zaprinast and vinpocetine

Within erythrocytes (RBCs), cAMP levels are regulated by phosphodiesterases (PDEs). Increases in cAMP and ATP release associated with activation of β-adrenergic receptors (βARs) and prostacyclin receptors (IPRs) are regulated by PDEs 2, 4 and PDE 3, respectively. Here we establish the presence of cytosolic PDEs in RBCs and determine a role for PDE5 in regulating levels of cGMP.. Purified cytosolic proteins were obtained from isolated human RBCs and western analysis was performed using antibodies against PDEs 3A, 4 and 5. Rabbit RBCs were incubated with dbcGMP, a cGMP analog, to determine the effect of cGMP on cAMP levels. To determine if cGMP affects receptor-mediated increases in cAMP, rabbit RBCs were incubated with dbcGMP prior to addition of isoproterenol (ISO), a βAR receptor agonist. To demonstrate that endogenous cGMP produces the same effect, rabbit and human RBCs were incubated with SpNONOate (SpNO), a nitric oxide donor, and YC1, a direct activator of soluble guanylyl cyclase (sGC), in the absence and presence of a selective PDE5 inhibitor, zaprinast (ZAP).. Western analysis identified PDEs 3A, 4D and 5A. dbcGMP produced a concentration dependent increase in cAMP and ISO-induced increases in cAMP were potentiated by dbcGMP. In addition, incubation with YC1 and SpNO in the presence of ZAP potentiated βAR-induced increases in cAMP.. PDEs 2, 3A and 5 are present in the cytosol of human RBCs. PDE5 activity in RBCs regulates cGMP levels. Increases in intracellular cGMP augment cAMP levels. These studies suggest a novel role for PDE5 in erythrocytes.

Topics: Animals; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 3; Cyclic Nucleotide Phosphodiesterases, Type 4; Cyclic Nucleotide Phosphodiesterases, Type 5; Cytosol; Erythrocytes; Humans; Isoenzymes; Isoproterenol; Male; Phosphodiesterase Inhibitors; Purinones; Rabbits; Spermine; Vinca Alkaloids

The effects of various selective phosphodiesterase (PDE) inhibitors on muscle contractility and cyclic nucleotide contents in the guinea pig gall bladder were investigated. Various selective PDE inhibitors, vinpocetine (type 1), erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, type 2), milrinone (type 3), Ro20-1724 (type 4), and zaprinast (type 5), inhibited CCh-induced contractions in a concentration-dependent manner. The rank order of potency for the gall bladder was Ro20-1724 > vinpocetine > EHNA > milrinone > zaprinast, which was different from that of the trachea, taenia coli, and aorta. In the presence of CCh (0.3 muM), vinpocetine, milrinone, and Ro20-1724 each increased cAMP content, but not cGMP. By contrast, zaprinast increased cGMP content, but not cAMP, and EHNA increased both cAMP and cGMP contents. These results suggest that vinpocetine-, milrinone-, and Ro20-1724-induced relaxation was correlated with cAMP, zaprinast-induced relaxation was correlated with cGMP, and that EHNA-induced relaxation was correlated with cAMP and cGMP in the guinea pig gall bladder. In conclusion, the effect of PDE inhibitors in the guinea pig gall bladder was different from those in smooth muscles, such as the trachea, taenia coli, and aorta.

Topics: 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; Adenine; Animals; Carbachol; Cyclic AMP; Cyclic GMP; Gallbladder; Guinea Pigs; Male; Milrinone; Muscle Contraction; Nucleotides, Cyclic; Phosphodiesterase Inhibitors; Purinones; Vinca Alkaloids

