Differential modulation of tissue function and therapeutic potential of selective inhibitors of cyclic nucleotide phosphodiesterase isoenzymes.

Since the discovery of cyclic nucleotide phosphodiesterase 30 years ago, there have been major advances in our knowledge of this group of isoenzymes. Five families, each composed of several isoforms and having differing tissue distributions, have been described. David Nicholson and colleagues compare the tissue distribution of phosphodiesterase isoenzymes and discuss the differential effects of inhibition of particular isoenzymes, with differing subcellular localization, on tissue function. They also review the potential use of isoenzyme selective phosphodiesterase inhibitors in a range of clinical disorders such as heart failure, asthma, depression and dementia.

Effects of selective phosphodiesterase inhibition on cyclic AMP hydrolysis in rat cerebral cortical slices.

1. The effects of selective inhibition of phosphodiesterase activities on the concentration and rate of hydrolysis of adenosine 3':5' cyclic-monophosphate (cyclic AMP) in rat cerebral cortical slices has been studied. 2. Isoprenaline caused a rapid, concentration-dependent increase in cyclic AMP concentration to new steady-state levels (basal: 7.1 +/- 0.7; 10 microM isoprenaline: 14.3 +/- 1.4 pmol mg-1 protein). Addition of a beta-adrenoceptor antagonist to isoprenaline-stimulated cerebral cortical slices caused a rapid decrease in cyclic AMP concentration to basal levels (t1/2: 58 +/- 18 s). 3. Preincubation of slices for 30 min with the phosphodiesterase inhibitors 1-methyl-3-isobutylxanthine, denbufylline, rolipram or Ro20,1724 caused concentration-dependent increases in basal and isoprenaline-stimulated cyclic AMP concentrations and decreased the rate of cyclic AMP hydrolysis measured after addition of a beta-adrenoceptor antagonist. However, SKF 94120 and zaprinast had none of these effects. 4. The results are discussed with respect to previous studies of phosphodiesterase isozymic activities isolated from cerebrum and it is suggested that the Ca2+/calmodulin-independent, low Km cyclic AMP phosphodiesterase isozyme, which is selectively inhibited by denbufylline, rolipram and Ro20,1724, and is present in cerebrum is of critical importance to the regulation of cyclic AMP concentration in this tissue.

Dissociation constants of isoprenaline and orciprenaline and their relative efficacies on guinea-pig isolated atria determined by use of an irreversible beta-adrenoceptor antagonist.

1. Dissociation constant (KA) of isoprenaline and orciprenaline were determined for the positive inotropic and chronotropic responses of guinea-pig isolated atria. Cumulative dose-response curves to the agonists were constructed before and after incubation with and washout of the irreversible beta-adrenoreceptor antagonist, Ro 03-7894 1-(5-chloracetylaminobenzfuran-2-yl)-2-isopropylaminoethanol). 2. After 20 min washout, the curves were displaced to the right with depression of the maxima. After 3 h washout, there was only depression of the maxima. 3. Dissociation constants were determined by plotting reciprocals of molar concentrations before Ro 03-7894 (1/A) against reciprocals of the equiactive concentrations after Ro 03-7894 (1/A'). KA = (slope - 1)/ intercept. 4. Isoprenaline had a greater affinity (KA) than orciprenaline on both rate and tension. The affinity for rate and tension was identical for both agonists, indicating that the beta-adrenoceptors were identical. 5. Isoprenaline and orciprenaline produced identical rate response maxima when compared in the same preparation but the orciprenaline tension maximum was only 92.0 +/- 0.3% that of isoprenaline. 6. These dose-response curves were replotted as response against -log RA/Rt (fraction of receptors occupied) for each agonist concentration, calculated from the equation RA/Rt = (A)/(KA + (A)). The antilogarithm of the distance along the -log RA/Rt axis gave the efficacy of orciprenaline relative to isoprenaline. It had a greater efficacy (2.24) for rate but a lower efficacy for tension responses (0.5).

Functional antagonism as a means of determining dissociation constants and relative efficacies of sympathomimetic amines in guinea-pig isolated atria.

