Whole-cell voltage patch clamp studies have shown that in bovine adrenal chromaffin cells held at -70 mV, a single 5-ns, 5 MV/m pulse activates a membrane conductance carried partially by Na[+] through TRPC4/5 channels and the sodium leak channel (NALCN). Here we used fluorescence imaging with the Na[+] indicator ING-2 to further investigate Na[+] influx pathways. A 5-ns, 5 MV pulse elicited a tetrodotoxin-insensitive rise in ING-2 fluorescence that exhibited a similar electric field dependency and response to pulse-pair stimulation as the inward current. Increases in ING-2 fluorescence were partially inhibited by the NALCN inhibitor CP96345, the TRPC4/5 channel inhibitor M084, or the broad spectrum TRP channel inhibitor La[3+], and fully blocked by the combination of these agents, suggesting involvement also of a La[3+]-sensitive Na[+] pathway. Cholesterol depletion with methyl-β-cyclodextrin or PIP2 synthesis inhibition with wortmannin also reduced the response. Full inhibition was achieved with the selective L-type voltage-gated Ca[2+] channel (VGCC) inhibitors nitrendipine, verapamil, or diltiazem but not the broad-spectrum inhibitor Cd[2+]. Inhibitors of N- and P/Q-type VGCC had no effect. Fluorescence Ca[2+] imaging in voltage-clamped GCaMP6f-expressing murine chromaffin cells held at -70 mV revealed a nitrendipine-sensitive, Cd[2+]-insensitive increase in intracellular Ca[2+] that accompanied the inward current. These results provide evidence that nanoelectropulse-induced Na[+] influx into chromaffin cells involves several Na[+] influx pathways that are facilitated by Ca[2+] influx via an L-type-like Ca[2+] channel associated with cholesterol-rich membrane domains. Investigating such membrane effects is essential for developing nanosecond electric pulse technologies for stimulating and/or modulating excitable cells.

To report the occurrence of transient atrial fibrillation in dogs with degenerative mitral valve disease (DMVD) American College of Veterinary Medicine (ACVIM) stage C/D, presenting with acute clinical signs.

Diltiazem (DTZ) is one of the most effective medications for treating cardiovascular diseases. It has been widely used for the treatment of angina pectoris, hypertension and some types of arrhythmia. The development and application of a modified carbon paste sensor with improved detection limits for the potentiometric determination of diltiazem are the main goals of the current study. Sensitivity, long-term stability, reproducibility and improving the electrochemical performance are among the characteristics that have undergone careful examination. A modified carbon paste sensor based on β-cyclodextrin (β-CD) as ionophore, a lipophilic anionic additive (NaTPB) and a ZnO-decorated polyaniline/coal nanocomposite (ZnO@PANI/C) dissolved in dibutyl phthalate plasticizer, exhibited the best performance and Nernstian slope. The ZnO@PANI/C based sensor succeeded in lowering the detection limit to 5.0 × 10[-7] through the linear range 1.0 × 10[-6] to 1.0 × 10[-2] mol L[-1] with fast response time ≤ 10.0 s. The prepared nanomaterial was characterized using X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy and scanning electron microscopy (SEM). The surface properties of the proposed sensor were characterized by electrochemical impedance spectroscopy (EIS). The selectivity behavior of the investigated sensor was tested against a drug with similar chemical structure and biologically important blood electrolytes (Na[+], K[+], Mg[2+], and Ca[2+]). The proposed analytical method was applied for DTZ analysis in pure drug, pharmaceutical products and industrial water samples with excellent recovery data.

