[Research advances on the mechanism of extracellular vesicles of adipose-derived mesenchymal stem cells in promoting wound angiogenesis].

Wound healing involves complex pathophysiological mechanism, among which angiogenesis is considered as one of the key steps in wound healing, and promoting wound angiogenesis can accelerate wound healing. In recent years, mesenchymal stem cell-derived extracellular vesicles have been proven to produce equivalent effects of wound healing promotion comparable to stem cell therapy, with the advantages of low antigenicity and high biocompatibility. The specific mechanism by which extracellular vesicles facilitate wound healing is still not fully understood and is thought to involve all stages of wound healing. This article focuses on the possible mechanism of extracellular vesicles of adipose-derived mesenchymal stem cells in promoting wound angiogenesis, so as to provide ideas for further study on the mechanism of extracellular vesicles to promote wound healing.

Subconductance states of the cardiac K(ATP) channel revealed by partial block with glybenclamide.

Currents carried by single ATP-sensitive potassium channels (K(ATP) channels) were recorded in membrane patches isolated from adult rat ventricular myocytes. Channel currents were blocked completely by ATP at millimolar concentrations and by glybenclamide at micromolar concentrations. However, at lower glybenclamide concentrations (1-1000 nM), a partial block, manifest as a subconductance state, was often seen. At concentrations of 100-300 nM the mean size of the subconductance state was 33+/-2.7 pS (175 mM potassium in the pipette; n=13). The size of the conductance substate varied slightly with the concentration of glybenclamide, (42 pS at 1 nM, 34 pS at 100 nM and 31 pS at 1 microM), while the open time of the subconductance state decreased with increasing glybenclamide concentration (n=4). ATP (4 mM) completely blocked both the main conductance state of the channel and the subconductance state induced by glybenclamide. Submaximal concentrations of ATP also appeared to induce subconductance states, but these could not be resolved into discrete conductance levels. The observation that subconductance states can be induced by low concentrations of glybenclamide may have implications for models of how the binding of glybenclamide is translated into closure of the Kir6.2 pore.

Does Ca2+ release from the sarcoplasmic reticulum influence heart rate?

1. The present review summarizes the evidence that Ca2+ release from the sarcoplasmic reticulum (SR) is an important contributor to the systolic rise in [Ca2+]i (the Ca2+ transient) and influences the pacemaker firing rate. 2. We believe that the mechanism whereby [Ca2+]i influences firing rate is through the dependence of the Na+-Ca2+ exchanger on [Ca2+]i. 3. Extrusion of Ca2+ by the electrogenic Na+-Ca2+ exchanger produces an inward current that contributes to the pacemaker currents. Confocal images of Ca2+ indicate the distribution of [Ca2+]i and Ca2+ sparks add to the evidence that Ca2+ release from SR is involved in pacemaker activity. 4. The normal pathway for increased heart rate is sympathetic activation; we discuss the evidence that part of the chronotropic effect of beta-adrenoceptor stimulation is through the modulation of SR Ca2+ release. 5. These studies show that Ca2+ handling by the pacemaker cells makes an important contribution to the regulation of pacemaker activity.

The mechanisms of sarcoplasmic reticulum Ca2+ release in toad pacemaker cells.