1 The positive inotropic and chronotropic responses to sympathomimetic amines were examined in guinea-pig isolated atria. 2 The order of potency measured from EC50 values was isoprenaline greater than orciprenaline greater than salbutamol greater than or equal to fenoterol greater than terbutaline. Terbutaline and salbutamol were partial agonists on rate and together with orciprenaline and fenoterol also on tension responses. 3 Functional antagonism by carbachol caused a rightwards shift of the dose-response curve and depression of the maximum response. The rate maxima for orciprenaline, fenoterol and terbutaline were above that of isoprenaline. All the tension maxima were below isoprenaline. 4 Dissociation constants (KA) and relative efficacies (er) were determined by analogy with irreversible antagonism. 5 The relative orders of affinity (KA) were isoprenaline greater than orciprenaline greater than fenoterol greater than salbutamol greater than terbutaline. Affinities were identical on rate and tension. 6 The relative efficacies were all greater than isoprenaline for rate responses. On tension they were the same or less than isoprenaline. 7 The implications of these results are discussed, in particular the fact that a partial agonist has a greater efficacy than a full agonist.

The ability of denbufylline to inhibit cyclic nucleotide phosphodiesterase and its affinity for adenosine receptors and the adenosine re-uptake site.

1. Denbufylline has been examined for its ability to inhibit cyclic nucleotide phosphodiesterase isoenzymes from rat cardiac ventricle and cerebrum, as well as for its affinity for adenosine A1 and A2 receptors and the re-uptake site. For comparison, SK&F 94120, theophylline and 3-isobutyl-1-methyl-xanthine (IBMX) were examined as phosphodiesterase inhibitors whilst N6-cyclohexyladenosine, R(-)-N6-(2-phenylisopropyl)-adenosine, 5'-N-ethylcarboxamido-adenosine, 2-nitrobenzylthioinosine, theophylline and IBMX were examined for their affinity for adenosine binding sites. 2. This investigation confirmed the presence of four phosphodiesterase activities in rat cardiac ventricle; in rat cerebrum only three were present. 3. Denbufylline selective inhibited one form of Ca2+-independent, low Km cyclic AMP phosphodiesterase. The form inhibited was one of two present in cardiac ventricle and the sole one in cerebrum. This form was not inhibited by cyclic GMP. The inotropic agent SK&F 94120 selectively inhibited the form of cyclic AMP phosphodiesterase which was inhibited by cyclic GMP present in cardiac ventricle. Theophylline and IBMX were relatively non-selective phosphodiesterase inhibitors. 4. Denbufylline was a less potent inhibitor of ligand binding to adenosine receptors than of cyclic AMP phosphodiesterase. This contrasted with theophylline, which had a higher affinity for adenosine receptors, and IBMX which showed no marked selectivity. Denbufylline, theophylline and IBMX all had a low affinity for the adenosine re-uptake site. 5. Denbufylline is being developed as an agent for the therapy of multi-infarct dementia. The selective inhibition of a particular low Km cyclic AMP phosphodiesterase may account for the activity of this compound.

Modulation of spasmogen-stimulated Ins(1,4,5)P3 generation and functional responses by selective inhibitors of types 3 and 4 phosphodiesterase in airways smooth muscle.