A Western-style diet (WD; high in fat and carbohydrates) increases vascular oxidative stress. We hypothesized that vascular cells adapt to a WD by developing resilience to oxidative stress. Male and female C57BL/6J mice (4 wk of age) were fed a control diet (CD) or a WD for 16-20 wk. Superior epigastric arteries (SEAs; diameter, ~125 µm) were isolated and pressurized for study. Basal reactive oxygen species production was greatest in SEAs from males fed the WD. During exposure to H2O2 (200 μM, 50 min), propidium iodide staining identified nuclei of disrupted endothelial cells (ECs) and smooth muscle cells (SMCs). For mice fed the CD, death of SMCs (21%) and ECs (6%) was greater (P < 0.05) in SEAs from males than females (9% and 2%, respectively). WD consumption attenuated cell death most effectively in SEAs from males. With no difference at rest, H2O2 increased intracellular Ca2+ concentration ([Ca2+]i) to the greatest extent in SEAs from males, as shown by fura 2 fluorescence. Selective disruption of the endothelium (luminal air bubble) increased [Ca2+]i and SMC death during H2O2 exposure irrespective of sex; the WD reduced both responses most effectively in males. Nonselective transient receptor potential (TRP) channel inhibition (ruthenium red, 5 μM) attenuated the rise of [Ca2+]i, as did selective inhibition of TRP vanilloid type 4 (TRPV4) channels (HC-067047, 1 μM), which also attenuated cell death. In contrast, inhibition of voltage-gated Ca2+ channels (diltiazem, 50 μM) was without effect. Thus, for resistance arteries during acute oxidative stress: 1) ECs are more resilient than (and can protect) SMCs, 2) vessels from females are inherently more resilient than those from males, and 3) a WD increases vascular resilience by diminishing TRPV4 channel-dependent Ca2+ entry.

Cyclic nucleotide-gated (CNG) channels are nonselective cation channels activated by cyclic AMP (cAMP) or cyclic GMP (cGMP). They were originally identified in retinal and olfactory receptors, but evidence has also emerged for their expression in several mammalian brain areas. Because cGMP and cAMP control important aspects of glial cell physiology, we wondered whether CNG channels are expressed in astrocytes, the most functionally relevant glial cells in the CNS. Immunoblot and immunofluorescence experiments demonstrated expression of the CNG channel olfactory-type A subunit, CNGA2, in cultured rat cortical astrocytes. In patch-clamp experiments, currents elicited in these cells by voltage ramps from -100 to +100 mV in the presence of the cGMP analogue, dB-cGMP, were significantly reduced by the CNG channel blockers, L-cis-diltiazem (LCD) and Cd(2+) . The reversal potentials of the LCD- and Cd(2+) -sensitive currents were more positive than that of K(+) , as expected for a mixed cation current. Noninactivating, voltage-independent currents were also elicited by extracellular application of the membrane permeant cGMP analogue, 8-Br-cGMP. These effects were blocked by LCD and were mimicked by natriuretic peptide receptor activation and inhibition of phosphodiesterase activity. Voltage-independent, LCD-sensitive currents were also elicited by 8-Br-cGMP in astrocytes of hippocampal and neocortical brain slices. Immunohistochemistry confirmed a broad distribution of CNG channels in astrocytes of the rat forebrain, midbrain, and hindbrain. These findings suggest that CNG channels are downstream targets of cyclic nucleotides in astrocytes, and they may be involved in the glial-mediated regulation of CNS functions under physiological and pathological conditions.

Alpha3-containing (alpha 3*) and alpha 7 nicotinic acetylcholine receptors (nAChRs) are expressed in human IMR-32 neuroblastoma cells and implicated in Ca(2+) signaling. In this study, we investigated the intracellular Ca(2+) transient generation evoked by selective activation of alpha 3* (agonist potency rank order: epibatidine>varenicline>nicotine approximately cytisine) and alpha 7 (rank order in the presence of alpha 7 positive allosteric modulator or PAM: A-795723>NS6784 approximately PNU-282987) using, respectively, varenicline and NS6784 (+alpha 7 PAM) by Ca(2+) imaging. Effects of inhibitors of nAChRs (MLA and mecamylamine), ER Ca(2+) ATPase pump (CPA and thapsigargin), Ca(2+)-induced Ca(2+) release (ryanodine and dantrolene), Ca(2+) channels (nitrendipine, diltiazem, and Cd(2+)), and removal of extracellular Ca(2+) were examined. alpha 7 PAMs, when tested in the presence of NS6784, were more active when added first, followed by the agonist, than in the reverse order. Removal of extracellular Ca(2+) - but not CPA, thapsigargin, ryanodine, dantrolene, nitrendipine, diltiazem, or Cd(2+) - diminished the alpha 7 agonist-evoked Ca(2+) transients. In contrast, only diltiazem and nitrendipine and removal of extracellular Ca(2+) inhibited the alpha 3*-mediated Ca(2+) transients. The differential effect of diltiazem and nitrendipine versus Cd(2+) was due to direct inhibition of alpha 3* nAChRs as revealed by Ca(2+) imaging in HEK-293 cells expressing human alpha 3 beta 4 nAChRs and patch clamp in IMR-32 cells. In summary, this study provides evidence that alpha 3* and alpha 7 nAChR agonist-evoked global Ca(2+) transient generation in IMR-32 cells does not primarily involve voltage-dependent Ca(2+) channels, intracellular Ca(2+) stores, or Ca(2+)-induced Ca(2+) release. These mechanisms may, however, be still involved in other forms of nAChR-mediated Ca(2+) signaling.

