Regulation of calcium channels in the heart.
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Animals↗
Biomedical subjects
H C Strauss
Publications and source records attributed to H C Strauss.
Time- and voltage-dependent modulation of a Kv1.4 channel by a beta-subunit (Kv beta 3) cloned from ferret ventricle.
In mammals, voltage-gated K+ channels can be made of complexes containing alpha-subunits similar to the Shaker K+ channel and smaller cytoplasmic beta-subunits. Recent studies have suggested that these ancillary beta-subunits can modulate K+ channel gating properties. We studied the effects of a K+ channel beta-subunit, Kv beta 3, coexpressed with a Kv1.4 alpha-subunit, FK1, on the time and voltage dependence of channel activation, inactivation, recovery from inactivation, and deactivation, using an oocyte expression system. Kv beta 3 was found to accelerate both the fast and the slow component of Kv1.4 inactivation. Kv beta 3 also altered the relative contributions of the two components of inactivation by increasing the contribution of the slow component to the inactivation process. Kv beta 3 slowed recovery from inactivation for Kv1.4, but not for a Kv1.4 deletion mutant lacking N-type inactivation. Finally, steady-state activation and the time course of Kv1.4 current activation were not strongly influenced by Kv beta 3; however, deactivation was slowed in the presence of Kv beta 3. This study suggests that Kv beta 3 alters channel states which follow activation.
Animals↗
Shear stress induces ATP-independent transient nitric oxide release from vascular endothelial cells, measured directly with a porphyrinic microsensor.
Shear stress causes the vascular endothelium to release nitric oxide (NO), which is an important regulator of vascular tone. However, direct measurement of NO release after the imposition of laminar flow has not been previously accomplished because of chemical (oxidative degradation) and physical (diffusion, convection, and washout) complications. Consequently, the mechanism, time course, kinetics, and Ca2+ dependence of NO release due to shear stress remain incompletely understood. In this study, we characterized these parameters by using fura 2 fluorescence and a polymeric porphyrin/Nafion-coated carbon fiber microsensor (detection limit, 5 nmol/L; response time, 1 millisecond) to directly measure changes in [Ca2+]i and NO release due to shear stress or agonist (ATP or brominated Ca2+ ionophore [Br-A23187]) from bovine aortic endothelial cells. The cells were grown to confluence on glass coverslips, loaded with fura 2-AM, and mounted in a parallel-plate flow chamber (volume, 25 microL). The microsensor was positioned approximately 100 microns above the cells with its long axis parallel to the direction of flow. Laminar flow of perfusate was maintained from 0.04 to 1.90 mL/min, which produced shear stresses of 0.2 to 10 dyne/cm2. Shear stress caused transient NO release 3 to 5 seconds after the initiation of flow and 1 to 3 seconds after the rise in [Ca2+]i, which reached a plateau after 35 to 70 seconds. Although the amount (peak rate) of NO release increased as a function of the shear stress (0.08 to 3.80 pmol/s), because of the concomitant increase in the flow rate, the peak NO concentration (133 +/- 9 nmol/L) remained constant. Maintenance of flow resulted in additional transient NO release, with peak-to-peak intervals of 15.5 +/- 2.5 minutes. During this 13- to 18-minute period, when the cells were unresponsive to shear stress, exogenous ATP (10 mumol/L) or Br-A23187 (10 mumol/L) evoked NO release. Prior incubation of the cells with exogenous NO or the removal and EGTA (100 mumol/L) chelation of extracellular Ca2+ blocked shear stress but not ATP-dependent NO release. The kinetics of shear stress-induced NO release (2.23 +/- 0.07 nmol/L per second) closely resembled the kinetics of Ca2+ flux but differed markedly from the kinetics of ATP-induced NO release (5.64 +/- 0.32 nmol/L per second). These data argue that shear stress causes a Ca(2+)-mediated ATP-independent transient release of NO, where the peak rate of release but not the peak concentration depends on the level of shear stress.(ABSTRACT TRUNCATED AT 400 WORDS)
Adenosine Triphosphate↗
Coordinate depression of bradykinin receptor recycling and microtubule-dependent transport by taxol.