1. The effects of isoenzyme-selective inhibitors of phosphodiesterases PDE3 and PDE4 on cyclic AMP concentration, two indices of phosphoinositide hydrolysis, and contractile responses to spasmogens have been investigated in bovine tracheal smooth muscle (BTSM). 2. Neither the PDE3-selective inhibitor ORG 9935, nor the PDE4-selective inhibitor rolipram increased cyclic AMP levels in BTSM. However, rolipram addition in the presence of PDE3 inhibition (ORG 9935; 1 microM) concentration-dependently (-log EC50 (M), 6.55+/-0.15; n = 3) increased cyclic AMP levels to about 70% of the maximal response to the beta-adrenoceptor agonist isoprenaline. 3. Rolipram per se inhibited histamine-stimulated [3H]-inositol (poly)phosphate ([3H]-InsP(X)) accumulation by > 80% (-log EC50 (M), 6.92+/-0.11; n = 3). Although ORG 9935 (1 microM) had little effect on histamine-stimulated [3H]-InsP(X) accumulation alone it greatly facilitated the inhibitory action of rolipram (-log EC50 (M), 8.82+/-0.39; n = 3). The effects of PDE3 and/or PDE4 inhibition on [3H]-InsP(X) accumulation stimulated by muscarinic acetylcholine (mACh) receptor activation were less marked. However, combined PDE3/4 inhibition significantly decreased this response at a submaximal concentration of mACh receptor agonist (carbachol; 1 microM). 4. The greater-than-additive effect of combined PDE3/4 inhibition was also observed at the level of contractile responses to histamine and carbachol. In experiments designed to investigate the effects of PDE3 and/or 4 inhibitors on the carbachol-mediated phasic contraction, additions of rolipram (10 microM) or ORG 9935 (1 microM) were without effect, whereas added together the inhibitors caused a significant (P < 0.01) 40% reduction in the peak phasic contractile response. 5. The effect on contraction correlated with a substantial inhibitory effect of PDE3/4 inhibition on the initial increase in inositol 1,4,5-trisphosphate (InsP3) accumulation stimulated by spasmogen. Thus, in the presence of ORG 9935 (1 microM) rolipram concentration-dependently inhibited carbachol-stimulated InsP3 accumulation by > or = 50% (-log EC50 (M), 6.77+/-0.21; n = 4). 6. Carbachol (100 microM) addition caused a rapid decrease (by 67% at 10 s) in BTSM cyclic AMP level in the presence of PDE3/4 inhibition. However, omission of Ca2+ from the incubation medium prevented the carbachol-evoked decrease in cyclic AMP and this coincided with a greater inhibition (> or = 80%) of the carbachol-stimulated InsP3 response. 7. These data indicate that combined PDE3 and PDE4 inhibition has greater-than-additive effects on second messenger and functional responses to spasmogens in BTSM. Furthermore, the ability of PDE3/4 inhibition significantly to attenuate mACh receptor-mediated contractile responses, may be, at least in part, attributed to an effect exerted at the level of InsP3 generation.

Multiple sigma binding sites in guinea-pig and rat brain membranes: G-protein interactions.

1. Evidence is accumulating for multiple sigma (sigma) sites in the mammalian CNS. 2. We have addressed this problem and have examined sigma site - G-protein coupling in guinea-pig and rat brain membranes. 3. Ditolylorthoguanidine (DTG), (+)-3-(3-hydroxyphenyl)-N-1-(propyl)piperidine (3PPP) and dextromethorphan displaced [3H]-DTG (3.4 nM) with low Hill slopes of 0.5, 0.6 and 0.6, respectively in guinea-pig brain membranes. 4. In the presence of 5'-guanylylimidodiphosphate (Gpp(NH)p; 100 microM), the specific binding of [3H]-DTG was reduced by 36.7%, the Hill slope of 3PPP was increased to near unity, the ability of dextromethorphan to displace DTG was virtually abolished and the Hill slope for DTG remained low (0.7), indicating the presence of at least two binding sites. These data indicate that although Gpp(NH)p removes a dextromethorphan high affinity site, two DTG selective sites remain in the presence of Gpp(NH)p. 5. The present study suggests that DTG binds to at least three sites in guinea-pig brain membranes, at least one of which is G-protein linked. 6. In rat brain membranes, DTG displaced itself (3.4 nM) with a Hill slope near 1. 3PPP displacement of [3H]-DTG was comparable with the guinea-pig (Hill slope 0.5) and displaced from more than 1 site. Dextromethorphan did not displace [3H]-DTG at concentrations below 10 microM. 7. The heterogeneity of sigma sites appears to be less in rat than in guinea-pig brain membranes.

