Structural Basis of the Modulation of the Voltage-Gated Calcium Ion Channel Cav 1.1 by Dihydropyridine Compounds*.

1,4-Dihydropyridines (DHP), the most commonly used antihypertensives, function by inhibiting the L-type voltage-gated Ca2+ (Cav ) channels. DHP compounds exhibit chirality-specific antagonistic or agonistic effects. The structure of rabbit Cav 1.1 bound to an achiral drug nifedipine reveals the general binding mode for DHP drugs, but the molecular basis for chiral specificity remained elusive. Herein, we report five cryo-EM structures of nanodisc-embedded Cav 1.1 in the presence of the bestselling drug amlodipine, a DHP antagonist (R)-(+)-Bay K8644, and a titration of its agonistic enantiomer (S)-(-)-Bay K8644 at resolutions of 2.9-3.4 Å. The amlodipine-bound structure reveals the molecular basis for the high efficacy of the drug. All structures with the addition of the Bay K8644 enantiomers exhibit similar inactivated conformations, suggesting that (S)-(-)-Bay K8644, when acting as an agonist, is insufficient to lock the activated state of the channel for a prolonged duration.

Cav1.2 channel current block by the PKA inhibitor H-89 in rat tail artery myocytes via a PKA-independent mechanism: Electrophysiological, functional, and molecular docking studies.

To characterize the role of cAMP-dependent protein kinase (PKA) in regulating vascular Ca2+ current through Cav1.2 channels [ICa1.2], we have documented a marked capacity of the isoquinoline H-89, widely used as a PKA inhibitor, to reduce current amplitude. We hypothesized that the ICa1.2 inhibitory activity of H-89 was mediated by mechanisms unrelated to PKA inhibition. To support this, an in-depth analysis of H-89 vascular effects on both ICa1.2 and contractility was undertaken by performing whole-cell patch-clamp recordings and functional experiments in rat tail main artery single myocytes and rings, respectively. H-89 inhibited ICa1.2 with a pIC50 (M) value of about 5.5, even under conditions where PKA activity was either abolished by both the PKA antagonists KT5720 and protein kinase inhibitor fragment 6-22 amide or enhanced by the PKA stimulators 6-Bnz-cAMP and 8-Br-cAMP. Inhibition of ICa1.2 by H-89 appeared almost irreversible upon washout, was charge carrier- and voltage-dependent, and antagonised by the Cav1.2 channel agonist (S)-(-)-Bay K 8644. H-89 did not alter both potency and efficacy of verapamil, did not affect current kinetics or voltage-dependent activation, while shifting to the left the 50% voltage of inactivation in a concentration-dependent manner. H-89 docked at the α1C subunit in a pocket region close to that of (S)-(-)-Bay K 8644 docking, forming a hydrogen bond with the same, key amino acid residue Tyr-1489. Finally, both high K+- and (S)-(-)-Bay K 8644-induced contractions of rings were fully reverted by H-89. In conclusion, these results indicate that H-89 inhibited vascular ICa1.2 and, consequently, the contractile function through a PKA-independent mechanism. Therefore, caution is recommended when interpreting experiments where H-89 is used to inhibit vascular smooth muscle PKA.

Modified cytoplasmic Ca2+ sequestration contributes to spinal cord injury-induced augmentation of nerve-evoked contractions in the rat tail artery.

In rat tail artery (RTA), spinal cord injury (SCI) increases nerve-evoked contractions and the contribution of L-type Ca2+ channels to these responses. In RTAs from unoperated rats, these channels play a minor role in contractions and Bay K8644 (L-type channel agonist) mimics the effects of SCI. Here we investigated the mechanisms underlying the facilitatory actions of SCI and Bay K8644 on nerve-evoked contractions of RTAs and the hypothesis that Ca2+ entering via L-type Ca2+ channels is rapidly sequestered by the sarcoplasmic reticulum (SR) limiting its role in contraction. In situ electrochemical detection of noradrenaline was used to assess if Bay K8644 increased noradrenaline release. Perforated patch recordings were used to assess if SCI changed the Ca2+ current recorded in RTA myocytes. Wire myography was used to assess if SCI modified the effects of Bay K8644 and of interrupting SR Ca2+ uptake on nerve-evoked contractions. Bay K8644 did not change noradrenaline-induced oxidation currents. Neither the size nor gating of Ca2+ currents differed between myocytes from sham-operated (control) and SCI rats. Bay K8644 increased nerve-evoked contractions in RTAs from both control and SCI rats, but the magnitude of this effect was reduced by SCI. By contrast, depleting SR Ca2+ stores with ryanodine or cyclopiazonic acid selectively increased nerve-evoked contractions in control RTAs. Cyclopiazonic acid also selectively increased the blockade of these responses by nifedipine (L-type channel blocker) in control RTAs, whereas ryanodine increased the blockade produced by nifedipine in both groups of RTAs. These findings suggest that Ca2+ entering via L-type channels is normally rapidly sequestered limiting its access to the contractile mechanism. Furthermore, the findings suggest SCI reduces the role of this mechanism.

