Changes in Aortic Pulse Wave Velocity in Hypertensive Postmenopausal Women: Comparison Between a Calcium Channel Blocker vs Angiotensin Receptor Blocker Regimen.

J Clin Hypertens (Greenwich). 2012;14:773-778. ©2012 Wiley Periodicals, Inc. Postmenopausal women are at greater risk for hypertension-related cardiovascular disease. Antihypertensive therapy may help alleviate arterial stiffness that represents a potential modifiable risk factor of hypertension. This randomized controlled study investigated the difference between an angiotensin receptor blocker and a calcium channel blocker in reducing arterial stiffness. Overall, 125 postmenopausal hypertensive women (age, 61.4±6 years; systolic blood pressure/diastolic blood pressure [SBP/DBP], 158±11/92±9 mm Hg) were randomized to valsartan 320 mg±hydrochlorothiazide (HCTZ) (n=63) or amlodipine 10 mg±HCTZ (n=62). The primary outcome was carotid-to-femoral pulse wave velocity (PWV) changes after 38 weeks of treatment. Both treatments lowered peripheral blood pressure (BP) (-22.9/-10.9 mm Hg for valsartan and -25.2/-11.7 mm Hg for amlodipine, P=not significant) and central BP (-15.7/-7.6 mm Hg for valsartan and -19.2/-10.3 mm Hg for amlodipine, P<.05 for central DBP). Both treatments similarly reduced the carotid-femoral PWV (-1.9 vs -1.7 m/s; P=not significant). Amlodipine was associated with a higher incidence of peripheral edema compared with the valsartan group (77% vs 14%, P<.001). BP lowering in postmenopausal women led to a reduction in arterial stiffness as assessed by PWV measurement. Both regimens reduced PWV to a similar degree after 38 weeks of treatment despite differences in central BP lowering, suggesting that the effect of valsartan on PWV is mediated through nonhemodynamic effects.

Changes in Aortic Pulse Wave Velocity in Hypertension Post-Menopausal Women: Comparison Between Calcium Channel Blocker (CCB) Vs Angiotensin Receptor Blocker (ARB) Regimen

Objective: To compare effects of a non-renin-angiotensin system (RAS) blocker, using a CCB, or a RAS blocker, using an ARB regimen on the arterial stiffness reduction in postmenopausal hypertensive women. Methods: In this prospective study, a total of 125 hypertensive women (age: 61.4_6 yrs; 98% Caucasian; BW: 71.9_14 kg; BMI: 27.3_5 kg/m2; SBP/ DBP: 158_11/92_9 mmHg) were randomized between ARB (valsartan 320mg_HCTZ) and CCB (amlodipine 10mg _ HCTZ). The primary outcome was carotid-femoral pulse wave velocity (PWV) changes after 38 weeks of treatment. Results: There were no significant differences in baseline demographic data between the two groups. Both treatments effectively lowered BP at the end of the study with similar (p>0.05) reductions in the valsartan (_22.9/_10.9 mmHg) and amlodipine based (_25.2/_11.7 mmHg) treatment groups. Despite a lower (p<0.05 for DBP) central SBP/DBP in the CCB group (_19.2/_10.3 mmHg) compared to the valsartan group (_15.7/_7.6 mmHg) at week 38, a similar reduction in carotid-femoral PWV (_1.7 vs _1.9 m/sec; p>0.05) was observed between both groups. The numerically larger BP reduction observed in the CCB group was associated with a much higher incidence of peripheral edema (77% vs 14%) than the valsartan group. Conclusion: In summary, BP lowering in postmenopausal women led to a reduction in arterial stiffness assessed by PWV measurement. Both regimens reduced PWV at 38 weeks of treatment to a similar degree, despite differences in BP lowering suggesting that the effect of RAS blockade to influence PWV may partly be independent of BP.

