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.

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.

SUMMARY1. 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+]

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 μM Rp-CPT-cAMPS and 9 μM 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-β-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 μM. Neither endothelium removal nor inhibition of neural transmission affected the action of Rp-CPT-cAMPS. The effect of 300 μM Rp-CPT-cAMPS was reduced by 82% after pretreatment of the vessel with 100 nM iberiotoxin, a blocker of calcium-activated potassium (KCa) channels. However, the effect of 300 μM Rp-CPT-cAMPS was not altered after pretreatment with 1 mM 4-aminopyridine, a blocker of delayed rectifier potassium channels, or 10 μM 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 μM MgATP increased KCa 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) KCa channels mediate, at least partly, this effect of PKA.

Brain death (BD) influences the quality of donor grafts in transplantation. To evaluate the impact of BD on pancreas grafts, we investigated the influence of BD on the microcirculation and histology of the pancreas in a rat model of explosive BD. A group of Wistar rats (n = = 7), rendered brain dead by inflating an intracranially inserted Fogarty catheter was compared with controls (CO) using intravital epifluorescence-microscopy over 4 h after BD induction; functional capillary density (FCD), leukocyte adherence (AL) in post-capillary venules, histology and pancreatic enzymes were investigated. Four hours after BD, FCD decreased (333 ± 11 vs. baseline 444 cm/cm 2 ± 5 SEM; p < 0.01) and showed lower values than CO (388 ± 9 p < 0.01). In BD, AL was increased (628 cells/mm 2 ± 110 SEM vs. baseline 123 ± 32, and vs. CO 180 ± 33; p < 0.001). BD caused increased histological damage (CO 1.6 scorepoints ± 0.7 SD vs. BD 8.3 ± 7.1; p < 0.05). Amylase was higher in BD (p < 0.05) but did not reach pathological values. We show for the first time that BD causes relevant changes in pancreatic microcirculation, histology and leukocyte endothelial interaction which might have a serious impact on the function of grafts. New strategies for preventing this damage are therefore highly desirable in order to improve the outcome of pancreas transplantation.