Entry Blockers Research Articles

Role of Elevated Heart Rate in the Development of Cardiovascular Disease in Hypertension

That elevated heart rate (HR) is a risk factor for cardiovascular morbidity and mortality in healthy people as well as in patients with cardiac diseases is supported by numerous epidemiological association studies.1–4 Increased HR has been recognized as a negative prognostic factor independent of many other clinical parameters that can influence the HR, including physical activity scores, left ventricular function, or use of β-blockers. Thus, HR appears to satisfy all epidemiological criteria for being considered as a true risk factor, and its predictive value for cardiovascular disease appeared to be as strong as that of most important cardiovascular risk factors. This is particularly true for the results obtained in hypertensive patients. Elevated HR is a common feature among hypertensive individuals.1 Among the young hypertensive subjects participating in the HARVEST study, >15% had a baseline resting HR ≥85 bpm and 27% had a HR ≥80 bpm.5 According to the Tensiopulse study, which evaluated 38 145 patients cared for by 2000 general practitioners all across Italy, >30% of the hypertensive patients had a resting HR ≥80 bpm.6 In a large French population, untreated hypertensive subjects had approximately a 6-bpm faster HR than normotensive individuals.7 Elevated HR is frequently associated with high blood pressure (BP) and metabolic disturbances and increases the risk of new onset hypertension and diabetes.1 Many experimental data obtained both in animals and in human beings support the importance of HR as a true risk factor for atherosclerosis and cardiovascular disease, providing convincing evidence for this pathogenetic mechanism.1–3 The pathogenetic connection between HR and cardiovascular disease has been discussed in several reports1–3,8,9 and is beyond the scope of this review. ### High HR as a Precursor of Hypertension, Obesity, and Diabetes Numerous studies have demonstrated that tachycardia is frequently associated with hypertension in …

Preliminary Finding on a New Calcium Channel Entry Blocker Chemotype: 5,6-Diamino-4-hydroxy-2-mercaptopyrimidine Derivatives

We report the preliminary in vitro characterization of a series of pyrimidines as a new chemotype that modulates cardiovascular parameters and relaxes ileum smooth muscle according to classical calcium entry blockers. Tested compounds showed an interesting negative inotropic selectivity. In patch-clamp experiments they block L- over T-type calcium currents. Two requisites seem essential for the activity: lipophilic substituents in positions 2 and 5 of the pyrimidine ring and the acetamidic function in position 6.

Orai1-Mediated Calcium Entry Controls the Cytosolic Calcium Concentration and SR Calcium Loading in Quiescent Myotubes

In the absence of store depletion plasmalemmal Ca2+ permeability in resting muscle is very low and its contribution in the maintenance of Ca2+ homeostasis at rest has not been studied in detail. Junctophilin 1 knockout myotubes (JP1 KO) have a severe reduction in Store Operated Ca2+ Entry (SOCE), presumably caused by physical alteration of the sarcoplasmic reticulum (SR) and T tubule junction leading to disruption of the SR signal sent by Stim1 to activate Orai1. Using JP1 KO myotubes as a model, we assessed the contribution of Orai1 mediated Ca2+ entry pathway on the overall Ca2+ homeostasis at rest with no store depletion. JP1 KO myotubes have decreased Ca2+ entry, myoplasmic [Ca2+]rest and SR Ca2+ content compared to Wt myotubes and unlike Wt myotubes, are refractory to BTP2, a Ca2+ entry blocker. JP1 KO myotubes show down regulation of Orai1 and STIM1 proteins suggesting that this pathway may be important in the control of resting Ca2+ homeostasis. Wt myotubes stably transduced with Orai1(E190Q) had similar alterations in their resting Ca2+ homeostasis as JP1 KO myotubes and were also unresponsive to BTP2. JP1 KO cells show decreased expression of TRPC1 and 3 but over-express TRPC4 and 6, on the other hand the TRPCs expression profile in Orai1(E190Q) myotubes was comparable to Wt. These data suggest that an important fraction of the resting plasmalemmal Ca2+ permeability is mediated by the Orai1 pathway, which contributes to the control of [Ca2+]rest and resting Ca2+ stores and that this pathway is defective in JP1 KO myotubes.

