PFI-3 induces vasorelaxation with potency to reduce extracellular calcium influx in rat mesenteric artery.

Background: PFI-3 is a small-molecule inhibitor that targets the bromodomains (BRDs) of Brahma-related gene 1 (BRG1). This monomeric compound, which has high selectivity and potent cellular effects, has recently been developed. Although PFI-3 has been reported as a potential therapeutic agent targeting thrombomodulin, its role in the regulation of vascular function remains unknown. Therefore, we aimed to investigate the impact of PFI-3 on arterial vessel tone. Methods: A microvascular tension measurement device (DMT) was utilized to identify alterations in vascular tension within the mesenteric artery. To detect variations in cytosolic [Ca2+]i, a Fluo-3/AM fluorescent probe and fluorescence microscope were employed. Additionally, whole-cell patch clamp techniques were utilized to evaluate the activity of L-type voltage-dependent calcium channels (VDCCs) in cultured arterial smooth muscle cells (A10 cells). Results: PFI-3 exerted a dose-dependent relaxation effect on rat mesenteric arteries with both intact and denuded endothelium after phenylephrine (PE)- and high-K+-induced constriction. PFI-3-induced vasorelaxation was not affected by the presence of L-NAME/ODQ or K+ channel blockers (Gli/TEA). PFI-3 abolished Ca2+-induced contraction on endothelium-denuded mesenteric arteries preincubated by PE in Ca2+-free solution. Incubation with TG had no impact on PFI-3-induced vasorelaxation pre-contracted by PE. PFI-3 reduced Ca2+-induced contraction on endothelium-denuded mesenteric arteries pre-incubated by KCl (60 mM) in Ca2+-free solution. PFI-3 declined extracellular calcium influx in A10 cells detected by Fluo-3/AM fluorescent probe and fluorescence microscope. Furthermore, we observed that PFI-3 decreased the current densities of L-type VDCC by whole-cell patch clamp techniques. Conclusions: PFI-3 blunted PE and high K+-induced vasoconstriction independent of endothelium on rat mesenteric artery. The vasodilatory effect of PFI-3 may be attributed to its inhibition of VDCCs and receptor-operated calcium channels (ROCCs) on vascular smooth muscle cells (VSMCs).

Synthesis of 6-alkoxy and 6-hydroxy-alkyl amine derivatives of braylin as vasorelaxing agents.

Braylin (10b) is a 8,8-dimethyl chromenocoumarin present in the plants of the family Rutaceae and Meliaceae and possesses vasorelaxing and anti-inflammatory activities. In this study, six 6-alkoxy (10b, 15-19), and twelve 6-hydroxy-alkyl amine (20a-20l) derivatives of braylin (11 and 12) were synthesized to delineate its structural requirement for vasorelaxing activity. The synthesized compounds were evaluated for vasorelaxation response in preconstricted intact rat Main Mesenteric Artery (MMA). The compounds showed l-type VDCC channel blockade depended and endothelium-independent vasorelaxation within the range of Emax < 50.00-96.70 % at 30 µM. Amongst all, 6-alkoxy derivatives were more active than 6-hydroxy-alkyl amine derivatives. The structural refinements about braylin showed that deletion of its methoxy group or homologation beyond ethoxy group presented deleterious effect on vasorelaxation response of braylin. Interestingly, substituting the ethoxy group in 10b presented the best activity and selectivity towards l-type VDCC channel blockade, a specific target cardiovascular function.

Potential binding modes of the gut bacterial metabolite, 5-hydroxyindole, to the intestinal L-type calcium channels and its impact on the microbiota in rats.

