Noriyoshi Teramoto

Regenerative component of slow waves in the guinea‐pig gastric antrum involves a delayed increase in [Ca2+]i and Cl− channels

Regenerative potentials were initiated by depolarizing short segments of single bundles of circular muscle isolated from the gastric antrum of guinea‐pigs. When changes in [Ca2+]i and membrane potential were recorded simultaneously, regenerative potentials were found to be associated with an increase in [Ca2+]i, with the increase starting after a minimum latency of about 1 s. Although the increase in [Ca2+]i was reduced by nifedipine, the amplitudes of the regenerative responses were little changed. Regenerative responses and associated changes in [Ca2+]i were abolished by loading the preparations with the Ca2+ chelator MAPTA‐AM. Regenerative potentials were abolished by 2‐aminoethoxydiphenyl borate (2APB), an inhibitor of IP3 induced Ca2+ release, by N‐ethylamaleimide (NEM), an alkylating agent which blocks activation of G‐proteins and were reduced in amplitude by two agents which block chloride (Cl−)‐selective channels in many tissues. The observations suggest that membrane depolarization triggers IP3 formation. This causes Ca2+ release from intracellular stores which activates Ca2+‐dependent Cl− channels.

Physiological roles of ATP‐sensitive K+ channels in smooth muscle

Potassium channels that are inhibited by intracellular ATP (ATPi) were first identified in ventricular myocytes, and are referred to as ATP‐sensitive K+ channels (i.e. KATP channels). Subsequently, K+ channels with similar characteristics have been demonstrated in many other tissues (pancreatic β‐cells, skeletal muscle, central neurones, smooth muscle). Approximately one decade ago, KATP channels were cloned and were found to be composed of at least two subunits: an inwardly rectifying K+ channel six family (Kir6.x) that forms the ion conducting pore and a modulatory sulphonylurea receptor (SUR) that accounts for several pharmacological properties. Various types of native KATP channels have been identified in a number of visceral and vascular smooth muscles in single‐channel recordings. However, little attention has been paid to the molecular properties of the subunits in KATP channels and it is important to determine the relative expression of KATP channel components which give rise to native KATP channels in smooth muscle. The aim of this review is to briefly discuss the current knowledge available for KATP channels with the main interest in the molecular basis of native KATP channels, and to discuss their possible linkage with physiological functions in smooth muscle.

Cyclic GMP-dependent but G-kinase-independent inhibition of Ca2+-dependent Cl- currents by NO donors in cat tracheal smooth muscle.

1 The effects of NO donors on Ca2+‐dependent Cl− currents (ICl(Ca)) were investigated in freshly isolated cat tracheal myocytes using the whole‐cell patch clamp technique. 2 With nystatin‐perforated whole‐cell recording, carbachol (CCh, ≥ 1 μm) induced a transient inward current (ICCh) with a reversal potential of about ‐20 mV. Activation of ICCh probably occurred through the M3 muscarinic receptor, since nanomolar concentrations of 4‐diphenylacetoxy‐N‐methylpiperidine methobromide (4‐DAMP) greatly inhibited this current, while 11‐(2‐(diethylamino)methyl)‐1‐piperidinylacetyl)‐5,11‐dihydro‐6H‐pyrido (2,3β) (1,4)benzodiazepine‐6‐one (AF‐DX 116) or pirenzepine at concentrations of up to 1 μm were almost ineffective. 3 Chloride channel/transporter blockers such as DIDS (100 μm), anthracene‐9‐carboxylic acid (9‐AC, 100 μm) and niflumic acid (100 μm) greatly inhibited ICCh, but cation channel blockers, such as nifedipine (10 μm), Zn2+ (500 μm) or Gd3+ (500 μm), were without effect. 4 Activation of ICCh was strongly attenuated by pretreatment with ryanodine (4 μm) plus caffeine (10 mM). Addition of neomycin (1 mM) into the bath or inclusion of heparin (3 mg ml−1) in the pipette abolished a substantial part of ICCh. These results suggest that ICCh is ICl(Ca), which is activated by inositol 1,4,5‐trisphosphate (IP3)‐mediated Ca2+ release. 5 The nitric oxide donor S‐nitroso‐N‐acetyl penicillamine (SNAP) reduced the amplitude of ICCh dose dependently (IC50, ≈10 μm). Similar inhibition was also exerted by other types of NO donor such as glyceryl trinitrate (GTN) and (±)‐E‐methyl‐2‐(E‐hydroxyimitol)‐5‐nitro‐6‐methoxy‐3‐hexeneamide (NO‐R). 6 SNAP‐induced ICCh inhibition was effectively antagonized by Methylene Blue (1‐100 nM), and mimicked by dibutyryl cGMP (db‐cGMP) (0.5‐1 mM), whereas two structurally distinct types of cGMP‐dependent (G)‐kinase inhibitor, N‐(2‐aminoethyl)‐5‐isoquinilinesulphonamide (H‐8, 2.5 μm) and KT5823 (1 μm), failed to counteract the inhibitory effects of SNAP or db‐cGMP. Another G‐kinase‐specific inhibitor Rp‐8‐(para‐chlorophenylthio)guanosine‐3′,5′‐cyclic monophosphorothioate (Rp‐8‐pCPT‐cGMPS; 1 μm) itself caused a marked reduction in ICCh. 7 SNAP (100 μm) or db‐cGMP (100 μm) exhibited no inhibitory actions, when caffeine (10 mM) or photolytically released IP3 were used instead of CCh to activate the inward current. 8 These results suggest that inhibition of ICCh by NO donors involves a cGMP‐dependent but G‐kinase‐independent mechanism, which may operate at a site(s) between the muscarinic (M3) and IP3 receptors.

