Serotonin (5-HT)2A/2C receptor agonist 2,5-dimethoxy-4-iodophenyl-2-aminopropane hydrochloride improves detrusor sphincter dyssynergia by inhibiting L-type voltage-gated calcium channels in spinal cord injured rats.

AIMS: To explore the role of voltage-gated calcium channels (VGCC) in 5-HT2A/2C receptor agonist 2,5-dimethoxy-4-iodophenyl-2-aminopropane hydrochloride's improvement of spinal cord injury (SCI) induced detrusor sphincter dyssynergia and the expressions of the 5-hydroxy tryptamine (5-HT) 2A receptors and VGCCs in lumbosacral cord after SCI. METHODS: Female Sprague-Dawley rats were randomized into normal control group and SCI group (N = 15 each). Cystometrogram (CMG), simultaneous CMG, and external urethral sphincter electromyography (EUS-EMG) were conducted in all groups under urethane anesthesia. Drugs were administered intrathecally during CMG and EUS-EMG. Rats were euthanized and L6-S1 spinal cord were acquired for immunofluorescence. RESULTS: In SCI rats, intrathecal administration of 2,5-dimethoxy-4-iodophenyl-2-aminopropane hydrochloride or L-type VGCC blocker, nifedipine, could significantly increase voiding volume, voiding efficiency, and the number of high-frequency oscillations. They could also prolong EUS bursting activity duration on EUS-EMG. Moreover, the effect of 2,5-dimethoxy-4-iodophenyl-2-aminopropane hydrochloride can be eliminated with the combined administration of L-type VGCC agonist, (±)-Bay K 8644. No significant differences were observed in CMG after intrathecal administration of T-type VGCC blocker TTA-P2. Additionally, immunofluorescence of the lumbosacral cord in control and SCI rats showed that the 5-HT2A receptor and Cav1.2 immunolabeling-positive neurons in the anterior horn of the lumbosacral cord were increased in SCI rats. CONCLUSIONS: Our study demonstrated that 5-HT2A/2C agonist 2,5-dimethoxy-4-iodophenyl-2-aminopropane hydrochloride may improve SCI-induced DSD by inhibiting the L-type voltage-gated calcium channel in lumbosacral cord motoneurons.

Differential Downregulation of ß1-Adrenergic Receptor Signaling in the Heart.

BACKGROUND: Chronic sympathetic stimulation drives desensitization and downregulation of ß1 adrenergic receptor (ß1AR) in heart failure. We aim to explore the differential downregulation subcellular pools of ß1AR signaling in the heart. METHODS AND RESULTS: We applied chronic infusion of isoproterenol to induced cardiomyopathy in male C57BL/6J mice. We applied confocal and proximity ligation assay to examine ß1AR association with L-type calcium channel, ryanodine receptor 2, and SERCA2a ((Sarco)endoplasmic reticulum calcium ATPase 2a) and Förster resonance energy transfer-based biosensors to probe subcellular ß1AR-PKA (protein kinase A) signaling in ventricular myocytes. Chronic infusion of isoproterenol led to reduced ß1AR protein levels, receptor association with L-type calcium channel and ryanodine receptor 2 measured by proximity ligation (puncta/cell, 29.65 saline versus 14.17 isoproterenol, P<0.05), and receptor-induced PKA signaling at the plasma membrane (Förster resonance energy transfer, 28.9% saline versus 1.9% isoproterenol, P<0.05) and ryanodine receptor 2 complex (Förster resonance energy transfer, 30.2% saline versus 10.6% isoproterenol, P<0.05). However, the ß1AR association with SERCA2a was enhanced (puncta/cell, 51.4 saline versus 87.5 isoproterenol, P<0.05), and the receptor signal was minimally affected. The isoproterenol-infused hearts displayed decreased PDE4D (phosphodiesterase 4D) and PDE3A and increased PDE2A, PDE4A, and PDE4B protein levels. We observed a reduced role of PDE4 and enhanced roles of PDE2 and PDE3 on the ß1AR-PKA activity at the ryanodine receptor 2 complexes and myocyte shortening. Despite the enhanced ß1AR association with SERCA2a, the endogenous norepinephrine-induced signaling was reduced at the SERCA2a complexes. Inhibiting monoamine oxidase A rescued the norepinephrine-induced PKA signaling at the SERCA2a and myocyte shortening. CONCLUSIONS: This study reveals distinct mechanisms for the downregulation of subcellular ß1AR signaling in the heart under chronic adrenergic stimulation.

