The effect of histamine on cyclic AMP levels in guinea-pig tracheal smooth muscle

Histamine (10(-5)--3 x 10(-4) M) increased the cylic AMP content of guinea-pig tracheal smooth muscle, the maximal effect being a 3-fold increase after 2-min incubation with 10(-4) histamine. Histamine-induced accumulation of cyclic AMP was not affected by propranolol or atropine, but was reduced by mepyramine. Aspirin and indomethacin abolished the cyclic AMP response to histamine and potentiated histamine-induced contractions of the smooth muscle. These results suggest that the elevation of cyclic AMP levels in response to histamine is mediated by prostaglandins, and represents an important negative feedback regulatory mechanism which modulates the contractile response of guinea-pig tracheal smooth muscle to histamine.     

Correlation between inhibition of a cyclic GMP phosphodiesterase and relaxation of canine tracheal smooth muscle

Inhibition of partially purified cyclic nucleotide phosphodiesterase activity as well as pharmacologically induced relaxation of respiratory airways smooth muscle was examined to determine whether any correlation between these two effects could be found. The phosphodiesterase in extracts of canine tracheal smooth muscle was chromatographed on a DEAE Bio-Gel A column and eluted with a sodium chloride gradient. The peak I activity hydrolyzed cGMP at a higher rate than cAMP although the apparent Km values for these two cyclic nucleotides were relatively close. Comparison of the Ki values for alkylxanthine inhibitors of peak I activity correlated remarkably well with the EC50 values of the same compounds as relaxants of canine tracheal smooth muscle strips. It is concluded that inhibition of the peak I enzyme may cause accumulation of an intracellular pool of cyclic nucleotide and thus produce or contribute to the muscle relaxant effects that were observed.     

Inhibition of a high affinity cyclic AMP phosphodiesterase and relaxation of canine tracheal smooth muscle

The cyclic nucleotide phosphodiesterase (PDE) activity of canine tracheal smooth muscle (CTS,) was examined. Column chromatography of soluble CTSM-PDE revealed five peaks of activity. One of these peaks (V) was examined further in this study and showed a high affinity for adenosine 3',5'-cyclic monophosphate (Km = 0.63 microM). Seven pharmacological PDE inhibitors were tested for their abilities to inhibit the peak V enzyme and also for their abilities to cause mechanical relaxation of CTSM strips in isolated tissue baths. A strong correlation (P greater than 0.001) between peak V PDE inhibition (-log Ki) and airway muscle relaxation (-log ED50) was found.     

Modulation of carbachol-induced inositol phosphate formation in bovine tracheal smooth muscle by cyclic AMP phosphodiesterase inhibitors.

An investigation was made of a range of agents capable of elevating tissue cyclic AMP levels, or acting as a stable analogue of cyclic AMP, upon carbachol induced inositol phosphate responses in bovine tracheal smooth muscle slices. Whereas the beta 2 adrenoceptor agonist salbutamol (1 microM) and the membrane permeable analogue of cyclic AMP, 8-bromo-cyclic AMP (1 mM) were without effect upon total [3H]inositol phosphate formation induced by carbachol, 3-iso-butyl-1-methylaxanthine (IBMX) (EC50 140 microM), the high Km, cyclic AMP selective phosphodiesterase inhibitor rolipram (EC50 41 microM) and theophylline (EC50 76 microM) all inhibited the inositol phosphate response to low (1 microM) concentrations of carbachol. IBMX (IC50 13 microM), rolipram (IC50 4.6 microM) and theophylline (IC50 180 microM) all relaxed bovine tracheal muscle strips precontracted with methacholine (1 microM). The adenylate cyclase activator forskolin (1 microM), produced a much smaller (10% inhibition) effect upon inositol phosphate formation induced by carbachol. Carbachol (1 microM-1 mM) did not inhibit forskolin induced [3H]cyclic AMP formation. An inhibitor of the cyclic GMP preferring phosphodiesterase isozyme, M&B 22948 (1-100 microM), was without effect upon either carbachol induced inositol phosphate formation or trachealis tone. It is concluded that IBMX, rolipram and theophylline inhibit carbachol stimulated inositol phosphate formation, possibly through a cyclic AMP independent mechanism.     

