microM Nifedipine Research Articles

Spontaneous Electrical Waveforms in Aging Guinea Pig Prostates

We characterized spontaneous electrical activity in the aging guinea pig prostate. Membrane potential recordings were made using conventional single microelectrode recording techniques. Three types of spontaneous waveforms were recorded, including spikes, slow waves and spontaneous transient depolarizations. Spikes were classified as hyperactive or active. Active cells showed a mean +/- SEM frequency of 5.06 +/- 0.63 minutes(-1), significantly different from that in hyperactive cells (362.05 +/- 151.82 minutes(-1), p <0.05). After hyperpolarization amplitude was also significantly different in the active and hyperactive groups (17.80 +/- 1.98 vs 9.96 +/- 1.05 mV). Spike activity was abolished by 1 microM nifedipine in 7 preparations (p <0.05). Slow wave activity occurred at a frequency of 5.2 +/- 0.5 minutes(-1). The spike component of slow wave activity was abolished by 1 microM nifedipine, although the depolarizing transient remained unaltered from control values (8.1 +/- 3.1 mV, paired Student's t test p >0.05). Spontaneous transient depolarizations were recorded in the presence of slow waves in 10 preparations and of spikes in 13, and in quiescent cells in 9. Spontaneous transient depolarization frequency was highest in otherwise quiescent cells (24.55 +/- 6.48 minutes(-1)) compared to that in the presence of slow waves or spikes. Adding 1 microM nifedipine in 5 preparations did not significantly affect any measured parameters (p >0.05). Pacemaker potentials were not recorded in the aging prostate. With increased age there is an increase in spike activity, which could conceivably explain the increased prostatic tone that accompanies aging. Spike activity and the spike component of the slow wave were abolished by nifedipine, suggesting a role for L-type channels. Finally, spontaneous transient depolarizations were unaffected by nifedipine, suggesting that mechanisms other than Ca(2+) entry via L-type channels are responsible for their generation and maintenance.

N-Type Ca2+-Channels in Murine Pancreatic β-Cells Are Inhibited by an Exclusive Coupling with Somatostatin Receptor Subtype 1

Somatostatin (SRIF) is a well-established inhibitor of insulin secretion, an effect in part mediated by a direct inhibition of voltage-operated Ca(2+)-channels. However, the identity of the somatostatin receptor subtypes (SSTRs) and voltage-operated Ca(2+)-channels involved in this process are unknown. Whole-cell perforated patch-clamp methods were applied to the murine pancreatic beta-cell line, MIN6, to explore the molecular pharmacology of this problem. SRIF-14 inhibited voltage-gated Ca(2+) currents (ICa(2+)) by 19 +/- 3% (n=24) with a pEC(50) = 9.05 (95% confidence limits 9-9.1). This action was mimicked solely by 100 nm CH-275, a selective agonist at the somatostatin type 1 receptor (SSTR1), but not by 100 nm BIM-23027, L-362855, or NNC-269100; agonists selective for the other four SSTRs known to exist in MIN6. The inhibition of ICa(2+) produced by SRIF and CH-275 was insensitive to pertussis toxin but was reversed by a prepulse to +100 mV. The inhibition of ICa(2+) by SRIF-14 was unaffected by 20 microm nifedipine, an inhibitor of L-type Ca(2+) channels. Application of the specific N-type Ca(2+) channel (Ca(v)2.2) inhibitor omega-conotoxin GV1A at 100 nm mimicked, and as a consequence abolished, the inhibitory effect of SRIF-14 on ICa(2+). SRIF selectively inhibits N-type Ca(2+)-channels in murine pancreatic beta-cells via exclusive coupling with SSTR1. These findings help explain how SSTR1 activation can inhibit insulin secretion in pancreatic beta-cells and suggest a possible new therapeutic lead for treatment of hyperinsulinemia.

Direct Effect of Electrical Stimulation on Induction of Brain-Derived Neurotrophic Factor from Cultured Retinal Müller Cells

