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Biomedical subjects

G Isenberg

Publications and source records attributed to G Isenberg.

At least 37 records · Page 2Linked to original sources
PubMed1991

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 degrees 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 and 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.

Animals↗
PubMed1991

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.

Animals↗
PubMed1991

Total and free myoplasmic calcium during a contraction cycle: x-ray microanalysis in guinea-pig ventricular myocytes.

1. At 36 degrees C and 2 mM [Ca2+]o single guinea-pig ventricular myocytes were voltage clamped with patch electrodes. With a paired-pulse protocol applied at 1 Hz, a first pulse to +5 mV was followed by a second pulse to +50 mV. When paired pulsing had potentiated the contraction to the maximum, the cells were shock-frozen for electron-probe microanalysis (EPMA). Shock-freezing was timed at the end of diastole (-80 mV) or at different times during systole (+5 mV). 2. The same paired-pulse protocol was applied to another group of myocytes from which contraction and [Ca2+]i was estimated by microfluospectroscopy (50 microM-Na5-Indo-1). Potentiation moderately reduced diastolic sarcomere length from 1.85 to 1.82 microns and increased diastolic [Ca2+]i from about 95 to 180 nM. In potentiated cells, during the first pulse, contraction peaked within 128 +/- 25 ms after start of depolarization. [Ca2+]i peaked within 25 ms to 890 +/- 220 nM (mean +/- S.E.M.) and fell within 100 ms to about 450 nM. 3. Sigma Camyo, the total calcium concentration in the overlapping myofilaments (A-band), was measured by EPMA in seventeen potentiated myocytes. During diastole, sigma Camyo was 2.6 +/- 0.4 mmol (kg dry weight (DW]-1 which can be converted to 0.65 mM (mmoles per litre myofibrillar space). Since [Ca2+]i was 180 nM, we estimate that 99.97% of total calcium is bound. 4. A time course for systolic sigma Camyo was determined by shock-freezing thirteen cells at different times after start of depolarization to +5 mV. Sigma Camyo was 5.5 +/- 0.3 mmol (kg DW)-1 (1.4 mM) after 15-25 ms, 4.6 +/- 0.5 mmol (kg DW)-1 (1.1 mM) after 30-45 ms, and 3.1 mmol (kg DW)-1 (0.8 mM) after 60-120 ms. The fast time course of sigma Camyo suggests that calcium binds to and unbinds from troponin C at a fast rate. Hence, it is the slow kinetics of the cross-bridges that determines the 130 ms time-to-peak shortening. 5. Mitochondria of potentiated cells contained during diastole a total calcium concentration, sigma Camito, of 1.3 +/- 0.2 mmol (kg DW)-1 (0.4 mM). During the initial 15-25 ms of systole, sigma Camito did not change, however, during 30-45 ms sigma Camito rose to 3.7 +/- 0.5 mmol (kg DW)-1 (1.2 mM). The data suggest that sigma Camito can follow sigma Camyo with some delay, thereby participating in both slow diastolic and fast systolic changes in total calcium (sigma Ca), at least under the given conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Ca2+ influx through ATP-gated channels increments [Ca2+]i and inactivates ICa in myocytes from guinea-pig urinary bladder.