R5X4 HIV-1 coreceptor use in primary target cells: implications for coreceptor entry blocking strategies

Entry coreceptor use by HIV-1 plays a pivotal role in viral transmission, pathogenesis and disease progression. In many HIV-1 infected individuals, there is an expansion in coreceptor use from CCR5 to include CXCR4, which is associated with accelerated disease progression. While targeting HIV-1 envelope interactions with coreceptor during viral entry is an appealing approach to combat the virus, the methods of determining coreceptor use and the changes in coreceptor use that can occur during disease progression are important factors that may complicate the use of therapies targeting this stage of HIV-1 replication. Indicator cells are typically used to determine coreceptor use by HIV-1 in vitro, but the coreceptors used on these cells can differ from those used on primary cell targets. V3 based genetic sequence algorithms are another method used to predict coreceptor use by HIV-1 strains. However, these algorithms were developed to predict coreceptor use in cell lines and not primary cells and, furthermore, are not highly accurate for some classes of viruses. This article focuses on R5X4 HIV-1, the earliest CXCR4-using variants, reviewing the pattern of coreceptor use on primary CD4+ lymphocytes and macrophages, the relationship between primary cell coreceptor use and the two principal approaches to coreceptor analysis (genetic prediction and indicator cell phenotyping), and the implications of primary cell coreceptor use by these strains for treatment with a new class of small molecule antagonists that inhibit CCR5-mediated entry. These are important questions to consider given the development of new CCR5 blocking therapies and the prognosis associated with CXCR4 use.

Identification of the minimal active sequence of an anti-influenza virus peptide.

The antiviral peptide, entry blocker (EB), inhibits influenza virus replication by preventing attachment to cells. Here, we identified the minimal and optimal EB sequence that retained antiviral activity with a 50% inhibitory concentration (IC(50)) and 50% effective concentration (EC(50)) similar to those of the full-length EB peptide and several truncated variants that possessed up to 10-fold lower IC(50)s. These data have implications for improving the antiviral efficacy of EB-derived peptides while decreasing production costs and easing synthesis.

Impaired Orai1-mediated Resting Ca2+ Entry Reduces the Cytosolic [Ca2+] and Sarcoplasmic Reticulum Ca2+ Loading in Quiescent Junctophilin 1 Knock-out Myotubes

In the absence of store depletion, plasmalemmal Ca(2+) permeability in resting muscle is very low, and its contribution in the maintenance of Ca(2+) homeostasis at rest has not been studied in detail. Junctophilin 1 knock-out myotubes (JP1 KO) have a severe reduction in store-operated Ca(2+) entry, presumably caused by physical alteration of the sarcoplasmic reticulum (SR) and T-tubule junction, leading to disruption of the SR signal sent by Stim1 to activate Orai1. Using JP1 KO myotubes as a model, we assessed the contribution of the Orai1-mediated Ca(2+) entry pathway on overall Ca(2+) homeostasis at rest with no store depletion. JP1 KO myotubes have decreased Ca(2+) entry, [Ca(2+)](rest), and intracellular Ca(2+) content compared with WT myotubes and unlike WT myotubes, are refractory to BTP2, a Ca(2+) entry blocker. JP1 KO myotubes show down-regulation of Orai1 and Stim1 proteins, suggesting that this pathway may be important in the control of resting Ca(2+) homeostasis. WT myotubes stably transduced with Orai1(E190Q) had similar alterations in their resting Ca(2+) homeostasis as JP1 KO myotubes and were also unresponsive to BTP2. JP1 KO cells show decreased expression of TRPC1 and -3 but overexpress TRPC4 and -6; on the other hand, the TRPC expression profile in Orai1(E190Q) myotubes was comparable with WT. These data suggest that an important fraction of resting plasmalemmal Ca(2+) permeability is mediated by the Orai1 pathway, which contributes to the control of [Ca(2+)](rest) and resting Ca(2+) stores and that this pathway is defective in JP1 KO myotubes.

