Calcium channel block by Diltiazem

Der Einstrom von Calcium durch spannungsabhängige Cav1.2 Kanäle reguliert Erregung und Kontraktilität der Muskulatur, die Freisetzung von Hormonen und Neurotransmittern aus sekretorischen Zellen oder Nervenzellen und eine Vielzahl anderer zellulärer Prozesse. Dieser Calciumeinstrom kann durch Calciumkanalantagonisten dreier verschiedener Klassen, Phenylalkylamine (PAA), 1,4 Dihydropyridine (DHPs), und Benzothiazepine (BTZs) inhibiert werden. Diltiazem (Dil), ein Vertreter der Benzothiazepine, wird bereits lange Zeit therapeutisch verwendet. Bislang konnten jedoch die molekularen Wirkungsmechanismen und der Zugangsweg („access path“) von Diltiazem zu seiner hochaffinen Bindungsstelle in Cav1.2 nicht geklärt werden. Um den extra- bzw. intrazellulären Zugang von Dil zu seiner Bindungsstelle zu untersuchen, wurde das quaternäre Diltiazem-Analogon qDil synthetisiert und entweder extrazellulär oder intrazellulär appliziert. Die intrazelluläre Applikation erzeugte einen konzentrationsabhängigen ‚Use-dependent Block‘, was einen intrazellulären Zugang zur Bindungsstelle nahelegt. Darüber hinaus konnte durch meine Studien mittels Mutationsanalyse und molekularem Modeling eine neue, hoch affine, Bindungsdeterminante von Dil identifiziert werden. Die Substitution von Threonin durch Alanin an der Position 1143 (T1143A) im Cav1.2 verringert den Block von qDil sowohl bei nieder- (0,2 Hz) als auch hochfrequenten (1 Hz) Depolarisationspulsen. Außerdem reduziert T1143A den Block von dem klinisch verwendetem tertiären Diltiazem und quarternären Phenylakylaminen (qDevapamil). T1143 hatte weder Einfluss auf die Aktivierung noch die Inaktivierung der Cav1.2 Kanäle. Die reduzierte Wirkung von Dil an der T1143A Mutante ist somit nicht über allosterische Mechanismen (Konformationsänderungen des Moleküls) zu erklären. Diese Befunde stützen die Hypothese, dass T1143 eine wichtige Bindungsdeterminante von Dil auf Cav1.2 darstellt.Calcium fluxes through CaV1.2 (L-type) channels determine cellular excitability and initiate contractions of muscle cells, release of hormones and neurotransmitters from secretory and nerve cells, gene expression, and many other cellular processes. A drug that blocks calcium influx through CaV1.2 is the benzothiazepinone (BTZ) diltiazem. Although diltiazem has been in clinical use for a long time, its molecular mechanisms and its access pathway to its binding site in CaV1.2 are not fully understood. To identify the access route of diltiazem to its putative binding site, the quaternary diltiazem analog qDil was synthesised and applied to either the extra- or intracellular site of the membrane. Intracellularly applied qDil induced a concentration- and use-dependent block suggesting an intracellular access path. During my studies a novel high affinity qDil binding site was identified by molecular modelling and mutational analysis. Substitution of threonine to alanine in position 1143 (T1143A) of the α1-subunit of CaV1.2 diminished the qDil block at low (0.2Hz) as well as high frequency (1Hz) depolarization pulses. Mutation T1143A also reduced channel block by the clinically used tertiary diltiazem and a quaternary PAA (qDevapamil). T1143A affected neither activation nor inactivation of CaV1.2, supporting the view that this residue forms part of the diltiazem binding pocket on CaV1.2

Apigenin and Structurally Related Flavonoids Allosterically Potentiate the Function of Human α7-Nicotinic Acetylcholine Receptors Expressed in SH-EP1 Cells

Phytochemicals, such as monoterpenes, polyphenols, curcuminoids, and flavonoids, are known to have anti-inflammatory, antioxidant, neuroprotective, and procognitive effects. In this study, the effects of several polyhydroxy flavonoids, as derivatives of differently substituted 5,7-dihydroxy-4H-chromen-4-one including apigenin, genistein, luteolin, kaempferol, quercetin, gossypetin, and phloretin with different lipophilicities (cLogP), as well as topological polar surface area (TPSA), were tested for induction of Ca2+ transients by α7 human nicotinic acetylcholine (α7 nACh) receptors expressed in SH-EP1 cells. Apigenin (10 μM) caused a significant potentiation of ACh (30 μM)-induced Ca2+ transients, but did not affect Ca2+ transients induced by high K+ (60 mM) containing solutions. Co-application of apigenin with ACh was equally effective as apigenin preincubation. However, the effect of apigenin significantly diminished by increasing ACh concentrations. The flavonoids tested also potentiated α7 nACh mediated Ca2+ transients with descending potency (highest to lowest) by genistein, gossypetin, kaempferol, luteolin, phloretin, quercetin, and apigenin. The specific binding of α7 nACh receptor antagonist [125I]-bungarotoxin remained unchanged in the presence of any of the tested polyhydroxy flavonoids, suggesting that these compounds act as positive allosteric modulators of the α7-nACh receptor in SH-EP1 cells. These findings suggest a clinical potential for these phytochemicals in the treatment of various human diseases from pain to inflammation and neural disease