CardioDPi: An explainable deep-learning model for identifying cardiotoxic chemicals targeting hERG, Cav1.2, and Nav1.5 channels.

The cardiotoxic effects of various pollutants have been a growing concern in environmental and material science. These effects encompass arrhythmias, myocardial injury, cardiac insufficiency, and pericardial inflammation. Compounds such as organic solvents and air pollutants disrupt the potassium, sodium, and calcium ion channels cardiac cell membranes, leading to the dysregulation of cardiac function. However, current cardiotoxicity models have disadvantages of incomplete data, ion channels, interpretability issues, and inability of toxic structure visualization. Herein, an interpretable deep-learning model known as CardioDPi was developed, which is capable of discriminating cardiotoxicity induced by the human Ether-à-go-go-related gene (hERG) channel, sodium channel (Na_v1.5), and calcium channel (Ca_v1.5) blockade. External validation yielded promising area under the ROC curve (AUC) values of 0.89, 0.89, and 0.94 for the hERG, Na_v1.5, and Ca_v1.5 channels, respectively. The CardioDPi can be freely accessed on the web server CardioDPipredictor (http://cardiodpi.sapredictor.cn/). Furthermore, the structural characteristics of cardiotoxic compounds were analyzed and structural alerts (SAs) can be extracted using the user-friendly CardioDPi-SAdetector web service (http://cardiosa.sapredictor.cn/). CardioDPi is a valuable tool for identifying cardiotoxic chemicals that are environmental and health risks. Moreover, the SA system provides essential insights for mode-of-action studies concerning cardiotoxic compounds.

Impact of quetiapine on ion channels and contractile dynamics in rat ventricular myocyte.

Antipsychotic drugs often lead to adverse effects, including those related to the cardiovascular system. Of these, quetiapine is known to cause significant changes in the QT interval although the underlying mechanism remains mysterious, prompting us to examine its effects on cardiac electrophysiological properties. Therefore, we investigated the effect of quetiapine on contraction, action potential (AP), and the associated membrane currents such as L-type Ca2+ and K+ using the whole-cell patch clamp method to examine its impacts on isolated rat ventricular myocytes. Our results showed that (1) quetiapine reduces cell contractility in a concentration-dependent manner and (2) leads to a significant prolongation in the duration of AP in isolated ventricular myocytes. This effect was both concentration and frequency-dependent; (3) quetiapine significantly decreased the Ca2+, transient outward K+, and steady-state K+ currents. However, only high concentration of quetiapine (100 µM) could significantly change the activation and reactivation kinetics of L-type Ca2+ channels. This study demonstrates that QT extension induced by quetiapine is mainly associated with the prolongation of AP. Moreover, quetiapine caused a significant decrease in contractile force and excitability of ventricular myocytes by suppressing Ca2+ and K+ currents.

Triclosan induces earlier puberty onset in female mice via interfering with L-type calcium channels and activating Pik3cd.

Triclosan (TCS) is a broad-spectrum antibacterial chemical widely presents in people's daily lives. Epidemiological studies have revealed that TCS exposure may affect female puberty development. However, the developmental toxicity after low-dose TCS continuous exposure remains to be confirmed. In our study, 8-week-old ICR female mice were continuously exposed to TCS (30, 300, 3000 µg/kg/day) or vehicle (corn oil) from 2 weeks before mating to postnatal day 21 (PND 21) of F1 female mice, while F1 female mice were treated with TCS intragastric administration from PND 22 until PND 56. Vaginal opening (VO) observation, hypothalamic-pituitary-ovarian (HPO) axis related hormones and genes detection, and ovarian transcriptome analysis were carried out to investigate the effects of TCS exposure on puberty onset. Meanwhile, human granulosa-like tumor cell lines (KGN cells) were exposed to TCS to further explore the biological mechanism of the ovary in vitro. The results showed that long-term exposure to low-dose TCS led to approximately a 3-day earlier puberty onset in F1 female mice. Moreover, TCS up-regulated the secretion of estradiol (E2) and the expression of ovarian steroidogenesis genes. Notably, ovarian transcriptomes analysis as well as bidirectional validation in KGN cells suggested that L-type calcium channels and Pik3cd were involved in TCS-induced up-regulation of ovarian-related hormones and genes. In conclusion, our study demonstrated that TCS interfered with L-type calcium channels and activated Pik3cd to up-regulate the expression of ovarian steroidogenesis and related genes, thereby inducing the earlier puberty onset in F1 female mice.

