Effect of the selective phosphodiesterase type 5 inhibitor sildenafil on erectile dysfunction in the anesthetized dog

The role of the sensitivity of bone marrow cells to, and the pharmacokinetics of granulocyte colony-stimulating factor (G-CSF) on the rhythm of leukocyte-increasing effect was investigated in ICR male mice housed under a standardized light-dark cycle (lights on at 0700, off at 1900). A significant circadian rhythm was demonstrated for leukocyte counts at 24 hr after G-CSF (250 microg/kg, s.c.) injection at six different circadian times (P < .01). The leukocyte counts of mice given G-CSF at 0500, 0900, 1300 or 1700 were significantly higher than those of mice given G-CSF at 2100 (P < .01, respectively). The rhythmic pattern resembled overall the rhythm occurring after saline injection. In the comparison between leukocyte counts after G-CSF injection at 0700 and 1900, the time when leukocyte counts are equal in nondrugged state, the leukocyte counts at 24 hr after G-CSF (250 microg/kg, i.v.) injection were approximately 50% higher in mice injected with the drug at 0700 than at 1900 (P < .01). Bone marrow cultures obtained at two times of day resulted in different numbers of myeloid colonies even when treated with the same concentrations of G-CSF in vitro. The colony-forming activity of G-CSF was significantly more potent at 0700 than at 1900 (P < .01). The plasma G-CSF concentrations after G-CSF (250 or 5 microg/kg, i.v.) injection were significantly higher in mice receiving injections with the drug at 0700 than at 1900 (P < .05, respectively). The area under the curve and mean residence time were significantly larger in mice injected with the drug at 0700 than at 1900 (P < .01, P < .05, respectively). Our suggests that the rhythm of G-CSF activity is caused by that of the sensitivity of bone marrow cells to, and the pharmacokinetics of the drug.     

Cicaprost and the type IV phosphodiesterase inhibitor, rolipram, synergize in suppression of tumor necrosis factor-alpha synthesis

Suppression of tumor necrosis factor-alpha (TNF) synthesis is one major target in pharmacological immunomodulation. We now showed the synergistic suppressive effect of the specific type IV phosphodiesterase inhibitor, rolipram, and of the stable prostacyclin analogue, cicaprost, on TNF synthesis. This effect was seen with lipopolysaccharide and Staphylococcus epidermidis as stimuli in human peripheral blood mononuclear cells and in whole blood. Lipopolysaccharide-induced TNF synthesis by mononuclear cells decreased from 3.4 ng/ml to 1.5 ng/ml in the presence of 100 nM rolipram and to 0.7 ng/ml in the presence of 10 nM cicaprost. The combination of both agents suppressed TNF synthesis more than 10-fold, to 0.3 ng/ml. Synergistic suppression was also demonstrated for TNF mRNA.     

Inhibition of eotaxin-mediated human eosinophil activation and migration by the selective cyclic nucleotide phosphodiesterase type 4 inhibitor rolipram

Epidemiological studies suggest that moderate consumption of alcoholic beverages may be beneficial for bone in postmenopausal women. To investigate prospectively these uncontrolled observations, female rats were divided in four groups of 10 animals each and treated with 1) ovariectomy (OVX) and 2.5% ethanol diet (OVX-ETOH group), 2) OVX and control diet (OVX-C group), 3) sham surgery and 2.5% ethanol diet (SHAM-ETOH group), or 3) sham surgery and control diet (SHAM-C group). Three weeks after surgery, bone histomorphometry revealed that the OVX-C group, as expected, had lower trabecular bone volume and higher parameters of bone formation and resorption than the SHAM-C group (p < 0.01). Intake of ethanol did not change these parameters in the SHAM rats, but in the OVX rats it was associated with sharp reduction in parameters of bone resorption (p < 0.01) without a concomitant effect on parameters of bone formation. The cytokines are believed to contribute to accelerated bone resorption during the early postmenopausal period. Indeed, the peripheral blood monocytic cells (PBMC) from the OVX-C rats produced higher amounts of TNF-alpha than the PBMC from the SHAM-C rats (p < 0.05) and administration of ethanol prevented this increase in OVX rats but had no effect in SHAM rats. In summary, short-term intake of moderate doses of ethanol was associated with markedly different effects in rats with and without ovarian function. Although ethanol had no significant effect on the bone tissue and TNF-alpha production of the SHAM rats, it was associated with markedly lower parameters of bone resorption and less TNF-alpha production in the OVX animals. This suggests that exposure to low-dose ethanol may protect from osteopenia following cessation of ovarian function.     