Intestinal microbiota and microbiota-derived metabolites play a key role in regulating the host physiology. Recently, we have identified a gut-bacterial metabolite, namely 5-hydroxyindole, as a potent stimulant of intestinal motility via its modulation of L-type voltage-gated calcium channels located on the intestinal smooth muscle cells. Dysregulation of L-type voltage-gated calcium channels is associated with various gastrointestinal motility disorders, including constipation, making L-type voltage-gated calcium channels an important target for drug development. Nonetheless, the majority of currently available drugs are associated with alteration of the gut microbiota. Using 16S rRNA sequencing this study shows that, when administered orally, 5-hydroxyindole has only marginal effects on the rat cecal microbiota. Molecular dynamics simulations propose potential-binding pockets of 5-hydroxyindole in the α1 subunit of the L-type voltage-gated calcium channels and when its stimulatory effect on the rat colonic contractility was compared to 16 different analogues, ex-vivo, 5-hydroxyindole stood as the most potent enhancer of the intestinal contractility. Overall, the present findings imply a potential role of microbiota-derived metabolites as candidate therapeutics for targeted treatment of slow intestinal motility-related disorders including constipation.

Vascular CaV1.2 channels in diabetes.

Diabetic vasculopathy is a significant cause of morbidity and mortality in the diabetic population. Hyperglycemia, one of the central metabolic abnormalities in diabetes, has been associated with vascular dysfunction due to endothelial cell damage. However, studies also point toward vascular smooth muscle as a locus for hyperglycemia-induced vascular dysfunction. Emerging evidence implicates hyperglycemia-induced regulation of vascular L-type Ca2+ channels CaV1.2 as a potential mechanism for vascular dysfunction during diabetes. This chapter summarizes our current understanding of vascular CaV1.2 channels and their regulation during physiological and hyperglycemia/diabetes conditions. We will emphasize the role of CaV1.2 in vascular smooth muscle, the effects of elevated glucose on CaV1.2 function, and the mechanisms underlying its dysregulation in hyperglycemia and diabetes. We conclude by examining future directions and gaps in knowledge regarding CaV1.2 regulation in health and during diabetes.

Rapid antidepressant-like effects of muscarinic receptor antagonists require BDNF-dependent signaling in the ventrolateral periaqueductal gray.

RATIONALE: Clinical reports reveal that scopolamine, an acetylcholine muscarinic receptor antagonist, exerts rapid antidepressant effects in depressed patients, but the mechanisms underlying the therapeutic effects have not been fully identified. OBJECTIVES: The present study examines the cellular mechanisms by which scopolamine produces antidepressant-like effects through its action in the ventrolateral midbrain periaqueductal gray (vlPAG). METHODS: We used a well-established mouse model of depression induced by chronic restraint stress (CRS) exposure for 14 days. Behaviors were tested using the forced swim test (FST), tail suspension test (TST), female urine sniffing test (FUST), novelty-suppressed feeding test (NSFT), and locomotor activity (LMA). Synaptic transmission in the vlPAG was measured by whole-cell patch-clamp recordings. IntravlPAG microinjection was used to pharmacologically verify the signaling cascades of scopolamine in the vlPAG. RESULTS: The results demonstrated that intraperitoneal injection of scopolamine produced antidepressant-like effects in a dose-dependent manner without affecting locomotor activity. CRS elicited depression-like behaviors, whereas intraperitoneal injection of scopolamine alleviated CRS-induced depression-like behaviors. CRS diminished glutamatergic transmission in the vlPAG, while scopolamine reversed the above effects. Moreover, intravlPAG microinjection of the L-type voltage-dependent calcium channel (VDCC) blocker verapamil, tropomyosin-related kinase B (TrkB) receptor antagonist ANA-12, mammalian target of rapamycin complex 1 (mTORC1) inhibitor rapamycin, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA) antagonist CNQX prevented scopolamine-induced antidepressant-like effects. CONCLUSIONS: Scopolamine ameliorated CRS-elicited depression-like behavior required activation of VDCC, resulting in activity-dependent release of brain-derived neurotrophic factor (BDNF), engaging the TrkB receptor and downstream mTORC1 signaling in the vlPAG.

Lysophosphatidic Acid Improves Human Sperm Motility by Enhancing Glycolysis and Activating L-Type Calcium Channels.