Vascular relaxing effect of Hydrocotyle umbellata L. is mediated by blocking of l-type Ca2+ channels.

ETHNOPHARMACOLOGICAL RELEVANCE: Hydrocotyle umbellata L. is a medicinal herb for the treatment of some health problems including hypertension, according to traditional medicine. Even so, its vascular effects and the pharmacological action mechanisms have not been analyzed. AIM OF THE STUDY: This experiment aimed to analyze the effects of hydroalcoholic extract of Hydrocotyle umbellata L. (HEHU) on isolated vessels and verify the interaction of hibalactone (chemical marker) against Cav1.2 channels using molecular docking. MATERIALS AND METHODS: Vascular reactivity experiments were performed using rat aortas with (E+) or without endothelium (E-) in an isolated organ bath. Computational molecular docking approaches were used to show the direct effect on L-type Ca2+ Channels. RESULTS: HEHU (0-560 µg/mL) induced relaxation of the pre-contracted arteries in a concentration-dependent manner. The maximum effect was higher in E+ (76.8 ± 4.1%) as compared to E- (47.3 ± 5.5%). Pre-treatment of E+ arteries with L-NAME or ODQ reduced the relaxation to similar level of E- arteries. The treatment of arteries with MDL-12,330 A, diclofenac, propranolol and atropine did not change the relaxation induced by HEHU. The contraction caused by internal Ca2+ release induced by caffeine was reduced after HEHU treatment. Moreover, the HEHU also impaired the contraction induced by Ca2+ influx stimulated with phenylephrine or high KCl. The docking study demonstrated the effectiveness of hibalactone in blocking the Cav1.2 channel. CONCLUSIONS: These findings show that HEHU induces vascular relaxation which is potentiated (but not dependent) by endothelial cells. Blocking of Ca2+ influx seems to be the main mechanism for the vascular effects of HEHU.

Calenduloside E protects against myocardial ischemia-reperfusion injury induced calcium overload by enhancing autophagy and inhibiting L-type Ca2+ channels through BAG3.

Calenduloside E (CE) is a saponin isolated from Aralia elata (Miq) Seem, which has anti-cardiovascular disease effects. This study aims to evaluate the anti-myocardial ischemia-reperfusion injury (MIRI) mechanisms of CE and regulation of BAG3 on calcium overload. We adopted siRNA to interfere with BAG3 expression in H9c2 cardiomyocytes and used adenovirus to interfere with BAG3 expression (Ad-BAG3) in primary neonatal rat cardiomyocytes (PNRCMs) to clarify the role of BAG3 in mitigating MIRI by CE. The results showed that CE reduced calcium overload, and Ad-BAG3 had a significant regulatory effect on L-type Ca2+ channels (LTCC) but no effects on other calcium-related proteins. And BAG3 and LTCC were colocalized in myocardial tissue and BAG3 inhibited LTCC expression. Surprisingly, CE had no regulatory effect on LTCC mRNA, but CE promoted LTCC degradation through the autophagy-lysosomal pathway rather than the ubiquitination-protease pathway. Autophagy inhibitor played a negative regulation of cardiomyocyte contraction rhythm and field potential signals. Ad-BAG3 inhibited autophagy by regulating the expression of autophagy-related proteins and autophagy agonist treatment suppressed calcium overload. Therefore, CE promoted autophagy through BAG3, thereby regulating LTCC expression, inhibiting calcium overload, and ultimately reducing MIRI.