To investigate the direct effect of electrical stimulation (ES) on the induction of brain-derived neurotrophic factor (BDNF) from cultured retinal Müller cells. Müller cells were isolated from rat retinas. ES was applied to passage 1 Müller cells with biphasic pulses (duration, 1 ms; frequency, 20 Hz; current, 10 mA) for 30 minutes. The changes in gene expression after ES were analyzed with microarrays. The mRNA and protein levels of BDNF were determined at each time point after ES by RT-PCR and ELISA, respectively. RT-PCR was also performed at 3 hours after ES of Müller cells that had been exposed to 1 microM nifedipine, a blocker of L-type voltage-dependent calcium channels (L-VDCCs). Microarray analyses showed an upregulation of 245 genes, including BDNF. The mRNA level of BDNF increased significantly (P < 0.05; by approximately 1.2-fold over that of the control) at 2 and 3 hours after ES. The intracellular protein level was upregulated significantly (by approximately 1.4-fold) at 6 hours after ES, whereas the extracellular level did not change at any time point. The total protein level of BDNF increased significantly ( approximately 1.3-fold) at 6 hours after ES. The increase in the mRNA level of BDNF was fully suppressed by exposure of the Müller cells to nifedipine. These results demonstrate that ES directly upregulates the transcriptional induction of BDNF through L-VDCCs in cultured Müller cells. The ES of Müller cells may be used to supply endogenous BDNF to the retina.

The Local Anesthetic Butamben Inhibits Total and L-Type Barium Currents in PC12 Cells

Butamben or n-butyl-p-aminobenzoate is a long-acting experimental local anesthetic for the treatment of chronic pain when given as an epidural suspension. We have investigated whether Cav1.2/L-type calcium channels may be a target of this butamben action. The effect of butamben on these channels was studied in undifferentiated rat PC12-cells with the whole-cell patch-clamp technique in voltage-clamp. Ba(2+) ions were used as the charge carriers in the calcium channel currents, whereas K(+) currents were removed using K(+) free solutions. Butamben 500 microM reversibly suppressed the total whole-cell barium current by 90% +/- 3% (n = 15), whereas 10 microM nifedipine suppressed this barium current by 75% +/- 7% (n = 6). Preexposure to butamben followed by washout decreased the inhibition by nifidepine to 47% +/- 5% (n = 10). These suppressive effects were not due to the measurement procedure and the drug vehicles in the solutions (<0.1% ethanol; n = 6). Butamben inhibits the total barium current through expressed calcium channel types in PC12 cells, including Cav1.2/L-type channels. Because Cav1.2 channels may also occur in human nociceptive C fibers, this result allows the possibility that these L-type channels are involved in the analgesic action of butamben.

Voltage-dependent Ca2+Current Identified in Freshly Isolated Interstitial Cells of Cajal (ICC) of Guinea-pig Stomach

The properties of voltage dependent Ca(2+) current (VDCC) were investigated in interstitial cells of Cajal (ICC) distributed in the myenteric layer (ICC-MY) of guinea-pig antrum. In tissue, ICC-MY showed c-Kit positive reactions and produced driving potentials with the amplitude and frequency of about 62 mV and 2 times min(-1), respectively, in the presence of 1 microM nifedipine. Single ICC-MY isolated by enzyme treatment also showed c-Kit immunohistochemical reactivity. These cells were also identified by generation of spontaneous inward current under K(+) -rich pipette solution. The voltage clamp experiments revealed the amplitude of - 329 pA inward current at irregular frequency. With Cs(+)-rich pipette solution at V(h)=-80 mV, ICC-MY produced voltage-dependent inward currents (VDIC), and nifedipine (1 microM) blocked VDIC. Therefore, we successfully isolated c-Kit positive single ICC from guinea-pig stomach, and found that ICC-MY potently produced dihydropiridine sensitive L-type voltage-dependent Ca(2+) currents (VDCC(L)).

Role of Ca2+ entry and Ca2+ stores in atypical smooth muscle cell autorhythmicity in the mouse renal pelvis

Electrically active atypical smooth muscle cells (ASMCs) within the renal pelvis have long been considered to act as pacemaker cells driving pelviureteric peristalsis. We have investigated the role of Ca2+ entry and uptake into and release from internal stores in the generation of Ca2+ transients and spontaneous transient depolarizations (STDs) in ASMCs. The electrical activity and separately visualized changes in intracellular Ca2+ concentration in typical smooth muscle cells (TSMCs), ASMCs and interstitial cells of Cajal-like cells (ICC-LCs) were recorded using intracellular microelectrodes and a fluorescent Ca2+ indicator, fluo-4. In 1 microM nifedipine, high frequency (10-30 min(-1)) Ca2+ transients and STDs were recorded in ASMCs, while ICC-LCs displayed low frequency (1-3 min(-1)) Ca2+ transients. All spontaneous electrical activity and Ca2+ transients were blocked upon removal of Ca2+ from the bathing solution, blockade of Ca2+ store uptake with cyclopiazonic acid (CPA) and with 2-aminoethoxy-diphenylborate (2-APB). STD amplitudes were reduced upon removal of the extracellular Na+ or blockade of IP3 dependent Ca2+ store release with neomycin or U73122. Blockade of ryanodine-sensitive Ca2+ release blocked ICC-LC Ca2+ transients but only reduced Ca2+ transient discharge in ASMCs. STDs in ASMCS were also little affected by DIDS, La3+, Gd3+ or by the replacement of extracellular Cl(-) with isethionate. ASMCs generated Ca2+ transients and cation-selective STDs via mechanisms involving Ca2+ release from IP3-dependent Ca2+ stores, STD stimulation of TSMCs was supported by Ca2+ entry through L type Ca2+ channels and Ca2+ release from ryanodine-sensitive stores.