Interrelationships between macro and microvascular structure and function

Macrovasculature and microvasculature are interrelated, and may influence each other. Microvascular structure is not only the site of vascular resistance but probably also the origin of most of the wave reflections generating increased central systolic blood pressure. Some index of large artery stiffness seems to be related with the media to lumen ratio of subcutaneous small resistance arteries of hypertensive patients. In addition, both microvascular structural alterations and changes in the mechanical properties of large arteries represent potent predictors of prognosis. In addition, drugs that may improve microvascular structure may also correct alterations in the mechanical properties of large arteries In fact, while β-blocking agents and diuretics have a negligible effect on microvascular structure, renin-angiotensin system antagonists and calcium entry blockers have favorable actions, improving also large artery mechanics and possibly reducing central wave reflections.

The Cardiovascular Nutrapharmacology of Resveratrol: Pharmacokinetics, Molecular Mechanisms and Therapeutic Potential

Red wine contains many compounds that may have therapeutic use, including resveratrol (3,4',5-trihydroxytrans-stilbene). Since resveratrol could be administered both in the diet and as a therapeutic agent, defining appropriate concentrations requires understanding of the pharmacokinetics. Resveratrol absorption is rapid but plasma concentrations are low as it is rapidly and efficiently converted into relatively hydrophilic phase-2 conjugates, and metabolites, which are then rapidly excreted via the urine and bile. Resveratrol is an effective antioxidant in vivo by increasing NO synthesis and also maintaining the reduced intracellular redox state via the thioredoxin system. Further, activation of sirtuins (one class of lysine deacetylases) may mediate the cardiovascular responses shown by resveratrol. Studies on animal models of human disease suggest that resveratrol has the potential to decrease cardiovascular symptoms in patients with myocardial infarction, arrhythmias, hypertension, cardiomyopathies, fibrosis, atherosclerosis, thrombosis and diabetes, but, as yet, human clinical trials are rare. Cardioprotection by resveratrol in rodent models may rely on mechanisms producing pharmacological preconditioning in the heart including reducing reactive oxygen species, improving vasorelaxation and angiogenesis, preventing inflammation and apoptosis, delaying atherosclerosis as well as decreasing cardiovascular remodelling. Interventional studies in humans need to be completed before resveratrol can be considered as a standard therapeutic agent. Therefore, future studies should focus on obtaining the level of evidence required to determine whether resveratrol can be added to the list of evidence-based therapies for cardiovascular diseases that includes renin-angiotensin system inhibitors, beta-adrenoceptor antagonists and calcium entry blockers.

Design, synthesis and pharmacological screening of hybrid molecules as antihypertensives

The synthesis and pharmacological profile of a series of hybrid compounds bearing the dihydropyrimidine moiety and substituted phenylthioureas joined via typical β-blocker aryloxypropanolamine group are described. Seven out of 10 new compounds tested proved to be endowed with negative inotropic and chronotropic effect as compared to Adrenaline on isolated perfused frog heart. Also ECG studies that were carried out on normotensive male rats showed that the most of the compounds exhibited significant reduction in Rmax value in the first 30 min although maintaining little change in heart rate, with hypotensive effect lasting for 1 h. We concluded that the most of the synthesized novel compounds exhibited hypotensive as well as antihypertensive effects which could be attributed to blockade of Ca2+ entry and β-adrenoreceptor blocking activities due to introduction of aryloxypropanolamine linked to N-1 substituted dihydropyrimidine moiety. Brady cardiac effects of compounds 5a–5d, 6c–6e resulting from Ca2+ entry and β-adrenoreceptor blocking attenuate the sympathetic activation-associated reflex tachycardia in the heart. We selected compound 5c as a promising lead for further detailed pharmacological and preclinical evaluation studies.

ChemInform Abstract: Synthesis and Preliminary Biological Evaluation of 4,6-Disubstituted 3-Cyanopyridin-2(1H)-ones, a New Class of Calcium Entry Blockers.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

A malignant hyperthermia-inducing mutation in RYR1 (R163C): alterations in Ca2+ entry, release, and retrograde signaling to the DHPR.