In bovine tracheal smooth muscle (TSM) strips, muscarinic antagonists (atropine, 4-DAMP, AFDX-116 and methoctramine) were able to increase simultaneously and a similar fashion the intracellular levels of cyclic nucleotides, with a cAMP/cGMP ratio higher than 2.0. These original pharmacological responses were time-and dose-dependent, exhibiting maximal values at 15 min, with a pEC(50) of 7.4 +/- 0.2 for atropine and 4-DAMP. These effects on cAMP and cGMP levels were similar to the ones obtained with isobutyl-methylxantine (IBMX, 10 microM), a non-selective cyclic nucleotide phosphodiesterase (PDE) inhibitor, suggesting the involvement of PDEs in these muscarinic antagonist responses. Neither, rolipram (10 microM), a specific PDEIV inhibitor, nor zaprinast (10 microM), a PDEV inhibitor, exhibited this "atropine-like" responses. Instead, atropine enhanced the increments of cAMP levels induced by rolipram and cGMP levels by zaprinast. However, vinpocetine (20 microM), a non-calmodulin dependent PDEIC inhibitor was able to mimic these muscarinic antagonist responses in intact smooth muscle strips. In addition, in cell free systems, muscarinic antagonists inhibited the membrane-bound PDEIC activity whereas soluble (cytosol) PDEIC activity was not affected by these muscarinic drugs. These results indicate that muscarinic antagonists acting possibly as inverse agonists on M(2)/M(3)mAChRs anchored to sarcolemma membranes can initiate a new signal transducing cascade leading to the PDEIC inhibition, which produced a simultaneous rise in both cAMP and cGMP intracellular levels in tracheal smooth muscle.

Topics: Animals; Atropine; Cattle; Cell-Free System; Cyclic AMP; Cyclic GMP; Muscarinic Antagonists; Muscle, Smooth; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Purinones; Rolipram; Trachea; Vinca Alkaloids

Effects of various selective phosphodiesterase (PDE) inhibitors on muscle contractility and cyclic nucleotide contents in guinea pig taenia coli were investigated. Forskolin and sodium nitroprusside inhibited carbachol (CCh)-induced contraction in a concentration-dependent manner. Various selective PDE inhibitors, vinpocetine (type 1), erythro -9-(2-hydroxy-3-nonyl)adenine (EHNA, type 2), milrinone (type 3), Ro20-1724(type 4) and zaprinast (type 5) inhibited CCh-induced contraction in a concentration-dependent manner, but the inhibition of milrinone was noticeably smaller than that of the other PDE inhibitors. The rank order of potency was zaprinast > vinpocetine > EHNA > Ro20-1724 > milrinone. In the presence of CCh (0.3 microM), vinpocetine and Ro20-1724 both increased cAMP content, but not cGMP. By contrast, EHNA and zaprinast both increased cGMP content, but not cAMP. Pretreatment with ODQ (30 microM), a soluble guanylyl cyclase inhibitor, decreased the inhibition of CCh-induced contraction by EHNA or zaprinast. Pretreatment with SQ22536 (100 microM), an adenylyl cyclase inhibitor, decreased the inhibition of CCh-induced contraction by vinpocetine or Ro20-1724. In conclusion, it was indicated that vinpocetine- or Ro20-1724-induced relaxation was correlated with cAMP but EHNA- or zaprinast- induced relaxation was correlated with cGMP.

Topics: Adenine; Animals; Carbachol; Colforsin; Colon; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Guinea Pigs; Male; Milrinone; Muscle Contraction; Nitroprusside; Nucleotides, Cyclic; Phosphodiesterase Inhibitors; Purinones; Vinca Alkaloids

Pharmacologic treatments are gaining widespread acceptance as first-line therapy for anal fissure. However, existing treatments have limited clinical usefulness because of side effects and incomplete healing rates.. Fresh human surgical resection specimens containing internal anal sphincter and rectal circular muscle were collected. Strips of smooth muscle were cut from each muscle group and mounted in a superfusion organ bath. The effects of increasing concentrations of phosphodiesterase inhibitors were evaluated.. All phosphodiesterase inhibitors tested caused a dose-dependent reduction in the tone of the internal anal sphincter, with potencies as follows: vinpocentine (phosphodiesterase-1 inhibitor; 50 percent maximum inhibition concentration = 0.87 +/- 0.10 microM), erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (phosphodiesterase-2 inhibitor; 32 +/- 4.8 microM), trequinsin (phosphodiesterase-3 inhibitor; 0.28 +/- 0.041 microM), rolipram (phosphodiesterase-4 inhibitor; 63 +/- 9 microM), zaprinast (phosphodiesterase-1,5,6,9,11 inhibitor; 3 +/- 0.69 microM), and dipyridamole (phosphodiesterase-5,6,8,10,11 inhibitor; 5.5 +/- 2 microM). Although all inhibitors were also effective on rectal circular muscle strips, erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride, trequinsin, and rolipram were at least an order of magnitude more potent in this tissue than in the internal anal sphincter.. There are several functionally important phosphodiesterases in the internal anal sphincter and rectal circular muscle. Both adenosine 3', 5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate appear to be important in the myogenic tone of the internal anal sphincter, and this study provides further evidence of the sphincteric specialization of this tissue. Phosphodiesterase inhibitors might represent a new therapy for the treatment of anal fissure.

