Enhanced bioavailability and antihypertensive activity of nisoldipine loaded nanoemulsion: optimization, cytotoxicity and uptake across Caco-2 cell line, pharmacokinetic and pharmacodynamic studies.

Objective: The present study explored the antihypertensive activity of nisoldipine in oil in water nanoemulsion to improve its oral bioavailability via intestinal lymphatic uptake.Methods: Nanoemulsion was prepared by ultrasonication technique using Peceol, Cremophor EL and Transcutol HP as oil, surfactant and cosurfactant respectively. Optimization was done employing 32 full factorial design. The developed formulation was assessed for in vitro,cell line, ex vivo and in vivo studies.Results: The experimental results indicated homogeneity of the nanoemulsion with globule size of 62.35 ± 2.55 nm and PDI value of 0.108 ± 0.01 with negative zeta potential (-26.2 ± 3.6 mV). Transmission electron microscopy showed spherical oil globules morphology. The in vitro diffusion study showed significant increase in drug release from NE formulations (98.51 ± 2.64%) as compared to plain drug dispersion (29.73 ± 2.15%) in 0.1 N HCl + 0.5% SLS medium. Moreover, higher quantitative and qualitative uptake of nanoemulsion via Caco-2 cells showed superior intestinal absorption and improved therapeutic activity of nisoldipine when compared to drug dispersion. Pharmacokinetic and pharmacodynamic study confirmed significantly (p ˂ 0.05) greater bioavailability and antihypertensive activity of nisoldipine nanoemulsion when compared to its dispersion. These results are visualized in abstract figure.Conclusion: Thus, prepared nanoemulsion showed potential as oral delivery system for nisoldipine with superior oral bioavailability and therapeutic efficacy over drug dispersion.

Effects of m-nisoldipine on the activity and mRNA expression of four CYP isozymes in rats.

1. For the first time, a systemic in vivo investigation was employed to evaluate the potential effects of m-nisoldipine on activities of rat cytochrome P450 enzymes (CYP1A2, CYP2C11, CYP2D1 and CYP3A1) by both cocktail probe drugs and the quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR). 2. m-Nisoldipine-treated and blank control groups were respectively administered m-nisoldipine at the dosage of 2.5, 5 and 12.5 mg/kg and CMC-Na solution for 15 days consecutively, then they were given the probe drugs of caffeine, diclofenac, dextromethorphan and midazolam (all probes were 5 mg/kg) by p.o. The blood samples were collected at different times for liquid chromatography coupled with mass spectrometry (LC-MS) analysis. The corresponding pharmacokinetic parameters were applied to evaluate the effects of m-nisoldipine on the four CYP isoforms in vivo. In addition, RT-qPCR was performed to determine the effects of m-nisoldipine on the mRNA expression of CYPs in rat liver. Results indicated that high dose and middle dose of m-nisoldipine showed significant effects on all four CYPs and CYP2C11, respectively. Moreover, for CYP2D1 and CYP1A2, there were no significant effects found at either low or middle dose of m-nisoldipine. 3. This study could provide not only experimental evidence for potential clinical application of m-nisoldipine but also a practical strategy for assessing CYP-mediated drug-drug interactions.

The L-type Ca(2+) channel facilitates abnormal metabolic activity in the cTnI-G203S mouse model of hypertrophic cardiomyopathy.