Until now, the molecular mechanisms underlining sperm motility defect causing male infertility are still poorly understood. Safe and effective compounds or drugs that can improve sperm motility are also very limited. Lysophosphatidic acid (LPA) is a naturally occurring phospholipid and a bioactive intermediate with multiple biological activities. It has been detected in various body fluids such as serum, plasma, saliva, tears, blister fluids, hen egg white, and ascites from patients with ovarian cancer. LPA is also abundant in seminal plasma and follicular fluid. It enhances follicle stimulation, improves oocyte fertilization, and promotes early embryonic development and embryo implantation. However, the physiological role of LPA in the male reproductive system remains unknown. Here, our study showed that LPA significantly improved the motility parameters of human sperm hyperactivation in a dose-dependent manner. The LPA-induced elevation of sperm motility is dependent on bovine serum albumin (BSA) but independent of the classical BSA-induced sAC/cAMP/PKA signaling pathway. The enhancement of sperm motility by LPA could not be blocked by CCCP, a respiratory inhibitor suppressing mitochondrial ATP production. Moreover, LPA improved the activity of triosephosphate isomerase in glycolysis. Meanwhile, LPA treatment significantly increased ATP and phosphoenolpyruvate levels and decreased ADP content during sperm glycolysis. Notably, none of known or identified LPA receptors was detected in human sperm. Further investigations showed that LPA promoted sperm motility through L-type calcium channels. In summary, this study revealed the involvement of LPA in the regulation for human sperm motility by enhancing glycolysis and activating L-type calcium channels. The current findings may shed new light on the understanding of causes of asthenozoospermia, and indicate that LPA could be used as a novel therapeutic agent to improve sperm function and fertilizing capacity.

Cellular mechanism underlying the facilitation of contractile response induced by IL-25 in mouse tracheal smooth muscle.

Asthma is a common heterogeneous respiratory disease characterized by airway inflammation and airway hyperresponsiveness (AHR) which is associated with abnormality in smooth muscle contractility. The epithelial cell-derived cytokine IL-25 is implicated in type 2 immune pathology including asthma, whereas the underlying mechanisms have not been fully elucidated. This study aims to investigate the effects of IL-25 on mouse tracheal smooth muscle contractility and elucidate the cellular mechanisms. Incubation with IL-25 augmented the contraction of mouse tracheal smooth muscles, which could be suppressed by the L-type voltage-dependent Ca2+ channel (L-VDCC) blocker nifedipine. Furthermore, IL-25 enhanced the cytosolic Ca2+ signals and triggered the upregulation of α1C L-VDCC (CaV1.2) in primary cultured mouse tracheal smooth muscle cells. Knocking down IL-17RA/IL-17RB receptors or inhibiting the transforming growth factor-ß-activated kinase 1 (TAK1)-tumor progression locus 2 (TPL2)-MAPK kinase 1/2 (MEK1/2)-ERK1/2-activating protein-1 (AP-1) signaling pathways suppressed the IL-25-elicited upregulation of CaV1.2 and hyperreactivity in tracheal smooth muscles. Moreover, inhibition of TPL2, ERK1/2 or L-VDCC alleviated the AHR symptom induced by IL-25 in a murine model. This study revealed that IL-25 potentiated the contraction of tracheal smooth muscle and evoked AHR via activation of TPL2-ERK1/2-CaV1.2 signaling, providing novel targets for the treatment of asthma with a high-IL-25 phenotype.

Schwarzinicine A inhibits transient receptor potential canonical channels and exhibits overt vasorelaxation effects.

