Autocrine and/or paracrine growth of aggressive ATLL cells caused by HGF and c-Met.

Adult T-cell leukemia/lymphoma (ATLL) is a neoplasia characterized by the massive invasion of various organs by tumor cells. Previously, we found that expression of the gene for c-Met, a receptor tyrosine kinase for hepatocyte growth factor (HGF), was specific to the acute type among 41 patients with ATLL by microarray. First in the present study, we analyzed the survival of the patients in relation to expression of c-Met and HGF in ATLL cells. Expression of the former but not the latter was associated with poor prognosis. Then, we analyzed the growth of ATLL cells caused by HGF and c-Met. c-Met was expressed in 0/7 chronic ATLLs, 12/14 acute ATLLs, 1/1 IL-2-independent ATLL cell line and 1/7 IL-2-dependent ATLL cell lines as assessed by flow cytometry. HGF induced the proliferation of primary cells from most acute cases examined as well as the c-Met-positive KK1 cell line in contrast to c-Met-negative cells. HGF induced autophosphorylation of c-Met in c-Met-positive cells from an acute case and KK1 cells. The plasma level of HGF was elevated in acute as compared to chronic cases. The levels of HGF and/or IL-6 which induces the production of HGF by stromal cells, were elevated in the supernatant of short-term cultured cells from certain patients with acute or chronic disease. Finally, infiltrated ATLL cells and adjacent stromal cells in liver were shown to be positive for c-Met/HGF and HGF, respectively, in acute cases. Autocrine and/or paracrine growth caused by HGF and c-Met was suggested in aggressive ATLL cells secreting HGF and/or IL-6, respectively.

Small number of HTLV-1-positive cells frequently remains during complete remission after allogeneic hematopoietic stem cell transplantation that are heterogeneous in origin among cases with adult T-cell leukemia/lymphoma.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) can provide long-term remission for patients with adult T-cell leukemia/lymphoma (ATLL) caused by human retrovirus, human T-lymphocyte virus (HTLV-1). To understand how HTLV-1-positive cells including ATLL cells were suppressed by allo-HSCT, we examined HTLV-1 provirus load and residual ATLL cells in peripheral blood of transplant recipients using PCR-based tests. We found that the copy number of HTLV-1 genome, called provirus, became very small in number after allo-HSCT; however, in most cases, provirus did not disappear even among long-term survivors. Tumor-specific PCR tests demonstrated that most of HTLV-1-positive cells that remained long after transplantation were not primary ATLL cells but donor-derived HTLV-1-positive cells. We also found a case having very low amount of residual disease in peripheral blood even long after transplantation. There was only one recipient in whom we failed to show the presence of HTLV-1 genome and antibody against HTLV-1 even with an extensive search, which strongly suggested the elimination of HTLV-1 after allo-HSCT. These results demonstrated that after allo-HSCT the small amount of residual HTLV-1-positive cells were heterogeneous in origin and that long-term disease control for ATLL could be obtained without the complete elimination of HTLV-1.

Preemptive Living Donor Kidney Transplantation and Kidney Function at the Initial Hospital Visit: A Single-Center Case-Control Study.

BACKGROUND: Studies have revealed that patients who undergo preemptive kidney transplantation (PKT) have favorable prognoses compared with those who undergo kidney transplantation after the initiation of dialysis. The number of PKT cases performed worldwide has been increasing. The goal of this study was to determine the clinical characteristics of patients who may successfully receive PKT. METHODS: A single-center, case-control study was conducted to determine the clinical factors that lead to referral for PKT. RESULTS: Between April 1, 2009, and August 1, 2015, a total of 118 patients underwent living donor kidney transplantation. Thirty of these patients had not undergone dialysis before their initial visit to the study hospital. Of these, 20 received kidney transplantation before and after dialysis initiation, respectively (group PKT+, successful PKT; group PKT-, failed PKT). The baseline characteristics at the primary visit were compared between groups. The median duration from the first visit to the study institution to PKT was 5.6 ± 0.7 months. Serum creatinine (Cr) levels differed significantly between groups (PKT+ vs PKT-, 6.0 ± 0.3 mg/dL vs 7.5 ± 0.5 mg/dL; P = .03). The receiver-operating characteristic curves revealed that a serum Cr level >5.7 mg/dL at the initial visit to the unit was a cutoff point for predicting the success of PKT (area under the curve, 0.721; P = .02). CONCLUSIONS: Our results indicate that PKT should be performed within ∼6 months of the initial visit to the transplant center. Serum Cr levels <5.7 mg/dL predict successful PKT.