Identification, characterization, and functional role of phosphodiesterase type IV in cerebral vessels: effects of selective phosphodiesterase inhibitors

Research on families of people with severe mental illness has identified significant caregiving burden. Although professional interventions generally assume that families have a major role in the client's progress, the focus of the consumer movement on recovery through empowerment, peer supports, and consumer-run services may have important, as yet unquantified, effects. Keeping in mind the functional heterogeneity of individuals with mental illness, it is suggested that consumer emphasis on autonomy may provide the major relief for family burden.     

Effect of DMPPO, a phosphodiesterase type 5 inhibitor, on hypoxic pulmonary hypertension in rats

PURPOSE: The study was done to improve quantification of multiple arterial stenoses and to investigate a new imaging technique for lower limb arteries. Three-dimensional power Doppler angiography was used to quantify in vitro arterial stenoses. METHODS: We built two types of artery phantoms containing multiple stenoses. One used stenotic porcine arteries, and the other was designed to control the proximal and distal stenoses while we assessed central stenosis of a wall-less agar lumen. Three-dimensional power Doppler angiograms of the flow lumens were generated at different flow rates under steady and pulsatile flow conditions with a PowerPC 8500 computer-based three-dimensional ultrasound imaging system. This experimental system works off-line, performs three-dimensional acquisition, reconstruction, and display of ultrasound images. Images of flow lumens were compared with the measured B-mode images or the true geometry. RESULTS: This technique produces good three-dimensional angiographic images of the flow lumen, and multiple stenoses do not affect the diagnosis of arterial stenoses. With this technique, the average errors for estimating 80% and 50% area reduction stenoses were -10% and 4%, respectively. CONCLUSIONS: Three-dimensional power Doppler angiography has the potential to quantitatively grade multisegmental stenoses in lower limbs and generate a map for vasculature surgery planning.     

Effects of RP 73401, a novel, potent and selective phosphodiesterase type 4 inhibitor, on contractility of human, isolated bronchial muscle

1. The aim of this study was to investigate the smooth muscle relaxant effects of the novel, selective phosphodiesterase (PDE) type 4 inhibitor, RP 73401 in comparison with the classical PDE 4 inhibitor, rolipram, the non-selective PDE inhibitor, theophylline and the beta-adrenoceptor agonist, isoprenaline on the human, isolated bronchus. 2. At resting tone, the rank order of potency (pD2) for the relaxants was RP 73401 > or = rolipram > or = isoprenaline > theophylline. In terms of maximum relaxation produced (Emax) the PDE 4-selective inhibitors were similar, but the maximal effects (70-75% of theophylline, 3 mM) were lower than that observed with isoprenaline (98% of theophylline, 3 mM) or theophylline itself (100%). 3. On the human isolated bronchus pre-contracted with acetylcholine (ACh, 0.1 or 1.0 mM), the rank order of potency remained the same. The maximal responses to RP 73401 and rolipram were however markedly reduced (Emax 39.9-46.6%) compared with isoprenaline (Emax 79-85%). 4. In tissues pre-contracted with ACh (0.1 mM), RP 73401 and rolipram (10(-9)-10(-7) M) significantly and concentration-dependently increased tissue sensitivity to isoprenaline. RP 73401 and rolipram were similar in potency. Both selective PDE 4 inhibitors also significantly increased the maximal relaxant effects of isoprenaline. These effects were not observed with the PDE 3 inhibitor, siguazodan. 5. In terms of retention by tissues (an index of duration of action), the onset of action of RP 73401 (2.11 +/- 0.53 min) and rolipram (1.70 +/- 0.45 min) was significantly slower than that of isoprenaline (0.33 +/- 0.06 min) or theophylline (1.17 +/- 0.25 min). The retention of RP 73401 (89.0 +/- 21.9 min) on the human isolated bronchial tissues after washing was however dramatically longer than that of rolipram (18.3 +/- 4.5 min), theophylline (3.43 +/- 0.58 min) or isoprenaline (2.81 +/- 0.31 min). 6. These data indicate that RP 73401 is a potent and long acting relaxant of human bronchial muscle in vitro. RP 73401 is more potent than the classical PDE 4-selective inhibitor rolipram and the non-selective PDE inhibitor theophylline and is retained in bronchial tissue for a much longer period of time.     