KEY POINTS: Genetic mutations in cardiac troponin I (cTnI) are associated with development of hypertrophic cardiomyopathy characterized by myocyte remodelling, disorganization of cytoskeletal proteins and altered energy metabolism. The L-type Ca(2+) channel is the main route for calcium influx and is crucial to cardiac excitation and contraction. The channel also regulates mitochondrial function in the heart by a functional communication between the channel and mitochondria via the cytoskeletal network. We find that L-type Ca(2+) channel kinetics are altered in cTnI-G203S cardiac myocytes and that activation of the channel causes a significantly greater increase in mitochondrial membrane potential and metabolic activity in cTnI-G203S cardiac myocytes. These responses occur as a result of impaired communication between the L-type Ca(2+) channel and cytoskeletal protein F-actin, involving decreased movement of actin-myosin and block of the mitochondrial voltage-dependent anion channel, resulting in a 'hypermetabolic' mitochondrial state. We propose that L-type Ca(2+) channel antagonists, such as diltiazem, might be effective in reducing the cardiomyopathy by normalizing mitochondrial metabolic activity. ABSTRACT: Genetic mutations in cardiac troponin I (cTnI) account for 5% of families with hypertrophic cardiomyopathy. Hypertrophic cardiomyopathy is associated with disorganization of cytoskeletal proteins and altered energy metabolism. The L-type Ca(2+) channel (ICa-L ) plays an important role in regulating mitochondrial function. This involves a functional communication between the channel and mitochondria via the cytoskeletal network. We investigate the role of ICa-L in regulating mitochondrial function in 25- to 30-week-old cardiomyopathic mice expressing the human disease-causing mutation Gly203Ser in cTnI (cTnI-G203S). The inactivation rate of ICa-L is significantly faster in cTnI-G203S myocytes [cTnI-G203S: τ1  = 40.68 ± 3.22, n = 10 vs. wild-type (wt): τ1  = 59.05 ± 6.40, n = 6, P < 0.05]. Activation of ICa-L caused a greater increase in mitochondrial membrane potential (Ψm , 29.19 ± 1.85%, n = 15 vs. wt: 18.84 ± 2.01%, n = 10, P < 0.05) and metabolic activity (24.40 ± 6.46%, n = 8 vs. wt: 9.98 ± 1.57%, n = 9, P < 0.05). The responses occurred because of impaired communication between ICa-L and F-actin, involving lack of dynamic movement of actin-myosin and block of the mitochondrial voltage-dependent anion channel. Similar responses were observed in precardiomyopathic mice. ICa-L antagonists nisoldipine and diltiazem decreased Ψm to basal levels. We conclude that the Gly203Ser mutation leads to impaired functional communication between ICa-L and mitochondria, resulting in a 'hypermetabolic' state. This might contribute to development of cTnI-G203S cardiomyopathy because the response is present in young precardiomyopathic mice. ICa-L antagonists might be effective in reducing the cardiomyopathy by altering mitochondrial function.

Contribution of ion currents to beat-to-beat variability of action potential duration in canine ventricular myocytes.

Although beat-to-beat variability (short-term variability, SV) of action potential duration (APD) is considered as a predictor of imminent cardiac arrhythmias, the underlying mechanisms are still not clear. In the present study, therefore, we aimed to determine the role of the major cardiac ion currents, APD, stimulation frequency, and changes in the intracellular Ca(2+) concentration ([Ca(2+)]i) on the magnitude of SV. Action potentials were recorded from isolated canine ventricular cardiomyocytes using conventional microelectrode techniques. SV was an exponential function of APD, when APD was modified by current injections. Drug effects were characterized as relative SV changes by comparing the drug-induced changes in SV to those in APD according to the exponential function obtained with current pulses. Relative SV was increased by dofetilide, HMR 1556, nisoldipine, and veratridine, while it was reduced by BAY K8644, tetrodotoxin, lidocaine, and isoproterenol. Relative SV was also increased by increasing the stimulation frequency and [Ca(2+)]i. In summary, relative SV is decreased by ion currents involved in the negative feedback regulation of APD (I Ca, I Ks, and I Kr), while it is increased by I Na and I to. We conclude that drug-induced effects on SV should be evaluated in relation with the concomitant changes in APD. Since relative SV was decreased by ion currents playing critical role in the negative feedback regulation of APD, blockade of these currents, or the beta-adrenergic pathway, may carry also some additional proarrhythmic risk in addition to their well-known antiarrhythmic action.

[m-Nisodipine inhibited 5-HT-induced proliferation of rat PASMCs through Rho/ROCK signal pathway].