Lacidipine Attenuates Symptoms of Nicotine Withdrawal in Mice.

Nicotine-withdrawal after daily exposure manifests somatic and affective symptom including a range of cognitive deficits. Earlier studies suggested participation of L-type calcium channels (LTCCs) in development of nicotine dependence and expression of withdrawal signs. An upsurge in Ca2+-induced oxidative stress in brain underlies the biochemical events and behavioral signs of nicotine-withdrawal. The present study is aimed to explore the effects of lacidipine (LTCC antagonist) against nicotine-withdrawal. Swiss albino mice were administered ( -)-nicotine hydrogen tartrate (3.35 mg/kg, t.i.d.) from days 1 to 7 and alongside lacidipine (0.3, 1, and 3 mg/kg, i.p.) given from days 1 to 14. Somatic withdrawal signs were noted 48 h after last dose of nicotine. Bay-K8644 (LTCC agonist) was administered in mice subjected to nicotine-withdrawal and lacidipine (3 mg/kg) treatments. Behavioral tests of memory, anxiety, and depression were conducted on days 13 and 14 to assess the effects of lacidipine on affective symptoms of nicotine-withdrawal. Biomarkers of oxido-nitrosative were quantified in the whole brain. Nicotine-withdrawal significantly enhanced somatic signs and symptoms of anxiety, depression, and memory impairment in mice. Lacidipine (1 and 3 mg/kg) attenuated nicotine-withdrawal induced somatic symptoms and also ameliorated behavioral abnormalities. Nicotine-withdrawal triggered an upsurge in brain lipid peroxidation, total nitrite content, and decline in antioxidants, and these effects were attenuated by lacidipine. Bay-K8644 significantly abolished improvement in somatic and affective symptoms, and antioxidant effects by lacidipine in mice subjected to nicotine-withdrawal. Lacidipine mitigated nicotine-withdrawal triggered somatic and affective symptoms owing to decrease in brain oxido-nitrosative stress.

Chronic methamphetamine-induced neurodegeneration: Differential vulnerability of ventral tegmental area and substantia nigra pars compacta dopamine neurons.

Methamphetamine (meth) increases monoamine oxidase (MAO)-dependent mitochondrial stress in substantia nigra pars compacta (SNc) axons; chronic administration produces SNc degeneration that is prevented by MAO inhibition suggesting that MAO-dependent axonal mitochondrial stress is a causal factor. To test whether meth similarly increases mitochondrial stress in ventral tegmental area (VTA) axons, we used a genetically encoded redox biosensor to assess mitochondrial stress ex vivo. Meth increased MAO-dependent mitochondrial stress in both SNc and VTA axons. However, despite having the same meth-induced stress as SNc neurons, VTA neurons were resistant to chronic meth-induced degeneration indicating that meth-induced MAO-dependent mitochondrial stress in axons was necessary but not sufficient for degeneration. To determine whether L-type Ca2+ channel-dependent stress differentiates SNc and VTA axons, as reported in the soma, the L-type Ca2+ channel activator Bay K8644 was used. Opening L-type Ca2+ channels increased axonal mitochondrial stress in SNc but not VTA axons. To first determine whether mitochondrial stress was necessary for SNc degeneration, mice were treated with the mitochondrial antioxidant mitoTEMPO. Chronic meth-induced SNc degeneration was prevented by mitoTEMPO thereby confirming the necessity of mitochondrial stress. Similar to results with the antioxidant, both MAO inhibition and L-type Ca2+ channel inhibition also prevented SNc degeneration. Taken together the presented data demonstrate that both MAO- and L-type Ca2+ channel-dependent mitochondrial stress is necessary for chronic meth-induced degeneration.

The mechanisms of calcium mobilization by procyanidins, flavonols and flavonoids from Cecropia glaziovii Sneth in pulmonary endothelial cell cultures endorse its popular use as vasodilator phytomedicine.