SR Ca2+ refilling upon depletion and SR Ca2+ uptake rates during development in rabbit ventricular myocytes

While it has been reported that a sparse sarcoplasmic reticulum (SR) and a low SR Ca(2+) pump density exist at birth, we and others have recently shown that significant amounts of Ca(2+) are stored in the neonatal rabbit heart SR. Here we try to determine developmental changes in SR Ca(2+) loading mechanisms and Ca(2+) pump efficacy in rabbit ventricular myocytes. SR Ca(2+) loading (load(SR)) and k(0.5) (Ca(2+) concentration at half-maximal SR Ca(2+) uptake) were higher and lower, respectively, in younger age groups. Inhibition of the L-type calcium current (I(Ca)) with 15 microM nifedipine dramatically reduced load(SR) in older but not in younger age groups. In contrast, subsequent inhibition of the Na(+)/Ca(2+) exchanger (NCX) with 10 microM KB-R7943 strongly reduced load(SR) in the younger but not the older age groups. Accordingly, the time integral of the inward NCX current (tail I(NCX)) elicited on repolarization was highly sensitive to nifedipine in the older groups and sensitive to KB-R7943 in the younger groups. Interestingly, slow SR loading took place in the presence of both nifedipine and KB-R7943 in all age groups, although it was less prominent in the older groups. We conclude that the SR loading capacity at the earliest postnatal stages is at least as large as that of adult myocytes. However, reverse-mode NCX plays a prominent role in SR Ca(2+) loading at early postnatal stages while I(Ca) is the main source of SR Ca(2+) loading at late postnatal and adult stages.

Spontaneous electrical and Ca2+ signals in typical and atypical smooth muscle cells and interstitial cell of Cajal‐like cells of mouse renal pelvis

Electrical rhythmicity in the renal pelvis provides the fundamental drive for the peristaltic contractions that propel urine from the kidney to bladder for storage until micturition. Although atypical smooth muscles (ASMCs) within the most proximal regions of the renal pelvis have long been implicated as the pacemaker cells, the presence of a sparsely distributed population of rhythmically active Kit-positive interstitial cells of Cajal-like cells (ICC-LCs) have confounded our understanding of pelviureteric peristalsis. We have recorded the electrical activity and separately visualized changes in intracellular Ca(2+) concentration in typical smooth muscle cells (TSMCs), ASMCs and ICC-LCs using intracellular microelectrodes and a fluorescent Ca(2+) indicator, fluo-4. Nifedipine (1-10 microm)-sensitive driven action potentials and Ca(2+) waves (frequency 6-15 min(-1)) propagated through the TSMC layer at a velocity of 1-2 mm s(-1). High frequency (10-40 min(-1)) Ca(2+) transients and spontaneous transient depolarizations (STDs) were recorded in ASMCs in the absence or presence of 1 microm nifedipine. ICC-LCs displayed low frequency (1-3 min(-1)) Ca(2+) transients which we speculated arose from cells that displayed action potentials with long plateaus (2-5 s). Neither electrical activity propagated over distances > 50 microm. In 1 microm nifedipine, ASMCs or ICC-LCs separated by < 30 microm displayed some synchronicity in their Ca(2+) transient discharge suggesting that they may well be acting as 'point sources' of excitation to the TSMC layer. We speculate that ASMCs act as the primary pacemaker in the renal pelvis while ICC-LCs play a supportive role, but can take over pacemaking in the absence of the proximal pacemaker drive.

