Serotonin contracts the rat mesenteric artery by inhibiting 4-aminopyridine-sensitive Kv channels via the 5-HT2A receptor and Src tyrosine kinase

Serotonin (5-hydroxytryptamine (5-HT)) is a neurotransmitter that regulates a variety of functions in the nervous, gastrointestinal and cardiovascular systems. Despite such importance, 5-HT signaling pathways are not entirely clear. We demonstrated previously that 4-aminopyridine (4-AP)-sensitive voltage-gated K+ (Kv) channels determine the resting membrane potential of arterial smooth muscle cells and that the Kv channels are inhibited by 5-HT, which depolarizes the membranes. Therefore, we hypothesized that 5-HT contracts arteries by inhibiting Kv channels. Here we studied 5-HT signaling and the detailed role of Kv currents in rat mesenteric arteries using patch-clamp and isometric tension measurements. Our data showed that inhibiting 4-AP-sensitive Kv channels contracted arterial rings, whereas inhibiting Ca2+-activated K+, inward rectifier K+ and ATP-sensitive K+ channels had little effect on arterial contraction, indicating a central role of Kv channels in the regulation of resting arterial tone. 5-HT-induced arterial contraction decreased significantly in the presence of high KCl or the voltage-gated Ca2+ channel (VGCC) inhibitor nifedipine, indicating that membrane depolarization and the consequent activation of VGCCs mediate the 5-HT-induced vasoconstriction. The effects of 5-HT on Kv currents and arterial contraction were markedly prevented by the 5-HT2A receptor antagonists ketanserin and spiperone. Consistently, alpha-methyl 5-HT, a 5-HT2 receptor agonist, mimicked the 5-HT action on Kv channels. Pretreatment with a Src tyrosine kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, prevented both the 5-HT-mediated vasoconstriction and Kv current inhibition. Our data suggest that 4-AP-sensitive Kv channels are the primary regulator of the resting tone in rat mesenteric arteries. 5-HT constricts the arteries by inhibiting Kv channels via the 5-HT2A receptor and Src tyrosine kinase pathway.

[ effect of block of the ultrarapid delayed rectifier potassium current [Ikur] by 50 micro M 4-amino pyrimidine on pacemaker activity in the mouse sinoatrial and atriovetricular nodes]

In the heart cells, ultrarapid delayed rectifier potassium current [IKur] is one of the important currents in action potential repolarization phase. Ultrarapid delayed rectifier potassium current is specifically blocked by low concentration of 4-Amino Pyrimidine [4-AP]. The aim of this study was to determine the effect of low concentration of 4-Amino Pyrimidine blocker on pacemaker activity of sinoatrial node [SAN] and atrioventricular node [AVN] of mouse heart. SAN and AVN were separated and the pacemaker activity of distinct intact SAN and AVN was recorded before and during 50 micro M 4-AP by two separate metal microelectrodes that were in contact with the endothelail surface of the nodes. Then the action potential cycle length [CL] was measured. 50 micro M 4-Amino Pyrimidine increased the action potential cycle length of SAN and AVN preparations respectively by 20.2 +/- 3.3% and 18 +/- 3%. These increases on the action potential cycle length were significant in both nodes. According to the results, IKur is present in both SAN and AVN nodes and the effect of 50 micro M 4-AP on action potential cycle length [CL] of the two nodes is the same

blocking activity of different toxins against potassium channels Kv3.4 in RLE cells

K[+] channel toxins are essential tools for the first purifications, analysis of subunit structures and brain localization of voltage-gated K[+] [Kv] channels. The effects of a lot of toxins on Kv are not fully known. Using whole-cell patch clamping technique the action of a series of toxins on Kv3.4 current in rat liver cells with expressed Kv3.4 channels [RLE] cloned cells was investigated. The cells were grown in Williams E medium and after 6-8 days, they were suitable for patch clamping. A family of currents was recorded during voltage-clamp steps to various potentials applied from a holding potential of-60 mV to 60-80 mV in 10 mV increments. Upon depolarization, all channels were opened with a sigmoidal time course, reached to the peak within a few 10[th] of milliseconds and then slowly inactivated. Bath application of tetraethyl ammonium [TEA] or 3, 4-diaminopyridine [DAP] reduced the current dose dependently and inhibited it completely at 3 mM and 25 micro M respectively. The Bunodosoma granulifera [BgK] and Heteractis magniflca [HmK] toxins at concentrations up to 30 and 10 micro M respectively could not completely inhibit the current. On the hand, toxins such as beta-bungarotoxin, corotoxin, novel toxin and dendrotoxins I [DIP] and K [DPK] even in high concentrations [up to 100 mM] had not any significant effect on Kv3.4 current. Comparison of chemical structures of these effective agents with other reported effective toxins such as blood depressing substances [BDS 1 and II] show no homology between them, but specially the potency of 3, 4-DAP is comparable with these toxins. These results showed that, the Kv3.4 is more sensitive than other K[+] channels to 3, 4-DAP. The sensitivity of this channel to the TEA is low [at mM concentration]. More investigation is necessary to find more selective and potent inhibitor of Kv3.4 channels

