous transient outward currents (STOCs). 5. Block of sarcolemmal Ca2+ fluxes by 1 mM La3+ increased [Ca2+]c by 250 +/- 100 nM and suppressed the spontaneous delta[Ca2+]c. However, La3+ did not significantly retard the rate of decay of STOCs which may therefore be limited by Ca2+ diffusion into the cytosol and not by Ca2+ extrusion. 6. The dissociation of IK(Ca) (or STOCs) and delta[Ca2+]c may indicate a Ca2+ concentration gradient during Ca2+ release directed from the sarcolemma towards the centre of the cell, which later reverses direction.

Contribution of Ca(2+)-induced Ca2+ release to the [Ca2+]i transients in myocytes from guinea-pig urinary bladder.

1. Smooth muscle cells from guinea-pig urinary bladder were studied at an extracellular Ca2+ concentration ([Ca2+]o) of 3.6 mM and 36 degrees C. Fluorescence of Indo-1 was used to monitor the cytosolic calcium concentration ([Ca2+]i) and its changes ([Ca2+]i transients) induced by step membrane depolarizations. 2. During a 6 s depolarization step from -60 to 0 mV [Ca2+]i increased from a resting 118 +/- 22 nM to 1150 +/- 336 nM and decayed to a sustained level of 295 +/- 62 nM. The experiments were designed to evaluate the contribution of the release of intracellularly stored Ca2+ to components of the depolarization-induced [Ca2+]i transient, i.e. 'phasic', which decayed during a maintained depolarization step, and 'tonic' which constituted the sustained elevation of [Ca2+]i above resting level. 3. A short (1 s) application of 10 mM caffeine mimicked the phasic component. After wash-out of caffeine, the subsequent depolarization induced a [Ca2+]i transient with reduced peak, the degree of suppression depending on the interval between wash-out of caffeine and depolarization. The phasic component of the depolarization and the caffeine-induced [Ca2+]i transients were not additive but saturative. 4. The phasic component was largely abolished in the continuous presence of 10 mM caffeine. It was also abolished by a 10 min cell dialysis of 10 microM ryanodine from the pipette solution and was strongly reduced by dialysis of 5 microM thapsigargin. Changes of the tonic component of the depolarization-induced [Ca2+]i transient were much less pronounced with all three interventions. 5. The tonic component of the depolarization-induced [Ca2+]i transient was increased when [Ca2+]o was elevated briefly before a depolarization close to 0 mV, whereas the phasic component was not significantly changed. Similarly, brief application of 1 microM Bay K 8644 increased the tonic component several-fold without modifying significantly the phasic component. 6. It is concluded that depolarization-induced influx of Ca2+ through L-type Ca2+ channels induces the release of Ca2+ from intracellular caffeine-sensitive stores which constitutes the major part of the phasic component. Ca2+ release superimposes on the effects of Ca2+ influx through L-type Ca2+ channels, the non-inactivating part of which constitutes the tonic component of the [Ca2+]i transient. Since the two processes interact, a dissection by simple subtraction is not possible.

Depolarization-mediated intracellular calcium transients in isolated smooth muscle cells of guinea-pig urinary bladder.