Sildenafil, a novel inhibitor of phosphodiesterase type 5 in human corpus cavernosum smooth muscle cells

In human corpus cavernosum, release of nitric oxide from the non-adrenergic, non-cholinergic nerves and/or the endothelium activates guanylyl cyclase and increases intracellular cGMP levels. The increase in intracellular cGMP modulates intracellular calcium and in turn regulates smooth muscle contractility and erectile function. Phosphodiesterases play an important physiological role by regulating the intracellular levels of cyclic nucleotides. In this study, we investigated the kinetic parameters of inhibition of phosphodiesterase (PDE) type 5 (E.C. 3.1.4.35 3',5'-cyclic GMP phosphodiesterase) by a novel, high affinity, selective PDE type 5 inhibitor, sildenafil, in soluble extracts of human corpus cavernosum smooth muscle cells. Sildenafil inhibited PDE type 5 cGMP-hydrolytic activity, in the crude extract (Ki=4-6 nM) and in partially purified preparations (Ki=2 nM) in a competitive manner, as determined by Dixon plots. Sildenafil (Ki=2-4 nM) was a more effective PDE type 5 inhibitor than zaprinast (Ki=250 nM). Stimulation of intracellular cGMP synthesis by the nitric oxide donor, sodium nitroprusside, resulted in less than a 5% increase in cGMP levels in the absence of sildenafil and a 35% increase in cGMP levels in the presence of sildenafil, in intact cells at physiological temperatures. These results are in accord with the clinical observations that sildenafil, taken orally, promotes penile erection through increased intracellular cGMP in response to sexual stimulation, potentiating smooth muscle relaxation.     

Quinazolines: combined type 3 and 4 phosphodiesterase inhibitors

A series of quinazolines has been prepared and evaluated for its ability to inhibit cyclic AMP phosphodiesterase type 3, type 4A, 4B and 4D. The most potent inhibitors showed IC50 values in the nanomolar range for type 3 and type 4 isoforms and bind with high affinity to the [3H]rolipram binding site. These quinazolines represent a new family of potent mixed PDE 3/4 inhibitors and are expected to have a therapeutic potential.     

High selectivity for inhibition of phosphodiesterase III and positive inotropic effects of MCI-154 in guinea pig myocardium

MCI-154 (0.3-100 microM) exerted a concentration-dependent positive inotropic effect in isolated guinea pig papillary muscles (EC50 0.8 microM). The efficacy of MCI-154 (253% of predrug value) was 1.7-fold higher than that of saterinone but comparable to that of milrinone. Carbachol markedly reduced the increase in force of contraction (FOC) of MCI-154. In intact contracting papillary muscles, the positive inotropic effect was accompanied by an increase in cyclic AMP content to 0.78 +/- 0.09 pmol/mg wet weight (n = 10), corresponding to 150% of the basal value (0.51 +/- 0.05 pmol/mg wet weight, n = 21) in the presence of submaximal cyclic AMP phosphodiesterase (PDE) isoenzyme III inhibiting concentrations of MCI-154 (30 microM). MCI-154 (1-1,000 microM) concentration-dependently inhibited the activity of PDE III from homogenates of guinea pig myocardium. The IC50 was 3.8 microM. PDE I, II, and IV were not significantly affected up to 100 microM (PDE I and IV) and up to 1,000 microM (PDE II). In comparison, milrinone and saterinone were PDE III/IV-selective PDE inhibitors. Rolipram inhibited PDE IV only. IBMX and theophylline were nonselective PDE inhibitors. MCI-154 had only a marginal positive chronotropic effect. The frequency of spontaneously beating right auricles from guinea pig heart was increased by 8.7% at most (n = 5). MCI-154 increased Ca2+ sensitivity in chemically skinned porcine ventricular muscle fibers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amrinone, a phosphodiesterase III inhibitor, and arachidonic acid metabolism in humans