Bidirectional signaling between the sarcolemmal L-type Ca2+ channel (1,4-dihydropyridine receptor [DHPR]) and the sarcoplasmic reticulum (SR) Ca2+ release channel (type 1 ryanodine receptor [RYR1]) of skeletal muscle is essential for excitation–contraction coupling (ECC) and is a well-understood prototype of conformational coupling. Mutations in either channel alter coupling fidelity and with an added pharmacologic stimulus or stress can trigger malignant hyperthermia (MH). In this study, we measured the response of wild-type (WT), heterozygous (Het), or homozygous (Hom) RYR1-R163C knock-in mouse myotubes to maintained K+ depolarization. The new findings are: (a) For all three genotypes, Ca2+ transients decay during prolonged depolarization, and this decay is not a consequence of SR depletion or RYR1 inactivation. (b) The R163C mutation retards the decay rate with a rank order WT > Het > Hom. (c) The removal of external Ca2+ or the addition of Ca2+ entry blockers (nifedipine, SKF96365, and Ni2+) enhanced the rate of decay in all genotypes. (d) When Ca2+ entry is blocked, the decay rates are slower for Hom and Het than WT, indicating that the rate of inactivation of ECC is affected by the R163C mutation and is genotype dependent (WT > Het > Hom). (e) Reduced ECC inactivation in Het and Hom myotubes was shown directly using two identical K+ depolarizations separated by varying time intervals. These data suggest that conformational changes induced by the R163C MH mutation alter the retrograde signal that is sent from RYR1 to the DHPR, delaying the inactivation of the DHPR voltage sensor.

A Small-Molecule Oxocarbazate Inhibitor of Human Cathepsin L Blocks Severe Acute Respiratory Syndrome and Ebola Pseudotype Virus Infection into Human Embryonic Kidney 293T cells

A tetrahydroquinoline oxocarbazate (PubChem CID 23631927) was tested as an inhibitor of human cathepsin L (EC 3.4.22.15) and as an entry blocker of severe acute respiratory syndrome (SARS) coronavirus and Ebola pseudotype virus. In the cathepsin L inhibition assay, the oxocarbazate caused a time-dependent 17-fold drop in IC(50) from 6.9 nM (no preincubation) to 0.4 nM (4-h preincubation). Slowly reversible inhibition was demonstrated in a dilution assay. A transient kinetic analysis using a single-step competitive inhibition model provided rate constants of k(on) = 153,000 M(-1)s(-1) and k(off) = 4.40 x 10(-5) s(-1) (K(i) = 0.29 nM). The compound also displayed cathepsin L/B selectivity of >700-fold and was nontoxic to human aortic endothelial cells at 100 muM. The oxocarbazate and a related thiocarbazate (PubChem CID 16725315) were tested in a SARS coronavirus (CoV) and Ebola virus-pseudotype infection assay with the oxocarbazate but not the thiocarbazate, demonstrating activity in blocking both SARS-CoV (IC(50) = 273 +/- 49 nM) and Ebola virus (IC(50) = 193 +/- 39 nM) entry into human embryonic kidney 293T cells. To trace the intracellular action of the inhibitors with intracellular cathepsin L, the activity-based probe biotin-Lys-C5 alkyl linker-Tyr-Leu-epoxide (DCG-04) was used to label the active site of cysteine proteases in 293T lysates. The reduction in active cathepsin L in inhibitor-treated cells correlated well with the observed potency of inhibitors observed in the virus pseudotype infection assay. Overall, the oxocarbazate CID 23631927 was a subnanomolar, slow-binding, reversible inhibitor of human cathepsin L that blocked SARS-CoV and Ebola pseudotype virus entry in human cells.