This paper is to report the exploration of the activation of Rho/ROCK signal pathway in 5-HT-induced proliferation of rat pulmonary artery smooth muscle cells (PASMCs) and the inhibitory effect of m-Nis on this pathway. PASMCs were cultured with the explant technique. MTT assay was used to explore the proliferation of PASMCs after 5-HT treated for different time and the intervening effect of m-Nis. RT-PCR and Western blot were used respectively to explore the mRNA expression of RhoA, ROCK1 and the protein expression of p-MYPT1 in 5-HT-treated PASMCs and intervening effect of m-Nis. The results of MTT assay suggested that 5-HT (1 µmol · L(-1)) treatment for 12-72 h significantly induced the proliferation of rat PASMCs (P<0.05 or P < 0.01), which were inhibited by m-Nis (1 x 10(-5), 1 x 10(-6), l x 10(-7), 1 x10(-8) mol · L(-1)) in dose-dependent manners (P < 0.05 or P < 0.01). Similarly, the mRNA expression of RhoA, ROCK1 and the protein expression of p-MYPT1 were also inhibited by m-Nis in different degrees (P < 0.05 or P < 0.01). Thus, the results of this study suggested that Rho/ROCK pathway played an important role in 5-HT-induced proliferation of rat PASMCs, m-Nis inhibited 5-HT-induced proliferation obviously, which may be related to the blockage of Rho/ROCK signal pathway.

Tetrodotoxin blocks L-type Ca2+ channels in canine ventricular cardiomyocytes.

Tetrodotoxin (TTX) is believed to be the most selective inhibitor of voltage-gated fast Na(+) channels in excitable tissues, including nerve, skeletal muscle, and heart, although TTX sensitivity of the latter is lower than the former by at least three orders of magnitude. In the present study, the TTX sensitivity of L-type Ca(2+) current (I (Ca)) was studied in isolated canine ventricular cells using conventional voltage clamp and action potential voltage clamp techniques. TTX was found to block I (Ca) in a reversible manner without altering inactivation kinetics of I (Ca). Fitting results to the Hill equation, an IC(50) value of 55 ± 2 µM was obtained with a Hill coefficient of unity (1.0 ± s0.04). The current was fully abolished by 1 µM nisoldipine, indicating that it was really I (Ca). Under action potential voltage clamp conditions, the TTX-sensitive current displayed the typical fingerprint of I (Ca), which was absent in the presence of nisoldipine. Stick-and-ball models for Cav1.2 and Nav1.5 channel proteins were constructed to explain the differences observed between action of TTX on cardiac I (Ca) and I (Na). This is the first report demonstrating TTX to interact with L-type calcium current in the heart.

Nisoldipine Inhibits Influenza A Virus Infection by Interfering with Virus Internalization Process.

Influenza virus infections and the continuing spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are global public health concerns. As there are limited therapeutic options available in clinical practice, the rapid development of safe, effective and globally available antiviral drugs is crucial. Drug repurposing is a therapeutic strategy used in treatments for newly emerging and re-emerging infectious diseases. It has recently been shown that the voltage-dependent Ca2+ channel Cav1.2 is critical for influenza A virus entry, providing a potential target for antiviral strategies. Nisoldipine, a selective Ca2+ channel inhibitor, is commonly used in the treatment of hypertension. Here, we assessed the antiviral potential of nisoldipine against the influenza A virus and explored the mechanism of action of this compound. We found that nisoldipine treatment could potently inhibit infection with multiple influenza A virus strains. Mechanistic studies further revealed that nisoldipine impaired the internalization of the influenza virus into host cells. Overall, our findings demonstrate that nisoldipine exerts antiviral effects against influenza A virus infection and could serve as a lead compound in the design and development of new antivirals.

Characterization of the rapidly activating delayed rectifier potassium current, I (Kr), in HL-1 mouse atrial myocytes.