The hypotensive and antihypertensive activities of the aqueous extract (AE) and butanolic fraction (ButF) isolated from Cecropia glaziovii Sneth have been demonstrated in previous studies in animal models. This study aimed to evaluate the molecular mechanism of action responsible for the vasodilatory effect of procyanidins, flavanols, and flavonoids found in C. glaziovii in endothelial cell culture. For this purpose, we analyzed the effect of procyanidin B2 and B3 compounds, catechin, epicatechin, orientin, isoorientin, and isovitexin in the mobilization of Ca2+ in rat endothelial cell cultures. Parallel associations with different antagonists were examined by considering the following in vivo hypotensive mechanisms: blockage of L-type calcium channels, action on ß-2 adrenergic receptors, and vasodilation via the nitric oxide pathway. All measurements of calcium mobilization were carried out by using the fluorescence measurement methodology in a Flexstation M3 spectrophotometer. The results indicate that some of the compounds have mixed actions, acting through different calcium mobilization pathways. The mobilization induced by such compounds significantly decreased when they were incubated with their corresponding antagonists. Taken together, our data suggest that the beneficial effects seen with the popular use of Cecropia glaziovii Sneth in pathological conditions, such as systemic arterial hypertension, seem to be related to the plant's hypotensive effect, very probably promoted by the actions of flavonols, flavonoids, and procyanidins, by different pathways of calcium mobilization.

N-Acetyl Neuraminic Acid (NANA) Activates L-Type Calcium Channels on Isolated Tentacle Supporting Cells of the Sea Anemone (Aiptasia pallida).

AbstractSensory receptors control nematocyst discharge on sea anemone tentacles. Micromolar N-acetylated sugars (e.g., N-acetyl neuraminic acid [NANA]) bind chemoreceptors on ectodermal supporting cells and predispose adjacent nematocyst discharge in response to mechanical contact via a cyclic adenosine monophosphate (cAMP)-dependent sensitization pathway, while higher NANA levels dose-dependently desensitize. Recent evidence implicates L-type calcium channels in desensitizing the pathway in aconitate sea anemones Aiptasia pallida (also known as Exaiptasia diaphana). We, therefore, hypothesize that NANA activates calcium influx via L-type calcium channels. We demonstrate a dose-dependent, NANA-activated 45Ca influx into dissociated ectodermal cells isolated from A. pallida tentacles, with maximal influx occurring at desensitizing concentrations of NANA. The L-type calcium channel inhibitors nifedipine, diltiazem, methoxyverapamil, and cadmium blocked NANA-stimulated 45Ca influx. Elevated extracellular KCl levels dose-dependently increased nifedipine-sensitive 45Ca influx to implicate voltage-gated calcium channels. Forskolin, 8-bromo-cAMP, and the protein kinase A inhibitor H-8 affect NANA-stimulated calcium influx in a manner consistent with activated cAMP-dependent pathway involvement. Because NANA chemoreceptors localize to supporting cells of cnidocyte supporting cell complexes, NANA activation of 45Ca influx into isolated tentacle ectodermal cells suggests that L-type calcium channels and NANA chemoreceptors co-localize to supporting cells. Indeed, a fluorescent marker of L-type calcium channels localizes to the apical ectoderm adjacent to nematocysts of live tentacles. We conclude that supporting cell chemoreceptors activate co-localized L-type calcium channels via a cAMP-dependent mechanism in order to initiate desensitization. We suggest that pathway desensitization may conserve nematocysts from excessive discharge during prey capture.

Questioning the renoprotective role of L-type calcium channel blockers in chronic kidney disease using physiological modeling.