Significant cardiovascular side effects have limited the use of taxol as an anticancer drug. A link between decreased plasma membrane dynamics and taxol has been implied because taxol can inhibit intracellular vesicle movements. Reduced membrane recycling caused by taxol could inhibit agonist-evoked Ca2+ signaling within endothelial cells, resulting in endothelium-dependent vasodilation. Bradykinin and ATP are two agonists that evoke Ca2+ transients in endothelial cells. Since the bradykinin receptor-agonist complex is internalized and recycled whereas the ATP agonist-receptor complex is not, we expected that a taxol inhibition of recycling would decrease bradykinin but not ATP receptor activity. We found that taxol depresses (i) the frequency (to 41% of control) and velocity (to 55% of control) of microtubule-dependent vesicle transport and (ii) bradykinin-evoked cytosolic Ca2+ transients (to 76% of control) in bovine aortic endothelial cells. In studying bradykinin receptor desensitization, which reflects receptor recycling, we demonstrate that taxol inhibits bradykinin-evoked Ca2+ transients by 50%. Taxol did not significantly alter ATP-evoked Ca2+ transients in either single-exposure or desensitization experiments. We suggest that taxol's reduction of bradykinin-evoked Ca2+ transients is due to altered microtubule-dependent membrane recycling. This report describes taxol's ability to alter plasma membrane composition through effects on vesicle transport and membrane trafficking pathways. This finding provides a possible mechanism by which taxol can substantially alter cardiovascular function.
Adenosine Triphosphate↗
Agonist-induced changes of [Ca2+]i and membrane currents in single bovine aortic endothelial cells.
Cultured bovine aortic endothelial cells (BAECs) possess an inward rectifier K+ current (IK1), a Ca(2+)-activated K+ current, a nonselective cation current (INS), and a Ca(2+)-activated Cl- current; however, their relative roles remain to be established. In single BAECs, cytosolic free Ca2+ concentration ([Ca2+]i) [K5-fura 2 (50 microM), ratio 340/380 nm] was measured simultaneously with whole cell currents at 22 degrees C. Bradykinin (BK, 2 microM), ATP (10 microM), ionomycin (100 nM), or 2,5-di-(tert-butyl)-1,4-benzohydroquinone (10 microM) were used as agonists. In physiological salt solution (PSS), agonist exposure caused a rapid [Ca2+]i increase, followed by an increase in outward current (greater than -50 mV) and a smaller increase in inward current (greater than -80 mV). Chelation of [Ca2+]i with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid attenuated agonist-induced [Ca2+]i and current responses. Inactivation of the cyclooxygenase pathway by acetylsalicyclic acid and ibuprofen (50 microM each) did not affect the BK-induced [Ca2+]i transient but abolished the current response. In K(+)-free solution, agonist-stimulated outward currents (at +50 mV) were 10 times smaller than in PSS and were consistent with the activation of both INS and a Cl- current. In Cl(-)-free solution, the outward current response following agonist exposure was virtually abolished; at the same time, a linear inward current component with a reversal potential near the equilibrium potential for Na+ was activated. The maximal amplitude of the agonist-induced outward current decreased with decreasing symmetrical Cl- concentrations. Our results suggest that 1) IKI is the dominant current in resting BAECs; 2) K+, Cl-, and nonselective cation conductances contribute to the agonist-induced current response; and 3) most of the agonist-induced activation of currents depends on increased [Ca2+]i and is sensitive to cyclooxygenase inhibitors.
Animals↗
Cloning and characterization of an Ito-like potassium channel from ferret ventricle.
