Pharmacokinetic and Pharmacodynamic Comparison of Sildenafil-Bosentan and Sildenafil-Ambrisentan Combination Therapies for Pulmonary Hypertension.

To elucidate whether the pharmacokinetics (PK) and pharmacodynamics (PD) of sildenafil are influenced differently when it is coadministered with bosentan (S+B) or with ambrisentan (S+A), we evaluated the PK and PD profiles of sildenafil before and after 4-5 weeks of S+A or S+B treatment in patients with pulmonary arterial hypertension. The area under the plasma concentration-time curve of sildenafil was significantly higher in S+A treatment than in S+B treatment (165.8 ng•h/mL vs. 396.8 ng•h/mL, P = 0.018) and the oral clearance of sildenafil was significantly lower after S+A treatment than after S+B treatment (120.6 L/h/kg vs. 50.4 L/h/kg, P = 0.018). In the PD study, incremental shuttle walking distance was superior during treatment with S+A than during treatment with S+B (S+B; 280 m vs. S+A; 340 m, P = 0.042). There were no concerns about safety with either combination therapy regime.

The effect of high sodium concentration on the action potential of the skate heart.

It already has been well documented that the maximum rate of depolarization and amplitude of action potentials are directly dependent on [Na+]o in the vertebrate myocardium. Almost all studies have been carried out at low sodium concentration ranges by substituting NaCl for other substances. Action potentials should be demonstrable in higher sodium concentrations, but cells are inevitably damaged by osmotic changes. The blood of elasmobranchs is nearly isosmotic with sea water, but NaCl accounts for 54.5% of the osmotic pressure and 38.7% of it is maintained by urea molecules. Utilizing this special situation in elasmobranchs, the effect of high sodium concentration was studied up to 170% of normal sodium concentration, while still retaining isosmotic condition. The rate of depolarization, amplitude, and duration of the myocardial action potential all increased in direct proportion to [Na+]o, and no depressant effect on transmembrane action potentials was observed in solutions of high sodium concentration. With regard to depolarization rate, the regression curve fitted by the least squares method passed through zero within two standard errors. At high sodium levels, the overshoot changed as expected theoretically, but at lower ranges it deviated from the theoretical values. [Na+]i and [K+]i in this tissue have been determined, and these data are explained on the basis of the Na theory.

N-ethylmaleimide uncouples muscarinic receptors from acetylcholine-sensitive potassium channels in bullfrog atrium.

The effect of N-ethylmaleimide (NEM), a sulphydryl alkylating agent, on the acetylcholine-activated K+ current, IK(ACh), has been studied in single cells from bullfrog atrium using a tight-seal, whole-cell voltage clamp technique. Addition of NEM (5 x 10(-5) M) produced a time-dependent complete block of IK(ACh). Dialysis of guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S, 5-10 x 10(-4) M), a nonhydrolyzable GTP analogue, into the myoplasm from the recording pipette gradually activated IK(ACh) even in the absence of acetylcholine. This effect is thought to be due to a GTP gamma S-induced dissociation of GTP-binding proteins (Gi and/or Go) into subunits that can directly activate these K+ channels. When NEM (5 x 10(-5) M) was applied after the GTP gamma S effect had fully developed, it failed to inhibit the GTP gamma S-induced K+ current, indicating that the NEM effect is unlikely to be on the dissociated subunits of the GTP-binding protein(s) or on the K+ channels. In contrast, pretreatment with NEM before GTP gamma S application markedly reduced the muscarinic K+ current, suggesting that NEM can block this K+ current by inhibition of the dissociation of the GTP-binding proteins into functional subunits. In NEM-treated cells the stimulatory effect of isoproterenol on ICa was present, but the inhibitory action of ACh on ICa was completely abolished. These results demonstrated that NEM can preferentially inhibit muscarinic receptor-effector interactions, probably by alkylating the GTP-binding proteins that are essential for these responses.