1. Whole-cell patch clamp was combined with microspectrofluometry (Indo-1) to study the effects of bath applied ATP on membrane currents and cytoplasmic Ca2+ concentration ([Ca2+]i) in single smooth muscle cells of the guinea-pig urinary bladder. Experiments were carried out at 22 degrees C and in 3.6 mM [Ca2+]o. Superimposed K+ currents were reduced by Cs+ dialysis from the patch electrode. 2. At -60 mV, ATP induced an inward current (Ins,ATP) that peaked within 0.4 s and then decayed. Ins,ATP was activated half-maximally by 1.1 microM-ATP and saturated at 50 microM-ATP to -1.1 +/- 0.2 nA (mean +/- S.E.M.). At 3.6 mM [Ca2+]o, Ins,ATP had a reversal potential (Erev) of -5 +/- 2 mV. From the shifts in Erev during changes in [Na+]o or [Ca2+]o we estimated that approximately 7% of Ins,ATP is carried by Ca2+ ions. 3. ATP (50 microM) increased [Ca2+]i transiently from resting 130 +/- 40 nM to 730 +/- 100 nM. At 22 degrees C, [Ca2+]i rose at a rate proportional to the instantaneous current amplitude of Ins,ATP. This relation was lost, however, after warming to 36 degrees C which increased the peak Ins,ATP (Q10 = 1.25) but reduced the peak of the ATP induced [Ca2+]i transient (Q10 = 0.75). We suggest that warming to 36 degrees C stimulated Ca2+ sequestration and Ca2+ efflux to such a degree that peak [Ca2+]i was attenuated significantly. 4. The contribution of Ca2+ ions to Ins,ATP was evaluated from a comparison of the increments in [Ca2+]i due to Ins,ATP and due to L-type Ca2+ channel current (ICa). For the same increment, Ins,ATP had to transport 19 times more charge than ICa. This number suggests that 5.8 +/- 0.8% of Ins,ATP is carried by Ca2+ ions which can be translated into a permeability ratio of PNa:PCa approximately 1:1. 5. During bath application of ATP, peak ICa was inhibited by 80 +/- 15%. Inhibition of ICa diminished to 20 +/- 8% after cell dialysis with 40 mM-EGTA, and it was 19 +/- 7% when extracellular Ca2+ had been substituted by Ba2+. These results are in agreement with the hypothesis of 'ICa inactivation by Ca2+'. Depletion of intracellular Ca2+ stores by pre-treatment with 20 mM-caffeine did not attenuate significantly the ATP-induced rise in [Ca2+]i or the ATP-induced inhibition of ICa. 6. The ATP-induced [Ca2+]i transients and the reduction of peak ICa recovered along a similar time course.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1991

Stimulation-induced potentiation of T-type Ca2+ channel currents in myocytes from guinea-pig coronary artery.

1. Whole-cell Ca2+ channel currents were studied in myocytes isolated from guinea-pig circumflex coronary artery at 36 degrees C and with 10 mM-Ba2+ (or Ca2+) as charge carrier. With 180 ms clamp steps from the holding potential of -100 mV, currents at -30 mV were carried mostly through the T-type calcium channels while at positive potentials currents were mostly of the L-type. 2. The increase in frequency of pulsing from 0.1 to 2.5 Hz resulted in a reduction of peak inward current ('negative staircase') with the 180 ms pulses to + 10 mV, but in a 2-fold potentiation ('positive staircase') with pulses to -30 mV. T-type currents and their frequency-mediated potentiation did not change significantly when Ba2+ was substituted by Ca2+ or Sr2+. 3. Potentiation of T-type currents was further analysed with a paired-pulse protocol: at a basal frequency of 0.1 Hz, a pre-pulse (inducing current I1) was followed by a 200 ms repolarization to -100 mV and a test pulse (inducing current I2). The potentiation could only be recorded using test pulses depolarizing the membrane to potentials between -40 and -10 mV; at more positive test potentials it was masked by the depressant effect of pre-pulses on the L-type current. 4. Potentiation of I2 by 200 ms pre-pulses started at pre-pulse potentials more positive than -60 mV and saturated at -20 mV (I2 potentiated by a factor 2.4). Between -20 and +130 mV the potentiation was not dependent on the pre-pulse potential suggesting that the influx of Ba2+ or Ca2+ is not required for this effect. Potentiation of I2 by a 10 s pre-pulse followed the voltage dependence of the steady-state inactivation curve of the T-type Ca2+ channel; potentiation became visible at potentials more positive than -80 mV and saturated at about -50 mV. 5. When changing the interval between two identical 200 ms pulses, the T-type current was found to recover completely from inactivation within 40 ms at -100 mV; at intervals of 160-320 ms maximal potentiation of I2 occurred. 6. With pre-pulses shorter than 200 ms, potentiation became attenuated when inactivation became less complete. When the potential during the interval between the pulses was -80 instead of -100 mV, maximal potentiation was reduced (I2 potentiated by a factor of 1.3 instead of 2.2) and occurred later (1.28 s). 7. Potentiated T-type currents inactivated faster.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Myocytes isolated from porcine coronary arteries: reduction of currents through L-type Ca-channels by verapamil-type Ca-antagonists.

