Characterization of ion currents of murine CD117(pos) stem cells in vitro and their modulation under AT2 R stimulation.

AIM: Hematopoietic stem cells, especially CD117(pos) cells, have been found to possess a regenerative potential in various tissues, in particular cardiac muscle. However, the characterization of the relevant ion currents of stem cells prior to implantation lacks documentation. Activation of angiotensin II type 2 receptor (AT2 R) can lead to further cell differentiation and receptor auto-expression and might thus influence electrophysiological properties of CD117(pos) stem cells. This study was designed to functionally characterize membrane currents of CD117(pos) cells under normal and AT2 R-stimulated conditions. METHODS: CD117(pos) murine bone marrow stem cells were isolated with MACS technique and stimulated for the AT2 R with angiotensin II and losartan for 3-5 days prior to patch-clamp measurements. RT-PCR was used to determine channel expression. Endothelial properties were analysed with immunocytochemistry and acLDL uptake assay. RESULTS: A well-expressed inward rectifying current (IKir ) was identified in cultured CD117(pos) cells. Furthermore, a ZD 7288 (HCN channel blocker)-sensitive current component was isolated. Voltage-dependent potassium currents and chloride currents were less expressed. A small fraction of cells demonstrated voltage- and time-dependent inward currents. In AT2 R-stimulated cells inward rectifying the hyperpolarization-induced inward currents were slightly attenuated on the translational level but showed increased mRNA expression. Cultured CD117(pos) cells express CD31 and VEGFR-2 and significantly increased the uptake of acLDL. CONCLUSIONS: CD117(pos) cells do not have properties of action potential-generating cells and moderately change their excitability during AT2 R stimulation. Electrophysiological and molecular properties of control and AT2 R-stimulated cells point to a differentiation to vascular endothelial cells. This could increase beneficial vascularization in injured tissues.

cAMP-dependent protein kinase is in an active state in rat small arteries possessing a myogenic tone.

The hypothesis that cAMP-dependent protein kinase (protein kinase A; PKA) is in an active state in small arteries possessing a myogenic tone was investigated in pressurized rat tail small arteries. At a pressure of 80 mmHg, these vessels constricted to 71.6 +/- 1.0% (n = 32) of the diameter of the fully relaxed state. The PKA inhibitors Rp-8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphothioate (Rp-CPT-cAMPS) and N-(2-([3-(4-bromophenyl)-2-propenyl]amino)-ethyl)-5- isoquinolinesulfonamide HCl (H-89) constricted these vessels dose dependently. For example, 300 microM Rp-CPT-cAMPS and 9 microM H-89 reduced vessel diameter by 11.0 +/- 1.2% (n = 8) and 14.3 +/- 3.6% (n = 5), respectively. The cGMP-dependent protein kinase (protein kinase G; PKG) inhibitor Rp-8-bromo-beta-phenyl-1,N(2)-etheno-guanosine 3', 5'-cyclic monophosphothioate (Rp-8-Br-PET-cGMPS) did not alter vessel diameter up to a concentration of 10 microM. Neither endothelium removal nor inhibition of neural transmission affected the action of Rp-CPT-cAMPS. The effect of 300 microM Rp-CPT-cAMPS was reduced by 82% after pretreatment of the vessel with 100 nM iberiotoxin, a blocker of calcium-activated potassium (K(Ca)) channels. However, the effect of 300 microM Rp-CPT-cAMPS was not altered after pretreatment with 1 mM 4-aminopyridine, a blocker of delayed rectifier potassium channels, or 10 microM ryanodine, a blocker of ryanodine receptor-generated calcium sparks. In inside-out patch-clamp experiments on cells isolated from rat tail small arteries, 10 U/ml of the catalytic subunit of PKA together with 100 microM MgATP increased K(Ca) channel activity 30.1 +/- 9. 8-fold (n = 9). Additionally, neither inhibition of PKA or PKG nor moderate activation of PKA or PKG altered the vessel response to a pressure step from 80 to 120 mmHg. These results suggest that in rat tail small arteries possessing a myogenic tone 1) PKA is in an active state modulating the level of the myogenic tone, and 2) K(Ca) channels mediate, at least partly, this effect of PKA.

Iloprost dilates rat small arteries: role of K(ATP)- and K(Ca)-channel activation by cAMP-dependent protein kinase.