Non Incretin Effects of DPP-4 Inhibitors: Comparative Study

Background: Diabetes mellitus is a metabolic syndrome that adversely affects all parts of the body especially the cardiovascular system. Dipeptidyl peptidase-4 (DPP-4) is a multifunctional protein and its inhibition has diverse effects. DPP-4 inhibition was shown to improve the survival rate after myocardial infarction in mice. Beneficial myocardial metabolic effects of DPP-4 inhibitors have been observed in experimental models. Cardiovascular outcome trials of DPP-4 inhibitors show variable adverse cardiovascular events. Objective: This experimental study was aimed to study the direct cardiovascular effects of DPP-4 Inhibitors on chronotropicity (Heart Rate, HR), inotropicity (Apical Force and; dP/dt(max), ECG and Coronary Flow(C.F) and detect its potential useful and harmful effects on cardiovascular parameters. Methods: The effects of graded doses (10-9 - 10-6M) of Sitagliptin (S) and Vildagliptin(V) were observed on retrograde perfused isolated rabbit hearts with warm Krebs-Henseliet solution on Radnoti working heart system. Fifty four(54) rabbits were grouped into nine groups i.e ; I(S1), II(S2), III(S3), IV(S4), V(S5), VI(S6), VII(V1), and VIII(V2) and IX(V3) each comprising of six animals(n=6).Effects of experimental drugs were observed on chronotropicity(HR), inotropicity (Apical Force and Peak rate of rise of LVP i.e; dP/dt(max) and Coronary flow(CF). The results were statistically analyzed with Graph Pad Grism and wherever necessary paired or unpaired “t” test was applied. Conclusion: Sitagliptin and Vildagliptin both have suppressant effects on HR. Sitagliptin has positive and Vildagliptin had negative inotropic effects. Both drugs (10-8 - 10-6M); decrease coronary flow but have no significant effect on ECG

Capsaicin Inhibits Multiple Voltage-Gated Ion Channels in Rabbit Ventricular Cardiomyocytes in TRPV1-Independent Manner

Capsaicin is a naturally occurring alkaloid derived from chili pepper which is responsible for its hot, pungent taste. It exerts multiple pharmacological actions, including pain-relieving, anti-cancer, anti-inflammatory, anti-obesity, and antioxidant effects. Previous studies have shown that capsaicin significantly affects the contractility and automaticity of the heart and alters cardiovascular functions. In this study, the effects of capsaicin were investigated on voltage-gated ion currents in rabbit ventricular myocytes. Capsaicin inhibited rapidly activated (IKr) and slowly activated (IKs) K+ currents and transient outward (Ito) K+ current with IC50 values of 3.4 µM,14.7 µM, and 9.6 µM, respectively. In addition, capsaicin, at higher concentrations, suppressed voltage-gated Na+ and Ca2+ currents and inward rectifier IK1 current with IC50 values of 42.7 µM, 34.9 µM, and 38.8 µM, respectively. Capsaicin inhibitions of INa, IL-Ca, IKr, IKs, Ito, and IK1 were not reversed in the presence of capsazepine (3 µM), a TRPV1 antagonist. The inhibitory effects of capsaicin on these currents developed gradually, reaching steady-state levels within 3 to 6 min, and the recoveries were usually incomplete during washout. In concentration-inhibition curves, apparent Hill coefficients higher than unity suggested multiple interaction sites of capsaicin on these channels. Collectively, these findings indicate that capsaicin affects cardiac electrophysiology by acting on a diverse range of ion channels and suggest that caution should be exercised when capsaicin is administered to carriers of cardiac channelopathies or to individuals with arrhythmia-prone conditions, such as ischemic heart diseases