Fas antigen (CD95) in pure erythroid cell line AS-E2 is induced by interferon-gamma and tumor necrosis factor-alpha and potentiates apoptotic death.

We investigated the expression of Fas antigen (CD95) in the pure erythroid cell line AS-E2 in the presence and absence of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). TNF-alpha induced apoptosis in AS-E2 cells, whereas IFN-gamma did not. In culture containing no IFN-gamma or TNF-alpha, AS-E2 cells expressed little Fas antigen. However, IFN-gamma and IFN-gamma and TNF-alpha both induced expression of Fas antigen and its mRNA within 24 hours after the stimulation. When anti-Fas monoclonal antibody (IgM) was added to AS-E2 cells after the induction of Fas expression, AS-E2 cells underwent apoptosis as shown by the induction of DNA fragmentation. This apoptotic change was inhibited by an inhibitor of caspase-3-like proteases (Ac-DEVD-CHO) and an inhibitor of CED-3/ICE family proteases (Z-Asp-CH2-DCB) but not by an inhibitor of caspase-1-like proteases (Ac-YVAD-CHO), suggesting a role for caspase-3-like proteases in Fas-receptor signaling. Although AS-E2 cells expressed Fas ligand mRNA, treatment with ZB4, an antibody that inhibits Fas-mediated cell death, failed to suppress IFN-gamma- or TNF-alpha-mediated cytotoxicity. These findings suggest that the late erythroid progenitor cells are negatively regulated by IFN-gamma and TNF-alpha, both of which are capable of inducing functional Fas expression.

Characteristic patterns of relapse after allogeneic hematopoietic SCT for adult T-cell leukemia-lymphoma: a comparative study of recurrent lesions after transplantation and chemotherapy by the Nagasaki Transplant Group.

Allogeneic hematopoietic SCT (allo-SCT) is a promising therapy that may provide long-term durable remission for adult T-cell leukemia-lymphoma (ATL) patients; however, the incidence of relapse associated with ATL remains high. To determine the clinical features of these patients at relapse, we retrospectively analyzed tumor lesions in 30 or 49 patients who relapsed following allo-SCT or chemotherapy (CHT), respectively, at three institutions in Nagasaki prefecture between 1997 and 2011. A multivariate analysis revealed that the development of abnormal lymphocytes in the peripheral blood of patients at relapse was less frequent after allo-SCT than after CHT (P<0.001). Furthermore, relapse with a new lesion only in the absence of the primary lesion was more frequent in allo-SCT (P=0.014). Lesions were more frequently observed in the central nervous systems of patients who relapsed with new lesions only (P=0.005). Thus, the clinical manifestation of relapsed ATL was slightly complex, especially in post-transplant patients. Our results emphasized the need to develop adoptive modalities for early and accurate diagnoses of relapsed ATL.

Spontaneous transient outward currents in smooth muscle cells.

Spontaneous transient outward currents (STOCs) lasting about 100 ms occur in single smooth muscle cells and represent the simultaneous opening of up to a hundred calcium-activated potassium (BK) channels. The recent observation of brief focal releases of sarcoplasmic reticulum (SR) calcium ('sparks') in smooth muscle cells has provided support for the original suggestion that STOCs arise due to the spontaneous releases of calcium from the SR close to the sarcolemma. However, it is possible that such releases occur in a region of close apposition of SR membrane and sarcolemma about 0.1 microns wide ('junctional space') in which case they would be detectable by endogenous calcium-sensitive molecules such as BK channels but, using present confocal microscopy technique, not by calcium-indicator dyes introduced into the cell; should calcium escape from the junctional space then it may be visualised as 'sparks' by the fluorescent emission from calcium-indicator dyes using confocal microscopy. Some STOCs seem too large to represent the effect of a single 'spark' and some form of calcium-induced calcium release or 'macrospark' may be involved in their generation. Depletion of calcium stores by caffeine, ryanodine, or by activation of receptors linked to the phospholipase C/inositol trisphosphate system abolishes STOCs. However, low concentrations of caffeine or inositol trisphosphate accelerate STOC discharge by an unknown mechanism and often decrease STOC size presumably by depleting store calcium; similar effects are produced by agents such as cyclopiazonic acid and thapsigargin which inhibit calcium storage mechanisms (largely the SR calcium pump).

