Effects of selective phosphodiesterase inhibitors on isolated coronary,lung and renal arteries from man and rat

The relaxant effects of some isozyme-selective phosphodiesterase inhibitors were evaluated in isolated human and rat coronary, lung and renal arteries. Milrinone and OPC 3911, inhibitors of a cGMP-inhibited cAMP phosphodiesterase (cGI-PDE), were shown to have distinct vasodilator actions. These agents were less potent as relaxants in rat lung and renal arteries than in the corresponding human tissues. OPC 3911, the more potent cGMP-inhibited cAMP phosphodiesterase inhibitor, was found to be the more potent vasorelaxant. Rolipram, a selective inhibitor of a cGMP-noninhibited cAMP phosphodiesterase, had small effects on coronary and lung arteries, but produced a slightly more pronounced relaxation of renal arteries from both man and rat. In human preparations contracted by 30 mm K⁺, milrinone and OPC 3911 had similar relaxant profiles, and were as potent in coronary as in renal arteries. These results do not support the notion that milrinone has reduced effects on renal vessels in man and show that there may be species differences in vascular responsiveness to cGMP-inhibited cAMP phosphodiesterase inhibitors.     

Effects of isozyme-selective phosphodiesterase inhibitors on rat aorta and human platelets: smooth muscle tone,platelet aggregation and cAMP levels

The inhibitors of the cGMP-inhibited, low-K_m cAMP phosphodiesterase—milrinone and OPC 3911-and an inhibitor of a non-cGMP-inhibited low-K_m cAMP phosphodiesterase—rolipram—were used to evaluate the functional importance of the two cAMP phosphodiesterase activities in vascular smooth muscle and in platelets. Vinpocetine, an inhibitor of a calcium-calmodulin-dependent phosphodiesterase was also studied. OPC 3911 and milrinone relaxed the contracted rat aorta, inhibited ADP-induced platelet aggregation and also enhanced isoprenaline-induced relaxation as well as the antiaggregatory effects of adenosine. In platelets, OPC 3911 and milrinone increased cAMP levels, but in the rat aorta the increase was significant only for milrinone (OPC 3911 P= 0.062). In both tissues OPC 3911 and milrinone enhanced the increase in cAMP caused by activators of adenylate cyclase (isoprenaline/adenosine). Rolipram had no effects on aggregation or cAMP levels in platelets and no overadditive effects in combination with adenosine. Rolipram had little effect on relaxation and cAMP levels, did not alter isoprenaline-induced relaxation of guanfacin-contracted rat aorta, but showed synergistic effects with isoprenaline in raising cAMP levels. In PGF_2α-contracted aorta rolipram enhanced relaxation caused by isoprenaline. Vinpocetine had a relaxant effect without affecting cAMP levels, but had no effect on platelets. These results support the concept that the cGMP-inhibited phosphodiesterase is an important modulator of vascular smooth muscle tone and platelet function. The role of the non-cGMP-inhibited phosphodiesterase in these tissues is less obvious.     

BRL 22321, a compound that has mast cell stabilizing activity similar to that of disodium cromoglycate and in addition has smooth muscle relaxing activity