FK1, a ferret ventricular full-length cDNA clone, encodes a 654-amino acid protein with 98% identity to human K+ transient outward current (Ito)-like HK1 (Tamkun et al. FASEB J.5: 331-337, 1991). FK1 is detectable in ferret brain, atrium, left and right ventricle, and kidney but not in skeletal muscle, endothelial cells, aorta, and liver. In Xenopus oocytes, FK1 cRNA gives rise to a rapidly activating and inactivating Ito-like current, which is highly K+ selective (Na(+)-to-K+ permeability ratio = 0.003). Activation occurs over an approximately 50-mV range (-40 to +10 mV) and displays a sigmoid delay in onset with potential-dependent time constants that decrease with depolarization. Steady-state activation can be described with either a simple Boltzmann relationship [half-activation potential (V1/2) = -25 mV, slope (k) = 10 mV] or a Boltzmann relationship raised to either the third or fourth power (alpha 3: V1/2 = -43 mV, kappa = 13.1 mV; alpha 4: V1/2 = -48 mV, kappa = 13.6 mV, where alpha is the activation variable). Inactivation kinetics are biexponential, with the main fast time constant becoming independent of membrane potential depolarized to 0 mV. Steady-state inactivation can be described with a single Boltzmann relationship (V1/2 = -57 mV, kappa = 5.0 mV). Fast inactivation is removed by NH2-terminal deletions. Recovery from inactivation (-90 mV) is quite slow (half-time = 4.8 +/- 2.5 s). In 2 mM extracellular K+ concentration ([K+]o), FK1 tail currents display conventional deactivation behavior; however, in 98 mM [K+]o the tail currents display "reopening" behavior. These results suggest a molecular basis for the electrophysiological similarities between ferret and human ventricular Ito (Campbell et al. J. Gen. Physiol. 101: 571-601, 1993; Näbauer et al. Circ. Res. 73: 386-394, 1993).
Amino Acid Sequence↗
Neuromodulation of calcium current by extracellular ATP in isolated ventricular myocytes.
The effects of extracellular ATP on the L-tpe Ca2+ current (ICa), action potential, and resting and intracellular Ca2+ levels were examined in enzymatically isolated myocytes from the right ventricles of ferrets. Extracellular ATP decreased the peak amplitude of ICa in a time- and concentration-dependent manner. The concentration-response relationship for ATP inhibition of ICa was well described by a conventional Michaelis-Menten relationship with a half maximal inhibitory concentration of 1 microM and a maximal effect of 50%. Extracellular ATP did not change the resting myoplasmic Ca2+ levels; however, it did decrease the Ca2+ transient. The effects of extracellular ATP were mediated independently of adenosine A1 receptors and a pertussis-toxin sensitive G protein. Pharmacological characterization of receptor subtype using ATP analogs was consistent with ATP binding to a P2Y type receptor.
Action Potentials↗
Voltage and temperature dependence of single K+ channels isolated from canine cardiac sarcoplasmic reticulum.
The temperature and voltage dependence of gating and conductance of sarcoplasmic reticulum K+ channels (S-R K+) isolated from adult canine hearts were studied using the reconstituted bilayer technique. Fusion of vesicles from this preparation frequently resulted in the incorporation of a single channel. Only bilayers into which a single S-R K+ channel had fused were studied. The three conductance states of the channel, fully open (O2), substate conductance (O1), and closed (C) were studied as a function of voltage (-50 to +50 mV) and temperature (16 to 37 degrees C). Permeation through the O1 state showed the same temperature dependence as the O2 state corresponding to an enthalpy of permeation of 4.1-4.2 kcal/mol, which is similar to that for K+ diffusion through water. As expected, increased temperature increased the frequency of gating transitions and shortened the average dwell time spent in any conductance state. Over the range of 25 to 37 degrees C, the average dwell time spent in the O1, O2, and C states decreased by 44 +/- 11, 36 +/- 13, and 78 +/- 7% (n = 3 to 4 channels), respectively. The ratio of probabilities between the various conductance states was not strongly temperature sensitive. Analysis of the voltage dependence of this channel was carried out at 37 degrees C and revealed that the dwell times of the O1 and O2 states were voltage insensitive and the probability ratio (PO2:PO1) was approximately 7 and was voltage insensitive. Nonlinear least-squares analysis of dwell times revealed that the closed state was biexponential and was thus composed of a fast (Cf) and a slow (C8) component.Tcf was voltage insensitive with an average value of 5.9 ms, whereas tau c was approximately two orders of magnitude slower and was voltage dependent. The voltage dependence of Cs was described by Tau c (ms) = exp(-0.025-(Vm (mV) - 250)).