The mechanisms of sarcoplasmic reticulum (SR) Ca2+ release in pacemaker cells from the sinus venosus of the cane toad (Bufo marinus) were studied. Single, isolated cells were voltage clamped using a nystatin-perforated patch. Ionic currents and intracellular Ca2+ concentration ([Ca2+]i) were recorded simultaneously. Depolarizations of 300 ms duration from a holding potential of -55 mV produced an inward current which had a bell-shaped relationship with voltage. Inward current first appeared at about -45 mV, reached a maximum of -343 +/- 46 pA at -15 mV and reversed at +45 mV. In contrast the amplitude of the increase in [Ca2+]i caused by depolarization (Ca2+ transient) increased monotonically with the increasing depolarization. At -15 mV the amplitude of the Ca2+ transient was 243 +/- 33 nM and at +45 mV it was 411 +/- 43 nM. The inward current produced by depolarizations to -5 mV was largely eliminated by the L-type Ca2+ channel blocker nifedipine (10 microM) while 37 +/- 7 % of the Ca2+ transient persisted. A significantly larger proportion of the Ca2+ transient (56 +/- 5 %) remained at +85 mV in the presence of nifedipine. The SR Ca2+ pump inhibitor 2, 5-di(tert-butyl)-1,4-hydroquinone (10 microM), which causes depletion of the SR Ca2+, reduced the amplitude of the Ca2+ transient to 34 +/- 1 % of control, irrespective of the voltage. Brief exposure to extracellular Ca2+-free solution abolished the Ca2+ transients caused by depolarization while the caffeine-induced Ca2+ release persisted. Tetrodotoxin (1 microM) had no effect on the amplitude of the depolarization-induced Ca2+ transient, although it reduced the fast component of the inward current. In contrast, Ni2+ (5 mM) abolished the Ca2+ transients at any given voltage. Ni2+ also abolished spontaneous Ca2+ transients. In conclusion, in toad pacemaker cells Ca2+ release from SR contributes approximately 66 % of the Ca2+ involved in the Ca2+ transient and requires extracellular Ca2+ influx to trigger its release. The L-type Ca2+ channels and Na+-Ca2+ exchange are major sources of Ca2+ influx under physiological conditions.

The distribution of calcium in toad cardiac pacemaker cells during spontaneous firing.

Isolated, spontaneously active pacemaker cells from the sinus venosus region of the toad heart were loaded with the calcium indicator fluo-3. The cells were examined with a confocal microscope to investigate the distribution of calcium during spontaneous activity. Three classes of calcium-related signals were present. First, intense, localised, time-invariant signals were detected from structures distributed across the cell interior. Based on the insensitivity to saponin and the distribution in the cell, these signals appear to arise from fluo-3 located in the sarcoplasmic reticulum and the nuclear envelope. Second, spatially uniform signals from the cytoplasm were present at rest and showed spontaneous increases in [Ca2+]i which propagated along the cell. These Ca2+ transients were uniform in intensity across the diameter of the cell and we could detect no significant delay in the middle of the cell compared to the edges. However, within the nucleus the Ca2+ transient showed a clear delay compared to the cytoplasm. Third, localised, transient increases in [Ca2+]i (Ca2+ sparks) which did not propagate were also detectable. These could be detected both near the surface membrane and in the interior of the cell and reduced in magnitude and increased in duration in the presence of ryanodine. The frequency of firing of Ca2+ sparks significantly increased in the 200-ms period preceding a spontaneous Ca2+ transient. These results suggest that pacemaker cells contain sarcoplasmic reticulum which is distributed across the cell. The Ca2+ transient is uniform across the cell indicating that near-synchronous release of Ca2+ from the sarcoplasmic reticulum is achieved. Ca2+ sparks occur in pacemaker cells though their role in pacemaker function remains to be elucidated.

Skeletal muscle hypertrophy is mediated by a Ca2+-dependent calcineurin signalling pathway.

Skeletal muscle hypertrophy and regeneration are important adaptive responses to both physical activity and pathological stimuli. Failure to maintain these processes underlies the loss of skeletal muscle mass and strength that occurs with ageing and in myopathies. Here we show that stable expression of a gene encoding insulin-like growth factor 1 (IGF-1) in C2C12 skeletal muscle cells, or treatment of these cells with recombinant IGF-1 or with insulin and dexamethasone, results in hypertrophy of differentiated myotubes and a switch to glycolytic metabolism. Treatment with IGF-1 or insulin and dexamethasone mobilizes intracellular calcium, activates the Ca2+/calmodulin-dependent phosphatase calcineurin, and induces the nuclear translocation of the transcription factor NF-ATc1. Hypertrophy is suppressed by the calcineurin inhibitors cyclosporin A or FK506, but not by inhibitors of the MAP-kinase or phosphatidylinositol-3-OH kinase pathways. Injecting rat latissimus dorsi muscle with a plasmid encoding IGF-1 also activates calcineurin, mobilizes satellite cells and causes a switch to glycolytic metabolism. We propose that growth-factor-induced skeletal-muscle hypertrophy and changes in myofibre phenotype are mediated by calcium mobilization and are critically regulated by the calcineurin/NF-ATc1 signalling pathway.

