Effect of KC399, a newly synthesized K+ channel opener, on acetylcholine-induced electrical and mechanical activities in rabbit tracheal smooth muscle.

1. Effects of KC399, an opener of ATP-sensitive K+ channels were investigated on membrane potential, isometric force and intracellular Ca2+ ([Ca2+]i) mobilization induced by acetylcholine (ACh) in smooth muscle from the rabbit trachea. 2. In these smooth muscle cells, ACh (0.1 and 1 microM) depolarized the membrane in a concentration-dependent manner, KC399 (1-100 nM) hyperpolarized the membrane whether in the presence or absence of ACh. When the concentration of ACh was increased, the absolute values of the membrane potential induced by the maximum concentration of KC399 were less negative. 3. ACh (0.1 to 10 microM) concentration-dependently produced a phasic, followed by a tonic increase in both [Ca2+]i and force. KC399 (above 3 nM) lowered the resting [Ca2+]i and attenuated the ACh-induced phasic and tonic increases in [Ca2+]i and force, in a concentration-dependent manner. The magnitude of the inhibition was greater for the ACh-induced tonic responses than for the phasic ones. Nicardipine (0.3 microM), a blocker of the L-type Ca2+ channel, attenuated the ACh-induced tonic, but not phasic, increases in [Ca2+]i and force. KC399 further attenuated the ACh-induced tonic responses in the presence of nicardipine. 4. In beta-escin-skinned strips, Ca2+ (0.3-10 microM) produced a contraction in a concentration-dependent manner. KC399 (0.1 microM) had no effect on the Ca(2+)-force relationship in the presence or absence of ATP with GTP. However, at a very high concentration (1 microM), this agent slightly shifted the relationship to the right and attenuated the maximum Ca(2+)-induced contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

A case of left atrial automatic tachycardia successfully treated with aprindine

Electrocardiogram in a 15-year-old girl showed persistent supraventricular tachycardia at rates of 130 to 140 bpm. Electrophysiological study confirmed left atrial automatic tachycardia, which was transferred to sinus rhythm by intravenous infusion of aprindine (100 mg/10 min). Therefore, aprindine (60 mg/day) was administered orally, and neither recurrence of left atrial automatic tachycardia nor side effects were observed during subsequent follow-up period of 16 months.     

Plasma lipoprotein(a) levels and expression of the apolipoprotein(a) gene are dependent on the nucleotide polymorphisms in its 5'-flanking region.

The apolipoprotein(a) (apo[a]) gene encodes a protein component of lipoprotein(a) [Lp(a)] whose plasma levels vary widely among individuals. Hyper-Lp(a)-emia constitutes a risk factor for thromboembolic disease. We previously subclassified the apo(a) gene into four allelic types (A-D) by polymorphisms in the 5''-flanking region. To elucidate whether these polymorphisms affect the expression of apo(a), we measured plasma Lp(a) concentrations in vivo by ELISA and examined expression of the gene by an in vitro assay using its 5''-flanking region. Homozygotes of type C had significantly higher Lp(a) levels than those of type D. The relative expression of type C was also about three times higher than that of type D, which was consistent with the in vivo results. Deletion analysis revealed that the substitution of C by T (+93) led to negative regulation in expression of the gene, while the change of G to A (+121) led to positive regulation. These results indicate that the polymorphisms in the 5''-flanking region of the apo(a) gene affect the efficiency of its expression and, in part, play a role in regulating plasma Lp(a) levels.     

Muscular involvement in lysosomal acid lipase deficiency in rats.

We investigated the pathological and biochemical changes of skeletal muscle in rats with lysosomal acid lipase deficiency, which is an animal counterpart of human Wolman's disease. In the affected rats, the acid lipase activity for three different substrates, 4-methylumbelliferyl-oleate (18.9% of the normal control level), [14C]cholesteryl oleate (23.5%), and [14C]triolein (26.9%), was similarly decreased in the lysosomal fraction of skeletal muscle which was obtained by differential centrifugation. Histochemical studies showed that acid phosphatase activity was high in the endomysium and perimysium and in some muscle fibers. Some fibers showed vacuolar degeneration resembling "rimmed vacuoles". Ultrastructural studies demonstrated many membrane-bound lipid droplets in the muscle fibers, especially in the subsarcolemmal space, indicating that a low density lipoprotein (LDL) uptake pathway apparently existed in the muscle cells. However, such lipid accumulation was much greater in the interstitial cells and the endothelial cells. This distribution also suggests that LDL/cholesterol is supplied to muscle cells predominantly through endothelial cells.     

Mobilization of free Ca2+ measured during contraction-relaxation cycles in smooth muscle cells of the porcine coronary artery using quin2

Ca²⁺ mobilization in dispersed smooth muscle cells of the porcine coronary artery was investigated using the fluorescent Ca²⁺ indicator, quin2. The resting [Ca²⁺]_i was 113±8 nM (a mean±SE), and was independent of intracellular quin2 concentrations. Acetylcholine (ACh; over 10 nM) or caffeine (over 3 mM) transiently increased the intensity of fluorescence, thereby reflecting the elevation of intracellular free Ca²⁺ (Ca²⁺ transient), while excess K⁺ gradually increased and maintained the intensity of fluorescence. Application of EGTA reduced the resting intensity of the fluorescence and blocked the K⁺-induced Ca²⁺ transient, but did not supress the Ach-or caffeine-induced ones. Nisoldipine (0.1 M) did not affect the resting intensity of the fluorescence. This agent blocked the K⁺ induced but not the ACh-or caffeine-induced Ca²⁺ transient. Thus, sources of Ca²⁺ contributing to the K⁺-induced Ca²⁺ transient differ from those evoked by other agents. The amount of Ca²⁺, as estimated from the increased Ca²⁺ transient by caffeine or ACh, was increased in proportion to the excess K⁺-induced influx of Ca²⁺.     