Animals↗
The calcium-independent transient outward potassium current in isolated ferret right ventricular myocytes. I. Basic characterization and kinetic analysis.
Enzymatically isolated myocytes from ferret right ventricles (12-16 wk, male) were studied using the whole cell patch clamp technique. The macroscopic properties of a transient outward K+ current I(to) were quantified. I(to) is selective for K+, with a PNa/PK of 0.082. Activation of I(to) is a voltage-dependent process, with both activation and inactivation being independent of Na+ or Ca2+ influx. Steady-state inactivation is well described by a single Boltzmann relationship (V1/2 = -13.5 mV; k = 5.6 mV). Substantial inactivation can occur during a subthreshold depolarization without any measurable macroscopic current. Both development of and recovery from inactivation are well described by single exponential processes. Ensemble averages of single I(to) channel currents recorded in cell-attached patches reproduce macroscopic I(to) and indicate that inactivation is complete at depolarized potentials. The overall inactivation/recovery time constant curve has a bell-shaped potential dependence that peaks between -10 and -20 mV, with time constants (22 degrees C) ranging from 23 ms (-90 mV) to 304 ms (-10 mV). Steady-state activation displays a sigmoidal dependence on membrane potential, with a net aggregate half-activation potential of +22.5 mV. Activation kinetics (0 to +70 mV, 22 degrees C) are rapid, with I(to) peaking in approximately 5-15 ms at +50 mV. Experiments conducted at reduced temperatures (12 degrees C) demonstrate that activation occurs with a time delay. A nonlinear least-squares analysis indicates that three closed kinetic states are necessary and sufficient to model activation. Derived time constants of activation (22 degrees C) ranged from 10 ms (+10 mV) to 2 ms (+70 mV). Within the framework of Hodgkin-Huxley formalism, Ito gating can be described using an a3i formulation.
4-Aminopyridine↗
The calcium-independent transient outward potassium current in isolated ferret right ventricular myocytes. II. Closed state reverse use-dependent block by 4-aminopyridine.
Block of the calcium-independent transient outward K+ current, I(to), by 4-aminopyridine (4-AP) was studied in ferret right ventricular myocytes using the whole cell patch clamp technique. 4-AP reduces I(to) through a closed state blocking mechanism displaying "reverse use-dependent" behavior that was inferred from: (a) development of tonic block at hyperpolarized potentials; (b) inhibition of development of tonic block at depolarized potentials; (c) appearance of "crossover phenomena" in which the peak current is delayed in the presence of 4-AP at depolarized potentials; (d) relief of block at depolarized potentials which is concentration dependent and parallels steady-state inactivation for low 4-AP concentrations (V1/2 approximately -10 mV in 0.1 mM 4-AP) and steady-state activation at higher concentrations (V1/2 = +7 mV in 1 mM 4-AP, +15 mV in 10 mM 4-AP); and (e) reassociation of 4-AP at hyperpolarized potentials. No evidence for interaction of 4-AP with either the open or inactivated state of the I(to) channel was obtained from measurements of kinetics of recovery and deactivation in the presence of 0.5-1.0 mM 4-AP. At hyperpolarized potentials (-30 to -90 mV) 10 mM 4-AP associates slowly (time constants ranging from approximately 800 to 1,300 ms) with the closed states of the channel (apparent Kd approximately 0.2 mM). From -90 to -20 mV the affinity of the I(to) channel for 4-AP appears to be voltage insensitive; however, at depolarized potentials (+20 to +100 mV) 4-AP dissociates with time constants ranging from approximately 350 to 150 ms. Consequently, the properties of 4-AP binding to the I(to) channel undergo a transition in the range of potentials over which channel activation and inactivation occurs (-30 to +20 mV). We propose a closed state model of I(to) channel gating and 4-AP binding kinetics, in which 4-AP binds to three closed states. In this model 4-AP has a progressively lower affinity as the channel approaches the open state, but has no intrinsic voltage dependence of binding.