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. 4. Rolipram was a potent relaxant of both histamine (1 pumol -') and methacholine (0.03 pmol -') precontracted preparations; (pD2 values; histamine 7.1 +/- 0.1, methacholine 6.8 /-+ 0.2 and 4.5 +/- 0.1, biphasic relaxation). IBMX also relaxed all preparations (pD2 values; histamine 5.6 +/- 0.1, methacholine 5.6 +/- 0.1) whilst zaprinast (pD2 values; histamine 5.2 +/- 0.1, methacholine 4.4 +/- 0.3), milrinone (pD2 values; histamine 5.2 + 0.1, methacholine 4.3 + 0.3) and Org 9935 (pD2 values; histamine 4.1 + 0.1, methacholine 4.1 +/- 0.2) did not completely relax preparations at concentrations up to 100 pImol I-. Org 30029 (pD2 values; histamine 6.2 +/- 0.1, methacholine 5.4 +/- 0.1) was a more effective relaxant than can be explained on the basis of PDE IV inhibition alone.5. We conclude that bovine tracheal smooth muscle contains five distinct PDE isoenzymes. PDE IV appears to be more important in the modulation of tissue function than PDE III and PDE V.

Human bronchial cyclic nucleotide phosphodiesterase isoenzymes: biochemical and pharmacological analysis using selective inhibitors.

1 The aims of the present study were to characterize the cyclic nucleotide phosphodiesterase (PDE) isoenzyme activities present in human bronchi and to examine the ability of selective isoenzyme inhibitors to relax histamine and methacholine precontracted preparations of human bronchi. 2 Three separations of pooled human bronchial tissue samples were performed. Ion-exchange chromatography showed that the soluble fraction of human bronchial preparations contains PDE I, II, III, IV and V isoenzyme activities. Multiple forms of PDE I and PDE IV were observed and PDE IV was the main cyclic AMP hydrolytic activity. 3 3-Isobutyl-l-methylxanthine (IBMX) non-selectively inhibited all separated isoenzyme activities. Zaprinast selectively inhibited PDE V, but also effectively inhibited one of the two PDE I isoforms identified. The PDE IV selective inhibitors rolipram and RO-201724, inhibited the PDE IV activities as did the dual PDE III/IV inhibitor, Org 30029. Org 9935, a PDE III selective inhibitor, potently attenuated part of the PDE IV activity peak in one of three separations performed, indicating that some PDE III activity may co-elute with PDE IV under the experimental conditions employed. 4 PDE IV-selective (rolipram), PDE III-selective (Org 9935) and dual PDE III/IV (Org 30029) inhibitors were effective relaxants of human bronchial smooth muscle. The PDE V/PDE I inhibitor, zaprinast was relatively ineffective. 5 The present study demonstrates in human bronchi, as in animal airways smooth muscle, that inhibitors of PDE III, PDEIV and dual PDE III/IV have potentially useful bronchodilator activity and are worthy of further consideration as anti-asthma drugs.

Pharmacology of nootropics and metabolically active compounds in relation to their use in dementia.

The development of effective drugs for the treatment of dementia is an important therapeutic target. Drugs which stop the progression of dementia have not been developed; however, nootropics and metabolically active compounds such as the vinca alkaloids and the ergot alkaloids as well as alkylxanthines are widely used to alleviate the symptoms. This review summarises animal studies investigating the mechanism of action of these compounds and highlights gaps in our knowledge of their pharmacology. Nootropics, such as piracetam, facilitate learning and retrieval of information and protect the brain from physical and chemical intoxication. Nootropics may produce these effects via an enhancement of acetylcholine or dopamine release; however, this postulate requires further evaluation. The pharmacology of vinca alkaloids is reviewed with particular reference to vinpocetine. This compound attenuates cognitive deficits, reduces ischaemia-induced hippocampal cell loss and increases cerebral blood flow and glucose utilisation. These effects may be induced by modulation of cyclic nucleotide levels and adenosine re-uptake inhibition. An extensively examined ergot alkaloid is co-dergocrine; this compound increases both the oxygen tension and the electrical activity of the ischaemic cerebral cortex. Alkylxanthines have a wide range of pharmacological activities, and in this review the pharmacology of pentoxifylline, propentofylline and denbufylline is contrasted with that of theophylline and caffeine. In particular, the pharmacology of propentofylline and the selective low Km cyclic AMP phosphodiesterase inhibitor denbufylline is summarised. Although more carefully controlled clinical trials in well defined patient collectives are required, present evidence suggests some therapeutic efficacy for nootropics and metabolically active compounds. Further studies to more closely evaluate their mechanism of action may lead to the development of more effective agents for the therapy of dementia.