Topics: Adenine; Aged; Aged, 80 and over; Anal Canal; Dipyridamole; Dose-Response Relationship, Drug; Female; Humans; In Vitro Techniques; Isoquinolines; Male; Middle Aged; Muscle Relaxation; Muscle, Smooth; Phosphodiesterase Inhibitors; Platelet Aggregation Inhibitors; Purinones; Rectum; Rolipram; Tetrahydroisoquinolines; Vinca Alkaloids

We examined the effect of 3-isobutyryl-2-isopropylpyrazolo[1,5-a]pyridine (ibudilast), which has been clinically used for bronchial asthma and cerebrovascular disorders, on cell viability induced in a model of reperfusion injury. Ibudilast at 10 - 100 microM significantly attenuated the H(2)O(2)-induced decrease in cell viability. Ibudilast inhibited the H(2)O(2)-induced cytochrome c release, caspase-3 activation, DNA ladder formation and nuclear condensation, suggesting its anti-apoptotic effect. Phosphodiesterase inhibitors such as theophylline, pentoxyfylline, vinpocetine, dipyridamole and zaprinast, which increased the guanosine-3',5'-cyclic monophosphate (cyclic GMP) level, and dibutyryl cyclic GMP attenuated the H(2)O(2)-induced injury in astrocytes. Ibudilast increased the cyclic GMP level in astrocytes. The cyclic GMP-dependent protein kinase inhibitor KT5823 blocked the protective effects of ibudilast and dipyridamole on the H(2)O(2)-induced decrease in cell viability, while the cyclic AMP-dependent protein kinase inhibitor KT5720, the cyclic AMP antagonist Rp-cyclic AMPS, the mitogen-activated protein/extracellular signal-regulated kinase inhibitor PD98059 and the leukotriene D(4) antagonist LY 171883 did not. KT5823 also blocked the effect of ibudilast on the H(2)O(2)-induced cytochrome c release and caspase-3-like protease activation. These findings suggest that ibudilast prevents the H(2)O(2)-induced delayed apoptosis of astrocytes via a cyclic GMP, but not cyclic AMP, signalling pathway.

Topics: Alkaloids; Animals; Animals, Newborn; Apoptosis; Astrocytes; Carbazoles; Cell Survival; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cytochrome c Group; Dipyridamole; DNA; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hydrogen Peroxide; Indoles; Mitochondria; Pentoxifylline; Peptide Hydrolases; Phosphodiesterase Inhibitors; Purinones; Pyridines; Rats; Rats, Wistar; Reperfusion Injury; Signal Transduction; Theophylline; Vinca Alkaloids

The effects of family selective inhibitors of phosphodiesterase (PDEI, PDE2, PDE3, PDE4, and PDE5) on the behavior of rats under either a differential-reinforcement-of-low-rate (DRL) 72-s schedule or a variable-interval (VI) 30-s schedule were determined; previous work has shown that antidepressant drugs increase reinforcement rate under long DRL schedules. The PDE4-selective inhibitor rolipram (0.03-0.1 mg/kg) reduced response rate and increased reinforcement rate under the DRL schedule in a dose-dependent manner; similar effects were observed with the tricyclic antidepressant drug desipramine (3-10 mg/kg). Both of these drugs produced biphasic effects on behavior maintained under the VI schedule, increasing response rate at the lower doses tested (rolipram: 0.003 mg/kg; desipramine: 0.03 mg/kg) and decreasing response rate at higher doses (rolipram: 0.1 mg/kg; desipramine: 0.3-18 mg/kg). Of the other PDE inhibitors tested, only the PDE5-selective inhibitor zaprinast (10 mg/kg) produced an antidepressant-like effect on DRL behavior. However, in contrast to the biphasic effects of rolipram and desipramine on VI behavior, zaprinast produced monotonic decreases in response rate (10-30 mg/kg). The PDE2-selective inhibitor trequinsin produced biphasic effects on response rate under the VI schedule, increasing rates at low doses (3-5.6 mg/kg) and decreasing rates at higher doses (18-30 mg/kg). Trequinsin also reduced response rate under the DRL schedule (30 mg/kg); however, the reduction in response rate was not accompanied by increased reinforcement rate. The PDE3-selective inhibitor milrinone (1-10 mg/kg) tended to increase response rates under both schedules while the PDE1-selective inhibitor vinpocetine did not affect behavior at the dose range tested (1-30 mg/kg). These findings suggest that inhibition of PDE4 results in a rather unique pattern of behavioral effects, most notably an antidepressant-like effect on DRL behavior. It remains to be determined if a similar effect produced by zaprinast also implicates PDE5 in the mediation of antidepressant activity or represents an effect of this drug on PDE4 activity at high doses.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Antidepressive Agents, Tricyclic; Conditioning, Operant; Cyclic Nucleotide Phosphodiesterases, Type 1; Desipramine; Isoquinolines; Male; Milrinone; Phosphodiesterase Inhibitors; Purinones; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Reinforcement Schedule; Rolipram; Tetrahydroisoquinolines; Vinca Alkaloids