The effect of cyclic AMP and cyclic GMP phosphodiesterase inhibitors on the superoxide burst of guinea-pig peritoneal macrophages.

1. The cyclic nucleotide phosphodiesterase (PDE) activity of guinea-pig peritoneal macrophages was partially characterized and the effects of selective and non-selective inhibitors of adenosine 3':5'-cyclic monophosphate (cyclic AMP PDE) and guanosine 3':5'-cyclic monophosphate (cyclic GMP PDE) phosphodiesterases on superoxide generation were investigated using peritoneal macrophages from horse-serum pretreated guinea-pigs. 2. The non-selective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX) and the PDE I/V selective inhibitor, zaprinast, inhibited spontaneous superoxide generation with IC50s of 30.7 +/- 11.3 microM and 145 +/- 17 microM respectively (n = 6 and 5). The concentration-response curves for the PDE IV selective inhibitors rolipram and Ro20-1724 were biphasic; mean maximum inhibitions were 56.9 +/- 5.9% and 66.8 +/- 10.5% respectively at 300 microM, but in 2 out of 6 (rolipram) and 2 out of 5 (Ro20-1724) experiments inhibition was < 50%. The PDE III inhibitor SK&F 94120 was without effect. Spontaneous superoxide generation was reduced 57 +/- 10% by 1 microM prostaglandin E2 (PGE2) and 62.6 +/- 3.76% by 1 microM salbutamol. 3. The increase in superoxide generation elicited by FMLP (10(-9)-10(-5)M) was unaffected by any of the PDE inhibitors studied. Inhibition of FMLP-stimulated superoxide generation by PGE2 was enhanced in the presence of 10 microM IBMX. 4. Macrophages were found to contain a predominantly membrane bound cyclic AMP PDE (90% of total activity) which was unaffected by cyclic GMP or calcium/calmodulin. The cyclic AMP PDE activity in the cytosolic fraction was enhanced in the presence of calcium/calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of cyclic GMP elevation on isoprenaline-induced increase in cyclic AMP and relaxation in rat aortic smooth muscle: role of phosphodiesterase 3.

1. In this study, the effects of a protein synthesis inhibitor, cycloheximide, and a soluble tumour necrosis factor (TNF) binding/IgG fusion protein, p55-sf2, on the priming and challenge stages of the local Shwartzman reaction (LSR) were assessed and compared with their effects on the acute inflammatory response induced by recombinant human tumour necrosis factor-alpha (rhTNF), lipopolysaccharide (LPS) and a reversed passive Arthus (RPA) reaction in rabbit skin. 2. The LSR was induced in skin by giving an intradermal (i.d.) priming injection of LPS followed by two i.v. challenge injections 20 h and 22 h later. Accumulation of 51-Cr-labelled red blood cells and [125I]-albumin were measured at 24 h as markers of haemorrhage and oedema formation, respectively. 3. The RPA reaction was induced in the rabbit by giving i.d. injections of Arthus anti-serum (anti-bovine-gamma-globulin, BGG) followed 5 min later by an i.v. injection of the antigen (BGG). Oedema formation and the accumulation of 111In-labelled neutrophils produced in the RPA reaction and in response to i.d. injection of rhTNF and LPS were measured over the 4 h period after inducing the responses. 4. A single local injection of cycloheximide (10 micrograms/site) did not inhibit neutrophil accumulation or oedema formation produced by 100% Arthus anti-sera. Although LPS injected i.d. induced a marked dose-dependent neutrophil accumulation, there was little associated plasma leakage. Cycloheximide (10 micrograms/site) did not significantly inhibit the neutrophil accumulation induced by LPS (0.1 microgram/site). In the LSR, priming i.d. injections of LPS caused a dose-dependent increase in haemorrhage and plasma leakage at skin sites after challenge with LPS (two injections of 100 micrograms, i.v.). Co-injection of a single dose of cycloheximide (10 micrograms/site) with LPS (30 micrograms/site) caused a marked reduction in the amount of haemorrhage. Local cycloheximide (10 micrograms/site) administered immediately before LSR challenge did not affect the responses produced in the LSR. 5. Neutrophil accumulation induced by TNF (0.17 micrograms/site) was abolished by co-administration of p55-sf2 (3 micrograms/site) whereas neutrophil accumulation induced by i.d. LPS and produced in the RPA reaction was not affected. In the LSR, haemorrhage and oedema formation were inhibited by p55-sf2 (3 micrograms/site) when it was administered i.d. with the LPS priming injection, but not when given i.d. immediately before LSR challenge. 6. These data suggest that the acute neutrophil accumulation produced in the RPA reaction and in response to i.d. LPS may not be dependent on local protein synthesis or TNF production. On the other hand, haemorrhage appears to be dependent on local protein synthesis during the priming phase but not during the challenge stage of the LSR. Importantly, haemorrhage and plasma leakage appear to be dependent on local TNF generation during the priming phase but not during the challenge stage of the LSR. Thus TNF appears to play a key role in the LSR in rabbit skin.     

