Characteristics of Ca Currents in Rabbit Basilar Arterial Smooth Muscle Cells

In order to determine the exact nature of Ca channels involved in various cerebrovascular contractile behaviour including vasospasm, we performed experiments to identify and characterize the types of Ca channels in rabbit basilar arterial smooth muscle cells by using kinetic and pharmacologic tools. Sngle smooth muscle cells were enzymatically isolated from rabbit basilar artery. Single cells were voltageclamped, and membrane currents were recorded using the whole-cell configuration of patch clamp technique. The measured cell capacitance (Cm) was 19.2 ± 0.65 pF ( n 21) and input resistance (Rmpu, ) was 2.04 ± 0.12 Gn(n 12), These passive membame properties are similar to other cerebraovascular smooth muscle cells. Inward Ca2 ' -channel current was recorded. Replacement of external Ca2+( 2 mM) with Ba" (10 mM) increased the amplitude of the current and did not shift the I-V relationship for lBa in comparison with that for Ie.. Changing the holding potential from -SO to -40 mV decreased the current amplitude but did not shift the voltage dependence. No detectable low-threshold, rapid inactivating inward current was observed. Steady-state activation and inactivation curves for lea(V, 210c') -4.4 mV; V, 2(inac,) -22.3 mV) and lBa(v, 2(oc') -7.5 mV; VI 2 {Inact } -20.5 mV) were determined. The theoritical 'window current's amplitude was calculated for lea and lBa• Calcium channel current was almost completely inhibited by l,uM nicardipine and enhanced by Bay K 8644, suggesting this is carried by 'L-type' but not by 'T-type' calcium channel. Bay K 8644 significantly shifted activation curve to the negative potential. Both 8-br-cAMPW.l ~ 1 mM) and 8-br-cGMP( 0.1 ~ 1 mM), a membrane permeable cyclic nucleotides, decreased the current amplitude. From the above results, it is suggested that only 'L-type' Ca-current(ICa_d exists in rabbit basilar arterial smooth muscle cells

Menthol Enhances an Antiproliferative Activity of 1α,25-Dihydroxyvitamin D3 in LNCaP Cells

1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], the most active form of vitamin D3, and its analogues have therapeutic benefits for prostate cancer treatment. However, the development of hypercalcemia is an obstacle to clinical applications of 1α,25(OH)2D3 for cancer therapy. In this study, we provide evidence that menthol, a key component of peppermint oil, increases an anti-proliferation activity of 1α,25(OH)2D3 in LNCaP prostate cancer cells. We found that menthol per se does not exhibit antiproliferative activity, but it is able to enhance 1α,25(OH)2D3-mediated growth inhibition in LNCaP cells. Fluorometric assays using Fura-2 showed that 1α,25(OH)2D3 does not induce acute Ca2+ response, whereas menthol evokes an increase in [Ca2+]i, which suggests that cross-talks of menthol-induced Ca2+ signaling with 1α,25(OH)2D3-mediated growth inhibition pathways. In addition, Western blot analysis revealed that 1α,25(OH)2D3 and menthol cooperatively modulate the expression of bcl-2 and p21 which provides the insight into the molecular mechanisms underlying the enhanced 1α,25(OH)2D3-mediated growth inhibition by menthol. Thus, our findings suggest that menthol may be a useful natural compound to enhance therapeutic effects of 1α,25(OH)2D3

Calpain-mediated proteolysis of polycystin-1 C-terminus induces JAK2 and ERK signal alterations

AbstractAutosomal dominant polycystic kidney disease (ADPKD), a hereditary renal disease caused by mutations in PKD1 (85%) or PKD2 (15%), is characterized by the development of gradually enlarging multiple renal cysts and progressive renal failure. Polycystin-1 (PC1), PKD1 gene product, is an integral membrane glycoprotein which regulates a number of different biological processes including cell proliferation, apoptosis, cell polarity, and tubulogenesis. PC1 is a target of various proteolytic cleavages and proteosomal degradations, but its role in intracellular signaling pathways remains poorly understood. Herein, we demonstrated that PC1 is a novel substrate for μ- and m-calpains, which are calcium-dependent cysteine proteases. Overexpression of PC1 altered both Janus-activated kinase 2 (JAK2) and extracellular signal-regulated kinase (ERK) signals, which were independently regulated by calpain-mediated PC1 degradation. They suggest that the PC1 function on JAK2 and ERK signaling pathways might be regulated by calpains in response to the changes in intracellular calcium concentration

The possible roles of hyperpolarization-activated cyclic nucleotide channels in regulating pacemaker activity in colonic interstitial cells of Cajal