1. Free intracellular calcium concentration ([Ca2+]i) was recorded in single smooth muscle cells of the guinea-pig urinary bladder held under voltage clamp at 36 degrees C and 3.6 mM-extracellular Ca2+. The Ca2+ indicator Indo-1 was loaded into the cells through patch electrodes. To separate Ca2+ currents (ICa), superimposed K+ currents were suppressed with a Cs(+)-containing electrode solution. 2. At a holding potential of -60 mV, resting [Ca2+]i was 114 +/- 22 nM (mean +/- S.D.). During 160 ms depolarization steps to 0 mV, [Ca2+]i rose to 885 +/- 140 nM. With steps of varied duration, peak [Ca2+]i increased with the time of depolarization up to about 1 s. Upon repolarization [Ca2+]i recovered to resting levels with a half-decay time of about 1 s; recovery was not significantly changed with repolarization potentials between -50 and -100 mV. 3. The potential dependence of the above peak [Ca2+]i transients was bell shaped, with a threshold around -40 mV and a maximum at 0 mV. During depolarization steps to potentials more positive than +80 mV [Ca2+]i did not significantly rise. 4. During step depolarizations to 0 mV lasting 10 s or longer, [Ca2+]i peaked within 814 +/- 18 ms and then decayed to a sustained level of 250 +/- 60 nM. The amplitude of the [Ca2+]i peak as well as the time course of the transient depended on the amplitude of ICa. The depolarizations increased [Ca2+]i to a sustained level with no clearly defined peak when ICa was reduced by partial inactivation or during steps close to the threshold of ICa (-40 mV). 5. The sustained level of [Ca2+]i with longer depolarizations of several seconds showed a bell-shaped voltage dependence with a maximum close to 0 mV. A bell-shaped voltage dependence for [Ca2+]i was also found during ramp-like depolarizations. However, when the rate of depolarization was low (7.5 mV s-1), the peak [Ca2+]i was found at more negative potentials (-15 mV). 6. The results are compatible with the idea that Ca2+ influx through voltage-operated Ca2+ channels is the key event in depolarization-mediated changes in [Ca2+]i in smooth muscle cells from urinary bladder.

Isolated guinea pig coronary smooth muscle cells. Acetylcholine induces hyperpolarization due to sarcoplasmic reticulum calcium release activating potassium channels.

Smooth muscle cells, dispersed from the circumflex coronary artery of the guinea pig, were studied with the whole-cell configuration of the patch-clamp. The resting potential of about -40 mV was superimposed by spikelike hyperpolarizations (SLHs) up to -20 mV amplitude. The SLHs resulted from spontaneous transient outward currents (spontaneous TOCs) measured under voltage-clamp (-40 or -50 mV). Acetylcholine (ACh; 10 microM) increased SLHs and TOCs in amplitude and frequency. Atropine blocked the ACh effects. ACh-induced SLHs or TOCs were suppressed by bath application of tetraethylammonium (1 or 10 mM) or by cell dialysis with cesium, suggesting that they result from induction of potassium currents. In cell-attached patches, induction of currents through 130-pS potassium channels was recorded when ACh was bath-applied. An ACh-induced increase in intracellular [Ca2+] is suggested as a second messenger since SHLs and TOCs were suppressed by cell dialysis of 10 mM EGTA. ACh induced SHLs and TOCs in the absence of extracellular calcium. Intracellular application of 5 mg/ml heparin blocked ACh-induced TOCs. When the intracellular calcium stores were depleted by pretreatment with caffeine, the ACh effects were suppressed. Similarly, ACh pretreatment reduced the caffeine-induced outward currents. The results suggested that ACh augments calcium release from the sarcoplasmic reticulum, and the released calcium activates maxi potassium channels. In the single cell, calcium-activated potassium channels generate TOCs and SLHs that sum up to a hyperpolarization of the multicellular tissue.

[Properties of synaptic currents during non-adrenergic inhibition of the smooth muscle cells of the large intestine in the guinea pig]. / Svoistva sinapticheskikh tokov pri neadrenergicheskom tormozhenii gladkomyshechnykn kletok tolstogo kishechnika morskoi svinki.

Inhibitory junctional currents (IJCs) were recorded under voltage clamp conditions in response to brief transmural stimulation of the circular muscle of the guinea pig colon using the double sucrose gap method in the presence of atropine. The time course of IJC decay was approximately exponential 100-150 ms after the peak value. The IJC amplitude depended linearly on the membrane potential with the reversal potential (-70 mV) near the potassium equilibrium potential. The time constant (tau) of the IJC decay depended exponentially on the membrane potential and became e-fold decreased when the membrane was hyperpolarized approximately by 120 mV. Varying the quantal content of IJC caused an increase of tau upon rising the amount or transmitter released and its decrease with the depression of IJC. Application of ATP (10(-3)M) caused a decrease of tau and IJC amplitude, while apamine reduced the amplitude of IJC without any changes in their time course. The results are discussed in terms of a buffered diffusion hypothesis supposing a cooperative action of transmitter released on junctional receptors.