Calcineurin inhibitors (CNI) are metabolized by cytochrome-P4503A (CYP3A) enzymes and extruded into the intestinal lumen by the drug efflux pump, P-glycoprotein (P-gp). The impact of single nucleotide polymorphisms (SNPs) of genes encoding CYP3A5 and P-gp on CNI dosing was examined among Asian renal transplant recipients. Frequencies of CYP3A5*1 versus *3 and MDR1-C3435T were correlated with tacrolimus (TAC) and cyclosporine (CSA) concentration-to-dose (C/D) ratios. Among 82 recipients (49% male; 88% Chinese), the majority were CYP3A5 expressors (*1*1 and *1*3, 11% and 40%, respectively) and 49% were nonexpressors (*3/*3). The prevalence of MDR-1-C3435T variants was 3435CC (41%), 3435CT (46%), and 3435TT (13%). Among 18 TAC-treated recipients, all receiving Diltiazem (DTZ), the median C/D ratio was lower for CYP3A5 *1/*1 versus *1/*3 versus *3/*3 (1.9, 4.6, and 13.5 ng/mL per 0.1 mg/kg/d, respectively; P = .001). The median C/D ratio was higher for TAC-treated patients with MDR-1-3435CC (14.1, 7.3, and 2.2 ng/mL per 0.1 mg/kg/d for CC, CT, and TT, respectively; P = .023). Neither CYP3A5 nor MDR-1-C3435T variants had an impact on CSA C/D ratios. Thus, CYP3A5 SNP has a significant impact on TAC dosing in Asian renal transplant recipients, which was likely to facilitate TAC metabolism. Although MDR-1-3435CC with higher P-gp expression should experience more TAC efflux and, therefore, lower TAC C/D ratios, all MDR-1-3435CC carriers were CYP3A5 nonexpressors; the latter ultimately contributed to the observed higher TAC C/D ratios in this population. This study advocates starting with a higher TAC dose for CYP3A5 expressors. Coadministration of DTZ may further optimize the TAC level through preferential P-gp binding and CYP3A4 inhibition.

Although cyclic nucleotide-gated (CNG) channels are expressed in numerous brain areas, little information is available on their functions in CNS neurons. The aim of the present study was to define the distribution of CNG channels in the rat medial vestibular nucleus (MVN) and their possible involvement in regulating MVN neuron (MVNn) excitability. The majority of MVNn expressed both CNG1 and CNG2 A subunits. In whole-cell current-clamp experiments carried out on brainstem slices containing the MVNn, the membrane-permeant analogues of cyclic nucleotides, 8-Br-cGMP and 8-Br-cAMP (1 mM), induced membrane depolarizations (8.9 +/- 0.8 and 9.2 +/- 1.0 mV, respectively) that were protein kinase independent. The cGMP-induced depolarization was associated with a significant decrease in the membrane input resistance. The effects of cGMP on membrane potential were almost completely abolished by the CNG channel blockers, Cd(2+) and L-cis-diltiazem, but they were unaffected by blockade of hyperpolarization-activated cyclic nucleotide-gated channels. In voltage-clamp experiments, 8-Br-cGMP induced non-inactivating inward currents (-22.2 +/- 3.9 pA) with an estimated reversal potential near 0 mV, which were markedly inhibited by reduction of extracellular Na(+) and Ca(2+) concentrations. Membrane depolarization induced by CNG channel activation increased the firing rate of MVNn without changing the action potential shape. Collectively, these findings provide novel evidence that CNG channels affect membrane potential and excitability of MVNn. Such action should have a significant impact on the function of these neurons in sensory-motor integration processes. More generally, it might represent a broad mechanism for regulating the excitability of different CNS neurons.