The presence of five cyclic nucleotide phosphodiesterase isoenzyme activities in bovine tracheal smooth muscle and the functional effects of selective inhibitors.

1. The profile of cyclic nucleotide phosphodiesterase (PDE) isoenzymes and the relaxant effects of isoenzyme selective inhibitors were examined in bovine tracheal smooth muscle. The compounds examined were the non-selective inhibitor 3-isobutyl-1-methylxanthine (IBMX), zaprinast (PDE V selective), milrinone and Org 9935 (4,5-dihydro-6-(5,6-dimethoxy-benzo[b]thien-2-yl)-5-methyl-1 (2H)-pyridazinone; both PDE III selective), rolipram (PDE IV selective) and Org 30029 (N-hydroxy-5,6-dimethoxy-benzo[b]-thiophene-2-carboximidamide HCl a dual PDE III/IV inhibitor). 2. Ion exchange chromatography showed three main peaks of PDE activity. The first peak was stimulated by Ca2+/calmodulin (PDE I), the adenosine 3':5'-cyclic monophosphate (cyclic AMP) hydrolytic activity of the second peak was stimulated by guanosine 3':5'-cyclic monophosphate (cyclic GMP) (PDE II) whilst that of the third peak was not significantly modified by any regulator (PDE IV). Calmodulin affinity chromatography revealed the additional presence of cyclic GMP-specific PDE (PDE V) in the first peak. A clearly distinct peak of cyclic GMP-inhibited PDE (PDE III) was not observed. However, Org 9935 inhibited the third activity peak more effectively in the presence, than in the absence, of rolipram (3 mumol l-1), indicating the presence of PDE III activity. 3. Rolipram was the most potent inhibitor of PDE IV. The mean -log50 IC50 values for rolipram, IBMX, milrinone, Org 30029, Org 9935 and zaprinast were 5.9 +/- 0.1, 4.9 +/- 0.1, 4.7 +/- 0.1, 4.6 +/- 0.1 and 4.6 +/- 0.1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic AMP-mediated regulation of vascular smooth muscle cell cyclic AMP phosphodiesterase activity