4-Aminopyridine↗
Modulation of basal L-type Ca2+ current by adenosine in ferret isolated right ventricular myocytes.
1. The whole-cell configuration of the gigaohm seal voltage clamp and an internal perfusion technique were used to study the effects of adenosine on the basal L-type Ca2+ current (ICa) in enzymatically isolated right ventricular myocytes of ferrets. Basal L-type ICa was isolated by using a Na(+)- and K(+)-free saline (replacement by N-methyl-D-glucamine+, Cs+ and TEA+, respectively). All experiments were conducted at room temperature (22-24 degrees C). 2. Basal ICa was markedly reduced during exposure to adenosine in a concentration-dependent manner with a half-inhibitory concentration (IC50) of 0.3 microM and maximum inhibition of 35%. This effect was completely abolished by 50 nM 8-cyclopentyl-1,3-dipropylxanthine (CPDPX), a specific A1 adenosine receptor antagonist with an inhibition constant, Ki = 0.48 nM. Inhibition was also observed in the presence of 1 microM atropine. 3. Adenosine decreased basal ICa by decreasing the peak amplitude of ICa without significantly altering (i) the voltage dependence of the current-voltage relationship, (ii) the apparent reversal potential, (iii) the voltage dependence of steady-state activation and inactivation, (iv) the kinetics of inactivation at 0 mV, and (v) the kinetics of recovery from inactivation at -70 mV. 4. Pretreatment of cells with 0.4 microns/ml pertussis toxin (PTX) for 4 h at 37 degrees C produced greater than 90% ADP ribosylation of PTX-sensitive G proteins. PTX pretreatment significantly attenuated the adenosine-mediated decrease in ICa (35% in control; 4.6% after PTX pretreatment). 5. The peptide inhibitor (PKI) of cyclic AMP-dependent protein kinase A at a concentration of 2 microM neither inhibited basal ICa nor attenuated the effects of adenosine on basal ICa. However, PKI decreased the stimulatory effects of 100 microM cAMP on ICa. 6. Increasing intracellular cAMP to a supra-saturable level by using 10 mM cAMP and 100 microM papaverine did not prevent adenosine from inhibiting ICa. 7. Consistent with the reduction of basal ICa, adenosine produced an inhibitory effect on the action potential under basal conditions, i.e. hyperpolarization of the plateau phase and marked shortening of action potential duration. These effects were concentration dependent. 8. These results demonstrate a reduction of the basal L-type ICa by adenosine in ferret ventricular myocardium. This reduction is not mediated by modification of voltage-dependent properties of macroscopic ICa. The shortening of action potentials may be explained in part by the reduction in ICa.(ABSTRACT TRUNCATED AT 400 WORDS)
Action Potentials↗
Modulation of L-type Ca2+ current by extracellular ATP in ferret isolated right ventricular myocytes.