Voltage-activated Calcium Currents in Octopus Cells of the Mouse Cochlear Nucleus

Octopus cells, neurons in the most posterior and dorsal part of the mammalian ventral cochlear nucleus, convey the timing of synchronous firing of auditory nerve fibers to targets in the contralateral superior paraolivary nucleus and ventral nucleus of the lateral lemniscus. The low input resistances and short time constants at rest that arise from the partial activation of a large, low-voltage-activated K(+) conductance (g(KL)) and a large mixed-cation, hyperpolarization-activated conductance (g(h)) enable octopus cells to detect coincident firing of auditory nerve fibers with exceptional temporal precision. Octopus cells fire conventional, Na(+) action potentials but a voltage-sensitive Ca(2+) conductance was also detected. In this study, we explore the nature of that calcium conductance under voltage-clamp. Currents, carried by Ca(2+) or Ba(2+) and blocked by 0.4 mM Cd(2+), were activated by depolarizations positive to -50 mV and peaked at -23 mV. At -23 mV they reached 1.1 +/- 0.1 nA in the presence of 5 mM Ca(2+) and 1.6 +/- 0.1 nA in 5 mM Ba(2+). Ten micromolar BAY K 8644, an agonist of high-voltage-activated L-type channels, enhanced I(Ba) by 63 +/- 11% (n = 8) and 150 microM nifedipine, an antagonist of L-type channels, reduced the I(Ba) by 65 +/- 5% (n = 5). Meanwhile, 0.5 microM omega-Agatoxin IVA, an antagonist of P/Q-type channels, or 1 microM omega-conotoxin GVIA, an antagonist of N-type channels, suppressed I(Ba) by 15 +/- 4% (n = 5) and 9 +/- 4% (n = 5), respectively. On average 16% of the current remained in the presence of the cocktail of blockers, indicative of the presence of R-type channels. Together these experiments show that octopus cells have a depolarization-sensitive g(Ca) that is largely formed from L-type Ca(2+) channels and that P/Q-, N-, and R-type channels are expressed at lower levels in octopus cells.

Intracellular signalling pathways in the vasoconstrictor response of mouse afferent arterioles to adenosine

Adenosine causes vasoconstriction of afferent arterioles of the mouse kidney through activation of adenosine A(1) receptors and Gi-mediated stimulation of phospholipase C. In the present study, we further explored the signalling pathways by which adenosine causes arteriolar vasoconstriction. Adenosine (10(-7) M) significantly increased the intracellular calcium concentration in mouse isolated afferent arterioles measured by fura-2 fluorescence. Pre-treatment with thapsigargin (2 microM) blocked the vasoconstrictor action of adenosine (10(-7) M) indicating that release of calcium from the sarcoplasmic reticulum (SR), stimulated presumably by IP(3), is involved in the adenosine contraction mechanism of the afferent arteriole. In agreement with this notion is the observation that 2 aminoethoxydiphenyl borate (100 microM) blocked the adenosine-induced constriction whereas the protein kinase C inhibitor calphostin C had no effect. The calcium-activated chloride channel inhibitor IAA-94 (30 microM) inhibited the adenosine-mediated constriction. Patch clamp experiments showed that adenosine treatment induced a depolarizing current in preglomerular smooth muscle cells which was abolished by IAA-94. Furthermore, the vasoconstriction caused by adenosine was significantly inhibited by 5 microM nifedipine (control 8.3 +/- 0.2 microM, ado 3.6 +/- 0.6 microM, ado + nifedipine 6.8 +/- 0.2 microM) suggesting involvement of voltage-dependent calcium channels. We conclude that adenosine mediates vasoconstriction of afferent arterioles through an increase in intracellular calcium concentration resulting from release of calcium from the SR followed by activation of Ca(2+)-activated chloride channels leading to depolarization and influx of calcium through voltage-dependent calcium channels.

Separation of early afterdepolarizations from arrhythmogenic substrate in the isolated perfused hypokalaemic murine heart through modifiers of calcium homeostasis

AimsWe resolved roles for early afterdepolarizations (EADs) and transmural gradients of repolarization in arrhythmogenesis in Langendorff-perfused hypokalaemic murine hearts paced from the right ventricular epicardium.MethodsLeft ventricular epicardial and endocardial monophasic action potentials (MAPs) and arrhythmogenic tendency were compared in the presence and absence of the L-type Ca2+ channel blocker nifedipine (10 nm–1 μm) and the calmodulin kinase type II inhibitor KN-93 (2 μm).ResultsAll the hypokalaemic hearts studied showed prolonged epicardial and endocardial MAPs, decreased epicardial-endocardial APD90 difference, EADs, triggered beats and ventricular tachycardia (VT) (n = 6). In all spontaneously beating hearts, 100 (but not 10) nm nifedipine reduced both the incidence of EADs and triggered beats from 66.9 ± 15.7% to 28.3 ± 8.7% and episodes of VT from 10.8 ± 6.3% to 1.2 ± 0.7% of MAPs (n = 6 hearts, P < 0.05); 1 μm nifedipine abolished all these phenomena (n = 6). In contrast programmed electrical stimulation (PES) still triggered VT in six of six hearts with 0, 10 and 100 nm but not 1 μm nifedipine. 1 μm nifedipine selectively reduced epicardial (from 66.1 ± 3.4 to 46.2 ± 2.5 ms) but not endocardial APD90, thereby restoring ΔAPD90 from −5.9 ± 2.5 to 15.5 ± 3.2 ms, close to normokalaemic values. KN-93 similarly reduced EADs, triggered beats and VT in spontaneously beating hearts to 29.6 ± 8.9% and 1.7 ± 1.1% respectively (n = 6) yet permitted PES-induced VT (n = 6), in the presence of a persistently negative ΔAPD90.ConclusionsThese findings empirically implicate both EADs and triggered beats alongside arrhythmogenic substrate of ΔAPD90 in VT pathogenesis at the whole heart level.