This study investigated the vasorelaxant effects of schwarzinicine A, an alkaloid recently reported from Ficus schwarzii Koord. Regulation of calcium homeostasis in vascular smooth muscle cells (VSMC) is viewed as one of the main mechanisms for controlling blood pressure. L-type voltage-gated calcium channel (VGCC) blockers are commonly used for controlling hypertension. Recently, the transient receptor potential canonical (TRPC) channels were found in blood vessels of different animal species with evidence of their roles in the regulation of vascular contractility. In this study, we studied the mechanism of actions of schwarzinicine A focusing on its regulation of L-type VGCC and TRPC channels. Schwarzinicine A exhibited the highest vasorelaxant effect (123.1%) compared to other calcium channel blockers. It also overtly attenuated calcium-induced contractions of the rat isolated aortae in a calcium-free environment showing its mechanism to inhibit calcium influx. Fluorometric intracellular calcium recordings confirmed its inhibition of hTRPC3-, hTRPC4-, hTRPC5- and hTRPC6-mediated calcium influx into HEK cells with IC50 values of 3, 17, 19 and 7 µM, respectively. The evidence gathered in this study suggests that schwarzinicine A blocks multiple TRPC channels and L-type VGCC to exert a significant vascular relaxation response.

The interactive effects of verapamil and CB1 cannabinoid receptor antagonist/inverse agonist, AM251 on passive avoidance learning and memory in rat.

There are reports regarding the effects of intracellular Ca2+ and synthesis and release of endocannabinoids. The secretion of endocannabinoids depends on the L-type calcium channel. The present study evaluated the involvement of the cannabinoid CB1 receptors in the effect of L-type calcium channel blocker verapamil on passive avoidance learning (PAL) in adult male rats. In this study, we examined the effects of an acute administration of the cannabinoid CB1 receptors antagonist/inverse agonist AM251 following a chronic administration of the Ca2+ channel blocker verapamil on PAL. Male Wistar rats were administered verapamil (10, 25 and 50 mg/kg) or saline intraperitoneally (i.p) daily for 13 days (n = 10/group). After this treatment period, a learning test (acquisition) was performed, and a retrieval test was performed the following day. The results indicated that chronic systemic administration of verapamil (in a dose-dependent manner) impaired memory acquisition and retrieval. Pre-training acute administration of a selective CB1 antagonist/inverse agonist, AM251 (5 mg/kg, i.p.) did not change memory acquisition and retrieval. Co-administration of the verapamil and AM251 significantly reversed verapamil-induced amnesia, suggesting a functional interaction between AM251 and verapamil. The results indicated the interactive effects of cannabinoid CB1 receptors and L-type calcium channel in passive avoidance learning and AM251 can counter the effects of verapamil on memory.

[Effects of chronic intermittent hypoxia on atrial electrical remodeling in rats].

OBJECTIVE: The aim of this study was to investigate the effects of chronic intermittent hypoxia (CIH) on atrial electrical remodeling in Sprague-Dawley (SD) rats, which provide the explication for the mechanisms of CIH promoting atrial fibrillation (AF). METHODS: Eighty SD rats were randomly divided into 2 groups: control group and CIH group (n=40). CIH rats were subjected to CIH 8 h/d for 30 days. After the echocardiography and hemodynamics examination, cardiac electrophysiological experiments, histological experiments, and molecular biological experiments were executed. AF susceptibility was measured by isolated heart electrophysiological experiments. Masson's trichrome stain was used to assess the degree of atrial fibrosis. The protein expression levels of sodium voltage-gated channel alpha subunit 5 (SCN5A/Nav1.5), calcium voltage-gated channel subunit alpha1 C (CACNA1C/Cav1.2) and potassium voltage-gated channel subfamily D member 3 (KCND3/Kv4.3) were measured by Western blot. In whole-cell patch clamp experiments, current clamp mode was used to record AP, and APD90 and APD50 were analyzed and compared between the two groups. In voltage clamp mode, INa, ICa-L, Ito and their kinetic parameters were recorded and compared between the two groups. RESULTS: Compared to the control rats, atrial interstitial collagen deposition (P＜0.01) and AF inducibility (P＜0.05) were increased in CIH rats, whereas the expression levels of Nav1.5, Cav1.2 and Kv4.3 were decreased (P＜0.05). APD90 and APD50 in CIH rats' atrial myocytes were longer than those of control rats, and CIH rats showed decreased current density of INa, ICa-L(P＜0.01) and Ito(P＜0.01). CONCLUSION: CIH-induced changes in the protein expression levels of ion channel subunits, current intensity, APD, and AF susceptibility, which may be the mechanisms of CIH promoting AF.