Myocytes were enzymatically isolated from large epicardial arteries of the pig. In the cell attached configuration, we studied currents through L-type Ca-channels. At 22 degrees C, open channel conductance was 9 pS with 110 mM Ca2+ and 24 pS with 110 mM Ba2+ as charge carrier. According to the life time of the open state, 2 'modes' of gating are distinguished; mode 1 contributed time constants shorter than 1 ms, mode 2 those longer than 6 ms to the open time distribution. Mode 2 openings appeared spontaneously, more frequently with Ba2+ than with Ca2+ as charge carrier. The Ca-agonist Bay K 8644 (0.5 microM) facilitated the appearance of mode 2. Bath application of the phenylalkylamine D600 (1 microM) did not change the gating modes, but it reduced the channel openness by increasing the percentage of blank records. With whole cell recordings, we studied reduction of ICa by 1 microM D 600 at 3.6 mM [Ca2+] and 35 degrees C. At a holding potential of -45 mV, D 600 induced an 'initial block' of 35% (10% at -65 mV). Upon repetitive 1 Hz pulsing (170 ms to 0 mV) an additional, 'use-dependent' block developed with time. More negative holding potentials attenuated reduction of ICa by D 600, hyperpolarizations to -100 mV had an 'unblocking' effect. In regard to reduction of ICa, we compared the partially uncharged D 600 (membrane permeable) with the completely charged compound D 890 (membrane impermeable). When applied with the bath, 1 or 10 microM D 600 reduced ICa dose-dependently whereas D 890 was ineffective. When D 890 was applied via the patch electrode to the cytosol, it reduced ICa. We discuss that D 600 enters the cell in the uncharged lipid soluble form and reaches form the inside its receptor associated with the Ca-channel.

Animals↗
PubMed1990

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

The 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 degrees C and 35 degrees 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 Q10's 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 Q10's 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.

Animals↗
PubMed1990

A Dictyostelium mutant lacking an F-actin cross-linking protein, the 120-kD gelation factor.

Actin-binding proteins are known to regulate in vitro the assembly of actin into supramolecular structures, but evidence for their activities in living nonmuscle cells is scarce. Amebae of Dictyostelium discoideum are nonmuscle cells in which mutants defective in several actin-binding proteins have been described. Here we characterize a mutant deficient in the 120-kD gelation factor, one of the most abundant F-actin cross-linking proteins of D. discoideum cells. No F-actin cross-linking activity attributable to the 120-kD protein was detected in mutant cell extracts, and antibodies recognizing different epitopes on the polypeptide showed the entire protein was lacking. Under the conditions used, elimination of the gelation factor did not substantially alter growth, shape, motility, or chemotactic orientation of the cells towards a cAMP source. Aggregates of the mutant developed into fruiting bodies consisting of normally differentiated spores and stalk cells. In cytoskeleton preparations a dense network of actin filaments as typical of the cell cortex, and bundles as they extend along the axis of filopods, were recognized. A significant alteration found was an enhanced accumulation of actin in cytoskeletons of the mutant when cells were stimulated with cyclic AMP. Our results indicate that control of cell shape and motility does not require the fine-tuned interactions of all proteins that have been identified as actin-binding proteins by in vitro assays.

Actins↗
PubMed1990

Sustained subthreshold-for-twitch depolarization in rat single ventricular myocytes causes sustained calcium channel activation and sarcoplasmic reticulum calcium release.