A 59-year-old patient who had photorefractive keratectomy (PRK) to correct high unilateral myopia developed a progressive nuclear cataract. Phacoemulsification and intraocular lens (IOL) implantation were performed. However, determination of IOL power using automated keratometry and computerized videokeratography was not successful in this case of high axial myopia because of a decentered ablation zone, resulting in too-steep keratometric readings. Postoperative hyperopia could only be corrected by an IOL exchange. Because it may not be possible to determine the exact keratometric values for IOL calculation after PRK, subtracting the change in refraction induced by PRK from the preoperative keratometric readings might have been more accurate in this patient.     

Antiasthmatic activity of the second-generation phosphodiesterase 4 (PDE4) inhibitor SB 207499 (Ariflo) in the guinea pig

We evaluated the airway activity of the novel phosphodiesterase type 4 inhibitor SB 207499 [Ariflo; c-4-cyano-4-(3-cyclopentyloxy-4-methoxyp henyl-r-1-cyclohexane carboxylic acid)], in the guinea pig. Ovalbumin (OA)-induced contractions of guinea pig isolated tracheal strips were inhibited by SB 207499 with an EC50 of 1 microM but had little or no effect on exogenous agonist-induced contraction, which suggests that its effect on OA-induced contraction in vitro is primarily due to inhibition of mediator release from mast cells. In anesthetized guinea pigs, SB 207499 inhibited OA-induced bronchoconstriction with i.v. and p.o. ID50 values of 1.7 and 17 mg/kg, respectively. At 1, 3 and 6 hr after SB 207499 (30 mg/kg p.o.), OA-induced bronchospasm was inhibited by 92%, 70% and 58%, respectively, corresponding to elevated plasma concentrations of 1.62 +/- 0.19, 1.65 +/- 0.29 and 0. 93 +/- 0.24 microg/ml, respectively, of SB 207499. SB 207499 also inhibited house dust mite-induced bronchoconstriction (ID50 = 0.9 mg/kg i.v. and 8.9 mg/kg p.o.). In contrast to its lack of bronchorelaxant activity in vitro, SB 207499 inhibited bronchospasm induced by i.v. leukotriene D4 (LTD4) [ID50 = 3 mg/kg i.v.]. The bronchorelaxant effect of i.v.-administered SB 207499 was at least additive with that of salbutamol in reversing infused histamine-enhanced airway tone, but it did not alter base line or enhance salbutamol-induced cardiovascular effects. In conscious guinea pigs, SB 207499 (10 or 30 mg/kg p.o.), 1 hr before antigen or LTD4 challenge, markedly reduced bronchospasm and subsequent eosinophil influx as measured by bronchoalveolar lavage 24 hr after provocation. SB 207499 administered after OA or LTD4 challenge also reduced airway eosinophilia measured at 24 hr after OA challenge or 96 hr after LTD4 challenge. These results, coupled with the broad anti-inflammatory activity of SB 207499 previously described (Barnett et al., 1998), suggest that SB 207499 will be useful in the treatment of asthma and other inflammatory disorders.     