Spontaneous Electrical Activity in Sheep Mesenteric Lymphatics

It has recently become apparent that the lymph pump is an electrical entity that rivals the heart in complexity. Many interesting currents have been demonstrated by voltage clamping isolated lymphatic smooth muscle cells, but until now the role of these currents in the intact syncitium has not been studied. Intracellular microelectrode recordings were made from smooth muscle of sheep mesenteric lymphatics to investigate the electrophysiological basis of lymphatic pumping. Approximately 50% of the vessels exhibited spontaneous electrical activity, varying from regular oscillations in membrane potential to spike complexes. Spike complexes generally consisted of one or more action potentials superimposed on a slower depolarization or 'plateau' phase and were often preceded by a slow diastolic depolarization or 'pre-potential'. Norepinephrine (5 microM) induced depolarizing events in quiescent preparations. Both agonist-induced oscillations and spike complexes were attenuated or completely abolished by 2-aminoethoxydiphenyl borate (2-APB); 10-100 microM). Cesium (1 mM) reduced the frequency of spontaneous firing by approximately 30% by flattening the pre-potential phase. In addition to having a negative inotropic effect, 10 mM Cs(+) also caused gradual membrane depolarization and prolonged the plateau. 1 microM nifedipine abolished spontaneous events while tetrodotoxin (TTX; 0.5-1 muM) decreased the amplitude and maximum dV/dt of the spike upstroke or stopped activity completely. Spontaneously active segments of lymphatic vessel were inhibited by the chloride channel blocker, anthracene-9-carboxylic acid (9-AC; 250 microM - 1 mM) suggesting that I(Cl(Ca)) plays a significant role in the generation of spontaneous activity in this tissue. Penitrem-A (0.1 microM) did not affect resting membrane potential but increased action potential amplitude and prolonged the plateau, suggesting that calcium-activated potassium current does not make a significant contribution to resting membrane conductance but is important in membrane repolarization following calcium influx during the action potential. In contrast 4-aminopyridine (4-AP; 5 microM) caused significant membrane depolarization, suggesting the existence of an active 4-AP-sensitive current at rest. These results demonstrate that the currents found in isolated voltage-clamped cells from sheep mesenteric lymphatics do play a significant role in the shaping of spontaneous electrical activity of the intact syncitium.

Effects of L‐type Ca2+ channel antagonism on ventricular arrhythmogenesis in murine hearts containing a modification in the Scn5a gene modelling human long QT syndrome 3