HL-1 is the adult murine cardiac cell line that can be passaged repeatedly in vitro without losing differentiated phenotype. The present study was designed to characterize the rapidly activating delayed rectifier potassium current, I (Kr), endogenously expressed in HL-1 cells using the whole-cell patch-clamp technique. In the presence of nisoldipine, depolarizing voltage steps applied from a holding potential of -50 mV evoked the time-dependent outward current, followed by slowly decaying outward tail current upon return to the holding potential. The amplitude of the current increased with depolarizations up to 0 mV but then progressively decreased with further depolarizations. The time-dependent outward current as well as the tail current were highly sensitive to block by E-4031 and dofetilide (IC(50) of 21.1 and 15.1 nM, respectively) and almost totally abolished by micromolar concentrations of each drug, suggesting that most of the outward current in HL-1 cells was attributable to I (Kr). The magnitude of I (Kr) available from HL-1 cells (18.1 +/- 1.5 pA pF(-1)) was sufficient for reliable measurements of various gating parameters. RT-PCR and Western blot analysis revealed the expression of alternatively spliced forms of mouse ether-a-go-go-related genes (mERG1), the full-length mERG1a and the N-terminally truncated mERG1b isoforms. Knockdown of mERG1 transcripts with small interfering RNA (siRNA) dramatically reduced I (Kr) amplitude, confirming the molecular link of mERG1 and I (Kr) in HL-1 cells. These findings demonstrate that HL-1 cells possess I (Kr) with properties comparable to those in native cardiac I (Kr) and provide an experimental model suitable for studies of I (Kr) channels.

[Effect of m-nisoldipine on the Ca2+/CaM/CaN signal pathway in 5-HT-induced proliferation of rat PASMCs].

This study is to explore the activation of the Ca2+/CaM/CaN signal pathway in 5-HT-induced proliferation of rat pulmonary artery smooth muscle cells (PASMCs) and the inhibitory effect of m-nisoldipine (m-Nis) on this pathway. PASMCs were cultured with the explant technique. The proliferation of PASMCs was evaluated by MTT assay. Confocal microscopy was used to measure the change of [Ca2+]i. The mRNA expression of CaM and CaN was evaluated by RT-PCR and the activity of CaN was measured according to the instruction of kits. The results of MTT assay suggested that 5-HT (1 micromol x L(-1)) significantly induced the proliferation of rat PASMCs (P < 0.01), which was inhibited obviously by m-Nis (P < 0.05 or P < 0.01). Similarly, m-Nis inhibited 5-HT-induced elevation of [Ca2+]i (P < 0.01). The mRNA expression of CaM, CaN and the activation of CaN were also inhibited by m-Nis at different degrees (P < 0.05 or P < 0.01). Thus, the results of this study suggested that Ca2+/CaM/CaN signal pathway played an important role in 5-HT-induced proliferation of rat PASMCs, the inhibition of m-Nis on proliferation of rat PASMCs may be related to the blockage of Ca2+/CaM/CaN signal pathway by inhibiting the elevation of [Ca2+]i.

Sodium current-induced release of calcium from cardiac sarcoplasmic reticulum.

The role of sodium-calcium exchange at the sarcolemma in the release of calcium from cardiac sarcoplasmic reticulum was investigated in voltage-clamped, isolated cardiac myocytes. In the absence of calcium entry through voltage-dependent calcium channels, membrane depolarization elicited release of calcium from ryanodine-sensitive internal stores. This process was dependent on sodium entry through tetrodotoxin-sensitive sodium channels. Calcium release under these conditions was also dependent on extracellular calcium concentration, suggesting a calcium-induced trigger release mechanism that involves calcium entry into the cell by sodium-calcium exchange. This sodium current-induced calcium release mechanism may explain, in part, the positive inotropic effects of cardiac glycosides and the negative inotropic effects of a variety of antiarrhythmic drugs that interact with cardiac sodium channels. In response to a transient rise of intracellular sodium, sodium-calcium exchange may promote calcium entry into cardiac cells and trigger sarcoplasmic calcium release during physiologic action potentials.

Omega-conotoxin GVIA blocks a Ca2+ current in bovine chromaffin cells that is not of the "classic" N type.