Single rat ventricular myocytes, voltage-clamped at -50 to -40 mV, were depolarized in small steps in order to define the mechanisms that govern the increase in cytosolic [Ca2+] (Cai) and contraction, measured as a reduction in myocyte length. Small (3-5 mV), sustained (seconds) depolarizations that caused a small inward or no detectable change in current were followed after a delay by small (less than 2% of the resting length), steady reductions in cell length measured via a photodiode array, and small, steady increases in Cai measured by changes in Indo-1 fluorescence. Larger (greater than -30 and less than -20 mV), sustained depolarizations produced phasic Ca2+ currents, Cai transients, and twitch contractions, followed by a steady current and a steady increase in Cai and contraction. Nitrendipine (or Cd, verapamil, or Ni) abolished the steady contraction and always produced an outward shift in steady current. The steady, nitrendipine-sensitive current and sustained increase in Cai and contraction exhibited a similar voltage dependence over the voltage range between -40 and -20 mV. 2 microM ryanodine in the presence of intact Ca2+ channel activity also abolished the steady increase in Cai and contraction over this voltage range. We conclude that when a sustained depolarization does not exceed about -20 mV, the resultant steady, graded contraction is due to SR Ca2+ release graded by a steady ("window") Ca2+ current. The existence of appreciable, sustained, graded Ca2+ release in response to Ca2+ current generated by arbitrarily small depolarizations is not compatible with any model of Ca2(+)-induced Ca2+ release in which the releasing effect of the Ca2+ channel current is mediated solely by Ca2+ entry into a common cytosolic pool. Our results therefore imply a distinction between the triggering and released Ca2+ pools.

Animals↗
PubMed1990

Effect of membrane potential on acetylcholine-induced inward current in guinea-pig ileum.

1. The whole-cell patch clamp technique with caesium aspartate internal solution was used with single isolated cells from the longitudinal muscle layer of guinea-pig ileum, to investigate the voltage-dependent gating of ACh-induced inward current. 2. In voltage clamp experiments, at holding potentials ranging from -80 to -30 mV, ACh (300 microM) produced a slow sustained inward current in physiological salt bath solution (PSS). The measurements of the reversal potentials on substituting Na+ by other monovalent and divalent cations showed that this current is through non-selective cation channels (Ins, ACh). 3. During hyperpolarizations, Ins, ACh instantaneously increased in amplitude and then relaxed to a new steady-state level. The I-V relationship of the instantaneous peak was linear with a reversal potential of 0 mV, while that of the steady state was bell-shaped. The time course of relaxation appeared to be monoexponential and its time constants were reduced by stronger hyperpolarizations. 4. These results were not affected by the organic Ca2+ antagonists D600 or nitrendipine (10 microM). Under this condition, maximal chord conductance of Ins, Ach which was observed at 0 mV was about 1.5 nS. The steady-state activation relationship was well fitted by Boltzmann's equation with a half-maximal activation (Vh) of -50 mV and a slope factor (k) of -15 mV at membrane potentials negative to 0 mV, but over 0 mV the degree of activation was again decreased. The time constants for relaxation also appeared to follow a sigmoid curve. 5. In current clamp experiments, superfusion of ACh (300 microM) depolarized the membrane up to -10 to 0 mV. Inward current injection resulting in the moderate hyperpolarization of the membrane (-70 to -80 mV) attenuated ACh-induced depolarization and stronger hyperpolarization (less than -80 mV) abolished it. 6. These results show that ACh-induced depolarization is controlled by the membrane potential, which is explained by the voltage-dependent gating of Ins, Ach.

Acetylcholine↗
PubMed1990

Intracellular calcium ions modulate acetylcholine-induced inward current in guinea-pig ileum.