Ventricular arrhythmogenesis in long QT 3 syndrome (LQT3) involves both triggered activity and re-entrant excitation arising from delayed ventricular repolarization. Effects of specific L-type Ca2+ channel antagonism were explored in a gain-of-function murine LQT3 model produced by a DeltaKPQ 1505-1507 deletion in the SCN5A gene. Monophasic action potentials (MAPs) were recorded from epicardial and endocardial surfaces of intact, Langendorff-perfused Scn5a+/Delta hearts. In untreated Scn5a+/Delta hearts, epicardial action potential duration at 90% repolarization (APD90) was 60.0 +/- 0.9 ms compared with 46.9 +/- 1.6 ms in untreated wild-type (WT) hearts (P < 0.05; n = 5). The corresponding endocardial APD(90) values were 52.0 +/- 0.7 ms and 53.7 +/- 1.6 ms in Scn5a+/Delta and WT hearts, respectively (P > 0.05; n = 5). Epicardial early afterdepolarizations (EADs), often accompanied by spontaneous ventricular tachycardia (VT), occurred in 100% of MAPs from Scn5a+/Delta but not in any WT hearts (n = 10). However, EAD occurrence was reduced to 62 +/- 7.1%, 44 +/- 9.7%, 10 +/- 10% and 0% of MAPs following perfusion with 10 nm, 100 nm, 300 nm and 1 mum nifedipine, respectively (P < 0.05; n = 5), giving an effective IC50 concentration of 79.3 nm. Programmed electrical stimulation (PES) induced VT in all five Scn5a+/Delta hearts (n = 5) but not in any WT hearts (n = 5). However, repeat PES induced VT in 3, 2, 2 and 0 out of 5 Scn5a+/Delta hearts following perfusion with 10 nm, 100 nm, 300 nm and 1 mum nifedipine, respectively. Patch clamp studies in isolated ventricular myocytes from Scn5a+/Delta and WT hearts confirmed that nifedipine (300 nm) completely suppressed the inward Ca2+ current but had no effect on inward Na+ currents. No significant effects were seen on epicardial APD90, endocardial APD90 or ventricular effective refractory period in Scn5a+/Delta and WT hearts following perfusion with nifedipine at 1 nm, 10 nm, 100 nm, 300 nm and 1 microm nifedipine concentrations. We conclude that L-type Ca2+ channel antagonism thus exerts specific anti-arrhythmic effects in Scn5a+/Delta hearts through suppression of EADs.

Mibefradil-sensitive component involved in the plateau potential in submucosal interstitial cells of the murine proximal colon

Submucosal interstitial cells of Cajal (ICC(SM)) produce plateau potentials comprised of initial fast and subsequent plateau components. The possible involvement of voltage-dependent Ca(2+) channels in plateau potentials was examined in ICC(SM) of the murine proximal colon. Increases in external K(+) concentration ([K(+)](o)) changed the rise rate of the initial component in a biphasic way, an increase in 10.6 or 15.3mM [K(+)](o) and a decrease in 20.0mM [K(+)](o). The rise rate of plateau potentials was significantly reduced by the application of 3 microM mibefradil or 100 microM Ni(2+) but not by 0.3 microM nifedipine. The inhibitory effect of mibefradil on the rise rate of plateau potentials was concentration-dependent with an IC(50) value of 1.0 microM. In conclusion, the initial phase of plateau potentials is partly due to the activation of T-type Ca(2+) channel in ICC(SM) from the murine proximal colon.

Extremely low frequency electromagnetic field exposure promotes differentiation of pituitary corticotrope‐derived AtT20 D16V cells

The pituitary corticotrope-derived AtT20 D16V cell line responds to nerve growth factor (NGF) by extending neurite-like processes and differentiating into neurosecretory-like cells. The aim of this work is the study of the effect of extremely low frequency electromagnetic fields (ELF-EMF) at a frequency of 50 Hz on these differentiation activities. To establish whether exposure to the field could influence the molecular biology of the cells, they were exposed to a magnetic flux density of 2 milli-Tesla (mT). Intracellular calcium ([Ca2+]i) and intracellular pH (pHi) were monitored in single exposed AtT20 D16V cells using fluorophores Indo-1 and SNARF for [Ca2+]i and pHi, respectively. Single-cell fluorescence microscopy showed a statistically significant increase in [Ca2+]i followed by a drop in pHi in exposed cells. Both scanning electron microscopy (SEM) and transmission microscopy of exposed AtT20 D16V cells show morphological changes in plasma membrane compared to non-exposed cells; this modification was accompanied by a rearrangement in actin filament distribution and the emergence of properties typical of peptidergic neuronal cells-the appearance of secretory-like granules in the cytosol and the increase of synaptophysin in synaptic vesicles, changes typical of neurosecretory-like cells. Using a monoclonal antibody toward the neurofilament protein NF-200 gave additional evidence that exposed cells were in an early stage of differentiation compared to control. Pre-treatment with 0.3 microM nifedipine, which specifically blocks L-type Ca2+ channels, prevented NF-200 expression in AtT20 D16V exposed cells. The above findings demonstrate that exposure to 50 Hz ELF-EMF is responsible for the premature differentiation in AtT20 D 16 V cells.