Molecular cloning of a novel gene involved in serotonin receptor-mediated signal transduction in rat stomach.

In Xenopus oocytes injected with small size mRNAs (500-700 b), obtained from rat stomach by fractionation, application of 10 microM 5-HT induced a substantial Ca2+-activated Cl- current (I(Cl-Ca)). I(Cl-Ca) was not elicited by 5-HT in native oocytes. Consistent results from this assay in the oocyte expression system motivated cDNA cloning experiments. A novel cDNA (named rat stomach serotonin receptor-related cDNA: RSS cDNA) which encodes a small protein involved in specific 5-HT receptor-mediated I(Cl-Ca) activation was identified. The molecular weight of RSS protein in the reticulocyte lysate translation system (approximately 10 kDa) is identical to that calculated from the amino acid sequence. Computer-aided analysis of the predicted protein does not show any obvious sequence homologies (< 18%) to any other proteins including G protein-coupled receptors. Northern analysis revealed that RSS mRNA is ubiquitously expressed at varying levels in a number of different tissues. Furthermore, the binding of [3H]spiperone, a 5-HT2 receptor antagonist, was examined in CHO cells, which highly expressed RSS transcripts (named CHO-RSS). Specific binding of [3H]spiperone was not clearly observed in native CHO but was detected in CHO-RSS. The dissociation constant was 10.3 nM in CHO-RSS. These results suggest that RSS protein may be a factor which facilitates 5-HT receptor expression or, alternatively, an enhancer of the affinity of native 5-HT receptor to 5-HT.

Molecular cloning and tissue distribution of an alternatively spliced variant of an A-type K+ channel alpha-subunit, Kv4.3 in the rat.

We describe here (1) the heterogeneous expression of Ca2+-independent transient (A-type) K+ channel alpha-subunits (Kv1.4, Kv3.3, Kv3.4, Kv4.2 and Kv4.3) in rat smooth muscle, heart and brain, (2) the molecular cloning and tissue distribution of a novel alternatively spliced variant of an A-type K+ channel alpha-subunit, Kv4.3, and (3) the functional expression of A-type K+ channels in HEK293 cells by the transfection with the novel splice variant of Kv4.3. A cDNA encoding this splice variant was identified from rat vas deferens by RT-PCR cloning. This cDNA clone contains a 1965 bp open reading frame that encodes for a protein of 655 amino acids. It has a 19 amino acid insertion in comparison with Kv4.3 previously reported in rat brain. RT-PCR analyses showed that the mRNAs of this longer variant are abundantly expressed in a number of smooth muscles of the rat, and that the mRNAs of the previously reported clones are absent. The longer splice variant is very weakly expressed in brain, but is the major product in heart.

Longitudinal analysis of mortality from breast cancer in Japan, 1950-1993: fitting Gompertz and Weibull functions.

Age-specific mortality rates from breast cancer (BC) in Japan from 1950 through 1993 were subjected to longitudinal Gompertzian and Weibull analyses. Age-specific BC mortality rate distributions between the ages of 30 and 75 years were Gompertzian in each year. Between 1950 and 1993, age-adjusted BC mortality rates increased by 65%. Gompertzian analysis might suggest that increasing mortality rates for BC can be attributed to worsening environmental influences during that period, especially after 1970. This suggests that the environmental factor has changed to account for increasing mortality from BC. The environmental factor increased 16% during that period and the competitive environmental factor was two times more contributory to mortality from BC in 1993 as compared with 1950. Age-specific mortality rates from BC were better fitted to the Weibull than the Gompertz function. The parameters of a and tau in the Weibull function ranged between 2.9 and 3.5 and between 8.5 and 13.0, respectively, during that period. The tau decreased 21% in 1993 as compared with 1950. As environmental factors of BC, the intake of animal protein and fat were discussed.