Synergism between Ca2+ and Rho‐kinase signaling during pulmonary arterial vasoconstriction in chronic hypoxic newborn lamb

Pulmonary artery (PA) contractility is mediated in large part by increases in cytosolic Ca2+, which activates myosin light chain (MLC) kinase and in turn phosphorylates myosin light chain20 (MLC20). Rho‐kinase further enhances phosphorylation of MLC20 and contractility through inhibition of MLC phosphatase. We hypothesized that Ca2+ and Rho‐kinase signaling synergistically regulate pulmonary vascular tone in chronically hypoxic (CH) newborn lambs. This hypothesis was tested by performing wire myography on serotonin (5‐HT) stimulated PA rings isolated from 12 to 17 day old lambs maintained at high altitude (3801 m) for the final 100 days of gestation and after birth (CH). 5‐HT elicited dose‐dependent contractions were attenuated with various Ca2+ entry or Rho‐kinase blockers. Voltage dependent and independent Ca2+ entry was inhibited with 30 μM KB‐R7943 (KBR), 100 μM flufenamate (FFA), 30 μM SN‐6, or 10 μM nifedipine (NIF) in the absence or presence of 50 μM SKF‐96365 (SKF). Rho‐kinase was inhibited with 10 μM fasudil or Y27632. The potency order for the Ca2+ entry antagonists was KBR ≥ NIF+SKF ≫ NIF > FFA ≫ SN‐6, whileY27632 > fasudil. Fasudil substantially augmented the inhibitory action of the Ca2+ entry blockers. These findings support the rationale for treating pulmonary hypertension with medications that target both Ca2+ and Rho‐kinase pathways. NIH P01HD031226, R01HD003807 (LDL), R01HL95973 (AB)

Genetic algorithm optimization in drug design QSAR: Bayesian-regularized genetic neural networks (BRGNN) and genetic algorithm-optimized support vectors machines (GA-SVM)

Many articles in "in silico" drug design implemented genetic algorithm (GA) for feature selection, model optimization, conformational search, or docking studies. Some of these articles described GA applications to quantitative structure-activity relationships (QSAR) modeling in combination with regression and/or classification techniques. We reviewed the implementation of GA in drug design QSAR and specifically its performance in the optimization of robust mathematical models such as Bayesian-regularized artificial neural networks (BRANNs) and support vector machines (SVMs) on different drug design problems. Modeled data sets encompassed ADMET and solubility properties, cancer target inhibitors, acetylcholinesterase inhibitors, HIV-1 protease inhibitors, ion-channel and calcium entry blockers, and antiprotozoan compounds as well as protein classes, functional, and conformational stability data. The GA-optimized predictors were often more accurate and robust than previous published models on the same data sets and explained more than 65% of data variances in validation experiments. In addition, feature selection over large pools of molecular descriptors provided insights into the structural and atomic properties ruling ligand-target interactions.

Crosstalk between adenylyl cyclase signaling pathway and Ca2+ regulatory mechanism under red blood cell microrheological changes

There are evidences that red blood cell (RBC) deformation and aggregation change under their incubation with catecholamines and it is connected with activation of intracellular signaling pathways. The present study was designed to explore the adenylyl cyclase signaling pathway and Ca2+ regulatory mechanism of RBCs together with their microrheological changes. The washed RBCs were resuspended in PBS. In each of the three research sessions RBC suspensions were divided into two aliquots: 1) control (without drug) and 2) with an appropriate drug. After cell incubation RBC deformability (RBCD) and aggregation (RBCA) were estimated. RBC incubation with catecholamines resulted in RBCD changes by 18-30%. RBCs incubation with forskolin facilitated an increase of RBCD by 17% (p < 0.05). A significant deformability rise under dB-AMP incubation was found by 27% (p < 0.01). Ca2+ cell influx, stimulated by A23187, was accompanied by an increase of RBCA; whereas red cell deformability was changed only slightly. On the other hand, Ca2+ entry blocking into the cells by verapamil has led to significant RBCA decrease and RBCD rise. The obtained results make us believe that RBCD change was closely associated with Ca2+ control mechanisms. An effect of Ca2+ concentration increase on RBC microrheology was removed, if it was preliminary added to incubation medium EGTA as Ca2+ chelator. It was found that all four PDE inhibitors: IBMX, vinpocetine, rolipram, pentoxifylline decreased RBCA significantly and, quite the contrary, they increased red cell deformability. Our data have shown that Ca2+ entry increase was accompanied by red cell aggregation rise, while adenylyl cyclase-cAMP system stimulation led to red cell deformability increase and its aggregation lowered. The crosstalk between two intracellular signaling systems is probably connected with phosphodiesterase activity.