1. The effects of extracellular adenosine triphosphate (ATP) on the basal L-type Ca2+ current (ICa) were investigated in ferret isolated right ventricular myocytes using the gigaohm seal voltage clamp in the whole-cell and cell-attached configurations. 2. Micromolar levels of extracellular ATP reversibly inhibited ICa in a concentration-dependent manner, without any significant changes in the voltage dependence of either the peak ICa I-V relationship or steady-state activation curve. 3. In contrast, micromolar levels of extracellular ATP did significantly alter the inactivation characteristics of ICa. Ten micromolar ATP: (i) increased the degree of steady-state inactivation of ICa; (ii) altered the time constants of ICa inactivation at 0 mV; and (iii) decreased the time constant of ICa recovery from inactivation at -70 mV. 4. The inhibitory effect of ATP on ICa was not blocked by atropine, a muscarinic cholinergic receptor antagonist, or CPDPX (8-cyclopentyl-3,4-dipropylxanthine), an A1 adenosine receptor antagonist. In contrast, the inhibitory effect of 10 microM ATP could be nearly completely antagonized by 100 microM suramin, a purinergic P2 receptor antagonist. 5. The potency order of ATP analogues in inhibiting ICa was 2-methyl-thio-ATP > ATP > alpha,beta-methylene-ATP, indicating involvement of a P2Y-type ATP receptor. 6. Pretreatment of cells with pertussis toxin (PTX) did not prevent the ATP-induced decrease in ICa. However, (i) ATP produced an irreversible decrease of ICa in the presence of intracellular GTP gamma S, and (ii) the inhibitory effect was significantly attenuated in the presence of intracellular GDP beta S, indicating the involvement of a PTX-insensitive G protein in the P2Y receptor-coupling process. 7. Neither (i) replacing extracellular Ca2+ with 1 mM Ba2+, nor (ii) intracellular perfusion of 10 mM BAPTA for at least 30 min attenuated the inhibitory effect of ATP on the current through Ca2+ channels, suggesting that the inhibitory effect was not obligatorily dependent upon influx of Ca2+ or changes in [Ca2+]i. 8. Ensemble-average current behaviour constructed from cell-attached patch recordings of single L-type Ca2+ channels (110 mM BaCl2) demonstrated that when 10 microM ATP was added to the superfusate on the outside of the patch electrode the inhibition of ICa was still observed, providing evidence for the involvement of intracellular diffusible second messenger(s).(ABSTRACT TRUNCATED AT 400 WORDS)
Adenosine↗
Effects of extracellular ATP on ICa, [Ca2+]i, and contraction in isolated ferret ventricular myocytes.
The effects of extracellular ATP on the voltage-activated "L-type" Ca current (ICa), action potential, resting and transient intracellular Ca2+ levels, and cell contraction were examined in enzymatically isolated myocytes from the right ventricles of ferrets. With the use of the whole cell patch-clamp technique, extracellular ATP (10(-7) to 10(-3) M) inhibited ICa in a time- and concentration-dependent manner. ATP decreased the peak amplitude of ICa without altering the residual current at the end of 500-ms clamp steps. The concentration-response relationship for ATP inhibition of ICa was well described by a conventional Michaelis-Menten relationship with a half-maximal inhibitory concentration of 1 microM and a maximal effect of 50%. Consistent with its inhibitory effect on ICa, ATP hyperpolarized the plateau phase and shortened the action potential duration. In fura-2-loaded myocytes, extracellular ATP did not change the resting myoplasmic Ca2+ levels; however, when current was elicited under voltage-clamp conditions, ATP both decreased the myoplasmic intracellular Ca2+ transient and inhibited the degree of cell shortening. Our results suggest that ATP could be a genuine and potent extracellular modulator of cardiac function in ferret ventricular myocardium.
Action Potentials↗
Intracellular calcium, currents, and stimulus-response coupling in endothelial cells.