Mechanism of acetylcholine-induced inhibition of Ca current in bullfrog atrial myocytes.

The mechanism of the anti-beta-adrenergic action of acetylcholine (ACh) on Ca current, ICa, was examined using the tight-seal, whole-cell voltage clamp technique in single atrial myocytes from the bullfrog. Both isoproterenol (ISO) and forskolin increased ICa dose dependently. After ICa had been enhanced maximally by ISO (10(-6) M), subsequent application of forskolin (50 microM) did not further increase ICa, suggesting that ISO and forskolin increase ICa via a common biochemical pathway, possibly by stimulation of adenylate cyclase. ACh (10(-5) M) completely inhibited the effect of low doses of forskolin (2 x 10(-6) M), as well as ISO, but it failed to block the effects of high doses of forskolin (greater than 5 x 10(-5) M). Intracellular application of cyclic AMP (cAMP) also increased ICa. ACh (10(-5) M) failed to inhibit this cAMP effect, indicating that the inhibitory action of ACh occurs at a site proximal to the production of cAMP. ACh (10(-5) M) also activated an inwardly rectifying K+ current IK(ACh). Intracellular application of a nonhydrolyzable GTP analogue, GTP gamma S (5 X 10(-4) M), activated IK(ACh) within several minutes; subsequent application of ACh (10(-5) M) did not increase IK(ACh) further. These results demonstrate that a GTP-binding protein coupled to these K+ channels can be activated maximally by GTP gamma S even in the absence of ACh. Intracellular application of GTP gamma S also strongly inhibited the effect of ISO on ICa in the absence of ACh. Pertussis toxin (IAP) completely prevented both the inhibitory effect of ACh on ICa and the ACh-induced activation of IK(ACh). GTP gamma S (50 microM-1 mM) alone did not increase ICa significantly; however, when ISO was applied first, GTP gamma S (5 x 10(-4) M) gradually inhibited the ISO effect on ICa. These results indicate that ACh antagonizes the effect of ISO on ICa via a GTP-binding protein (Gi and/or Go). This effect may be mediated through a direct inhibition by the alpha-subunit of Gi which is coupled to the adenylate cyclase.

Inhibition of the muscarinic receptor-activated K+ current by N-ethylmaleimide in rabbit heart.

The effects of N-ethylmaleimide (NEM), a sulfhydryl alkylating agent, on the ACh-activated K+ current were examined in single cells from rabbit hearts using whole-cell and single channel patch clamp techniques. Bath application of NEM (50 microM) or the muscarinic antagonist, atropine (1 microM) completely inhibited the ACh-activated K+ current in whole-cell recordings. In cell-attached patch conditions, the inhibitory effect of NEM was still observed; in contrast, atropine was ineffective when the agents were bath applied, indicating that the site of action of NEM is different from that of atropine. Inside-out patch recordings confirmed that GTP was required on the internal side of the membrane for activation of this K+ channel when ACh was present in the pipette. NEM abolished this GTP-activated K+ channel activity. GTP gamma S, a non-hydrolysable GTP analogue, was able to activate this K+ channel in the absence of a muscarinic agonist, an effect thought to be due to the direct activation of GTP-binding proteins. Pretreatment with NEM almost completely prevented this effect of GTP gamma S. In contrast, after the activation of the K+ channel by GTP gamma S had reached a steady-state, NEM failed to show a significant inhibitory effect. These results demonstrate that NEM prevents the activation of muscarinic receptor-regulated K+ channel and suggest an involvement of alkylation of the GTP-binding proteins which are coupled to this type of K+ channel.

Electrophysiology of single cardiac cells.

Whatever techniques of isolation one may use, it is certainly true that the isolated single heart cells are very useful in various physiological experiments. For electrophysiology, the application of the single cell serves to test findings previously obtained in the multicellular preparation. Therefore, summary and comparison of the data will become necessary in the near future. Furthermore, the application of the single cell opens new areas for electrophysiology of the heart, because when using only the single cell preparation, one can dialyse intracellular millieu and can also measure single channel analyses.