Membrane properties and inhibitory innervation of the circular muscle cells of guinea-pig caecum

1. The membrane properties of the circular muscle cells of guinea-pig caecum and nervous factors influencing the muscle activity were studied with micro-electrodes using partition and field stimulating methods.2. The mean membrane potential was -52 mV. Spontaneous discharges appeared as regular bursts between silent periods, as regular spikes without silent period, or as regular slow potential changes with superimposed spikes.3. Spontaneous spikes with overshoot were frequently observed. The mean maximum rate of rise was 5.2 V/sec. The mean conduction velocity of evoked spikes was 5.4 cm/sec.4. The amplitude of the elctrotonic potential was linearly proportional to the current applied by the partition stimulating method. The spatial decay of the electrotonic potential along the tissue was exponential, with a mean length constant of 1.7 mm.5. The time constants of the membrane calculated from the electrotonic potential, and from the conduction velocity, length constant and time course of the foot of the spike were about 200 and 100 msec respectively. These results indicate that the circular muscle of guinea-pig caecum possesses cable like properties.6. Field stimulation (0.3 msec pulse duration) to the circular muscle evoked three different responses successively, i.e. initial depolarization (initial excitatory junction potential) with or without spike, hyperpolarization (inhibitory junction potential) and delayed depolarization (delayed excitatory junction potential) with or without spikes.7. These three different potential changes were completely blocked by treatment with tetrodotoxin (5 x 10(-6) g/ml.), and both the initial and late excitatory junction potentials were blocked by treatment with atropine (5 x 10(-5) g/ml.).8. The distribution of inhibitory nerves in the circular muscle cells was investigated. The results indicate that inhibitory nerves arise from Auerbach's plexus situated just beneath the taenia coli and the nerve branches spread over the whole distance from one taenia coli to the next along the ciruclar muscle cells, a width of about 3 mm.9. The mean conduction velocity of excitation of the inhibitory nerves was 16.0 cm/sec. Hexamethonium, in a concentration of 5 x 10(-6) g/ml. depolarized the circular muscle membrane and lowered the rate of rise and fall of spike, but did not block the generation of inhibitory junction potentials.     

In vivo depletion of macrophages by desulfated iota-carrageenan in mice

Almost 90% of the sulfate groups of iota-carrageenan (CGN) was removed with acid-methanol in an attempt to obtain a product which would selectively eliminate macrophages in mice. Desulfated CGN(DS-CGN) failed to induce in vivo polyclonal antibody production in DBA/2 mice. However, the number of phagocytes in the peritoneal cavity, spleen, thymus and lymph node of DBA/2 mice was reduced stringently by DS-CGN treatment. The number of Mac-1 positive cells(macrophages) in DS-CGN-treated mice gradually decreased for at least 7 days after the last injection of DS-CGN. In contrast, the relative proportion of T and B lymphocytes in the lymphoid organs was unaffected by DS-CGN treatment. DS-CGN suppressed antibody responses to SRBC, a T cell and macrophage-dependent antigen, but no such suppressive effect was observed in the polyclonal antibody responses to LPS, a T cell and macrophage-independent B cell activator. Furthermore, the impaired SRBC antibody responses in DS-CGN-treated mice were restored following transfer of adherent cells but not T cells. These experimental results indicate that DS-CGN selectively eliminates macrophages without influencing lymphocyte function in vivo.     

Lattices of Type I Collagen Select Invasive Variants of K-ras Oncogene-Transfected NIH3T3 with Less Cellular fibronectin

A clone of NIH3T3 transformant (H3) can yield subcutaneous tumors and experimental pulmonary metastasis in nude mice. Compared to H3 in culture, the cells after in vivo tumor growth (H3-N) acquired enhanced tumorigenicity and metastatic ability. Also, indirect immunofluorescence revealed that cellular fibronectin (c-FN) of H3-N was decreased remarkably. We have studied the interactions between H3 and extracellular matrices to elucidate these phenomena. In the present study, we observed the effect of NIH3T3, H3, and H3-N cultured in type I collagen gel. Morphologically in the collagen gel, NIH3T3 assumed an extensive elongated fiber-like shape, H3 assumed a moderately elongated shape, and H3-N assumed a round or spindle shape with short pseudopodia. Compared to conventional cultures on dishes, cell proliferation of all three types was suppressed in collagen gel, but the degree of the suppression was least in H3-N. As a result, H3-N grew fastest in collagen gel. The variants which acquired growth advantage in the subcutaneum of mice also kept it in collagen gel. H3 cells were cultured in type I collagen gel for 4 weeks, a period comparable to that of tumor formation in nude mice. The cells after this long-term culture (H3-C) acquired enhanced tumorigenicity and metastatic ability nearly equal to that of H3-N. FACS analysis revealed that the c-FN of H3-C had decreased to a value comparable to that of H3-N. This means that type I collagen gel as well as subcutaneous tissues could select variants of H3 with less c-FN through proliferation. Moreover, it is suspected that lattices of type I collagen regulate cell proliferation of fibroblast via c-FN. This revised version was published online in August 2006 with corrections to the Cover Date.