To determine whether phosphodiesterase (PDE) is involved in the degradation of cGMP in human erythrocytes, we studied the cell cGMP content in the presence of different PDE inhibitors: zaprinast and dipyridamole, specific inhibitors of cGMP-binding, cGMP-specific PDE (cG-BPDE); vinpocetine, a specific inhibitor of Ca2+, calmodulin-dependent phosphodiesterase (CaM-PDE); an unspecific inhibitor, 3-isobutyl-1-methylxanthine (IBMX). IBMX, zaprinast, and dipyridamole at 30 microM did not affect the intracellular cGMP content. However, vinpocetine at this concentration increased the cGMP content by 102 +/- 14% (p < 0.05). The effect of vinpocetine was dose-dependent, reached the maximal level after 1 min of incubation and flattened at the same level. Ca2+ (10 microM) in the presence of the Ca(2+)-ionophore, A23187 (5 microM), decreased the cGMP content (-23% +/- 4; p < 0.05), which can be explained by the CaM-PDE activation. The Ca(2+)-induced decrease in cGMP was completely inhibited by the CaM antagonist, W-7 (100 microM). These data suggest that erythrocytes contain Ca2+, CaM-PDE.

Topics: 1-Methyl-3-isobutylxanthine; 3',5'-Cyclic-GMP Phosphodiesterases; Calcium; Calmodulin; Cyclic GMP; Erythrocytes; Humans; Hydrolysis; Phosphodiesterase Inhibitors; Purinones; Sulfonamides; Vinca Alkaloids

In the coronary circulation, endothelin-1 (ET-1) evokes spasms which are difficult to treat when the endothelial integrity is compromised. This study compares several classes of relaxing agents on already established contractions to ET-1 in an in vitro model using ring segments of the porcine left descending coronary artery (pLAD). All segments were precontracted with 10 nmol/L ET-1. The calcium channel blocker isradipine was 300 times more potent than verapamil, but was only a partial relaxant; the maximal relaxation obtained was 52 +/- 2% (n = 6). Atrial natriuretic peptide (ANP) was an equally potent relaxant of the ET-1 contraction; however, it too was an incomplete relaxant, maximal relaxation being < 60%. A 50% relaxation of the ET-1 contraction was obtained with 0.28 +/- 0.24 mumol/L ANP, n = 4 (IC50). Comparison of cyclic nucleotide analogues revealed a 30 times higher potency for 8-bromo-cyclic guanosine monophosphate (8-Br-cGMP)(IC50 44 +/- 11 mumol/L, n = 6) than for 8-bromo-cyclic adenosine monophosphate (8-Bi-cAMP) (IC50 1600 mumol/L, n = 6). The cyclic nucleotide phosphodiesterase (PDE) inhibitor milrinone, a PDE 3-inhibitor with an IC50 2.4 +/- 1.8 mumol/L, (n = 6) was 10 times more potent than rolipram (PDE 4-inhibitor), zaprinast (PDE 5-inhibitor) and vinpocentine (PDE 1-inhibitor). Withdrawal of these analogues and inhibitors from segments continuously exposed to 10 nmol/l ET-1 revealed that vinpocentine and 8-Br-cGMP were irreversible relaxants, in contrast to milrinone and 8-Br-cAMP. In conclusion, this study has demonstrated that cGMP-enhancing agents, such as the naturally occurring ANP, the calcium channel blocker isradipine, and the synthetic inhibitor of PDE 3, were the most effective relaxants of ET-1 evoked contractions in pLAD in vitro.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Atrial Natriuretic Factor; Caffeine; Calcium Channel Blockers; Calcium Channels; Calcium Channels, L-Type; Colforsin; Coronary Vessels; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Endothelin-1; In Vitro Techniques; Isradipine; Milrinone; Muscle, Smooth, Vascular; Papaverine; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Purinones; Pyrrolidinones; Rolipram; Swine; Vasoactive Intestinal Peptide; Vasoconstriction; Vasodilation; Verapamil; Vinca Alkaloids

