Shunichi Kajioka

Calcium-associated mechanisms in gut pacemaker activity.

•  Introduction ‐  Voltage‐operated Ca2+ channels ‐  Non‐selective cation channels ‐  Cl− channels ‐  [Ca2+]i oscillations ‐  Intracellular Ca2+ release channels ‐  ICC‐like cells in auxiliary digestive organs

Pinacidil inhibits the ryanodine‐sensitive outward current and glibenclamide antagonizes its action in cells from the rabbit portal vein

Pinacidil, a potassium‐channel opener, inhibited the ryanodine‐sensitive oscillatory outward potassium current induced by Ca released from an intracellular store. Glibenclamide, a blocker of the ATP‐sensitive K‐channel, prevented the action of pinacidil, suggesting the presence of an additional site (to K channels) for the vasodilator actions of pinacidil at which glibenclamide can act as an antagonist.

Tyrosine kinase inhibitors and ATP modulate the conversion of smooth muscle L-type Ca2+ channels toward a second open state

Properties of smooth and cardiac L‐type Ca2+ channels differ prominently in several physiological aspects, including sympathetic modulation. To assess the possible underlying mechanisms, we applied the whole cell patch‐clamp technique to guinea pig detrusor smooth muscle cells, in which only L‐type Ca2+ channel currents are observed in practice. During depolarization to large positive potentials, the conformation of the majority of L‐type Ca2+ channels is converted from the normal (O1) to a second open state (O2), which undergoes little inactivation during depolarization. Extracellular application of genistein, a known tyrosine kinase inhibitor, significantly attenuated the voltage‐dependent conversion of Ca2+ channels to O2, accompanied by reduction of availability, whereas genistin, an inactive analog, had little effect. In the absence of ATP in the patch pipette, intracellular application of either genistein or tyrphostin‐47 suppressed the conversion to O2. Computer calculation revealed that the acceleration of the O1 to an inactivated state qualitatively reconstructs the unique effects of PTK inhibitors antagonized by ATP. We concluded that under normal conditions smooth muscle L‐type Ca2+ channels are already modulated by tyrosine‐kinase and ATP‐related mechanism(s) and thereby easily achieve the second conversion, which yields voltage‐dependent modulation of L‐type Ca2+ current analogous to that in cardiac myocytes during β‐adrenoceptor stimulation.—Nakayama, S., Ito, Y., Sato, S., Kamijo, A., Liu, H.‐N., Kajioka, S. Tyrosine kinase inhibitors and ATP modulate the conversion of smooth muscle L‐type Ca2+ channels toward a second open state. FASEB J. 20, E652–E663 (2006)

Endogenous Cardiac Troponin T Modulates Ca2+-Mediated Smooth Muscle Contraction

Mechanisms linked to actin filaments have long been thought to cooperate in smooth muscle contraction, although key molecules were unclear. We show evidence that cardiac troponin T (cTnT) substantially contributes to Ca2+-mediated contraction in a physiological range of cytosolic Ca2+ concentration ([Ca2+]i). cTnT was detected in various smooth muscles of the aorta, trachea, gut and urinary bladder, including in humans. Also, cTnT was distributed along with tropomyosin in smooth muscle cells, suggesting that these proteins are ready to cause smooth muscle contraction. In chemically permeabilised smooth muscle of cTnT+/− mice in which cTnT reduced to ~50%, the Ca2+-force relationship was shifted toward greater [Ca2+]i, indicating a sizeable contribution of cTnT to smooth muscle contraction at [Ca2+]i < 1 μM. Furthermore, addition of supplemental TnI and TnC reconstructed a troponin system to enhance contraction. The results indicated that a Tn/Tn-like system on actin-filaments cooperates together with the thick-filament pathway.

A target K+ channel for the LP-805-induced hyperpolarization in smooth muscle cells of the rabbit portal vein

SummaryThe resting membrane potential of smooth muscle cells of the rabbit portal vein was −51.2 mV. LP-805 (8-tert-butyl-6,7-dihydropyrrolo[3,2-e] 5-methylpyrazolo [1,5-a] pyrimidine-3-carbonitrile) hyperpolarized the membrane to −62.3 mV (10 μM) and inhibited the burst spike discharges as measured using the microelectrode method. In dispersed smooth muscle cells, LP-805 (10 μM) generated an outward-current with a maximum amplitude of 68 pA at a holding potential of −40 mV in experiments using the voltage-clamp procedure. The reversal potential of the outward current evoked by LP-805 was −82 mV and this value was close to the equilibrium potential for K+ (−80 mV) in the present ionic conditions, suggesting that LP-805 activated the K+ channel. Generation of both the hyperpolarization and the outward c urrent by LP-805 was inhibited by glibenclamide (≥ 1 μM). Using the cell-attached and cell-free patch-clamp (in the presence of GDP) procedures, the maxi-K+ channel current (150 pS) could be recorded in the absence of LP-805; application of LP-805 additionally opened a small conductance K+ channel current (15 pS) without change in the activity of the maxi-K+ channel. The maxi-K+ channel was sensitive to charybdotoxin (0.1 μM) and to intracellular Ca2+ ([Ca2+]i) concentration. The 15 pS channel was insensitive to [Ca2+]i and charybdotoxin, but sensitive to intracellular ATP concentration. Glibenclamide (> 1 μM) inhibited the 15 pS K+ channel activated by LP-805. These actions of LP-805 on the maxi-K+ and 15 pS K+ channels are the same as those previously observed for nicorandil and pinacidil. Thus, LP-805 is a K+ channel opener with a chemical structure different from those of the known openers.