Does adrenaline modulate the Na+-Ca2+ exchanger in isolated toad pacemaker cells?

beta-Adrenergic stimulation of pacemaker cells from the sinus venosus of the cane toad (Bufo marinus) increases intracellular calcium ([Ca2+]i) and firing rate. The increase in [Ca2+]i could contribute to the increased firing rate by increasing the inward Na+-Ca2+ exchange current (INa-Ca) during diastole. In this study we measured [Ca2+]i and membrane currents in single, isolated, voltage-clamped pacemaker cells. We show that INa-Ca increases during beta-adrenergic stimulation. To test whether this increase in INa-Ca is caused by elevated [Ca2+]i or by changes in the properties of the Na+-Ca2+ exchanger, we made rapid applications of caffeine and plotted the INa-Ca against [Ca2+]i. This relationship was linear during the declining phase of the [Ca2+]i signal caused by caffeine and was not significantly different in the presence or absence of beta stimulation. These results show that INa-Ca is increased during beta-adrenergic stimulation and will contribute to the increased firing rate. However the increase in INa-Ca appears to be a consequence of the increase in [Ca2+]i and is not caused by changes in the intrinsic properties of the Na+-Ca2+ exchanger.

How does beta-adrenergic stimulation increase the heart rate? The role of intracellular Ca2+ release in amphibian pacemaker cells.

1. The mechanism by which sympathetic transmitters increase the firing rate of pacemaker cells was explored in isolated cells from the sinus venosus of the cane toad Bufo marinus. Intracellular calcium concentration ([Ca2+]i) was measured with indo-1 and membrane potential and currents were recorded with the nystatin perforated-patch technique. 2. Adrenaline or isoprenaline (2 microM) increased the transient rise in [Ca2+]i and increased the firing rate; these effects were blocked by propranolol (2 microM). 3. To determine whether the changes in [Ca2+]i might influence the firing rate we studied agents which affect either the loading or the release of Ca2+ from the sarcoplasmic reticulum (SR). Rapid application of caffeine (10 mM) to spontaneously firing cells caused a large Ca2+ release from the SR and the cells were then quiescent for 24 s. In the presence of beta-adrenergic stimulation the caffeine-induced [Ca2+]i was 14 % larger but the period of quiescence after application was reduced to 12 s. 4. Ryanodine, at either low (1 microM) or high (> 10 microM) concentration, stopped firing. However, when the SR store content of Ca2+ was tested with caffeine, at low ryanodine concentration the SR Ca2+ store was empty whereas at the high concentration the SR store was still loaded with Ca2+. beta-Adrenergic stimulation was not able to restore firing at the low concentration of ryanodine but did restore firing at the high ryanodine concentration. 5. An SR Ca2+ pump blocker, 2, 5-di(tert-butyl)-1,4-hydroquinone (TBQ) which depletes the SR store of Ca2+, also rapidly and reversibly stopped spontaneous firing. 6. The relation between the amplitude of the [Ca2+]i transient and firing rate established in the presence of ryanodine was similar when firing was restored by beta-stimulation. 7. In both spontaneously firing and voltage-clamped cells, depleting the SR store with either ryanodine or TBQ suggested that about half of the Ca2+ which contributes to the calcium transient is released from the SR. 8. These results show that the amplitude of the [Ca2+]i transient is an important factor in the firing rate of toad pacemaker cells and consequently agents which modify SR Ca2+ release influence firing rate. The effects of beta-stimulation on firing rate seem to be largely mediated by changes in amplitude of the [Ca2+]i transient.

Intracellular calcium and Na+-Ca2+ exchange current in isolated toad pacemaker cells.