Longitudinal Gompertzian analysis of mortality from pancreatic cancer in Japan, 1955-1993.

Age-specific mortality rates from pancreatic cancer (PanC) in Japan from 1955 through 1993 were subjected to longitudinal Gompertzian analysis. Age-specific PanC mortality rate distributions between age 30 and 70 years were highly Gompertzian for each and every year for both men and women. Between 1955 and 1993, age-adjusted PanC mortality rates increased 5.1 times for men and 4.3 times for women. The environmental factor for PanC mortality increased 68% for men and 89% for women over this period. Gompertzian analysis suggests that rising mortality from PanC in Japan may be related to rapidly changing lifestyles among Japanese. Pure alcohol consumption was the identified environmental factor most strongly correlated with PanC mortality.

Longitudinal gompertzian analysis of mortality from stomach cancer in Japan, 1950-1993.

Age-specific mortality rates from stomach cancer (SC) in Japan from 1950 through 1993 were subjected to longitudinal Gompertzian analysis. Age-specific SC mortality rate distributions between ages 45 and 75 years were highly Gompertzian for each- and every year for both men and women. Between 1950 and 1993, age-adjusted SC mortality rates decreased 48.6% for men and 59.6% for women. Gompertzian analysis might suggest that declining SC mortality rates can be attributed to beneficial environmental influences during that period, especially after 1970. This suggests that the environmental factor has changed to account for the dramatic decline in the incidence of, and mortality from SC. The environment was 32.3 and 14.4 times less conducive to mortality from SC in 1993 as compared to 1950 for men and women respectively. Reduced salt consumption was considered as the decline in the SC mortality rate. Intake of green-yellow vegetables, fruits, and vitamin A were also considered as factors for reducing SC mortality. The mass-screening for SC may effectively enhance survival for SC patients, which might partially contribute to a dramatic decline in SC mortality.

Longitudinal Gompertzian analysis of Parkinson's disease mortality in Japan, 1950-1993.

Age-specific mortality rates from Parkinson's disease (PD) in Japan from 1950 through 1993 were subjected to longitudinal Gompertzian analysis. Age-specific PD mortality rate distributions between age 45 and 75 years were determined by a common fixed intersection point and a variable competitive factor. The intersection point for PD occurred at age 65.36 years and mortality rate 2.45 per 100,000 for men, and at age 65.49 years and mortality rate 2.12 per 100,000 for women from 1950-1951 to 1992-1993. The increase in PD mortality is due entirely to rapidly increasing age-specific mortality rates at ages greater than the intersection points for both sexes. Longitudinal Gompertzian analysis suggests that the rising mortality from PD has been the consequence of competitive influences upon PD mortality dynamics.

Cyclopiazonic acid, an inhibitor of Ca(2+)-ATPase in sarcoplasmic reticulum, increases excitability in ileal smooth muscle.

1. Effects of cyclopiazonic acid (CPA), a specific inhibitor of Ca(2+)-ATPase in endo- and sarcoplasmic reticulum (ER/SR), on contractile responses, cytosolic Ca2+ concentration and spontaneous electrical activity were examined in ileal longitudinal smooth muscle strips. 2. After intracellular stored Ca2+ in intact ileal strips was depleted by application of 25 mM caffeine in Ca(2+)-free solution, Ca(2+)-loading was performed in the absence or presence of 10 microns CPA in a standard solution containing 2.2 mM Ca2+. Subsequent application of caffeine in Ca(2+)-free solution induced a phasic contraction which was significantly smaller in the strip pretreated with CPA than that in the control. 3. Spontaneous and 20 mM K(+)-induced contractions in the presence of 1 microM atropine were markedly enhanced by 1-30 microM CPA, whereas that induced by 80 mM K+ was not. The magnitude of repetitive transient elevation of cytosolic Ca2+ concentration ([Ca2+])i) and concomitant phasic contractions were markedly enhanced by CPA. The effects were abolished by 10 microM verapamil and restored by 10 microM Bay K 8644. 4. Application of 10 microM CPA depolarized the cell by about 5 mV, decreased the action potential (AP) afterhyperpolarization and markedly increased the frequency of spontaneous AP. These effects were mimicked by 100 nM charybdotoxin. 5. The rate of decay of [Ca2+]i and tension after the bathing solution was changed from one containing 140 mM K+ and 2.2 mM Ca2+ to one containing 5.9 mM K+ and 0 mM Ca2+ was significantly slowed when 10 microM CPA was added to the latter solution. 6. These results indicate that CPA enhances ileal smooth muscle excitability and increases Ca2+-influx through voltage-dependent Ca2+ channels. The effect may be consistent with the hypothesis that CPA-induced decrease in stored Ca due to Ca-pump inhibition reduces the Ca2+-dependent K+ current and indirectly enhances Ca2+-influx through membrane activity resulting from the increased excitability.Direct evidence for the regulation of Ca2+ channel activity by intracellular Ca storage sites was not obtained in the present study.