1. The modulatory effect of internal Ca2+ on the current through the ACh-activated non-selective cation channels (Ins, ACh) was investigated by the whole-cell patch clamp technique in single isolated cells of guinea-pig ileum. 2. Ins, ACh was isolated with caesium aspartate internal solution of low Ca2(+)-buffering capacity (10 microM-EGTA). With preceding depolarizations which evoked voltage-operated Ca2+ currents (ICa), Ins, ACh increased in amplitude and decayed more rapidly. The extent of this 'facilitating' effect depended on the number and duration of the depolarizations. 3. When depolarizing pulses were applied during the sustained phase of Ins, ACh, they were followed by large inward tail currents. These tail currents (tail Ins, ACh) resembled the non-facilitated Ins, ACh recorded without the depolarizing pulse, in regard to voltage-dependent gating and dependence on the extracellular Na+ concentration, thus suggesting that the currents are flowing through the same class of channels. 4. The tail Ins, ACh was apparently composed of two components distinguished by the insensitivity to organic Ca2+ antagonists. The minor component (about 20% of tail Ins, ACh) showed a rapid decay (about 150 ms at -60 mV) which could be attributed to voltage-dependent kinetics. The major component decayed slowly within 5 s and appeared to be related to changes in the intracellular Ca2+ concentration. The latter component was not recorded when Ba2+ or Sr2+ were used as a charge carrier for ICa and was blocked by 10 microM-D600 or nitrendipine, or Cd2+ 0.2-0.5 mM). 5. The tail Ins, ACh increased in proportion to Ca2+ influx when the duration of depolarizing pulses were prolonged from 15 to 200 ms, but this 'facilitating' effect was greatly suppressed when the cell was perfused with 40 mM-EGTA. 6. When the pCa in the pipette was varied using 40 mM-Ca-EGTA, the conductance through Ins, ACh increased in a manner dependent on intracellular Ca2+ concentration. Half-maximal and submaximal activation occurred at about 200 nM and 1 microM, respectively. 7. These results show that the activity of Ins, ACh is very sensitive to the intracellular Ca2+ concentration in the physiological range.

Acetylcholine↗
PubMed1990

Contribution of two types of calcium channels to membrane conductance of single myocytes from guinea-pig coronary artery.

1. Whole-cell and single-channel current recordings were used to study calcium channels in single smooth muscle cells isolated from guinea-pig coronary artery. Potassium currents were blocked by intracellular Cs+ ions. 2. Whole-cell currents were recorded with 10 mM-barium in the bath. Step pulses of 200 ms from a holding potential of -90 mV activated calcium channel current when the depolarization reached -55 to -50 mV. All cells showed a current component which inactivated slowly and incompletely. About half of the cells showed an additional current component with a rapid inactivation time course. Both components were abolished by Cd2+ ions (1 mM) and were reduced by changing the holding membrane potential to -40 mV or by addition of 0.1 mM-Ni2+. 3. Single calcium channel currents were measured in cell-attached patches with 110 or 10 mM-Ba2+ as a current carrier. Two different types of single calcium channel activity were observed. 4. A high-conductance calcium channel was activated near -30 mV with 110 mM-Ba2+ and this threshold was changed to about -60 mV with 10 mM-Ba2+ in the patch pipette. The conductance was 28.0 +/- 1.5 pS (mean +/- S.D.) in 110 mM-Ba2+ and 16.0 +/- 1.0 pS in 10 mM-Ba2+. Dependence of the conductance on the concentration of Ba2+ in the patch pipette followed a Langmuir curve: the apparent dissociation constant of Ba2+ was 8 mM. It was concluded that this channel type corresponds to L-type calcium channels. 5. Another calcium channel was found in these experiments. It had a low conductance and was activated at around -50 mV with 110 mM-Ba2+, and this threshold was shifted to about -70 mV when 10 mM-Ba2+ was the charge carrier. The conductance of this calcium channel was 7.5 +/- 0.6 pS in 110 mM-Ba2+ and 5.5 +/- 1.0 pS in 10 mM-Ba2+. With 10 mM-Ba2+, inactivation of the mean current was slow at potentials -70 to -50 mV, but fast and complete (within 100 ms) at more positive potentials. It was concluded that this type of calcium channel corresponds to T-type calcium channels. 6. With the membrane potential continuously held at -50 to -40 mV (with 10 mM-Ba2+ in the patch pipette), i.e. close to the usual resting potential of these cells, T-type calcium channels were completely inactivated whereas rare openings of L-type calcium channels could be detected.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1990

Acetylcholine activates nonselective cation channels in guinea pig ileum through a G protein.

