A new type of Ca-channel modulation by a novel class of 1,4-dihydropyridines.

Both Ca-antagonistic as well as Ca-agonistic 1,4-dihydropyridines (DHPs) have become extremely important tools to investigate the role of Ca-channels under various physiological and pathophysiological conditions. While Ca-antagonists stabilize the inactivated state of the Ca-channel without influencing the voltage dependent open and closed times, Ca-agonists prolong the mean open time of the channel. We here report for the first time the effects of a novel DHP, BAY Y 5959, which modulates Ca-channel gating in a unique manner: It increases both the mean open time and the mean closed time of the Ca-channel by binding to the DHP receptor. This results in a reduced rate of Ca-current activation, an increased peak current, and a strongly prolonged tail current decay. All these effects are strongly voltage dependent. Therefore it depends on resting membrane potential and shape of the action potential whether and how the Ca-influx into the cell is influenced. This novel mode of action of BAY Y 5959 results in an interesting pharmacological profile: It has a strong positive inotropic effect in the heart without influencing vessel tone. Therefore the term Ca-promoter is suggested; it could become a new approach for the drug treatment of congestive heart failure.

Induction of c-fos expression in rat vascular smooth muscle reporter cells by selective activation of the thrombin receptor.

The rat vascular smooth muscle cell (VSMC) line A10 (ATCC CRL 1476) was stably transfected with a human c-fos promoter-driven luciferase reporter gene to monitor thrombin receptor activation and subsequent induction of c-fos expression. Selective activation of the endogeneous thrombin receptor by the thrombin receptor activating peptide (TRAP1-6), SFLLRN, is shown here to result in a significant transient increase of intracellular [Ca2+], dose-dependent induction of c-fos promoter-mediated luciferase activity, and stimulation of DNA synthesis. These data demonstrate that A10 cells and reporter line derivatives thereof possess a functional thrombin receptor very similar or identical to that previously described. Results obtained with various signal transduction modulating or inhibiting agents support previous notions showing that thrombin receptor activation by SFLLRN is coupled to events involving p21ras activation, protein tyrosine kinase, and activation of PKC. The A10 reporter line described here proved to be a helpful and reliable tool to study alpha-thrombin and TRAP1-6-mediated intracellular events, since it retained most of the spectrum of biological responses found in primary VSMC cultures.

Monitoring of Ca(2+)-transients in electrically stimulated A7r5 vascular smooth muscle cells fills the experimental gap between KCL-induced depolarization and patch-clamp studies.

The effects of electrical field stimulation on [Ca2+]i in the A7r5 vascular smooth muscle cell line have been monitored with the Ca(2+)-sensitive dye fura-2. The experimental set-up allowed high-temporal resolution of the [Ca2+]i-measurements and fast application of test solutions. Electrical field stimulation of A7r5 cells in the confluent growth state resulted in a transient increase in [Ca2+]i from resting values below 100 nM to values in the range of some hundred nM. For a given cell, the electrically induced Ca(2+)-transients were highly reproducible. The requirement for the presence of extracellular Ca2+ and the sensitivity to the Ca(2+)-antagonist nifedipine and the Ca(2+)-agonist BAY K 8644 suggest that the Ca(/+)-transients reflect [Ca2+]i-changes based on Ca(2+)-influx through voltage-dependent L-type Ca(2+)-channels. Therefore, electrical field stimulation of confluent A7r5 cells provides an easy-to-establish and highly reproducible method for the investigation of the physiology and pharmacology of voltage-dependent Ca(2+)-channels in intact vascular smooth muscle cells, which fills the gap between KCl-induced depolarization and the patch-clamp technique.

The molecular mode of action of the Ca agonist (-) BAY K 8644 on the cardiac Ca channel.