Adrenergic Regulation of Calcium Channels in the Heart.

Each heartbeat is initiated by the action potential, an electrical signal that depolarizes the plasma membrane and activates a cycle of calcium influx via voltage-gated calcium channels, calcium release via ryanodine receptors, and calcium reuptake and efflux via calcium-ATPase pumps and sodium-calcium exchangers. Agonists of the sympathetic nervous system bind to adrenergic receptors in cardiomyocytes, which, via cascading signal transduction pathways and protein kinase A (PKA), increase the heart rate (chronotropy), the strength of myocardial contraction (inotropy), and the rate of myocardial relaxation (lusitropy). These effects correlate with increased intracellular concentration of calcium, which is required for the augmentation of cardiomyocyte contraction. Despite extensive investigations, the molecular mechanisms underlying sympathetic nervous system regulation of calcium influx in cardiomyocytes have remained elusive over the last 40 years. Recent studies have uncovered the mechanisms underlying this fundamental biologic process, namely that PKA phosphorylates a calcium channel inhibitor, Rad, thereby releasing inhibition and increasing calcium influx. Here, we describe an updated model for how signals from adrenergic agonists are transduced to stimulate calcium influx and contractility in the heart.

Single-pill combination of cilnidipine, an l-/n-type calcium channel blocker, and valsartan reduces the day-by-day variability of morning home systolic blood pressure in patients with treated hypertension: A sub-analysis of the HOPE-combi survey.

We examined the effects of a fixed-dose single-pill combination of cilnidipine (10 mg), an L-/N-type calcium channel blocker, and valsartan (80 mg) (SPC of Cil/Val) on the day-by-day variability of morning home systolic blood pressure (MHSBP) in 616 patients with treated hypertension for 12 months as a sub-analysis of the HOPE-Combi survey, multicentral, post-marketing, and prospective observational survey. The SPC of Cil/Val was administrated once a day in the morning. The SPC of Cil/Val decreased the standard deviation (SD, from 6.3 ± 4.8 to 5.1 ± 3.8 mmHg, p < .01), coefficient of variation (from 4.3 ± 3.2 to 3.8 ± 2.9%, p < .05), average real variability (ARV, from 7.9 ± 6.6 to 6.3 ± 5.1 mmHg, p < .01), and the difference between maximum and minimum (MMD, from 11.9 ± 9.2 to 9.7 ± 7.2 mmHg, p < .01) of MHSBP. The variability of MHSBP increased with age; however, this was not increased in patients ≥70 years at the baseline. In elderly patients (≥70 years, N = 283), the SPC of Cil/Val decreased the SD (from 6.9 ± 5.6 to 5.6 ± 4.4 mmHg, p < .01), ARV (from 8.6 ± 7.7 to 6.9 ± 5.7 mmHg, p < .05), and MMD (from 13.2 ± 10.7 to 10.7 ± 8.3 mmHg, p < .01) of MHSBP at 12 months; the reduction in these MHSBP variability parameters was comparable to that in adults <70 years. These results suggest that the SPC of Cil/Val is effective in reducing day-by-day variability of MHSBP in elderly patients.

Single-pill combination of cilnidipine, an L-/N-type calcium channel blocker, and valsartan effectively reduces home pulse pressure in patients with uncontrolled hypertension and sympathetic hyperactivity: The HOPE-Combi survey.