Effect of a calcium-channel blocker (verapamil) on the morphology, cytoskeleton and collagenase activity of human skin fibroblasts

The effects of verapamil modulating collagen biosynthesis have prompted us to study the role of this drug in cultured fibroblasts. In this article, we describe the effects of verapamil on fibroblast behaviour, with special emphasis to phenotypic modifications, reorganisation of actin filaments and secretion of MMP1. Human dermal fibroblasts treated with 50-mu M verapamil changed their normal spindle-shaped morphology to stellate. Treated cells showed discrete reorganisation of actin filaments, as revealed by fluorescein isothiocyanate (FITC)-phalloidin staining and confocal microscopy. We hypothesised that these effects would be associated to lower levels of cytosolic Ca(2+). Indeed, short time loading with calcium green confirmed that verapamil-treated fibroblasts exhibited lower intracellular calcium levels compared to controls. We also observed that verapamil increases the secretion of MMP1 in cultured fibroblasts, as demonstrated by zymography, specific substrate assays and immunoblot. The morphological alterations induced by verapamil are neither cytotoxic nor associated with other dramatic cytoskeleton alterations. Thus we may conclude that this drug enhances collagenase secretion and does not disrupt the major tracks necessary to deliver these enzymes in the extracellular space. The present results suggested that verapamil could be used at physiological levels to enhance collagen I breakdown, and maybe considered a potential candidate for intralesional therapy of wound healing and fibrocontractive diseases. (C) 2010 Elsevier Ltd and ISBI. All rights reserved.

Looking over Toxin-K+ Channel Interactions. Clues from the Structural and Functional Characterization of alpha-KTx Toxin Tc32, a Kv1.3 Channel Blocker

alpha-KTx toxin Tc32, from the Amazonian scorpion Tityus cambridgei, lacks the dyad motif; including Lys27, characteristic of the family and generally associated with channel blockage. The toxin has been cloned and expressed for the first time. Electrophysiological experiments, by showing that the recombinant form blocks Kv1.3 channels of olfactory bulb periglomerular cells like the natural Tc32 toxin, when tested on the Kv1.3 channel of human T lymphocytes, confirmed it is in an active fold. The nuclear magnetic resonance-derived structure revealed it exhibits an alpha/beta scaffold typical of the members of the alpha-KTx family. TdK2 and TdK3, all belonging to the same alpha-KTx 18 subfamily, share significant sequence identity with Tc32 but diverse selectivity and affinity for Kv1.3 and Kv1.1 channels. To gain insight into the structural features that may justify those differences, we used the recombinant Tc32 nuclear magnetic resonance-derived structure to model the other two toxins, for which no experimental structure is available. Their interaction with Kv1.3 and Kv1.1 has been investigated by means of docking simulations. The results suggest that differences in the electrostatic features of the toxins and channels, in their contact surfaces, and in their total dipole moment orientations govern the affinity and selectivity of toxins. In addition, we found that, regardless of whether the dyad motif is present, it is always a Lys side chain that physically blocks the channels, irrespective of its position in the toxin sequence.

Design, synthesis and pharmacological characterization of analogs of 2-aminoethyl diphenylborinate (2-APB), a known store-operated calcium channel blocker, for inhibition of TRPV6-mediated calcium transport

2-Aminoethyl diphenylborinate (2-APB) is a known modulator of the IP3 receptor, the calcium ATPase SERCA, the calcium release-activated calcium channel Orai and TRP channels. More recently, it was shown that 2-APB is an efficient inhibitor of the epithelial calcium channel TRPV6 which is overexpressed in prostate cancer. We have conducted a structure-activity relationship study of 2-APB congeners to understand their inhibitory mode of action on TRPV6. Whereas modifying the aminoethyl moiety did not significantly change TRPV6 inhibition, substitution of the phenyl rings of 2-APB did. Our data show that the diaryl borinate moiety is required for biological activity and that the substitution pattern of the aryl rings can influence TRPV6 versus SOCE inhibition. We have also discovered that 2-APB is hydrolyzed and transesterified within minutes in solution.