The primary drug action of (-) BAY K 8644 on whole-cell Ca current in atrial myocytes was measured under conditions where secondary Ca-mediated changes of Ca channel activity were minimized. The most direct action of (-) BAY K 8644 is the change of gating kinetics which results in a strictly voltage-dependent increase of the peak current in the voltage range between -40 and 0 mV. Peak currents were increased dose dependently in the concentration range from 1 to 30 nM. Analysis of peak current/voltage relations revealed a linear shift of the current activation by approximately 23 mV to more negative membrane potentials, without any change in its voltage dependence and in the current reversal potential or the maximum whole-cell conductance. Measurement of Ca current activation and deactivation time constants suggests that (-) BAY K 8644 prolongs the single-channel open time without affecting the closed time. From the shift of the open time function to more negative voltages by about 50 mV the energy transferred to the gating process is calculated to be 5.4 kJ/mol (1.3 kcal/mol). The drug-induced slow component of tail current has been used to estimate the true dose/response relation for (-) BAY K 8644. A KD value of 4.3 nM and a Hill coefficient of 1.25 were determined. Flash-induced competition experiments with the Ca antagonist nifedipine allowed the measurement of binding kinetics of (-) BAY K 8644. The association rate constant is estimated to about 5 x 10(6) mol-1.s-1 and dissociation time constant is approximately 50-70 s; both are in close agreement with receptor binding studies. Results are discussed in relation to models for drug action of dihydropyridine-type compounds and to implications for the structure of the Ca channel protein.

Kinetics of ATP-induced Ca2+ transients in cultured pig aortic smooth muscle cells depend on ATP concentration and stored Ca2+.

1. Single cultured pig aortic smooth muscle cells were studied using fura-2 and dual excitation wavelength microfluometry. 2. Extracellular ATP in micromolar concentrations induced a transient increase of [Ca2+]i due to Ca2+ release from internal stores. In the same concentration range application of ATP resulted in an increase of intracellular inositol phosphate level. 3. In a medium range of ATP concentrations (2-10 microM) the Ca2+ signal was oscillating, whereas at higher and lower concentrations only a Ca2+ transient with a single peak was elicited. 4. The rank order of potency for the tested purine and pyrimidine nucleotides was: UTP > ATP > ADP >> AMP = adenosine = alpha,beta-methylene ATP = 0. The response to the nucleotides could be abolished by the P2-purinoceptor antagonist suramin. 5. The latency between agonist application and onset of the Ca2+ transients as well as their amplitude and rate of rise are dependent on ATP concentration. 6. Removal of Ca2+ from the extracellular solution led to a progressive decrease of amplitude and prolonged latency of the Ca2+ transients. This shows that depletion of the Ca2+ stores affects kinetics of the ATP-induced Ca2+ release. 7. The inorganic Ca(2+)-influx blockers Ni2+ and Co2+ affected amplitude and latency in a manner similar to Ca2+ removal, while the Ca2+ antagonist nifedipine was ineffective up to a concentration of 10(-6) M. 8. These results reveal a dual dependency of the InsP3-induced Ca2+ release on agonist concentration and filling state of the Ca2+ stores, which supports the hypothesis of a feedback amplification between InsP3 and released Ca2+.

Spontaneous and experimentally evoked [Ca2+]i-transients in cardiac myocytes measured by means of a fast Fura-2 technique.

A setup for dual wavelength-excitation fluorescence measurements is introduced which permits a temporal resolution of up to 1 KHz, using the Ca2(+)-sensitive fluorescent dye Fura-2. The system makes use of a novel technical solution for chopping between two excitation wavelengths which does not move any optical components. Two beams, which are alternatively opened or shut by a rotating chopper wheel, are united by a dichroic mirror and are used for low-noise epifluorescence microscopy. The system includes a device for fast changes of extracellular solution that can be used for studying various components of [Ca2+]i-regulation in excitable and non-excitable cells. Sample recordings of spontaneous and experimentally-evoked [Ca2+]i-transients from cardiac myocytes are presented. Cardiac myocytes are a cell species that produces particularly fast [Ca2+]i-transients and therefore, a high temporal resolution is required in order to study physiological and/or pharmacological properties of these transients.

Ca-agonists: a new class of inotropic drugs.

The basic pharmacology of dihydropyridine Ca-agonists published so far (BAY k8644, CGP 28-392, H 160/51, YC 170, and 202-791) is described. The importance of the potency of the enantiomeres for the effect of a racemic compound is underlined. The Ca agonist prototype BAY k8644 leads to an increase of the maximal rate of rise of left ventricular pressure (LV(dP/dt)) and an increase of left ventricular stroke work in conscious dogs. When the vascular effects of BAY k8644 are counterbalanced by intravenous injection of sodium-nitroprusside, the left ventricular functions curves show markedly increased stroke work against the same mean arterial blood pressure at the same filling pressure. BAY k8644 stimulates the heart economically: the net efficiency in isolated working guinea-pig hearts is about 20%, identical to a stimulation by calcium or ouabain. Cardiotonic drugs acting via cAMP-dependent mechanisms like isoprenaline, amrinone, or pimobendane however, stimulate the heart about 1/3 less economically. The mechanism of action of Ca-agonists is explained from electrophysiological findings: Ca-agonistic dihydropyridines increase the open probability of the Ca-channels by a shift of the open-probability curve to more negative membrane potentials. As a consequence, the steady-state inactivation curve of the Ca-channel is also shifted in the same direction. While the effect on open-probability is the underlying mechanism for Ca-agonism, the latter effect results in Ca-antagonism. Therefore, depending on drug concentration and on membrane resting potential, a single chemical compound can act either as a Ca-agonist or a Ca-antagonist. A kinetic model of dihydropyridine action on the Ca-channel is described.