SB 207499 (Ariflo), a second generation phosphodiesterase 4 inhibitor, reduces tumor necrosis factor alpha and interleukin-4 production in vivo

The ability of the second generation phosphodiesterase 4 inhibitor SB 207499 (Ariflo), [c-4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)-r-l-cyclohexane carboxylic acid], to inhibit inflammatory cytokine production in vivo was evaluated and compared to that of rolipram, a first generation phosphodiesterase 4 inhibitor. To examine human tumor necrosis factor alpha (TNFalpha) production, human monocytes were adoptively transferred into Balb/c mice and challenged with lipopolysaccharide (LPS). In this model, SB 207499 inhibited human TNFalpha production with oral ED50 of 4.9 mg/kg. Similarly, R-rolipram inhibited human TNFalpha production with an ED50 of 5.1 mg/kg, p.o. In contrast to their equipotent activity against TNFalpha production, SB 207499 (ED50 = 2.3 mg/kg, p.o.) was 10-fold less potent than R-rolipram (ED50 = 0.23 mg/kg, p.o.) in reversing reserpine-induced hypothermia, a model of antidepressant activity. In time course studies, SB 207499 (30 mg/kg, p.o.) inhibited TNFalpha production for at least 10 hr; substantial plasma concentrations of SB 207499 were detected over the same interval. The ability of SB 207499 to modulate interleukin-4 production in vivo was assessed in a chronic oxazolone-induced contact sensitivity model in Balb/c mice. In this model, topical administration of SB 207499 (1000 microgram) inhibited intralesional concentrations of interleukin-4 (55%; P <.01). The results demonstrate that SB 207499 is a potent inhibitor of inflammatory cytokine production in a variety of settings in vivo. Moreover, although it is as potent as R-rolipram in inhibiting TNFalpha production, it has substantially less central nervous system activity. Thus SB 207499 represents an excellent candidate with which to evaluate the antiinflammatory potential of PDE4 inhibitors.     

Effect of cilostazol, a phosphodiesterase type III inhibitor, on histamine-induced increase in [Ca2+]i and force in middle cerebral artery of the rabbit

1. The effect of cilostazol, an inhibitor of phosphodiesterase type III (PDE III), on the contraction induced by histamine was studied by making simultaneous measurements of isometric force and the intracellular concentration of Ca2+ ([Ca2+]i) in endothelium-denuded muscle strips from the peripheral part of the middle cerebral artery of the rabbit. 2. High K+ (80 mM) produced a phasic, followed by a tonic increase in both [Ca2+]i and force. Cilostazol (10 microM) did not modify the resting [Ca2+]i, but it did significantly decrease the tonic contraction induced by high K+ without a corresponding change in the [Ca2+]i response. 3. Histamine (3 microM) produced a phasic, followed by a tonic increase in both [Ca2+]i and force. Cilostazol (3 and 10 microM) significantly reduced both the phasic and tonic increases in [Ca2+]i and force induced by histamine, in a concentration-dependent manner. 4. Rp-adenosine-3':5'-cyclic monophosphorothioate (Rp-cAMPS, 0.1 mM), a PDE-resistant inhibitor of protein kinase A (and as such a cyclic AMP antagonist), did not modify the increases in [Ca2+]i and force induced by histamine alone, but it did significantly decrease the cilostazol-induced inhibition of the histamine-induced responses. 5. In Ca2+-free solution containing 2 mM EGTA, both histamine (3 microM) and caffeine (10 mM) transiently increased [Ca2+]i and force. Cilostazol (1-10 microM) (i) significantly reduced the increases in [Ca2+]i and force induced by histamine, and (ii) significantly reduced the increase in force but not the increase in [Ca2+]i induced by caffeine. 6. In ryanodine-treated strips, which had functionally lost the histamine-sensitive Ca2+ storage sites, histamine (3 microM) slowly increased [Ca2+]i and force. Cilostazol (3 and 10 microM) lowered the resting [Ca2+]i, but did not modify the histamine-induced increase in [Ca2+]i, suggesting that functional Ca2+ storage sites are required for the cilostazol-induced inhibition of histamine-induced Ca2+ mobilization. 7. The [Ca2+]i-force relationship was obtained in ryanodine-treated strips by applying ascending concentrations of Ca2+ (0.16-2.6 mM) in Ca2+-free solution containing 100 mM K+. Histamine (3 microM) shifted the [Ca2+]i-force relationship to the left and increased the maximum Ca2+-induced force. Under the same conditions, whether in the presence or absence of 3 microM histamine, cilostazol (3-10 microM) shifted the [Ca2+]i-force relationship to the right without producing a change in the maximum Ca2+-induced force. 8. It is concluded that, in smooth muscle of the peripheral part of the rabbit middle cerebral artery, cilostazol attenuates the histamine-induced contraction both by inhibiting histamine-induced Ca2+ mobilization and by reducing the myofilament Ca2+ sensitivity. It is suggested that the increase in the cellular concentration of cyclic AMP that will follow the inhibition of PDE III may play an important role in the cilostazol-induced inhibition of the histamine-contraction.     