The home blood pressure (BP) control by a single-pill combination of cilnidipine (an L-/N-type calcium channel blocker; CCB) and valsartan (HOPE-Combi) survey is a multicenter, post-marketing, prospective observational study of a single-pill combination of cilnidipine 10 mg and valsartan 80 mg (SPC of Cil/Val) in patients with uncontrolled hypertension. We examined the effects of the SPC of Cil/Val on morning home systolic BP (MHSBP) and morning home pulse pressure (MHPP) of 1036 patients with hypertension over 12 months. MHSBP decreased by 14.0 mm Hg (P < .01), and MHPP decreased by 6.6 mm Hg (P < .01). Moreover, morning home pulse rate (MHPR) decreased by 2.1 bpm (P < .01). A more progressive and greater decrease in MHSBP (-17.2 vs -10.3 mm Hg, P < .01) and MHPP (-7.6 vs -4.9 mm Hg, P < .01) was observed in patients with higher MHPR (≥70 bpm) than in those with lower MHPR (<70 bpm) over the treatment period. In particular, in patients with a wide MHPP (≥70 mm Hg), the difference in the MHPP reduction was greater in patients with higher MHPR than in those with lower MHPR (-17.9 vs -13.6 mm Hg, P < .01). These results suggested that the SPC of Cil/Val, which possesses the unique sympatholytic characteristics of an L-/N-type CCB, was particularly effective in patients with uncontrolled hypertension and sympathetic hyperactivity.

The role of Na(+), K(+)-ATPase in the hypoxic vasoconstriction in isolated rat basilar artery.

Hypoxia-induced cerebrovascular dysfunction is a key factor in the occurrence and the development of cerebral ischemia. Na(+), K(+)-ATPase affects the regulation of intracellular Ca(2+) concentration and plays an important role in vascular smooth muscle function. However, the potential role of Na(+), K(+)-ATPase in hypoxia-induced cerebrovascular dysfunction is unknown. In this study, we found that the KCl-induced contraction under hypoxia in rat endothelium-intact basilar arteries is similar to that of denuded arteries, suggesting that hypoxia may cause smooth muscle cell (SMC)-dependent vasoconstriction in the basilar artery. The Na(+), K(+)-ATPase activity of the isolated basilar artery with or without endothelium significantly reduced with prolonged hypoxia. Blocking the Na(+)-Ca(2+) exchanger with Ni(2+) (10(-3)M) or the L-type Ca(2+) channel with nimodipine (10(-8)M) dramatically attenuated KCl-induced contraction under hypoxia. Furthermore, prolonged hypoxia significantly reduced Na(+), K(+)-ATPase activity and increased [Ca(2+)]i in cultured rat basilar artery SMCs. Hypoxia reduced the protein and mRNA expression of the α2 isoform of Na(+), K(+)-ATPase in SMCs in vitro. We used a low concentration of the Na(+), K(+)-ATPase inhibitor ouabain, which possesses a high affinity for the α2 isoform. The contractile response in the rat basilar artery under hypoxia was partly inhibited by ouabain pretreatment. The decreased Na(+), K(+)-ATPase activity in isolated basilar artery and the increased [Ca(2+)]i in SMCs induced by hypoxia were partly inhibited by pretreatment with a low concentration of ouabain. These results suggest that hypoxia may educe Na(+), K(+)-ATPase activity in SMCs through the α2 isoform contributing to vasoconstriction in the rat basilar artery.

The Physiology, Pathology, and Pharmacology of Voltage-Gated Calcium Channels and Their Future Therapeutic Potential.