Irreversible beta-adrenoceptor blockade of atrial rate and tension responses.

The competitive reversible beta-adrenoceptor antagonist activity of Ro 03-5255 [1-(5-acetylaminobenzfuran-2-yl)-2-isopropylaminoethanol] upon isoprenaline-induced increases of the rate and tension of guinea-pig isolated atria is described. The chlorinated derivative [Ro 03-7894; 1-[5-chloracetylaminobenzfuran-2-yl)-2-isopropyl-aminoethanol] in contrast exhibited concentration-dependent non-competitive irreversible blocking activity as measured by depression of the maximum responses which were not restored by a washout period that successfully reversed Ro 03-5255. When orciprenaline was used as a weak agonist of low efficacy, the maximum responses were depressed to a greater extent. The blockade by Ro 03-7894 was relatively specific for beta-adrenoceptors since it did not antagonize histamine or calcium chloride. The depression of the maximum responses to orciprenaline was reduced by the presence of sodium thiosulphate. Sodium thiosulphate was ineffective in reversing an established blockade. The blockade by Ro 03-7894 was therefore assumed to involve irreversible binding to the beta-adrenoceptor after conversion to an appropriate electrophilic ligand. The significance of this is discussed.

The lack of utility of the rat vas deferens as a functional bioassay for sigma ligands.

The present study examined the utility of the rat vas deferens preparation as a bioassay for sigma site ligands. sigma Ligands such as (+/-)-pentazocine, phencyclidine (PCP) and (+)-SK&F 10047 potentiated neurogenic twitch contractions. However, neither the order of potency nor the absolute potency of (+/-)-pentazocine and (+)-SK&F 10047 correlated with their affinity at central sigma sites. Furthermore, another potent sigma ligand, ditolyl-ortho guanidine (DTG) neither affected neurogenic twitch contractions nor inhibited twitch potentiation by PCP or (+)-SK&F 10047 at concentrations up to 30 mumol/l. These data indicate that the rat vas deferens is not a useful bioassay for the evaluation of sigma ligands. PCP, (+)-SK&F 10047 and (+/-)-pentazocine probably enhance neurogenic contractions in rat vas deferens primarily by inhibition of the neuronal uptake of noradrenaline.

The sigma ligand 1,3-di-o-tolylguanidine depresses amino acid-induced excitation non-selectively in rat brain.

The sigma ligand 1,3-di-o-tolylguanidine (DTG) has been applied by microiontophoresis to neurones in the rat hippocampal slice and to neurones in the neocortex and hippocampus of rats anaesthetised with urethane. DTG depressed the excitatory responses of cells to both N-methyl-D-aspartate (NMDA) and quisqualate on a majority of the units tested, in no case causing an enhancement. Haloperidol had no consistent effect of its own and did not prevent the depressant effects of DTG. It is concluded that in the preparations used, DTG did not selectively modify neuronal sensitivity to NMDA.

Comparison of the effects of cromakalim, a potassium conductance enhancer, and nimodipine, a calcium antagonist, on 5-hydroxytryptamine responses in a variety of vascular smooth muscle preparations.