Identification of inhibitor specificity determinants in a mammalian phosphodiesterase

Mammalian phosphodiesterase types 3 and 4 (PDE3 and PDE4) hydrolyze cAMP and are essential for the regulation of this intracellular second messenger in many cell types. Whereas these enzymes share structural and biochemical similarities, each can be distinguished by its sensitivity to isozyme-specific inhibitors. By using a series of chimeric enzymes, we have localized the region of PDE4 that confers sensitivity to selective inhibitors. This inhibitor specificity domain lies within a short sequence at the carboxyl terminus of the catalytic domain of the protein, consistent with the competitive nature of inhibition by these compounds. Surprisingly, the identified region also includes some of the most highly conserved residues among PDE isoforms. A yeast-based expression system was used for the isolation and characterization of mutations within this area that confer resistance to the PDE4-specific inhibitor rolipram. Analysis of these mutants indicated that both conserved and unique residues are required for isoform-specific inhibitor sensitivity. In some cases, combined point mutations contribute synergistically to the reduction of sensitivity (suppression of IC50). We also report that several mutations display differential sensitivity changes with respect to distinct structural classes of inhibitors.     

Effect of the phosphodiesterase inhibitor Pentoxyfylline on human sperm motility

Tyrode fluid and Tyrode fluid plus Pentoxifylline were individually added to aliquots of semen samples obtained from 6 normal men and 6 infertile patients considered to have idiopathic normogonadotropic oligoasthenozoospermia. Pentoxifylline was added to final concentrations of 0.15, 0.30 and 0.60 mM. One aliquot with no addition served as control. Samples were incubated in 37 degrees C and observed by light microscopy at 30 minutes and at 1, 2 and 4 hours after obtaining the material. At observation time, semen quality was evaluated by determining the percentages of forwardly progressive spermatozoa, slowly progressive spermatozoa, "in situ" motile spermatozoa, live and non-motile spermatozoa and dead spermatozoa. Results reported included only the first and last category. Tyrode fluid did not affect significantly the motility and the duration of activity of spermatozoa. Ejaculated human spermatozoa both from normal and asthenozoospermic men added the Pentoxifylline at 0.30 and 0.60 mM showed a longer lasting activity than those of control semen and semen added only with Tyrode fluid.     

Reversal of pulmonary capillary ischemia-reperfusion injury by rolipram, a cAMP phosphodiesterase inhibitor