Calcium-agonists.

In contrast to nifedipine-like calcium antagonists, calcium agonistic 1,4-dihydropyridines have vasoconstricting and positive inotropic properties. BAY K 8644 has become the prototype of this new class. Its enantiomers show opposite effects on the calcium channel: one acts as a calcium agonist with the pharmacological profile of the racemic compound, its antipode has calcium antagonistic effects at 10 times higher concentrations. Voltage clamp studies reveal calcium current increasing effects at 3 X 10(-8) mol/l BAY K 8644, while the current is reduced at 3 X 10(-6) mol/l. Analysis of the calcium current activation and deactivation kinetics shows that BAY K 8644 leaves the mean closed times of the calcium channel unchanged while it increases the mean open times. From these data a reaction model of drug action is derived, suggesting that BAY K 8644 binds only to the open state of the Ca-channel.

The optical isomers of the 1,4-dihydropyridine BAY K 8644 show opposite effects on Ca channels.

The optical isomers of the 1,4-dihydropyridine BAY K 8644 were studied in isolated rabbit aorta and heart preparations. The (-)-enantiomer has the known vasoconstricting and positive inotropic properties of the Ca agonistic compound. In contrast, its antipode shows at about 10-50 times higher concentrations the vasodilating and negative inotropic effects of Ca antagonistic drugs. It is concluded that neither simple chemical nor physical actions can be responsible for the opposite effects of Ca antagonistic and Ca agonistic dihydropyridines.

Removal of Ca current inactivation in dialysed guinea-pig atrial cardioballs by Ca chelators.

Ca currents flowing during voltage clamp depolarizations were studied in cultured guinea-pig atrial cardioballs by means of single low resistance patch clamp pipettes. The pipettes were filled with solutions containing Cs+ as major cation in order to block K+ currents and high concentrations of various Ca chelating agents (EGTA, nitrilotriacetic acid, citrate, dipicolinic acid) to prevent rises of the intracellular Ca-activity by Ca-entry. Ca currents of myocytes loaded with 20 mM of either EGTA [(ethylenedioxy)-diethylenedinitrilo)tetra-acetic acid] or NTA (nitrilotriacetic acid) display a biphasic time course of inactivation at membrane potentials between -25 and +45 mV. The fast phase is reduced with increasingly positive membrane potentials. In cells loaded with either citrate or DPA (dipicolinic acid, pyridine-2,6-dicarboxylic acid) inactivation is negligible or absent for small depolarizations. In the range of membrane potentials where maximum current flows (0-+10 mV) a monophasic slow time course of inactivation is observed. At more positive membrane potentials inactivation is slowed. The amount of inactivation under this condition is related to the current density of the cell. Conditions, which for a given membrane potential reduce the amplitude of ICa such as extracellular application of blocking ions (Co2+, Cd2+), a conditioning depolarization, or 'rundown' of Ca-channels lead to a slowing or a complete removal of inactivation in cells dialysed with citrate or DPA respectively. Cells loaded with these Ca chelators did not show any symptom of voltage dependent inactivation of ICa. Under the conditions described action potentials were recorded in the current clamp mode. Upon dialysis with EGTA the typical 'triangular shaped' atrial action potential develops a plateau of 500 to 800 ms in duration. With citrate-containing pipette solutions the action potential duration usually is several seconds. The results for the first time demonstrate that inactivation of cardiac ICa can be considerably slowed or even removed. They provide further strong support for the hypothesis that inactivation of this current depends on Ca entry rather than membrane potential. The fast phase of inactivation observed with EGTA (NTA) possibly reflects the slow kinetics of the binding reaction of this type of Ca chelators.

Evidence for Ca-mediated inactivation of ICa in dialysed guinea-pig atrial cardioballs.