Effects of various intracellular Ca ion concentrations on the calcium current of guinea-pig single ventricular cells.

The effects of changing the intracellular Ca concentration ([Ca]i) on the calcium current (iCa) were studied in isolated single ventricular cells of the guinea-pig. [Ca]i was varied by an intracellular perfusion technique using a suction pipette. iCa measured from internally perfused cells at a pCa lower than 9.0 was dependent on the extracellular Ca concentration ([Ca]o). Increasing [Ca]o from 1.8 to 5.4 mM increased the amplitude of iCa, and reduction of [Ca]o from 1.8 to 0.01 mM decreased the amplitude. The inactivation time course of iCa became faster as [Ca]o was increased and slower as [Ca]o was reduced. By decreasing the pCa of the internal perfusate from 9.0 to 6.8, the amplitude of iCa was decreased markedly, but no significant change was observed in its time course. Analysis of the I-V curve led to the conclusion that a change in the driving force was not a major factor in the reduction of iCa. The "steady state inactivation" of iCa was measured by a double-pulse method. The amplitude of iCa elicited by the test pulse was smaller at pCa 7.4 than at pCa 9.0 at potentials of between -27 and +33 mV. By normalizing the iCa amplitude, however, the "steady state inactivation" curve in the control and at high [Ca]i overlapped. Similar results were obtained in Sr-Tyrode solution. The degree of "steady state inactivation" of iCa at the potentials positive to +10 mV was larger in Ca-Tyrode than in Sr-Tyrode solution. It is proposed that the reduction in amplitude of iCa at higher [Ca]i is caused by a reduction of the maximum conductance of iCa (gCa) and that Ca ions passing through iCa channels have a remarkable effect on its inactivation.

Effects of Na+ and K+ on the resting membrane potential of the rabbit sinoatrial node cell.

To estimate the "resting potential" of the sinoatrial node cell, membrane potentials in quiescent states under various conditions were compared with the holding potential at which no net membrane current flowed during the voltage clamp. The membrane potential of the rabbit sinoatrial node cell was recorded using a conventional microelectrode method. The single sucrose gap method and the double microelectrode method were employed for the voltage clamp. The membrane potential immediately before the resumption of the spontaneous activity in normal Tyrode solution after a quiescence in high K+ concentration, in low Na+ concentration or in low temperature was approximately - 37 m V and was similar to that of the temporarily quiescent sinoatrial node cells after dissection. The holding potential at which no net membrane current flowed was - 38.4 m V, which coincided with the membrane potential during quiescence. The resting potential in quiescence increased by approximately 17 m V for a tenfold decrease in the extracellular Na+ concentration, and 22 m V for a tenfold increase in the K+ concentration. The relative membrane conductance decreased transiently on removing Na+ from the bathing medium. These findings suggest that a potential equivalent to the resting potential existss in the S-A node cell approximately 20 m V positive to the maximum diastolic potential, and this low value might be due to the high Na+ conductance of the cell membrane.

The effect of polarization on the action potentials of the rabbit AV nodal cells.

The resting potential of various parts of the AV nodal regions of an excised rabbit heart was changed by applying constant electrical current with a suction electrode. The membrane potential was recorded with an intracellular microelectrode and the maximum rate of rise of depolarization was computed. The action potential recorded from the AV nodal cell responded to constant current in a manner qualitatively similar to other myocardiums. The relationship between resting potential and amplitude of action potential was expressed by a straight line, while a sigmoidal relationship was observed between the resting potential and the maximum rate of rise of depolarization. Most of the initial current system in alpha region was inactivated at the control membrane potential. The maximum value of the maximum rate of rise of depolarization of alpha region was 30 V/sec which was lower than that of other parts of the nodal region.

The effect of sodium ion on the initial phase of the sinoatrial pacemaker action potentials in rabbits.