BACKGROUND: Hyperpolarization-activated cyclic nucleotide (HCN) channels are pacemaker channels that regulate heart rate and neuronal rhythm in spontaneously active cardiac and neuronal cells. Interstitial cells of Cajal (ICCs) are also spontaneously active pacemaker cells in the gastrointestinal tract. Here, we investigated the existence of HCN channel and its role on pacemaker activity in colonic ICCs. METHODS: We performed whole-cell patch clamp, RT-PCR, and Ca(2+)-imaging in cultured ICCs from mouse mid colon. RESULTS: SQ-22536 and dideoxyadenosine (adenylate cyclase inhibitors) decreased the frequency of pacemaker potentials, whereas both rolipram (cAMP-specific phosphodiesterase inhibitor) and cell-permeable 8-bromo-cAMP increased the frequency of pacemaker potentials. CsCl, ZD7288, zatebradine, clonidine (HCN channel blockers), and genistein (a tyrosine kinase inhibitor) suppressed the pacemaker activity. RT-PCR revealed expression of HCN1 and HCN3 channels in c-kit and Ano1 positive colonic ICCs. In recordings of spontaneous intracellular Ca(2+) [Ca(2+)](i) oscillations, rolipram and 8-bromo-cAMP increased [Ca(2+)](i) oscillations, whereas SQ-22536, CsCl, ZD7288, and genistein decreased [Ca(2+)](i) oscillations. CONCLUSIONS: HCN channels in colonic ICCs are tonically activated by basal cAMP production and participate in regulation of pacemaking activity

The Possible Role of Nitric Oxide on Enteric Nerves-Mediated Relaxation of the Gastric Smooth Muscle of the Guinea Pig

The influence of the enteric nerves stimulation on the contractility of gastric circular muscle was studied in guinea pig stomachs. The enteric nerves were activated by electric field stimulation(EFS; 90 V, 1 rns, 32 Hz square pulses for 1 s), EFS produced initial transient contraction followed by relaxation and slow recovery. The initial contraction was sensitively blocked by treatment with atropine. The following relaxation still occurred in nonadrenergic noncholinergidNANC) state. EFSinduced relaxation was reduced by LG-nitro-L-arginine(L-NNA), a nitric oxide(NO) synthase inhibitor. The relaxation was restored from the suppressed state after the application of L-arginine(L-arg), a substrate of nitric oxide synthase. With conventional intracellular recording, slow wave and inhibitory junction potential(IJP) were recorded. L-NNA had no effect on IJP, while apamin blocked it. In conclusion, it is suggested that NO may playa major role in EFS-induced relaxation. The exact mechanism of this relaxation is unknown, but the relaxation does not result from the IJP induced by the activation of apamin-sensitive potassium channels

The Effect of Stimulation Frequency on the Ionic Currents in Single Atrial Cells of the Rabbit

In single atrial cells isolated from rabbit hearts the calcium current and [Caj-dependent transient outward current were recorded using the whole-cell clamp technique and the effect of stimulation frequency on these currents was investigated. Voltage dependent transient outward current, which contributes the initial, rapid repolarization phase of the action potential and is frequency-dependent, was also investigated. Increasing the stimulation frequency from O. 025 Hz to 1 Hz had no effect on the calcium current and [Caj-dependent transient outward current and greatly inhibited voltage-dependent transient outward current. The amplitude of voltage dependent transient outward current increased as the membrane potential became depolarized, its steady-state inactivation spans the voltage range -70 mV to -10 mVand steady-state activation curve -30 mV to 30 mV. Within the range of the resting membrane potential (at -70 mV), the voltage-dependent recovery time constant was 1. 3 s. The reversal potential was about -50 mV. Voltage-dependent transient outward current was inhibited by K-channel blockers and not inhibited by modulation of [Cali. From the above findings, it is concluded that due to the amplitude and voltage-dependent recovery time constant which were the basic mechanisms for frequency-dependency, the voltage- dependent transient outward current contributes the initial, rapid repolarization phase and changed the action potential configuration according to stimulation frequency in the rabbit atrium

A comprehensive manually curated protein–protein interaction database for the Death Domain superfamily

The Death Domain (DD) superfamily, which is one of the largest classes of protein interaction modules, plays a pivotal role in apoptosis, inflammation, necrosis and immune cell signaling pathways. Because aberrant or inappropriate DD superfamily-mediated signaling events are associated with various human diseases, such as cancers, neurodegenerative diseases and immunological disorders, the studies in these fields are of great biological and clinical importance. To facilitate the understanding of the molecular mechanisms by which the DD superfamily is associated with biological and disease processes, we have developed the DD database (http://www.deathdomain.org), a manually curated database that aims to offer comprehensive information on protein–protein interactions (PPIs) of the DD superfamily. The DD database was created by manually curating 295 peer-reviewed studies that were published in the literature; the current version documents 175 PPI pairs among the 99 DD superfamily proteins. The DD database provides a detailed summary of the DD superfamily proteins and their PPI data. Users can find in-depth information that is specified in the literature on relevant analytical methods, experimental resources and domain structures. Our database provides a definitive and valuable tool that assists researchers in understanding the signaling network that is mediated by the DD superfamily