Isoproterenol (ISO) and forskolin, agents that increase adenosine 3',5'-cyclic monophosphate (cAMP) via adenylyl cyclase activation, reverse lung injury associated with increased microvascular permeability. We studied the role of rolipram, a relatively isozyme-selective cAMP phosphodiesterase (PDE) inhibitor, in reversing increased capillary permeability due to ischemia-reperfusion (I/R), a form of oxidant injury in the lung, by using the isolated perfused rat lung model. Rolipram (2 microM) administered after 45 min of ischemia and 45 min of reperfusion reduced I/R-increased permeability as measured by the capillary filtration coefficient to control lung values. Computer image analysis of air space edema and perivascular cuffing, as well as wet-to-dry weight ratios, confirms the permeability reversal by rolipram administration. Rolipram inhibition of cAMP PDE in the lung was assessed by using [3H]adenine prelabeling adapted for the whole lung and perfusate [3H]cAMP accumulation. Rolipram failed to increase perfusate cAMP alone but dramatically increased perfusate cAMP above ISO alone. Dose-response relationships of ISO or rolipram show a close correlation of the half-maximal effective dose (ED50) for injury reversal and perfusate cAMP production. The combination of rolipram and ISO produced synergistic reversal of I/R injury. We conclude that reversal of I/R-induced increased microvascular permeability can be achieved with rolipram and that the mechanism of action of rolipram is probably through PDE isozyme-selective inhibition. The similarity of the ED50 values for cAMP efflux and reversal of permeability increases also supports a close coupling between cAMP accumulation and endothelial cell permeability.     

The effect of zardaverine, an inhibitor of phosphodiesterase isoenzymes III and IV, on endotoxin-induced airway changes in rats

Zardaverine is a novel phosphodiesterase III/IV inhibitor, developed as a potential therapeutic agent for asthma. In this study we evaluated the effect of zardaverine in an in vivo animal model of airway inflammation and hyperresponsiveness. Endotoxin exposure in rats causes a transient increase in airway responsiveness and a neutrophilic inflammation of the bronchi, which are both at least partly mediated through the secondary release of tumour necrosis factor alpha (TNF alpha). Groups of 10 animals each were pretreated with placebo or zardaverine (1, 10, 30 mumol/kg) i.p., 30 min prior to exposure to aerosolized endotoxin (LPS) or saline. Ninety minutes later, airway responsiveness to 5-HT was assessed and bronchoalveolar lavage (BAL) performed. Zardaverine did not influence baseline lung resistance (RL), but inhibited dose dependently the 5-HT induced increase in RL in control animals. In placebo pretreated animals LPS exposure caused a significant decrease in PC50RL5-HT (provocative concentration of 5-HT causing a 50% increase in RL), compared to the saline exposed control group (1.1 +/- 0.1 vs 2.7 +/- 0.4 micrograms/kg) (P < 0.01). This decrease in PC50RL5-HT was significantly inhibited by zardaverine 30 mumol/kg (5.4 +/- 1.8 vs 1.1 +/- 0.1 micrograms/kg) (P < 0.05). Compared to placebo pre-treated, LPS exposed animals, zardaverine 30 mumol/kg also significantly inhibited to LPS induced neutrophil increase (193.0 +/- 50.0 vs 915.6 +/- 181.3 x 10(3)) (P < 0.01), increase in elastase activity (23 +/- 11 vs 54 +/- 9 nmol substrate/h/ml) (P < 0.05) and TNF alpha release in BAL fluid (93.1 +/- 19.5 vs 229.5 +/- 24.8 U/ml BAL fluid) (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Vasorelaxant effect of olprinone, an inhibitor of phosphodiesterase 3, on mesenteric small artery and vein of rabbits

An augmented synthesis of tetrahydroisoquinolines, such as salsolinol (SAL) or an increased N-methylation of these compounds has been addressed by various investigators as putative pathophysiologic mechanisms in Parkinson's disease (PD). Aim of this study was (1) to investigate putative relations between plasma levels of dopamine and R- and S-enantiomers of SAL and (2) whether these metabolic precursors of the neurotoxic N-methylated-SAL (NMSAL) are elevated in untreated "de-novo" Parkinsonian patients compared to age- and sex-matched healthy controls. Plasma levels of R- and S-SAL and dopamine did not significantly (R-SAL: p=0.61, S-SAL: p=0.51, dopamine: p=0.84) differ in both groups. Parkinsonian patients' R-SAL plasma levels were inversely related to intensity (p=0.03, r =-0.42) and duration of PD (p=0.03, r=-0.43) in contrast to S-SAL and dopamine. Dopamine levels were not associated to R-SAL (p=0.88, r2=0.0008) and S-SAL (p=0.088, r2=0.12) neither in Parkinsonian patients nor in controls. We conclude, that an upregulation of N-methylation of tetrahydroisoquinolines takes place in PD by enzymes such as neutral N-methyltransferase specific for R-SAL. The activity of this enzyme has been found elevated in parkinsonian lymphocytes. This increased N-methylation by the N-methyltransferase specific for R-SAL leads to the known augmented levels of neurotoxic R-NMSAL in Parkinsonian patients compared to controls in the cenral nervous system especially in the beginning of PD.     