The maximum rate of rise of depolarization of the isolated rabbit S-A node cells was studied with an intracellular microelectrode. Both the amplitude and the maximum rate of rise of depolarization reduced in proportion to the extracellular sodium concentration, but the reduction in the maximum rate of rise of depolarization was greater than that in the amplitude of action potentials. The frequency of spontaneous activities and the duration of action potentials were increased during application of depolarizing current, while they were both reduced during application of hyperpolarizing current. Both the amplitude of the action potential and the maximum rate of rise of depolarization showed voltage dependent characteristics. The maximum value of the maximum rate of rise of depolarization in pacemaking cells ranged from 2 to 11 V/sec and was quantitatively lower than other myocardial fibers. The maximum value of the maximum rate of rise of depolarization in 50% Na+ solution was reduced to approximately 1/2 of the control in normal Na+ solution. A signoidal relationship was observed between the membrane potential and the maximum rate of rise of depolarization. When the temperature of the perfusate was lowered, the signoidal curve shifted toward the negative direction. It is concluded that sodium ion is responsible for the generation of the initial phase of the S-A node action potentials, as in other myocardial cells. However, the amount of ionic current required for spike generation is quantitatively smaller than other myocardial cells.

Contracture and hyperpolarization of the rabbit sinoatrial node cells in Na-depleted solution.

A study was made on both hyperpolarization and contracture which developed in the rabbit sinoatrial node cell during perfusion with Na-depleted solution. The membrane was hyperpolarized in 19 out of 27 specimens (-69.5 +/- 6.0 mV), while in the remaining 8 specimens the value of the membrane potential remained low (-39.4 +/- 2.7 mV). Slope conductance increased as the membrane was hyperpolarized in Na-depleted solution, but not when the value of the membrane potential remained at low. The amplitude of hyperpolarization increased with decrease in Na concentration, increase in Ca concentration and increase in perfusate temperature. A marked increase in diastolic tension was observed in 23 out of 29 specimens but in the 6 remaining specimens the increase in tension was small. Tension increased with decrease in Na concentration, increase in Ca concentration, and increase in perfusate temperature. Pretreatment with ouabain caused a consistent increase in contracture tension, while its effect on the membrane hyperpolarization was variable among different specimens. These results may be explained by assuming that intracellular Ca concentration increased with Na-Ca exchange in Na-depleted solution. In addition to contracture, increase in intracellular Ca concentration might have caused an increase of membrane K conductance and produced marked hyperpolarization.

Effect of procaine amide on the membrane currents of the sino-atrial node cells of rabbits.

Using small rabbit sino-atrial node preparations, the effects of procaine amide in concentrations from 0.01 to 2 mg/ml on the membrane potentials currents were studied by both current-clamp and voltage-clamp experiments. Procaine amide in concentrations over 0.1 mg/ml reduced the peak of the action potential, maximum diastolic potential and the maximum rate of depolarization. The action potential duration was prolonged, the resting potential was decreased and the heart rate was reduced. In the voltage-clamp experiments, procaine amide (0.1 mg/ml) reduced the slow inward current (is), the outward current (iK) and the inward current activated by hyperpolarization (ih). The major effect, however, was the reduction of the outward current. Sine the degree of the steady-state activation of iK and its time constant were unchanged, the observed reduction of iK could have been caused by a reduction of iK.

Effects of calcium ion on the rising phase of the action potential in rabbit sinoatrial node cells.

To clarify the role of Ca ion in the rising phase of the sinoatrial (S-A) node action potential, the sigmoidal relationship between the maximum rate of rise of the action potential and the maximum diastolic potential was examined at various concentrations of Ca. The membrane potential was changed by applying a current across a single sucrose gap. The sigmoidal curve shifted toward the positive potential without a change in maximum value when the Ca concentration was increased from nominal "zero" to 10 mM. Therefore, it is concluded that Ca ion modifies the inactivation process of Na current which is responsible for the rapid rising phase of the S-A node action potential. The duration of the action potential and the maximum diastolic potential were decreased with an increase in Ca concentration. The observation that the overshoot of the action potential increased by 12 mV for a tenfold increase in concentration of Ca (within the range of 0.1-5.0 mM) suggests that the inward current of Ca ion may be responsible for the overshoot of the S-A node action potential.