1. Single pacemaker cells were isolated from the sinus venosus of cane toad (Bufo marinus) in order to study the mechanisms involved in the spontaneous firing rate of action potentials. Intracellular calcium concentration ([Ca2+]i) was measured with indo-1 to determine whether [Ca2+]i influenced firing rate. A rapid transient rise of [Ca2+]i was recorded together with each spontaneous action potential. [Ca2+]i at the peak of systole was 655 +/- 64 nM and the minimum at the end of diastole was 195 +/- 15 nM. 2. Reduction of extracellular Ca2+ concentration from 2 to 0.5 mM caused a reduction in both systolic and diastolic [Ca2+]i and the spontaneous firing rate also gradually declined. 3. Application of the acetoxymethyl (AM) ester of BAPTA (10 microM), in order to increase intracellular calcium buffering, caused a decline in systolic and diastolic [Ca2+]i. The firing rate declined progressively until the cells stopped firing after 10-15 min. At the time that firing ceased, the diastolic [Ca2+]i had declined by 141 +/- 38 nM. 4. In the presence of ryanodine (2 microM), which interferes with Ca2+ release from the sarcoplasmic reticulum, the systolic and diastolic [Ca2+]i both declined and the firing rate decreased until the cells stopped firing. At quiescence diastolic [Ca2+]i had declined by 93 +/- 20 nM. 5. Exposure of the cells to Na+-free solution caused a rise in [Ca2+]i which exceeded the systolic level after 4.8 +/- 0.3 s. This rise is consistent with Ca2+ entry on a Na+-Ca2+ exchanger. 6. Rapid application of caffeine (10-20 mM) to cells clamped at -60 mV caused a rapid increase in [Ca2+]i which then spontaneously declined. An inward current with a similar time course to that of [Ca2+]i was also generated. Application of Ni2+ (5 mM) or 2,4-dichlorobenzamil (25 microM) reduced the amplitude of the inward current produced by caffeine by 96 +/- 1 % and 74 +/- 10 %, respectively. In a Na+-free solution the caffeine-induced current was reduced by 93 +/- 7 %. 7. Under a variety of circumstances the diastolic [Ca2+]i showed a close association with pacemaker firing rate. The existence of a Na+-Ca2+ exchanger and its estimated contribution to inward current during the pacemaker potential suggest that the Na+-Ca2+ exchange current makes a contribution to pacemaker activity.

Hypoxia increases persistent sodium current in rat ventricular myocytes.

1. A persistent inward current activated by depolarization was recorded using the whole-cell, tight seal technique in rat isolated cardiac myocytes. The amplitude of the inward current increased when cells were exposed to a solution with low oxygen tension. 2. The persistent inward current had the characteristics of the persistent Na+ current described previously in rat ventricular myocytes: it was activated at negative potentials (-70 mV), reversed close to the equilibrium potential for Na+ (ENa), was blocked by TTX and was resistant to inactivation. 3. Persistent single Na+ channel currents activated by long (200-400 ms) depolarizations were recorded in cell-attached patches on isolated ventricular myocytes. Hypoxia increased the frequency of opening of the persistent Na+ channels. 4. Persistent Na+ channels recorded during hypoxia had characteristics similar to those of persistent Na+ channels recorded at normal oxygen tensions. They had a null potential at ENa, their amplitude varied with [Na+], they were resistant to inactivation and their mean open time increased with increasing depolarization. 5. The persistent Na+ channels in cell-attached patches were blocked by TTX (50 microM) in the patch pipette and by lidocaine (100 microM). 6. It was concluded that hypoxia increases the open probability of TTX-sensitive, inactivation-resistant Na+ channels. The voltage dependence of these channels, and their greatly increased activity during hypoxia, suggest that they may play an important role in the generation of arrhythmias during hypoxia.

Tetrodotoxin-sensitive inactivation-resistant sodium channels in pacemaker cells influence heart rate.

There is currently some uncertainty about whether cardiac pacemaker cells contain tetrodotoxin (TTX)-sensitive Na+ channels although TTX is known to slow heart rate. We have recorded transient and persistent single-channel currents activated by depolarization in myocytes isolated from the toad sinus venosus. The myocytes were identified as pacemaker cells by their characteristic morphology, spontaneous action potentials that were blocked by cobalt but not by TTX, and lack of an inwardly rectifying K+ current. The voltage dependence of the single-channel currents, their presence in solutions containing no K+ or Ca2+, or in solutions to which Cs+ and Co2+ had been added, their dependence on [Na+] and their sensitivity to TTX indicated that they were Na+ channel currents. The persistent Na+ channel currents were resistant to inactivation and were activated over the range of potentials that occur during diastole in pacemaker cells: they would therefore contribute to the pacemaker current that sets heart rate. It was concluded that TTX slows heart rate by blocking these channels in pacemaker cells.