Inhibition of platelet function by A02131-1, a novel inhibitor of cGMP-specific phosphodiesterase, in vitro and in vivo

The effect of A02131-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzyl thieno (3,2-c)pyrazole], a cGMP-specific phosphodiesterase (PDE) inhibitor, on platelet function was investigated. The compound was found to inhibit the aggregation of and adenosine triphosphate (ATP) release from human platelet-rich plasma and washed platelets that were induced by aggregation inducing drugs such as arachidonic acid (AA), collagen, U46619, platelet-activating factor (PAF), adenosine diphosphate (ADP) and A23187, and the inhibitory effect was concentration-dependent. A02131-1 also disaggregated the performed platelet aggregates induced by these inducers. Thromboxane B2 (TXB2) formations caused by collagen, PAF, ADP, and A23187 were inhibited by A02131-1 at concentrations that did not affect the AA-induced formation of TXB2 and prostaglandin D2 (PGD2). A02131-1 suppressed both the generation of inositol 1,4,5-triphosphate (IP3) and the increase of intracellular Ca2+ concentration stimulated by these aggregation inducers. A02131-1 was shown to increase the cAMP and cGMP levels in platelets and the extent was found to be dependent on concentration as well as time. A02131-1 increased the cAMP level much more slowly than the cGMP level. Activities of adenylate cyclase, guanylate cyclase, and PDEs (type I and III) were not altered by A02131-1. However, the activity of cGMP-specific PDE (type V) was inhibited by A02131-1. The antiplatelet aggregation activity and the effect on raising cAMP level of A02131-1 were both potentiated by prostaglandin E1 (PGE1). In the mouse tail bleeding test, A02131-1 was clearly shown to be more effective than dipyridamole in prolonging the tail bleeding time of conscious mice. These data indicate that A02131-1 is a cGMP-specific PDE (type V) inhibitor in human platelets.     

Phosphorylation and activation of hormone-sensitive adipocyte phosphodiesterase type 3B

Conducted vasomotor responses are important for the effective distribution of blood flow, although the mechanism by which these responses are initiated is not well understood. ATP, a substance which is released from circulating red blood cells in response to low PO2 and low pH, two conditions which are associated with decreased supply relative to demand, has been shown to initiate conducted vasodilation following its intraluminal application in first and second order arterioles. Since such low PO2 and low pH conditions would most likely occur on the venous side of the vasculature, we evaluated the response of the arteriolar and capillary networks to application of ATP into venules in the Saran-covered hamster cheek pouch retractor muscle using in vivo video microscopy. Intraluminal application of 40 and 400 pl of 10(-6) M ATP resulted in dose-dependent increases in arteriolar diameter > 450 microm upstream from the site of application. These changes in arteriolar diameter were accompanied by significant increases in red blood cell flux. In capillaries, red blood cell flux doubled in response to ATP administration. Since NO was previously determined to be involved in the vascular response to intraluminal ATP in arterioles, we evaluated its role in these responses. We found that systemic administration of l-NAME prior to ATP application eliminated any conducted response and this effect of l-NAME was reversed by the systemic administration of l-arginine. These data suggest that ATP, which is released from red blood cells in response to low PO2 and low pH, conditions which would be found in the venular microvasculature, may serve a role in distributing perfusion in response to alterations in supply.