1. Rat cultured aortic vascular smooth muscle cells (VSMC) express both cyclic GMP-inhibited cyclic AMP phosphodiesterase (PDE3) and Ro 20-1724-inhibited cyclic AMP phosphodiesterase (PDE4) activities. By utilizing either cilostamide, a PDE3-selective inhibitor, or Ro 20-1724, a PDE4-selective inhibitor, PDE3 and PDE4 activities were shown to account for 15% and 55% of total VSMC cyclic AMP phosphodiesterase (PDE) activity.
2. Treatment of VSMC with either forskolin or 8-bromo-cyclic AMP caused significant concentration- and time-dependent increases in total cellular cyclic AMP PDE activity. Using cilostamide or Ro 20-1724, we demonstrated that both PDE3 and PDE4 activities were increased following forskolin or 8-bromo-cyclic AMP treatment, with a relatively larger effect observed on PDE3 activity. The increase in cyclic AMP PDE activity induced by forskolin or 8-bromo-cyclic AMP was inhibited by actinomycin D or cycloheximide, demonstrating that new mRNA synthesis and protein synthesis were required. An analogue of forskolin which does not activate adenylyl cyclase (1,9-dideoxyforskolin) or an analogue of cyclic GMP (8-bromo-cyclic GMP) did not affect total cyclic AMP PDE activity.
3. Incubation of VSMC with 8-bromo-cyclic AMP for 16 h caused a marked rightward shift in the concentration-response curves for both isoprenaline- and forskolin-mediated activation of adenylyl cyclase. A role for up-regulated cyclic AMP PDE activity in this reduced potency is supported by our observation that cyclic AMP PDE inhibitors (IBMX, cilostamide or Ro 20-1724) partially normalized the effects of isoprenaline or forskolin in treated cells to those in untreated cells.
4. We conclude that VSMC cyclic AMP PDE activity is increased following long-term elevation of cyclic AMP and that increases in PDE3 and PDE4 activities account for more than 70% of this effect. Furthermore, we conclude that increases in cyclic AMP PDE activity contribute to the reduced potency of isoprenaline or forskolin in treated VSMC. These results have implications for long-term use of cyclic AMP PDE inhibitors as therapeutic agents.

     

Characterization and selective inhibition of cyclic nucleotide phosphodiesterase isozymes in canine tracheal smooth muscle

Cyclic nucleotide phosphodiesterases (PDEs) from canine trachealis were characterized with respect to their kinetic properties, sensitivity to selective inhibitors, and subcellular distribution. Extracts from whole tissue homogenates were applied to DEAE-Sepharose anion exchange columns and eluted with a linear sodium acetate gradient. Three major peaks of PDE activity were resolved. The first (PDE I), which eluted at 0.2 M sodium acetate, was applied to a calmodulin (CaM)-Sepharose affinity column and resolved into CaM-insensitive and CaM-sensitive PDEs. The CaM-insensitive isozyme (PDE Ia) had apparent Km values of 135 microM (cAMP) and 4 microM (cGMP) and was potently inhibited by zaprinast (Ki = 0.1 microM). The CaM-sensitive isozyme (PDE Ic) had apparent Km values of 1 microM (cAMP) and 2 microM (cGMP) and was inhibited by zaprinast with an apparent Ki of 35 microM. The second peak of activity (PDE II) from the anion exchange column eluted at 0.3 M sodium acetate and had apparent Km values of 93 microM (cAMP) and 60 microM (cGMP). The enzyme displayed positive cooperativity with respect to the hydrolysis of cAMP (nH = 1.7). Low concentrations of cGMP (0.1-1 microM) reduced cooperativity (nH = 1.1) and increased the hydrolysis of 1 microM cAMP. The third peak of activity from the anion exchange column eluted at 0.6 M sodium acetate and displayed anomalous kinetics that suggested the presence of two isozymes. This was supported by the observation that enzyme activity was only partially inhibited by SK&F 94120 or Ro 20-1724 but was abolished by the combination of the two PDE inhibitors. Subsequent studies confirmed the existence of two isozymes. The first, PDE III, had apparent Km values of 0.3 microM (cAMP) and 8 microM (cGMP) and was inhibited by cGMP (IC50 = 0.1 microM), SK&F 94120 (Ki = 7.8 microM), and SK&F 94836 (Ki = 0.4 microM). The second, PDE IV, had apparent Km values of 4 microM (cAMP) and 40 microM (cGMP) and was inhibited by Ro 20-1724 (Ki = 5.2 microM) and rolipram (Ki = 0.5 microM) but not by cGMP. Assessment of the 100,000 x g soluble and particulate PDE activity revealed that all five isozymes were present in the soluble fraction, but only four isozymes (PDEs Ia, Ic, III, and IV) were present in the particulate fraction. These results indicate that five distinct PDE isozyme exist in canine trachealis and that these isozymes differ in their kinetic characteristics, sensitivity to activators and inhibitors, and subcellular distribution.     