A novel open channel blocker of GABA-A receptors

GABA-A receptors are chloride ion channels composed of five subunits, mediating fast synaptic and tonic inhibition in the mammalian brain. 19 different subunit isoforms have been identified, with the major receptor type in mammalian adult brain consisting of α1, β2, and γ2 subunits. GABA-A receptors are the target of numerous sedating and anxiolytic drugs such as benzodiazepines. The currently known endogenous ligands are GABA, neurosteroids and the endocannabinoid 2- arachidonoyl glycerol (2-AG). The pharmacological properties of this chloride ion channel strictly depend on receptor subunit composition and arrangement. GABA-A receptors bind and are inhibited by epileptogenic agents such as picrotoxin, and cyclodiene insecticides such as dieldrin. We screened aromatic monovalent anions with five-fold symmetry for inhibition of GABA-A receptors. One of the anions, PCCPinhibited currents elicited by GABA with comparable potency as picrotoxin. This inhibition showed all characteristics of an open channel block. The GABA-A receptor ion channel is lined by residues from the M2 membrane-spanning segment. To identify important residues of the pore involved in the interaction with the blocking molecules PCCP-, a mutation scan was performed in combination with subsequent analysis of the expressed mutant proteins using electrophysiological techniques. In a second project we characterised a light-switchable modulator of GABA-A receptors based on propofol. It was my responsibility to investigate the switching kinetics in patch clamp experiments. After its discovery in 1980, propofol has become the most widely used intravenous general anaesthetic. It is commonly accepted that the anaesthesia induced by this unusually lipophilic drug mostly results from potentiation of GABA induced currents. While GABA-A receptors respond to a variety of ligands, they are normally not sensitive towards light. This light sensitivity could be indirectly achieved by using modulators that can be optically switched between an active and an inactive form. We tested an azobenzene derivative of propofol where an aryldiazene unit is directly coupled to the pharmacophore. This molecule was termed azopropofol (AP2). The effect of AP2 on Cl- currents was investigated with electrophysiological techniques using α1β2γ2 GABA-A receptors expressed in Xenopus oocytes and HEK-cells. In the third project we wanted to investigate the functional role of GABA-A receptors in the liver, and their possible involvement in cell proliferation. GABA-A receptors are also found in a wide range of peripheral tissues, including parts of the peripheral nervous system and non-neural tissues such as smooth muscle, the female reproductive system, liver and several cancer tissues. However their precise function in non neuronal or cancerous cells is still unknown. For this purpose we investigated expression, localization and function of the hepatocytes GABA-A receptors in model cell lines and healthy and cancerous hepatocytes.

A pentasymmetric open channel blocker for Cys-loop receptor channels.

γ-Aminobutyric acid type A receptors (GABAA receptors) are chloride ion channels composed of five subunits, mediating fast synaptic and tonic inhibition in the mammalian brain. These receptors show near five-fold symmetry that is most pronounced in the second trans-membrane domain M2 lining the Cl- ion channel. To take advantage of this inherent symmetry, we screened a variety of aromatic anions with matched symmetry and found an inhibitor, pentacyanocyclopentdienyl anion (PCCP-) that exhibited all characteristics of an open channel blocker. Inhibition was strongly dependent on the membrane potential. Through mutagenesis and covalent modification, we identified the region α1V256-α1T261 in the rat recombinant GABAA receptor to be important for PCCP- action. Introduction of positive charges into M2 increased the affinity for PCCP- while PCCP- prevented the access of a positively charged molecule into M2. Interestingly, other anion selective cys-loop receptors were also inhibited by PCCP-, among them the Drosophila RDL GABAA receptor carrying an insecticide resistance mutation, suggesting that PCCP- could serve as an insecticide.

Efficacy and safety of calcium channel blocker/diuretics combination therapy in hypertensive patients: a meta-analysis.

Although recent guidelines recommend the combination of calcium channel blockers (CCBs) and thiazide (-like) diuretics, this combination is not widely used in clinical practice. The aim of this meta-analysis was to assess the efficacy and safety of this combination regarding the following endpoints: all-cause and cardiovascular mortality, myocardial infarction, and stroke. Four studies with a total of 30,791 of patients met the inclusion criteria. The combination CCB/thiazide (-like) diuretic was associated with a significant risk reduction for myocardial infarction (risk ratio [RR], 0.83; 95% confidence interval [CI], 0.73-0.95) and stroke (RR, 0.77; CI, 0.64-0.92) compared with other combinations, whereas it was similarly effective compared with other combinations in reducing the risk of all-cause (RR, 0.89; CI, 0.75-1.06) and cardiovascular (RR, 0.89; CI 0.71-1.10) mortality. Elderly patients with isolated systolic hypertension may particularly benefit from such a combination, since both drug classes have been shown to confer cerebrovascular protection.

An N-methyl-d-aspartate receptor channel blocker with neuroprotective activity

Excitotoxicity, resulting from sustained activation of glutamate receptors of the N-methyl-d-aspartate (NMDA) subtype, is considered to play a causative role in the etiology of ischemic stroke and several neurodegenerative diseases. The NMDA receptor is therefore a target for the development of neuroprotective agents. Here, we identify an N-benzylated triamine (denoted as NBTA) as a highly selective and potent NMDA-receptor channel blocker selected by screening a reduced dipeptidomimetic synthetic combinatorial library. NBTA blocks recombinant NMDA receptors expressed in Xenopus laevis oocytes with a mean IC50 of 80 nM; in contrast, it does not block GluR1, a glutamate receptor of the non-NMDA subtype. The blocking activity of NBTA on NMDA receptors exhibits the characteristics of an open-channel blocker: (i) no competition with agonists, (ii) voltage dependence, and (iii) use dependence. Significantly, NBTA protects rodent hippocampal neurons from NMDA receptor, but not kainate receptor-mediated excitotoxic cell death, in agreement with its selective action on the corresponding recombinant receptors. Mutagenesis data indicate that the N site, a key asparagine on the M2 transmembrane segment of the NR1 subunit, is the main determinant of the blocker action. The results highlight the potential of this compound as a neuroprotectant.