Vascular remodeling, macro- and microvessels: Therapeutic implications

Macrovasculature and microvasculature are deeply interrelated, since microvascular structure is not only the site of vascular resistance but probably also the origin of most of the wave reflections generating increased central systolic blood pressure. In fact, preliminary data suggest that some index of large artery stiffness is related with the media to lumen ratio of subcutaneous small resistance arteries of hypertensive patients. Microvascular structural alterations and changes in the mechanical properties of the macrovessels represent potent predictors of prognosis. Hypertension-related damage to the micro- and macrovascular system may be corrected by pharmacological agents. Among them, beta-blocking agents and diuretics have a negligible effect on microvascular structure, while renin–angiotensin system antagonists and calcium entry blockers have favorable actions, improving large artery mechanics and possibly reducing central wave reflections.

Effect of capsaicin on Ca2+ fluxes in Madin‐Darby canine renal tubular cells

AbstractThe effect of capsaicin, a transient receptor potential vanniloid‐1 (TRPV1) receptor agonist, on cytosolic free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells is unclear. This study explored whether capsaicin changed basal [Ca2+]i levels in suspended MDCK cells by using fura‐2 as a Ca2+‐sensitive fluorescent dye. Capsaicin at concentrations between 10–100 µM increased [Ca2+]i in a concentration‐dependent manner. The Ca2+ signal was reduced by 80% by removing extracellular Ca2+. Capsacin induced Mn2+ influx, leading to quench of fura‐2 fluorescence suggesting Ca2+ influx. This Ca2+ influx was inhibited by phospholipase A2 inhibitor aristolochic acid and the non‐selective Ca2+ entry blocker La3+, but not by store‐operated Ca2+ channel blockers nifedipine, econazole, and SK&amp;F96365, and protein kinase C/A modulators. In Ca2+‐free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished capsaicin‐induced Ca2+ release. Conversely, pretreatment with capsaicin partly reduced thapsigargin‐induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not alter capsaicin‐induced [Ca2+]i rise. The TRPV1 receptor antagonist capsazepine also induced significant Ca2+ entry and Ca2+ release. Collectively, in MDCK cells, capsaicin induced [Ca2+]i rises by causing phospholipase C‐independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via phospholipase A2‐regulated, La3+‐sensitive Ca2+ channels in a manner dissociated from stimulation of TRPV1 receptors. Drug Dev Res, 2009. © 2009 Wiley‐Liss, Inc.

The Metabolism of a Calcium Entry Blocker, Arylalkylamine Analogue, in the In-Vitro Rat Hepatic System

The In vitro metabolism of an arylalkylamine analogue (McN-5691), a calcium entry blocker, was conducted after 0 min and 60 min incubations with the rat hepatic S9 fraction in the presence of an NADPH-generating system. Unchanged McN-5691 (31% of the sample) plus 15 metabolites from the 60min incubation were profiled, quantified, and tentatively identified on the basis of API-MS and MS/MS data. The formation McN-5691 metabolites are via the following four metabolic pathways: A. N-demethylation, B. O-demethylation, C. phenylhydroxylation, and D. oxidative N-dealkylation. Pathways A to C formed 9 major/moderate/minor metabolites, N-desmethyl (MI, 42% of the sample), 4-O-desmethyl (M2, 6%), 4'-O-desmethyl (M3, 6%), OH-phenyl (M4, 1%), O,O-didesmethyl (M5, 1%), and OH-phenyl-M1 (M6, 1%) McN-5691s, and 3 other minor phenylhydroxyl/N-desmethyl/O-desmethyl-combined McN-5691 metabolites (M7-M9, ≤ 1%). Pathway C produced a N-dealkylated metabolite, M10 (2%); in conjunction with pathways A to C yielded 5 minor oxidized N-dealkylated metabolites M11-M15 (each, ≤ 1-2%). Rat appeared to metabolize McN-5691 extensively in this hepatic system.