SAMMA Induces Premature Human Acrosomal Loss by Ca2+Signaling Dysregulation

SAMMA is licensed for development as a contraceptive microbicide. Understanding mechanisms of its biological activity is prerequisite to designing more active second generation products. This study examined Ca(2+) involvement in SAMMA-induced premature acrosomal loss (SAL) in noncapacitated human spermatozoa. SAMMA causes acrosomal loss (AL) in a dose-dependent manner (ED(50) = 0.25 microg/mL). SAL requires extracellular Ca(2+) (ED(50) = 85 microM). SAL is inhibited by verapamil (nonspecific voltage-dependent Ca(2+) channel blocker; IC(50) = 0.4 microM), diphenylhydantoin and NiCl(2) (T-type [Ca(v)3.x] channel blockers; IC(50) 210 microM and 75 microM, respectively). Verapamil blockade of L-type (Ca(v)1.x) channels is use-dependent; activated channels are more sensitive to inhibition. However, verapamil inhibition of SAL does not increase after repeated SAMMA stimulation. SAL is unaffected by 10 microM nifedipine (selective L-type channel blocker). This contrasts to 40% inhibition (P < .001) of AL induced by 1 microM thapsigargin (Ca(2+)-ATPase inhibitor; releases intracellular Ca(2+) stores, promotes capacitative Ca(2+) entry). SAL is unaffected by 1 microM BAPTA-AM (intracellular Ca(2+) chelator), and 50 microM 2-APB (blocks InsP3 receptors and store-operated channels). This contrasts with thapsigargin-induced AL, inhibited nearly 65% by BAPTA-AM (P < .005) and 91% by 2-APB (P, .001). The results suggest that SAL is mediated by Ca(2+) entry through channels pharmacologically similar to the T-type (Ca(v)3.2) class. This process appears distinct from that caused by physiological stimuli such as progesterone or zona pellucida-derived proteins. SAMMA's contraceptive activity may be caused by induction of premature AL through dysregulation of Ca(2+) signaling.

Calcium currents in interstitial cells from the guinea‐pig bladder

To determine whether there are inward currents in interstitial cells (IC) isolated from the guinea-pig detrusor and if so, to characterise them using the patch-clamp technique and pharmacological agents. Using the whole-cell patch-clamp technique, inward currents were studied in IC enzymatically isolated from the detrusor of the guinea-pig bladder. Currents were evoked by stepping positively from a holding potential of - 80 mV. Outward K+ currents were blocked by Cs+ internal solution to reveal inward currents, which activated at voltages more positive than - 50 mV, peaked at 0 mV, reversed near + 50 mV and were half-maximally activated at - 27 mV. The inward currents showed voltage-dependent inactivation and were half-maximally inactivated at - 36 mV. Fitting the activation and inactivation data with a Boltzmann function revealed a window current between - 40 mV and + 20 mV. The decay of the current evoked at 0 mV could be fitted with a single exponential with a mean time-constant of 88 ms. Replacing external Ca2+ with Ba2+ significantly increased this to 344 ms. The current amplitude was augmented by Ba2+, and by Bay K 8644. Inward currents were significantly reduced by 1 microm nifedipine, across the voltage range, but the blockade was more effective on the current evoked at 0 mV than that evoked by a step to - 20 mV, perhaps indicating voltage-dependence of the action of nifedipine or another component of inward current. Increasing the concentration of the drug to 10 microm caused no further significant reduction either at 0 mV or at -20 mV. However, in the presence of 1 microm nifedipine the latter current was significantly reduced by 100 microm Ni2+. Both currents were significantly reduced in Ca2+-free solution. IC from the guinea-pig detrusor possess inward currents with typical characteristics of L-type Ca2+ current. They also have a component of inward Ca2+ current, which was resistant to nifedipine, but sensitive to Ni2+. Further work is needed to characterise the latter conductance.

Differential Modulation of Cav1.2 and Cav1.3-Mediated Glucose-Stimulated Insulin Secretion by cAMP in INS-1 Cells: Distinct Roles for Exchange Protein Directly Activated by cAMP 2 (Epac2) and Protein Kinase A