BRL 22321 is 4,9-dihydro-6,7-dimethyl-4,9-dioxo-1H-naphtho[2,3-d]-v-triazole sodium salt. It combines some of the chemical structural features of disodium cromoglycate (DSCG) and theophylline. It has mast cell stabilizing activity similar to that DSCG and in addition it has some smooth muscle relaxant activity. BRL 22321 was more potent than DSCG as an inhibitor of rat passive cutaneous and peritoneal anaphylaxis (PCA and PPA) and histamine release by antigen from passively sensitized rat peritoneal cellsin vitro. In the rat PCA test it was active when given orally and a large intravenous dose of DSCG reduced the potency of subsequent intravenous doses of either DSCG or BRL 22321, suggesting that the compounds were active by a common pathway. The compounds had similar potencies as inhibitors of the release, by antigen, of histamine and slow reacting substance of anaphylaxis (SRS-A) from passively sensitized human lung fragments. BRL 22321 was more potent than DSCG and theophylline as an inhibitor of a cyclic adenosine and a cyclic guanosine monophosphate phosphodiesterase. BRL 22321 inhibited histamine release in systems in which DSCG was inactive, for example it inhibited histamine release by antigen from chopped lung taken from hyperimmune guinea pigs and by anti-IgE from human leucocytes; in these tests it was more potent than theophylline. BRL 22321 had smooth muscle relaxant activity not shown by DSCG. It relaxed histamine elevated tone in guinea pig lung strip and tracheal spiral preparations at doses at which theophylline produced a dose dependent response. It was difficult to compare potencies since there was a difference in the nature of the responses to the drugs. DSCG had little activity over the same dose range or at higher doses. BRL 22321 also reduced increased airways resistance in the anaesthetized guinea pig produced by 5-hydroxytryptamine (5-HT) or histamine but at somewhat higher doses than those at which theophylline was active. BRL 22321 was not an antagonist of acetylcholine, histamine, 5-HT or SRS-A on the isolated guinea pig ileum.     

Relaxin signalling links tyrosine phosphorylation to phosphodiesterase and adenylyl cyclase activity

The relaxin receptor has so far avoided molecular cloning and characterization. We have therefore characterized the signalling events activated by relaxin (RLX), using two different cell culture-based bioassay systems: primary human endometrial stromal cells from the cycle (ESC) and the human monocyte cell line THP-1. Upon RLX stimulation, both cell types showed a rapid increase in cAMP accumulation, which could be inhibited by an inhibitor of G-protein activation, GDP-beta-S. However, evolutionarily one would expect the RLX receptor, like those for the structurally related hormones insulin and insulin-like growth factor-I, to involve tyrosine kinase activity. The specific tyrphostins AG 1478, AG 527 and AG 879 inhibited the RLX-stimulated cAMP response in human ESC and THP-1 cells in a dose-dependent manner, though the potent broad range tyrphostin AG 213 had no effect. Also, treatment of THP-1 cells with the potent phosphotyrosine phosphatase inhibitors bpV(phen) and mpV(pic) increased RLX-stimulated cAMP accumulation in a dose-dependent manner. The effect of the general tyrosine kinase inhibitor genistein (which can also inhibit some phosphodiesterases) on RLX-mediated cAMP accumulation strongly depended on the activity status of phosphodiesterase. In the absence of a phosphodiesterase inhibitor, genistein enhanced RLX-stimulated cAMP accumulation in both bioassays. When phosphodiesterase was inhibited by isobutylmethylxanthine, this effect was not observed. The results imply that activation of the RLX receptor uses tyrosine kinase signalling to control phosphodiesterase activity, and hence to up-regulate intracellular cAMP.     

The inotropic mechanism of the phosphodiesterase inhibitor OPC 3911 alone and in combination with rolipram,studied in papillary muscles of ferret and guinea–pig and isolated myocytes of guinea–pig ventricular muscle