Effects of cyclopiazonic acid, a novel Ca(2+)-ATPase inhibitor, on contractile responses in skinned ileal smooth muscle.

1. Effects of cyclopiazonic acid (CPA), a specific inhibitor of the Ca(2+)-ATPase in sarcoplasmic reticulum (SR) of skeletal and cardiac muscles, on contractile responses induced by Ca(2+)-release from intracellular storage sites were examined in the longitudinal smooth muscle strip of the guinea-pig ileum skinned with beta-escin. 2. Ca(2+)-loading of storage sites (Ca(2+)-uptake) was performed in pCa 6.3 solution. The amount of Ca2+ taken up was monitored by use of the amplitude of contraction following application of 25 mM caffeine or 25 microM inositol 1,4,5-trisphosphate (IP3). 3. Contractile responses to caffeine or IP3 were reduced or abolished when the preceding Ca(2+)-uptake was performed in the presence of 0.1-10 microM CPA. The dose of CPA required to inhibit the contraction induced by caffeine or IP3 by 50% was approximately 0.6 microM. The CPA-sensitive Ca(2+)-uptake completely depended upon the presence of ATP in the solution during Ca(2+)-uptake. 4. When 1 microM CPA was added after Ca(2+)-uptake, the subsequent caffeine- or IP3-induced contraction was not significantly affected by the presence of CPA. 5. Acetylcholine-induced contraction was also almost abolished when the preceding Ca(2+)-uptake was performed in the presence of 10 microM CPA. 6. The relationship between pCa and contraction was not affected by the presence of 10 microM CPA in skinned fibres where Ca2+ storage sites had been destroyed by treatment with A23187. The enhancement of contraction in pCa 6.0 solution by calmodulin was not affected by 10 microM CPA.7. These results suggest that CPA selectively inhibits ATP-dependent Ca2"-uptake into intracellular storage sites in skinned ileal smooth muscle strips. CPA appears to be a potent, reversible, and very specific inhibitor of the Ca2+-pump in the storage sites of smooth muscle, and is an extremely valuable pharmacological tool.

Modulation of calcium channel currents by arachidonic acid in single smooth muscle cells from vas deferens of the guinea-pig.

1. Effects of arachidonic acid (AA) on voltage-dependent Ca channel currents were investigated by whole-cell-clamp methods in single smooth muscle cells freshly isolated from vas deferens of the guinea-pig. 2. Ca channel current was decreased by application of 1-30 microM AA in a concentration-dependent manner. When Ca2+ or Ba2+ was the charge carrier, Ca channel current (ICa or IBa) was reduced by AA to a similar extent (IC50 = 10 and 6 microM, respectively). Addition of 15 mM BAPTA to the pipette solution did not affect the reduction of IBa by 10 microM AA. 3. The effect of AA on IBa was not prevented by internal application of 1 mM nordihydroguaiaretic acid (NDGA) and 1 mM indomethacin (Indo). When the pipette solution contained 0.1 mM guanosine-5'-triphosphate (GTP), IBa was decreased slightly but significantly by application of 30 microM prostaglandin F2 alpha (PGF2 alpha) but not by PGE2. This effect of PGF2 alpha was irreversible or not observed when the pipette solution contained 0.3 mM guanosine-5'-(3-thiotriphosphate) (GTP gamma S) or both GTP or guanosine-5'-O-(2-thiodiphosphate) (GDP beta S), respectively. 4. External application of 100 units ml-1 superoxide dismutase slightly but significantly attenuated the inhibition of IBa by 1-30 microM AA. Intracellular application of 1 mM GDP beta S or 0.3 mM GTP gamma S did not significantly change the effect of AA. Intracellular application of 0.1 mM 1-(5-isoquinolinesulphonyl)-2-methylepiperazine (H-7) also did not change the effect of AA. 5. These results indicate that the decrease in Ca channel currents in vas deferens smooth muscle cells is mainly due to AA itself, as opposed to its metabolites. The effect of AA may be due to AA itself, as opposed to its metabolites. The effect of AA may be due to its direct action on Ca channels or membrane phospholipids, but may not be mediated by activation of GTP binding proteins or protein kinase C. The inhibition of Ca channel current by AA may be partly induced by superoxide radicals derived from AA oxidation. PGF2A also reduces Ca channel currents but probably by a separate mechanism via activation of a GTP binding protein.