Previous studies have identified two components of whole-cell Ca2+ current in bovine chromaffin cells. The "standard" component was activated by single depolarizations, while "facilitation" could be activated by large prepulses or repetitive depolarizations. Neither current component was sensitive to changes in holding potential between -100 and -50 mV; thus neither appeared to be carried by N-type Ca2+ channels. We now report that the facilitation Ca2+ current is insensitive to omega-conotoxin GVIA (omega-CgTx), but that the toxin blocks approximately 50% of the standard Ca2+ current. In some cells the toxin blocks all of the standard Ca2+ current, in others about half of the current, while in others it has no effect. Kinetic differences in current activation are observed after toxin application. These results suggest that the standard component of chromaffin cell Ca2+ current is composed of two pharmacologically distinct channels-one is omega-CgTx sensitive and the other is not. Two kinetically distinct types of 14 pS Ca2+ channels that may correspond to the omega-CgTx-sensitive and -insensitive components were observed in single-channel experiments. Because omega-CgTx blocked Ca2+ channels that were not inactivated by a depolarized holding potential, the commonly used Ca2+ channel categorization scheme may be inadequate to describe the Ca2+ channels found in chromaffin cells.

Effects of three metabolites of propiverine on voltage-dependent L-type calcium currents in human atrial myocytes.

The non-selective muscarinic receptor antagonist propiverine impairs L-type Ca(2+) currents (I(Ca,L)) in human detrusor smooth muscle cells and atrial cardiomyocytes. Here, we have investigated the effects of three metabolites of propiverine on human cardiac I(Ca,L). Propiverine reduced I(Ca)(,L) with a -logIC(50) [M] value of 4.1, M-5 only showed minor effect on I(Ca)(,L) at high concentrations, M-6 did not influence I(Ca)(,L) at all. Like the parent compound M-14 also reduced I(Ca)(,L) (-logIC(50) [M]=4.6). We conclude, that propiverine and M-14 reduce cardiac I(Ca)(,L) at higher concentrations than in detrusor cells and therefore preferentially reduce I(Ca)(,L) in the urinary bladder than in the heart.

m-Nisoldipine attenuates monocrotaline-induced pulmonary hypertension by suppressing 5-HT/ERK MAPK pathway.

Effect of new calcium antagonist m-nisoldipine (m-Nis) on MCT-induced PH in rats and its mechanisms were investigated. Rats were injected with a single dose (60 mg x kg(-1)) of MCT subcutaneously to induce PH. Pulmonary haemodynamic measurement and lung tissue morphological investigations were undertaken. The MDA production and SOD activity in the serum were tested. PCNA, ERK1 and p-ERK expressions were analyzed by Western blotting. The expressions of 5-HT and PCNA were observed with immunohistochemistry. Results suggested that the PAP, right ventricular index and the degree of muscularization of small pulmonary artery were elevated markedly in MCT group, which was attenuated by m-Nis treatment. A significant reduction in MDA production and an increase in the SOD activity in the serum were also observed in all three m-Nis groups. The number of PCNA and 5-HT positive smooth muscle cells increased significantly in MCT group, and m-Nis treatment attenuated the expression obviously. Western blotting results suggested that the protein expression of PCNA and the ratio of p-ERK/ ERK1 increased markedly in MCT group and decreased by m-Nis. In conclusion, m-Nis protected against MCT-induced PH by decreasing PAP, right ventricular index, PAMSCs proliferation and pulmonary artery remodelling, which may be related to the reduction of 5-HT and the suppression of the ERK/MAPK signal pathway.

The inhibitor of connexin Cx36 channels, mefloquine, inhibits voltage-dependent Ca2+ channels and insulin secretion.