OPC 3911 is a potent inhibitor and PDE III is a specific inhibitor in cardiac muscle. The effects of the drug alone and in combination with the non–inotropic PDE IV inhibitor rolipram were analysed using hearts from guinea–pigs and ferrets. OPC 3911 had an EC₅₀ value of 0.1μM. At 0.1 fiM peak force was increased by 50.7 ± 7.6% (n = 6, P< 0.001), time to peak tension (TPT) reduced by 18,7±5.6% (n = 6, P < 0.05). Time to half relaxation (THR) was prolonged by 19±4.2% (n= 6, P < 0.001). After addition of rolipram (30 μM) there was a potentiation of peak force at all concentrations of OPC 3911. At 0.1 μM OPC rolipram increased peak force by 82.8±8.9% (n= 6, P < 0.001), reduced TPT by 73 ± 6% (n = 6, P < 0.005) and increased THR by 27±5% (P<0.01). OPC 3911 shortened action potential duration (APD) at 50% repolarization by 5.3 ±2.5% (n= 6, P < 0.05). Addition of rolipram prolonged APD by 3.7 ± 2.5% (n = 6, P < 0.05). Second inward current (I_8l) was increased at 3μM OPC 3911 by 46 ± 6% (n = 6, P< 0.05). The combination of OPC 3911 and rolipram intensified the I_8l to 101 ± 5% (n= 3). Rolipram slowed the rate of restitution and the onset of restitution was prolonged. Relative maximum post–extrasystolic potentiation was reduced in the presence of OPC 3911 from 67 ± 5% to 45 ±6%. Adding rolipram caused potentiation of 55 ± 6%. OPC 3911 increased the recirculation fraction of activator calcium from 0.36 to 0.42 (n = 10, P < 0.05). After addition of rolipram the recirculation fraction was 0.41 ± 0.04 (n =10, P < 0.05). The results suggest that rolipram exerts its potentiating effect on OPC 3911 via an increased I_sl.     

Antagonism between parathyroid hormone and norepinephrine on cyclic adenosine-3′:5′-monophosphate (cAMP) levels in isolated tubules from rat kidney cortex

Isolated rat kidney cortex tubules were used as a model system to study the hormonal regulation of cyclic adenosine-3':5'-monophosphate (cAMP) levels in vitro. When incubated over 15 min, parathyroid hormone increased cAMP levels 4-fold in the absence of inhibitors of cAMP phosphodiesterase. Norepinephrine in a concentration of 5-10-7 M which had itself no effect on cAMP levels under this condition, inhibited the effect of parathyroid hormone by 50%. This effect of the catecholamines could be completely abolished by the addition of an alpha-receptor blocking agent, phentolamine. The addition of an inhibitor of cAMP phosphodiesterase, in a concentration sufficient to prevent the breakdown of extratubular cAMP, potentiated hormone effects on cAMP levels. The antagonism between catecholamines and parathyroid hormone on cAMP levels was however not abolished by this treatment. This indicated that catecholamines probably inhibited parathyroid hormone stimulated cAMP formation. Since most of the cAMP was found to be intratubular, it can be assumed that norepinephrine and parathyroid hormone interact in the same cell. Proximal tubular sodium reabsorption and renal gluconeogenesis are discussed as possible events of this hormone interaction.     

Effects of milrinone on inflammatory response-related gene expressions in cultured rat cardiomyocytes

Congestive heart failure (CHF) is defined as a cardiac dysfunction leading to low cardiac output and inadequate tissue perfusion. Intravenous positive inotropes are used to increase myocardial contractility in hospitalized patients with advanced heart failure. Milrinone is a phosphodiesterase Ⅲ inhibitor and used most commonly for inotropic effect. The well-known PROMISE study investigated the effects of milrinone on mortality in patients with severe CHF, and concluded that long-term therapy with milrinone increased morbidity and mortality among patients with advanced CHF. Previous studies have suggested that phosphodiesterase inhibitors can have potential effects on inflammatory pathways. Hence, we hypothesized that milrinone may alter inflammatory gene expressions in cardiomyocytes, thus leading to adverse clinical outcomes. We used rat cardiomyocyte cell line H9C2 and studied the impact of exposing cardiomyocytes to milrinone (10 μmol/L) for 24 hours on inflammatory gene expressions. RNA extracted from cultured cardiomyocytes was used for whole rat genome gene expression assay (41 000 genes). The following changes in inflammatory response-related gene expressions were discovered. Genes with increased expressions included: THBS2 (+ 9.98), MMP2 (+3.47), DDIT3 (+2.39), and ADORA3 (+3.5). Genes with decreased expressions were: SPP1 (?5.28) and CD14 (?2.05). We found that the above mentioned gene expression changes seem to indicate that milrinone may hinder the inflammatory process which may potentially lead to adverse clinical outcomes. However, further in vivo and clinical investigations will be needed to illustrate the clinical relevance of these gene expression changes induced by milrinone.     