Chronic kidney disease (CKD) is characterized by the progressive functional loss of nephrons and hypertension (HTN). Some antihypertensive regimens attenuate the progression of CKD (blockers of the renin-angiotensin system). Although studies have suggested that calcium channel blocker (CCB) therapy mitigates the decline in renal function in humans with essential HTN, there are few long-term clinical studies that have determined the impact of CCBs in patients with hypertensive CKD. Dihydropyridine (DHP) or L-type CCBs preferentially vasodilate the afferent arteriole and have been associated with glomerular HTN and increases in proteinuria in animal models with low renal function. Small clinical studies in vulnerable populations with renal disease such as African Americans, children, and diabetics have also suggested that DHP CCBs exacerbate glomerular injury, which questions the renoprotective effect of this class of antihypertensive drug. We used an established integrative mathematical model of human physiology, HumMod, to test the hypothesis that DHP CCB therapy exacerbates pressure-induced glomerular injury in hypertensive CKD. Over a simulation of 3 yr, CCB therapy reduced mean blood pressure by 14-16 mmHg in HTN both with and without CKD. Both impaired tubuloglomerular feedback and low baseline renal function exacerbated glomerular pressure, glomerulosclerosis, and the decline in renal function during L-type CCB treatment. However, simulating CCB therapy that inhibited both L- and T-type calcium channels increased efferent arteriolar vasodilation and alleviated glomerular damage. These simulations support the evidence that DHP (L-type) CCBs potentiate glomerular HTN during CKD and suggest that T/L-type CCBs are valuable in proteinuric renal disease treatment.NEW & NOTEWORTHY Our physiological model replicates clinical trial results and provides unique insights into possible mechanisms that play a role in glomerular injury and hypertensive kidney disease progression during chronic CCB therapy. Specifically, these simulations predict the temporal changes in renal function with CCB treatment and demonstrate important roles for tubuloglomerular feedback and efferent arteriolar conductance in the control of chronic kidney disease progression.

[8]-Gingerol exerts anti-myocardial ischemic effects in rats via modulation of the MAPK signaling pathway and L-type Ca2+ channels.

Myocardial ischemia (MI) remains the leading cause of mortality worldwide. Therefore, it is urgent to seek the treatment to protect the heart. [8]-Gingerol (8-Gin), one of the most active ingredients in ginger, has antioxidant, cardiotonic, and cardiovascular protective properties. The present study elucidated the cardioprotection effects and underlying mechanisms of 8-Gin in isoproterenol (ISO)-induced MI. ISO (85 mg/kg/d) was subcutaneously injected for 2 consecutive days to induce acute MI model in rats. Electrocardiography, oxidative stress levels, calcium concentrations, and apoptosis degree were observed. The effects of 8-Gin on L-type Ca2+ current (ICa-L ), contraction, and Ca2+ transients were monitored in rat myocytes via patch-clamp and IonOptix detection systems. 8-Gin decreased J-point elevation and heart rate and improved pathological heart damage. Moreover, 8-Gin reduced the levels of CK, LDH, and MDA, ROS production, and calcium concentrations in myocardial tissue, while increased the activities of SOD, CAT, and GSH. In addition, 8-Gin down-regulated Caspase-3 and Bax expressions, while up-regulated Bcl-2 expression. 8-Gin produced a marked decrease in the expression of p38, JNK, and ERK1/2 proteins. 8-Gin inhibited ICa-L , cell contraction, and Ca2+ transients in isolated rat myocytes. The results indicate that 8-Gin could exert anti-myocardial ischemic effects, which may be associated with oxidative stress reduction, cardiomyocytes apoptosis inhibition through MAPK signaling pathway, and Ca2+ homeostasis regulation via ICa-L modulation.

Molecular mechanism of apelin-13 regulation of colonic motility in rats.

Apelin is a novel neuropeptide identified as the endogenous ligand for the apelin receptor. Apelin and its receptor are widely distributed in the gastrointestinal tract. Studies have reported that apelin-13 is involved in modulating gastrointestinal motility; however, the evidence is insufficient and the relevant mechanism is still not fully clear. Consequently, our study designed to explore the effect induced by exogenous apelin-13, to analyze the mechanism of action in isolated rat colons and colonic smooth muscle cells. The spontaneous contractions of colonic smooth muscle strips from rat were measured in an organ bath system. L-type calcium currents and large conductance Ca2+-activated K+ (BKCa) currents in rat colonic smooth muscle cells were investigated using the electrophysiological patch-clamp technique. Apelin-13 decreased the spontaneous contractile activity of colonic smooth muscle strips in a dose-dependent manner, and the inhibitory effect was not abolished by tetrodotoxin. The electrophysiological recordings revealed that apelin-13 reduced the crest currents of L-type calcium in a concentration-dependent manner in colonic smooth muscle cells at the test potential of 0 mV. Moreover, apelin-13 moved the current-voltage (I-V) curves of L-type calcium channels upward, but did not change their contour. Furthermore, the characteristics of L-type calcium channels with steady-state activation and steady-state inactivation were not significantly changed. Similarly, application of apelin-13 also significantly decreased BKCa currents in a concentration-dependent manner. In conclusion, apelin-13 inhibited the spontaneous contractile activity of isolated rat colons via the suppression of L-type calcium channels and BKCa channels in colonic smooth muscle cells.