The antimalarial agent, mefloquine, inhibits the function of connexin Cx36 gap junctions and hemichannels and has thus become a tool to investigate their physiological relevance in pancreatic islets. In view of earlier reports on a KATP channel-block by mefloquine, the specificity of mefloquine as a pharmacological tool was investigated. Mouse pancreatic islets and single beta cells were used to measure membrane potential, whole cell currents, Ca2+ channel activity, cytosolic Ca2+ concentration ([Ca2+]i) and insulin secretion. Mefloquine was tested in the concentration range of 5-50 µM 25 µM mefloquine was as effective as 500 µM tolbutamide to depolarize the plasma membrane of beta cells, but did not induce action potentials. Rather, it abolished tolbutamide-induced action potentials and the associated increase of [Ca2+]i. In the range of 5-50 µM mefloquine inhibited voltage-dependent Ca2+ currents in primary beta cells as effectively as 1 µM nisoldipine, a specific blocker of L-type Ca2+ channels. The Ca2+ channel opening effect of Bay K8644 was completely antagonized by mefloquine. Likewise, the increase of [Ca2+]i and of insulin secretion stimulated by 40 mM KCl, but not that by 30 mM glucose was antagonized by 50 µM mefloquine. Neither at 5 µM nor at 50 µM did mefloquin stimulate insulin secretion at basal glucose. In conclusion, mefloquine blocks KATP channels and L-type Ca2+ channels in pancreatic beta cells in the range from 5 to 50 µM. Thus it inhibits depolarization-induced insulin secretion, but in the presence of a stimulatory glucose concentration additional effects of mefloquine, possibly on intracellular Ca2+ mobilization, and the metabolic amplification by glucose permit a sustained rate of secretion.

Intracellular calcium and ventricular function. Effects of nisoldipine on global ischemia in the isovolumic, coronary-perfused heart.

Ischemia-induced ventricular dysfunction has been shown to be associated with increased diastolic and systolic intracellular concentrations of free, ionized calcium ([Ca2+]i). The present study was designed to determine the effects of the Ca2+ antagonist nisoldipine on the relationship between [Ca2+]i and left ventricular contraction and relaxation during ischemia and reperfusion on a beat-to-beat basis. Nine isovolumic coronary-perfused ferret hearts were made globally ischemic for 3 min and reperfused for 10 min. Ischemia and reperfusion were repeated during perfusion with a buffer containing 10(-8) M nisoldipine. From left ventricular developed pressure, time to peak pressure and time to 50% pressure decline were obtained. [Ca2+]i was determined with the bioluminescent protein aequorin. Global ischemia caused a rapid decline in contractile function and a significant increase in diastolic [Ca2+]i, from 0.35 to 0.81 microM, and in systolic [Ca2+]i, from 0.61 to 0.96 microM. During reperfusion, [Ca2+]i returned to baseline while ventricular function was still impaired. Relaxation was more affected than systolic contractile function. Nisoldipine significantly reduced the ischemia-induced rise in diastolic [Ca2+]i to 0.62 microM, and in systolic [Ca2+]i to 0.77 microM, and lessened the decrease in contractile function. Nisoldipine significantly accelerated the decline in [Ca2+]i during reperfusion and improved recovery of contractility and relaxation. These effects were associated with a significant diminution in ischemic lactate production. Taken together, our results provide direct quantitative evidence on a beat-to-beat basis that the calcium antagonist nisoldipine can ameliorate ischemia-induced abnormalities in [Ca2+]i handling, an effect that was associated with improved myocardial function during early reperfusion.

Role of nitric oxide in Ca2+ sensitivity of the slowly activating delayed rectifier K+ current in cardiac myocytes.