Roles of inositol trisphosphate and protein kinase C in the spontaneous outward current modulated by calcium release in rabbit portal vein

We examined the effects of heparin, guanosine nucleotides, protein kinase C (PKC) modulators, such as phorbol 12,13-dibutylate (PDBu) and H-7 on Ca2+-dependent K+ currents in smooth muscle cells of the rabbit portal vein using the whole-cell patch-clamp technique, to explore the effects of PKC on the oscillatory outward current (Ioo). Neomycin (30 μM), an inhibitor of phospholipase C, and intracellular applications of heparin (10 μg/ml) and guanosine 5′-O-(2-thiodiphosphate) (GDP[βS]; 1 mM) partly but consistently inhibited the generation of Ioo, whereas a higher concentration of heparin (100 μg/ml) transiently enhanced then suppressed the generation of Ioo. Inhibition of Ioo generation by heparin was more powerful at the holding potential of + 20 mV than at −20 mV. Inositol 1,4,5-trisphosphate (InsP3; 30 μM) continuously generated Ioo at holding potentials more positive than −60 mV. Noradrenaline (10 μM) and caffeine (3–20 mM) transiently augmented, then reduced the generation of Ioo. Heparin (10 μg/ml) completely inhibited responses induced by InsP3 and noradrenaline, but not those induced by caffeine. Intracellular application of guanosine 5′-triphosphate (GTP; 200 μM) or low concentrations of guanosine 5′-O-(3-thiotriphosphate) (GTP[γS]; ⩽ 3 μM) continuously augmented the generation of Ioo. High concentrations of GTP[γS] (⩾10 μM) transiently augmented, then inhibited Ioo. Neither GTP[γS] nor noradrenaline induced the transient augmentation or the subsequent inhibition of Ioo when applied in the presence of GDP[βS] (1 mM), neomycin (30 μM) or heparin (10 μg/ml). PDBu (0.1 μM) reduced the generation of Ioo but failed to produce an outward current following application of caffeine (3–5 mM). This action of PDBu was inhibited by pretreatment with H-7 (20 μM). In the presence of H-7, GTP[γS] continuously enhanced the generation of Ioo. The suppression of the generation of Ioo during application of noradrenaline (10 μM) was reduced by pretreatment with H-7. Thus both InsP3 and protein kinase C contribute to the generation of Ioo in smooth muscle cells of the rabbit portal vein and heparin is not a specific InsP3 antagonist on the InsP3-induced Ca2+-release channel (PIRC). InsP3 opens PIRC and protein kinase C may deplete the stored Ca2+ by either inhibiting the reuptake of Ca2+ or by enhancement of the releasing actions of InsP3.

The effects of caffeine on ATP-sensitive K+ channels in smooth muscle cells from pig urethra

The effects of caffeine on both levcromakalim‐induced macroscopic and unitary currents in pig proximal urethra were investigated by the use of patch‐clamp techniques (conventional whole‐cell configuration and cell‐attached configuration). The effects of caffeine were also examined on currents in inside‐out patches of COS7 cells expressing carboxy terminus truncated inwardly rectifying K+ channel (Kir6.2) subunits (i.e. Kir6.2ΔC36) which form ATP‐sensitive K+ channels (KATP channels). In conventional whole‐cell configuration, the levcromakalim (100 μM)‐induced inward current (symmetrical 140 mM K+ conditions) was inhibited by caffeine (1 mM) at a holding potential of −50 mV. In contrast, ryanodine (10 μM) caused no significant inhibitory effect on the gradual decay of the levcromakalim‐induced current at −50 mV. The amplitude of the 30 μM levcromakalim‐induced current was enhanced by 3‐isobutyl‐1‐methylxanthine (IBMX, 100 μM). In cell‐attached configuration, the levcromakalim‐induced K+ channel openings were inhibited by subsequent application of 10 mM caffeine, decreasing the channel open probability at −50 mV. Reverse transcriptase‐polymerase chain reaction (RT–PCR) analysis revealed the presence of Kir6.2 transcript in pig urethra. Caffeine (3 mM) inhibited the channel activity of Kir6.2ΔC36 expressed in COS7 cells (3 mM caffeine, 65±6%, n=4; 10 mM caffeine, 29±2%, n=4). These results suggest that caffeine can inhibit the activity of KATP channels through a direct blocking effect on the pore‐forming Kir subunit.