Reconstruction of sino-atrial node pacemaker potential based on the voltage clamp experiments.

The pacemaker activity of the S-A node cell was explained by reconstructing the pacemaker potential using a Hodgkin-Huxley type mathematical model which was based on the reported voltage clamp data. In this model four dynamic currents, the sodium current iNa, the slow inward current, is, the potassium current, iK, and the hyperpolarization-activated current, ih, in addition to a time-dependent leak current, i1 were included. The model simulated the spontaneous action potential the current voltage relationship, and the voltage clamp experiment in normal Tyrode solution of the rabbit S-A node. Furthermore, the changes of activity induced by the potassium current blocker Ba2+, by applying constant current, acetylcholine, and epinephrine were also reconstructed. It was strongly suggested that the pacemaker depolarization in the S-A node cell is mainly due to a gradual increase of iS during diastole, and that the contribution of iK is much less compared to the potassium current iK2 in the Purkinje fiber pacemaker depolarization. The rising phase of the action potential was due to iS and the plateau phase is determined by both the inactivation of iS and activation of iK.

Resting K conductances in pacemaker and non-pacemaker heart cells of the rabbit.

Currents through the inward rectifier K channel (iK X rec) and the ACh-operated K channel (iK X ACh) were recorded in isolated heart cells of rabbit using the patch clamp technique with electrodes having 0.5-1 micron tip inner diameter. The maximum number of overlaps of open iK X rec channels per patch was measured over 347 experiments. An average of 2.3 was found in ventricular cells and 0.03-0.06 in sinoatrial (S-A) and atrioventricular (A-V) node cells. The estimated total number of the iK X rec channels for each ventricular cell was great enough to supply the resting K conductance of the cell. The iK X ACh channel was present in S-A and A-V node cells, but was never observed in the ventricular cells. The resting conductance of the nodal cells measured with whole cell clamp recordings was about 16 times smaller than that of the ventricular cells, and was hardly decreased at all by the removal of K+ from the bath solution. Thus, the lower membrane potential of the nodal cells compared with that of the ventricular cells was attributed to the smaller K conductance of the resting membrane, which is due to the very low density of the iK X rec channel. On the other hand, the iK X ACh channel, when activated by neural regulation, may play a major role in generating the resting K conductance of the nodal cells.

The spontaneous action potential of rabbit atrioventricular node cells.

The rabbit atrioventricular (A-V) node, with dimensions of approximately 5 X 3 mm was dissected into 15 small specimens (0.5 X 0.5 mm). A majority of the specimens continued to discharge spontaneous action potentials, the action potential configuration being almost identical in different specimens. The amplitude (98 mV) and the maximum rate of rise of the action potential (11 V/sec) were similar to those recorded from the intact A-V node (amplitude 98 mV, maximum rate of rise of action potential 12 V/sec). In these small specimens, the "resting membrane potential (-44 mV)" was approximately 20 mV less negative than that in the intact A-V node preparation (-62 mV) before the dissecting procedure. The spontaneous discharge in these small A-V node specimens was attributed to the low resting membrane potential. The small specimen became inexcitable under the effects of blockers of is, though TTX had no significant effect on the action potential. The after-hyperpolarization was observed after cessation of the depolarizing current pulse. It is concluded that the slow inward current and the slow kinetics of the outward current contribute to the generation of the spontaneous A-V node action potentials as in the sinoatrial node.

Kinetics and rectification of the slow inward current in the rabbit sinoatrial node cell.