Rolipram was previously reported to elevate plasma cyclic adenosine 3',5'-monophosphate (cAMP) and inhibit serum tumor necrosis factor-alpha (TNF-alpha) production in mice. CP-80,633, a new cyclic nucleotide phosphodiesterase (PDE4) inhibitor, has been shown to augment intracellular cAMP levels and to inhibit TNFalpha release from human monocytes in vitro. This study was undertaken to determine the effect of p.o. CP-80,633 on plasma cAMP levels and lipopolysaccharide-induced TNFalpha production in mice with and without adrenal glands. CP-80,633 dose-dependently (3-32 mg/kg p.o.) elevated plasma cAMP levels and decreased systemic TNFalpha production in response to i.p. injection of lipopolysaccharide. Elevated plasma cAMP levels can be detected for up to 4 hr. CP-80,633 (10 mg/kg p.o.) caused a 6-fold increase in the plasma cAMP level, a 2-fold increase in the plasma epinephrine level and a greater than 95% reduction in TNFalpha production. Unlike CP-80,633, neither vinpocetine, dipyridamole, SKB-94,120 nor zaprinast, at 100 mg/kg p.o., modified the cAMP response, which suggests that this response is mediated by inhibition of PDE4. Adrenalectomy reduced the cAMP response and completely blocked the epinephrine response; however, the levels of plasma cAMP in the CP-80,633-treated mice (10 mg/kg p.o.) remained elevated (vehicle: 47.3 +/- 6.8 vs. CP-80,633: 98.4 +/- 10.3 pmol/ml, n = 7, P < .05). This effect is mimicked by treatment of control mice with propranolol, which demonstrates that beta adrenoreceptors contribute to the cAMP response. Removal of adrenal glands significantly increased the LPS-induced elevation of serum TNFalpha. The ability of CP-80,633 to block the TNFalpha response was only slightly affected by adrenalectomy (ED50 = 1.2 mg/kg in controls vs. 3.9 mg/kg in adrenalectomized mice). Taken together, these results show that CP-80,633, when given p.o. to mice, is capable of elevating plasma cAMP and inhibiting TNFalpha production and that adrenal catecholamines contribute significantly to the effect of CP-80,633 on the cAMP response but only slightly to its effect on the systemic TNFalpha response.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adrenalectomy; Animals; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 4; Dipyridamole; Epinephrine; Humans; Kinetics; Lipopolysaccharides; Male; Mice; Mice, Inbred BALB C; Monocytes; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Piroxicam; Propranolol; Purinones; Pyrimidinones; Thromboxane B2; Time Factors; Tumor Necrosis Factor-alpha; Vinca Alkaloids