Sarcolemmal Ca2+ entry is a vital step for contraction of cardiomyocytes, but Ca2+ overload is harmful and may trigger arrhythmias and/or apoptosis. To maintain the amount of Ca2+ entry within an appropriate range, cardiomyocytes have feedback systems that tightly regulate ion channel activities in response to the changes in intracellular Ca2+ concentration ([Ca2+]i), thereby regulating Ca2+ entry. In guinea pig ventricular myocytes, Ca2+ ionophore, A23187, induced suppression of the L-type Ca2+ currents (I(Ca,L)) and enhancement of the slowly activating delayed rectifier K(+) currents (I(Ks)). At a low stimulation rate, I(Ca,L) suppression and I(Ks) enhancement contributed to the A23187-induced APD shortening with a similar magnitude, whereas at a high stimulation rate, I(Ks) enhancement dominantly contributed to APD shortening. I(Ks) enhancement induced by A23187 was attributable to actions of nitric oxide (NO), because they were inhibited by an inhibitor of NO synthase (NOS) and by a NO scavenger. A23187-induced alterations of APD and I(Ks) were strongly suppressed by a NOS3 inhibitor, but barely affected by a NOS1 inhibitor, suggesting that NOS3 was responsible for NO release in this phenomenon. Inhibition of calmodulin (CaM), but not Akt, blocked the enhancement of I(Ks) by A23187. Thus, CaM-dependent NOS3 activation confers the selective Ca2+-sensitivity on I(Ks). Ca2+-induced I(Ks) enhancement and resultant APD shortening potentially act as a physiological regulatory mechanism of Ca2+ recycling, because they were observed at a physiological range of [Ca2+]i in cardiac myocytes and are induced by physiologically relevant Ca2+ loading, such as digitalis application and rise in extracellular Ca2+ concentration.

Adenoviral-mediated expression of dihydropyridine-insensitive L-type calcium channels in cardiac ventricular myocytes and fibroblasts.

Cardiac voltage-gated Ca2+ channels regulate the intracellular Ca2+ concentration and are therefore essential for muscle contraction, second messenger activation, gene expression and electrical signaling. As a first step in accessing the structural versus functional properties of the L-type Ca2+ channel in the heart, we have expressed a dihydropyridine (DHP)-insensitive CaV1.2 channel in rat ventricular myocytes and fibroblasts. Following isolation and culture, cells were infected with adenovirus expressing either LacZ or a mutant CaV1.2 channel (CaV1.2DHPi) containing the double mutation (T1039Y & Q1043M). This mutation renders the channel insensitive to neutral DHP compounds such as nisoldipine. The whole-cell, L-type Ca2+ current (ICa) measured in control myocytes was inhibited in a concentration-dependent manner by nisoldipine with an IC50 of 66 nM and complete block at 250 nM. In contrast, ICa in cells infected with AdCaV1.2DHPi was inhibited by only 35% by 500 nM nisoldipine but completely blocked by 50 microM diltiazem. In order to study CaV1.2DHPi in isolation, myocytes infected with AdCaV1.2DHPi were incubated with nisoldipine. Under this condition the cells expressed a large ICa (12 pA/pF) and displayed Ca2+ transients during field stimulation. Furthermore, addition of 2 microM forskolin and 100 microM 3-isobutyl-1-methylxanthine (IBMX), to stimulate protein kinase A, strongly increased IBa in the AdCaV1.2DHPi-infected cells. A Cd2+-sensitive IBa was also recorded in cardiac fibroblasts infected with AdCaV1.2DHPi. Thus, expression of CaV1.2DHPi will provide an important tool in studies of cardiac myocyte and fibroblast function.

Pharmacologic agents in the management of hypertension--nisoldipine coat-core.

Nisoldipine coat-core (CC), a 1,4-dihydropyridine calcium antagonist, is indicated for the treatment of hypertension and may be used alone or in combination with other antihypertensive agents. The CC technology allows for extended delivery of the drug and once-daily dosing. Nisoldipine CC tablets are absorbed across the entire gastrointestinal tract, including the colon. Eighty percent of the total dose is in the slow-release outer coat, while the core has immediate-release characteristics suitable for absorption in the distal gastrointestinal tract. Numerous double-blind, randomized studies of this agent have been done in patients with hypertension. The use of nisoldipine CC reduced both clinic and ambulatory blood pressure to a similar degree when compared with angiotensin-converting enzyme (ACE) inhibitors, beta-blockers, and the calcium antagonists amlodipine and felodipine. The drug has also been studied in hypertensive African Americans and demonstrated equivalent efficacy to amlodipine. Tolerability of the drug is good, with the most common side effect of edema at a rate similar to other dihydropyridine calcium antagonists. Thus, results of more than a decade of clinical trial data support the use of nisoldipine CC as once-daily therapy for the treatment of hypertension.