8-Bromo-cyclic inosine diphosphoribose: towards a selective cyclic ADP-ribose agonist

cADPR (cyclic ADP-ribose) is a universal Ca2+ mobilizing second messenger. In T-cells cADPR is involved in sustained Ca2+ release and also in Ca2+ entry. Potential mechanisms for the latter include either capacitative Ca2+ entry, secondary to store depletion by cADPR, or direct activation of the non-selective cation channel TRPM2 (transient receptor potential cation channel, subfamily melastatin, member 2). Here we characterize the molecular target of the newly-described membrane-permeant cADPR agonist 8-Br-N1-cIDPR (8-bromo-cyclic IDP-ribose). 8-Br-N1-cIDPR evoked Ca2+ signalling in the human T-lymphoma cell line Jurkat and in primary rat T-lymphocytes. Ca2+ signalling induced by 8-Br-N1-cIDPR consisted of Ca2+ release and Ca2+ entry. Whereas Ca2+ release was sensitive to both the RyR (ryanodine receptor) blocker RuRed (Ruthenium Red) and the cADPR antagonist 8-Br-cADPR (8-bromo-cyclic ADP-ribose), Ca2+ entry was inhibited by the Ca2+ entry blockers Gd3+ (gadolinium ion) and SKF-96365, as well as by 8-Br-cADPR. To unravel a potential role for TRPM2 in sustained Ca2+ entry evoked by 8-Br-N1-cIDPR, TRPM2 was overexpressed in HEK (human embryonic kidney)-293 cells. However, though activation by H2O2 was enhanced dramatically in those cells, Ca2+ signalling induced by 8-Br-N1-cIDPR was almost unaffected. Similarly, direct analysis of TRPM2 currents did not reveal activation or co-activation of TRPM2 by 8-Br-N1-cIDPR. In summary, the sensitivity to the Ca2+ entry blockers Gd3+ and SKF-96365 is in favour of the concept of capacitative Ca2+ entry, secondary to store depletion by 8-Br-N1-cIDPR. Taken together, 8-Br-N1-cIDPR appears to be the first cADPR agonist affecting Ca2+ release and secondary Ca2+ entry, but without effect on TRPM2.

The Effect of Long-Term Verapamil Treatment on the Secretion of Cortisol and Aldosterone in Subjects with Normal and High Blood Pressure

Based on their previous observations of the stimulating action of verapamil on the secretory reserve of cortisol after one-week oral administration to healthy volunteers, the author investigated the effect of this calcium entry blocker on the adrenocortical function (cortisol and aldosterone secretion) during therapeutic administration to patients with mild arterial hypertension and normotensive patients suffering from Raynaud's syndrome. Verapamil, 3-4 X 80 mg per day by the oral route, did not lead to significant changes of cortisol levels at rest nor after ACTH stimulation when assessed at the end of the first month of treatment in both groups. However, normotensive subjects with Raynaud's syndrome showed a decline of ACTH stimulated cortisolaemia during the 3rd month (p less than 0.05) and 4th month (p less than 0.01) and a reduction of cortisol secretory reserve (delta cortisol) during the 3rd month of treatment (p less than 0.05). In aldosterone secretion of normotensives with Raynaud's syndrome no significant changes were observed. In hypertensive subjects, the decline of ACTH stimulated cortisolaemia at the end of the 3rd and 4th month was not significant and delta cortisol did not change. The initial aldosterone levels before and after ACTH and delta aldosterone before treatment of hypertensives was lower as compared with normotensives (p less than 0.01, p less than 0.01 and p less than 0.05, respectively), and increased gradually during treatment in the 4th month significantly (p less than 0.05, p less than 0.01 and p less than 0.01, respectively). The hypotensive effect of verapamil persisted throughout the treatment.(ABSTRACT TRUNCATED AT 250 WORDS)