Antiallergic activity profile in vitro of RHC 2963 and related compounds

RHC 2963 (7-methyl-pyrido (3',2':4,5)-thieno (3,2-d)-1,2,3 triazine-4(3H)-one and 20 related compounds have been investigated for their antiallergic activities in 3 in vitro models of anaphylaxis and for their effects on cyclic nucleotide phosphodiesterases (cNUC-PDE) from purified rat mast cells (RMC). Nine compounds were potent (I50 less than or equal to 80 microM) inhibitors of antigen-induced release of histamine (AIR) from RMC, 2 compounds inhibited anti-IgE-induced release of histamine from human basophils (I50 less than or equal to 60 microM) and one compound inhibited AIR from guinea pig lung slices (I50 = 55 microM). RHC 2963 was 18 times more potent than disodium cromoglycate (DSCG) as inhibitor of AIR from RMC and had an activity profile identical to that of DSCG in the following respects: loss of inhibitory activity with increasing preincubation time, tachyphylactic properties and inability to inhibit non-immunologic release of histamine induced by compound 48/80. Neither RHC 2963 nor DSCG had any effect on anti-IgE-induced release of histamine from human basophils or IgG1-mediated release of histamine from guinea pig lung. Twelve of the compounds in this chemical series were more potent than theophylline as inhibitors of cyclic AMP and/or cyclic GMP phosphodiesterase (PDE) from RMC. Paired regression analysis of the I50 values for inhibition of AIR and cNUC-PDE from RMC revealed no statistically significant correlation between the inhibition of AIR and inhibition of cAMP- or cGMP-PDE. We conclude: (1) RHC 2963 and some of the related compounds are potent inhibitors of immunologic release of histamine from RMC with a mechanism of action similar to that of DSCG, and (2) inhibition of cAMP- or cGMP-PDE by these compounds is not the biochemical mechanism by which they inhibit AIR from RMC.     

Acute hypoxia induces vasodilation and increases coronary blood flow by activating inward rectifier K+ channels

We examined the effects of acute hypoxia on vascular tone and coronary blood flow (CBF) in rabbit coronary arteries. In the pressurized arterial preparation of small arteries (<100 μm) and the Langendorff-perfused rabbit hearts, hypoxia induced coronary vasodilation and increased CBF in the presence of glibenclamide (K_ATP channel blocker), Rp-8-Br-PET-cGMPs [cyclic guanosine monophosphate (cGMP)-dependent protein kinase inhibitor, Rp-cGMPs], and methionyl transfer RNA synthetase (MRS) 1334 (adenosine A₃ receptor inhibitor); these increases were inhibited by the inward rectifier K⁺ (Kir) channel inhibitor, Ba²⁺. These effects were blocked by the adenylyl cyclase inhibitor SQ 22536 and by the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) inhibitors Rp-8-CPT-cAMPs (Rp-cAMPs) and KT 5720. However, cGMP-dependent protein kinase was not involved in the hypoxia-induced increases of the vascular diameter and CBF. In summary, our results suggest that acute hypoxia can induce the opening of Kir channels in coronary artery that has small diameter (<100 μm) by activating the cAMP and PKA signalling pathway, which could contribute to vasodilation and, therefore, increased CBF.     

