G. Isenberg

Endothelin depolarizes myocytes from porcine coronary and human mesenteric arteries through a Ca-activated chloride current.

The effect of endothelin (ET) on membrane potential and current was studied in myocytes isolated from porcine coronary or from human mesenteric arteries at 3.6 mM extracellular Ca2+ concentration and 37° C. ET (1–100 nM) induced cell shortening and membrane depolarization from a resting potential of −50 mV to about −15 mV. Ca currents (ICa, L-type) were transiently reduced by ET. At −50 mV, ET induced an inward current that peaked within 2 s and fell within 10 s to a sustained level. The current could be enlarged by reducing bath extracellular Cl− ion concentration, but removal of extracellular Na+ ions had no effect. The voltage dependence suggests that the ET-induced current is a Cl current (ICl) at potentials negative to −30 mV; at more positive potentials K currents (IK, Ca) are superimposed. The effects of ET on ICa, ICl, IK, Ca contraction were prevented by intracellular Ca chelators, suggesting a Ca-dependent activation mechanism. The ET effects were abolished by pretreatment with 20 mM caffeine or prior cell-dialysis with heparin [thought to block inositol triphosphate-induced sarcoplasmic reticular Ca release]. The results suggest that ET releases Ca from the SR through a phosphoinositol response and that the released Ca acts as second messenger in modulating the membrane currents.

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.

Stretch-activated nonselective cation channels in urinary bladder myocytes: importance for pacemaker potentials and myogenic response

Filling of the bladder with urine stretches the myocytes in the wall. Stretch activates nonselective cation channels (SACs) thereby constituting a pacemaking mechanism. Once action potentials are triggered, Ca2+ influx through nifedipine-sensitive Ca2+ channels provides activator Ca2+ for the stretch-induced increase in wall tension (myogenic response). An additional component of myogenic response is independent of nifedipine and membrane potential; Ca2+ influx through SACs is large enough to induce Ca2+ release from intracellular stores.

ATP suppresses activity of Ca2+ -activated K+ channels by Ca2+ chelation

Ca2+-activated maxi K+ channels were studied in inside-out patches from smooth muscle cells isolated from either porcine coronary arteries or guinea-pig urinary bladder. As described by Groschner et al. (Pflügers Arch 417:517, 1990), channel activity (NPo) was stimulated by 3 μM [Ca2+]c (1 mM Ca-EGTA adjusted to a calculated pCa of 5.5) and was suppressed by the addition of 1 mM Na2ATP. The following results suggest that suppression of NPo by Na2ATP is due to Ca2+ chelation and hence reduction of [Ca2+]c and reduced Ca2+ activation of the channel. The effect was absent when Mg ATP was used instead of Na2ATP. The effect was diminished by increasing the [EGTA] from 1 to 10 mM. The effect was absent when [Ca2+]c was buffered with 10 mM HDTA (apparent pKCa 5.58) instead of EGTA (pKCa 6.8). A Ca2+-sensitive electrode system indicated that 1 mM Na2ATP reduced [Ca2+]c in 1 mM Ca-EGTA from 3 μM to 1.4 μM. Na2ATP, Na2GTP, Li4AMP-PNP and NaADP reduced measured [Ca2+]c in parallel with their suppression of NPo. After the Na2ATP-induced reduction of [Ca2+]c was re-adjusted by adding either CaCl2 or MgCl2, the effect of Na2ATP on NPo disappeared. In vivo, intracellular [Mg2+] exceeds free [ATP4−], hence ATP modulation of maxi K+ channels due to Ca2+ chelation is without biological relevance.

L-type Ca-channels: similar Q10 of Ca-, Ba- and Na-conductance points to the importance of ion-channel interaction

SummaryThe temperature-dependence of currents through L-type Ca-channels was studied in myocytes isolated from the urinary bladder of the guinea pig. Currents were measured at 22 °C and 35 °C with Ca-, Ba- and Na-ions as charge carrier. The higher temperature increased the open channel conductance for Ca-ions from 8.5 to 16 pS (Q10 =1.63±0.07, mean ± S.D.), for Ba-ions from 24 to 43 pS (Q10=1.55±0.06), and for Na-ions (pH 9) from 74 to 131 pS (Q10 of 1.55±0.09). The differences in the Q10s are not significant, the activation energy approximates a common high value of 34.8±2.5 kJ/mol. A three barrier model with intra-channel binding predicts high Q10s for Ca and Ba but not for Na. To fit the results we postulate that the temperature-dependence reflects multiple ion-channel interactions within a central permeability barrier, e.g. polar groups substituting part of the ionic water shell.

Nonselective Cation Channels in Cardiac and Smooth Muscle Cells

In cardiac and smooth muscle cells, nonselective cation channels can be activated by hormones and neurotransmitters, by cell stretch, and by changes in membrane potential. Activation of nonselective cation channels can depolarize the cell membrane, induce Ca2+ influx through voltage-gated Ca2+ channels and contraction. Activation of nonselective cation channels may trigger contraction even when membrane depolarization is absent or when voltage-gated Ca2+ channels are blocked, provided the Ca2+ permeability of these channels is sufficiently high.