Pharmaco-mechanical coupling in the response to acetylcholine and substance P in the smooth muscle of the rat iris sphincter.

In the rat iris sphincter muscle contractile responses to transmural stimulation consisted of two components, a fast cholinergic followed by a slow non-adrenergic, non-cholinergic (NANC) one. The magnitude of the latter varied widely and was on average 5% of that of the cholinergic component. Exogenous substance P (1 nM-1 microM) produced a concentration-dependent contraction, the maximum amplitude of which was as large as that produced by acetylcholine (ACh). Capsaicin (10 microM) induced a transient contraction only once in each preparation. After the treatment with capsaicin the NANC component disappeared. Neither nerve nor direct electrical stimulation with short pulses elicited any active change in the membrane potential under physiological conditions, but an action potential was triggered by direct stimulation when the extracellular Ca ion was totally replaced by Ba ion. Under the latter conditions spontaneous spike potentials occurred repetitively. ACh and substance P produced a large contraction without modifying the membrane potential. This was also the case in the presence of 5 mM Ba. These results suggest that substance P-ergic innervation may have a far lesser physiological significance than that which has been described in rabbits and that pure pharmaco-mechanical coupling is characteristic of the responses to acetylcholine, substance P, and nerve stimulation in the rat iris sphincter muscle.

Mechanisms of long-lasting effects of benidipine on Ca current in guinea-pig ventricular cells.

1. Benidipine (KW-3049), a new derivative of 1,4-dihydropyridine (DHP), showed dose-dependent inhibition of Ca current (ICa) which was elicited by depolarization from -40 mV to +10 mV at 0.2 Hz in single cardiac cells isolated from guinea-pig ventricle under whole cell voltage clamp. Half inhibition doses (IC50) of benidipine and nifedipine for the peak ICa at +10 mV were 2.7 nM and 63.1 nM, respectively. 2. A change in holding potential from -40 to -75 mV partially removed the block induced by both 10 nM benidipine and 100 nM nifedipine. The block of ICa by benidipine strongly depended upon holding potentials as did that induced by nifedipine. 3. The effect of 100 nM nifedipine was mostly removed when the cells were kept quiescent at holding potentials negative to -75 mV for 5 min after withdrawal of nifedipine. In contrast, hyperpolarization for several minutes did not significantly accelerate the removal of benidipine-induced block after withdrawal of the drug. Effects of 10 nM benidipine could not be washed out for up to 30 min regardless of the holding potentials. 4. It is suggested that the dissociation of benidipine from the DHP binding site, like that of nifedipine, is greatly accelerated by hyperpolarization. Benidipine but not nifedipine may have an additional interaction with the channel or lipid membrane and cannot be washed away even after the dissociation. Alternatively, the dissociation of benidipine from the DHP binding site may be too slow to occur substantially during the limited period of hyperpolarization in the present study (less than 30 min). In that case, however, the Ca channel bound by benidipine must become unblocked and available to be opened during the hyperpolarization.

Cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum Ca(2+)-pump, reduces Ca(2+)-dependent K+ currents in guinea-pig smooth muscle cells.