We have examined the effects of the potassium conductance enhancer cromakalim (BRL34915) and the calcium entry blocker nimodipine upon 5-hydroxytryptamine (5-HT) induced contractions in ring preparations from rabbit basilar and mesenteric arteries, and from pig coronary arteries. Cumulative concentration-response (CR) curves to 5-HT were biphasic in basilar and mesenteric arteries, and monophasic in coronary arteries. Coronary artery 5-HT CR curves and the first component of the mesenteric artery 5-HT CR curve were antagonized by ketanserin (pKB values 8.9 and 8.8, respectively), whereas basilar artery CR curves were not. Prazosin antagonized the second component of the mesenteric 5-HT CR curve, but not that of the basilar artery. Cromakalim (0.1-10 mumols/l) and nimodipine (0.001-1 mumol/l) both caused reductions in resting tension in basilar and coronary arteries denuded of their endothelia, but this effect was not seen with mesenteric arteries. Procaine (5 mmol/l) abolished this vasorelaxant effect of cromakalim in basilar artery. Both agents concentration-dependently depressed the 5-HT CR curve in coronary artery, the effect of cromakalim was antagonized by lidocaine (100 mumols/l). In basilar artery, only the first component was cromakalim sensitive unlike nimodipine which depressed both components of the CR curve. In mesenteric artery, 5-HT CR curves were depressed by cromakalim, but only slightly affected by nimodipine (1 mumol/l). It is concluded that cromakalim, like nimodipine, possesses anti-vasospastic activity; however, differences exist in the sensitivity of the 5-HT mediated contractions of the three arterial preparations to the agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement of muscle blood cell flux and pO2 by cromakalim (BRL 34915) and other compounds enhancing membrane K+ conductance, but not by Ca2+ antagonists or hydralazine, in an animal model of occlusive arterial disease.

The effect of Ca2+ antagonists, hydralazine and agents which enhance membrane K+ conductance (cromakalim, pinacidil and nicorandil) in smooth muscle cells, was compared on normal and hypoxic skeletal muscle blood cell flux and pO2. The K+ conductance enhancers and verapamil, diltiazem and nifedipine increased blood cell flux in normally perfused muscle. At equieffective blood pressure lowering dosages, the Ca2+ antagonists produced greater increases than the K+ channel openers. Hydralazine did not elevate blood cell flux in the normal muscle. In hypoxic skeletal muscle, the K+ conductance enhancers produced a marked increase in blood cell flux and in tissue oxygen tension, indicating that they had increased the nutritive blood flow in the muscle. The Ca2+ antagonists and hydralazine either did not change hypoxic muscle blood cell flux and pO2 or reduced them. The dissimilarity in the activity of the compounds may be due to differences in their site of action in the vascular bed. Ca2+ antagonists and hydralazine are known to reduce arteriolar vessel resistance and do not increase blood flow in hypoxic skeletal muscle. The positive effect of cromakalim, pinacidil and nicorandil may be due to relaxant activity on larger arterial blood vessels including collaterals. This effect could be related to their ability to enhance membrane K+ conductance in vascular smooth muscle cells.

The effect of denbufylline on the viscosity of rat whole blood and on the deformability (filterability) of rat blood cell suspensions.

The novel alkylxanthine, denbufylline [1,3-di-n-butyl-7-(2-oxopropyl)-xanthine] has been examined, in vitro, for effects on the viscosity of rat whole blood and on the filterability of rat blood cell suspensions. For comparison, pentoxifylline was also examined for rheological activity. Denbufylline reduced the viscosity of whole blood at all shear rates utilised, up to 128.5 s-1. The effect was, however, more pronounced at a low (0.7 s-1) than at a high (94.5 s-1) shear rate indicating that the compound reduces blood cell aggregation and increases blood cell deformability. Denbufylline also increased the filterability of blood cell suspensions. This is further evidence that the compound increases the deformability of blood cells. Denbufylline elevated the filterability of both pure erythrocyte and mixed erythrocyte/leucocyte suspensions, the effect being greatest with the latter. This suggests that denbufylline may influence the deformability of both red and white blood cells. However, the effect on white blood cells, under the experimental conditions employed, is apparently more marked. Pentoxifylline also reduced the viscosity of rat whole blood and increased the filterability of rat blood cell suspensions. However, denbufylline was 10-100-fold more potent in these tests than pentoxifylline.

Comparison of cyclic nucleotide phosphodiesterase isoenzymes in rat and rabbit ventricular myocardium: positive inotropic and phosphodiesterase inhibitory effects of Org 30029, milrinone and rolipram.