1. We have examined the effects of the isoenzyme-selective phosphodiesterase (PDE) inhibitors, vinpocetine (type 1), siguazodan (type 3), rolipram (type 4) and zaprinast (type 5) and the non-selective PDE inhibitor enprofylline on methacholine (MCh) contractile concentration-response curves on guinea-pig and rat isolated ileum. 2. In guinea-pig ileum, vinpocetine (10-300 microM), zaprinast (1-300 microM) and enprofylline (100-1000 microM) produced a concentration-dependent depression of the maximum response (Emax) to MCh only without effect on the MCh EC50 values (rank order of potency: zaprinast > vinpocetine > enprofylline). In contrast, siguazodan (10-300 microM) and rolipram (10-300 microM) produced a rightward displacement of the MCh concentration-response curve (increase in EC50: rank order; rolipram > siguazodan), with effects on the MCh maximum seen only at higher concentrations. 3. In the rat ileum, vinpocetine (10-300 microM), zaprinast (0.1-300 microM) and enprofylline (100-1000 microM) caused depression of the MCh maximum contraction (rank order: zaprinast > vinpocetine > enprofylline). Low concentrations of rolipram and siguazodan had no significant effect on the MCh maximum. In the presence of higher concentrations (> 100 microM) of rolipram and siguazodan, a maximum response was not achieved at the highest concentration of MCh tested. As in the guinea-pig ileum, only rolipram (10-300 microM) and siguazodan (10-300 microM) produced a significant, concentration-dependent, rightward displacement of the MCh concentration-response curve (increase in EC50: rank order: rolipram > siguazodan). 4. In the guinea-pig ileum, isoprenaline (0.1 microM) produced a rightward displacement (approximately 3 fold) of the MCh concentration-response curve, accompanied by a significant depression of the maximum response. Increasing the isoprenaline concentration (1 microM) had no further effect on either parameter. Sodium nitroprusside (SNP, > or = 10 microM) produced a concentration-dependent depression of the MCh maximum without an effect on the EC50. 5. In the rat ileum, isoprenaline (1 microM) produced a concentration-dependent rightward displacement (approximately 2.8 fold) of the MCh concentration-response curve with depression of the MCh maximum at higher (> or = 100 microM) concentrations. SNP produced depression of the MCh maximum at a concentration of 10 microM and above. Effects on the MCh EC50 were seen only at 100 and 300 microM. 6. In guinea-pig ile

Topics: Animals; Dose-Response Relationship, Drug; Guinea Pigs; Ileum; Isoproterenol; Male; Methacholine Chloride; Muscle Contraction; Phosphodiesterase Inhibitors; Purinones; Rats; Rats, Sprague-Dawley; Vinca Alkaloids

This study was undertaken to characterize adenosine 3'5'-cyclic monophosphate (cAMP) and guanosine 3'5'-cyclic monophosphate (cGMP) phosphodiesterases (PDEs) in porcine detrusor smooth muscle and to define their possible role in tension regulation.. PDEs were isolated from porcine detrusor homogenate by Q-Sepharose anion exchange and calmodulin affinity chromatography. The effects of selective inhibitors of cAMP and cGMP PDEs were investigated on isolated PDEs and on carbachol (1 microM) precontracted detrusor strips.. Six PDE isoenzymes were isolated by Q-Sepharose anion exchange and calmodulin affinity chromatography: one calmodulin-stimulated PDE (PDE I) which hydrolyzed mainly cGMP, one cGMP-stimulated cAMP PDE (PDE II), two cAMP-specific PDE (PDE IV alpha and IV beta), and two cGMP-specific PDE (PDE V alpha and V beta). PDE I was potently inhibited in a dose-dependent fashion by papaverine, vinpocetine, and zaprinast; the PDE IVs were potently inhibited by papaverine and rolipram; and the PDE Vs were weakly inhibited by papaverine. In organ bath studies, inhibitors of PDE III (milrinone), IV (rolipram), and V (zaprinast) caused only minor relaxations at high concentrations (200 microM), whereas papaverine and vinpocetine caused relaxations of more than 50%.. Our findings support the involvement of cyclic nucleotide metabolism in the regulation of the detrusor smooth muscle tone in the pig and its regulation by PDEs. The weak action of PDE IV and V inhibitors in vitro may be explained by a possible intracellular compartmentalization of such PDEs and the low cyclic nucleotide turnover rate at the conditions used.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Calmodulin; Carbachol; Chromatography, Affinity; Chromatography, Agarose; In Vitro Techniques; Isoenzymes; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Papaverine; Phosphodiesterase Inhibitors; Purinones; Pyrrolidinones; Rolipram; Swine; Urinary Bladder; Vinca Alkaloids

The effect of selective PDE isozyme inhibitors including vinpocetine (PDE-I), CI-930 and milrinone (PDE-III), rolipram and nitraquazone (PDE-IV) and zaprinast (PDE-V) on monocyte viability and production of tumor necrosis factor (TNF alpha) and interleukin-1 beta (IL-1 beta) elicited from endotoxin-stimulated human monocytes was investigated. None of the inhibitors affected monocyte viability at 10 microM or lower concentrations. PDE-IV inhibitors and to a lesser extent, PDE-III inhibitors suppressed TNF alpha production. Only high concentrations of PDE-IV inhibitors modestly suppressed IL-1 beta. Zaprinast stimulated IL-1 beta and to a lesser extent TNF alpha production. These data show that TNF alpha and IL-1 beta production are differentially regulated, and that PDE III, PDE-IV and PDE-V isozymes are functional in endotoxin-stimulated monocytes. Clinical trials will be needed to ascertain if PDE-IV inhibitors are able to suppress TNF alpha levels in man.