雾化吸入米力农对烟雾吸入性损伤大鼠肺的保护作用

目的探讨米力农对大鼠烟雾吸入性损伤肺保护作用。方法雄性Sprague-Dawley大鼠32只,随机分为正常组（Ⅰ组）、生理盐水组（Ⅱ组）、低剂量米力农组（Ⅲ组）、高剂量米力农组（Ⅳ组）,每组8只。Ⅱ、Ⅲ、Ⅳ组建立吸入性损伤模型,伤后30min开始分别给予雾化生理盐水、0.4mg/ml米力农、1mg/ml米力农,每次10min,间隔50min,共4次,Ⅰ组不予处理。最后一次雾化治疗结束后30min后,检测4组大鼠血清白细胞介素-6（IL-6）、白细胞介素-10（IL-10）、肿瘤坏死因子-α（TNF-α）含量,以及肺组织匀浆中丙二醛（MDA）含量、髓过氧化物酶（MPO）和超氧化物歧化酶（SOD）活性,取左肺组织经做病理HE染色切片光镜观察。结果Ⅲ组以及Ⅳ组的IL-6、TNF-α、MDA、MPO均比Ⅱ组降低,IL-10、SOD活性增高;Ⅳ组相对于Ⅲ组IL-6、MDA、MPO降低,SOD活性增高（P〈0.05）。光镜下Ⅲ组和Ⅳ组较生理盐水组肺组织炎细胞浸润减少,肺水肿减轻。结论吸入性损伤后,早期雾化吸入米力农可以调节炎症/抗炎症以及氧化/抗氧化的平衡,对肺损伤早期起到肺组织保护作用。     

Milrinone, a phosphodiesterase III inhibitor, antagonizes the neuromuscular blocking effect of a non-depolarizing muscle relaxant in vitro

Phosphodiesterase (PDE) inhibitors are occasionally used in patients receiving non-depolarizing muscle relaxants during anesthesia and intensive care. However, little is known about the influence of PDE III inhibitors on the effects of non-depolarizing muscle relaxants. The aim of this study was to elucidate the effects of milrinone, a PDE III inhibitor, on d-tubocurarine (dTc)-induced muscle relaxation in vitro and then to compare its effects with those of other activators of the adenylate cyclase (AC) system (aminophylline, a non-selective PDE inhibitor; forskolin, a direct AC activator; and isoproterenol, a beta-adrenoceptor agonist). Isometric twitch tensions of rat nerve-hemidiaphragm preparations elicited by indirect or direct stimulation (0.1 Hz) were measured. Indirectly elicited twitch tension partially depressed by dTc (1 microM) was antagonized by milrinone, aminophylline, and forskolin but was attenuated by isoproterenol. Directly elicited twitch tension was increased by aminophylline, forskolin, and isoproterenol but was not altered by milrinone. The results indicate that milrinone antagonizes dTc-induced muscle relaxation by recovering the neuromuscular transmission. It is noteworthy that PDE inhibitors and a beta-adrenergic agonist affect non-depolarizing muscle relaxation in opposite direction.     

Role of cyclic nucleotide phosphodiesterase isoforms in cAMP compartmentation following β2-adrenergic stimulation of ICa,L in frog ventricular myocytes

The role of cyclic nucleotide phosphodiesterase (PDE) isoforms in the β₂-adrenergic stimulation of the L-type Ca²⁺ current ( I _Ca,L) was investigated in frog ventricular myocytes using double patch-clamp and double-barrelled microperfusion techniques. Isoprenaline (ISO, 1 nM to 10 μM) was applied on one half of the cell, either alone or in the presence of PDE inhibitors, and the local and distant responses of I _Ca,L were used to determine the gradient of local vs. distant cAMP concentration (α). IBMX (100 μM), a non-selective PDE inhibitor, reduced α from 40 to 4.4 indicating a 9-fold reduction in intracellular cAMP compartmentation when all PDE activity was blocked. While PDE1 and PDE2 inhibition had no effect, PDE3 inhibition by milrinone (3 μM) or PDE4 inhibition by Ro 20-1724 (3 μM) reduced α by 6- and 4-fold, respectively. A simultaneous application of milrinone and Ro 20-1724 produced a similar effect to IBMX, showing that PDE3 and PDE4 were the major PDEs accounting for cAMP compartmentation. Okadaic acid (3 μM), a non-selective phosphatase inhibitor, or H89 (1 μM), an inhibitor of cAMP-dependent protein kinase (PKA), had no effect on the distant response of I _Ca,L to ISO indicating that PDE activation by PKA played a minor role in cAMP compartmentation. Our results demonstrate that PDE activity determines the degree of cAMP compartmentation in frog ventricular cells upon β₂-adrenergic stimulation. PDE3 and PDE4 subtypes play a major role in this process, and contribute equally to ensure a functional coupling of β₂-adrenergic receptors with nearby Ca²⁺ channels via local elevations of cAMP.     