Rosuvastatin Reduces L-Type Ca2+ Current and Alters Contractile Function in Cardiac Myocytes via Modulation of ß-Adrenergic Receptor Signaling.

Rosuvastatin is one of the most used statins to lower plasma cholesterol levels. Although previous studies have reported remarkable cardiovascular effects of rosuvastatin (RSV), the mechanisms of these effects are largely unknown. In this study, we investigated the acute effects of RSV on L-type Ca2+ currents and contractile function of ventricular myocytes under basal conditions and during ß-adrenergic stimulation. The effects of RSV were investigated in freshly isolated adult rat ventricular myocytes. L-type Ca+2 currents and myocyte contractility were recorded using patch-clamp amplifier and sarcomere length detection system. All experimental recordings were performed at 36 ± 1 °C. L-type Ca+2 currents were significantly reduced with the administration of 1 µM RSV (~ 24%) and this reduction in Ca2+ currents was observed at almost all potential ranges applied. Suppression of L-type Ca2+ current by RSV was prevented by adenylyl cyclase (AC) and protein kinase A (PKA) inhibitors SQ 22536 and KT5720, respectively. However, inhibition of Rho-associated kinases (ROCKs) by Y-27632 or nitric oxide synthase (NOS) by L-NAME failed to circumvent the inhibitory effect of RSV. Finally, we examined the effect of RSV during ß-adrenergic receptor stimulation by isoproterenol and observed that RSV significantly suppresses the ß-adrenergic responses in both L-type Ca2+ currents and contraction parameters. In conclusion, RSV modulates the ß-adrenergic signaling cascade and thereby mimics the impact of ß-adrenergic receptor blockers in adult ventricular myocytes through modulation of the AC-cAMP-PKA pathway.

Orthograde signal of dihydropyridine receptor increases Ca2+ leakage after repeated contractions in rat fast-twitch muscles in vivo.

The purpose of this study was to investigate the mechanism underlying sarcoplasmic reticulum (SR) Ca2+ leakage after in vivo contractions. Rat gastrocnemius muscles were electrically stimulated in vivo, and then mechanically skinned fibers and SR microsomes were prepared from the muscles excised 30 min after repeated high-intensity contractions. The mechanically skinned fibers maintained the interaction between dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs), whereas the SR microsomes did not. Interestingly, skinned fibers from the stimulated muscles showed increased SR Ca2+ leakage, whereas Ca2+ leakage decreased in SR microsomes from the stimulated muscles. To enhance the orthograde signal of DHPRs, SR Ca2+ leakage in the skinned fiber was measured 1) under a continuously depolarized condition and 2) in the presence of nifedipine. As a result, in either of the two conditions, SR Ca2+ leakage in the rested fibers reached a level similar to that in the stimulated fibers. Furthermore, the increased SR Ca2+ leakage from the stimulated fibers was alleviated by treatment with 1 mM tetracaine (Tet) but not by treatment with 3 mM free Mg2+ (3 Mg). Tet exerted a greater inhibitory effect on the DHPR signal to RyR than 3 Mg, although their inhibitory effects on RyR were almost similar. These results suggest that the increased Ca2+ leakage after muscle contractions is mainly caused by the orthograde signal of DHPRs to RyRs.

L-type calcium channel blocker use and proteinuria among children with chronic kidney diseases.