Sodium currents in toad cardiac pacemaker cells.

Cells in the pacemaker region of toad (Bufo marinus) sinus venosus had spontaneous rhythmic action potentials. The rate of firing of action potentials, the rate of diastolic depolarization and the maximum rate of rise of action potentials were reduced by TTX (10 nM to 1 microM). Currents were recorded with the whole cell, tight seal technique from cells enzymatically dissociated from this region. Cells studied were identified as pacemaker cells by their characteristic morphology, spontaneous rhythmic action potential activity that could be blocked by cobalt but not by TTX and lack of inward rectification. When calcium, potassium and nonselective cation currents (If) activated by hyperpolarization were blocked, depolarization was seen to generate transient and persistent inward currents. Both were sodium currents: they were abolished by tetrodotoxin (10 to 100 nM), their reversal potential was close to the sodium equilibrium potential and their amplitude and reversal potential were influenced as expected for sodium currents when extracellular sodium ions were replaced with choline ions. The transient sodium current was activated at potentials more positive than -40 mV while the persistent sodium current was obvious at more negative potentials. It was concluded that, in toad pacemaker cells, TTX-sensitive sodium currents contributing both to the upstroke of action potentials and to diastolic depolarization may play an important role in setting heart rate.

Inactivation-resistant channels underlying the persistent sodium current in rat ventricular myocytes.

Single-channel sodium currents that could be blocked with TTX were elicited by depolarizing voltage pulses in either cell-attached or inside-out patches from rat ventricular myocytes. A transient burst of channels was followed by late-opening (persistent) channels with low open probability. Conditioning depolarizing pre-pulses that inactivated transient channels and 'chattering' late-opening channels had no effect on persistent channels. The open probability of persistent channels reached a maximum at more negative potentials than transient channels. Between -70 mV and -40 mV, the average open time of persistent channels increased, whereas the average open time of transient channels did not change significantly, so the open times of the two channels diverged as the potential became more positive. The conductance of transient and persistent channels was similar, and the conductance of both kinds of channel increased at more depolarized potentials.

Effects of lignocaine and quinidine on the persistent sodium current in rat ventricular myocytes.

1. The effects of the Class 1 antiarrhythmic agents lignocaine and quinidine on action potentials, and on sodium currents and potassium currents activated by depolarization, were examined in rat isolated ventricular myocytes by the whole cell, tight seal recording technique. 2. Tetrodotoxin and lignocaine shortened, whereas quinidine prolonged, the duration of the plateau phase of action potentials. 3. At low concentrations, lignocaine and quinidine blocked a persistent sodium current that was resistant to inactivation but they had only a small effect on the transient sodium current. At higher concentrations, they also blocked the transient sodium current. 4. Quinidine, but not tetrodotoxin or lignocaine, depressed potassium currents activated by depolarization and this could account for the prolongation of the plateau phase caused by quinidine. 5. It is suggested that block of the persistent sodium current may be responsible, at least in part, for the antiarrhythmic action of lignocaine and quinidine.

A persistent sodium current in rat ventricular myocytes.

1. The tight seal, whole-cell, voltage-clamp technique was used to record currents from single ventricular myocytes acutely dissociated from adult rat hearts. Subtraction of currents recorded in the presence and absence of tetrodotoxin (TTX, 50 microM) revealed a small, persistent, inward current following a much larger, transient, inward current. 2. Both currents were sodium currents because they reversed close to the sodium equilibrium potential and were depressed when choline was substituted for extracellular sodium. 3. The persistent sodium current could be recorded when the transient current had been inactivated with conditioning depolarization. Only slight inactivation of the persistent current occurred during depolarizing pulses lasting up to 900 ms. 4. A lower concentration of TTX (0.1 microM) blocked the persistent sodium current while having little effect on the transient sodium current. 5. The persistent sodium current was activated at more negative potentials than the transient sodium current. It cannot have been a window current because it was recorded at positive potentials when the transient current was completely inactivated. 6. Because the persistent and transient sodium currents had a different voltage dependence and sensitivity to TTX, it was concluded that different channels are responsible for the two currents.