1. Effects of cyclopiazonic acid (CPA), a specific inhibitor of the Ca(2+)-ATPase in sarcoplasmic reticulum (SR), on membrane ionic currents were examined in single smooth muscle cells freshly isolated from ileal longitudinal strips and urinary bladder of the guinea-pig. 2. Under whole-cell clamp, CPA (1-10 microM) reduced peak outward current elicited by depolarization in a concentration-dependent manner. The concentration of CPA required for 50% decrease in the peak outward current was approximately 3 microM in ileal cells under these conditions. The current reduced by CPA recovered by more than 70% after washout. 3. The transient outward current elicited by application of 5 mM caffeine at a holding potential of -50 mV in Ca2+ free solution was almost abolished, when the preceding Ca(2+)-loading of the cell in a solution containing 2.2 mM Ca2+ was performed in the presence of 3 microM CPA. 4. When the Ca(2+)-dependent K+ current (IK-Ca) and Ca2+ current (ICa) were inhibited by addition of Ca2+, the remaining delayed rectifier type K+ current was not affected by 10 microM CPA. When outward currents were blocked by replacement of K+ by Cs+ in the pipette solution, the remaining ICa was not affected by 10 microM CPA. 5. CPA (10 microM) did not affect the conductance of single maxi Ca(2+)-dependent K+ channels or the Cd(2+)-dependence of their open probability in both inside- and outside-out configurations. 6. These results indicate that IK-Ca is selectively and strongly suppressed by CPA.Its effects may be attributed to a decrease in Ca2"-uptake into SR, resulting in a decrease in Ca2"-induced Ca24 release which is triggered by Ca24 entering through voltage-dependent Ca24 channels and therefore less activation of these K channels.7. CPA may be extremely valuable pharmacological tool for investigating intracellular Ca24 mobilization and ionic currents regulated by intracellular Ca24.

Effects of KRN2391 on ionic currents in rabbit femoral arterial myocytes.

The effects of KRN2391, an ATP-sensitive K+ channel opener (KCO) which also acts as a nitrate, on ionic membrane currents in rabbit femoral arterial myocytes were examined. Under whole-cell clamp conditions where cells were superfused with physiological salts solution containing 5.9 mM K+, KRN2391 elicited an outward current at a holding potential of -30 mV. KRN2391-induced current had a reversal potential of -78 mV and was abolished by glibenclamide (glib). KRN2391 was approximately 25 times more potent than nicorandil to activate an ATP-sensitive K+ current (I:(KATP)). On the other hand, 10 microM KRN2391 did not affect either voltage-dependent Ca(2+) or delayed rectifier K+ channel currents. In the inside-out patch configuration, KRN2391 activated 47 pS K+ channels in the presence of nucleotide diphosphates (NDPs) under the symmetrical 140 mM K+ conditions. Glib and intracellular ATP reversibly inhibited the activity of the 47 pS K+ channels. The 47 pS K+ channels activated by KRN2391 are similar in their conductance and other properties to NDP-sensitive K+ channels (K(NDP) channels) described in other smooth muscles and the cloned channels. KRN2391 is a potent activator of the 47 pS K+ channels and the activation can contribute to the KRN2391-induced vasodilation in arterial muscles.

Levcromakalim causes indirect endothelial hyperpolarization via a myo-endothelial pathway.

1. Effects of K+ channel opener, levcromakalim, on vascular endothelial cells were examined. Under voltage- and current-clamp conditions, application of acetylcholine to dispersed endothelial cells isolated from rabbit superior mesenteric artery (dispersed RMAECs) produced hyperpolarization and outward currents. On the other hand, dispersed RMAECs did not respond to levcromakalim. 2. When membrane potential was recorded from endothelium in a mesenteric arterial segment, exposure to levcromakalim in a concentration range of 0.1 to 3 microM caused concentration-dependent hyperpolarization. The hyperpolarization was observed in the absence of external Ca2+ and was inhibited by 10 microM glibenclamide. 3. The presence of 1 mM heptanol did not affect the levcromakalin-induced hyperpolarization, whereas treatment of the mesenteric arterial segment with 20 microM 18 beta-glycyrrhetinic acid significantly reduced the hyperpolarization. The response to acetylcholine of RMAECs in an arterial segment with 18 beta-glycyrrhetinic acid was, however, similar to that without 18 beta-glycyrrhetinic acid. 4. These suggest that although RMAECs themselves are functionally insensitive to levcromakalim, those in an arterial segment are hyperpolarized by levcromakalim via myo-endothelial electrical communication.