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.

Arginine-vasopressin induces mode-2 gating in L-type Ca2+ channels (smooth muscle cells of the urinary bladder of the guinea-pig)

The effect of arginine-vasopressin (AVP, 0.1 μM) on elementary Ca2+ channel currents (L-type) was studied in cell-attached patches with 10 mM BaCl2 as the charge carrier. At a constant potential of −30 mV, bath applied AVP increased the channel openness (NPo) by a factor of 4.7±3.0 (mean±SD, n=9), the effect resulted from an increase in the frequency of opening (factor 2.5±0.8) and from a longer mean open time. Under control, openings longer than 5 ms contributed only 4% of the total, however, with the application of AVP this contribution increased to 29%. Under control, the open times were distributed along a single exponential (τo1=0.8±0.4 ms), a double exponential distribution was obtained during AVP (τo1=0.8±0.5 ms, τo2=7.5±0.7 ms). The Ca2+ agonist BAYk8644 (1 μM) changed the open time distribution similarly to AVP (τo1=1.0±0.5 ms, τo2=9±2.8 ms). With 1 μM BAYk8644 in the bath, AVP did not significantly increase the relative contribution of long openings, however, AVP increased the frequency of openings by a factor of 2.0±1 (n=6). The results are compatible with the idea that AVP can change the gating of L-type Ca2+ channels from mode 1 to mode 2.

Ca2+ Influx Through Voltage- and Purinoceptor-Operated Channels Estimated from [Ca2+]C Signals (Myocytes from Guinea-Pig Urinary Bladder)

The rise in free cytosolic calcium was studied by a combination of the techniques of microspectrofluorometry and whole-cell patch clamp. By comparing the membrane currents with their effect on [Ca2+]c, the relative importance of Ca2+ influx could be quantified for both L-type Ca2+ channels and non-selective channels activated by extracellular ATP.

Potentiation of Contraction as Related to Changes in Free and Total Intracellular Calcium

In voltage-clamped guinea-pig ventricular myocytes, we studied the potentiation of contraction in dependence on the concentration of intracellular calcium; ionized calcium [Ca2+]c was measured by Indo-1 microfluospectroscopy and total calcium (sigma Ca) by electronprobe microanalysis (EPMA). After a 15 min rest period, [Ca2+]c was approx. 90 nM and sigma Ca was below the detection limit (80 microM) in myoplasm (sigma Ca(myo)), junctional sarcoplasmic reticulum (sigma CaSR) and mitochondria (sigma Ca(Mito)). Post rest, repetitive clamp steps (1 Hz) potentiated extent and rate of shortening by 300%. In the literature, post-rest potentiation is attributed to the replenishment of SR with releasable calcium; by EPMA the postulated increase in sigma CaSR was measured directly. Post-rest, the peaks of systolic [Ca2+]c transients increased, however only by 40%. In addition, a moderate increase of end-diastolic [Ca2+]c was measured. In an other series of experiments, contraction was potentiated by 800% increase by means of paired voltage-clamp pulses (1 Hz, 36 degrees C, 2 mM [Ca2+]o). In the potentiated state, end-diastolic [Ca2+]c was 180 nM and sigma Ca(myo) was 0.65 mM. During systole, [Ca2+]c peaked within 20 ms to 950 nM. sigma Ca(myo) rose within 20 ms to 1.4 mM and fell within 40 ms to 1.1 and within 90 ms to 0.8 mM. In contrast, the time course of contraction was slow and peaked at a time (130 ms) when the [Ca2+]c and sigma Ca(myo) transients were finished. We suggest that Ca2+ bound to troponin C (TnC) controls only the onset but not the time course of myofilament interaction. From [Ca2+]c and sigma Ca(myo) we estimated a Ca2+ buffering capacitance of 1.5 mmol sigma Ca(myo) per pCa change, only a fraction of which can be attributed to Ca2+ binding sites on TnC. A model explaining the results requires the assumption of 0.6 mM additional slow, high affinity Ca2+ sites and 2 mM fast, low affinity Ca2+ sites. We discuss that end-diastolic Ca2+ binding to these sites contributes to the potentiation of contraction. Junctional SR. At the end of diastole sigma CaSR was 2.4 mM which is 4 times larger than sigma Ca(myo). This difference disappeared 20 ms after depolarization (sigma CaSR 1.1 mM), within another 20 ms it largely recovered (sigma CaSR 2.0 mM). These properties suggest that the junctional SR is a compartment suitable not only for Ca2+ release but also for rapid Ca2+ reuptake. Mitochondria. Paired-pulse potentiation increased end-diastolic sigma Ca(Mito) significantly (0.4 mM).(ABSTRACT TRUNCATED AT 400 WORDS)