Combined Effect of Nicotine and Caffeine on Orthodontic Tooth Movement in Rats

Background: The individual effects of nicotine and caffeine have been reported in previous studies but their combined effect on tooth movement needs to be elucidated. The objective of this study was to evaluate the combined effect of nicotine and caffeine on the magnitude of orthodontic tooth movement (OTM) in rats. Material and Methods: This experimental study was conducted on Sprague-Dawley rats (Animal House and Pathology Laboratory; Post Graduate Medical Institute, Lahore) in the department of Orthodontics, de’Montmorency College of Dentistry, Lahore from 8th July 2014 to 8th January 2015. Forty male Sprague-Dawley rats were divided into four equal groups: Control group (CR), nicotine group (NT), caffeine group (CF) and combined nicotine and caffeine group (CNC). Closed coil nickel titanium (NiTi) spring was placed between incisor and maxillary molar. Nicotine group (NT) was treated by intraperitoneal injections of nicotine. Caffeine was given to caffeine group and Combined nicotine and caffeine group (CNC) was treated in the same way as individual nicotine and caffeine groups daily for 14 days. All the rats were sacrificed on 15th day. Magnitude of the orthodontic tooth movement was measured using digital Vernier caliper. Means and standard deviation were calculated for orthodontic tooth movement. One-way ANOVA was used to determine the mean difference in OTM. Post hoc Tukey test was used for multiple comparisons among the groups. Results: The mean orthodontic tooth movement (OTM) was 0.32 mm ± 0.05 in control group, 0.56 mm ± 0.04 in nicotine group, 0.52 mm ± 0.034 in caffeine group and 0.8 mm ± 0.06 in combined NC group, respectively. The difference between mean OTM among the groups was statistically significant (P-value <0.001). The mean OTM in CNC group was significantly higher as compared to other groups (CR, NT, CF, NT) (P-value <0.001). Conclusions: In rats, the combined use of nicotine and caffeine results in greater orthodontic tooth movement as compared to their individual use. Key words: Bone remodeling, Caffeine, Nicotine, Orthodontic tooth movemen

Reasons for seeking orthodontic treatment in Lahore population: A cross-sectional survey in a low-income country

ABSTRACT Introduction: Poor esthetics, dysfunction and discomfort are the key reasons for seeking orthodontic treatment across the world as reported by many researchers. This paper presents the causative factor for seeking orthodontic treatment in the patients who are visiting Punjab Dental Hospital of a populous city Lahore (de\u27 Montmorency College of Dentistry) in local settings and associating these reasons with demographic characteristics. Objective: Aim of this cross-sectional survey was to explore the reasons for seeking orthodontic treatment among individuals who are visiting PDH. Materials and methods: This study was carried out in Punjab Dental Hospital (PDH) after the approval of the Institutional Review Board (IRB) on a sample of 98 individuals having malocclusion assessed with Angle\u27s classification of the malocclusion. We chose simple random sampling. A self-structure questionnaire was designed to get data by the principal investigator after taking verbal and written consent. Descriptive statistics were calculated using SPSS 21. Chi-square test of association was applied to associate reasons with different demographic variables. P-value <0.05 was taken as significant. Results: Female respondents were more in number than males. Around one-third of respondents (30.6 %) had a monthly income of less than 25000 PKR ($ 170). Esthetics was the primary reason for seeking orthodontic treatment. The most common type of malocclusion was the Class II malocclusion. Statistically significant factors that emerged in this study that turned into reasons for seeking orthodontic treatment were hurdles in marriage, referral by a general dentist, motivation by parents, self-esteem and speech problems. Conclusion: In conclusion, patients seek orthodontic treatment mainly to enhance facial esthetics and self-confidence, motivation by the parents, and social acceptability

Phytochemical screening and antioxidant evaluation of millet varieties of Pakistan

The current research was planned to measure the comparative phytochemical and anti-oxidative potential of aqueous and methanolic extracts of two indigenously grown millet varieties of Pakistan. The locally available millet varieties, i.e. MB-87 and AF-POP flours were chemically characterized through the proximate and mineral analysis. The antioxidant extract was prepared in water and methanol. The extracts were screened for saponins, terpenoids, flavonoids and tannins; methanolic extract of MB-87 and AF-POP showed flavonoids in an average quantity (++), whilst aqueous extract had only trace (+) values. The phytochemical screening showed the presence of saponins only in the aqueous extract of both varieties. However, terpenoids and tannins were present in both methanolic and aqueous extracts. During antioxidant evaluation, millet methanolic extracts showed higher level of TPC and DPPH of MB-87 and AF-POP as 0.30±0.001 & 0.23±0.0012 mg GAE/g and 48±0.96 &46±1.09%, respectively. However, the β-carotene activity of the aqueous extract of both varieties (MB-87 and AF-POP) was recorded higher. In a nutshell, the methanolic extract of MB-87 has an appreciable atioxidant profile. Furher research should be planned to screen the important constituents of Pakistani millet varieties

Potassium Activates mTORC2-dependent SGK1 Phosphorylation to Stimulate ENaC: Role in Rapid Renal Responses to Dietary Potassium