An analysis of inhibitory junction potentials in the guinea‐pig proximal colon

Intracellular recordings were made from either sheets or isolated bundles of the circular muscle layer of guinea‐pig proximal colon and the responses evoked by stimulating inhibitory nerve fibres were analysed. Inhibitory junction potentials (IJPs), evoked by single stimuli, had two components which could be separated on their pharmacological and temporal characteristics and their voltage sensitivities. The initial component, which was abolished by apamin and reduced in amplitude by pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), had a brief time course: its amplitude was changed when the external concentration of potassium ions ([K+]o) was changed. The second component of the IJP had a slower onset than the first component, was abolished by l‐nitroarginine (NOLA) and oxadiazolo quinoxalin‐1‐one (ODQ), an inhibitor of soluble guanylate cyclase: its amplitude was little affected by changing [K+]o and was increased when the membrane potential of the circular layer was hyperpolarized. The observations suggest that the initial component of the IJP results from the release of ATP which triggers an increase in membrane conductance to K+ and that the second component results from the release of nitric oxide which suppresses a background inward current.

EVALUATION OF TRANSFECTION EFFICIENCY IN SKELETAL MUSCLE USING NANO/MICROBUBBLES AND ULTRASOUND

Recent studies have revealed that ultrasound contrast agents with low-intensity ultrasound, namely, sonoporation, can noninvasively deliver therapeutic molecules into target sites. However, the efficiency of molecular delivery is relatively low and the methodology requires optimization. Here, we investigated three types of nano/microbubbles (NMBs)-human albumin shell bubbles, lipid bubbles and acoustic liposomes-to evaluate the efficiency of gene expression in skeletal muscle as a function of their physicochemical properties and the number of bubbles in solution. We found that acoustic liposomes showed the highest transfection and gene expression efficiency among the three types of NMBs under ultrasound-optimized conditions. Liposome transfection efficiency increased with bubble volume concentration; however, neither bubble volume concentration nor their physicochemical properties were related to the tissue damage detected in the skeletal muscle, which was primarily caused by needle injection.

Actions of two main metabolites of propiverine (M-1 and m-2) on voltage-dependent L-type Ca2+ currents and Ca2+ transients in murine urinary bladder myocytes

The anticholinergic propiverine (1-methyl-4-piperidyl diphenylpropoxyacetate), which is used for the treatment of overactive bladder syndrome, has functionally active metabolites [M-1 (1-methyl-4-piperidyl diphenylpropoxyacetate N-oxide) and M-2 (1-methyl-4-piperidyl benzilate N-oxide)], but the site of actions of these metabolites is uncertain. Propiverine is rapidly absorbed after oral administration and is extensively biotransformed in the liver, giving rise to several active metabolites (M-1 and M-2). This study determines the effect of M-1 and M-2 on voltage-dependent nifedipine-sensitive inward Ca2+ currents (ICa) using patch-clamp techniques and fluorescent Ca2+ imaging [after electrical field stimulation (EFS) and acetylcholine (ACh)] in the murine urinary bladder. In conventional whole-cell recording, propiverine and M-1 but not M-2 inhibited the peak amplitude of ICa in a concentration-dependent manner at a holding potential of –60 mV (propiverine, Ki = 10 μM; M-1, Ki = 118 μM). M-1 shifted the steady-state inactivation curve of ICa to the left at –90 mV by 7 mV. Carbachol (CCh) reversibly inhibited ICa. This inhibition probably occurred through muscarinic type 3 receptors, coupling with G-proteins, because nanomolar concentrations of 4-diphenylacetoxy-N-methyl-piperidine greatly reduced this inhibition, whereas pirenzepine or 11-([2-[(diethylamino)methyl]-1-piperdinyl]acetyl)-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116) at concentrations up to 1 μM was almost ineffective. In the presence of M-2, the CCh-induced inhibition of ICa was blocked. In fluorescent Ca2+ imaging, M-2 inhibited EFS-induced and ACh-induced Ca2+ transients. These results suggest that M-1 acts, at least in part, as a Ca2+ channel antagonist (as it inhibited ICa), whereas M-2 has more direct antimuscarinic actions.

Actions of ZD0947, a novel ATP‐sensitive K+ channel opener, on membrane currents in human detrusor myocytes

ATP‐sensitive K+ channels (KATP channels) play important roles in regulating the resting membrane potential of detrusor smooth muscle. Actions of ZD0947, a novel KATP channel opener, on both carbachol (CCh)‐induced detrusor contractions and membrane currents in human urinary bladder myocytes were investigated.

Actions of veratridine on tetrodotoxin-sensitive voltage-gated Na+ currents, NaV1.6, in murine vas deferens myocytes

Background and purpose:  The effects of veratridine, an alkaloid found in Liliaceae plants, on tetrodotoxin (TTX)‐sensitive voltage‐gated Na+ channels were investigated in mouse vas deferens.