The species dependent variation in the cardiotonic activity of selective cyclic nucleotide phosphodiesterase (PDE) isoenzyme inhibitors was examined by comparing the inotropic and PDE inhibitory effects of Org 30029 (N-hydroxy-5,6-dimethoxy-benzo[b]thiophene-2-carboximidamide HCl), 3-isobutyl-1-methyl-xanthine (IBMX), milrinone and rolipram in rat and rabbit ventricular myocardium. The relative activities of PDE isoenzymes in rat and rabbit cardiac ventricle were also examined to assess the role of the different PDE subtypes in modulating contractile force in the two species. In rabbit papillary muscles, IBMX, Org 30029 and milrinone increased contractile force whilst rolipram was inactive. The rank order of potency of the active compounds was Org 30029 greater than IBMX greater than milrinone. Only Org 30029 and IBMX produced significant positive inotropic responses in rat papillary muscles, milrinone and rolipram being inactive. However, large positive inotropic responses were obtained in rat papillary muscles when milrinone and rolipram were tested in combination. In rabbit papillary muscles, the positive inotropic action of milrinone was markedly potentiated by rolipram. Four main types of PDE (I, II, III, IV) isoenzymes were resolved, by DEAE-sepharose or Mono-Q ion-exchange chromatography, from both rat and rabbit cardiac ventricular tissue. In rabbit, Ca2+/calmodulin dependent PDE (PDE I) and cyclic GMP inhibited PDE (PDE III) were the dominant cAMP activities. In contrast, cyclic GMP stimulated PDE (PDE II), PDE III and cGMP insensitive PDE (PDE IV) represented the main cAMP activities in rat cardiac ventricle. The inhibitory effects of Org 30029, IBMX, milrinone and rolipram on PDE isoenzymes from rat and rabbit cardiac ventricle were essentially similar.(ABSTRACT TRUNCATED AT 250 WORDS)

The analysis and assay of cyclic nucleotide phosphodiesterase isoenzyme activity.

Greater knowledge over the past decade on the biochemical properties, as well as the identification of specific pharmacological tools has led to a marked improvement in the methods employed for the analysis and assay of PDE isoenzymes. A major message has been the marked species and tissue-dependent variation in the distribution of the various isoenzymes and their subtypes. This has great implications not only in terms of extrapolating animal data to the human situation, but also from one tissue to another. Thus, it is critical, in particular for drug discovery efforts, to characterize human PDEs in the relevant tissue. Molecular cloning is probably the best and most direct route to achieve this objective since access to disease-free human tissue is heavily limited. Alternatively, well-characterized animal tissues showing PDE and pharmacological profiles similar to human tissues may be utilized. The methods described in this chapter have been successfully applied to study to the biochemistry and pharmacology of PDEs in both animal and human tissues, and in the discovery of novel selective inhibitors for these proteins.

Species-dependent differences in the properties of particulate cyclic nucleotide phosphodiesterase from rat and rabbit ventricular myocardium.

The ability of cyclic nucleotide phosphodiesterases (PDEs) to hydrolyse cyclic (c)AMP in rat and rabbit ventricular myocardium has been compared. The PDE activity of rabbit, but not rat, cardiac homogenate and supernatant fraction was potentiated by Ca2+/calmodulin and attenuated by cGMP. Both rabbit and rat ventricular myocardium were shown to have a membrane bound PDE. However, rabbit membrane-bound PDE was inhibited by cGMP and low concentrations of milrinone (IC50 2.7 microM). In contrast, rat membrane-bound PDE was not inhibited by either cGMP or low concentrations of milrinone (IC50 19 microM), but it was potently inhibited by rolipram (IC50 2.2 microM). Thus, in rabbit the particulate PDE is milrinone sensitive (PDE III) whilst in rat it is the rolipram sensitive (PDE IV) isoenzyme. There are clearly species differences in the intracellular localization and relative activities of PDE isoenzymes in cardiac tissue. This may explain the species differences already found in the activity of selective PDE isoenzyme inhibitors as inotropic agents.