Antagonistic actions of S(-)-Bay K 8644 on cyclic nucleotide-induced inhibition of voltage-dependent Ba(2+) currents in guinea pig gastric antrum.

(+/-)-Bay K 8644, a conventional racemic mixture of Bay K 8644, is widely used as an L-type Ca(2+) channel agonist. Although interactions between Bay K 8644 and cyclic nucleotide have been described, they have not been properly characterized. We have investigated whether two optical isomers of Bay K 8644 (i.e., R(+)- and S(-)-Bay K 8644) modify cyclic nucleotide (cAMP and cGMP)-induced inhibitory effects on nifedipine-sensitive voltage-dependent Ba(2+) currents (I (Ba)) recorded from guinea pig gastric myocytes. Conventional whole-cell recordings were used to compare the effects of R(+)-Bay K 8644 and S(-)-Bay K 8644 on I (Ba). S(-)-Bay K 8644 enhanced the peak amplitude of I (Ba) evoked by depolarizing pulses to +10 mV from a holding potential of -70 mV in a concentration-dependent manner (EC(50) = 32 nM), while R(+)-Bay K 8644 inhibited I (Ba) (IC(50) = 975 nM). When R(+)-Bay K 8644 (0.5 microM) was applied, I (Ba) was suppressed to 71 +/- 10% of control. In the presence of R(+)-Bay K 8644 (0.5 microM), additional application of forskolin and sodium nitroprusside (SNP) further inhibited I (Ba). Conversely, in the presence of S(-)-Bay K 8644 (0.5 microM), subsequent application of forskolin and SNP did not affect I (Ba). Similarly, in the presence of 0.5 microM S(-)-Bay K 8644, db-cAMP and 8-Br-cGMP had no effect on I (Ba). These results indicate that S(-)-Bay K 8644, but not R(+)-Bay K 8644, can prevent the inhibitory actions of two distinct cyclic nucleotide pathways on I (Ba) in gastric myocytes of the guinea pig antrum.

Using dihydropyridine-release strategies to enhance load effects in engineered human bone constructs.

We report on the development of a novel biodegradable scaffold capable of enhancing mechanical signals for tissue-engineering applications. It has been shown that mechanotransduction enhances bone formation in vitro and in vivo; in tissue-engineering applications, this phenomenon is exploited through the use of mechanical bioreactors to generate bone tissue. The dihydropyridine agonist Bay K8644 (Bay) acts to increase the opening time of mechanosensitive voltage-operated calcium channels (VOCCs), specifi- cally L-type VOCCs, which are known to play a fundamental role in the early mediation of mechanotransduction. We have produced porous 3-dimensional, Bay-encapsulated biodegradable poly(L-lactide) acid scaffolds using a solvent-casting and salt-leaching technique. The effects of the released Bay on osteoid production and mineralization in human bone cell-seeded constructs following incubation in a perfusion-compression bioreactor in vitro was investigated using Western blotting techniques and a calcium assay protocol developed in our lab. Our newly developed scaffolds act by slowly releasing the calcium channel agonist Bay K8644 as observed using ultraviolet spectroscopy, maintaining the open state of mechanosensitive VOCCs responding to load, which augments the load signal at sites of strain across the scaffold. Our results demonstrate that, in the presence of physiological loading regimes in vitro, release of Bay enhances collagen I protein production and osteoid calcification more than non-Bay control constructs do. Osteopontin and alpha2delta1 VOCC subunit protein levels were also higher as a result of perfusion-compression conditioning. These results indicate that Bay-encapsulated scaffolds can be used in the presence of load to enhance the production of load-bearing engineered tissue.