The slow inward current (is) in the rabbit sinoatrial node cell was studied by the conventional two-microelectrode voltage clamp technique. When is was measured as the difference between two records obtained before and after blocking is with D 600, the fully activated current (is)-voltage relation was non-linear; the conductance decreased in the negative potential range resulting in an almost constant amplitude of is negative to -10 mV. The degree of steady-state activation was about 1 at -5mV and 0 at -65mV. The recovery time course of is during repolarization was measured by varying the interval between two sequential depolorizing pulses with various holding potentials. The time constant of the exponential recovery time course was about 120 msec at -40 mV and decreased to about 40 msec at -70 mV. The steady-state conductance of is, calculated from the activation and inactivation curves, produced a large hump in the steady-state current voltage relation between -60 and -20 mV, which was not observed in the experiment. When the above kinetics were incorporated, the S-A node model failed to discharge the spontaneous activity. The activation and inactivation curves which can simulate the experimental I-V curve and the action potential were proposed.

Spontaneously active cells isolated from the sino-atrial and atrio-ventricular nodes of the rabbit heart.

Single cells or cell clusters composed of 3-10 cells were isolated from the S-A and A-V nodes of the rabbit heart by coronary perfusion of collagenase dissolved in Ca-free Tyrode solution (0.04%, for 1 hr). For comparison, atrial and ventricular cells were also isolated from the same heart. Shapes of the isolated nodal cell were either rod or round and nodal cells were slightly smaller than ventricular cells. Spontaneous activity was observed in both rod and round nodal cells. The action potentials had the configurations similar to those recorded from larger conventional preparations. The membrane current recorded from the small nodal cell clusters (5-10 cells) by the two-microelectrode voltage clamp technique showed a time course similar to that of previous recordings from conventional preparations, but the amplitude of the currents was 5-10 times smaller. The isolated cells showed normal sensitivities to both acetylcholine and epinephrine. Findings given in this study indicate that the isolated cells maintain the typical membrane characteristics of the nodal cells and that they are suitable for electrophysiological studies of the cardiac pacemaker cell.

Effect of hemolysin produced by Vibrio parahaemolyticus on membrane conductance and mechanical tension of rabbit myocardium.

The mechanisms of the action of hemolysin extracted from Vibrio parahaemolyticus in the S-A node and right atrium cells of rabbit were studied by means of the single sucrose gap and isometric tension recording methods. Hemolysin caused the membrane to depolarize reversibly without affecting the action potential generating mechanism. Lowering of [Na+]o inhibited membrane depolarization in the presence of hemolysin while the readmission of normal Tyrode solution induced depolarization. Tetrodotoxin (TTX) barely antagonized the depolarizing action of hemolysin but slowed the rate of development of depolarization. Therefore, this depolarization is considered to be primarily due to the increase in conductance to Na which TTX may not block. The dose-response relationship was obtained by measuring a change in membrane resistance. The concentration necessary to yield one-half of the maximum reduction of the membrane was determined to by 7.5 micrograms/ml. Accumulation of Na within the cell may be responsible for an increase of twitch tension observed during the action of a low concentration of hemolysin. On the other hand, a higher concentration of hemolysin seemed to promote exchange of intracellar Na with extracellular Ca, especially when the Na concentration of the perfusing solution was reduced, and led to stronger contracture.

Automated microanalysis of creatinine by coupled enzyme reactions.

Hydrogen peroxide was generated from creatinine by the sequential enzyme reactions of creatinine amidohydrolase, creatine amidinohydrolase and sarcosine oxidase. Hydrogen peroxide was, in turn, used stoichiometrically for the condensation of 4-aminoantipyrine and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine catalyzed by horse-radish peroxidase, resulting in the formation of quinone dye with maximum absorbance at 546 nm. The optimized assay conditions for the enzymatic determination of creatinine in a HITACHI 7250 autoanalyzer was established. This system, which requires less than 5 microliters of sample, was found to be the most economical for laboratories equipped with autoanalyzers.