Telmisartan improves insulin sensitivity in nondiabetic patients with essential hypertension.

Hypertension is a cardiovascular risk factor commonly associated with insulin resistance, the metabolic syndrome, and type 2 diabetes mellitus. Recent in vitro data indicate that certain angiotensin receptor antagonists, for example, telmisartan, activate peroxisome proliferator-activated receptor gamma (PPAR-gamma) and increase adiponectin protein content in adipocytes. By this means, they may improve insulin sensitivity in vivo. To investigate the effect of antihypertensive treatment on insulin sensitivity and fasting adiponectin serum levels, 37 nondiabetic patients with essential hypertension were randomized to receive telmisartan, the calcium channel blocker nisoldipine, or their combination for 6 weeks in a prospective, parallel group study. Fasting serum glucose, insulin, and adiponectin were evaluated before, 3 weeks (low dose), and 6 weeks (high dose) after initiation of treatment. Furthermore, the effect of telmisartan on PPAR-gamma receptor activity was investigated in vitro using a PPAR-gamma reporter gene assay. As reported previously, telmisartan significantly enhanced PPAR-gamma receptor activity in vitro. At baseline, a positive correlation between insulin serum levels and body mass index of investigated subjects was observed, whereas body mass index and serum adiponectin levels were negatively associated. High-dose treatment with telmisartan but not with nisoldipine reduced serum insulin levels as well as the homeostasis model assessment of insulin resistance, but did not affect serum adiponectin levels. In conclusion, in our study cohort of nondiabetic patients with essential hypertension, telmisartan improved insulin sensitivity by mechanisms apparently not involving adiponectin induction. Future studies will demonstrate whether these telmisartan-induced effects may contribute to a blood pressure-independent reduction in cardiovascular morbidity.

Coupling of Ca(2+) to CREB activation and gene expression in intact cerebral arteries from mouse : roles of ryanodine receptors and voltage-dependent Ca(2+) channels.

Pathological changes of the vasculature are characterized by changes in Ca(2+) handling and alterations in gene expression. In neurons and other cell types, [Ca(2+)](i) often drives changes in gene expression. However, the relationship between Ca(2+) signaling and gene expression in vascular smooth muscle is not well understood. This study examines the ability of Ca(2+) influx through voltage-dependent, L-type Ca(2+) channels (VDCCs) and Ca(2+) release through ryanodine receptors (RyRs) to activate the transcription factor, cAMP-responsive element binding protein (CREB), and increase c-fos levels in intact cerebral arteries. Membrane depolarization increased the fraction of nuclei staining for phosphorylated CREB (P-CREB) and levels of c-fos mRNA in intact mouse cerebral arteries. Ryanodine, which inhibits RyRs, increased P-CREB staining and c-fos levels. Forskolin, an activator of adenylyl cyclase, and sodium nitroprusside, an NO donor, increased P-CREB and c-fos levels. Nisoldipine, an inhibitor of VDCCs, reversed the effects of depolarization and ryanodine on P-CREB and c-fos levels, but not the effects of forskolin or sodium nitroprusside. Inhibition of Ca(2+)/calmodulin-dependent protein kinase (CaM kinase) blocked increases in P-CREB and c-fos levels seen with membrane depolarization, suggesting that CaM kinase has an important role in the pathway leading from Ca(2+) influx to CREB-mediated changes in c-fos levels. Our data suggest that membrane depolarization increases [Ca(2+)](i) through activation of VDCCs, leading to increased P-CREB and c-fos, and that RyRs have a profound effect on this pathway by indirectly regulating Ca(2+) entry through VDCCs. These results provide the first evidence of Ca(2+) regulation of CREB and c-fos in arterial smooth muscle.