Characterizing the efficacy of calcium channel agonist-release strategies for bone tissue engineering applications.

We have previously reported on the use of Bay K8644-release strategies in combination with perfusion-compression bioreactor systems for up regulating bone formation in three-dimensional PLLA scaffolds. Here we report on the analysis of Bay activity following its release from our PLLA scaffolds over the culture period imposed in our tissue engineering protocol using UV spectroscopy in combination with whole cell patch clamping techniques. Bay was released continually from scaffolds within the physiological range required for agonist activity (1-10 microM). Patch clamping allowed for the effects of Bay released from scaffolds to be monitored directly with respect to osteoblast electrophysiology. A characteristic shift in the current-voltage (I-V) relationship of L-type VOCC currents was observed in rat osteoblast sarcoma (ROS) cells patched in a solution with Bay released from scaffolds following 14 and 28 days incubation, with statistically significant differences observed in peak currents compared to non-Bay controls. An increase in the magnitude of the peak inward currents was also noted. The electrophysiological response of osteoblasts in the presence of Bay released from scaffolds demonstrates that the released Bay is stable and maintains its bioactivity following culture of up to 28 days.

Optimising the EVA descriptor for prediction of biological activity.

EVA is a multivariate molecular descriptor for use in QSAR studies. It is constructed from vibrational eigenvalues derived from either a quantum theoretical or molecular mechanical treatment of molecular structure. This paper applies the method to biological-activity data using measures of the inotropic potential of a range of Calcium channel agonists. The performance of the descriptor, as both an explanatory and a predictive tool, is analysed in relation to the way in which it is constructed using a rigorous statistical treatment. Its capabilities are examined in relation to those of previously published methodology which used a composite descriptor. It is shown to have improved performance and several procedural advantages, such as ease of calculation and operation. It is a 3-D structural descriptor which does not require prior co-alignment of structures for a QSAR study.

Effects of the enantiomers of BayK 8644 on the charge movement of L-type Ca channels in guinea-pig ventricular myocytes.

The effects of the agonist enantiomer S(-)Bay K 8644 on gating charge of L-type Ca channels were studied in single ventricular myocytes. From a holding potential (Vh) of -40 mV, saturating (250 nm) S(-)Bay K shifted the half-distribution voltage of the activation charge (Q1) vs. V curve -7.5 +/- 0.8 mV, almost identical to the shift produced in the Ba conductance vs. V curve (-7.7 +/- 2 mV). The maximum Q1 was reduced by 1.7 +/- 0.2 nC/microF, whereas Q2 (charge moved in inactivated channels) was increased in a similar amount (1.4 +/- 0.4 nC/microF). The steady-state availability curves for Q1, Q2, and Ba current showed almost identical negative shifts of -14.8 +/- 1.7 mV, -18.6 +/- 5.8 mV, and -15.2 +/- 2.7 mV, respectively. The effects of the antagonist enantiomer R(+)BayK 8644 were also studied, the Q1 vs. V curve was not significantly shifted, but Q1max (Vh = -40 mV) was reduced and the Q1 availability curve shifted by -24.6 +/- 1.2 mV. We concluded that: a) the left shift in the Q1 vs. V activation curve produced by S(-)BayK is a purely agonistic effect; b) S(-)BayK induced a significantly larger negative shift in the availability curve than in the Q1 vs. V relation, consistent with a direct promotion of inactivation; c) as expected for a more potent antagonist, R(+)Bay K induced a significantly larger negative shift in the availability curve than did S(-)Bay K.

Development of a 'mechano-active' scaffold for tissue engineering.