BACKGROUND: Increasing evidence implicates the signaling kinase mTOR complex-2 (mTORC2) in rapid renal responses to changes in plasma potassium concentration [K+]. However, the underlying cellular and molecular mechanisms that are relevant in vivo for these responses remain controversial. METHODS: We used Cre-Lox-mediated knockout of rapamycin-insensitive companion of TOR (Rictor) to inactivate mTORC2 in kidney tubule cells of mice. In a series of time-course experiments in wild-type and knockout mice, we assessed urinary and blood parameters and renal expression and activity of signaling molecules and transport proteins following a K+ load via gavage. RESULTS: A K+ load rapidly stimulated epithelial sodium channel (ENaC) processing, plasma membrane localization, and activity in wild-type but not in knockout mice. Downstream targets of mTORC2 implicated in ENaC regulation (SGK1 and Nedd4-2) were concomitantly phosphorylated in wild-type but not knockout mice. We observed differences in urine electrolytes within 60 minutes, and plasma [K+] was greater in knockout mice within 3 hours of gavage. Renal outer medullary potassium (ROMK) channels were not acutely stimulated in wild-type or knockout mice, nor were phosphorylation of other mTORC2 substrates (PKC and Akt). CONCLUSIONS: The mTORC2-SGK1-Nedd4-2-ENaC signaling axis is a key mediator of rapid tubule cell responses to increased plasma [K+] in vivo. The effects of K+ on this signaling module are specific, in that other downstream mTORC2 targets such as PKC and Akt are not acutely affected, and ROMK and BK channels are not activated. These findings provide new insight into the signaling network and ion transport systems that underlie renal responses to K+in vivo

Physicochemical properties of pore residues predict activation gating of CaV1.2: A correlation mutation analysis

Single point mutations in pore-forming S6 segments of calcium channels may transform a high-voltage-activated into a low-voltage-activated channel, and resulting disturbances in calcium entry may cause channelopathies (Hemara-Wahanui et al., Proc Natl Acad Sci U S A 102(21):7553–7558, 16). Here we ask the question how physicochemical properties of amino acid residues in gating-sensitive positions on S6 segments determine the threshold of channel activation of CaV1.2. Leucine in segment IS6 (L434) and a newly identified activation determinant in segment IIIS6 (G1193) were mutated to a variety of amino acids. The induced leftward shifts of the activation curves and decelerated current activation and deactivation suggest a destabilization of the closed and a stabilisation of the open channel state by most mutations. A selection of 17 physicochemical parameters (descriptors) was calculated for these residues and examined for correlation with the shifts of the midpoints of the activation curve (ΔVact). ΔVact correlated with local side-chain flexibility in position L434 (IS6), with the polar accessible surface area of the side chain in position G1193 (IIIS6) and with hydrophobicity in position I781 (IIS6). Combined descriptor analysis for positions I781 and G1193 revealed that additional amino acid properties may contribute to conformational changes during the gating process. The identified physicochemical properties in the analysed gating-sensitive positions (accessible surface area, side-chain flexibility, and hydrophobicity) predict the shifts of the activation curves of CaV1.2

Post-intervention Status in Patients With Refractory Myasthenia Gravis Treated With Eculizumab During REGAIN and Its Open-Label Extension

OBJECTIVE: To evaluate whether eculizumab helps patients with anti-acetylcholine receptor-positive (AChR+) refractory generalized myasthenia gravis (gMG) achieve the Myasthenia Gravis Foundation of America (MGFA) post-intervention status of minimal manifestations (MM), we assessed patients' status throughout REGAIN (Safety and Efficacy of Eculizumab in AChR+ Refractory Generalized Myasthenia Gravis) and its open-label extension. METHODS: Patients who completed the REGAIN randomized controlled trial and continued into the open-label extension were included in this tertiary endpoint analysis. Patients were assessed for the MGFA post-intervention status of improved, unchanged, worse, MM, and pharmacologic remission at defined time points during REGAIN and through week 130 of the open-label study. RESULTS: A total of 117 patients completed REGAIN and continued into the open-label study (eculizumab/eculizumab: 56; placebo/eculizumab: 61). At week 26 of REGAIN, more eculizumab-treated patients than placebo-treated patients achieved a status of improved (60.7% vs 41.7%) or MM (25.0% vs 13.3%; common OR: 2.3; 95% CI: 1.1-4.5). After 130 weeks of eculizumab treatment, 88.0% of patients achieved improved status and 57.3% of patients achieved MM status. The safety profile of eculizumab was consistent with its known profile and no new safety signals were detected. CONCLUSION: Eculizumab led to rapid and sustained achievement of MM in patients with AChR+ refractory gMG. These findings support the use of eculizumab in this previously difficult-to-treat patient population. CLINICALTRIALSGOV IDENTIFIER: REGAIN, NCT01997229; REGAIN open-label extension, NCT02301624. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that, after 26 weeks of eculizumab treatment, 25.0% of adults with AChR+ refractory gMG achieved MM, compared with 13.3% who received placebo