Control of cyclic AMP levels in primary cultures of human tracheal smooth muscle cells.

1. [3H]-adenosine 3':5'-cyclic monophosphate ([3H]-cyclic AMP) responses were studied in primary cultures of human tracheal smooth muscle cells derived from explants of human trachealis muscle and in short term cultures of acutely dissociated trachealis cells. 2. Isoprenaline induced concentration-dependent [3H]-cyclic AMP formation with an EC50 of 0.2 microM. The response to 10 microM isoprenaline reached a maximum after 5-10 min stimulation and remained stable for periods of up to 1 h. After 10 min stimulation, 1 microM isoprenaline produced a 9.5 fold increase over basal [3H]-cyclic AMP levels. The response to isoprenaline was inhibited by ICI 118551 (10 nM), (apparent KA 1.9 x 10(9) M-1) indicating the probable involvement of a beta 2-adrenoceptor in this response in human cultured tracheal smooth muscle cells. However, with 50 nM ICI 118551 there was a reduction in the maximum response to isoprenaline. Prostaglandin E2 also produced concentration-dependent [3H]-cyclic AMP formation (EC50 0.7 microM, response to 1 microM PGE2 6.4 fold over basal). 3. Forskolin (1 nM - 100 microM) induced concentration-dependent [3H]-cyclic AMP formation in these cells. A 1.6 fold (over basal) response was also observed following stimulation with NaF (10 mM). 4. The nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) (0.1 mM) and the type IV, cyclic AMP selective, phosphodiesterase inhibitor rolipram (0.1 mM) both elevated basal [3H]-cyclic AMP levels by 1.8 and 1.5 fold respectively. IBMX (1-100 microM) and low concentrations of rolipram (< 10 microM), also potentiated the response to 1 microM isoprenaline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contribution of phosphodiesterase isoenzymes and cyclic nucleotide efflux to the regulation of cyclic GMP levels in aortic smooth muscle cells.

Involvement of phosphodiesterase isoenzymes (PDEs) in guanosine-3',5'-cyclic monophosphate (cGMP) hydrolysis was analyzed in aortic smooth muscle cells. Four families of PDEs were separated from pig aorta: PDE1 (calcium-calmodulin-activated), PDE3 (cGMP-inhibited), PDE4 (adenosine 3',5'-cyclic monophosphate [cAMP]-specific), and PDE5 (cGMP-specific). Within this PDE complement, PDE1 and PDE5 mostly contributed to the hydrolysis of cGMP both in the presence and absence of calcium-calmodulin. The role of these isoenzymes in cGMP degradation was analyzed in primary cultures of porcine aortic smooth muscle cells after stimulation with sodium nitroprusside (SNP) or atrial natriuretic factor (ANF). Pretreatment with 10 microM zaprinast, a concentration that selectively inhibits PDE5, did not potentiate the SNP- or ANF-induced rise of cGMP, questioning the widespread opinion that only PDE5 accounts for cGMP hydrolysis in this tissue. Further evidence came from experiments assessing the effect of zaprinast or 3-isobutyl-1-methylxanthine at concentrations inhibiting both type 1 and type 5 isoenzymes, in which this potentiation was clearly seen. Contribution of cGMP egression to the control of intracellular cGMP levels after SNP or ANF stimulation was also investigated. Shortly after guanylate cyclase activation, extracellular cGMP levels surpassed intracellular levels. However, comparison of the amounts of cGMP extruded to the extracellular medium with those degraded by PDEs leads to the conclusion that efflux is of relatively minor importance in regulating intracellular cGMP levels. In cells made tolerant to SNP, selective PDE5 inhibition synergistically increased intra- and extracellular cGMP amounts after SNP stimulation. These results indicate a previously undescribed greater relevance of PDE5 after tolerance development in aortic smooth muscle cells.     