Voltage-gated calcium channels are required for many key functions in the body. In this review, the different subtypes of voltage-gated calcium channels are described and their physiologic roles and pharmacology are outlined. We describe the current uses of drugs interacting with the different calcium channel subtypes and subunits, as well as specific areas in which there is strong potential for future drug development. Current therapeutic agents include drugs targeting L-type Ca(V)1.2 calcium channels, particularly 1,4-dihydropyridines, which are widely used in the treatment of hypertension. T-type (Ca(V)3) channels are a target of ethosuximide, widely used in absence epilepsy. The auxiliary subunit α2δ-1 is the therapeutic target of the gabapentinoid drugs, which are of value in certain epilepsies and chronic neuropathic pain. The limited use of intrathecal ziconotide, a peptide blocker of N-type (Ca(V)2.2) calcium channels, as a treatment of intractable pain, gives an indication that these channels represent excellent drug targets for various pain conditions. We describe how selectivity for different subtypes of calcium channels (e.g., Ca(V)1.2 and Ca(V)1.3 L-type channels) may be achieved in the future by exploiting differences between channel isoforms in terms of sequence and biophysical properties, variation in splicing in different target tissues, and differences in the properties of the target tissues themselves in terms of membrane potential or firing frequency. Thus, use-dependent blockers of the different isoforms could selectively block calcium channels in particular pathologies, such as nociceptive neurons in pain states or in epileptic brain circuits. Of important future potential are selective Ca(V)1.3 blockers for neuropsychiatric diseases, neuroprotection in Parkinson's disease, and resistant hypertension. In addition, selective or nonselective T-type channel blockers are considered potential therapeutic targets in epilepsy, pain, obesity, sleep, and anxiety. Use-dependent N-type calcium channel blockers are likely to be of therapeutic use in chronic pain conditions. Thus, more selective calcium channel blockers hold promise for therapeutic intervention.

Decreased myocardial injury and improved contractility after administration of a peptide derived against the alpha-interacting domain of the L-type calcium channel.

BACKGROUND: Myocardial infarction remains the leading cause of morbidity and mortality associated with coronary artery disease. The L-type calcium channel (IC a-L) is critical to excitation and contraction. Activation of the channel also alters mitochondrial function. Here, we investigated whether application of a alpha-interacting domain/transactivator of transcription (AID-TAT) peptide, which immobilizes the auxiliary ß2 subunit of the channel and decreases metabolic demand, could alter mitochondrial function and myocardial injury. METHODS AND RESULTS: Treatment with AID-TAT peptide decreased ischemia-reperfusion injury in guinea-pig hearts ex vivo (n=11) and in rats in vivo (n=9) assessed with uptake of nitroblue tetrazolium, release of creatine kinase, and lactate dehydrogenase. Contractility (assessed with catheterization of the left ventricle) was improved after application of AID-TAT peptide in hearts ex vivo (n=6) and in vivo (n=8) up to 12 weeks before sacrifice. In search of the mechanism for the effect, we found that intracellular calcium ([Ca(2+)]i, Fura-2), superoxide production (dihydroethidium fluorescence), mitochondrial membrane potential (Ψm, JC-1 fluorescence), reduced nicotinamide adenine dinucleotide production, and flavoprotein oxidation (autofluorescence) are decreased after application of AID-TAT peptide. CONCLUSIONS: Application of AID-TAT peptide significantly decreased infarct size and supported contractility up to 12 weeks postcoronary artery occlusion as a result of a decrease in metabolic demand during reperfusion.

Early and late calcium waves during wound healing in corneal endothelial cells.

Immediately after wounding, bovine corneal endothelial cells develop a fast calcium wave that propagates from the wound border to the rest of the monolayer and extinguishes in approximately 5 minutes. One hour after wounding, a late, slow calcium wave (SCW) develops concomitantly to the depolarization of the plasma membrane potential of the border cells. The incorporation of inhibitors of the epithelial sodium channel and of the sodium-calcium exchanger produces inhibition of the membrane depolarization and the SCW, and diminishes the rate of wound healing. The L-type calcium channel blocker nimodipine does not have any effect on the SCW. The reversible inhibition of the fast calcium wave does not affect the SCW and only slightly decreases the velocity of healing. Our results suggest that the SCW is at least partially produced by the coupling of the epithelial sodium channel and the sodium-calcium exchanger functioning in reverse mode. They also suggest that the SCW may play a role in the overall healing process.

Effects of chloroacetaldehyde in 2-chloroethanol-induced cardiotoxicity.