BACKGROUND: Hypertension is common among children with chronic kidney disease (CKD), and dihydropyridine calcium channel blockers (dhCCBs) are frequently used as treatment. The impact of dhCCBs on proteinuria in children with CKD is unclear. METHODS: Data from 722 participants in the Chronic Kidney Disease in Children (CKiD) longitudinal cohort with a median age of 12 years were used to assess the association between dhCCBs and log transformed urine protein/creatinine levels as well as blood pressure control measured at annual visits. Angiotensin-converting enzyme inhibitor (ACEi) and angiotensin receptor blocker (ARB) use was evaluated as an effect measure modifier. RESULTS: Individuals using dhCCBs had 18.8% higher urine protein/creatinine levels compared to those with no history of dhCCB or ACEi and ARB use. Among individuals using ACEi and ARB therapy concomitantly, dhCCB use was not associated with an increase in proteinuria. Those using dhCCBs had higher systolic and diastolic blood pressures. CONCLUSIONS: Use of dhCCBs in children with CKD and hypertension is associated with higher levels of proteinuria and was not found to be associated with improved blood pressure control.

Gut bacteria-derived 5-hydroxyindole is a potent stimulant of intestinal motility via its action on L-type calcium channels.

Microbial conversion of dietary or drug substrates into small bioactive molecules represents a regulatory mechanism by which the gut microbiota alters intestinal physiology. Here, we show that a wide variety of gut bacteria can metabolize the dietary supplement and antidepressant 5-hydroxytryptophan (5-HTP) to 5-hydroxyindole (5-HI) via the tryptophanase (TnaA) enzyme. Oral administration of 5-HTP results in detection of 5-HI in fecal samples of healthy volunteers with interindividual variation. The production of 5-HI is inhibited upon pH reduction in in vitro studies. When administered orally in rats, 5-HI significantly accelerates the total gut transit time (TGTT). Deciphering the underlying mechanisms of action reveals that 5-HI accelerates gut contractility via activation of L-type calcium channels located on the colonic smooth muscle cells. Moreover, 5-HI stimulation of a cell line model of intestinal enterochromaffin cells results in significant increase in serotonin production. Together, our findings support a role for bacterial metabolism in altering gut motility and lay the foundation for microbiota-targeted interventions.

T1143 essential for CaV1.2 inhibition by diltiazem.

Careful analysis of previously published reports and some new insights into the structure activity studies revealed an important role of Threonine 1143 in drug binding. Substituting T1143 by alanine and other residues significantly reduced channel inhibition by qDil and Dil. Mutation T1143A did not affect channel activation or inactivation while almost completely diminishing channel block by Dil or qDil. These findings support the view that T1143 serves as drug binding determinant. Other mutations in this position than T1143A (T1143L/Y/S/N/C/V/E) diminished channel inhibition by qDil but additionally affected channel activation and inactivation and may therefore affect channel block allosterically. Collectively, our data suggest that T1143 is an essential diltiazem binding determinant.

The Use of Voltage Sensitive Dye di-4-ANEPPS and Video-Based Contractility Measurements to Assess Drug Effects on Excitation-Contraction Coupling in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

ABSTRACT: Because cardiotoxicity is one of the leading causes of drug failure and attrition, the design of new protocols and technologies to assess proarrhythmic risks on cardiac cells is in continuous development by different laboratories. Current methodologies use electrical, intracellular Ca2+, or contractility assays to evaluate cardiotoxicity. Increasingly, the human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are the in vitro tissue model used in commercial assays because it is believed to recapitulate many aspects of human cardiac physiology. In this work, we demonstrate that the combination of a contractility and voltage measurements, using video-based imaging and fluorescence microscopy, on hiPSC-CMs allows the investigation of mechanistic links between electrical and mechanical effects in an assay design that can address medium throughput scales necessary for drug screening, offering a view of the mechanisms underlying drug toxicity. To assess the accuracy of this novel technique, 10 commercially available inotropic drugs were tested (5 positive and 5 negative). Included were drugs with simple and specific mechanisms, such as nifedipine, Bay K8644, and blebbistatin, and others with a more complex action such as isoproterenol, pimobendan, digoxin, and amrinone, among others. In addition, the results provide a mechanism for the toxicity of itraconazole in a human model, a drug with reported side effects on the heart. The data demonstrate a strong negative inotropic effect because of the blockade of L-type Ca2+ channels and additional action on the cardiac myofilaments. We can conclude that the combination of contractility and action potential measurements can provide wider mechanistic knowledge of drug cardiotoxicity for preclinical assays.