Correlation of cyclic AMP accumulation and relaxant actions of salmeterol and salbutamol in bovine tracheal smooth muscle.

1. The ability of salmeterol to stimulate cyclic AMP accumulation and relaxation has been compared with that of salbutamol in bovine tracheal smooth muscle. In addition, the anti-spasmogenic effects of these agents and their abilities to modulate histamine-stimulated [3H]-inositol phosphate accumulation have also been investigated. 2. In tissue strips, a close temporal correlation was found to exist between salmeterol (0.1 microM)-induced relaxation of methacholine (500 nM)-induced tone and cyclic AMP accumulation, both maximal reversal of induced tone (26.2 +/- 6.0%) and maximal levels of cyclic AMP accumulation being achieved after 30-40 min. In contrast to salmeterol, salbutamol exerted greater and more rapid effects on both parameters. Maximal reversal of methacholine-induced tone (79.3 +/- 14.0%) and maximal levels of cyclic AMP accumulation were produced within 5 min. 3. Salmeterol-induced cyclic AMP accumulation (EC50 = 5.3 [1.8 - 15.2] nM) and inhibition of histamine (0.1 mM)-stimulated [3H]-inositol phosphate accumulation (IC50 = 1.4 [0.3-6.3] nM) were both more potent than those induced by salbutamol (EC50 = 169 [99 - 290] nM; IC50 = 13.8 [7.0 - 27.4] nM). However, maximal effects exerted by each of these agents were similar in magnitude. 4. Anti-spasmogenic effects were examined by beta-adrenoceptor agonist application to tissue strips prior to construction of spasmogen concentration-effect curves. Both salmeterol and salbutamol exerted more marked inhibition of the contractile response induced by histamine than that induced by methacholine, salmeterol being the more potent agent, while salbutamol produced a greater maximal inhibitory effect. 5. The results demonstrate that salmeterol is a more potent agent than salbutamol and have highlighted a close temporal correlation between promotion of cyclic AMP accumulation and tissue relaxation stimulated by each agent when both parameters are measured under identical conditions.     

Pulmonary effects of type V cyclic GMP specific phosphodiesterase inhibition in the anaesthetized guinea-pig.

1. We have investigated the bronchodilator potential of type V phosphodiesterase (PDE V) inhibitors in anaesthetized ventilated guinea-pigs using the potent and selective PDE V inhibitor, SK&F 96231. We have compared its activity to that of salbutamol, the PDE III inhibitors, siguazodan and SK&F 95654 and to the PDE IV inhibitor rolipram. 2. Administered as an i.v. infusion SK&F 96231 (0.6 and 1 mg kg-1 min-1, i.v.) caused a slowly developing inhibition of histamine (100 nmol kg-1, i.v.)-induced bronchoconstriction and elevated tracheal cyclic GMP levels in the anaesthetized guinea-pig. SK&F 96231 (0.1 and 0.3 mg kg-1 min-1, i.v.) was without effect on histamine-induced bronchoconstriction. In the presence of a sub-threshold infusion of SNP (0.1 mumol kg-1 min-1, i.v.) there was a marked enhancement of SK&F 96231-induced inhibition of histamine responses such that at infusion rates that were ineffective alone, SK&F 96231 caused a > 50% inhibition of histamine responses. The stimulation of tracheal cyclic GMP accumulation by SK&F 96231 was also potentiated. 3. Administered directly into the airway, SK&F 96231 (300 micrograms in 5 mg lactose carrier) was largely without effect on histamine-induced bronchoconstriction (4.9 +/- 1.9% inhibition). In the presence of SNP (0.1 mumol kg-1 min-1, i.v.) or isosorbide dinitrate (200 micrograms administered by insufflation into the trachea) there was a marked potentiation of the inhibitory activity of SK&F 96231 (40 +/- 4% and 62 +/- 1.8% respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Vascular smooth muscle cell phosphodiesterase (PDE) 3 and PDE4 activities and levels are regulated by cyclic AMP in vivo