Acetylcholine (ACh) depolarizes the membrane of mammalian intestinal myocytes by activating a nonselective cation channel (G. D. Benham, T. B. Bolton, and R. J. Lang. Nature Lond. 316: 345-347, 1985; R. Inoue, K. Kitamura, and H. Kuriyama. Pfluegers Arch. 410: 69-74, 1987). Here, we present evidence that occupation of the muscarinic receptor by ACh couples to channel activation via a G protein; the coupling can be blocked by pertussis toxin or by intracellular guanosine 5'-O-(2-thio-diphosphate) (GDP beta S), whereas intracellular guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) activates the channel in the absence of ACh. The currents, activated by either ACh or GTP gamma S, are nonadditive, conduct sodium ions, and are similar in their voltage dependence and facilitation by submicromolar calcium ions in the cytosol.

Acetylcholine↗
PubMed1990

Force measurements from voltage-clamped guinea pig ventricular myocytes.

We describe the first observations of isolated mammalian guinea pig ventricular myocytes that combine measurements of contractile force with the voltage-clamp method. The myocytes were attached by poly-L-lysine to the beveled ends of a pair of thin glass rods having a compliance of 0.76 m/N. The contractile force of a cell caused a 1- to 3-microm displacement of the rods; the motion of which was converted to an output voltage by phototransistors. By the use of the whole cell patch-clamp technique, the cells were depolarized at 1 Hz with 200-ms-long clamp pulses from -45 to +5 mV (35 degrees C, 3.6 mM CaCl2). Isometric force began after a latency of 7 +/- 2 ms, peaked at 93 +/- 21 ms, and relaxed (90%) at 235 +/- 63 ms. The time course of force was always faster than that of isotonic shortening (time to peak 154 +/- 18 ms). With 400-ms-long depolarizations, a tonic component was recorded as either sustained force or sustained shortening that decayed on repolarization. Substitution of Ca by Sr in the bath increased the inward current through Ca channels but slowed down the time course of force development. The results are consistent with the hypothesis that activator calcium derives mainly from internal stores and that Ca release needs Ca entry through channels.

Administration, Topical↗
PubMed1990

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.

Acetylcholine↗
PubMed1989

A Dictyostelium mutant deficient in severin, an F-actin fragmenting protein, shows normal motility and chemotaxis.

A severin deficient mutant of Dictyostelium discoideum has been isolated by the use of colony immunoblotting after chemical mutagenesis. In homogenates of wild-type cells, severin is easily detected as a very active F-actin fragmenting protein. Tests for severin in the mutant, HG1132, included viscometry for the assay of F-actin fragmentation in fractions from DEAE-cellulose columns, labeling of blots with monoclonal and polyclonal antibodies, and immunofluorescent-labeling of cryosections. Severin could not be detected in the mutant using these methods. The mutation in HG1132 is recessive and has been mapped to linkage group VII. The mutant failed to produce the normal severin mRNA, but small amounts of a transcript that was approximately 100 bases larger than the wild-type mRNA were detected in the mutant throughout all stages of development. On the DNA level a new Mbo II restriction site was found in the mutant within the coding region of the severin gene. The severin deficient mutant cells grew at an approximately normal rate, aggregated and formed fruiting bodies with viable spores. By the use of an image processing system, speed of cell movement, turning rates, and precision of chemotactic orientation in a stable gradient of cyclic AMP were quantitated, and no significant differences between wild-type and mutant cells were found. Thus, under the culture conditions used, severin proved to be neither essential for growth of D. discoideum nor for any cell function that is important for aggregation or later development.

Actins↗