The involvement of L-type Ca(2+) channels in the relaxant effects of the ATP-sensitive K(+) channel opener ZD6169 on pig urethral smooth muscle

1. The effects of ZD6169, a novel ATP-sensitive K(+) channel (K(ATP) channel) opener, were investigated on membrane currents in isolated myocytes using patch-clamp techniques. Tension measurement was also performed to study the effects of ZD6169 on the resting tone of pig urethral smooth muscle. 2. Levcromakalim was more potent than ZD6169 in lowering the resting urethral tone. Relaxation induced by low concentrations of ZD6169 (⩽3 μM) was completely suppressed by additional application of glibenclamide (1 μM). In contrast, glibenclamide (1 – 10 μM) only partially inhibited the relaxation induced by higher concentrations of ZD6169 (⩾10 μM). 3. Bay K8644 (1 μM) reduced the maximum relaxation produced by ZD6169 (⩾10 μM). 4. In whole-cell configuration, ZD6169 suppressed the peak amplitude of voltage-dependent Ba(2+) currents in a concentration- and voltage-dependent manner, and at 100 μM, shifted the steady-state inactivation curve of the voltage-dependent Ba(2+) currents to the left at a holding potential of −90 mV. 5. In cell-attached configuration, open probability of unitary voltage-dependent Ba(2+) channels (27 pS, 90 mM Ba(2+)) was inhibited by 100 μM ZD6169 and by 10 μM nifedipine. 6. Reverse transcriptase-polymerase chain reaction (RT – PCR) analysis revealed the presence of the transcript of the α(1C) subunit of L-type Ca(2+) channels in pig urethra. 7. These results demonstrate that ZD6169 causes urethral relaxation through two distinct mechanisms, activation of K(ATP) channels at lower concentrations and inhibition of voltage-dependent Ca(2+) channels at higher concentrations (about 10 μM)

The effects of flavoxate hydrochloride on voltage-dependent L-type Ca(2+) currents in human urinary bladder

1. The effects of flavoxate hydrochloride (Bladderon®, piperidinoethyl-3-methylflavone-8-carboxylate; hereafter referred as flavoxate) on voltage-dependent nifedipine-sensitive inward Ba(2+) currents in human detrusor myocytes were investigated using a conventional whole-cell patch-clamp. Tension measurement was also performed to study the effects of flavoxate on K(+)-induced contraction in human urinary bladder. 2. Flavoxate caused a concentration-dependent reduction of the K(+)-induced contraction of human urinary bladder. 3. In human detrusor myocytes, flavoxate inhibited the peak amplitude of voltage-dependent nifedipine-sensitive inward Ba(2+) currents in a voltage- and concentration-dependent manner (K(i)=10 μM), and shifted the steady-state inactivation curve of Ba(2+) currents to the left at a holding potential of −90 mV. 4. Immunohistochemical studies indicated the presence of the α(1C) subunit protein, which is a constituent of human L-type Ca(2+) channels (Ca(V)1.2), in the bundles of human detrusor smooth muscle. 5. These results suggest that flavoxate caused muscle relaxation through the inhibition of L-type Ca(2+) channels in human detrusor

Serum anti-DIDO1, anti-CPSF2, and anti-FOXJ2 antibodies as predictive risk markers for acute ischemic stroke

Abstract Background Acute ischemic stroke (AIS) is a serious cause of mortality and disability. AIS is a serious cause of mortality and disability. Early diagnosis of atherosclerosis, which is the major cause of AIS, allows therapeutic intervention before the onset, leading to prevention of AIS. Methods Serological identification by cDNA expression cDNA libraries and the protein array method were used for the screening of antigens recognized by serum IgG antibodies in patients with atherosclerosis. Recombinant proteins or synthetic peptides derived from candidate antigens were used as antigens to compare serum IgG levels between healthy donors (HDs) and patients with atherosclerosis-related disease using the amplified luminescent proximity homogeneous assay-linked immunosorbent assay. Results The first screening using the protein array method identified death-inducer obliterator 1 (DIDO1), forkhead box J2 (FOXJ2), and cleavage and polyadenylation specificity factor (CPSF2) as the target antigens of serum IgG antibodies in patients with AIS. Then, we prepared various antigens including glutathione S-transferase-fused DIDO1 protein as well as peptides of the amino acids 297–311 of DIDO1, 426–440 of FOXJ2, and 607–621 of CPSF2 to examine serum antibody levels. Compared with HDs, a significant increase in antibody levels of the DIDO1 protein and peptide in patients with AIS, transient ischemic attack (TIA), and chronic kidney disease (CKD) but not in those with acute myocardial infarction and diabetes mellitus (DM). Serum anti-FOXJ2 antibody levels were elevated in most patients with atherosclerosis-related diseases, whereas serum anti-CPSF2 antibody levels were associated with AIS, TIA, and DM. Receiver operating characteristic curves showed that serum DIDO1 antibody levels were highly associated with CKD, and correlation analysis revealed that serum anti-FOXJ2 antibody levels were associated with hypertension. A prospective case–control study on ischemic stroke verified that the serum antibody levels of the DIDO1 protein and DIDO1, FOXJ2, and CPSF2 peptides showed significantly higher odds ratios with a risk of AIS in patients with the highest quartile than in those with the lowest quartile, indicating that these antibody markers are useful as risk factors for AIS. Conclusions Serum antibody levels of DIDO1, FOXJ2, and CPSF2 are useful in predicting the onset of atherosclerosis-related AIS caused by kidney failure, hypertension, and DM, respectively