Prolonged incubation of several cell types, including cultured vascular smooth muscle cells (VSMC), with cyclic AMP-elevating agents increases cAMP phosphodiesterase (PDE) activity and levels. In this work, we describe for the first time an increase in arterial VSMC cAMP PDE activity and levels caused by cAMP-elevating agents when these agents are administered to rats in vivo. Injections of rats with dibutyryl cAMP (dbcAMP) or forskolin increased both PDE3 and PDE4 activities in aortic and femoral artery VSMC. Consistent with the idea that cAMP-elevating agents increased PDE3 and PDE4 activities by acting directly on VSMC, local delivery of dbcAMP or forskolin to femoral arteries using a pluronic gel-based approach increased femoral artery VSMC PDE3 and PDE4 activities to levels similar to those observed after injection of these agents. Consistent with a role for de novo mRNA and protein synthesis in the cAMP-elevating agent induced increase in PDE3 and PDE4, 1) systemic administration of forskolin increased PDE3A, PDE3B, and PDE4D mRNA levels in aortic VSMC and femoral artery VSMC, 2) local delivery of dbcAMP increased PDE3A, PDE3B, and PDE4D3 protein levels in femoral artery VSMC, and 3) local administration of either actinomycin D or cycloheximide attenuated the effect of dbcAMP. In addition, our results indicate that the PDE3 and PDE4 variants increased by cAMP-elevating agents in arterial VSMC in situ were distinct from those elevated by these agents in cultured arterial VSMC. Consistent with the effect of increased VSMC cAMP PDE on blood vessel function, inhibition of PDE3 and PDE4 activities potentiated the relaxant effect of forskolin in dbcAMP-treated femoral artery rings to a greater extent than in untreated control blood vessels. We propose that our findings are consistent with the concept that cAMP regulates VSMC cAMP PDE activity and levels in vivo and that VSMC phenotype influences the choice of cAMP PDE variant that is elevated. Our findings are discussed in the context that agents aimed at specific PDE3 or PDE4 variants could perhaps allow greater control of cAMP-mediated regulation of VSMC behaviors that are phenotype-dependent.     

Cromakalim-induced membrane current in guinea-pig tracheal smooth muscle cells

The characteristics of the cromakalim-induced membrane current were examined in single tracheal myocytes of the guinea-pig under voltage-clamp conditions. When K(+) concentrations in the pipette and bathing solutions were approximately 140 mM, cromakalim activated a membrane current (I(crom)) which was inward at -60 mV and reversed at -2 mV. I(crom) was blocked by 10 microM glibenclamide and potentiated when the ATP concentration in the pipette solution was decreased. The K(d) and Hill coefficient of glibenclamide for I(crom) block were 200 nM and 1.05, respectively. Application of the tyrosine kinase inhibitors, genistein and alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamid (ST638), reduced I(crom) in a concentration-dependent manner. Daidzein, which does not inhibit tyrosine kinase, was about 10 times less effective than genistein. Herbimycin A had no effect on I(crom). Internal application of these inhibitors from the pipette did not affect I(crom). In conclusion, cromakalim is a potent activator of the ATP-sensitive K(+) channel (K(ATP) channel) in guinea-pig tracheal myocytes. The inhibition of I(crom) by genistein and ST638 may be due to the direct block of the channel from outside.