The effect of the stable prostacyclin analog iloprost and its mechanism of action were investigated with the use of pressurized rat tail small arteries with a spontaneous myogenic tone. Iloprost concentration dependently dilated these vessels with a half-maximal effective dose of 5.0 +/- 0.5 x 10(-8) M. Application of 10(-7)-10(-6) M glibenclamide, a blocker of ATP-sensitive potassium (K(ATP)) channels, inhibited the iloprost-induced dilation. Glibenclamide did not affect the basal vessel diameter. The application of 5 x 10(-5)-10(-3) M tetraethylammonium (TEA) and 5 x 10(-9)-10(-7) M iberiotoxin, blockers of calcium-activated potassium (K(Ca)) channels, decreased vessel diameter in the presence of iloprost. Both TEA and iberiotoxin reduced the basal vessel diameter. Glibenclamide at 10(-6) M inhibited the dilation produced by 5 x 10(-5) M Sp-5,6-DCl-cBIMPS, an activator of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. Iberiotoxin at 10(-7) M decreased vessel diameter in the presence of Sp-5,6-DCl-cBIMPS. H-89 and Rp-8-CPT-cAMPS, blockers of cAMP-dependent protein kinase A (PKA), inhibited the iloprost-induced dilation of these vessels. With use of the whole cell configuration of the patch-clamp technique, it was observed that 5 x 10(-7) M iloprost enhanced an outward current, determined largely by K(Ca) channels, 1.79 +/- 0.17-fold in freshly isolated smooth muscle cells from rat tail small artery. These data show that iloprost dilates rat tail small arteries with a spontaneous myogenic tone and suggest that K(ATP) as well as K(Ca) channels are involved in this effect, which is mediated, at least partly, by PKA.

Iloprost activates KCa channels of vascular smooth muscle cells: role of cAMP-dependent protein kinase.

The patch-clamp technique was used to investigate the effect of iloprost on activity of calcium-activated potassium (KCa) channels of freshly isolated rat tail artery smooth muscle cells. In the whole cell configuration, outward current, determined largely by KCa channels, was enhanced 1.73 +/- 0.11-fold by 5 x 10(-7) M iloprost, 1.80 +/- 0.12-fold by 10(-4) M 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3', 5'-cyclic monophosphothioate (Sp-5,6-DCl-cBIMPS), a specific activator of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA), and 2.78 +/- 0.95-fold by 10 U/ml of the catalytic subunit of PKA + 10(-4) M MgATP, whereas the heat-inactivated catalytic subunit of PKA + MgATP was without effect. Iloprost at 5 x 10(-7) M increased this current 1.70 +/- 0.27-fold after pretreatment of cells with 10(-6) M okadaic acid, a specific phosphatase inhibitor, but did not alter this current after pretreatment of cells with 2 x 10(-4) M Rp-8-(4-chlorophenylthio)-adenosine-3',5'-cyclic monophosphorothioate, a specific PKA inhibitor. In the cell-attached configuration, activity of KCa channels was enhanced 2.48 +/- 0.44-fold by 5 x 10(-7) M iloprost and 2.09 +/- 0.07-fold by 10(-4) M Sp-5,6-DCl-cBIMPS. Iloprost at 5 x 10(-7) M did not alter intracellular calcium concentration in these cells measured using indo 1. In the inside-out configuration, activity of KCa channels was increased 87.12 +/- 45.04-fold by 10 U/ml of the catalytic subunit of PKA together with 10(-4) M MgATP, whereas no effect was observed after application of the catalytic subunit of PKA together with its regulatory subunit and MgATP, MgATP, cAMP, or the catalytic subunit of PKA alone also did not change KCa channel activity. Thus these results show that iloprost is able to activate KCa channels of freshly isolated rat tail artery smooth muscle cells and suggest that this effect is mediated by a PKA-induced phosphorylation of the channel.

Analysis of pressurized resistance vessel diameter changes with a low cost digital image processing device.

A low cost digital image processing device (frame grabber) together with a program running under MS_WINDOWS for automatic on-line analysis of diameter changes of in vitro pressurized blood vessels with an inner diameter of 80-400 microns is presented. The frame grabber is designed to receive light microscopic images either from a video camera or from a VCR and to present the digitized image on the computer monitor. The special software allows to manipulate the image, e.g. filtering, calibrating, storing of vessel images, and detects the outer and inner border of the two vessel walls with a new, simple algorithm. The inner diameter and the vessel wall thickness are calculated and the diameter is presented in a diameter versus time diagram on the monitor screen. Further, these data are stored in an ASCII-file for later import into calculation and presentation programs like MS-EXCEL.

Ca2+ influx through ATP-gated channels increments [Ca2+]i and inactivates ICa in myocytes from guinea-pig urinary bladder.