In this study. we investigate the potential for manipulating bone cell mechanotransducers in tissue engineering. Membrane ion channels such as voltage operated calcium channels (VOCC) have been shown to be a critical component of the bone cell transduction pathway with agonists and inhibitors of this pathway having profound effects on the load signal. By encapsulating a calcium channel agonist with slow release within a poly(L-lactide) (PLLA) scaffold, we can generate a 'mechano-active' scaffold for use in skeletal tissue engineering. PLLA scaffolds with and without a calcium channel agonist, BAY K8644, were seeded with primary human bone cells or the human MG63 bone cell line and cultured for 13 weeks followed by mechanical stimulation with a four-point bending model. Our results show that addition of the agonist for slow release is sufficient to enhance the load-related responses in bone cells within the scaffolds. Specifically, collagen type I expression and the ratio of alkaline phosphatase to protein are elevated in response to cyclical mechanical stimulation of approximately 1000 microstr which is then further enhanced in the mechano-active' scaffolds. As the agonists only act when the calcium channels are open by attenuating the calcium flux, the stimulation is specifically targeted to scaffolds subjected to load either in vitro or ultimately in vivo. Our results suggest that manipulating the VOCC and attenuating the opening of the calcium channels may be an effective technique to amplify matrix production via mechanical stimulation which may be applied to bone tissue engineering and potentially engineering of other load-bearing connective tissues.

[Antiradical activity and membranotropic action of cardio- and vasotropic compounds of varying chemical nature]. / Antiradikal'naia aktivnost; i membranotropnoe deistvie kardio- i vazotropnykh soedinenii razlichnoi khimicheskoi prirody.

Antiradical activity and ability to interaction with phosphatidylcholine bilayer of the physiological active compounds (PhAC), concerning the classes of phenylalkilamines (dobutamine, verapamil), dihydro pyridines (BAY-K-4688, nifedipine), analogues of crown ether (carbicyle, diol) was studied. By means of the method of microcalorimetry and spectrophotometry it was shown the complexing ability PhAC with the phospholipid bilayer of the model membrane. It was stated the simbasity in the changes of the thermal effect of the compounds with the negative inotropic activity during its reaction with phosphatidylcholine bilayer and antiradical activity. That show on the presence of antiradical component in the mechanism that compounds' action.

Allosteric interactions at L-type calcium channels between FPL 64176 and the enantiomers of the dihydropyridine Bay K 8644.

Functional interactions between the enantiomers of the dihydropyridine 1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)-phenyl]-3-pyridi ne carboxylic acid methyl ester (Bay K 8644) and the benzoylpyrrole methyl 2,5-dimethyl-4-[2(phenylmethyl)benzoyl]-H-pyrrole-3-carboxylate (FPL 64176) were investigated on L-type Ca++ channels in guinea pig ileal longitudinal smooth muscle. The effects of these drugs, when applied individually, were as described in earlier studies. For instance, both (-)-(S)-Bay K 8644 and FPL 64176 caused concentration-dependent contraction, which is consistent with Ca++ channel activation, whereas (+)-(R)-Bay K 8644 gave concentration-dependent relaxation, which is consistent with Ca++ channel inhibition. The activities of the different drugs were dependent on the extracellular levels of KCI. When applied in combination, however, the responses evoked were not those predicted from the effects of the drugs applied individually. Contractions produced by FPL 64176 (25 nM to 1 microM) were abolished in the presence of 100 nM (-)-(S)-Bay K 8644 but were potentiated by 10 to 150 nM (+)-(R)-Bay K 8644 and inhibited by 1 microM (+)-(R)-Bay K 8644. Conversely, contractile responses to (-)-(S)-Bay K 8644 were abolished by 100 nM FPL 64176. In the presence of 1 microM FPL 64176, however, (-)-(S)-Bay K 8644 gave concentration-dependent relaxation of the muscle, which is consistent with Ca++ channel inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

The long-term down-regulation of dihydropyridine receptors by Bay K 8644 in PC12 cells.

Treatment of PC12 cells with Bay K 8644 for 12 hr or more leads to an almost 80% decrease in the subsequent ability of Bay K 8644 to stimulate the uptake of radioactive calcium into the cells. This effect is a property of the S(-)isomer of Bay K 8644; pre-treatment with the R(+)isomer, now known to be a calcium channel blocker, has the opposite effect. This treatment is specific in that it does not interfere with the stimulation of calcium uptake by potassium, ATP, or nerve growth factor. Such treatment is accompanied by a 90% decrease in the ability of Bay K 8644 to stimulate the release of norepinephrine. The characteristics of the binding of [3H]isradipine to control and to treated cells indicates that the decrease in the effect of dihydropyridines is accompanied by a marked decrease in the number of dihydropyridine binding sites with no apparent change in the affinity of the remaining sites. The continued ability of depolarizing levels of potassium to stimulate calcium uptake and the induction of the protooncogene c-fos in Bay K 8644-treated cells indicates that the L-type calcium channels are still intact, but are simply unresponsive to dihydropyridine agonists.