Heterogeneity in the response to dopamine of monkey cerebral and peripheral arteries

In helical strips of monkey cerebral and mesenteric arteries contracted with prostaglandin F2 alpha, dopamine in low concentrations produced a moderate relaxation but in high concentrations produced a contraction from the level of relaxation. On the other hand, coronary, renal, and femoral arterial strips responded to dopamine with only a concentration-dependent contraction. Treatment with phenoxybenzamine or phentolamine potentiated the dopamine-induced relaxation seen in cerebral and mesenteric arteries and reversed the contraction in the other arteries to a relaxation. After treatment with phenoxybenzamine, relaxant responses to dopamine of cerebral, mesenteric, and renal arteries were almost identical, and, compared with those, the responses of coronary and femoral arteries were appreciably less. Relaxations induced by dopamine were not influenced by propranolol, atropine, aminophylline, cimetidine, and aspirin but were markedly attenuated by droperidol. Adenosine-induced relaxations were not affected by droperidol. It is concluded that dopamine preferentially relaxes monkey cerebral and mesenteric arteries, possibly via dopaminergic receptors. It appears that the dopamine-induced contractions mediated by alpha-adrenoceptors predominate over the relaxation in coronary, renal, and femoral arteries, and dopaminergic receptor function is greater in cerebral, mesenteric, and renal arteries than in coronary and femoral arteries.     

Anti-allergic effects of milrinone and sulmazole in isolated rat lungs in comparison with theophylline

Milrinone and sulmazole, two recently developed drugs, inhibit specific fractions of the phosphodiesterase (PDE) isozyme system. Since theophylline aspecifically inhibits the PDE complex, we compared the effects of milrinone and sulmazole with those of theophylline on antigen-induced bronchoconstriction, vasoconstriction, mediator release and leukotriene production. In the isolated perfused and ventilated lung of actively sensitized rats, we elicited antigen-induced bronchoconstriction, vasoconstriction and release of mediators like histamine, 5-hydroxytryptamine (5-HT) and slow-reacting substance of anaphylaxis (SRS-A). Milrinone, sulmazole and theophylline inhibited antigen-induced bronchoconstriction and vasoconstriction in a dose-dependent manner with minor differences in potency. Antigen-induced release of preformed mediators like histamine and 5-HT was inhibited only at high concentrations of milrinone, whereas sulmazole failed to inhibit mediator release. Theophylline also failed to inhibit 5-HT release. However, SRS-A synthesis was markedly reduced by these drugs in relatively low concentrations. It is concluded that milrinone and sulmazole have anti-allergic effects similar to those of theophylline and that all three PDE inhibitors reduce SRS-A synthesis.     

Theophylline interferes with the modulatory role of endogenous adenosine on cholinergic neurotransmission in guinea pig ileum