Topics: Cell Survival; Cells, Cultured; Endotoxins; Humans; Interleukin-1; Isoenzymes; Milrinone; Monocytes; Phosphodiesterase Inhibitors; Purinones; Pyridones; Pyrrolidinones; Quinazolines; Rolipram; Tumor Necrosis Factor-alpha; Vinca Alkaloids

Five phosphodiesterase isozymes were separated from the supernatant of pig aortic smooth muscle homogenates, using DEAE-Toyopearl 650S chromatography in the presence of 0.1 mM Ca2+ followed by re-chromatography in the absence of Ca2+ and affinity chromatography on immobilized rolipram or cGMP. Type I (calmodulin-dependent family) preferentially hydrolysed cGMP and its activity was stimulated by calmodulin. Type II (cGMP-stimulated family), which had not yet been identified in aortic smooth muscle, hydrolysed both cGMP and cAMP. Its cAMP hydrolysis was stimulated by 10 microM cGMP. Type III (cGMP-inhibited family) and IV (cAMP-specific family) preferentially hydrolysed cAMP. The cAMP hydrolytic activity of Type III was inhibited by cGMP, but that of Type IV was not. Type V (cGMP-specific family) preferentially hydrolysed cGMP and its activity did not depend on calmodulin. The inhibition of all five phosphodiesterase isozymes by various phosphodiesterase inhibitors was investigated, and the potency and selectivity of each phosphodiesterase inhibitor discussed.

Topics: Animals; Aorta; Calmodulin; Chromatography, Affinity; Cyclic AMP; Cyclic GMP; Dipyridamole; Isoenzymes; Muscle, Smooth, Vascular; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Purinones; Swine; Vinca Alkaloids

Two subclasses of cyclic guanosine monophosphate (GMP)-specific phosphodiesterases were identified in vascular tissue from several beds. The activity of one subclass (phosphodiesterase IB) was stimulated severalfold by calmodulin and selectively inhibited by the phosphodiesterase inhibitor TCV-3B. The activity of the other subclass (phosphodiesterase IC) was not stimulated by calmodulin and was selectively inhibited by the phosphodiesterase inhibitor M&B 22,948. To assess the involvement of both subclasses in regulating cyclic GMP-dependent responses, the ability of TCV-3B and M&B 22,948 to potentiate the in vitro and in vivo responses to the endogenous guanylate cyclase stimulator atrial natriuretic factor (ANF) was evaluated. Both TCV-3B and M&B 22,948 relaxed isolated rabbit aortic and pulmonary artery rings and also potentiated the relaxant effect of ANF. In addition, both inhibitors produced small increases in urine flow and sodium excretion in anesthetized rats and potentiated the diuretic and natriuretic responses to exogenous ANF. M&B 22,948 (30 micrograms/kg/min) produced a threefold increase in the natriuretic response to simultaneously administered ANF, and TCV-3B (10 micrograms/kg/min) produced a twofold increase in the response to ANF. The results of the present experiments suggest that both the calmodulin-sensitive and calmodulin-insensitive subclasses of cyclic GMP-specific phosphodiesterase play a role in regulating the in vitro and in vivo response to ANF.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Atrial Natriuretic Factor; Blood Vessels; Dogs; Drug Synergism; Kinetics; Male; Phosphodiesterase Inhibitors; Purinones; Rabbits; Rats; Vinca Alkaloids