Photochemical generation of nitric oxide from nitro-containing compounds: possible relation to vascular photorelaxation phenomena.

We examined the production of nitric oxide (NO) from various nitro-containing compounds (500 microM and 100 microM solutions in Krebs buffer, pH 7.4). Sealed vials containing solutions of NaNO2, N-nitro-L-arginine, 4-nitrophenol, BAY K 8644, or N-nitro-L-arginine methyl ester were stored in the dark, under normal room light, or were exposed to ultraviolet light (365 nm), for 30 min (24 degrees C). NO was measured in the vial headspace after 30 min, using a sensitive assay previously established in our laboratory. Production of NO was found to be dependent on the intensity of light exposure for all compounds, and the highest degree of light-induced production of NO was found for NaNO2 and BAY K 8644 solutions. Since NO is a relaxant of smooth muscle, these results help explain the increased sensitivity to relaxation by UV light of vascular and other types of smooth muscle in the presence of NaNO2, BAY K 8644 and N-nitro-L-arginine, as observed by other investigators.

(+)-isradipine but not (-)-Bay-K-8644 exhibits voltage-dependent effects on cat adrenal catecholamine release.

1. Catecholamine release from cat adrenal glands perfused at a high rate (4 ml min-1) at 37 degrees C with polarizing (1.2 or 5.9 mM K+) or depolarizing (17.7, 35, 59 or 118 mM K+) solutions, was triggered by 5 or 10 s pulses of Ca2+ (0.5 or 2.5 mM) in the presence of various concentrations of K+. 2. In polarized glands, secretion was greater the higher the K+ concentration present during the secretory K+/Ca2+ test pulse. Thus, in 17.7 mM K+, catecholamine released was 162 +/- 27 ng per pulse, while in 118 mM K+ secretion rose to 1839 +/- 98 ng per pulse. In depolarized glands, secretion reached a peak of around 1000 ng per pulse in 35-59 mM K+; in 118 mM K+, secretion did not increase further, suggesting that voltage changes are implicated in the control of the secretory process. 3. Blockade of secretion by increased concentrations of (+)-isradipine was much more manifest in polarized glands. The higher the degree of depolarization was (35, 59 or 118 mM K+), the lower the IC50 s were. So, the ratios between the IC50 s in polarized and depolarized glands rose from 3.92 in 35 mM K+ to 26.7 in 118 mM K+. 4. In contrast, the Ca2+ channel activator (-)-Bay K 8644 potentiated catecholamine release evoked by K+/Ca2+ pulses equally well in polarized or depolarized glands. The ratios between EC50 s in polarized or depolarized glands were, respectively, 0.30, 0.59 and 0.69 for 17.7, 35 and 118 mM K+. 5. In simultaneous experiments, the two enantiomers of Bay K 8644 exhibited opposite effects on secretion. (+)-Bay K 8644 (a Ca21 channel blocker) inhibited secretion better in depolarized than in polarized glands, whilst (-)-Bay K 8644 potentiated secretion in a voltage-independent manner. 6. Potentiation of secretion by (-)-Bay K 8644 was concentration-dependent from 10-8 to 10-6M. At 10- 5M, such potentiation largely disappeared in both polarized and depolarized glands. However, this dual effect of (-)Bay K 8644 was better seen in depolarizing conditions, suggesting that using the same enantiomer, the voltage-dependence is only seen when blockade of secretion dominates. 7. In the presence of increasing concentrations of (-)Bay K 8644 (3 x 10-9, 3 x 10-8 and 3 x 10-7M), the concentration-response curves for (+)isradipine to inhibit secretion were displaced to the right. However, a Schild plot of (dose ratio - 1) against (-)-Bay K 8644 concentrations gave a slope of 0.6, suggesting that the interactions between (+)-isradipine and (-)Bay K 8644 were non-competitive in nature. The pA2 for (-)-Bay K 8644 was 9.13. 8. Overall, the results suggest that potentiation of secretion by (-)Bay K 8644 (a voltage-independent phenomenon), and blockade by (+)-isradipine or (+-Bay K 8644 (a voltage-dependent phenomenon) might be exerted through binding of the dihydropyridines activators and blockers to separate sites on chromaffin cell L-type Ca2 + channels.