The ability of theophylline and other phosphodiesterase inhibitors to alter contractile responses to cholinergic nerve stimulation was investigated in isolated longitudinal muscle of the guinea pig ileum. Theophylline in low concentrations (10–100 μM), having no or little effect on measured phosphodiesterase activity, antagonized inhibitory effects of exogenous adenosine. In higher concentrations (0.1–10 mM), shown to be effective in inhibiting phosphodiesterase, theophylline as well as a “pure” cAMP phosphodiesterase inhibitor, ZK 62, 711, inhibited contractile responses. Dipyridamole and dilazep, inhibitors of adenosine inactivation, and also selective inhibitors of cAMP and cGMP phosphodiesterase, respectively, were found to enhance effects of exogenous adenosine and to cause a marked leftward shift of adenosine threshold dose. When dipyridamole and dilazep by themselves had inhibitory effects these could be antagonized by theophylline, suggesting an action through increased levels of endogenous adenosine. As a further indication of endogenous adenosine modulating neurotransmission low concentrations of theophylline enhanced responses to transmural stimulation. Endogenous purine concentrations in tissues and bath media were measured by HPLC. Because of tissue and microbial adenosine inactivation direct estimates of extracellular adenosine concentration could not be obtained. However, adenosine levels increased during transmural stimulation, and during inhibition of adenosine inactivation were sufficient, even in the bath medium, to interfere with the cholinergic neurotransmission.     

Inhibition of antigen-induced histamine release from rat mast cells by a cyclic GMP-phosphodiesterase inhibitor and sodium nitrite

Antianaphylactic properties have been attributed to cyclic nucleotide phosphodiesterase inhibitors through increase of cyclic AMP levels, according to the concept that increases in cyclic AMP reduce release and increases in cyclic GMP enhance release. However,Coulson et al. [3] showed that the inhibition of histamine release from human lung is correlated to the inhibition of cyclic GMP hydrolysis. We studied the effect of specific inhibitors of cyclic AMP and cyclic GMP hydrolysis on the antigen-induced mediator release from rat mast cells and human basophils and on airways relaxation [4]. The results suggested that the modulation of mediator release was different from one cell type to the other, enhancement of cyclic AMP levels leading to the inhibition of release from basophils, while cyclic GMP appears to be predominantly involved in mast cells. The present paper shows that high concentrations of sodium nitrite, a stimulating agent of guanylate cyclase, inhibit histamine release from rat mast cells in the presence or absence of M&B 22948, a selective cyclic GMP phosphodiesterase inhibitor.     

Comparative effects of some calcium-channel blockers on human peripheral arteries and veins

We investigated the effects of five different calcium-channel blockers (CCBs), verapamil, nifedipine, diltiazem, flunarizine and lidoflazine, on contractions evoked in vitro by noradrenaline (NA) in small human arteries and veins from the epigastric region. Vessels were obtained from patients without obvious vascular diseases undergoing surgery because of inguinal hernias. The human superficial epigastric artery has previously been shown to contain mainly alpha 1-adrenoceptors, whereas in the vein alpha 2-adrenoceptors predominate. In experiments where NA (10(-5) M) was added non-cumulatively, it was found that nifedipine was the most potent relaxant agent in both arteries and veins, but that this drug showed no preference for any type of vessel. In contrast verapamil (10(-6) M) and (10(-5) M) diltiazem, flunarizine and lidoflazine inhibited the NA-induced contractions to a significantly greater extent in the arteries than in the veins. Comparison between diltiazem and nifedipine on contractions induced by cumulative addition to NA showed that both drugs had significantly more depressive effects on arteries than on veins if the vessels were contracted by relatively high concentrations of NA (10(-6) and 10(-5) M). The results thus confirm the clinical finding that CCBs have more pronounced effects on the arterial than on the venous side of the circulation. They do not support the view that CCBs are more effective inhibitors of alpha 2- than alpha 1-adrenoceptor mediated contraction in isolated human blood vessels.     

Iloprost inhibits neutrophil-induced lung injury and neutrophil adherence to endothelial monolayers.