1. The mechanism by which M&B 22,948, MY-5445, vinpocetine and 1-methyl-3-isobutyl-8-(methylamino)xanthine (MIMAX), which have been described as selective cyclic GMP phosphodiesterase (PDE) inhibitors, relax rat aorta was investigated. 2. Three cyclic nucleotide PDEs were identified in the soluble fraction of rat aorta; a Ca2+-insensitive form exhibiting substrate selectivity for cyclic GMP (cGMP PDE), a Ca2+/calmodulin-stimulated form which also preferentially hydrolyzed cyclic GMP (Ca2+ PDE), and a form demonstrating substrate selectivity for cyclic AMP (cAMP PDE). 3. M&B 22,948 and MIMAX inhibited cGMP PDE (Ki = 0.16 microM and 0.43 microM, respectively) and Ca2+ PDE (Ki = 9.9 microM and 0.55 microM, respectively), but exhibited weak activity against cAMP PDE (Ki = 249 microM and 42 microM, respectively). MY-5445 selectivity inhibited cGMP PDE (Ki = 1.3 microM) and vinpocetine selectively inhibited Ca2+ PDE (Ki = 14 microM). 4. M&B 22,948 and MIMAX induced dose-dependent increases in the accumulation of cyclic GMP, but not cyclic AMP, in rat aorta pieces. These effects were greatly reduced by endothelial denudation and by methylene blue (5 microM) which blocks the actions of endothelium-derived relaxant factor. MY-5445 and vinpocetine had no effect on rat aorta cyclic GMP or cyclic AMP accumulation. 5. All four compounds caused dose-related relaxation of 5-hydroxytryptamine (10 microM) contracted, endothelium-intact rat aorta, the effects of M&B 22,948 and MIMAX being greatly reduced by methylene blue (5 microM). Methylene blue also caused 10 fold and 100 fold rightward shifts in the dose-response curves of MY-5445 and vinpocetine, respectively. 6. The results are consistent with the smooth muscle relaxant actions of M&B 22,948 and MIMAX, but not vinpocetine and MY-5445, being mediated through a mechanism involving inhibition of cyclic GMP hydrolysis.

Topics: 1-Methyl-3-isobutylxanthine; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Aorta, Thoracic; Cyclic AMP; Cyclic GMP; In Vitro Techniques; Kinetics; Male; Methylene Blue; Muscle Relaxants, Central; Nitric Oxide; Phthalazines; Purinones; Pyridazines; Rats; Rats, Inbred Strains; Theophylline; Vinca Alkaloids

In this study three forms of cyclic nucleotide phosphodiesterase (PDE) isolated from rabbit aorta were pharmacologically characterized, and the consequence of selective inhibition of calmodulin-stimulated PDE (CaM-PDE) and cGMP specific PDE (cG-PDE) was evaluated using PDE inhibitors. The cG-PDE (F1) was selectively inhibited by M&B 22948 (IC50 = 0.5 microM) and dipyridamole (IC50 = 7 microM). The cAMP-PDE (cA-PDE, F3) was inhibited more effectively by the cA-PDE inhibitor milrinone than by other PDE inhibitors. The cA-PDE preparation appeared to contain both cG-inhibited PDE and cG-insensitive PDE based on an additive inhibition of the activity by milrinone and SQ 65442, respective inhibitors of these enzymes. Vinpocetine, 8-methoxymethyl isobutylmethylxanthine (8-MeOMeMIX) and M&B 22948 effectively inhibited CaM-PDE (F2). Vinpocetine was a more selective inhibitor of CaM-PDE than M&B 22948 or 8-MeOMeMIX. CaM-PDEs isolated from rabbit aorta and bovine brain exhibited a similar sensitivity to these inhibitors. Seventy-two percent of the cGMP-hydrolyzing activity of this rabbit aortic CaM-PDE preparation was immunoadsorbed to monoclonal antibody (ACC-1) against CaM bound to brain CaM-PDE. Vinpocetine, 8-MeOMeMIX and M&B 22948 at concentrations (30 and 100 microM) which inhibit CaM-PDE greater than 60% increased cGMP but not cAMP levels in l-norepinephrine (NE) preincubated rabbit aortic slices. At concentrations selectively inhibiting cG-PDE, dipyridamole and M&B 22948 increased cGMP levels in untreated slices but failed to increase cGMP levels significantly in NE-treated slices. By contrast, vinpocetine failed to increase cGMP significantly in untreated slices, although it increased cGMP levels in NE or KCl preincubated slices. These data indicate that, in activated (precontracted) aorta, CaM-PDE is a major enzyme, whereas in untreated aorta cG-PDE is a predominant enzyme for the hydrolysis of cGMP. This study also shows a usefulness of selective inhibitors in identifying different forms of PDE and similar drug sensitivities and immunoadsorption of aortic and brain CaM-PDEs by a monoclonal antibody.

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Animals; Aorta; Brain; Calmodulin; Male; Milrinone; Nucleotides, Cyclic; Phosphodiesterase Inhibitors; Purinones; Pyridones; Rabbits; Vinca Alkaloids