Toward our goal to better understand the physiological parameters that mediate olfactory information processing on the cellular level, voltage-activated calcium currents (I(Ca)) in olfactory interneurons of the antennal lobe from adult cockroaches were analyzed under two conditions: 1) in acutely dissociated cells (in vitro) and 2) in an intact brain preparation (in situ). The study included an analysis of modulatory effects of potential inorganic and organic Ca(2+) channel blockers. I(Ca) was isolated and identified using pharmacological, voltage, and ion substitution protocols. I(Ca) consisted of two components: transient and sustained. The decay of the transient component was largely Ca(2+) dependent. In vitro, I(Ca) had an activation threshold of -50 mV with a maximal peak current at -7 mV and a half-maximal voltage (V(0.5act)) for tail-current activation of -18 mV. In situ these parameters were significantly shifted to more depolarized membrane potentials: I(Ca) activated at -40 mV with a maximal peak current at 8 mV and a V(0.5act) for tail-current activation of -11 mV. The sensitivity of I(Ca) to the divalent cations Cd(2+), Co(2+), and Ni(2+) was dose dependent. The most effective blocker was Cd(2+) with an IC(50) of 10(-5) M followed by Ni(2+) (IC(50)=3.13 x 10(-3) M) and Co(2+) (IC(50)=1.06 x 10(-3) M). The organic channel blockers verapamil, diltiazem, and nifedipine also blocked I(Ca) in a dose-dependent way and had differential effects on the current waveform. Verapamil blocked I(Ca) with an IC(50) of 1.5 x 10(-4) M and diltiazem had an IC(50) of 2.87 x 10(-4) M. Nifedipine blocked I(Ca) by 33% at a concentration of 10(-4) M.

Acetylcholine (ACh) induces hyperpolarization and dilation in a variety of blood vessels, including the cochlear spiral modiolar artery (SMA) via the endothelium-derived hyperpolarization factor (EDHF). We demonstrated previously that the ACh-induced hyperpolarization in the SMA originated in the endothelial cells (ECs) by activating a Ca(2+)-activated K(+) channel (K(Ca)); the hyperpolarization in smooth muscle cells was mainly an electrotonic spread via gap junction coupling. In the present study, using intracellular recording, immunohistology, and vascular diameter tracking techniques on in vitro SMA preparations, we found that 1) ACh-induced hyperpolarization was suppressed by intermediate-conductance K(Ca) (IK) blockers clotrimazole (IC(50) = 116 nM) and nitrendipine and by the calmodulin antagonist trifluoperazine, but it was not suppressed by the big-conductance K(Ca) blocker iberiotoxin. The immunoreactivity to anti-SK4/IK1 antibody was localized mainly in ECs. 2) The three dihydropyridines--nifedipine, nitrendipine, and nimodipine--all concentration-dependently inhibited the ACh-induced hyperpolarization, with an IC(50) value of 455, 34, and 3.2 nM, respectively. 3) Among other L-type Ca(2+) channel (I(L)) blockers, 10 microM verapamil exerted a 20% inhibition on ACh-induced hyperpolarization, whereas diltiazem and the metal ion Ca(2+) channel blockers Cd(2+) and Ni(2+) had no effect. 4) Nitrendipine and charybdotoxin abolished ACh-induced dilation in the SMA. We conclude that ACh-induced hyperpolarization in the SMA is generated mainly by activation of the IK in the ECs, and dihydropyridines suppress the EDHF-mediated hyperpolarization by blocking the IK channel, not the I(L) channel. The clinical relevance of this dihydropyridine action is discussed.