Vascular endothelium appears to be a unique organ. It not only responds to numerous hormonal and chemical signals but also senses changes in physical parameters such as shear stress, producing mediators that modulate the responses of numerous cells, including vascular smooth muscle, platelets, and leukocytes. In many cases, the initial response of endothelial cells to these diverse signals involves elevation of cytosolic Ca2+ and activation of Ca(2+)-dependent enzymes, including nitric oxide synthase and phospholipase A2. Both the release of Ca2+ from intracellular stores, most likely the endoplasmic reticulum, and the influx of Ca2+ from the extracellular space contribute to the [Ca2+]i increase. The most important trigger for Ca2+ release is inositol 1,4,5-trisphosphate, which is generated by the action of phospholipase C, a plasmalemmal enzyme activated in many cases by the receptor-G protein cascade. Ca2+ influx appears to be related to the activity of receptor-G protein-enzyme complex and to the degree of fullness of the endoplasmic reticulum but does not involve voltage-gated Ca2+ channels. The magnitude of the Ca2+ influx depends on the electrochemical gradient, which is modulated by the membrane potential, Vm. Under basal conditions, Vm is dominated by a large inward rectifier K+ current. Some stimuli, e.g., acetylcholine, have been shown to hyperpolarize Vm, thus increasing the electrochemical gradient for Ca2+, which appears to be modulated by activation of Ca(2+)-dependent K+ and Cl- currents. However, the lack of potent and specific blockers for many of the described or postulated channels (e.g., nonselective cation channel, Ca(2+)-activated Cl- channel) makes an estimation of their effect on endothelial cell function rather difficult. Possible future directions of research and clinical implications are discussed.
Animals↗
'Reverse use-dependent' effects of 4-aminopyridine on the transient outward potassium current in ferret right ventricular myocytes.
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4-Aminopyridine↗
Ionic current mechanisms generating vertebrate primary cardiac pacemaker activity at the single cell level: an integrative view.
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Amphibians↗
Regulation of extracellular calcium entry in endothelial cells: role of intracellular calcium pool.
We have investigated the role of the intracellular Ca2+ pool in regulating Ca2+ entry into vascular endothelial cells. The intracellular Ca2+ pool was mobilized using either thapsigargin (TG) or 2',5'-di(tert-butyl)-1,4-benzohydroquinone (BHQ), inhibitors of the endoplasmic reticulum Ca(2+)-adenosinetriphosphatase (ATPase). Mobilization of intracellular Ca2+ stores with either inhibitor depleted intracellular Ca2+ and greatly reduced subsequent mobilization of the inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular Ca2+ pool by bradykinin. However, bradykinin-induced mobilization of the IP3-sensitive intracellular Ca2+ pool only partially reduced the subsequent response of cells to TG and BHQ. Mobilization of the intracellular Ca2+ pool by either TG or BHQ led to a concentration-dependent elevation of cytosolic Ca2+ concentrations ([Ca2+]i) without initiating inositol polyphosphate formation. In contrast to the rapidly developing, transient rise in Ca2+ concentration initiated by bradykinin, maximal concentrations of TG and BHQ stimulated a slowly developing, prolonged elevation of [Ca2+]i that required extracellular Ca2+ and could be blocked by extracellular Ni2+. Extracellular Ca2+ entered the cell through an activated cation entry pathway, since bradykinin, TG, and BHQ stimulated Mn2+ and 45Ca2+ entry. Bradykinin-stimulated 45Ca2+ uptake reached a peak within 2 min, whereas 45Ca2+ influx initiated by TG or BHQ continued for at least 8 min. Importantly, the [Ca2+]i response after low concentrations of BHQ was more transient than that seen after TG. The return of [Ca2+]i to basal values after low concentrations of BHQ was associated with reversal of Ca(2+)-ATPase inhibition and refilling of the IP3-sensitive Ca2+ pool. The continued elevation of [Ca2+]i and prolonged Ca2+ entry seen with TG was associated with continued Ca(2+)-ATPase inhibition and an empty IP3-sensitive Ca2+ pool. We conclude that mobilization of intracellular Ca2+ stores induces Ca2+ entry in endothelial cells which continues until the intracellular Ca2+ pool is refilled.
Animals↗
Cellular and molecular biology of the cardiovascular system.
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Animals↗