1. Whole-cell patch clamp was combined with microspectrofluometry (Indo-1) to study the effects of bath applied ATP on membrane currents and cytoplasmic Ca2+ concentration ([Ca2+]i) in single smooth muscle cells of the guinea-pig urinary bladder. Experiments were carried out at 22 degrees C and in 3.6 mM [Ca2+]o. Superimposed K+ currents were reduced by Cs+ dialysis from the patch electrode. 2. At -60 mV, ATP induced an inward current (Ins,ATP) that peaked within 0.4 s and then decayed. Ins,ATP was activated half-maximally by 1.1 microM-ATP and saturated at 50 microM-ATP to -1.1 +/- 0.2 nA (mean +/- S.E.M.). At 3.6 mM [Ca2+]o, Ins,ATP had a reversal potential (Erev) of -5 +/- 2 mV. From the shifts in Erev during changes in [Na+]o or [Ca2+]o we estimated that approximately 7% of Ins,ATP is carried by Ca2+ ions. 3. ATP (50 microM) increased [Ca2+]i transiently from resting 130 +/- 40 nM to 730 +/- 100 nM. At 22 degrees C, [Ca2+]i rose at a rate proportional to the instantaneous current amplitude of Ins,ATP. This relation was lost, however, after warming to 36 degrees C which increased the peak Ins,ATP (Q10 = 1.25) but reduced the peak of the ATP induced [Ca2+]i transient (Q10 = 0.75). We suggest that warming to 36 degrees C stimulated Ca2+ sequestration and Ca2+ efflux to such a degree that peak [Ca2+]i was attenuated significantly. 4. The contribution of Ca2+ ions to Ins,ATP was evaluated from a comparison of the increments in [Ca2+]i due to Ins,ATP and due to L-type Ca2+ channel current (ICa). For the same increment, Ins,ATP had to transport 19 times more charge than ICa. This number suggests that 5.8 +/- 0.8% of Ins,ATP is carried by Ca2+ ions which can be translated into a permeability ratio of PNa:PCa approximately 1:1. 5. During bath application of ATP, peak ICa was inhibited by 80 +/- 15%. Inhibition of ICa diminished to 20 +/- 8% after cell dialysis with 40 mM-EGTA, and it was 19 +/- 7% when extracellular Ca2+ had been substituted by Ba2+. These results are in agreement with the hypothesis of 'ICa inactivation by Ca2+'. Depletion of intracellular Ca2+ stores by pre-treatment with 20 mM-caffeine did not attenuate significantly the ATP-induced rise in [Ca2+]i or the ATP-induced inhibition of ICa. 6. The ATP-induced [Ca2+]i transients and the reduction of peak ICa recovered along a similar time course.(ABSTRACT TRUNCATED AT 400 WORDS)

Possible relationship between mechanical response, action potential repolarization and Na/Ca exchange in myocardium of normotensive (NWR) and spontaneously hypertensive rats (SHR).

Electrical and mechanical restitution was tested in ventricular preparations of adult NWR and SHR. As in atrial preparations we found a positive correlation between force and action potential duration in the late repolarization phase (APD90), though the effect of test interval prolongation on APD90 was less in papillary muscle compared to atrial preparations. These relationships as well as the differences between NWR and SHR-responses may be interpreted in part by the altered activity of the Na/Ca exchange.

The effect of stimulus interval variation on action potential repolarization of atrial muscle preparations in normotensive (NWR) and spontaneously hypertensive rats (SHR).

Action potential (AP) repolarization was tested in atrial preparations of adult NWR and SHR. Prolongation of pause interval interposed in the basic driving frequency of 2 Hz caused a shortening of the early phase and a prolongation of the final phase of repolarization in both NWR and SHR. AP's in SHR exhibited longer durations compared to NWR. However, at pause prolongation the relative increment of AP-duration at the 70% repolarization level (APD70) was less in SHR. This might indicate a positive correlation between electrical and mechanical restitution kinetics. The Na-Ca-exchange could be a possible link for an interpretation of this correlation.

The dependence of trout's electroretinographic response on temporal gradient of luminance. II. The off-response.

In this study trout's electroretinographic off-responses, elicited by ramp-like off-stimuli of variable decay time were recorded and evaluated. In the off-response of the trout, at least two different subwaves could be separated. As the stimulus decay time was increased stepwise from 10 ms to 3000 ms, the earlier wave d1 showed a pronounced amplitude attenuation and only little increase of the peak time, while at the second, later wave d2 a less marked effect on the amplitude but a distinct increase of the peak time could be observed. Possible relations of the subwaves d1 and d2 to the photopic or the scotopic system, comparison with the on-response at similar stimulating conditions, and correlations with psychophysical findings are discussed. The parallels to the observations on mammals' ERG including the human being are demonstrated.