We hypothesized that Iloprost, a long-acting prostacyclin analog, would inhibit neutrophil (PMN)-induced lung injury and decrease PMN adherence to vascular endothelium. Human PMNs infused into isolated buffer-perfused rat lungs subsequently stimulated with phorbol myristate acetate (PMA) resulted in lung injury as assessed by the accumulation of [125I]bovine serum albumin (125I-BSA) in lung parenchyma and alveolar lavage fluid. Addition of Iloprost to the lung perfusate, prior to activation of the PMNs, reduced lung injury as assessed by a decrease in the accumulation of 125I-BSA in the lung. This protective effect was not due to the vasodilatory effect of Iloprost. Protection by Iloprost was not linked to a reduction in PMA-induced PMN superoxide production since Iloprost did not reduce the amount of superoxide released into lung perfusate. In vitro, Iloprost caused a dose-dependent inhibition of PMA-stimulated PMN adherence to endothelial cells. Iloprost did not affect the number of Mo1 adhesion molecules constitutively expressed or the number of receptors expressed on the PMNs following PMA. Addition of cAMP or dibutyryl cAMP to the endothelial cells mimicked the effects of Iloprost, diminishing PMA-stimulated PMN adhesion. In separate experiments, addition of the phosphodiesterase inhibitor IBMX to Iloprost resulted in a greater inhibition of PMA-stimulated PMN adherence, while addition of an adenylate cyclase inhibitor, SQ 22,536, or cAMP antibodies with the Iloprost abolished Iloprost's inhibitory effect on PMN adhesion. Thus, Iloprost inhibits PMA-activated PMN-induced lung injury despite continued superoxide production. Iloprost inhibition of PMN adhesion is dependent on cAMP.     

Effects of selective phosphodiesterase inhibitors on the polymorphonuclear leukocyte respiratory burst

Modulation of the human polymorphonuclear leukocyte (PMN) respiratory burst by selective cyclic 3',5' adenosine monophosphate (cAMP) phosphodiesterase (PDE) inhibitors was studied with respect to PDE isozyme characteristics. Zaprinast, an inhibitor of a cyclic guanosine monophosphate (cGMP)-specific PDE (PDE I), at concentrations up to 100 mumol/L, had no significant effect on the respiratory burst. Milrinone and imazodan, inhibitors of cAMP-metabolizing, cGMP-sensitive PDE (PDE III), reduced the respiratory burst to 60% of control magnitude but only had significant effects when they were introduced at high (100 mumol/L) concentrations. In contrast, rolipram and RO 20-1724, inhibitors of a cAMP-metabolizing, cGMP-insensitive PDE (PDE IV), had significant effects at low concentrations (0.1 mumol/L) and caused marked reduction of the respiratory burst at higher concentrations (25% of control at 10 mumol/L). The selective PDE IV inhibitors significantly potentiated PMN inhibition by isoproterenol. Diethylaminoethyl (DEAE)-Sepharose chromatography demonstrated a predominant PDE isozyme with high affinity and selectivity for cAMP that was insensitive to cGMP and was completely inhibited by rolipram, a PDE IV inhibitor. These results are consistent with the conclusion that the PMN respiratory burst is inhibited by an elevation of cAMP induced by PDE IV inhibition.     

Staurosporine and herbimycin a augment agonist-induced elevation of cAMP in jurkat T-lymphoblasts

Cross-talk between signalling pathways appears to play an important role in T-lymphocyte activation. In the present work, we have studied the effects of different inhibitors of protein tyrosine kinases or protein serine/ threonine kinases on the agonist-induced cAMP accumulation in the human T-lymphoblast cell line Jurkat. Staurosporine, a potent but nonspecific inhibitor of protein kinases, produced a ten-fold enhancement of the response to PGE2. No significant effect was obtained with two specific protein kinase C inhibitors (GF 109203X and H7), whereas herbimycin A, a specific protein tyrosine kinase inhibitor, markedly enhanced the PGE2-induced cAMP accumulation: its effect was approximately 60% that of staurosporine. It was confirmed that both staurosporine and herbimycin A inhibited by more than 90% the release of IP3 induced by ligation of the T-cell receptor, a known protein tyrosine kinase-dependent mechanism. To our knowledge, this study provides the first indication of a protein tyrosine kinase-mediated inhibition of agonist-induced cAMP accumulation. The possible targets of this inhibition are discussed.