Using insulin-secreting cell line (INS)-1 cells stably expressing dihydropyridine-insensitive mutants of either Cav1.2 or Cav1.3, we previously demonstrated that Cav1.3 is preferentially coupled to insulin secretion and [Ca2+]i oscillations stimulated by 11.2 mM glucose. Using the same system, we found that insulin secretion in 7.5 mM glucose plus 1 mM 8-bromo-cAMP (8-Br-cAMP) is mediated by both Cav1.2 and Cav1.3. Treatment of INS-1 cells or INS-1 cells stably expressing Cav1.2/dihydropyridine-insensitive (DHPi) channels in the presence of 10 microM nifedipine, with effector-specific cAMP analogs 8-(4-chlorophenylthio)-2'-O-methyladenosine-cAMP [8-pCPT-2'-O-Me-cAMP; 100 microM; Exchange Protein directly Activated by cAMP 2 (Epac2)-selective] or N6-benzoyl-cAMP [50 microM; Protein Kinase A (PKA)-selective] partially increased insulin secretion. Secretion stimulated by a combination of the two cAMP analogs was additive and comparable with that stimulated by 1 mM 8-Br-cAMP. In INS-1 cells stably expressing Cav1.3/DHPi in the presence of 10 microM nifedipine, N6-benzoyl-cAMP, but not 8-pCPT-2'-O-Me-cAMP, significantly increased glucose-stimulated insulin secretion. However, the combination of N6-benzoyl-cAMP and 8-pCPT-2'-O-Me-cAMP significantly increased glucose-stimulated secretion compared with N6-benzoyl-cAMP alone. In INS-1 cells, 8-Br-cAMP potentiation of insulin secretion in 7.5 mM glucose is blocked by thapsigargin (1 microM) and ryanodine (0.5 microM). In contrast, ryanodine has no effect on insulin secretion or [Ca2+]i oscillations stimulated by 11.2 mM glucose in INS-1 cells. Our data suggest that both Cav1.2 and Cav1.3 mediate insulin secretion stimulated by 7.5 mM glucose and cAMP via a mechanism that requires internal stores of Ca2+. Furthermore, cAMP modulation of secretion mediated by Cav1.2 seems to involve both Epac2 and PKA independently. In contrast, cAMP modulation of Cav1.3-mediated secretion depends upon PKA activation, whereas the contribution of Epac2 is dependent upon PKA activation.

Role of L‐type Ca2+ channels in neural stem/progenitor cell differentiation

Ca(2+) influx through voltage-gated Ca(2+) channels, especially the L-type (Ca(v)1), activates downstream signaling to the nucleus that affects gene expression and, consequently, cell fate. We hypothesized that these Ca(2+) signals may also influence the neuronal differentiation of neural stem/progenitor cells (NSCs) derived from the brain cortex of postnatal mice. We first studied Ca(2+) transients induced by membrane depolarization in Fluo 4-AM-loaded NSCs using confocal microscopy. Undifferentiated cells (nestin(+)) exhibited no detectable Ca(2+) signals whereas, during 12 days of fetal bovine serum-induced differentiation, neurons (beta-III-tubulin(+)/MAP2(+)) displayed time-dependent increases in intracellular Ca(2+) transients, with DeltaF/F ratios ranging from 0.4 on day 3 to 3.3 on day 12. Patch-clamp experiments revealed similar correlation between NSC differentiation and macroscopic Ba(2+) current density. These currents were markedly reduced (-77%) by Ca(v)1 channel blockade with 5 microm nifedipine. To determine the influence of Ca(v)1-mediated Ca(2+) influx on NSC differentiation, cells were cultured in differentiative medium with either nifedipine (5 microm) or the L-channel activator Bay K 8644 (10 microm). The latter treatment significantly increased the percentage of beta-III-tubulin(+)/MAP2(+) cells whereas nifedipine produced opposite effects. Pretreatment with nifedipine also inhibited the functional maturation of neurons, which responded to membrane depolarization with weak Ca(2+) signals. Conversely, Bay K 8644 pretreatment significantly enhanced the percentage of responsive cells and the amplitudes of Ca(2+) transients. These data suggest that NSC differentiation is strongly correlated with the expression of voltage-gated Ca(2+) channels, especially the Ca(v)1, and that Ca(2+) influx through these channels plays a key role in promoting neuronal differentiation.

Embryonic Stem Cells in Predictive Cardiotoxicity: Laser Capture Microscopy Enables Assay Development

Embryonic stem (ES) cells offer unprecedented opportunities for in vitro drug discovery and safety assessment of compounds. Cardiomyocytes derived from ES cells enable development of predictive cardiotoxicity models to increase the safety of novel drugs. Heterogeneity of differentiated ES cells limits the development of reliable in vitro models for compound screening. We report an innovative and robust approach to isolate ES-derived cardiomyocytes using laser microdissection and pressure catapulting (LMPC). LMPC cells were readily applied onto 96-well format in vitro pharmacology assays. The expression of developmental and functional cardiac markers, Nkx 2.5, MLC2V, GATA-4, Connexin 43, Connexin 45, Serca-2a, cardiac alpha actin, and phospholamban, among others, was confirmed in LMPC ES-derived cardiomyocytes. Functional assays exhibited cardiac-like response to increased extracellular calcium (5.4 mM extracellular Ca2+) and L-type calcium channel antagonist (1 microM nifedipine). In conclusion, laser microdissection and pressure catapulting is a robust technology to isolate homogeneous ES-derived cell types from heterogeneous populations applicable to assay development.