Obstruction enhances rho-kinase pathway and diminishes protein kinase C pathway in carbachol-induced calcium sensitization in contraction of α-toxin permeabilized guinea pig detrusor smooth muscle†‡

We investigated the relative important role of rho kinase (ROK) and protein kinase C (PKC) pathways in carbachol (CCh)‐induced Ca2+ sensitization in α‐toxin permeabilized Guinea pig detrusor smooth muscle (DSM) following bladder outlet obstruction (BOO).

Involvement of IP3-receptor activation in endothelin-1-induced Ca2+ influx in rat pulmonary small artery

We examined the endothelin-1 (ET-1)-induced increase in the intracellular free Ca(2+) concentration ([Ca(2+)]i) in fura-2-loaded rat pulmonary small arteries. ET-1 (30 nM) elicited a long-lasting increase in [Ca(2+)]i in physiological salt solution (PSS). In subsequent experiments, arteries were pretreated with BQ-788, an ETB-specific blocker, to allow us to focus on responses mediated via the ETA receptor, the existence of which was confirmed by immunohistochemistry. In Ca(2+)-free PSS, ET-1 evoked a small transient increase in [Ca(2+)]i, indicating Ca(2+) release from the SR (sarcoplasmic reticulum). After a switch to PSS (containing 2mM CaCl2), ET-1 elicited a long-lasting increase in [Ca(2+)]i that was not inhibited by 1 μM nicardipine, an L-type Ca(2+)-channel inhibitor, suggesting involvement of a Ca(2+)-influx pathway independent of that channel. In arteries preincubated with 30 μM cyclopiazonic acid (CPA) or 2 μM thapsigargin (TG), the ET-1-induced Ca(2+)-release was greatly reduced, and the induced Ca(2+)-influx was attenuated. U-73122, a phospholipase C (PLC) inhibitor, had inhibitory effects similar to those of CPA and TG on the ET-1-induced Ca(2+)-release and Ca(2+)-influx, whereas U-73343, an inactive analogue of U-73122, had no such effects. Two putative membrane-permeable IP3-receptor blockers, 2-aminoethoxydiphenyl borate (2APB, 50 μM) and Xestospongin C (20 μM), (a) almost completely inhibited the ET-1-induced Ca(2+)-release and Ca(2+)-influx, and (b) reduced the ET-1-induced contraction. These results indicate that in rat pulmonary small arteries, ET-1 induces receptor-operated Ca(2+) influx via the ETA receptor, and that this influx interacts with InsP3-receptor activation.

The profiles and patterns of detrusor overactivity and their association with overactive bladder symptoms in men with benign prostatic enlargement associated with detrusor overactivity

To investigate the association of both the urodynamic profiles and patterns of detrusor overactivity (DO) with the severity of the symptoms related to overactive bladder (OAB) in men with benign prostatic enlargement (BPE) associated with DO.

Equol inhibits prostate cancer growth through degradation of androgen receptor by S-phase kinase-associated protein 2

Chemopreventive and potential therapeutic effects of soy isoflavones have been shown to be effective in numerous preclinical studies as well as clinical studies in prostate cancer. Although the inhibition of androgen receptor signaling has been supposed as one mechanism underlying their effects, the precise mechanism of androgen receptor inhibition remains unclear. Thus, this study aimed to clarify their mechanism. Among soy isoflavones, equol suppressed androgen receptor as well as prostate‐specific antigen expression most potently in androgen‐dependent LNCaP cells. However, the inhibitory effect on androgen receptor expression and activity was less prominent in castration‐resistant CxR and 22Rv1 cells. Consistently, cell proliferation was suppressed and cellular apoptosis was induced by equol in LNCaP cells, but less so in CxR and 22Rv1 cells. We revealed that the proteasome pathway through S‐phase kinase‐associated protein 2 (Skp2) was responsible for androgen receptor suppression. Taken together, soy isoflavones, especially equol, appear to be promising as chemopreventive and therapeutic agents for prostate cancer based on the fact that equol augments Skp2‐mediated androgen receptor degradation. Moreover, because Skp2 expression was indicated to be crucial for the effect of soy isoflavones, soy isoflavones may be applicable for precancerous and cancerous prostates.

Spatial Analysis of Slowly Oscillating Electric Activity in the Gut of Mice Using Low Impedance Arrayed Microelectrodes

Smooth and elaborate gut motility is based on cellular cooperation, including smooth muscle, enteric neurons and special interstitial cells acting as pacemaker cells. Therefore, spatial characterization of electric activity in tissues containing these electric excitable cells is required for a precise understanding of gut motility. Furthermore, tools to evaluate spatial electric activity in a small area would be useful for the investigation of model animals. We thus employed a microelectrode array (MEA) system to simultaneously measure a set of 8×8 field potentials in a square area of ∼1 mm2. The size of each recording electrode was 50×50 µm2, however the surface area was increased by fixing platinum black particles. The impedance of microelectrode was sufficiently low to apply a high-pass filter of 0.1 Hz. Mapping of spectral power, and auto-correlation and cross-correlation parameters characterized the spatial properties of spontaneous electric activity in the ileum of wild-type (WT) and W/Wv mice, the latter serving as a model of impaired network of pacemaking interstitial cells. Namely, electric activities measured varied in both size and cooperativity in W/Wv mice, despite the small area. In the ileum of WT mice, procedures suppressing the excitability of smooth muscle and neurons altered the propagation of spontaneous electric activity, but had little change in the period of oscillations. In conclusion, MEA with low impedance electrodes enables to measure slowly oscillating electric activity, and is useful to evaluate both histological and functional changes in the spatio-temporal property of gut electric activity.