Inhibition of the Gravitropic Response of Snapdragon Spikes by the Calcium-Channel Blocker Lanthanum Chloride1

The putative Ca2+-channel blocker LaCl3 prevented the gravitropic bending of cut snapdragon (Antirrhinum majus L.) spikes (S. Philosoph-Hadas, S. Meir, I. Rosenberger, A.H. Halevy [1996] Plant Physiol 110: 301–310) and inhibited stem curvature to a greater extent than vertical and horizontal stem elongation at the bending zone. This might indicate that LaCl3, which modulates cytosolic Ca2+, does not influence general stem-growth processes but may specifically affect other gravity-associated processes occurring at the stem-bending zone. Two such specific gravity-dependent events were found to occur in the bending zone of snapdragon spikes: sedimentation of starch-containing chloroplasts at the bottom of stem cortex cells, as seen in cross-sections, and establishment of an ethylene gradient across the stem. Our results show that the lateral sedimentation of chloroplasts associated with gravity sensing was prevented in cross-sections taken from the bending zone of LaCl3-treated and subsequently gravistimulated spikes and that LaCl3 completely prevented the gravity-induced, asymmetric ethylene production established across the stem-bending zone. These data indicate that LaCl3 inhibits stem curvature of snapdragon spikes by preventing several gravity-dependent processes. Therefore, we propose that the gravitropic response of shoots could be mediated through a Ca2+-dependent pathway involving modulation of cytosolic Ca2+ at various stages.

Calcium Channel Blocker Use and Risk of Prostate Cancer by TMPRSS2:ERG Gene Fusion Status

BACKGROUND: Calcium channel blockers (CCBs) may affect prostate cancer (PCa) growth by various mechanisms including those related to androgens. The fusion of the androgen-regulated gene TMPRSS2 and the oncogene ERG (TMPRSS2:ERG or T2E) is common in PCa, and prostate tumors that harbor the gene fusion are believed to represent a distinct disease subtype. We studied the association of CCB use with the risk of PCa, and molecular subtypes of PCa defined by T2E status.

METHODS: Participants were residents of King County, Washington, recruited for population-based case-control studies (1993-1996 or 2002-2005). Tumor T2E status was determined by fluorescence in situ hybridization using tumor tissue specimens from radical prostatectomy. Detailed information on use of CCBs and other variables was obtained through in-person interviews. Binomial and polytomous logistic regression were used to generate odds ratios (ORs) and 95% confidence intervals (CIs).

RESULTS: The study included 1,747 PCa patients and 1,635 age-matched controls. A subset of 563 patients treated with radical prostatectomy had T2E status determined, of which 295 were T2E positive (52%). Use of CCBs (ever vs. never) was not associated with overall PCa risk. However, among European-American men, users had a reduced risk of higher-grade PCa (Gleason scores ≥7: adjusted OR = 0.64; 95% CI: 0.44-0.95). Further, use of CCBs was associated with a reduced risk of T2E positive PCa (adjusted OR = 0.38; 95% CI: 0.19-0.78), but was not associated with T2E negative PCa.

CONCLUSIONS: This study found suggestive evidence that use of CCBs is associated with reduced relative risks for higher Gleason score and T2E positive PCa. Future studies of PCa etiology should consider etiologic heterogeneity as PCa subtypes may develop through different causal pathways. 

The Analgesic Effect Of Dipyrone In Peripheral Tissue Involves Two Different Mechanisms: Neuronal K(atp) Channel Opening And Cb(1) Receptor Activation.