Cardiovascular effects have often been found in 2-chloroethanol (2-CE) intoxicated patients, but the 2-CE elicits cardiovascular toxicity mechanism is not clear. Recently, we have found that chloroacetaldehyde (CAA) accumulation in 2-CE-intoxicated rat's blood and play an important role in 2-CE intoxication. In this study, we used an isolated rat atrium model to examine the cardiotoxicity of 2-CE and CAA. Results indicated that 2-CE did not cause tension arrest in isolated rat right atria, but CAA did. 2-CE caused tension inhibition in the isolated rat left atria. In addition, CAA caused significant tension inhibition and contracture in the isolated rat left atria. Nifedipine, an L-type calcium channel blocker, decreased CAA-induced tension inhibition and contracture. Meanwhile, atrial nNOS and calmodulin (CaM) had significantly greater expression in the 2-CE group and the CAA group than control group. Nifedipine could decrease CAA-induced nNOS and CaM expression. 2-CE-induced cardiovascular toxicity might be due to its metabolite CAA. CAA-induced cardiovascular toxicity might be mediated by calcium channel and nifedipine protected against nNOS-triggered cardiovascular effects.

Effect of ageing on CA3 interneuron sAHP and gamma oscillations is activity-dependent.

Normal ageing-associated spatial memory impairment has been linked to subtle changes in the hippocampal network. Here we test whether the age-dependent reduction in gamma oscillations can be explained by the changes in intrinsic properties of hippocampal interneurons. Kainate-induced gamma oscillations, but not spontaneous gamma oscillations, were reduced in slices from aged mice. CA3 interneurons were recorded in slices from young and aged mice using Fura-2-filled pipettes. Passive membrane properties, firing properties, medium- and slow-afterhyperpolarisation amplitudes, basal [Ca(2+)](i) and firing-induced [Ca(2+)](i) transients were not different with ageing. Kainate caused a larger depolarisation and increase in [Ca(2+)](i) signal in aged interneurons than in young ones. In contrast to young interneurons, kainate increased the medium- and slow-afterhyperpolarisation and underlying [Ca(2+)](i) transient in aged interneurons. Modulating the slow-afterhyperpolarisation by modulating L-type calcium channels with BAY K 8644 and nimodipine suppressed and potentiated, respectively, kainate-induced gamma oscillations in young slices. The age-dependent and stimulation-dependent increase in basal [Ca(2+)](i), firing-induced [Ca(2+)](i) transient and associated afterhyperpolarisation may reduce interneuron excitability and contribute to an age-dependent impairment of hippocampal gamma oscillations.

Oxidant stress evoked by pacemaking in dopaminergic neurons is attenuated by DJ-1.

Parkinson's disease is a pervasive, ageing-related neurodegenerative disease the cardinal motor symptoms of which reflect the loss of a small group of neurons, the dopaminergic neurons in the substantia nigra pars compacta (SNc). Mitochondrial oxidant stress is widely viewed as being responsible for this loss, but why these particular neurons should be stressed is a mystery. Here we show, using transgenic mice that expressed a redox-sensitive variant of green fluorescent protein targeted to the mitochondrial matrix, that the engagement of plasma membrane L-type calcium channels during normal autonomous pacemaking created an oxidant stress that was specific to vulnerable SNc dopaminergic neurons. The oxidant stress engaged defences that induced transient, mild mitochondrial depolarization or uncoupling. The mild uncoupling was not affected by deletion of cyclophilin D, which is a component of the permeability transition pore, but was attenuated by genipin and purine nucleotides, which are antagonists of cloned uncoupling proteins. Knocking out DJ-1 (also known as PARK7 in humans and Park7 in mice), which is a gene associated with an early-onset form of Parkinson's disease, downregulated the expression of two uncoupling proteins (UCP4 (SLC25A27) and UCP5 (SLC25A14)), compromised calcium-induced uncoupling and increased oxidation of matrix proteins specifically in SNc dopaminergic neurons. Because drugs approved for human use can antagonize calcium entry through L-type channels, these results point to a novel neuroprotective strategy for both idiopathic and familial forms of Parkinson's disease.