Ca current (ICa) was studied in cultured myocytes from right atria of adult guinea-pigs (cardioballs) by means of single low-resistance patch pipettes. K currents were blocked by dialysis of the cells with solutions containing Cs+ as main cation and extracellular TEA (70 mM). Under this condition membrane currents elicited by voltage clamp pulses of 200 ms in duration from -45 mV (holding potential) are net inward for depolarizations up to +55 mV without detectable contamination by outward current components. The peak inward current (ICa) has a maximum between +5 and +10 mV and reverses around +60 mV. Measurements of ICa tail currents obtained after clamp pulses of increasing duration to more and more positive membrane potentials suggest that ICa inactivation is not genuinely voltage-dependent.

Guinea-pig atrial cardioballs.

Myocytes from atria of adult guinea-pigs were isolated by means of a previously described enzyme perfusion with some modifications. The problem of Ca-intolerance of the dispersed cells was circumvented by (i) avoiding cooling of the cells below 25 degrees C and (ii) increasing the Ca concentration slowly already during the enzyme perfusion. Isolated atrial myocytes were taken in long-term cell culture. Under this condition they become spherical within about 2 days. The rounded stage (cardioballs), which is found in the cultures for a period of ca. 10 days is highly suited for electrophysiological studies using the different recording configurations of the patch clamp technique, including 'tight-seal whole-cell recording' with simultaneous cell dialysis.

Properties of an inward rectifying K channel in the membrane of guinea-pig atrial cardioballs.

Single channel outward current fluctuations are recorded in excised (outside-out) membrane patches of isolated atrial cells in culture (cardioballs) from hearts of adult guinea-pigs. The ionic channel displays a high selectivity to K ions. Accordingly the reversal potential of the single channel current is close to the K equilibrium potential. The open channel conductance is unaffected by the membrane potential but depends on the K concentration of the outside solution (19.7pS at 2 mM Ko to 30.7pS at 20 mM Ko). The open state probability (Po) of the channel shows a marked voltage dependence. Po amounts to c.0.9 at -40 mV and decreases to c.0.1 at +40 mV. Under the assumption of no channel interaction a macroscopic steady state current voltage relationship is reconstructed from the single channel data. The relationship displays inward-going rectification. The rectification is due to the voltage dependence of Po. The I-V curve displays a negative slope at membrane potentials positive to -15 mV. In bathing solutions containing Ba ions (0.2 mM) Po is reduced by rapid closures which interrupt the open state events. The unit channel conductance is unaffected by Ba ions. The channel block exerted by Ba ions is augmented with increasing membrane hyperpolarization. The results suggest that the channel studied may represent a background K conductance.

Atrial muscle cells from hearts of adult guinea-pigs in culture: a new preparation for cardiac cellular electrophysiology.

By means of a modified Langendorff perfusion technique using collagenase and elastase cell suspensions of viable myocytes from atria of adult guinea-pigs can be obtained. If the cell isolation is performed aseptically the myocytes can be kept in long term cell culture. Under these conditions the cells attach to the bottom of the culture dish within 12 to 24 h after plating. Thereafter they round up forming spherical 'cardioballs'. These cardioballs are highly suitable for electrophysiological experiments using different configurations (cell-attached and cell-free) of the patch-clamp technique. They can be employed for these experiments for up to 8 days after isolation. Thereafter they tend to flatten resembling embryonic heart cells in tissue culture.

Facilitation of acetylcholine release from cardiac parasympathetic nerve endings. Effect of stimulation pattern and Mn ions.

The influence of the stimulus intervals and the effect of Mn ions on facilitation of acetylcholine (ACh) release from parasympathetic nerve terminals were studied in quiescent guinea-pig auricles by electrophysiological methods. A maximum facilitation occurs at intervals of about 50ms. The half time of decay of facilitation after a conditioning stimulus is about 500ms. When conditioning trains of stimuli were applied, a second much longer lasting component of facilitation was found (t1/2 congruent to 4s). Mn ions, after exerting an inhibitory effect, cause an increase of ACh release, the development of which is dependent on frequent stimulation of the nerve fibres. This potentiation is accompanied by an apparent loss of facilitation. A further increase in ACh release occurs when superfusion is changed from Mn containing to normal Tyrode's solution. The decay to the control level displays a half time of about 20 min and can also be accelerated by frequent stimulation of the parasympathetic nerve fibres. It is suggested that Mn ions not only inhibit a Ca inward current but may also act on intracellular Ca2+ bindings sites in the nerve terminal. When these sites are blocked even a reduced Ca influx can be more effective in the process of transmitter release.