Dipyrone (metamizole) is an analgesic pro-drug used to control moderate pain. It is metabolized in two major bioactive metabolites: 4-methylaminoantipyrine (4-MAA) and 4-aminoantipyrine (4-AA). The aim of this study was to investigate the participation of peripheral CB1 and CB2 cannabinoid receptors activation in the anti-hyperalgesic effect of dipyrone, 4-MAA or 4-AA. PGE2 (100ng/50µL/paw) was locally administered in the hindpaw of male Wistar rats, and the mechanical nociceptive threshold was quantified by electronic von Frey test, before and 3h after its injection. Dipyrone, 4-MAA or 4-AA was administered 30min before the von Frey test. The selective CB1 receptor antagonist AM251, CB2 receptor antagonist AM630, cGMP inhibitor ODQ or KATP channel blocker glibenclamide were administered 30min before dipyrone, 4-MAA or 4-AA. The antisense-ODN against CB1 receptor expression was intrathecally administered once a day during four consecutive days. PGE2-induced mechanical hyperalgesia was inhibited by dipyrone, 4-MAA, and 4-AA in a dose-response manner. AM251 or ODN anti-sense against neuronal CB1 receptor, but not AM630, reversed the anti-hyperalgesic effect mediated by 4-AA, but not by dipyrone or 4-MAA. On the other hand, the anti-hyperalgesic effect of dipyrone or 4-MAA was reversed by glibenclamide or ODQ. These results suggest that the activation of neuronal CB1, but not CB2 receptor, in peripheral tissue is involved in the anti-hyperalgesic effect of 4-aminoantipyrine. In addition, 4-methylaminoantipyrine mediates the anti-hyperalgesic effect by cGMP activation and KATP opening.

Solution structure and proposed binding mechanism of a novel potassium channel toxin kappa-conotoxin PVIIA

Background: kappa-PVIIA is a 27-residue polypeptide isolated from the venom of Conus purpurascens and is the first member of a new class of conotoxins that block potassium channels. By comparison to other ion channels of eukaryotic cell membranes, voltage-sensitive potassium channels are relatively simple and methodology has been developed for mapping their interactions with small-peptide toxins, PVIIA, therefore, is a valuable new probe of potassium channel structure. This study of the solution structure and mode of channel binding of PVIIA forms the basis for mapping the interacting residues at the conotoxin-ion channel interface. Results: The three-dimensional structure of PVIIA resembles the triple-stranded beta sheet/cystine-knot motif formed by a number of toxic and inhibitory peptides. Subtle structural differences, predominantly in loops 2 and 4, are observed between PVIIA and other conotoxins with similar structural frameworks, however. Electrophysiological binding data suggest that PVIIA blocks channel currents by binding in a voltage-sensitive manner to the external vestibule and occluding the pore, Comparison of the electrostatic surface of PVIIA with that of the well-characterised potassium channel blocker charybdotoxin suggests a likely binding orientation for PVIIA, Conclusions: Although the structure of PVIIA is considerably different to that of the alpha K scorpion toxins, it has a similar mechanism of channel blockade. On the basis of a comparison of the structures of PVIIA and charybdotoxin, we suggest that Lys19 of PVIIA is the residue which is responsible for physically occluding the pore of the potassium channel.

Prior calcium channel blockade and short-term survival following acute myocardial infarction

There is concern over the safety of calcium channel blockers (CCBs) in acute coronary disease. We sought to determine if patients taking calcium channel blockers (CCBs) at the time of admission with acute myocardial infarction (AMI) had a higher case-fatality compared with those taking beta-blockers or neither medication. Clinical and drug treatment variables at the time of hospital admission predictive of survival at 28 days were examined in a community-based registry of patients aged under 65 years admitted to hospital for suspected AMI in Perth, Australia, between 1984 and 1993. Among 7766 patients, 1291 (16.6%) were taking a CCB and 1259 (16.2%) a betablocker alone at hospital admission. Patients taking CCBs had a worse clinical profile than those taking a beta-blocker alone or neither drug (control group), and a higher unadjusted 28-day mortality (17.6% versus 9.3% and 11.1% respectively, both P < 0.001). There was no significant heterogeneity with respect to mortality between nifedipine, diltiazem, or verapamil when used alone, or with a beta-blocker. After adjustment for factors predictive of death at 28 days, patients taking a CCB were found not to have an excess chance of death compared with the control group (odds ratio [OR] 1.06, 95% confidence interval [CI]; 0.87, 1.30), whereas those taking a beta-blocker alone had a lower odds of death (OR 0.75, 95% CI; 0.59, 0.94). These results indicate that established calcium channel blockade is not associated with an excess risk of death following AMI once other differences between patients are taken into account, but neither does it have the survival advantage seen with prior beta-blocker therapy.