Isolation of 4-aminopyridine resistant mutants affecting alkali-insoluble glucan content of cell walls in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells when grown on synthetic medium plates containing 10 mM of 4-aminopyridine (4-AP) undergo cell lysis. Using an ethylmethane sulfonate mutagenesis (EMS) screen, 4-AP resistant mutants (apr) were isolated which could grow on inhibitory concentration of 4-AP. Eighty mutants were obtained that were recessive, monogenic and formed two complementation groups. To identify genes, whose products might be interacting with the apr loci, extragenic suppressors were isolated, which reverted 4-AP resistance phenotype of apr mutants. The suppressors, when genetically characterized, were found to be recessive and represented two loci with overlapping functions. Representative alleles from apr mutants were analyzed for cell wall composition. They were found to have a higher amount of alkali-insoluble glucan signifying the role of alkali-insoluble glucan in cell wall maintenance.

Inhibitory Effects of Potassium Channel Blockers on Carbachol-induced Contraction in Rat Detrusor Muscle

We present accidental findings that potassium channel blockers, such as tetraethyl-ammonium (TEA) or 4-aminopyridine (4-AP), inhibit the sustained tonic contraction induced by carbachol in rat detrusor muscle strips. The relatively lower concentrations (5 mM) potentiated phasic contractions. The potentiation of phasic contraction was not observed in nicardipine pretreated condition. In nicardipine pretreated condition, the concentration-response curves for the negative inotropic effect of potassium channel blockers were shifted to the right by the increasing concentration of carbachol from 0.5 micrometer to 5 micrometer. IC50 was changed significantly from 0.19 to 0.64 mM (TEA) and from 0.21 to 0.96 (4-AP). Such inhibitory effects were also observed in Ca2+ depleted condition, where 0.1 mM EGTA and 1 micrometer thapsigargin were added into Ca2+ free solution. In conclusion, inhibitory effects of potasssium channel blockers on carbachol-induced contraction may be ascribed to the direct inhibition of receptor-agonist binding.

4-Aminopyridine (4-AP) augments Ca(2+)-dependent action potential and changes oscillatory firing patterns in rat cerebellar Purkinje cells

Intracellular recordings in cerebellar slice preparation showed that applications of 4-AP altered the pattern of oscillatory firing activity in Purkinje cells (PCs), especially yielding pronounced changes in action potential shape. 4-AP increased the amplitude and duration of action potential significantly and decreased the spike frequency. After 4-AP application, the duration of bursting was prolonged and the duration of after-burst hyperpolarization was progressively shortened. In all PCs tested, the rhythmicity of oscillatory firing activity was abolished completely at the steady state. These results suggest that 4-AP-sensitive currents determine the shape and frequency of individual Ca(2+)-dependent action potentials as well as maintaining oscillatory firing activity in PCs.

A review on drugs and toxins affecting presynaptic K+ currents and phasic quantal transmitter release at motor nerve terminals

Esta revisión reúne la información disponible sobre los agentes farmacológicos y toxina sque bloquean los diferentes tipos de corrientes de K+ presinápticas, y discute la importancia relativa de estas corrientes en el control de la liberación fásica y cuántica del transmisor. Los agentes farmacológicos y toxinas que bloquean la corriente rápida de K+ voltaje-dependiente (IKf), aumentan la liberación fásica de actilcolina evocada por el impulso nervioso. Este efecto es debido al aumento del influjo de Ca2+ durante la despolarización de la membrana presináptica. El bloqueo selectivo de la correinte de K+ calcio-dependiente IK(Ca) no produce ningún cambio en la liberación fásica del transmisor, lo cual indica que en condiciones fisiológicas esta corriente no tiene un papel significaivo en la repolarización de la membrana presináptica. La contribución de la corriente lenta de K+ voltaje-dependiente (IKs) en la liberación fásica de acetilcolina no ha sido aún dilucidada. En conclusión, IKf, IK(Ca) e IKs pueden modular la entrada de Ca2ñ en las terminaciones nerviosas motoras; sin embargo, en condiciones fisiológicas solamente IKf tiene una importancia clave, ya que controla el indlujo transitorio de Ca2+ que es responsable d ela liberación fásica del neurotransmisor